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tryp is the least abundant amino acid in mammalian organisms, and accounts for only 11.5% of the total protein amino acid content. however, it was not until the late 1970s and 1980s that kp generated considerable interest among neuroscientists, since it was discovered that quin and kynurenine acid (kyna), (two metabolites of the kp) exhibited significant and opposing actions on neuronal cells. quin is a selective agonist at the n-methyl d-aspartate (nmda) site of the excitatory nmda (glutamate subtype) receptor, while kyna is an antagonist at both the nmda and glycine site of this ionotropic receptor (guillemin, 2012). activation of the nmda receptor has been shown to permeate cells to ca, na and k ions. increased intracellular ca influx has been shown to activate several secondary messenger signalling pathways leading to synaptic alterations. moreover, increased intracellular ca influx due to excessive nmda receptor activation can induce excitotoxicity and neuronal cell death in several neurodegenerative diseases (braidy et al., 2010). in humans, cns quin levels are increased in several neurological disorders including ad, depression, epilepsy, autism, schizophrenia, and patients are more susceptible to suicide risk (brundin et al., 2016). this has led to the hypothesis that the increase in cns levels of quin is pivotal to the pathogenesis of these disorders through its mode of action at the nmda receptor. quin may also induce toxicity to brain cells via exogenous free radical production. on the contrary, reduced levels of kyna have been reported in neurological disorders such as huntington's disease (hd), and ad. it has been suggested that the development of inflammatory mediated neuropathology is correlated to changes in the ratio of kyna to quin rather than quin levels alone. given the extent that the kp influences neuronal function, identifying the site of tryp metabolite production during cns inflammation is beneficial to gain a sound understanding of inflammatory mediated neuropathology. these metabolites may have originated either from upregulated tryp catabolism within the cns during inflammation, or may cross the blood-brain barrier after being systemically produced. it has been reported that cns levels of kp metabolites increase independently of their systemic concentration during neuroinflammation, thus suggesting that kp metabolites can be present at upregulated levels locally within the cns. quin is converted to nicotinic acid mononucleotide (namn) by the enzyme quinolinic acid phosphoribosyl transferase (qprt) in the presence of mg2 +. further transformation leading to the synthesis of the parent molecule of the pyridine nucleotide, nad, appears to be nuclear, mitochondrial, and golgi specific, by nicotinamide mononucleotide adenyl transferase (nmnat1, 2, and 3) in the presence of atp to produce desamido-nad. in the presence of glutamine desamido-nad is amidated to the parent pyridine nucleotide, nad, as the final product of the kp in addition to its de novo synthesis for tryp, nad can also be synthesised from either one of three routes: (1) nicotinic acid (na), which is then converted to nad via the three-step preiss-handler pathway; (2) the enzyme nicotinamide phosphoribosyl transferase (nampt) converts nm to nicotinamide mononucleotide (nmn) and then to nad by the action of nmnat1, 2, and 3 in the presence of atp, or (3) phosphorylation of nicotinamide riboside (nr) to nmn by nr kinases (nrks) (ratajczak et al., 2016) (figure 1a and b). in spite of the potential for nad production from vitamins, the de novo synthesis of nad from tryp appears to be more important than nad production from vitamins under normal physiological conditions. (a) the de novo synthesis of nad from tryp appears to be more important than nad production leading to the production of several neuroreactive compounds such as the neurotoxin and nmda receptor agonist, quinolinic acid, and the nmda receptor antagonist, kynurenine acid. (b) nad can also be produced by the salvage of nicotinic acid, nicotinamide and nicotinamide riboside. (c) parp activity following dna damage utilized nad as the essential substrate to repair damaged dna. hyperactivation of parp due to accumulation of free radicals can lead to cell death by nad depletion, and energy restriction. nmda: n-methyl d-aspartate; parp: poly(adp-ribose) polymerase; ros: reactive oxygen species; tryp: tryptophan. the kp is not fully expressed in all brain cells. to date, the only cells in the cns demonstrated to possess the enzyme 3-hydroxyanthranilic acid oxygenase (3-hao) which generates quin are astroglial and microglia/macrophages/dendritic cells (braidy et al., 2016). therefore, the increase in tryp catabolism observed during neuroinflammation must necessarily involve these two cell types. ifn- is the primary activating factor of macrophage/microglial/dendritic cells in the cns and elsewhere, increasing their antimicrobial activity through the modulation and upregulation of a variety of activities including, enhanced production of reactive oxygen species (ros), increased nitric oxide synthase activity, upregulation of mhc antigens, secretion of cytokines such as interleukin (il)-1, il-6, tumour necrosis factor- (tnf-), platelet activating factor (paf), macrophage chemotactic protein (mcp-1), and secretion of other biologically active proteins such as complement pathway components. indoleamine 2,3 dioxygenase (ido) is the primary enzyme of the kp, and is potently induced by ifn- in both astrocytes and inflammatory cells leading to a marked increase in kp metabolites in these cells. ifn- activated microglial/macrophages/dendritic cells will readily catabolize tryp through induction of ido, producing significant amounts of metabolic neuroreactive intermediates such as kyn, anthranilic acid (aa), 3-hydroxykynurenine (3-hk), 3-hydroxyanthranilic acid (3-haa), and quin. the kp also leads to the production of the metal chelating agent, picolinic acid (pic) (schwarcz and stone, 2016). astrocytes also readily degrade tryp in response to ifn- treatment through the induction of ido. kyn appears to be the main metabolite found in the supernatants of ifn- treated astroglial cells. however, significant but trace amounts of aa from an astrocytoma cell line, and quin, from human foetal brain cultures have also been reported (guillemin et al., 2000). it has been suggested that tryp catabolism increased in some cells to decrease tryp concentrations in the microenvironment thereby reducing the availability of this essential amino acid for microbial metabolism. one of the products of tryp catabolism, 3-haa, can function as an effective antioxidant, and potential nitric oxide synthase inhibitor. production of this metabolite may therefore serve to reduce non-specific oxidative damage at the site of neuroinflammation induced by activated mononuclear phagocytes, and may explain the increased tryp catabolism under these conditions (krause et al., 2011). however, increased secretion of kyn and quin under these circumstances has not been confirmed. moreover, ifn- induced ido tryp catabolism has been shown to increase cellular nad concentrations in an astroglioma cell line (grant et al., 1999). despite the consolidated role of the classic kp metabolites (e.g., kyn, kyna, 3-haa, and quin) during immune responses, it has recently emerged that cinnabarinic acid (ca) is crucial for immune system functions (hiramatsu et al. ca can also be produced via non-enzymatic reactions under oxidative conditions. in inflammatory cells such as neutrophils, co-expression of ido and other enzymes involved in the formation of free radicals may divert the kp away from the production of quin, and instead towards ca over pic or quin (lowe et al., 2014). poly(adp-ribose) polymerase and nad depletion: double strand dna breaks caused by excessive oxidative damage activate the parp enzyme. neuronal cells exposed to pathological concentrations of the excitotoxic neurotransmitter, glutamate show both an increase in intracellular oxidative stress and parp activity. parp is a protein modifying nucleotide polymerising enzyme found abundant in the nucleus, with approximately one molecule of enzyme per 1,000 base pairs. parp, along with dna dependent protein kinases appears to play an important role in maintaining genomic integrity (massudi et al., 2012). however, the precise physiological roles of parp are not completely understood. as a dna nick sensor, parp rapidly binds to dna strand breaks and is activated. activated parp uses up nad (and nadp+), exclusively as substrate to poly(adp-ribosylate) itself and a number of nuclear proteins that are involved in the repair of dna, releasing nicotinamide as a by-product. recent evidence suggests that polyadp-ribosylation of histones or transcription factors may also be involved in nuclear receptor signalling (morales et al., 2014) a significant decrease in intracellular nad levels has been reported in the brain and a variety of cell types as a result of dna strand breaks and parp activation following exposure to free radical generators, excitotoxins, infections, ad, and during inflammation or ageing (abeti and duchen, 2012; braidy et al., 2014). increased parp activity resulting in decreased nad has been shown to decrease atp and neurotransmitter levels in the brain, as well as cell lysis and death. inhibition of parp activity following oxidant injury has been shown to preserve nad and atp levels, prevent cell lysis, although damage to the dna was not prevented. additionally, at low levels of oxidant, parp cells survived better than parp cells, suggesting that loss of nad may be a cause of cell death. elevated levels of free radicals, pro oxidants, and excitotoxins have been reported in inflammatory brain disorders, and in most cases, dna damage has been observed. this suggests that nad depletion through parp activation may play a crucial role in cns dysfunction and pathology under these conditions (massudi et al., 2012). immune activation of macrophage/microglial/dendritic cells results in a marked increase in their generation of ros. elevated extracellular fluid levels of these free radicals have been implicated in the cause of tissue damage during inflammation in several disorders. damage to surrounding tissue, including astrocytes and neurons in the cns, may occur in response to inflammation in the cns. as well, the non-discriminating nature of these free radicals may induce damage to the activated macrophage itself. at least one study has reported an increase in parp activity in ifn- activated macrophages, suggesting that dna damage has occurred with a corresponding increase in the rate of nad catabolism. the cellular immune response potentially increases nad catabolism in cells at the site of inflammation. a mechanism by which this important nucleotide could be regenerated appears to be essential to the continued viability of cells within this environment. therefore therapeutic strategies targeted at inhibiting kp metabolism alone may not be sufficient for the resolution of symptomatic disease. however, inhibition of quin production may need to be coupled to the administration of a suitable nad precursor.

immune-mediated activation of tryptophan (tryp) catabolism via the kynurenine pathway (kp) is a consistent finding in all inflammatory disorders. several studies by our group and others have examined the neurotoxic potential of neuroreactive tryp metabolites, including quinolinic acid (quin) in neuroinflammatory neurological disorders, including alzheimer's disease (ad), multiple sclerosis, amylotropic lateral sclerosis (als), and aids related dementia complex (adc). our current work aims to determine whether there is any benefit to the affected individuals in enhancing the catabolism of tryp via the kp during an immune response. under physiological conditions, quin is metabolized to the essential pyridine nucleotide, nicotinamide adenine dinucleotide (nad+), which represents an important metabolic cofactor and electron transporter. nad+ also serves as a substrate for the dna nick sensor and putative nuclear repair enzyme, poly(adp-ribose) polymerase (parp). free radical initiated dna damage, parp activation and nad+ depletion may contribute to brain dysfunction and cell death in neuroinflammatory disease.

PMC5319230

pubmed-702

a number of studies have documented the potential benefits of probiotic bacteria which prevent and ameliorate human t-helper-2-cell- (th2-) related diseases, such as atopic dermatitis and asthma. at the helper t-cell level, a number of probiotic lactobacilli promote the production of t-helper-1-cel-l (th1-) skewing cytokines, such as interleukin (il-) 12 and interferon (ifn-), and suppress the production of ige and th2 cytokines. other studies have demonstrated that bifidobacteria suppress th2 cytokine and ige production, without significant induction of th1 cytokines. the th2-suppressive effect of probiotic bacteria is mediated by regulatory t (treg) cells linked to the induction of regulatory cytokines, including il-10 and transforming growth factor (tgf-) [4, 5]. it has been reported that macrophages stimulated with lactococcus lactis w58 produce il-10 and that tgf- is induced by lactobacillus paracasei ncc246 in mixed lymphocyte reactions. in addition, recent clinical studies have proven the prevention and curative features of probiotics in some intestinal disorders, such as inflammatory bowel diseases (ibd) including crohn's disease and ulcerative colitis. it was demonstrated that administration of bifidobacterium longum bb536 was effective in inducing remission of patients with ulcerative colitis. multiple mechanisms of action have been suggested to explain the protective effects of probiotics in intestinal inflammation. these can be broadly classified as follows: (1) suppression of growth or epithelial binding/invasion by pathogenic bacteria, (2) improved epithelial barrier function, and (3) immunoregulatory activities. as for immunoregulation, several probiotics can induce protective cytokines, including il-10 and tgf-, and suppress pro-inflammatory cytokines, such as tnf-, in the mucosa of patients with paucities and crohn's disease. besides th1, th2, and treg, t helper 17 cells (th17) have received considerable recent attention as they exhibit effector functions distinct from th1 and th2 cells [9, 10]. differentiation factors (tgf- plus il-6 or il-21), growth and stabilization factor (il-23), and transcription factors (signal transducer and activator transcription 3 (stat3), retinoic acid receptor-related orphan receptor (ror-) t and ror-) involved in the development of th17 cells have been identified. notably, th17 cells produce the pleiotropic cytokine il-17, which potently induces tissue inflammation and is associated with the pathogenesis of many human diseases, such as ibd and rheumatoid arthritis. thus, modulation of th17 cells is currently viewed as a potentially positive pharmacological outcome. although some studies have revealed that lactic acid bacteria and bifidobacteria may have potential use as anti-inflammatory agents in certain chronic inflammatory diseases, such as ibd, it is unclear whether these bacteria are able to suppress excess th17 activity. we recently reported that bifidobacterium longum infantis jcm 1222 (type strain) suppresses il-17 production in murine splenocytes in vitro and in inflamed intestinal cultures ex vivo. to identify other bacterial strains with potential th17 modulating activity, we previously performed an in vitro assay with 200 strains of streptococci and found that streptococcus thermophilus st28 exhibited potent inhibitory activity towards il-17 production. the main objective of this study was to evaluate the effects of oral administration of st28 on il-17 production by murine lamina propria lymphocytes (lpls) and on the expression of th17-related surface markers in inflamed intestines. we first evaluated and compared in vitro cytokine production patterns in th17-skewed conditions following the exposure of splenocytes to st28 and atcc 19258 (type strain). the in vivo effects of st28 were also evaluated using flow cytometry and real-time pcr in dextran-sodium-sulphate- (dss-) induced colitis mice, a human ibd model in which il-17 plays a pivotal role in pathogenesis. two strains of s. thermophilus, atcc 19258 (type strain) and st28, were obtained from the american type culture collection and glico dairy products (tokyo, japan), respectively. each strain was cultured in m17 broth (merck, whitehouse station, nj, usa) at 37c for 1765 hours. the cultured cells were washed with sterile distilled water and incubated at 100c for 50 min. following the incubation, the heat-killed bacteria were lyophilized and used for subsequent experiments. six-week-old female balb/c mice were obtained from charles river japan (kanagawa, japan), and all experimental protocols involving animals were approved by the animal care committee, graduate school of biosphere science, hiroshima university. to harvest splenocytes, mice were sacrificed by cervical dislocation, spleens were removed, and suspensions of splenocytes (1.2 10 cells) were incubated with 2 ng/ml tgf- (r&d systems, minneapolis, minn, usa) plus 20 ng/ml il-6 (r&d systems) at 37c for 72 hours in 120 l rpmi 1640 medium (life technologies, foster city, california, usa) supplemented with 10% fetal bovine serum (fbs, icn biomedicals, osaka, japan), 10 m 2-mercaptoethanol, 10 mm hepes, 5 units/ml penicillin (life technologies), and 5 g/ml streptomycin (life technologies) in a 96-well cell culture plate (thermo fisher scientific, waltham, ma, usa). heat-killed bacteria (1.2 10 cells) were then added to the cell cultures. a culture to which no tgf- plus il-6 or heat-killed bacterial cells were added was included as a control. following the incubation, cells were applied to flow cytometry (figure 1), and culture supernatants were assayed for cytokine concentrations (table 1) as described below. the splenocytes (1 10 cells/ml) were incubated at 37c for 4 hours in rpmi 1640 medium containing 0.5 g/ml phorbol-12-myristate-13-acetate (mp biomedicals, aurora, ohio, usa), 1 g/ml ionomycin (wako pure chemical industries, osaka, japan), and 3 g/ml brefeldin-a (wako pure chemical industries). then, anti-mouse cd32/16 antibody was added and incubated 4c for 30 min. for analysis of surface markers, fluorescein isothiocyanate (fitc) anti-mouse cd11c antibody (biolegend, san diego, calif, usa), allophycocyanin (apc) anti-mouse cd11b antibody (ebioscience, san diego, calif, usa), or apc anti-mouse cd4 antibody (ebioscience) was added to the cell suspension, which was then incubated in the dark at 4c for 30 min. for isotype controls, fitc-armenian hamster igg, apc rat igg2a (biolegend), or apc rat igg2b (ebioscience), the cells were permeabilized by intraprep (beckman coulter, marseille codex, france) for intracellular cytokine staining. phycoerythrin (pe) anti-mouse il-17 antibody (ebioscience) or pe rat igg2a (ebioscience) was added to the cell suspension and incubated in the dark at 4c for 30 min. following the incubation, the cells were washed twice with 5% fbs-hanks ' balanced salt solution (hbss) and suspended in 300 l of 5% fbs-hbss. fluorescence intensity was measured on a guava easycyte flow cytometry system (millipore, billerica, mass, usa). cytokine (ifn-, il-2, il-4, il-5, il-10, il-12(p70), tumor-necrosis-factor- (tnf-), and granulocyte macrophage colony-stimulating factor (gm-csf)) concentrations of the culture supernatants were determined by the microbead method using the bio-plex suspension array system (biorad laboratories, hercules, calif, usa), according to the manufacturer's instructions. briefly, culture supernatants were incubated with beads conjugated with anti-ifn-, -il-2, -il-4, -il-5, -il-10, -il-12(p70), -tnf-, and -gm-csf antibodies (biorad laboratories) followed by sandwich immunoassay using biotinylated secondary antibodies. the beads were washed three times after each incubation, and pe-streptavidin was used as a reporter. the relative fluorescence units were determined by counting 100 beads using the bio-plex system. data were evaluated with the bio-plex manager software 6.0 (biorad laboratories). separately, the il-17 concentration of culture supernatants was measured using a duoset sandwich elisa kit (r&d systems) following the manufacturer's instructions. six-week-old female balb/c mice were housed in an air-conditioned room under a 12 hours light-dark cycle and were allowed ad libitum access to tap water and a standard diet (mf; oriental yeast, tokyo, japan). acute colitis was induced in mice by adding 3.5% (w/v) dss (molecular weight, 36,00050,000; mp biomedicals) to their drinking water for 5 days. the experimental protocols (figure 1(a)) were approved by the animal care committee, graduate school of biosphere science, hiroshima university. group 1 (control group) mice were treated orally with phosphate-buffered saline (pbs) from days 1 to 3, while group 2 (control group) received drinking water containing 3.5% (w/v) dss and were orally administered pbs. groups 3 and 4 also received drinking water containing 3.5% (w/v) dss, and, 24 hours after the start of dss administration, the mice were orally administered 10 cfu/day of heat-killed st28 (group 3) or atcc 19258 (group 4) in pbs for 3 consecutive days (figure 2(a)). the severity of intestinal inflammation was assessed on day 5 by assigning a score for each of the 2 stool scores, consistency, and bleeding (figure 2(b)). the scale used for consistency was 0 (normal stool), 1 (loose stool), 2 (diarrheal stool), and 3 (watery stool), and that used for bleeding was 0 (normally colored stool), 1 (blood visibly present in the stool), 2 (adherent of blood on anus), and 3 (gross bleeding on anus). then, the total score was expressed as the sum of the scores of the 2 items (total score, 6). after the experimental period, the entire colon was removed from mice (figure 2(c)), and lamina propria from the excised colons was removed. pooled (n=4) colon tissue was suspended in hbss containing 10 mm dithiothreitol (wako pure chemical industries) and incubated at 20c for 10 min with shaking. the tissue was further treated with 2.1 mg/ml collagenase d (wako pure chemical industries) at 37c for 20 min in a shaking water bath [19, 20]. only live cells were collected from single-cell suspensions from the lpls by dead cell removal kit (miltenyi biotec, auburn, calif, usa), and used for subsequent experiments. portion of lpls (1 10 cells) were suspended in the same rpmi 1640 medium as described above and incubated at 37c for 48 hours in the presence of 1 g/ml anti-cd3 (ebioscience) and 1 g/ml anti-cd28 (ebioscience). following the incubation, cells were applied to flow cytometry, and culture supernatants were assayed for il-17 and ifn- concentrations (figure 3). for analysis of intracellular il-17 (figure 4(a)), lpls (1 10 cells/ml) were incubated at 37c for 4 hours in rpmi 1640 medium containing 0.5 g/ml phorbol-12-myristate-13-acetate, 1 g/ml ionomycin, and 3 g/ml brefeldin a. then, apc anti-mouse cd4 antibody or apc rat igg2a was added to the cell suspension. after the incubation, the cells were permeabilized by intraprep, and pe anti-mouse il-17 antibody or pe rat igg2a was added to the cell suspension. following the incubation, the cells were applied to flow cytometry. for analysis of surface markers (figure 4(b)), freshly prepared lpls were incubated with fitc anti-mouse cd11c antibody and apc anti-mouse cd86 antibody (ebioscience), or with fitc armenian hamster igg and apc rat igg2a, were added to the cell suspension and applied to flow cytometry. pooled (n=4) colon tissue from other dss colitis mice was suspended in hbss containing 5 mm edta and incubated at 37c for 20 min in a shaking water bath. the tissue was further treated with 1 mg/ml collagenase d (roche applied science, nonnenwald, germany) and 3 mg/ml dispase ii (life technologies) at 37c for 30 min in a shaking water bath. briefly, tissue was resuspended in 40% percoll (ge healthcare biosciences ab, uppsala, sweden) and overlaid on 80% percoll, after which density gradient was performed by centrifugation at 1,000 g for 20 min at 25c. lpls were subsequently collected from the interface fraction between the 40% and 80% percoll's layers [19, 20]. rna was extracted from the lpls using trizol (life technologies) following the manufacturer's instructions. reverse transcription reactions were performed with high-capacity cdna reverse transcription kit (life technologies) at 25c for 10 min and 37c for 120 min. the reaction was terminated by heating at 85c for 5 sec, followed by cooling at 4c. real-time pcr was performed using kapa sybr fast abi prism qpcr kit (kapa biosystems, woburn, mass, usa) and the primer sequences listed (/see/ in supplementary material available on line at doi: 10.1155/2011/378417). reactions were performed at 95c for 2 min, followed by 40 cycles of 95c for 5 sec and 60c for 30 sec. the dissociation stage was analyzed at 95c for 15 sec, followed by 1 cycle of 60c for 15 sec and 95c for 15 sec. the fluorescence of the sybr green dye was determined as a function of the pcr cycle number, giving the threshold cycle number at which amplification reached a significant threshold. data were analysed by the ct method and presented as fold changes in gene expression after normalization to the internal control -actin gene expression level (supplementary figure 1). briefly, bone marrow cells were collected from femurs of six-week-old female balb/c mice. bone marrow mononuclear cells (2 10 cells/ml) were cultured at 37c for 7 days in rpmi1640 medium containing mouse 200 units/ml granulocyte macrophage colony-stimulating factor (miltenyi biotec), 10% fbs, 10 m 2-mercaptoethanol, 10 mm hepes, 5 units/ml penicillin, and 5 g/ml streptomycin in a tissue culture flask. on day 7, bmdc (1 10 cells/ml) were seeded in 24-well cell culture plate and matured by 1 g/ml lipopolysaccharide (lps, sigma-aldrich, st. louis, mo, usa) for 24 hours. on day 8, heat-killed bacteria (1 10 cells/well) were added to the cell cultures. after 24-hour incubation, il-6 and il-10 concentrations of culture supernatants were measured using a duoset sandwich elisa kit (r&d systems) following the manufacturer's instructions. statistical analysis was performed using one-way anova followed by tukey's post hoc test. when live st28 was added to murine splenocytes and coincubated at 37c for 72 hours, st28 grew in rpmi 1640 medium, and it became impossible to properly examine the effect of st28 on splenocytes. thus, in subsequent experiments, st28 and atcc 19258 were used after heat killing. prior to the experiment, we first purified t cells from splenocytes by magnetic bead-based cell separation (macs), and stimulated the macs-purified t cells with tgf- plus il-6. however, il-17 production was not detectable (data not shown), which suggested that il-17 production from th17 cells in splenocytes required the coexistence of non-t cells. therefore, we alternatively stimulated whole splenocytes with tgf- plus il-6 for further analyses, after which the stimulated splenocytes were applied to flow cytometry in order to analyze cd4, cd11b, and cd11c cells separately. as a result, most of the il-17 cells were cd4 t cells, which indicated that the major source of il-17 was th17 cells. more importantly, it was confirmed that st28 decreased the percentage of cd4 il-17 cells (figure 1). as shown in table 1, st28 significantly (p<0.01, () versus st28) repressed il-17 production (78% reduction). on the other hand, atcc 19258 failed to repress its production. based on these results, st28 was judged to be a suitable candidate strain for the treatment of th17-mediated diseases, and its suppressive effects were further examined in vivo using dss-induced colitis mice. in regard to other cytokines than il-17, the productions of all cytokines tested were changed by the stimulation with tgf- plus il-6. among them, ifn- and tnf- productions in the st28 group were significantly higher than those in the atcc 19258 group, and il-2 and il-4 productions were significantly lower. on the other hand, there were not significantly differences in il-10 (anti-inflammatory cytokine) and il-12 (proinflammatory cytokines) productions between the two groups. acute colitis was induced in mice by exposure to 3.5% dss for 5 days, which resulted in an increase in colon inflammation score compared to the control group (score, 2.5 1.5 versus 0.5 0.3) (figure 2). however, oral treatment with st28 ameliorated the inflammation (score, 1.0 0.4). atcc 19258 exerted the same ameliorative effect on the intestinal inflammation, but the effect was weaker (figures 2(b) and 2(c)). next, lpls from the mice was applied to ex vivo culture experiment (figure 3). although differences in ifn- concentrations in lpls culture were not prominent among four groups, lpls from dss colitis mice produced substantial level of il-17. however, st28 markedly suppressed il-17 production from lpls culture (86% suppression), while atcc 19258 failed to suppress il-17 production. from the analyses of mrna expression in lpls, it was revealed that the expression of both il-17 and ror-t, the master regulator of th17, in lpls from the dss-administered group was drastically upregulated, compared to control mice (supplementary figure 1). notably, st28 markedly inhibited both expressions, which indicated that st28 suppressed the differentiation of nave t cells towards th17 in lpls. cd11c and cd86 were also found to be downregulated by the oral administration of st28. it was also confirmed by flow cytometry that cd4il17 cells (th17 cells) disappeared in lpls by the oral administration of both st28 and atcc 19258 (figure 4(a)). on the other hand, the effects of dss and those of the administration of st28 and atcc 19258 on cd11c cd86 cells, inflammatory dendritic cells (dc), were unclear, although atcc 19258 seemed to decrease the percentage of cd11c cd86 cells. to clarify this point the effect of st28 on lps-matured bmdc was evaluated (figure 5). il-10 production was not significantly decreased by the addition of st28 and atcc 19258. however, both st28 and atcc 19258 significantly suppressed il-6 production. accordingly, the percentage of cd86cells, which is a hallmark of maturation, was also decreased by st28 and atcc 19258. therefore, it is highly probable that st28 suppressed th17 cells via a dc-dependent mechanism. in medical therapy for ibd, the modulation of immune responses and the regulation of intestinal inflammation is essential. although the pathogenesis of ibd is complex, involving environmental, microbial, genetic, and immunological factors, several recent studies have shown that th17 cells play a significant role in ibd pathology [23, 24]. here, we demonstrated that s. thermophilus st28 repressed il-17 production in mouse splenocytes under th17-skewed conditions in vitro (table 1). moreover, oral treatment of dss-induced colitis mice with st28 suppressed inflammatory th17 cells in lpls. although s. thermophilus is not commonly considered a probiotic bacterium, the findings of the present study imply that this species would be useful in the treatment of ibd by suppressing exaggerated th17 activity in inflamed intestines. th1/th2 balance can influence the direction and outcomes of immune responses, due to the mutually antagonistic nature of th1 and th2 responses. in this regard, the modulating activity of probiotic and commensal bacteria on th1/th2 balance is commonly evaluated [3, 4]. here, as ifn-/il-4 production ratio in st28 group was high even in th17-skewed conditions (table 1), it is expected that st28 would suppress not only th17, but also th2 cells. this response would also affect ige production in allergic conditions, because th2 cells drive ige class switching by allergen-stimulated b cells. suppressing both th2 and th17 cells is desirable for the repression of ige-mediated reaction, because these cell types are closely related with each other in allergic patients. to confirm that st28 also suppressed th17 cells in vivo, acute colitis was induced by dss treatment of mice. dss induces acute inflammation and recruitment of immune cells, whose subsequent activation directly causes epithelial cell. in dss-induced colitis mice, which serve as a useful model of human ibd, st28 administration improved inflammation score (figure 1), repressed il-17 production (figure 3), and decreased th17 cells (figure 4) in lpls isolated from inflamed intestines. moreover, st28 decreased the mrna expressions of both rort and il-17 in lpls (supplementary figure 1). considering all of the data, although several lines of evidence indicate probiotic bacteria have beneficial effects on dss colitis [7, 14, 26], the mechanisms by which bacteria mitigate intestinal inflammation have not been fully clarified. we propose that st28 ameliorates dss-induced colitis in mice by suppression of inflammatory th17 cells. it should be noted that even heat-killed bacteria could suppress il-17 production in this study. therefore, some heat-resistant components of st28 might exert th17 supprressive activity. it is generally accepted that bacterial components are recognized by members of the pattern recognition receptor family, such as toll-like receptors (tlrs). in regard to the epithelial barrier, some studies have shown that the tlr2 ligand pcsk ameliorated tnf--induced intestinal barrier impairment in the human epithelial caco-2 cells [28, 29] and dss colitis mice. it is likely that the recognition of heat-resistant bacterial components by these kinds of receptors is also involved in the suppression of th17 cells. it has been recently suggested that cd86 plays a critical role in the initiation of t-cell responses including th17 cells. for example, gene silencing of cd40, cd80, and cd86 protected collagen-induced arthritis, one of the th17-related inflammation. in the effector cd4 t-cell responses inducing antigen-induced arthritis, blockade of cd86 significantly suppressed il-17 production in splenocytes, and cd86 enhanced disease severity by upregulating il-17 production. also, high cell surface expression of cd86 was observed in patients with ibd and in mice with cd4 t-cell-induced colitis. these findings lead us to examine the effect of st28 on the expression of cd86. the mrna expressions of cd86 and cd11c were drastically upregulated in lpls from dss colitis mice, but they were downregulated by the oral administration of st28 (supplementary figure 1). st28 also significantly decreased the percentage of cd86cells in matured bmdc (figure 5). collectively, it is highly probable that st28 ameliorated dss induced colitis by suppressing cd86 dc, which needs to be clarified in detail. it has been described that il-17 is involved in the pathogenesis of dss-induced colitis in mice, and il-17 receptor signaling plays a critical role in the development of trinitrobenzenesulfonic acid-induced acute colitis in mice, yet another report has suggested that il-17 might offer an inhibitory role in the development of dss-induced colitis. o'connor et al. also demonstrated the data on a protective function for il-17 in t-cell-mediated intestinal inflammation. for example, buonocore et al. have recently suggested that a novel innate lymphoid cell population accumulates in the inflamed colon and induces il-17 and ifn-. further investigations on th17/th1 in the intestinal inflammation are required to clarify the beneficial functions of st28 on th17-mediated diseases. in conclusion, we have demonstrated that st28 ameliorates intestinal inflammation in dss-induced colitis mice at least partially through suppression of inflammatory th17 cells. the use of probiotic bacteria in ibd therapy has been investigated in a number of clinical studies. although the efficacy of this treatment approach was demonstrated for ulcerative colitis, the results for crohn's disease are not yet clear owing to conflicting results and a paucity of trials. in any case, the majority of the demonstrated curative and palliative effects of probiotic bacteria appear to be mediated by modulation of the intestinal immune system. clinical approaches using probiotics are appealing due to a lack of toxicity and patient desire for the use of natural physiological approaches to treating disease. since s. thermophilus has a long history of being safely consumed in yogurt, its further application to the treatment and/or prevention of th17-mediated diseases such as ibd

the effects of streptococcus thermophilus st28 on cytokine production by murine splenocytes stimulated with transforming growth factor- plus interleukin- (il-) 6 were evaluated. the addition of st28 significantly repressed il-17 production compared to atcc 19258 (type strain). st28 also decreased the number of th17 cells in the stimulated splenocytes. the anti-inflammatory effects of st28 administration were evaluated in mice with colitis induced by dextran sodium sulphate (dss). oral treatment of mice with st28 ameliorated the intestinal lesions by dss. upon dss treatment, il-17 production in lamina propria lymphocytes (lpls) was induced, but st28 significantly decreased its production. st28 also decreased the percentage of th17 cells in lpl from dss-induced colitis. the present results imply that st28 suppresses the th17 response in inflamed intestines and would be useful in the treatment of th17-mediated diseases, such as inflammatory bowel disease.

PMC3196311

pubmed-703

epidermolysis bullosa (eb) is a rare genetic disorder characterized by abnormal fragility of skin and mucosal surface. the separation of skin layers occurs after application of friction or shearing forces and results in intradermal fluid accumulation and bullae formation. in addition to considerations associated with positioning, monitoring, infection, and prevention of skin and mucosal trauma, anesthetic management of eb is uniquely challenging because of the effects on the airway. this case report describes the successful anesthetic management of a patient with eb presenting for syndactyly release. a 6-year-old male child, weighing 14 kg; a known case of eb since birth presented with congenital left hand syndactyly [figure 1]. significant preoperative findings were generalized scars, bed sores, pustules, and joint contractures. airway assessment revealed mallampatti class iii, microstomia, loose teeth, and poor oral hygiene with thyromental distance 3 cm. left hand shows syndactyly and right shows iv canula secured with nonadhesive technique airway assessment shows anticipated difficult airway oral antibiotics were administered preoperatively. intravenous (iv) line 22-g secured with use of emla and fixed with vaseline gauze. premedication with iv fentanyl 20 g, midazolam 0.4 mg, and dexamethasone 2 mg was given. were given with iv ranitidine 1 mg/kg and cefotaxime 50 mg/kg, respectively. intraoperative heart rate, rhythm, and oxygen saturation were monitored with lubricated clip on pulse oximeter. noninvasive blood pressure (nibp) and electrocardiogram monitoring were avoided [figure 3]. patient was positioned supine and pressure points were padded with cotton. due to anticipated difficult airway, a difficult airway cart was prepared with various size masks, laryngoscope, endotracheal tubes (ets), ventilating bougie, and stylet. though supraglotic airway devices were inappropriate in this patient they were kept ready for an emergency airway situation. minimal monitoring was used intraoperatively preoxygenation was followed by induction with iv propofol 20 mg along with oxygen and sevoflurane under gentle mask holding with vaseline gauze. after confirming ventilation, with use of lubricated macintosh laryngoscope trachea was intubated with uncuffed et no. 5 and tube secured with nonadhesive lubricated bandage [figure 4]. et secure with nonadhesive technique anesthesia was maintained with oxygen, nitrous oxide, and sevoflurane with manual ventilation using jackson rees circuit. as rectal suppositories, subcutaneous and intradermal routes are generally not recommended; postoperative analgesia was provided with iv diclofenac sodium 25 mg 8 hourly. regional block was avoided due to joint contracture and scars, which made location of anatomical landmarks difficult. neuromuscular blockade was antagonized at the end of the procedure, and trachea extubated when adequate signs of spontaneous recovery were evident. postoperatively, oral examination revealed small intra-oral blister formation not requiring active management. eb encompasses an array of autosomal dominant and recessive conditions that may have either localized or generalized dermatological manifestations. the loss or absence of normal intracellular bridges is due to a collagen abnormality, which makes patient susceptible for blister formation by friction/shearing forces and subsequent scarring. equipment and techniques routinely used in the induction and maintenance of general anesthesia can be the source of serious postoperative complications. anesthesia is frequently required for multiple surgeries like daily dressings, dental procedure, esophageal dilatation, gastrostomy, contracture release, and syndactyly release. malnutrition, anemia, and decreased immunity are present in patients due to decrease oral intake secondary to oropharyngeal and esophageal lesions. malnutrition can leads to hypoprotienemia, anemia and electrolyte imbalance which may affect pharmacokinetic effects of anesthetic agents. infection is common as patients often have both poor immunity and long-term corticosteroid treatment. antacid prophylaxis is required due to history of reflux, regurgitation, or esophageal stricture. patients with eb are prone for ophthalmic complications like corneal erosion, conjunctival symblepharon, and ectropion. dental problems like cleaning difficulty, poor eating patterns, enamel dysplasia, carious, and loose teeth are frequently associated. iv or intra-arterial access should be secured with bandage, cotton wrap, or suture. all persons involved in handling these children must be aware of the extreme vulnerability of skin. during transport or mobilization of the patient, the most important task is to maintain the integrity of the skin and avoid friction and trauma. direct pressure to the skin is not as damaging as frictional or shearing forces, so nibp and tourniquet can be used with pressure and duration limitations. as our case was of a short duration with no expected major blood loss and hemodynamic shifts, use of nibp and tourniquet therefore, clinical use of nibp and tourniquet in such patients depends on its merits and demerits. microstomia, neck contracture, oropharyngeal lesion, ankyloglossia (decrease mobility of the tongue), thickened epiglottis, and possible tracheal stenosis make maintenance of a patent airway challenging. fiberoptic intubation is less traumatic to the mucosa than direct laryngoscopy and should be the first choice in eb patients with a difficult airway. laryngeal, tracheal involvement in eb is rare because that tissue is pseudostratified, columnar, ciliated epithelium and whereas oropharyngeal, esophageal mucosa is stratified squamous. supraglottic airway increases bullae formation but can be used in difficult airway scenario with appropriate care. after application of moisturizing ophthalmic gel, eyes should be covered with moistened gauze to protect from mechanical trauma. regional anesthesia should be considered whenever possible but contractures, scars, and infections are the issues associated with regional anesthesia. perioperative blister should be treated with liquid paraffin, silver sulphadiazine, or steroids. in ambulatory careful monitoring, transport and positioning was instituted in the present case to avoid undue skin trauma. atraumatic ventilation and intubation was possible with use of vaseline gauge and gentle mask holding. minimal mandatory monitoring and no touch principle remains the key to successful anesthesia management of eb cases. patients with eb present a unique challenge for all anesthesia-care providers. with maximal skin and mucous membrane protection, anesthesia in children with eb can be conducted with few sequelae. hence, meticulous execution of preoperative planning is indispensable for ensuring a favorable intraoperative and postoperative course.

epidermolysis bullosa (eb) is a rare genetic mechanobullous disorder, with excessive fragility of the skin and mucous membranes. avoiding mechanical injury to the skin and mucous membranes is essential in the anesthetic management. shearing forces applied to the skin result in bullae formation, while compressive forces to the skin are tolerated. the challenge is to use monitoring technology without damaging the epithelial surface. difficult airway, positioning issues, nutritional deficiencies, poor immunity, and carcinogenic potential add to the comorbidities. we managed a child with eb undergoing syndactyly release. ensuring maximal skin and mucous membrane protection, anesthesia in children with eb can be conducted with few sequelae.

PMC3788243

pubmed-704

with the number of sequenced genomes at various stages of completion being in the tens of thousands and the number of genomic features (genes, rnas, etc.) identified across these genomes in the millions, the need for accurate and consistent genomic annotation is paramount. the gene ontology (go) was created in 1998 by researchers at flybase, the saccharomyces genome database (sgd), and the mouse genome database as a collaborative effort to address the need for consistent descriptions of gene products across different databases. this group has since grown to include 26 consortium members and associates and the go is a key member of the open biological and biomedical ontologies (obo) community. in this context ontologies provide a controlled vocabulary for representing and communicating knowledge about a topic and a set of relationships that hold among the terms of the vocabulary. the topic for the go is genes and gene products such as transcripts, proteins, or rnas that are described in three related subontologies (also called namespaces), biological process, the broad biological system in which a gene product is involved; molecular function, the specific role a gene product has or potentially has within a biological process; and cellular component, the location in a cell where the gene product performs its molecular function. each ontology is composed of nodes (terms) and edges (relationships) and is structured as a directed acyclic graph (dag). as one moves down the nodes of the graph from a parent to a child, terms become increasingly more specific. a dag allows for child terms to have more than one parent and this enables complex relationships to exist between them. the go currently contains approximately 38 thousand terms that have been used by consortium members to annotate almost 25 million gene products. each annotation is accompanied by an evidence code to denote the method by which the annotation was made. computationally sourced annotations, such as the evidence code iea (inferred from electronic annotation), are regarded as less confident annotations than those that are assigned on the strength of experimental evidence. all data is publicly available for download and the go team provides a number of entry points to query the data including an application programming interface (api) to permit custom queries to either a go database or an ontology flat file. the results of gene expression microarrays can be enriched with terms pertaining to biological process, which helps to discover if entire pathways are upregulated as opposed to simply individual genes. subcellular location can be predicted for an unknown gene sequence by performing a similarity search such as blast and inferring from the cellular component go terms of top hits. one such limitation is that, by design, there is no link between the three subontologies. this means one can not directly answer questions such as what are all the biological processes occurring in the cellular component mungall exploited the high degree of regularity in phrase structure of obo term definitions and converted tokenized definitions from the go, biochemical ontology, and the cell ontology into a language (obol) that can be parsed computationally with a reasoner. this was used, amongst other things, to find missing relationships within the go and between ontologies. similarly, wroe et al. converted the go to a daml+oil framework to enable reasoners to parse the ontology. bada and hunter used regular expressions to find patterns in go definitions and create an assertional model to integrate the three go subontologies, the chemical entities of biological interest ontology (chebi), and the cell type ontology (cto). interrogated the go, compiling groups of terms cooccurring in annotations of gene products, in order to suggest potential biologically relevant terms to annotators. describe functionality within the quickgo tool from the go annotation (goa) group that provides information on how many times a query term cooccurs with other go terms specifically across the uniprot knowledgebase (uniprotkb) database. this allows researchers to input a query term such as nucleus and view all terms that are frequently annotated alongside this term. however, there are limitations to this method, such as not being able to explore custom subsets of data within the uniprotkb database and a hard limit on the number of terms returned to the user. this paper presents golink, an alternative tool for finding go terms from across the three go namespaces that cooccur with a given query term. golink differs from existing tools by using the go api to mine the full complement of the go database, using methods that take into account the namespace of a query term when assessing cooccurrence. the key advantages of using this particular method and source data are described along with other practical uses for the tool. these include predicting the most likely biological process for genes by using just their assigned molecular function terms, where golink achieves levels of specificity, sensitivity, and accuracy above 80%. golink is written in perl and requires the go database api (go-db-perl) module (go::apphandle), available from the comprehensive perl archive network (cpan). golink requires access to a go database (ideally locally installed). for the analyses in this paper, the go database v201212 was downloaded from http://www.geneontology.org/go.downloads.database.shtml and installed using the instructions at http://archive.geneontology.org/latest-full/readme. all analyses were run under 64-bit linux on a single 2.3 ghz core of a multicore amd server; however, golink can be run on both windows and macos platforms provided perl, the required cpan modules, and a go database are installed. golink is made freely available from the project website (http://bioinformatics.childhealthresearch.org.au/software/golink/) under the gnu general public license (gpl). all usage instructions are detailed in the software manual also available from the project website. a user provides golink with a go term such as go:0005634 representing nucleus, which is referred to as the query term, along with any desired filters (evidence codes or annotation source databases see below). golink will then retrieve all genes that have been annotated with the query term or any of its child terms (figure 1(a)). each gene is assessed in turn (figure 1(b)) and its full complement of go annotation is assigned to one or more terms list (figure 1(c)) based on whether or not the annotation fulfills the list's specific criteria as follows.all_list: stores all the gene's annotated go terms.query_list: stores all the gene's annotated go terms only if the query term is one of those terms. this method is similar to quickgo.parchild_list: stores all the gene's annotated go terms only if all terms within the same namespace as the query term are either one of the query term's parents or children. query_list: stores all the gene's annotated go terms only if the query term is one of those terms. parchild_list: stores all the gene's annotated go terms only if all terms within the same namespace as the query term are either one of the query term's parents or children. in an iterative process, each list is gradually formed of cooccurring terms from all namespaces that fit the criterion above and that can subsequently be confidently associated with the initial query term. once the lists are compiled, scores are calculated for terms in each list to allow them to be ranked. pr empirical probability ratio: (1)(t/q)(c/a). s%empirical probability similarity ratio: (2)(t(q+ct))100, where, given term x in the context of a single list, q=the number of gene products fulfilling the criteria of the list; t=the number of gene products fulfilling the criteria of the list that contain term x; c=the number of gene products annotated with term x in the entire go database, given any filters; and a=the number of gene products in the entire go database, given any filters. the empirical probability ratio is a measure of how similar t is with c. while being a useful score this penalises interesting candidates that have high c but a slightly lower value of t. this discrepancy is addressed in the empirical probability similarity ratio, which not only accounts for the relationship between t and c but gives higher scores where t and q are also similar in value. users can explore subsets of go annotation data by optionally applying a choice of three filters. the namespace filter controls the nature of the terms output to the final terms list. unfiltered, golink will return terms from all namespaces that cooccur with the query term. however, users can request that only terms from a limited set of the three namespaces are returned. when a go term is assigned to a gene product by a go consortium member (e.g., flybase, sgd) an evidence code golink provides a database filter to target annotations made by a particular consortium member and an evidence code filter to allow fine-grained control on the level of quality of terms returned. for convenience a can be specified to not include terms from a particular database or evidence code. golink will only consider terms that are not attributed with the evidence code iea (inferred from electronic annotation). given that the iea evidence code suggests a less confident term assignment, using! golink terms lists can be ordered as required; however, it is recommended that results are sorted by descending empirical probability similarity ratio (s%) and then descending empirical probability ratio (pr). this serves to place the terms most strongly linked to the query term at the top of the list and also mirrors the ordering method used by quickgo. golink outputs three terms lists, each with related yet contrasting results due to the differing criteria used to compile each list. when reviewing these lists it is important to take the initial query term used for the analysis into account. if the query term originates from towards the top of go dag, it will be more likely to have a large number of child terms, which inevitably results in many more terms being returned to all the golink terms lists than for more specific query terms at lower levels of the go graph. in this case conversely, lower level terms are more likely to not return any results to the more stringent golink terms lists due to there being fewer annotations where specific criteria are met. here golink terms lists were generated using the biological process query term regulation of gene expression comparisons of golink terms lists were performed using a three-way venn diagram of all terms in each list using venny. a perl script was created that, given an initial go term, pulls all genes annotated with that term from the go database along with their other annotated go terms. using this script a positive and a negative list of genes and their go annotation (go:0010468) for the positive list and a combination of lipid metabolic process (go:0006629) and cell motility (go:0048870) for the negative list. genes were only included in each list if they had at least one molecular function term as part of their annotation. golink terms lists were then generated using the biological process query term regulation of gene expression (go:0010468), opting to only output molecular function terms. in all cases the evidence code filter! a gene in the positive or negative list was deemed as having a role in the regulation of gene expression if any of its molecular function terms matched either the first 10, 20, 30, 40, or 50 (separately) molecular function terms in each of the three golink terms lists. those genes in the negative list providing positive results were manually checked in uniprotkb to ensure that they did not also have a role in the regulation of gene expression as well as either lipid metabolic process or cell motility. a statistical assessment of performance was calculated as follows: (3)sensitivity=tp(tp+fn),specificity=tn(fp+tn),positive predictive value (ppv)=tp(tp+fp),negative predictive value (npv)=tn(tn+fn),accuracy=(tp+tn)(tp+tn+fp+fn), where tp=number of genes correctly predicted to be involved with the regulation of gene expression; tn=number of genes correctly predicted to be not involved with the regulation of gene expression; fp=number of genes incorrectly predicted to be involved with the regulation of gene expression; and fn=number of genes incorrectly predicted to not be involved with the regulation of gene expression. in this context, sensitivity refers to how likely golink predicts a role in the regulation of gene expression for those genes in the positive list (tp), specificity measures how well golink predicts no role in the regulation of gene expression for genes in the negative list (tn), ppv assesses the chance that a gene is involved with regulation of gene expression given a prediction that it is involved, and npv calculates the chance that a gene is not involved with regulation of gene expression given a prediction that it is not involved in that process. mixed bar and line graphs displaying the results of this assessment were produced using r. (saccharomyces genome database), pombase (main resource for the fission yeast schizosaccharomyces pombe), mgi (mouse genome informatics), zfin (the zebrafish model organism database), and uniprotkb, separate golink terms lists were generated using the biological process query term regulation of gene expression using data from each respective all_list, pairwise comparisons were made between terms from uniprotkb and each of the other source databases using venny. a quickgo (released on friday, 04 january 2013) terms list was exported by using the query term regulation of gene expression and selecting from the! iea an equivalent golink dataset was generated by using the query term regulation of gene expression and applying the! the position at which each of the 100 quickgo terms fell in the sorted golink list was then obtained. terms list was used to compare against the quickgo terms list as the term assignment criteria for both lists are the most comparable. the exported quickgo terms list was also subjected to a separate similar statistical assessment to that described above, whereby a gene in the positive or negative list was deemed as having a role in the regulation of gene expression if any of its molecular function terms matched any of the molecular function terms in the quickgo terms list. tables 1(a)1(c) show the top 10 golink cooccurring terms and their scores in each terms list generated using the query term regulation of gene expression (go:0010468) and applying the evidence code filter! the complete lists can be found in supplementary file 1 (see supplementary material available online at http://dx.doi.org/10.1155/2013/594528). overall the all_list, query_list and parchild_list, returned 6513, 419, and 322 terms, respectively. as expected there are a large number of unique terms in the all_list given the comparatively large number of terms returned and there are no unique terms found within either the query_list or parchild_list. the 360 terms overlapping the all_list and query_list in the venn diagram (figure 2) represent terms from gene products that are annotated with the query term but have other terms in the biological process namespace that are not either a parent or a child of the query. the 263 terms (figure 2) overlapping the all_list and parchild_list represent terms from gene products that were not directly annotated with the query term but with one of its child terms. overall, there were 59 consensus terms across the three lists, which are listed in supplementary table 1. a useful application of the golink term lists is, for example, to use a gene's existing molecular function go terms to establish whether it has a role in a particular biological process, such as the regulation of gene expression. if a gene has been annotated with only molecular function terms, one can compare these terms to a golink terms list for a biological process query term of interest. if the molecular function terms are found in the golink terms list, one can infer that the gene's function may be associated with the initial biological process query term. figure 3(a) and supplementary file 2 show the sensitivity, specificity, ppv, npv, and accuracy of such an assessment matching only the molecular function terms from the three golink terms lists with a positive and negative list of genes and their annotated go terms. they also show the proportion of genes found in each of the positive and negative lists by the three golink terms lists. the positive list contained 41 genes (1163 terms, 154 mf terms) annotated as having a role in the regulation of gene expression, while the negative list contained 154 individual genes (2723 terms, 503 molecular function terms). the genes contained in the positive and negative lists and their associated go annotation can be found in supplementary file 2. the top 50 molecular function terms from each of the three golink terms lists that were used in the matching process are also shown in supplementary file 2 and a venn diagram showing their overlap can be found in supplementary figure 1. it should be noted that the rationale behind using only up to the first 50 molecular function terms from the golink terms lists in the matching process was simply that each list contained at least 50 terms. overall the query_list outperformed the other two golink terms lists in predicting genes with a role in the regulation of gene expression using only their assigned molecular function terms. for this list, sensitivity, specificity and accuracy all averaged between ~80% and 90% with the highest values occurring with comparisons using the top 30 golink molecular function terms. using these top 30 terms in the query_list yielded 33/41 true positives and only 12/154 false positives giving a ppv of 73% and a npv of 94%. the all_list and parchild_list showed increasingly higher sensitivity as more terms were added in to the comparison, but this was at the expense of specificity, which remained above 50% but much lower than that of the query_list. eleven of the 154 negative genes were deemed to be actually true positives (supplementary table 2) with a role in the regulation of gene expression as well as either cell motility or lipid metabolic process despite all but one not being directly annotated with these genes (such as sterol regulatory element-binding proteins 1 and 2 and coup transcription factor 2) were removed from their respective negative lists and the statistics recalculated, which served to increase values marginally across the board (table (a) in supplementary file 3). most of the false positive genes predicted by the golink terms lists were annotated with the go term protein binding (go:0005515). this fairly generic term has relevance for both the negative and positive lists so users of the software are cautioned to consider this when performing analyses of this nature and may opt to remove terms from the golink lists where appropriate. indeed removing both the false positives from the negative list and removing the go term protein binding from the golink terms lists have a substantial impact on the statistical assessment across the board as can be seen in figure 3(b). figure 4 shows the overlap of golink terms obtained using four species specific annotation source databases (mgi, pombase, zfin, and sgd) in comparison with terms obtained from the more broad uniprotkb database, using the same query term (regulation of gene expression) and evidence code filter (! iea). for each of the species specific databases figure 4 shows that, as expected, the majority of terms (between 53% and 87%) in their respective lists were also in the uniprotkb list. this group represents those terms that cooccur with the query term in annotation within both the species specific database and in annotation by uniprotkb for other species. the remaining terms are either unique to the species specific database or to uniprotkb. in the context of the species specific database these unique terms are either highly relevant to the species and only ever likely to have been used in annotation by the curators of the species specific database (e.g., ascospore formation within pombase and sgd, fin development within zfin) or represent terms that only cooccur with the query term in this database compared to uniprotkb. conversely, those terms unique to uniprotkb represent terms highly irrelevant to the species specific database (e.g., viral reproduction in all cases) or similarly only cooccur with the query term within uniprotkb assigned annotation. tables (a)(c) in supplementary file 5 show the full golink query_list and quickgo terms lists used in the comparison (( b) and (c)) and a combined summary detailing the positions of the top 100 quickgo terms as found in the golink query_list (a). overall the vast majority (72%) of quickgo terms are confined to the top 200 golink terms and figure 5 shows that there is a 64% overlap between the top 100 quickgo and golink terms. while all 100 quickgo terms are represented somewhere in the golink query list, table (a) in supplementary file 5 shows that there are distinct differences in the ordering of the results within the more significant first 100 terms. this can be explained by the way protein identifiers are mapped within the database underlying quickgo. the database used by golink is maintained by the go consortium. within this database, the database for quickgo is maintained by the goa team and provides high quality annotation for all proteins within uniprot. here, single protein identifiers are first mapped to uniprot accessions, and in many cases one identifier can map to several uniprot accessions (since there is likely to be several matches in trembl, the nonreviewed section of uniprot). in these cases the same or similar annotation is assigned to each of the uniprot accessions. while appropriate in the context of the goa project this leads to more redundancy in annotation in the database used by quickgo than within that used by golink. consequently, when considering cooccurring terms in quickgo, terms from those genes that have multiple entries in trembl will appear to cooccur more often than is the case within the go database, which may not be desired. for example, in table (a) in supplementary file 5, quickgo reports that the term forebrain development, which appears second in the quickgo terms list, has been used to annotate 308 proteins within the goa database. this term appears tenth in the golink list, where golink reports its assignment to 200 gene products within the go database. furthermore quickgo and golink report that this term cooccurs in annotation with the query term on 56 and 26 occasions respectively. on closer examination, multiple uniprot accession numbers exist that map to single mgi identifiers annotated with the term forebrain development and this has the effect of inflating the number of cooccurrences with the query term, thus influencing the s% calculation and positioning it higher in the quickgo terms list. a more extreme example can be seen for the term spermatid differentiation which appears at position 11 in the quickgo list but position 531 in the golink list. in addition, the result of the pr calculation is altered as a consequence of the increased number of proteins within the goa database. as both quickgo and golink order their results by s% and then by pr and quickgo only provides the first 100 hits, this explains why equivalent search parameters on these similar but different source databases lead to similar but differing results for golink compared to quickgo. in order to test quickgo's ability to predict a gene's biological process from its molecular function a similar matching process to that undertaken for golink was performed with the top 100 cooccurring terms from quickgo. only 9 of the 41 positive genes were found using these terms, which consequently gave lower comparative performance scores to golink (data not shown), however it should be noted that as seen in table (c) in supplementary file 5 this list of terms only contained 4 molecular function terms to use in the comparison process. the gene ontology is a dynamic, growing resource for the annotation of genes and gene products. it is organized into three subontologies describing the cellular location of action of a gene or gene product, the main process it is involved in, and its role in this process. in order for the three subontologies to remain orthogonal, there are no designed links between them. however, this prevents legitimate attempts to answer queries involving data from more than one of the subontologies. this paper discusses a new tool, golink, which uses the go perl api to explore the go database and implement three increasingly stringent methods to compile lists of go terms cooccurring in gene product annotations with a provided query term. when given an initial query term, the three methods store either all terms cooccurring with the query term or any of its child terms (all_list), only those terms cooccurring with query term (query_list) or only those terms cooccurring in annotations with the query term or its child terms where other annotated terms in the same namespace (cellular component, molecular function, and biological process) as the query term are limited to the query term and any of its parent or child terms (parchild_list). however, the key difference between the remaining golink lists and quickgo is that the latter uses a pool of terms from genes directly annotated with the query term, whereas the golink all_list and parchild_list use a pool derived from genes annotated with the query term and/or its child terms. the main reason golink uses this extended pool is to not penalize the partonomic/taxonomic relationship between a term and its child terms. furthermore, this allows golink to apply further logical stringency in the parchild_list to specifically exclude occurrences where the query term is annotated together with terms in the same namespace that are neither a parent nor a child of the query term. for example, if a gene performs a particular function and operates both in the cytoplasm and the nucleus it may well be annotated with 2 cellular component terms (nucleus and cytoplasm) and at least 1 molecular function term. in this case the molecular function term can not be exclusively linked with either of the cellular component terms as the molecular function term may only be relevant for the gene's function in the cytoplasm. if this distinction is required then the parchild_list will take this into account, whereas this functionality is not possible using quickgo. the other main advantages of golink over quickgo lie in the distinct differences between the underlying databases of the two software and the availability of flexible methods to filter the terms assessed and those returned. the process of identifier mapping in the goa database that provides the annotation used by quickgo leads to an increased redundancy of annotation across this database as single gene product identifiers commonly map to multiple uniprot accessions. this results in a potentially inflated cooccurrence of go terms as demonstrated in this paper. quickgo provides two results sets; one unfiltered list of cooccurring terms and one with a! filter applied to discount associations from purely computationally assigned terms. for both, only the first 100 cooccurring terms are returned. golink is much more flexible in that not only does it produce multiple terms lists of varying stringency, but it also provides fine-grained customizable filtering. the database filter allows a user to identify cooccurring terms within the context of a particular species that could be diluted if searching within a broader context such as the full uniprot database. the evidence code filter gives precise control over the quality of annotation considered during an analysis using the wide variety of evidence codes provided within the go database. finally, applying the golink namespace filter will only return results from a defined set of the three available namespaces. if an analysis only called for examining cooccurring molecular function terms then this assessment can only be confidently made using golink as the full complement of cooccurring molecular function terms may not be available within the static top 100 results gleaned from quickgo. this limitation of quickgo also hinders its use in the types of analyses undertaken in the statistical assessment of golink to predict, for example, a gene's biological process from its assigned molecular function terms, as there may not be sufficient molecular function terms returned. quickgo cooccurring terms are immediately available via the quickgo web interface, whereas the golink algorithm can take a number of hours to run for some analyses. there are a number of sections of the golink algorithm; however, that lend themselves to parallel processing. this will be available in future versions of the software; however, its absence in the current version does not hinder the tool's utility. the golink method performs very well in predicting the biological process from a gene's assigned molecular function terms, showing both high sensitivity and specificity. this application of golink is equivalent to first predicting molecular function terms for an unknown gene using, for example, interproscan then using golink to generate terms lists for a biological process query term of interest and then predicting the likelihood of the unknown gene being involved in that particular biological process. similarly using a cellular component query term and comparing the golink terms with interproscan or otherwise derived go terms, a researcher can predict the cellular component of a gene. in cases where a more direct prediction of, for example, biological process is required, multiple molecular function query terms could be provided to golink with the most likely associated biological process being the highest scoring consensus term found in all terms lists. in addition, as cellular component, molecular function, and biological process terms can be incorporated into the golink terms lists, in a similar method to bada et al., golink can be used to help annotators choose potential terms to assign to a gene. furthermore, it was discovered that a number of false positives found in the statistical analysis were not specifically assigned the term regulation of gene expression but clearly have a role in this process. golink is a perl based tool that finds terms cooccurring with a given query term in annotation across the full complement of the gene ontology database. it has advantages over other existing tools and can be used in a variety of applications.

the gene ontology (go) provides a resource for consistent annotation of genes and gene products that is extensively used by numerous large public repositories. the go is constructed of three subontologies describing the cellular component of action, molecular function, and overall biological process of a gene or gene product. querying across the subontologies is problematic and no standard method exists to, for example, find all molecular functions occurring in a particular cellular component. golink addresses this problem by finding terms from all subontologies cooccurring with a term of interest in annotation across the entire go database. genes annotated with this term are exported and their go annotation is assigned to three separate golink terms lists based on specific criteria. the software was used to predict the most likely biological process for a group of genes using just their molecular function terms giving sensitivity, specificity, and accuracy between 80 and 90% across all the terms lists. golink is made freely available for noncommercial use and can be downloaded from the project website.

PMC3892482

pubmed-705

periodontitis (pd) is a chronic inflammatory disease where resident cells and preformed mediators induce leukocyte infiltration and progressive destruction of the tooth supporting tissues as a result of interaction between bacterial products, cell populations, and mediators in disease-susceptible individuals [1, 2]. this is also influenced by genetic and environmental risk factors and is characterized as a complex disease with multifactorial etiology [3, 4]. in this context, environmental factors, including oral hygiene/bacterial plaque, smoking, and stress, play an important role in the expression of pd. furthermore, it has been evidenced by some authors that there is a joint influence of polymorphisms in multiple genes, such as the genes of il-10 and il-6. polymorphonuclear neutrophils (pmns) represent the first line of defense to protect the host from periodontal pathogens in the gingival sulcus and junctional epithelium. pmns are a critical arm of defense against periodontitis, but bacterial evasion of the neutrophil microbicidal machinery coupled with delayed neutrophil apoptosis may transform the neutrophil from defender to perpetrator. actually, these cells can release a variety of factors, such as reactive oxygen species, collagenases, and other proteases, [1, 9], such as stimulation from a wide range of cytokines. in this scenario, macrophages can act as antigen-presenting cells, promoting the activation of lymphocytes. therefore, the cellular concentration of neutrophils in the inflammatory infiltrates decreases during the transition between gingivitis and periodontitis, in which there is a predominance of lymphocytes. it has been described that proinflammatory cytokines, prostaglandin e2, matrix metalloproteinase (mmp), nitric oxide (no), and other inflammatory mediators play a crucial role in the pathogenesis of pd [1012]. moreover, an increase of tnf-, il-1, il-6, il-11, and il-17 can induce osteoclastogenesis by increasing the expression of receptor activator of nf-b ligand (rankl) and by reducing the osteoprotegerin (opg) production in osteoblasts and stromal cells. in fact, it was demonstrated that il-17 and rankl were overregulated and il-10, an anti-inflammatory cytokine, and tgf-1 were downregulated in active periodontal lesions compared with inactive lesions [14, 15] (figure 1(a)). considering that an imbalance between bone formation and resorption is also linked to various diseases, studies suggest that pd may be a risk factor for other diseases, such as rheumatoid arthritis (ra), but without consensus. although pathogenesis of ra is not completely understood, it is recognized that the activation of the complement system is important in disease development, the abnormal response of circulating lymphocytes from patients, and an alteration in the structure of these cells, which contribute to the autoimmunity, immunosuppression, and the genesis of the disease. studies report there is a correlation between both pd and ra since the mechanisms for the development of ra have consonance with the pathogenesis of chronic pd. in fact, ra is defined as an inflammatory and autoimmune disease characterized by accumulation of leukocyte inflammatory infiltrate in the synovial membrane, as well as mediators such as pge2, tnf-, il-1, il-6, il-12, il-17, il-18, il-33, granulocyte macrophage colony-stimulating factor (gm-csf), monocyte colony-stimulating factor (m-csf), rankl, mmps, and no, all being found in the synovial fluid [2024], and leading to synovitis and joint architecture destruction. some studies have suggested that the susceptibility of ra may be associated with genetic or environmental factors. one of the most important genetic factors is the human leukocyte antigen (hla) class ii. certain alleles of this antigen are often associated with the development of rheumatoid arthritis (hla-drb10101, hla-drb10102, hla-drb10401, hla-drb10404, hla-drb10405, hla-drb10408, hla-drb10410, hla-drb11001, and hla-drb11402). other factors include the allele of 620w of ptpn22 (protein tyrosine phosphatase nonreceptor type 22), a gene encoding tyrosine phosphatase that is involved in controlling the intracellular signaling triggered through t and b receptors; c5-traf1, which can interfere with disease susceptibility and severity of the alteration in the structure, function, and levels of complement component c5/factor 1 associated with the tnf receptor; gene encoding the ctla4 (cytotoxic t lymphocyte antigen-4), the protein responsible for the regulation of t lymphocyte activation; peptidylarginine deiminase (pad2), the enzyme responsible for the generation of citrullinated proteins, which are related to the formation of anticyclic citrullinated peptide autoantibodies (figure 1(b)). with regard to environmental factors, smoking is a risk factor that duplicates the risk of developing ra, but its effect is limited to those with antibodies to citrullinated peptides [30, 31]. other factors refer to the excessive consumption of coffee (more than 10 cups daily) which can be related to the development of the disease and bacterial microbiota, including oral bacterial species which can participate in the etiopathogenesis of ra. on the other hand, the intake of alcohol may exert a protective effect in rheumatoid arthritis in a dose-dependent manner. the literature shows that the basic difference between both diseases is that ra is an inflammatory autoimmune disease, while pd is an immunoinflammatory disease of bacterial origin. however, it is noteworthy that many epidemiological studies seem to dilute the subtle differences expressed by some parameters, though clinically important. indeed, analyses of inflammatory mediators and other molecular markers are examples where the differences found in a trial with few participants could disappear in a large and diverse sample. in this sense, this review is a critical appraisal of studies that address potential associations of periodontitis with ra and with an overall comprehensive approach. for this review, the us national library of medicine national institutes of health pubmed was searched by two independent researchers who agreed with the search criteria of studies with patients with both pd and ra and checked by a third researcher separately. the keywords periodontitis and rheumatoid arthritis were used and 367 articles published in english were found. the time period was limited from january 2012 to march 2015, and 162 references were found. then, a critical reading based on titles and abstracts was made and 136 papers were excluded, such as reviews, assays in vitro and animal studies, articles that were not in english, studies not related to both pd and ra, case study, workshop, or unavailable and incomplete articles. then, 26 articles were finally included for this review, which related to pd and ra, considering epidemiological aspects, mechanical periodontal treatment, mediators of inflammation, oral microbiota, and antibodies as seen in figure 2. table 1 shows demographic data, such as gender, age and habits, comorbidities and medications, and the relationship between both diseases investigated through clinical and epidemiological associations, presence of oral bacterial dna in patients with ra, proinflammatory mediators, antibodies against bacteria, and autoantibodies, as well as the effects of mechanical periodontal treatment, related to the 26 selected articles. in most articles (92.3%), the analyzed groups were mainly composed of women. regarding age, most patients were 40 years old, except for the study of dev et al. (2013) and ranade and doiphode (2012), whose patients were above 20 and 30 years old, respectively. among the 26 articles, 57.7% [3436, 38, 39, 42, 5057, 59] used samples with smoker patients, while 30.8% established smoking as criteria for excluding [37, 41, 43, 4549]. 11.5% did not mention smoking status of patients [40, 44, 58]. comorbidities such as diabetes, sjgren's syndrome, hypertension, cardiovascular disease, hyperlipidemia, renal disease, and osteoporosis/osteopenia have only been reported in studies of mikuls et al. 50% [3537, 40, 41, 44, 45, 49, 50, 52, 53, 55, 58] of the articles did not specify the pharmacological treatment. in the remainder of the studies, the most frequently reported treatment for rheumatoid arthritis included disease-modifying antirheumatic drugs (methotrexate, sulfasalazine, and leflunomide) [38, 39, 42, 43, 46, 51, 54, 56, 57, 59], biologic therapy (anti-tnf-) [34, 38, 39, 42, 59], corticosteroids (prednisolone) [38, 42, 43, 46, 51, 54, 56, 59], and/or nonsteroidal anti-inflammatory drugs [43, 4648, 51, 54, 57]. among the selected trials, eight studies broached the epidemiological and clinical relationship of patients with pd and ra [38, 41, 44, 45, 49, 54, 56, 59], indicating a higher prevalence of pd in patients with ra, which have worse periodontal parameters. the effect of mechanical removal of foci of infection in the oral cavity on the severity of ra and periodontal clinical parameters were shown by four studies [37, 42, 46, 51], which demonstrated the beneficial effects of the mechanical treatment in the improvement of clinical parameters of ra. two studies were related to the oral bacteria influence of the pathogenesis of ra [40, 52]. seven trials highlighted the presence of citrullinated proteins and their antibodies, antibodies to p. gingivalis in patients with ra and periodontitis, and also the association between anti-p. gingivalis and periodontal parameters, and the titers of rheumatoid factor and antibodies anticyclic citrullinated peptide, which were also related to the severity of pd [35, 36, 39, 50, 53, 55, 57]. regarding the inflammation in both diseases, five trials considered the mediators of inflammation to the pd and ra [34, 43, 47, 48, 58], such as mmp-9, tnf-, il-17, rankl, and opg. considering the relationship between rheumatoid arthritis and periodontitis, only two articles showed no statistical significant association, while 24 studies have established this association, either by descriptive (3 studies) or statistical analysis (21 studies). in this review, demographic data and other aspects that can modify one or both diseases were presented, as well as the relationship between both diseases investigated through clinical and epidemiological associations, effects of mechanical periodontal treatment, presence of oral bacterial dna in patients with ra, proinflammatory mediators, antibodies against bacteria, and autoantibodies. this aspect was interesting, as a possible relationship between female sex hormones and susceptibility of rheumatoid arthritis had been reported in the literature, so that low levels of those hormones at menopause promote the risk of developing the disease early. however, a protective role of oral contraceptives on the risk for rheumatoid arthritis in women is still controversial [6163]. on the other hand, there is strong evidence that estrogen deficiency influences the severity of periodontitis, since worse periodontal parameters were observed as bleeding on probing, gingival recession, and clinical attachment loss in postmenopausal women with osteoporosis. cigarette smoking is considered an important risk factor for the development of rheumatoid arthritis, since it was demonstrated that lifelong cigarette smoking was positively associated with the risk of ra even among smokers with a low lifelong exposure. moreover, it has been related that smoking interacts with hla-dr se genes and increases the risk of anti-ccp antibodies in patients with rheumatoid arthritis. regarding the periodontium, it was shown that smokers presented greater probing depths, when compared to the probing depths of patients who never smoked. the literature shows that pd does not usually require pharmacological treatment, except for mechanical periodontal treatment as routine. in this review, this fact was also observed, while half of the studies had shown that rheumatoid arthritis involved some pharmacological approach. the use of disease-modifying antirheumatic drugs (dmards) aims to reverse the symptoms of the disease, reduce the progression of joint damage, and consequently improve the quality of life of patients. the conventional synthetic dmards include methotrexate, sulfasalazine, and leflunomide; the available tumor necrosis factor inhibitors (adalimumab, etanercept, and infliximab), the t cell costimulation inhibitor (abatacept), the anti-b cell agent (rituximab), and the interleukin-6 receptor blocking monoclonal antibody are included in biological dmards. these medications may be associated with glucocorticoids (gc) or nonsteroidal anti-inflammatory drugs (nsaids). the long-term, low-dose glucocorticoid and nsaids therapy were shown to reduce joint symptoms, pain, and other systemic manifestations [70, 71]. although these benefits are present, the long-time treatment with gc and methotrexate decreased immune response and promoted oral changes, such as candidiasis, periodontitis, and oral ulceration besides impaired saliva secretion. indeed the literature demonstrated that patients on corticosteroids exhibited higher levels of candidiasis, clinical attachment loss, and probing pocket depth. these aspects, at least in part, may contribute to the worse periodontal status of ra patients when compared to healthy patients. moreover, the use of medications referred to in half of the articles could compromise the evaluation of this review. however, it is noteworthy that the other half of the articles did not use any medication [3537, 40, 41, 44, 45, 49, 50, 52, 53, 55, 58]. analysing the articles, it was observed that most patients with ra showed a significant increase in the incidence of pd as compared to healthy individuals, while only few articles concluded the opposite, probably due to the lack of standardization of parameters in evaluating the different types of periodontitis. although epidemiological studies outlined by dev et al. (2013) have not observed a significant ra incidence in subjects with periodontitis where these authors suggested that periodontitis is an independent factor for ra, several other studies have shown that patients with ra were more susceptible to the development of periodontitis [38, 44], since these patients had worse periodontal parameters, such as clinical attachment level [37, 56], alveolar bone loss [56, 59], probing depth [37, 49], plaque index, and bleeding on probing [37, 41, 54]. indeed, the mechanical periodontal treatment as scaling and root planning in the control of periodontal infection interfered not only with the severity of ra but also with the periodontal clinical parameters. this result can be explained by a reduction in the foci of oral bacteria, and therefore the low levels of inflammation demonstrated a decrease of das28 (disease activity score in 28 joints) and serum levels of il-1, tnf-, c-reactive protein, and erythrocyte rate sedimentation [37, 42, 46, 51]. in this sense, studies have defended the hypothesis that oral infections play an important role in the pathogenesis of ra, promoting the citrullination of proteins, which can be based on the detection of bacterial dna using the techniques of dna isolation (pcr and dna-dna hybridization) and high titers of antibodies against bacteria in synovial fluid and serum samples from patients with ra [40, 51, 52]. most of the studies have shown the presence of oral bacteria in patients with ra, highlighting p. gingivalis and f. nucleatum [40, 52]. markedly, p. gingivalis is the most elucidated in the development of ra, and studies using animal models have demonstrated the potential of this proinflammatory bacterium promoting the development of experimental arthritis and increased serum levels of c-reactive protein, tnf-, il-1, il-17, mmp-13, and rankl. furthermore, ra is an autoimmune disease characterized by autoantibodies specific for citrullinated peptide antigen (anticyclic citrullinated peptide), which are synthetized by peptidylarginine deiminase and characterized as the most specific markers for the diagnosis of the disease [76, 77]. considering that the p. gingivalis is regarded as being capable of expressing this enzyme (pad), it is suggested that infection with this microorganism could influence the pathogenesis of ra [78, 79]. these citrullinated proteins were also found in periodontal tissues, indicating a link between these peptides generated in the oral cavity and those observed in articular tissues [36, 80]. additionally, the presence of antibodies to p. gingivalis was investigated. (2015) have not detected this, antibody titres significantly differ between early rheumatoid arthritis and healthy controls. other studies observed the antibodies to p. gingivalis in patients with ra and severe periodontitis and were associated with probing depth and clinical attachment level and the titers of rheumatoid factor and anticyclic citrullinated peptide autoantibodies [35, 50], which may be found in patients with ra and related to the severity of periodontitis. in summary, the studies suggested that p. gingivalis might play a role in the pathogenesis of ra. the response in periodontitis was related to uncitrullinated peptide, suggesting that these peptides break tolerance and can be involved in pathogenesis of ra (figure 1(c)). most of the studies have found high levels of proinflammatory cytokines and other mediators of inflammation, such as mmp-9, tnf-, il-17, rankl, and opg. moreover, it was demonstrated that the hypomethylated status, a single region of the il-6, may contribute to elevated serum levels of this cytokine, implying a role in the pathogenesis of these diseases, while the anti-inflammatory cytokines in the gcf, such as il-4 and il-10, showed no consensus among studies regarding the differences observed among individuals with pd and ra. in addition, hypotheses have been proposed to explain the relationship between periodontitis and systemic diseases, such as rheumatoid arthritis. in the literature, studies have suggested that chronic periodontitis generates local constant high levels of microparticles, which have been considered inflammatory biomarkers or mediators responsible for distant cell signalling and regulation. moreover, it has been reported that these microparticles play an important role in thrombosis and angiogenesis and mediate cellular communication by transferring mrnas and micrornas from the cell of origin to target cells. thus, the microparticle participation and its spread into the bloodstream could constitute the explanation to the increased risk for systemic disease in patients with periodontitis. despite these evidences showing a link between rheumatoid arthritis and periodontitis, the exact mechanisms involving this association thus, well-designed longitudinal multicentre clinical trials and further studies with sufficient sample sizes are required to determine the biochemical processes and clinical relationships between these chronic inflammatory conditions. moreover, these studies should consider other potential confound factors such as the drugs administered for the treatment for each disease or differences in oral hygiene or smoking habits in these patients. the majority of the articles have confirmed that there is a correlation between pd and ra, since both disorders have characteristics in common and result from an imbalance in the immunoinflammatory response. although it is necessary to highlight the importance of the mechanical treatment for periodontitis and pharmacological treatments mainly for ra patients, more research is needed to assess whether the coexistence of both diseases can affect the clinical signs of periodontitis and systemic markers of rheumatoid arthritis and strengthen the capacity of oral bacteria to stimulate an autoimmune response, thus establishing that cell constituents or mediators could share common pathophysiological pathways for both diseases and therefore define the best therapy.

periodontitis (pd) and rheumatoid arthritis (ra) are immunoinflammatory diseases where leukocyte infiltration and inflammatory mediators induce alveolar bone loss, synovitis, and joint destruction, respectively. thus, we reviewed the relationship between both diseases considering epidemiological aspects, mechanical periodontal treatment, inflammatory mediators, oral microbiota, and antibodies, using the keywords periodontitis and rheumatoid arthritis in pubmed database between january 2012 and march 2015, resulting in 162 articles. after critical reading based on titles and abstracts and following the inclusion and exclusion criteria, 26 articles were included. in the articles, women over 40 years old, smokers and nonsmokers, mainly constituted the analyzed groups. eight studies broached the epidemiological relationship with pd and ra. four trials demonstrated that the periodontal treatment influenced the severity of ra and periodontal clinical parameters. nine studies were related with bacteria influence in the pathogenesis of ra and the presence of citrullinated proteins, autoantibodies, or rheumatoid factor in patients with pd and ra. five studies investigated the presence of mediators of inflammation in pd and ra. in summary, the majority of the articles have confirmed that there is a correlation between pd and ra, since both disorders have characteristics in common and result from an imbalance in the immunoinflammatory response.

PMC4539505

pubmed-706

according to the who, over 3 000 000 children under the age of five die each year due to causes related to environmental risk factors. identification of recognized environmental health risk factors is considered to be one of the most important objectives of health policy, as it relates to the everyday work of medical health-care professionals including family doctors, pediatricians, and nurses. determination of mentioned risk factors will allow implementation of proper preventive measures, and a system of providing parents and teachers with information about health risks will, in turn, decrease the risks which may improve the effectiveness of health care. the who green page questionnaire was designed as a potential tool to determine and monitor children s environmental conditions in all places where they live and develop. it can be a valuable source of supplemental information acquired in the course of children s and parents appointments with family physicians, and it may also be a great support for doctors in determining children s environmental risks at home and school. the who green page questionnaire has not yet been analyzed in terms of its practical utility with respect to medical diagnostics. the expected goal of this research was to assess the possibility of implementation of the who green page as a tool to supplement basic medical interviews with potential environmental health risk factors for children and determination of real risk factors currently existing in home and school environments. the who green page questionnaire was implemented with parents of children from urban, suburban, and rural environments who visited a family practice doctor. due to exclusion of questionnaires missing the majority of data (more than 50% of the answers were missing), the responses of 159 patients were analyzed. when we analyzed the questionnaires with less than 50% of the questions unanswered, the denominator for percentage calculation was lower than that for the 159 parents. wilk test was carried out. for measurable (quantitative) variables, arithmetic means, and standard deviations were calculated, while for qualitative variables, the frequency (percentage) was determined. the analysis of qualitative variables was based on contingency tables and the test. to compare quantitative variables in two non-related and related groups, wallis test was conducted for means of variables that did not meet the criteria for variance analysis. in total 159 parents took part in the study, including 87 parents of girls (58% of the examined) and 63 parents of boys (42% of the examined). the average age of the children was 11.2 years (sd 6.2, median 11). the children mostly lived with both parents (91.3%, 136), while 6.7% (10) of them lived with their mothers only. the majority of the examined cases came from urban areas (56.2%, 87), while 38.3% (59) and 5.2% (8) were from rural and suburban areas respectively (p<0.05). overpopulation at home was noted only among 4.4% (7) of the respondents. domestic animals were present around the homes of 74.5% (117) of the respondents, and the distribution of answers was statistically significant (p<0.05), as it relates to the child s place of living. contact with domestic animals was predominant in rural areas (animals were present in 91.53% of cases) as opposed to urban and suburban areas (63.95% and 57.14% respectively; p<0.05). it was noted that 24.3% (34) of caregivers expressed concern about their children s environment (vs 75.7%, 106), which did not maintain statistically significant dependency with the children s living environment (p>0.05). knowledge and awareness of particular existing environmental risks were noted in 23.7% (32) of the subjects. most of the respondents (96.2%, 153) were aware of the presence of disease-transmitting factors: 42.5% of the subjects knew disease-transmitting factors. the distribution of the answers to this question was dependent on the children s living environment it was distinctively different in rural areas (disease-transmitting factors were identified by 64.3% of the examined) as opposed to urban and suburban areas (correct answers were given by 31.4% and 33.3% the examined respectively), p<0.05. in addition 7.0% (11) of the surveyed stated that their children had sustained injuries in connection with road traffic prior to the questionnaire study, and one child had sustained injuries in connection with fire. the distribution of the answers to those questions was not dependent on the children s living environment (p>0.05). existing exposure to chemical substances (pesticides and detergents) was confirmed by 5.2% (8) of the respondents, and 6.4% (10) of the respondents reported that their children had been poisoned before the questionnaire as a result of contact with chemical substances. furthermore, 9.6% (15) maintained that there existed a threat from poisonous animals. the distribution of the answers to the questions about contact with chemical substances and threat from poisonous animals was not dependent on the children s living environment (p>0.05). following analysis of the respondents answers, it was determined that the examined children lived in a densely built-up area in 79.6% (125) of the cases and in a low-risk geographical zone in 76.3% (119) of the cases. the food they ate was of appropriate quality according to 94.9% (149) of the respondents, the indoor air quality was considered to be good by 81.5% (119) of the respondents, and the outdoor air quality was considered to be average by 51.0% (80) of the respondents. the land they lived on was seen as appropriate by 72.9% (113) of the respondents, and 92.3% (143) of the examined thought that sewage was disposed of in an appropriate way. appropriate disposal of waste was confirmed by 87.9% (138) of the respondents, and the noise level was considered low by 68.2% (107) of the examined. exposure to chemical substances was low according to 70.7% (111) of the respondents. road traffic was seen as low by 44.6% (70) of the examined (table 1table 1abc of environmental conditions homeschoolabcabcbuilt-up environment79.6% (125)20.4% (32)0.0% (0)60.8% (90)38.5% (57)0.7% (1)geographical zone76.3% (119)23.1% (36)0.6% (1)71.3% (102)28.0% (40)0.7% (1)food94.9% (149)4.5% (7)0.6% (1)83.5% (111)15.8% (21)0.8% (1)indoor air quality81.5% (119)17.1% (25)1.4% (2)64.1% (84)34.4% (45)1.5% (2)outdoor air quality40.8% (64)51.0% (80)8.3% (13)35.0% (50)55.9% (80)9.1% (13)water-drinkable86.5% (128)13.5% (20)0.0% (0)82.1% (110)17.9% (24)0.0% (0)land/soil72.9% (113)25.8% (40)1.3% (2)63.2% (91)35.4% (51)1.4% (2)sewage disposal92.3% (143)7.7% (12)0.0% (0)88.0% (125)11.3% (16)0.7% (1)waste disposal87.9% (138)11.5% (18)0.6% (1)79.2% (114)20.1% (29)0.7% (1)noise68.2% (107)28.7% (45)3.2% (5)28.6% (42)51.7% (76)19.7% (29)exposure to chemical substances70.7% (111)28.7% (45)0.6% (1)65.3% (96)32.7% (48)2.0% (3)road traffic44.6% (70)43.9% (69)11.5% (18)25.2% (37)56.5% (83)18.4% (27)environmental risk factors: a, proper; b, average; c, potential. differences between answers are statistically significant (p<0.05).). environmental risk factors: a, proper; b, average; c, potential. (90) of cases, the children schools were in very well-built areas, and the schools were situated in low-risk geographical zones in 71.3% (102) of cases. the food offered at school was of proper quality according to 94.4% (149) of the examined. indoor air quality at schools was good according to 64.1% (84) of the respondents, and outdoor air quality was considered to be average by 55.9% (80). the land where their schools stand was considered appropriate by 63.2% (91) of the examined, and 88.0% (125) of the examined thought that sewage was disposed of in an appropriate way. appropriate waste disposal was confirmed by 79.2% (114) of the respondents, and the noise level was considered average by 51.7% (76) of the examined. exposure to chemical substances was low according to 65.3% (96) of the respondents. road traffic was seen as moderate by 56.5% (83) of the examined (table 1). the majorities of the children involved in the environmental assessment were of school age, lived with both parents, and came from urban areas. the caregivers expressed minor concerns about the children s living and study environments as well as low knowledge and awareness of existing environmental risks. a very low percentage of parents stated that their children sustained injuries in connection with road traffic, and in one case, a child sustained injuries in connection with fire; these were not statistically significantly dependent on where they lived, exposure to chemical substances, or threat from poisonous animals. both the places they lived and studied were located in low-risk geographical zones. in the opinions of the majority of the respondents, the food, indoor and outdoor air, tap water, land, sewage and waste, noise level, exposure to chemical substances, and road traffic were appropriate quality in both places. this picture is positive and does not fully correspond with reality from the hygienic point of view and after performing an environmental interview, which emphasizes people s low awareness of environmental risks. the questionnaire may prove to be a useful tool in determining the kind of potential environmental risk factors that exist in children s living and study places. the who green page supplements the information of the basic medical interview by being a part the patient s medical history.

background: the objective of this study was to assess the possibility of implementation of the who green page as a tool to supplement basic medical interviews with environmental health risk factors for children. methods: the who green page questionnaire was tested on parents of children who visited family practice doctors. results: a total of 159 parents took part in the study. it was noted that 24.3% of caregivers expressed concern about their children s environment without naming the risk factors. it was also found that 23.7% of the parents demonstrated knowledge and awareness of existing real environmental risks, and 7.0% of them stated that their children had sustained injuries in connection with road traffic prior to the questionnaire study. conclusions: the who green page will provide additional information to the basic medical interview and, if regularly updated, will allow for monitoring of changing environmental conditions of children.

PMC4310053

pubmed-707

precise localization of the iac is important and successful implant placement or orthognathic surgery depends on the exact determination of the location of the inferior alveolar nerve (ian). permanent or temporary ian damage can cause numbness of the lower lip and chin in orthognathic surgery [1]. pressure on this nerve during implant placement is one of the common causes of treatment failure and postoperative pain. on some radiographs, the iac has a cortical boundary, but in others, the canal may be mistaken for bone marrow especially in osteoporotic patients [2]. in addition, the anatomical variation of the ian may be a factor that leads to the failure of block injections [3]. recently, by use of cbct, high anatomical variability of this nerve was discovered [4]. although the morphology and the position of the canal vary in different ethnic groups and in different types of jaws, these variations are ignored in many cases and cause problems in dental treatments [5]. a detailed understanding of the factors affecting the anatomical variations of canal shape and morphology can minimize this problem. therefore, in this study, we investigated the relationship between the ga size and course of the iac in human dry mandibles using cbct. the results may be useful for more accurate localization of the iac on radiographs before dental treatment. in this in vitro study, we obtained dry mandibles from the anatomy department of mashhad university of medical sciences. samples with no anomaly or bone defects were assessed, and those in primary or mixed dentition were excluded [3, 6 8]. a total of 25 dry adult human mandibles and 6 left and 5 right hemi mandibles (a total of 61 hemi mandibles) of unknown gender or origin were studied. to increase the accuracy and for easy tracing of the canal, a 0.5 mm diameter orthodontic wire was inserted into the iac before imaging [7]. the cbct scans were obtained (promax 3d, planmeca, helsinki, finland) with 8080 mm field of view. as the image field did not cover the entire mandibular bone, the stitch option of cbct was used, and an oral and maxillofacial radiologist, using romexis software 2.4.2.r (planmeca inc. intra-observer error was calculated by re-measuring a random sample of 30 images after an interval of two weeks. both readings were then analysed by paired t-test. in the sagittal view of each ramus, line b was traced tangential to the most prominent point on the posterior border of the ramus and condyle. the angle between lines b and d was measured as ga (fig. 1) [9 14]. gonial angle measurements the scans were divided into the following two ga groups: low angle (125) and high angle (> 125) [15]. to evaluate the shape and position of the canal in coronal views, the distance between the mandibular foramen and the mental foramen was divided into three equal segments and the central cuts (c1, c2, and c3) were measured in each segment [16, 17]. in order to evaluate the canal shape, the superior-inferior and buccolingual diameters of the canal were measured in each slice. to evaluate the buccolingual position of the iac, we measured the distances from the center of the iac to the tangential lines with buccal and lingual borders and to the lowest point of the inferior border (fig. 2) [17, 18]. in each slice, the distance from the buccal plate to the lingual plate was measured as buccolingual width of the mandibular body [16, 19]. consequently, in order to define the buccolingual position of the canal in each section, the ratio of the distances between the center of the iac and the buccal and lingual plates to the width of mandible in each section was calculated [16]. evaluation of the buccolingual position of the iac by calculating the distance from the center of iac to buccal, lingual, and inferior borders in c1, c2, and c3 slices to investigate the total length of iac, the nerve was traced in coronal views. by using the serial measurements option, the total length of the nerve was exactly measured on the sagittal view (fig. 3). to investigate the canal course on the sagittal view, the nerve path was classified into three types of a, b, and c [7, 17]. in type a, the canal course had a straight path and was positioned at the same level of the mental foramen. in type b, the canal had a curved path, and in type c, the canal had a forward path and then ran superiorly to reach the mental foramen with a sharp ascent (fig. to evaluate the canal course on the axial view, the nerve path was traced and classified according to the mental foramen angle. if the canal course made an acute angle with mental foramen (90), it was defined as type a1; otherwise, it was type a2 (fig. 5) [7]. different types of canal course on the sagittal view type a: the canal course has a straight path at the same level as the mental foramen. type c: the canal has a forward path and then runs up to reach the mental foramen with a sharp ascent. the canal course forms an acute angle with the mental foramen on the right side (a1 type) and an obtuse angle on the left side (a2 type) statistical analysis was done using pasw version 18 (spss inc., paired-sample t-test was used for right and left symmetry and calculation of intra-observer agreement between the two readings. pearson s correlation coefficient was used to examine the correlation between the right and left gas. also, independent-samples t-test was used for quantitative comparison between different canal types on different views. pearson s chi-square test was applied for evaluation of the correlation of canal course in the sagittal plane and ga. a p-value less than 0.05 intra-observer reproducibility for all variables was calculated by re-examining 30 scans. the differences between measurements in the first and second readings were not significant (p=0.160.35). the mean size of ga was 121.87.05 at the right side and 123.86.32 at the left side (table 1). the results showed that there was a statistically significant correlation in the ga size at both sides (p=0.000, r=0.9); however, a significant difference was noted between them (p=0.03). the mean standard deviation of canal length at the right and left sides were 63.035.48 mm and 62.75.51 mm respectively. there was no significant correlation in the mean total canal length in the right and left sides. no statistically significant difference was found between the two sides in terms of canal lengths (p=0.53). descriptive findings for gas sd: standard deviation after evaluating different sagittal views of the mandibular canal, it was obvious that the most common type of iac course on the sagittal view was type b (53.8%), followed by type c (26.2%) and type a (20%). in 70% of the cases, there was no right-left symmetry in the course of the canal on the sagittal view. in addition, it was clear that there was no significant difference between the canal length on the sagittal view, according to canal course (p=0.59). the samples were grouped according to ga size as high angle (> 125) and low angle (125). the gas were smaller than 125 and larger than 125 in 60.7% and 39.3% of samples, respectively. as shown in table 2, there was a significant relationship between different types of canal courses on the sagittal view and the different ga groups (p=0.04). therefore, in the low ga group, type b was the more common canal course (73.5%), while type a was dominant in the high ga group (66.7%) as shown in table 2. cross tabulation between different canal course types and ga groups on the sagittal view. after evaluating the canal course on the axial view, type a1 was found to be more common (73.43%) than type a2 (26.56%). according to the data in 10.34% of the cases, the canal type was different at both sides of the mandible. there was no statistically significant relationship between the canal course on the axial view and ga group (p>0.05). the total canal length was 63.255.4 mm in type a1 and 60.744.7 mm in type a2. no significant difference existed in the canal length in different types of canal course on the axial view (p=0.09). evaluation of the iac course on the coronal view in segments c1, c2, and c3 showed that on the right side, the canal was closer to the lingual cortex in 86% of the cases in c1, 100% in c2, and 90% in c3; on the left side the corresponding percentages were 77%, 93%, and 93% of the cases, respectively. according to our results, the canal is positioned lingually throughout its path up to the mental foramen, and then reaches the mental foramen with an acute or an obtuse angle. analysis of the diameters of iac showed that the greatest mean diameter in the superior-inferior and buccolingual dimensions was in c1 and the smallest was in c2. in this study, the gender of the individuals from whom the dried human mandibles were obtained was unknown. many previous studies, including those conducted by de oliveira-santos et al, [4] ozturk et al, [7] kisser et al, [8] liu et al, [17] apinhasmit et al, [20] angel et al, [21] and raustia and salonen [22] demonstrated that the position of some anatomic landmarks, such as iac and ga, is not related to gender or age. the mean size of ga in this study was similar to that found in earlier studies [9 13, 23]. the mean ga of the right side was 2 smaller than that in the left side, and this difference was statistically significant. the difference in the mean size of the right and left ga was mentioned in the study by raustia and salonen [22], who stated that the right ga was significantly smaller than the left one. when we analysed the canal shape in the coronal sections, we found that ovoid shape was more common than other shapes. similar to ozturk et al, [7] we found that type b was the most common type of canal course on the sagittal view, followed by types c and a. liu et al. [17] classified the canal course into four groups (types 1, 2, 3, and 4) using panoramic radiography [17]. the shape of the canal in type 1 was similar to type a in the current study and the study carried out by ozturk et al [7]. types 2 and 3, which had a catenary like path, were similar to type b, and type 4 was similar to type c. in the study by liu et al, [17] the frequency sum of types 2 and 3 was greater than that of type b, but the frequency of types 1 and 4 was lower than that of types a and c [17]. the difference in the prevalence rate of iac course shape in the study by liu et al, [17] compared to that observed by ozturk et al, [7] and in the current study, may be due to the difference in the type of imaging system and methodology of the studies, and may also be because of the difference in ga [7, 17]. there was no difference in the total canal length between types a and b; however, there was a high prevalence (66.7%) of type a in the high-angle group, while type b was dominant (73.5%) in the low-angle group. it is analogous to a situation in which you have two identical ropes; if you tie the ends of one of the ropes, its path will be bent, whereas the other rope would have a more direct route. there was no difference in the canal length observed in our study and that found by liu et al [17]. although the method to obtain the total canal length used by liu et al. was via the tracing cord with a 5 mm arch, while we calculated the length using a specific type of software; the average length found in both studies was very similar [17]. introduced four types of canal course on the axial view, and two of these (types b and c) were introduced for the first time [7]. similar to liu et al, [17] we did not observe these types in the current study, but type a1 was the most commonly found, which is in accordance with the results of ozturk et al [7]. in the current study, the iac was located near the lingual plate throughout its path, and then travelled to the buccal plate with a sharp ascent near the mental foramen in the majority of samples. ozturk et al, [7] hwang et al, [19] som and curtain [24], kim et al, [25] and fabian [26] also found that the iac is close to the lingual plate, and then runs to the buccal plate near the mental foramen [7, 19, 24 hwang et al. showed that the iac is close to the lingual plate in the posterior two thirds of the mandible and runs towards the buccal side in the anterior third [19]. in our study, the entire canal course was of a1 and a2 types on the axial view, which shows that the canal is in the lingual plate, then in front of the mental foramen, it reaches with a sharp turn to the buccal side [7, 19]. the canal was located almost 1 cm above the inferior border of the mandible in the second premolar and the first molar region, which appears to provide the greatest superior-inferior dimension for implant placement. showed that the least distance to the lower border was almost 1 cm in the first molar region [7]. according to the results of the current study, the iac has the greatest diameter in the ramus region (behind the third molar) and the smallest diameter in the molar region. de oliveira-santos et al. studied canal diameter in the first molar region and their conclusion was in accordance with our findings [4]. no other study has calculated iac diameter on the coronal view. in this study we were not able to identify the sex of subjects whose dry mandibles were evaluated; thus further studies on human mandibles are suggested taking into account the sex and age parameters. we concluded that: 1-the ga size has a correlation with the iac course. in subjects with small gas but in cases with large gas, the canal is more straight and at the same level as the mental foramen.2-the alveolar crest has the maximum distance from the iac in the second premolar and first molar region. the ga size has a correlation with the iac course. in subjects with small gas but in cases with large gas, the canal is more straight and at the same level as the mental foramen. the alveolar crest has the maximum distance from the iac in the second premolar and first molar region. since the iac is located in the lingual side of the mandible, the safe zone for implant placement and pre-prosthetic surgery is believed to be the buccal side. in addition, the greatest height of bone can be obtained in the second premolar and first molar regions.

objectives: accurate localization of the inferior alveolar canal (iac) is extremely important in some dental treatments. anatomical variation of the canal means that it can be difficult to locate. the purpose of this study was to assess the relationship of the gonial angle (ga) size and iac position using cone beam computed tomography (cbct). materials and methods: in this in vitro study, 61 dry adult human hemi-mandibles were used. the cbct scans were taken of all samples and ga was measured on all cbct scans. the samples were divided into two groups of low angle (125) and high angle (> 125). the canal dimensions, length and course were evaluated. on the sagittal view, the iac path was classified as type a, b or c. on the axial view, canal course was defined as a1 or a2 according to the mental foramen angle. results:the average ga size was 121.87.05 at the right side and 123.86.32 at the left side. on the sagittal view, there was a significant correlation between the ga size and the canal course (p=0.04). in the high-angle group, type a was dominant; whereas in the low-angle group, type b was more common. on the axial view of iac course, type a1 was more common (73.43%). conclusion: the results showed that ga size was associated with iac course. in cases with a larger ga, the canal runs in a more straightforward path, and at the same level as the mental foramen.

PMC4888162

pubmed-708

worldwide, the prevalence of overweight and obesity has reached epidemic proportions and includes not only adults but also children and adolescents [2, 3]. for example, it has been reported that the worldwide prevalence of overweight and obesity includes approximately 110 million children while in the united states (us), an estimated 12.5 million children and adolescents are either overweight or obese. this is problematic because overweight and obese youth have been shown to be at an increased risk of becoming overweight and obese adults, and thus placing them at an increased risk for premature all-cause mortality. based on 2005 data, overweight and obesity as well as physical inactivity in adults were reported to be the third leading causes of preventable death in the us (about 1 in 10 deaths each) behind cigarette smoking and high blood pressure. the issue of obesity has become so problematic that it has recently been recognized as a disease by the american medical association. exercise, a nonpharmacologic intervention that is available to the vast majority of the general public, may play a pivotal role in the treatment of overweight and obese children and adolescents. systematic reviews with meta-analysis, a quantitative approach for combining the results of different studies on the same topic, are considered by many to be the most important type of evidence for determining the efficacy and effectiveness of various treatments on selected outcomes [9, 10]. unfortunately, with the proliferation of systematic reviews on the same topic, it becomes difficult to make informed decisions regarding the effects of various interventions on selected outcomes. for example, a recent systematic review identified 22 previous meta-analyses examining the effects of exercise on blood pressure. given the proliferation of reviews, a need now exists to systematically review these previous reviews in order to provide decision-makers with the information they need to make evidence-based decisions regarding the efficacy and effectiveness of various interventions on selected outcomes as well as provide direction for future research. given the former, the purpose of the current study was to conduct a systematic review of previous meta-analyses addressing the effects of exercise (aerobic, strength training, or both) in the treatment of overweight and obese children and adolescents. the a priori inclusion criteria for this study were as follows: (1) previous systematic reviews with meta-analysis of randomized controlled trials or data reported separately for randomized controlled trials, (2) children and adolescents 5 to 18 years of age, (3) aerobic exercise and/or progressive resistance training intervention(s) lasting for an average of at least 4 weeks, (4) published and unpublished (dissertations and master's theses) studies in any language from 1990 forward, and (5) exercise minus control group difference in one or more of the following variables that were primary outcomes in the original meta-analysis: body weight, body mass index, body mass index percentile, body mass index z-score, percent body fat, fat mass, and fat-free mass. post hoc, percentage overweight, adjusted for height as well as waist-to-hip ratio were also included as outcomes. meta-analyses were limited to randomized controlled trials because they are the only way to control for unknown confounders as well as the fact that nonrandomized controlled trials tend to overestimate the effects of treatment in healthcare interventions [13, 14]. potentially eligible meta-analyses were also limited to those that included studies in which exercise was an intervention, defined here as planned, structured, and repetitive and purposive in the sense that the improvement or maintenance of one or more components of physical fitness is the objective. while somewhat arbitrary, 4 weeks was chosen as the minimum length of exercise since one should expect some type of change in overweight/obese outcomes during this period of time. based on a pubmed search, 1990 was chosen as the starting point for searching because it was the first year in which a potentially eligible study was identified for review. any studies that did not meet all of the above criteria were excluded from our review. ineligible studies were broadly categorized as excluded based on one or more of the following reasons: (1) inappropriate population (adults, animals, etc.), (2) inappropriate intervention (nutrition, pharmacologic, etc.), (3) inappropriate comparison (exercise versus diet), (4) inappropriate outcome (blood pressure, lipids, etc.), and (5) inappropriate study type (meta-analysis that included nonrandomized controlled trials, systematic review without meta-analysis, etc.). using the graphical-user interfaces for each database, the following electronic sources were searched: (1) pubmed, (2) sport discus, (3) web of science, (4) scopus, (5) proquest, (6) cochrane database of systematic reviews (cdsr), (7) physiotherapy evidence database (pedro), (8) database of abstract of reviews of effects (dare), and (9) health evidence canada (hec). all searches were conducted during the month of april, 2013 with the last searches conducted on april 20. scopus was included in our database searches because it has been reported to provide coverage of embase. with the exception of pubmed, which was searched from its inception in order to identify a starting year for searching, a list of all search strategies for each database is shown in supplementary file 1 (supplementary material available online at http://dx.doi.org/10.1155/2013/783103). in addition to electronic database searches, cross-referencing for potentially eligible meta-analyses from retrieved reviews was also conducted. the coding sheets could hold up to 253 items from each included meta-analysis. both authors coded all studies independent of each other. upon completion of coding, all coding sheets using cohen's kappa statistic (), the overall agreement rate prior to correcting discrepancies was 0.68. methodological quality for each included meta-analysis was assessed using the assessment of multiple systematic reviews (amstar) instrument [2225]. amstar was chosen over other instruments [26, 27] because of its reported interrater reliability (= 0.70), construct validity (intraclass correlation coefficient=0.84) and feasibility (average of 15 minutes per study to complete). no, ca n't answer, or not applicable. the response ca n't answer is chosen when an item is relevant but not described. the response not applicable is chosen when an item is not relevant (meta-analysis of data not possible, etc.) when summing responses, the following question was modified from was the status of publication (i.e. grey literature) used as an inclusion criterion? to was the status of publication (i.e. grey literature) as an inclusion criterion avoided? in addition, we considered the question regarding conflict of interest as adequately met if the authors of the systematic review provided a statement on conflict of interest versus the reporting of conflict of interest by both the authors of the systematic review and the original studies included in the meta-analysis. using cohen's kappa statistic (), the overall agreement rate prior to correcting discrepancies was 0.82. a priori, the overall results from each meta-analysis were extracted, with a focus on random effects models since they incorporate between-study heterogeneity into the model and should almost always be the model of choice regardless of whether or not significant heterogeneity exists [28, 29]. overall point estimates and 95% confidence intervals (cis) along with the q statistic, a measure of heterogeneity, were extracted for each outcome. an alpha value 0.10 was considered to represent statistically significant heterogeneity. however, because of issues surrounding the power of the q statistic, the i statistic was also reported if it was provided in the meta-analysis. if it was not provided, it was calculated if sufficient data existed to do so. the i statistic=100% (q df)/q, where q is cochran's heterogeneity statistic and df, the degrees of freedom. negative values of i are set to zero (0) so that i falls between 0% and 100%. a value of 0% indicates no observed heterogeneity while larger values indicate increasing heterogeneity. while somewhat arbitrary, values of 25%, 50%, and 75% were considered to represent low, moderate, and high amounts of heterogeneity. since it was assumed that none of the eligible meta-analyses would include 95% prediction intervals (pis), these were calculated if the overall findings were statistically significant and the results from each study included in each meta-analysis were provided [3234]. prediction intervals are used to estimate the treatment effect in a new trial [3234] and are calculated as follows: (1)meantdf(se2+ 2), where t is the centile point (95%) of the t distribution with k 2 degrees of freedom, se is the squared standard error, and is the between-study variance [34, 35]. all pis were calculated using the user-written metan command in version 11.0 of stata. in order to enhance application, the number-needed-to treat (nnt) was calculated for any overall findings that were reported as statistically significant. in addition, the nnt was used to provide gross estimates of the number of obese children and adolescents in the us who could benefit from exercise, based on 12.5 million obese children and adolescents as well as the number of overweight and obese children worldwide who could benefit from exercise, based on 110 million overweight or obese children [2, 38]. it was assumed that none of the children and adolescents included in the original estimates were exercising regularly. because neither of the included meta-analyses assessed publication bias or conducted influence analysis with each outcome deleted from the model once, a post-hoc decision was made to test for both if sufficient data were provided. publication bias was assessed using the regression-intercept approach of egger et al.. both publication bias and influence analysis were conducted using comprehensive meta-analysis (version 2.2). for those outcomes that were reported using the standardized mean difference (smd), values of 0.2, 0.5, and 0.80, were considered to represent small, medium, and large effects. with the exception of heterogeneity, tests with two-tailed alpha levels 0.05 were considered to be statistically significant. two-tailed alpha levels>0.05 but 0.10 were considered as a trend towards statistical significance. precision of estimates was considered robust if 95% confidence intervals for continuous outcomes did not cross zero (0). dispersion statistics were reported as either standard deviations (sd) or standard errors (se). with the exception of fat-free mass, negative values for all other outcomes were indicative of improvement. of the 511 citations initially identified, 308 (60.3%) remained after removing duplicates. of the 308 articles that were screened, the full text from 25 articles (8.1%) was retrieved and assessed for potential eligibility. upon completion of the review, two aggregate data meta-analyses met the criteria for inclusion [42, 43]. the major reasons for exclusion of the other studies were an inappropriate study design (49.8%) followed by an inappropriate population (21.2%), outcome (15.9%), intervention (12.0%), and comparison (1.6%). a flow diagram that depicts the search process can be found in figure 1 while a list of excluded studies, including the reasons for exclusion, is shown in supplementary file 2. for the two included meta-analyses [42, 43], one focused specifically on exercise while the other focused on nonsurgical interventions, including exercise. both meta-analyses included overweight and obese children and adolescents according to the criteria described by each of the original studies they included [42, 43]. a general description of the characteristics of each meta-analysis is provided in table 1. satisfied 7 of the 11 amstar criteria (64%) while the study by mcgovern et al. both meta-analyses were judged as (1) not avoiding the status of publication as an inclusion criterion, (2) not providing a list of excluded studies, and (3) not assessing for potential publication bias [42, 43]. in addition, the meta-analysis by atlantis et al. was judged as not providing a conflict of interest statement. amstar results for each question from each meta-analysis are shown in supplementary file 3. a description of the overall findings from each meta-analysis is shown in table 2. a statistically significant reduction in percent body fat along with nonoverlapping 95% cis was observed for both the atlantis et al. meta-analysis while nonoverlapping 95% pis were observed for the mcgovern et al. an examination for publication bias indicated no statistically significant publication bias for either the atlantis et al. (b0, 0.32, 95% ci, 4.0 to 3.4, p=0.84) or mcgovern et al. (b0, 1.09, 95% ci, 3.4 to 1.2, p=0.27) meta-analyses. with each outcome in each meta-analysis deleted from the model once, results remained statistically significant or trended towards statistical significance (p<0.001 to 0.07) for both, ranging from an smd of 0.31 to 0.50 in the atlantis et al. no statistically significant changes in bmi or bmi-related outcomes were found for either the atlantis et al. in addition, overlapping confidence intervals were observed for both meta-analyses [42, 43]. for the mcgovern et al. meta-analysis, no statistically significant heterogeneity or publication bias (b0, 0.62, 95% ci, 3.2 to 2.0, p=0.60) was observed. with each outcome deleted from the model once, changes remained nonsignificant (p=0.29 to 0.99), ranging from a smd of 0.11 to 0.02. insufficient bmi data were available to test for heterogeneity, publication bias, and influence analysis in the atlantis et al. meta-analysis also reported outcome results for body weight and central obesity (waist circumference and waist-to-hip ratio). a trend for statistically significant reductions in body weight in addition, publication bias was also found to be statistically significant (b0, 1.7, 95% ci, 3.3 to 0.05, p=0.05). with each outcome deleted from the model once, results were statistically significant, and all heterogeneity was removed when one outcome from one study was deleted from the model (x-se, 3.7 1.4 kg, 95% ci, 6.4 to 0.9, p=0.009; q=7.0, p=0.63, i=0%). for central obesity, a trend for statistical significance (p=0.07) was reported but 95% cis were overlapping. no statistically significant heterogeneity or publication bias (b0, 0.90, 95% ci, 4.7 to 2.9, p=0.41) was observed. results were in the direction of benefit (smd, 0.19 to 0.29) but remained nonsignificant (p=0.12 to 0.23) when each outcome was deleted from the model once. the atlantis et al. meta-analysis conducted several additional analyses beyond the overall findings for percent body fat and body weight. for percent body fat, reductions were greater (smd, 0.6, 95% ci, 0.8 to 0.3, p<0.001) when studies were limited to higher (x-sd, 177 23 minutes per week) versus lower (x-sd, 153 25 minutes per week, <3 days per week) doses of exercise as well as when strength training studies were removed from the model (smd, 0.5, 95% ci, 0.8 to 0.1, p=0.003). results remained stable when separate analyses were conducted with studies that reported changes in dietary intake (p=0.02) and exercise-only studies (p=0.005) deleted from the models. results were no longer statistically significant when studies that did not report exercise compliance or changes in exercise were deleted from the analysis (smd, 0.3, 95% ci, 0.7 to 0.2, p=0.11). for changes in body weight, reductions were greater (x-, 4.9 kg, 95% ci, 9.1 to 0.7, p=0.01) when studies were limited to higher (x-sd, 156 25 minutes per week) versus lower (x-sd, 117 46 minutes per week, <3 days per week) doses of exercise as well as when strength training studies were removed from the model (x-, 3.1 kg, 95% ci, 6.1 to 0.1, p=0.02). with studies that reported dietary intake removed, reductions in body weight increased (x-, 5.1 kg, 95% ci, 8.6 to 1.6, p=0.002). in contrast, results were no longer statistically significant when studies that did not report exercise compliance or changes in exercise were deleted from the analysis (x-, 2.3 kg, 95% ci, 6.8 to 2.1, p=0.20). for dose-response, no statistically significant associations were observed for changes in percent body fat, body weight, and central obesity when correlated with the volume of prescribed exercise (minutes per week) and total dose (minutes per week length of study in weeks). a trend was observed for greater reductions in body weight and exercise interventions that occurred over a greater number of weeks. meta-analysis, changes in percent body fat were reported to be greater than changes in bmi-related measures (p for interaction=0.0007). however, when limited to trials that assessed both, results were no longer statistically significant (p=0.28). the nnt and gross estimates of the number of overweight and obese children and adolescents who might reduce their percent body fat from participation in an exercise program are shown in table 3. as can be seen, the 95% cis for the nnt and subsequent estimates from the atlantis et al. depending on the meta-analysis, approximately 2.8 to 3.6 million of the 12.5 million overweight and obese children in the us could reduce their percent body fat (ideally) by participating in a regular exercise program. the purpose of the current study was to conduct a systematic review of previous meta-analyses addressing the effects of exercise (aerobic, strength training, or both) in the treatment of overweight and obesity in children and adolescents. overall, it appears that exercise reduces percent body fat in overweight and obese children and adolescents. this interpretation is further supported by the robustness of results across both meta-analyses [42, 43] with respect to magnitude of effect, nonoverlapping confidence intervals, influence analysis (each study deleted from the model once) and publication bias. in contrast, heterogeneity and overlapping 95% pis were found for the atlantis et al. in addition, while the absolute nnt was similar across both meta-analyses [42, 43], the 95% cis were wider for the atlantis et al. study. given that the atlantis et al. most notably, greater reductions in percent body fat were found with higher exercise doses as well as when strength training studies were deleted from the model. while the former results appear plausible, the latter may be questioned. however, it is feasible that the potentially increased caloric expenditure from aerobic exercise may have resulted in greater reductions in percent body fat. the former notwithstanding, these results need to be interpreted with caution for at least two reasons. first, given the large number of statistical tests conducted, these findings could have been nothing more than the play of chance. second, because studies are not randomly assigned to covariates, they are considered to be observational in nature. consequently, the results of moderator and regression analyses conducted in a meta-analysis do not support causal inferences. nonetheless, these findings are probably important as they support the need for addressing these potential associations in future, well-designed, randomized controlled trials. while improvements in percent body fat were observed, there is currently insufficient evidence that exercise improves bmi-related measures, body weight, and central obesity in overweight and obese children and adolescents. however, it is important to understand that a lack of evidence of effect does not mean evidence of no effect. as additional evidence accumulates, one may gain a better understanding regarding the effects of exercise on these outcomes in overweight and obese children and adolescents. the results of the current systematic review of previous meta-analyses on the effects of exercise in the treatment of overweight and obese children and adolescents have several implications for future research. first, while the overall quality of the two meta-analyses was considered adequate, there are several areas that might be improved upon in future meta-analytic work. these include (1) avoiding the use of publication status as an inclusion criterion, (2) documenting and providing a list of not only included studies but also excluded studies, including the reasons for exclusion, and (3) assessing publication bias. the former notwithstanding, avoiding the use of publication status as an inclusion criterion could be questioned. for example, van driel et al. concluded that (1) the difficulty in retrieving unpublished work could lead to selection bias, (2) many unpublished trials are eventually published, (3) the methodological quality of such studies is poorer than those that are published, and (4) the effort and resources required to obtain unpublished work may not be warranted. second, both of the included studies were aggregate data meta-analyses [42, 43]. while this continues to be the most common type of meta-analysis, individual-participant data meta-analyses (ipd) are considered to be the gold standard when attempting to quantitatively thus, future meta-analysts may want to consider using the ipd approach when addressing the effects of exercise in the treatment of overweight and obese children and adolescents. however, the use of the ipd approach needs to be considered with respect to the ability to retrieve ipd from investigators as well as the increased costs associated with the conduct of such, although methods to address the former have recently been developed. third, given the apparent lack of available data in the original studies included in the two meta-analyses [42, 43], there is a need for future randomized controlled trials to examine and report the safety and cost-effectiveness of their exercise intervention(s) in the treatment of overweight and obesity among children and adolescents. in addition, since the average length of studies in the included meta-analyses was only 16 and 23 weeks, a need exists for longer intervention studies, including follow-up studies, to more fully understand the longitudinal effects of exercise on adiposity. similarly, the apparent focus on per-protocol versus intention-to-treat analyses in the original trials allows one to draw conclusions regarding the efficacy (does the treatment work ?) but not the effectiveness (does the treatment work in the real world ?) of exercise in the treatment of overweight and obese children and adolescents. fourth, the dose-response effects of exercise on measures of adiposity remain elusive. while the atlantis et al. meta-analysis concluded that 155 to 180 minutes per week of moderate to high intensity exercise is effective for reducing body fat in overweight and obese children and adolescents, additional research on this topic is needed, especially with respect to body weight, bmi-related measures, and central obesity. fifth, percent body fat, but not bmi, appeared to be a more sensitive indicator of exercise-induced changes in adiposity among overweight and obese children and adolescents. thus, future researchers may want to focus on percent body fat as their primary outcome despite the finding that bmi has been shown to correlate well with body fatness in children and adolescents. however, while numerous methods exist for the assessment of percent body fat (skinfold calipers, hydrostatic weighing, whole body air-displacement plethysmography, dilution, dual energy x-ray absorptiometry, computerized tomography, magnetic resonance imaging) [45, 51], none may be practical in many settings, including the community-based setting. thus, the use of bmi-related measures such as bmi z-score and bmi percentile may need to be considered but interpreted with the realization that they may not be very sensitive to change. in addition, given its simplicity, bmi is currently the universally accepted method for assessing adiposity in children and adolescents. sixth, because neither meta-analysis reported nnt [42, 43], it is suggested that future meta-analytic work includes such. from the investigative team's perspective, the reporting of such information is important because it provides practically relevant information to decision-makers (practitioners, policy-makers, etc.) along those lines, formulas now exist for calculating nnt from continuous data. finally, the most recent meta-analysis that met our inclusion criteria, published in 2008, included studies published up to february 2006, more than 7 years ago. while there is no definitive consensus regarding when to update a systematic review, with or without a meta-analysis, recent research by pattanittum et al. concluded that three practical statistical methods could be applied to examine the need to update systematic reviews, with or without meta-analysis. such updated work is critical with respect to providing guidelines based on the most recent evidence available. first, while there is a lack of cost-effectiveness and safety data, the use of exercise appears to be efficacious for improving adiposity, specifically percent body fat, in overweight and obese children and adolescents. the relatively low nnt observed as well as the potential number of overweight and obese children and adolescents who may benefit lends further support for this recommendation. second, while the dose-response effects of exercise in the treatment of overweight and obese children and adolescents have not been fully elucidated, it would appear prudent to recommend that practitioners follow the general recommendations for exercise in children and adolescents, that is, 60 minutes or more of physical activity each day. the majority of the 60 minutes should be comprised of moderate to vigorous aerobic activity (bicycling, running, etc.) as well as muscle strengthening (pushups, etc.) and bone strengthening (jumping rope, etc.), 3 days per week. given the initial difficulty that overweight and obese children and adolescents may have in meeting these requirements, an individual exercise prescription that gradually progresses them to this level of effort seems appropriate. third, given the apparent lack of sensitivity of bmi, it is recommended that practitioners assess and track changes in adiposity using one of the numerous methods available for assessing percent body fat. if not possible, then the assessment of adiposity using bmi z-score or percentile can be used with the understanding that the true effects of exercise on adiposity in overweight and obese children and adolescents may not be fully realized with this approach. finally, given the observed magnitude of response of exercise on percent body fat in overweight and obese children and adolescents, exercise combined with other lifestyle and/or pharmacological interventions may be necessary for eliciting a health-improving impact on percent body fat in overweight and obese children and adolescents. along those lines, an evaluation and treatment algorithm currently exists for addressing this issue. first, to the best of the authors ' knowledge, this is the first systematic review of previous meta-analyses that has examined the effects of exercise in the treatment of overweight and obese adolescents, an increasingly important approach for addressing the effects of various healthcare interventions. second, the additional analyses conducted based on the available data (influence analysis, publication bias, etc.) helped strengthen the validity and findings of the two included meta-analyses [42, 43]. for example, the finding of no apparent publication bias for those studies that examined changes in percent body fat helped to strengthen the conclusions regarding the effects of exercise on percent body fat in overweight and obese children and adolescents. while conducting additional analyses beyond those reported in an original meta-analysis does not appear to be common when conducting systematic reviews of previous meta-analyses, future investigators may want to incorporate this methodology into their reviews while at the same time considering the additional time and effort involved in such an endeavor. third, the nnt and gross estimates of the absolute number of overweight and obese children and adolescents who might reduce their percent body fat by participating in a regular exercise program were provided. in the authors ' opinion, fourth, the calculation and inclusion of pis for statistically significant outcomes in the current study provide investigators with information that can aid them in planning future randomized controlled trials. in addition to the strengths of the current study, there are several potential limitations. first, the investigative team established fairly strict eligibility criteria for the current systematic review. as a result, only two previous meta-analyses met all eligibility criteria [42, 43]. while more focused and applicable, other relevant issues such as the effects of exercise on quality-of-life in overweight and obese children and adolescents were not captured. second, the gross population estimates for the number of children who could reduce their percent body fat by participating in an exercise program assumed that none of the overweight and obese children and adolescents were exercising regularly. finally, as with any systematic review, many of the biases inherent in both the included meta-analyses as well as the original trials that comprise each meta-analysis may also be present in a systematic review of previous meta-analyses. the results of the current systematic review of previous meta-analyses suggest that exercise is efficacious for reducing percent body fat in overweight and obese children and adolescents. however, there is currently insufficient evidence to suggest that exercise reduces bmi-related measures, body weight, and central obesity in overweight and obese children and adolescents.

purpose. conduct a systematic review of previous meta-analyses addressing the effects of exercise in the treatment of overweight and obese children and adolescents. methods. previous meta-analyses of randomized controlled exercise trials that assessed adiposity in overweight and obese children and adolescents were included by searching nine electronic databases and cross-referencing from retrieved studies. methodological quality was assessed using the assessment of multiple systematic reviews (amstar) instrument. the alpha level for statistical significance was set at p 0.05. results. of the 308 studies reviewed, two aggregate data meta-analyses representing 14 and 17 studies and 481 and 701 boys and girls met all eligibility criteria. methodological quality was 64% and 73%. for both studies, statistically significant reductions in percent body fat were observed (p=0.006 and p<0.00001). the number-needed-to treat (nnt) was 4 and 3 with an estimated 24.5 and 31.5 million overweight and obese children in the world potentially benefitting, 2.8 and 3.6 million in the us. no other measures of adiposity (bmi-related measures, body weight, and central obesity) were statistically significant. conclusions. exercise is efficacious for reducing percent body fat in overweight and obese children and adolescents. insufficient evidence exists to suggest that exercise reduces other measures of adiposity.

PMC3886589

pubmed-709

mucormycosis is an aggressive invasive opportunistic fungal infection belonging to the order mucorales, usually found in immunocompromised individuals. it is a life and limb threatening fungal infection with high mortality rate of 40%.1 though mucormycosis presents as a spectrum of disease, it is very rare in the musculoskeletal system. almost all the immunocompromised patients with osteomyelitis of long bones due to mucormycosis require aggressive debridement and may sometimes need amputation.2 we present a patient with acute myeloid leukemia, who was diagnosed to have mucormycosis osteomyelitis of right proximal femur and was treated with limb salvage surgery successfully. a 41-year-old gentleman presented with sudden onset of pain and swelling over his right groin and upper thigh for a period of 10 days. he was a known patient with type ii diabetes mellitus and acute myeloid leukemia m2 diagnosed on the basis of bone marrow morphology and immunophenotyping 2 years ago. he received induction chemotherapy with cytosine and daunorubicin, followed by consolidation chemotherapy and peripheral autologous bone marrow transplant. fourteen months post bone marrow transplantation, he had remission and developed the above complaints. examination revealed mild tenderness over the right proximal femur with no significant restriction of movements. magnetic resonance imaging (mri) of the right hip revealed altered signal intensity in the right proximal femur with no cortical breach or evidence of abscess. his complaints gradually increased in the next 3 weeks and he had difficulty in weight bearing and walking. on clinical examination, he had severe tenderness over the right proximal femur with painful restriction of movements at the hip joint. plain radiographs revealed a 6 3 cm, ill-defined osteolytic lesion in the proximal shaft of right femur extending into the greater trochanter with wide zone of transition and adjacent soft tissue swelling [figure 1a]. mri showed altered signal intensity in proximal metadiaphysis of right femur, including greater trochanter, with focal collection of 5 2.6 cm with cortical breach. there were two small abscesses measuring 1.5 and 0.7 cm, respectively, in the soft tissues [figure 1b]. (a) plain radiograph revealing an osteolytic lesion involving the right proximal femur (b) mri showing altered signal intensity in the proximal metadiaphysis of right femur with abscesses and cortical breach (c) bone scan demonstrating increased tracer uptake in the metadiaphyseal region of right proximal femur bone scintigraphy with technetium 99 m methylene diphosphonate (tc 99 m mdp) demonstrated intense increase in tracer uptake in the proximal third of right femur with normal tracer distribution in rest of the skeleton, suggestive of an infective pathology [figure 1c]. intraoperatively, black discoloration of the right proximal femoral metaphysis with foul smelling discharge was noticed. the tissue was smeared with 10% koh solution and calcofluor white solution initially and then cultured in sabraoud's dextrose agar (sda) and lactophenol cotton blue (lpcb) mount which revealed broad aseptate fungal hyphae suggestive of mucormycosis. histopathology of the cancellous bone and soft tissue exhibited large areas of necrosis and hemorrhage, replaced by inflammatory granulation tissue with dense infiltrates of lymphocytes, plasma cells, histiocytes, neutrophils, and few multinucleate giant cells. there were many broad, twisted, and few branching fungal filaments suggestive of mucormycosis [figure 2]. histopathology with broad, twisted, and few branching fungal elements after the diagnosis of mucormycosis was confirmed, patient was evaluated for primary infection. there was no history of gastrointestinal infections and previous biopsy from gastric mucosa was negative. hence, primary source of infection could not be traced. in the immediate postoperative period, the patient developed pathological fracture at the same site and was put on skeletal traction for 19 days before definitive fixation. he was treated with liposomal amphotericin 3 mg/kg/day for 19 days, following which radical debridement of the lesion proceeded by skeletal stabilization with 95 condylar blade plate for the right proximal femur. considering the location of the pathological fracture (proximal femur) and the need for aggressive debridement and stable fixation, a 95 condylar blade plate fixation was achieved after adequate debridement. to prevent the usual complications of external fixation instead of infection such as pin tract infection, difficulty in mobilization and also considering adequate preoperative antifungal therapy and aggressive debridement, internal fixation was decided. 300 mg of fungitericin (amphotericin b) in powdered form was mixed with 40 g of simplex cement polymer and the mixture was then added with monomer, and while setting, cement beads were made into chains using stainless steel wire (two strings with 15 beads each) [figure 3a]. (a) postoperative plain radiograph with proximal femur stabilized with condylar blade plate and augmented with antifungal cement beads (b) three years followup plain radiograph with no evidence of osseous lesion with abundant callus intraoperatively, he had torrential blood loss amounting to 2.53 litres and went into hypovolemic shock. he was resuscitated with five units of packed red cells, eight units of cryoprecipitate, and eight units of fresh frozen plasma. radical debridement, inability to use the tourniquet, and difficulty in differentiating infected and uninfected marrow were the causes for unexpected blood loss. after aggressive debridement, bone to bone cortical contact was achieved for healing which led to loss of metaphyseal bone loss of 3 cm. postoperatively, liposomal amphotericin 3 mg/kg/day (180 mg/day) was given for 3 weeks and downgraded to 1 mg/kg/day (60 mg/day) for the next 9 weeks. patient had received a total dosage of 7560 mg of liposomal amphotericin for a total period of 12 weeks. he was advised postoperatively physiotherapy and toe-touch weight bearing crutch walking for 12 weeks. bone grafting was done along with beads removal and the tissue sent during surgery for histopathology and microbiological review was negative for mucormycosis. at 3-year followup, there was 3 cm limb shortening noticed for which a shoe raise was given. plain radiographs revealed healed fracture with no residual/recurrent lesion in the metadiaphysis region of right proximal femur [figure 3b]. the common predisposing factors are uncontrolled diabetes, hematological malignancies like leukemia, lymphoma, etc., organ transplantation, severe burns, diseases like tuberculosis and aids, post bone marrow transplantation, neutropenia, renal failure, immunosuppressive medications, etc. mucormycosis has been described in various sites like cranium, hands, and feet, humerus, tibiae, femur, vertebrae, and joints.1 the etiopathogenesis of mucormycosis follows after entry into the host via ingestion, inoculation, or inhalation. in the immunocompromised host, the spores undergo angioinvasion leading to local necrosis and necrotizing infection, ultimately leading to systemic inflammatory response syndrome, multiorgan dysfunction, and death. surgery includes radical debridement, mostly amputation, followed by high doses of amphotericin b. role of antifungal cement beads is limited mainly due to its cement elution properties and high local concentration leading to toxicity. multiple in vitro studies have reported amphotericin b impregnated pmma beads to have a successful role in the management of fungal infections.37 goss et al. in their study on elution and mechanical properties of amphotericin concluded that amphotericin was released from cement at a clinically insignificant level.8 in vitro studies on amphotericin beads have shown to provide adequate release of its concentration (1.75-2.0 microgm/ml) up to 110 days from all bone cements.3 excessive local concentration of amphotericin b above 100 microgm/ml is lethal and concentration between 5-10microgm/ml causes abnormal morphology and reduced proliferation.4 of the seven cases of mucormycosis of tibia described in the literature, five were immunocompromised and 60% of those patients underwent amputation.9 the causes of amputation may be related to delayed diagnosis of mucormycosis, compromised immune status, inadequate debridement, or inadequate antifungal therapy. we present this case in view of the unusual site of presentation, limb-threatening osteomyelitis which was treated with radical debridement, liposomal amphotericin therapy, and using antifungal beads to enable limb salvage. high index of clinical suspicion, early diagnosis, aggressive debridement, and adequate liposomal amphotericin b are the key treatment modalities in the successful management of musculoskeletal mucormycosis.

mucormycosis is one among the aggressive, invasive fungal infections usually seen in immunocompromised patients. mucormycosis osteomyelitis is very rare. we present a patient with acute myeloid leukemia who complained of pain over the right proximal thigh. plain radiograph revealed ill defined osteolytic lesion of proximal femur. mri showed altered signal in proximal femur with focal collection and cortical breach. biopsy and tissue culture diagnosed mucormycosis both histologically and microbiologically. he was treated with aggressive debridement, skeletal stabilization, and amphotericin antifungal cement beads. he recovered with no residual pain, minimal limb shortening, and no clinical or radiological evidence of recurrence at 3 years followup. the high index of suspicion, early diagnosis, aggressive surgical debridement, and adequate antifungal therapy play a significant role in the treatment of musculoskeletal mucormycosis.

PMC3377153

pubmed-710

in europe, prostate cancer (pca) is the most common malignancy in males and the third most common cause of cancer mortality. currently, more extensive and earlier diagnosis has led to a decrease in pca related mortality [1, 2]. despite an extended search for a more specific biomarker than prostate specific antigen (psa), psa still remains the only widely employed diagnostic and follow-up marker for pca. the predictive capability of the 4 ng/ml psa threshold as a biopsy indicator is deficient since 2040% of pca cases are thereby missed. thus, other accurate diagnostic pca biomarkers, especially for aggressive and potentially life-threatening pca, are needed. since increased aerobic glycolysis or the warburg effect has been identified as common to neoplastic cells, this mechanism promises potential for diagnostic and therapeutic targets. the enzyme transketolase-like 1 (tktl1), therefore, comes into investigational focus since it is a crucial enzyme for sugar fermentation, linking glucose and fat metabolism without pyruvate dehydrogenase [6, 7]. when overexpressed, tktl1 activates the pentose phosphate pathway (ppp), accelerating tumor cell growth and supporting tumor survival and systemic dissemination. accordingly, an assay was developed to evaluate tktl1 based on the fluorometric epitope detection of specific antibodies in cd14/cd16 positive monocytes, following tumor cell phagocytosis and digestion (edim-test). still, the role of tktl1 as a cancer biomarker is controversial [10, 11] and the edim-test has not been approved for routine clinical application. nevertheless, though tktl1 does not play a role in conventional medicine, it has gained high popularity in alternative/complementary medicine, not only as a diagnostic but also as a prediction marker to assess the risk of metastatic progression. the goal of the current investigation was to compare the tktl1 serum level in patients with a clinically localized pca to that in healthy controls. furthermore, the investigation was directed towards establishing whether the tktl1 serum level correlates with clinical and histologic parameters of the tumor, thus facilitating identification of patients harboring life-threatening disease requiring definitive treatment. for this purpose, the serum concentration of tktl1 in pca patients and healthy controls was analyzed by means of an enzyme-linked immunosorbent assay (elisa), which is a highly standardized detection system, and correlated to clinical and histologic parameters. patients (n=66) undergoing curative radical prostatectomy (rpe) for biopsy-proven pca in the department of urology, goethe-university, frankfurt am main, germany, were included in the investigation. controls (n=10) were healthy, age-matched, male volunteers. firstly, 10 ml peripheral blood was drawn from patients several days before surgery and from controls. blood samples were allowed to coagulate and then centrifuged at 3000 rpm at+4c for 10 minutes. the concentration of tktl1 was determined in pca patient and control serum using a commercially available elisa kit (seh018hu, cloud-clone corp, houston, tx, usa; sensitivity:<0.055 ng/ml with no significant cross-reactivity or interference between tktl1 and analogues). all assays were done in duplicate and the concentration was calculated from a standard curve using a 4-parameter curve fit (magellan software, tecan). univariate analysis was performed by the wilcoxon-man-whitney-test for comparison between two groups and the kruskal-wallis-test with the iman-conover-method (bonferroni-holm-corrected) for more than two groups. hanns ackermann, epsilon-publishers, frankfurt, germany). the null hypothesis (tktl1 concentration in serum of pca patients does not differ from that of healthy volunteers) was rejected if p-values were less than 0.05. the clinical tumor stage was classified according to the 7 edition of the ajcc and the pathologic tumor stage was determined according to the 6 edition of the tnm classification. imaging was carried out according to the currently valid guidelines of the european association of urology. the median age at tumor diagnosis was 66 years (range 4688) and the median serum psa was 8.0 ng/ml (range 1.857.0) (median age control group: 60 years (5572); control psa: 2.8 ng/ml (2.04.0). nearly all pcas submitted to surgery were clinically significant. none of the patients had evident clinical signs of infection or acute or chronic inflammation at surgery. clinical and histopathological demographics of 66 pca patients values expressed as median with range or number (%). rpe radical prostatectomy; dre digital rectal examination, psad psa density univariate analysis of the elisa investigation demonstrated that serum tktl1 was significantly lower in the serum of pca patients, compared to healthy controls (p=0.0001, effect size indicator r=z/sqr(n)=0.4179, figure 1). however, correlation between serum tktl1 and serum psa (p=0.38), biopsy and prostatectomy gleason sum (p=0.79 and 0.89, respectively), prostate volume (p=0.23), psa density (p=0.80), clinical and pathologic t-stage (p=0.66 and 0.65), highest gleason pattern in the biopsy and prostatectomy specimen (p=0.83 and 0.74, respectively), upgrade of gleason sum from biopsy to prostatectomy (p=0.86), pn-, l-, v-, n- and r status (p=0.32, 0.88, 0.30, 0.90 and 0.32, respectively), extracapsular extension, seminal vesicle invasion as well as d`amico classification (p=0.75, 0.89 and 0.34, respectively) did not reach statistical significance. tktl1 serum concentration (ng/ml) in pca patients and controls. box: lower line quartile q1 (25% quantile); middle line tktl1, as evaluated by the edim-test, has been propagated as a reliable cancer biomarker, since a positive correlation between tktl1 expression and tumor progression has been reported. however, investigations negating the reliability of tktl1 have also been published [11, 13], making a definitive assessment regarding tktl1 reliability as a biomarker questionable. indeed, the decreased serum tktl1 found in pca patients in the present investigation stands in opposition to the increase in monocyte associated tktl1 claimed by the proponents of the edim-test. however, the different methods and localities, where tktl1 was measured, in serum and in macrophages, could account for the differing results. speculatively, assuming the accuracy of the edim test together with the theoretical background proposed by coy and colleagues, an inverse correlation between tktl1 detected in macrophages and extracellular tktl1 in serum could occur, since tumor cells undergo phagocytosis. tktl1 could therefore be sequestered in macrophages and the tktl1 level in serum be reduced. still, this hypothesis is speculative and requires further evaluation. although, in the present investigation, serum tktl1 levels differed in cancer patients and healthy persons, no correlation was apparent between serum tktl1 and clinical or pathologic parameters. conflicting reports about tktl1 expression associated with various cancer entities and clinical outcome have been published by several investigators [10, 13, 14]. tktl1, determined with the same immunohistochemical method in colorectal cancer tissues, has been positively associated with tumor progression and tktl1-expression level in one study, whereas another study pointed to a significant decrease in tktl1-expression associated with metastasis. recently, the dna demethylating agent 5-aza-2'-deoxycytidine (5-aza) has been reported to augment expression of tktl1 in melanoma cells and was associated with enhanced invasion of the tumor cells, whereas others have demonstrated reduced invasion of melanoma cells under 5-aza treatment. grimm et al. correlated an increase of edim scores with a metabolic shift from aerobic to anaerobic conditions, whereas this correlation could not be confirmed by others. possibly, these inconsistencies have led to the recommendation of combining tktl1 quantification with a standardized panel of established blood biomarkers. the ambivalence of tktl1 expression in so many different investigations shows that the role of tktl1 may be more complex than initially thought. this study was designed as a pilot investigation and thus includes a limited number of patients. since patients only underwent a general health check, this could contribute to a potential, though unlikely, bias for the high serum tktl1 expression found in this cohort. the study was designed to assess the diagnostic potential of tktl1, not its prognostic ability. since pca, in most cases, is associated with slow progression, long-term follow-up would be required to follow the course of serum tktl1 during the course of the disease. serum tktl1 was decreased in patients with clinically localized pca, but failed to facilitate identification of patients with aggressive disease, who might particularly benefit from definitive cancer treatment. based on these results, we can not currently advise introducing serum tktl1 into clinical practice. further long-term studies including larger patient cohorts and simultaneous measurement of serum tktl1 and macrophage sequestered tktl1 are warranted to clarify the role of tktl1 in pca and resolve its applicability as a pca biomarker.

introductionmonocyte associated transketolase-like 1 (tktl1) as a cancer biomarker has become popular with alternative practitioners, but plays no role in conventional medicine. this investigation evaluates the potential of serum tktl1 as a biomarker for prostate cancer. material and methodspatients (n=66) undergoing curative radical prostatectomy (rpe) for biopsy-pro-ven pca were included in the study. controls (n=10) were healthy, age-matched, male volunteers. 10 ml of peripheral blood was drawn from patients several days before surgery and from controls. serum tktl1 was measured using the elisa method. resultsthe median age at tumor diagnosis was 66 years and median serum psa was 8.0 ng/ml. nearly 96% of pcas submitted to surgery were clinically significant. compared to healthy controls, serum tktl1 was significantly lower in pca patients (p=0.0001, effect size indicator r=z/sqr(n)=0.4179). no correlation was apparent between serum tktl1 and serum psa, gleason sum, tumor stage or further clinical and pathologic parameters. conclusionsreduced serum tktl1 in pca patients stands in opposition to tktl1 epitope detection in monocytes (edim) based studies, whereby increased tktl1 in monocytes of tumor patients has been reported. since serum tktl1 does not correlate with clinical parameters in the current investigation, further research is needed to clarify whether serum tktl1 has potential as a biomarker for pca.

PMC5057049

pubmed-711

recurrent aphthous stomatitis (ras) is one of the most frequently encountered oral mucosal disorders. despite extensive amount of research, the etiology of ras remains unclear. the aim of the study was to assess the levels of anxiety and salivary cortisol levels in patients with ras and also to determine the association and relationship of salivary cortisol levels to variations of stress. a total of 30 patients suffering with ras, along with the same number of age and sex matched healthy controls were included in the study. saliva was collected from all the subjects at 9.00 am to avoid diurnal variations of cortisol levels. student's t-test was used to compare the anxiety and salivary cortisol levels between both groups. the mean salivary cortisol level of the ras group showed a very highly significant difference (p=0.000) from the controls. the mean anxiety scores of the ras group showed a very highly significant difference (p=0.000) from the controls. the values of pearson correlation coefficient between anxiety and salivary cortisol was 0.980 and one with a p value of 0.000 showing that there is a highly positive correlation between anxiety and salivary cortisol. thus besides traditional treatment of ras patients, our findings suggest that psychological support is also needed. recurrent aphthous stomatitis (ras) is the most common type of ulcerative disease of the oral mucosa, and it affects approximately 20% of the general population with a range from 5% to 66%. highest prevalence of 66% was found by ship et al. on dental and medical students. minor ras, which makes up more than 80% of all ras cases, is a small (up to 1 cm in diameter), shallow, painful, well-circumscribed, and round-shaped ulceration that is covered with a yellow-grayish pseudo membrane and surrounded by an erythematous halo. major ras is characterized by ulcers that are typically larger and deeper than minor ras. herpetiform ulcers manifest as multiple recurrent clusters of small ulcers (less than 4 mm in diameter) that are scattered throughout the oral mucosa. previous studies have suggested that psychological disturbances such as stress and anxiety could play a role in the onset and recurrence of ras lesions. the worldwide distribution, high frequency and decreased quality-of-life generated by ras have resulted in a great deal of research into the etiology and efficient therapy of this disease. however, the etiology of ras still remains unclear, and the currently available therapy remains inadequate. several studies have reported a relationship between ras and various causes, but the results are conflicting. it is suggested that stress with its presumed effects on the immune system, constitutes one of the major causative agents of ras. an insight into a patient's psychological status can be estimated from both serum free and salivary cortisol levels. cortisol, also called as a stress hormone, has been used as an indicator in the stress evaluation studies. salivary cortisol may actually provide a better measure than serum cortisol of the stress response as it more accurately measures the amount of unbound cortisol compared to serum measures. salivary cortisol exhibits a clear diurnal variation and circadian rhythmicity with a time course closely parallel to that of plasma cortisol. various scales were used in the psychological assessment of patients in previous studies, some were self-reported and others assessed by a psychiatrist. though stress and anxiety have been mentioned as possible factors related to the development of ras; this association somewhat remains controversial. the aim of this cross sectional study was to conduct an investigation in assessing the relationship between anxiety and salivary cortisol in patients with ras by using both psychological testing instrument (hamilton's anxiety scale [has ]) and physiological testing instrument (salivary cortisol). the study was conducted in the department of oral medicine and radiology, kamineni institute of dental sciences, india, after approval by institutional ethics committee. thirty patients suffering from ras (17 females 57%, 13 males 43%) were taken for the study after an informed consent. the inclusion criteria for patients were as follows: minor form of ras, non-smokers, and a minimum of 2 years of ras history to rule out from other types of acute recurrent ulcers like recurrent herpes. patients suffering from systemic diseases including endocrine and metabolic diseases, hematinic deficiencies, patients using steroids/oral contraceptive pills, pregnant patients, smokers were excluded from the study. saliva samples were obtained from the study group when aphthous stomatitis was absent after healing. control and study group samples were collected between 9 and 9:15 am, before a meal without stimulation by passive drooling directly into a sterile glass tube until 5 ml is collected. the collected salivary samples were centrifuged for 15 min at 3000 rpm and frozen at -20c until shortly before assay. during assay, salivary cortisol was measured by competitive enzyme linked immunosorbent assay method, by using cortisol eia (diametra kit, korea). the normal cortisol concentrations that were given as a guide line according to the kit are in the ranges from 8.2 to 27.59 nmol/l (0.3-1 g/dl) at morning time and 1.65-6.89 nmol/l (0.06-0.25 g/dl) at evening collected samples. after saliva collection anxiety levels were measured by using has that provides the measures of overall anxiety, psychic anxiety (mental agitation and psychological distress) and somatic anxiety (physical complaints related to anxiety). this scale consists of 14 questions, among which seven address the psychic and the remaining seven addresses the somatic anxiety. the individuals are rated on a five point scale for each of the 14 items ranging from 0 to 4. the total anxiety score ranges from 0 to 56, where<17 indicates mild severity, 18-24 mild to moderate severity and 25-30 moderate to severe. t-test was used to compare the anxiety and salivary cortisol levels between patients with ras and the control group as the base line data was normally distributed. pearson's correlation analysis was used to study the correlation among anxiety and salivary cortisol levels in patients with ras. logistic regression analysis was used for calculation of each variable's independent contribution to dependent variable. ras was a dependent variable, and salivary cortisol and anxiety levels were independent variables in this model. for this trail 60 patients were enrolled. the ras group comprised of 13 males and 17 females and the mean age was 29.2 years (range 18-60 years). the control group comprised of the same number of age and sex matched individuals (mean age 29.2 years). the mean salivary cortisol levels were 47.73 8.80 nmol/l (1.73 0.319 g/dl) in ras patients and 13.905 3.55 nmol/l (0.504 0.129 g/dl) in controls with a p value of 0.000. the mean anxiety levels in ras group were 27 4.76 and 10.2 3.27 in the control group (p=0.000) as indicated in table 1. comparison of salivary cortisol levels and anxiety scores (meansd) of patients we found that salivary cortisol and anxiety levels were significantly higher in ras group as compared to control group. there was a highly significant positive correlation (p=0.000) between salivary cortisol levels and anxiety. a logistic regression model in which the ras was taken as dependent variable and salivary cortisol and anxiety levels were taken as independent variables were performed. salivary cortisol levels and anxiety scores were found significantly related with ras [table 2]. logistic regression analysis, in which the group membership (ras or control) was dependent variable the impact of oral disorders on quality of life has been increasingly recognized as an important outcome measure for clinical trials, especially since oral disorders frequently have detrimental effects on speech, nutrition, physical appearance, self-esteem and social interaction. ras frequently affects patient quality of life as a result of long lasting and recurrent episodes of burning pain. the etiopathogenesis of ras appears to be complex; interactions with genetic, nutritional and hematological factors are reported. much has now been clarified about the mechanisms involved, interesting new associations such as the involvement of t-cell mediated immunologic reaction have emerged. several reviews have, however, reported little objective evidence to support such an association. there is good evidence that stress and anxiety are related to increased resting levels of cortisol. the present study was undertaken in an attempt to gain a better understanding of the role that has been attributed to stress, anxiety and salivary cortisol in the development of ras and also to assess the relationship of salivary cortisol levels with stress and anxiety in patients with ras. a previous study mccartan et al. investigated the possible association between anxiety, measured by hospital anxiety and depression scale, and salivary cortisol in patients with ras; and concluded that stress may play a role in the etiology of ras. albanidou-farmaki et al. conducted a case control study and compared the association between state and trait anxiety, measured by spielberger's state-trait anxiety inventory; and serum and salivary cortisol levels in patients with ras; concluding that stress may be involved in the pathogenesis of ras. conducted a case control study to assess the influence of psychological stress on manifestations of ras; by means of a questionnaire developed by the psychology institute of sao paulo university (symptoms of stress list; vas visual analog scale questionnaire) and concluded that stress may play a role in the manifestation of ras. present study results also showed statistically significant increase in salivary cortisol and anxiety levels in ras patients during inactive stage compared to control group. our study in contrast to previous studies (2 and 5) used has, which provides the measures of overall anxiety, psychic anxiety (mental agitation and psychological distress) and somatic anxiety (physical complaints related to anxiety). stress and anxiety may play a significant role in the onset and recurrence of ras lesions. ras is typically observed during stressful situations such as school exam periods, dental treatments and periods of significant changes in life. stress alters the regulation of both the sympathetic and parasympathetic branches of the nervous system, with consequential alterations in hpa-hypothalamic-pituitary-adrenal axis. autonomic activation and elevation of hormones, including those produced by hpa axis play pivotal roles in regulating immune surveillance mechanisms. this immune regulatory activity with an increased number of leukocytes at the sites of inflammation induced by psychological stress is characteristic, often observed during the pathogenesis of ras. in conclusion, the present study showed a positive association between salivary cortisol levels and anxiety in ras patients during inactive stage. therefore, measurement of the salivary cortisol and anxiety which reflect response to stress seems a promising parameter in the investigation of ras. in conclusion, we suggest that stress management interventions may be beneficial along with conventional treatment methods in ras patients.

background and objectives: recurrent aphthous stomatitis (ras) is one of the most frequently encountered oral mucosal disorders. despite extensive amount of research, the etiology of ras remains unclear. psychological-emotional factors were considered as one of the major predisposing factors. the aim of the study was to assess the levels of anxiety and salivary cortisol levels in patients with ras and also to determine the association and relationship of salivary cortisol levels to variations of stress. materials and methods: a total of 30 patients suffering with ras, along with the same number of age and sex matched healthy controls were included in the study. saliva was collected from all the subjects at 9.00 am to avoid diurnal variations of cortisol levels. salivary cortisol levels were measured by competitive enzyme linked immunosorbent assay. anxiety levels of both groups were measured by using hamilton's anxiety scale. student's t-test was used to compare the anxiety and salivary cortisol levels between both groups. results:the mean salivary cortisol level of the ras group showed a very highly significant difference (p=0.000) from the controls. the mean anxiety scores of the ras group showed a very highly significant difference (p=0.000) from the controls. the values of pearson correlation coefficient between anxiety and salivary cortisol was 0.980 and one with a p value of 0.000 showing that there is a highly positive correlation between anxiety and salivary cortisol. conclusion:results suggest that anxiety may be involved in the pathogenesis of ras. thus besides traditional treatment of ras patients, our findings suggest that psychological support is also needed.

PMC4287781

pubmed-712

animal models utilizing human recombinant activated factor vii (rfviia; novoseven; novo nordisk a/s, bagsvrd, denmark) are limited by interspecies differences in terms of coagulation molecule interactions. the interaction between tissue factor and factor human factor vii has been shown to have reduced activity when it is exposed to porcine thromboplastin. these considerations are compounded by the fact that the majority of standard in vitro coagulation assays are performed with rabbit brain thromboplastin. both rabbit and human thromboplastin induce coagulation more efficiently in human plasma than in swine plasma. therefore, changes in coagulation parameters seen with the use of human rfviia in animals may not correlate with hemostatic end-points. because of these interspecies differences with regard to human rfviia activity, minimal conclusions concerning optimal dosing in humans can be drawn from animal experiments. in pharmacologic models the dose of rfviia needed for effect varies substantially between animal species, including the dog (60 g/kg), rabbit (2 mg/kg), and mouse (10 mg/kg). animals given 2 mg/kg rfviia had decreased time of bleeding and blood loss compared with control animals. the efficacy of reversing a bolus of low-molecular-weight heparin (lmwh) was studied in a rabbit ear puncture model. animals were given 1800 anti-factor x units/kg of lmwh, which raised the primary bleeding time approximately fourfold. the absence of efficacy was hypothesized to be secondary to very high anti-factor xa levels produced by bolus dosing of lmwh. the efficacy of rfviia in reversing the effects of coumadin and the direct thrombin inhibitor melagatran has also been studied in a rat tail bleeding model. the use of rfviia resulted in decreased blood loss in animals given warfarin once but it did not reduce blood loss significantly in animals given warfarin twice. in rats given melagatran, 1 mg rfviia resulted in a decreased bleeding time but no significant reduction in blood loss. the effect of rfviia administration on dissemination of clotting and thrombogenicity was investigated in the rabbit. in a model of stasis-induced thrombosis, administration of rfviia at a dose that enhanced clot formation at the site of injury resulted in no change in platelet count or the plasma concentration of antithrombin, indicating that rfviia was not associated with systemic activation of coagulation. in an experimental model of arterial thrombosis, rfviia given in concentrations that decreased ear bleeding time and blood loss from incision sites in the liver and spleen did not affect arterial thrombosis. currently, three swine trauma models exist in the literature for the study of the efficacy and safety of rfviia. these models include a grade v liver injury model, a liver avulsion model, and an infrarenal aortotomy model. in addition to differences in the mechanism and site of injury, these models also differ with respect to animal temperature and coagulation status, timing and rate of resuscitation, timing and dose of therapy, and use of conventional therapies. the first of these studies, reported by martinowitz and coworkers, utilized a specially designed clamp to create a grade v liver injury based on american association for the surgery of trauma organ injury scaling criteria (fig. 10 animals underwent a 60% isovolemic exchange transfusion with 6% hydroxyethyl starch and cooling to 33c. thirty seconds after injury, blinded therapy, consisting of either 180 g/kg rfviia or saline, was given and liver injuries were packed with gauze. resuscitation with 40c lactated ringer's was initiated at 250 ml/min 5.5 min after injuries. animals were resuscitated and maintained at their baseline blood pressure for the 1-hour study period. treatment with rfviia resulted in a statistically significant reduction in blood loss from 976 cc to 527 cc. treatment also resulted in significant reduction in prothrombin time, and elevation in the fvii clotting activity (fvii: c) was observed. postmortem analysis revealed no evidence of large clots in the hepatic veins or inferior vena cava and no evidence of microthrombosis. clamp designed to create a grade v liver injury based on organ injury scaling criteria from the american association for the surgery of trauma [12, 13]. in a follow-up study, schreiber and coworkers repeated the analysis with minor differences in the model used. the 60% isovolemic hemodilution was performed with 5% albumin instead of hydroxylethyl starch to avoid the potential coagulopathic effects of starch solutions. the creation of the dilutional coagulopathy resulted in a hypotensive state with a starting mean arterial pressure of 45 mmhg. blinded therapy, packing, and initiation of resuscitation were performed simultaneously at 30 s and animals were resuscitated with lactated ringer's at 100 ml/min. three groups of 10 animals were given either 180 g/kg rfviia, 720 g/kg rfviia, or an equivalent amount of buffer solution. similar to the study conducted by martinowitz and coworkers, animals treated with rfviia had significantly less blood loss than did controls (control animals 2187 ml, 180 g/kg group 1085 ml, and 720 g/kg group 1086 ml). nadir blood pressures following injury were significantly lower in control animals than in treated animals. treated groups also had significantly lower prothrombin times and higher thrombin antithrombin complexes, suggesting activation of thrombin. death occurred in four out of 10 controls, three out of 10 in the 180 g/kg group, and two out of 10 in the 720 g/kg group. these differences did not achieve statistical significance. the only difference between the animals that received 720 g/kg rfviia and 180 g/kg rfviia was a dose-dependent increase in fvii: c levels following delivery of drug. the same investigators also tested the efficacy of rfviia as sole therapy in noncoagulopathic, normothermic animals. in this study, 30 animals were randomly assigned to receive either 150 g/kg rfviia or an equivalent volume of buffer solution 30 s after injury. animals were resuscitated with warmed lactated ringer's solution at 100 ml/min initiated 15 min after injury to maintain their baseline blood pressure and normothermia (38c) for the 2-hour study period. liver injuries were not treated in this model. despite documentation of a significant increase in fvii: c and decrease in prothrombin time, there was no difference in blood loss between groups. blood pressure curves were similar and thrombin antithrombin complexes became equally elevated between groups throughout the study. potential explanations for the failure of rfviia to reduce blood loss in this model included the use of the drug as a sole agent in this large venous injury model and the normotensive state of the animals at the start of the study compared with the hypotensive state produced in the dilutional coagulopathy model. lynn and coworkers described a reproducible grade iv liver injury whereby a clamp was utilized to crush and avulse the left median liver lobe and major vessels of the left lateral lobe. in the first study using this model, 13 animals were randomly assigned to receive 180 g/kg rfviia or blinded placebo given when the mean arterial blood pressure dropped by 10% of baseline. blood loss in the treatment group was 33 ml/kg versus 27 ml/kg in the control group (p=0.2). mortality was 43% in the control group and 0% in the treatment group (p=0.08). these investigators used the same model to study 24 animals divided into three groups: control (group 1); 180 g/kg rfviia (group 2); and 720 g/kg rfviia (group 3). the study drug was again given following a 10% drop in mean arterial pressure following grade iv liver injury. death occurred in four out of eight in group 1, two out of eight in group 2, and none of eight in group 3 during the first hour (p=0.02 for control versus 720 g/kg). all deaths in the first two groups occurred within 22 min and the only death in the third group occurred at 116 min. there was no evidence of microthrombosis or macrothrombosis within viewed sections of kidney, mesentery of small intestine, small intestine wall, lung, heart, or brain in the treated groups. the final animal study described in this review was designed by sondeen and coworkers to test the efficacy of rfviia in producing stronger clots and elevating the blood pressure at which rebleeding occurs in a pig aortotomy model. by elevating the blood pressure at which rebleeding occurs, rfviia would theoretically permit more effective resuscitation without increasing blood loss. thirty pigs were randomly assigned to a control group, a 180 g/kg group, or a 720 g/kg group. blinded treatment was administered 5 min before injury. a skin biopsy punch was used to make a 2 mm hole in the infrarenal aorta. resuscitation was initiated 10 min after injury with lactated ringer's at 100 ml/min. if rebleeding did not occur before the mean arterial pressure achieved a plateau after administration of 4 l resuscitation fluid, animals were given epinephrine (adrenaline) to raise the blood pressure to a maximum of 200 mmhg. the rebleeding blood pressure in the control group was significantly lower (45 mmhg) than that in the 180 g/kg group (69 mmhg) and the 720 g/kg group (66 mmhg). there was a trend toward greater rebleed volume in the control group (39 ml/kg in controls versus 22 ml/kg and 26 ml/kg for the 180 g/kg and 720 g/kg groups, respectively). four of 10 animals required epinephrine in the high-dose group versus one out of 10 in each of the other two groups. significantly more animals in the control group (nine out of 10 animals) rebled at mean arterial pressures below baseline than in the rfviia groups (two out of 10 animals). rfviia-treated animals received significantly more fluid resuscitation before rebleeding than did control animals. similar to prior studies, fvii: c exhibited a dose-related increase in the two treatment groups. cohort studies in humans have suggested that there is a population of patients who do not respond to treatment with rfviia. this group of patients includes those extremely ill individuals with the lethal triad of hypothermia, coagulopathy, and acidosis who are on pressor therapy. it also includes those with irreversible hemorrhagic shock, who are unlikely to respond to any therapy. based on these findings, meng and coworkers attempted to determine the effects of temperature and acidosis on factor viia activity utilizing blood from healthy adults. factor viia activity on phospholipid vesicles and on platelets was measured by determining factor xa generation in the presence and absence of tissue factor. tissue factor dependent factor xa generation increased with an increase in temperature from 24c to 37c, whereas tissue factor-independent factor xa generation decreased to an equal degree. alternatively, reduction in ph resulted in marked diminution in factor viia activity from both tissue factor dependent and independent mechanisms. the authors concluded that acidosis effects overall factor viia activity but that hypothermia does not. the efficacy and safety of rfviia has been demonstrated in multiple small and large animal models. because of species specific differences in the interaction between human rfviia and animal tissue factor, comments regarding dosing remain guarded. however, there does appear to be a maximum dose above which no additional benefit is achieved. published ex vivo data suggest that rfviia may not be effective in severely acidotic environments. preclinical data fvii: c=factor vii clotting activity; lmwh=low-molecular-weight heparin; rfviia=recombinant activated factor vii.

preclinical studies in animals and ex vivo human blood have provided a solid rationale for conducting prospective randomized trials in trauma patients. small animal models have been utilized to study the efficacy of recombinant activated factor vii (rfviia; novoseven) in treating thrombocytopenic rabbits and for the reversal of anticoagulation. safety models in the rabbit also exist to test for systemic activation of clotting and pathologic thrombosis. animal models simulating traumatic injuries in humans have primarily been performed in pigs because of species similarities in terms of coagulation characteristics and the larger internal organs. the pig studies, utilizing human rfviia, have shown increased strength of clot formation, decreased bleeding, and improved survival. however, these findings are not uniform and are dependant on the model chosen. all of the animal models described have provided good safety data and suggest that the use of rfviia is not associated with systemic activation of coagulation or microthrombosis of end organs.

PMC3226120

pubmed-713

fish viscera are one of the sources of digestive enzymes that may have some unique properties of fascinate with both basic research and industrial applications. their survival in waters required adaptation of their enzyme activity to low temperatures of their habitats. that is to say, fish proteinases have higher catalytic efficiency at low temperatures than those from warm-blooded animals [1, 2]. in addition, the strong positive correlation between the habitat temperature of marine fish and the thermostability of its trypsin has been demonstrated [311]. high activity at low temperatures and instability against heat, low ph, and autolysis of fish proteinases are interesting for some industrial applications. cod trypsin is already practically used in food production and cosmetics [13, 14]. furthermore, pacific cod and atlantic cod trypsins were utilized as catalyst of enzymatic peptide synthesis [9, 15]. on the other hand, lipids in the tissue conventional methods for the removal of lipids from materials involve cooking, pressing, and liquid extraction. on liquid extraction for enzyme preparation, it is usually used with organic solvents, such as hexane, ethanol, and acetone, and so forth [16, 17]. however, the removal of lipids with organic solvents causes protein denaturation and/or loss of functional properties. organic solvents are also highly flammable and are toxic for human health. consideration of such factors has led investigators to apply supercritical fluid extraction techniques to the lipid separation. carbon dioxide (co2) is a popular supercritical extractant particularly in food processing, flavor and aroma isolation, and pharmaceuticals manufacture, because co2 is nontoxic and does not leave a residue. supercritical co2 (sco2) has been used for extraction of oils from some marine organisms [2022]. but, the aims of these studies were mainly the gain of oils rich in polyunsaturated fatty acids, especially epa and dha. so, the application of sco2 for isolation of enzymes and production of quality protein meal from different sources should be examined. recently, we prepared a defatted powder of squid viscera treating with sco2 and detected protease, lipase, and amylase activities in crude extract from the powder. next, we purified a phospholipase a2 from the starfish pyloric ceca defatted by sco2 extraction process. in this study, with the aim of utilization of fish trypsin for food industry, we purified a trypsin (sc-t) from the mackerel viscera powder treated by sco2 defatting process and compared its enzymatic properties with those of other fish trypsins purified from the viscera defatted by acetone. mackerel (scomber sp.) were caught off busan, republic of korea. the mackerel viscera were collected from f&f co., busan, republic of korea, and the visceral waste was brought to the laboratory in iced condition. the co2 (99.99% pure) was supplied by kosem, korea. n-p-tosyl-l-arginine methyl ester hydrochloride (tame) and ethylenediaminetetraacetic acid (edta) were obtained from wako pure chemicals (osaka, japan). 1-(l-trans-epoxysuccinyl-leucylamino)-4-guanidinobutane (e-64), soybean trypsin inhibitor, n-p-tosyl-l-lysine chloromethyl ketone (tlck), and pepstatin a were purchased from sigma chemical co. (st. louis, mo, usa). the defatted powder of mackerel viscera was prepared as described by chun et al. the lipid extraction by sco2 was performed at temperature of 45c and pressure of 25 mpa. the total extraction time was 2.5 h. the sco2 defatted powder was stored at 60c until further analysis. trypsin was extracted by stirring from 10.0 g of defatted powder in 50 volumes of 10 mm tris-hcl buffer (ph 8.0) containing 1 mm cacl2 at 5c for 3 h. the extract was centrifuged (h-200, kokusan, tokyo, japan) at 10,000 xg for 10 min, and then the supernatant was concentrated by lyophilization and used as crude trypsin (50 ml). ten milliliters of crude trypsin was applied for four times to a column of sephacryl s-200 (3.9 64 cm) pre-equilibrated with 10 mm tris-hcl buffer (ph 8.0) containing 1 mm cacl2,and proteins were eluted (0.8 ml/min) with the same buffer. each main trypsin fractions were gathered and concentrated by lyophilization. then the concentrated fraction (10 ml) was applied to a sephadex g-50 column (3.9 64 cm) pre-equilibrated with the above buffer, and proteins were eluted (0.7 ml/min) with the same buffer. a single trypsin fraction was pooled and used as purified trypsin (sc-t). trypsin activity was measured by the method of hummel using tame as a substrate. one unit of enzyme activity was defined as the amount of the enzyme hydrolyzing one micromole of tame in a minute. the effect of inhibitors on trypsin was determined by incubating trypsin with an equal volume of proteinase inhibitor solution to obtain the final concentration designated (0.1 mm e-64, 1 mg/ml soybean trypsin inhibitor, 5 mm tlck, 1 mm pepstatin a and 2 mm edta). after incubation of the mixture at 25c for 15 min, the remaining activity was measured, and percent inhibition was then calculated. the ph dependencies of trypsin were determined in 50 mm buffer solutions [acetic acid-sodium acetate (ph 4.07.0), tris-hcl (ph 7.09.0), and glycine-naoh (ph 9.011.0)] at 30c. the temperature dependencies of trypsin were determined at ph 8.0 and at various temperatures. the temperature and ph stabilities of trypsin were found by incubating enzyme at ph 8.0 for 15 min at a range of 2080c and by incubating the enzyme at 30c for 30 min at a range of ph 4.011.0, respectively. the effect of cacl2 on trypsin activity was found by incubating the enzyme at 30c and at ph 8.0 in the presence of 10 mm edta or 10 mm cacl2. sds-page was carried out using a 0.1% sds-13.75% polyacrylamide slab gel by the method of laemmli. the gel was stained with 0.1% coomassie brilliant blue r-250 in 50% methanol-7% acetic acid, and the background of the gel was destained with 7% acetic acid. to analyze the n-terminal amino acid sequence of sc-t, the enzyme was electroblotted to a polyvinylidene difluoride (pvdf) membrane after sds-page. the amino acid sequence of the enzyme was analyzed by using a protein sequencer, procise 492 (perkin elmer, foster city, calif, usa). the viscera of mackerel (scomber sp.) were treated by sco2 to separate lipids on the condition of 40c, 25 mpa, and 2.5 h. since sco2 extracted almost all oil from the squid viscera in the previous study, we adopted the condition to remove lipids from the mackerel viscera. trypsin was then extracted from 10.0 g of defatted powder by sco2, and the crude enzyme was prepared. as shown in table 1, the crude enzyme contained 1,390 mg of total protein and 1,049 u of total trypsin activity. previously, we extracted trypsin from the pyloric ceca powder (13.9 g) of spotted mackerel defatted by acetone, and 3,633 mg of total protein and 3,270 u of total trypsin activity were detected in the crude enzyme solution. although these data were not compared directly, the yields of protein and trypsin activity per weight of acetone powder were approximately two times higher than those of sco2 powder. however, the specific activity (0.8 u/mg) of crude enzyme in this study is almost the same as that (0.90 u/mg) in the previous study. so, it is thought that the difference of total activity might come from the variation of specimen, and the defatting condition with sco2 in this study would not cause significant denaturation of fish trypsin. the sc-t was purified from the crude enzyme solution by two steps of chromatographies including sephacryl s-200 and sephadex g-50, which is the same purification procedure as that in the previous study. the sc-t was consequently purified 48-fold with a high recovery (50%) from the crude enzyme solution (table 1) and had a specific activity of 36 u/mg which is fairly higher than that of spotted mackerel trypsin. in addition, the sc-t was found nearly homogeneous on sds-page (figure 1), and its molecular weight was estimated as approximately 24,000, which is similar to that of spotted mackerel trypsin. furthermore, the n-terminal twenty amino acids sequence of sc-t was analyzed to be ivggyectpysqpwtvslns that accords with that of spotted mackerel trypsin. these results also show that the sco2 defatting process in this study removes lipids in fish viscera as effectively as acetone defatting process for preparation of fish trypsin. crude enzyme extract usually contains various proteins, and sometimes fish trypsin is composed of some isozymes. in general, the purification of fish trypsin was carried out by the combination of some types of chromatography [2830]. however, we achieved a high purification of a trypsin from the mackerel viscera powder defatted by acetone using only two steps of gel filtration. so, in this study, we purified the sc-t by gel filtration according to the previous study. the sc-t was strongly inhibited by specific trypsin inhibitors (soybean trypsin inhibitor and tlck), but e-64 (cysteine proteinase inhibitor), pepstatin a (aspartic proteinase inhibitor), and edta (metalloproteinase inhibitor) had no inhibitory effect on the activity of sc-t (table 2). the influence of ph on the sc-t activity is shown in figure 2(a). the enzyme hydrolyzed tame substrate effectively between ph 7.0 and 9.0, with an optimum around ph 8.0. the optimum ph of sc-t was the same as those of other fish trypsins [311, 3138], but lower than those of bluefish (ph 9.5) and atlantic bonito (ph 9.0). the sc-t was active over a broad temperature range (2070c) with the optimum at about 60c. because mackerel is a temperate-zone fish, the sc-t possesses similar optimum temperature with other trypsins from temperate-zone fish, such as anchovy, true sardine, yellow tail, and jacopever. the optimum temperature sc-t is slightly lower than those of tropical-zone fish (around 65c) [3335, 39, 40] but is evidently higher than those of frigid-zone fish trypsins (around 50c) [4, 610]. the ph stability of sc-t is shown in figure 3(a). the sc-t was stable at 30c for 30 min in the ph range from ph 6.0 to 11.0. unlike mammalian trypsins, diminished stability of the trypsin was more pronounced after exposure at acidic ph. instability at acidic ph was also observed for other fish trypsins [1, 311, 3235, 37, 39, 40]. for temperature stability, the sc-t was stable below 40c, but the activity quickly fell over 50c (figure 3(b)). while the sc-t and other temperate-zone fish trypsins are relatively less stable than tropical-zone fish trypsins, they are obviously stable than frigid-zone fish trypsins [8, 10]. as described previously, there is a strong relationship between habitat temperature of marine fish and thermostability of their trypsins [8, 10]. the effect of calcium ion on the stability of sc-t was then investigated. the stability of sc-t was enhanced by calcium ion (figure 4). similar results have been reported for various fish trypsins [1, 311, 3335, 39, 40]. bovine trypsinogen has two calcium-binding sites, and the primary site, with a higher affinity for calcium ions, is common in trypsinogen and trypsin and the secondary site is only in the zymogen [41, 42]. occupancy of the primary calcium-binding site stabilizes bovine trypsin toward thermal denaturation or autolysis [41, 42]. in the previous paper, we described trypsin of arabesque greenling which also has the primary calcium-binding site. the sc-t was stabilized by calcium ion from denaturation in this study. so, the result suggests that the sc-t may possess the primary calcium-binding site like bovine and arabesque greenling trypsins. with the aim of utilization of fish trypsin for food industry, we purified a trypsin (sc-t) from mackerel (scomber sp.) viscera powder treated by the sco2 defatting process and compared its enzymatic properties with those of other fish trypsins purified from the viscera defatted by acetone. in this study, we adopted the condition of 40c, 25 mpa, and 2.5 h to separate lipids from the viscera. consequently, we could remove most of the lipids from the viscera and could extract considerable amount of trypsin from the defatted powder. the characteristics of purified sc-t were nearly the same as those of other fish trypsins, especially spotted mackerel trypsin. therefore, we concluded that the sco2 defatting process is useful as a substitute for the organic solvent defatting process.

viscera of mackerel (scomber sp.) were defatted by supercritical carbon dioxide (sco2) treatment. trypsin (sc-t) was then extracted from the defatted powder and purified by a series of chromatographies including sephacryl s-200 and sephadex g-50. the purified sc-t was nearly homogeneous on sds-page, and its molecular weight was estimated as approximately 24,000 da. n-terminal twenty amino acids sequence of sc-t was ivggyectahsqphqvslns. the specific trypsin inhibitors, soybean trypsin inhibitor and tlck, strongly inhibited the activities of sc-t. the ph and temperature optimums of sc-t were at around ph 8.0 and 60c, respectively, using n-p-tosyl-l-arginine methyl ester as a substrate. the sc-t was unstable below ph 5.0 and above 40c, and it was stabilized by calcium ion. these enzymatic characteristics of sc-t were the same as those of other fish trypsins, especially spotted mackerel (s. borealis) trypsin, purified from viscera defatted by acetone. therefore, we concluded that the sco2 defatting process is useful as a substitute for organic solvent defatting process.

PMC3268041

pubmed-714

twenty-one animals (16 cattle and 5 sheep) showing clinical signs suggestive of bluetongue were sampled by the federal agency for the safety of the food chain on august 18, 2006, at 11 farms in northeastern belgium. two serologic tests that detect antibodies against the major serogroup antigen vp7 (bluetongue virus antibody competitive elisa [celisa]; veterinary medical research and development inc., pullman, wa, usa and competitive vp7 bluetongue kit; idvet, montpellier, france) identified 21 virus-positive animals. two newly developed and validated reverse transcription quantitative pcrs (rt-qpcrs) that detected btv strains representing the 24 serotypes (4) were then conducted to determine whether these seropositive animals also had viral rna. the first assay (rt-qpcr_s1), which amplified a 357-nt fragment in segment 1, detected virus in erythrocytes of the 21 seropositive animals (mean cycle threshold [ct] value 29.0). the second assay (rt-qpcr_s5), which amplified a 94-nt fragment in segment 5, detected virus in the same 21 seropositive animals (mean ct value 26.5). the 2 serologic tests and the 2 molecular assays detected btv in 21 animals from 11 belgian farms within 14 hours. virus isolation was conducted on august 18, 2006, by injection of blood from infected sheep into 11-day-old embryonated chicken eggs, followed by passage on bhk-21 cells (atcc-ccl10) as previously described (5). the specificity of the cytopathic effect observed 48 hours after passage on bhk-21 cells was confirmed by rt-qpcr and electron microscopy (figure 1) after fixation and negative staining as previously described (6). two virus neutralization tests were conducted on 2 virus strains isolated by the belgian and the french reference laboratories at 2 belgian farms 30 km apart. each strain was also partially neutralized by reference serum against serotype 18, which confirmed cross-neutralization between serotypes 8 and 18 (7). to september 14, 2006, the study farms were screened for animals with clinical signs of bluetongue. blood samples were tested by serologic tests or rt-qpcr. for cattle, 97 (68%) of 142 samples had antibodies to btv and 32 (78%) of 41 samples contained viral rna (table 1). however, for sheep, only 23 (29%) of 79 samples had antibodies to btv and 15 (45%) of 33 samples contained viral rna. other diseases that cause similar signs might explain this lower frequency in sheep. contagious ecthyma was diagnosed by using pcr and electron microscopy for several sheep that showed bluetonguelike signs but did not have antibodies to btv or viral rna. agreement between celisa and rt-qpcr results was analyzed for 124 animals (table 2). one sample negative by rt-qpcr_s1 and rt-qpcr_s5 was positive by celisa. although this result might reflect lack of specificity of the celisa, elimination of btv rna from the animal several weeks after being infected can not be ruled out. a false-negative result in the rt-qpcr is unlikely because 1) 2 different rt-qpcrs that amplified 2 different segments were used, 2) the quality of the rna was confirmed by a third rt-qpcr that quantified mrna of -actin, and 3) both rt-qpcrs are highly sensitive (they can detect 0.01 infectious doses of virus) (4). false-positive results were not observed with the idvet celisa when we analyzed 650 negative serum samples from artificial insemination centers and field samples collected from belgian livestock in 2004 and 2005. seven animals with bluetonguelike clinical signs were positive according to each rt-qpcr but negative according to the celisa (table 2). these results support the finding that rt-qpcr can be used to detect viral rna in infected animals before antibodies are detectable. celisa, competititve elisa; rt-qpcr, reverse transcription quantitative pcr. samples with different results in celisa and rt-qpcr_s5 were retested with rt qpcr_s1 (4). this latter test always confirmed the result of the rt-qpcr_s5. on september 14, 2006 (4 weeks after the first identification of btv in belgium) as many as 84 belgian farms had at least 1 btv-infected animal. the maximal distance between herds in this study was 110 km (figure 2a). most outbreaks were confirmed in the area where the disease was initially detected (area i, figure 2). most (64%) infected animals showed a high virus load with individual ct values<30. of the remaining animals, 30% had moderate virus loads (ct values 3035) and 6% had ct values>35. the high ct values for the latter animals might have remained undetected had pooled blood samples been analyzed. thus, results of pooled samples need to be validated before being used for diagnosis. none of the animals from zones iii and iv showed a ct value<30, whereas all animals from zone ii showed low ct values, which are indicative of high virus loads. lower virus loads and acute clinical signs in animals from zones iii and iv might indicate onset of infection. however, we can not rule out decreased infection in these animals because they also had positive serologic results that indicated infection for at least 45 days. further epidemiologic studies are required before conclusions can be drawn on the evolution of these epidemics. a) distribution of outbreaks of bluetongue (shaded areas) reported in belgium from august 18 through september 14, 2006. b) cycle threshold (ct) values observed in different zones as a result of conducting reverse transcription btv has been detected and its isolation and characterization have considerably progressed in the first weeks of the epidemics. results of virus neutralization tests for 2 belgian isolates and molecular characterization of the dutch btv strain by the community reference laboratory (8) indicate that btv serotype 8 is present in belgium and the netherlands. although this observation suggests 1 serotype circulating in northern europe after a common virus introduction, it must be confirmed by detailed epidemiologic studies. northward spread of bluetongue in europe has been correlated with climate warming (9). however, one characteristic of the current epidemics of bluetongue is the severity of clinical signs reported in cattle (10). the present results also demonstrate that clinical signs observed in cattle are more specific than those observed in sheep. confusing clinical signs in sheep underline the need for developing diagnostic tests to discriminate between bluetongue and other confounding diseases such as contagious ecthyma, border disease, and foot-and-mouth disease. our results also indicate the usefulness of rt-qpcr, which detected viral rna in recently infected animals with clinical signs of bluetongue but no detectable antibodies to btv. the rt-qpcr and elisa are independent but complementary tests because they detect viral rna and virus-specific antibodies, respectively. these tests indicated that an outbreak of bluetongue was occurring in belgium. despite high sensitivity of rt-qpcr (4), our results suggest that using this test with pooled samples might not detect animals with low viral loads. rt-pcr positive results in animals that are no longer infectious (11) should also be considered before deciding whether pooled samples are acceptable.

bluetongue has emerged recently in belgium. a bluetongue virus strain was isolated and characterized as serotype 8. two new real-time reverse transcription quantitative pcrs (rt-qpcrs) that amplified 2 different segments of bluetongue virus detected this exotic strain. these 2 rt-qpcrs detected infection earlier than a competitive elisa for antibody detection.

PMC2725968

pubmed-715

within the last half century, there has been remarkable progress in the treatment of psychiatric disease. the first chemical antidepressants were discovered fortuitously in clinical trials designed to treat tuberculosis.1 since then, researchers in both academia and industry have developed an impressive array of related compounds, which work through similar mechanisms but treat a host of different psychiatric conditions.2 these developments have led to an abundance of pharmacotherapies that have dramatically increased the number of available treatment options for psychiatrists. unfortunately, currently available drugs still produce numerous deleterious side effects and, for a substantial number of patients, are largely ineffective in alleviating their disease symptoms. consequently, there remains a strong need for more effective, targeted pharmacotherapies to treat psychiatric disease. a better understanding of the neurobiological processes that underlie psychiatric disease states is expected to help drug development efforts. accordingly, there has been a considerable effort to identify biological variables that consistently differ between healthy individuals and those with disease.3 studies in humans have implicated various circuits in specific psychiatric disease states using neuroimaging, genomic, and post mortem tissue analyses.4 however, data produced from these studies are largely post hoc in nature and can not be used to conclusively infer cause and effect. the attribution of causality to specific neuronal circuitry or reliable biomarkers has been hampered by technological limitations and overall etiological complexities.5 in order to determine if a specific biological process is integral to a psychiatric condition, it must be experimentally manipulated in preclinical models of the disease. to this end, numerous animal models of psychiatric disease states have been developed.6 one approach to modeling psychiatric disease in an animal is to use a behavioral task in which an animal s performance is sensitive to drug treatments that have already proved to be effective in humans.7 when new drugs produce behavioral effects that are similar to those elicited with proven pharmacotherapies, they are likely to have similar mechanisms of action to the old drugs and be similarly effective in treating the disease. this strategy provides strong predictive validity in modeling treatment of disease states and has been successfully used to screen novel compounds. however, its utility may be limited to compounds that act via the same mechanisms as those used to initially develop the behavioral model. an additional caveat to this line of research is that predictive validity in modeling treatment does not necessarily mean that the etiology or mechanisms underlying the disease in humans are relevant to the given model. thus, while animal models of psychiatric disease offer a powerful approach to understand symptomatology, they may be less able to provide information about the neural processes that are ultimately responsible for the initiation of disease states. the unclear neuronal substrates of psychiatric diseases and correspondingly slow progress of novel drug development have resulted in pharmaceutical companies recently abandoning development of psychiatric compounds.8 to reinvigorate research, it may be necessary to take a more refined technological approach to the study of disease symptoms and treatment. the maladaptive behaviors associated with complex psychiatric disorders are likely the result of specific aberrant processes in discrete, albeit multiple, neural circuits. the development of optogenetics has provided researchers with a powerful set of tools to manipulate neural activity in genetically defined populations of cells in a pathway-specific manner.9 in applying optogenetic strategies to established animal models of psychiatric disease, investigators have made rapid progress in defining the neural circuitry responsible for both psychiatric disease and symptom relief. optogenetic approaches enable researchers to activate or inhibit groups of neurons, which can be defined by genetic identity and/or projection target. this is made possible through the targeted expression of light-sensitive proteins known as rhodopsins, which are responsive to specific wavelengths of light. rhopdopsins are protein complexes that contain an opsin (channel) and a light-sensitive cofactor (retinal).10 the opsins used in optogenetic strategies are generally derived from the type 1 class of prokaryotic rhodopsin proteins that support a covalent bond with an internalized retinal molecule.11 the ionic environment around this covalent bond influences the spectral sensitivity of the protein, whereas the amino acid residues around it play a significant role in channel kinetics.12 photon absorption causes a conformational change in the rhodopsin protein, via retinal isomerization, which allows for ion translocation across the channel. opsins are broadly categorized based on their depolarizing or hyperpolarizing effects. depolarizing opsins, or channelrhodopsins (chrs), permit the translocation of cations into the intracellular space upon photon absorption and retinal isomerization. channelrhodopsin-1 (chr1) was the first of this type to be characterized, but it produces relatively small photocurrents and is not widely used.13 channelrhodopsin-2 (chr2) has a higher photocurrent than its predecessor and has proved to be quite effective at altering neuronal firing patterns in mammalian tissue.14,15 it is maximally activated by 470 nm wavelength light and can sustain reliable firing up to 40 hz. multiple variants of chr1 and chr2 have recently been developed through point mutations and chimeric complexes in an attempt to improve or alter ion permeability (catch), channel kinetics (cheta), and wavelength selectivity (c1v1)12,16 (see table 1 for a list of notable variants). the creation of red-shifted opsins, such as c1v1, has enabled independent excitation of two depolarizing opsins in the same tissue.17,18 in addition, longer wavelengths mitigate light scatter and absorption, thus promoting deeper tissue penetration.19 in general, the photocurrent generated from opsin activation decays quickly following light cessation. this feature is an asset for most experimental designs, but certain behavioral paradigms benefit from prolonged manipulations. variants known as stabilized-step function opsins significantly extend the open-state of the channel from a single pulse of blue light, which promotes a sustained depolarized state. this state increases the likelihood of firing in response to endogenous input, and can be inactivated with a single pulse of yellow light.20 recent step function opsin variants can induce depolarized states that last as long as 30 minutes following a single activating pulse of light.21 hyperpolarizing opsins, in contrast, are more limiting in both their overall characteristics and variety. most hyperpolarizing opsins have an activation profile that is significantly red-shifted in comparison with the standard depolarizing chrs. potent inhibition can be achieved through the pumping of chloride ions into the neuron through halorhodopsin (eg, enphr3.022) or via expelling protons from the intracellular space using archaerhodopsin (eg, earch3.016). however, despite their effectiveness, pumps are not energetically efficient, owing to their one proton to one ion photocycle,23 and do not alter the cell s input resistance.24 additionally, sustained halorhodopsin activation can disrupt chloride gradients, leading to a change in gamma-aminobutyric acid type a (gabaa) receptor reversal potential, thus eliciting a post-inhibitory excitatory state.25 recently, however, it has been shown that point mutations in a chimeric chr protein can convert it into a chloride conducting channel.26,27 the slow mutant variant, known as slow chloc, possess a 10-second long open-state during which neurons are effectively silenced.26 longer periods of neuronal inhibition can be achieved with a pharmacogenetic approach that utilizes designer receptors that are exclusively activated by designer drugs.28 in addition to the advances in opsin-mediated excitatory and inhibitory neuronal activity, there has been success in engineering chimeric opsin proteins that directly alter intracellular signaling cascades through g-protein coupled receptors.29 this class of proteins are known as optoxrs, and they have been designed to incorporate bovine rhodopsin (type ii opsin) into endogenously expressed adrenergic30 and serotonergic31 receptors. as a result of these developments, optogenetics provides a temporally refined and multifaceted toolkit of opsins allowing various controls on neuronal activity within genetically defined neuronal cell types. to achieve selective opsin expression, dna constructs encoding the opsins, often fused with a fluorescent protein marker (eg, yellow fluorescent protein), are introduced into neurons in an anatomically restricted manner, commonly by transgenic introduction, viral injection, or in vivo electroporation.3235 expression of opsins within neurons can be further restricted with the use of selective promoters or dna recombination systems. for example, one popular expression protocol involves the use of cre-recombinase, for its ability to invert gene orientations based on cre-identifiable tags (lox sites) that flank opsin genes. thus, cell-specific opsin expression can be achieved using transgenic animals that express cre under a cell-specific gene promoter (eg, tyrosine hydroxylase), followed by viral infection carrying a payload with the opsin gene in an inverted orientation. as a result, only neurons expressing cre will be able express the opsin gene since it will have been inverted to its correct orientation. once the proteins are expressed, they will diffuse within the membranes of cells and traffic to distal neural processes. this allows for the direct manipulation of spiking activity, either through stimulation of cell bodies or their distal processes. thus, there are multiple dimensions by which photostimulation can be restricted, ie, anatomical location of dna delivery, cell-type specific expression, and localized light delivery. when these approaches are used together, anatomically localized, genetically defined neural pathways can be repeatedly stimulated or inhibited, in vitro and in vivo. current treatments for clinical depression include pharmacotherapies that directly increase synaptic levels of serotonin and/or norepinephrine via reuptake inhibition. these two neurotransmitter systems have diffuse projections throughout the brain and are involved in a variety of functions, which may be the reason why individual patients respond to different antidepressant drugs in a highly variable manner. while antidepressant drugs act on these entire systems, it is presumed that only a subset of these projections are ultimately responsible for the therapeutic actions.36,37 this appears to be an area ripe for optogenetic analyses. however, antidepressant drugs typically require weeks of medication before symptoms alleviate, which complicates experimental designs and raises questions about the direct role of serotonin/norepinephrine in the therapeutic effects of antidepressant medications. the initial optogenetic explorations of depression have instead attempted to dissect a nonpharmacological treatment for depression known as deep brain stimulation, a treatment that has been explored as an alternative therapeutic option for severely depressed individuals. deep brain stimulation via implanted electrodes that target frontal cortical areas and the nucleus accumbens has been demonstrated to relieve symptoms of intractable depression in human subjects.3842 it is unclear if there is a common neurobiological substrate mediating the therapeutic effects of electrical brain stimulation and antidepressant medication. unlike drug-induced antidepressant effects, symptom relief following deep brain stimulation has a rapid onset. however, interpretation of the immediate effects of electrical stimulation is problematic, because it is not obvious how neural activity is altered by this manipulation. electrical stimulation elicits nonspecific activation of all neuronal cell types and processes in a small area around the electrode. since frontal cortical areas integrate information from throughout the brain and have projections that are similarly diffuse, it is difficult to attribute therapeutic effects to specific neural pathways. as such, electrical stimulation lacks the resolution needed to precisely define which local networks and cell types are responsible for the observed therapeutic changes. researchers attempts to dissect stimulation-induced antidepressant effects with optogenetic tools have focused on alleviating depression in animal models of the disease. one of these animal models is known as the forced swim test, where laboratory rodents are placed in tanks of water and scored for time spent actively struggling versus passively floating.43,44 antidepressant treatments increase the amount of time that animals spend struggling. other models of interest expose animals to stressful events, which creates a behavioral phenotype that is sensitive to chemical antidepressant treatments.45 optogenetic studies employing these animal models (summarized in table 2) demonstrate that photostimulation of neurons in the medial prefrontal cortex can elicit antidepressant-like behavior in both forced swim and chronic social defeat models of depression.4649 additional research has demonstrated that optogenetic stimulation directed to the specific medial prefrontal cortex projections that innervate the dorsal raphe nucleus is sufficient to produce antidepressant-like effects.48 the dorsal raphe nucleus is the principal source of ascending serotonin projections and the neurotransmitter system that most antidepressant pharmacological treatments act upon.50 thus, convergent lines of evidence implicate the dorsal raphe nucleus and its regulation by the medial prefrontal cortex in the treatment of depression. despite the strong evidence implicating the medial prefrontal cortex-dorsal raphe nucleus circuit in treating symptoms of depression, many ambiguities remain regarding its precise role in this process. this pathway appears to preferentially excite gabaergic interneurons in the dorsal raphe nucleus,46,5153 suggesting that excitatory medial prefrontal cortex input to the dorsal raphe nucleus may act to depress serotonin levels via feed-forward inhibition. consistent with this idea, pharmacological inhibition of the medial prefrontal cortex has been shown to enhance both stress-induced serotonin activity and learned helplessness behavior.54 collectively, these optogenetic and pharmacological studies support the provocative conclusion that acute decreases in serotonin function are capable of producing antidepressant-like effects. similar behavioral effects have been demonstrated by reducing serotonin function via dorsal raphe nucleus negative-feedback autoreceptor activation55 or overexpression.56 these findings go against studies in humans in which acute depletion of serotonin synthesis decreases mood in people with a history of major depressive disorder. responses in some behavioral models might be produced by any perturbation of serotonin signaling, regardless of directionality of effect. in contrast with the forced swim test, optogenetic research exploiting the chronic social defeat model of depression has supported the more conventional conclusion that enhancing serotonin neuron activity in the dorsal raphe nucleus reduces depression-like behavior. specifically, social defeat experiences have been demonstrated to sensitize gabaergic interneuron function in the dorsal raphe nucleus, and photoinhibition of this population of neurons prevents the acquisition of defeat-induced social avoidance.57 further, bidirectional manipulations of medial prefrontal cortex-dorsal raphe nucleus circuitry can modulate defeat behavior in positive or negative ways, accordingly.46 a greater understanding of these behavioral models and the microcircuitry of the dorsal raphe nucleus will help explain how different neural inputs are integrated in this region of the brain. other medial prefrontal cortex projection targets, in addition to the dorsal raphe nucleus, have recently been explored as well. optogenetic stimulation to the medial prefrontal cortex projections to the nucleus accumbens have been shown to produce antidepressant-like effects in mice that exhibited depressive symptomatology following cholecystokinin infusions into the medial prefrontal cortex.58 this work highlights a potential role for the nucleus accumbens in depression, which is a region that has been somewhat overlooked in relation to this disease state. while the nucleus accumbens does receive serotonin and norepinephrine input,59,60 it is most prominently innervated by dopaminergic fibers. photoactivation of midbrain dopamine neurons that innervate the nucleus accumbens has been shown to elicit antidepressant-like effects in both the tail suspension and forced swim tests.61 on the other hand, activation of accumbens-projecting dopamine cell bodies exacerbates the effects of social defeat stress,62 so this system may not have a simple role in depressive symptomatology. indeed, midbrain dopamine neuron stimulation has been reported to produce opposite effects on sucrose preference in different studies, which suggests that specific conclusions may be more attributable to experimental parameters than depression in general.6163 more research is needed to clarify how dopamine and its downstream effectors regulate mood in relation to chronic depression. anxiety disorders are generally characterized by excessive feelings of apprehension in the absence of any immediate threat. this category of disorders is broad, as it includes generalized anxiety, obsessive-compulsive, and post-traumatic stress disorders.64 modern medications used to treat anxiety disorders largely target serotonin and gabaergic neurotransmitter systems; however, drugs that block norepinephrine function appear to have particular utility in post-traumatic stress disorder.65,66 research into the neurobiology of anxiety has largely focused on the amygdala and its extended compartment in the bed nucleus of the stria terminalis (bnst), as these regions are critical for the manifestation of fear. early studies in laboratory animals found that electrical stimulation of the amygdala produced behavioral and autonomic responses that resemble a state of fear, whereas lesion or inactivation of this region inhibited the expression of experimentally induced anxiety and conditioned fear.67 the basolateral amygdala, where multiple pathways converge, appears to be the locus of associative fear learning. neural activity here is acutely sensitive to the presentation of previously learned, fear-evoking stimuli.68 neurons in the basolateral complex innervate the central nucleus of the amygdala, which is the primary output structure of the amygdala nuclei. this region regulates the behavioral and physiological manifestations of fear.69,70 the bnst, which primarily receives input from within the amygdala, serves as an additional output relay station.71 recent optogenetic studies have extended this model of amygdala circuitry and highlighted a role for this structure in behavioral measures of anxiety (figure 1). photostimulation of the basolateral amygdala is sufficient to induce fear, and any stimuli paired with this stimulation can become fear-inducing.72 in addition, activity in this region is critical for the consolidation of fear memories.73 these studies have supported the classical view of the amygdala as a critical site of fear learning. other optogenetic studies have broadened this view by showing that neighboring projections out of the basolateral amygdala can mediate opposing effects on fear and anxiety. photostimulation of basolateral complex projections to the central lateral amygdala reduces anxiety in the elevated plus maze,74 whereas projections to the ca1 region of the hippocampus increase these and other measures of anxiety.75,76 this work underscores the importance of characterizing the role of adjacent structures and discrete projections. indeed, the hippocampus is similarly nonuniform, as photostimulation of the ventral, but not dorsal, dentate gyrus area elicits a robust anxiolytic effect.77,78 anxiety regulation within the extended amygdala is also complex. the bnst contains two parallel output pathways to the ventral tegmental area (vta), ie, a gabaergic pathway that is anxiolytic and a glutamatergic pathway that is anxiogenic.79 the behavioral and physiological components of anxiety can be dissociated via other bnst projections as well. stimulation of bnst to lateral hypothalamus projections produces avoidance of open arms in an elevated plus maze, but has no effect on respiration rate. conversely, stimulation of bnst to parabrachial nucleus projections increases respiratory rate without altering plus maze behavior.80 stimulation of bnst cell bodies directly can produce various responses that depend on the exact location and genetic cell type targeted by the stimulation, factors that likely influence which output pathways are preferentially recruited. beyond the extended amygdala, a role for the lateral septum in anxiety has also been demonstrated with optogenetics. although this structure has been classically associated with anxiolytic responses,81 optogenetic stimulation of cells expressing corticotropin-releasing factor receptor type 2 produces anxiogenic effects.82 this effect was demonstrated to occur via gabaergic projections to the anterior hypothalamus. the hypothalamus contains oxytocin-producing neurons that project to the amygdala and participate in the regulation of anxiety. selective stimulation of this pathway reduced expression of conditioned fear by directly activating gabaergic neurons in the central lateral amygdala.83 obsessive-compulsive (ocd) disorder is a specific subset of anxiety disorders that is characterized by repetitive behaviors aimed at relieving anxiety related to intrusive, irrational thoughts. as with general anxiety disorders, research into ocd has revealed a role of striatal circuitry in the expression of stereotyped, repetitive actions. a genetic mouse model of ocd has been developed in which mice lack the postsynaptic scaffolding protein sapap3.84 this gene is expressed strongly in the striatum, and its deletion results in defective corticostriatal circuitry. mice lacking the gene exhibit high levels of anxiety and excessive grooming behavior, both of which are reversed with chronic antidepressant treatment. dorsal striatal medium spiny neurons in these mutant mice exhibit exaggerated responses to the presentation of cues associated with grooming.85 photostimulation of lateral orbitofrontal cortical projections to the dorsal striatum attenuated this striatal neuron hyperactivity by activating fast-spiking interneurons in the striatum. other recent studies that have examined the orbitofrontal projections to the ventral striatum in wild-type mice found that photostimulation of this pathway can increase grooming.86 while this phenomenon was only produced following chronic stimulation, it persisted for 2 weeks and could be reversed by chronic antidepressant treatment. these studies highlight the importance of corticostriatal circuitry in ocd, but future studies need to clarify where stimulation could be targeted to ultimately alleviate symptoms of the disorder. indeed, a recent study implicates the more lateral orbitofrontal cortex in controlling the shift from habitual to goal-directed actions.87 drug abuse disorders are characterized by compulsive drug use in the face of adverse consequences. highly addictive substances seem to disrupt the processes of self-control associated with normal reward-seeking behavior. initial research mapping out regions of the brain involved in reward processing and motivation began over a half century ago with electrical intracranial self-stimulation experiments.88 recent studies that have used optogenetic techniques to extend this research have already confirmed that stimulation of midbrain dopamine neurons in the vta and substantia nigra pars compacta is highly reinforcing.8992 although the predominant effect of vta dopamine neuron stimulation is to reinforce and motivate operant behavior, an aversive subcircuit has also been identified, that consists of vta dopaminergic neurons receiving input from the lateral habenula and projecting to the medial prefrontal cortex.93 in addition, photoactivation of gabaergic interneurons in the vta has proved to be aversive.94,95 this research has largely confirmed ideas that have been developed over the preceding years, but a new level of detail is starting to emerge. the reinforcing effects of midbrain dopamine neuron activity are believed to be mediated primarily via projections to the striatum,9698 although it has been recently shown that vta-habenula projections also contribute to this effect.99 within the striatum, multiple glutamatergic inputs, in addition to the dopaminergic pathway, can reinforce instrumental behavior.100102 projections out of the striatum are segregated into two populations based on the expression of certain receptor proteins in the efferent neurons as well as projection targets.103105 optogenetic activation of these neuronal populations produces opposing behavioral effects, as photostimulation of dopamine d1 receptor-expressing neurons (direct pathway neurons) produces reward-related behavior and photostimulation of dopamine d2 receptor-expressing neurons (indirect pathway neurons) produces aversion.106 more refined behavioral studies are necessary to expand upon this simple reward/aversion dichotomy. as individuals repeatedly abuse addictive substances, addictive behaviors develop that are marked by a loss of self-control. research into the mechanisms of this process has focused on the neural plasticity that addictive drugs produce within reward circuitry.107110 within the striatum, drug exposure appears to preferentially potentiate glutamatergic inputs onto direct pathway striatal neurons.109,111 individual animals with greater potentiation onto the other projection neurons in the striatum, ie, the indirect pathway neurons, show resistance to compulsive drug use.111 following prolonged withdrawal, potentiation of formerly silent basolateral amygdala-striatum synapses is prominent.112 pharmacological reversal of this potentiation reverses behavioral manifestation of incubation of drug craving. numerous pathways, however, appear to be sensitive to drug exposure. within the dorsomedial nucleus accumbens, drug-induced synaptic potentiation has also been shown to occur within the glutamatergic hippocampus-striatum pathway.101 in addition, direct pathway striatal neurons (dopamine d1 receptor-expressing), which project back to the vta and target gabaergic interneurons, are also potentiated by repeated drug use.113 several studies have focused on plasticity occurring within the medial prefrontal cortex. individual rats that exhibit compulsive drug-seeking show intrinsic hypoactivity of medial prefrontal cortex neurons,114 while presynaptic enhancement occurs in the prefrontal cortex-accumbens projection.115 the causal role of cortical areas in addiction appears to be highly complex. whereas 1 hz stimulation of prefrontal cortex cell bodies inhibits compulsive drug-seeking,114 inhibition of the prefrontal cortex-accumbens pathway reduces both reinstatement of cocaine-seeking116 and compulsive alcohol self-administration.117 furthermore, depotentiation of the prefrontal cortex-accumbens pathway reverses cocaine-induced psychomotor sensitization.109 thus, while optogenetics have allowed for the ability to study pathway specificity in drug-induced plasticity, future studies are crucial for determining the role of parameters such as drug type and schedule in mediating this plasticity. schizophrenia and autism involve a constellation of symptoms that are not easy to model in rodents. one avenue of research into schizophrenia has focused on neurobiological markers seen within human populations, rather than attempting to recapitulate behavioral models of specific symptoms.118 for example, cortical gamma oscillations (3080 hz), which are altered in schizophrenic patients, can be driven by optogenetic stimulation of fast-spiking cortical interneurons.119,120 this manipulation alters the gating of sensory information, which is a common symptom of schizophrenia. a related line of research examines how disruptions in the cellular balance of excitation and inhibition within neural networks underlie deficits in social behavior.121 recent research has found that increasing excitatory, but not inhibitory, input to pyramidal neurons of the prefrontal cortex causes social and cognitive disturbances in rodents.21 this experiment utilized a step function opsin in conjunction with red-shifted opsins to simultaneously alter excitatory and inhibitory components in cortical microcircuits. autism spectrum disorders can manifest as repetitive actions and reduced social interaction, which are symptoms that animal studies have tried to address in relation to ocd8587 and depression.4649 as the symptomatology of many psychiatric diseases overlap, some rodent behavioral studies have the potential to address common underlying circuit disturbances. however, there are numerous and obvious differences between people suffering from autism spectrum disorders and those with ocd. for example, people with autism are generally not bothered by their repetitive behaviors, whereas compulsions in ocd are a significant source of anxiety. additional studies are needed to delineate the neural circuit disruptions that may be common across disorders versus those that are unique to specific psychiatric diseases. significant progress has also been made with optogenetic tools in counteracting neural circuit disruptions that underlie certain neurological disorders, such epilepsy and parkinson s disease, which are comparatively easy to model in animals. epileptiform activity can be completely shut down with photoinhibitions targeted to excitatory neurons as well as with photostimulations targeted to inhibitory neurons.122124 this work is comparable with the advances seen in parkinson s disease, in which movement deficits have been minimized in animal models through photostimulations and inhibitions targeted to discrete pathways and groups of neurons.125127 the research on neurological disorders benefits from having straightforward behavioral assays that are directly relevant to the treatment of human disease. the use of genetically-encoded, light-sensitive proteins, which can activate and inhibit discrete neural circuits, is revolutionizing behavioral neuroscience. when integrated with established techniques, ie, brain imaging, genetic manipulations, behavioral assays, and electrophysiology, optogenetics can help uncover the circuitry responsible for complex psychiatric disease states. to date, these tools have been used preclinically to advance our understanding of these phenomena in both rodent models and in nonhuman primates. there has been early success with this research in delineating previously overlooked neural pathways that should be a target in future drug discovery efforts. however, a great deal of clarification is still needed in this work, particularly in the cases where different publications have supported disparate conclusions. as our understanding of the neurobiology of various psychiatric disease states advances, optogenetic tools may eventually be used as therapeutic agents in human patients. this research promises to advance therapeutic drug development and to help refine the groupings of diverse symptomatology present within many psychiatric diseases.

there have been significant advances in the treatment of psychiatric disease in the last half century, but it is still unclear which neural circuits are ultimately responsible for specific disease states. fortunately, technical limitations that have constrained this research have recently been mitigated by advances in research tools that facilitate circuit-based analyses. the most prominent of these tools is optogenetics, which refers to the use of genetically encoded, light-sensitive proteins that can be used to manipulate discrete neural circuits with temporal precision. optogenetics has recently been used to examine the neural underpinnings of both psychiatric disease and symptom relief, and this research has rapidly identified novel therapeutic targets for what could be a new generation of rational drug development. as these and related methodologies for controlling neurons ultimately make their way into the clinic, circuit-based strategies for alleviating psychiatric symptoms could become a remarkably refined approach to disease treatment.

PMC4114904

pubmed-716

in recent years, various strategies have been adopted for specific drug delivery to well-defined sites of the gastrointestinal (gi) tract, the colon being the most important one [15]. enteric polymers are used for this purpose, as they are able to release the drug at a particular ph. the ph-sensitive copolymers, such as methacrylic acid/methyl methacrylate copolymers and eudragit types l and s, dissolve in aqueous media at ph 6 and 7, respectively, which may be equivalent to drug release in the distal ileum. similarly, chitosan-based polyelectrolyte complexes have been employed as potential carrier materials in drug delivery systems. furthermore, a growing interest in polyelectrolyte complexes has led to the formulation and characterization of systems involving a variety of anionic and cationic polymers: eudragit l 30 d-55 and gelatin, eudragit l 100-eudragit s 100, eudragit e-eudragit l [10, 11], eudragit e-sodium alginate, chitosan-alginate/chitosan-carrageenan (mainly kappa-carrageenan with low amounts of lambda-carrageenan), chitosan-polygalacturonic acid, chitosan-carboxymethylcellulose, and chitosan-alginate. conventional drug delivery is unfavourable to special cases where drug targeting is applied, that is, when avoidance of gastric dissolution or targeting to the colon is desirable. colon-targeted drug delivery differs from ordinary enteric coatings (that are designed to merely avoid drug release in the stomach) in that the tablet or capsule is specially formulated to channel greater quantity of drug release to the colonic compartment, thus preventing or highly reducing drug release until the dosage form reaches the colon. although the large intestine is difficult to access through peroral delivery, it is still favoured as the appropriate site to tackle local colon-related diseases. colon-targeted delivery could be achieved by the use of ph-dependent systems, time-dependent systems, colonic microflora-activated systems and use of prodrugs. anti-inflammatory, antibacterial, antiamebic, protein drugs, are a few out of other drugs that can be targeted for site-specific delivery to the colon. ibuprofen is a nonsteroidal anti-inflammatory agent belonging to the group of propionic acid derivatives; it presents a plasmatic half-life of 1.82.0 h; as a result, it has to be administered three to six times a day, making this drug a suitable candidate for a controlled release formulation. the swellability properties of ipecs prepared from chitosan (cs) and eudragit l 100-55 (l 100-55) have been evaluated for their possible pharmaceutical application as new carrier for oral colon-specific drug delivery systems (ddss). similarly, a comparative study of ipecs of chitosan with eudragit l 100 and eudragit l 100-55 as potential carriers for controlled oral delivery of diclofenac sodium has been undertaken. however, to the best of our knowledge, there is no scientifically reported study on chitosan-eudragit rl-100 (cs-el) polyelectrolyte complexes of ibuprofen. thus, this study was designed to investigate the formation of ipec between cs and el, to characterize the product formed, and to evaluate its performance as a matrix for controlled release of drugs, using ibuprofen (ibf) as a model. ibuprofen (basf, germany), acetic acid, acetone, ammonium acetate, maize starch, magnesium stearate, lactose, concentrated hydrochloric acid (bdh, england), sodium hydroxide (merck, germany), and monobasic potassium phosphate (sigma chemical co., usa) were used as purchased from the manufacturers without further purification. chitosan of low viscosity nd was fines were retaine (fluka, switzerland) and eudragit rl 100 (mw 135,000) (rohm pharma, germany) were preliminarily dried at 40c under vacuum for two days. the ipec of cs and el was prepared following the standard procedures with slight modifications [12, 2327]. chitosan 300 mg was accurately weighed and dissolved in 15 ml of 3% v/v acetic acid followed by the addition of 8 ml volume of 5 m ammonium acetate. similarly, eudragit rl 100 (300 mg) was separately dissolved in 7 ml ethanol and was covered to prevent evaporation. the mixture was poured in a petri plate and was dried at 50c for 48 h. films with a total polymer content of 2.5% w/v containing 60: 40, 50: 50, and 40: 60 (i.e., 3: 2, 1: 1, and 2: 3) ratios of chitosan: eudragit rl 100 were prepared using this method. a control batch (el) containing only eudragit rl 100 was also prepared. ibf granules (average weight 297.3 mg) containing 200 mg of ibf were prepared by wet granulation technique using cs: el interpolymer complexes (50: 50 w/w) as binder. 10 (1.7 mm mesh) and was dried at 50c for about 1 h until all the moisture was removed. 16 (1.0 mm mesh) and was stored in a desiccator until used. magnesium stearate (1% w/w) (lubricant) and lactose (bulking agent) were added to the granules. tablets were compressed using 4 mm biconvex punches in a single station tablet compression machine (manesty, england) at a pressure of 50 kg/cm. the formulated ibf tablets containing cs: el (50: 50 w/w) as binder were coated with aqueous solutions containing (50: 50, 60: 40, and 40: 60 w/w) of cs: el ratio as ipecs films. the coating solution was sprayed at a rate of 5 ml/min with the help of peristaltic pump using a spray gun of 1 mm nozzle in a coating pan (12 diameter) being rotated at 18 rpm. the inner surface of coating pan was modified by attaching inert tubes (8 mm diameter) from the centre to the periphery for easy rolling of tablets, thereby ensuring efficient mass transfer of polymer. a control batch coated with eudragit rl 100 was also prepared. the degree of swelling of films of the ipec was investigated simulating the physiological conditions of the gastrointestinal tract [2327]. for this purpose, the films were placed in a preweighted basket of the dissolution equipment and immersed for 2 h in 30 ml of 0.1 m hydrochloric acid, then 10 ml of 0.20 m tribasic sodium phosphate was added to ph of 6.8 0.05, and after additional 3 h, another 10 ml of phosphate buffer ph 7.4 was added and the experiment was allowed to continue for another 19 h, giving a total of 24 h. the temperature of the medium was 37 0.5c. the measurements consisted in removing the basket from the medium, blot-drying by filter paper, and weighing in an analytical balance (mettler al 204, mettler-toledo int. inc. the degree of swelling was calculated using the formula (1)h(%)=(m2m1)m1100, where m1 is the initial weight of the film (g) and m2 is the final weight of the swollen film (g). twenty tablets from each batch were weighed together and individually, and the mean weight and percentage deviation were calculated according to british pharmacopoeia. the tablets were set to rotate at 25 rpm for 10 min in an erweka friabilator. the friability was calculated according to the formula (2)friability=(w1w2)w1, where w1 is the initial weight and w2 is the final weight. the force required to break each tablet was determined using a monsanto-stokes tablet tester. the average force of the ten tablets was taken as the crushing strength (kgf). three tablets were randomly selected from each batch and were placed in the inner compartment of a disintegration apparatus (which was tied with a thermoresistant thread to the clamp of a retort stand) of the disintegrating apparatus containing 500 ml of distilled water maintained at 37 1c. the medium was stirred at 150 rpm and the time taken for the tablets to disintegrate was recorded. the test was performed in triplicate for each batch, and the average time for each batch was calculated. in vitro release of ibuprofen from the tablets was performed using usp (dissolution apparatus 1-basket method) at 37 0.5c and 100 rpm in three release media (ph 1.2, 6.8, and 7.4). each tablet was placed in the cylindrical basket of a dissolution apparatus (veego, india) attached to the rotating spindle suspended in the dissolution medium of volume of 900 ml (ph 1.2). the rectangular glass container into which the one-litre cylindrical plastic container was immersed was filled with sufficient water to get more than half of the cylindrical container immersed in the water. the heating element in it the equipment was switched on to rotate at a speed of 100 rpm. at predetermined time intervals, 5 ml samples of the dissolution medium were withdrawn and were assayed spectrophotometrically (uv/vis, unico, usa) after appropriate dilution and filtration. meanwhile, two hours were chosen to mimic the average gastric emptying time. at the end of the 2 h, the equipment was switched off the rotating spindle attached to the basket-bearing tablet was unscrewed out and properly rinsed of the previous medium after carefully removing the tablet. the cylindrical plastic material containing the dissolution medium was also disposed of the ph 1.2 medium and adequately rinsed with purified water. then, 900 ml of a second dissolution medium, ph 6.8, was emptied into the 900 ml plastic container and the temperature allowed to attain 37 0.5c. the spindle was screwed back in place and dissolution run as before but for 3 h. the average time for change of dissolution medium was about 20 min. three hours was chosen because the reported average intestinal transit time is 3-4 h. at the end of 3 h, the medium was again removed and replaced with a third medium of ph 7.4 to mimic the ileocecal ph and the same process was repeated but this time until the tablet released all or nearly all the drug. previous studies indicate that polymers did not interfere with the determination of the model drug, ibf [2327]. the withdrawn samples were immediately analyzed using a spectrophotometer at 221 nm, 272 nm, and 281 nm for the release study in the ph 1.2, 6.8, and 7.4 medium, respectively. administration of nsaids such as ibuprofen is usually associated with gastrointestinal disturbances [12, 2327]. thus, research efforts have been directed to solve, or at least improve, this inconveniences, through various techniques of protection of the gastric mucosa or alternatively of preventing the release of nsaids in the gastric region. the site-specific delivery of drugs to the colon can be highly advantageous for various applications including the local treatment of inflammatory bowel diseases (ibds). in this study, various ipecs, formed between el and cs, were obtained and evaluated as potential colon-targeted oral controlled release matrices for ibf, a model nsaid. the ipecs films were formulated by nonstoichiometric method, and tablets containing ibf and ipecs were prepared by wet granulation technique. the formulations were evaluated in terms of friability, hardness, disintegration, swellability, and drug dissolution. here, the liquid ethanol was employed for dissolving the el so as to enable its proper incorporation into the cs to form the ipecs. lactose was selected as the bulking agent, maize starch as the disintegrant, and magnesium stearate as the lubricant. the mean weight of the various batches of the tablets (table 1) ranged from 296.32 0.30 mg to 301.57 0.93 mg. this shows that all the batches met compendial requirement for weight variation [28, 29], implying that these tablets were uniform in weight. table 1 equally indicates that average times of 55.97 2.84, 70.25 1.63 and 60.81 3.87 min each was required for tablets containing respectively 1: 1, 2: 3 and 3: 2 ratios of cs and el, and 35.79 2.45 min for el only-based tablets to disintegrate at the experimental conditions. this (disintegration time) test was performed to determine the ease with which ibf is released from the tablets at a controlled temperature of 37 1c. the results indicate that the adhesive force existing between the components of the tablets of batch 2: 3 is more than that in the tablets of batch 3: 2 and lowest in the tablets of batch 1: 1. the reason for this is uncertain, but may be attributed to greater concentration of the el on the tablets of batch 2: 3 than tablets of batch 3: 2. this implies that el exerted significant effect on the force of adhesion of the tablet ingredients, thereby increasing the disintegration time. the data equally revealed that the tablets of batches 2: 3 and 3: 2 demonstrated greater sustained release effect than tablets of batch 1: 1. it is also likely that high concentration of el and cs in the tablets of batch 2: 3 and 3: 2, respectively, was responsible for this. more so, the low disintegration time of the tablets of batch 3: 2 suggests that these tablets have prospects of dose dumping. furthermore, the friability test was carried out to determine the ability of the tablets to withstand mechanical shock or abrasion. low values of friability indicate high resistance to abrasion and good binding/adhesion properties [28, 29]. the friability test result revealed that all the batches met compendial requirement for resistance to abrasion, with tablets of batch cs: el (2: 3) and batch el having the greatest (0.95 0.01%) and least (0.72 0.03%) resistance to abrasion, respectively. in addition, the crushing strength test was undertaken to determine the level of resilience of the tablets to crushing when a force is applied. the crushing strength results show that tablets of batch cs: el (2: 3) possessed the highest mean crushing strength of 4.71 0.32 kgf followed by tablets of batch cs: el (1: 1), which is 4.62 0.09 kgf. the lowest crushing strength of 4.15 0.27 kgf was observed in tablets of batch el. the implication is that tablets of batch cs: el (2: 3) have higher adhesive force than tablets of batch cs: el (1: 1), and this force holds the components of these tablets together such that they are not easily broken. tablets of batch el have the least force of adhesion, and thus these tablets are easily broken. for compressed tablets, a crushing strength 5 kgf is considered the upper limit of acceptance and since none of the batches of the tablets exceeded this value; then they are acceptable. it is well known that the potential of polymeric carriers to be used as controlled release materials can be predicted by determination of their swelling characteristics. in a previous study, a group of researchers evaluated the swelling behavior of polycomplex matrices made from cs and el 100 in simulated gastro-intestinal tract (git) and all systems used were stable in ph 1.2 (1 h) and ph 6.8 (2 h). according to the specifications of degussa, the dissolution of el depends on the copolymer structure and is well regulated by the ratio between methyl methacrylate or ethyl acrylate and methacrylic acid. figure 1 shows degree of swelling at equilibrium and time of swelling for the different ipec films. in figure 1, h1.2 and t1.2 represent the degree of swelling at equilibrium at ph 1.2 and the time of swelling, respectively. similarly h6.8 and t6.8 also represent the degree of swelling at equilibrium at ph 6.8 and the time of swelling, respectively. the swelling profiles are similar: increasing degree of swelling in acidic medium due to a progressively increasing number of ionized nh3 groups of cs and decreased swellability for systems containing el, probably due to leaching of undissolved particles of el. the swelling behavior of ipecs films is completely different from that of the el only-based films (figure 1). in these systems, the electrostatic repulsion of free ionized amino groups is responsible for swelling. in case of ipec made up of cs: el 2: 3, the degree of swelling was 150% at ph 1.2, but afterwards a two-fold increase in swelling at ph 6.8 could be observed. on immersing the polycomplex matrix into the acidic medium (ph 6.8), free amino groups got protonated and their hydration increased the degree of swelling within the first part of the experiment. later, full ionization of all amino groups turned it into a polyelectrolyte with a relatively high charge density. as a result, the structure of the ipec is changed because the ionic bonds are not fixed and they could move from one electrostatic site to another [30, 31]. the protonated carboxylic acid groups of el (weak polyacid) became charged by ionized amino groups of cs to form new interpolymer contacts. comparable observations were made with ipec prepared from two types of eudragit [911] and eudragit e 100 and alginate sodium. after transferring the matrix to the second medium of ph 6.8, carboxylic groups of el became more ionized giving rise to an increase in the degree of swelling. however, previously protonated amino groups began to lose their charge and may be responsible for the increase in the hydrophobic units in the ipec structure. as a result, the swelling slightly decreased at the end of the second medium (ph 6.8) but began to increase in the third buffer (ph 7.4) due to a progressive increase in the number of carboxylate group, in spite of the solubility of cs which decreased at higher ph values. the formulated ipecs, as many of the investigated stoichiometric polycomplexes, would have a more or less homogenous network structure in the swollen state, which could be changed during swelling. completely different changes were observed in the cs: el (3: 2) system. the polycomplexes showed the highest degree of swelling; increasing the cs content led to an increase in the swellability of the ipecs. this system is stable in the first acidic medium, but with a relatively low degree of complexation, and completely destructive to individual polymers afterwards. this system is very sensitive to ph and is not stable in simulated intestinal tract (sit) conditions. the reason is that polycomplexes with participation of el (consisting of more hydrophobic methacrylate chains) are simply destroyed in neutral media. similar results of high ph sensitivity were observed in polycomplex systems made up of cs-pectin and cs-dextran sulfate. in order to assess the potential of the ipecs to be used in matrix controlled drug delivery systems, we evaluated the release of the model drug (ibf) from all investigated matrix systems. based on the results of the previous studies from dissolution behavior of ibf, as a model drug, from the polycomplex matrix systems based on cs and el in gastrointestinal simulated conditions, we decided to use three release media (ph 1.2, 6.8, and 7.4) in the present study. d1.2 and t1.2 represent the cumulative amount of drug released at ph 1.2 and the time of release respectively. similarly, d6.8 and t6.8 also represent the cumulative amount of drug released at ph 6.8 and the time of release, respectively. as expected, very low ibf release occurred in a ph-gradient (from 6.8 to 7.4) medium showing that, below solubility of the enteric copolymers, no drug release occurred (el). as shown in figure 2, polycomplex matrices made up of cs and el showed a release behavior that is somehow slower than that of the tablets coated with only el. the reason is that due to high swelling properties at all ph values, these polycomplexes form gel-like matrices, which can sustain ibf release. in case of cs: el (3: 2) polycomplex, the release of ibf was slowest, with the most constant drug release rate as well as swelling properties when compared to all the other systems. this means that an excess of cs in the ipec structure led to formation of a well-equilibrated polycomplex (with a high degree of complexation) which is not so ph sensitive and stable in sit conditions. it is evident that general retardation and low amount of drug release took place at ph 1.2 and 6.8, respectively; that is, all the ipecs batches released negligible quantity of drug in the first two dissolution media when compared to ph 7.4. it has been reported that a successful colon-targeted delivery system should be able to retard or withhold drug release in the upper part of the gastrointestinal region but release the drug promptly on entry into the colon, since the ph gradient ranges from 1.2 in the stomach through 6.6 in the proximal small intestine to a peak of up to 7.5 in the distal small intestine. the control batch (el) coated with only el recorded the lowest cumulative drug release at both ph 1.2 and ph 6.8. however, a closer look showed that the cumulative percent drug release (d7.4) at ph 7.4 for the three (ipec) batches were between 80 and 95%. this means that it has the potential of making sufficient quantity of drug available in the colon, but then how long (t7.4) it would take the drug to be released in the colon is more important. although it is good for a colon-specific delivery system to withhold drug release at both ph 1.2 and 6.8 for some reasonable hours, but being able to promptly release drug at the ascending colon, it is much better if release spreads throughout the colon. the longer the time (t7.4) the higher the probability that release would continue all through the colonic transit period. the ipecs presented the possibility of having a greater contact time in the colon, greater duration of action, and larger area of action. it has been reported that gastrointestinal (gi) absorption of orally administered drugs is determined by not only the permeability of gi mucosa but also the transit rate in the gi tract. this envisaged that improved drug release by the ipecs may likely cause the ibf to impinge on infected cells as in colitis and colorectal cancer, consistent with an earlier report. overall, the release profiles of the tablets based on the ipecs are characterized by a constant and slow release behavior (sustained-release systems). more so, the release profiles are in agreement with the results obtained in the swelling studies. it is pertinent to draw attention to some advantages of our coated tablets, which have sustained release property may have in common with multiparticulate dosage forms. actually some reporters have favoured multiparticulate dosage forms as presenting better advantages over single dosage forms. this is because the use of single unit dosage forms for colon-targeted delivery has been found to be fraught with some shortcomings such as premature disintegration due to production flaws or sudden change in git physiology, which could lead to reduced bioavailability or therapeutic efficacy. on the other hand, some advantages of multiparticulate dosage forms for colon targeting include reduced risk of systemic toxicity, increased bioavailability, low propensity to cause local irritation, and predictable gastric emptying. our dosage form design is composed of multiparticulates within a unit dosage form from where gradual release took place. this may likely enable the coated tablets to enjoy many if not all the advantages of multiparticulates enumerated previously. in order to understand the mechanism and kinetics of drug release, the results of the in vitro drug release study were fitted into various kinetic equations like zero order (cumulative percent drug released versus time), first order (log cumulative percent drug retained versus time), higuchi (cumulative percent released versus t), and peppas (log of cumulative percent drug released versus log time) as depicted in table 2. the kinetic model that best fits the dissolution data was evaluated by comparing the coefficient of determination (r) values obtained in various models. in the peppas (fickian diffusion) model, mechanisms of drug release are characterized using the release exponent (n value) n value of 1 corresponds to zero-order release kinetics (case ii transport); 0.5<n<1 means an anomalous (non-fickian) diffusion release model; n=0.5 indicates fickian diffusion, and n>1 indicates a super case ii transport relaxational release. results of the kinetic analysis of drug release (table 2) indicates that the most predominant release mechanism was zero order. n values of between 1.00 and 1.16 which implies super case ii release kinetics (a strong indication of zero order). zero-order release is the ideal in controlled drug release and has been reported not to be common with matrix systems, this being attributed to time-dependant changes in drug depleted matrix surface area and diffusional path length. therefore, to achieve linear or zero-order release with matrix systems, several manipulative strategies would be inevitably required to impart geometric and structural adjustments on the tablets [4143]. zero-order release has a lot of advantages including ability to deliver drug at a constant rate, thus providing a predictable bioavailability status. the differences between the different ipecs that were observed during the swelling experiments as well as during the drug release studies show that drug release could be tuned based on the composition of the ipec, with cs: el (3: 2) ipecs as the best formulation. this study has shown that ipecs based on cs and el could be exploited successfully for colon-targeted delivery of ibf in the treatment of ibds.

colon-targeted drug delivery systems (ctddss) could be useful for local treatment of inflammatory bowel diseases (ibds). in this study, various interpolyelectrolyte complexes (ipecs), formed between eudragit rl100 (el) and chitosan (cs), by nonstoichiometric method, and tablets based on the ipecs, prepared by wet granulation, were evaluated as potential oral ctddss for ibuprofen (ibf). results obtained showed that the tablets conformed to compendial requirements for acceptance and that cs and el formed ipecs that showed ph-dependent swelling properties and prolonged the in vitro release of ibf from the tablets in the following descending order: 3: 2>2: 3>1: 1 ratios of cs and el. an electrostatic interaction between the carbonyl (co) group of el and amino (nh3 +) group of cs of the tablets formulated with the ipecs was capable of preventing drug release in the stomach and small intestine and helped in delivering the drug to the colon. kinetic analysis of drug release profiles showed that the systems predominantly released ibf in a zero-order manner. ipecs based on cs and el could be exploited successfully for colon-targeted delivery of ibf in the treatment of ibds.

PMC3748778

pubmed-717

a 67-year-old male (height, 165.4 cm; weight, 69.1 kg) with a history of hypertension and hyperthyroidism was scheduled to excise a laryngeal mass because of right vocal fold leukoplakia. the patient's preoperative laboratory test, chest x-ray, pulmonary function test results were unremarkable. transnasal fiberoptic laryngoscopy taken 2 months prior to the surgery showed right vocal cord palsy (fig. the initial vital signs of patient in the operating room were systolic/diastolic blood pressure of 150/70 mmhg and oxygen saturation of 98% on room air. general anesthesia was induced with 120 mg propofol and 40 mg rocuronium, followed by preoxygenation with 5 l oxygen. two min after injecting the rocuronium, the patient was intubated with a 6.5 mm endotracheal laser tube (medtronic, laser-shield ii 6.5 mm i.d. anesthesia was maintained with 67 vol% desflurane in an oxygen-air mixture under a 0.3 fraction of inspired oxygen (fio2). total gas flow was 4 l/min. at the end of the surgical procedure, 15 mg pyridostigmine and 0.4 mg glycopyrrolate were administered after irregular spontaneous breathing of the patient. five min after administering the anticholineasterase, we removed the tube after confirming clear consciousness of the patient and inspiratory pressure less than 40 mmhg. the patient responded well to commands and 5 sec grip strength was good on both hands. the patient was placed on a face mask for manual ventilation and inhaled racemic salbutamol sulfate (100 g as salbutamol) twice through the face mask to facilitate bronchodilation. laryngeal edema, right vocal cord palsy, and paradoxical adduction of the left vocal cord during inspiration were detected by laryngoscopy. hydrocortisone sodium succinate (100 mg) was administered intravenously to alleviate the laryngeal edema and 1 mg midazolam was administered intravenously twice for anxiolysis. the patient still complained of dyspnea and inspiratory stridor after 5 min so we applied 5 cmh2o manual ventilation by face mask to support the patient's self-ventilation and the dyspnea and inspiratory stridor improved immediately. 2). immediately after applying cpap, the dyspnea and inspiratory stridor were relieved. we detected left vocal fold abduction from the midline on inspiration and widening of the glottic gap on the exam. we decided to maintain 5 cmh2o cpap applied via a face mask during transport to the intensive care unit (icu). an arterial blood gas analysis (abga) was done immediately in the icu and showed ph 7.4, paco2 39.8 mmhg, pao2 104.2 mmhg and sao2 97.7%. he had maintained oxygen saturation>95% on a high-flow nasal oxygen cannula system (optiflow, fisher&paykel healthcare limited, auckland, new zealand) under settings of fio2 0.35 and flow 35 l/min (fig. another transnasal fiberoptic laryngoscopy was performed 9 hours after extubation and no left vocal fold adduction movement was detected on inspiration. the results of an abga at that time were ph 7.411, paco2 43.0 mmhg, pao2 65.6 mmhg, and sao2 93.6%. no left vocal fold adduction movement was detected on inspiration during transnasal fiberoptic laryngoscopy (fig. pvfm is a disorder that presents paradoxical adduction of the vocal fold during inspiration and abduction during expiration. it was reported first by patterson et al. in 1974. at that time, pvfm was considered as neurologic or psychiatric disorder, therefore it was described as " munchausen's stridor ". the symptoms of pvfm are inspiratory stridor, shortness of breath, choking sensation, voice changes, and cough due to the limited airway opening associated with paradoxical vocal fold adduction. the major differential diagnoses are asthma, bilateral vocal cord paralysis, unilateral vocal cord paralysis, vocal cord granuloma, subglottic or glottis stenosis, laryngomalacia, tracheomalacia, benign or malignant neoplasm of the upper airway, and palatine or lingual tonsil hypertrophy. pvfm is often misdiagnosed as asthma because its clinical symptoms are similar with those of asthma. patients with pvfm are resistant to asthmatic medical therapy, therefore, they tend to be exposed to unnecessary long-term steroid therapy and escalated drug use. features that help distinguish pvfm from asthma are inspiratory stridor heard loudly over the larynx, rare sputum production, and associated voice changes. the organic causes of pvfm are brain stem compression, upper or lower motor neuron injury, and a movement disorder similar to parkinson's disease. the functional causes of pvfm include conversion disorder, anxiety disorder, depression, personality disorder, and stress disorder. according to a 2002 study by forrest et al. patients with psychologically based pvfm present with conversion disorder alone or accompanying diseases, such as asthma, gastroesophageal reflux disease, laryngeal sicca, chronic laryngitis, and laryngeal sensory neuropathy. neurologic factors consist of focal respiratory dystonia with or without spasmodic dysphonia, multiple sclerosis, and autonomic dysfunction. the gold standard for diagnosing pvfm is direct visualization of paradoxical inspiratory adduction of the vocal fold by laryngoscopy while the patient is complaining of symptoms, such as inspiratory stridor and dyspnea.. a pulmonary function test (pft) can be helpful when there is a cut-off or flattening of the inspiratory limb on spirometry that suggests an extrathoracic obstruction. clinical symptoms and laryngoscopic findings are essential for diagnosing pvfm but a pft is not. typical findings on transnasal fiberoptic laryngoscopy are adduction of all parts of the true vocal cord and posterior diamond-shaped glottal chinking on inspiration. if the patient is asymptomatic at the time of laryngoscopy, some specific examination, such as metacholine challenge, exercise, histamine, and pungent smell, should be performed to trigger pvfm. treatments for pvfm include reassurance, speech therapy, psychotherapy, biofeedback, benzodiazepines, and inhaled anticholinergics. if the patient does not improve with these therapies, positive pressure ventilation, a bag-valve-mask, or cpap should be considered. in an emergency situation, such as postoperative pvfm, invasive procedures, such as endotracheal intubation or tracheostomy, are performed under a falling oxygen saturation condition due to near complete vocal cord obstruction. in this case, the patient was self-medicating with a benzodiazepine preoperatively because of concern about the right hand tremor. the spo2 of the patient was maintained>97% after extubation, therefore, we decided to apply cpap rather than performing invasive procedures, such as re-intubation or a tracheostomy. applying cpap in a patient with pvfm lowers expiratory flow and increases lung volume, which helps the glottis to open and relieves dyspnea. cpap also reduces the effort needed for inspiration by establishing a favorable pressure gradient for inhalation. however, few patients with pvfm encounter an emergency situation, such as severe dyspnea. therefore, rapid diagnosis and treatment are very important for a patient's prognosis. pvfm is very difficult for anesthesiologists to diagnose in the post-anesthesia care unit because the gold standard for pvfm is transnasal fiberoptic laryngoscopy. however, pvfm should be considered when patients complain of dyspnea and stridor after endotracheal extubation. in conclusion, oxygen supply and positive pressure ventilation could be applied first in a case of pvfm when the patient complains of severe inspiratory dyspnea after endotracheal extubation. if the patient continues to suffer from prolonged dyspnea after supplying oxygen and positive pressure ventilation, cpap could be effective before performing invasive procedures, such as endotracheal intubation or a tracheostomy.

paradoxical vocal fold movement (pvfm) is an uncommon upper airway disorder defined as paradoxical adduction of the vocal folds during inspiration. the etiology and treatment of pvfm are unclear. the physician should manage this condition because of the possibility of near complete airway obstruction in severe case of pvfm. we report a case of successful airway management in a patient with pvfm by applying continuous positive airway pressure (cpap). in this case, pvfm was detected after removing an endotracheal tube from a 67-year-old male who underwent excision of a laryngeal mass. the patient recovered without complications in 1 day with support by cpap.

PMC4754275

pubmed-718

signaling between nervous and immune systems is in part due to the fact that these two systems share ligands and receptors. the cellular components involved in these interactions within the central nervous system (cns) are mainly mastocytes, also called mast cells, and glia. in human brain, mastocytes are very scarce and are preferentially located in perivascular territories. by contrast, glial cells comprise about 90% of the total cell content in the cns and are classified as microglia and macroglia (astrocytes, oligodendrocytes, and ependymal cells). representative of the immune system in the cns are mastocytes and microglia, two cell types derived from hematopoietic cells of the bone marrow that migrate to the brain before closure of the blood brain barrier (bbb) [2, 3]. the cns challenged by different aggressions frequently elicits immune and inflammatory responses [4, 5]. mastocytes and microglia are efficient sensors of adverse endogenous or exogenous conditions of the cns [2, 6]. moreover, stress conditions induce rapid mastocyte degranulation via the hypothalamic peptide corticotropin-releasing hormone (crh) and exogenous danger molecules like polyinosinic-polycytidylic acid (poly (i: c)), bacterial lipopolysaccharide (lps), and peptidoglycan (pgn), which are detected by mastocytes and microglia via toll-like receptors (tlrs) [8, 9]. also, glucocorticoids (gcs) play a relevant role in stress-induced potentiation of neuroinflammatory responses by sensitizing microglia to proinflammatory stimuli. as part of these responses, glial tlrs, connexin hemichannels (cx hcs), pannexin (panx) channels might be key players in acute and chronic neurodegenerative diseases characterized by open bbb, demyelinization, and neuronal degeneration. the causes of various chronic diseases that affect the cns, such as alzheimer's disease (ad), parkinson's disease (pd), and multiple sclerosis (ms), are complex and can be related to multiple factors. notably, the innate host defense has been demonstrated to play an active role in promoting neurodegeneration [12, 13]. however, the possible role of these cellular and molecular elements during brain ontogenesis and the consequences in the adult cns remain unknown. this review presents possible implications of glial toll-like receptors (tlrs) and cx hc and panx channels activation after potentiation by stress in cns dysfunctions. during pregnancy, viral infections are common and emerge to predispose the offspring to develop psychiatric diseases [14, 15]. viral mimic polyinosinic: polycytidylic acid [poli (i: c)] resembles the structure of double-stranded rna (dsrna) generated in host cells during viral replication, and it is recognized by tlr3 that activates the innate immune response. the administration of poly (i: c) is a way to trigger the innate immune response, which mimics the early phase of viral infections, and avoids the use of infectious agents, and treatments can be standardized and experiments may be easily compared. all together, they represent an interesting area because perinatal infections, particularly those of viral etiology, are frequent and have been associated with diverse alterations of adult cns, including schizophrenia and autism [19, 20]. tlrs are highly conserved germ line-encoded pattern-recognition receptors that initiate innate immune responses via recognition of pathogen-associated molecular patterns (pamps) as well by recognition of danger-associated molecular patterns (damps) that correspond to endogenous ligands released after tissue injury or cellular stress, such atp, histones, heat-shock proteins, mrna, high-mobility group box-1 protein (hmgb1), surfactant proteins a and d, and mitochondrial proteins. activation of tlrs triggers a cascade of intracellular events leading to activation of several transcription factors, including nf-b, activator protein-1 (ap-1), and ifn-regulatory factor-3 (irf-3) and -7 that regulate the expression of various cytokines and chemokines, responses that are performed in the cns mainly by mastocytes and microglia. in addition, activation of innate immune responses via tlrs is a prerequisite for the generation of adaptive immune responses that become relevant in autoimmune diseases such as experimental autoimmune encephalomyelitis (eae). the number of molecular members that comprise the tlr family is ten in humans (tlrs 110) and twelve in mice (tlrs 19; tlrs 1113). some tlrs can be expressed on the cell surface (tlrs 1, 2, 4, 5, 6, and 10) or in intracellular compartments (tlrs 3, 7/8, and 9), but others can be found in both the cell membrane and intracellular compartments (tlr3 and tlr7; endosomes and endoplasmic reticulum). each tlr detects distinct pamps derived from viruses, bacteria, mycobacteria, fungi, or parasites. for example, tlr3 and tlr7/8 detect ds and single-stranded (ss) rnas from virus, respectively; tlr4 responds to lps from gram-negative bacteria; and tlr9 senses bacterial dna that contains unmethylated cytosine-guanosine dinucleotides (cpg) [2225]. in the adult brain, mastocytes are mainly found in leptomeninges and thalamus close to the bbb [26, 27], but they are also present early in brain ontogeny [28, 29]. mastocytes can be activated by antigens that induce crosslinking of ige bound to mast cells, cd47 recognition, calcium ionophore, atp, compound 48/80, and also by recognition of damps or pamps [26, 27]. if these activators bind to mastocytes for a short period of time (from seconds to a few minutes), they lead to rapid degranulation and release bioamines, proteoglycans, proteases, atp, tnf- and chemokines stored in preformed granules, whereas activations of longer durations lead to the release of newly formed cytokine (tnf-, il1, and granulocyte macrophage colony-stimulating factor (gm-csf)), and chemokine (c c motif) ligand 3 (ccl3), enzymes (tryptase, chymase, carboxypeptidase), lipid mediators (prostaglandins, leukotrienes, thromboxanes, and platelet-activating factor), and nitric oxide (no), mediating the recruitment of effector cells, fluid extravasation, and tissue inflammation [30, 31]. murine mastocytes express the mrna of tlrs 14 and 69 but not tlr5 [3236]. moreover, human mastocytes express the mrna of tlrs 110 with the exception of tlr8 [9, 3739]. in mastocytes, tlr ligands, such as poly (i: c), lps, r-848, and cpg oligodeoxynucleotide, promote il-6 and tnf- secretion as well as regulated upon activation, normal t cell expressed and secreted (rantes) and macrophage inflammatory protein (mip) without significant degranulation [35, 38, 40, 41]. more specifically, in rodent mastocytes, binding of lps to tlr4 induces the release of de novo expressed (without degranulation) and secreted tnf-, il-5, il-10, and il-13 but not gm-csf, il-1, or leukotriene c4 (ltc4), while binding of pgn to tlr2 induces degranulation that includes histamine release [9, 34, 37]. in three different mouse models, where tlr3, tlr4, and tlr7 were specifically deleted in mastocytes, the recruitment of effector cd8 t cells, neutrophils, and dendritic cells, respectively, this implies that mastocytes recognize, respond, and coordinate immune responses, features that are suppressed by trls 3, 4, and 7. not only ligands, but also immunological host environments are decisive for mastocyte activity. in human mastocytes, prolonged lymphotoxin-alpha (lta) and pgn exposure downregulate fcri, decreasing degranulation products after an antigen crosslinking reaction. poly (i: c) treatment also decreases degranulation in an in vitro allergic model, affecting mastocyte adhesion to fibronectin and vitronectin through conformational inactivation of cd29, the receptor of fibronectin. moreover, lps and pgn induce mastocytes migration in vitro after brief treatment with il-6 and ccl5/rantes, respectively. the activation and migration of mastocytes occur in several neurologic disorders including ms [46, 47], pd, amyotrophic lateral sclerosis (als) [49, 50], ad, traumatic injury, ischemic and hemorrhagic stroke [53, 54], and viral infections. mastocytes activation and migration are critical for the increased bbb permeability and progression of neuroinflammation. additionally, proteases released during mastocyte degranulation can also degrade myelin components, contributing to myelin damage in the cns and peripheral nervous system. microglia can rapidly respond to pathogens through their tlrs but do not sense apoptotic cells through the same mechanism [58, 59]. moreover, levels of tlrs expressed by microglia vary depending on the stages of development or pathological conditions. tlr activation induces a cascade of intracellular events leading to the activation of several transcription factors, including nf-b, ap-1, irf-3, and irf-7 that regulate the expression of many molecular elements of inflammatory responses. in human microglia, activation of tlr3 by agonists such as poly (i: c) induces a strong proinflammatory response that allows microglia to mediate the development of t-helper 1 (th1) cells. moreover, infection with the west nile virus (a retrovirus that produces dsrna) in mice lacking tlr3 shows reduced microglial activation and more resistance to lethal infection with reduced viral load and inflammatory responses in the brain compared to wild-type mice. mastocytes release several cytokines in response to tlr2 activation including tnf-, il-4, il-5, il-6, and il-13. meanwhile, the activation of tlr4 causes release of tnf-, il-6, il-13, il-5, il-10, and eotaxin [34, 6365]. also, numerous chemokines including ccl5/rantes, can also induce a proinflammatory profile in microglia [37, 38, 59, 66]. il-33 derived from microglia modulates the activation of p2 receptors on mastocytes inducing secretion of il-6, il-13, and monocyte chemoattractant protein-1 (mip-1), which in turn can modulate the microglia activity. it induces microglia to secrete tnf-, il-6, and ros and activate in microglia proteinase-activated receptor-2 (par-2), a g protein-coupled receptors widely expressed in neurons, astrocytes, and microglia that are implicated in the pathogenesis of ischemia and neurodegeneration, because it induces widespread inflammation [7173]. the activation of microglial par-2 also upregulates p2x4 receptors and promotes release of brain-derived neurotrophic factor, tnf-, and il-6 that upregulate the expression mastocyte of par-2, which results in activation and release of tnf-. it is interesting to note that mastocytes but not microglia have been described to be the first responder in cns injuries, such as perinatal hypoxia-ischemia. many cells produce tnf- in response to several stimuli, but mastocytes store tnf- in granules, and thus they can release it before other cells including microglia and endothelial cells. additionally, the recruitment and activation of mastocytes occur previous to responses elicited by neurons, glia, and endothelial cells. therefore, mastocytes initiate acute inflammations in response to a stimulus, and when inhibited, the brain damage decreases, as observed when the early mastocyte response is inhibited with cromolyn (a mastocyte stabilizer), and then significant neuroprotection is observed. a strong link between lps, the tlr4 agonist, and brain injury both in fetal and newborn animals has been demonstrated. lps injected into developing mouse and rat brains has been shown to induce injury in white matter. moreover, systemic lps administration to preterm fetal sheep induces cerebellar white matter injury, and in vitro assays demonstrate that tlr4 gene deletion prevents lps-induced oligodendrocyte death. in astrocytes, the expression of tlrs is limited in astrocytes, probably because of the neuroectodermal origin of astroglia. these cells express tlr2, which increases in response to proinflammatory stimuli [22, 80]. they also express tlr3 that responds to poly (i: c), hence producing among other cytokines il-6 that contributes to inflammation in humans and mice [8082]. the gene profile of astrocytes activated via tlr3 shows neuroprotective mediators and cell growth factors, that is, differentiation and migration molecules comprising a neuroprotective response rather than a proinflammatory phenotype [83, 84]. tlr4 has been shown to participate in stroke-caused brain damage [8587] and in ad [88, 89]. likewise, tlr4 could play a pivotal role in demyelinating diseases, such as ms. both tlr agonists and cytokines induce the expression of chemokines ccl2, ccl3, ccl5, intercellular cell adhesion molecule-1 (icam-1), and vascular cell adhesion molecule-1 (vcam-1). moreover, lps and poly (i: c) induce the production of il-6, tnf-, ifn-4, ifn-, and inos. lps and dsrna in parallel induce astrocyte activation, which leads to il-1, il-1, il-6, tnf-, gm-csf, lt, and tgf-3 secretion, although macrophage migration inhibitory factor (mif) secretion is inhibited. however, no effect has been found on anti-inflammatory cytokines such as il-2, il-3, il-4, il-5, il-10, tgf-1, tgf-2, and tnf- [11, 93]. recently, in addition to tlr2, tlr3, and tlr4, tlr1, tlr5, tlr6, and tlr7/8 have been found in astrocytes, but their functional roles remain unknown [22, 84]. therefore, the understanding of the detailed mechanisms of tlr signaling in astrocyte activation in cns inflammatory conditions still needs further investigation. the expression and function of tlrs in oligodendrocytes, unlike other glial cells, have been poorly studied. only tlr2, -3, and -4 have been evaluated, being these receptors related to the regulation of inflammatory processes, gliosis, and remyelination after injury [95, 96]. knockout mice for tlr2 and tlr4 exposed to spinal cord injuries show a lower remyelination capacity, and thus it is believed that these receptors would have a key role in the formation of myelin. astrocyte dysfunction triggers primary microglial activation, which induces demyelination [78, 97]. furthermore, injection of lps in the bone marrow induces a rapid oligodendrocyte loss, followed by an increase in oligodendrocyte number. after acute demyelination induced by lps, a more widespread distribution of oligodendrocyte precursor cells is triggered by the activation of microglia/macrophages, which is an event that accelerates remyelination [99, 100]. rats treated with zymosan, a tlr2 agonist, show oligodendrocyte and axonal loss without regeneration. in addition, rats treated with lps, that is, a tlr4 agonist, show oligodendrocyte death and demyelination [76, 101]. also, lps-induced spinal cord damage shows significant demyelinization associated with an important reduction in the amount of oligodendrocytes. other researchers have shown that tnf- and tnfr1 play a relevant role in oligodendrocyte death induced by tlr activation [103105]. however, bsibsi et al. showed that zymosan and lps reduce survival, differentiation, and myelin-like membrane formation, while poly (i: c) triggers apoptosis in rat oligodendrocyte cultures. these findings suggest that tlrs play a pivotal role in oligodendrocyte differentiation and myelination, both in physiological and pathological conditions. compared to other cell types, tlrs play direct roles in regulating various aspects of oligodendrocyte's behavior. however, the apparent contradiction between the effects of lps and zymosan on oligodendrocytes in different models has not been clarified. future research could help to determine the functionality of tlr receptors in oligodendrocytes under physiological and pathological conditions. with regard to the neuroendocrine modulation of the activity of tlrs, this can take local, regional, and systemic routes. local components include neuropeptides such as substance p, crh, calcitonin gene-related peptide (cgrp), and endogenous opioids released by peripheral nervous system. among the regional components, the sympathetic and parasympathetic innervations release neurotransmitters (adrenaline and acetyl choline), and neuropeptides (neuropeptide y or vasoactive intestinal peptide (vip)) play a relevant role. also at a regional level, a neuronal component regulates immunity through the innervation of immune organs and release of noradrenaline, and also a hormonal component regulates immunity systemically by means of adrenaline released from the medulla of the adrenal glands, whereas the systemic factors include the neuroendocrine system through the hypothalamic-pituitary-adrenal (hpa) axis and the anti-inflammatory effects of gcs. furthermore, neuropeptides including cholecystokinin (cck), somatostatin, melanocyte-stimulating hormone (msh), vip, and gastrin also reduce the inflammatory response. additionally, il-1 participates in several aspects of the immune response to infections such as regulation of inflammation and modulation of adaptive immune responses against viral infections [108, 109]. the inflammasome is a multiprotein complex that activates a platform for caspase-1 and caspase-1-dependent proteolytic maturation and secretion of interleukin-1 (il-1). the signal 1 corresponds to tlr ligands or tnf-, and the signal 2 includes atp, amyloid- (a), k efflux, pore-forming toxins, and silicic and uric acid crystals [111113]. after tlr2 and tlr4 activation, secretion and maturation of cytokines il-1 and il-18 depend on caspase-1 cleavage of their premature forms. in both cases, inflammasome complex proteins mediate caspase-1 activation in the presence of high concentrations of extracellular atp through activation of p2x7 receptors [114, 115]. activation of p2x7 receptor leads to a large membrane pore formation identified as panx1 channels [116, 117], which recently has been found critical for caspase-1 activation [116, 118]. not only in immune cells but also in neurons and astrocytes, panx1 recruitment mediates caspase-1 activation, suggesting that during infections, overall tlrs and panx1 channels could enhance inflammatory responses. one hc corresponds to one-half of a gap junction channel and is located at unapposed cell surfaces serving as communication pathway between the intra- and extracellular compartments. two types of hcs are formed in most cells, and they are generally coexpressed. one of them is formed by connexins (cxs, 21 in humans) and the other by panxs 13. hcs provide a membrane pathway for releasing signaling molecules (e.g., atp, glutamate, pge2, and nad) and thus are recognized as paracrine/autocrine communication pathways under normal and pathological conditions [121, 122]. inflammation is a key condition in neurodegeneration that occurs in postischemic brain, diabetes, ms, pd, ad, and possibly in various other neurodegenerative diseases [123, 124]. in neuroinflammatory conditions, the successive activation of different glial cells via hcs has been partially demonstrated [125, 126], and mastocytes are likely to be involved in early steps of different pathological conditions (figure 1). as mentioned previously, the degranulation response of mastocytes is an early and rapid response and might require precise coordination where hcs could be essential. mastocytes express cxs 32 and 43, but to our knowledge, it remains unknown whether they form functional hcs. in addition, no clear evidence of panx1 expression in mastocytes has been published, but activation of p2x7 receptors leads to the formation of membrane pores permeable to molecules up to about 900 kda with single currents, similar to what has been described for panx1 channels, along with histamine release [117, 128]. since the degranulation process depends on influx of extracellular ca, it is possible that panx1 channels participate in atp release, and then atp activates p2x7 receptors, which are ca permeable, allowing the influx of ca required for the mastocyte degranulation response. then, glial cells become involved and microglial cells respond before astrocytes (within several minutes to few hours). in the normal cns, they express the macrophage marker cd11b, low levels of cd45, and practically undetectable levels of major histocompatibility complex (mhc) class ii molecules, cd40, and cd86. in vitro, the microglia activation process is characterized by an upregulation of cd45, mhc class ii, and the costimulatory molecules cd40 and cd86 [130, 131]. the expression of mhc ii antigens is a characteristic feature of antigen-presenting cells, and their coexpression with costimulatory molecules is a hallmark of microglial cells ' ability to interact with other cells, such as t cells. activated, microglia proliferate and migrate to the injury site where they form cell aggregates and secrete pro- and anti-inflammatory cytokines and chemokines, no, and growth factors. the activation of microglia can be acute or chronic, and this would depend not only on the duration of an external stimulus but also on the quality of the stimulus (stress, infection, inflammation, and signals from damaged neurons). in fact, they show differences when activation is induced by stress or inflammation. for instance, acute stress induces morphological activation of microglia and increased c-fos expression in the periaqueductal gray matter but not in the surrounding midbrain. if activation is chronic, it can lead to microglial overactivation followed by microglial degeneration. therefore, activated microglia secrete tnf- and il-1, which in astrocytes induce opening of cx43 hcs leading to the release of atp and glutamate by astrocytes, which can kill neurons through the activation of panx1 channels, p2x7 receptors, and nmda receptors in neurons. another way of cell-cell interaction used by activated microglia can be found in cx- and panx-based channels. microglia express low to undetectable levels of cx32, cx36, cx43, and cx45 [136139]. they also express panx1, and treatment with a2535 has been shown to increase its surface levels. similarly, the expression of cx43 is upregulated in cultured rat/mouse microglia treated with lps or tnf- plus ifn-, calcium ionophore plus phorbol 12-myristate 13-acetate, or pgn derived from staphylococcus aureus. however, the possible functional role of cx-based hcs expressed by activated microglia remains to be elucidated. under normal conditions, astrocytes are highly coupled with each other, forming intercellular networks, through which ca waves propagate. extracellular atp acts as a paracrine messenger in these waves, since it activates purinergic receptors (p2x and p2y) in astrocytes of surrounding cells, thus resulting in an increase of [ca]i. the mechanisms for atp release from astrocytes may include vesicle-mediated exocytosis and diffusion through cx43 hcs [125, 145, 146] and/or channels formed by panx1. astrocytes also release several transmitters called gliotransmitters, including glutamate, gaba, atp, and adenosine. increases in [ca]i can induce the release of gliotransmitters that promote increases in [ca]i in neighboring neurons, for example, through atp- and glutamate receptor-dependent pathways. the increased [ca]i occurs in local astroglia as well as in astrocytes located more distantly. gliotransmitters might affect diverse neuronal functions including arborization and neuronal plasticity as well as more complex functions such as fear memory. thus, astrocytic cx hcs and panx1 channels might be molecular targets to prevent undesired effects induced by stress. most astrocytes also express cx30 and cx43, and at least cx43 forms hcs that are activated by proinflammatory cytokines, hypoxia-reoxygenation, and high glucose. for instance, lps does not induce cell permeabilization to fluorescent dyes in primary cultures highly enriched with astrocytes of newborn brains, but astrocytes cocultured with microglia respond to lps with a large increase in cx43 hc activity. moreover, the effect of lps is mimicked by exogenous applied tnf- and il-, indicating that astrocytes do not respond to lps in the absence of microglia. moreover, astrocytes previously exposed for 24 h to medium conditioned by a-treated microglia (cm-a) are permeabilized via cx43 hcs. as part of the mechanism, tnf- and il-1 have been shown to mimic the effect of cm-a, and neutralizing tnf- with soluble receptors and il-1 antagonists abrogated this effect. recent in vivo studies have demonstrated that cx43 hcs are critical mediators of postischemic white and gray matter dysfunction and injury. moreover, upregulation of astroglial panx1 channels and cx43 hcs has been found using an experimental model of brain abscess, suggesting that both channel types could play an orchestrated function in some inflammatory responses. cx43 hcs of reactive astrocytes favor the release of excitotoxic compounds, atp, and glutamate, which activate neuronal p2x7 receptors, nmda receptors, and panx1 channels, hence promoting neurodegeneration. activation of neuronal panx1 channels by atp and glutamate released through cx43 hcs from astrocytes exposed to cm-a was shown to induce neuronal death. therefore, it has been proposed that blockade of astroglia and/or neuronal cx hcs and panx1 channels of the inflamed nervous system may represent a strategy to reduce neuronal loss in various pathological states [157159]. additionally, the effect of the maternal environment on the developing cns in the offspring has been analyzed in fetal nonhuman primates. to this end, mothers were subject to a high-fat diet (hfd), and the cns of the fetuses showed increased levels of il-1 and il-1 type 1 receptor, as well as a rise in microglia activation markers, suggesting the activation of the local inflammatory response. under the previous conditions, it is possible that microglia and astrocytes also present upregulation of hc activity, but this needs experimental demonstration. oligodendrocytes might respond within the same time frame as astrocytes, since they can communicate via gap junctions as previously described herein. these cells are responsible for producing and maintaining myelin from the earliest stages of embryonic development to adulthood. like other cells of the cns, oligodendrocytes have low renewal capacity. however, oligodendrocyte precursor cells induce remyelination, following the loss of myelin as a consequence of an injury. many of their functions are accomplished by the expression of a variety of interactions between cx- and pannexin-based channels. oligodendrocytes form gap junction channels with cell bodies of adjacent oligodendrocytes and between layers of myelin, called reflective gap junctions; oligodendrocytes form gap junctions with astrocytes as well. collectively, this gap junction communicated network helps to absorb and remove extracellular k and glutamate released during neuronal activity, thus generating a spatial buffer where ions and molecules are diluted among cell communicated via gap junction channels [165167]. the study of demyelinating diseases, consisting of loss or destruction of myelin, has revealed panx1 channels, cx hcs, and gap junction channels as key factors in oligodendrocyte survival, as well as neuroprotection and myelin maintenance. moreover, by means of the qpcr technique, the mrna of panxs 1 and 2 was detected in primary cultures of oligodendrocytes obtained from optic nerves of 12-day-old rats. both were located in somas as well as in the layers of the myelin sheath. extracellular atp mediates the ischemic damage to oligodendrocytes and is partially explained by the activation of panx1 channels. both genetic and/or inflammatory diseases triggered by viral or toxic sources may affect myelin formation (hypomyelinating diseases) or its maintenance (demyelinating diseases) as it has been found in human diseases associated with hcs formed by mutated cxs. the first event in pathological manifestations of demyelinating disease of the cns is the disruption of the bbb that leads to access of demyelinating antibodies [161, 171174]. also, activated t cells entering the cns mediate the release of inflammatory cells, which together with activated microglia release proinflammatory cytokines that promote oligodendrocyte death in vitro [175178]. tnf- and ifn- can activate microglia and/or macrophage that destroy oligodendrocytes by oxidative stress [180, 181]. this repair is called remyelination, and its process, mediated by oligodendrocyte progenitor cells, is associated with functional recovery. it has been shown that chemokine- (cxcl-) 2 and proinflammatory cytokines, such as il-1 and il-6, promote oligodendrocyte progenitor cell proliferation, differentiation, and remyelination. under inflammatory conditions, oligodendrocytes show upregulation of mhc i molecules, which are constitutively expressed, as well as fas, ifn-, and tnf- receptors (tnfri-ii), transforming them into targets for cd8 cells [175, 176, 182185]. under control conditions there is no expression of mhc ii molecules in these cells [186, 187]. however, cultured oligodendrocytes treated with ifn- in the presence of the synthetic gc (dexamethasone) express mhc ii molecules, suggesting that under stress they could interact with cd4 t lymphocytes and either activate immune reactions or become the targets of t-cell-mediated cytotoxic attack. an excess of extracellular atp is an activator of both innate and acquired immunities, acting as a damp that is chemotactic factor for neutrophils, and a strong regulator of activation, death, and survival of microglial cells [189191]. pathway for atp release is highly variable and includes connexin hcs, panx1 channels, volume-regulated anion channel (vrac), purinergic p2x7 receptor, and/or vesicular exocytosis [192195]. moreover, mastocytes represent an abundant source of atp stored in granules that are released under activation conditions [196198] such as specific (e.g., ige+antigen) and nonspecific stimulation (e.g., stress, mechanic stimulation, and osmotic swelling). with regard to the participation of mastocytes in cns alterations, atp can be released by trauma-induced degranulation and thus stimulates adjacent neurites via p2x and p2y receptors. additionally, the neuropeptide sp released from nerve terminals upon bradykinin stimulation participates in nerve mastocyte communication. this enables interactions between nerve and mast cells and initiates and represents the development of neuroimmunological synapses. also, glial cells are involved in neuroimmune cross-communication, and atp induces glial cells to release il-1, tnf-, and il-33. therefore, atp released from mastocytes is an important autocrine/paracrine/exocrine factor that mediates cross-communication between different cell types. moreover, human lad2 mast cells stimulated with ige, anti-ige, or substance p (sp) secrete mitochondrial particles, mitochondrial dna (mtdna), and atp in absence of cell death. furthermore, mitochondria added to mast cells trigger degranulation and release of histamine, pgd2, il-8, tnf-, and il-1, and this response is partially inhibited by dnase and atp receptor antagonists. only 30 min of immobilization stress can stimulate the hpa axis and cause degranulation in ~70% of rat dura mastocytes. this response could be triggered by neurotensin (nt) and crh acting on mastocytes increasing the permeability of the bbb [203206]. as mentioned previously, activated mastocytes release proinflammatory cytokines and atp among other bioactive compounds that promote microglia, and astrocyte activation and both reactive glia promote neuronal damage [123, 124]. related to this, acute or chronic stress through gcs sensitizes microglia to a subsequent proinflammatory challenge, suggesting that stress should worsen the outcome of neuroinflammation. to our knowledge, it remains unknown if signal transduction of proinflammatory agents via tlrs and activity of hcs is enhanced by gcs or stress. related to the issue presented previously, various neurodegenerative disorders present activation of microglia in different brain regions and restraint combined with water immersion induces massive microglial activation in the hippocampus, hypothalamus, thalamus, and periaqueductal gray matter [207, 208]. although the precise mechanism of microglia activation induced by stress remains unknown, it is likely that bioactive molecules released by activated mastocytes (see what is mentioned previously) lead to the activation of microglia and, therefore, induce progression of neurodegenerative changes. in an ex vivo approach, rats were first pretreated in vivo with ru486 (gc receptor antagonist) and then exposed to an acute stressor (inescapable tail shock; is), and 24 h later, hippocampal microglia were isolated and stimulated with lps. microglia obtained from rats not treated with a gc receptor antagonist showed an increase in gene expression of proinflammatory cytokines (il-1 and il-6). however, in rats pretreated with ru486, the sensitization of microglial to proinflammatory stimuli did not occur. astrocytic signaling is potentiated by gcs (i.e., methylprednisolone and dexamethasone) via long-range calcium waves, and an increase is observed in resting cytosolic ca levels, as well as the extent and amplitude of calcium wave propagation (twofold) compared to control conditions. furthermore, it is known that stress affects microglial function and viability during adulthood and early postnatal life. experiments both in vitro and in vivo have shown that stress hormones can affect the function and viability of microglia. however, little is known if stress during pregnancy affects microglia of the offspring. in a recent report, prenatal stress effects on microglia of the offspring were studied. in this model, prenatal stress during embryonic days 1020 consisted of 20 min of forced swimming. in the offspring, a reduction in the number of immature microglia in the two main brain reservoirs of amoeboid microglia, corpus callosum, and internal capsule was observed. moreover, accelerated microglial differentiation into ramified forms in the internal capsule and brain regions, such as the entorhinal cortex, parietal lobe neocortex, thalamus, and septum, was seen in the neonates in relation to an increase in plasma corticosterone in the pregnant dam. the stimulation of microglial tlr3 with its ligand leads to the release of il-6, il-12, tnf-, and ifn- among others (figure 1). in connection to this, the importance of tlr in various cns diseases (i.e., infection, trauma, stroke, neurodegeneration, and autoimmunity) has been described. this is how viral infections have been implicated in the onset of ms by stimulation of tlr3. additionally, in an animal model of schizophrenia, the stimulation of pregnant mothers with poly (i: c) results in reduced neuronal arborization of the offspring, which is correlated with a status of higher activation. interestingly, cx hcs participate in neurite outgrowth and release of atp and glutamate, which also affect neuronal arborization [214, 215]. it is interesting to note that sensitivity to drug abuse behavior, as well the neuroinflammatory response to a subsequent proinflammatory challenge (as noted previously), is associated with stress and stress-induced release of gcs. neuroinflammatory mediators derived from glia have an important role in the development of drug abuse. this is how neuroinflammatory mediators, such as proinflammatory cytokines, are induced by opioids, psychostimulants, and alcohol, all of which modulate many effects including drug reward, dependence, tolerance, and analgesic properties. an interesting aspect is that drugs of abuse may directly activate microglial and astroglial cells via tlrs, which mediate the innate immune response to pathogens. a key aspect is the timing of stress exposure relative to inflammatory challenge, and if a proinflammatory stimulus (e.g., lps) is added immediately before stress exposure, stress induces an anti-inflammatory effect, which is reflected in the inhibition of the increase in brain il-1 levels. the importance of stress associated with infections is given by the fact that the acute or chronic stress sensitizes the inflammatory responses of the cns to immunological challenges. microglia show an increase in expression of mhc ii, tlr4, and the f4/80 antigens. therefore, stress changes the microenvironment of the cns to a phenotype with inflammatory characteristics. one explanation to this phenomenon is that gcs sensitize microglia to infections [10, 218]. in peripheral blood monocytes from individuals under chronic stress, an increase in the expression of genes with promoter response elements for nf-b is observed as well as allows expression of genes that have promoter elements for gc receptors. otherwise, in older stressed or chronically depressed adults, an increase in inflammatory response occurs when they are challenged with antigens, showing depressive characteristics and elevated levels of il-6 after immunization with influenza vaccines. further evidence that supports this notion comes from observations in older caregivers of patients with dementia, who also presented an elevation of il-6 for over four weeks after vaccination with influenza vaccines, whereas this elevation was not observed in non stressed individuals. furthermore, stress worsens immunity and brain inflammation, which is important in ms and neuropsychiatric disorders [221226]. under stress, the neuropeptides crh and nt are secreted and thus can activate microglia and mast cells, which in turn release molecules with proinflammatory properties. this results in maturation and activation of th17 autoimmune cells and disruption of the bbb that leads to t cells entry into the cns enhancing the brain inflammation, which might support the pathogenesis of ms. nt also stimulates secretion of vascular endothelial growth factor (vegf) and induces expression of crh receptor-1 in mast cells [20, 206, 227]. several lines of evidence associate microglia with the pathogenesis of ms because activation of microglia is prominent and precedes t-lymphocyte infiltration and demyelination. activated microglia release glutamate and no causing neuronal death and bbb disruption [228, 229]. with regard to the participation of mastocytes in the pathogenesis of ms, patients with this disease show elevated levels of tryptase (that activate microglia) and histamine in cerebrospinal fluid (csf) [68, 230] therefore, several lines of evidence suggest an important role of mastocytes and microglia in neuroinflammatory diseases. therefore, both cell types represent therapeutic targets to be considered for treatment of ms and other neuroinflammatory diseases. among the factors relevant to the development of autism spectrum disorders (asd), stress during pregnancy and the first 6 months of postnatal life has been associated with increased risk of asd. as mentioned previously, crh also activates mast cells, resulting in the release of several proinflammatory cytokines including il-6, which in turns may increase the bbb permeability [222, 234, 235]. recently, a decrease in the mitochondrial function in approximately 60% of patients with autism has been demonstrated [236238]. the brain of these patients shows lines of evidence of neuroinflammation [239242], with high levels of mitochondrial dna. additionally, elevated levels of nt that could activate mast cells have been detected in children with autism. the involvement of mast cells and brain inflammation is related to mitochondrial fission and translocation to the cell surface during degranulation, which leads to release of atp and mitochondrial dna. the importance of atp is that it can maintain inflammation by activating mast cells [225, 246]. this is how prenatal stress modifies the phenotype, distribution, and activation statuses of microglia in the offspring. different stressors, together with the activation of the inflammatory immune response, enhance the effects of proinflammatory molecules or conditions, showing synergistic effects. viral infections are the most common causes of infection during prenatal life, and maternal respiratory infection can also increase the risk of the offspring to develop certain mental disorders. the most direct evidence for this comes from a prospective study of pregnant women with medically documented respiratory infections, where the risk for schizophrenia in the offspring is increased 3-fold by infection in the second trimester. evidence that supports this phenomenon comes from models of cocultures between astroglia and microglia treated with dexamethasone. in these experiments, functional membrane properties of astrocytes in cocultures are differentially regulated, which might reflect steroid effects in adjacent glial components in vivo. in cocultures with 30% microglia, dexamethasone-treated cocultures show significant increased gap junctional intercellular communication, which could facilitate the propagation of inflammatory signal along astrocytic networks. therefore, if a stressor is sufficiently sustained, this may reflect neurochemical processes that can make the organism more vulnerable to pathological stimuli producing behavioral and neurochemical responses [250, 251]. this can be reflected in an increased susceptibility to diseases of the nervous system, such as the progression of depressive disorders and anxiety, and can even affect the course of neurological diseases [250, 251]. furthermore, activated microglia affect the expression of cx hcs in astrocytes, which in turn increases the astrocytic atp and glutamate release with deleterious consequences on neurons. therefore, these lines of evidence represent an aspect to be addressed in a model of stress in pregnant animals, in which one can analyze the effects of stress on microglia of the offspring in terms of activation and its effect on astrocytes, which could promote neuronal damage, with cx hcs and panx1 channels being possible therapeutic targets. additionally, the synergistic effect of stress and stimulation with viral infection (for which rna viral mimics poly (i: c)) has not been studied in offspring of pregnant females subjected to stress, which is also a novel approach and can be correlated with a possible susceptibility of offspring to diseases of the nervous system. an important aspect is that when microglia are strongly activated, they remain in a preactivate state for years, which means that microglia are excessively responsive to even slight stimuli. this fact also has been linked to the activation of microglia by viral infections early in life and that can be later reactivated more rapidly compared to microglia in normal state [252, 253]. therefore, the possibility of having microglia (using minocycline) and mastocytes activation (with grh-r antagonists) as therapeutic targets opens the possibility of their modulation as treatment for various neuropsychiatric disorders, viral infections, and other neuroinflammatory pathologies of the cns. in summary, parental stress is proposed to induce potentiation of neuroinflammatory responses by first: activating directly mast cells through crh recognition. second: mast cells proinflammatory mediators prime microglia, astrocytes and olygodendrocytes, modifying their phenotype, distribution, and activation statuses in the offspring, but mainly promoting hc expression. third: sensitized microglia exposed to inflammatory stimuli (i.e., tlr3 ligands) (figure 1) are activated and secrete cytokines (tnf-, il-1). they also show increased functional expression of panx1 channels and cx hcs through which atp and glutamate are released to the extracellular milieu. astrocyte and oligodendrocyte become activated and release atp and glutamate in an hc depending way, and thus they promote neurodegeneration (figure 2).

in the central nervous system (cns), mastocytes and glial cells (microglia, astrocytes and oligodendrocytes) function as sensors of neuroinflammatory conditions, responding to stress triggers or becoming sensitized to subsequent proinflammatory challenges. the corticotropin-releasing hormone and glucocorticoids are critical players in stress-induced mastocyte degranulation and potentiation of glial inflammatory responses, respectively. mastocytes and glial cells express different toll-like receptor (tlr) family members, and their activation via proinflammatory molecules can increase the expression of connexin hemichannels and pannexin channels in glial cells. these membrane pores are oligohexamers of the corresponding protein subunits located in the cell surface. they allow atp release and ca2+influx, which are two important elements of inflammation. consequently, activated microglia and astrocytes release atp and glutamate, affecting myelinization, neuronal development, and survival. binding of ligands to tlrs induces a cascade of intracellular events leading to activation of several transcription factors that regulate the expression of many genes involved in inflammation. during pregnancy, the previous responses promoted by viral infections and other proinflammatory conditions are common and might predispose the offspring to develop psychiatric disorders and neurological diseases. such disorders could eventually be potentiated by stress and might be part of the etiopathogenesis of cns dysfunctions including autism spectrum disorders and schizophrenia.

PMC3713603

pubmed-719

serious complications, such as renal scarring, hypertension, and chronic renal failure can result following a delay in diagnosis and treatment. the prevalence of renal scarring following febrile uti has been reported as 10% to 65%. risk factors, including sex, not being circumcised, constipation, and vesicoureteral reflux (vur), increase the incidence of uti. vur is the backflow of urine from the bladder to the ureter and, in some cases, to the pelvis and calyces. previous studies suggest that the prevalence of vur in children ranges from 25% to 40%. although voiding cystourethrography (vcug) is currently used as a reliable imaging method for diagnosing vur, it is painful and expensive and exposes the patient to radiation. considering the side effects of vcug, the lack of vur in more than 50% of children with uti, and the spontaneous recovery from low-grade vur, researchers have sought cost-effective noninvasive markers for predicting vur. soylu et al. reported that fever higher than 38 and c-reactive protein (crp) of more than 50 mg/dl were suitable predictive markers for the presence of vur and high-grade vur, respectively. in this regard, the present study was conducted to determine the predictive value of clinical, laboratory, and imaging findings in the diagnosis of vur in children with their first febrile uti. this prospective cross-sectional study examined 153 children aged 1 month to 12 years with their first diagnosed febrile uti in qazvin's children's hospital, qazvin, iran, in 2012 through 2013. this hospital is the only referral hospital for children in qazvin province that is affiliated with the qazvin university of medical sciences. the sample size was calculated on the basis of p=58% (sensitivity for feve r>38.5 to discriminate patients to correct groups), d=0.08, =0.05, 1-=0.95, =0.2, and 1- (statistical power of study)=0.8 and by using the following equation: consecutive sampling continued until the desired sample size was reached. the inclusion criteria for children with febrile uti were as follows: (1) first febrile uti; (2) having symptoms of febrile uti, such as fever, chills, vomiting, diarrhea, and irritability in infants, and fever, vomiting, abdominal and flank pain, dysuria, and frequency in children; (3) abnormal urinalysis (the presence of leukocyturia, a positive urinary nitrite test, etc.); (4) positive urine culture (urine culture more than 110 colonies of a single pathogen in a midstream urine sample or clean catch method or 110 colonies of a single pathogen via urinary catheterization, or presence of any number of colonies of organism in urine culture taken by suprapubic method); (5) performance of renal ultrasound, dimercaptosuccinic acid (dmsa) renal scanning, and vcug. children meeting the following criteria were excluded from the study: (1) using antibiotics; (2) failing to undergo vcug; (3) having accompanying and underlying disease, such as septicemia and immune disorders; and (4) having structural abnormalities of the urinary system (such as ureteropelvic junction obstruction, neurogenic bladder, etc.) except vur. first, the symptoms of the disease were recorded, and, before the start of antibiotic therapy, serum samples were delivered to the laboratory to test white blood cell count, neutrophil count, platelet count, erythrocyte sedimentation rate (esr), and crp quantitative level. all laboratory examinations were performed by use of standard methods in the laboratory department of qazvin children hospital. the renal ultrasound was performed within the first 48 hours of admission, the renal vcug was performed at the end of treatment when the patients were discharged from the hospital, and the dmsa renal scan was done in the first week of admission. the ultrasound and vcug were carried out by a radiologist, and the dmsa renal scan was performed and interpreted by a nuclear medicine specialist. any report of hydronephrosis or hydroureteronephrosis without evidence of mechanical obstructions, such as ureteropelvic junction obstruction, ureterovesical junction obstruction, and posterior urethral valves, in the renal ultrasound, and any report of reduced uptake on the dmsa renal scan for pyelonephritic changes in the kidneys were considered as suspicious for vur. according to the results of the vcug, the patients were divided into two groups: a group with vur and a group without vur. the severity of vur was graded according to the international study of reflux in children. grades 1 and 2 were regarded as low-grade vur, and grades 3, 4, and 5 were regarded as high-grade vur. the sensitivity, specificity, positive (ppv) and negative predictive value (npv), positive (lrp) and negative likelihood ratio (lrn), and accuracy of the clinical, laboratory, and imaging variables for diagnosis of vur were determined. chi-square test, exact test, t-test, and nonparametric tests (mann-whitney test) were applied to analyze the obtained data. the children were included in the study after their parents agreed and signed the informed consent form. of the 153 studied patients, the male-to-female ratio was 18:135. the most frequent symptoms in decreasing frequency were fever (100%), chills (62.7%), and dysuria (42.5%). the most frequently grown microorganism in the urine culture was escherichia coli (80.3%). of the 153 studied patients, vur was observed in 60 (39.2%). comparisons of the different variables between children with and without vur and also between the low-grade and high-grade vur groups are shown in tables 1, 2, 3, 4. by use of receiver operating characteristic curve analysis, it was shown that for predicting vur in children with febrile uti, crp20 mg/dl had a sensitivity of 61% (95% confidence limit [cl], 49-74), specificity of 57% (95% cl, 46-67), lrp of 1.43, and accuracy of 58%. in addition, fever38.2 had a sensitivity of 60% (95% cl, 47-72), specificity of 53% (95% cl, 42-62), lrp of 1.26, and accuracy of 55.5% (table 5). the sensitivity, specificity, lrp, and accuracy of the dmsa renal scan for predicting vur were 63% (95% cl, 51-75), 96% (95% cl, 91-99), 14.7, and 79.5%, respectively. also, those of the renal ultrasound were 30% (95% cl, 18-41), 96% (95% cl, 85-97), 3.4, and 60.5%, respectively. the multivariate logistic regression analysis revealed significant positive correlations between fever>38.2 and dmsa renal scan and vur, and also between esr, positive urinary nitrite test, hyaline cast, and ultrasound and high-grade vur (table 6). this study showed that the best predictive markers for the presence of vur in children with their first febrile uti are fever>38.2 and dmsa renal scan. in addition, for high-grade vur, esr, positive urinary nitrite test, hyaline cast, and ultrasound were the best predictive markers. given than only 25% to 40% of children with uti have vur and that vcug is invasive and expensive and exposes the gonads to radiation, researchers have looked for noninvasive markers for predicting vur to avoid unnecessary vcug. 's study on 88 children with febrile uti revealed a significant difference between two groups with and without vur in terms of fever38.5, pyuria25/high power field, and crp23.5 mg/l. however, logistic regression analyses showed that only fever38.5 was an appropriate predictor of the presence of vur. moreover, the above researchers revealed that crp50 mg/dl was a suitable predictor for the presence of high-grade vur. performed a study on 140 children less than 5 years old with their first febrile uti and assessed variables including age, sex, and family history of uti, crp, and renal ultrasound. their clinical approach yielded a sensitivity of 100% and specificity of 17% for predicting all vur grades and a sensitivity of 100% and specificity of 38% for predicting vur gradeiii. a similar study conducted by leroy et al. on 149 children aged 1 month to 4 years with their first febrile uti the results of the present study were somewhat similar to those of the studies by soylu et al. and oostenbrink et al.. although the quantitative crp level, rbc count in urine, results of renal ultrasound, and dmsa renal scan showed a significant difference between the groups with and without vur in the present study, the multivariate logistic regression analysis revealed a significant positive correlation between fever>38.2 and renal dmsa scan and vur. also, although there was a significant difference between the low-grade vur and high-grade vur groups regarding the neutrophil count, esr, leukocyturia, positive urinary nitrite, and ultrasound results, the multivariate logistic regression showed a significant positive correlation of high-grade vur with esr, positive urinary nitrite test, hyaline cast, and renal ultrasound. in a study by tseng et al on 142 children less than 2 years old with their first febrile uti, the authors reported that the sensitivity, specificity, positive predictive value, and negative predictive value of dmsa renal scan in predicting vur were 88%, 37%, 36%, and 88%, respectively. the above authors concluded that children with a normal dmsa renal scan rarely had vur and never had high-grade vur. a study conducted by camacho et al. on 152 children with their first febrile uti showed that vur was more frequent in children with an abnormal dmsa renal scan than in children with a normal dmsa renal scan (48% vs. 12%). the above researchers concluded that the risk of renal damage was very low in children with their first febrile uti and a normal dmsa renal scan. another study pointed out the predictive value of dmsa renal scan and renal ultrasound in the diagnosis of high-grade vur. that study reported the detection rate of ultrasound for low- and high-grade vur to be 86% and 41.7%, respectively, and that of the dmsa renal scan for low- and high-grade vur to be 88.4% and 37.5%, respectively. on the contrary, sorkhi et al. reported that the dmsa renal scan alone or along with renal ultrasound could not predict vur. therefore, they argued that vcug must be done to diagnose vur. in the present study, the highest lrp was respectively related to the dmsa renal scan, renal ultrasound, crp20 mg/dl, and fever higher than 38.2. the dmsa scan had high sensitivity and specificity for the diagnosis of vur. in this respect,, it can be concluded that in the case of a normal dmsa scan, the risk of vur is very low, and performing vcug is unnecessary. although some studies mentioned procalcitonin as a predictor of vur, the relevant test is more costly than other routine tests, such as crp and esr, and is not available everywhere. we hope that the results of the present study will help to avoid unnecessary vcug in children affected by their first febrile uti. given that the present study was conducted in one educational hospital, further multicenter studies are recommended. this study revealed that the best predictive markers for the presence of vur in children with their first febrile uti are the fever>38.2 and dmsa renal scan. esr, positive urinary nitrite test, hyaline cast, and renal ultrasound are best predictive markers for the presence of high-grade vur.

purposethis study was conducted to determine the predictive value of clinical, laboratory, and imaging variables for the diagnosis of vesicoureteral reflux in children with their first febrile urinary tract infection. materials and methodsone hundred fifty-three children with their first febrile urinary tract infection were divided into two groups according to the results of voiding cystourethrography: 60 children with vesicoureteral reflux and 93 children without. the sensitivity, specificity, positive and negative predictive value, likelihood ratio (positive and negative), and accuracy of the clinical, laboratory, and imaging variables for the diagnosis of vesicoureteral reflux were determined. resultsof the 153 children with febrile urinary tract infection, 60 patients (39.2%) had vesicoureteral reflux. there were significant differences between the two groups regarding fever>38, suprapubic pain, c-reactive protein quantitative level, number of red blood cells in the urine, and results of renal ultrasound and dimercaptosuccinic acid renal scanning (p<0.05). there were significant positive correlations between fever>38.2 and dimercaptosuccinic acid renal scanning and vesicoureteral reflux. also, there were significant positive correlations between the erythrocyte sedimentation rate, positive urinary nitrite test, hyaline cast, and renal ultrasound and high-grade vesicoureteral reflux. conclusionsthis study revealed fever>38.2 and dimercaptosuccinic acid renal scanning as the best predictive markers for vesicoureteral reflux in children with their first febrile urinary tract infection. in addition, erythrocyte sedimentation rate, positive urinary nitrite test, hyaline cast, and renal ultrasound are the best predictive markers for high-grade vesicoureteral reflux.

PMC4131083

pubmed-720

aging is the most prominent risk factor for the occurrence of neurodegenerative diseases among others, including oxidative stress (keller et al., 2005; jain et al., 2011), telomere length (harris et al., 2006), genetic mutations (anderton et al., 2002) and head injury (maiese et al., 2008). in the united states there are over 35 million of people with a mean age of 65 years and even older, that mainly die from age-related diseases (drago et al., aging increases susceptibility of people to environmental stressors, thereby increasing the chance to develop neurodegenerative conditions, most likely because the self-repair ability is compromised and tissues and/or organs undergo a progressive decline (musumeci et al., 2014a). the aging process is associated with a number of structural, biochemical, functional and neurocognitive changes in the brain. the structural changes include expansion of cerebral ventricles, regional decreases in cerebral volume (raz et al., 2005), loss of neural circuits and reduced brain plasticity (burke and barnes, 2006; kolb and gibb, 2011), thinning of the cortex (shahani et al., 2006), decrease in both of the grey and the white matter volume (bartzokis, 2011), changes in neuronal morphology (sowell et al., 2003) and formation of neurofibrillary tangles (hedden and gabrieli, 2004; neill, 2012). among the age-related biochemical changes significant decreases in dopamine receptors d1, d2, and d3 (wang et al., 1998; kaasinen et al., 2000) and decreasing levels of different serotonin receptors and their transporters such as 5-hydroxytryptamine transporters (5-htts) (chang and martin, 2009; chang et al., 2009) have been repeatedly reported. among the neuropsychological changes, alterations in orientation (benton et al., 1981) and memory (hof and morrison, 2004) are the most common ones. moreover, many age-related neurodegenerative diseases are characterized by accumulation of disease-specific misfolded proteins in the central nervous system (cns) (van ham et al., 2009). these include -amyloid peptides and tau/phosphorylated tau proteins in alzheimer's disease (ad), -synuclein in parkinson's disease (pd), superoxide dismutase (sod) in amyotrophic lateral sclerosis (als) (durham et al., 1997), and mutant huntingtin in huntington's disease (hd) (scherzinger et al., 1997). the association between age and protein misfolding is not clear yet, but it is probably related to alterations of molecular mechanisms triggered by aging cells, such as telomere shortening, cells shrinkage and decline of quality control over protein synthesis mechanisms (hung et al., 2010; thanan et al., 2014) schematic representation illustrating some of the most common risk factors that contribute to the onset and/or progression of neurodegenerative diseases and the related mechanisms driving the neurodegenerative process. telomeres are an evolutionarily conserved repetitive nucleotide sequences (ttaggg) localized at the end of each chromosome, that are folded into a t loop structure by a protein complex called shelterin (stewart et al., 2012). telomeres play four fundamental roles: protecting genetic information from erosion during dna replication; protecting dna from damage; serving as a binding site for dna repair proteins; and providing information about the cell proliferation history (stewart et al., 2012; musumeci et al., 2015; giunta et al., the telomere length is the sand glass of the cell since it specifies the number of divisions a cell can undergo before it finally dies; thus, it indicates the cell proliferative potential. telomere shortening leads to the attainment of the so-called hayflick limit, which indicates the transition of cells to the state of senescence. following this step, cells progressively enter a state of crisis, which is accompanied metabolic disturbances that culminate in massive cell death. telomerase plays a pivotal role in the pathology of aging and cancer by maintaining genome integrity, controlling cell proliferation, and regulating tissue homeostasis. telomerase is essentially composed of an rna component, the telomerase rna or terc, which serves as a template for telomeric dna synthesis, and a catalytic subunit, telomerase reverse transcriptase (tert). the canonical function of tert is the synthesis of telomeric dna repeats, and the maintenance of telomere length. however, accumulating evidence indicates that tert may also exert some fundamental functions that are independent of its enzymatic activity (verdun and karlseder, 2007) (please refer to figure 2). a reduction in telomerase expression contributes to telomere shortening in mitotic cells, while high levels of the enzyme in mesenchymal stem cells (mscs) contribute to their the phenomenon of telomere shortening is closely associated with aging itself, but it has been widely demonstrated that cells can also undergo premature aging due to several factors such as oxidative stress, inflammation and infections, which are able to speed up this process and determine age-related dysfunctions (hung et al., 2010; jenny, 2012; kong et al., 2013; kota et al., therefore, given the involvement of these factors (and in particular of oxidative stress) in the development of neurodegenerative/age-associated diseases, it becomes of primary importance to also gain more insights on the underlying mechanisms triggered by these stressors, as this could serve to improve current therapeutic strategies based on the use of mscs to treat neurodegenerative conditions. the telomerase is composed of an rna component, telomerase rna or telomerase rna component (terc), which serves as a template for telomeric dna synthesis, and a catalytic subunit, tert. tert besides its canonical function in telomere elongation has also a role as a transcriptional modula-tor of the wnt--catenin (-cat) signalling pathway. tert acts as a cofactor in the -cat transcription complex; in this complex, tert interacts with brg1, a chromatin remodeling factor, to regulate the wnt/-cat signalling pathway. tert is not only acti-vated by the wnt/-cat pathway, but -cat could also be directly regulated by tert induction, which results in maintenance of telomere length. in the mitochondria, tert also plays a role in regulating apoptosis in-duced by oxidative damage of mitochondrial dna (mtdna). cen: centromere. a reason why telomeres are the preferred targets of oxidative insult seems to be primarily related to their dna composition, which tends to be rich in guanine residues (coluzzi et al., indeed, the high incidence of guanine bases promotes the generation of alterations to dna bases to species called 8-oxoguanine (8-oxog), which, if not repaired, may lead to single or double strand breaks, mutations or even genomic instability (grollman et al., 1993). of interest, genomic instability, oxidative stress and ageing are not to be considered as independent causative factors in telomere shortening, but need to be considered as interconnected phenomena. consistent with this theory, convergent data has identified an accelerated wnt/-catenin cascade activation as a common denominator triggered by these insults. activation of this pathway reduces mscs proliferation potential, hampers telomerase activity and drives a cellular shift of mscs towards a differentiated/senescent phenotype (as elegantly reviewed by fukada et al.,, it is auspicable that strategies aimed at dampening the occurrence of these detrimental events in neurons or to block the wnt/-catenin intracellular pathways could have the potential to significantly impact the senescent process, including premature telomere shortening. early neuronal cell death is a feature of neurodegenerative disorders and reduced telomere length has been associated with premature cellular senescence. studies have shown that reduced telomere length in peripheral blood is associated with the incidence of illnesses associated to the aging phenotypes, such as dementia (thomas et al., 2008), neurodegenerative disorders such as hd and genetic neurovascular diseases such as ataxia telangiectasia (at) (metcalfe et al., 1996; kota et al., since ltl is reflective of global cellular morbidity and mortality, it has been proven that it could be used as a useful tool to screen neurodegenerative disorders (sahin and depinho, 2012). it is worth emphasizing, however, that leukocytes include diverse cell populations that play complementary roles in tissue homeostasis and responses to infections and diseases, and the possibility exists that simply monitoring ltl may lead to misleading results. indeed, the three major classifications of leukocytes are granulocytes, lymphocytes, and monocytes. these populations have different telomere lengths and erosion rates as a result of differences in telomerase activity, proliferation history, and telomere trimming (stewart et al., 2012). these differences have to be carefully taken into account when considering a possible study of age-related processes in neurodegenerative disorders. however, a recent study has consistently showed that the ltl was reduced in individuals suffering from neurodegenerative disorders as described above, suggesting that the phenomenon of telomere shortening could at least be partly implicated or could contribute to the triggering of pathological pathways activated in these diseases (kota et al., the reduced telomere length has been attributed to oxidative stress, aberrations in mitochondrial homeostasis, deficient dna repair mechanisms, and decreased dna methylation status (von zglinicki, 2002; blasco, 2007; gackowski et al., 2008; copped and migliore, 2009; van groen, 2010; sahin and depinho, 2012). interestingly, a review of data from literature concerning the potential use of ltl as a biomarker in ad and pd showed to be inconsistent in both cases, since the number of studies reporting no association between ltl and disease states almost overlapped the ones indicating a correlation between ltl shorthening and neurodegeneration (eitan et al., 2014). interestingly enough, a recent study reported an even longer ltl in pd patients, associating short telomeres with reduced risk of pd (schrks et al., 2014). the reason of these inconsistencies could be dependent on the population number used in these studies, as the low number of patients together with the inter-individual variability may often result in significantly reduced statistical power, and purportedly to unreliable results. it has been shown that variability in ltl in individuals can be induced by different factors such as chronic stress, diet, lifestyle, chronic inflammation state and hormone levels (liu et al., 2010; broer et al., the interaction between these factors and genotype can also play a role in ltl variability (takata et al., 2012). certainly, further investigation in this field are needed to clarify the precise role and diagnostic or therapetic potential of ltl in neurodegeneration. the limited regeneration power of the cns represents a major challenge for the development of new therapeutic strategies efficacious to promote its functional repair. mscs have been proposed as a viable therapeutic tool for degenerative disorders as they possess high proliferative ability and they are able to differentiate into multiple lineages (mobasheri et al., 2014; musumeci et al., 2014c; tanna and sachan, 2014). mscs can differentiate into neuron-like cells and determine a paracrine effect by modulating the plasticity of damaged host tissues; by secreting neurotrophic and survival-promoting growth factors that inhibit apoptosis and promote neurogenesis, glial scar formation, immunomodulation, angiogenesis and neuronal and glial cell survival; by restoring synaptic transmitter release; by integrating into existing neural and synaptic networks; and by re-establishing functional afferent and efferent connections (siniscalco et al. in addition, low immunostimulating and high immunosuppressive properties make mscs a suitable source for cellular therapy (abumaree et al., 2012; kwon et al., 2014). another point in favor to mscs employment in therapy is that cells can be transplanted directly without any prior genetic modification or reprogramming, and are able to migrate to the tissue injury sites (amado et al., 2005). mscs have also been proven to be useful for the treatment of pathologies in which tissue damage is caused by oxidative stress and thus in those pathologies linked to stress-induced telomere shortening and premature aging, where mscs are likely to be more resistant to oxidative insult than normal somatic cells (benameur et al., 2015). this feature is particularly important since it makes mscs an interesting and testable model for the treatment of age-related neurodegenerative disorders. currently, there is a great interest towards the use of mscs in pioneering therapies aimed at treating chronic and progressive neurodegenerative diseases, which are currently incurable and whose attempts to find disease-modifying therapies have failed, such as ad, pd, als and hd. it has been shown that after transplantation into the brain, mscs promote neuronal growth, decrease apoptosis, reduce the levels of free radicals, stimulate the formation of new synaptic networks from damaged neurons by supporting axonal outgrowth, modulate neuroinflammatory activities and promote proteosomal degradation of ubiquitinated misfolded proteins (caplan and dennis, 2006; mezey, 2007; uccelli et al., 2011). through paracrine mechanisms, mscs are also able to interact with neighbouring damaged host cells and influence their microenvironment, by sharing proteins, rnas and even mitochondria (spees et al., 2006; olson et al., 2012). as a proof-of-concept, mazzini et al. demonstrated that mscs can decrease motor neuron cell death through paracrine actions when implanted into the cns of als patients (mazzini et al. recently, the paracrine properties of bone marrow-derived mscs (bm-mscs) have been also shown in rat model of ad, suggesting their potential therapeutic role in this disease (salem et al., 2014). the potential efficacy of human mscs (hmscs) has been also confirmed recently, as treatment succeeded to ameliorate some behavioral defects observed in a rodent model of hd, hence demonstrating that xenologous transplantation of hmscs could be considered a potentially successful approach to counteract neurodegeneration caused by hd, and perhaps other cns disorders (hosseini et al., 2014). mscs can be readily isolated from various tissues, show high plasticity and are capable to differentiate into many functional cell types (woodbury et al., 2000; numerous studies have shown that bm-mscs can differentiate into cells that display neuronal or even dopaminergic characteristics both in vitro and in vivo (ni et al., 2010; zeng et al., 2011) a recent study reported that mouse bm-mscs provided neuroprotection by secreting a key factor, prosaposin, a molecule capable of rescuing mature neurons from apoptotic death. the secretome of bm-mscs showed to reduce toxin-induced cell death in cultures of rat pheochromocytoma cells, human rencell cortical neurons, and rat cortical primary neurons (li et al., 2010). unfortunately, the medical procedure to obtain bm-mscs from the bone marrow is invasive and definitely painful to patients. therefore, efforts have been made to find more practical alternatives. indeed, recently other mscs sources have gained clinical interest for use in regenerative medicine; and adipose tissue represents one of these sources with a broad spectrum of benefits. human adipose tissue represents a readily available autologous source of mscs (ghasemi and razavi, 2014). human adipose tissue-derived mscs (hat-mscs) retain morphological, phenotypic and functional characteristics resembling those of bm-mscs (zuk et al., 2002), are stable over long term culture, expand efficiently in vitro and possess multi-lineage differentiation potential (zuk et al., 2001; musumeci et al., 2011; choudhery et al., 2013; latest observations suggest that transplantation of hat-mscs into the brains of elderly mice improved both locomotor activity and cognitive functions. transplanted cells rapidly differentiated into neurons and in part, into astrocytes, and produced choline acetyltransferase proteins, restoring acetylcholine levels in thebrain. moreover, transplantation of hat-mscs restored neuronal integrity by stimulating the release of neurotrophic factorsby neighbouring cells (park et al., 2013). in this regard, an aspect to be considered in mscs therapies is that it is now well-recognized that many pleiotrophic molecules endowed with neuroprotective potential, including some neuropeptides produced locally by resident glial cells or neurons (i.e., pituitary adenylyl cyclase activating polypeptide and/or vasoactive intestinal peptide), when stimulated by neighbouring cells (i.e., implanted mscs) may prevent cognitive decline caused by aging (pirger et al., 2014), facilitate nerve recovery after injury both in the cns (reviewed by waschek, 2013) and the periphery (tamas et al., 2012), stimulate remielination processes and glial regenerative support to neurons (castorina et al., 2014, 2015) and are even capable to prevent retinal damage and mantain retinal barrier properties (giunta et al., 2012; scuderi et al., 2013) or impede oxidative insults (castorina et al., 2012), a broad spectrum of physiopathological events that, at different degrees, are negatively impacted by senescence. even more interesting, combinatorial administration of these molecules with mscs has been suggested to support spinal cord recovery after damage (fang et al., 2010), inferring on the mutual reciprocity between the two, especially desirable to complement the existing gaps determined by the single therapeutic employment of mscs in aged patients affected by neurodegenerative disorders. another source of mscs that has captured minor scientific interest is represented by dental pulp stem cells (dpscs). dpscs have also been recognized as capable to differentiate into a variety of cell lineages (zhang et al., 2006; huang et al., 2009), but more studies are required to better define their potential. other sources of stem cells are that obtained from human-exfoliated deciduous teeth (shed), which have been shown to contain multipotent stem cells (miura et al., 2003). the importance of shed is that they are derived from a tissue similar to the umbilical cord. notably, both kinds of dscs can be induced to differentiate into neuron-like cells and be transplanted in brain injury and/or neurodegenerative disease animal models to conduct neuroregeneration studies (sakai et al., 2012; based on these findings, it is plausible to believe that the extracted teeth, considered a common waste product from dental extraction procedures, could be employed in the future to exploit in tissue engineering strategies as a promising substitute of bm-mscs. a major issue that has significantly limited the use of mscs-based therapy is the low yielding of viable mscs from donor tissue. in fact, in order to harvest sufficient mscs to procure some clinical benefits cells need to replicate several times in vitro. unfortunately, a number of studies have demonstrated that mscs from various animals undergo spontaneous transformation when cultured for long terms, posing a limit to this approach. indeed, transformed mscs show some of the features of senescent cells, with a progressive shortening of telomers and consequently, cell death (ahmadbeigi et al., 2011; ren et al., 2011; he et al., 2014). such an aging process occurring in mscs appears to be tissue-specific and has been shown to be regulated by evolutionarily conserved signaling pathways. more recently, a signaling pathway that has shown to be tightly associated to age-related cellular changes is the wnt/-catenin signaling cascade (decarolis et al., 2008; hiyama et al., 2010; stevens et al., wnt/-catenin signaling plays a functional role as a key regulator of self-renewal and differentiation properties in mscs. (2014) found that activation of the wnt/-catenin pathway delays the progression of cellular senescence as shown by the decrease in senescence effectors p53 and phospho-retinoblastoma (prb), lowered senescence-associated -galactosidase (sa--gal) activity, and increased telomerase activity. in contrast, suppression of the wnt pathway promoted senescence in mscs (jeoung et al., 2014). 2012) also showed that wnt/-catenin pathway is connected and regulates tert expression through the interaction with kruppel-like factor 4 (klf4), a core component of the pluripotency transcriptional network (a schematic representation is depicted in figure 2). unfortunately, to date, the mechanism through which the wnt/-catenin signaling pathway regulates age-related neurogenic differentiation in mscs still remains unclear and needs further investigations. it has been assumed that aging is presumably linked to diminished organ repair capacity due to reduced functionality of mscs. for this reason, it should be taken into account that the effectiveness of mscs-based therapies are highly influenced by donor age. it was observed that progressively aging murine bm-mscs exhibit a decline in mscs number, proliferation, differentiation, angiogenic and wound healing properties, along with enhanced apoptotic and senescent features (kretlow et al., 2008; choudhery et al., 2012a, b). in a study using adipose tissue-derived mesenchymal stem cells (at-mscs) from both young and old donors, it was observed that both were able to form colonies, but at-msc from younger donors produce more colonies containing larger numbers of cells and increased proliferative rate than those obtained from older donors (alt et al., 2012). moreover, at-mscs obtained from aged donors displayed increased senescent features, as indicated by the greater expression levels of p16 and p21 genes, which have been indicated as markers of senescence (stolzing et al., 2008). in the latter study, the expression of sa--gal was measured and it was also found at higher levels in aged at-mscs cultures, while sod activity was decreased (stolzing et al., 2008). it was further identified that mscs from elderly donors became more granular and developed a more flat and larger morphology at passages 56, indicating the appearance of typical morphological signs of replicative senescence (khan et al., recently, in a study conducted on hbm-mscs from young and old donors used to differentiate and promote neurite outgrowth from dorsal root ganglia neurons (drgn), brohlin and coworkers observed that treatment of hbm-mscs with growth factors induced protein expression of the glial cell marker s100 in cultures from young but not old donors. however, exogenous administration of growth factors enhanced the levels of brain-derived neurotrophic factor (bdnf) and of vascular endothelial growth factor (vegf) transcripts in both donor cell groups and partly recovered stemness properties of mscs from elderly, supporting the hypothesis stated above. finally, in the same study it was demonstrated that mscs co-cultured with drgn significantly enhanced total neurite length only when obtained from young but not old donors. moreover, mscs from young donors maintained their proliferation rate while those from the old ones showed increased population doubling times (brohlin et al., 2012). these observations suggest that mscs isolated from either young or old donors may benefit of a combinatorial approach to retain, at least in part, their regenerating properties on neurons. nevertheless, to date mscs from young donors are still to be considered the first choice mscs source to use for cns repair (figure 3). the fact that mscs can be conveniently obtained from different accessible tissues (such as bone marrow, blood, adipose and dental tissue) and demonstrate neuroprotective effects, immunomodulatory properties and self-migratory activity, makes them an attractive therapeutic tool for potential application in neurodegenerative disorders. however, there are some critical points that still need to be clarified before msc-based therapy can be adopted in clinical practice. these include the reduced stemness properties of mscs isolated from elderly or caused by long-term expansion in vitro, which could result in reduced efficacy for regenerative cellular therapy. the complex pathways involved in neurodegenerative disorders, should be evaluated with care, in the attempt to extend the current understanding of the pathogenesis of these diseases and identifying targets for intervention. to be suitable for use inneuroregenerative therapy, the mechanisms that govern the self renewal capacity of mscs, it is proposed that scientific effort should focus more on finding the appropriate microenvironment (culture conditions) that more likely will allow to yield sufficient number of functional mscs. as previously discussed, amolecular mechanism worthy of attention could be represented by the wnt/-catenin signaling pathway, whose involvement in triggering the shift of mscs towards a senescent phenotype appears to be clear. these findings, together with the evidences obtained with combinatorial approaches using neuroprotective agents, support the idea that trophic molecules, including some neuropeptides, may elicit a regulatory function on the wnt signaling cascade, which in turn, could be the key element in controlling mscs senescence (jeoung et al., 2014). alternatively, another critical mechanism to target could be telomere regulation, but this strategy has already reach general consensus, since studies on the mechanisms controlling telomere status and regulation in these cells have progressively gained importance in the last years. in fact, strategies to prevent telomere loss or to increase telomere length of mscs may prevent or delay degeneration and hence the onset of symptoms in neurodegenerative disorders, improving the results of mscs-based therapetic approaches. finally, a further and reasonable method to expand mscs validity in therapy could be represented by banking younger adipose tissue for later use. preservation of mscs at a younger age, when their biological utility is maximal, could provide a usable source of functional mscs with full regenerative potential for future applications in regenerative medicine.

aging is the most prominent risk factor contributing to the development of neurodegenerative disorders. in the united states, over 35 million of elderly people suffer from age-related diseases. aging impairs the self-repair ability of neuronal cells, which undergo progressive deterioration. once initiated, this process hampers the already limited regenerative power of the central nervous system, making the search for new therapeutic strategies particularly difficult in elderly affected patients. so far, mesenchymal stem cells have proven to be a viable option to ameliorate certain aspects of neurodegeneration, as they possess high proliferative rate and differentiate in vitro into multiple lineages. however, accumulating data have demonstrated that during long-term culture, mesenchymal stem cells undergo spontaneous transformation. transformed mesenchymal stem cells show typical features of senescence, including the progressive shortening of telomers, which results in cell loss and, as a consequence, hampered regenerative potential. these evidences, in line with those observed in mesenchymal stem cells isolated from old donors, suggest that senescence may represent a limit to mesenchymal stem cells exploitation in therapy, prompting scholars to either find alternative sources of pluripotent cells or to arrest the age-related transformation. in the present review, we summarize findings from recent literature, and critically discuss some of the major hurdles encountered in the search of appropriate sources of mesenchymal stem cells, as well as benefits arising from their use in neurodegenerative diseases. finally, we provide some insights that may aid in the development of strategies to arrest or, at least, delay the aging of mesenchymal stem cells to improve their therapeutic potential.

PMC4498333

pubmed-721

successful outcomes of endodontic treatment depend on the identification of all roots and root canals which in turn guarantees complete extirpation of pulp tissue, proper chemomechanical cleaning and shaping and three-dimensional obturation of the root canal system with an inert filling material. failure of at least one of these stages entails high risk of unsuccessful root canal treatment of the tooth with a subsequent development or persistence of a periapical lesion. normally, mandibular first molars have one mesial root and one distally. the mesial root has two canals (mesiobuccal and mesiolingual), ending mainly in two distinct apical foramina. the distal root typically has one root canal, although if the orifice is particularly narrow and round, a second distal canal may be present. anatomical variations in the number of roots as well as canal configuration in mandibular molars are not uncommon [3, 4]. one of the major anatomical variations is the presence of an additional third root, also called the radix entomolaris (re) which is located distolingually in mandibular molars. in very rare cases, when this additional root is located mesiobuccally, it is called radix paramolaris [5, 6]. anatomical studies have reported an association between the presence of a separate re in the first mandibular molar and certain ethnic groups. in populations with mongoloid traits, such as chinese, eskimos, and american-indians, it occurs with a frequency of 5 to more than 30% [7, 8]. in african population, a maximum frequency of 3% was found [9, 10], whereas in europeans the incidence was even less. using full-mouth periapical radiographs, investigated the incidence of radix entomolaris in german population. seven patients were found to have a three-rooted mandibular first molar with an overall incidence of 1.35%. in indian population, garg et al. examined 1054 periapical radiographs and reported 5.97% of occurrence of re in mandibular first molars. the same method was used by karale et al. who reported a higher incidence (6.67%) of re. knowledge of occurrence, location, and incidence of any tooth anatomical variation is important as it has a significant role in clinical dentistry. many epidemiological studies have highlighted the importance of watching re while performing root canal treatment on mandibular first molars. dental schools have been very recently established in palestine and there is no single research dealing with teeth anatomy and the incidence of anatomical variation in our country. with a huge number of dentists and very few specialists in endodontics, this research has been conducted so as to provide information about various anatomical variations that could be encountered during endodontic therapy. the purpose of this study was to evaluate the percentage of permanent mandibular first molar teeth with three roots in a palestinian population using conventional digital x-rays in two different angles. three hundred and twenty two mandibular first molars from 185 females and 137 males of different ages scheduled for root canal treatment at the dental center of the arab american university were included in this clinical investigation. the study sample represents all patients who needed primary root canal treatment or referred for retreatment over a 2-year period. the age of the 322 participants ranged from 11 to 62 (mean=37) years. this study was approved by the ethics committee of the school of dentistry research centre. the criteria used to indicate the presence of re were clear distinction of an extra root, indicated by the crossing of translucent lines defining the pulp space and periodontal ligaments, originating in the upper half of the distal root. one endodontist and one paediatric dentist (both instructors of undergraduate dental students) served as examiners in this study. disagreement in the interpretation of the radiographs was discussed between the two investigators until a consensus was reached. at least two preoperative radiographs were taken for each tooth undergoing root canal treatment using a digital x-ray sensor (dr. suni, san jose, california, usa). one radiograph was taken from orthoradial position and the other taken either 30 mesially or distally. when the radiographs revealed a case of re, another radiograph for the opposing side was taken. after obtaining adequate anaesthesia, the tooth was isolated with rubber dam and root canal treatment was initiated. the pulp chamber was irrigated with 3% sodium hypochlorite and carefully examined with an endodontic probe (dg-16, dentsply, gloucester, uk). all canals were scouted using k-file number 10 (dentsply, maillefer, ballaigues, switzerland). working length was estimated using an apex locator (novapex, forum technologies, rishon le-zion, israel) and confirmed with a working length radiograph with k-files introduced into the canals. ah plus (dentsply detrey, konstanz, germany) was used as a sealer. a postoperative radiograph was taken to assess the technical quality of root canal filling. when satisfactory, a permanent filling was placed. figures 1, 2, and 3 show an example of a mandibular first molar with three roots. the incidence of re and comparison of the occurrence between males and females and between the right and left sides of the mandible were recorded. comparison of the incidence and the correlations between males and females and left- and right-side occurrences were analyzed by using the pearson chi-square test with spss (15.0; spss inc., three hundred and twenty two patients comprising 185 females and 137 males formed the study sample. there was no significant difference in the incidence of three-rooted mandibular first molars between females (7/185) and males (5/137) (table 1). however, there was a significant difference between the right side (8/12) and the left side (4/12) (p<0.05). knowledge of both normal and abnormal anatomy of teeth dictates the parameters for execution of root canal therapy and can directly affect the probability of success. therefore, practitioners should be familiar with the existence as well as the prevalence of teeth abnormalities. mandibular first molars seem to be the most frequent teeth in need of root canal treatment as they are the first permanent teeth to erupt. nonetheless, anatomical variations of the root canal system in molars are not appreciated by a great number of general practitioners. the presence of a third root (re) may complicate the endodontic treatment and lead to failure as a result of canal missing. while conducting root canal treatment in mandibular first molars, clinicians should be aware of this morphological abnormality. de moor et al. type ii refers to an initially curved entrance which continues as a straight root/root canal. type iii refers to an initial curve in the coronal third of the root canal and a second curve beginning in the middle and continuing to the apical third. the infrequent occurrence of re requires that the clinician be cautious in diagnosis and management of the lower molar teeth. although it is not necessary, an additional root is often associated with an increased number of cusps and an increased number of root canals with a more prominent occlusodistal or distolingual lobe. radiographs taken at different angulations reveal the basic information regarding the anatomy of a tooth and can thus help to detect any aberrant anatomy such as extra canals and/or roots. when the outline of the distal root or the root canal seems unclear on the preoperative radiograph, the presence of a hidden third root should be suspected. studies have shown that a second radiograph should be taken from a more mesial or distal angle (30 degrees) which could probably reveal the presence of re. periapical radiographs were used in this study because they are routinely used in the dental school of the arab american university throughout endodontic steps. this technique is noninvasive and inexpensive and allows for interstudy comparisons relating to gender and bilateral occurrence difference for three-rooted mandibular first molars. on the other hand, this method has some disadvantages. as radix entomolaris is mostly situated in the same buccolingual plane as the distobuccal root, a superimposition of both roots can appear on the radiograph resulting in an inaccurate diagnosis. the digital system offers many advantages over the conventional radiography such as ease and speed of use, reduction in time between exposure and image interpretation, less radiation dosage to the patient, elimination of chemical waste hazard, and the ability to digitally manipulate the captured image. unfortunately, a more advanced technology represented by the cone beam computed tomography (cbct) is not available. cone beam computed tomography scans were recently shown to be a valuable tool in several stages of endodontic treatment as they provide an immediate and accurate three-dimensional radiographic image. preoperatively, these images give information about the internal and external tooth anatomy which include number and location of roots and canals, root and canal curvatures, size of the pulp chamber, and the degree of calcification. cbct images allow a complete elimination of the superimposition of structural images outside the area of interest and provide a high-contrast resolution and data from a single computed tomography imaging process. they also provide three dimensional images in the axial, coronal, or sagittal planes. in cases of re, cbct shows the exact position of distolingual root and hence it helps in tracking the curvature and prevents iatrogenic event that might occur in relation to canal curvature like instrument separation, perforation, ledge formation, and so forth. when the occurrence of re is confirmed or suspected on the radiograph, the access cavity preparation should be modified from the classic triangular access to a more rectangular or trapezoidal outline. the orifice of re is mainly located disto- to mesiolingually from the main distal canal. if the entrance of re canal is not clearly visible after removal of the pulp chamber roof, a more thorough inspection of the pulp chamber floor and wall, especially in the distolingual region, is necessary. the introduction of dental operating microscope (dom) has changed the face of endodontics. although the operating microscope is not used in our clinics, its use is recommended in routine endodontic practice as it offers an excellent illumination and magnification of the operating field and provides a tremendous advantage in locating and treating extra canals. coelho de carvalho and zuolo reported that the dom had enabled them to locate 8% more canals in mandibular molars. al-nazhan has examined 251 mandibular first molars of saudi patients (clinically and radiographically). he reported an incidence of 6% of re amongst saudi population. in their clinical investigation conducted on chinese population, yu et al. screened 378 cases of mandibular first molars with root canal therapy and reported an incidence of 27% of teeth with re. in our study, the overall incidence of patients with three-rooted mandibular first molars was 3.73%. this finding was in a range of previous reports on middle easterners [24, 26]. however, it was low when compared with data reported for asian races: 24.5% in koreans, 32% in chinese, and 25.6% in taiwanese. in the present study, the same result was reported by other studies [11, 12, 28, 30]. when considering the right and left sides of the mandible, three-rooted mandibular first molars occurred more frequently on the right side than on the left side. these findings were in accordance with some previous studies [12, 28] and different from some others [11, 31] which reported that re can occur more on the left side. loh report a bilateral occurrence of three rooted mandibular permanent first molars from 50 to 67%; however, in our study the bilateral occurrence was only 33.3%. this percentage was higher than the study on german population and much lower than the studies [2729] involving asian subjects (koreans, chinese, and taiwanese, resp.). general practitioners as well as specialists in endodontics should always think about a possible third root (re) when planning a root canal treatment for a mandibular first molar. careful clinical and radiographic examination is indispensable in the diagnosis of any anatomic variation in the root canal system of any tooth prior to initiating treatment. as this study revealed that the incidence of a third root in palestinian population was within the range of previous reports from the middle east but considerably lower than the percentage from the far east, the use of conventional two-dimensional radiographs for the assessment of re would be probably considered as a limitation in the clinical approach and methodology of this study. hopefully, future research in palestine would be able to study a larger and more varied population utilizing cone beam computed tomography.

purpose. the aim of this investigation was to evaluate clinically the percentage of permanent mandibular first molar teeth with three roots amongst palestinian population. patients and methods. three hundred twenty-two mandibular first molars from 185 females and 137 males scheduled for root canal treatment at the dental center of the arab american university were examined over a 2-year period. the incidence of a third root revealed by periapical radiographs and the comparison of the occurrence between males and females and between the right and left sides of the mandible were recorded. statistical analysis. it was performed using the chi-square test with a significant level set at p<0.05%. results. of the 322 treated mandibular first molars, twelve teeth were found to have a third root with an overall incidence being 3.73%. more teeth with a third root were treated on the right side of the mandible compared to the left side. conclusion. the incidence of a third root in palestinian population was within the range of previous reports from the middle east but considerably lower than the percentage from the far east.

PMC4897562

pubmed-722

heart failure (hf) is the final consequence of different heart diseases and it represents a prominent cause of morbidity and mortality worldwide. at the cellular level, the occurrence of hypertrophy of cardiac muscle cells represents a common feature of failing myocardium [2, 3] and is considered as an adaptive response to increased external load in the presence of pathological situations such as hypertension or myocardial infarction. however, sustained overload eventually leads to contractile dysfunction and hf through incompletely understood mechanisms [46]. dysfunctional vascular regulation is an important component of the pathophysiology of hf, and reduced levels of vascular endothelial growth factor (vegf) have been observed in myocardium models of advanced hf. angiogenesis was enhanced during the acute phase of adaptive cardiac growth but reduced as hearts underwent pathological remodelling and it has been demonstrated that inhibition of vegf signalling at the myocardium level leads to the transition from compensatory hypertrophy to cardiac failure, since both heart size and cardiac function are angiogenesis-dependent. a large number of preclinical studies have raised hope that increasing the expression of vegf and/or other proangiogenic cytokines at the myocardial level show beneficial effects especially in animal models of postmyocardial infarction [7, 8]. nevertheless, the clinical trials based on the proangiogenesis hypothesis have failed to provide conclusive results on the therapeutic benefits of clinical approaches aimed at improving myocardial angiogenesis especially in the early phases after myocardial infarction. it should also be noticed that although most experimental studies point to a positive role of angiogenic cytokines at the cardiac level, few studies have addressed the potential role of circulating proinflammatory and proangiogenic cytokines in patients with hf. since human serum contains a myriad of cytokines, a major limitation to the study of the proangiogenic capability of human serum is also due by the fact that most in vitro and in vivo angiogenic tests are complex and not easy to be reproduced [9, 10]. on these bases, in the present study, we have adopted a simple and reproducible in vitro endothelial cell proliferation assay in order to investigate the proangiogenic effects of human sera obtained from both healthy individuals and from a limited group of hf patients. the differential capability to promote in vitro endothelial cell proliferation was correlated with the presence and level of a variety of cytokines, analysed with the multiplex technology, and, for the hf patients, with relevant clinical parameters, such as ntpro-bnp levels and occurrence of cardiovascular events in the follow-up. the healthy group was represented by 66 subjects (age range: 2560 years). the study approval was granted by the ethics review board of the azienda ospedaliero-universitaria, arcispedale sant'anna, ferrara, and informed consents were obtained in accordance with the declaration of helsinki of 1975. the main demographic and clinical parameters of patients were abstracted from clinical records and are reported in supplementary table 1 (see supplementary material available online at http://dx.doi.org/10.1155/2014/257081). hf diagnosis was based on history of hf of at least six months duration, reduced exercise tolerance, objective left ventricular functional impairment (lvef), and raised level of n-terminal pro-brain natriuretic peptide (ntpro-bnp) above the normal range at hospital entry. hf staging was performed by the new york heart association (nyha) classification and on the basis of ntpro-bnp value. serum of healthy individuals and of hf patients was obtained from blood samples collected from an antecubital vein. after clot formation, samples were centrifuged at 3000 rpm for 15 min and serum was immediately stored frozen at 80c in single-use aliquots. human umbilical vein endothelial cells (huvec) were isolated from umbilical cords as previously described, with some modification [1214]. briefly, after cannulation and rinsing with cord buffer (pbs supplemented with 0.011 m glucose), the umbilical vein was infused with type 1 collagenase solution (0.4 mg/ml; worthington, lakewood, nj) and the umbilical cord was placed for 20 min at 37c for enzymatic digestion. vein was flushed with warm egm-2 medium (lonza, walkersville, md) and the resulting endothelial cell suspension was centrifuged for 10 min at 150 g. primary cultures of huvecs were seeded into 25 cm flasks precoated with fibronectin (bd bioscience, becton dickinson, san jos, ca) at 5 g/cm and cultured in egm-2 medium at 37c in a humidified atmosphere with 5% co2. primary cultures of huvec were dissociated with 0.025% trypsin/0.025% edta (gibco brl, grand island, ny) and collected by centrifugation for cell banking and/or to perform in vitro experiments. cell viability was monitored by light microscopic analysis of the cell monolayers after hematoxylin-eosin staining or by quantitative examination after detachment of the monolayers by means of trypan blue dye exclusion, as described in [15, 16]. for this study, 80% of confluent huvec monolayers (passages 25) the purity of primary huvec cultures was evaluated by flow cytometry analysis performed on a bd facsaria ii (bd), as previously described [14, 17]. briefly, detached cells were resuspended in 200 l of pbs containing 1% bsa (sigma-aldrich, saint luis, mo) and incubated 30 min at 4c with the following antibodies (ab): fitc-conjugated anti-cd146 (miltenyi biotec, gladbach, germany, clone 541-10b2), horizon v450-a-conjugated anti-cd144 (bd, clone 55-7h1), pe-a-conjugated anti-cd31 (miltenyi biotec, clone ac128), alexa fluor 647-a-conjugated anti-cd105 (bd, clone 266), pe-cy7-a-conjugated anti-cd34 (bd, clone 581), apc-h7-a-conjugated anti-cd45 (bd, clone 2d1), and horizon v500-a-conjugated anti-cd14 (bd, clone m5e2). data files were collected and analysed using the facsdiva software program (version 6.1.3; bd). only cell suspensions characterized by endothelial cell purity equal to or greater than 98% and low levels of apoptosis, evaluated by annexin-v/pi double staining [18, 19], were used for the in vitro assays. cell proliferation experiments were performed using the xcelligence real-time cell analyzer (rtca-dp version; roche diagnostics, mannheim, germany), which monitors continuously the cellular events recording label-free changes in electrical impedance (reported as cell index). briefly, the background impedance was performed using the standard protocol provided in the software with 100 l egm-2 complete medium (supplemented with 2% of fetal bovine serum and specific endothelial growth factors) per well, in 16-well plates. in each assay, four thousand endothelial cells (huvec) were seeded in 100 l of complete egm-2 in quadruplicate in fibronectin-precoated wells and left to equilibrate at room temperature for 30 min before data recording. cultures were grown at 37c in a humidified atmosphere with 5% co2 over-night until preestablished cell index. then cultures were washed two times with fresh rpmi medium before the addition of rpmi medium supplemented only with 20% human serum derived from the control subjects or from the hf patients. in selected experiments, human serum was added to the cell cultures after 30 min of preincubation with neutralizing ab anti-human vegf (peprotech inc., rocky hill, nj) or with control ig (sigma). in parallel, as control, endothelial cell cultures were exposed to recombinant vegf165 (peprotech). data were analysed using the xcelligence software (version 1.2.1) and expressed as mean sd of cell index normalized to the last cell index recorded before the time of cells treatment (addition of human serum). serum samples were frozen and thawed only once before performing the milliplex map human cytokine/chemokine panel (merck millipore, billerica, ma), a bead-based multiplex immunoassay, which allows the simultaneous detection and quantification of the following 29 human cytokines/chemokines: egf, eotaxin, g-csf, gm-csf, ifn-2, ifn-, il-10, il-12(p40), il-12(p70), il-13, il-15, 1l-17, il-1 receptor antagonist (ra), il-1, il-1, il-2, il-3, il-4, il-5, il-6, il-7, il-8, ip-10 (cxcl10), mcp-1, mip-1, mip-1, tnf-, tnf-, and vegf. samples were processed following the manufacturer's recommended protocol and analyzed by using a magpix instrument provided with the milliplex-analyst software that uses a five-parameter nonlinear regression formula to calculate cytokine/chemokine concentrations from the standard curves. for each set of experiments, values were reported as median and/or means sd. the results were evaluated by using student's t- and the mann-whitney rank-sum tests, when appropriate. one of the purposes of this study was to set up a reliable and reproducible biological assay using huvec as cellular target for monitoring the overall presence of circulating cytokines with a proangiogenic activity. for this purpose, purity of endothelial cell cultures was determined by flow cytometry analysis as cells expressing (98%) cd146, cd144, cd31, cd105, and cd34 and negative for cd45 and cd14 (figure 1(a)). in order to monitor and reproducibly measure endothelial cell proliferation, we adopted a system which measures the impedance of the cell monolayer (cell index, ci) in real-time and in label-free manner without disturbing/altering the culture (figures 1(b) and 1(c)). for this assay, endothelial cells were seeded and let to adhere and proliferate in the presence of complete medium (supplemented with 2% of fetal bovine serum and specific endothelial growth factors) until reaching a preestablished cell index value, settled in the range from 2.5 to 3.5. subsequently, to assess and comparatively measure the effects of human serum samples on endothelial cell proliferation, medium was removed and cultures were extensively washed before the addition of medium supplemented only with 20% human serum (figures 1(b) and 1(c)) collected from healthy subjects and hf patients. cell index was recorded for up to 48 hours after treatment and analysis was carried out after normalization (figures 1(b)1(d)). in the first group of experiments, we analysed the ability of sera obtained from healthy subjects in promoting/affecting endothelial cell proliferation. in this analysis, we observed that, at the time points examined, sera obtained from older (mean age: 52 7.6) subjects induced a significantly (p<0.05) higher huvec proliferation compared to sera obtained from younger (mean age: 29 8.6) healthy subjects (figures 1(c) and 1(d)). this unexpected finding suggested differences between the sera of young and older healthy individuals in terms of angiogenic and/or angiostatic cytokine levels. therefore, to check this hypothesis, we next evaluated the levels of several circulating cytokines and chemokines using the luminex technology that allows the simultaneous detection and quantification of a panel of 29 analytes (table 1). sera obtained from older individuals showed significantly (p<0.05) higher levels of eotaxin and ifn-2 together with significantly (p<0.001) higher levels of il-7, il-8, mip-1, and vegf (table 1 and figure 2). on the other hand, sera from younger healthy individuals showed significantly (p<0.05) higher levels only of egf as compared to older individuals (table 1 and figure 2). we next analysed whether the huvec proliferation assay might also be useful to stratify sera of hf patients in relationship to relevant clinical parameters. for the purpose of this pilot study, we have enrolled 29 hf patients whose main characteristics, including cardiac functionality parameters and cardiovascular risk factors and therapy, are reported in supplementary table 1. twenty-three patients (79.3%) had ischaemic aetiology, whereas 6 (20.7%) satisfied the criteria for idiopathic dilated cardiomyopathy or had hf because of hypertension and valvular disorders. all patients were receiving guidelines pharmacological therapy consisting of ace inhibitors (55.2%), angiotensin ii receptors blockers (34.5%), -blockers (82.8%), antialdosterone drugs (41.4%), and diuretics (89.7%). we did not observe significant differences between nyha class groups as for hf aetiology and the most common cardiovascular risk factors: age, diabetes, hypercholesterolemia, smoking habits, history of hypertension, and coronary diseases familiarity. based on the normalized cell index values, determined as previously described for sera from healthy subjects, we observed that sera obtained from the hf patients exhibited different effects on huvec proliferation, as exemplified in figure 3(a). the results of this assay allowed us to subdivide hf samples into two groups (figure 3(b)): a group (n=18) with a high proliferation index (referred to as high endothelial cell index) and another group (n=11) with a significantly lower proliferation index (referred to as low endothelial cell index). of note, the observed differences in the ability to promote endothelial cell proliferation between the 2 hf patient subgroups were not due to differences of ages (age of the high ci hf patients: 69.8 12.2; age of the low ci hf patients: 73.1 8.7). in parallel, we have measured the levels of the same panel of cytokines/chemokines previously analysed in the sera of healthy subjects. as shown in table 2, sera from hf patients belonging to the high ci endothelial proliferation group showed higher levels of several cytokines/chemokines with respect to patients ' samples of the low ci endothelial proliferation group, including the angiogenic cytokines vegf (p<0.05), il-8 and mip-1 (figure 4(a)). it is noteworthy that the levels of vegf observed in the group low of hf patients (table 2 and figure 4(a)) were comparable to those previously observed in younger healthy donors (table 1 and figure 2). the important, but not exclusive, contribution of vegf to the in vitro endothelial cell proliferation in response to hf patients ' sera was underscored by experiments carried out using neutralizing ab anti-vegf (supplementary figure 1). of interest, within the cytokines/chemokines analyzed in hf patients, the levels of il-12p70, il-8, mcp-1, mip-1, and vegf correlated (p<0.05) positively with the endothelial proliferation index assessed in two distinct time points (36 and 48 hours), with il-8 and vegf showing a higher correlation (figure 4(b)). the evaluation of potential correlation with key clinical parameters revealed no significant correlations between these cytokines and the left ventricular ejection fraction, while a significant correlation was observed between il-12p70 and ntpro-bnp levels (r=0.25, p<0.05). comparison of the cytokine levels between the whole hf population and healthy individuals showed significantly (p<0.05) higher levels of few cytokines, including il-12p70 and il-8, in the hf patients (supplementary table 2). although the vegf levels in the hf patients ' sera were higher than the levels measured in healthy controls (254.9 287.1 pg/ml versus 155.4 24.6 pg/ml, resp.) the difference was not statistically significant. anyhow, it has to be underlined that the small numbers of subjects limited the overall statistical analysis. in order to understand the potential clinical relevance of our in vitro endothelial proliferation assay, pointing to a subdivision of the hf patients into two groups (high versus low endothelial cell proliferation index), we first analysed the distribution of nyha classes within the two groups. as shown in figure 5(a), although we did not observe a significant correspondence between the classification of the hf patients in high/low and the nyha classes, the patients group was represented by a higher percentage of patients belonging to classes i-ii (52% in low ci versus 29% in high ci). moreover, the low ci patients showed lower levels of ntpro-bnp (median: 510.1 pg/ml; mean sd: 687.6 357.6 pg/ml) as compared to high ci patients (median: 1141.5 pg/ml; mean sd: 1402.5 743.1 pg/ml), although the difference did not reach statistical significance (p=0.07). on the other hand, the two groups were very similar for the left ventricular ejection fraction (low ci patients: median 33%, mean sd 31.2% 7; high ci patients: median 34%, mean sd 32.7% 8.2, p=0.3). next, we analysed the subdivision of the hf patients into the two groups, in relation to the clinical events they experienced in two years of follow-up. for this purpose, we considered as outcome the occurrence of major cardiovascular events: mortality (of cardiac origin), acute myocardial infarction, percutaneous transluminal coronary angioplasty, implant of defibrillator, and surgery of aortic aneurysm. of interest, we observed a marked different distribution of the event-free hf patients between the high ci and low ci groups. in particular, while the hf patients that did not experience any major clinical cardiac events constitute the 90.1% of the group low ci, the majority (72.2%) of the group high ci was constituted by patients that experienced major events in the follow-up (figure 5(b)). therefore, from these observations, it emerges a trend indicating that sera from patients with severe pathology are prone to enhance endothelial cell proliferation and, in particular, serum exhibiting high endothelial proliferation activity could indicate a higher risk for the hf patient of worsening the disease. in our study, by employing a standardized in vitro endothelial proliferation assay, we have demonstrated for the first time the following: (i) endothelial cell proliferation in response to serum samples from healthy individuals increased with age and was coupled to different serum levels of proangiogenic cytokines, including vegf and il-8; (ii) the endothelial cell proliferation index determined in response to serum samples from hf patients was correlated with circulating levels of several proinflammatory/proangiogenic cytokines (il-12p70, mcp-1, mip1, il-8, and vegf), with il-12p70 showing a positive correlation also with the levels of levels of ntpro-bnp; (iii) hf patient sera exhibiting high endothelial proliferation activity could indicate a higher risk for the patient of worsening the disease. our current findings are unprecedented and somewhat counter intuitive if one considers the important role of cardiac angiogenesis in counteracting hf. indeed, dysfunctional blood vessel formation is a major problem in advanced hf, regardless of the aetiology. however, clinical trials of vegf gene therapy in patients with coronary artery disease or peripheral artery disease have not, to date, demonstrated clinical benefit. in this respect, only few studies have tried to evaluate the levels of proangiogenic cytokines in cardiovascular patients, and mostly were carried out in patients with acute myocardial infarction (ami) [2026]. after an initial attempt to evaluate the predictive role of serum cytokines in patients presenting at the emergency room with chest pain and patients with ami [2224], korybalska et al. demonstrated that serum vegf measured in patients with ami (n=106) was significantly higher in patients than in healthy controls and correlated with clinical and angiographic parameters. moreover, iribarren et al. demonstrated that median serum concentration of vegf was significantly higher (260 pg/ml) in a large cohort of patients with ami (n=695) with respect to age-matched healthy controls. thus, although in our pilot study we have analysed only 29 hf patients, our data are in line with these previous studies obtained in patients with ami, extending the notion that elevated levels of circulating proangiogenic cytokines are predictive of a poor prognosis, not only in patients with ami but also in patients with established hf. in this respect, it is of interest that other studies have clearly demonstrated that elevated levels of proangiogenic cytokines, and in particular of vegf, have a well-established pathological clinical significance in different clinical settings, such as in patients affected by different types of cancer [27, 28]. moreover, although the role of vegf in vascular diseases, such as atherogenesis, still remains controversial [29, 30], it has been recently demonstrated that vegf-a gene transfer induced proatherogenic changes in lipoprotein profiles in a apo mouse model. finally, the levels of circulating vegf have also been associated to adhesion and inflammation markers in normal healthy population. it is noteworthy that the in vitro endothelial proliferation assay, we have applied on sera derived from both healthy subjects and hf patients, represents a reproducible and reliable tool able to summarize the overall biological effects on the endothelium driven by the cytokines/chemokines milieu present in the peripheral blood. although we are aware that the endothelial cell proliferation assessed in our assay could be the result of the contribution of additional cytokines beside those measured by our multiplex assay, our findings confirm the key role of the circulating vegf in the promotion of endothelial cell proliferation and also suggest the potential contribution of other circulating cytokines, including the il-12p70. it is of interest the correlation observed between il-12p70 and ntpro-bnp that has not been previously reported in hf patients and that could deserve further investigation. moreover, although it will be necessary to assess the in vitro endothelial proliferation assay in a higher number of clinical cases, our pilot study on the hf patients ' sera suggests its potential prognostic value.

although myocardial angiogenesis is thought to play an important role in heart failure (hf), the involvement of circulating proinflammatory and proangiogenic cytokines in the pathogenesis and/or prognosis of hf has not been deeply investigated. by using a highly standardized proliferation assay with human endothelial cells, we first demonstrated that sera from older (mean age 52 7.6 years; n=46) healthy donors promoted endothelial cell proliferation to a significantly higher extent compared to sera obtained from younger healthy donors (mean age 29 8.6 years; n=20). the promotion of endothelial cell proliferation was accompanied by high serum levels of several proangiogenic cytokines. when we assessed endothelial cell proliferation in response to hf patients ' sera, we observed that a subset of sera (n=11) promoted cell proliferation to a significantly lesser extent compared to the majority of sera (n=18). also, in this case, the difference between the patient groups in the ability to induce endothelial cell proliferation correlated to significant (p<0.05) differences in serum proangiogenic cytokine levels. unexpectedly, hf patients associated to the highest endothelial proliferation index showed the worst prognosis as evaluated in terms of subsequent cardiovascular events in the follow-up, suggesting that high levels of circulating proangiogenic cytokines might be related to a worse prognosis.

PMC3981563

pubmed-723

it is well established that the transition of vascular smooth muscle cells (vsmcs) from a differentiated phenotype to a dedifferentiated state plays a critical role in the pathogenesis of atherosclerosis. the phenotypic modulation in vsmcs is accompanied by accelerated migration, proliferation and production of extracellular matrix components. eventually, these cellular events result in the formation of atherosclerotic lesions. however, the molecular mechanisms involved in phenotypic control are still unclear. micrornas (mirnas) are a class of endogenous, small, non-coding rnas that pair with sites can in 3 ' untranslated regions in mrnas of protein-coding genes to downregulate their expression. more importantly, one mirna is able to regulate the expression of multiple genes because it can bind to its mrna targets as either an imperfect or a perfect complement. thus, a mirna can be functionally as important as a transcription factor. as a group, mirnas it is therefore not surprising that mirnas are involved in the regulation of all major cellular functions. recently, the role of mirnas in cardiac cell differentiation has been described. in this study, kwon et al. demonstrated that mir-1 plays important roles in modulating cardiogenesis and in maintenance of muscle-gene expression by targeting transcripts encoding the notch ligand delta. the biological roles of mir-145 in diverse cancer cells have been recently identified (reviewed in). in a recent article reported by xu et al., mir-145 was found to be a critical switch for embryonic stem cell differentiation by repressing some core pluripotency factors. whether mirnas participate in the phenotypic control of vsmcs was unknown until recently. in this respect, three independent groups have reported exciting new discoveries regarding the critical role of the vsmc-enriched mirna, mir-145, in vsmc phenotypic modulation [8-10]. they identified that mir-145 plays a role not only in the differentiation of multipotent neural crest stem cells into vsmcs, but also in the differentiation of adult vsmcs. other mirnas that may participate in the phenotypic modula tion of vsmcs are mir-143 and mir-221 [9-12]. this minireview summarizes the current research progress regarding the roles of mir-145 in the vsmc phenotype and the potential therapeutic opportunities of mirnas in atherosclerotic vascular disease. ji et al. demonstrated that mir-145 is the most abundant mirna in arteries. its expression is significantly downregulated in rat balloon-injured arteries with neointimal lesion growth. a recent nature article by cordes et al. showed that mir-145 expression was also decreased in mouse carotid arteries after ligation injury. more interestingly, transcripts of mir-145 were down regulated to nearly undetectable levels in atherosclerotic lesions containing neointimal hyperplasia. our own unpublished data also revealed that mir-145 is largely downregulated in atherosclerotic mouse and human arteries, although the downregulation is less pronounced compared with that from cordes ' study. if the selected atherosclerotic tissue had fewer vsmcs, the expression level of mir-145 could be lower. recently, cheng et al. found that mir-145 is the most abundant mirna in differentiated vsmcs. also, its expression is quickly downregulated in subcultured dedifferentiated vsmcs and in dedifferentiated vsmcs induced by stimulation with platelet-derived growth factor (pdgf). our unpublished data also indicate that mir-145 expression in vsmcs isolated from balloon-injured rat carotid arteries and atherosclerotic apoe-knockout mouse aortas is significantly decreased compared with that in vsmcs isolated from normal control arteries.. found that, during the development of arteries, the expression of mir-145 is associated with the state of vsmc differentiation. mir-145 expression is notably absent in the aorta and pulmonary arteries during later cardiogenesis, during which vsmcs and arteries are developing. in contrast, high transcript levels of mir-145 in vsmcs of the arteries are demonstrated postnatally, after vsmcs and arteries have completed their development. cordes et al. demonstrated that mir-145 was necessary and sufficient to induce differentiation of multipotent neural crest stem cells into vsmcs. in addition, cheng et al. identified for the first time that mir-145 is a critical modulator for the vsmc phenotype both in vitro and in vivo. vsmc differentiation marker genes such as sm -actin, calponin, and sm-mhc were downregulated by mir-145 downregulation, and upregulated by mir-145 upregulation. the regulatory effect of mir-145 on the vsmc phenotype was further verified by cordes and colleagues. in contrast, another co-expressed mirna, mir-143, had no significant effect on marker genes for vsmc differentiation, although it had a strong effect on vsmc proliferation.. demonstrated that the expres sion of the mir-143/145 cluster is confined to vsmcs during development. they found that mir-143 and mir-145 are required for vsmc acquisition of the contractile phenotype because the vsmcs from mir-143/145-deficient mice are locked in the synthetic state. the regulatory effects of mirnas on the vsmc phenotype are not limited to mir-143 and mir-145. two recent studies revealed that mir-221 is also related to the vsmc phenotype that affects vsmc proliferation and migration. it is well known that phenotypic modulation of vsmcs is an initial cellular event in the development of atherosclerotic vascular disease. to determine the therapeutic potential of mir-145 in vascular disease ,. demonstrated that restoration of mir-145 in rat balloon-injured carotid arteries via adenovirus-mediated gene transfer significantly inhibited neointimal lesion growth. in contrast, knockout of mir-143 and mir-145 resulted in the formation of neointima in mouse arteries. the roles of mir-145 in the vsmc phenotype and atherosclerotic vascular disease are summarized in figure 1. expression modulation of mir-145 in the vascular walls and the potential roles of mir-145 in vascular smooth muscle cell phenotype and atherosclerosis. its expression is significantly downregulated in developing arteries and in adult arteries containing intimal hyperplasia. the downregulated mir-145 in adult arteries will increase vsmc dedifferentiation and result in the development of atherosclerotic lesions. mirna-based therapy may have some advantages compared with that for other molecular targets because one endogenous mirna can target its multiple target genes. although the recent studies have demonstrated that targeting mirnas, and mir-145 in particular, may represent a new therapy for atherosclerosis, there is still a long road before mirna-based technology can be translated to clinical therapy. first, the critical mirnas responsible for the development of atherosclerosis must be further identified, especially in human atherosclerotic arteries. second, the detailed cellular and molecular mechanisms of these critical mirnas in the prevention and treatment of atherosclerosis should be studied. third, in addition to the biological effects of these mirnas on vsmcs, their effects on other atherosclerosis-related cellular events should be identified. fourth, although methods are available to downregulate a mirna in vivo, technology for upregulating a mirna in the vascular walls in vivo requires development. finally, the potential side-effects of mirna-based therapy should be studied before application in the clinic. mirna: microrna; mir-145: microrna-145; pdgf: platelet-derived growth factor; vsmc: vascular smooth muscle cell. this work was supported by a national institutes of health grant hl080133 and a grant from the american heart association 09grnt2250567.

micrornas (mirnas) represent a class of small, non-coding rnas that negatively regulate gene expression via degradation or translational inhibition of their target mrnas. recent studies have identified that mir-145 is the most abundant mirna in normal arteries and vascular smooth muscle cells (vsmcs), and its expression is significantly downregulated in dediffer-entiated vsmcs and atherosclerotic arteries. mir-145 plays a critical role in modulating vsmc phenotype. because phenotypic modulation of vsmcs is an initial cellular event in the development of atherosclerosis, mirnas, and mir-145 in particular, may represent new therapeutic targets for atherosclerosis.

PMC2768992

pubmed-724

the app is a type i transmembrane protein with characteristics of an orphan receptor, which shares with other members of its class a particular signaling mechanism termed regulated intramembrane proteolysis (rip). the first occurs outside the transmembrane domain, usually in response to ligand binding, inducing the release of the extracellular domain. this first cleavage event elicits a conformational change that triggers the second proteolytic cleavage which takes place on the transmembrane segment. this mechanism controls several cellular processes, such as the unfolded protein response, cholesterol synthesis, and cell fate instruction. rip of the app is mediated by three different proteases. while-and -secretases catalyze extracellular cleavage, the -secretase complex cuts at the intramembrane domain and leads to the generation of two peptides: an app active fragment, termed aicd and the a. in contrast, two independent groups indicate that aicd is produced mainly from the 695 aminoacids isoform of app through the amyloidogenic pathway (dependent on -secretase activity) [6, 7] and is therefore generated in equimolar quantities with a. the last one accumulation and the formation of various aggregates and deposits in the brain have been the main hypothesis to explain the neuropathological development of ad for almost 20 years. initially, the study of the functions associated with the aicd was limited by the hindrance in its detection. however, recent studies showing that the levels of the aicd are increased in brains of ad patients and murine models reproducing the disease, open up the possibility that this fragment participates in the molecular mechanisms contributing to ad. the aicd is the most evolutionarily-conserved region of the app, accounting for its functional importance. despite its relatively small size (59 aminoacids or less), it acts as a docking site for a particularly large group of intracellular proteins. amongst this group of proteins are pin1, the x11 protein family, disabled (dab)-1, shc, jnk-interacting protein (jip)-1, and the fe65 protein family [1719], which includes fe65 itself and two closely related homologues, fe65l1 and fe65l2. fe65 family members contain three protein-protein interaction domains: a ww domain at the n-terminal involved in interactions with proline-rich sequences and two phosphotyrosine binding domains (ptb1 and ptb2) located at the c-terminal. the second ptb domain (ptb2) is responsible for the interaction between fe65 and the sequence 682yenpty687 of the app (following the numbering of the app695 isoform). the possibility of aicd to form multiprotein complexes through its association with fe65 and its multiple ligands (table 1) has unexpectedly expanded the potential roles of aicd. aicd binds to fe65 in a region that is essential for a production, making fe65 a good candidate for regulating app processing. this could occur via two mutually-exclusive pathways: the amyloidogenic pathway, leading to a production mediated by the -secretase and the nonamyloidogenic pathway leading to the production of a large extracellular fragment (sapp), which is mediated by the -secretase and prevents the generation of a. fe65 acts as a potent modulator by altering the balance between the two pathways. the overexpression of fe65 in cell lines induces a dramatic increase in a secretion, whereas a secretion was decreased in fe65 knockdown cells and in hippocampal neurons of fe65/fe65l1 knockout (ko) mice. the effect on the a secretion appears to be dependent on the interaction between fe65 and app, because the knock-in mice carrying the y682 g mutation, that inhibits aicd binding to fe65, show decreased levels of a and a massive increase in sapp, as a consequence of the nonamyloidogenic pathway this is in agreement with a study showing that fe65 is a potent suppressor of the nonamyloidogenic pathway in primate cells. the mechanism by which fe65 modulates a secretion is related to its interaction with the apolipoprotein e (apoe) receptors: the low density lipoprotein receptor-related protein (lrp) and apoe receptor 2 (apoer2). related to the participation of the aforementioned receptors, the effect of fe65 in the secretion of soluble app fragments is lost in cells lacking lrp. the functional relation with apoer2 is more complex and depends on the presence of its extracellular ligand, reelin, and its intracellular adapter, dab-1. reelin reduces a secretion by promoting the binding of dab1 to the app and displacing fe65, because they share the same binding region. a decrease in reelin expression in the entorhinal cortex (the first region of the brain where a deposits can be observed), displayed in pdapp transgenic mice (which carry human app with mutations swedish (swe) and indiana) and in ad patients, could seriously affect the balance of dab1 and fe65 in their binding to aicd, increasing a secretion. this has been observed in transgenic mice which lack reelin expression (reeler) and carry the mutations swedish and arctic in app. a decade ago, a possible role for the rip of app was first suggested. since app processing seems to be similar to notch processing, it has been suggested that rip of app could be involved in transcriptional regulation. in fact, the fusion of the dna binding domain of yeast gal4 (gal4db) to the c-terminal of app induced a strong transactivation of a luciferase reporter dependent on the formation of a trimeric complex with the adapter protein fe65 and the histone acetyltransferase tip60. a reciprocal experiment using tip60 or fe65 fused to the gal4db gave rise to some contradictory results [49, 50]. nevertheless, a consensus model can be generated including the vast majority of observations derived from these studies (figure 1). the app acts as an anchor for fe65 and fe65-associated proteins that is,: tip60, inducing its association with membrane compartments. membrane recruitment seems to be essential for the activation of the complex, since the overexpression of soluble aicd has no effect on transactivation. the autoinhibited conformation of fe65, produced by the association of the ww domain with a region flanked by the ptb1 and ptb2 domains. the association with the plasma membrane allows the activation of the complex, induced by the phosphorylation of tip60 by cyclin-dependent kinases (cdks). an excellent prospective candidate is cdk-5, that can be found associated with plasma membranes through its activator p35 and displays high activity in the brain. the release of the complex from the plasma membrane may be produced by the app cleavage by -secretase or additionally by the app phosphorylation at thr668 which induces a conformational change in the region recognized by fe65, decreasing the affinity for each other. although some groups have observed aicd in the nucleus, particularly in nuclear domains such as transcriptional factories, the splicing factor compartment or directly at promoters of some genes [5961], apparently in the artificial transactivation system, the nuclear translocation of aicd is not essential to enhance luciferase expression. the n-terminal region of fe65 that includes the ww domain is necessary for nuclear translocation and therefore for its activity as a transactivating protein. although this region lacks a nuclear localization sequence (nls), it could be directed to the nuclei by association with a protein carrying a functional nls. a good candidate to perform this function would be the nucleosome assembly protein set that binds the ww domain and is required for transactivation mediated by the fe65gal4db fusion protein. the phosphorylation of tyr547 in the fe65 ptb2 domain mediated by the abl kinase stimulates its transactivational activity, possibly preventing the association of fe65 with dexras, a ras family gtpase, that acts as an inhibitor of the complex. the search for target genes regulated by the aicd has been complex and has yielded conflicting findings. it has been reported that the aicd/fe65 complex regulates the app expression itself, glycogen synthase kinase (gsk)-3 [63, 64], tip60, the -secretase (bace1), the primate-specific caspase 4, the a degrading enzyme neprylisin [61, 65, 66], the tetraspanin kai1 [26, 63], the lipoprotein receptor-related protein (lrp1), the epidermal growth factor receptor (egfr), and the tumor suppressor p53. nevertheless, many of these studies have been refuted by others, which using different strategies for modulating the aicd/fe65 complex did not produce changes in the expression of the aforementioned genes [6973]. the possible origin of the reported differences is unclear, but regarding the most intensively discussed target, neprilysin, recent data may shed light on the controversy. it was shown that the aicd-binding to neprilysin gene promoter is cell type-dependent [61, 74]. furthermore, aicd-dependent gene regulation is influenced by the passage number and cell density, providing two likely experimental explanations for this disagreement. the majority of the evidence pointing to a role of aicd in transcriptional responses derives from the use of artificial reporter systems that in fact measure the release of components from the membrane, without monitoring endogenous transcriptional activity. besides the potential participation of fe65 in promoting the expression of several genes described above, fe65 has been also proposed to perform other nuclear functions such as the repair of dna damage. fe65 ko mice are more sensitive to dna damage, and this can be overcome by increasing the availability of nuclear fe65. moreover, genotoxic damage produces a rapid translocation of fe65 to the nuclear matrix and stimulates app processing by the -secretase complex and app phosphorylation in thr668, two mechanisms that allow translocation to the nucleus of the complexes associated with aicd. fe65 is required for efficient repair of dna double strand breaks (dsb), a function that depends on its interaction with tip60 and aicd. the fe65-dependent recruitment of tip60 to dsb sites is essential because the histone acetyltransferase activity leads to chromatin opening at the injury site, enabling the access of the complexes involved in repair. on the other hand, tip60 acetylates and activates the ataxia telangiectasia mutated (atm) kinase which in turn phosphorylates a histone h2a variant, called h2ax, which acts as a mark for the recruitment of the reparation machinery. changes in h2ax phosphorylation could be also dependent on the stability of p53 in a mechanism that requires the accumulation of fe65 in the nuclei [81, 82]. however, the fact that phosphorylated h2ax may be also increased in fe65 ko cells under genotoxic damage suggests that complementary mechanisms may regulate this behavior. fe65 is highly enriched in the brain where it is expressed as two isoforms produced by the alternative splicing of a 6 bp miniexon. the isoform that includes this exon (which encodes arg-glu inserted in the ptb1 domain) is expressed exclusively in neurons, whereas the isoform lacking these two aminoacids is expressed only in nonneuronal cells. fe65 protein expression may change during development and also in pathological conditions such as ad, opening up the possibility that it participates in plastic processes in neurons, which is reflected in the phenotype of fe65 and fe65l1 double ko mouse. these mice exhibit defects in the positioning of cortical neurons characterized by the presence of ectopic neurons that break the pialmeningeal basement membrane and displace cajal-retzius neurons and also have serious defects in axonal projections. many of these phenotypical features are shared by mice lacking some of the fe65-binding partners such as the app family and the mammalian homolog of drosophila enabled (mena). mena belongs to a family of proteins that regulate actin dynamics and thereby modulate cell motility and morphology. mena is located in areas of dynamic actin remodeling such as lamellipodia and growth cones and interacts with the actin-binding protein, profilin. mena interacts with the fe65 ww domain, assembling a macromolecular complex with app that regulates axonal branching, cell motility, and possibly the dynamics of actin at the growth cone and synapsis. in a previous attempt to generate a fe65 ko, it was expressed a truncated protein lacking the n-terminal domain and translated from met261. this 60 kda variant does not contain the ww domain and does not display the transactivation activity of the larger isoform. in spite of the expression of this smaller protein, the animal shows defects in hippocampal-dependent learning and long-term potentiation (ltp) [93, 94]. however, it is difficult to assess whether these defects are due to the 97 kda isoform loss or the appearance of this new 60 kda isoform acting as a dominant negative protein. although the amyloid cascade hypothesis has become the mainstream in the study of ad neurobiological mechanisms, several groups have recently suggested that this should be at least reevaluated in the light of new findings [9597]. transgenic mice that overexpress the aicd and the adapter fe65 in the forebrain (under the control of the camkii promoter) display several neuropathological features observed in various transgenic models and in the ad patients brains, with the exception that they do not show a accumulation in the brain. the expression of aicd together with fe65 seems to be essential to induce an ad-like phenotype in the transgenic model, since a single aicd transgenic mouse developed by an independent group does not present the characteristics of the double transgenic, indicating that the functional relationship between both proteins, discussed in the previous sections, is indeed essential. as in the brain of patients with ad and several other transgenic models used to study ad, the aicd/fe65 mice show an increase in gsk-3 activity. interestingly, the double aicd/fe65 transgenic does not affect the gsk-3 mrna or protein levels, as would be expected from a previous study which suggests that the kinase should be transcriptionally regulated by the aicd/fe65 complex. kinase activation in the double transgenic is indeed correlated with an increase in the tyr216 activating phosphorylation and a decrease in the ser9 inhibitory phosphorylation. a molecular explanation for this may be related with the fact that fe65, through its ww domain, interacts and promotes gsk-3 phosphorylation on tyr216. increased gsk-3 activity in the aicd/fe65 mice produces hyperphosphorylation of two direct targets: the microtubule-binding proteins, collapsin-response mediator protein (crmp)-2 and tau [11, 98]. increased crmp-2 phosphorylation is also found in transgenic mice expressing mutated forms of app and presenilin (ps)-1 and also in the cerebral cortex of ad patients. increased crmp-2 phosphorylation is an early event that precedes the formation of amyloid plaques and neurofibrillary tangles. interestingly, this posttranslational modification seems to be specific for ad, since it has not been reported in other neurodegenerative conditions like the frontotemporal dementia and pick's disease [100, 101]. hyperphosphorylation of tau is the initial event in the pathway to tau self-aggregation, forming the paired helical filaments (phfs). phfs are found at the core of the highly insoluble intraneuronal neurofibrillary tangles, one of the two neuropathological lesions (another is the senile plaques) that characterize the ad patients brains. the aicd/fe65 mouse shares with 3xtg mice the capacity to promote the formation of tau insoluble aggregates, which are not observed in most mouse models for ad. the aicd/fe65 double transgenic mouse has nonconvulsive seizures with aging, abnormal electroencephalogram (eeg) spiking, and a greater sensitivity to seizures induced by kainic acid (ka) in young animals. it also presents several alterations in hippocampal neural circuits, characterized by abnormal sprouting of the mossy fiber terminals with increased neuropeptide y (npy) expression and loss of calbindin-positive neurons. alterations in the eegs and seizures have been observed in ad patients and in mouse models for this pathology, such as mice r1.40 (with appswe), appps1, and pdapp [105, 106]. aged aicd/fe65 animals (> 18 months) show neurodegeneration in the ca3 hippocampal area, although the defects in working memory (evaluated by the y maze paradigm) start at a young age (8 months). since most of the mouse models for ad are based on the expression of mutant variants of the human app or presenilin found in cases of familiar ad, the identity of neurotoxic app fragments has not been clearly discerned yet. several studies have shown that a deposition in senile plaques does not correlate with neuronal death and cognitive deficits present in different transgenic models [107, 108]. for example, the overexpression of wild type happ in mice produces memory deficits, tau hyperphosphorylation, synaptic loss, and neurodegeneration without inducing an increase in a levels. surprisingly, overexpression of happ together with -secretase in mice induces a decrease in a levels and plaque deposition, but the animals suffer severe neurodegenerative disorders and learning defects. in both models, an accumulation of c-terminal fragments of app including the aicd is it therefore possible that this fragment generated along with the a may be responsible for the alterations in transgenic models of ad? interestingly, the ad model termed pdapp, when combined with a mutated form of the aicd (d664a), shows a complete reversion of the neuropathological hallmarks of the disease, including synaptic loss, the dentate gyrus atrophy, the astrogliosis, the deficits in synaptic transmission and memory, and the behavioral abnormalities without affecting the a levels or the plaque accumulation [111114]. these results strongly suggest that the causal relationship between the a accumulation and the neuropathological defects usually associated with ad may be challenged and position the aicd as a good candidate to explain the effects observed in various transgenic models based on mutations in app and ps1. the two hallmarks of ad, the amyloid plaques, and neurofibrillary tangles, which are elegantly related through the amyloid cascade hypothesis, are the main components in the current research on the molecular mechanisms leading to this pathology. since its origin, the amyloid cascade hypothesis has accumulated substantial evidence in its support, which has virtually overshadowed the fact that clinical trials based on this hypothesis have been shown to be unsuccessful. one of many possibilities to explain the failure of clinical trials could be related with the fact that several mouse models express the human-mutated app found in familial ad, so it is unclear which abnormalities detected in these models are product of specific a species (like oligomers) or another toxic metabolites of app (like aicd) or simply due to effects of overexpression of happ. however, the evidence collected from the transgenic models here reviewed could help to discern whether the a species or the aicd are the key elements triggering neurodegeneration. three independent transgenic mice lines (a single transgenic of happ, a double aicd/fe65 transgenic, and the double happ/-secretase transgenic) recapitulate the neuropathological alterations of the disease without any increase in a secretion. all of these models have an accumulation of the app c-terminal fragments. moreover, the introduction of a point mutation in the aicd in transgenic mice expressing the happ with the swe and indiana mutations, the ad-like phenotype is reversed, in spite of increased a production. all of these evidences suggest that the aicd could be acting as the bona fide toxic intermediate in the ad progression and could become a target for future therapeutic interventions against this devastating disease.

since its proposal in 1994, the amyloid cascade hypothesis has prevailed as the mainstream research subject on the molecular mechanisms leading to the alzheimer's disease (ad). most of the field had been historically based on the role of the different forms of aggregation of -amyloid peptide (a). however, a soluble intracellular fragment termed amyloid precursor protein (app) intracellular domain (aicd) is produced in conjunction with a fragments. this peptide had been shown to be highly toxic in both culture neurons and transgenic mice models. with the advent of this new toxic fragment, the centerpiece for the ethiology of the disease may be changed. this paper discusses the potential role of multiprotein complexes between the aicd and its adapter protein fe65 and how this could be a potentially important new agent in the neurodegeneration observed in the ad.

PMC3296194

pubmed-725

its onset and progression cause deterioration of sensory, motor, and autonomic nerve functions, markedly reducing patients ' quality of life (qol). the polyol pathway is a side pathway metabolizing excess (or unused) glucose to sorbitol. it is thus suggested that intracellular sorbitol production (which is increased by accelerated metabolism in the hyperglycemic condition) may trigger the development and progression of dpn. aldose reductase (ar) is a rate-limiting enzyme that controls the polyol pathway. ar inhibitors (aris), expected to ameliorate dpn, have been extensively developed. a promising ari zenarestat not only reduced sorbitol production and improved nerve conduction velocity (ncv), but also significantly increased myelinated nerve fiber density in the sural nerve via reducing the sorbitol concentration by 80% or more. it has already been demonstrated that ranirestat orally administered for 12 weeks significantly inhibited accumulation of sorbitol within the sural nerve: a dosage of 20 mg/day reduced accumulation by 83.5%. furthermore, the 12-week treatment improved sensory ncv (the change from baseline reached 1 m/s). even after an additional 48-week treatment, the improved sensory ncv was long maintained and associated with ameliorated peroneal motor ncv [3, 4]. on the basis of these results, we carried out the present clinical trial to explore the effectiveness and safety of ranirestat in japanese dpn patients. this study was a multicenter (20 sites in japan), double-blind, randomized, placebo-controlled study in which patients with dpn were assigned to either ranirestat 20 mg/day or placebo administered after breakfast as a once-daily dose for 26 weeks. the 20 mg/day dose was selected because it was associated with an 83.5% inhibition of sorbitol accumulation in the 12-week biopsy study. the following procedures were performed at entry for each patient: medical history, physical examinations, nerve conduction studies (ncs), and both the toronto clinical neuropathy score (tcns) and modified tcns (mtcns) [57]. adverse events were recorded. at weeks 12 and 26, ncs, tcns, and mtcns were repeated. the primary end point was the summed change in sensory ncv from baseline of the bilateral sural and proximal median sensory nerves. secondary end points were the changes for individual ncvs, amplitudes, minimum f-wave latencies (mfwl), tcns, and mtcns. we enrolled patients who met the following entry criteria: age 2070 years, either sex, type 1 or 2 diabetes for at least 6 months, glycemic control stable for at least 6 weeks before entry, and hba1c (7.4% but 11.5%) [8, 9]. dpn was diagnosed when two of the following four modified san antonio criteria were present: (1) symptoms of dpn, (2) signs of dpn, (3) abnormal results of ncs with at least two abnormal nerves (meeting this criterion was mandatory), and (4) abnormal vibration perception threshold (< 10 seconds using a 128 hz tuning fork). the requirement for both sural nerves potential amplitude responses of at least 1.0 v insured the presence of viable nerve fibers to allow accurate measurements and avoided inclusion of patients with severe neuropathy who would not be expected to respond. since a sural nerve generally shows symmetrical responses, the difference in sural nerve potential amplitude and conduction velocity between the right and left legs should be limited (amplitude<6.0 v, ncv<7.0 m/s). patients with nondiabetic neuropathy were excluded, as well as those with any clinically significant abnormal clinical laboratory parameter or any abnormal liver function test. the study was performed in accordance with the guidelines expressed in the declaration of helsinki. testing was standardized for measurement of temperature, side of testing, stimulation protocol, averaging of sensory potentials, and measurement of latencies and amplitudes. standardized techniques with temperature controlled and distal distances fixed were used for ncs. the minimum temperature was maintained at 31c in the forearm and 30c in the lower calf. if limb temperature was lower than specified, the limbs tested were warmed in a heating water bath before starting the test. it was recommended to warm cold legs in hot water at approximately 40c for at least 20 min before performing the test. unilateral ncss were performed on the nondominant median motor, dominant tibial motor, and nondominant median sensory nerves. the fixed distal surface electrode distances for motor ncs were 60 mm for the median nerve and 80 mm for the tibial nerve. corresponding distances for sensory ncs were 20 mm proximal to the distal wrist crease for the median nerve and 140 mm for the sural nerve. measurements of distances, response latencies, and amplitudes were performed in a standard fashion using onset latencies and baseline-to-peak amplitudes. f-waves were generated for all motor nerves with 16 supramaximal stimuli per nerve, and the minimal reproducible latency of at least three responses was measured. the examiners had access to the previous temperatures, distances, and results through this trial. results of the screening ncs for each patient were reviewed and the eligibility of each patient was decided by the nerve conduction study assessment committee before randomization. this central supervision ensured consistency of study procedures and high quality of data under blinding. the tcns has been modified to better capture sensory test results reflecting early dysfunction in dpn, also to improve the sensitivity and specificity of the original tcns [6, 7]. the mtcns includes a symptom domain and a sensory test domain. in the symptom domain, pain, numbness, tingling, weakness, and ataxia in foot and upper limb is separated into 4 stages: 0=absent, 1=present but not interfering with the sense of well-being or activities of daily living, 2=present and interfering with the sense of well-being but not with activities of daily living, and 3=present and interfering with both the sense of well-being and activities of daily living. in the sensory test domain, pinprick, temperature, light touch, vibration, and position sense were assessed as 0=normal, 1=reduced at the toes only, 2=reduced to a level above the toes, but only up to the ankles, and 3=reduced to a level above the ankles and/or absent at the toes. the mtcns scale varies from 0 (no signs or no symptoms of dpn) to 33 (all symptoms and signs of dpn present with a maximum score of 18 symptom points and 15 sensory test points). the full analysis set was used in the efficacy analysis and included all randomized patients but excluded those receiving no investigational drug and those with no efficacy data (figure 1). changes from baseline to the last observation in efficacy variables were compared between the treatment groups. the last observation was recorded at week 26. when no data were available at week 26, the data were compared between groups by analysis of covariance using group as a factor and baseline values as a covariate. the changes were determined by group at each visit for which summary statistics were calculated and plotted against visit. within-group differences were tested using the paired student t-test by group and visit. for binary data, the number and percentage were determined by group and visit and compared between the groups using the fisher exact test. for ordinal data, the number and percentage were determined by group and visit and compared between the groups using the mantel test. we screened 130 patients and excluded 57 patients for not meeting the inclusion criteria or meeting the exclusion criteria at screening (n=54) and for withdrawing their consent prior to randomization (n=3). seventy-three patients were randomized to either ranirestat or placebo (40: 33), and all 73 received an investigational drug (figure 1). some differences in hba1c between the ranirestat and placebo groups were observed at baseline. because the magnitude of change in the individual ncv varied, the sensory ncvs were summed to comprehensively evaluate each sensory nerve's function. the summed sensory ncv (primary endpoint) was the sum of the ncv in the bilateral sural sensory nerves and proximal median sensory nerves. distal median sensory ncv was not included in the summed sensory ncv in order to avoid the possible influence of carpal tunnel syndrome. the change from baseline to the last observation was 7.28 1.27 m/s (least squares mean [lsm] se) in the ranirestat group and 1.92 1.39 m/s in the placebo group (table 3). analysis of covariance of the changes in the summed sensory ncv at the last observation using drug group as a factor and the summed sensory ncv at baseline as a covariate detected a significant improvement in the ranirestat group compared with the placebo group (p=0.006). in order to investigate how the imbalance of baseline hba1c between the two groups influences the results, analysis of covariance (ancova) was conducted to assess change in summed sensory ncv from the baseline to the last observation, controlling for hba1c by adding baseline hba1c as a covariate, in reference to the ich e9 guideline. the changes in summed sensory ncv were 7.54 1.29 m/s in the ranirestat group and 1.60 1.42 m/s in the placebo group, indicating significant difference between the two groups (p=0.003). there was no significant effect of baseline hba1c because the changes before and after adding the covariate of baseline hba1c were similar. table 3 also shows that the improvement of ncv from baseline to the last observation in the individual nerves was consistently significant in the ranirestat group (p<0.0010.030) for all except the median motor ncv. the between-group differences in proximal median sensory ncv were significant (p=0.019). there was a tendency of significant between-group difference in median motor ncv (p=0.051). analysis of covariance of the change at the last observation detected a significant difference for the proximal (p=0.026) and distal (p=0.019) median motor nerves between the ranirestat group and the placebo group. the improvement in the tibial motor mfwl was significant (50.12 4.69 msec versus 50.98 5.28 msec, p=0.007). however, analysis of covariance for each nerve detected no significant improvement for the ranirestat group compared to the placebo group. the total score improved at 12 weeks in the two groups, and no between-group difference was observed in the change from baseline at the final evaluation. by domain, similar time-course changes were seen in the symptom domain in the two groups, while ranirestat change tended to increase at the final evaluation in the sensory test domain. an additional analysis in a subgroup of patients with mild to severe neuropathy according to the tcns severity classification revealed significant improvements in the sensory test domain in the ranirestat group (p=0.037), although there was no between-group difference in total score change at the final evaluation. the prevalence of adverse events was similar in both groups: 33 of 40 patients (82.5%) in the ranirestat group and 29 of 33 (87.9%) in the placebo group. in the ranirestat group, 3 serious adverse events (appendicitis perforated, peritonitis, and spinal compression fracture) were noted, but all were judged by the investigator to be unrelated to ranirestat (table 5). this clinical trial has demonstrated that oral administration of ranirestat at 20 mg/day for 26 weeks, as compared with placebo, significantly improved the primary endpoint of summed sensory ncv: summed sensory ncvs in the ranirestat group increased after 12-week treatment and were significantly higher than that in the placebo group at the final evaluation (p=0.006). in a proof-of-concept study conducted in north america, 12-week treatment with ranirestat 20 mg/day reduced the sorbitol concentration in the sural nerve by more than 80% and ameliorated sensory nerve ncv. these study results were reproduced in the present study carried out in japanese patients with dpn. blood glucose control is critical for the treatment of dpn, as indicated by the results of a large-scale clinical trial. the blood glucose control status may affect the results of our study. since an imbalance in hba1c was found at baseline in the present study, we investigated whether the blood glucose control status at baseline affected the study results. using baseline hba1c as another covariate on the basis of the ich e9 guideline, we performed an additional analysis of summed sensory ncv and found the significance of differences between the ranirestat and placebo groups had remained unchanged (p=0.0034): the robustness of our results was thereby confirmed. the changes from baseline to last observation in hba1c were+0.26% in the ranirestat group versus+0.07% in the placebo group; there were no significant changes from baseline in the two groups. furthermore, we performed a subgroup analysis of summed sensory ncv, via dividing participants into well-controlled (improved or unchanged hba1c [hba1c 0% ]) and poorly controlled (deteriorated hba1c [hba1c>0%]). the change from baseline in summed sensory ncv was 9.4 m/s for ranirestat (n=23) versus 4.1 m/s for placebo (n=12) in well-controlled participants and 3.8 m/s for ranirestat (n=14) versus 0.5 m/s for placebo (n=19) in poorly controlled participants. in either subgroup, ranirestat produced greater changes, indicating that difference in blood glucose control during the study period had no effect on the results or conclusions in this study. thus, we consider that baseline hba1c imbalance has no relevant effect on study conclusions. nevertheless, as the present study is a small-scale trial with limited subgroup analysis, a further study involving a larger number of participants is desired for a valid conclusion. dpn is a systemic neuropathy that damages both the sensory and motor nerves as well as both the upper and lower limbs. the landmark diabetes control and complication trial followed up patients receiving intensive treatment and those receiving conventional treatment for 5 years and reported that ncv at 5 years was lower by more than 1 m/s in the conventional treatment group than the intensive treatment group. in the present study, we examined and evaluated a sensory nerve and a motor nerve in both the upper and lower limbs. compared with placebo, ranirestat significantly increased proximal-median sensory ncv (p=0.019). these findings imply that the effect of ranirestat is not limited to a particular nerve but extends to all peripheral nerves. in this study, ncv of each nerve tested was higher by 0.83 to 2.17 m/s in the ranirestat group than in the placebo group, indicating that ranirestat and strict control of blood glucose play equally potent roles in maintaining nerve function. a clinical trial using median motor ncv (mmncv) as a parameter for long-term treatment with epalrestat (the only ari in clinical use) reported that epalrestat significantly reduced mmncv deterioration by 0.78 m/s at one year, by 1.21 m/s at 2 years, and by 1.60 m/s at 3 years as compared with the control. in this study, the difference in mmncv between the ranirestat and placebo groups was 1.06 m/s (p=0.051). in regard to ncv, these findings indicate that ranirestat can be expected to exert an effect as potent as the existing therapeutic epalrestat. in parallel to the present trial, a phase iii clinical trial of ranirestat was carried out in north america. reported that summed motor ncv was significantly improved by ranirestat, but summed sensory ncv was not. on the other hand, in our study, summed sensory ncv was significantly improved, whereas motor ncv was not significantly changed, although there was a tendency of significant between-group difference in median motor ncv (p=0.051). it is difficult to clearly elucidate the reason for the different results between two trials. the number of participants in their trial was more than three times as large as ours. it may be one of reasons that summed motor ncv was significantly improved in their trial. as for summed sensory ncv, measurement of sensory ncv using surface electrodes is affected by a variety of conditions such as skin temperature and measurement site condition, because the amplitude of sensory nerve action potential (measured in microvolts) is markedly lower than that of compound muscle action potential (measured in millivolts). to overcome these difficulties, measurement in ncs was performed more precisely by standardization of measuring methods, use of common procedures, and intensive evaluation in the core laboratory to increase data reproducibility in the phase iii trial and our trial. however, a large difference in demographic characteristics of patients such as bmi might partially affect condition of measurement sites such as subcutaneous tissue; bmi (mean sd) was 25.0 3.5 in our study versus 33.1 6.8 in the north america study. dpn is a nerve-degenerative disease that progresses slowly and is characterized by a variety of clinical manifestations including subjective symptoms (such as spontaneous pain; positive symptoms) and sensory deterioration (negative symptoms) associated with progression of nerve destruction. in this study, these various clinical symptoms were evaluated with the use of an mtcns (with symptom domain dedicated to positive symptoms and sensory test domain dedicated to negative symptoms). both the original mtcns and japanese version are recognized as valid and reliable evaluation tools [6, 7]. in this study, the total score of mtcns improved at 12 weeks in both groups and no between-group difference in change from baseline was found at the final evaluation: no obvious effect of ranirestat was observed on clinical symptoms. since the mtcns used in this study is based on the tcns, all patients were divided into two subgroups, based on tcns severity. one subgroup of patients with tcns total score 5 (9 in the ranirestat group and 4 in the placebo group) was excluded in order to perform an additional analysis. in the other subgroup of patients with tcns total score 6, ranirestat elicited significant improvement in the sensory test domain, as compared with placebo (p=0.037). as sensory test results have been reported to well correlate with risk of foot ulcers, improvement in negative signs is important for preventing foot ulcers and avoiding limb amputation, which are targets of dpn treatment. nevertheless, the efficacy of ranirestat on clinical symptoms remains to be elucidated, probably because this trial was limited by short treatment duration and presence of a placebo effect. evaluation using mtcns in the phase iii trial of ranirestat in north america also demonstrated that mtcns scores were improved in the all groups including placebo at 12 weeks and no efficacy of ranirestat was detected at 52 weeks. placebo effects were also noted in the recent phase ii/iii studies of ranirestat with 2-year treatment duration (in asia, europe, north america, and russia) and resulted in a failure to demonstrate significant efficacy on clinical symptoms. for evaluation of clinical symptoms of dpn, different scales have been used in different clinical trials. placebo effects have been observed in multiple trials, perhaps not only in trials using the mtcns [16, 17]. in our and other studies, blood glucose was relatively well controlled and maintained, which might be attributable to lack of deterioration in the placebo group. because dpn is slowly progressive, it may be necessary to design a study with longer duration of more than 2 years and with a more sensitive tool for assessing or detecting clinical symptoms. regarding the adverse events in this study, there was no particular difference between the two groups; adverse effects on hepatic and/or renal function associated with use of other drugs were also undetectable. these findings assure the safety of the 26-week treatment with ranirestat at a daily dose of 20 mg. as compared with placebo, ranirestat administered to japanese dpn patients at a once-daily dose of 20 mg for 26 weeks significantly improved summed sensory ncv. a subgroup analysis revealed that treatment with ranirestat led to significant improvement in clinical signs (i.e., increased the sensory test domain score of the mtcns). however, this study aiming at proof-of-concept was limited by its short-term treatment duration and small number of patients. further studies are needed to establish the efficacy of ranirestat in the treatment of dpn.

we conducted a 26-week oral-administration study of ranirestat (an aldose reductase inhibitor) at a once-daily dose of 20 mg to evaluate its efficacy and safety in japanese patients with diabetic polyneuropathy (dpn). the primary endpoint was summed change in sensory nerve conduction velocity (ncv) for the bilateral sural and proximal median sensory nerves. the sensory ncv was significantly (p=0.006) improved by ranirestat. on clinical symptoms evaluated with the use of modified toronto clinical neuropathy score (mtcns), obvious efficacy was not found in total score. however, improvement in the sensory test domain of the mtcns was significant (p=0.037) in a subgroup of patients diagnosed with neuropathy according to the tcns severity classification. no clinically significant effects on safety parameters including hepatic and renal functions were observed. our results indicate that ranirestat is effective on dpn (japic cti-121994).

PMC4736957

pubmed-726

attempting to motivate our children to go to bed at a reasonable time, parents have for generations invoked these three time-honored rewards. health is the absence of something, namely sickness, and is only fully appreciated when we no longer have it. yet, when we are really sick, health becomes our paramount concern. few of us would choose to be a wealthy, wise man or woman if the price was advanced alzheimer's disease, terminal cancer, or crippling arthritis. in this context, we have a major problem as a society. despite our amazingly advanced technologies in such areas as communication, space travel, and transportation why is it that our capacity to innovate in biomedical sciences seems to lag so dramatically behind our innovation capacity in aircraft design, for example? we serve as directors of the california institute for quantitative biology (qb3), which seeks to promote innovation in the biomedical sciences. at qb3, we look closely at the impediments to improving health through technology and explore solutions to this problem. we are convinced that many answers could be found in our research universities if they were restructured. they need to be innovative in how they manage their science, as well as in how they perform it. universities are without peer in their ability to discover the fundamental principles of science and are responsible for much innovation in our society. the creativity and nimbleness of their science is unfortunately not matched by equal creativity and nimbleness in administration and management. although service innovation is now widely seen as contributing to economic growth and addressing societal needs as effectively as science innovation, universities in general continue to operate using time-honored, unchallenged principles. this must change. we need to evaluate how effectively we perform our public mission, and whether our exclusive focus on specialization precludes our usefulness. to know what to change, we have to go back to the way our biomedical industrial complex is constructed. we have come a long way from the days when science was done by a gentleman scientist in a room in his mansion, supported by his own funds or those of a generous friend. nowadays, governments put a significant part of their gross domestic product into supporting research and development. much of this investment, about $50 billion per year in the united states alone, goes to our research universities. governments justify this use of taxpayers money by pointing out, correctly, that unfettered research activities are the most effective way of fostering the innovation needed to address current and future challenges to society. scientists also are motivated by the sheer beauty, elegance, and excitement of fundamental discovery, but this is an acquired taste, not usually shared by the general public! what society does expect are results that meet its needs. to get societal value from university research if investors and manufacturers see a potential market for an innovation, then the discovery moves efficiently out of the lab and into the marketplace. not only does society get something it needs, as measured by its willingness to pay for it, but the manufacturing and marketing process creates jobs and economic growth. the social contract of science, therefore, is that university scientists are given the freedom to pursue their research interests, supported by taxpayers funds, because the taxpayers have been convinced that potentially commercializable discoveries will be made, which will result in new products and economic growth. the contract specifies roles for the three stakeholders: government, universities, and the private sector. in the life sciences, the system is not working well for all three stakeholders. government is questioning whether it is getting sufficient value when its investment in university research generates one start-up company per $100 million and one patent license for every $12 million (battelle technology partnership practice, 2010). university scientists are being told that they must stop pursuing fundamental biological principles and direct their research toward specific societal needs. venture capital, especially seed funds for life sciences innovation, is drying up (phillippidis, 2011). finally, the powerhouses of the biomedical industry, the large pharmaceutical companies, have few new products coming to market and are looking at a drop in income of over $100 billion in the next few years (begley and carmichael, 2010; wilson, 2011). the crisis in the biomedical innovation world is what is frequently called a wicked problem, one that is challenging to solve because it involves the interaction of disconnected entities. to solve this crisis requires stakeholders to interact with one another more productively. we believe that research universities are ideally suited to solve wicked problems but need to adjust their operating structures to do so. a few years ago, the petroleum giant bp awarded a consortium of universities led by berkeley the largest grant in university of california history, $50 million per year for 10 yr. despite the well-publicized travails of bp, that commitment remains firmly in place. graham fleming, now vice chancellor for research at berkeley but earlier a qb3 leader and one of the architects of the european bioinformatics institute consortium, explains it this way. manufacture of biofuels is a classic wicked problem, requiring for its solution the expertise of many stakeholders with disparate backgrounds and nonoverlapping goals. manufacturing biofuels requires economists to verify a market, chemical engineers to design refineries, industrial microbiologists to optimize the enzymatic breakdown of biomass, botanists to select the optimum biomass, agronomists to define the growing regions, and hydrologists to provide them with adequate water. even a company with the resource base of bp does not have quality expertise in all these fields. in contrast, universities do have the requisite talent, but the trick is to network the faculty, creating a team. the berkeley leadership skillfully assembled a biofuel ecosystem and so deservedly won the bp competition for the $500 million. this example demonstrates that, with inspired leadership, universities can assemble teams to address wicked problems. an obvious challenge is whether creation of an analogous bio-innovation ecosystem might help address the current troubles in the life sciences industries. insight into how a bio-innovation ecosystem might impact the difficulties faced by the life sciences industries can be garnered by examining the aviation industry. the pharmaceutical and aircraft industries both invest huge amounts of money and time in creating new products. aeronautical engineers may not completely understand the physics of wing lift, but they can predict what will fly with remarkable accuracy. a plane is designed by engineers, built to their specifications, rolled out on a runway, and takes off perfectly. we have such trust in our aviation knowledge and our engineers that we are not surprised. in contrast, over 90% of candidate drugs fail completely to do any good when administered to patients during phase i trials (paul et al., 2010). if manufacturing new aircraft were similar to designing new drugs, 9 out of every 10 newly designed planes would crash on takeoff! an airplane cockpit is crammed with indicators that monitor the status of almost every important function. if something begins to go wrong, it is quickly detected and the pilot can take corrective actions and determine if they are indeed working. how do we know whether a drug for alzheimer's or schizophrenia or cancer is having an effect? lacking quantitative biomarkers that reflect the progress of a disease makes it a huge challenge to measure drug efficacy. as much as the industry needs new drugs, it yearns for biomarkers to precisely measure efficacy and sensitively warn of unwanted toxicities (editorial, 2010). creating such biomarker sets to monitor a single disease (e.g., alzheimer's progression) we may scoff at the old theories from galen's time of balancing the humors by medical intervention, but in truth we are not much more sophisticated now. to continue the airplane analogy, it is like repairing a defect in an airplane by breaking something else! the explanation of this paradox is emerging, albeit slowly, as we move from reductionism to looking at the human body as a set of interlocking systems. aircraft engineers have followed a systems approach for decades and biologists are beginning to follow in their footsteps. until biologists have assembled a systems approach to replace current molecular reductionism, we may have to settle on an empirical strategy to guide drug development, using biomarkers as indicators of benefits and dangers. successful innovation in the life sciences will require more than assembling teams of academic scientists to develop predictive biomarkers. it will also require that three entities with fiercely independent cultures and largely nonoverlapping goals have meaningful dialogues with one another. government, academia, and the private sector need to reconcile their separate goals and work together more cohesively. a dialogue with the private sector is the obvious way to find out whether a university discovery has practical implications (silber, 2010).. universities have poorly defined mechanisms for determining what society, or even the life sciences industry, really needs and so, by default, let research programs be driven by faculty interest, not societal need. for a successful dialogue, the private sector must also have something to learn from the university. what the life sciences industries want is the identification of novel drug targets, disease biomarkers, and disruptive technologies for a start, but few faculty do research pertinent to those wants, even if they are aware of them. the investment community wants to prowl the halls looking for a juicy academic project that will give impressive returns on investment in a small number of years a biological google! the manufacturing companies and the investors are keenly sensitive to regulatory and reimbursement hurdles imposed by government, to tax incentives, and to grants for small businesses. in return, government wants to hear about the link between scientific innovation and tax revenue, economic growth, and job creation (national economic council, 2011). finally, the universities need to confer with government about what fraction of research funding should be targeted for applied versus basic research (usdin, 2011). our contention is that the dialogue can be dramatically optimized by creating a university-centered bio-innovation ecosystem. the structures of the ecosystem will facilitate three-way conversations among the three stakeholders and make those conversations disciplined and productive. (companies advise government on university funding; universities give government input on small business grants, innovation zones, and regulatory science; companies help universities identify unmet needs.) we argue that this much-needed innovation in the way research universities carry out their mission will pay off in generating more user-driven innovation in the universities, more efficient use of clinical trials data, a marked increase in efficacious drugs coming to markets, more evidence-based regulatory frameworks, enhanced economic growth through job creation, and consolidation of america's place as a leader in bio-innovation. the paucity of new therapeutic drugs coming to market can not be attributed exclusively to weaknesses in our pharmaceutical industries. several concrete steps are suggested: acknowledge that academia must interact more collegially with the two other stakeholders, government and the private sector.recognize that the shortage of new drugs is a wicked problem and that universities are ideal places to solve wicked problems.commit to solving wicked problems by creating networks run by academic generalists who can extract value from our silos of specialization.recruit, reward, and promote academic generalists to coordinate the activities of specialists in the solution of wicked problems.set up criteria that can assess the value of academic generalists. if successful, universities should: foster partnerships between clinical and basic scientists;link academic scientists to industry partners with complementary skills, allowing them to work together to address pressing societal problems;link academics to the resources they need to start up companies;advocate effectively in a nonpartisan manner for improvements in government funding of research and in reimbursement and regulatory policies; andalert their academic colleagues, both faculty and students, to opportunities that match their research specialties to pressing needs of society. acknowledge that academia must interact more collegially with the two other stakeholders, government and the private sector. recognize that the shortage of new drugs is a wicked problem and that universities are ideal places to solve wicked problems. commit to solving wicked problems by creating networks run by academic generalists who can extract value from our silos of specialization. recruit, reward, and promote academic generalists to coordinate the activities of specialists in the solution of wicked problems. if successful, universities should: foster partnerships between clinical and basic scientists; link academic scientists to industry partners with complementary skills, allowing them to work together to address pressing societal problems; link academics to the resources they need to start up companies; advocate effectively in a nonpartisan manner for improvements in government funding of research and in reimbursement and regulatory policies; and alert their academic colleagues, both faculty and students, to opportunities that match their research specialties to pressing needs of society. besides supporting research and education in the quantitative biosciences, qb3 has led an aggressive effort to create a small academy of generalists devoted to solving wicked problems and to creating a bio-innovation ecosystem that includes government and the private sector. although it is still more a work in progress than a blueprint for success, our experience to date suggests there is a role in universities for scientists who are willing to go beyond the comfort zone of their own special expertise. linking the expertise of others to find solutions to pressing problems can be equally rewarding.

academia should be willing to shoulder some of the responsibility for the current dearth of new therapeutic drugs. our research funding is predicated on the assumption that it will bring value to society, but our emphasis on scientific specialization hinders our ability to add value when a broader vision is required. a solution is the creation of an academy of science generalists motivated to bring together clinical and basic scientists, academia and the private sector, government legislators and industry. a small investment in academic generalists could yield benefits far beyond its modest cost.

PMC3103397

pubmed-727

generalized convulsive status epilepticus (gcse) and non-convulsive status epilepticus (ncse) are important neurological conditions potentially associated with significant morbidity and mortality.1 even with the best current practice, the mortality of patients with refractory gcse is up to 50%. therefore, there is an urgent need for new treatment options that can treat these seizures safely and more effectively than the current standard drugs.2 the most common treatment protocols for status epilepticus include an intravenous benzodiazepine, either lorazepam (lzp) or diazepam, as the initial antiepileptic drug (aed) therapy, followed by phenytoin (dph) or fosphenytoin. phenobarbital (pb) is added if the seizures continue.3 a pharmacologically induced coma, using the barbiturates, propofol or midazolam, is also a frequently used therapy for refractory status epilepticus (rse),2,4,5 which is defined as the persistence of discrete seizures without return to baseline, despite treatment with benzodiazepines and at least two adequate aeds.6,7 however, it requires artificial ventilation and hemodynamic support, and is associated with significant complications and increased mortality.2,4 topiramate (tpm) is an aed with demonstrated efficacy for a broad spectrum of seizure types8; it has multiple activities at receptors and ion channels that may be more effective than conventional anticonvulsants in treating rse.7,9 we report our experience with the use of tpm for the treatment of benzodiazepine-refractory status epilepticus (se) or recurrent seizures in 16 patients who are under severe medical complications such as systemic infection, renal dysfunction, hepatic dysfunction, and pancytopenia from bone marrow suppression. we retrospectively identified patients with gcse or ncse who were treated with tpm at our hospital between july 15, 2006 and august 10, 2008. the medical records were reviewed for information regarding patient characteristics, associated medical conditions, type of se (gcse or ncse), duration, abnormal laboratory findings, treatment history prior to administration of tpm, and outcome. hepatic dysfunction was defined as an ast and alt>200 mg/dl and renal dysfunction was defined as a serum creatinine>2.0 mg/dl and a creatinine clearance<0.6. anemia was defined as a hemoglobin<12.0 g/dl, thrombocytopenia as a thrombocyte count<150,000/mm and pancytopenia as a leukocyte<4,000/mm with anemia and thrombocytopenia. administration of aeds, other than tpm, followed our hospital s protocol: lzp (4 mg) was injected intravenously and dph (15 mg/kg) or vpr (20 mg/kg) were infused intravenously for 30 minutes. the mixture was allowed to sit for several minutes to avoid clumping and then administered via syringe into a nasogastric tube. the loading dose was individualized by patient; 612 mg/kg/day, up to 1,000 mg/day. the resolution of recurrent gtc and gcse was determined by cessation of clinical seizures, and confirmed by follow-up electroencephalopgrahy (eeg). for ncse, from the database on se patients, 16 patients were identified for inclusion in this study. no patient had a history of seizures or was receiving tpm or other aeds prior to admission. the most common previous, co-morbid disease at the onset of seizures was sepsis (n=8). the laboratory studies showed that 12 patients had hematological problems (pancytopenia 8, anemia alone 2, and anemia with thrombocytopenia 2). seven patients had renal dysfunction, three patients had hepatic dysfunction only and another four patients had both renal and hepatic dysfunction. the majority of patients had experienced gcse (n=6) or ncse (n=7). the mean tpm treatment dose was 637.5244.6 mg (3001,000 mg). within a few days, 13 patients could experience their seizure control (the mean duration, 3.72.6 days; range 18 days), but the seizures of the other 3 subjects did not be terminated in spite of all efforts. none of the patients had a worsening of their cbc or blood chemistry profiles with the tpm treatment. for seven patients, the tpm was administrated after the failure of the loading of high doses of dph or vpr. the administration of tpm was within 2 hours after the infusion of dph or vpr. the overall outcome was described as follows: patient death (n=11), improved (n=4), transferred to another hospital (n=1). the causes of 11 deaths were sepsis (n=7), fulminant hepatitis, sah, hypoxic brain damage, and herpes encephalitis. we now represent two illustrative cases using tpm for controlling recurrent seizures from a variety of medical derangement. a 48-year-old woman with no history of seizures was admitted to the hospital in poor health. the patient was diagnosed with paroxysmal nocturnal hemoglobinuria (pnh) 20 years prior to admission and had been treated as an outpatient with oral steroids. the routine blood tests on admission revealed pancytopenia (hemoglobin 4.4 g/dl, hematocrit 13.3%, wbc 730/mm, and a platelet count of 52,000/mm) and acute renal failure (bun 126.5 mg/dl and creatinine 9.23 mg/dl). continuous renal replacement therapy (crrt) was performed in the intensive care unit. on the third hospital day, gcse developed. a loading dose of tpm (800 mg) was administrated via a nasogastric tube. by the next day, however, intermittent brief seizures occurred several times under a maintenance dose of tpm (300 mg bid) during the subsequent two days. the diffusion-weighted mri revealed multifocal high signal intensity involving the occipital lobes, the right basal ganglia and the subcortical white matter of the prefrontal gyri on the diffusion and t2-weighted images. the identified lesions showed increased values on the adc map, suggesting vasogenic edema or a posterior reversible encephalopathy (pres). the tpm was maintained at a dose of 200 mg bid, and there was no further seizure activity. however, aspiration pneumonia and sepsis developed, and the patient died on the 15th hospital day. a 56-year-old woman was admitted to the hospital with impaired mental status. the patient had a history of multiple myeloma (stage iiia) diagnosed three years prior to the admission vad chemotherapy using vincristine, doxorubicin and dexamethasone was started. however, generalized seizures developed during the third cycle of chemotherapy, and the patient became unresponsive with her eyes deviated to the right side. the routine blood tests revealed pancytopenia (hemoglobin 5.2 g/l, hematocrit 14.4%, wbc 4,570/mm, and a platelet count of 15,000/mm), and septic shock developed. however, the blood culture revealed staphylococcus epidermidis, and she died on the 49th hospital day from septic shock. we described 16 patients with gcse, ncse or recurrent gtc in whom tpm was administered alone or together with other aeds. most patients with overt, convulsive status epilepticus respond to the first or second aed. however, when the patient fails to respond to this standard protocol during the initial treatment, the patient is considered refractory and requires additional, more aggressive treatment.3 rse is a life-threatening condition that carries a high mortality risk. aggressive, early intervention permitting avoidance of a pharmacological coma can reduce the overall morbidity and mortality.4 current recommendations for the treatment of refractory status epilepticus include midazolam (mdz), pentobarbital and propofol.10 however, respiratory depression and/or hypotension may result from this treatment, which may necessitate endotracheal intubation and/or vasopressor support .10,11 oral aeds such as tpm or lev, however, have much to offer if they can pre-empt these more aggressive managements.12 tpm has at least five independent actions at the cellular level13: inhibition of kainate-evoked currents, enhancement of -aminobutyric acid (gaba)-evoked currents, blockage of voltage-activated sodium channels, blockage of voltage-activated calcium channels, and inhibition of carbonic anhydrase isoenzymes. the multiple mechanisms of action confer broad-spectrum efficacy against different seizure types and make tpm an attractive choice in the treatment of both partial and generalized se.14 since tpm has pleiotropic effects on neuronal excitability, a favorable pharmacokinetic profile and appears to have few side effects in the setting of rse, it is a reasonable therapeutic option in patients with se.3 tpm is also considered appropriate for treatment of patients with of hepatic or renal disease. it does not significantly increase the risk of hepatotoxicity and renal disease is not a contraindication to the use of tpm.15 there are some reports on the use of tpm in se patients. 16 reported a patient with drug-resistant complex partial se who responded to tpm. towne and colleagues described six adult patients with a variety of types of se who were unresponsive to conventional treatment, including two who also failed to respond to treatment with pentobarbital.7 a suspension of tpm administered by a nasogastric tube was effective in aborting the rse in all cases. remarkably, no adverse events were observed and all six patients who survived and were discharged from the hospital. bensalem et al. also described the use of tpm via a nasogastric tube in three patients with rse (two with generalized seizures and one with partial seizures).14 tpm appeared to be very effective in stopping the rse; in two of the patients that were resistant to either pentobarbital or propofol, and no adverse effects were noted. patients who had se after therapeutic doses of at least two antiepileptic medications were given tpm, 10 mg/kg/d for consecutive days, followed by maintenance doses of 5 mg/kg/d. in each case, the se was stopped within 21 hours of the initial dose of tpm. in our study, the seizures of subjects were terminated both clinically and electrographically in 13 out of 16 patients. the timing of improvement in our patients was within 18 days of starting the administration of tpm. these findings are consistent with the pharmacokinetic properties of tpm and previous reports. in six cases, given tpm after loading with dph or vpr failed, tpm successfully stopped the seizures, even in medically complicated patients. although seizures were stopped clinically and electrographically in the majority of our patients, the overall outcome was not very favorable in our study.. the high mortality rate of the patients studied reflects the large number of leukemia and lymphoma patients treated in our hospital. the majority of the patients studied had serious and often life threatening hematological problems such as pancytopenia or thrombocytopenia. they had a variety of medical comorbidities, including systemic infection, hepatic or renal dysfunction and hematologic/oncologic disorders. however, our results convincingly support that tpm is safe and effective in a special group of seriously ill patients. we could find that tpm was not only safe but very effective for the control of recurrent epileptic seizures or se in patients with serious medical co-morbidities. tpm may be considered as another treatment option when conventional protocols fail. a large prospective, randomized, controlled study is warranted to investigate the efficacy and safety of tpm for the treatment of se .

background and purpose: the conventional therapeutic regimen for status epilepticus (se) may require artificial ventilation and hemodynamic support, and is associated with significant complications and increased mortality. we investigated the safety and effectiveness of topiramate (tpm) in patients with refractory se, who had medical complications such as systemic infection, renal dysfunction, hepatic dysfunction, and bone marrow suppression. methods:we analyzed the clinical features and therapeutic outcome in 16 patients with gcse, ncse or recurrent gtc in whom tpm was administered for its control. results:the majority of our patients had gcse (n=6) or ncse (n=7). the common co-morbid diseases at the onset of seizures were hematological disorders (pancytopenia 8, anemia 2, anemia with thrombocytopenia 2) and sepsis (n=8). twelve patients were under the renal and/or hepatic dysfunction. within a few days, 13 patients could experience their seizure control (the mean duration, 3.7 2.6 days), but the seizures of the other subjects did not be terminated in spite of all efforts. no patients experienced a worsening of their cbc or blood chemistry profiles with the tpm treatment. conclusions:we could confirm that tpm was not only safe but very effective for the control of recurrent epileptic seizures or se in patients with serious medical co-morbidities. tpm may be considered as another treatment option when conventional protocols are ineffective.

PMC3952331

pubmed-728

poly(vinyl alcohol) (pva) is a biocompatible polymer with a great variety of biomedical and pharmaceutical applications. pva in aqueous solution is able to form chemical or physical gels under a variety of conditions. the physical gelation capacity of pva solutions has been well known for a long time; however, the preparation of hydrogels by repeated freezingthawing cycles has attracted renewed interest. the high degree of swelling in water, the rubbery elasticity, the chemical and mechanical stability, the porous fibrilar network structure, and the lack of toxicity makes freezethaw pva hydrogels an attractive polymer matrix for biotechnological applications. to understand the origin of these properties and therefore the bioapplicability of this material, it is necessary to study the physical gelation process produced by the freezingthawing cycles as well as the formed network structure. komatsu el al.(13) investigated the phase diagram of the water/pva binary system. according to this diagram, gelation occurs with or without spinodal decomposition according to the polymer concentration and temperature. the process seems to start with a clustering of chains, which is primarily caused by the association of polar groups of the dissolved polymer, followed by polymer crystallization. this means that the physical cross-links, which are responsible for the network formation, could be formed by different processes, such as hydrogen bonding, polymer crystallization, and in some cases (depending on the gelation conditions), phase separation.(14) in addition, the application of freezingthawing cycles to pva solutions leads to the formation of heterogeneous networks with different morphology and properties as compared with nonfrozen gel systems. were the pioneers in the development of this type of polymer cryogels.(7) in earlier work, they attribute the gel formation to a partial crystallization of chain segments to microcrystalline structures. however, the intensity of the diffraction maximum that corresponds to the 101 reflection is clear for only pva hydrogels with high polymer content. for hydrogels with 10 to 15% polymer, solid-state nmr was used to identify and quantify the crystalline phase, although x-ray analysis is also possible.(29) most recent works show that gelation by freezethaw processing forms heterogeneous networks of interconnected micro- and macropores. willcox et al.,(28) in an exhaustive study using transmission electron microscopy (tem) images and nmr, showed the formation of networks with rounded pore morphology, fibrillar network morphology, or both depending on the number of cycles as well as aging. they attribute the formation of cross-links to a kinetically frustrated crystallization in the first freezing cycle. after this process, subsequent cycles (or aging processes) lead to the creation of new (secondary) crystallites and the growing of the primary crystals; however, the mesh spacing slightly changes. this fact seems to indicate that the average distance between the primary crystallites that are formed during the first thermal treatment constitutes the main controlling factor for the network structure. in addition, auriemma and coworkers in a series of works showed that the porous structure originates from freezing in the first step because of the incomplete crystallization of water. the polymer concentration in this unfrozen water phase is higher than that in the original solution, and the polymer network is formed within this microphase because of pva crystallization. the cross-link points are therefore constituted of polymer crystallites, and the formed gels could be described by a bicontinuous structure of polymer-rich and polymer-poor regions. on the basis of dsc measurements, also inferred the possibility of the existence of covalent cross-links between the polymer chains brought about by the formation of radicals due to strong local shear during water crystal growth.(33) in summary, the pva hydrogels obtained by freezingthawing cycles appear to be constituted by a very complicated network structure that is based on different phenomena (i.e., crystallization, hydrogen bonding, liquidliquid phase separation, and covalent bonds). therefore, a complete analysis of this structure requires the use of different characterization techniques. however, most of them require the manipulation of the hydrogel sample, provoking changes in the original structure. for example, the use of chromatographic techniques or scattering techniques to measure molecular weight or cluster size requires the dilution of the gel(24) (although it was shown that it is possible). mechanical measurements have the limitation of water evaporation at higher temperatures.(9) the cryo-tem technique has the inconvenience of handling difficulties to avoid artifacts.(28) for the study of the crystallite content, very useful techniques such as differential scanning calorimetry (dsc) as well as x-ray diffraction are challenged by a sensitivity problem in dilute samples; for this reason, measurements were sometimes made in dry samples.(34) in this vein, solid-state nmr is a powerful tool for studying polymer networks, but it has not been extensively used in this field. the focus of this work is the use of a low-field nmr spectrometer to analyze the structure of the pva gel in detail as a function of the freezingthawing cycles, checking all of the structural models suggested in the literature. in addition, a melting process that transforms these physical gels into isotropic solutions of pva in water was also studied. importantly, we focus on detecting protons so that sensitivity, even at the low concentrations under study, is not a serious issue. the use of an inexpensive low-field nmr spectrometer on as-prepared samples to study this complex matrix has the advantage that several of the cited inconveniences in sample characterization are avoided. by using different nmr methodologies, we are able to carry out a complete and quantitative study of the complex structure exhibited by the pva gels: multiple-quantum (mq) nmr allows us to gain direct access to residual dipolar coupling constants that persist because of the existence of cross-links and other topological constrains. therefore, this experimental procedure is a powerful tool for quantifying the gelation process(41) and gives us quantitative detail on not only the microstructure(42) but also the dynamics(43) of the gel, that is, a complete picture of the network structure evolution depending on, for example, the number of freezingthawing cycles. in addition, we used pulsed mixed magic-sandwich echo (mse) experiments that provide a near-quantitative refocusing of the rigid contribution to the initial part of the free induction decay (fid)(44) and allow for an essentially quantitative determination of the crystallinity. to our knowledge, this is the first time that advanced nmr pulse sequences performed on a low-field spectrometer were applied to the study of the network structure and phase composition of physical hydrogels. the sample was>99% hydrolyzed, had a weight-average molecular weight of 94 000 g/mol, and had syndio-, hetero-, and isotacticities of 17.2, 54.1, and 28.7%, respectively. pva was dissolved in deuterated water (with polymer concentrations of 10 and 20% w/w) at 80 c under continuous stirring to avoid inhomogeneities and local gelation. solutions were stored at 80 c overnight; then, they were cooled to room temperature for 1 h. then, pva solutions were introduced to 10 mm od nmr tubes and were flame sealed to avoid variations in the water content. two technical considerations are important at this point: (i) the quantity of sample inside the tube has to be 8 mm in height and located in the center of the radio frequency (rf) coil to ensure good rf field (b1) homogeneity. (ii) the length of the nmr tube should be as short as possible for good control of the temperature. this is important because in this way, the whole tube can be inserted in the probe head, thus avoiding possible temperature gradients due to heat conduction. pva hydrogels were obtained by repetition of freezingthawing cycles of solutions inside the sealed tubes that consisted of 1 h of cooling to 32 c and 1 h of thawing at room temperature. after this process, samples were immediately inserted in the nmr spectrometer and measured to avoid the effect of aging. in some cases (specified below), the first freezing cycle was extended to 12 h (overnight), and subsequent cycles were 3 h long. solid-state h nmr spectroscopy is a powerful and widely used experimental technique in the field of polymer science.(35) in recent years, the use of inexpensive and easy-to-use low-field spectrometers has gained the attraction of scientists because it certainly produces quantitative results on polymer structure and dynamics, albeit, of course, without chemical resolution. on the basis of different experiments, which will be extensively described in the next sections, applications include quantitative studies on polymer network structure(38) and polymer crystallization,(44) that is, the main topics of this study. in this work, experiments were carried out on a bruker minispec mq20 spectrometer operating at 0.5 t with 90 pulses of 1.7 s length and a dead time of 12 s. mq spectroscopy is one of the most versatile and robust quantitative techniques for investigating not only the structure but also the dynamics of polymer networks.(38) in solution, the fast segmental motion of the polymer chains through the different accessible conformations is isotropic and completely averages the dipolar coupling interaction that is typical for solid-state spectra. however, constraints, irrespective of their nature (e.g., cross-links, entanglements, or chain packing), lead to nonisotropic segmental fluctuations and therefore to the persistence of a weak residual dipolar coupling (dres), which is our central nmr observable. in other words, the measured effect relies on the orientation dependence of the fluctuating dipolar coupling tensor with respect to the magnetic field, which can be described by an orientation autocorrelation function of the individual chain segments. fast segmental dynamics (in the range of nanoseconds to microseconds) lead to a loss of correlation to a plateau value that is related to the existence of preferential local orientation generated by the existence of cross-links. in fact, the measurable dres is directly proportional to a local dynamic order parameter of the polymer backbone,(38) which is defined as a time average over the fluctuations of the segment-fixed dipolar tensor over the time until the plateau region is reached. it connects the experimental observable with the network parameters: here dstat is the segmental averaged dipolar coupling constant in the static limit and k is a rescaling factor that takes into account the fast dynamics (in the range of picoseconds) inside the statistical (kuhn) segments. the nmr observable is related to r, that is, the ratio of the end-to-end vector to its average unperturbed melt state (r=r/r0), and to n, the number of statistical segments between constraints. a variety of nmr experiments was used to detect residual dipolar couplings to monitor gelation processes, and recently, h mq nmr has evolved as the most powerful tool for obtaining a direct measurement of dres and even its distribution. details of the pulse sequence as well as the data analysis are already published elsewhere. here temperature usually plays an important role in mq measurements(43) because as previously mentioned, the order parameter should be obtained as an average of the segmental fluctuations over all of the possible conformations on the experimental time scale (less than microseconds). therefore, the temperature is required to be far above the polymer glass-transition temperature tg to ensure that the segmental dynamics is fast enough to sweep out the whole conformational space between topological constraints and to achieve full averaging. if this is not the case, then the obtained dres (and therefore the number of junctions in the network) will be overestimated.(41) in this work, the studied samples are hydrogels; therefore, the dissolved state of the polymer chain ensures that the segmental dynamics is indeed fast enough to detect the order parameter in the plateau regime, even at room temperature. similar to the traditional transverse relaxation experiments, the analyzed result of the mq experiment is a time-dependent but normalized double-quantum (dq) signal function, indq, that has the advantage of being independent of any temperature-dependent true relaxation (decay) effect. it is dominated by the dipolar interactions and independent of the polymer dynamics. for this purpose, the directly measured experimental dq build-up curve (idq) must be relaxation-corrected by the use of the also measured reference intensity (iref). initially, iref contains a signal from not only the dipolar-coupled network chains but also the uncoupled, isotropically mobile components such as sol, dangling chains, and so on. the total mq magnetization (imq) needed for normalization is the sum of idq and iref but only after subtracting the non-network contributions. this fraction has a slower relaxation and can be easily identified (figure 1a). in the studied hydrogels, even though we used deuterated water to dissolve the polymer, we found a non-negligible water signal resulting from actual residual water and exchanged oh protons. therefore, we had to identify and subtract two exponential long-time contributions to imq (i.e., the noncoupled fraction of polymer chains (b fraction in eq 2) and the solvent signal (fraction c)), which relax with longer relaxation times (t2c* t2b *) mq nmr build-up and decay data for 10% pva sample after one freezingthawing cycle. (a) as-acquired idq and imq and (b) imq (after extraction of noncoupled contributions) and normalized dq build-up curves (idq). the solid line in a represents the exponential fit for water subtraction, and the dashed line is the exponential fit representing the subtracted noncoupled polymeric defects. in b, solid and dashed lines represent the fits assuming a gamma distribution of dres and a single residual dipolar constant, respectively. (see figure 1b.) finally, it is important to point out that in rubber networks the distribution effects of dres (related to different end-to-end separations and polydispersities of network chains) usually do not play any role.(56) therefore, the normalized dq build-up curve of a bulk rubber can be analyzed in the quasi-static limit in terms of a single dres(38) however, the complex and heterogeneous pva network structure, in addition to specific effects related to the swollen state of the sample (which is proven to be heterogeneous in polymer networks(57)), leads to large deviations from the inverted gaussian shape in the studied pva gels. the actual coupling distribution (related to a microstructurally heterogeneous distribution of constraints) the insert in figure 1b shows the broad distribution of dipolar interactions in pva networks. it resembles a gamma distribution and is consistent with observations of very heterogeneous network structures proposed in the literature. all analyzed samples showed a similar distribution shape independently of the number of freezingthawing cycles. because regularization analysis is subject to some limitations(42) (further work about this point is in progress), we have analyzed all build-up curves under the assumption of the gamma distribution of dipolar couplings that is predicted by the gaussian distribution of end-to-end separations (but which is screened in bulk elastomers) upon fitting, the integral is numerically evaluated within the curve fitting environment of the origin software. in using this distribution, we do not want to imply that this distribution is of any significance in the studied structure; the resulting build-up curves merely describe all of our experimental build-up data very well and have the benefit of not introducing an additional fit parameter, yet they provide a well-defined average residual coupling davg. (the width of the gamma distribution is directly related to the average.) finally, note that the mq analysis pertains to the mobile fraction of the cryogels only; this fraction always constitutes the major part of the sample (> 80%); the rest is rigid crystallites, and the signal of which rapidly relaxes and is not observable under the conditions of the mq experiment. according to previous papers, polymer crystallites act as junctions in pva networks obtained by freezingthawing cycles. solid-state nmr is a useful method for characterizing polymer crystallinity or phase composition in this type of system. the main concept is that the crystalline signal usually decays in the first 20 s of the free-induction decay; therefore, the signal detected after the dead time (12 s in our case) partially conceals this information. nevertheless, in some works, the fraction of protons in a glassy state in pva gels was estimated by monitoring the fid intensity during the first 20 s, which renders these studies qualitative. perhaps the most important is the use of solid echoes with different echo delays combined with back extrapolation(64) to correct for its inability to refocus the dipolar interactions in a multiple-spin system fully. certainly, the best approach is the use of a spectrometer with short (1 to 2 s) dead time.(65) the use of an mse was recently proposed(44) as a more effective method for refocusing the multiple dipolar interactions that lead to the fast decay of the initial part of the fid, therefore removing the dead-time problem and obtaining a near-quantitative rigid fraction determination in polymer systems. under the same experimental conditions, differences of 40% in the quantity of rigid phase in pva compounds figure 2 shows the mse-fids for different total echo duration (realized by increasing the interpulse spacings). the signal decay data are well represented by a gaussian function (parabolic initial decay), and its extrapolation to t=0 allows us to conclude that the signal loss is negligible. (it could be estimated to be 2% on the absolute scale, which corresponds to 10% on the relative scale.) parabolic (gaussian) extrapolation of the maximum mse-refocused fid intensities to t=0. it indicates only minor losses of rigid signal at the shortest echo time (10% on a scale relative to the total crystallinity). the inserted graph shows mse-refocused fids of 10% pva gel after six cycles measured at different echo times at room temperature (304 k). however, note that even for the mse the refocusing efficiency breaks when there is molecular motion on its time scale. in particular, this may be the case if the observed rigid component is glassy rather than crystalline and close to tg. under any condition irrespective of temperature, however, the typical decay time upon incrementing the echo was never significantly shorter than the (relatively weak) decay seen in figure 2, which is ultimately associated with the breakdown of the pulse sequence efficiency because of higher-order imperfections. we thus conclude that all observed rigid signals are associated with crystal-like domains; otherwise, a significant signal loss upon going through tg would have been observed. therefore, mse-fid curves were used to quantify the fraction of fast-decaying rigid polymer present in pva gels. the first fast decay (caused by the rigid polymer fraction) in the mse-fids (insert in figure 2) has a quasi-gaussian decay, whereas the slower signal decay could be fitted with an exponential function. therefore, the first 140 s of the normalized mse-fid curves (unity signal for zero time) were fit according to eq 6 in this expression, a is the fraction of detectable rigid phase in the sample and b is an adjustable parameter for a better fit of the fast decay shape. in all of the cases, b 2 (i.e., the crystalline fraction is described by a gaussian function). in addition, to reference the hard polymer phase to the total amount of polymer (instead of the sample fraction), we corrected the a parameter according to the actual fraction of polymer in the sample obtained from saturation recovery experiments (note again that the samples contain residual water) on the basis of the fact that water has a much longer t1 relaxation time. from such experiments, and assuming no significant noe transfer between the species on the same time scale, the water content can be obtained from a biexponential fit where d is the water fraction in the sample and t1w and t1p are the longitudinal relaxation time constant of water and polymer, respectively (with t1w t1p). for example, at t=304 k, the relaxation times were t1w=1520 ms and t1p=60 ms for a network with 10% pva after seven freezingthawing cycles. the fast-decaying rigid crystallite signal was not detected in these experiments (the fid was evaluated at a time beyond 50 s) such that d is determined relative to the mobile polymer fraction (i.e., (1 a) in eq 6). finally, the rigid polymer fraction discussed below was obtained according to obviously, for these corrections, it is essential to use a recycle delay that is long enough to observe the full equilibrium magnetization of the sample (5(t1w) was used as a recycle delay in all experiments, i.e., 720 s depending on the temperature). the aim of this work is to study the structure and phase composition of pva cryo-gels by using a low-field nmr spectrometer. to revise the structural models suggested in the literature, we will separately analyze the effects of three factors on the pva network structure: (i) the number of applied freezingthawing cycles, (ii) the temperature and melting process, and (iii) the aging process. to achieve this objective, in every point, dq experiments were performed to quantify the proportion of noncoupled network defects, and the molecular weight between cross-links and mse-fid pulse sequence were applied to determine the rigid fraction of the polymer. the starting point of our study is the characterization of two isotropic solutions of 10 and 20% (w/v) of pva in water. the 10% pva solution does not give any detectable dq signal, but for the decay (iref) data, two different processes can be identified (i.e., the decay of the polymer magnetization and the water relaxation). this result demonstrates that under these preparation and measurement conditions, the dilute solution can be considered to be nonentangled and above the gelation threshold. this is not a trivial result because dq experiments are, in principle, sensitive to transient links (entanglements or hydrogen bonds), and this indeed appears indicated by the 20% pva solution; under the same preparation conditions, dq experiments did yield a detectable build-up curve. however, this is in our case due to a nondissolved or partially gelled pva subphase. to avoid this problem, 20% pva solutions were prepared under more vigorous stirring as well as higher temperatures up to 90 c. in this way, a totally isotropic solution of polymer was obtained according the dq experiments. dq experiments were performed on pva samples subject to different numbers of freezingthawing cycles to determine the evolution of noncoupled network defects as well as the network mesh size. in figure 3, we can see that after the first freezingthawing cycle, 50% of the polymer chains are coupled and are therefore part of the network, whereas the rest are not linked to it or at least belong to elastically inactive dangling chains or loops. the number of pva chains incorporated in the gel network increases with the successive cycles until it reaches a plateau after the sixth cycle, where the network has the minimum defect content. this important result shows that independent of the number of freezingthawing cycles applied to a pva solution 25% of the polymer is not coupled and therefore should not be elastically active. this is a very significant observation that is usually not taken into consideration when mechanical, dynamic-mechanical, or rheological properties of this material are analyzed. urushizaki et al.(66) have related the viscoelastic properties of this type of gel to the nonincorporated pva chains, and they have estimated their amount from the difference in weight between the polymer gel before and after immersion in distilled water. they found that 10% of the polymer was not incorporated after the first freezingthawing cycle. similar results were also indirectly inferred from the measure of intradiffusion coefficients of micellar aggregates of surfactant molecules dissolved in these types of gels.(67) obviously, our experimental values are much larger because we detect not only the non-cross-linked chains but also the dangling chains and other nonelastic network defects (e.g., loops). the effect of d2o in measured noncoupled network defects is another important point to take into consideration when this fraction of polymer is analyzed. pva monomers have an exchangeable proton (oh), which, in presence of d2o, could be replaced by deuterium that is undetectable by h nmr, whereas the proton will become detectable as water (hdo). free water signal (h2o or hdo) is subtracted from the long-time decay of the signal because it can be identified by its own longer t2. however, the number of noncoupled network defects could be slightly overestimated because of the protondeuterium exchange. variation of noncoupled network defects (i.e., non-cross-linked polymer chains, dangling chains, and loops) as a function of the number of freezingthawing cycles for the studied pva solutions. in addition, the decrease in the number of defects is correlated with an increase in dres that is proportional to the network chain order parameter (eq 1), see figure 4. the formation of cross-links (independent of their nature) renders the polymer segmental motion nonisotropic, whereby the chain segments become ordered with respect to the end-to-end distance, and residual dipolar couplings arise. according to this basic principle, the increase in dres is directly related to the (inverse) length of the chains between the constraints. similar to the observation for the noncoupled network defects, the network chain length appears to approach a plateau after cycle number six, yet the tendency is not as clear as the former result. even after 16 cycles, the cross-link density still appears to increase. variation of the average residual dipolar couplings (directly related to 1/n and therefore the cross-link density or the inverse mesh size) extracted from dq experiments as a function of the number of freezingthawing cycles for the studied pva solutions. represents a sample containing 20% pva after 3 h of freezing, and is from the same sample after 16 h of freezing. with this observation, it is important to note that samples with different polymer concentration appear to exhibit the same behavior during gelation. a possible explanation could be that in the framework of a heterogeneous scenario cross-linking occurs via partial crystallization in the nonfrozen parts of the sample; it simply means that in less concentrated samples more water freezes. this fact could explain the dependence of the dimension and shape of pores on polymer concentration and the regimes of cryogenic treatment.(20) in this context, it appears to be worthwhile to study the influence of the actual freezing temperature, which should affect the polymer concentration under the conditions at which the crystallites are formed. however, the freezing time (especially in the first cycle when the network is formed) seems to have some influence in the gel network structure. as is shown in figure 4, the apparent dipolar coupling constant is very low (48 hz) after 1 h of freezing at 32 c. if the freezing period is increased to 3 h, then the cross-link density of the gel almost doubles (82 hz), whereas longer freezing (e.g., 16 h) does not lead to further variation this may indicate that 1 h of freezing is not long enough to complete the process, and for this reason, further analyses are based on samples with a first freezing cycle of 12 h and subsequent cycles of 3 h each. returning to the origin of the pva networks produced via freezingthawing cycles from homogeneous polymer solutions in water, it appears to be well demonstrated by now that during the first freezing cycle, freezing of some water(32) increases the concentration of pva in the still unfrozen phase.(31) crystallization of pva takes place in this concentrated microphase,(68) but it is kinetically frustrated by the gelation of the polymer solution. this fact could explain the small size of these primary crystallites (5 nm), which then act as physical junctions between the amorphous and mobile polymer chains. results extracted from the mse-fid curves of the pva gels after the first freezingthawing cycle (figure 5) indicate that 8% of the polymer behaves like a rigid solid. this rigid fraction increases with the number of cycles until it reaches a plateau close to cycle number six, just as our other reported network properties. the maximum amount of rigid phase estimated by our nmr methodology (20% of the total polymer) is substantially higher than the crystallinity of similar samples deduced by dsc as well as some other h nmr methods. variation of the rigid (crystalline) polymer fraction extracted from mse-fid curves as a function of the number of freezingthawing cycles for the studied pva solutions. in all cases, the crystallinity of pva cryo-gels obtained in dsc studies is estimated to be 5% (not taking into account studies of dry networks(34) because the structure should be different). however, as was pointed out by willcox et al.,(28) this magnitude could very well be underestimated because the heat of fusion of the crystals in gels may be substantially lower than the assumed pva bulk value because of their small size and large surface effects (hydrogen bonds to surrounding water, etc.). in addition, another important point is the difficulty in measuring small quantities of crystal phase in dilute gels via calorimetric methods in general; for example, a crystallinity of 8% in a gel with a pva concentration of 20% represents only a sample fraction of 0.016. nevertheless, this difficulty seems to be reduced by the use of microcalorimetry. in this way, similar results were obtained by performing this type of measurement on gels with low pva concentrations,(8) although the uncertainties related to the obtained results are still large. for these reasons, the use of solid-state nmr seems to be a better methodology for quantifying the crystallinity in dilute pva gels. however, there are still some discrepancies in the total amount of rigid phase measured by the use of different nmr methodologies. ricciardi et al.(18) estimated the crystallinity of pva gels after 11 freezingthawing cycles to be 8% by analyzing the first microsecond of the h fid. clearly, as was pointed out in the, this experimental procedure falls victim to the dead-time problem. depending on the spectrometer, the amount of rigid signal lost during this time could be more or less significant. (ref (15) does not specify the dead time; therefore, it is not possible to estimate the related error.) for example, our low-field spectrometer has a dead time of 12 s; therefore, the fid analysis would lead to an underestimation of the rigid polymer fraction by 50%. by using different c nmr experiments (direct polarization as well as hc cross polarization), willcox et al.(28) established that after the first freezingthawing cycle, pva gel contains a rigid polymer phase of 5 2%, increasing up to 12 4% after cycle number 12. as was pointed out by the authors,(28) the relative fraction comparing different samples could be estimated with a high accuracy, but the used methodology (in particular, the use of the intrinsically nonquantitative cross polarization) does not give a reliable absolute crystallinity. in conclusion, the key to obtaining the most accurate crystal fraction in these dilute polymer samples is to use a solid-state nmr method that allows us to detect the rigid part fully by refocusing the multispin dipolar interactions with the mse, thus solving the dead-time problem and minimizing the signal loss. of course, detecting the more-sensitive and 100% abundant protons instead of the naturally abundant c is an additional advantage, which more than compensates for the use of an intrinsically less-sensitive low-field spectrometer. summarizing all results discussed so far, the network structure of pva cryo-gels is very apparently based on the formation of rigid polymer areas (most probably polymer crystallites) during the cooling process. after the first cycle, almost half (8%) of the final amount (20%) of the rigid phase is already formed. in this stage, subsequent freezingthawing cycles increase the rigid polymer fraction, which has two consequences: first, the noncoupled polymer fraction is further reduced (i.e., more pva segments become elastically active), and second, the network mesh size is further reduced but only slightly during further freezingthawing cycles. therefore, we believe that subsequent freezingthawing cycles not only increase the size of the crystals formed during the first freezing process but also lead to the creation of other more imperfect crystals. independent of the polymer concentration of the gel, the melting of smaller and more imperfect secondary crystals takes place at lower temperature and over a large range, leading to a continuous decrease in the detected rigid polymer phase with the temperature (figure 6). at 7580 c (in good agreement with the main endotherm observed in dsc measurements), this quantity drops below the detection limit. this should be correlated with the melting of the main (primary) crystals (estimated to 5% of the polymer fraction) that support the network structure of the gel. these results are consistent with dsc measurements because they show an increase in the melting endotherm that corresponds to the initial crystals as well as to the appearance of a second endotherm at lower temperatures when the number of freezingthawing cycles was increased for a given pva solution. variation of the rigid polymer fraction (extracted from mse-fid experiments) as a function of temperature for pva gels formed after seven freezingthawing cycles. a network with 10% pva was first heated until 324 k () and then kept at room temperature for 12 h before the second heating procedure was performed (). a solution with 20% pva was submitted to two different treatments, that is, one freezingthawing cycle and seven freezingthawing cycles, respectively. all samples were frozen for 12 h in the first cycle and 3 h in the subsequent cycles. the effect of the number of freezingthawing cycles on the crystallinity is easily understandable by comparing the evolution of the rigid polymer fraction of a pva gel obtained after seven cycles with that of a pva gel after only one freezingthawing cycle (figure 6). the onset of melting after only one freezing cycle is estimated to be 50 c, leaving the rigid polymer fraction constant at lower temperatures. therefore, subsequent cycles seem to create more, yet imperfect, secondary crystallite structures. above 50 c, all samples exhibit a similar behavior related to the partial melting of primary crystals or to the melting of less-perfect secondary structures. importantly, the temperature above which it is not possible to detect any more signal of rigid polymer remains invariant, yet the final fraction seems to increase slightly with the number of cycles. therefore, according to this result, freezingthawing cycles have a direct influence on not only the number of crystals formed during the first freezing process but also the creation of other more imperfect secondary crystallites. to ensure that the results shown in figure 6 are not artifacts produced by different magnetization relaxation at different temperatures that could possibly arise from regions that undergo a glass-to-liquid rather than a melting transition, the evolution of the signal at any given temperature was studied as a function of echo delay, see figure 7. at all of the measured temperatures, similar behavior was found, with a loss of intensity estimated to between 0.7 and 1.8% for the two extreme cases, which corresponds to a constant 10% loss on a relative scale, as is usually found. importantly, the drop in overall signal corresponds, within the error margins, to the expected drop due to the curie factor (temperature-dependent spin polarization). in addition, a direct check is the control of the total intensity, comparing the pva gels to the corresponding pva solutions (insert in figure 7). it is seen that no significant signal is lost/undetectable (we observe almost all protons in the sample), and from this, it is clear that the loss of intensity caused by the mse in pva gels is at a minimum, with no other (hidden) phenomena that could be related to an intermediately mobile (almost glassy) rigid fraction. given this experimental evidence, the decay of the rigid h fraction with temperature can be related to only a melting of pva crystallites. variation of the initial signal intensity of mse-fids with increasing echo delay at different temperatures for a pva gel containing 20% polymer after one freezingthawing cycle (12 h of freezing and 1 h thawing). the insert presents the mse-fid of the pva gel measured at 304 k with the minimum echo delay (0.0022 ms) as well as the estimated intensity at zero echo time in comparison with the mse-fid of the isotropic solution of the same sample measured under the same conditions after complete melting at 90 c. obviously, the temperature-dependent variation of the rigid polymer phase has a direct effect on the network structure, as shown in figure 8. the melting of secondary crystals leads to an almost exponential increase in the number of network defects (obviously, in a reversible pathway, comparing the phenomenon with the effect of successive freezingthawing cycles). however, the network mesh size (as measured by the residual dipolar coupling) remains almost constant. this means that secondary crystallization is very important in the network organization, reducing the number of network defects and therefore increasing the number of polymer segments that are elastically active. however, the molecular weight between the constraints appears to be dictated mostly by the primary crystallites formed during the first freezing cycles. for this reason, the network is completely destroyed at temperatures of 80 c when the primary crystal phase is molten, leaving no observable dq signal (i.e., an isotropic pva solution is formed). on the side, we note that the differences in the davg values given in figures 4 and 8b, which indicate a 50% decreased mesh size for the samples studied in figure 8, are related to the increase in the freezing time (12 h in the first step and 3 h in the successive cycles instead of 1 h) used in each cycle, as pointed out above. variation of (a) the network defect fraction and (b) the inverse mesh size (given by davg measured by mq nmr) with temperature for the 20% pva gel prepared after seven freezingthawing cycles (12 h first freezing cycle, 3 h subsequent cycles). error bars represent the fitting uncertainty, whereas lines are only guides to the eye. these nmr results on the mesh size perfectly agree with the trends observed in studies of mechanical properties reported in the literature. clearly, the elastic behavior of pva gels depends on the number of freezingthawing cycles. therefore, the main factor that determines the improvement of the mechanical properties upon further cycles should be associated with the number of elastically active chains. the polymer concentration in the original aqueous solution has an important effect on the elastic properties; that is, the elastic modulus increases. however, in the view of our data, this does not appear to have important consequences for the actual network structure; that is, there are no large variations in the network mesh size. therefore, the reported variations are simply related to differences in polymer concentration. in addition, different works demonstrate a slight decrease in the elastic modulus with the temperature, until a major drop occurs at 50 c.(65) this is obviously related to the partial melting of secondary and more imperfect crystallites that leads to an increasing amount of network defects (elastically inactive chains). at temperatures close to 80 c, the complete loss of crystallinity essentially liberates all chains, and the system drops below the gel point. in view of the important role of the substantial polymer amount that is not elastically active (free and dangling chains, loops), it may be worthwhile to reconsider detailed frequency-dependent mechanical measurements, where these components should contribute to an increased loss modulus over the frequency range covering their relaxation. the capacity of our investigated systems to be transformed from an isotropic aqueous solution to a physical cryo-gel by the application of freezingthawing cycles, including thermal reversibility back to an isotropic pva solution, is in clear contradiction with the assertion that covalent bonds could form between the polymer chains during gelation. if covalent cross-links were an important contribution to the final gel properties, then residual couplings should persist even above the melting temperature of the crystallites, which is not the case. however, the presence of (rapidly tumbling) microgels above the main melting point can not be ruled out and is in fact indicated by the relatively high viscosity (as checked by a simple tilt test). however, we point out that even the pristine 20% pva solution exhibited some detectable dq signal, indicating extended gel-like structures, unless it was heated to>90 c. therefore, an undetectably small fraction of crystallites (or extended hydrogen-bonded aggregates) may always be present, even at>80 c, and hold the system close to the gel point, depending on the sample and its thermal history. from our perspective, permanent bonds can be ruled out as a major factor determining the cryo-gel structure. finally, we address aging phenomena that are a part of the data shown in figure 6 and were not commented on until now. after heating a sample containing 10% pva to 50 c and recording the loss of crystallinity, the sample was stored for 12 h, and another measurement was performed at room temperature. the amount of rigid polymer fraction after 12 h of storage was again considerably increased, as can be seen in figure 6. yet, this new crystalline fraction melts at rather low temperatures, which again indicates the growth of secondary crystallites. similar prominent aging effects have been reported in the literature. for more clear evidence, a sealed sample containing 20% pva was submitted to seven freezingthawing cycles (with 12 h of freezing in the first cycle) and was characterized after different storage times at room temperature. according to figure 9, aging clearly increases the secondary population of imperfect crystals. it also increases the amount of elastically active polymer segments but has no significant effect on the network mesh size. therefore, aging effects on pva gel properties are again mainly related to an increasing fraction of polymer incorporated into the network structure and not to a decrease in mesh size. effect of aging on (a) the rigid polymer fraction, (b) the noncoupled defects fraction, and (c) the average residual dipolar coupling of a gel with 10% pva obtained after seven freezingthawing cycles (12 h of freezing for the first cycle and 3 h for the following cycles). this physical gel is supported by rigid polymer areas (pva crystallites) that act as junctions between the remaining mobile and entropically elastic polymer chains and thus determine the network mesh size. in our study, the crystallites are quantitatively detected as a rigidlike fraction of quickly relaxing (and fully refocused to overcome the dead-time problem) magnetization, and the mesh size is deduced from mq experiments that measure residual dipolar couplings, the latter arising from constraints on the motion of the mobile chains (i.e., cross-links) that render the segmental motion locally anisotropic. the most important crystal fraction forms in the first freezing cycle, and subsequent freezingthawing cycles produce only a slight increase in the initial crystal size as well as induce the formation of more imperfect, secondary crystallites. this secondary crystal fraction (which constitutes around two-thirds of the total crystal phase in samples submitted to a large number of cycles, extended aging, or both) plays an important role by increasing the number of elastically active polymer chains, but it does not appreciably affect the mesh size. pva cryo-hydrogels are shown to be totally thermoreversible; that is, pva physical networks can be transformed into an isotropic aqueous solution by increasing the temperature. during this process, the more imperfect crystals are molten first, and the number of network defects increases exponentially. in contrast, the network mesh size does not undergo significant variations until the primary crystal phase is molten at temperatures of 80 c. our experiments did not reveal any evidence of the possible formation of covalent cross-links between pva chains during gelation; no residual couplings were detected after the melting of this primary crystal fraction. independent of the number of cycles, the aging, or the polymer concentration in the original pva solution, a considerable fraction of polymer (at least 25%) is not elastically active, which is a result that is not yet not taken into account in the large body of literature addressing the formation and the properties of pva cryo-hydrogels. this finding is central to the interpretation and understanding of the elastic properties of this useful material and must be taken into account in a reinterpretation of the origin of the changes of the elastic moduli of pva cryogels. finally, it is important that the in-depth study of both processes (i.e., the pva gelation by the application of freezingthawing cycles) as well as the destruction of the network structure by heating were, for the first time, entirely based on the use of an inexpensive low-field solid-state nmr spectrometer. we show that the combination of different advanced nmr strategies, that is, mq spectroscopy and component analysis of mse-refocused free-induction decays, is vital for obtaining quantitative information on network structure and phase composition (crystallinity), respectively, to arrive at conclusions that can be matched with information extracted from other diverse techniques. in addition, the use of low-field solid-state nmr spectroscopy not only is useful for extracting structural information but also could be a powerful tool for investigating the polymer dynamics of these complex systems.

the network structure of poly(vinyl alcohol) (pva) hydrogels obtained by freezingthawing cycles was investigated by solid-state 1h low-field nmr spectroscopy. by the application of multiple-quantum nmr experiments, we obtain information about the segmental order parameter, which is directly related to the restrictions on chain motion (cross-links) formed upon gelation. these measurements indicate that the network mesh size as well as the relative amount of nonelastic defects (i.e., non-cross-linked chains, dangling chains, loops) decrease with the number of freezingthawing cycles but are independent of the polymer concentration. the formation of the pva network is accompanied by an increasing fraction of polymer with fast magnetization decay (20 s). the quantitative study of this rigid phase with a specific refocusing pulse sequence shows that it is composed of a primary crystalline polymer phase (5%), which constitutes the main support of the network structure and determines the mesh size, and a secondary population of more imperfect crystallites, which increase the number of elastic chain segments in the polymer gel but do not affect the average network mesh size appreciably. correspondingly, progressive melting of the secondary crystallites with increasing temperature does not affect the network mesh size but only the amount of network defects, and melting of the main pva crystallites at 80 c leads to the destruction of the network gel and the formation of an isotropic pva solution.

PMC2756085

pubmed-729

lentiviral (lv) vectors, such as those derived from hiv-1, show exceptional promise as gene transfer agents and have been proven to be effective vehicles for transduction of epithelial cells of various organs, including airway epithelial cells in the lung. the epithelium lining the bronchi/bronchioli is the target cell compartment for a therapeutic approach based on gene delivery in cystic fibrosis (cf), a chronic autosomal recessive disorder due to mutation in the cf transmembrane conductance regulator (cftr) gene. lv vectors bear some fundamental characteristics which could be useful for treating the cf lung disease, such as: (1) they integrate into the host genome and determine a long-term expression of either marker or cftr gene in animal and human xenograft models [37]; (2) they can be repeatedly administered without loss of efficiency; and (3) they do not elicit a gross inflammatory response in vitro and in vivo [4, 10]. the mechanism(s) of viral vector interaction with the apical plasma membrane and internalization has been an intensely studied question, in particular for adenoviral and adeno-associated viral vectors [1116]. the expression of receptors for these viral vectors and oncoretroviruses is more abundant on the basolateral membrane than on the apical side of the respiratory epithelium and they are hardly accessible because of the airway tight junctions [12, 17, 18]. hiv-1-derived lv vectors expressing vesicular stomatitis virus g glycoprotein (vsv-g) on their capsid have been shown previously to transduce a polarized airway epithelium only in the presence of preconditioning agents with disruptive, although transient, effects on tight junctions [4, 5, 19]. these studies indicated entry block due to absence or low numbers of vsv-g receptors on the apical membrane of the airway epithelium, as it has been identified for other viral vectors, or postentry block, concerning the endocytosis route and nuclear import of viral genomes. lv vectors have not been investigated in so much detail in respect to their attachment to and internalization by the cells. building on the previoulsy published work by guibinga et al., we observed that glycosaminoglycans (gags) are involved in lv vector-mediated transduction of polarized airway epithelial cells. in that study, a good transduction efficiency of polarized cellular monostrates with a last-generation lv vector pseudotyped with vsv-g was obtained, but only at high virus to cell ratios. given that no studies have tested directly the effect of lv vectors on the tight junction (tj) stability and organization in airway epithelial cells, we evaluated the transduction efficiency as well as acute cytotoxicity, transepithelial resistance (ter), and occludin localization at the tj level in a bronchial-derived cell line grown in polarized fashion. we find that the vsv-g-pseudotyped lv vector infection at high viral loads is cytotoxic and determines occludin loss from the apical plasmamembrane and reduction of ter. in order to rule out the toxic effect of lv particles, we used lv virions noncovalently complexed with a cationic vector (i.e. polyethylenimine). the vsv-g pseudotyped lv vector stock was prepared as previously described [21, 22]. briefly, the lentivirus-based gene delivery system comprises four components: (1) the packaging plasmid pmdlg/prre, which contains elements such as structural proteins and enzymes involved in the formation of the viral particles, derived from the gag-pol genes; (2) the prsv-rev plasmid, which contains posttranscriptional regulator for gag and pol expression, as well as nuclear rna export encoded by the rev gene; (3) the transfer vector prrlsin18.cppt.cmv.egfp.wpre carrying the transgene gfp with the insertion of the ppt and the woodchuck post-transcriptional regulatory element (wpre); and (4) the pmd2.g plasmid containing the heterologous glycoprotein vsv-g. cotransfection of the four plasmid vectors was performed on 293 t cells by calcium phosphate precipitation. the supernatant containing lv particles was concentrated by two rounds of ultracentrifugation and assayed for p24 gag antigen by enzyme-linked immunosorbent assay. the viral titer was determined by hela cell infection and subsequent flow cytometry analysis. the yield of the concentrated virus was typically 10 transducing units (tu)/ml and the specific activity ranged between 1.56 and 4.17 10 tu/ng of p24. 16hbe41o- cells, derived from human bronchial epithelium (a kind gift of professor d. gruenert, university of california at san francisco, ca, usa), were grown in mem supplemented with earle's salt, 10% fetal bovine serum, l-glutamine and penicillin/streptomycin. they were routinely grown on plastic flasks coated with an extracellular matrix containing fibronectin/vitrogen/bovine serum albumin. the extracellular matrix coating is prepared in the laboratory as follows: 10 g/ml fibronectin (bd biosciences, ca, usa), 100 g/ml albumin from bovine serum (sigma-aldrich, milan, italy), and 30 g/ml bovine collagen type i (bd biosciences) are dissolved in mem. cells were seeded on 6.5-mm diameter snapwell, 0.4-m pore size (corning, acton, ma, usa) at 1 10 per filter coated with the same extracellular matrix. under these conditions, cells grow as a polarized sheet of cells and develop a transepithelial resistance of 550 ohm cm on average, as measured by a voltohmmeter (millicell-ers; millipore, vimodrone, italy). polarized cells were incubated with the lv-gfp vector at different multiplicities of infection (moi) for 4 or 24 hours and then either immediately studied for propidium iodide staining and cell viability or incubated for further 48 hours for evaluation of gfp expression. moi refers to the number of tu per one cell. because we seeded 1 10 cells per well, a moi of 10 is equivalent to 10 tu; and a moi of 100 refers to 10 tu, and so forth. branched polyethylenimine (mw 25,000 da) was obtained from sigma as 50% w/v solution. the solution was titrated with hcl solution to a ph of 7.4 and used as a 4.5 mg/ml stock solution (100 mm; stoichiometrically, this solution corresponds to 10.8 10 molecules of pei per l of solution). ten l of saline containing different amounts of pei stock solution were added to 10 l of saline containing 50 moi (5 10 tu) of lv in order to obtain pei molecules/tu ratios ranging from 5 10 to 1 10(corresponding to a range of 0.062512.5 g/l as final concentration of pei). spermidine (sigma-aldrich) stock solution (1 m) was diluted in order to obtain a final concentration ranging from 0.08 m to 8 mm (corresponding to spermidine molecules/tu ratios ranging from 1 10 to 1 10). the suspension containing either pei/lv or spermidine/lv was incubated for 15 minutes at room temperature and then added to cells. in another experimental setting, cells were preincubated with spermidine (final concentration ranging from 0.08 mm to 8 mm) for 1.5 hours, washed, and then infected with pei/lv at the ratio of 1 10 with 50 moi lv. the medium was changed 24 hours later and after further 48 hours, a preliminary evaluation of gfp expression was carried out by epifluorescence and confocal microscopy (see below). the cells were washed twice with phosphate-buffered saline (pbs), harvested by digesting with trypsin/ethylenediaminetetracetic acid (edta), and fixed in 2% paraformaldehyde. the cells were analyzed by fluorescence-activated cell sorting (facs) with a epics xl mcl flow cytometer (beckman coulter fullerton, ca, usa). the percentage of gfp-positive cells was determined after setting the gating on 99% of an untransfected control population of cells and by subtracting the fluorescence of the untransfected control cells. analysis of gfp production was performed by plotting the flh-1 channel (512537 nm, with peak at 525 nm) against the flh-3 channel (608632 nm, with peak at 620 nm). propidium iodide is an effective stain to identify nonviable cells since the dye is excluded by intact cell membranes and passes through damaged cell membranes and intercalates with dna and rna to form a bright red fluorescent complex [23, 24]. briefly, cells on transwells were incubated with 25 g/ml propidium iodide (sigma-aldrich) for 20 minutes on ice, were washed with pbs, harvested by digesting with trypsin/edta, and resuspended in pbs. in each experiment, as a toxicity control, cells were incubated with 0.1% triton x-100 (sigma-aldrich) for 5 minutes at room temperature. the percentage of propidium iodide positive cells was determined after setting the gating on 99% of an untreated control population of cells and by subtracting the fluorescence of untreated control cells. analysis of propidium iodide positive cells was performed by plotting the red channel (flh-2; 562588 nm, with peak at 575 nm) against the flh-1 channel. mtt (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is a water-soluble yellow dye that is readily taken up by viable cells and reduced by the action of mitochondrial dehydrogenases. the reduction product is a water-insoluble blue formazan, that must then be dissolved for colorimetric measurement. briefly, a stock solution of mtt (sigma-aldrich) in phosphate buffered saline (pbs) (5 mg/ml) was added to the upper compartment of each well reaching a final concentration of 0.5 mg/ml (in 200 l of complete medium). after 4 hours the formazan crystals were dissolved in a 10% sds/50% dimethyl-formamide solution, and 100 l of the solution were transferred in a 96-well plate and measured spectrophotometrically by an elisa reader (powerwave ht, bio-tek, milan, italy) at a wavelenght of 570 nm with a reference wavelenght of 690 nm. the relative viability was calculated in respect to untreated cells (considered as 100%). peripheral blood mononuclear cells (pbmcs) were isolated from the whole blood of three normal donors using lymphoprep (axis-shield, oslo, norway), according to the published protocol, and pooled. total rna was extracted from polarized 16hbe14o- cells and pbmcs using trizol reagent (invitrogen, s. giuliano milanese, italy) following the protocol suggested by the manufacturer. the concentration of rna was estimated by nanodrop 1000 spectrophotometer (thermo scientific, waltham, ma, usa) at 260 nm wavelength and the purity was confirmed by measuring the absorbance ratio at 260/280 nm wavelengths. 1 g rna was used to prepare cdna by using a revertaid first strand cdna synthesis kit (fermentas, burlington, canada). retrotranscription was performed under the following conditions: incubation for 60 minutes at 37c followed by 5 minutes at 70c. rna were retrotranscripted in the presence of 200 units of revertaid m-mulv reverse transcriptase, 0.4 m of oligo (dt)18 primer, 1 mm of nucleotide mix, 20 units of ribolock rnase inhibitor, and commercial buffer (fermentas). for the pcr reaction, 100 ng of cdna were amplified in the presence of 1 unit taq polymerase (fermentas), 0.4 m of each primer (eurofins mwg operon/m medical srl, milan, italy), 0.2 mm of nucleotide mix, and commercial buffer containing 2 mm mgcl2 (fermentas). the primers for occludin amplification were forward 5'-agt gag tgc tat cct ggg cat- 3 ' and reverse 5'-cct ttg cag gtg ctc ttt ttg-3 ' which produced a dna segment of 600 bp. pcr was performed under the following conditions: initial denaturation for 2 minutes at 94c, followed by 25 cycles of denaturation (15 seconds, 94c), annealing (30 seconds, 58c) and extension (1 minute, 72c). as control for rna integrity, we performed the actin pcr reaction using the following primers: forward 5'-caa ctg gga cga cat gga-3 ' and reverse 5'-acg tca cac ttc atg atg ga-3 ', which produced a dna segment of 610 bp. pcr was performed under the following conditions: initial denaturation for 2 minutes at 94c, followed by 35 cycles of denaturation (15 seconds, 94c), annealing (30 seconds, 56c) and extension (1 minute, 72c). the identity of amplified products was confirmed by determination of molecular size on agarose gel electrophoresis (1.5% agarose in buffer containing 40 mm tris/acetate and 1 mm edta) and visualized by ethidium bromide staining (0.5 g/ml) under ultraviolet light. for occludin immunolocalization, polarized 16hbe14o- cells were washed three times with pbs, fixed in 3% paraformaldehyde, 2% sucrose, and permeabilized with ice cold triton hepes buffer (20 mm hepes, 300 mm sucrose, 50 mm nacl, 3 mm mgcl2, 0.5% triton x-100, ph 7.4) for 5 minutes at room temperature. cells were incubated with blocking solution (2% bovine serum albumin [bsa], 2% fbs) for 15 minutes at 37 c, then with fluorescein isothiocyanate (fitc)-conjugated mouse anti-occludin antibody (zymed laboratories inc., san francisco, ca, usa) (dilution 1: 100) for 30 minutes at 37 c. filters were excised and placed side up on a glass slide, and overlayed with a drop of fluorescent mounting medium (dako, milan, italy) followed by a coverslip. cells were analyzed using nikon te2000 microscope coupled to a radiance 2100 confocal dual-laser scanning microscopy system (bio-rad, segrate, italy). the microscope was equipped with a fitc filter (excitation 395 nm, emission 509 nm). digital images were processed using the program laser sharp 2000 (bio-rad). for gfp detection, cells were fixed and permeabilized, incubated with propidium iodide (diluted at 1: 5,000 of 1 mg/ml stock solution) for 5 minutes at 37c, and washed. filters were mounted and observed through the fitc and tritc (excitation 488 nm, emission 620 nm) filters. statistical significance of differences was evaluated by a two-tailed unpaired student's t-test. we have previously demonstrated lv-mediated transgene delivery and expression in polarized airway epithelial cells at high lv: cell ratios, necessitating at least 2000 moi (multiplicity of infection). thus, we interrogated the transduction efficiency and the cytotoxicity of lv vectors in polarized 16hbe41o- cells. cells were incubated with different mois of lv particles for 24 hours, and then either immediately studied for propidium iodide staining (to assess membrane permeability) and viability (by means of the mtt assay), or incubated for further 48 hours for evaluation of gfp expression. as shown in figure 1(a), the higher the moi the higher the percentage of gfp-positive cells. only cells with altered plasmamembrane permeability will intake propidium iodide which will bind nuclear dna. the higher the moi the higher the percentage of propidium iodide-stained cells (figure 1(b)). the viability was decreased only at mois 500 and 2000 (figure 1(c)). to investigate acute cytotoxicity of lv particles, membrane permeability and viability were assayed also after incubation of cells with lv particles for 4 hours. at any moi, lv particles did not exert any direct permeabilizing effect on polarized cells (figure 1(e)) and decreased the viability only at moi 2000 (figure 1(f)). under these conditions, the percentage of transduced cells was lower than in the 24-hour protocol (figure 1(d)). these data show a dose-dependent cytotoxic effect of the lv vector, in terms of both alteration of membrane permeability and cell viability. since the lv vector shows a disturbing effect on membrane permeability, the impact of lv particles on the tightness of the epithelial monostrates was initially investigated by measuring transepithelial resistance (ter). ter was decreased in a moi-dependent fashion when cells were incubated with lv vectors for 24 hours (figure 2(a)), whereas was not affected at 4 hours (figure 2(b)). the effect observed with 2000 moi was similar to that achieved by ethylene glycol-bis(2-aminoethyl)-n, n, n,n-tetra-acetic acid (egta) (figure 2), a ca chelator known to transiently disrupt epithelial tight junctions. tight junctions (tjs) are multiprotein complexes composed of integral proteins (claudins, occludin, and jam [junctional adhesion molecule ]) that associate with cytoplasmic plaque proteins (zo-1, zo-2, and zo-3). the former mediate cell-cell adhesion, while the latter function as a bridge between the tj and the actin cytoskeleton [27, 28]. since occludin has been shown to be internalized upon infection with group b coxsackievirus (cvb) and hepatitis c virus (hcv), we chose to study occludin expression and localization in cells infected with the lv vector. reverse transcription-pcr revealed the presence of an occludin specific band (figure 3(a)). freshly isolated and unstimulated lymphocytes and monocytes have been shown to not express occludin at the mrna and protein level [31, 32]. pbmcs obtained from normal donors were negative for occludin mrna expression (figure 3(a)), confirming the specificity of the occludin amplification in 16hbe14o- cells. polarized 16hbe14o- cells were incubated with the lv vector at different mois and analyzed by immunofluorescence and confocal microscopy 24 hours postinfection. untreated cells displayed sharp circumferential organization of occludin at the lateral membrane between neighbouring cells (figure 3(b)). in egta-treated cells, high lv mois (500 and 2000) determined discontinuity in the occludin pattern at tj location (figures 3(d) and 3(e), with 2000 moi causing stronger disorganization of tjs. on the other hand, 50 moi did not cause any alteration in occludin localization at the cell periphery (figure 3(c)). taken together, these results show that high but not low viral-to-cell ratios determine disruption of tjs when tjs are probed at 24 hours postinfection. these results prompted us to investigate if lv-mediated transduction at low moi could be enhanced by the polycation polyethylenimine (pei). cationic lipids and polymers have been used to increase retrovirus titer and to enhance transduction of target cells [3336]. various amounts of branched 25 kda pei molecules were mixed with 50 moi of lv particles to obtain different pei molecules/tu ratios. cells were incubated with lv alone or pei/lv for 24 hours and gfp expression was evaluated 48 hours later by cytofluorimetry. the percentage of transduced cells did not change with low pei: lv ratios as compared with lv alone, and only the 10 ratio produced a significant 2.5-fold increase in gfp-positive cells as compared to plain lv (figure 4(a)). epifluorescence and confocal microscopy analysis of transduced cells confirmed that pei increased by approximatively 3 fold the efficiency of lv-mediated transduction, as visualized by the number of gfp-positive cells (figure 5). to see whether the enhancing effect of pei could be universal to polyamines, we sought to determine the effect of native spermidine, a polyamine which is cationic at physiological ph. a wide range of spermidine molecules/tu ratios was tested (corresponding to a molar concentration range from 0.08 m to 8 mm), but no effect on the transduction rate given by plain lv particles (used at 50 moi) was observed (figure 4(b)). thus, the enhancing effect seems to be unique to pei. in alternative, we investigated whether spermidine could inhibit the pei/lv-mediated transduction. in this case preincubation of polarized 16hbe14o- cells with native spermidine (0.088 mm) before addition of pei/lv formulated at the highest ratio (1 10 with 50 moi lv) did not exert any alteration in the efficiency of pei/lv (figure 4(c)). in these experiments, pei/lv particles and free pei were interrogated for their cell toxicity using the propidium iodide staining and the mtt assay. cells were incubated with vectors for 24 hours, stained with propidium iodide for 30 minutes, and analyzed by cytofluorimetry. as shown in figure 6(a), only cells incubated with the highest pei: lv ratios with 50 moi lv, (10 pei/tu) showed a significant increase in nuclei stained with propidium iodide (from 0% up to 6.5 2.4%). intriguingly, the same amount of pei alone (1 10 molecules) caused the uptake of propidium iodide by 9.9 4.9% of cells. figure 6(b) shows that pei/lv and free pei exerted a small toxic effect on cells at highest doses. these results suggest that the membrane permeabilizing effect of pei/lv vectors could be attributed to free pei, as previously shown by the toxicity exerted by pei alone. ter was measured at 4 and 24 hours postinfection and was not affected by pei/lv particles (not shown). occludin localization was not altered by incubation of cells with pei/lv at 24 hours (figures 7(b)7(d)). also free pei (10 molecules) did not exert any effect on tj integrity (figure 7(e)). positively charged polycations such as polybrene are known to be required for efficient infection of cells with retroviruses and retrovirus vectors, possibly by stabilizing the interaction between negatively charged virus particles and target cellular membranes [20, 39]. indeed, polybrene has been used also in lv-mediated transduction of polarized epithelial cells obtained from the airways. in our hands, polybrene was toxic to the cells and thus it was withdrawn from the transduction protocol. for this reason, we had to use high mois (i.e. at least 2000) to achieve a meaningful transduction of polarized airway bronchial and tracheal epithelial cells. in this study, we show that high lv mois are toxic to polarized airway epithelial cells, which are considered a good approximative model for native airway epithelium [26, 4143]. high mois determined an increase in membrane permeability at 24 hours but not at 4 hours, suggesting that the direct interaction of lv particles with cells is not harmful, rather it is so for entry, transport, and viral transcription within the cytosol. interestingly, the damage caused by lv particles was reflected also by the decrease of ter and loss of occludin at the tjs between cells. the mtt assay shows that only high lv doses decreased the cellular viability both at 4 and 24 hours. these results strongly suggest that lv particles exert their toxic action through direct interaction with tj proteins, an effect visible only at 24 hours. some recently acquired data indicate that proficient viral infection is dependent on the interaction of viral envelope glycoproteins with tj proteins. the primary cvb receptor, the coxsackievirus and adenovirus receptor (car), is a transmembrane component of the tj and cvb enters polarized epithelial cells from the tj, causing a transient disruption of tj integrity. cvb does not induce major reorganization of the tj, but stimulates the specific internalization of occludin within macropinosomes. hcv envelope glycoproteins induce a loss of claudin-1, zo-1, and occludin-delineated junctional accumulation and occludin is required for late entry step of hcv into cells. overall, based on our previous publication, we speculate that vsv-g-pseudotyped lv virions are concentrated on the apical surface of polarized airway epithelial cells by initial attachment to gags. the binding of lv particles with gags is based on nonelectrostatic interactions. in a further step, lv virions should then bind to entry receptors (not identified yet) and eventually to occludin for internalization. loss of occludin from the tjs and their opening could be a disadvantage in lung diseases such as cf, because of the presence of bacteria and bacterial products in the airways. in order to find a transduction protocol with less viral loads, minimal toxicity and eventually no delocalization of occludin from the tjs, we have combined lv particles with the polycation pei, based on the rationale that a cationic component would charge associate with lv particles, which carry a net negative surface charge. several previous studies have reported the use of cationic molecules to enhance viral uptake and subsequent transgene expression in vitro and in vivo, mainly for adenoviral [4752] and retroviral vectors [3336]. in particular, pei facilitated transduction efficiency by adenoviral vectors in cultured mouse myotubes and in 9l gliosarcoma cells and enhanced retroviral transduction in nih3t3 cells. to our best knowledge, ours is the first study showing that pei enhanced lv-mediated transduction of airway epithelial cells. pei/lv particles increased membrane permeability only at the highest pei/lv ratio, an effect likely due to excess free pei, with a little effect on the viability. indeed, pei is known to induce the formation of transient, nanoscale holes in the membranes of living cells and these holes allow a greatly enhanced exchange of materials across the cell membrane, including propidium iodide. although we have not investigated the membrane permeability with spermidine/lv vectors, the fact that spermidine had no enhancing effect on lv-mediated transduction supports the notion that the pei's action is due to a membrane destabilizing effect. pei/lv particles and free pei did not cause any alteration on ter and in occludin localization, indicating that pei-induced nanoscale holes in the plasma membrane do not affect tj integrity. in conclusion, pei/lv vectors are more efficient than lv alone used at low viral load in transducing polarized epithelial cells without so pronounced cytotoxicity, and, more importantly, without disrupting tight junctions. because the transduction efficiency mediated by pei/lv is still low it needs further refinement for obtaining higher transduction rates of polarized airway epithelia, a goal which could be achieved by testing other pei architectures. for these reasons the pei/lv vector warrants further characterization for being considered as a valid tool in gene therapy of genetic lung diseases.

lentiviral (lv) vectors are promising agents for efficient and long-lasting gene transfer into the lung and for gene therapy of genetically determined pulmonary diseases, such as cystic fibrosis, however, they have not been evaluated for cytotoxicity and impact on the tightness of the airway epithelium. in this study, we evaluated the transduction efficiency of a last-generation lv vector bearing green fluorescent protein (gfp) gene as well as cytotoxicity and tight junction (tj) integrity in a polarized model of airway epithelial cells. high multiplicities of infection (moi) showed to be cytotoxic, as assessed by increase in propidium iodide staining and decrease in cell viability, and harmful for the epithelial tightness, as demonstrated by the decrease of transepithelial resistance (ter) and delocalization of occludin from the tjs. to increase lv efficiency at low lv: cell ratio, we employed noncovalent association with the polycation branched 25 kda polyethylenimine (pei). transduction of cells with pei/lv particles resulted in 2.53.6-fold increase of percentage of gfp-positive cells only at the highest pei: lv ratios (1107 pei molecules/transducing units with 50 moi lv) as compared to plain lv. at this dose pei/lv transduction resulted in 6.5 2.4% of propidium iodide-positive cells. on the other hand, pei/lv particles did not determine any alteration of ter and occludin localization. we conclude that pei may be useful for improving the efficiency of gene transfer mediated by lv vectors in airway epithelial cells, in the absence of high acute cytotoxicity and alteration in epithelial tightness.

PMC2896616

pubmed-730

cervical spondylosis is a common cause of chronic neck pain, radiculopathy and/or myelopathy resulting in significant disability. when patients do not respond to conservative therapy, anterior cervical discectomy and fusion (acdf) usually is used to achieve neural decompression, maintain cervical lordosis, and provide segmental stabilization. in our practice, it was often found that patients with cervical spondylosis tended to have concomitant cervical vertigo symptoms such as tinnitus, headache, blur vision, and palpitation. recent studies have indicated that cervical spondylosis patients with cervical vertigo symptoms could be managed successfully with acdf. in this study, we report 2 cases of cervical spondylosis with concomitant cervical vertigo and hypertension that were treated successfully with acdf. this study obtained the approval of the medical ethics committee of our hospital (general hospital of armed police force, beijing, china). case 1, a 49-year-old female, was admitted with 2 years of history of chronic neck pain, headache, and episodic vertigo and tinnitus. episode occurred 1 to 2 times every day, and lasted 1 to 2 h each time. she felt numbness of both upper extremities and weakness of all 4 extremities for 1 year. physical examination showed that limited neck motion, slightly diminished sensation in both arms, and brisk deep tendon reflexes. radiographic examination of the cervical spine showed mild diminution in the height of the c5/6 disc space (figure 1). magnetic resonance imaging (mri) scan showed cord compression by a large central herniation of c5/6 disc with signs of myelomalacia (figure 2). lateral radiograph of cervical spine before operation showed slight narrowing of the c5/6 disc space. t2-weighted sagittal magnetic resonance imaging (mri) revealed a large herniation of c5/6 disc before operation, with marked compression of cervical cord, resulting in myelomalacia. the patient had a 2-year history of hypertension, blood pressure was 160/100 mmhg at onset, and then controlled in the normal range (110130/7080 mmhg) by oral medications (betaloc, 100 mg, once daily; enalapril, 10 mg, once daily). case 2, a 60-year-old male, was admitted with 10 years of history of chronic neck pain and stiffness. he also experienced vertigo, palpitation, nausea and vomiting with noted numbness of upper limbs, and heaviness of 4 limbs for over 6 months before admission. radiographic examination of the cervical spine revealed large anterior and posterior spur formation at the c5/6 and c6/7 disc levels, severe diminution in the height of the c5/6 disc space, and moderate diminution in the height of the c6/7 disc space. mri scan showed cervical canal stenosis with marked cord compression secondary to spondylotic changes in c5/6 and c6/7 discs. the patient had a 1-year history of hypertension (maximum at 180/120 mmhg), which was controlled within the normal range by oral medications (irbesartan, 150 mg, once daily; nifedipine sustained release tablets, 20 mg, once daily; amlodiping besylate, 5 mg, once daily). cervical spondylotic myelopathy was diagnosed and subsequently cervical anterior surgery was recommended for the 2 patients preoperatively based on clinical findings consistent with imaging study. case 1 underwent acdf at the c5/6 disc level (figures 3 and 4). after surgery, strength in all 4 limbs was significantly recovered, and symptoms of neck pain, headache, vertigo, and tinnitus disappeared. interestingly, her blood pressure also dropped to within the normal range after surgery even without oral medications. lateral radiograph of cervical spine 3 months after operation showed anterior cervical plate fixation with cage fusion at c5/6 disc level. t2-weighted sagittal magnetic resonance imaging (mri) 7 days after operation showed disappearance of cord compression at c5/6 disc level. his symptoms of cervical vertigo completely disappeared, numbness of both upper limbs significantly released, and muscle strength of 4 limbs obviously increased. similarly to case 1, her cervical vertigo never recurred, and her blood pressure remained normal without the use of antihypertensive medications. case 2 has undergone a follow-up of 14 months, the patient never had cervical vertigo anymore, and his blood pressure remained normal throughout (120130/7585 mmhg). clinical results indicated that acdf can eliminate the concomitant vertigo symptoms in the 2 patients. however, the exact mechanisms of resolution of symptoms can not be elucidated precisely. because cervical spondylosis is a common disease, vertigo occurred in patients with cervical spondylosis is of particular importance. one previous study showed that vertigo was present in 50% of patients with cervical spondylosis, whereas another study identified cervical spondylosis as the cause of dizziness in 65% of elderly patients. it has been shown that the patients with cervical spondylosis complaining of vertigo have significant lower blood flow parameters than non-vertigo patients with cervical spondylosis. insufficient blood supply to posterior circulation is called vertebrobasilar insufficiency. the most common complaint in patients with vertebrobasilar insufficiency insufficient blood supply does not necessarily cause symptoms if there is sufficient collateral circulation, whereas a full range of symptoms commonly occur as a result of an insufficient terminal vessel. the vascular supply to the vestibulocochlear organ, being an end artery, makes this organ more susceptible to vertebrobasilar insufficiency. neurons, axons, and hair cells in the vestibulocochlear system are known to respond to ischemia by depolarizing, causing transient hyperexcitability with ectopic discharges, manifesting as tinnitus, vertigo, and dizziness. it was hypothesized that vertebral artery insufficiency secondary to cervical spondylosis could result in vertigo. however, the mechanisms of vertigo caused by cervical spondylosis are not clear so far. the mechanical compression on vertebral artery from spurs of the luschka joint was considered as a main mechanism. the reduction of vertebral artery flow can be more obvious with the rotation and hyperextension of the head. in our current study in addition, mechanical compression can not explain some other symptoms such as palpitation, nausea, vomiting, etc. recent clinical studies by hong et al and li et al demonstrated that the stimulation of the sympathetic nerve fibers other than the compression of the vertebral artery induces these symptoms such as vertigo, tinnitus, nausea, and vomiting. current study clearly indicates that hypertension can be associated with cervical spondylosis, as a secondary condition. we speculate that the mechanisms of hypertension induced by cervical spondylosis could be the same as that of cervical vertigo. it is well known that cervical disc, dura mater, and posterior longitudinal ligament are rich in sympathetic fibers. sympathetic nerve fibers distributed around the vertebral artery have been implicated in the autoregulation of vertebrobasilar artery blood flow and cerebral blood flow. it has been evidenced that sympathetic nerve activity withdraws the blood flow, whereas sympathectomy can increase it. a previous study investigated sympathetic and parasympathetic changes incited by the stimulation of nerves around the proximal vertebral artery in cat model and found subsequent papillary changes, pulse and blood pressure changes. elevation in levels of inflammatory cytokines has been detected in painful discs of humans, and thought to be related to degeneration and pain. the pathogenic change in the painful disc is featured with the formation of zones of vascularized granulation tissue and extensive innervation extending from the outer layer of the annulus fibrosus into the nucleus pulposus. degenerative changes in the disc, such as loss of the normal structure and a mechanical load, can lead to abnormal motion, which can provoke mechanical stimulation. mechanical stimuli, which are normally innocuous to disc nociceptors can, in certain circumstances such as inflammation, generate an amplified response termed peripheral sensitization. the vertebral arteries are mainly innervated by nerve fibers from the cervical sympathetic ganglia (13). innervation in cervical disc is analogous to that in the lumbar spine, receiving innervation posteriorly from the sinuvetebral nerves, laterally from the vertebral nerve, and anteriorly from the sympathetic trunks. recently, an animal study revealed a reciprocal neural connection between cervical spinal and sympathetic ganglia. it seems likely that stimulation of sympathetic nerve fibers in the pathologically degenerative disc and surrounding tissues produce sympathetic excitation, and induce a sympathetic reflex to cause vertebrobasilar insufficiency and hypertension. improvement of neurological function, resolution of vertigo, and recovery of blood pressure following surgery could be attributed to the excision of degenerative and herniated cervical disc, the decompression of the dura mater and posterior longitudinal ligament, and the stabilization of degenerated segment. chronic neck pain is one of the major symptoms in patients with cervical spondylosis, whereas some studies have indicated that resting blood pressure levels may be elevated in patients with persistent pain. in healthy individuals, these interactions are believed to reflect a homeostatic feedback loop that helps restore arousal levels in the presence of acutely painful stimuli. a role for baroreceptors in this feedback loop appears likely, with pain triggering sympathetically driven blood pressure increases, resulting in increased stimulation of baroreceptors, which in turn activates descending pain inhibitory pathways. previous studies suggested that blood pressure/pain regulatory relationship may be substantially altered in chronic pain conditions. a retrospective study by bruehl et al suggested that chronic pain could be associated with increased risk of hypertension. their study found that over 39% of patients with chronic pain were diagnosed with clinical hypertension, compared with only 21% of pain-free internal medicine patients. they thought that chronic pain-related impairments in overlapping systems modulating both pain and blood pressure would result in a higher prevalence of clinically diagnosed hypertension in patients with chronic pain than in comparable pain-free patients. our study indicates a possible relation between chronic neck pain and hypertension. if chronic neck pain could lead to hypertension through sympathetic arousal and failure of normal homeostatic pain regulatory mechanisms, early treatment for resolution of chronic neck pain may have a beneficial impact on cardiovascular disease risk in patients with cervical spondylosis we report here 2 patients of cervical spondylosis with concomitant cervical vertigo and hypertension that were treated successfully with anterior cervical discectomy and fusion. stimulation of sympathetic nerve fibers in pathologically degenerative disc could produce sympathetic excitation, and induce a sympathetic reflex to cause cervical vertigo and hypertension. in addition, chronic neck pain could contribute to hypertension development through sympathetic arousal and failure of normal homeostatic pain regulatory mechanisms. early treatment for resolution of symptoms of cervical spondylosis may have a beneficial impact on cardiovascular disease risk in patients with cervical spondylosis.

abstractcervical spondylosis and hypertension are all common diseases, but the relationship between them has never been studied. patients with cervical spondylosis are often accompanied with vertigo. anterior cervical discectomy and fusion is an effective method of treatment for cervical spondylosis with cervical vertigo that is unresponsive to conservative therapy. we report 2 patients of cervical spondylosis with concomitant cervical vertigo and hypertension who were treated successfully with anterior cervical discectomy and fusion. stimulation of sympathetic nerve fibers in pathologically degenerative disc could produce sympathetic excitation, and induce a sympathetic reflex to cause cervical vertigo and hypertension. in addition, chronic neck pain could contribute to hypertension development through sympathetic arousal and failure of normal homeostatic pain regulatory mechanisms.cervical spondylosis may be one of the causes of secondary hypertension. early treatment for resolution of symptoms of cervical spondylosis may have a beneficial impact on cardiovascular disease risk in patients with cervical spondylosis.

PMC4602471

pubmed-731

although herpes simplex virus (hsv) infections are very common worldwide, herpes simplex encephalitis (hse) is a rare disease with an incidence of one case per 250,000500,000 individuals per year. approximately 50% of patients with hse are older than 50 years.1 cytomegalovirus (cmv), another member of the herpesvirus family, is the most common cause of congenital infection, with a prevalence rate of 0.2%2.5% in all live newborns.2,3 in adults, most reported cmv infections are seen in immunocompromised patients such as those with hiv (human immunodeficiency virus)4,5 and after chemoradiotherapy.6 in these cases, the patients were also co-infected with hsv. here, we present a patient with coinfection of cmv and hsv type ii (hsv-ii), who was admitted to the hospital for acute psychotic symptoms, but never treated with immunosuppressants before this admission. for acute psychotic symptoms of delirium, delusion of persecution, fidget, over-alertness, and aggressive behaviors, a 67-year-old chinese man was sent to the outpatient department of the shantou central hospital, guangdong province, people s republic of china, on october 15, 2013. on october 14, he became suspicious, had delusion of persecution, and ran around. on the same day, he was sent to the local hospital where a magnetic resonance imaging was performed showing multiple hyperintensities in the left occipital lobe and temporal lobes in the t2-weighted images (figure 1a). in addition, multiple hyperintensity lesions were seen in the temporal and parietal lobes and demyelination lesions in the white matter surrounding the lateral ventricles in the fluid-attenuated inversion recovery images (figure 1b). then, the patient was referred to the shantou central hospital, affiliated shantou hospital of sun yat-sen university on october 15. on examination, but, physical examinations showed no abnormalities (normal blood pressure and there was no fever). the positive and negative syndrome scale score was 109 (positive syndrome score of 36, negative of 14, and general of 59). the patient had no history of head trauma and hospitalization for mental disorders or any tumors, according to the family members. emergency treatment was started with the administration of the tranquilizer sodium phenobarbital (100 mg) to reduce dysphoria and aggressive behaviors, the mood stabilizer sodium valproate (100 mg, administered in two fractions) to stabilize the mental state of the patient, and the antipsychotic olanzapine (10 mg, administered in four fractions) for the delusion of persecution. in addition, along with supportive treatments (piracetam 20 grams in 100 ml 0.9% sodium chloride), empirical administration of antiviral treatment (ganciclovir 250 mg in 0.9% sodium chloride) was instituted under suspicion of viral encephalitis. on october 16, a blood examination was performed which showed no anemia and leukocytosis, but presented a higher percentage of neutrophil number (80.60%; reference range 40%75%), a lower percentage of lymphocytes (11.70%; reference range 20%50%), very high levels of creatine kinase (ck; 619 u/l, reference range 0174 u/l) and its isoenzyme (ckmb; 39 u/l, reference range 024 u/l). in addition, a higher level of c-reactive protein (crp, 113 mg/l; 08 chest radiographs (october 17) showed stale tuberculous lesions in the upper lobe of right lung. on october 18, while the psychotic symptoms were improved, rough breath sounds were heard in the lungs, suggesting the presence of an infection there. therefore, the antibiotic treatment with piperacillin-tazobactam (4,500 mg in 0.9% sodium chloride) was initiated while the other treatments continued. on the same day, an electroencephalogram was performed showing slow waves characterized by strong peaks of delta and theta frequencies in the frontal lobe of the right hemisphere (figure 2). on october 19, a susceptibility-weighted imaging was performed, which more clearly showed the stale hemorrhages in the frontal lobe of the right hemisphere (figure 1c), in addition to those seen in the left occipital lobe and temporal lobes of both sides. the outcomes of cerebrospinal fluid (csf) examination came out on october 20, 21, and 22, respectively, showing blood cells in normal ranges, mild lower levels of chloride (117 mmol/l; reference range 120132 mmol/l), and glucose (2.41 mmol/l; reference range 2.84.5 mmol/l). no bacteria were found. but both anti-cmv igg and anti-hsv-ii igm were positive, supporting a diagnosis of coinfection with hsv-ii and cmv in the brain. on october 28, as required by the family members, the patient was discharged. his psychotic symptoms were significantly improved as evidenced by the reduction of positive and negative syndrome scale from 109 to 71 (positive syndrome score of 14, negative of 13, and general of 44). the patient continued the antiviral and antipsychotic treatments at home after the discharge from the hospital. he died of progressive cachexia 4 months after the discharge (february 26, 2014), although his psychotic symptoms did not relapse. the ethics committee of the shantou central hospital did not require approval for this case study. except for one early case report,7 all other double infections of the central nervous system (cns) with cmv and hsv were reported in immunocompromised patients.46,8 the patient reported here had no history of being treated with immunosuppressants. his blood examination showed normal cell count despite of the presence of anti-cmv igg and anti-hsv-ii igm in csf, which is in accordance with the reported evidence for increased production of antibodies against several neurotropic infectious pathogens including hsv-i, hsv-ii, and cmv, in the csf of individuals with bipolar disorder9 or autism spectrum disorders.10,11 the mild signs of cns inflammation in this patient may account for the absence of seizures and focal neurological signs although he presented primarily acute psychotic symptoms and mental status changes. in line with this patient with hsv-ii infection, neurological sequelae were more frequent among patients with hsv-i compared with those infected with hsv-ii.12 in addition, cmv encephalitis in immunocompetent individuals has been associated with mild signs of cns inflammation, including absent to moderate pleocytosis.1315 in contrast, cmv in immunocompromised patients was featured with more severe clinical manifestations.5,6,16 the pathological hallmark of hse is hemorrhagic necrosis in the medial part of the temporal lobes. in addition, adjacent areas such as the orbital surface of the frontal lobe and cingulate gyrus may be involved. in line with these, magnetic resonance imaging of this patient showed hemorrhages in multiple regions of the brain, including the right frontal lobe, left occipital lobe, and the temporal lobes of both sides. the hemorrhage in the right frontal lobe may be related to the peaks of delta and theta frequencies seen in electroencephalogram of this brain region. in addition, this patient showed multiple small infarctions and demyelination in the white matter surrounding the lateral ventricles. the multiple small infarctions may be the consequence of cmv infection. in line with this speculation, animal, and human studies1720 have shown the cmv-induced damage of microvascular endothelium, vasculitis, thrombotic occlusions, and hemorrhages. another feature of this patient is a very high level of crp in his blood. the co-existence of high crp level and the antibodies to cmv and hsv-ii reminded us of a recent human study, in which the crp level was strongly associated with viral detection rate and mixed viral/bacterial detection rate; whereas the rate of bacterial detection was not associated with the crp level.21 more significantly, plasma crp was an independent predictor of mortality in patients with chronic obstructive pulmonary disease caused by virus infections.21,22 in summary, we present a case of a 67-year-old male with coinfection of cmv and hsv-ii and without history of being treated with immunosuppressants. the presence of his psychosis symptoms and absence of neurological manifestations may represent the clinical features of coinfection of cmv and hsv-ii.

herpes simplex encephalitis is a rare disease. in adults, most of the reported cytomegalovirus (cmv) infections are seen in immunocompromised patients. we present a case of 67-year-old chinese male with the coinfection of cmv and herpes simplex virus type ii (hsv-ii). he had no history of being treated with immunosuppressants, showed symptoms of psychosis and was scored 109 on the positive and negative syndrome scale. this patient presented with a rare case of coinfection of cmv and herpes simplex virus type ii with psychotic symptoms.

PMC4636174

pubmed-732

the estimated incidence of bnc after open retropubic radical prostatectomy (rrp) ranges from 3% to 26% [1-8]. robot-assisted laparoscopic radical prostatectomy (ralp) is becoming a more frequently performed procedure because it clearly has a lower rate of postoperative complications than rrp. in particular, the incidence of bnc after ralp ranges from 0.6% to 3% [9-12]. several case reports have suggested that rrp, pure laparoscopic radical prostatectomy (lrp), and ralp are associated with complications related to the use of surgical clips, including bnc and the formation of bladder stones [13-19]. the purpose of this study was to evaluate the surgical clip-related complications that occurred after rrp, lrp, and ralp in our institution. in our institution, rrp was the method of choice for radical prostatectomy until august 2005, when the first case of lrp was performed. thereafter, lrp was frequently performed between august 2005 and april 2008. from may 2008 we retrospectively reviewed a database that has been maintained in our institution of the clinical, surgical, and pathological parameters of these procedures. we reviewed a total of 641 cases who underwent radical prostatectomy at our institution between january 2006 and april 2009. of the 641 patients, 439, 49, and 153 underwent rrp, lrp, and ralp, respectively. in all cases, the median follow-up time for the entire cohort was 19.0 months (range, 1-42 months). of the rrp, lrp, and ralp patients, 25 (5.7%), 1 (2.0%), and 2 (1.3%) had bnc, respectively, and 2 of the rrp patients developed a bladder stone. in total, 6 patients with complications related to the use of surgical clips were identified in the cohort. cystoscopic examination of patient 2 indicated bnc and the presence of a metal clip protruding into the urethra through the urethrovesical anastomosis at 4 o'clock (fig. patients 3 and 4 were both found to have a hem-o-lok clip in the bladder neck (fig. their bncs were resolved after a single urethral dilatation, after which the symptoms disappeared. patients 5 and 6 were found to have a bladder stone that had formed around the metal clip in the bladder (fig. retrospective re-evaluation of the kidney, ureter and bladder x-ray (kub) revealed the metal clip in the bladder region (patient 5: fig. 4). during the follow-up, neither patient exhibited signs of bladder stone recurrence. in all patients, one study based on a patient self-reported questionnaire, which revealed that 25.9% of rrp patients reported at least one episode of bnc that required treatment, was unable to identify factors that could predict the occurrence of bnc. in contrast, surya et al reported that many factors promote the occurrence of bnc after rrp, including urinary extravasation, excessive blood loss, and previous bladder neck surgery. similarly, thiel et al found that 17.5% of rrp patients required stricture dilatation by a mean time of 6 months after surgery and revealed that increased age and increased blood loss were statistically associated with stricture formation. recently, erickson et al suggested that improved surgical technique and increased surgeon experience appear to be the most important factors that reduce the incidence of bnc. huang and lepor added a history of previous transurethral resection of the prostate, pelvic external beam radiotherapy, and a hypertrophic healing response to the risk factors for bnc after rrp. another study found that of the 11.1% of patients who developed bnc after rrp, the biggest risk factor was current smoking (26%), with comorbidities such as hypertension, coronary artery disease, and diabetes also being significantly associated with the formation of bnc. the investigators hypothesized that local ischemia of the urethra and bladder neck caused by microvascular disease may lead to poor anastomotic healing and scar formation. similarly, the incidence of bnc in our cohort of 153 consecutive ralp patients was 1.3%. it is not clear why the rate of bnc after ralp is significantly lower than that quoted in the rrp literature, but msezane et al suggested that it may be due to the running anastomosis, better visualization, improved instrument maneuverability, and decreased blood loss. moreover, a comprehensive review of the literature comparing rrp, lrp, and ralp revealed that higher estimated blood loss was a significant factor in the development of bnc. recently, a study comparing rrp and ralp suggested that the predisposing causes of bnc are likely to be the combination of a fixed circular bladder stomatization with the subsequent healing of the stoma anastomosis to the urethra. the present study shows that bnc may also arise as a result of clip migration. this has also been observed in the literature (table 2), although it should be noted that most reported complications with surgical clips relate to hemorrhage. long et al reported the first case of metal clip migration-induced bnc after rrp in a patient who had suffered multiple episodes of urinary retention and had undergone several failed urethrotomies. later, blumenthal et al reported the first case report of hem-o-lok migration into the vesicourethral anastomosis after ralp. those authors believe that the relationship between surgical clip migration into the anastomosis and bnc formation is not coincidental; they postulated that the physical disruption of the anastomosis by the surgical clip contributed to poor healing and may have elicited an inflammatory reaction. tunnard et al then described a case of a hem-o-lok clip-related complication following lrp: during bladder neck dilatation for a tight bladder neck, a hem-o-lok was found in the bladder, having migrated from the urethrovesical anastomosis. it was removed successfully but a repeat cystoscopy 3 months later revealed another hem-o-lok device that had eroded through the vesicourethral anastomosis. this was removed successfully with the aid of a holmium laser. in the present study, stones developed around the surgical clip in 2 patients after rrp. other studies have also reported surgical clip-related complications other than bnc (table 2). these include the case described by palou et al of severe perineal pain after rrp due to the protrusion of a metal clip into the urethra at the urethrovesical anastomosis, and the case of banks et al, in which the migration of a hem-o-lok clip into the bladder led to stone formation after lrp. there was also a recent report by kadekawa et al of a case in which a metal clip had migrated into the urinary bladder after rrp. it was hypothesized that inflammation had arisen around the urinary bladder or vesicourethral anastomosis and that this also involved the metal clip, which then eroded the bladder wall and eventually migrated into the bladder. in addition, mora et al reported a case of intravesical migration and subsequent calculus formation with the spontaneous expulsion of a hem-o-lok clip after lrp. these findings suggest that foreign bodies in the bladder can act as a nidus for bladder stone formation owing to the presence of persistent chronic inflammation. the mechanism underlying the migration of a surgical clip into the urinary tract is unclear. in our series, the rate of bnc after ralp was significantly lower than that after rrp. because improvements in outcomes are related to temporal improvements in particular procedures, this trend might account for some improvement in outcome in the ralp group. it appears that surgical clips are prone to migration and may cause, or significantly contribute to, bnc or the formation of bladder stones after radical prostatectomy. these findings raise questions regarding the use of foreign bodies in close proximity to the vesicourethral anastomosis during radical prostatectomy. at the very least, they indicate that care must be taken with the surgical clips used for inducing hemostasis near the apex of the prostate in radical prostatectomy. in addition, because the incidence of bnc after ralp is low, when unexplained voiding difficulty occurs after ralp, one should suspect that a hem-o-lok clip has migrated, especially because hem-o-lok clips can not be detected on x-rays.

purposethe aim of this study was to describe the surgical clip-related complications that can occur after open retropubic prostatectomy (rrp), pure laparoscopic prostatectomy (lrp), and robot-assisted laparoscopic radical prostatectomy (ralp). materials and methodsa database of 641 patients who underwent rrp (n=439), lrp (n=49), and ralp (n=153) at our institution between january 2006 and april 2009 was reviewed to identify patients with complications related to the use of surgical clips. the median follow-up time for the entire cohort was 19.0 months (range, 1-42 months). resultsof the 641 patients, 25 (5.7%), 1 (2.0%), and 2 (1.3%) had a bladder neck contracture after rrp, lrp, and ralp, respectively. two rrp patients had a bladder stone. in total, 6 patients had surgical clip-related complications. metal clip migration was associated with 2 (8%) of the 25 rrp cases of bladder neck contracture and both (100%) of the rrp cases of bladder stone. moreover, both (100%) of the ralp cases of bladder neck contractures were associated with hem-o-lok clip migration into the anastomotic site. conclusionssurgical clips are prone to migration and may cause, or significantly contribute to, bladder neck contracture or the formation of bladder stones after radical prostatectomy. these findings also suggest that because the incidence of bladder neck contracture after ralp is low, the migration of hem-o-lok clips should be suspected when voiding difficulty occurs after ralp.

PMC2963780

pubmed-733

over the past few decades continuous progress has been made in the development of insulin therapy. the need for basal insulin was felt around 60 years ago, and so longer-acting insulins like lectin and neutral protamine hagedorn (nph) were developed. studies have also shown that missing two basal insulin injections per week can lead to a 0.20.3% increase in glycated hemoglobin (hba1c). at the same time, existing basal insulins were found to have limitations. in order to counter these limitations, insulin analogues were developed. there are three kinds of insulin analogues: rapid-acting insulin analogues, biphasic insulin analogues, and basal insulin analogs. the primary structure of insulin is based on a specific sequence of amino acids in the protein. the same structure slight changes were made in this insulin structure to produce different analogs with desired pharmacokinetic properties. the need of insulin analogues arises from the fact that the human insulin injections have a lag period of around 1/2 h between administration and onset of action. as a result, endogenous insulin, after secretion from the pancreas, enters the portal circulation, after which it reaches the systemic circulation. on the other hand, exogenous human insulin, after injection into sub-cutaneous tissue, enters the systemic circulation, and then about 10% of the originally administered insulin reaches the portal circulation. thus, the portal and systemic gradient of insulin is inverted in the case of exogenous insulin. it is because of this reason that exogenous human insulin is not the best physiological way of using insulin therapy. rapid-acting insulin analogs overcome this by being more in tune with the physiological rise and fall of glucose values after each meal. the basal human insulin that is available is nph, which lacks the desired 24 h effect. this limitation highlights the need for the development of basal insulin analogues including insulin detemir, insulin glargine and insulin degludec with duration of action of at least a day. in addition, a research study has established that insulin detemir has better glycaemic control with fewer hypoglycaemic epsiodes in children when compared to nph insulin. longer duration of action, a flat time-action profile, low day-to-day glycemic variability, and the potential for flexible dosing. hypoglycemia in children and adults is still a concern, but with the advent of better devices, it is easier to detect and manage hypoglycemia. the physicians thus aim to achieve tighter glucose control, as a result of which the hba1c targets are improved. insulindetemir, for instance, produce better hba1c control with less hypoglycemia than nph insulin. research studies have shown that in children/adolescents, an intramuscular instead of subcutaneous injection of insulin therapy leads to faster absorption and action, and also shorter duration of action. it has also been found that the rigidity of the insulin regimen interferes with the patient's lifestyle (diet/exercise), which is an integral part of any child or adolescent's life. all the basal insulin analogues have a similar onset of action (between 1 and 2 h) but the duration of action varies (glargine: 20-30 h, detemir: 24 h and degludec: 42 h). both detemir and degludec have a 50 amino acid chain while glargine has a 53 amino acid chain. in glargine, there is the addition of two, and substitution of one amino acid. in detemir, there is a lack of b30 amino acid and an addition of acylated fatty acid side chain at b29 position. in degludec, there is a lack of b30; addition of glutamic acid spacer and diacylated fatty acid side chain at b29. the mechanism of prolongation of action of glargine involves precipitation at acidic ph, while the mechanism of prolongation of action of detemir and degludec involves binding to albumin and multi-hexamer formation respectively. both glargine and degludec have been classified as pregnancy category c molecules while detemir is classified as pregnancy category b. glargine is a pro-drug that requires conversion from its monomeric soluble form to a microcrystalline precipitate in the subcutaneous tissue to obtain its long-acting pharmacokinetics. this process may be disturbed by its rapid access to the circulation after intramuscular injection, leading to hypoglycemia. a study has shown that accidental intramuscular injection of glargine can happen in 30% of children with type 1 diabetes mellitus (t1 dm). the definition of hypoglycemia is debatable. according to the 2009 and 2014 ispad guidelines,<3.6 mmol/l (65 mg/dl) has been used most often for clinical definition, and (70 mg/dl) is the recommended lower target for blood glucose, whereas the ada working group suggests 70 mg/dl as hypoglycemia. the who guidelines for neonatal hypoglycemia suggest that the newborn/infant/child with signs of illness would be diagnosed with hypoglycemia if blood glucose is<2.5 mmol/l or 45 mg/dl; for healthy term/pre-term newborn feeding well, the hypoglycemia cut-off value is<1.1 mmol/l or 19.8 mg/dl, and for an infant/child with severe malnutrition the value is<3.0 mmol/l or 54 mg/dl. insulin degludec is neutral, soluble ultra-long-acting insulin that forms large multi-hexamer assemblies at physiological ph following subcutaneous injection. its molecular structure is similar to the human insulin amino acid sequence, apart from deletion of threonine at position b30 and the addition of a 16-carbon fatty acid chain attached to lysine at position b29 via a glutamic acid spacer. these subsequently release monomers that are absorbed at a slow and steady rate into the circulation. pharmacokinetic/pharmacodynamic studies show that insulin degludec has a very long duration of action of 42 h, with a half-life exceeding 25 h, and variability of 25% with respect to glargine. a study was recently conducted which investigated once-daily insulin degludec versus insulin detemir, both in combination with bolus insulin aspart in a 26 week trial, followed by another 26 week extension, in children and adolescents with t1 dm. this trial (n=350) was the first to look into the long-term safety of insulin degludec in children and adolescents (age: 118 years). the results showed that at 26 weeks, insulin degludec in combination with insulin aspart was noninferior to insulin detemir in combination with insulin aspart. the mean basal insulin doses at the end of treatment were reduced by 31% in the insulin degludec group. in the 26-week extension, a lower insulin dose and a significantly greater reduction in fasting plasma glucose versus insulin detemir both regimens had similar rates of overall and nocturnal hypoglycemia, the rate of severe hypoglycemia was numerically higher with insulin degludec plus insulin aspart. of note, patients on insulin degludec had significantly lower rates (59% less) of hyperglycemia with ketosis. insulin degludec, however, is currently not approved in patients below the age of 18 years. new formulations of basal insulin that are under development are u300 glargine, which has completed phase iii and pegylated insulin lispro, which is in phase iii, insulin degludec/insulin aspart (idegasp), which is a soluble co-formulation of insulin degludec and insulin aspart, and ideglira, which is a fixed-ratio combination of insulin degludec and liraglutide for t2 dm.

over the past few decades, continuous progress has been made in the development of insulin therapy. basal insulins were developed around 60 years ago. however, existing basal insulins were found to have limitations. an ideal basal insulin should have the following properties viz. longer duration of action, a flat time-action profile, low day-to-day glycaemic variability, and the potential for flexible dosing. basal insulins have advanced over the years, from lectin and neutral protamine hagedorn to the currently available insulin degludec. currently, the focus is on developing a basal insulin that can give coverage for the entire day, with lesser variability and flexible administration. insulin degludec has been a significant leap in that direction. in addition, u300 insulin glargine and pegylated lispro represent further developments in basal insulin pharmacotherapeutics.

PMC4413397

pubmed-734

six cases were reported from 2007 to 2012 to the oral and maxillofacial unit of annaswamy mudaliar general hospital, bangalore, out of which four patients were males with the lesion occurring between the second decade of life and sixth decade of life [figures 1a3c]. in all cases, the mandible was involved with the greatest size of tumor seen being 11 cm 9.6 cm [figure 1a]. all cases exhibited enlargement of cortical plates with perforation, pain and tenderness with regional lymph node enlargement, with three cases showing fixed submandibular groups of lymph nodes (level ib) and in one case parasthesia of inferior alveolar nerve. five cases showed unilocular radiolucency and one case showed multilocular radiolucency with erosion of bone, including inferior border of mandible in one case [figure 1c]. maximum follow-up was for 39 months with one patient being lost to the follow-up [table 1]. all patients had a chest x-ray and an abdomen ultrasound done to avoid missing distant metastasis as we were aware of the potential of this lesion to metastasize. incisional biopsy was done for diagnosis for all cases and was diagnosed as features suggestive of ameloblastoma with areas showing dysplastic changes in the epithelium suggestive of a neoplastic change. (a) patient exhibiting large extra oral tumor with draining sinus, (b) intra oral view of tumor: (c) orthopantomogram showing bicortical involvement of mandible in one patient, wide excision was done as the lesion was small and the patient being young did not wish aggressive measures that may have had postsurgical cosmetic deformity [figure 2a c]. he chose the wait and watch policy and there is no evidence of disease after 32 months. (a) preoperative view showing tumor involving left mandible and sulcus, (b) preoperative orthopantomogram, (c) intra oral view of tumor being excised two patients underwent hemimandibulectomy and neck dissection because of increased involvement of mandible with palpable level ib nodes where fine-needle aspiration cytology showed positive for carcinoma, as also one more patient who had underwent a wide excision with hemimandibulectomy and neck dissection due to skin involvement and palpable fixed nodes at ib level [figure 1a c]. one patient had a segmental resection of the mandibleone patient had a hemimandibulectomy [figure 3a c]all the resected specimens were sent for histological examination. one patient had a segmental resection of the mandible one patient had a hemimandibulectomy [figure 3a c] all the resected specimens were sent for histological examination. (a) preoperative view of tumor involving left mandible, (b) intra oral view of tumor, (c) orthopantomogram showing involvement of left mandible the largest diameter of the tumors was 10 cm 9 cm [figure 1a] showing increased vascularity with numerous cystic spaces and areas of necrosis. typical features found were multiple pieces of tissue with predominant tumor islands within connective tissue seen under scanner. under higher magnification tumor islands appearing epithelial odontogenic in origin showed infiltrative pattern with each island showing peripheral columnar cells with central stellate reticulum exhibiting feature of ameloblastoma, but with atypical features such as bizarre mitosis, altered nuclear chromatin ratio, hyperchromatisim, and mild pleomorphism. individual cell keratinization with keratin pearl formation with stroma showed predominant inflammatory component and endothelial lined blood vessels [figure 4]. immunohistochemical findings showed that ameloblastic carcinomas reacted strongly with antibodies directed against cytokeratin chm and aei and ae3. histopathological picture showing bizarre mitosis, altered nuclear chromatin ratio, hyperchromatisim, mild pleomorphism, and central stellate reticulum confirming ameloblastic carcinoma no evidence of disease or metastasis was seen postsurgery in any of the patients. it is interesting to note that up to date of completion of this study 71 cases of ameloblastoma had reported to our department between february 2007 and may 2012. in this series, ameloblastic carcinoma is a lesion with histologic behavior that dictates a more aggressive surgical approach than that of a simple ameloblastoma. mean age for occurrence is 30.5 years, male: female ratio is 1.5:1, and 80% are located in the mandible posterior region. involvement of the maxilla is less frequent than that of the mandible. the clinical symptoms of ameloblastic carcinoma are more aggressive than conventional ameloblastoma, with swelling, pain, rapid growth, trismus, dysphonia, expansion of the jaws, and frequently perforation of the cortex. these were mirrored in our series also. radiographic appearance of ameloblastic carcinoma is consistent with that of ameloblastoma except for occasional presence of some focal radiopacities, reflecting dystrophic calcification. histologically, ameloblastic carcinoma does not show uniform proliferation, and has pleomorphic and hyperchromatic cells arranged in the form of sheets and chords with reversed polarity. an additional consideration in the differential diagnosis is the squamous cell carcinoma arising in the lining of an odontogenic cyst. histologically, this lesion tends to more closely resemble oral squamous cell carcinoma ameloblastic carcinoma. the squamous odontogenic tumor may also be mistaken for ameloblastic carcinoma, being composed of islands of squamous epithelium that lack stellate reticulum like zones, peripheral palisading, microcystic changes and dystrophic calcifications. however, the epithelium of the squamous odontogenic tumor lacks any cytologic evidence of malignant disease. thus, the term ameloblastic carcinoma can be applied to our series, where all showed evidence of malignant disease including cytologic atypia and mitoses with indisputable features of classic ameloblastoma. whether ameloblastoma may transform biologically and histologically from a classic ameloblastoma to a malignant lesion is controversial. many authors have shown that metastasizing ameloblastomas are histologically indistinguishable from classic ameloblastomas and others have identified malignant features in the tumor, usually after repeated surgical excisions. the high rate of recurrence maybe due to the mode of growth and surgical mismanagement rather that any inherent malignant properties and metastases are exceedingly rare. wide local excision is the treatment of choice as most investigators have recommended, which we had followed in all our cases. regional lymph node dissection should be performed selectively as we did in three of our cases where in the regional lymph node was palpable. the efficacy of adjuvant radiation or chemotherapy as a postsurgical treatment is not clear and seems to have a limited value. lanham treated a patient with doxorubicin, cisplatin, cyclophosphamide, dacarbazine, and 5 fu; but, the tumor failed to respond. yoon et al. have reported distant metastasis as early as 4 months and as late as 47 months after surgery. none of our patients showed recurrence, but one patient was lost to follow-up. none of our patients were sent for radiotherapy or chemotherapy. in our case series, least follow-up until date is 22 months and maximum follow-up being 39 months. this small series of cases illustrates the malignant portion in the spectrum of ameloblastomas and it is possible that ameloblastoma may show a wide variety of histologic and biologic behavior ranging from benignity to frank malignancy. cases of ameloblastoma should be studied carefully, correlating their histologic pattern with biologic behavior to detect changes in histology that may predict aggressive behavior. when a case is diagnosed as ameloblastic carcinoma, assessment of nodal metastasis and evidence of distant metastasis treatment of ameloblastic carcinoma is wide surgical resection and if evidence of nodal metastasis is present, neck dissection should also be considered.

ameloblastic carcinoma is a rare odontogenic tumor exhibiting not only features of ameloblastoma, but also features of carcinoma. clinical dissemination of this lesion is more aggressive and rapid than that of ameloblastoma and it can metastasize to the lung or regional lymph node. histologically, there are features of both ameloblastoma and carcinoma.<50 cases have been reported until 2011. we report a series of six cases with our treatment modalities.

PMC4157272

pubmed-735

fat embolism syndrome (fes) is a rare and potentially lethal complication of long bone fractures. the incidence of clinically significant fes occurs only in 0.9-2.2% of long-bone fractures. hypoxia or respiratory distress, deteriorating mental status, and petechial hemorrhage are the main diagnostic criteria; secondary diagnostic signs include tachycardia, fever, anemia, and thrombocytopenia typically seen in the context of long-bone fracture. neurologic symptoms can be transient and widely varies from a diffuse encephalopathy to focal deficits. fat emboli can pass through the pulmonary vasculature, resulting in systemic embolization, most commonly in the brain and kidneys. in this article, we aimed to highlight the diagnosis and treatment of cerebral fat embolism through an illustrative a case developing change of consciousness, respiratory failure and epileptic seizures after long-bone fracture caused by gunshot injury. a 20-year-old male patient was admitted to the emergency department with sudden change in consciousness and muscle contractions after 1 day follow-up in a hospital for long bone fractures of femur and tibia due to gun-shot injury. on admission, he had generalised tonic clonic seizures and his glascow coma scale was seven with localization of painful stimuli, incomprehensible muttering, and no eye opening. in the physical examination, blood pressure was 130/80 mmhg, pulse was 128/min, breath rate was 22/min and body temperature was 38c. no lateralising motor deficit was found. there was a long bone splint on the left lower extremity. the results of hematologic and biochemical parameters of the patient with the range of normal values of our laboratory in the paranthesis were as follows at first admission: hemoglobine: 11.1 gr/dl (13,6-17,2), platelet count: 75000/l (156000-373000), glucose: 189 mg/dl (70-110), aspartate aminotransferase (ast): 143 iu/l (0-46), alanine aminotransferase (alt): 53 iu/l (0-46), and creatine phosphokinase (cpk): 7866 iu/l (35-195). other biochemical findings and blood gas analysis were normal. there were no abnormalities on a postero-anterior chest x-ray cervical and thoracic computed tomography (ct). an initial ct of the head was unremarkable for intracranial abnormalities. magnetic resonance imaging (mri) was performed diffusion weighted mri trace images with a 2 mm slice gap, 5 mm slice thickness, echoplanar imaging (epi) sequence type, b value of 1000 mm/s, signal intensity dots on a dark background. diffusion coefficient map slice of the patient revealed the hypointense punctuate to lesions in centrum semiovale and periventricular regions [figures 1b and 2b]. diffusion weighted cranial magnetic resonance image reveals starfield pattern; bilaterally localised multiple hyperintense punctate lesions in centrum semiovale regions (arrows). the slice thickness of the images was 5 mm, echoplanar imaging was used and the b value of the slices was 500 diffusion weighted magnetic resonance image reveals starfield pattern; multiple milimetric sized hyperintense lesions (arrows) due to restricted diffusion areas in periventricular regions. echoplanar imagings were taken with slice thickness of 5 mm and b value of 1000 diffusion coefficient map slice of the patient reveals the hypointense punctuate to lesions (arrows) in centrum semiovale region. the slice thickness of the images was 5 mm, echoplanar imaging was used and the b value of the slices was 500 diffusion coefficient map slices of the patient reveals the hypointense punctate lesions (arrows) in periventricular region. the slice thickness was 5 mm, echoplanar imaging with b value of 500 was used an electroencephalogram was performed and diffuse bioelectrical delay on the right hemisphere was found. with the above findings, the diagnosis of the case was cerebral diffuse fes. antiepileptic therapy with phenytoin (2 150 mg) and low molecular weighted heparin therapy with enoxaparin sodium (2 4000 u) were administered. the patient had hypoxemia and the arterial blood gas analysis revealed pao2 as 65 mmhg, and saturation of arterial oxygen as 82% requiring mechanical ventilation, disturbance of conscioussness and sudden drop in hemoglobin and platelet count. hemoglobin was 7.1 gm/dl and platelet count had decreased to 53000/l at that time. the patient was rehydrated and administered with sulbactam/ampicillin (4 1 g i.v.) for fever and a possible infection due to open injuries. three units of erhthrocyte suspension and 3 units of fresh frozen plasma were given to patient to replace blood loss. on the 5 day of hospitalization, the level of consciousness of the patient improved and mechanical ventilatory support could be discontinued. fes was first described in 1862 after an autopsy identified fat in the pulmonary vasculature following a crush injury. pinney et al., reported a fes rate of only 4% in a study of 274 consecutive patients with isolated femoral shaft fractures. fat globules generated within the systemic circulation may cause pulmonary dysfunction, neurological changes, dermal symptoms, and dysfunction of several other organs. the incidence of symptomatic fat embolism was found much lower (0.9%) than the incidence of fat globules within the circulation (22.0%). long bone fractures are known as recognized to be important risk-factors for fes, especially femur fracture, followed by tibia fracture. most of the fes were reported 24-72 h after long bone fractures. in the present case neurological symptoms can be transient and widely varied from a diffuse encephalopathy to focal deficits. the most common clinical symptoms were reported as hypoxemia (pao2<70 mmhg), change of consciousness, infiltrates infiltration findings on chest plain film, and sudden drop in hemoglobin and platelets. the present patient also had hypoxemia requiring mechanical ventilation, disturbance of conscioussness and a sudden drop in platelet count. gregorakos et al., described two teenage males suffering from prolonged coma secondary to fes due to lower extremity fractures from a motor vehicle collision. mri showed multiple areas of increased signal intensity in the cerebral white matter in the first patient and diffuse high signal intensity in periventricular and subcortical white matter in the second one. in a patient with fes, chang et al., reported multifocal high signal intensity changes in bilateral cerebral hemispheres and cerebellum in diffusion weighted magnetic resonance images. the majority of case reports on fes found that cerebral dysfunction associated with fes to be reversible. parizel et al., described a teenage female who fractured her left tibia secondary to motor vehicle injury. diffusion weighted mri showed punctate foci of high signal intensity in subcortical white and gray matter and the patient had full neurological recovery by 1 week and repeated magnetic resonance images 4 weeks after the accident revealed disappearance of signal abnormalities. the outcome of patients with fes who receive supportive care is generally complete resolution of pulmonary, neurological, and dermatological lesions with a mortality of less than 10%. cerebral fat embolism is extremely rare but it should be suspected in trauma patients with long-bone fractures accompanied by unexplained neurological or respiratory deterioration.

cerebral fat embolism syndrome is a lethal complication of long-bone fractures and clinically manifasted with respiratory distress, altered mental status, and petechial rash. we presented a 20-year-old male admitted with gun-shot wounds to his left leg. twenty-four hours after the event, he had generalized tonic clonic seizures, decorticate posture and a glascow coma scale of seven with localization of painful stimuli. subsequent magnetic resonance imaging of the brain showed a star-field pattern defining multiple lesions of restricted diffusion. on a 4-week follow-up, he had returned to normal neurological function. despite the severity of the neurological condition upon initial presentation, the case cerebral fat embolism illustrates that, cerebral dysfunction associated with cerebral fat embolism illustrates reversible.

PMC3963200

pubmed-736

approximately 1-5% of peripheral blood t cells express the t-cell receptor instead of the conventional t-cell receptor. the versus t-cell lineage commitment during intrathymic t-cell development seems to be controlled by the signal strength provided to the t-cell receptor. in healthy donors, most blood t cells carry a specific t-cell receptor composed of v9 and v2 elements. in addition to effector functions shared with t cells, the v9v2 t cells can acquire professional antigen-presenting capacity characteristic of dendritic cells. in contrast to t cells, v9v2 t cells do not see processed antigenic peptides presented by major histocompatibility complex molecules, but rather recognize small phosphorylated non-peptide molecules (phosphoantigens) produced by many microorganisms but also by transformed eukaryotic cells. while microbial phosphoantigens are active at pico- to nanomolar concentrations, micromolar concentrations of the eukaryotic phosphoantigen isopentenyl pyrophosphate (ipp) are required for t-cell activation. such high concentrations are not achieved in the mevalonate pathway of isoprenoid synthesis used in non-transformed cells. interestingly, human v9v2 t cells can kill a broad variety of epithelial tumor and leukemia/lymphoma cells. the sensitivity of tumor cells to t-cell-mediated killing is increased upon treatment of tumor cells with aminobisphosphonates (n-bps), drugs that are used in clinical practice for the treatment of osteoporosis and bone metastasis in cancer patients. n-bps inhibit the ipp-processing enzyme farnesyl diphosphate synthase (fpps), thereby leading to an accumulation of ipp, which is then sensed by the t cells. t cells are poor producers of interleukin-2 (il-2), which is required for expansion of t cells. therefore, attempts to activate tumor-reactive t cells endogenously by treating patients with n-bps must take into consideration an appropriate supply of il-2. alternative strategies consider the adoptive transfer of in vitro expanded tumor-reactive t cells [10-14]. the critical role of fpps in the control of intracellular ipp levels, and thus of the sensitivity of tumor cells toward t-cell killing, has been recently demonstrated using short hairpin rna-mediated knock-down of fpps. knock-down of fpps caused tumor cells, which otherwise were not recognized by t cells, to be susceptible to t-cell killing. therefore, v9v2 t cells recognize and kill tumor cells on the basis of the unbalanced isoprenoid metabolic pathway in transformed cells, a pathway that is stable in non-malignant cells. the discovery that n-bps activate t cells by inhibiting fpps, thereby leading to accumulation of ipp, has paved the way for proof-of-principle studies to activate t cells in patients with advanced cancer. in a phase i clinical trial, dieli and colleagues treated patients with hormone-refractory prostate cancer with a standard application of the n-bp zoledronate (4 mg intravenous infusion every 21 days) either with or without additional low-dose (6 10 iu) subcutaneous application of il-2. various parameters, including subset analysis of t cells, and serum levels of prostate-specific antigen and cytokines, were monitored over time. although the two cohorts comprised only a few patients, statistically significant effects of zoledronate plus il-2 on the mobilization and effector cell maturation of t cells were recorded. very importantly, the two cohorts showed distinct clinical outcomes, with clinical responses seen in six of nine patients treated with zoledronate plus il-2 but only in one of nine patients treated with zoledronate alone. interestingly, a correlation between favorable outcome at 12 months and t-cell numbers or functional status (or both) was observed. similarly, wilhelm and colleagues had previously shown that the combined application of n-bp plus low-dose il-2 can induce objective tumor responses in patients with lymphoid malignancies. together, these studies support the view that application of n-bps plus il-2 is safe, induces in vivo activation/maturation of t cells, and may have beneficial effects in advanced cancer (figure 1a). (a) n-bps inhibit farnesyl diphosphate synthase (fpps), thus preventing processing of isopentenyl pyrophosphate (ipp) to farnesyl diphosphate (fpp). the combined application of n-bp plus il-2 leads to in vivo activation of t cells. (b) alternatively, t cells can be activated in vitro with n-bp or synthetic phosphoantigens in the presence of antigen-presenting cells (apc) and can be subsequently expanded to large cell numbers by an exogenous supply of il-2 for subsequent adoptive transfer into cancer patients. the cell preparation can be performed under gmp (good manufacturing practice) conditions. in a case study reported by laggner et al., regression of lung and bone metastases was observed in a patient with advanced stage melanoma upon systemic treatment with zoledronate and localized radiotherapy. although t-cell subsets were analyzed, it is difficult to ascertain a substantial role of t-cell activation in the resolution of metastases in this single case, particularly since il-2 was omitted in the treatment of this patient. in addition to their t-cell activating properties, n-bps also exhibit direct anti-tumor activities by both inhibiting proliferation and inducing apoptosis in tumor cells. while zoledronate seems to be the most potent t-cell-activating substance among the n-bps licensed for clinical application, derivatives of zoledronate with further improved t-cell-stimulating capacity and enhanced direct anti-tumor activity are under development. such modified n-bps might also exert improved in vivo activation of t cells when given to patients together with il-2. t-cell-based immunotherapeutic strategy is the adoptive transfer of in vitro expanded v9v2 t cells from tumor patients (figure 1b). recently, efficient protocols for the large-scale in vitro expansion of v9v2 t cells based on stimulation with synthetic phosphoantigens or zoledronate have been established. first results indicate that the repetitive adoptive transfer of in vitro expanded t cells is well tolerated and may induce anti-tumor responses in patients with solid tumors, including renal cell carcinoma and myeloma. the protocol developed by dieli et al. for the in vivo activation of t cells based on zoledronate plus low-dose il-2 application is ready to be explored in larger clinical trials and in other tumor entities with poor prognosis, for example, pancreatic ductal adenocarcinoma where it might be combined with standard regimens such as gemcitabine. it is conceivable that this protocol can be further improved, for instance, by combination with tumor-targeting monoclonal antibodies. along this line, it has been shown that b-cell lymphoma or breast tumor cell killing by fc receptor-expressing t cells is enhanced in the presence of targeting antibodies rituximab (cd20) or trastuzumab (her2/neu), respectively. moreover, a t-cell-stimulating synthetic phosphoantigen was found to enhance the depletion of cd20 b cells by rituximab in a non-human primate model in vivo, pointing to the possible use of phosphoantigen plus anti-cd20 antibodies in the treatment of cd20 leukemias and lymphomas. furthermore, cytokines promoting homeostatic proliferation and survival of t cells, such as il-15, or cytokines potentiating the cytolytic activity and pro-inflammatory response, such as il-21, might be combined with il-2 or used instead of il-2. this could be considered both for in vivo application together with n-bps and for optimization of in vitro expansion of t cells. in addition, future study protocols might include the combination of in vivo activation of t cells (by n-bp or phosphoantigen plus il-2) followed by the adoptive transfer of in vitro expanded t cells, finally, it should be stressed that t-cell-based immunotherapy is not expected to replace established therapeutic protocols. rather, it might offer additional benefit to the patient, for instance, in combination with conventional chemotherapy.

t lymphocytes are a numerically small subset of t cells with potent cytotoxic activity against a variety of tumor cells. human t cells expressing the v9v2 t cell antigen receptor recognize endogenous pyrophosphate molecules that are overproduced in transformed cells. moreover, the intracellular accumulation of such pyrophosphates is strongly enhanced by aminobisphosphonates used in the treatment of osteoporosis and bone metastasis in certain cancer patients. a new concept of cancer immunotherapy is based on the endogenous activation of t cells with aminobisphosphonates plus low-dose interleukin-2.

PMC2950047

pubmed-737

nasolacrimal duct obstruction (nldo) is a common congenital abnormality that occurs in 30% of neonates (range 6 to 84%).1 only 2 to 4% of these children become symptomatic, and most cases of nldo resolve spontaneously in the first year of life.1 2 3 4 dacryocystocele or congenital mucocele of nasolacrimal duct is a relatively rare variant of nldo, representing 0.1% of children with congenital obstruction, and results from the coexistence of a distal and a proximal obstruction.1 5 nasolacrimal duct mucocele typically presents as a bulging in the lower medial canthus of the eye, associated with epiphora., we report a case of congenital nasolacrimal duct mucocele, which recurred after surgery and was treated with endoscopic approach, followed by a literature review. the patient was a 30-year-old man with a history of bilateral congenital cyst of nasolacrimal duct diagnosed by the presence of a bulge in both lacrimal sac topographies since birth, without associated symptoms. he underwent surgery on the left side at 9 years of age and on the right side at 21 years of age, but the tumor recurred on the right side, without tearing, pain, discharge, or other symptoms. in subsequent evaluation with an ophthalmologist, a lack of upper and lower right lacrimal ducts was identified and indicated reconstruction surgery lacrimal spot, which was done in december 2007. however, he developed ipsilateral epiphora later, requiring another two procedures dacryocystectomy in july 2011 and august 2011but without success, leading to recurrent local infections. after the last procedure, computed tomography (ct) showed a cystic expansion in the right lacrimal sac topography and dilatation of the bony canal of the nasolacrimal duct (figs. 1 and 2). computed tomography of the paranasal sinuses, axial section, in bone window, showing hypodense cystic lesion in the right nasolacrimal duct topography. computed tomography of the paranasal sinuses, coronal section, in bone window, showing hypodense cystic lesion in the right nasolacrimal duct topography. the patient was then referred for evaluation by the rhinology group of hospital universitrio professor edgard santos (salvador, brazil); nasal endoscopy showed no alterations. he had endoscopic dacryocystorhinostomy in december 2011, with confection of mucosal flap and osteotomy of nasolacrimal bone, noting that the lacrimal sac was already opened with drainage of thick purulent secretion. the patient remains asymptomatic and without clinical signs 1 year and 8 months after surgery. the nasolacrimal duct is formed by canalization of the caudal extremity of an epithelial cord derived from the ectoderm in the naso-optic fissure, which is often not completed at birth. nldo at birth is common and usually asymptomatic or presents with epiphora in neonates and infants, which resolves spontaneously in most cases.2 generally, it results in blockage in the distal end of the nasolacrimal system, at the hasner valve level, although the blockage can also occur at the lacrimal spot.2 3 5 nasolacrimal mucocele, on the other hand, occurs in a small proportion of children with nldo, when there is a distal obstruction (distal membrane perforation failure) associated with a proximal obstruction, which can be functional or mechanical.2 3 5 it is characterized by a cystic mass at the medial canthus with dilation of the nasolacrimal duct that can, rarely, extend into nasal cavity.2 3 6 patients with dacryocystoceles may present with local infection or difficult breathing or breast-feeding in the breast ipsilateral to the mucocele.5 although dacryocystoceles are rare in adults, recurrent chronic keratitis of bacterial etiology (chlamydia trachomatis) has been reported in patients with trachoma, which, due to repeated infections, can lead scarring of the conjunctiva and even to lacrimal obstruction.7 it is believed that a mixture of mesodermal cells, mucus, amniotic fluid, tears, and colonizing bacteria compose the contents of the lacrimal sac, causing distention of the lacrimal system seen in dacryocystocele.7 encephalocele, hemangioma, dermoid cysts, and nasal gliomas may present similarly and must be considered in the differential diagnosis.5 dacryoceles appear as rounded, well-circumscribed lesions centered in the region of lacrimal sac in ct and magnetic resonance imaging (mri). in ct, if infected, it may show a peripheral contrast uptake.8 conservative treatment of dacryocystocele is based on a short course of topical antibiotics, warm compresses, and local massage three times a day, with a reported resolution rate of 76%. dacryocystitis may occur within a few days or weeks and requires intravenous antibiotics to prevent sepsis. most ophthalmologists recommend early surgical intervention in cases of respiratory compromise, dacryocystitis, cellulitis, large dacryocystoceles inducing astigmatism, or recurrent dacryocystoceles and in cases of failed conservative strategies.1 9 10 in cases of infection or respiratory compromise, drainage is required 24 to 48 hours after the start of antibioticoterapia.6 dacryocystorhinostomy is a surgery commonly performed, in which a fistula is created between the lacrimal sac and the nasal cavity to relieve the epiphora caused by nldo. the success rates vary from 75 to 95% in external access and 60 to 90% in endoscopic approach. the most common cause of failure in endoscopic surgery is obstruction of the new ostium by granulation or scar tissue.11 12 13 however, recent studies have shown better results with endoscopic dacryocystorhinostomy with confection mucosal flap in front of the middle turbinate and subsequent lateral wall osteotomy, with similar rates compared with the external access. the endoscopic technique has advantages such as no scars, less surgical trauma, less bleeding, a quicker return to work, and preservation of the medial canthus structure, providing sustainability of lacrimal pump mechanism.11 14 the success of surgery depends on creating a large bony ostium and preventing closure of this stoma. use of mucosal flaps after wide resection of bone surrounding the sac is one technique used to prevent granulation tissue and narrowing of the canal, with good results according to the literature.11 14 15 the main advantage of external dacryocystorhinostomy (dcr) is visualization of the anatomy that facilitates the precise removal of bone in the lacrimal fossa and enables the exact anastomosis of the nasal mucosa and lacrimal sac. nevertheless, an intranasal component that is not recognized prior to the external access increases the chance of treatment failure if the catheter does not pass over the wall of the cyst.5 11 nasolacrimal duct mucocele is a rare occurrence; however, it carries a risk of major complications (local infection, cellulitis, respiratory distress, etc.). surgery (dacryocystorhinostomy) is considered as the definitive treatment, and external access is still the most widespread and commonly used approach by ophthalmologists. however, the interest in an endoscopic nasal approach has increasingly grown as recent studies have shown good results with this technique, with similar success rates between the two types of procedures.

introduction mucoceles are benign expansive cystic formations, composed of a mucus-secreting epithelium (respiratory or pseudostratified epithelium). nasolacrimal mucocele occurs in a small proportion of children with nasolacrimal duct obstruction and is characterized by a cystic mass in the medial canthus with dilation of the nasolacrimal duct; although dacryocystoceles are rare in adults, they have been reported in patients with trachoma. objective discuss clinical aspects, diagnosis, and therapeutic management of mucocele of nasolacrimal duct based on literature review. resumed report the authors report a case of bilateral congenital nasolacrimal duct cysts in a 30-year-old man, identified as a tumor in the topography of both lacrimal sacs since birth without associated symptoms. the patient underwent successive surgical treatments, leading to recurrence of the tumor at the right side and recurrent local infections. conclusion endoscopic dacryocystorhinostomy has been increasingly used with good results and success rates similar to the external access.

PMC4392515

pubmed-738

a 61-year-old man with a history of impaired glucose tolerance, hypertension, and chemical pneumonitis without chronic lung disease sought care at a community hospital in northwestern minnesota after experiencing 2 days of fever, productive cough, and exertional dyspnea. during recent travel through parks in the western united states, he had been exposed to animal antlers and hides, wild bison, and donkeys. on hospital admission, the patient s temperature was 37.3c, blood pressure 148/72, pulse 100, respiratory rate 20, and room air oxygen saturation 89% (median 95%, range 65%100%). laboratory tests (table 1) and chest imaging (figure 1) were performed. blood samples for culture were obtained, and ceftriaxone (2 g) and azithromycin (500 mg) (table 2) were administered intravenously (iv) for presumed community-acquired pneumonia (cap). initial admission was to a community hospital; on day 4, the patient was transferred to a tertiary referral center. chest x-ray and computed tomographic scan images for a patient with inhalation anthrax, minnesota, usa. a) on hospital day 1, the x-ray image revealed a right upper lobe infiltrate and widening of the mediastinum. b) on hospital day 2, computed tomographic scan of the chest with intravenous contrast showed dense consolidation of the right upper lobe, mediastinal adenopathy (small arrow), and bilateral pleural effusions (large arrows). c) by hospital day 4, progressive infiltrates in the right lung were present.*iv, intravenous; hospital day, number of days in hospital including day of admission; ph, post-hospitalization days. iv medication was discontinued and oral medication was started on day of discharge (hospital day 26) and continued for 35 additional days to complete 60 days of therapy. on hospital day 2, the patient had increasing tachycardia and higher oxygen requirements. a bacillus species was isolated from blood cultures and sent to the minnesota department of health public health laboratory (mdhphl) for identification, and iv ciprofloxacin was initiated. on hospital day 3, the patient s condition continued to decline (table 1). mdhphl identified b. anthracis in the blood cultures, and meropenem and vancomycin were added to the treatment regimen. the isolate was sent to the centers for disease control and prevention (cdc, atlanta, ga, usa). on hospital day 4, the patient was transferred to a referral center with progressive respiratory failure requiring endotracheal intubation. iv ciprofloxacin was continued, and iv rifampin and clindamycin were administered (table 2). a chest tube was placed in the right pleural space, and 550 ml of serosanguineous fluid was drained during the initial 24 hours. pleural fluid analysis showed a leukocyte count of 3,389 cells/ml (neutrophils 38%, lymphocytes 56%, monocytes 6%), a lactate dehydrogenase level of 352, and negative gram stain results. on day 5, anthrax immune globulin (aig) was requested from cdc on day 4 and administered on day 5 without adverse reaction. the patient s disease course was complicated by nonoliguric renal failure; serum creatinine peaked at 1.5 mg/dl. on day 8, rifampin was discontinued; meropenem, which had been discontinued on day 5, was resumed for prophylaxis against nosocomial infection and improved central nervous system coverage of b. anthracis infection. after stabilization, the patient was maintained on volume control ventilation: tidal volume 500 ml, positive end the patient was extubated on day 11, and the chest tube was removed on day 13; left-sided pleural effusion did not recur. he completed a 10-day course of clindamycin and a 14-day course of meropenem. upon discharge on day 26, cdc performed susceptibility testing using broth microdilution (technical appendix). in compliance with the investigational new drug protocol for aig administration, we obtained serial serum samples to assess levels of lethal factor (lf) and anti protective antigen (pa) igg. we used an elisa to determine serum anti-pa igg levels before and after aig administration (4). the lower limit of quantification (lloq) for this assay is 3.7 g/ml. antimicrobial susceptibility testing (technical appendix) performed on the b. anthracis isolate showed a mic of penicillin of<0.015 g/ml and mic of ciprofloxacin of 0.12 g/ml. the patient s initial plasma lf level was 58.0 ng/ml, which declined to 1.5 ng/ml before aig administration: pleural fluid lf was 16.2 ng/ml at initial drainage and declined steadily (figure 2). before aig administration, no anti-pa igg was discernable because these quantifications were below the lloq (figure 2). immediately after aig administration, anti-pa igg reached maximal value of 160.5 g/ml and maintained a plateau thereafter. plasma and pleural fluid lethal factor levels and anti-protective antigen igg (aig) levels for a patient from the time of examination in the community hospital emergency department to discharge from the tertiary referral center. asterisks indicate that anti-protective aig levels obtained before anthrax immune globulin administration were below the lower limit of quantification. the patient s initial plasma lethal factor level was 58.0 ng/ml and declined to 1.5 ng/ml before aig administration. we describe the second us case of naturally acquired inhalation anthrax since the bioterrorism-related infections of 2001 and the third known case worldwide in which the patient received aig (2,6). before this case, the most recent case in the united states had occurred in pennsylvania during 2006 (2). that patient had a 3-day prodromal illness, and initial plasma lf (294.3 ng/ml) and pleural fluid lf (543.2 ng/ml) levels substantially higher than those reported here. seroconversion to anti-pa igg occurred before aig administration in the 2006 case, possibly because of a longer interval between symptom onset and aig infusion (10 vs. 6 days). the patient, who appears to have been in the fulminant phase of illness when tailored antimicrobial drug therapy was initiated on hospital day 4, died on day 7 despite aig treatment (6). (7) proposed a scoring system derived from multivariate analysis to distinguish inhalation anthrax from cap on the basis of clinical features at disease onset. in the case-patient described here, 3 of 5 identifying variables were present: elevated alanine aminotransferase/aspartate aminotransferase, normal leukocyte count, and tachycardia. the 2 remaining possible variables, low serum sodium level and nausea/vomiting were not present. this correlated with a sensitivity of 82% and specificity of 81% for diagnosing anthrax rather than cap. mediastinal widening, exhibited in the patient in this report, has also been proposed as a characteristic that can distinguish anthrax from cap. when anthrax patients were compared with an age-, sex-, and race-matched control population, mediastinal widening occurred in 82% of anthrax patients and 8% of cap patients (8). a systematic review of inhalation anthrax cases showed improved survival if antimicrobial drugs were initiated during the prodromal rather than fulminant phase of illness: 75% of patients who survived and 10% of those who died were administered antimicrobial drugs in the prodromal phase (9). a new staging system for inhalation anthrax was proposed that divides the prodromal period into early and intermediate progressive stages (10). early diagnosis facilitated implementation of multi antimicrobial drug therapy during the intermediate progressive stage, which is associated with increased the survival rate (67% vs. 21%) (9). for the case discussed here, systems to facilitate cooperation between a community hospital and state health agency enabled definitive identification of b. anthracis within 24 hours of culture becoming positive, leading to specific interventions including combination antimicrobial therapy, pleural drainage, and aig administration. drainage of pleural fluid has also been associated with increased survival (83% vs. 9%) (9); it was performed shortly after the current patient s illness reached the late fulminant stage. besides elevated lf levels in pleural fluid of recent us case-patients, patients with bioterrorism-related cases had large quantities of b. anthracis cell wall antigens, which further supports use of drainage procedures (11). although no clinical studies have reported on efficacy of passive immunization of humans against anthrax as treatment, mortality rates have been reduced in studies of inhalation anthrax in which animals were given polyclonal antibodies against pa (12). aig administration in the current case was associated with a reduction in toxemia, although the role of passive immunization in anthrax treatment needs further evaluation.

bacillus anthracis was identified in a 61-year-old man hospitalized in minnesota, usa. cooperation between the hospital and the state health agency enhanced prompt identification of the pathogen. treatment comprising antimicrobial drugs, anthrax immune globulin, and pleural drainage led to full recovery; however, the role of passive immunization in anthrax treatment requires further evaluation.

PMC3901492

pubmed-739

gait is the most natural motion performed by humans in their ordinary life and has the highest frequency in human activities. numerous musculoskeletal muscles and nerves of the lower extremities respond together during gait1, 2. during gait, movement occurs through a series of interactions among the heel bones, the soles, and the ends of the feet. damages to the feet is associated with impact force, control force, and the distribution of plantar pressure during grounding of the heel bones3. flat-arched feet, in particular, have been associated with altered foot function, including prolonged calcaneal eversion, increased tibial internal rotation, increased forefoot abduction, reduced efficiency of gait, and reduced shock absorption4. the medial longitudinal arch (mla) plays an important role in shock absorbance and energy transfer during walking5. although the etiology of this deformity can be arthritic or traumatic in nature6, it is most commonly associated with posterior tibial tendon dysfunction7. load during gait increases the number of steps and the double support time, decreases step length, raises energy consumption, and makes the repulsive force against the earth or the lower extremity joints greater8, 9. during gait, the kinds or directions of loading change the distribution of pressure delivered to the center of the body and the feet, which may trigger abnormal gait by causing fatigue fracture or affecting muscle activity or postural alignment9, 10. based on the fact that individuals with the flatfeet more easily feel muscle fatigue of the lower extremities and have a higher risk of damages to the musculoskeletal system than individuals with normal feet, the aim of this study was to examine differences between flatfeet and normal feet while subjects walked on an ascending slope like when climbing a mountain. the subjects in the present study, people with normal feet (n=15) or flatfeet (n=15), were between the ages of 21 and 30. sufficient explanations of this study s intent and the overall purpose were given, and voluntary consent to participate in this study was obtained from all of the subjects. all procedures were reviewed and approved by the institutional ethics committee of eulji university hospital. flatfoot was confirmed by posture analysis (gps400, redbalance, italy). as described by clarke11 strake s line is the line that passes between the medial border of the forefoot and the medial border of the hindfoot, and marie s line is the line that passes between the center of the 3rd toe and the center of the hindfoot. there is also a bisector line between strake s line and marie s line. we categorized subjects into the normal foot group if their medial soles passed to the lateral side of marie s line, and we categorized subjects into the flat foot group if their medial soles passed between the bisector line and strake s line. all subjects received a sufficient explanation about the research and provided consent to participation. a treadmill (ac5000 m, scifit, uk) was used to examine kinematic features during gait. the average gait velocities of the men at slow, normal, and fast rates are 3, 4, and 5 km/h using a slope of 10%, respectively, and those of women are 2.7, 3.7, 4.7 km/h using a slope of 10%, respectively12. subjects walked for one minute to determine their natural gait velocity before the experiment. muscle activity data were collected and analyzed using a wireless surface electromyograph (telemyo 2400 t, noraxon co., usa). the electrode diameter was 11.4 mm, and the distance between the electrodes was 20 mm. the sampling rate for the emg signal was set at 1000 hz, the bandwidth was set between 20450 hz, and the notch filter was set at 60 hz. emg was conducted after removing the horny layer with sand paper, and cleansing the areas with an alcohol swab. to measure muscle activations in the lower extremities during gait, electrodes were attached to the abductor hallucis, tibialis anterior, medial gastrocnemius, lateral gastrocnemius, peroneus longus, vastus medialis, vastus lateralis, and biceps femoris muscles. the frequency range of the emg signal was band-pass filtered between 20 and 500 hz, and the sampling frequency was 1024 hz. we normalized the signals of muscles to the maximal voluntary isometric contraction (mvic). the general subject characteristics (age, height, and weight) were tested for homogeneity using the independent t-test. data were analyzed by repeated measures anova in spss for windows (version 17.0), and the differences between groups at the different gait velocities were examined with the independent t-test. statistical significance was accepted for p values less than 0.05. the general characteristics of the subjects are shown in table 1table 1.general characteristics of each group (meanse)eg (n=15)cc (n=15)number of individuals (male/female)5/106/9age (years)21.41.322.10.6height (cm)164.21.6167.42.1body weight (kg)61.2.357.22.4foot length (mm)254.24.2257.22.7ankle width (cm)5.60.36.21.2. muscle activities of the flat-footed subjects were significantly different from those of the normal-footed subjects at all of the different gait velocities (p< 0.05) (table 2table 2. comparison of variable with different velocity on a 10% slope (% mvc)groupslownormalfastrectus femoriseg*20.83.224.13.230.63.7cg*21.42.523.73.929.94.7vastus medialiseg*19.92.325.15.838.06.2cg*21.71.624.21.431.84.1vastus lateraliseg*20.51.823.81.629.53.6cg*20.72.424.31.929.13.7tibialis anterioreg*23.40.724.81.430.24.5cg*22.51.825.22.130.03.4peroneus longuseg*20.83.127.02.031.00.7cg*22.11.327.63.432.94.3medial gastrocnemiuseg*32.24.534.24.635.83.2cg*33.31.034.61.337.11.5lateral gastrocnemiuseg*33.41.734.41.636.22.3cg*32.71.234.11.338.71.7abductor halluciseg*14.64.217.34.020.23.6cg*15.23.421.45.026.43.2*p<0.05, eg: experimental group; cg: control group), especially those of the vastus medialis and abductor hallucis muscles (p <0.05) (table 3table 3. results of between-subject comparisons of the effects of muscle activation during treadmill gait on a 10% slopetype iii ssdfmsrectus femorisgroup0.210.2error225.2288.0vastus medialis*group23.9123.9error155.7285.5vastus lateralisgroup0.010.0error68.4282.4tibialis anteriorgroup0.410.4error70.7282.5peroneus longusgroup11.6111.6error111.7283.9medial gastrocnemiusgroup6.316.3error311.12811.1lateral gastrocnemiusgroup1.711.7error63.5282.2abductor hallucis*group98.2198.2error381.12813.6*p<0.05). in the subjects with the flatfeet, the function of their feet may did not activate properly as a result of overuse when conducting activities that put repetitive loads on the feet13. when the subtalar joints are excessively pronated, the medial tibia areas of the knees slip backward at a rapid speed, rotate and enter under the medial femoral condyles, which may cause damage to the medial side of the knees6. the results of analysis of the muscle activities of the subjects during gait on an ascending slope showed that overall, their muscle activities increased according to the rise in gait speed, and there were significant differences between the two groups in the vastus medialis and abductor hallucis muscles. in the subjects with flatfeet, extensor muscles, in particular, the vastus medialis, play a role in lowering speed and absorbing impact through eccentric contraction. the subjects with flatfeet received more loads on the vastus medialis than those with normal feet due to weakened plantar flexion muscles. drawing in a slant line of the patellas by the vastus medialis muscles has an important meaning in stabilization and direction of the patellas when they pass or slip through the intercondylar areas of the femurs14. the abductor hallucis muscles, which are situated below the medial longitudinal arches of the feet, stop at the sesamoid bones of the distal phalanxes from the heel bones and provide dynamic stability to the medial longitudinal arches15. in this study, the changes in activity of the abductor hallucis muscles according to the changes in speed and gradient were smaller in the subjects with flatfeet than those with normal feet. this indicates that the abductor halluces muscles of individuals with flatfeet do not properly function as muscles for dynamic stability of the medial longitudinal arches. the present study verified that in individuals with flatfeet, the abductor hallucis muscles affected descent of the navicular bones by inducing blockage16 and fatigue of the tibial nerves17.

[purpose] this study determined the difference between flatfeet and normal feet in humans on an ascending slope using electromyography (emg). [subjects] this study was conducted on 30 adults having normal feet (n=15) and flatfeet (n=15), all of whom were 21 to 30 years old. [methods] a treadmill (ac5000 m, scifit,) was used to analyze kinematic features during gait. these features were analyzed at slow, normal, and fast gait velocities on an ascending slope. a surface electromyogram (telemyo 2400 t, noraxon co., usa) was used to measure muscle activity changes. [results] the activities of most muscles in the subjects with flatfeet were significantly different from the muscle activities in the subjects with normal feet at different gait velocities on an ascending slope. there were significant differences in the vastus medialis and abductor hallucis muscles. [conclusion] because muscle activation of the vastus medialis in relation to stability of the lower extremity has a tendency to increase with an increase in gait velocity on an ascending slope, we hypothesized that higher impact transfer to the knee joints occurs in subjects with flatfeet due to the lack of a medial longitudinal arch and that the abductor halluces muscles, which provide dynamic stability to the medial longitudinal arches, do not activate well when they are needed in subjects with flatfeet.

PMC4047230

pubmed-740

cell invasion is important in many biological processes, particularly in the invasive stage of cancer and in embryonic development [23, 32]. there has recently been some interest in how to mathematically model cell cell and cell extracellular matrix adhesions in the context of cancer invasion, since the latter is believed to be characterized by a number of processes that include loss of cell cell adhesion and enhanced cell matrix adhesion in addition to active migration, cell proliferation and the secretion of matrix degrading enzymes. accurate modelling of cell adhesion is, therefore, important, but it is challenging to do so using continuous mathematical models because in such an approach one uses continuous variables for cell densities. individual cells are not recognized as such and, therefore, there is no representation of cell boundaries. a modelling approach that aims to address these difficulties was proposed by armstrong et al.. the ideas have been taken further and applied to various contexts by, for example, [3, 12, 15, 23, 31]. for a cell density p(x, t) the basic model in one spatial dimension is for x (,), t>0 with initial condition p(x, 0)=0(x). equation (1) incorporates a direct representation of cell-cell contact via a non-local flux term, the integral term in equation (1). spatially structured models of tumour invasion that incorporate other aspects such as haptotaxis and age structure were considered in [911]; see also [34, 35]. the purpose of the present paper is to formulate and analyse an extension of equation (1) to the case of n spatial dimensions, considering only cell-cell adhesion. again letting p(x, t) denote the cell density at (x, t), but now with x, t>0 and b denoting the n-dimensional ball centred at 0 and of radius, we propose the equation with initial condition the function f(p) models cell loss and gain, and for biological reasons we assume that f(0)=0 and that there is a number p1>0 such that f(p)>0 for p (0, p1), and f(p)<0 for p>p1. these assumptions imply that at lower densities, there is cell gain, but at large enough densities cell loss occurs more rapidly than the generation of new cells via division, due to the effects of crowding. motion of cells is assumed to be partly due to fickian diffusion, which gives rise to the laplacian term. note from simulations of their more complex cell matrix model that, in the absence of a fickian term, and unusually for a continuous model, non-invasive growth is possible for some parameter regimes while invasive growth takes place in others. the other component to cell motion is the movement of cells due to adhesion as modelled by the cell adhesion term in equation (2). it is an advective flux term and models motion of cells that is caused by mechanical forces attributable to adhesive bonds that cells have formed with their closer neighbours. cells are able to sense their surroundings over some radius, which is believed to be of the order of several cell diameters, due to their ability to deform and extend protrusions. these protrusions cause adhesive bonds to form with other cells, and the resulting forces cause cell movement. in fact, the cell adhesion term in equation (2) is an advective flux term of the form (p(x, t)u(x, t)), where u(x, t) is the velocity of the cells at x at time t. if we imagine the cells as tiny spheres moving through a viscous fluid, then stokes ' law leads us to suppose that they are subject to a resistive force which is directly proportional to the velocity. this analogy leads us to suppose that the velocity of a cell is proportional to the net adhesive force on it due to the bonds formed with nearby cells. so, the velocity u(x, t) can effectively also be thought of as the adhesive force on a cell due to bonds with these other nearby cells within the sensing radius (the range over which a cell can detect its surroundings). this force is taken to be of the form we think of expression (4) as a force, but it is really a velocity. expression (4) is assumed to contain dimensional constants such as the viscosity of the medium (perhaps embodied within the functions g and h), to ensure that it has the units of velocity. viewing expression (4) as a force, we are asserting that the total force on a cell at position x is the sum, over all cells within the sensing radius, of the local forces attributable to bonds formed with the cells at the nearby points x +, b. the function h: describes how the magnitude of the force depends on ||, and is a positive function since adhesive forces are always directed towards cell centres. the vector in front of h(||), which is not present in equation (1), gives direction to the force on cells at x due to bonds with cells at x +. another formulation would be to make this direction vector a unit vector and write (/||)(||) rather than h(||), as some other authors have done, see for example painter et al.. there is no real distinction; we are simply taking h(||)=(||)/||. the magnitude of the adhesive forces from cells at the nearby location x+ will depend on the number of adhesive attachments made by cells at x+ to a cell at x, and hence on the cell density at x +. this explains the term g(p(x +, t)) in the integrand of expression (4), and the function g(p) describes how the forces depend on the local cell density. we anticipate that g(p) should increase linearly with p if cell density is not too great, because in this situation a doubling of the number of cells at x+ should roughly double the number of adhesive attachments made to cells at x. however, at higher densities, particularly as close cell packing is approached, the tendency to form attachments drops off and so g(p) in fact decreases once p has risen above a certain threshold, and is zero for all p above a critical cell density p2>0 corresponding to close cell packing. the shift in balance between cell division and cell loss should occur at an achievable cell density.. there is unquestionably a need for analytic study of equations of the form (2) in spatial domains of dimension n recent interest in this kind of equation has been largely in the areas of cancer cell invasion and wound healing. gerisch and chaplain and painter et al. both consider systems containing a term of the same form as the non-local term in equation (2), and with the same interpretation cell velocity due to adhesive bonds with other cells with an emphasis on two dimensions so that b becomes a disk of radius. these studies have been largely numerical, which is time-consuming in the 2d case. in addition to their numerical simulations, gerisch and chaplain examine the possibility of expanding the non-local term, using taylor series in the case when is small. this analytical approach does yield some useful insight, although the approximated equations can allow unrealistic phenomena such as blow up. two- and three-dimensional studies are extremely important in the cancer modelling context because of the manner in which cancer cells invade tissue and the possibility of phenomena such as fingering at the invasive front (see figure 5 in). the shape of a tumour-host boundary is an important diagnostic indicator (, and the references therein). straight and/or sharp boundaries tend to imply non-invasive tumours, whereas if a tumour has a diffuse and/or wavy boundary it is likely to be invasive. another important reason for considering equations of the form (2) in higher space dimensions is that the inclusion of another space dimension can be an important test of the stability and robustness of a one-dimensional pattern, and therefore of the plausibility of a type of mathematical model in a situation where many details are unknown. this was an important issue in armstrong et al. who considered a system with the same type of adhesive flux term as in equation (2) as a model for somite formation. segmentation in a number of organisms proceeds through the formation of somites, which will eventually become repeated structures such as the vertebrae. somitogenesis is, therefore, seen as a one-dimensional process (along the anterior-posterior axis of the presomitic mesoderm) and an important step in the model validation process can be to include a second spatial dimension and investigate whether the model is still capable of forming somites, as was done in. using fractional powers of the diffusion operator, we establish results about the existence and uniqueness of solutions and their positivity and boundedness in spaces of uniformly continuous functions. we also look at solutions in l spaces and show that under suitable conditions the non-zero uniform state is asymptotically stable in l and that under more stringent conditions all solutions converge to this uniform state. initially, we will work in the spaces in either case, take the norm ||u||k=||k ||du||, where for i=1,, n, di: (di) so di is a closed linear operator. set d=(d1,, dn), with domain (d)=(d1) (dn). if =(1,, n), d=d1 dn. we define ||=1++n and say for n-tuples and if the inequality holds component-wise. note that from page 33, u buc0() implies that, for || k, du 0 as |x|. we write when results apply to either x or x0 and set x=x. we define: () x x, by for >0 set a=+i, (a)=() so that the spectrum of a is contained in the open right half plane. we rewrite the problem (2)(3) abstractly as where p(t): [0,) x, t 0, f :, g :, and with ith component ki()(x). let t(t) be the analytic semigroup in x generated by, so and t(t): x0 x0. let tk(t) be the analytic semigroup which is t(t) restricted to the space (k an integer); the corresponding generators k are given by the operator but restricted to different domains. it is easy to see that for x, k>0, || k so we also have t(t): x x, set ak=k +. we will use the fractional powers ak of ak, and exploit the result that, for suitable, the operator diak: x x is bounded, see. we will write =(ak) in x, 0=(ak) in the space x0 and =(ak) in the space. thus the mild form of (2)(3) in x is observe that for (d), g c(), h l(0,), we have k() (), note that in our proofs of existence, we will take f, g: but we will then prove that if 0 0 then p(t) 0. thus, provided there is enough smoothness at 0 it is only the properties of f and g on [0,) that are relevant as we can then define f and g appropriately for p<0. we also have [17, 18, 22, 24] if >0, then for every t>0 the operator aktk(t) is a bounded linear operator in and for any >0 there exists c1>0 such that if 0 1, then ak: is a bounded linear operator so there exists a constant c2 1 such that for all u x, if <<1, then diak: is a bounded linear operator so there exists a constant c3 1 such that for all u x, the following lemma is proved in very much the same way as lemma 1 in suppose that for some k 0, f c(), g c() and h l(0,), <<1 and f(0)=0. then g: and (a)if ||ak||k r1 there exists a constant k1(r1) such that(b)if ||i||k r2, i=1, 2, then there exists a constant k2(r2) such that(c)if k 2, and ||||k2 r3 if ||ak||k r1 there exists a constant k1(r1) such that if ||i||k r2, i=1, 2, then there exists a constant k2(r2) such that if k 2, and ||||k2 r3 suppose that for some k 0, f c(), g c(), f(0)=0, that h l(0,), and that 0 q 0, where <<1, and either q=0 or there exists p1>0 such that f(p1)=0 and q=p1. then the problem (2) and (3) has a unique mild solution p(t) such that p(t) q c([0, t0]; 0), for t0>0 small enough. in addition p(t) is the classical solution on [0, t0] of the problem (2) and (3) in the sense that and for 0<t t0, p(t) (ak), and also, under the above conditions, if k 2 m, in particular if k=2, p(t) q c([0, t0]; x0) and equation (23) also holds at t=0. let [0, 0) be the maximal interval of existence of the solution p(t). now let pn(t) be the solution of equation (2) with initial data pn(x, 0)=n(x) where n q 0. suppose that ||ak(n 0)||k 0 as n. then, given any t1 [0, 0), pn(t) is defined on [0, t1] for large enough p(t))||k 0 as n, uniformly on [0, t1]. to deal with the case q=p1 we set p(x, t)=w(x, t)+p1 in equations (2) and (3) to get where f(w)=f(w+p1), and g(w)=g(w+p1). if we set the abstract form of equation (25) is if g()=d (( a+p1)k(a)) f(a), then it is easy to see that g satisfies lemma 1 and we can apply the same methods to the resulting equation in w. we can now obtain positivity and boundedness. suppose that h c [0,], f c(), g c(), f(0)=0, and that 0 q d0, where <<1 and q=0 or q=p1>0 where f(p1)=0. let 0(x) 0 for all x. if p(t) is the unique classical solution of equations (2) and (3) on [0, t0], then p(x, t) 0 for all 0 t t0, x. proof take m1>0 and m2 0 such that and, using the mean value theorem, choose c sufficiently large that where n is the surface area of b1, and define u(x, t)=p(x, t) e, so we now prove that p(x, t) 0 on [0, t0]. suppose not, then u(x, t) will be strictly negative somewhere in this set and hence attains a global minimum at some (x0, t0) (0, t0]. thus for all i in the ith integral in the second term in the right-hand side of equation (31), we may replace /xi by /i and therefore an integration by parts shows that the sum at the point (x0, t0) equals the above quantity is bounded in absolute value by evaluating equation (31) at (x0, t0), and making use of (32) and the above bound, we obtain using inequality (29), the fact that u(x0, t0)<0 and that c satisfies inequality (30). suppose that h c[0,], h 0, f c(),g c(), f(0)=0 and that 0 q d0 where <<1, and q=0 or q=p1>0 where f(p1)=0. suppose there exists p2>p1 such that g(p) 0 for p [0, p2], f(p2)<0 and that let 0 0(x) p2 for all x. if p(t) is the unique classical solution of equations (2) and (3) on [0, t0], then 0 p(x, t) suppose that h c [0,], h 0, g bounded, f c(), g c(), f(0)=0, and that 0 q 0 where <<1, and q=0 or q=p1>0 where f(p1)=0. suppose there exists p3>p1 such that for p>p3, f(p)<0 and let 0 0(x) p3 for all x. if p(t) is the unique classical solution of equations (2) and (3) on [0, t0], then 0 p(x, t) the method uses proposition 2 or 3 to show first that p(t) is bounded on bounded intervals of time. thus by lemma 1(a) ||g(ap(t))|| is bounded linearly, so we can use the gronwall-type inequality in 1.2.1 to show that ||ap(t)|| is suitably bounded. then we can use lemma 1(c) to get a linear bound on ||g(a2p(t))||2, so, using the gronwall-type inequality again, ||a2p(t))||2 is also bounded as required suppose that h c[0,], h 0, f c(), g c(). 0 where <<1, and q=0 or q=p1>0 where f(p1)=0 and 0(x) 0 for all x. suppose that either (a)there exists p2>p1 such that 0(x) p2 for all x, g(p) 0 for p [0, p2], f(p2)<0 and inequality (34) holds. p1 such that 0(x) p2 for all x, g(p) 0 for p [0, p2], f(p2)<0 and inequality (34) holds. or g is bounded and there exists p3>p1 such that 0(x) p3 for all x, and for p>p3, f(p)<0 and inequality (35) holds. p< define the operator, l: (l) l() l() by similarly, we can also define l: (l) l() l(), for a suitable domain (l1). for 1 p <, >0, set al=l+i, (al)=(l). it is well known that l generates a positive analytic semigroup {tl(t)}t0, with ||tl(t)|| 1. further, in the case 1<p <, from propositions 4.1.7 and 1.1.4 and example 1.3.9, if <<<1, thus, if we define di: (di) l() l(), then inequality (18) holds in l(). to show that inequality (18) also holds in l(), it is sufficient to show that (al) w(). to do this note first that it follows from example 1.3.9, example 1.3.11 and remark 1.3.7 that if 1<s<2 and 0<<<1, then now, by proposition 4.1.7, theorem 1.7 and prop 4.8, and prop 1.1.4, for 1<s<2 (the definition and properties of the besov spaces bp, q, and their relationship to the sobolev-slobodeckii spaces w, can be found in [1, 16, 20].) as before, from, tl(t) satisfies equation (8), hence if x l(), tl(t)=tk(t). equally (al) (al) implies al=ak. we now have the following local existence theorem in l(): suppose that f c(), g c(), that h l(0,), f(0)=0 and that 0 q (al), where <<1, and either q=0 or there exists p1>0 such that f(p1)=0 and q=p1. then, for n 3, the problem (2) and (3) has a unique mild solution p(t) such that p(t) q c([0, t0]; (al)), for t0>0 small enough. in addition p(t) is the classical solution on [0, t0] of the problem (2) and (3) in the sense that and for 0<t t0, p(t) (al), and also there is continuous dependence on the initial data. let [0, 0) be the maximal interval of existence of the solution p(t). now let pn(t) be the solution of equation (2) with initial data pn(x, 0)=n(x) where n q (al). suppose that ||al(n 0)||l 0 as n. then, given any t1 [0, 0), pn(t) is defined on [0, t1] for large enough p(t))||l 0 as n, uniformly on [0, t1]. finally, if in addition 0 q (al), then also p(t) q c([0, t0]; (al)). the proof of the first part follows from theorems 3.3.3 and 3.4.1 in provided we can show that g: l() l() and g: l() l() are locally lipschitz. each product has just one term of the form dial. the rest are functions of al d(al). but, for <<<1, and n 3 (see 7.34) and the local lipschitzness follows. similarly for g. note further that, also using inequality (18) in l (), g, g: l () l() l () l() and are locally lipschitz continuous in l(). for 0 (al) (al), we set up the iteration: 0=al 0, then the iteration converges in l() and l() and by considering the restrictions to bounded subsets of, it can be seen that the limits are equal almost everywhere. similarly for g, and we now consider the local asymptotic stability of the uniform state p=p1. for simplicity, we will consider the case where f and g are logistic. each of f and g in equation (2) can be of the form rp(1 p/k) with different r and k. after suitable non-dimensionalization, without loss of generality, they can be taken as f(p)=p(1 p) and g(p)=p( p), with >1. the uniform steady state is then p=1. to examine its stability, set p=w+1, 0=w0+1 to obtain the following equation for w: take n 3. suppose that h l(0,), and that 0 1 (al), where <<1. for define then if k()<0 for all the zero solution of equation (40) is uniformly asymptotically stable in (al). precisely, if >0 is such that k()< for all, then there exist >0, m 1 such that if ||alw0||l, then moreover, k()<0 for all holds if conversely if there exists 0 such that k(0)>0, then the stationary solution is unstable. (al) l(), such that and the operator h: l() l() such that h()=g() bal. using the same reasoning as in theorem 3 it can be seen that if ||||l r, then there exist constants k(r) and k(r) such that in the case of stability, we note that this implies that h()=o(||||l) as ||||l 0. we have already seen that g: l() l() is locally lipschitz continuous. thus theorem 5.1.1 of holds so that if the solutions of the linearized problem are uniformly asymptotically stable then so too are those of the nonlinear problem. the right-hand side of equation (44) is linear and generates an analytic semigroup r(t), say. so, for all u0 l(), equation (44) has a global classical solution u(t)=r(t)u0. first take u0 l() l(), so that, as in theorem 3, u(t) l() l(), and we may take fourier transforms. if we denote the fourier transform of u by thus, using the plancherel identity, as required. thus by density ||r(t)u0||l ||u0||l2 exp(t) for all u0 l(), and the result follows. the inequality (42) follows from completing the square in k(), while inequality (43) follows from the fact that |sin( x)| | x| |||x|. for instability, we apply corollary 5.1.6 of. from inequality (45) h()=o(||||l) as ||||l 0, so now we require that there exists in the spectrum of the generator of r(t) such that re >0. suppose not, so that there exists m such that, for all l(), but, using the expression for (, t) in (46), there exists >0 such that if we now choose u0 such that b|0(+0)| d>0, then using the plancherel identity gives a contradiction to inequality (47). the next result follows in a similar fashion to theorem 3 and theorem 3. suppose that f c(), g c(), h c[0,]. let 0 q 0, where <<1, and either q=0 or q=p1>0 with f(p1)=0. suppose also that the hypotheses of proposition 1 and of either proposition 2 or proposition 3 on f, g, h, and 0 hold. take also 0 q (al). q c([0,); 0) be the unique classical solution of equations (2) and (3). then, for each t 0, p(t) q (al)=w(). furthermore p(t) q c((0,); l()). let h c[0,] be such that h 0, and let f(p)=p(1 p) and g(p)=p( p) with >1. let 0 1 0 (al(n)), where <<1, and let p(x, t) be the solution of the problem (2) and (3). (a)if 0 0(x) for all x, where satisfiesthen 0 p(x, t) for all x, t 0.(b)if 0(x) for all x, where 0< p(x, t) for all x, t 0.(c)if 0(x)< for all x, where >0, satisfies inequalities (48), (49), andthen p(x, t) 1 exponentially as t in the sense that for all t>0 if 0 0(x) for all x, where satisfies then 0 p(x, t) for all x, t 0. if 0(x) for all x, where 0< <, satisfies inequality (48), and then p(x, t) for all x, t 0. if 0(x)< for all x, where >0, satisfies inequalities (48), (49), and then p(x, t) 1 exponentially as t in the sense that for all t>0 first, to prove (a), note that inequality (48) implies inequality (34) with p2 =, f(p)=p(1 p), and thus, solutions remain in the closed interval [0,] for all positive times. next, to prove (b), suppose that there exists t *>0 and a corresponding x*with p(x *, t *) =, p(x *, t*)/xi=0, p(x *, t*)/xi 0, then note that 0 g(p) /4 when p [0,]. therefore by inequality (49). hence p(x, t) for t>0 and (b) is proved. last, to prove (c), define w(x, t) by p(x, t)=1+w(x, t), so that w(x, t) satisfies equation (40) and 1 w 1. multiplying equation (40) by w(x, t) and integrating with respect to x over, and then integrating by parts on the laplacian term and the /xi term (this is justified by theorem 5), the integrand of the middle term in the right-hand side will now be estimated by using the inequality a b this bounds the integral by a sum of two integrals, one of which cancels with the first term on the right-hand side to give since >1, it is easy to show that recalling that 0 1+w(x, t), so using inequality (54) and theorem iv.15 from, therefore inequality (53) becomes note that w(x, t) (1 )w(x, t), so that inequality (56) becomes and (c) follows from inequality (50). we illustrate in figures 14 the results of the previous two sections with simulations that can be applied to in vitro wound closure experiments. in these experiments, cell cultures are scored in a thin line that closes as the cell population proliferates. a review of mathematical models of wound healing is given in and other references are found in [57, 13, 14, 21, 2529, 33, 36]. in previous work, we modelled in vitro wound healing experiments for the model (2) with n=1, and examined the dependence of solutions on the diffusion parameter and the sensing radius. in numerical simulations demonstrated that for larger values of the diffusion parameter and smaller values of the sensing radius the wound closed completely across the wound opening, but for smaller values of and larger values of complex patterns arose across the wound opening with incomplete closing. either of the alternative conditions (42) or (43) is sufficient for the stability of the uniform steady state p=1, and note that both of these conditions are largeness conditions on and smallness conditions on both h() and on the sensing radius. similarly conditions (48), (49) and (50) are sufficient for convergence to the uniform steady state and conditions (48) and (49) are smallness conditions on h() and while (50) is in addition a largeness condition on. in our simulations here, we examine the dependence of the wound healing model with respect to the parameter, which is the non-dimensionalized cell density corresponding to close cell packing, in equation (2) with g(p)=p( =0.1, =0.2, f(p)=p(1.0 p), g(p)=p( p), h(x)=arctan (100.0x)/(10.0x arctan 100.0) and initial data 0(x)=1.0 0.8 exp((0.1x)). the red plane and lines correspond to =0.2 and the green plane and lines correspond to 0.0. (a) graph of p(x, t), 0 t<2.0. the wound does not close, and instead the solution exhibits an evolving pattern of peaks and valleys which do not converge to 1.0. (b) graphs of p(x, .0), p(x, .5), p(x, 1.0), p(x, 2.0). simulation with =1.0, =1.5 (a), =17.0 (b), =18.0 (c), =19.0 (d), =1.0, =0.2, f(p)=p(1.0 p), g(p)=p( p), h(x)=arctan(100.0x)/(9.0xarctan 100.0), and initial data 0(x)=1.0 0.8 exp((0.1x)). for the higher values of, the wound healing simulation wave patterns as in figure 2 with adhesion strength parameters (a) =17.0, (b) =18.0, and (c) =19.0. for =17.0, the solution is shown at times t=100.0 (blue), t=95.0 (red) and t=90.0 (green). the amplitude envelope of the oscillation packet decreases to a minute value for the times shown. for =18.0 the solution is shown at times t=200.0 (blue), t=150.0 (red) and t=100.0 (green). for =19.0, the solution is shown at times t=150.0 (blue), t=125.0 (red), t=100.0 (green), t=75.0 (yellow) and t=50.0 (cyan). the simulations indicate convergence to the uniform equilibrium 1.0 in (a) <17.62, but not in (b) and (c), >17.62. wound healing simulations with parameters =2.0, =1.0, =3.0, f(p)=p(1.0 p), h(x)=9.0 arctan(100.0x)/(4.0x arctan 100.0), and initial data 0(x)=1.0 0.4 exp((0.1x)) (top left), 0(x)=1.0 0.95 exp((3x)) (top right), 0(x)=1.0 0.5 exp((0.2x)) (bottom left) and 0(x)=1.0 0.95 exp((0.1x)) (bottom right). the wounds top left and top right both close but the wound bottom right which has the same width as the first and the same depth as the second fails to close. the widest wound, bottom left, also closes. in figure 1, n=1, p), =0.1, =0.2, =1.0, h(x)=arctan(100.0x)/(10.0x arctan 100.0), and 0(x)=1.0 0.8 exp((0.1x)). in this case, theorem 6 (48) is satisfied if <18.94, (49) is satisfied if <1.457, and (50) is satisfied if <2.823. we take =19.0, so that none of the conditions (48), (49), (50) of theorem 6 are satisfied. the simulation in figure 1 shows that for this value of, the solution does not remain bounded below by and does not converge to the equilibrium 1.0. on the other hand, if condition (48) were satisfied, then the solution p would be bounded (0 p), and theorem 2 would give global existence of the solution. indeed, if we take =18, then condition (48) holds, and we find that the solution behaves qualitatively the same as in figure 1. this provides an example where the theory gives global existence of the solution, but the solution does not converge to the equilibrium but instead forms a series of peaks. in figures 2 and 3, n=1, p), =1.0, =0.2, h(x)=arctan(100.0x)/(9.0x arctan 100.0), and 0(x)=1.0 0.8 exp((0.1x)). we consider different values of to illustrate the sensitivity of this parameter for the convergence of the solutions to the equilibrium 1.0. the hypotheses of theorem 6 require that <16.94 (48), <2.683 (49), and <2.597 (50). for <2.597 all three conditions are satisfied, and theorem 6 implies the solution remains in the interval [0.2,] and converges to 1.0 for all initial data such that 0.2 0(x). on the other hand if one looks at the stability condition from theorem 4, then the equilibrium 1.0 is asymptotically stable for <17.62 with instability if >17.62. in figure 2 (a) =1.5 and there is convergence to 1.0 (the solution appears to converge monotonically to 1.0 from below, never rising above 1.0). in figure 2(b)(d) =17.0, 18.0, 19.0, respectively, and the solutions develop a series of peaks, symmetric to the right and left of 0.0, and rising above and falling below 1.0. further examination of this wave behaviour is given in figure 3 for the cases =17.0 (a), =18.0 (b), and =19.0 (c). for =17, the amplitude of the waves decreases as t increases and the solution appears to converge slowly to 1.0. for =18 and =19, the wave propagates with amplitude dependent on (the greater the value of, the greater the amplitude). in general, for the roles of the model parameters, we postulate that cells concentrate in interior and exterior regions of the wound in a regular pattern depending on the strength of adhesion, the sensing radius, and the diffusion coefficient. in figure 4, we show that the convergence to the equilibrium 1.0 corresponding to the healed wound depends on the initial conditions. in these simulations, =3.0, f(p)=p(1.0 p), g(p)=p( p), and h(x)=9.0 arctan(100.0x)/(4.0x arctan 100.0). in this figure, the only change in the simulations is the initial data the parameters are the same for all. for initial conditions corresponding to a shallow, wide wound, and to a narrow, deep wound, closure occurs, but for an initial condition corresponding to a wound with the same width as the first and the same depth as the second, closure does not occur. we note that the function k() defined in equation (41) is negative for all, which by theorem 4 implies that the equilibrium 1.0 is stable in this example. we have chosen to illustrate the behaviour of solutions for the model using the simple case of an equilibrium corresponding to wound healing experiments, in which wound closure corresponds to convergence to a constant function in the x-direction, perpendicular to the direction of the scoring .

a model for cell cell adhesion, based on an equation originally proposed by armstrong et al. [a continuum approach to modelling cell cell adhesion, j. theor. biol. 243 (2006), pp. 98113], is considered. the model consists of a nonlinear partial differential equation for the cell density in an n-dimensional infinite domain. it has a non-local flux term which models the component of cell motion attributable to cells having formed bonds with other nearby cells. using the theory of fractional powers of analytic semigroup generators and working in spaces with bounded uniformly continuous derivatives, the local existence of classical solutions is proved. positivity and boundedness of solutions is then established, leading to global existence of solutions. finally, the asymptotic behaviour of solutions about the spatially uniform state is considered. the model is illustrated by simulations that can be applied to in vitro wound closure experiments.ams classifications: 35a01; 35b09; 35b40; 35k57; 92c17

PMC3957472

pubmed-741

although ascites has been reported in cases with cervical cancer, it is due to other causes such as ovarian metastasis. a 78-year-old diabetic woman who presented with ascites and abdominopelvic mass was misdiagnosed with ovarian cancer and treated with neoadjuvant chemotherapy followed by radical hysterectomy and adjuvant radiotherapy. however, pathology confirmed locally advanced cervical cancer stage iv in this patient. considering all signs and symptoms to reach a verdict would reduce such malpractices and consequently lead to select the best management and treatment. the initiating event in cervical cancer is hpv infection and immunosuppression is a risk factor (2). several studies proposed that diabetes mellitus (dm) could increase the risk of developing cervical cancer (35). the most common symptoms are abnormal vaginal bleeding and discharge; patients with advanced disease may present with pelvic pain and bowel or urinary symptoms (6). although ascites has been reported in the cases with cervical cancer, it is due to other causes such as ovarian metastasis (7, 8). many studies were done to compare different treatments of locally advanced cervical cancer. finally, primary cisplatin-based chemoradiotherapy was recommended as the best current treatment of it (9, 10). recently, a new study has reported that neoadjuvant chemotherapy followed by radical hysterectomy (nac+rh) improves survival of patients with locally advanced cervical cancer in comparison to concurrent chemoradiotherapy (ccrt) (11). in this study, a case was reported who was consulted by gynecologic oncology service because of pelvic mass and ascites. our case was a menopausal woman who had diabetes mellitus and was a new case of cirrhosis. neoadjuvant chemotherapy was suggested for her because she had comorbidity and suffered from cirrhosis and it was presumed that she had ovarian cancer. during laparotomy after chemotherapy, our misdiagnosis was discovered because she had a pelvic mass in upper part of cervix that was fixed to bladder. therefore, radical hysterectomy was done and she received several courses of radiotherapy after surgery. our case was a 78-year-old diabetic woman who presented with ascites and abdominopelvic mass and was admitted into firoozgar hospital in tehran, iran on june 2010. she also complained of weight loss about 8 kg in the last 3 months, nausea, vomiting, anorexia, dysuria, and fever. she had diabetes mellitus since ten years ago and took metformin, glibenclamide, losartan, and atorvastatin. on physical examination, her abdomen was distended with fluid wave and there was a palpable 25 cm 20 cm mass in hypogartric part. during bimanual pelvic examination, a large, solid, irregular, fixed pelvic mass was found which occupied the whole pelvis. laboratory data revealed a normocytic anemia, pyuria, hematuria, mild hyponatremia, hypoalbu-minemia, increased prothrombin time, and elevated serum level of cancer antigen 125 (ca125). abdominal ultrasound (us) reported a small-sized liver (4 cm) with increased paranchymal echogenicity, bilateral hydronephrosis, huge amount of free fluid in abdominopelvic area, and a large pelvic mass with irregular border which us was not able to identify its origin. abdominopelvic computed tomography (ct) scan showed ascites and frozen pelvis with no evidence of metastasis and lymphadenopathy (figures 12). large mass was shown in pelvic ct scan cirrhosis was revealed by abdominal ct scan our first diagnosis was ovarian cancer; therefore, three cycles of neoadjuvant chemotherapy with taxol 175 mg/m and carboplatin 300 mg/m were performed. laparotomy was performed 3 weeks after the last chemotherapy cycle. during exploration of pelvis, the disease was at stage 4 and therefore radical hysterectomy was prepared for the patient and performed removing parameters and 1/3 of proximal vagina. she was in menopause and bilateral salpingo-oophorectomy was done and the tissue samples were sent for pathologic evaluations. the histopathology of removed cervix showed localized advanced squamous cell carcinoma, stage iv (figures 34). pathology: invasion to myometrium pathology: invasion to the bladder staging of ovarian cancer was surgicopathological but staging cervical cancer is clinical. there was a misdiagnosis about our case and after pelvic exploration during laparotomy, correct diagnosis was reached and radical hysterectomy was performed. the patient was discharged three weeks later with no complication. she has received twelve courses of epr (external pelvic radiotherapy) after surgery till now. our patient was successfully treated with nac+rh+epr (neo adjuvant chemotherapy+radical hysterectomy+external pelvic radiotherapy). she was followed up with serial clinical examination, pap smear, and abdominopelvic ct scan. in this case presentation, an old diabetic and newly-diagnosed cirrhotic woman was reported who presented with abdominopelvic mass, ascites, and elevated ca125. findings during laparotomy and pathologic evaluation of specimens confirmed locally advanced cervical cancer, stage iv in this patient. the most common symptoms are abnormal vaginal bleeding, postcoital bleeding, and vaginal discharge. in advanced stages of the disease, it presents with pelvic or lower back pain, and urinary and bowel involvement symptoms (6, 12). while abdulhathi et al. reported cervical cancer cases presented with ascites, they were due to ovarian metastasis (7, 8). no study could be found to report ascites due to cervical cancer alone or with concomitant cirrhosis. approximately, 5 percent of ascites cases have more than one cause, such as cirrhosis plus peritoneal carcinomatosis (13). in ultrasonography, the presence of solid non hyperechoic ovarian mass and ascites together could be suggestive of ovarian malignancy but metastases to the ovary should be considered as a differential diagnosis. metastatic disease accounts for 6 to 9 percent of ovarian malignancies and the uterus is also a common primary site for ovarian metastases (14, 15). therefore, if there is any doubt in diagnosis, image-guided biopsy of the peritoneum or omental cake may help to exclude a non ovarian malignancy (16). in literature review, zhan et al., jee et al., and also kuriki et al. showed that dm increased the risks for developing cervical cancer but there is no evidence to offer, such an association with ovarian cancer (35). similarly, immunosuppression is one of the risk factors of cervical cancer that can be caused by cirrhosis and dm in this case. human papillomavirus (hpv) infection is the most common causal agent of cervical cancer and can be detected in 99.7 percent of cervical cancers (17, 18). it is also increased in adenocarcinoma of the cervix, liver disease and cirrhosis, ascites, diabetes, and in approximately 1 percent of healthy women (19). if the patient was diagnosed correctly with cervical cancer stage iv, she needed to be treated by ccrt rather than nac+rh, especially in this case (9, 10). she presented with ascites and had comorbid disease and accordingly was not a good candidate for initial surgery (20). although yin has recently reported that nac+rh improve the long-term disease-free survival (dfs) and overall survival (os) of patients with locally advanced cervical cancer compared with ccrt, the patients in his sample were all in stage ib2-iib and this case was in stage iv (11). in conclusion, the combination of abdominopelvic mass, ascites, elevated ca125, and negative human papillomavirus infection in conjunction with lack of attention to epidemiologic hints, dm history, bilateral hydronephrosis, and newly diagnosed cirrhosis misled us to diagnosis of ovarian cancer in this patient. it seems that considering all signs and symptoms to reach a verdict would reduce such malpractices and consequently lead to selecting the best management and treatment. it seems necessary to refine our differential diagnosis using complementary workup and confirm our final diagnosis before choosing the appropriate treatment for each patient.

background: cervical cancer is the second most common malignancy in women worldwide. vaginal bleeding and vaginal discharge are the most common symptoms. although ascites has been reported in cases with cervical cancer, it is due to other causes such as ovarian metastasis. case presentation: a 78-year-old diabetic woman who presented with ascites and abdominopelvic mass was misdiagnosed with ovarian cancer and treated with neoadjuvant chemotherapy followed by radical hysterectomy and adjuvant radiotherapy. however, pathology confirmed locally advanced cervical cancer stage iv in this patient. she was discharged from the hospital three weeks after surgery with no serious complications.discussion:considering all signs and symptoms to reach a verdict would reduce such malpractices and consequently lead to select the best management and treatment.

PMC4508358

pubmed-742

multiple surveillance systems are used in the united states each year to characterize seasonal influenza epidemics and to detect unusual events such as infections with novel viruses or those with pandemic potential. these systems track a variety of outcomes, including laboratoryconfirmed influenza hospitalizations, outpatient visits for influenzalike illness (ili), pneumonia and influenzacoded deaths for all ages, pediatric laboratoryconfirmed deaths, and positive laboratory samples. despite the utility of these existing systems, additional data to estimate disease severity and track illness at the state level were needed during the 2009 h1n1 pandemic, as timely and representative information describing 2009 pandemic influenza a (h1n1) (ph1n1) activity the centers for disease control and prevention (cdc) and the council of state and territorial epidemiologists (cste) established the aggregate hospitalization and death reporting activity (ahdra) as part of an overall national surveillance strategy implemented to collect timely and representative data describing ph1n1 infections in the united states. the ahdra was designed to (i) track severe disease within states and territories to better characterize the focal nature of the pandemic, (ii) track disease trends over brief periods of time to facilitate rapid public health responses to changes in ph1n1 epidemiology, and (iii) accommodate variation in local resources by providing a simple, flexible method that allowed reliable reporting by all states and territories without overwhelming health departments during the pandemic response. in this report, we describe the methods and implementation of ahdra and provide preliminary results from this new surveillance activity. from august 30, 2009, through april 6, 2010, cdc requested weekly reporting of influenzaassociated hospitalizations and deaths from all 50 us states, the district of columbia, new york city, and six us territories. states and territories were asked to identify hospitalizations and deaths in their jurisdictions according to either a laboratoryconfirmed or syndromic surveillance definition and could use either definition to report hospitalizations or deaths. polymerase chain reaction (rtpcr) testing, direct fluorescent antigen testing (dfa), immunofluorescent antigen testing, or viral culture; identification of influenza type or subtype was not required. syndromic reports included cases of pneumonia and influenza based on clinical syndrome, admission or discharge data, or a combination of data elements that could include diagnostic laboratory test results. prior to the first reporting period, 33 jurisdictions indicated they intended to submit laboratoryconfirmed hospitalizations, and 20 indicated they would submit syndromic hospitalizations. thirtysix jurisdictions intended to submit laboratoryconfirmed death reports, and 17 indicated they would submit syndromic deaths; information describing method of reporting was unavailable for one state and four territories. jurisdictions were instructed to submit aggregate counts each week by age group (04, 518, 1924, 2549, 5064, and 65 years). aggregate counts were used to calculate agespecific weekly and cumulative rates per 100 000 according to 2008 postcensal us population estimates. laboratoryconfirmed and syndromic data were analyzed for relative increase or decrease by state each week, and laboratoryconfirmed cumulative rates were used to describe the age distribution of ph1n1 influenzaassociated hospitalizations and deaths. owing to differences between laboratoryconfirmed and syndromic reporting definitions, we calculated two national incidence estimates of ph1n1 influenzaassociated hospitalizations and deaths: one extrapolating reports from laboratoryconfirmed jurisdictions to the entire country and one extrapolating reports from syndromic jurisdictions to the entire country. calculation of rates involving laboratoryconfirmed influenzaassociated hospitalizations and deaths used the populations of states reporting laboratoryconfirmed cases as a denominator; calculations involving syndromic influenzaassociated hospitalizations and deaths used the populations of states reporting syndromic cases as a denominator. laboratoryconfirmed reports from ahdra were used to estimate weekly, age groupspecific national influenzaassociated deathtohospitalization ratios. these ratios were incorporated into a model used to estimate the national illness burden of influenzaassociated cases, hospitalizations and deaths during the pandemic, accounting for variation in medical careseeking, laboratory practice and detection capability, and underreporting of confirmed cases. all data were maintained in a database on a secure server at cdc, and all analyses were performed using microsoft excel and sas v 9.1 (sas institute, cary, nc, usa). this activity was determined by cdc to be part of routine public health practice and was not subject to institutional review board approval for human research protections. the median number of jurisdictions reporting laboratoryconfirmed hospitalizations each week was 36 (range 2938), and the median number of jurisdictions reporting syndromic hospitalizations each week was 18 (range 1219). the median number of jurisdictions reporting laboratoryconfirmed deaths each week was 39 (range 3040), and the median number of jurisdictions reporting syndromic deaths each week was 14 (range 816). with the exception of 12 weeks at the beginning and end of the surveillance period, only two jurisdictions changed their surveillance definition during the reporting period (one from laboratory confirmed to syndromic and one from syndromic to laboratory confirmed), and only two jurisdictions failed to report for more than 1 week during the reporting period. number of jurisdictions reporting to the aggregate hospitalization and death reporting activity by surveillance definition and by week august 30, 2009 to april 6, 2010. in 27 of 36 jurisdictions reporting laboratoryconfirmed hospitalizations for which information was available, the median proportion of hospitals under surveillance was 100% of all hospitals within the jurisdiction (range 18100%). for 16 of 18 jurisdictions using a syndromic hospitalization definition, the median proportion of hospitals under surveillance was 45% of all hospitals within the jurisdiction (range 9100%). information regarding the type of diagnostic test used to identify cases was available for 24 jurisdictions reporting laboratoryconfirmed hospitalizations and 22 jurisdictions reporting laboratoryconfirmed deaths from september 8 to october 6, 2009. sixteen of 24 (67%) jurisdictions employed rtpcr, viral culture, or dfa testing to identify the majority of reported hospitalizations (at least 75% of reported cases in each jurisdiction), and 18 of 22 (82%) jurisdictions used one of these methods to identify the majority of reported deaths. laboratoryconfirmed cases not identified by rtpcr, dfa, or viral culture were identified using rapid antigen testing or an unspecified diagnostic test. a total of 41 689 laboratoryconfirmed hospitalizations and 2096 laboratoryconfirmed deaths were reported from august 30, 2009, through april 6, 2010. weekly laboratoryconfirmed hospitalizations peaked at>5000 during the last week of october 2009 and declined from that date to<200 by the end of march 2010 (figure 2). weekly laboratoryconfirmed deaths peaked at nearly 200 during the same week as the laboratoryconfirmed hospitalization peak, before declining to<20 per week by the end of march 2010 (figure 2). the highest laboratoryconfirmed hospitalization rate was observed in the 0 to 4yearold age group, which had a rate 2 to 3fold higher than those observed in the other age groups (figure 3). the majority of laboratoryconfirmed hospitalizations (> 70%) reported to ahdra were in patients<50 years of age, and fewer than 10% were in patients 65 years of age or older. the ahdra weekly laboratoryconfirmed death rate peaked in october 2009 at 0078 and fell to<0001 per 100 000 persons by march 2010. the highest laboratoryconfirmed death rate was seen in the 5064 year old age group, and 69% of laboratoryconfirmed deaths occurred in patients between 25 and 64 years of age (figure 3). weekly laboratoryconfirmed and syndromic ph1n1 hospitalizations and deaths reported to the aggregate hospitalization and death reporting activity august 30, 2009 to april 6, 2010. estimated rates per 100 000 persons of laboratoryconfirmed and syndromic ph1n1 hospitalizations and deaths reported to the aggregate hospitalization and death reporting activity, by age group, august 30, 2009 to april 6, 2010. a total of 134 441 syndromic hospitalizations and 13 983 syndromic deaths were reported to ahdra. weekly syndromic hospitalizations peaked at nearly 7000 during the last week of october 2009 and were distributed in a pattern similar to the weekly laboratoryconfirmed hospitalization curve. weekly syndromic deaths peaked at 605 approximately 1 month later but did not show a pattern resembling the weekly laboratoryconfirmed death curve (figure 2). the highest rates of syndromic hospitalizations were reported in patients 65 years of age (399 per 100 000), and in patients 04 years of age (255 per 100 000). greater than 80% of all syndromic deaths reported were in patients 65 years of age, and fewer than 2% were in patients<25 years of age (figure 3). extrapolating ahdra reports to the entire country yielded cumulative counts of hospitalizations and deaths that estimate what may have been observed had all jurisdictions reported using either a laboratoryconfirmed or syndromic surveillance definition (table 1). observed and extrapolated*estimates of ph1n1associated hospitalizations and deaths in the united states reported to the aggregate hospitalization and death reporting activity from august 30, 2009 to april 6, 2010*extrapolated counts were calculated using the direct method of standardization and represent the number of hospitalizations and deaths that would have occurred in the united states if all states had used either a laboratoryconfirmed or a syndromic surveillance definition. laboratoryconfirmed hospitalization and death rates were calculated by dividing the number of cases by the sum of the state populations for states using a laboratoryconfirmed definition (207 654 216 for hospitalizations; 245 351 708 for deaths). syndromic hospitalization and death rates were calculated by dividing the number of cases by the sum of the state populations for states using a syndromic definition (96 405 508 for hospitalizations; 58 708 016 for deaths). both laboratoryconfirmed and syndromic hospitalization and death rates were then applied to the standard population (u.s. census, july 2008; 304 059 724) to derive extrapolated counts. although the weekly laboratoryconfirmed deathtohospitalization ratio demonstrated considerable variability especially during the latter part of the surveillance period (3081376%), the cumulative ratio quickly stabilized near its mean of 502% in october 2009 and remained within 1% of this value throughout the remainder of the surveillance period (figure 4). the cumulative age groupspecific laboratoryconfirmed deathtohospitalization ratio was substantially lower for 0 to 18yearolds compared to older age groups and the overall ratio for all age groups (figure 4). weekly deathtohospitalization ratio from laboratoryconfirmed reports submitted to the aggregate hospitalization and death reporting activity august 30, 2009 to april 3, 2010. laboratoryconfirmed data collected by ahdra helped characterize the epidemiology of ph1n1associated influenza hospitalizations and deaths in the united states, revealing a time course and illness distribution for ph1n1 that were substantially different from those seen in seasonal influenza epidemics. aggregate hospitalization and death reporting activity laboratoryconfirmed peak hospitalizations and deaths occurred much earlier than the typical peak for seasonal influenza activity, which most often occurs during january or february each year., furthermore, the age distribution of laboratoryconfirmed hospitalizations reported to ahdra was markedly different from typical influenza seasons when hospitalizations are more common among persons over 65 years of age.,, other recent studies corroborate this finding, showing that nearly half of all patients in the united states hospitalized with ph1n1 influenza infections were under the age of 25 years, and<10% were over the age of 65., overall, the age distribution of laboratoryconfirmed death rates determined from ahdra data was also markedly different from that seen in typical influenza seasons. in contrast to typical influenza seasons, when 90% of deaths occur in the elderly,, 86% of laboratoryconfirmed deaths reported to ahdra were in persons<65 years of age, with the highest rate found in persons aged 5064 years. laboratoryconfirmed ahdra data were also useful in monitoring trends in the distribution of illness and age groups over time in specific jurisdictions. aggregate hospitalization and death reporting activity laboratoryconfirmed data helped define the beginning and end of the 20092010 influenza season and accurately depicted the second wave of ph1n1 illness seen in the fall of 2009; similar doublewave patterns have been seen in previous pandemics.,, ahdra was also instrumental in the detection of and response to a minor third wave of ph1n1 activity in the southeast united states in early 2010. ahdra reporting by state and local health departments allowed tracking of trends in severe disease with greater geographic representativeness than would have been possible with existing systems alone and informed decisionmaking at the state and national levels. for example, although the emerging infections program (eip) has conducted populationbased surveillance for laboratoryconfirmed influenzaassociated hospitalizations in the united states since 1995, the eip network of hospitals conducts surveillance in certain counties in only 16 states. laboratoryconfirmed surveillance via ahdra during the pandemic was implemented in more than twice the number of states as in the eip network. aggregate hospitalization and death reporting activity laboratoryconfirmed hospitalization and death surveillance was also consistent with data from existing influenza surveillance systems. emerging infections program hospitalizations peaked at approximately the same time in october 2009 as did ahdra laboratoryconfirmed reports, and the age distribution of ph1n1 hospitalizations described by the two systems was similar, with each identifying the highest rate in the 0 to 4yearold age group and a similar distribution of rates in other age groups. aggregate hospitalization and death reporting activity data also accurately reflected outpatient influenza illness during the pandemic. outpatient influenzalike illness surveillance network (ilinet) collects data from over 3000 healthcare providers each week on the proportion of patient visits for ili.*the ilinet weekly percentage peak for the 20092010 season (77% of all patient visits) occurred 1 week prior to the ahdra laboratoryconfirmed and syndromic hospitalization peaks in october 2009. this approximate 1week lag between ili onset and severe outcome (hospitalization or death) has been noted consistently during both annual influenza seasons and during pandemics. despite its usefulness during the pandemic, ahdra first, jurisdictions were permitted to report according to different surveillance criteria (e.g., use of a laboratoryconfirmed or syndromic case definition, multiple diagnostic testing methods) and results therefore did not measure identical outcomes. this disparity is evident in the age distributions of laboratoryconfirmed and syndromic hospitalization rates although both reporting methods show similar peaks in the hospitalization rate for the youngest age group, laboratoryconfirmed rates thereafter generally decline with increasing age, while syndromic rates initially decline but then show a dramatic increase for the 65yearold age group. presumably, the difference is largely because of the relatively low specificity of a syndromic compared to a laboratoryconfirmed definition of influenza infection. syndromic reporting likely captures many hospitalizations associated with noninfluenza respiratory illness, which often occur with greater frequency in young children and the elderly,, ,, while laboratoryconfirmed reporting is much more likely to identify only cases of influenza illness. thus, it is inappropriate to make comparisons between reporting jurisdictions in ahdra without adjusting for differences in reporting methods and practices. instead, ahdra s best use may have been to track the progression of the epidemic within each state, a goal consistent with the original intent of the system. a more important consequence of the decision to allow two surveillance definitions in ahdra is the inherent limitation of using a syndromic definition to conduct national surveillance for influenzaassociated infections. although ahdra syndromic data were useful to track trends of disease within those jurisdictions submitting syndromic reports, they may not otherwise accurately reflect the burden or severity of influenzaassociated hospitalizations and deaths in the united states. because the proportion of syndromic respiratory hospitalizations and deaths attributable to influenza is small, it is unclear whether syndromic findings in ahdra represent influenza infections or hospitalizations and deaths caused by other respiratory illnesses. interpretation of syndromic data was further complicated by the limited number of hospitals included in syndromic reports (a median of 45% of all hospitals within each reporting jurisdiction). a further limitation of ahdra is that the system does not collect several potentially useful data elements, such as population denominator information and influenza type or subtype (although>99% of circulating influenza viruses during the surveillance period were ph1n1). also, the additional effort required of reporting jurisdictions to conduct ahdra surveillance may be high and may exhaust state and local health department resources during a pandemic. finally, extrapolated burden estimates derived from ahdra data do not account for variations in medical careseeking, laboratory practice and detection capability, degree of underreporting of confirmed cases, and other population differences across jurisdictions. however, ahdra laboratoryconfirmed data were important components of modelbased estimates that do account for these sources of underestimation. understanding the impact of ph1n1 influenza hospitalizations and deaths was important to guide the pandemic response and will be important to inform preparedness and response plans for future public health crises. the ahdra was an important component of us influenza surveillance efforts during the pandemic and provided a level of geographic representativeness and timeliness for reporting of severe influenzaassociated outcomes that was not available from existing national surveillance systems. laboratoryconfirmed reporting in ahdra supplied valuable information to public health practitioners during the pandemic and should inform refinements to seasonal surveillance activities in the coming seasons, as well as revisions of pandemic surveillance plans. although useful in monitoring trends within jurisdictions, ahdra syndromic reports, as a measure of influenzaassociated hospitalizations and deaths, were difficult to interpret. these data were complicated by limited representativeness and a low specificity for detecting influenzaattributable hospitalizations and deaths among events associated with respiratory illness. using only syndromic surveillance data to monitor epidemic or pandemic influenza is thus not recommended, particularly in a setting like the 2009 h1n1 pandemic when elderly persons were largely immune because of prior exposures to antigenically related influenza viruses. because ahdra was implemented within a few weeks time, the system may prove particularly useful as a prototype for a pandemic or epidemic respiratory infection surveillance system that needs to be implemented quickly and efficiently on a national scale. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc.

please cite this paper as: jhung et al. (2011) preliminary results of 2009 pandemic influenza surveillance in the united states using the aggregate hospitalization and death reporting activity. influenza and other respiratory viruses 5(5), 321327. background to augment established influenza surveillance systems in the united states, the centers for disease control and prevention and the council of state and territorial epidemiologists implemented the aggregate hospitalization and death reporting activity (ahdra) in august 2009. the ahdra was designed to meet increased demands for timely and detailed information describing illness severity during the 2009 h1n1 influenza a (ph1n1) pandemic response. objectives we describe the implementation of ahdra and provide preliminary results from this new surveillance activity. methods all 50 us states were asked to report influenzaassociated hospitalizations and deaths to ahdra each week using either a laboratoryconfirmed or syndromic surveillance definition. aggregate counts were used to calculate agespecific weekly and cumulative rates per 100 000, and laboratoryconfirmed reports were used to estimate the age distribution of ph1n1 influenzaassociated hospitalizations and deaths. results from august 30, 2009, through april 6, 2010, ahdra identified 41 689 laboratoryconfirmed influenzaassociated hospitalizations and 2096 laboratoryconfirmed influenzaassociated deaths. aggregate hospitalization and death reporting activity rates peaked earlier than hospitalization and death rates seen in previous influenza seasons with other surveillance systems, and the age distribution of cases revealed a tendency for hospitalizations and deaths to occur in persons<65 years for age. conclusions aggregate hospitalization and death reporting activity laboratoryconfirmed reports provided important information during the 2009 pandemic response. aggregate hospitalization and death reporting activity syndromic reports were marked by low representativeness and specificity and were therefore less useful. the ahdra was implemented quickly and may be a useful surveillance system to monitor severe illness during future influenza pandemics.

PMC4942043

pubmed-743

the baylisascaris genus of nematodes (family: ascarididae) is comprised of nine recognized species, each parasitizing distinct definitive hosts and a vast array of intermediate hosts (bauer, 2013, kazacos, 2001, sorvillo et al., 2002). the most widely studied of these is b. procyonis, which primarily utilizes the common raccoon (procyon lotor) as its principal definitive host (kazacos, 2001). embryonated eggs of baylisascaris species are infectious to over 90 species of mammals and birds including humans. larval infections with baylisascaris species can lead to irreversible neural, optical and visceral damage, in both wild animals as well as humans (gavin et al. the severity of pathology of baylisascaris infection in the intermediate host is known to vary depending on the species of the infecting parasite (tiner, 1953). understanding which species of baylisascaris a host is infected with may provide valuable information concerning prognosis and treatment, as well as the source of the infection. as with other nematodes, baylisascaris species have been traditionally identified through morphometric data. however, distinguishing larval nematodes to the species level is problematic (graeff-teixeira et al., 2016). with the current widespread availability of molecular tools, the use of genetic analysis to rapidly and accurately identify organisms to the species level is increasingly standard. however, differentiation of closely related species such as b. procyonis and b. columnaris has proven difficult (dangoudoubiyam et al., 2009, gatcombe et al., 2010). extensive previous research has documented the life-cycle, distribution and prevalence of b. procyonis (graeff-teixeira et al., 2016, additionally, the complete mt genome of b. procyonis, has been sequenced (xie et al. in contrast to the relative abundance of studies on b. procyonis, little is known of its closest relative, b. columnaris. previous research using copromicroscopic detection has shown b. columnaris infection prevalence of 25% in a population of eastern spotted skunks (spilogale putorius) (lesmeister et al., 2008) and 25% prevalence in captive striped skunks in europe (d'ovidio et al., 2017). to our knowledge, the prevalence of b. columnaris in wild populations of striped skunks has not previously been reported. (2013) reported the cloning and sequencing of partial sequences of b. columnaris for the cox1 (413 bp) and cox2 (483 bp) genes. additionally, in this work franssen et al. identified three single nucleotide polymorphisms (snps) in the partial cox1 sequence and one snp found in the partial cox2 sequence which are useful in differentiating b. procyonis from b. columnaris. the partial sequence of these mitochondrial genes has provided a reference which has facilitated the differentiation of b. procyonis from b. columnaris by dna sequencing. in addition to the four snps which allow for the molecular differentiation of b. procyonis and b. columnaris, franssen et al. also reported a number of intragenic differences in b. columnaris mitochondrial sequences suggesting there is a high degree of genetic diversity in this species. in this study we sought to determine the prevalence of b. columnaris in a wild population of striped skunks (mephitis mephitis) in salt lake county, utah, usa. in addition to determining the prevalence of these parasites in a native skunk population we report the complete sequence of 11 mitochondrial genes (cox1, cox2, nd2 and 8 trna genes) comprising 3638 bp of the b. columnaris mitochondrial genome. results revealed several novel snps in these genes, which further facilitates and improves the molecular distinction between b. procyonis and b. columnaris. additionally, several intragenic snps were identified in b. columnaris worms. in summary, these results demonstrate the prevalence of b. columnaris in a wild population of skunks and extend the number of known genetic differences between b. procyonis and b. columnaris. skunks were acquired through nuisance animal calls from the public in salt lake county, utah, usa during the fall and winter of 2013. institutional animal care and use committee (iacuc) or ethics committee approval was not necessary, as animals were not sacrificed for research purposes. skunks were captured in live traps (tru-catch, belle fourche, south dakota, usa). skunks were euthanized by chemical immobilization with 5/1 ketamine/xylazine followed by intracardiac injection of potassium chloride. the presence of nematode parasites was determined by emptying the small intestinal contents through manual extrusion using finger/grip pressure and the visual examination of intestinal contents. species determination of collected worms was performed by extracting dna from one or more worms from each skunk and performing dna sequencing of the cox1 gene. sequences were aligned with the previously published cox1 to determine worm genus and species (genbank: kc543474.1) (https://blast.ncbi.nlm.nih.gov). a total of 34 worms were collected and the cox1 gene of 22 of these worms was sequenced. a single worm from several animals was then used for more extensive dna sequencing of a number of genes as described below. pcr primers were designed based on the mitochondrial genome of b. procyonis (ac. no. primers were designed to flank the gene of interest by 100300 bp from the 5 and 3 ends. this primer design resulted in the amplification of segments of dna which contained the complete gene of interest (cox1, cox2, nd2) as well as several small trna genes contained at the 5 and 3 ends of each amplicon (table 1). three separate amplicons were generated with amplicon 1 containing the sequences for the trna genes q, r, i, s, and l, as well as the complete nd2 gene. amplicon 2 contained the complete cox1 gene and amplicon 3 contained the trna genes d, g, and h as well as the complete cox2 gene. primer sequences for each amplicon are contained in table 1. upon collection of parasites (described above), individual specimens were preserved by immediately freezing each worm at 80 c. for genetic analysis, specimens were thawed and a 2.0 mm portion of each worm macerated with a razor blade. the macerated tissue samples were then placed in a 1.5 ml microcentrifuge tube and dna extracted using the qiagen dneasy blood&tissue kit (qiagen inc., pcr reactions were carried out in a final reaction volume of 50 l containing 2 l of dna, 25 l of onetaq dna polymerase (new england biolabs, ipswich, ma usa), 0.5 l of each primer with 22 l of molecular grade water. polymerase chain reaction conditions used to amplify the cox1 and cox2 containing amplicons (amplicons 2 and 3), involved a 30 s initial denaturation at 94 c followed by 30 cycles of denaturation at 94 c for 30 s, annealing at 55 c for 30 s, and elongation at 68 c for 80 s, followed by a 10-min final extension at 68 c. the pcr amplification of the nd2 target region (amplicon 1) was identical to the aforementioned profile, with the exception of the annealing temperature being 63 c. the pcr amplified products of interest for each amplicon were then extracted and gel purified using the wizard sv gel and pcr clean-up system (promega madison, wi). the sequencing of each amplicon was performed using primers designed to bind approximately every 500 bp (supplemental fig. 1). cycle sequencing was performed by the brigham young university dna sequencing center using an abi 3730xl automated sequencer. primers were designed using the forward dna strand and sequencing was performed on a single strand. contigs for each sample were assembled by mapping sample reads to the mitochondrial genome of b. procyonis (ac. sequence reads were determined using geneious software (biomatters limited, san francisco, ca, usa). snp analysis, multiple alignments, and prediction of transmembrane and cytoplasmic regions were performed using geneious software. we first sought to determine the prevalence of b. columnaris in a wild population of skunks. the intestinal tracts of 16 skunks were collected and the prevalence of nematode infection determined by gross examination of the intestinal contents. ten of the 16 skunks examined were found to be infected with roundworms, with numbers of worms ranging from 1 to 10 in infected animals (table 2). all worms were identified as b. columnaris based on gross morphology and confirmed by detailed sequence homology to the published partial sequence of b. columnaris cox1 (genbank: kc543474.1). due to the essential function of cox1, this gene is present in a wide variety of organisms yet has sufficient variation to distinguish closely related species (hebert et al., 2003). consequently, the sequence of the cox1 gene is a frequently used marker for population genetic and phylogenetic studies (ai et al., 2011, xie et al., 2011b). in this study, the complete cox1 gene (1578 bp) of eight baylisascaris worms isolated from eight different hosts our analysis revealed 11 novel loci, which consistently distinguished b. columnaris from b. procyonis, including the three previously reported by franssen et al. the majority of these species specific snps are found in the 3 portion of the gene with 8 of the 11 being found between nucleotides 1002 and 1506 of the cox1 gene. previously published work has shown a relatively high degree of intraspecies heterogeneity in the cox1 gene of b. columnaris (franssen et al., 2013). in our analysis of cox1, 11 total intraspecies snps were identified 10 of these having not previously been reported (fig. 1). previous sequencing of a partial sequence of the b. columnaris cox2 gene has shown one specific snp which was useful in differentiating b. procyonis from b. columnaris as well as three intragenic snps (franssen et al., 2013). these previous studies were done in a european population of skunks. in our sequencing analysis of a north american population, we found six intragenic snps within the cox2 gene. importantly, our data showed that none of these nucleotide variations were species specific, whereas previous analysis had suggested the snp at position 168 was useful in differentiating b. columnaris from b. procyonis (fig. 2). there have been no previous reports of sequencing of the b. columnaris nd2 gene. in our sequencing of this gene and subsequent analysis, in addition, two intraspecies snp were identified in the nd2 gene of b. columnaris (fig. alignment of eight of the b. columnaris concatenated sequences and homologous regions of b. procyonis were used to determine the sequence of several trna genes from b. columnaris. this analysis revealed two snps and an indel within mt-trna genes which differentiated b. procyonis from b. columnaris. in addition, five intragenic snps were identified (one of them being in the same position as the indel of trna s (fig. 4)). based on the high degree of similarity between these organisms, we were initially skeptical of this degree of variation in trna genes. therefore, we next compared the trna genes from all mt-dna genomes of sequenced baylisascaris species. this comparison demonstrated a relatively high degree of sequence variation in the trna genes of several closely related baylisascaris species (fig. this level of heterogeneity in trna sequences from numerous baylisascaris species lends confidence in this unanticipated level of heterogeneity if trna genes. additionally, concatenated sequences were used to generate maximum likelihood relationships of b. columnaris with other ascarid species. results of these analyses demonstrated maximum likelihood relationships in agreement with previous findings (franssen et al., 2013). both skunks as well as raccoons commonly live in urban areas facilitating human contact and potential ingestion of embryonated eggs which can cause visceral, ocular and neural larval migrans (roussere et al., 2003, the molecular identification of b. columnaris was not possible due to the lack of any dna sequences in public databases. in 2009, a partial (529 bp) sequence of the b. columnaris cox2 gene was generated (dangoudoubiyam et al., 2009). in 2013 work by franssen et al. resulted in partial sequences for b. columnaris cox1 (413 bp) and cox2 (483 bp) genes (franssen et al., the availability of these sequences has facilitated the molecular identification of these parasites for us as well as other researchers (d'ovidio et al., 2017). in this study, we utilized gene sequencing to identify roundworms as b. columnaris. in this population of wild skunks we then extended earlier findings by sequencing a total of 3638 bp of the mitochondrial genome of b. columnaris. in so doing several novel snps were identified, facilitating the molecular discrimination of b. columnaris from b. procyonis. these snps provide additional species specific targets for the molecular differentiation of b. columnaris from b. procyonis. importantly this data demonstrate that three previously reported species specific snps do not accurately differentiate b. procyonis from b. columnaris (168 of cox2 and 804 and 834 of cox1). all of these snps involved transitions between the purines g and a. this unique finding, compared to previously published work, is likely due to the previous study being done in the netherlands, while skunks (and by extension parasites of skunks) are native to the americas. parasites infecting european skunks are likely from a relatively small founder population of nematodes infecting skunks transported outside of north america. it is logical to assume that the nematodes (as well as the skunks) would have less genetic diversity than a native, free roaming population of animals. a recent study on the prevalence of b. columnaris in captive european skunks found that 25% of skunks tested were infected with b. columnaris (d'ovidio et al. data indicates that in our study area there is a much higher infection rate of striped skunks than reported in studies of captive striped skunks in europe. this is likely due to wild animals encountering other wild skunks as well as infected intermediate hosts more commonly than captive skunks. as expected, more intragenic variation of parasites was observed from this population of native wild skunks compared to the imported non-native population studied previously (franssen et al., 2013). the higher prevalence of parasites in our study compared to a previous study of spotted skunks (lesmeister et al., 2008) is likely due to the difficulty in accurately identifying infected animals through fecal analysis compared to our method of visual inspection of the small intestine. additionally, several of the animals in our study had very few worms infecting them. animals infected with a single male worm would not be detected by microscopic analysis of feces but would easily be identified through visual inspection on the intestinal contents. differences in the susceptibility to baylisascaris infection by these two types of skunks, as well as other environmental factors may also play a role in variations in infection prevalence. in summary, in this study several novel intragenic snps were identified. additionally, nine novel polymorphisms are identified which aid in the molecular differentiation of b. procyonis from b. columnaris. three polymorphisms, which were previously thought to differentiate these two species were shown to in fact be intragenic rather than species specific snps.

members of the genus baylisascaris utilize omnivores or carnivores as their definitive hosts. the best known member of this genus is baylisascaris procyonis, which is an intestinal parasite of raccoons. the closest relative of b. procyonis is b. columnaris, which utilizes the common skunk as its definitive host. although b. procyonis has been extensively studied, relatively little is known of b. columnaris. for example, the mitochondrial genome of b. procyonis has been sequenced in its entirety. conversely, the mitochondrial genome of b. columnaris remains largely unexplored. likewise, the prevalence of this parasite in its wild host has not been documented. in this study, we collected parasites from a wild population of skunks in the state of utah, united states. the cytochrome c oxidase subunit 1 and 2 genes, nadh dehydrogenase 2 and several trna genes were sequenced from the mitochondrial genome of these parasites. we also determined the prevalence of b. columnaris in a wild population of skunks. in this work we identify several novel polymorphic genetic loci between b. procyonis and b. columnaris. these findings provide additional molecular targets for the differentiation of baylisascaris species through clarification of genetic differences between b. columnaris and b. procyonis.

PMC5403792

pubmed-744

the epithelial ovarian carcinoma is one of the most fatal gynecological cancers across the globe. in spite of early recovery by surgical and chemotherapy treatments, the database globcan related to the world health organization (who) has reported incidence of about 192000 cases in the world, in the year of 2000. 6000 cases of the mentioned cases have occurred in the uk, and 21000 cases in the u.s. for treating the disease, the tumor will be removed by surgical procedures and then chemotherapy would be started with platinum-based chemotherapy (cisplatin and carboplatin), which treating regime includes cisplatin and carboplatin with the drugs such as paclitaxel, docetaxel, cyclophosphamide, and doxorubicin. in some of the patients, the disease relapses after 6 months of chemotherapy; this condition is defined as platinum resistant, in which treatment would be continued with drugs such as topotecan and etoposide. etoposide, as other chemotherapy agents, has many side effects such as bone marrow suppression, granulocytopenia, thrombocytopenia, mucositis, moderate to severe esophagitis, hepatotoxicity, metabolic acidosis, and anemia. the complications of anticancer drugs have caused scientists to try two approaches to solve the problem: developing new drugs with fewer side effects and application of new drug delivery systems with high specificity to cancerous tissues; the second approach has lower costs and more attention nowadays. solid lipid nanoparticles (slns) are one of the most important nanosized drug delivery systems that were introduced about two decades ago. slns that are often considered for intravenous application are colloidal submicron carriers sized 50 to 1000 nm and composed of solid lipids dispersed in water or surfactant aqueous solution. these nanoparticles have particular features like small size, high surface area, and high loading of drug that makes them potent and beneficial carriers for improving drug efficacy [5, 6]. slns are similar to o/w emulsions used for total parenteral nutrition; the difference is that emulsion liquid lipid has been replaced with a solid lipid. slns have advantages such as controlled drug release in considered site, excellent biocompatibility, increase in drug stability, high drug content, easy industrialization and sterilization, better control of drug release kinetics, high bioavailability for bioactive drugs, chemical protection of sensitive drugs, easier producing rather than biopolymeric nanoparticles, producible by common emulsification methods, long-time stability, and various applications [4, 7, 8]. for parenteral administration, slns with appropriately small particle size less than 200 nm can be sterilized using filtration. autoclaving the finished dispersion is not practical as the lipids melt at sterilizing temperatures and the molten lipid droplets coalesce. therefore just aseptic manufacturing processes following sterilization of the starting materials by gamma irradiation of the final dispersion or exposure to ethylene oxide (eo) gas are applicable for their sterilization. bacterial endotoxins in raw materials need to be monitored, especially when raw materials are of natural origin. it may be possible to lyophilize the sln dispersions, and this lyophile can be irradiated or exposed to eo. slns are used in transdermal applications, as gene vector carriers, for topical uses, as cosmeceuticals, as targeted carriers of anticancer drugs to solid tumors, in breast cancer and lymph node metastases and in antitubercular chemotherapy. for example, the study of yadav et al. was performed in the survey of poly(lactic-coglycolic acid)-monomethoxy-poly(polyethylene glycol) and poly(lactic-coglycolic acid)-pluronic block copolymers and the study of reddy et al. on nanoparticles produced by tripalmitin could be mentioned. hyaluronan (figure 1), available in the market as sodium hyaluronate (ha), is a high molecular weight glycosaminoglycan present in extracellular matrix and is necessary for cellular growth and structural stability of organs and tissue structure. ha regulates cell proliferation and movements by interacting with cd44 receptors and receptor for ha mediated motility (rhamm). because of overexpression of cd44 receptors by cancer cells, interfering in cd44-ha interaction by targeting drugs at cd44 is an effective strategy to treat cancers. ha bound to nanoparticles, in addition to its targeting role, may act as a protecting agent of nanoparticles against body phagocytosis system [1113]. the mentioned method has been used to deliver agents such as doxorubicin, epirubicin, paclitaxel, mitomycin c, sirna, and dna. to our knowledge there is not any report on the application of the hyaluronate targeted slns in drug delivery of etoposide in sk-ov-3 cells although there are some studies on the hyaluronate targeted slns. this study alongside with thousands of similar ones could help to introduce new clinically applicable drug delivery systems with appropriate physicochemical properties, successful targeting, and enhanced cytotoxicity in the future. this study was performed in order to evaluate cytotoxicity of ha targeted slns containing etoposide, prepared and optimized in our previous study in sk-ov-3 cells. stearylamine (sa), dodecylamine (dda), cetyl alcohol, dialysis bags with molecular weight cut-off of 12400 da, and thiazolyl blue tetrazolium bromide (mtt) were from sigma-aldrich company (us). acetone, dichloromethane, and tween 80 were from merck chemical company (germany). rpmi 1640 culture medium, penicillin-streptomycin, and fetal bovine serum were from paa company, austria. sodium hyaluronate (mw=6,400 da) was from lifecore biomedical (us) and sk-ov-3 cells were from pasteur institute (iran). slns were produced by emulsification-solvent evaporation method. according to the results of our previous study, the lipid phase including 30 mg etoposide, 30 mg cetyl alcohol, and 30 mg sa was dissolved in 1.8 ml of 1: 1 mixture of acetone-dichloromethane. then the mentioned solution was added during 3 minutes to the 18 ml of tween 80 solution (1% w/v) in deionized water, while stirring in 1200 rpm. ultimately, produced nanoemulsion was stirred in 600 rpm in room temperature for 75 minutes to evaporate the solution. after 15 minutes of adding organic phase to aqueous phase, ha dissolved in deionized water containing tween 80 (1% w/v) was added to nanoparticles mixture during 5 minutes, while stirring at 600 rpm, in order to produce targeted nanoparticles. nonbound ha was separated from nanoparticles mixture by dialyzing versus 100 ml deionized water containing tween 80 (1% w/v) using dialysis bag with molecular weight cut-off of 12,400 da for 40 minutes so that the deionized water containing tween 80 (1% w/v) was replaced every 10 minutes. to determine the amount of ha bounded to slns after separation of unbound ha, some part of the targeted nanoparticles mixture was dried under vacuum and subjected to elemental analysis (chn) (chns-932, leco, usa) and, by subtracting the total amount of ha from gaining value, the amount of ha bound on the slns surface was calculated. the particle size, polydispersity index, and zeta potential of nanoparticles were measured by a zetasizer (zetasizer 3000; malvern instruments, malvern, uk), after 1: 10 diluting the samples with deionized water. the loading efficiency percent was determined by centrifugation (eppendorf 5430 centrifuge, germany). the dispersion of nanoparticles was poured in centrifugal filter tubes (amicon ultra, ireland) with a 10 kda molecular weight cutoff to separate the aqueous medium. the concentration of free etoposide in the filtrate was determined by measuring its absorption in 276.4 nm (uv-vis spectrophotometer, shimadzu scientific instruments, japan) and converting the absorbance to concentration using the calibration equation of etoposide in aqueous phase containing 1% w/v of tween 80. the amount of encapsulated drug was computed indirectly by calculating the difference between the total amounts of drug used in preparation of nanoparticles and the free drug. ultimately, loading efficiency percent was computed by the following equation: (1)loding efficiency percent =(total drug weightfree drug weight)total drug weight100. drug release profiles from the npls were determined in phosphate buffer saline (pbs, 0.01 m, ph 7.4 containing 1% w/v tween 80) at 37c. a total of 2 ml of npls suspension was placed in dialysis bag with molecular weight cut-off of 12,400 da and suspended in a beaker containing 50 ml of pbs on a magnetic stirrer with a speed of 200 rpm. samples were withdrawn periodically and replaced with the same volume of pbs at the same temperature. the content of etoposide in the samples was determined spectrophotometrically at 268.7 nm. to determine cell proliferation, a total of 180 l of the cell suspension (5 10 cells/ml) were placed in each well of a 96-well plate except for one row for blank that was filled by an equal amount of medium. after a 24 h period of incubation at 37c in a co2 incubator with 5% co2 and 95% humidity, all 4 wells of cells were treated with 20 l of one of the concentrations of etoposide as much as 0.475, 0.95, 1.9, and 3.8 m of etoposide. the ic50 of etoposide for sk-ov-3 cells was determined to be 1.9 m. in order to assure that microorganisms would not be able to contaminate the slns and interfere with cytotoxicity results, preparation of solution of free drug and also preparation and dilution of slns suspensions it should be pointed out that solutions of organic and aqueous phases were presterilized by ultraviolet germicidal irradiation method. treated groups included either a solution of free drug in 1 w/v% aqueous solution of tween 80 or encapsulated drug in nontargeted and targeted nanoparticles, with blanks of nontargeted and targeted nanoparticles, while culture medium and tween 80 1 w/v% (each one in 8 wells) serve as control groups. the cells were incubated for further 48 h. after the treatment, 20 l/well of the mtt solution (5 mg/ml of pbs) was added to the cells and incubated for 3 h; then the supernatant was removed carefully and the formazan crystals were dissolved by adding 150 l of dmso. finally, the absorbance of each well was measured at 570 nm by an eliza plate reader (stat fax 2100 microplate reader, awareness technology, inc., us). the effect of each treatment on cell viability was calculated by comparing the relative absorbance of treated cells against the respective controls, using the following equation: (2)cell survival% =(mean absorbance of each group mean absorbance of blank) (mean absorbance of negative control mean absorbance of blank)1 100. first, 2700 l of the cellular suspension with the concentration of 10 cells/ml was poured into 10 wells of a 12-well plate containing lamels at the bottom and then incubated for 48 h in co2 incubator. then the nontargeted and targeted nanoparticles were loaded with sodium fluorescein instead of etoposide by the same method as mentioned above for drug-loaded slns. the final concentration of loaded sodium fluorescein in nanoparticles was 1 mg/ml. blank nanoparticles were also prepared but without sodium fluorescein. to prepare free sodium fluorescein solution, 10 l of stock solution (100 mg/ml) was diluted to 1 ml to provide the final concentration of 1 mg/ml. finally, 300 l of each sample was added to 2 wells (one for imaging in the 1st hour and the other for imaging in 4th hour) and was incubated. lamels were withdrawn and imaging was performed by visible fluorescence microscope (olympus, ix71, japan). all data are the results of three separate experiments, and the results are expressed as the mean standard deviation (n=3). statistical analysis was performed using one-way analysis of variance (anova) and an independent student's t-test with the spss software (version 18, us). the particle size of nontargeted and targeted slns was 179.6 16.31 and 416.42 31.85, respectively. zeta potential of nontargeted slns was 11.82 0.52 that changed to 12.65 0.49 after coating with ha. drug loading efficiency was about 64.92 3.76% and release efficiency percent in 24 h was 65.47 4.68% which is an acceptable value. slns have generally long-term stability (about 13 years) as small particle size and density close to unity of slns mean that the gravity has little effect on particles in dispersion and the brownian motion is sufficient to maintain colloidal dispersions without creaming or sedimentation. in the present study the presence of physically bound ha and the negative zeta potential of targeted slns may seem to threaten stability, but our unpublished results showed that properties of the mentioned slns suspension did not change significantly within 10 days. however, as freeze-drying is a suitable method to prevent the ostwald ripening and avoid aggregation of slns, we also dried the nontargeted and targeted slns under vacuum with 5% glycerol serving as cryoprotectant and then recovered them by adding deionized water. the results showed that nontargeted slns only needed 5 minutes of stirring at 800 rpm and targeted slns needed twice the stirring at 800 rpm each time for 3 minutes and then 10 seconds of sonication at a power of 30 w, to retrieve their primary properties.. the observed release rate (64.1% in the first 6 hours and 73.1% in 24 hours) could provide appropriate serum concentrations for routine chemotherapy schedules in which the drug (with an iv half-life of 612 hours) is administered once daily. also the mean diameter of typically 200400 nm is well below the size of the smallest blood capillaries in the range of 5-6 m. furthermore, because of the heterogeneity of tumors and dynamic status of each tumor, it will be very difficult to assume any maximum single value for particles to exploit the enhanced permeation and retention (epr) effect. however, the study of bae and park suggests that the porosity of the blood vessels in tumors is around 400 nm. a tumor-dependent functional pore cutoff size ranges from 200 nm to 1.2 m, but the pore cutoff size of porous blood vessels in the majority of tumors is known to be 380780 nm. sterically stabilized liposomes of 400 nm in diameter were able to penetrate into tumor interstitium. accumulation of hyaluronic acid-coated self-assembled nanoparticles with particle size of 400 nm has been reported in the tumor tissue too. the obtained results of mtt cytotoxicity assay have been illustrated in table 2 and figure 3. all drug-loaded nanoparticles caused higher cytotoxicity compared to the free etoposide at the same concentration and their respective blank slns. the mechanism of enhanced cytotoxicity of drug-loaded lipid nanoparticles has been previously reported [30, 31]. it is well understood that improvement in the cytotoxicity is because of the elevated drug concentrations within the cells. as we can see in figure 3, nontargeted drug-loaded slns have lower cell survival compared to the free etoposide solution. for example, the observed cell survival after treatment with targeted nanoparticles was 36.08 0.88%, while it was 42.73 1.49% and 48.57 1.61% for nontargeted slns and free drug solution, respectively, at the concentration of 1.9 m (p<0.05). the results verified that targeted and nontargeted slns of etoposide have reduced ic50 to 52% and 83% of free drug, respectively (table 2). in a study, saliou et al. reported that lipid nanocapsules of etoposide reduced the ic50 of the drug from 100 to 2.5 m in h209 cells these lipid nanocapsules also could reduce the ic50 of etoposide to about 430 times in glioma cell lines. in an experiment conducted by nasti et al. chitosan/triphosphate nanoparticles coated with ha showed the ic50 of about half of the noncoated nanoparticles on murine fibroblasts of l929 and macrophage cells of j774.2.. successfully overcame on drug resistance of mcf-7/adr cells with 4.3-fold reduction in ic50 of doxorubicin by sirna polyamidoamine-hyaluronic acid complex. it could be concluded that the internalization of the drug into cells was enhanced when the drug was encapsulated in slns. this phenomenon might be the result of the high affinity of lipid materials of slns for the cell membrane and the nanoscaled size of slns. the correlation between nanoparticles size and intracellular concentration has been observed in the study performed by zhang et al. and their results indicated that the less the particle size is, the more the intracellular drug concentration and cytotoxicity is. in addition, comparing the targeted and nontargeted nanoparticles determines that the cytotoxicity in the targeted nanoparticles has been increased, probably due to the presence of ha on targeted nanoparticles which could interact with cd44 receptors and make them internalized into cells more easily. cho et al. have surveyed npls containing docetaxel targeted by ha upon cancer cell line mcf-7 and showed that they were endocytosed by cd44 receptors. after incubating for 1 hour, only targeted nanoparticles made a slight fluorescence in the cells (figure 4). after 4 hours of incubation, the fluorescence was remarkably higher in the cells which were treated by targeted slns rather than those which were treated by nontargeted nanoparticles, and there was no observable fluorescence in cells incubated with pure sodium fluorescein (figure 4). therefore, it could be concluded that increased cytotoxicity in results obtained from mtt assay has resulted from special uptake of targeted nanoparticles due to presence of ha as targeting agent. hyaluronate targeted slns containing etoposide increase the cytotoxicity of etoposide in sk-ov-3 cells and could be a valuable method for reducing the prescribed dose and also systemic side effects.

the epithelial ovarian carcinoma is one of the most fatal gynecological cancers. etoposide is used in treating platinum-resistant ovarian cancer. sodium hyaluronate is a substance that binds to the cd44 receptors overexpressed in sk-ov-3 cells of epithelial ovarian carcinoma. the aim of the present work was to study the cytotoxicity effect of hyaluronate targeted solid lipid nanoparticles (slns) of etoposide on sk-ov-3 cells. the cytotoxicity of the targeted and nontargeted slns of etoposide was compared to free drug on the sk-ov-3 cells by mtt assay method. the cellular uptake of the targeted and nontargeted nanoparticles containing sodium fluorescein was also studied. the difference of cell vitality between nontargeted nanoparticles and also targeted nanoparticles with free drug was significant. targeted nanoparticles also caused more toxicity than nontargeted nanoparticles (p<0.05). after 4 hours of incubating, the fluorescence was remarkably higher in the cells treated by targeted slns rather than nontargeted ones, and there was no observable fluorescence in cells incubated with pure sodium fluorescein. hyaluronate targeted slns containing etoposide increased the cytotoxicity of etoposide on sk-ov-3 cells which may be a worthwhile potential method for reducing the prescribed dose and systemic side effects of this drug in epithelial ovarian carcinoma.

PMC4020396

pubmed-745

simple enones are known to undergo ni-catalyzed cycloaddition with diynes (eq 2). however, these conditions fail to provide any cycloadduct product with tropone as shown in eq 3. thus, we focused our investigation on discovering an alternative ni-catalyzed cycloaddition protocol.2 diyne 1 and tropone a were used as model substrates and were subjected to a catalytic amount of ni(0) and a variety of phosphine and n-heterocyclic carbene (nhc) ligands (eq 4). reactions run with monodentate and bidentate phosphines mostly afforded dimerization of diyne along with traces or low amounts of the desired cycloadduct (table 1, entry 110). however, reactions run with nhcs resulted in good to excellent yields of cycloadduct 1a, which couples the diyne with a single c further optimization led to our final reaction conditions: diyne (1 equiv), tropone (1.1 equiv), 3 mol% of ni(cod)2, 6 mol% of sipr, thf, 60 c, and 5 h. reaction conditions: 10 mol% of ni(cod)2, 20 mol% of l, diyne (1 equiv, 0.1 m), tropone (1.1 equiv), toluene, 60 c, 5 h. determined by gc using naphthalene as an internal standard. the model substrates afforded the desired product 1a along with another isomer 1a in excellent yield and>90% selectivity for 1a (eq 5). similarly, cycloaddition of sulfonamide diyne 2 afforded a mixture of major and minor isomers 2a and 2a respectively, which on subsequent treatment with p-bromobenzoyl chloride/net3/dmap afforded major isomer 2a in 74% yield and p-bromobenzoyl derivative of minor isomer (2b) in 13% yield (eq 6) as crystalline solids (figure 2). surprisingly, 2b (and, therefore, 2a as well) has [576] ring fusion compared to [567] in the case of the major isomer, 2a.5 ortep diagram of 2a and 2b. with optimized reaction conditions in hand, the substrate scope was explored (table 2). the cycloaddition occurred smoothly with the diyne bearing a sulfone backbone to form 3a along with minor isomer 3a. notably, this diyne is completely unreactive in several reported ni-catalyzed cycloadditions. although ni catalysts have been reported to catalyze the cycloaddition of nitriles and diynes to form pyridines, diyne 4, which has a nitrile group in the backbone, selectively reacted with tropone to afford the desired cycloadducts (4a and 4a) in excellent yield. inspired by carreira s work, we performed the cycloaddition reaction with 5, a diyne with a metabolically stable backbone, to give cycloadduct 5a in good yield along with minor isomer 5a. aryl substituted internal diynes are one of the most challenging substrates in ni/nhc-catalyzed cycloaddition reactions. nevertheless, the reaction of aryl substituted symmetrical diynes with tropone afforded 6a and 7a in good yields. interestingly, no minor cycloadduct (6a or 7a) was obtained in these cases. to investigate the effect of electronics on the regioselectivity, we subjected unsymmetrical diynes 8 and 9 to standard reaction conditions; remarkably, exclusive formation of one regioisomer was detected (8a and 9a). the use of different aryl groups (i.e., 3,4-dimethoxyphenyl and naphthyl) on alkyne terminals is also possible (10a and 11a). due to recent interest in indole bearing novel compounds, diynes 12 and 13 were investigated. biaryl cycloadducts (12a, 13a) were formed in very good yield and high regioselectivity. interestingly, the regioselectivity was higher in the case of 3-substituted indole diyne than 5-substituted indole diyne (13a vs 12a). cycloaddition of phenyl ethyl diyne 14 and phenyl silyloxymethyl 15 afforded regioisomers 14a and 15a, where the carbonyl resides next to the phenyl ring, exclusively, while a diyne with covalently bound -tocopherol can also be easily clicked together with tropone to afford regioselective cycloadduct 16a. an unsymmetrical diyne bearing an internal gem-dimethyl group reacted with tropone to afford an exclusive regioisomer 17a, which suggests that regioselectivity is highly dependent on the substituents on the alkyne units of a diyne rather than backbone. the cycloaddition of unsymmetrical isopropyl methyl diyne (18) afforded products 18a and 18a, where the bulkier group is next to the carbonyl of tropone. cycloaddition of phenyl isopropyl diyne 19 affords a product where the isopropyl group is away from the carbonyl group (19a), suggesting that electronic factors override the steric factors (19a). unfortunately, terminal diynes did not afford any cycloaddition product with tropone due to their high propensity to oligomerize under our reaction conditions. reaction conditions: diyne (1 equiv, 0.1 m), tropone (1.2 equiv), 3 mol% of ni(cod)2, 6 mol% of sipr, thf, 60 c, 5 h. isolated yields (in black), ratio of major and minor cycloadducts (in blue), ratio of major and minor regioisomers (in red). the ratios were determined by h nmr of crude reaction mixture. the lack of general methods to access troponoids prompted our investigation on the ability to convert the cycloadduct to a fully aromatized product. we found that compound 2a can be consistently converted to tropone 20a by a three-step protocol. the hydrogenation of alkene 2a led to a saturated cycloheptanone, which was then subjected to dibromination. specifically, we studied the catalytic cycles for [ni(ipr)2]-catalyzed cycloaddition of nona-2,7-diyne and tropone with dft calculations. homocoupling, where two alkynes undergo initial oxidative coupling, and heterocoupling, where an alkyne and the tropone undergo initial oxidative coupling, were both investigated. the free energy changes for the homocoupling pathway are shown in figure 3. from [ni(ipr)2] complex 21, the coordination of diyne to form intermediate 22 is endergonic by 7.0 kcal/mol. subsequent intramolecular oxidative cyclization via ts23 requires a 13.4 kcal/mol barrier with respect to 22, generating the metallacyclopentadiene intermediate 24. this intermediate undergoes a facile 8 insertion (instead of 2 insertion, vide infra) of tropone via ts25, with a barrier of only 9.8 kcal/mol. the 8 insertion produces the eight-membered ring intermediate 26 with the tropone oxygen coordinated to nickel. the tropone piece of complex 26 can coordinate to nickel in four different fashions, generating the complexes 26 to 29. the four isomers have similar stabilities, and complex 29 undergoes reductive elimination via ts30 to give the product-coordinated complex 31. product extrusion from 31 is exergonic by 2.8 kcal/mol to release the product and regenerate the nickel diyne complex 22. the tautomerization of intermediate complex 29 to 29-enol is endergonic by 5.8 kcal/mol, and subsequent reductive elimination from 29-enol will form the 576 tricyclic product; however, the irreversible reductive elimination via ts30 suggests that the 576 tricyclic product arises from ni-free tautomerization. free energies (298 k) with respect to 21 are shown in kcal/mol. the calculations indicate that the resting state of pathway a is [ni(ipr)2] complex 21, and oxidative cyclization through ts23 is the rate-limiting step of the catalytic cycle. the overall reaction barrier is 20.4 kcal/mol, which is consistent with the experimental conditions (60 c, 5 h). in contrast, the heterocoupling pathway of [ni(ipr)]-catalyzed cycloaddition between nona-2,7-diyne and tropone displays higher free energies (figure 4). specifically, from nickel diyne complex 22, the intermolecular oxidative cyclization between alkyne and tropone can occur via ts34, with the carbonyl group of tropone distal to the forming c c bond. this step requires a barrier of 42.5 kcal/mol with respect to the [ni(ipr)2] complex 21, which is much less favorable as compared to the homocoupling pathway discussed above (figure 4). alternatively, the intermolecular cyclization can occur with the tropone carbonyl group proximal to the forming c c bond, as in ts36. ts36 is 31.6 kcal/mol higher in free energy than the resting state 21, which is also less favorable than the productive homocoupling pathway. therefore, unlike other ni-catalyzed couplings between alkynes and carbonyls, a heterocoupling mechanism is not operative in the [ni(ipr)]-catalyzed cycloaddition between diyne and tropone. free energies (298 k) with respect to 21 are shown in kcal/mol. next, insertion of the tropone was investigated, and two probable pathways emerged: a traditional 2 insertion and a distinctive 8 insertion. the transition states of 8 (ts25) and 2 insertion (ts37) of tropone were both located, and their free energies and structures are shown in figure 5. interestingly, the 8 insertion is found to be more favorable by 12.3 kcal/mol. optimized structures, gibbs free energies, and distortion and interaction energies of transition states of 8 insertion (ts25) and 2 insertion (ts37) of tropone. the gibbs free energy changes (298 k) with respect to 24 are shown in kcal/mol. we studied the origins of this preference by employing the distortion/interaction model on ts25 and ts37. the distortion energy reflects the structural changes from nickel complex 24 or tropone to the corresponding geometries in the transition states, and the interaction energy is the energy of interactions between the distorted fragments, computed as the difference between the activation energy and the total distortion energy. the difference between the distortion energies of ts25 and ts37 is the major reason for the preference for 8 insertion. the distortion energy is 17.8 kcal/mol energy for 24 and 13.7 kcal/mol energy for tropone to achieve the distorted geometries in ts25, while it requires much larger distortions (24.3 kcal/mol for 24 and 16.6 kcal/mol for tropone) in ts37. stronger steric repulsions are generated between the nickelacyclopentadiene moiety and tropone in ts37 as compared to those in ts25. this difference between the steric repulsions eventually leads to the preference of the unconventional 8 insertion, and this is the highest order of poly- insertion so far. the transition states for the 8 insertion of tropone with unsymmetrical diynes were also studied. for the isopropyl methyl diyne (figure 6), the transition state ts38-c1 that contains the bulky isopropyl group on the diyne next to tropone is more stable than ts38-c2 by 0.7 kcal/mol to avoid steric repulsions between the isopropyl group and ipr ligand. for phenyl methyl diyne, the transition state ts39-c1 is 4.7 kcal/mol more stable than ts39-c2 mainly due to the steric repulsions between the phenyl group and the bulky nhc ligand in ts39-c1. also, for phenyl isopropyl diyne, the phenyl group is more sterically demanding, and a 4.0 kcal/mol preference to the ts40-c1 is found. overall, our data suggest that ni-catalyzed cycloaddition occurs via the mechanism shown in figure 7. the homo-oxidative coupling of diyne on ni(0) forms ni(ii)cyclopentadiene intermediate i that undergoes 8 insertion of tropone to afford seven-membered ring complex ii. intermediate ii isomerizes from oxygen coordination to -coordination resulting in intermediate iii, which can subsequently isomerize to an -coordinated-ni(ii) complex v through intermediates iv. finally intermediate v reductively eliminates to give vi, which releases the tricyclic product, vii, and regenerates the ni(0) catalyst. at this point, compound vii can preferentially aromatize via sigmatropic shifts to afford major product viii. however, a minor pathway involves tautomerization of vii to cycloheptatrienol ix, which undergoes further 6 electrocyclization to afford an interesting bis(divinyl)cyclopropane intermediate, x. intermediate x can either revert to ix or irreversibly rearrange to [576] fused intermediate xi, which undergoes further sigmatropic shifts to yield the observed minor product, xii. this sigmatropic shift could be catalyzed by a trace amount of water through the bridge of one or multiple molecules of water. due to the uncertainty of the catalyst, we did not perform computational studies on the isomerization of the tricyclic product vii. proposed mechanism for the ni-catalyzed cycloaddition of diynes and tropone. in conclusion, we have discovered a nickel catalyst that can effectively and selectively incorporate a single c c -bond of tropone in the cycloaddition with diynes. the mechanism of this novel cycloaddition reaction has been investigated using dft calculations. it involves a unique 8 insertion of tropone to form the observed major and minor products. the mechanistic studies to further understand this unique reactivity of tropone and application of this chemistry are underway in our laboratories.

a ni/n-heterocyclic carbene catalyst couples diynes to the c()c() double bond of tropone, a type of reaction that is unprecedented for metal-catalyzed cycloadditions with aromatic tropone. many different diynes were efficiently coupled to afford [567] fused tricyclic products, while [576] fused tricyclic compounds were obtained as minor byproducts in a few cases. the reaction has broad substrate scope and tolerates a wide range of functional groups, and excellent regioselectivity is found with unsymmetrical diynes. theoretical calculations show that the apparent enone cycloaddition occurs through a distinctive 8 insertion of tropone. the initial intramolecular oxidative cyclization of diyne produces the nickelacyclopentadiene intermediate. this intermediate undergoes an 8 insertion of tropone, and subsequent reductive elimination generates the [567] fused tricyclic product. this initial product undergoes two competing isomerizations, leading to the observed [567] and [576] fused tricyclic products.

PMC4291811

pubmed-746

focal epilepsies account for about two-thirds of all adult epilepsy patients, and temporal lobe epilepsy (tle) is the most common type of focal epilepsy (1,2). temporal lobe epilepsy is subcategorized as mesial (i.e., amygdalohippocampal) and neocortical (i.e., lateral) temporal. mesial temporal lobe epilepsy (mtle) with hippocampal sclerosis (hs) is one of the most common types of epilepsy referred for surgical treatment; it is often refractory to antiepileptic drugs (aeds), but responds favorably to surgery (1,4). patients with mtle-hs and intractable seizures often experience progressive behavioral changes including increasing memory deficit with the passage of time. in addition, surgical outcome may be worse with longer duration of epilepsy or increasing age at surgery, which suggests that mtle-hs is a progressive disorder (5). therefore, early detection of this syndrome and its distinction from other tle syndromes has important practical implications with regard to planning an optimal treatment strategy for the affected patient. in the current study, we tried to compare the clinical characteristics of patients with mtle-hs with those who had tle due to other etiologies in order to identify potentially differentiating clinical characteristics between these two groups of patients. in this retrospective analytic study all patients with a clinical diagnosis of tle were recruited in an outpatient epilepsy clinic at shiraz university of medical sciences, which is the only referral epilepsy clinic in south iran. the diagnosis of tle was made exclusively by one epileptologist working at this institution (the first author) and based on clinical grounds (semiology), electroencephalographic (eeg) findings and imaging, such as magnetic resonance imaging (mri). all patients were followed up by the epileptologist at our institution for at least one year (i.e., until may 2014). routine eeg was requested for all patients at the time of referral. in difficult-to-diagnose or difficult-to- treat patients we often order video-eeg monitoring to ascertain the diagnosis and formulate an individualized treatment plan, accordingly. for all patients, a 1.5 tesla brain mri (epilepsy protocol) was performed. we classified patients as having mts (if they had clear signs of mesial temporal sclerosis and/or atrophy in their mri) and those who had any other mri abnormality. patients with dual pathology (i.e., mesial temporal sclerosis associated with other structural lesions) were excluded from the study. age, gender, age at seizure onset (i.e., the first afebrile seizure), seizure type(s), epilepsy risk factors (including pregnancy complications, history of febrile seizure, cns infection, significant head trauma, positive family history of epilepsy), eeg findings (the most informative eeg), and mri findings of all patients were registered routinely. seizure types were categorized as generalized tonic clonic seizures (gtcs), complex partial seizures (cps) and auras. pearson chi-square, fisher's exact, and mann-whitney u tests were used for statistical analyses. until may 2014, 2890 patients with epilepsy were registered at our epilepsy clinic. four hundred twenty-seven patients (14.7%) were diagnosed as having tle. of these, 174 patients were eligible to enter the study (105 patients with mtle-hs and 69 patients with tle due to other etiologies). demographic and clinical characteristics of patients with mtle-hs and those who had tle due to other etiologies are shown and compared in table 1. frequency of seizure types (i.e., gtcs vs. cps vs. auras) was not significantly different between these two groups (table 1). specific types of auras were significantly different between these two groups (table 1), however most auras were reported by both groups, similarly. epilepsy risk factors were reported to be as follows (mtle-hs vs. others): parental consanguinity in 41/30 (p= 0.6), family history of epilepsy in 18/10 (p= 0.6), significant head trauma in 15/7 (p= 0.4), cns infection in 2/2 (p= 0.6) and pregnancy complications in 2/3 (p= 0.3). mtle-hs: mesial temporal lobe epilepsy (mtle) with hippocampal sclerosis (hs). video-eeg monitoring was available in 155 patients; and 19 patients had a routine eeg only. eleven patients (10.4%) with mtle-hs and four (5.8%) with other etiologies had normal eeg (p= 0.2). right sided, left sided or bilateral focal epileptiform discharges (i.e., temporal seizures, spikes or sharp waves) were observed in 36, 38, and 20 patients with mtle-hs and 31, 21 and 13 patients with other etiologies, respectively (p= 0.4). focal polymorphic delta activity or temporal intermittent rhythmic delta activity (tirda) was seen in 60 and 7 patients with mtle-hs, respectively. these figures were 32 and 4 in patients with other etiologies (p= 0.3). magnetic resonance imaging (mri) in patients with other etiologies (non- mtle-hs group) showed temporal lobe tumors in 27 patients (39.1%), non-specific white matter mri abnormalities in 11 (15.9%), cavernoma in seven (10.1%), sequels of head injury in six (8.7%), arachnoid cysts in five (7.2%) and other etiologies in 13 (18.8%) patients. twelve patients had dual pathology (mesial temporal sclerosis in addition to another lesion). mesial temporal lobe epilepsy with hippocampal sclerosis is a common type of epilepsy referred for surgical treatment due to medical refractoriness. as a matter of fact however, it has been suggested that early surgical intervention after seizure onset is an important precondition for achieving seizure-free status after surgery (6). therefore, early detection of this syndrome has important practical implications with regard to planning an optimal treatment strategy for the affected patient, particularly those with medically-refractory seizures. in the current study, we observed that earlier age at epilepsy onset (i.e., teenagers compared to young adulthood), a past history of febrile seizures and reporting affective auras (e.g., fear, anxiety, depression, joy, and anger) were commonly seen in patients with mtle-hs, while auditory auras were more frequently reported by those with tle due to other etiologies. in previous studies dominantly represented by patients with hs on mri, it has been observed that age at onset is variable (7). the finding that age at onset is a potential clinical variable to distinguish mtle-hs from tle due to other etiologies should be further explored in future studies. several studies have shown a significant relationship between a history of febrile seizures in early childhood and mesial temporal sclerosis. in one study, it was observed that hippocampal t2 hyperintensity after febrile status epilepticus represents acute injury often evolving to a radiological appearance of hs after one year (8). having a past history of febrile seizure is a useful historical clue in favor of mtle-hs compared to tle due to other etiologies. as the first ictal symptoms, auras can provide important localizing and lateralizing information useful in determining the location of the epileptogenic zone (9). the observation that, affective auras (e.g., fear, anxiety, depression, joy, and anger) were commonly seen in patients with mtle-hs, while auditory auras were more frequent among those with tle due to other etiologies, has very important clinical implications. in one study, the authors examined the relationship between presence of different types of auras and post-surgical outcomes in 157 patients with medically intractable mesial temporal lobe epilepsy (mtle) with unilateral hippocampal sclerosis (hs). the occurrence of multiple auras was not associated with post-surgical outcome (p=0.7). but, the presence of extratemporal auras (e.g., somatosensory, visual and dysphasic auras) was significantly higher in patients with poor outcome (10). designing future studies to investigate the relationship between presence of mesial temporal lobe auras (e.g., psychic symptoms, cognitive and affective auras, autonomic auras such as epigastric sensation, olfactory and gustatory auras) versus lateral temporal lobe auras (e.g., vertiginous and auditory auras) and surgery outcome in patients with mtle-hs is necessary. the mainstay for making a correct diagnosis, when evaluating a patient with seizure, is having a standardized approach, particularly with regard to taking a detailed clinical history. one may find important clues in the clinical history (e.g., age at onset, detailed seizure description and past history) to make a syndromic diagnosis. the syndromic diagnosis forms the basis for the treating physician to decide upon an appropriate management plan. this was a clinic-based series and may not represent the full spectrum of tles. we would like to thank neurosciences research center, shiraz university of medical sciences, shiraz, iran for supporting this study.

background: this study compares the clinical characteristics of patients with mesial temporal lobe epilepsy with hippocampal sclerosis (mtle-hs) with those who have temporal lobe epilepsy (tle) due to other etiologies. methods: in this retrospective study all patients with a clinical diagnosis of tle were recruited in a referral outpatient epilepsy clinic at shiraz university of medical sciences from september 2008 to may 2013. we classified the patients with tle as having mesial temporal sclerosis if they had clear signs of mesial temporal sclerosis and/or atrophy in their mri and others who had any other mri abnormality. results: a total of 174 patients were studied (including 105 patients with mtle-hs and 69 patients with tle due to other etiologies). frequency of seizure types was not significantly different between these two groups. earlier age at epilepsy onset (p= 0.005), a past history of febrile seizures (p= 0.010) and presence of affective auras (p= 0.008) were commonly seen in patients with mtlehs, while auditory auras (p= 0.020) were more frequent in those with tle due to other etiologies. conclusion: the mainstay for making a correct diagnosis, when evaluating a patient with seizure, is having a standardized approach, particularly with regard to taking a detailed clinical history. one may find important clues in the clinical history (e.g., age at disease onset, detailed seizure description and past history) to make a correct diagnosis.

PMC4715408

pubmed-747

leukocytospermia is a kind of common disease of male reproductive tract infection, which is usually caused by bacterial infection. an increase of white blood cells in seminal plasma can lead to the decrease of sperm quality and the increasing number of inflammatory cytokines such as interleukin il-6 is the main factor involved in the process. according to current studies, paf is a kind of cytokine which has many kinds of biological activities, such as inflammation factors and sperm cell activation factors. how does the paf change and what role does it play the role during the reproductive tract infection? the purpose of our study was to observe the expression of paf and tnf- in seminal plasma of patients with leukocytospermia. male patients age from 23 to 40 years old, who came to the urology surgical department of our hospital from april to september 2011, were enrolled. according to who directives, 22 cases with leukocytospermia and 15 normal males were chosen. all the patients did not have any urinary, reproductive, blood, or endocrine diseases. the patients who have ever suffered from mumps, cryptorchidism and sexual dysfunction and ever accepted radiotherapy or chemotherapy were excluded. the study was admitted by the local ethical committee, and written informed consent was obtained from all participants. the semen should be obtained in one time by using masturbation method and be placed into disposable sterile dry plastic container with cover. after the full liquefaction of semen plasma, the computer-assisted semen analysis (casa) carried out a routine analysis. the rest of the seminal plasma was collected and accepted centrifugal test for 10 minutes at 4000 r/min, the upper layer of seminal plasma was placed into eppendorf tube and stored at 20c for reservation. density count of seminal plasma white blood cell in seminal plasma refer to peroxidase dyeing method, recommended by the who. human platelet activation factor (paf) enzyme league immune kit and human tumor necrosis factor (tnf-) enzyme league immune kit were bought from jiahui biotechnology company (rd import repacking), beijing weili wjy9000 sperm analyzer, low temperature centrifuge (sigma, usa), 37c constant temperature water-bath box, full-wavelength enzyme standard instrument (finland leibo company). the operator should operate strictly in accordance with the manual of elisa kit for inspecting tnf- and paf. the od value should be read on enzyme standard instrument within 15 minutes at 450 nm wavelength. normal characteristic test and homogeneity test of variance would be carried out before intragroup comparison of paf and tnf- expression, and t-student test was adopted. there are two kinds of correlation analyses here, straight line related analysis and spearman rank correlation analysis, which one should be adopted, it depends on whether the expression of paf or tnf- on normal distribution or not in the seminal plasma. significant difference was found in the aspects of sperm survival rate and vigor between leukocytospermia group and normal group, p<0.01, while the difference in sperm density and liquefaction time was not obvious, p>0.01 (table 1). paf concentration in leukocytospermia group was (2.14 0.43 ng/ml), which was significantly lower than the normal group (6.21 1.38 ng/ml); the difference was statistically significant (p<0.01). wbc count in leukocytospermia group was (1.33 0.18) 10/ml, and tnf- concentration was (5.51 1.46 ng/ml), which was significantly higher than that of normal group wbc (0.43 0.19) 10/ml and tnf- (3.48 1.08 ng/ml), and the difference was statistically significant (p<0.01) (table 2). in this group, paf has no significant correlation with sperm density and liquefaction time. and what's more, it has positive correlation with sperm survival rate (r=0.452, p<0.01) and sperm vigor (r=0.642, p<0.01). (p>0.01). while it has negative correlation with sperm survival rate (r=0.415, p<0.01) and sperm vigor (r=0.725, p<0.01) (table 3). linear correlation analysis shows that wbc count has negative correlation to paf concentration in leukocytospermia group (r=0.62, p<0.01) (figure 1), however, it has positive correlation with tnf- concentration (r=0.77, p<0.01); paf concentration had significantly negative correlation to tnf- (r=0.68, p<0.01) (figure 2). leukocytospermia refers to the increase of white blood cell (wbc) density in seminal plasma which is more than 1 10/l, and it is mainly caused by the reproductive tract infection. the white blood cells in seminal plasma can produce many inflammatory cell factors. with the affection of these cell factors, the sperm quality, density and its vigor get lower; sperm capacitation and acrosomal reaction would be affected as well, which lead to male infertility. paf is a kind of cell factors found in recent years, and it is involved in various physiological and pathological processes. sperm movement ability is the precondition of fertilization, wherein paf with certain concentration is considered to be one of the conditions to obtain certain movement ability. when the exogenous paf concentration is between 1 10 and 1 10 mol/l, the sperm's motility is prominently increased after 120 min culture time. in animal experiments, the sperm motility can be immediately improved after exogenous paf treatment on freeze-thaw boar sperm; paf concentration in seminal plasma of squirrel monkeys in the breeding season is significantly higher than that during the nonbreeding season, thereby, it is believed that paf plays an important role in the reproductive process. paf also participates in sperm capacitation and acrosomal reaction (ar); sperm capacitation can be affected through culturing with sperm together, thereby increasing the acrosomal reaction rate. ar% is different when paf concentration changes, relatively, high density (1 10 mol/l) can induce ar better. in addition, paf can obviously increase the pregnancy rate of accepting artificial insemination treatment due to unexplained infertility. the seminal plasma of dog stored in low temperature has prominently increased sperm movement performance through treatment with different concentrations of exogenous paf. research shows that the sperm movement performs best when the concentration is 1 10 m with 120 min of cultivating time, at the same time, atp concentration improves obviously as well. we also found that the mitochondrial function of the sperm through paf processing remains unchanged, and the integrity of sperm has not been damaged either. at the same time, it promotes the platelets and neutrophils to gather together under the pathologic state participate in super oxide formation, and it also promotes the protein phosphorylation process. it can also induce submucosal vasoconstriction, and induce neutrophils and other inflammatory cells to produce a large number of active oxygen and free radicals. it is confirmed that paf involves in gastric ulcer, alcoholic stomach injury, necrotizing enterocolitis, ischemic stomach, and small intestinal mucosal injury. in addition, it also plays an important role in acute pancreatitis and acute lung injury development [9, 10]. as a result, the role paf which plays in reproductive tract infection is worth discussing. alpha subtype of tumor necrosis factor (tnf-) is a kind of important cell factor involved in the inflammation reaction progress. it is mainly produced by macrophages, and widely involved in inflammation response, immune response, endotoxin sex shock, and other pathologic process. at the same time, tnf- is a kind of cell factor, which has close relations with sterility occurrence and development. a certain amount of tnf- can significantly inhibit the activity of sperm acrosomal enzyme and acrosomal reaction. tnf- has certain influence on sperm mitochondrial function, it can interfere sperm energy metabolism and lower sperm movement ability. it may also promote the sperm apoptosis through the mitochondria apoptosis way, which causes infertility. tnf- also can reduce the amount of nitrate in normal sperm and affects no synthesis, which is necessary for sperm movement. studies have reported that paf and tnf- can mutually cooperate with each other to promote the progress of inflammatory response, such as acute lung injury, monilia infection, and necrotizing enterocolitis. how is the paf expression in the condition of reproductive tract infection? and how does the interaction happens between paf and tnf-? elisa method was adopted to observe the expression of paf and tnf- in seminal plasma of patients with leukocytospermia. the results show that paf level is lower, while tnf- is higher compared with that in the normal seminal plasma. after analyzing the expressions of paf and tnf-, the relation between them, and the white blood count, we found that the expression of paf and tnf- have negative correlation with each other (r=0.68, p<0.01figure 1); paf is negatively correlated with white blood cell count (r=0.62, p<0.01figure 2); tnf- has positive correlation to white blood cell count (r=0.77, p<0.01). the existing research results showed that paf is favorable for sperm function activities, while tnf- could inhibit sperm function activities. so we believe that, under condition of reproductive tract infection, the organism increase the composition of cell factors which can inhibit sperm activity on one hand, such as tnf-, and decrease the composition of cell factors which can promote sperm on the other hand, such as paf, thereby affect sperm vigor. however paf level is lowered under the state of reproductive tract infection, this phenomenon is of great significance and worth being studied. the adjustment of paf is affected by several factors, wherein it is related to platelet activating factor acetylhydrolase (paf-ah). paf-ah is specific to paf, its concentration is negatively correlated with paf. paf-ah level has significant negative correlation with sperm motility in panda seminal plasma and has negative correlation with the state of sperm movement too. the concentration of paf-ah in seminal plasma of male patient, whose spinal column was injured, is prominently higher than normal people; meanwhile, it has negative correlation to sperm motility. in a study investigating the relation between paf-ah level and sperm vigor, the researchers found that when sperm motility was equal to or above 50%, paf-ah density was 442.03 14.37 iu/l, while when the vigor is below 50%, the paf-ah density is 882.16 18.45 this research also shows that paf-ah is an important factor to adjust paf. whether paf-ah plays an important role in lowering paf level in leukocytospermia or not, it is still worthy of further study. in conclusion, low expression of paf or high expression of tnf- in leukocytospermia affects the sperm quality, both of which contributed to sterility. the reason why paf shows a low expression during reproductive tract infection is still not clear now, and it is of important significance, which deserves further study.

objective. discuss the changes and roles of paf in the reproductive tract infection by observing the expression of platelet activating factor (paf) and tumor necrosis factor (tnf-) in seminal plasma of patients with leukocytospermia. methods. the seminal plasma was obtained from 22 cases of leukocytospermia and 15 cases of normal males; the peroxidase dyeing method was adopted for seminal plasma white blood count; the elisa was adopted to test paf and tnf- concentration in seminal plasma. result. paf concentration (2.14 0.43 ng/ml) of leukocytospermia group was significantly lower than the normal group (6.21 1.38 ng/ml, p<0.01) while tnf- (5.51 1.46 ng/ml) was significantly higher than that of normal group (3.48 1.08 ng/ml). there was negative correlation between paf and tnf-, (r=0.68, p<0.01); the same situation existed in paf and wbc (r=0.62, p<0.01); but tnf- was positively correlated to wbc (r=0.77, p<0.01). conclusion. (1) low expression of paf and high expression of tnf- in leukocytospermia affect the sperm motility, which is one of the reasons that leads to infertility. (2) lower expression of paf has its particularity during the reproductive tract infection.

PMC3504446

pubmed-748

focal myositis is an idiopathic inflammatory myopathy and a rare inflammatory pseudotumor of the skeletal muscle with unknown etiology (1). the disease typically disappears spontaneously, does not last more than 4 years, and has no recurrence (1,2). this myositis can be treated successfully with steroids or nonsteroidal anti-inflammatory drugs (nsaids) (1,2). we herein describe the case of a man with recurrent episodes of bilateral focal myositis. a 38-year-old man presented with myalgia of the right gastrocnemius muscle in may 2005. a needle biopsy of the muscle was performed in another hospital and indicated no malignancy. the patient did not receive any treatment at this time. as his symptom did not improve, he visited our hospital in august 2005 with tenderness of the right gastrocnemius muscle. on physical examination, no significant signs were detected on the skin, cardiovascular, respiratory, or abdominal examinations. manual muscle testing indicated full scores except in the right gastrocnemius, which demonstrated grade 4 (of 5) because of myalgia. evaluations of his mental status, sensory, and cerebellar systems were normal. laboratory data indicated that the patient's serum creatine kinase (ck), aldolase, myoglobin, erythrocyte sedimentation rate (esr), and c-reactive protein (crp) were within normal limits, and anti-jo-1 autoantibody, anti-ars autoantibody, and antinuclear antibody were negative (table). hepatitis b surface antigen (hbs ag) was positive (cut-off>2,000), and hb virus dna was negative (< 3.7 leg/ml). human immunodeficiency virus (hiv) and hepatitis c virus antibody tests were negative (table). magnetic resonance imaging (mri) revealed very high signal intensity in the right gastrocnemius medial head muscle on a short-tau inversion recovery (stir) image. 1a and b). a muscle biopsy of the right gastrocnemius revealed interstitial muscle infiltration by mononuclear inflammatory cells without findings of vasculitis, along with muscle fiber necrosis and regeneration (fig. 2a and b). immunohistochemical staining of the muscle biopsy showed that the infiltrating cells predominantly consisted of cd4+t cells, cd68+macrophages, and cd20+b cells accompanied by a few cd8+t cells (fig. focal myositis was diagnosed. magnetic resonance imaging (mri) of the lower limbs. a, b: august 2005; c, d, e: june 2006; f, g, h: january 2008. a, b, e, f, h: axial (a, f) and coronal (b, e, h). a-f: unenhanced mri image and g: gadolinium-enhanced mri image. stir images show high signal intensity in the affected muscles of the lower legs (white arrowhead). c, d: axial and coronal stir images show high signal intensity in the affected femoral muscles (white arrowhead). f, g: axial gadolinium-enhanced mri (g) shows no enhanced area in the thickened fascia of the right gastrocnemius, which showed high signal intensity on the unenhanced mri image (f) (white arrow). muscle biopsy. a, b: hematoxylin and eosin staining, 400; c-f: immunohistochemical staining, 400; c: cd4; d: cd8; e: cd68; f: cd20; g: electron microscopy. a muscle biopsy of the right gastrocnemius reveals mononuclear inflammatory cells (a, b: black arrowhead) that infiltrated the interstitial spaces of the muscle without vasculitis findings, along with muscle fiber necrosis and regeneration (a, b: white arrowhead). infiltrating cells predominantly consisted of cd4+t cells, cd68+macrophages, and cd20+b cells accompanied by a few cd8+t cells (c-f: white arrow). electron microscopy of the muscle biopsy specimens reveals nemaline rods (g: black arrows). as the patient was hbs ag-positive, lamivudine treatment was initiated for the prevention of de novo hepatitis under immunosuppressive conditions. prednisolone (psl) (30 mg/day) was administered to treat the focal myositis in september 2005, and his symptoms and mri findings were immediately improved. the clinical course is shown in fig. the patient's muscle pain worsened gradually at around this time, and walking became difficult without a cane in june 2006. although his crp level was 0.4 mg/dl, the serum myogenic enzyme level was not re-elevated. mri revealed multiple high-intensity areas in the lower limbs that included the femoral muscle (both sides of the vastus lateralis, the left vastus intermedius, the right biceps femoris, and the left gracilis) (fig. the psl dosage was increased to 60 mg/day for relapse of myositis, and the patient's symptoms immediately improved. psl: prednisolone, mtx: methotrexate, aza: azathioprine, ivig: high-dose intravenous immunoglobulin after tapering the psl dosage to 17.5 mg/day, a second myositis relapse occurred in october 2007. 1f and h), left popliteus muscle, and right semimembranosus on stir images (data not shown), and mild edema was evident in the subcutaneous tissue in both lower limbs in january 2008. as thickening of the fascia of the right gastrocnemius on the stir image (fig. 1 g, white arrow), it was considered to result from edema. because the patient was unusually resistant to the treatment for focal myositis, we performed a muscle biopsy of the left gastrocnemius to re-confirm the diagnosis, which showed a similar result to the first biopsy (data not shown). electron microscopy of the muscle biopsy specimens in february 2008 revealed nemaline rods (fig. the dosage of psl was again increased to 60 mg/day for the third relapse of myositis. thereafter, we administered azathioprine (max 100 mg/day) for a steroid-sparing effect, but it failed to bring the patient persistent relief from his myalgia and gait disturbance. therefore, we were forced to continue to administer 15 mg/day or more of the steroid in order to maintain remission. in september 2011, the relapse of myositis involved myalgia of both lower limbs and arthralgia of both foot joints. the patient's psl dosage was increased from 15 mg/day to 30 mg/day, however, the effect was incomplete. as he had a compression fracture of the vertebrae due to steroid-induced osteoporosis, high-dosage psl treatment was avoided. methotrexate (mtx) (7.5 mg/week) was initiated with psl 20 mg/day in october 2011, as hepatitis b virus (hbv) infection had been controlled by lamivudine. the dosage of mtx was gradually increased to 16 mg/week by august 2012.. the patient can maintain standing on his heels, but not on his tiptoes. the differential diagnoses of inflammatory myopathy were postulated to be polymyositis, dermatomyositis, inclusion body myositis, eosinophilic myositis, and sarcoidosis. he had no past medical history and no other organ disorders including those affecting the skin and lung. the lesion originated from the distal muscle, with no eosinophilia or increase of ck or crp in the peripheral blood. the pathology of the muscle biopsy revealed myositis and did not indicate granulomas, eosinophilic infiltration, or vacuoles. focal myositis is a type v idiopathic inflammatory myopathy, classified by bohan and peter as miscellaneous myopathies (3). the levels of acute phase reactants and myogenic enzymes, such as ck, and the site of the involved muscle also vary in myositis (1,2,4-10). in this case, the patient's myalgia began in the right gastrocnemius and spread to both lower limbs. morevoer, sekiguchi et al. reported that normal serum crp and ck levels might be associated with mild muscle inflammation in patients with focal myositis (11). although the mechanism of muscle pain is not fully understood, inflammatory muscle pain was related to molecules including interleukin (il)-6, tumor necrosis factor-alpha, il-1beta, and kinin in experiments using animal models (12,13). dina et al. reported a novel experimental model of chronic muscle pain induced by mild acute muscle inflammation (14). psl is presumed to have led to the alleviation of symptoms by suppressing muscle inflammation. some reports have reported an association between chronic hbv infection and myositis (15-17). lamivudine or entecavir, antiviral therapies for hbv infection, improved symptoms in patients with myositis (18,19). reported that the investigation of possible viral etiology via polymerase chain reaction was negative in focal myositis (20). the present patient was an hbv carrier, however, antiviral therapy did not affect his myositis. although electron microscopy of specimens from the second muscle biopsy revealed nemaline rods, we judged the finding to be a non-specific change because his clinical syndrome was not characteristic of nemaline myopathy (24), and there have been several reports concerning the non-specific finding of nemaline rods in inflammatory myopathies (polymyositis and dermatomyositis) (22-25). the majority of focal myositis cases involve a limited lesion, and bilateral lesions are rare. in our case, the patient demonstrated myositis progression from the right gastrocnemius to both sides approximately 1 year after onset and recurrent myalgia, and the myositis had temporary femoral muscle involvement. heffner jr et al. reported six patients with polymyositis beginning as a focal process that rapidly progressed 3-6 months from the onset, and some cases of bilateral focal myositis have been reported (26). additionally, some reports have discussed the difference between focal myositis and polymyositis (27,28). conversely, there are also some reports of polymyositis cases with distal muscle involvement or without elevation of serum ck (29-34). the muscle biopsy in our case showed mononuclear inflammatory infiltrates that predominantly consisted of cd4+t cells. previous studies also reported cd4+-dominant cell infiltration in the muscle in focal myositis (37,38) and dermatomyositis (35,36), but not in polymyositis (39). in our patient, muscle weakness derived from myalgia was present but not progressive, and his immunohistochemical findings indicated focal myositis rather than polymyositis. it appears that focal myositis and polymyositis have different pathophysiological mechanisms; however, whether focal myositis is a subtype of polymyositis remains to be determined. focal myositis usually shows a good response to treatment with psl and nsaids (2). it has a good prognosis and might be a self-limiting disease (1-3). furthermore, the response to nsaids is often good even in cases of repeated relapse (41-43), and some cases of spontaneous remission have occurred (8,49). although the present patient demonstrated a good response to steroid treatment, psl dose reduction induced the recurrence of myalgia. because he experienced repeated myositis relapses when the psl dose was reduced to 15-17.5 mg, it became necessary to use immunosuppressive agents for a steroid-sparing effect. azathioprine and ivig did not affect the patient's myositis. because his hbv infection was successfully controlled with lamivudine, we used mtx and were able to reduce the psl dose. reports of immunosuppressive therapy for focal myositis are limited (6,47,51,52), and only one previous case report, in a child, described mtx treatment for this condition (47). further studies are needed in order to clarify the sparing effect of treatment via mtx in steroid-resistant cases of focal myositis.

this report describes a rare case of recurrent bilateral focal myositis and its successful treatment via methotrexate. a 38-year-old man presented myalgia of the right gastrocnemius in may 2005. magnetic resonance imaging showed very high signal intensity in the right gastrocnemius on short-tau inversion recovery images. a muscle biopsy revealed inflammatory cd4+cell-dominant myogenic change. focal myositis was diagnosed. the first steroid treatment was effective. tapering of prednisolone, however, repeatedly induced myositis relapse, which progressed to multiple muscle lesions of both lower limbs. initiation of methotrexate finally allowed successful tapering of prednisolone, with no relapse in the past 4 years.

PMC5173511

pubmed-749

juvenile idiopathic arthritis (jia) is the most common inflammatory rheumatic disease in childhood, affecting one in 1000 children. jia is characterized by severe joint inflammation in one or more joints, which persists for at least six weeks, with disease onset before the age of 16. this heterogeneous group of diseases can be divided into several subtypes on the basis of clinical symptoms, medical history, and abnormalities in laboratory measures. a biomarker is a small component which is easily measurable in accessible patient material, e.g. blood, urine or saliva, and is ideally obtained using a relatively non-invasive approach. ideally, the component used as a biomarker should be stable over time within the sample, and would be able to be measured by an accurate, reproducible assay, at a relatively affordable cost to health service providers. in addition, the ideal biomarker for paediatric use would not be affected by age-related development of children, avoiding the need for age-specific normal-range data sets. biomarkers are already used in many areas of clinical practice, but most biomarker studies focus on adults rather than children. data from these studies are sometimes extrapolated to children without considering differences in disease pathogenesis, age-dependent changes in reference ranges for biological laboratory measures, growth and development of children over time, effect of ontogeny on disease evolution and response to treatment, and changes in phenotypic gene expression [3, 4]. despite the huge potential of paediatric biomarkers, for jia there are currently no validated paediatric biomarkers available to help in setting up a tailored or a more tailored approach would be beneficial for patients because it could facilitate disease remission at an earlier disease stage, which would reduce burden of disease, limit side effects, and improve quality of life. in this review we will discuss recent developments in the potential use of biomarkers for jia, for predicting the type, severity, and progression of disease after onset and the development of complications linked to jia, and their effect on our ability to predict response to treatment and determine when stable disease remission has been reached. jia is a heterogeneous group of disorders, and its classification relies on both clinical findings and a small number of biomarkers used to divide cases into relatively homogeneous subtypes. for example, the two polyarticular forms of jia, involving five or more joints in the first six months of disease, are distinguished by the absence or presence of serum autoantibody, known as rheumatoid factor (rf), on two occasions three months or more apart. these two clinical subgroups of jia are distinct in their genetics, age of onset, and prognosis [6, 7]. similarly, the presence of positive serum anti-nuclear antibody (ana) has been revealed in several studies to be associated with an increased risk of chronic anterior uveitis in jia. this is a serious comorbidity of jia, involving painless but potentially very damaging inflammation of the anterior chamber of the eye, which requires assiduous screening to prevent permanent visual loss [8, 9]. a recent large cohort study has confirmed earlier studies demonstrating that ana positivity is a risk factor for developing jia-associated uveitis; current uk clinical guidelines for how long screening should be continued include the use of ana positivity to direct clinical practice. thus, both of these antibody biomarkers (rf and ana), which are stable proteins and are easily measured in a small volume of serum, form part of routine clinical care and treatment pathway decisions for jia. at the mild end of the jia clinical spectrum, oligoarticular jia, which presents with involvement of four or fewer joints in the first six months of disease, can lead to widely divergent outcomes, ranging from complete remission off medication, to a more severe, extended form of jia which spreads to involve many joints. extended oligoarticular jia can be highly erosive and destructive and may be difficult to control with conventional disease-modifying antirheumatic drugs (dmards), frequently requiring long-term treatment with biological therapy. several studies have revealed that immunological differences between these two outcomes (persistent versus extended oligoarticular jia) can be observed in the inflamed joint. for example, t cell types (e.g. regulatory t cells, or highly proinflammatory th17 cells) and their frequencies differ significantly between these two clinical types [13, 14]. in addition, differences in cell frequencies, inflammatory protein levels, and gene expression can be detected in children who will develop more severe disease, before extension occurs: for example, in the so-called extended-to-be group of cases, the cd4:cd8 ratio in synovial fluid is lower and the levels of the chemokine ccl5 are higher in extended-to-be oligoarticular jia compared with persistent oligoarticular jia [15]. in this study, analysis of differentially expressed genes at mrna level also provided novel insights into the pathological mechanisms involved in severity, indicating the importance of the complement pathway and of activated monocytes in extension to more severe disease. studies analysing the proteome within synovial fluid have also revealed differences between these subtypes: gibson et al. demonstrated that the proteome and post-translational modifications of proteins differed between those whose disease remained mild and those who went on to develop severe disease. validation of these findings will be required before development of a predictive biomarker test, but this field may well yield valuable biomarkers by which to identify children with a poor prognosis. assessment of disease activity in jia typically includes measurement of inflammatory markers in peripheral blood, including either the erythrocyte sedimentation rate (esr) or c-reactive protein (crp) [17, 18]. both of these inflammatory indices have low sensitivity and specificity, because they can be raised for many reasons other than jia activity and in some children do not closely mirror disease activity. recently, the pro-inflammatory s100 proteins, s100a8/9 (also known as calprotectin or myeloid-related protein (mrp) 8/14) and s100a12, have been described to be sensitive measures for disease activity in jia, and both correlate well with physicians assessment of disease or with actively inflamed joints [5, 19]. s100 proteins can be measured in serum by enzyme-linked immune-sorbent assay (elisa). however, the standardisation of the measurements and the detailed procedures for assay and dilution of serum samples from a wide range of patients, with varying disease activity, are non-trivial, complicating implementation of such a test in routine clinical care. the s100 proteins are released into serum at highly elevated levels in the most severe form of jia, systemic onset jia (sjia), where they correlate well with disease activity as assessed by physicians global assessment of disease activity (r=0.62), childhood health assessment questionnaire (r=0.56), and active joint count (r=0.46)and with crp (r=0.71) and esr (r=0.72) (for all p<0.001). a recent study which included measurement of serum s100 proteins has proposed a biomarker panel for predicting flare in sjia, compared with quiescent disease. the same group also tested the feasibility of measuring such biomarkers in urine for sjia, which could have many advantages for the paediatric population, especially if testing kits could be designed for use in the clinic office or even at home [22]. one of the most severe complications of sjia, known as macrophage activation syndrome (mas) or secondary hemophagocytic lymphohistiocytosis (hlh), remains a cause of mortality in jia and may be difficult to distinguish from infection [23, 24]. a gene expression profiling study, of sjia patients with or without mas compared with control patients, identified clusters of genes that correlated with sjia activity and with mas. if validated, these gene clusters could lead to an mrna-expression biomarker panel for predicting this potentially life-threatening complication, or to distinguish it from other complications. interestingly, some of the differentially expressed mrna species identified, which differed between children with mas and those without, were transcripts of genes known to be involved in other causes of hlh, for example rab27a and sh2d1a. thus, several autoantibodies are already in widespread use as biomarkers in routine care of jia, and some newer biomarkers (protein, cellular, or mrna) are under development, for use in defining disease subtype, probable disease course, or comorbidity. recommendations for treatment of jia are made by the american college of rheumatology (acr), and are regularly reviewed. depending on the number of joints affected and after failure of monotherapy with non-steroidal anti-inflammatory drugs (nsaids) and/or glucocorticoids joint injection in cases of less severe disease the first-line treatment approach for active disease is administration of methotrexate (mtx). in cases of no or poor response to mtx, biological agents, for example drugs which block tumour necrosis factor alpha (tnf), are added to the treatment strategy. although many patients respond well to mtx and reach stable disease remission, approximately 3050% [2729] of patients treated with mtx either do not respond or respond poorly. many studies analysing response to mtx used the core set variables defined by giannini et al., in which levels of response in terms of acr30, 50, or 70 are calculated. those patients who fail to respond to mtx are first exposed to mtx, and experience side effects associated with non-response to mtx, before more effective biological agents can be offered. a biomarker (or set of biomarkers) predicting which patients will respond to treatment would be beneficial for the patient and prevent side effects caused by ineffective drugs. the pro-inflammatory s100 proteins have been shown to correlate well with disease activity [5, 19, 20, 30], and recently the level of s100 proteins in serum has been shown to also be correlated with response to treatment. six months after starting treatment, mrp8/14 levels were lower in sjia patients responding to mtx treatment (defined as reaching at least acr70), whereas mrp8/14 levels in sjia patients not responding to mtx treatment (acr30) were increased or slightly decreased. furthermore, sjia patients treated with anti-il1 or anti-tnf had reduced mrp8/14 levels and disease activity. patients who did respond to mtx had more active joints, higher crp levels, and higher serum cytokine levels before the start of mtx, which suggests that higher disease activity is positively correlated with good response to mtx. in addition, jia patients with high levels of mrp8/14 before starting mtx treatment had a higher chance of good response to mtx and had better disease outcomes (at least acr50) after six months of treatment than non-responders (acr30 or below). differences between patients and response to treatment could be genetic, and therefore genetic factors could be potential biomarkers. one study identified two single nucleotide polymorphisms (snps) in the 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (atic) gene and one snp in the inosine triphosphate pyrophosphatase (itpa) gene which were found to be associated with a higher risk of poor response to mtx treatment. using a validation cohort, one of the snps in the atic gene had a trend towards association with response to mtx. genotyping of snps in genes involved in the polyglutamylation process of mtx and in cellular uptake and efflux of mtx have also shown association with mtx response. one snp in the adenosine-triphosphate-binding cassette transporter b1 (abcb1) and one snp in abcc3 were found to be associated with good response to mtx, whereas an snp in the solute carrier 19a1 was associated with poor response to mtx. in another approach, snp genotyping of genes found to be differentially expressed in a gene expression profiling study, of a uk cohort of jia patients before and after treatment with mtx, identified three snps in the solute carrier family 16 member 7 (slc16a7) gene associated with response to mtx. validation of these snps in a validation cohort revealed significant association of one of the snps with non-response to mtx. a prediction model was recently developed combining clinical and genetic variables to predict non-response of jia patients to mtx. in this study, response was defined as reaching acr70 in at least two out of three visits during the first year of treatment. such a model is important, because it could help in preventing unnecessary treatment of patients who will benefit from monotherapy with mtx and do not need additional treatment with biologicals, which are expensive and might have side effects. the clinical variable included in the model is the esr. genetic variables are snps in the genes coding for methionine synthase reductase, multidrug resistance 1 (mdr-1/abcb1), multidrug resistance protein 1 (mrp-1/abcc1), and proton-coupled folate transporter (pcft). the model had a moderate predictive power of 65% for the validation cohort, which might be because of the small number of patients included in the validation cohort [34]. thus, evidence suggests that the pro-inflammatory s100 proteins have the potential to serve as biomarkers for predicting response to treatment. furthermore, several snps have been found to be associated with response to treatment, indicating that genetic factors can also provide such biomarkers. it is probable that a combination of genetic, biological, and clinical variables will be required to develop accurate and robust predictive algorithms with which to predict response to drug treatment of jia, and these may need to be disease-subtype specific. clinical remission in jia can be reached with the use of medication, and is ideally maintained after treatment is stopped (criteria for clinical remission on and off treatment are described by wallace et al.). however, after discontinuation of mtx treatment, approximately 3050% of patients relapse [2729], suggesting the presence of subclinical disease activity. this subclinical disease activity is not detectable using clinical and standard laboratory tests, and these patients will not reach stable remission off medication [37]. subclinical disease activity makes it difficult to determine when jia patients have reached stable remission and treatment can be stopped. therefore, a biomarker identifying patients at risk of disease relapse on the basis of the inflammatory status of their disease would be extremely valuable for improving paediatric medical care. it has been suggested that continuation of mtx treatment for a longer period of time after clinical remission has been reached might reduce the risk of relapse after stopping treatment. therefore, a randomised clinical trial was conducted to investigate whether longer treatment with mtx, for those who achieved clinical remission, could reduce relapse after mtx withdrawal, and which biomarkers could identify patients at higher risk of relapse after withdrawal. patients were continuously treated with mtx for either six or 12 months after clinical remission was reached, and then followed after withdrawal of mtx. however, there was no significant difference in relapse between the two groups [37]. on the basis of previously published results from this group, which revealed that mrp8/14 is a marker for subclinical disease activity [38, 39], they determined mrp8/14 levels in serum in both groups. interestingly, patients with a high relapse rate after discontinuation of mtx had higher levels of mrp8/14 before stopping mtx than patients who did not flare. thus, serum mrp8/14 levels could be a potential biomarker for predicting which patients will achieve stable remission after withdrawal of mtx, on the basis of inflammatory disease status [37]. similar results regarding the potential of mrp8/14 as a biomarker to identify patients at risk of relapse were described specifically for sjia patients. patients with new onset of active disease or with relapse had higher levels of serum mrp8/14, compared with patients who had reached stable remission. measurements of mrp8/14 levels at time of stopping treatment revealed that, within up to six months, mrp8/14 levels were higher in patients with disease relapse than patients with no disease relapse. prediction of patients at risk of relapse was highly accurate, with a sensitivity of 92% and specificity of 88% using mrp8/14 levels>740 ng ml as a cut-off. another study investigated whether s100a12 and high sensitivity (hs) crp could identify patients who have reached clinical remission on treatment, and are at risk of disease relapse after withdrawal of medication. high s100a12 and mrp8/14 levels were observed in patients who suffered disease relapse within six months of discontinuation of treatment. hscrp levels did not differ between patients with disease relapse and those in remission, but this could be caused by the use of inclusion criteria based on normal hscrp levels, because patients with high hscrp levels are more prone to disease relapse. the patient group which relapsed within three months of stopping treatment had higher median levels of s100a12 and mrp8/14 compared with the group which relapsed later. s100a12, mrp8/14, and hscrp were separately tested for their performance as biomarkers, and s100a12 was the best single biomarker for predicting disease relapse. treatment strategies for jia have the objective of full clinical remission, but only a very small number of patients remain in clinical remission, off medication, for long periods of time. a recent study compared the transcriptional profiles of patients with active disease or those in clinical remission on medication (achieved by treatment with mtx or mtx plus tnf blockade) with the profiles of healthy children. differences in transcriptional profile were observed between patients with active disease and those in clinical remission on medication, and also between those in remission off treatment and healthy children. interestingly, the same group also demonstrated differences in transcriptional profile between patients in clinical remission induced by treatment with mtx or with mtx plus tnf inhibitor, and healthy individuals. this study confirmed previous findings from the group that the inflammatory status of patients in remission does not return to normal. network analysis suggested a function for hepatocyte nuclear factor 4 alpha (hnf4), which is expressed by t cells and granulocytes, in controlling genes associated with remission. such tests need to be reliable, simple to perform, economically feasible, and robust. in addition, they need to be tested and validated for large cohorts of children with jia. where proved reliable, biomarkers may assist with predicting disease type, course or severity, with predicting response to medication and therefore aiding treatment choices, and with accurate identification of children who can safely stop medication once apparent clinical remission is reached. the accurate choice of medicines most likely to work for each child, to enable the achievement of rapid remission for all, while avoiding unnecessary exposure to toxic drugs for those who do not need them and reducing unopposed inflammation to a minimum, is now the ultimate objective of modern management of arthritis. to move rapidly towards such ambitious objectives, multi-centre and international collaborations are needed to give every child with jia the opportunity to take part in biomarker and cohort studies.

use of biomarkers in clinical practice has proved extremely valuable and is a rapidly expanding field. however, despite the huge potential of biomarkers, for juvenile idiopathic arthritis (jia) there are currently no validated paediatric biomarkers available to help with setting up a more tailored approach on which drug choice could be based, to achieve remission early in the course of disease. early remission reduces burden of disease, limits side effects from toxic and unnecessary medication, and, most importantly, enhances quality of life. several studies have suggested promising biomarkers: these may be a protein, cellular component, mrna, or genetic component, for example a single nucleotide polymorphism (snp). here we describe recent developments in the use of biomarkers for jia and their potential to assist in management of disease by predicting disease phenotype, severity, progression, and response to treatment, and determining when patients have reached stable remission and can safely discontinue treatment.

PMC3930839

pubmed-750

a 29-year-old man (contact 1) transported 8 dromedaries from oman to united arab emirates on may 7, 2015 (table 1). the same day, as part of a national policy for controlling mers, samples were collected from the dromedaries at a screening center located at the united arab emirates border. the samples were tested by reverse transcription pcr (rt-pcr) on may 10 and found to be positive for the mers-cov open reading frame (orf) 1a and upstream of e genes (10). this finding led local public health authorities to conduct active surveillance on humans who had contact with the infected dromedaries.*contacts 1 and 2, humans who had direct physical contact with infected dromedaries; mers-cov, middle east respiratory syndrome; rt-pcr, reverse transcription pcr. a sputum sample collected from contact 1 on may 10, 2015, was tested by rt-pcr on may 12 and found to be positive for mers-cov; the man was admitted to a hospital the same day. follow-up respiratory samples obtained on may 13 and 14 were still rt-pcr positive, but a sample obtained on may 18 was negative. the patient was asymptomatic at hospital admission and throughout his hospital stay (technical appendix). contact 2 was a 33-year-old man who worked at the screening center mentioned above. he had direct contact with the same group of infected dromedaries during the sampling procedures. a nasal aspirate sample was obtained from the man on may 14, 2015, and found to be rt-pcr positive for mers-cov. a follow-up sample obtained on may 18 was rt-pcr negative for mers-cov. samples from 32 other persons were also tested by rt-pcr (technical appendix). seven days later (may 14), follow-up nasal swab samples from 5 dromedaries were still positive by rt-pcr (table 2); the animals also had mucopurulent nasal discharge. the animals were tested for the presence of mers-cov specific neutralizing antibodies (11); all were seropositive. two 4-month-old calves (adfca-hku1 and adfca-hku2) had the highest virus loads by real-time rt-pcr and the lowest neutralizing antibody titers (table 2). positive by rapid antigen testing (12), which suggests the calves were still shedding virus 7 days after the first detection of virus. the 2 calves were rt-pcr negative for mers-cov, and the whole group of camels was released from quarantine.*ct, cycle threshold; i d, identification; mers-cov, middle east respiratory syndrome; neg, negative; pos, positive; rt-pcr, reverse transcription pcr. results were determined by open reading frame 1a and upstream of e gene rt-pcr assays; ct values were from the open reading frame 1a assay. results were determined by immunochromatographic tests for mers-cov nucleocapsid protein (12). respiratory specimens from the 2 infected humans and the 5 dromedaries that were still positive at the second sampling were analyzed by dideoxy sequencing as previously described (13); the nucleocapsid gene sequences of all dromedary samples were found to be identical. samples from dromedaries adfca-hku13 were selected for further analysis, and a sequence contig encompassing the 3 end of the orf1ab gene through the 3 untranslated region of the mers-cov genome (8,900 nt; sequence coverage 4) was obtained from each sample. contigs from the 3 samples were identical, with the exception of a v221i (gttatt) mutation in the orf4b protein of the sample from dromedary adfca-hku2. the viral rna content of the 2 human samples available for analysis was too low to provide long pcr amplicons (cycle threshold 35.5 and 36.9 by upstream of e gene assay). however, partial sequences of mers-cov spike (466 nt, contacts 1 and 2), orf34a (273 nt, contact 1), and nucleocapsid (451 nt, contacts 1 and 2) gene regions could be detected from the samples. the 3 sequence contigs obtained from the dromedary samples were phylogenetically closely related to those of viruses detected in humans in the saudi arabia, china, and south korea in 2015 (figure). all sequences from this cluster, together with the partial orf34a sequence detected in the sample from contact 1, shared 2 cluster-specific mutations, 79s (tcatct) and p86l (cct ctt), in the orf3 protein, suggesting that these viruses may share a common lineage. apart from the unique v221i mutation, the sequences for viruses from the 3 dromedaries shared a unique orf4a-q102e (gagcag) mutation that was not found in any published mers-cov genomes. other than those mutations, all of the orfs (nonstructural protein 13, spike, orf3, orf5, envelope, membrane, nucleocapsid, and orf8b) of these virus sequences were unremarkable. phylogenetic analyses of partial middle east respiratory syndrome coronavirus (mers-cov) genomic sequences for viruses detected in dromedaries imported from oman to united arab emirates, may 2015. a partial viral rna sequence spanning the 3 end of the open reading frame 1ab gene through the 3 untranslated region of the mers-cov genome (8,900 nt) was used in the analysis. the phylogenetic tree was constructed with mega6 software (http://www.megasoftware.net/) by using the neighbor-joining method. symbols indicate mers-covs detected from dromedaries s. scale bar indicates the estimated genetic distance of these viruses. we report 2 cases of mers-cov infection in men who had direct contact with the same group of infected dromedaries. neither man had a concurrent medical condition or a history of exposure to human mers cases in the 14 days before their first mers-cov positive test results. genomic sequences for the viruses derived from the men and dromedaries and findings from the epidemiologic investigation suggest possible zoonotic transmission of mers-cov from dromedaries to humans. although it is unlikely, we can not exclude the possibility that the men and dromedaries were independently infected by other sources. both infected humans were kept in the hospital for 2 incubation periods and were asymptomatic during this period. clinical observations and positive rt-pcr results suggest that the men were asymptomatically infected with mers-cov. our findings provide further evidence that asymptomatic human infections can be caused by zoonotic transmission. nonetheless, our findings highlight the importance of systematic surveillance of persons who have frequent contact with dromedaries. a recent study demonstrated that persons who have frequent exposure to camels are more likely than the general population to be seropositive for mers-cov (4). the unique border screening program and multisectoral collaborations highlighted in this investigation serve as a model for effective mers-cov surveillance at the animal human interface. we did not test serum samples from the human contacts; such testing would be of interest for follow-up investigation of the patients serologic responses. we also obtained limited rna samples from these persons, which prevented us from conducting more extensive viral sequence analyses. mers-cov genomic sequences determined in this study are similar to those of viruses detected in 2015 in patients in saudi arabia and south korea with hospital-acquired infections. the infected dromedaries in this study were imported from oman, which suggests that viruses from this clade are widely circulating on the arabian peninsula. sequence analyses of mers-covs found in south korea and china do not suggest that viruses from this clade are necessarily more transmissible variants (15). however, given that a single introduction of mers-cov from this clade caused>180 human infections in hospital settings (2) and that viruses of this clade are causing other human infections in saudi arabia, further phenotypic risk assessment of this particular mers-cov clade should be a priority. additional information regarding 2 persons with asymptomatic mers-cov infection and other persons tested in the study.

in may 2015 in united arab emirates, asymptomatic middle east respiratory syndrome coronavirus infection was identified through active case finding in 2 men with exposure to infected dromedaries. epidemiologic and virologic findings suggested zoonotic transmission. genetic sequences for viruses from the men and camels were similar to those for viruses recently detected in other countries.

PMC4672428

pubmed-751

immune (idiopathic) thrombocytopenic purpura (itp) is an autoimmune disorder characterized by persistent thrombocytopenia due to antibody binding to platelet antigen(s) and causing their premature destruction by the reticuloendothelial system, particularly in the spleen. the american society for hematology guidelines define itp as isolated thrombocytopenia with no clinically apparent associated conditions or other causes of thrombocytopenia. therefore, itp is a condition generally diagnosed by exclusion of the numerous other causes of thrombocytopenia, such as infections, medications, hematological malignancies, disseminated intravascular coagulation, and other autoimmune conditions. in itp, it is estimated that thrombocytopenia (defined as a platelet count less than 150 10/l) occurs in approximately 7% of pregnant women, with 74% of those with low platelet counts having incidental thrombocytopenia of pregnancy that can be managed routinely and in which the platelet count remains more than 70 10/l. additional causes of thrombocytopenia include complications of hypertensive disorders in pregnancy (21%) and immunological disorders of pregnancy, including itp, systemic lupus erythematosus, and other secondary causes of immune thrombocytopenia (4%). itp occurs in 1 to 2 of every 1000 pregnancies, which in the united states represents about 3000 to 6000 cases of itp in pregnancy per year. several studies have examined pregnancy outcomes for women with itp and found that most pregnancies were uneventful, with successful outcomes for both mothers and children [68]. the majority of studies used data from single medical centers [6, 9, 10], by employing retrospective analyses of medical charts [1015]. most studies have not differentiated between incidental thrombocytopenia, itp, and citp in women who have low platelet counts during pregnancy, and, to our knowledge, there are few published reports regarding the outcomes of pregnant women with citp. the primary objective of this study was to examine the pregnancy and birth outcomes of women with itp and citp identified from medical claims and abstracted medical records from a large health plan in the united states. our hypothesis was that there would be no significant difference in adverse pregnancy outcomes, including congenital anomalies, between the two groups of women (itp versus citp). the method of data ascertainment enabled the inclusion of data from multiple medical centers, thus increasing the size of the patient cohort and extending the study to a broader population. we studied pregnancy and birth outcomes among women with itp or citp over 16 years of age from 1994 to 2009 inclusive. the data for this study were derived from a large us health insurance database based on eligibility, pharmacy claims, and medical claims data. this study utilized stork (systematic tracking of real kids) to identify pregnancies and link the health experience of mothers with that of their infants within an administrative claims database in order to study the effects of maternal exposures and health conditions on pregnancy outcomes [16, 17]. further details about stork are provided in supplementary material available online at http://dx.doi.org/10.1155/2016/8297407. after obtaining institutional review board (irb) approval and waiver of patient authorization from the affiliated privacy board, the study population consisted of women with at least one diagnostic claim for itp (icd-9 code 287.31 or 287.3 if used before 01/12/2005) and at least one diagnostic or procedure code related to pregnancy (live birth, spontaneous abortion, stillbirth, or therapeutic abortion) in the database between 1995 and 2009. to be eligible for the study, women with at least one claim for itp had to fulfill the following criteria: have complete medical and pharmacy benefit coverage, have at least 280 days of continuous enrollment before the pregnancy outcome date, and have a 9-month baseline period prior to the estimated date of conception. if a woman had more than one pregnancy during the study period, analyses included the first pregnancy after the first itp claim, ignoring any subsequent pregnancies. if a woman's first itp claim was later than the end date of her last pregnancy, she was not eligible for this study. the database was then searched for infants associated with the deliveries and linked them to the mother's claims. linkage between mother's and newborn's claims could not be performed if the infant was enrolled in another health insurance plan, as may happen if the partner has a different health insurer. patients were classified as having citp if they met at least one of these requirements: two or more claims for itp separated by at least 6 months; a claim for itp and treatment occurring at least 6 months later with one or more of these medications: corticosteroids, anti-d antibody, rituximab, danazol, colchicine, and dapsone; and/or a claim for splenectomy after the first diagnostic itp claim. indicators for the timing of the itp or citp diagnosis relative to pregnancy were based on the estimated date of conception and the end of pregnancy. patients were classified as having itp or citp before pregnancy if they met the full diagnostic criteria before the estimated date of conception. patients were classified as having itp or citp during pregnancy if they met the full diagnostic criteria before the end of the pregnancy. women who had an initial claim for itp prior to delivery or termination of pregnancy but met the full diagnostic criteria for citp after pregnancy were considered to have itp during pregnancy, but not citp, since they did not meet the full criteria until after the pregnancy ended. the primary outcome of interest was the prevalence of major congenital anomalies (mcas) at birth (i.e., anomalies that cause significant functional or cosmetic impairment, require surgery, or are life-limiting). other birth outcomes evaluated included 3 or more minor congenital anomalies, preterm birth (< 37 weeks of gestation), low birthweight (< 2,500 grams), and measurements consistent with small for gestational age (sga) status (weight, length, or head circumference below the 10th percentile for sex and gestational age) among infants born to mothers with itp or citp. pregnancy outcomes in this study included live born infant, spontaneous abortion, elective termination, and stillbirth. if the claims search did not identify a pregnancy outcome, the pregnancy outcome was classified as unknown. claims were reviewed to identify medical providers and facilities to query from them medical records to confirm pregnancy outcomes and to collect additional data on pregnancy characteristics and infant follow-up for the first year of life. each patient's pregnancy medical records were abstracted to obtain relevant covariate data from their prenatal and pregnancy outcome history, including estimated date of conception and date and type of pregnancy outcome. for infants with evidence of a congenital anomaly diagnosed within the first 12 months after birth, as identified through icd-9 diagnosis or procedure codes in the medical claims, the medical record was sought from the physician or hospital where the anomaly was diagnosed. a standardized medical record abstraction form was used to record elements from each mother's chart, including the reported estimated date of conception, type of pregnancy outcome, and other pregnancy characteristics from relevant clinician notes, inpatient records, and/or hospital discharge summaries. patient information drawn directly (without abstraction) from deidentified medical records of infants with claims-based congenital anomalies was combined with claims data and reviewed by a dysmorphologist to validate the diagnosis. following medical record review, all pregnancies were classified according to covariates describing maternal characteristics, pregnancy characteristics, and maternal comorbidities and according to whether they were identified through claims or medical chart abstraction. maternal characteristics included age and year at conception, geographic region of health plan, and ethnicity. pregnancy characteristics included use of prenatal vitamins, amniocentesis, chorionic villus sampling, fetal monitoring, ultrasonography, alpha-fetoprotein (afp) testing, obstetric panel, multiple gestation, high-risk pregnancy supervision, antepartum hemorrhage, abruptio placentae, placenta previa, excessive vomiting, early or threatened labor, late pregnancy, measurements consistent with sga, disorders relating to short gestation and unspecified low birthweight, and total cost of care for up to 280 days before delivery. maternal comorbidities such as diabetes, hypertension, and infections, along with substance abuse and receipt of teratogenic drugs during pregnancy, were identified using diagnostic codes and medication claims. frequency analyses were carried out for categorical variables and means and standard deviations were calculated for continuous variables, such as the number of physician visits. data were analyzed separately for women who fulfilled the criteria for itp or citp prior to pregnancy and those who fulfilled the criteria during pregnancy. for infants with claims of congenital anomalies, data were categorized according to maternal age at conception as follows: 14 years and under, 5-year intervals from 15 years through 49 years, and over 50 years. we identified 585 women with at least one claim of itp and claims indicating pregnancy during the study period (january 1, 1994, through december 31, 2009). the remaining 446 women made up the claims-based itp cohort for this study. medical records were sought to provide more detailed information on the patients ' pregnancy characteristics not captured in the claims data, and charts for 311 of 446 women (69.7%) were obtained. figure 1 provides a schematic view of the claims-based pregnancy outcomes observed in this cohort of 446 women with claims for itp and the results of chart-based review. among the 311 women with charts, outcome data were unavailable for 42 of them. of the remaining, 260 charts had a confirmed live birth and 9 (3.3%) indicated a fetal loss with no further details. among the 446 pregnancies in women with a claims-based diagnosis of itp the claims-based diagnosis was available in 432 cases before or during the pregnancy (table 1). approximately half of the women reside in the south/southeast regions of the united states and, of those with ethnicity reported in the enrollment data, approximately 70% are caucasian. of the 446 women, 84 (18.8%) were identified as having citp before or during pregnancy. of all 446 pregnancies with claims-based diagnosis of itp, 346 (77.6%) indicated a live birth (table 2). when stratified by timing of itp diagnosis, live births were more frequent among women diagnosed with itp during pregnancy (290 of 357 or 81.2%) than in pregnant women diagnosed with itp before pregnancy (56 of 89 or 62.9%). of those who were diagnosed with itp during pregnancy, 14/357 (3.9%) were fetal losses compared with 10/89 (11.2%) of those diagnosed with itp prior to pregnancy. the magnitude and trend of this difference (7.3%) were not found among women with citp (9/66 or 13.6% of those diagnosed during pregnancy resulting in fetal loss compared to 2/18 or 10.2% among those diagnosed prior to pregnancy). the prevalence of low birthweight was higher among women with a diagnosis of itp before pregnancy (10 of 56 or 17.9%) compared to women with a diagnosis of itp during pregnancy (28 of 290 or 9.7%). the prevalence of low birthweight in women with citp was similar in both groups, but the sample size was too small to reach a conclusion. table 3 presents the prevalence of major congenital anomalies (mcas) among the 346 infants of mothers with claims for itp. there were 27 infants (n=27/346; 7.8%) who had a claim for at least one major malformation and charts were available for 17 of them. the most frequent claims were for ostium secundum type atrial septal defects [n=10/346 (2.9%)], hypospadias [n=8/151 (5.3%)], patent ductus arteriosus [n=6/346 (1.7%)], and ventricular septal defect [n=4/346 (1.2%)]. a total of 4 infants (1.2%) had claims for 3 or more major malformations. of all infants of mothers with claims for itp that were assumed to have no mca and whose charts were obtained (n=336), 10 (3.0%) had at least one chart-confirmed major malformation (table 4). the most frequent of these confirmed mcas were hypospadias [n=4/336 (1.2%)], ventricular septal defect [vsd, n=3/336 (0.9%)], and patent ductus arteriosus [pda, n=2 (0.6%)]. two infants (0.6%) had 3 or more confirmed major malformations. among the subgroup of 68 infants of women with claims for citp, 7/68 (10.3%) the most frequent claims were for hypospadias [n=4/30 (13.3%)], ostium secundum type atrial septal defects [n=2/68 (2.9%)], and patent ductus arteriosus [n=2/68 (2.9%)]. two of these 68 infants (2.9%) had claims for 3 or more major malformations. of all infants of mothers with claims for citp that were assumed to have no mca and whose charts were obtained (n=66), 4 (6.1%) had at least one chart-confirmed major anomaly (table 4). the most frequent of these major congenital anomalies was hypospadias [n=3/66 (4.5%)]. there was no statistically significant difference between the prevalences of malformations in the infants of mothers with itp compared to those with citp, regardless of whether the data was claims-based or chart-based (table 4). although severe maternal or neonatal bleeding is rare when pregnant women with itp are managed by an expert team, a recent questionnaire of women with itp revealed that 14/50 women were advised to avoid becoming pregnant. our study is unique because of the availability of data on congenital anomalies in infants born to mothers with itp and citp. in addition, the sample size of the population we evaluated is larger than previously published. the findings of this study are consistent with other published reports. in one prior publication, preterm birth was present in 16/58 (27.6%) infants born to mothers with itp diagnosed prior to pregnancy and in 15/75 (20%) infants born to mothers with itp diagnosed during pregnancy, with an overall prevalence of 23.3%. premature birth affects 510% of newborns in most developed countries and approximately 12% of live births in the united states. therefore, a prevalence of prematurity of 23.3% among pregnancies complicated by itp represents approximately a 2- to 4-fold increased frequency. in a study by debouverie et al. of 50 women with citp who had platelet counts below 150 10/l for at least one year, there were no fetal deaths in 62 pregnancies but 9 (14%) were premature, 6 (9%) were small for gestational age, and 2 (3%) demonstrated evidence of hemorrhage. in a group of women treated for severe thrombocytopenia (platelet count<10 10/l) during pregnancy, there were 29 live births from 31 pregnancies, with a mean gestational age of 36.5 weeks (range, 743 weeks). there was one missed abortion at 7 weeks and one termination because of intrauterine death at 16 weeks of gestation. this child was born to a mother who had complications of a bleeding gastric ulcer due to severe thrombocytopenia (platelet count<20 10/l) and who died from acute pulmonary edema following a caesarean section. the remaining 28 newborns had no complications. thrombocytopenia attributed to aplastic anemia or myelodysplasia was associated with a 53.8% rate of premature birth compared to incidental thrombocytopenia in pregnancy (11.3%) and itp (16.7%). contrasted the characteristics of infants born to 57 mothers with itp diagnosed before pregnancy to 75 women diagnosed with itp during pregnancy. in the group of mothers with itp diagnosed prior to pregnancy, there were 2 intrauterine fetal deaths at 224 and 247 days of pregnancy and a higher frequency of infants requiring admission to the neonatal intensive care unit (20/57 or 34.48% versus 12/75 or 16%; p=0.01). maternal age and platelet count, gestational age at delivery, 5-minute apgar scores<7, rate of caesarean deliveries, mean neonatal birthweight, and the mean neonatal platelet count did not differ between the two groups. there were three neonates (3/84; 2.3%) with platelet counts<50 10/l that were all born to mothers with itp diagnosed prior to pregnancy, but there were no severe bleeding complications and no intracranial hemorrhage in the infants. however, maternal itp refractory to splenectomy has been correlated with a higher risk of intracranial hemorrhage in the infants. conducted a retrospective study of women with itp in pregnancy, most of whom (83/92) had itp prior to pregnancy. there were 119 pregnancies and two fetal deaths: one stillbirth at 39 weeks of gestation and a stillbirth at 27 weeks of gestation that had extensive hemorrhage throughout the brain, born to a mother with a 4-year history of severe itp (postsplenectomy) and platelets counts<50 10/l. there were no reports of fetal malformations and the authors estimated that the fetal loss rate was approximately 1-2%. no case of hydrocephalus was found in the present study. hydrocephalus can occur as a rare complication of intracranial hemorrhage in fetuses born to mothers with itp. kim and choi described a neonate with severe thrombocytopenia (platelet count 1 10/l), multiple bruises on the face and scalp, widespread petechiae, cleft palate, and moderate to severe hydrocephalus without evidence of intraventricular hemorrhage. computerized tomography confirmed severe hydrocephalus without significant compression of the brain parenchyma, diffuse ischemia, and encephalomalacia of both cerebral hemispheres. ostium secundum, a type of atrial septal defect (asd), is found in less than 1% of newborns in the general population. in our study, this congenital heart defect occurred approximately three times more frequently among infants born to mothers with itp. in infants born to mothers with citp, the incidence of ostium secundum was 2/68 (2.9%) and there were two additional infants with ostium secundum plus other cardiac malformations (table 3). ventricular septal defect and patent ductus arteriosus were also noted in other babies born to mothers with citp (table 3). however, the numbers in the group of citp pregnancies were small and the significance of this finding is unclear. this study provides a claims-based and chart-based evaluation of pregnancy outcomes among women with itp and citp in the united states. the wide range inclusion criteria and large source population (more than 1.2 million pregnancies) allowed us to obtain results that reflect pregnancy outcomes broadly among women with itp and citp. however, the database has some limitations and valuable information may be missing or misdiagnosed. however, there were a small number of pregnancy cases in women with itp and citp with no linkage to births. in addition, data from women with different medical or pharmacy coverage and from those without medical coverage could not be assessed by this study. anomalies documented in live newborns and those requiring billable medical intervention were assessed in this study. however, minor or major anomalies that may have occurred in stillborn infants and in fetuses of elective or spontaneous abortions were not available for analysis. in addition, clinical validation of the diagnoses of itp and citp by a specialist was not feasible in this study. while referral to a maternal-fetal specialist is likely for these patients, the documentation from specialists was generally not included in the primary obstetrical records. finally, the dataset did not include maternal medical conditions other than itp, nor medical treatments performed during the pregnancy for itp or other conditions. therefore, it was not possible to evaluate their potential effects on the pregnancy and birth outcomes. based on the evaluation of 446 pregnant women with itp, a diagnosis of itp or citp prior to their estimated date of conception may indicate a higher risk for stillbirth or fetal loss, premature delivery, and infants with specific congenital anomalies than an itp diagnosis during pregnancy. therefore, the results of this study provide further evidence that the duration of maternal itp may be an important determinant of the outcomes of pregnancy.

objective. to examine pregnancy and birth outcomes among women with idiopathic thrombocytopenic purpura (itp) or chronic itp (citp) diagnosed before or during pregnancy. methods. a linkage of mothers and babies within a large us health insurance database that combines enrollment data, pharmacy claims, and medical claims was carried out to identify pregnancies in women with itp or citp. outcomes included preterm birth, elective and spontaneous loss, and major congenital anomalies. results. results suggest that women diagnosed with itp or citp prior to their estimated date of conception may be at higher risk for stillbirth, fetal loss, and premature delivery. among 446 pregnancies in women with itp, 346 resulted in live births. women with citp experienced more adverse outcomes than those with a pregnancy-related diagnosis of itp. although 7.8% of all live births had major congenital anomalies, the majority were isolated heart defects. among deliveries in women with citp, 15.2% of live births were preterm. conclusions. the results of this study provide further evidence that cause and duration of maternal itp are important determinants of the outcomes of pregnancy.

PMC4820621

pubmed-752

a 2-year-old girl presented with partial seizures that manifested as twitching of the left angle of the mouth and jerking of the left upper limb but usually did not result in a loss of consciousness. the child s motor and cognitive development were within the normal ranges. at the age of 3 years, she was referred to our center with the aim of achieving control of her seizures. brain magnetic resonance imaging (mri) showed focal cortical dysplasia in the right centroparietal region (figure 1a), while interictal tc-ethyl cysteinate dimer single-photon emission computed tomography (spect) did not detect any abnormalities (data not shown). a, at the age of 3 years, a t2-weighted mri scan showed focal cortical dysplasia in the right centroparietal region (). b, on day 2 after admission, a t2-weighted mri image showed a high signal intensity lesion () adjacent to the focal cortical dysplasia lesion. c, apparent diffusion coefficient mapping also showed a high signal intensity lesion in the same region. d, the high signal intensity lesion () was located close to the right insular cortex (left). on day 69, the high signal intensity lesion had diminished in size, and the patient s cluster seizures had improved (right). dimer spect detected hyperperfusion in a region containing the high signal intensity lesion seen on mri, which was located close to the right insular cortex. interictal tc-ethyl cysteinate dimer spect performed on day 68 did not detect any abnormalities. mri indicates magnetic resonance imaging; spect, single-photon emission computed tomography. at the age of 10 years and 5 months, the patient was admitted because of a sudden worsening of her seizures. on admission, she was taking phenytoin (250 mg/d) in combination with valproate (400 mg/d). an ictal eeg showed rhythmic fast waves arising from the right central region, which sometimes evolved into diffuse slow waves corresponding to asymmetric bilateral tonic seizures (left>right). the patient s seizures did not respond to the intravenous administration of midazolam, lidocaine, and phenobarbital. on the second day after admission, t2-weighted mri and apparent diffusion coefficient mapping revealed a high signal intensity lesion adjacent to the focal cortical dysplasia lesion (figure 1b-d, left). diffusion-weighted imaging also revealed a high signal intensity lesion in the same region (data not shown). an eeg performed at the onset of the seizure showed rhythmic fast waves arising from the right central region. ten seconds after the onset of the seizure, when the epileptic activity evolved into diffuse slow waves, the patient s baseline sinus heart rate of about 100 beats/min suddenly dropped to 40 to 50 beats/min, and she started to exhibit an idioventricular rhythm. immediately after the seizure activity disappeared, the patient s cardiac rhythm returned to a normal sinus rhythm (figure 2). oxygen desaturation (an oxygen saturation value of<92%) due to apnea (defined as breath arrest lasting>10 seconds) also occurred after the appearance of ictal bradycardia. cardiac assessments, including continuous 24-hour ambulatory electrocardiography (ecg) monitoring and echocardiography, did not detect any abnormalities between the seizures. a, an eeg performed at seizure onset showed rhythmic fast waves arising from the right central region. b, about 10 seconds after seizure onset, when the patient s epileptic activity evolved into diffuse slow waves, her baseline sinus heart rate of about 100 beats/min suddenly dropped to 40 to 50 beats/min and then exhibited an idioventricular rhythm. c, after the seizure activity disappeared (68 seconds after onset), the patient s cardiac rhythm returned to a normal sinus rhythm. between 3 and 5 days after the patient s admission, the frequency of her seizures increased to 5 to 10 an hour. the patient s ictal bradycardia and apnea were usually accompanied by seizures, despite the prompt discontinuation of phenytoin and lidocaine. the idioventricular rhythm was observed in about half of the seizures. on the sixth day after admission, when a brief period of ictal asystole occurred, barbiturate (sodium thiopental) therapy was started while the patient was under mechanical ventilation. the frequency of the patient s seizures decreased to once or twice an hour after the initiation of the barbiturate therapy (5 mg/kg/h), and her ictal bradycardia subsided. after the continuous intravenous infusion of sodium thiopental for 48 hours, the dose was gradually reduced. however, the patient s bradycardia was less severe than it had been previously, her heart rate only fell to around 60 to 70 beats/min, and she did not develop cardiac arrhythmia. on the 25th day after admission, ictal spect performed on day 27 showed hyperperfusion in the area containing both the focal cortical dysplasia lesion and the high signal intensity lesion seen on mri (figure 1e). the frequency of the patient s seizures was reduced via the continuous infusion of midazolam and high-dose phenobarbital, and hence, her ictal bradycardia subsided again. interictal spect performed on day 68 did not reveal any abnormalities (figure 1e). on day 69, the high-intensity lesion adjacent to the focal cortical dysplasia lesion was found to have diminished in size on follow-up mri (figure 1d, right). interictal f-fluorodeoxyglucose positron emission tomography/computed tomography was performed 2 months after discharge and detected hypometabolism in the right centroparietal region but not in the right insular cortex (data not shown). she underwent surgery at the age of 13 years and 1 month, and the lesion was partially resected. after surgery, the patient had infrequent partial seizures that were similar to those observed before surgery but no longer developed cluster seizures. ictal bradycardia is considered to be rare in children because there have only been a few reported pediatric cases. however, a recent study found that ictal bradycardia occurred in 8.2% of the pediatric patients with epilepsy admitted to a pediatric epilepsy monitoring unit. another comparative study reported that ictal bradycardia occurred more often in children (23.8% of pediatric seizures) than in adults (7.3% of adult seizures). in adults, ictal bradycardia was found to be more strongly associated with seizures originating from the temporal lobe, while in children it was more prevalent in patients having seizures of extratemporal origin. the risk factors for ictal bradycardia include seizure clustering, having suffered with epilepsy for a long period, taking multiple antiepileptic drugs, a younger age at seizure onset, and refractory epilepsy. the propagation of seizure activity into the autonomic centers that regulate the cardiovascular system is assumed to be the leading mechanism of ictal bradycardia. in 1990, oppenheimer and cechetto demonstrated that stimulation of the left insular cortex evoked bradycardia in rats. the same authors later showed that perioperative stimulation of the insular cortex tended to result in heart rate changes (left insular cortex: bradycardia and right insular cortex: tachycardia) in patients with epilepsy. although conflicting results have been obtained regarding the left- or right-sided predominance of ictal bradycardia, several other studies have suggested that the insular cortex plays an important role in cardiac regulation in humans. however, to the best of our knowledge, there has only been 1 case report of ictal bradycardia due to an insular cortex lesion, which involved a 3-year-old girl with focal cortical dysplasia in her right insular cortex. some antiepileptic drugs, such as carbamazepine, lamotrigine, phenytoin, and lidocaine, are known to act as sodium channel blockers and can have side effects that affect the cardiac conduction system. our patient was receiving phenytoin and lidocaine for her cluster seizures when the first episode of ictal bradycardia occurred. however, phenytoin and lidocaine are unlikely to have caused the bradycardia experienced in the present case due to (1) the prolonged (> 2 weeks) persistence of the bradycardia, despite the prompt discontinuation of the above-mentioned drugs and (2) the absence of cardiac arrhythmia between seizures according to continuous 24-hour ambulatory ecg monitoring. focal cortical dysplasia constitutes the most important cause of intractable localization-related epilepsy in childhood. a recent study reported that ictal spect is useful for defining the epileptic zone in a high proportion of children with focal cortical dysplasia who undergo surgical evaluation. in our patient, the high signal intensity lesion seen on t2-weighted mri image appeared when the patient had cluster seizures and diminished in size as her seizures improved, suggesting that these mri changes were related to seizure clustering. ictal spect detected hyperperfusion in the area containing the high signal intensity lesion depicted on mri, which was located very close to the right insular cortex. the ictal bradycardia experienced by our patient might have been caused by the following mechanism: repetitive seizure activity might have expanded from the hyperperfusion zone into the right insular cortex, which controls cardiac rhythm, resulting in ictal bradycardia. the autonomic alterations and heart rate changes seen during epilepsy are complex and are not fully understood. although the presence of hyperperfusion on ictal spect might represent zones of seizure propagation, there have not been any reports about the ictal spect findings of patients with ictal bradycardia. in our patient, ictal spect demonstrated hyperperfusion in an area that was close to the insular cortex but did not involve the insular cortex itself. subtraction ictal spect coregistered to mri analysis also failed to detect ictal hyperperfusion in the right insular cortex (data not shown). one possible reason for this is that the patient s bradycardia was less severe at the time of the ictal spect study than it had been previously. although the seizures experienced by the patient after the injection of the radiotracer used for the spect produced similar symptoms and paroxysmal eeg discharges to the previous seizures, the bradycardia induced was mild (the patient s heart rate only fell to 60-70 beats/min) and did not lead to cardiac arrhythmia. thus, seizures involving more severe ictal bradycardia might have been found to involve the insular cortex on spect. in this study, ictal and interictal spect were performed during the patient s cluster seizures (day 27), when ictal bradycardia was observed, and on day 68, when the ictal bradycardia had gone into remission. however, no ictal spect scans were performed after the patient s ictal bradycardia went into remission because she did not experience seizures often during the follow-up period. comparing the 2 ictal spect scans might have helped us to clarify the origin of the patient s ictal bradycardia and why her ictal bradycardia was transient. further studies with ictal spect are necessary to elucidate the pathophysiology of ictal bradycardia in patients with epilepsy, providing that clinical conditions allow it.

ictal bradycardia, which is considered to be one of the causes of sudden unexplained death in epilepsy, is rare. a 10-year-old girl with focal cortical dysplasia in her right centroparietal region developed transient ictal bradycardia during cluster seizures. brain magnetic resonance imaging demonstrated a high signal intensity lesion adjacent to the focal cortical dysplasia lesion. ictal 99mtc-ethyl cysteinate dimer single-photon emission computed tomography (spect) detected hyperperfusion in an area containing the high signal intensity lesion, which was located close to the insular cortex. since the hyperperfusion zone observed on spect was considered to reflect seizure propagation, it is possible that the ictal bradycardia experienced in the present case was caused by the following mechanism: the repetitive seizure activity caused the high-intensity lesion seen on mri to expand into the right insular cortex, which controls cardiac rhythm, resulting in ictal bradycardia.

PMC5417018

pubmed-753

attention has widespread effects on brain activity. from thalamus and colliculus to many regions of cortex, for example, responses to visual input are enhanced when this input is relevant to behavior (oconnor et al., 2002; ignashchenkova et al., 2004; roelfsema et al., 1998; moran and desimone, 1985). often, attentional modulations grow over time from stimulus onset as the appropriate attentional focus is established (roelfsema et al., 1998; schall et al., 1995). in some cases, inputs appear to compete for control of neuronal activity, for example, when two stimuli fall within the receptive field of a visual cell (chelazzi et al. in such cases, directing attention to one or the other stimulus determines how closely neural activity resembles the response to that stimulus presented alone (moran and desimone, 1985; reynolds et al., 1999; see also reynolds and heeger, 2009). such competition for control of neural activity resembles classic attentional models, in which concurrent stimuli or cognitive events compete for processing resources (e.g., broadbent, 1958; kahneman, 1973). this form of competition is best established in early visual areas, where it is predominantly local. when two stimuli fall within a cell s spatial receptive field, moving attention from one to the other determines which of the two drives activity. competition and attentional modulation are much weaker when stimuli are widely separated (moran and desimone, 1985; lee and maunsell, 2010). in behavior, however, there are global limits on attentional capacity, such that even very dissimilar tasks can be hard to carry out together (bourke et al., 1996; neurophysiologically, attentional modulations are strong in prefrontal cortex (rainer et al., 1998; lennert and martinez-trujillo, 2011), even with stimuli in opposite visual hemifields (everling et al., 2002), and it is commonly proposed that prefrontal cortex plays a central role in attentional competition and control (norman and shallice, 1980; dehaene et al., 1998; botvinick et al., 2001; miller and cohen, 2001 according to adaptive coding proposals (duncan, 2001; duncan and miller, 2002), prefrontal neurons have highly flexible response properties, allocated to coding different information in different task contexts. functional brain imaging shows that similar regions of prefrontal cortex are active during many different kinds of cognitive activity (duncan and owen, 2000; miller and cohen, 2001), providing a plausible basis for global limits on attentional capacity (e.g., dehaene et al., 1998; marois and ivanoff, 2005; bourke et al., 1996). on such a view, processing activity in prefrontal cortex would be flexible but limited, allocated to a currently attended stimulus or task, and providing a critical prefrontal mechanism for attentional competition and its resolution. here we examined the dynamics of attentional allocation in prefrontal cortex with widely separated visual stimuli. in the behaving monkey, we used time-resolved measures of neural population activity (e.g., buschman et al., 2012; kaping et al., 2011; stokes et al., 2013) to track development of the attentional focus under varying levels of attentional competition. attentional competition was manipulated using a simple form of visual search, in which the animal detected and later responded to a cued target object (t). in some trials, t was presented alone, while in others, competition was introduced by an additional nontarget (n) in the opposite visual hemifield. it is well known from human search experiments that processing conflict in such a task is determined by training history, with strong competition from a nontarget that has often previously been experienced as a target (inconsistent nontarget or ni), but much less from a nontarget that can always be ignored (consistent nontarget or nc) (schneider and shiffrin, 1977; schneider and fisk, 1982). analogous effects of training history have been shown in the frontal eye field, with a relative enhancement of response to stimuli previously trained as targets (bichot et al., 1996; bichot and schall, 1999). in our task (figure 1a), each trial began with a central cue indicating this trial s target object. based on preexperimental training,, there followed a choice display containing either a single object, to left or right of fixation, or one object to either side. for single-object displays, the choice stimulus could be either the cued target (t), the stimulus associated with the other cue and thus serving as a target on other trials (ni), or a fixed nontarget object never serving as a target (nc). in the two-object case, a target t could be accompanied by either ni or nc, or ni and nc could appear together. following the 500 ms choice display and a subsequent brief delay, the monkey was rewarded for a saccade to the t location, or if no t had been presented, maintained fixation (no-go response) for later reward (see experimental procedures). in line with the proposal of adaptive coding, our results show how, in a prefrontal cell population, activity resembles the limited processing resource of classic attentional models. as previously described (e.g., freedman et al., 2001; kusunoki et al., 2010), many cells were devoted to making task-relevant stimulus discriminations. with attentional competition, processing capacity was initially divided between competing display objects, with different neurons responding to different objects. specifically, neural events in each hemisphere were initially dominated by response to the display item in the contralateral visual field, whether t or n. the result was that neurons failed to make critical stimulus discriminations in the ipsilateral visual field, resembling poor information coding when processing resources are diverted in classic attentional models (broadbent, 1958; kahneman, 1973). subsequently, this initial, incoherent state was replaced by transition to a global focus on the behaviorally critical target, with this target now controlling neural activity in both hemispheres. the speed and extent of transition between these states reflected the strength of attentional competition, being more rapid and complete for t +nc than for t +ni displays. the results track dynamic allocation of processing resources in prefrontal cortex, with gradual establishment of a coherent and global attentional focus. behavioral data (figure 1b) showed high accuracy for singly presented t and nc stimuli. the comparative difficulty of ignoring ni was confirmed by much reduced accuracy for this stimulus presented alone, with the majority of errors (72.2%) being saccades to the stimulus location at the time of the go signal. saccades to the ni location were also common (59.2% of errors) for t +ni displays, while for t +nc displays, the most common form of error (79.1%) was a no-go response. during performance of the task, responses of 461 single neurons were recorded on the lateral frontal surface of three hemispheres, two in monkey a (n =192 right, 113 left) and one in monkey b (n =156, right). pooled results are presented here, as similar patterns were seen in all three recorded hemispheres. recording locations (figure 1c; figure s1 available online) were located in dorsal and ventral regions of the posterior lateral prefrontal cortex, including the posterior third of the principal sulcus. to ask how prefrontal cortex represents task events, we examined responses to the 12 possible single-object displays (3 stimulus categories [t, ni, nc] 2 visual fields [contralateral or ipsilateral to recording location] 2 cues). for each neuron, data were examined by anova with factors stimulus category, visual field, and cue. analyses were separately conducted on firing rates from early (50250 ms from display onset) and late (300500 ms) response periods. in both analysis periods, many cells (79/461 early, 118/461 late) showed significant (p <0.05) main effects of stimulus category. two examples are shown on the left of figure 2a. in the first cell (top row), responses were strongest to t and weakest to nc, this pattern arising much earlier for contralateral stimuli. a complementary pattern is illustrated by the second cell (figure 2a left, bottom row), with a late, selective response to nontargets, especially nc. in both analysis periods, there were also many cells (155/461 early, 114/461 late) showing significant main effects of visual field. an early preference for contralateral stimuli and a late preference for ipsilateral stimuli are illustrated by the two cells on the right of figure 2a. main effects of cue were less common (47/461 cells early, 43/461 late). numbers of cells showing different patterns of interaction are listed in table s1. as shown in figure s1, both category- and location-selective cells were broadly distributed across recording locations, including dorsolateral and ventrolateral surfaces, as well as the posterior recording area lying between arcuate and principal sulci. though these results suggest many cells coding the behavioral category of stimuli, the data suggested little direct role in the saccadic response. of 58 cells showing an interaction of stimulus category and visual field in the late period (see table s1), there were 33 with a strong, sustained response for targets in one location (figure s2). though such a pattern might plausibly reflect oculomotor preparation, even these cells showed little evidence of activity linked to the saccadic response (figure s2). these results match prior findings from similar tasks, suggesting prefrontal activity linked largely to behavioral categorization rather than motor output (everling et al., 2002; kusunoki et al., 2009). to examine stimulus coding across the whole cell population, neural activity at each time point from stimulus onset (see experimental procedures) was represented as a vector of firing rates across the full sample of 461 recorded cells. twelve such vectors were obtained for each of the 12 separate single-object displays, and to measure separation of population activity for any two displays, we used euclidean distance between their activity vectors. a two-dimensional representation of the resulting similarity space, derived using multidimensional scaling (mds), is shown in figure 2b. separate similarity spaces are shown for early (175 ms from stimulus onset) and late (450 ms) stages of processing. even early in processing, the prefrontal representation already showed discrimination of both stimulus category and hemifield. in particular there was clear separation of t, ni, and nc categories, especially in the contralateral hemifield. representations of a given behavioral category were similar for the two cues, despite actual stimuli exchanging roles as t or ni. it is noteworthy that hemifield coding was strong for both targets and nontargets, though for the latter it had no behavioral significance. at both stages of processing, neural representations for ni were intermediate between those for t and nc, in agreement with previous data (kusunoki et al., 2010) and the example cells on the left side of figure 2a. as usual in lateral prefrontal cortex, these data show many cells coding current task events, with strong but not exclusive emphasis on behaviorally relevant stimulus categorizations. to examine attentional competition, we turned to two-object displays and the dynamics of information coding as the choice display is processed. on a resource model of prefrontal activity, a plausible hypothesis is that, just as attentional competition impairs behavioral accuracy, it might impair neural discrimination. such results would match classic attentional models, in which division of attentional resource reduces processing efficiency (broadbent, 1958; kahneman, 1973). to address this question, we used euclidean distance to measure discrimination between critical stimulus pairs, t versus ni and t versus nc. in each case, we measured discrimination either for the critical stimuli presented alone (no-competition case), or in the presence of an additional nontarget in the opposite hemifield (attentional competition). the design of our displays (figure 1) allowed us to examine four such cases (figure 3): discrimination of t versus ni in the hemifield contralateral to the recording location (tcon versus nicon), either presented alone or with a concurrent stimulus (nc) in the ipsilateral hemifield; discrimination of tcon versus nccon, either presented alone or acompanied by niips; and similarly for discrimination of tips/niips, presented alone or with nccon, and discrimination of tips/ncips, presented alone or with nicon. in each case, we selected for analysis a population of cells that were most relevant to the critical discrimination (e.g., tcon versus nicon), ensuring that this selection was unbiased for comparison of no-competition and competition cases (see experimental procedures). we predicted that critical t/n discriminations might be impaired when an additional stimulus is added in the opposite hemifield. the pattern of results was strikingly different for contralateral and ipsilateral discriminations (figure 3). for single stimuli in the contralateral hemifield, t/n discrimination became significant at around 100 ms from onset and remained throughout the duration of the choice stimulus (figure 3, left panels; cf. data for two-object displays closely tracked those for single objects, suggesting little change in contralateral t/n discrimination with attentional competition. ipsilateral discriminations, in contrast, showed evidence of impairment by competing contralateral stimuli (figure 3, right panels). again, t/n discrimination began at around 100 ms for single-object displays. with addition of nccon, discrimination of tips from niips remained close to zero until around 200 ms and then rapidly increased toward single-object values (figure 3, top right). this delay led to a period of strongly significant difference between discrimination strength in one- and two-object displays (figure 3, top right, dark gray bars). a larger and more extended impairment in discriminating tips from ncips was created by addition of nicon, lasting throughout most of the stimulus duration (figure 3, bottom right). the results confirm that critical neural discriminations can be impaired by competing stimuli, especially early in stimulus processing, and when training history makes a competing stimulus (ni) hard to ignore. the impairment, however, takes an intriguing form impairment of ipsilateral discrimination by a contralateral competitor, but not vice versa. how might the idea of flexible resource allocation, in particular competition of inputs to drive neural responses, explain these discrimination data? the results suggest a critical role of visual field, with a tendency at the start of processing the choice display to ignore the ipsilateral field. anatomical connections from visual cortex to frontal lobe are much stronger within than between hemispheres (ungerleider et al., 1989), and if competing stimuli are presented in opposite hemifields, neural activity in inferotemporal cortex is dominated by the contralateral input (chelazzi et al., 1998). at least early in processing a choice display, prefrontal activity may show a similar contralateral dominance, meaning that in each hemisphere, there is little information concerning a competing ipsilateral event. when a target is present, however, an accompanying nontarget has no relevance to behavior. we considered the hypothesis that, across time of processing a choice display, activity evolves to a coherent attentional state, with responses in both hemispheres controlled by the behaviorally critical target. a neural mechanism of this sort would be analogous to progressive focus of processing resources in classical attention modes (kahneman, 1973). on this hypothesis, responses to a t +n display should be predictable from the separate responses produced by the component t and n presented alone. for tcon +nips, activity should follow response to tcon alone throughout choice stimulus processing. early in processing, tcon dominates because it is contralateral, while late in processing, the same stimulus dominates because it is the target, but in either case, response to tcon +nips follows response to tcon alone. for tips now, an early response to ncon should be replaced by later activity based on tips. the top left panel shows responses to tcon +nips displays and their component single stimuli for a cell with very different tcon and nips responses. throughout choice stimulus processing, the bottom left panel shows a complementary pattern, where again, response to the two-object display resembled the suppression produced by tcon alone. the right panels show very different dynamics for tips +ncon displays. in the top panel is a cell showing a strong, sustained response to tips alone. in the two-object display, this strong tips response was initially suppressed by the accompanying ncon. later, suppression tended to be released but earlier and more rapidly when the suppressing stimulus was nc. the bottom right shows a complementary example, with strong response to a single ncon but little response to tips. in the two-object display, again, the early response resembled that to ncon alone, but, especially when the nontarget was nc, later activity was dominated by tips. to confirm this pattern at the population level in the first, we examined mean responses in selected cell groups (figure 4b) with clear differences in response to the two component stimuli of a two-object display. to examine responses to tcon +nips displays, we selected all cells with significantly different responses to tcon versus nips (see experimental procedures). for tcon>nips cells (figure 4b, top left), strong responses to tcon alone were matched by closely similar responses to both tcon +niips and tcon +ncips displays.<nips cells, with similar suppression to tcon presented alone or with an accompanying nontarget (figure 4b, bottom left). a contrasting pattern was seen for tips>ncon cells responding to the tips +ncon display (figure 4b, top right). early responses to the tips +ncon display resembled the suppression produced by ncon alone, then, beginning before 200 ms, departed to approach tips responses. for tips +nccon, activity rapidly approached the response to tips alone, while for tips +nicon, some suppression compared to tips alone remained throughout stimulus processing. a corresponding pattern of results was seen for tips <ncon cells (figure 4b, bottom right). in a second analysis, we turned to the whole recorded cell population and asked how well single neuron responses to each two-object display were predicted by responses to the two component objects individually. results for three different analysis windows early (100200 ms from choice stimulus onset), middle (250350 ms) and late (400500 ms)are shown in figure 5. responses of each neuron to the different stimulus displays were first normalized (division by mean response to all displays; see experimental procedures), and responses to two-object displays were then plotted against responses to each of the two component single stimuli. for tcon +nips displays, results were similar at all time points from stimulus onset. across neurons, there was a strong tendency for response to the two-object display to match the response given by tcon rather than nips. for tips +ncon displays dynamics were more complex. in the early window, response to the two-object display tended to follow response to ncon alone, with response to tips unpredictive. in the middle window, the ncon response retained some influence for the case of ni, though this influence had already disappeared for nc. in contrast, response to the two-object display was increasingly predicted by response to tips. by the late window finally, to ask how closely population responses to a two-object display approached those to component single stimuli, we again used a euclidean distance measure calculated across the whole recorded sample of 461 cells. across the time course of choice stimulus processing, we measured population discrimination of each two-object display (e.g., tcon +niips; figure 6, top left) from its component single stimuli (tcon, niips). a simple pattern of results emerged when t was contralateral. throughout stimulus processing, the population response to the two-object display was barely discriminable from response to the contralateral t presented alone (figure 6, left panels). beginning<100 ms from stimulus onset, in contrast, response to the two-object display diverged rapidly from response to the component ipsilateral n (figure 6, left panels). when t was ipsilateral, however, events followed a more complex time course, in particular for the highest-competition case (tips +nicon; figure 6, top right). in the first phase of processing, the two-object display was strongly discriminated from tips presented alone, but not from nicon. only toward the end of the stimulus presentation did the curves cross, indicating a response to the two-object display that more closely resembled response to its component target. for the lower-competition case (tips +nccon), strong discrimination from the component target was short lived, with a correspondingly rapid increase in discrimination from the component nontarget (figure 6, bottom right). these results show how, as prefrontal processing evolves, there is large-scale reallocation of processing resources. early in choice stimulus processing, neural activity in each hemisphere is dominated by response to the contralateral stimulus. in different groups of neurons (figure 4), this response can be either an increase or decrease from baseline; in either case, the response to a competition display resembles response to the contralateral stimulus alone. later, this separation between hemispheres resolves to a coherent state of activity based on the critical t stimulus, especially when the accompanying stimulus is nc. across both hemispheres, the final, global state is close to the state produced by the t stimulus alone. in further analyses, we examined the generality of attentional reallocation across recording locations and cell types. to produce an index of reallocation for each cell, we defined dt as the absolute difference between firing rates for a tips +ncon display and for tips alone and, similarly, dn as the absolute difference between firing rates for the tips +ncon display and its component ncon alone. in an early analysis window (100200 ms from stimulus onset), we defined a dominance index as(dtdn)(dt+dn). following the results shown in figure 6, this early index was generally positive, reflecting response to tips +ncon that was closer to ncon than to tips alone. the same dominance index calculated for a late analysis window (400500 ms) was generally negative, indicating response to tips +ncon that was closer to tips than to ncon alone. subtracting the late index from the early index gave us a final reallocation index, separately calculated for each cell and for tips +nicon and tips +nccon displays. across the whole recorded cell population, the mean reallocation index for tips +nicon displays was 0.147. by t test, this value was significantly greater than zero (p <0.001). for tips +nccon displays, the mean reallocation index was 0.124, again significantly greater than zero (p <0.001). to examine generality across anatomical regions, we divided the full recorded cell sample into three groups, a smaller group (n =42) recorded in a posterior region between the principle and arcuate sulci (see figure s1) and larger groups recorded in more anterior dorsolateral (n =145) and ventrolateral (n =274) regions, divided by the fundus of the principal sulcus. both for tips +nicon and tips +nccon displays, the mean reallocation index was positive for all three cell groups. anova showed no difference between cell groups, for tips +nicon f(2, 456) =1.17, for tips +nccon f(2, 455) =1.29 (missing data for cases in which dt =dn =0). in a second analysis, we examined generality across cell types, defined by coding of stimulus category and/or location in single-stimulus displays. to examine category-selective cells, we took all those cells (n =162) with a main effect of stimulus category in either early- or late-period anovas on single-stimulus activity (see earlier section, coding of single choice stimuli). to examine location-selective cells, we took all cells (n =210) with a main effect of location in either analysis period. again, the mean reallocation index was significantly positive in both groups (category-selective cells: mean index =0.222, p <0.005 for tips +nicon, mean index =0.226, p <0.001 for tips +nccon; location-selective cells: mean index =0.233, p <0.001 for tips +nicon, mean index =0.096, p <0.06 for tips +nccon). these results show substantial generality in the overall pattern of attentional reallocation for tips +ncon displays. for all regions in our recording area, and whatever stimulus feature a cell coded, early response was determined largely by the contralateral n, while later response was determined largely by t. finally, we found no evidence for attentional reallocation on error trials. combining data for all cells, and for both major error types (saccade to wrong location, no-go), mean reallocation index on error trials was 0.006, t test against zero p =0.56 for tips +nicon displays, and mean reallocation index 0.014, p =0.49 for tips +nccon displays. only correct trials, evidently, were associated with reallocation of prefrontal processing resources from contralateral nontarget to ipsilateral target. when stimuli or other cognitive events compete for attention, processing resources must be allocated to the most important (broadbent, 1958; kahneman, 1973). our results track development of an attentional focus in the population activity of prefrontal cortex. in line with the proposal of adaptive prefrontal coding of task-relevant information, we found that many prefrontal cells discriminated task-critical stimulus categories and locations. when two stimuli were present in the display, attentional competition resolved through widespread reallocation of neural resources. early processing lacked attentional coherence, with different neurons responding to different items in the display. specifically, neural activity in each hemisphere was dominated by the contralateral display item a pattern (figure 2) coding both hemifield and behavioral category of that stimulus. accordingly, critical stimulus discriminations within one visual field were impaired in the ipsilateral hemisphere, matching the classical proposal of reduced processing efficiency when processing resources are withheld (broadbent, 1958; kahneman, 1973). construction of this global attentional focus resembles the classical proposal that processing resources are allocated to the most important cognitive events (broadbent, 1958; kahneman, 1973). we found that the time course of transition depends on the attentional weight of nontargets. for nc, a stimulus never serving as a target, control of the contralateral hemisphere was released quickly and easily. for ni, a target stimulus on other trials, release was slow and incomplete. again, these results match comparable findings from human studies, showing how processing resources are rapidly allocated to targets when nontargets have been extensively practiced as task irrelevant (schneider and shiffrin, 1977; schneider and fisk, 1982). it is commonly proposed that, in early visual areas, stimuli within or close to the receptive field of a cell compete to control its activity (moran and desimone, 1985; reynolds et al., 1999; reynolds and heeger, 2009). accordingly, moving attention from one stimulus to another can have large effects when the two are close together; with widely separated stimuli, the effect is much smaller, with only modest enhancement of response to the attended input (moran and desimone, 1985; lee and maunsell, 2010). in prefrontal cortex, instead, we found widespread target dominance by the end of display processing, reflecting global allocation of processing resources to the behaviorally critical stimulus. global division of prefrontal processing capacity is a plausible neural basis for many cases of attentional competition, including interference between widely separated visual stimuli and even between very dissimilar tasks (marois and ivanoff, 2005; dehaene et al., 1998 ;. in prefrontal cortex, global processing competition, and its dynamic resolution, probably reflect the breadth of inputs from other brain regions (pandya and yeterian, 1996) and the strong interconnectivity between one prefrontal region and another (pucak et al., 1996). our finding of early activity dominated by the contralateral visual field resembles results previously reported for inferotemporal cortex (chelazzi et al., 1998). in that study, pairs of stimuli were presented either within one hemifield or one to each hemifield. as in the current study, animals searched for a prespecified target, responding with an immediate saccade or lever release. when both stimuli fell in the same hemifield, neural activity was dominated by the target, but with stimuli in opposite hemifields, activity was dominated by the contralateral stimulus, whether target or nontarget. because responses in that study were immediate, data were only available for the brief period before the response was made. it is not known whether, with a longer stimulus presentation, global dominance by the attended target might develop in inferotemporal cortex, as we have shown here for prefrontal cortex. the neural mechanisms of visual search have also been examined in the frontal eye field, with some similarities to the current results. in the frontal eye field, as in early visual areas, there is response enhancement when the stimulus within the receptive field is the current target (schall et al., 1995). enhancement reflects training history, with earlier enhancement after long practice in searching for a given target (bichot et al., 1996) and enhancement for stimuli that share features with a previous target (bichot and schall, 1999). as in early visual areas, however, such target enhancements are far from the widespread reallocation of processing activity we observed in prefrontal cortex. even when the target is well outside the receptive field, the nontarget within the receptive field drives strong activity up to the time of the response (schall et al., 1995; thompson et al., 1997 a likely mechanism is communication between the two frontal lobes (tomita et al., 1999), allowing processing on both sides to be dominated by the same, critical stimulus event. on this model, target information from one hemisphere displaces nontarget information in the other; this happens rapidly and relatively completely when nontarget status is fixed throughout training but only more slowly and partially when the nontarget to be displaced is a target on other trials. evidently, the competitive mechanisms allowing nontarget displacement must be influenced both by current behavioral status allowing t to dominate even ni but also by long-term learning. on the current trial, some context signal initiated by the cue (stokes et al., 2013) must determine which stimulus is t and which is ni, thus directing the outcome of competition for control of population activity. across learning, in contrast, nc is always irrelevant, resulting in long-term reduction of competitive weight. an even stronger separation of competitive weights may be obtained with spatial cues, directly indicating which visual field should be attended in a subsequent visual display. with advance spatial cueing, information from the attended field may dominate some cells even from the outset of visual processing (everling et al., 2002), though even in this case, there is some response to the unattended side (everling et al., again, the strength of such attentional modulations reflects the variable strength of spatial cues (lennert and martinez-trujillo, 2011). an enduring debate in the search literature rests on the distinction between serial and parallel processing. behavioral (egeth and dagenbach, 1991; kyllingsbaek and bundesen, 2007) and neurophysiological (buschman and miller, 2009) arguments can be assembled on both sides of this debate and, in the present case, data are not easily explained by a simple serial model. instead, processing begins with parallel coding of both display inputs, one dominating each hemisphere, then resolves over several hundred milliseconds (figures 5 and 6) to a global state of target dominance. it is an open question how this conclusion relates to other kinds of task and search display, e.g., displays containing larger numbers of stimuli (buschman and miller, 2009). a second enduring question in the cognitive literature is the extent to which the two hemispheres act as separate pools of processing capacity (pashler and obrien, 1993; alvarez and cavanagh, 2005; see also buschman et al., 2011). our data suggest that the answer may be dynamic, evolving with construction of the attentional focus. early in processing a two-object display, we indeed found that the two hemispheres were focused on different stimuli, like parallel processing pools. later, we found coherence, with both hemispheres focused on the same, behaviorally critical stimulus. though here we examined attentional competition between visual fields, more generally, similar processing principles may apply to many different cases of processing competition. in many such cases, prefrontal activity may move from an early, unfocused state to attentional coherence. attentional coherence is critical to organized cognition, as multiple brain systems must converge to process the stimuli, responses, reward, etc. of current behavior. through feedback to multiple brain systems (dehaene et al., 1998; miller and cohen, 2001; desimone and duncan, 1995; moore and armstrong, 2003), the construction of globally consistent prefrontal activity patterns may be critical in assembly of distributed yet coherent attentional episodes. subjects were two male rhesus monkeys (macaca mulatta) weighing 11 (monkey a) and 10 (monkey b) kg. all experimental procedures were approved by the uk home office and were in compliance with the guidelines of the european community for the care and use of laboratory animals (euvd, european union directive 86/609/eec). task events were controlled by a pentium pc running cortex software, with displays presented on a 19 inch led screen placed in front of the monkey s chair. each trial began with onset of a red dot at screen center, which the animal was required to fixate (window 5 5 for monkey a, 4 4 for monkey b) until the final saccadic response at the end of the trial. a premature saccade away from screen center immediately terminated the trial without reward (trials discarded from all data analyses). once fixation had been held for 1,000 ms, a central cue stimulus (500 ms) indicated the target for the current trial. based on preexperimental training, each of two alternative cue stimuli was associated with a different target (see figure 1a inset for cue-target pairs for monkey a; different cue, target, and nontarget images were used for monkey b). a randomly varying delay of 400600 (monkey a) or 400800 (monkey b) ms was followed by a 500 ms choice display. the display contained either a single object, centered on the horizontal meridian randomly 6 to left or right of fixation, or two objects, one to either side. for single-object displays, the stimulus object was either the cued target t, the object associated with the alternative cue (inconsistent nontarget, ni), or a third object never used as a target (consistent nontarget, nc). for two-object displays, major trial types were t +ni, t +nc, and ni +nc (in randomly varying left-right or right-left configuration), though in some sessions, small numbers of ni +ni trials were also included (data not shown). to avoid response biases, we adjusted frequencies of individual trial types to ensure that t was present in half of all single-object and half of all two-object displays; otherwise, frequencies of all major trial types were the same. following choice stimulus offset, there was a further random delay of 100150 (monkey a) or 300500 (monkey b) ms, after which the fixation point turned green to indicate the monkey s response interval. for go trials (t present in choice display), the monkey was immediately rewarded with a drop of liquid for a saccade to the remembered t location (target window 6 6 for monkey a, 3.5 3.5 for monkey b). for no-go trials (t absent), the monkey was required to hold fixation for the whole 1,000 ms response interval and was then either given immediate reward (monkey b) or rewarded for a further saccadic response (monkey a). for monkey a, some sessions had cues randomly varying between trials, while others had alternating brief (1520 trials) blocks of fixed cues. physiological data were very similar in the two cases and were combined. for monkey b, cues always varied randomly between trials. each monkey was implanted with a custom-designed titanium head holder and recording chamber(s) (max planck institute for biological cybernetics), fixed on the skull with stainless steel screws. chambers were placed over the lateral prefrontal cortex of the left (ap =25.3, ml =20.0; ap, anterior-posterior; ml, medio-lateral) and right (ap =31.5, ml =22.5) hemispheres for monkey a and the right hemisphere (ap =30.0, ml =24.0) for monkey b. recording locations for each animal are shown in figure s1. we used arrays of tungsten microelectrodes (fhc) mounted on a grid (crist instrument) with 1 mm spacing between adjacent locations inside the recording chamber. the electrodes were independently controlled by a hydraulic, digitally controlled microdrive (electrodes drive, nan for monkey a; multidrive 8 channel system, fhc for monkey b). neural activity was amplified, filtered, and stored for offline cluster separation and analysis with the plexon map system (plexon). eye position was sampled using an infrared eye tracking system (120 hz, asl for monkey a; 60 hz, iscan for monkey b) and stored for offline analysis. we did not preselect neurons for task-related responses; instead, we advanced microelectrodes until we could isolate neuronal activity before starting the task. at the end of the experiments, animals were deeply anaesthetized with barbiturate and then perfused through the heart with heparinized saline followed by 10% formaldehyde in saline. the brains were removed for histology and recording locations confirmed on dorsal and ventral frontal convexities and within the principal sulcus. physiological data were analyzed just from successfully completed trials, on average including 17 repetitions for each combination of cue, choice stimulus type, and hemifield/spatial arrangement. for all analyses, spike data were smoothed with a gaussian kernel of sd 20 ms, cutoffs 1.5 sd. to measure neural discrimination between any two choice stimuli x and y for each cell in the population, we measured the difference in absolute firing rate for x and y. as is standard, euclidean distance was defined as the square root of the sum of these squared differences. for mds (figure 2b), we used raw euclidean distances. for quantitative analysis (figures 3 and 6), we used a correction for the fact that euclidean distance must always be positive and scales with absolute firing rate. for each distance measure, we calculated the expected chance value by randomly permuting x, y labels across trials, then subtracted the median permuted value (across 1,000 permutations) from the obtained raw value. analyses were repeated at each 1 ms time point from 100 ms to 600 ms from choice stimulus onset. to measure discrimination between two-object displays and their component single stimuli (figure 6), we used activity vectors based on the full cell sample (n =461). to compare discrimination of the same stimulus pairs (x, y) in single- versus two-object displays (figure 3), we selected just those cells most sensitive to the critical discrimination, giving equal weight to single- and two-object data. for this purpose we used anova on activity of each cell over the full (0500 ms) period of choice stimulus presentation, with factors critical stimulus (x, y) accompanying stimulus (absent, present) cue, and selected just those cells with a significant (p <0.05) effect of critical stimulus. permutation testing was used to compare distances in single- and two-object cases (figure 3). on each permutation, for each cell we randomly maintained or switched single- and two-object labels; when labels were switched, they were switched for all of that cell s data. after this permutation of labels the true difference in distances (figure 3) was compared with the distribution of permuted values across 1,000 permutations. for comparison of mean neural activities for two-object displays and their component single stimuli (figure 4b), we selected all cells with a significant difference between responses to singly presented targets on one side and nontargets on the other. significance (p <0.05) was again determined by anova on activity across the full period of choice stimulus presentation, with factors stimulus (e.g., tcon, nips) cue.<nips; tips>ncon; tips <ncon) as shown in figure 4b. for calculation of mean activity across cells (figure 4b, figure s2), responses of each cell were first normalized by dividing by mean firing rate across all choice displays, calculated across the full 0500 ms display period. the same normalization was used to create scatterplots of response to each two-object display as a function of response to component single stimuli (figure 5).

summaryprefrontal cortex has been proposed to show highly adaptive information coding, with neurons dynamically allocated to processing task-relevant information. to track this dynamic allocation in monkey prefrontal cortex, we used time-resolved measures of neural population activity in a simple case of competition between target (behaviorally critical) and nontarget objects in opposite visual hemifields. early in processing, there were parallel responses to competing inputs, with neurons in each hemisphere dominated by the contralateral stimulus. later, the nontarget lost control of neural activity, with emerging global control by the behaviorally critical target. the speed of transition reflected the competitive weights of different display elements, occurring most rapidly when relative behavioral significance was well established by training history. in line with adaptive coding, the results show widespread reallocation of prefrontal processing resources as an attentional focus is established.

PMC3791408

pubmed-754

subclassification and study of cancer patients based on mutational status presents opportunities to learn the significance of genomic alterations (and their combinations) and to develop additional therapies (and combinations of therapies). however, the large number of mutations known to be important in cancer development and the presence of multiple mutations in any individual patient combines to create a great diversity in populations of patients with a specific tumor type. shrager and tenenbaum note that cancer is in effect, a large number of rare diseases occupying a very high dimensional space with very few opportunities for action and observation in each subtype. to efficiently search a space of this nature, one needs to capture the learnings from as many patients and treatment experiments as possible in a continuously updated knowledge base.1 the stratification of cancer patients by mutational status and resultant decrease in the proportion of patients available for study enrollment presents serious problems for observational and interventional research. the low prevalence (12%) of many driver mutations in solid tumors precludes recruitment of sufficient numbers of subjects from traditionally sized research consortia and has led to new models of clinical research based on interinstitution collaboration, community outreach, and broad data sharing, with the goal of learning from every treatment encounter. this article reviews the challenges and opportunities of such collaborations from the perspective of the department of veterans affairs healthcare system, the largest integrated healthcare system in the united states. creating generalizable knowledge as well as informing current individual patient care is therefore enabled by learning from all available previous treatment experiences of every relevant patient in the entire system, aggregating data across many medical centers. familiar challenges to this approach include technical issues, such as data element provenance, data quality, and database variability across institutions, ensuring patient protections in data sharing related to informed consent, privacy/confidentiality, and health insurance portability and accountability act (hipaa) authorization, scalability and sustainability of aggregated databases, and cultural and financial barriers to data sharing in a research community. the ability to commoditize healthcare data has created opportunities for new consortium models that enable data sharing and patient access (for clinical trials). data sharing is an important element of the collaborations exemplified by orien,2 medc,3 tapur,4 and apollo5 (see table 1). the national cancer institute (nci) has created the genomic data commons, a unified data repository6 that enables data sharing across these and other cancer genomic studies, in support of precision medicine. the repository houses clinical health record and genomic data (fastq file format) and complements the cancer imaging archive,7 another ncisponsored repository that contains radiographic and pathology images for cancer patients. sharing with the research community the department of veterans affairs (va) has begun to move consented and hipaaauthorized patient data from the va electronic medical records to these nci data repositories for subsequent sharing with the research community (see table 1). this approach replaces the need for cancer patients to sign multiple forms consenting to data sharing with a single broad consent that satisfies the requirements of the various project institutional review boards (irbs). it also provides data collection and curation that meets requirements of the individual project aims. is a joint effort by the va and the nci to provide these data to early career scientists to develop analytics and other tools in support of clinical and research objectives.8 as discussed above, traditional recruitment approaches from single or limited groups of institutions do not provide sufficient numbers of eligible study subjects to fulfill inclusion criteria with specific tumor mutation combination requirements. furthermore, thirdparty payers seldom reimburse for mutational analysis required for patient screening prior to entry into research, thus shifting to the research enterprise the cost of screening large numbers of patients, of whom only a small fraction will be found eligible. the lack of data on the mutational status of patients is the major bottleneck for clinical trial execution and slows progress in precision oncology. in the era of precision oncology treatment, the new standard of care requires clinical reimbursement for expanded panel testing in cancer patients, with subsequent recruitment in clinical trials when appropriate for the individual patient. that standard then leads to sharing and reuse of patient data for clinical trials and observational research. major efforts underway to solve these and related problems are exemplified by the medc and orien initiatives. the medc program offers insurance coverage of testing for institutions and patients who agree to contribute clinical data to the n1 registry for subsequent analysis. the oncology research information exchange network (orien) is a research collaboration founded by the moffitt cancer center in tampa and the ohio state university comprehensive cancer center to match patients to targeted treatments and promote datasharing activities. the group engages industry partners on sponsored projects across the clinical trials continuum and serves as a broker between research and healthcare communities. these and other programs (particularly ncisponsored clinical trial consortia) that foster collaboration between clinical care and research communities represent new models to advance precision oncology. membership of va sites in nci consortia and programs such as medc, orien, and tapur makes data sharing and clinical trial participation opportunities available for veterans at participating va medical centers but leaves behind patients at facilities that lack oncology research programs and infrastructure. this structural problem is not unique to the va, as opportunities for patients to participate in cancer clinical trials are similarly limited in communities not located near cancer centers. if participation in clinical trials is considered a new standard of care, then this phenomenon exposes a new and important access disparity in a healthcare system. the distributed enrollment program under development at the va cooperative studies program presents a model to move clinical trials to patients remote from va cancer centers by seeking preapproval of cancer protocols by a central irb, reduction of researchspecific training requirements imposed on participating clinicians, and centralized trial management (such as data collection and submission). indeed, much of the administrative overhead built into the clinical research apparatus in the name of quality assurance, while highly appropriate for research designed primarily to benefit the broader community (such as registration studies for drugs where effective alternatives exist), may have reduced relevance in a precision oncology setting, where the patient's motivation to participate is first and foremost to obtain study drug, whether or not alternatives exist. by its nature, precision oncology is a biomarkerdriven field critically dependent on acquisition of clinical biosamples and electronic medical record data for discovery and validation with lack of access to both resources as a central limitation to the pace of discovery. collaborations to costshare for data and tissue procurement between biotech, pharma, and healthcare systems, in a precompetitive fashion, are emerging.4 to reach full potential, such collaborations require more complete integration within healthcare systems, as exemplified in the apollo program, a partnership between the nci, dod, and va. in apollo, tumor tissue is made available for biomarker analysis (proteomics in this case) and the results transmitted back to healthcare providers if they are determined to offer incremental value to patient care, beyond mutational analysis. while observational data is useful in this regard, randomization accelerates learning, and results in more certain knowledge. for example, demonstration that patients randomized to genomic augmented with proteomic analysis had superior treatment outcomes to those randomized to genomic analysis alone supports a compelling argument to adopt this new biomarker of response to targeted therapy. problematically, the introduction of randomization (to biomarkers) into clinical care using traditional clinical trial methods is costprohibitive. the department of veterans affairs continues to make progress in this area through the pointofcare research9 and precision oncology10 programs whereby patients are randomized to minimal risk alternatives with relaxed regulatory requirements appropriate with the degree of risk (riskbased monitoring). data generated from these embedded studies are derived exclusively from the ehr (realworld evidence) and require fda acceptance if used for registration of a new companion diagnostic.

cancer genomic research reveals that a similar cancer clinical phenotype (e.g., nonsmall cell lung cancer) can arise from various mutations in tumor dna. thus, organ of origin is not a definitive classification. further, targeted therapy for cancer patients (precision oncology) capitalizes on knowledge of individual patient mutational status to deliver treatment directed against the protein products of these mutations with the goal of reducing toxicity and enhancing efficacy relative to traditional nontargeted chemotherapy.

PMC5414893

pubmed-755

pluripotent embryonic stem cells (escs) have the potential to differentiate into any cell in the adult body, making them an ideal source of cells for tissue regeneration when other options are absent. since escs display robust teratoma forming potential in vivo, differentiated products are thought to be a better option for cellular replacement of diseased or damaged tissues. however, differentiated esc derivatives are often short-lived and are undetectable after transplantation in vivo, leading us to question the developmental compatibility and possible immune rejection of esc derivatives in the adult host [24]. syngeneic hematopoietic stem cells (hscs) from the bone marrow (bm) or umbilical cord blood have been used therapeutically to treat blood diseases and allogeneic bm transplantation has been used to induce tolerance to other nonhematopoietic tissues. embryonic stem cell-derived hematopoietic progenitors (eshps), as well as a variety of terminally differentiated hematopoietic cells, can be cultured in vitro [6, 7], and eshps express markers commonly found on natural adult and embryonic hematopoietic stem and progenitor cell populations [8, 9]. however, eshps often fail to engraft at high levels after transplantation in vivo, even in immunodeficient mouse models [8, 1012]. investigation of the adult host immune response to escs and their derivatives has resulted in some controversy. several groups have described eshps as immune-privileged [13, 14], while others have described their ability to induce responses by t cells and natural killer (nk) cells [3, 12, 1517]. macrophages have been observed to respond and phagocytose cells of the embryonic inner cell mass, escs, and cultured esc derivatives [1821]. we previously showed that macrophages from the 129 and balb/c mouse strains preferentially phagocytosed eshps in vitro. here, we extend those findings and present evidence which supports that host macrophages are an innate immune barrier to eshp engraftment in vivo. d3 esc lines (derived from 129 mice, h-2) were purchased from atcc (manassas, va, usa). escs were maintained in an undifferentiated state on mitomycin-c treated sto cells (atcc) in dmem supplemented with 15% fbs (atlanta biologicals, norcross, ga), 0.15 mm monothioglycerol (sigma-aldrich, st. louis, mo), 1x penicillin-streptomycin (pen/strep) (invitrogen, san diego, ca), and 1000 u/ml leukemia inhibitory factor (lif). escs were passaged every two to three days by trypsinization (0.25% trypsin-edta (invitrogen)). prior to differentiation, escs were transferred to 0.1% gelatin-coated plates to wean them from the feeder layer in imdm (invitrogen) media supplemented as described above. cells were incubated in a humidified incubator with 5% co2 at 37c [21, 22]. eshps were differentiated from escs using coculture on op9 stromal cell monolayers (atcc), as published. briefly, op9 cells were cultured in alpha-mem media (invitrogen) supplemented with 20% fbs and 1x pen/strep. one hundred thirty thousand escs were plated on op9 monolayers at 80% confluency in 150 mm tissue culture dishes in the presence of 5 ng/ml flt3l and il-7 (peprotech, rocky hill, nh) in 20 ml of media. at days 4 and 11, 10 ml of media was added with 10 ng/ml flt3l and il-7. at day 7, all cells were harvested using cell lifters, filtered through 64 m nylon mesh (small parts, inc., miami lakes, fl), and replated onto fresh monolayers in the presence of 5 ng/ml flt3l and il-7. eshps were harvested and digested in medium 199 (invitrogen) containing 0.125% w/v collagenase d and 0.1% v/v dnase i (both from roche, south san francisco, ca), for 60 minutes at 37c, followed by dissociation by vigorous pipetting. m199+media containing 2% fbs in medium 199 and centrifuged at 2000 rpm for 10 minutes. the bm from tibiae and femora from adult mice were also collected as described as a source of adult hematopoietic progenitors. to obtain lineage-negative (lin) cells from the adult bm, cells were stained with a biotinylated anti-lineage (lin) cocktail (anti-cd3, cd4, cd8, cd11b, cd19, nk1.1, gr1, and ter119), and lin cells were positively selected from the whole bm using the easysep biotin positive selection kit (stem cell technologies, vancouver, canada) and two rounds of magnetic selection per the manufacturer's instructors. lin bm cells were used in phagocytosis assays and transplantation assays. for sorting eshps, cultured cells were harvested and then blocked with 2.4g2 (anti-cd16/cd32) hybridoma supernatant to block fc receptors. then, one of two strategies was used for preenrichment and sorting of eshps. in the first strategy, all harvested cells were stained with biotinylated anti-lineage (lin) cocktail (anti-cd3, cd4, cd8, cd11b, cd19, nk1.1, gr1, and ter119), pe-anti-cd41 (clone mwreg30, biolegend), and apc-anti-cd45 (clone 30f11, biolegend) for 30 minutes and then washed. in a second incubation, the cells were then stained with streptavidin-pacific blue (invitrogen) to develop the anti-lineage cocktail and dapi (as a viability marker). sorting was then performed to obtain cd41 cd45 and cd41 cd45 cells together and/or cd41 cd45 cells on a bd facs aria ii or aria iii flow cytometer in two steps. first, sorting was performed on yield mode, which allowed for rapid enrichment of the target cells, and then the enriched cells were resorted in purity, cd41 progenitors were preenriched by staining with cd41-biotin and easysep-streptavidin coated magnetic beads (stem cell technologies, inc., three rounds of positive selection on the magnet were performed to collect cd41 cells per the manufacturer's instructions, and the final fraction was then costained with streptavidin-pe, apc-anti-cd45, and dapi as a viability marker. these cells were then sorted on the flow cytometer using purity mode. sorted progenitors with either strategy were 8595% pure after sorting (supplemental figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/2414906). the uc merced institutional animal care and use committee approved all animal procedures. mice of the 129 (h-2, cd45.2, stock #002448) and nsg (h-2, cd45.1, stock #005557) strains were purchased from the jackson laboratory (west sacramento, ca). mice were housed in specific pathogen-free conditions with autoclaved food and sterile water. mice were given 200 rads of irradiation (a sublethal dose in nsg mice) using a cs-source (jl shepherd and associates, san fernando, ca). after irradiation, mice received 2 mg/ml neomycin sulfate in their drinking water for 2 weeks (sigma). control mice were transplanted with 5 10 lin bm progenitors or 510 10 whole adult bm cells. recipients were analyzed at beginning at day 17 after transplantation to assess chimerism in the bm, spleen, and thymus by flow cytometry. cells were stained at 4c for 30 minutes in 2.4g2 (anti-cd16/cd32) hybridoma supernatant to block fc receptors. cells were then stained with specific antibody cocktails for 30 minutes in a total volume of 100 l in facs buffer, using antibodies to the lineage markers cd3 (clone 2c11), cd4 (gk1.5), cd8 (2.43), b220 (ra3-6b2), igm (rmm-1), gr-1 (rb6-8c5), cd11b (m1/70), and f4/80 (bm8). cells were further costained with cd45.1 (a20) and cd45.2 (104) to mark host and donor hematopoietic cells, respectively. cells were analyzed by gating on live, singlet cell populations on bd facs aria ii or aria iii flow cytometers and data were analyzed using flowjo software. for histological analysis, half of the spleens from eshp and bm recipients were frozen in tissue-tek optimal cutting temperature compound (sakura finetek, inc., seven m thick sections were cut using a cryostat, flash fixed in acetone for 1015 seconds, and followed by fixation in 4% paraformaldehyde for 10 minutes. f4/80 or rat igg2a isotype control antibodies at 1: 50 dilution (biolegend) were used to stain the tissue section. macrophages were enriched by adherence to plastic tissue culture dishes overnight in a humidified incubator at 37c with 5% co2. adherent cells were removed by trypsinization for 5 minutes at 37c followed by mechanical lifting using a cell lifter. fifty thousand macrophages were plated per well with 1 10 cd41 eshps or lin bm target cells labeled with cfse (molecular probes). to label target cells, eshps or lin bm cells were washed twice with 1x pbs prewarmed to 37c and then resuspended at 1 10/ml in pbs. one l/ml of 5 mm cfse was added to the cells, which were then incubated at 37c for 10 minutes, and then washed twice with m199 +. phagocytosis assay cultures were then harvested and stained with anti-f4/80 apc (biolegend) and dapi. nsg mice were depleted of macrophages by treatment with clodronate-loaded liposomes (cll), whereas control mice were treated with phosphate-buffered saline-loaded liposomes (pll), obtained through clodlip bv (http://clodronateliposomes.com/). at day 3, mice were treated with 0.04 mg of liposomes per gram of mouse weight, and on days 1,+5,+10, and+15 mice were treated with 0.02 mg per gram of mouse weight via intraperitoneal injection, with day 0 representing the day that mice received irradiation and hematopoietic transplant. spleens and bm cells were analyzed by flow cytometry after animals were sacrificed. to determine the group sizes for transplantation, we utilized data from previous studies in which eshps were derived in vitro and transplanted into similar genetic backgrounds to the nsg mice [8, 11] to estimate the average expected engraftment success rate in vivo and the minimum number of animals per group required to achieve meaningful and statistically sound results. two-tailed unpaired t-tests were performed to test differences in the means between groups using graph pad prism (san diego, ca). transplantation of in vitro-derived eshps into adult mouse hosts has not led to high levels of donor chimerism or long-term engraftment without transgenesis [8, 12]. our previous work suggested that immune responses to eshps might be partly responsible for their poor engraftment. to test this further, we utilized mice on the nsg background which lack t, b, and nk lymphocytes but still develop myeloid lineage cells, such as granulocytes, dendritic cells, and, importantly, macrophages. previous studies have shown the definitive mouse hematopoietic progenitors in the embryo express cd41 and transition through cd41 cd45, then cd41 cd45, and cd41 cd45 stages of maturation [25, 26], and we previously showed that eshps with these phenotypes could be generated using a coculture system on the op9 bone marrow stromal cell line. eshps were sorted based on cd41 (least mature) or cd45 (most mature) expression after 16 days of in vitro differentiation, as shown in figure 1(a), in order to compare their levels of engraftment in vivo. one hundred thousand to 5 10 purified eshps were injected into sublethally irradiated nsg hosts. since 129 and nsg mice differ at the cd45 locus, cd45.1 (expressed by the nsg host strain) and cd45.2 (expressed by 129 donor strain) specific antibodies were used to determine relative levels of donor engraftment (figure 1(b)). the frequency of host cd45.2 cells was low in eshp nsg recipients, as compared to 129 bm nsg recipients. controls to distinguish nonspecific background staining of anti-cd45.1 versus anti-cd45.2 antibodies were performed using tissues from untransplanted nsg mice (which do not express cd45.2) and untransplanted 129 mice. these results showed that the cd45.2 signal observed in eshp nsg recipients was clearly distinguishable from that in untransplanted nsg controls. donor hematopoietic chimerism averaged 5% or less in the spleen and bone marrow in eshp recipients, which was low compared to whole adult bmt controls, which averaged about 90% (figure 1(c)). no significant difference in the level of donor chimerism in recipients of cd41 eshps and cd41 cd45 eshps was observed. the low level of donor chimerism in eshp recipients is consistent with the results from other groups [8, 10]. eshps were capable of multilineage differentiation, as shown by myeloid differentiation in vitro and lymphoid and myeloid differentiation in the bone marrow and spleen in vivo (supplemental figure 1). donor chimerism was also evident in the thymus (figure 1(b)), with signs of t cell development into cd4 cd8 and cd4 cd8 thymocytes in 50% of eshp recipients (supplemental figure 2). donor chimerism in eshp recipients was not observed in any tissues after 34 days after transplant. based on our previous findings, we tested the hypothesis that eshps were actively rejected by the host innate immune cells. enlarged spleens in eshp recipients were consistently observed compared to both untransplanted nsg and adult whole bmt controls (figure 2(a)). to quantify this observation, the spleens in adult bm-transplanted controls displayed a 2.62-fold increase in mean weight compared to untransplanted nsg mice (figure 2(b)), consistent with their increased donor hematopoietic chimerism (figure 1(c)). similarly, the mean spleen weights in eshp recipients were increased 3.71-fold compared to untransplanted nsg controls (figure 2(b)). although some donor eshp-derived cells were observed in the spleen (figure 2(c)), a higher absolute number of host-derived cells in eshp recipients were present (figure 2(d)). this number of host-derived cells in the spleen was significantly higher than that of adult bm recipients (figure 2(d)). since nsg mice lack nk, t, and b lymphocytes, we reasoned that only host myeloid cell populations (which include cd11b, gr-1, and f4/80 macrophages could be increased in the eshp recipients). indeed, a significant increase in host-derived cd11b or gr-1 cells was observed between eshp recipients versus adult bmt controls (figures 3(a) and 3(b)); but the numbers of cd11b and gr-1 cells did not account for the observed enlargement of spleen size (figures 2(a), 3(a), and 3(b)). remarkably and in contrast, eshp recipients displayed a statistically significant increase in host f4/80 macrophages compared to both untransplanted nsg and bmt controls (figure 3(c)), and f4/80 cells were significantly increased in both percentage and absolute number in eshp nsg mice compared to 129 bm nsg (supplemental figure 3). the prevalence of f4/80 macrophages was also visible by immunohistochemical staining (figure 3(d)). we hypothesized that eshps were actively phagocytosed by host f4/80 macrophages, but the low levels of donor-derived cells in eshp recipients precluded our ability to test this hypothesis directly in vivo. instead, we used an in vitro phagocytosis assay developed in our laboratory, in which phagocytosis of labeled targets can be quantified by flow cytometry. macrophages from the 129 mouse strain, which is syngeneic to the eshps, and macrophages from nsg mice, which are allogeneic to the eshps, were used. regardless of their source, macrophages phagocytosed eshp targets at a higher rate than control adult lin bm targets isolated from the respective strains (11.81- and 24.09-fold higher by syngeneic 129 and allogeneic nsg macrophages, resp. in addition, nsg macrophages were 1.65-fold more efficient in eshp phagocytosis than 129 macrophages (figure 4), suggesting that allogeneic macrophages may react more robustly toward eshps than their syngeneic counterparts. these in vitro data corroborate the increase in host f4/80 splenic macrophages in the spleens and the low donor hematopoietic chimerism in eshp recipients observed in vivo. furthermore, flow cytometric measures of forward scatter properties of f4/80 macrophages indicated that host f4/80 macrophages in eshp nsg mice were larger in size than f4/80 macrophages in 129 bm nsg controls, consistent with phagocytosis of eshps (supplementary figure 4). the increased phagocytosis of eshps by allogeneic nsg macrophages provided further support to our hypothesis that macrophages were indeed responsible for poor engraftment of eshps after transplantation in vivo. to directly test if macrophages were a barrier to eshp engraftment in vivo, clodronate-loaded liposomes (cll) [24, 28] were used to deplete nsg mice of macrophages prior to and on days 5, 10, and 15 after transplant. cll treatment specifically depleted f4/80 macrophages subsets in the spleens and bm (supplemental figure 5(a)). donor hematopoietic chimerism was 6.4-fold higher in the spleens of eshp recipient mice treated with cll compared to control eshp-transplanted mice treated with pbs-loaded liposomes (pll+eshp) (p=0.0020, figures 5(a) and 5(b) and supplementary figure 5(d)), even though the spleen size of cll+eshp-treated mice was smaller (figure 5(c) and supplementary figure 5(c)). in contrast, in the bm, no significant differences in donor chimerism were observed between pll+eshp and cll+eshp groups (figure 5(b) and supplementary figure 5(e)). cll treatment did not affect the high level of donor engraftment attained in mice transplanted with whole adult bm cells or enriched lin bm progenitors (supplemental figure 5(b)). our previous work demonstrated indirect recognition of eshps by macrophages could stimulate t cell proliferation in vitro. in this report, we extend this work to demonstrate that depletion of host macrophages can improve the tissue-specific engraftment of eshps in vivo. taken together, we conclude that f4/80 macrophages are a specific immune barrier for eshps after transplantation. a working model that summarizes our results we observed a specific increase in host f4/80 cd11b macrophages in eshp recipients and have strong evidence that these macrophages phagocytose eshps in vitro. although it is possible that local inflammation in the host could result in the macrophage increases in vivo, we have not observed any evidence of contamination in our eshp cultures or pathogenic infection in our eshp or control transplanted mice. f4/80cd11b red pulp macrophages have roles in both filtering the blood and removing damaged erythrocytes [29, 30], so we favor the possibility that host red pulp macrophages are induced to expand specifically in response to eshps in vivo. we hypothesize that macrophage recruitment and phagocytosis of eshps may be related to three aspects of their maturation state: (1) eshps secrete products that recruit monocytes and macrophages or induce differentiation or proliferation of macrophages, (2) eshps express activating ligands [31, 32], and/or (3) eshps lack macrophage inhibitory ligands [3337]. with regard to the latter point, major histocompatibility complex class i (mhc-i) has been characterized as a macrophage inhibitory ligand [29, 38], and eshps express mhc-i at moderate levels compared to adult hematopoietic cells. in addition, in preliminary studies, we observed that eshps express minimal levels of the macrophage inhibitory ligands cd47 and cd200 (supplemental figure 6). whether induced expression of macrophage inhibitory ligands can improve eshp engraftment, and whether the host macrophage response is directed to a particular cell subtype within eshps, will require further experimentation. the observation of higher eshp numbers in the host spleen versus the bm naturally leads to the question of why this is the case. one explanation could be that the spleen is the natural niche for eshps. in support of this idea, extramedullary hematopoiesis is common in the red pulp region of the spleen in fetal and neonatal mice and decreases in adults as hematopoiesis moves to the bm. therefore, we speculate that poor engraftment of eshps in vivo may be caused by a developmental incompatibility between the adult spleen microenvironment and the eshps. this is supported by recent evidence that embryonic hscs demonstrate a propensity to engraft better in neonatal recipients than adult recipients and also populate the niches that best match their developmental stage. another possibility is that eshps may harbor defects in homing that prevents their sufficient migration to the bm cavity and these homing defects reduce their engraftment [10, 41]. further experimentation to test different routes of eshp transfer, such as intrafemoral injection [12, 42], is necessary to test this hypothesis. furthermore, macrophages have been implicated as niche cells that promote retention of hscs in the bm and our data showed that cll treatment increased donor hematopoietic chimerism in the spleen but not in the bm of eshp recipients. if f4/80 host macrophages serve as a bm niche cell population for eshps, we posit that their depletion may prevent engraftment of eshps in the bm. there are some caveats of the clodronate liposome system that should be taken into consideration when interpreting our results. since there is evidence in the literature of toxicity of clodronate liposomes and the liposomes alone can have some nonspecific effect on macrophages, we empirically determined the appropriate dosage of pll and cll in untransplanted nsg animals to find the optimal dose of liposomes that would deplete the macrophages but not kill the animals. similar levels of donor chimerism in the eshp only group and the pll+eshp group were expected. however, donor chimerism in the pll+eshp group (figure 5) was observed to be lower than the eshp only groups (figure 1(c)), suggesting nonspecific detrimental effects of the control empty since we can not rule out this possibility, we are of the opinion that comparison of donor chimerism in pll+eshp versus cll+eshp-treated animals is appropriate and better than comparison of donor chimerism in cll+eshp-treated mice versus mice that received eshp without liposomes. we conclude that albeit low, there is clearly a higher level of donor chimerism in the cll+eshp-treated mice compared to the pll+eshp-treated mice. furthermore, there is evidence that cll can deplete cell types other than macrophages, including dendritic cells, osteoclasts, and neutrophils. additional studies to target specific subsets of phagocytes are necessary to ascertain their roles on eshp engraftment. the derivation and transplantation of eshps that resemble adult hscs are an important goal for the field of hematopoietic stem cell biology. although transplanting eshps using the adult definition of hsc seems straightforward (lineage-negative, ckit, and sca-1), it is well documented in the literature that embryonic and adult hematopoietic progenitors expressed different surface markers in vivo and in vitro. in particular, cd41 is expressed on definitive embryonic hematopoietic progenitors in vivo, before the expression of the classic cd45 hematopoietic cell marker [25, 26]. subfractionated different eshp populations from their esc cultures and found superior engraftment by cd41 eshps after transplantation in vivo (regardless of ckit or sca-1 expression) and more recently demonstrated that these cells are not definitive hscs by transcriptional analysis. in line with these previous studies, our results do not support the assumption that eshps share the same markers and behavior as their adult hsc counterparts. we have only observed short-term hematopoietic engraftment with multilineage differentiation from eshps transplanted into immunodeficient mice. eshp engraftment was not expected to reach the same level of bm engraftment, as previous work showed that esc-derived hematopoietic progenitors with a similar surface phenotype to our eshps achieved a wide range of donor chimerism levels in lethally irradiated immunodeficient hosts, but reconstitution was not achieved in 100% of the animals. since it is technically difficult to obtain sufficient eshps in culture for in vivo transplantation and there was a high probability of mouse deaths due to lack of reconstitution, we opted to use sublethal irradiation in our studies and optimized the cll dosage to reduce nonspecific toxicity in the animals. the observation that mice treated with eshps do not achieve the same levels of donor chimerism as bmt controls demonstrates that eshps may not compete well with host nsg bm progenitors, whereas bm cells from 129 adult donors are able to do so. treatment with cll increased the level of donor chimerism in mice that received eshps compared to pll-treated mice. cll+eshp-treated mice also showed decreased spleen weights compared to pll+eshp-treated mice (supplemental figure 3(c)), which could be attributed to the presence of fewer host macrophages in the former, delaying the innate immune rejection of eshps. pll or cll treatment in bmt recipients did not prevent high levels of engraftment in nsg hosts (data not shown). taken together, we interpret these data as a demonstration that low engraftment from eshps is, in part, due to an innate immune response by host macrophages. several groups have reported strong recruitment of macrophages after transplantation of esc or esc derivatives undergoing active rejection [19, 20]. observed that adult macrophages could destroy the inner cell mass (which contain escs) from early blastocysts but that the trophoblast repelled these macrophages, suggesting a possible biological mechanism to protect the inner cell mass cells from the maternal (host) immune system macrophages during early embryonic development. it is possible that this natural embryonic trophoblast mechanism, which prevents adult macrophages from rejecting embryonic cells, might be leveraged to protect in vitro esc-derived tissues after transplantation in adult hosts. current treatments for hemophagocytic lymphohistiocytosis and macrophage activation syndromes include broad immunosuppressive therapy [4951] which could accompany any eshp transplantation in humans. recently, the janus kinase inhibitor ruxolitinib was shown to improve the symptoms of hlh in murine models and this is another possible direction for future human eshp transplants. strategies to skew polarization of macrophages toward proinflammatory m1 versus regulatory m2 phenotypes are currently being explored for clinical applications (although further studies are required to determine the m1 versus m2 phenotype of the f4/80 macrophages in the eshp nsg chimeras). to our knowledge, a comparison of the expression levels and functional abilities of macrophage inhibitory ligands and in the nsg versus 129 strains has not been performed. however, there is evidence of mouse strain differences in the binding of the ability of the nod, balb/s, and b6 forms of sirp to bind to the human macrophage inhibitor receptor cd47 and prevent phagocytosis of xenogeneic cells in mice. our observations that eshps are phagocytosed by host macrophages in vitro and that clodronate treatment promotes higher donor chimerism from eshps in mice in vivo strongly suggest that eshps stimulate innate immune responses and that control of macrophage-induced immune rejection should be considered in the field as new hematopoietic derivatives are produced from escs or esc-like induced pluripotent stem cells for in vivo transplantation.

understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. murine embryonic stem cell-derived hematopoietic progenitors (eshps) were generated via coculture with the bone marrow stromal cell line, op9, and then transplanted into nod.scid.common gamma chain (nsg) knockout mice, which lack b, t, and natural killer cells. compared to control mice transplanted with adult lineage-negative bone marrow (lin bm) progenitors, eshp-transplanted mice attained a low but significant level of donor hematopoietic chimerism. based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of eshps in vivo. enlarged spleens were observed in eshp-transplanted mice and found to contain higher numbers of host f4/80+macrophages compared to bm-transplanted controls. in vivo depletion of host macrophages using clodronate-loaded liposomes improved the eshp-derived hematopoietic chimerism in the spleen but not in the bm. f4/80+macrophages demonstrated a striking propensity to phagocytose eshp targets in vitro. taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic eshp engraftment in vivo.

PMC5107259

pubmed-756

although spinal cord abnormalities in patients with acquired immunodeficiency syndrome (aids) have been infrequently reported in the literature, myelitis is a known complication of aids and is occasionally the initial complaint. the incidence of myelopathy may be as high as 20%, with 50% of the cases reported post-mortem [3, 5]. toxoplasmosis is the most common cause of intracranial lesions responsible for neurological deficits in aids patients, occurring in 310% of patients in the united states and in up to 50% of aids patients in europe, latin america, and africa [6, 10]. a review of existing literature suggests that although toxoplasmic myelitis is uncommon, it should be suspected in immunocompromised patients who present with symptoms of acute or sub-acute myelopathy. the initial evaluation should aid in differentiating between other reported causes of myelopathy (such as vacuolar myelopathy, lymphoma, tuberculosis, and viral infections including cytomegalovirus infection, herpes zoster, and herpes simplex) in aids patients [2, 4]. since 1986, 18 cases of apparent toxoplasmosis of the spinal cord have been described [4, 7, 11, 12]. a case report and pertinent literature were reviewed, leading to the diagnosis and management options discussed below. a 40-year-old hispanic man was admitted to the hospital after being found unconscious. he had a 2-day history of disorientation that manifested itself as his being unable to recognize family members. upon admission he regained consciousness, becoming alert and oriented, but developed urinary retention and was unable to move or feel his lower extremities. he had no history of systemic illness, but mentioned having been treated for herpes zoster approximately 3 years prior. he denied having a history of fever, chills, nausea, vomiting, rash, seizures, or other constitutional symptoms. he had not traveled recently, and his hiv status was unknown at the time of admission. physical examination revealed fever, nuchal rigidity, and a distended bladder that required catheterization. a neurological examination confirmed that he had intact cranial nerves and normal upper extremity strength. both lower extremities exhibited flaccid paralysis and reduced response to pain, touch, and temperature (bilaterally from l1). cerebellar examination was normal in the upper extremities, with an adequate finger-to-nose exam. the patient s wbc, hemoglobin, platelets, electrolytes, bun, and creatinine were within normal ranges on admission. the initial brain ct (without contrast) revealed a focal area of vasogenic edema in the left frontal lobe (without significant mass effect) and abnormal white matter hypo-density seen along the lateral aspect of the right frontal horn of the internal capsule. a brain mri (with and without gadolinium enhancement) revealed multiple bilateral ring-enhancing intra-axial brain lesions, and an ill-defined cortical and sub-cortical enhancement, with diffuse edema in the frontal lobe. spinal mri (with and without gadolinium) showed the spinal cord to be abnormally diffuse, with swelling and edema in the cervicothoracic region. along with the diffuse abnormal hyperintense swelling, the cauda equina had an edematous appearance, and signal intensity was abnormally increased, which is compatible with transverse myelitis (figs. 1 and 2). finally, imaging also revealed a focal area of abnormal signal intensity in the ventral inferior pons. 1initial mri: notice the abnormal spinal cord swelling cervical and at cauda equinafig. 2normal f/u study initial mri: notice the abnormal spinal cord swelling cervical and at cauda equina he was initially given broad spectrum antibiotics (ceftriaxone, vancomycin, and ampicillin) and acyclovir at admission for a presumptive cns infection. hiv serology was recommended, though the patient denied ever having engaged in high-risk behavior or having had a blood transfusion at any time in his past. analysis of csf showed abnormal values including a wbc count of 11/mm and a protein level of 184.5 mg/dl, with a glucose concentration of 51 mg/dl. stain and cultures for bacteria, fungi, and acid-fast bacilli were negative. a csf cryptococcal antigen titer and htlv i an anti-toxoplasma immunoglobulin (igg) immune titer was positive at 8.99 (< 0.9=negative; 1.10=positive). at this point, the patient disclosed having engaged in homosexual behavior and accepted hiv testing, which was reactive by elisa. empiric treatment for toxoplasmosis with sulfadiazine, pyrimethamine, folinic acid, and haart therapy with lamivudine/zidovudine and indinavir were started. intravenous dexamethasone was administered for 7 days, and oral methylprednisolone for another week. biopsy was deferred due to possible complications and the evidence of improvement with therapy. within 4 days of empiric treatment for toxoplasmosis, the patient s neck rigidity resolved. he progressively developed discrimination to touch and his vibratory sensation improved, as did his bilateral strength (1/5) of the lower extremities. follow-up consisted of a brain and spinal cord mri (with and without gadolinium), 14 days after treatment with anti-toxoplasmosis therapy combined with steroids, which demonstrated resolved swelling and residual t2 hyper-intensity in the mid-thoracic spinal cord, without an interval change in the brain mri. aids-related spinal cord disorders include neoplasms, infections (including hiv itself), vascular disease, and other undefined etiologies. toxoplasmosis and lymphoma are the two most common intracranial lesions, and both have been reported in increasing frequency in the spinal cord. myelopathy is usually under-diagnosed, probably because of the occurrence of coexisting conditions such as aids dementia complex (adc), cerebral lesions of varying etiologies, vacuolar myelopathy, lumbosacral myelopathy, or peripheral neuropathy that may mask the clinical signs suggestive of myelopathy [5, 9, 10]. other causes of myelopathy appear to be less common than toxoplasmosis and include tuberculoma, cytomegalovirus, varicella-zoster virus, and, possibly, lymphoma [4, 10]. postmortem histopathology examination has resulted in a definitive diagnosis of myelopathy in the majority of patients. spinal cord lesions often manifest with a variety of symptoms, such as leg weakness, progressive paraparesis with spasticity, absent reflexes, ataxia, incontinence, and paresthesias. in the 14 toxoplasmosis cases with spinal cord involvement reviewed by vyas and ebright, they found the most common presentations of acute or sub-acute symptoms were paraparesis, urine retention, sensory level deficits, fever, and local pain. csf cytology showed increased protein-level elevation and a cd4 count of less than 50/mm. in the patients evaluated by spinal cord mri imaging, localized intramedullary lesions or spinal cord edema were found in more than 90% of cases associated with positive t. gondii igg antibody. no cases of associated transverse myelitis, as in this case, were described. in our case, evaluation in a symptomatic patient should include serum and csf cytology and antibody (immunological) studies as they remain the gold standard for identifying an infectious agent. complete radiographic imaging of the entire neuroaxis [3, 15] is key in clearly defining inflammatory lesions of the brain and spine, and in the visualization of typical lesions that allow for rapid diagnosis. this helps determine whether the lesion is extramedullary or an intrinsic spinal cord disease, which in turn provides information to determine whether surgical or medical therapy is needed. in opportunistic diseases, the imaging studies also have a crucial role in diagnosis and monitoring of the therapeutic response. neuroimaging findings, along with other clues, help to narrow the differential diagnosis. on an mri, findings of a normal-sized spinal cord but with an abnormal signal should signal the possibility of vacuolar myelopathy and adc-related hiv myelitis. lesions of viral myelitis can potentially cause spinal cord enlargement; however, laboratory data are required for confirmation. if spinal cord enlargement is present, toxoplasma myelitis and lymphoma should be strongly considered. lymphoma has been associated with positive csf polymerase chain reaction (pcr) for epstein-barr virus. if both spinal cord enlargement and an abnormal signal are associated with meningeal enhancement, then cmv, m. tuberculosis, lymphoma, and toxoplasmosis should be considered along with other less likely infectious causes. mri (plain and contrast-enhanced) is currently considered appropriately sensitive for detecting brain and spinal cord lesions; however, equal sensitivity has been reported using delayed-contrast ct scanning. the current guidelines for diagnosis of intracranial lesions state that t1 spect is an option when available. when positive (marked uptake in contrast to toxoplasma), it appears to be highly specific for primary cns lymphoma [6, 8]. these guidelines have been described as helpful in spinal cord lesions and suspected lymphoma, although specific guidelines for spinal cord lesions are not presently available. the newer and more available mri techniques of diffusion-weighted imaging (dwi) may be of help in the differentiation of lymphoma, showing no restriction in water diffusion. if a diagnosis of solitary or atypical lesions can not be made with noninvasive methods, a biopsy (open or stereotactic) may be warranted in patients with concomitant negative toxoplasmosis serology since a negative serology does not exclude diagnosis of toxoplasmosis nor differentiate from lymphoma. a biopsy may also be considered necessary if a patient experiences a rapid decline in function or, alternately, fails to improve despite therapy. brain biopsies have been associated with hemorrhage risk and an increased mortality (2%) and morbidity rate (12%) in patients with hiv/aids. empiric treatment for toxoplasmosis with oral pyrimethamine and sulfadiazine (with folinic acid) has been recommended in all cases of intracranial mass lesions in patients with hiv/aids (except in a solitary mass with negative toxoplasma serology). steroids promote radiological improvement in about 80% of patients, and improvement can be seen in about 1 week, supporting the diagnosis. patients are usually monitored clinically and radiographically for response to treatment over a 10- to 14-day period following empiric therapy. if responsive, anti-toxoplasmosis therapy is continued indefinitely, and reevaluation in the absence of steroid treatment is mandated. if the lesions remain unchanged or progress, the diagnosis has to be reconsidered and the therapeutic strategy reevaluated. in cases of atypical large solitary toxoplasma lesions resembling lymphoma (that also present with spinal cord enlargement), additional diagnostic modalities should be performed [3, 6]. due to the immunosuppressant effects of steroids, the optimal dosage and period of treatment for spinal cord lesions must be addressed in future studies. in this case, the brain and spinal cord lesions were found in an hiv-positive man who had not previously been diagnosed. for this reason, other causes of cns infection and lymphoma were first considered. after the positive serology, empiric treatment for cerebral toxoplasmosis was initiated. the patient s clinical and radiographic improvement led to the final diagnosis of toxoplasmic myelitis and encephalitis, similar to other cases documented in the literature by vyas et al. in 1996. furthermore, toxoplasmic myelopathy has been described in patients without hiv, albeit with severe immunosuppression [2, 3, 11, 12], and in patients with isolated spinal cord lesions [1, 2, 11]. new neurological deficit in any patient should raise a high index of suspicion of hiv infection. appropriate diagnostic methods and management that are practical for all settings, including those with limited technologies, should be sought. newer methods of diagnosis and management for all neurological complications of hiv should be addressed. we established ours based on the clinical manifestations, history, and general improvement of the patient's condition. although spinal cord toxoplasmosis is uncommon, it has been suggested that most patients with aids that present with evolving myelopathy, characterized by extremity weakness, sensory involvement, spinal cord enlargement, and enhancing lesions in brain or spinal cord ct or mri, have toxoplasmic myelitis [3, 5]. the likelihood of diagnosis increases if serum titers for toxoplasma antibodies are positive or where the initiation of early treatment with empiric anti-toxoplasmosis therapy and steroids improves both the patient s clinical and radiographic manifestations.

approximately 10% of patients with aids present with some neurological deficit as their initial complaint, and up to 80% will have cns involvement during the course of their disease. toxoplasmosis is the most common cause of cerebral mass lesions in patients with aids, but appears to be an uncommon cause of spinal cord disease. the incidence of myelopathy may be as high as 20%, with 50% of the cases reported post-mortem. we present a unique case of spinal cord disease as the initial presentation of aids. we also present a comprehensive literature review of this topic, its diagnosis and treatment. this is a retrospective chart review case report. after a detailed case presentation, several diagnostic and therapeutic aspects of this unique case are thoroughly discussed. although spinal cord toxoplasmosis is uncommon, it has been suggested that most patients with aids that present with evolving myelopathy, characterized by extremity weakness, sensory involvement, spinal cord enlargement, enhancing lesions in brain or spinal cord ct or mri, have toxoplasmic myelitis.

PMC3047888

pubmed-757

soft tissue recessions frequently cause esthetic disharmony and dissatisfaction. compared with soft tissue coverage around a tooth, the coverage of an implant site is obviously unpredictable. particularly in the cases of thin mucosa, a significant greater amount of recession takes place compared to thick mucosa. to overcome this problem, this case report demonstrates a two-step mucosal dehiscence coverage technique for an endosseous implant. a 33-year-old female visited us with the chief complaint of dissatisfaction with the esthetics of an exposed implant in the maxillary left cental incisor region. a subepithelial connective tissue graft was positioned in the apical site of the implant and covered by a mucosal flap with normal tension. at 12 months after surgery, the recipient site was partially covered by keratinized mucosa. however, the buccal interdental papilla between implant on maxillary left central incisor region and adjacent lateral incisor was concave in shape. to resolve the mucosal recession after the first graft an esthetically satisfactory result was achieved and the marginal soft tissue level was stable 9 months after the second graft. this two-step approach has the potential to improve the certainty of esthetic results. soft tissue recessions around dental implants have frequently been observed. a recession during the early phase after implant crown placement originates from the process of modeling of the peri-implant mucosa. reported that bone resorption and soft tissue recession were manifested as 0.7 and 0.6 mm at the buccal aspect of the implants during the period between implant placement and abutment connection. muller et al. has suggested that thin mucosa is friable and recesses more readily following mechanical stress and surgical procedures than does thick mucosa. furthermore, the quantity, quality, and position of the existing peri-implant bone also affects soft tissue recession. burkhardt et al. surgically covered soft tissue recessions using a coronally advanced flap (caf) in combination with a free connective tissue graft although the implant sites revealed a substantial, clinically significant improvement following coronal mucosal displacement, clinically significant soft tissue shrinkage was observed after one month of healing. we hypothesized that a two-step approach is useful in acquisition of esthetic results because the first graft can increase the thickness of the peri-implant mucosa. the present case demonstrates a two-step mucosal dehiscence coverage technique in an endosseous implant. sufficient soft tissue regeneration was achieved and the marginal soft tissue level was stable with an esthetically satisfactory result. the patient was a 33-year-old female who visited us on october 2008 with a chief complaint of esthetic dissatisfaction in the exposed implant of the maxillary left cental incisor region. periodontal examination revealed a healthy peri-implant condition with a probing depth ranging from 2 to 3 mm and satisfactory oral hygiene was observed. there was 3 mm abutment exposure in the buccal mucosa around the implant (fig. an intraoral periapical radiograph and cone beam computed tomography were taken to evaluate peri-implant bone resorption. because only 1 mm of marginal peri-implant bone resorption was observed (fig. 2) and the implant was a bit buccally angulated, no bone graft or removal of the implant was performed, but it was decided to carry out only soft tissue augmentation to restore esthetics. following local anesthesia, a circumferential partial-thickness incision was performed using a ck3 stainless steel blade (blade round tip angled 10 deg, swan analytical usa inc., wheeling, il, usa) to achieve a 17 mm wide12 mm height pouch around the dehiscence (fig. 3). a 106 mm subepithelial connective tissue graft (sctg) was harvested from the palate in the right second premolar to second molar region. the graft tissue was trimmed to fit the formerly prepared recipient bed. a horizontal incision to the bone was made 5 mm from the palatal gingival margin and the blade (razor blade, feather safety razor co., osaka, japan) was subsequently placed parallel to the long axis of the roots. another horizontal incision was made 2 mm coronal to the first incision and the periosteum was dissected before removing the wedge of soft tissue. then, the sctg was inserted into the recipient pouch and sutured with 7.0-nylon (nicho kogyo ltd., the graft was positioned and fixed by a 7.0-nylon suture in apical site of the implant. after covering the sctg graft with a mucosal flap, the flap was coronally stretched with a 7.0-nylon suture (coronally positioned flap, cpf). the donor site was sutured with 5-0-nylon (look suture 774b, angiotech, vancouver, bc, canada) (fig. 4). at 12 months after surgery, the recipient site was partially covered by keratinized mucosa (fig. 5a). however, the buccal interdental papilla between implant on maxillary left cental incisor region and adjacent lateral incisor was in a concave shape. to achieve an esthetic result, we planned a second graft. following local anesthesia, an sctg was harvested from the palate in the left second premolar to second molar region. 5c). because proper vertical mucosal gain was achieved at 8 months after the second graft, prosthetic treatment was started. after an impression of implant on maxillary left central incisor region and prepared adjacent lateral incisor the all-ceramic crowns (zirconia framework and glass-ceramic veneer material) were fixed with resin cement. the intra-oral picture shows that the peri-implant mucosa is harmonious with the right central incisor at 9 months after graft (fig. although the interdental papilla between implant and adjacent tooth was more apical than the contralateral side, it was esthetically acceptable to the patient. a two-step split pouch technique using an sctg demonstrated that it was possible to achieve a substantial area of soft tissue dehiscence coverage around the endosseous implant. burkhardt et al. evaluated soft tissue dehiscence coverage using a cpf in combination with a free connective tissue graft around the implants. the soft tissue recessions were covered with a coronal overcompensation at a mean of 0.5 mm after the graft. after 1 and 6 months, shrinkage of 75% and 66%, respectively, was observed. compared with the percentages of soft tissue coverage around the tooth, the implant sites clearly could not be covered consistently. a folding process further hampers vascularization of the graft and could induce extensive shrinkage. to overcome these problems, we considered the anatomical and physiological properties of the buccal peri-implant mucosa. burkhardt et al. reported that the preoperative mucosal thickness in the implant site was positively correlated with the height of recession coverage. nozawa et al. also reported that marginal soft tissue dimensions indicated that there may be a relationship between the thickness and the height of about 1.5:1 at the platform level. the results may support the approach of acquiring thick mucosa to improve vertical mucosal regeneration. thick soft tissue, which is obtained from the first graft, can increase the blood supply to the graft; thus, it may prevent necrosis and recession after the second graft. although pedicle grafts, such as the laterally positioned flap, double pedicle flap, oblique rotational flap and cpf have an advantage over free soft tissue grafts, there is only a limited increase in the tissue thickness and width of the keratinized gingiva. on the other hand, free soft tissue grafts, such as the free gingival graft (fgg) and sctg can increase the width of the keratinized gingiva and tissue thickness. the sctg was statistically superior in achieving root coverage when compared to the fgg, cpf alone, guided tissue regeneration, and allogeneic tissue grafts. the caf with envelope technique has the advantages of increasing keratinized mucosa, a better postoperative course, and a more positive esthetic evaluation than caf with vertical releasing incisions (vris). the longer surgical time to complete the caf with vris furthermore, vris often cause unesthetic visible scars after healing that patients can find unsatisfactory. from a biologic standpoint, the blood supply to soft tissue grafts is critical for the success of the surgery. site-related factors such as the dimensions of the recession defect, vestibulum depth, and the level of interdental or interimplant papilla should be considered. miller classified the oral mucosal recession by the marginal soft tissue level and interproximal bone loss. based on the literature, complete root coverage can be expected in class 1 and 2. however, there are no reports on the classification of soft tissue recession in dental implants therefore, site related factors, surgical methods, and the patient's expectations should be cautiously taken into account.

purposesoft tissue recessions frequently cause esthetic disharmony and dissatisfaction. compared with soft tissue coverage around a tooth, the coverage of an implant site is obviously unpredictable. particularly in the cases of thin mucosa, a significant greater amount of recession takes place compared to thick mucosa. to overcome this problem, this case report demonstrates a two-step mucosal dehiscence coverage technique for an endosseous implant. methodsa 33-year-old female visited us with the chief complaint of dissatisfaction with the esthetics of an exposed implant in the maxillary left cental incisor region. a partial-thickness pouch was constructed around the dehiscence. a subepithelial connective tissue graft was positioned in the apical site of the implant and covered by a mucosal flap with normal tension. at 12 months after surgery, the recipient site was partially covered by keratinized mucosa. however, the buccal interdental papilla between implant on maxillary left central incisor region and adjacent lateral incisor was concave in shape. to resolve the mucosal recession after the first graft, a second graft was performed with the same technique. resultsan esthetically satisfactory result was achieved and the marginal soft tissue level was stable 9 months after the second graft. conclusionsthe second graft was able to resolve the mucosal recession after first graft. this two-step approach has the potential to improve the certainty of esthetic results.

PMC3394995

pubmed-758

older adults with hoarding behaviour are often at a high risk of being homeless making aging in place extremely complex. this paper reports on a study that examines the value of a community-based planning approach that responds to the needs of this population, a population that is both increasing in number and that is very seldom studied. it synthesizes new research about the complexities associated with remaining in one's own home when he/she is over 55 and has compulsive hoarding behaviour. and it examines how a collaborative community response promotes successful aging in place for this population. not only are community-based services necessary to better understand because they are central to all health sectors, but also current research surrounding those with hoarding behaviour is mostly focussed on methods addressing individual-level behavioural characteristics of hoarding through cognitive behavioural therapy (cf., the extensive work of frost and steketee). missing are descriptions of community-based planning approaches for health and social service sectors working hard to make aging in place a possibility. for older individuals with hoarding behaviour, aging in place is complex because hoarding behaviour is multifaceted; it touches on social, environmental, familial, and personal issues. aging in place is also not simple for those with hoarding behavior because they want to remain living in their own homes, neighbourhoods and communities which seem to require that a collection of agencies, often representing different sectors, understand their needs in order to help them stay in the community and age in place. in this paper, aging in place refers to an ideal where people can age in the familiarity of their homes, neighbourhoods, and communities where their quality of life is maximized by the availability and accessibility of supports and services that respond to their needs and capacities [4, 5]. aging in place, in addition, is about belonging to a community that supports one's many needs, for example, physically, socially, mentally, environmentally, and so forth. to understand the varying aspects of aging in place in late life, older individuals with hoarding behaviour need to be further understood so their aging journey is successful; collaborative approaches by community support agencies can help to make that possible. in our case, social and health related organizations from different sectors, that in some way supported people with hoarding behaviour in edmonton, ab, canada, were brought together in 2007 through the leadership of the social worker of a seniors after having visited a number of individuals with hoarding behaviour (age 55 +) in her professional role, she noticed, as did those members of the imminent community collaborative, that those with hoarding behaviour were at a high risk for being evicted from their homes, and they experienced shame associated with their hoarding resulting in isolation, as well as depression; they were at risk of falling in their own homes, and generally, they were living in unsanitary conditions. to approach this highly vulnerable population, to respond to some of their needs, especially to prevent their potential eviction, a concerted effort by a broad representation of social, health, and other agencies was necessary. the collaborative met together regularly (e.g., once every 2 months over the years and as of june 2011, it continues to meet) to continually plan for and improve the support of older adults with hoarding behaviour in the edmonton area. although little is still known about the effects of one's neighbourhood on the mental health of older adults, positive mental health in later life may be influenced by the way in which older people feel about their neighbourhood. it is fair to conclude, therefore, that for older adults with hoarding behaviour, place matters and the role of place as locality is key when making meaning at both the individual and the collective levels. in particular, local place is an important factor in identity, in ones sense of community, and attachment; this is crucial to the determinants of attachment, satisfaction, and behaviour. also important is place and the person-place relationship are very much grounded in a context of fear over loss of place and the obliteration of locality. aging and compulsive hoarding behaviour need to be examined in concert with one another. as our aging population rapidly increases, the number of older adults with hoarding behaviour will also quickly increase. although hoarding behaviour usually begins in early adolescents, its severity increases with age. results from the hopkins epidemiology of personality disorder study found that the odds of hoarding were over two times as great in the oldest compared with the youngest age group, and in a study of hoarding-related complaints to public health departments in massachusetts, 40% of individuals that hoarded were involved with elder service agencies. referring to frost and hartl when defining hoarding behaviour, there are key characteristics: the acquisition of and failure to discard a large number of possessions that seem to be of little use or limited value; cluttered living spaces that can not be used as intended; significant distress and/or impairment associated with the clutter. hoarded possessions clutter living spaces until rooms are difficult or impossible to use for their designed purpose, causing significant stress to the individuals themselves. possessions may be purchased through compulsive buying and/or through acquiring things like newspapers or discarded items from dumpsters. household clutter may interfere with and prevent daily activities, like food preparation, moving freely in one's home, and using the bathroom [11, 17]. and as clutter increases, falling, fire, sanitation issues, depression, and isolation are possible, creating significant risk for a number of outcomes but especially eviction and thus homelessness. community collaboration is one way to respond to social and health needs of a population. in fact, genuine collaborate is say to be the very thing that successfully reforms health systems. although there are many ways to define collaboration, most definitions emphasize the importance of shared responsibility and a team approach, and using a collaborative approach can significantly increase the available pool of resources from which team members can draw. a response to the multiple challenges of older adults with hoarding behaviour requires a comprehensive and far-reaching approach, more than one single agency can provide alone [22, 23]. philosophically, collaboration is rooted in systems theory which says that entities in a system are dependent on one another and ecological theory in particular proposes that causes and solutions of health and/or social problems are beyond the individual and are associated with such determinants as the health and social services that we receive. service recipients can benefit from a collaborative approach to the provision of community health services as can the agencies participating in the collaborative [2628]. using a collective made up of representative agencies to support this vulnerable population between january 2007 and january 2010, approximately 75 older adults (ages 55 +) with hoarding behaviour in edmonton, ab, canada, were provided with community supports to prevent them from being evicted from their homes. the seniors association of greater edmonton (sage) offers support through a program referred to as this full house. and this full house is a direct outcome of the work of the community collaborative. the aim of this full house for older individuals with hoarding behaviour is to prevent eviction from their home, improve their health and well-being, maintain positive social contacts, and contribute to the building of a healthy community. as the population of potential participants with hoarding behaviour in edmonton is not particularly large, a small-n approach was used in an attempt to create heuristic generalizations which tsoukas defines as opportunities to refine analytic understanding and to make more incisive distinctions than were previously possible. small-n studies are not designed to support or refute a theory, but rather, to further refine it. as such, the purpose of this study is to further examine the role of a collaborative planning approach in a community setting when seeking to help those over 55 years with issues relevant to having compulsive hoarding behaviour and wanting to age well in one's community. to further understand the value of collaboration and, in particular, its role and value in this community support, interviews and a focus group were conducted with seniors with hoarding issues involved with this full house and with the community collaborative. all interviews were semistructured and conducted by a third party researcher (i.e., research assistant). ethics approval for this research was received from the university of alberta, research ethics board. (n=5) individuals with hoarding behaviour involved with this full house, all of whom were over the age of 55. the semistructured, face to face interviews took place at a location expressed as being most comfortable to the interviewee, for example, at their homes, at a university office, at a nearby coffee shop. these study participants were first contacted by the social worker from sage who directs this full house. the individuals with hoarding behaviour had either referred themselves to this full house or had been referred to it by one of their health care practitioners or family members. at an appointment with the individuals, the social worker informed them about the study asking if they might be interested in being interviewed by a researcher about their experience with this full house. if they agreed (which all five did), she gave them a one-page written description of the study. she then went through the summary of the study ensuring they understood what was being requested from them. with their agreement, their telephone numbers were provided to the researcher (i.e., research assistant) who contacted them and established a location and time to meet for the interview. the aim of the one-hour interviews was to gather information regarding their experience with this full house. in particular, the interview was used to understand their perceptions of the impact of or value of their association with this full house, that is, the aspects of the program they benefitted from most. a community collaborative made up of social and health related agency representatives providing insights into the ongoing development of this full house the focus group interview included ten members (n=10) (of the possible 11 members in total) and was conducted in a face-to-face manner for approximately 1.5 hours. members of this collaborative represented a number of expert groups: social workers, home care nurses, geriatric neuropsychologists, geriatric nurses, fire and safety investigators, public health practitioners, and environmental health and safety officers. the focus group questions centered on the nature of the working relationship between and amongst the members and their observations about the value and impact their work may have had on the service users, that is, those with hoarding behaviour. the questions guiding the semistructured interview were generated from key themes highlighted in the literature (i.e., health services, collaboration, community support, etc.) that aligned with the purpose of the study. grounded theory means that the data analysis essentially, is grounded in the data. therefore, the concepts and themes we describe in our results have evolved from and are embedded in the data collected and have been mined through a process of conceptual ordering. grounded theories are said to provide further insight, to enhance understanding, and to be used as a guide to inform action (i.e., acting on the results). as strauss and corbin describe in more detail, our interview data, in the form of pages of the exact words from the interview, was organized into categories that were not predetermined but that evolved after reading and rereading this data many times. and that, mainly described ideas and offered explanations from interviewees that had commonalities to each other. the themes described here are those that were mentioned frequently and carried the same meaning. because our aim in this study was to further our understanding of how older adults with hoarding behaviour were supported by a particular community-based planning approach, grounded theory provided the most effective means of organizing, reducing, and understanding the data. suggested below is a picture that describes how the work of the community collaborative, because of its high level of collaboration, resulted in many important benefits for older people with hoarding behaviour that align well with and facilitate the goals and ideals associated with an aging in place model. the picture also describes how the community collaborative members valued their experience on the team. as a result of this group working together to respond to the needs of older adults with hoarding behaviour, several themes evolved from the data demonstrating direct benefits for these individuals: being able to remain in their own homes; reducing their potential for harm, and minimizing their isolation all which allowed them to experience a feeling of empowerment which also helped them to generate insight into issues surrounding their hoarding behaviour. there are considerable challenges associated with aging in one place for older individuals with hoarding behaviour. they can be at significant risk of being evicted from their homes and their behaviour can be a major public health concern leading to eviction as a result of violating building, fire, or property maintenance codes. it may not be until a particular emergency occurs (i.e., water leakage, fire, and pest infestation) that a landlord is notified. one focus group member describes their role, as a member of the community collaborative, in minimizing evictions for this population: our legislation says that we do have the right to go into any public or private place if we believe there may be a public health nuisance and if that means we have to order their suite cleaned out, we'll do it. um, because you ca n't get control of bedbugs and cockroaches unless you treat all the suites and if somebody's hoarding, you ca n't get rid of them so, so they have to clean up. the five interviewees in this study were all in a situation where eviction from their homes was a potential, but they were able to remain at home as a result of a community-based approach that addressed some of their needs. prior to the existence of this community collective, one focus group member describes how the health inspector had to play all roles and visit clients once a week and nag people into cleaning up, which was mostly unsuccessful. for example, when there is a potential home eviction for individuals with hoarding behaviour, the social worker and a public health worker, together with the client and other members of the collaborative, that is, where necessary such as a fire and safety representative, provide input into the problem-solving process. a professional organizer usually assists with the practical aspects associated with cleaning up including heavy lifting, removing garbage, and reorganizing resulting in [these clients being able to] stay living at home without being on the street and [being] homeless. being able to age in one's own home, one's neighbourhood, and community fosters independence significantly impacts a more positive relationship in the person-place relationship. the five people with hoarding behaviour that we interviewed spoke about the value of being able to remain living in their own homes. one person found it motivating to have someone help him/her to clean up his/her apartment, it's the motivation of having someone there plus the helper doing the heavy lifting and heavy carrying making 76 thousand trips to the garbage bin its stressful but helpful [and] i certainly would not have been able to hire a company on my own [] if the program had not been in effect [and] i would be in deep do-do with capital health. for another individual, what helped him remain in his own home was having the home care worker put him in touch with the social worker and the cleaning person who helped him find ways to deal with issues of parting with his stuff. and for this gentleman, the social worker supported him by suggesting options: she made life more convenient for me by offering me options. and for another person, the social worker and the cleaner reminded her that the condition of her house, the bugs, the mouse droppings, the make up from years ago, the shiny covered magazines all over, and the infestation that she had, was not her fault and she could probably face organizing and cleaning it with encouragement and the help of a plan. hoarding behaviour creates a significant safety risk for the individual him/her self and for the community. harm reduction is a core principle that is essential to address the needs of those with compulsive hoarding behaviour because promoting safety is foremost. in this case, focusing on harm reduction by the community collective ensured that safety was embedded within all the actions, initiatives, and supports they provided. one focus group member talks about the value of taking on this strategic focus: we subsequently learned the value of focusing on a harm reduction approach wherein we address issues of harm first so that the person [with hoarding behaviour], at least, will be safe. even though they may be living with a significant amount of stuff every day of their lives, but at least they are safe. a harm reduction philosophy considers behaviour change to be incremental and assumes that people will maintain their behaviour change when they have decision-making power to influence their goals and put them into action. this focus on safety and reducing harm or the potential for harm helped the individuals with hoarding behaviour buy into the larger process at hand, that is, to contribute to the building of healthy neighbourhoods, supporting their well-being, and helping them stay in their homes as long as possible. instead of suddenly or immediately removing the person from a potentially unsafe environment or situation, the aim instead is first to reduce the potential for harm and create a safe place to live. in the case of these five individuals, it meant such things as: hiring a person to help them remove and reorganize their excessive items, getting help to fumigate their apartments, openly talking about their hoarding situation to help them reflect upon it, receiving nonjudgemental support, and establishing a plan to minimize household items. one interviewee describes the value of setting goals and generating a plan, we set a plan and the social worker would come back and generally we would accomplish that goal whatever it was. it also meant helping the person at risk of potential eviction, for example, to respond to requests made by the public health inspector. one person with hoarding behaviour describes the role of this service (i.e., this full house): they sort of mediate [between varying agencies] and rub off the sharp corners. reducing harm by promoting safety enables an aging in place philosophy and model as both can facilitate positive and long-term aging in one location. the community collaborative, used in this study as a planning approach helped to address the problem of isolation for these older adults. several of the representative organizations of the community collaborative, that is, a social worker, public health nurse, geriatric nurse, and so forth, offer home visitation to many of their clients and observe their living environment. a lot of these seniors are very lonely, very isolated, and so the fact that they have someone that's coming to their home often, helps. another member talked about the impact of the support group (which is provided for people with hoarding behaviour on a monthly basis as part of this full house services) on minimizing their sense of loneliness and connecting with people that have similar experiences: [there is] value of that coming together, meeting with other people and seeing and hearing that you're not alone. two interviewees with hoarding behaviour confirmed this same sentiment, about the importance of feeling connected to a group: the group has helped me and [i realized] you are not the odd one out. as a result of not feeling alone and part of a group, interviewees with hoarding behaviour said that they felt empowered. empowerment can be an outcome of collaborative relationships; it offers a catalyst for new community programs and other supports, changes in policies, and advancing health practices [35, 36]. the members of the community collaborative intentionally aimed to facilitate empowerment using it as a principle to guide their work that addressed the needs of older individuals with hoarding behaviour in the greater edmonton area. during the focus group interview, one member describes how empowerment as a guiding principle was translated into action benefitting a particular individual with hoarding behaviour: we [the community collaborative] have entertained some really creative approaches in terms of dealing with management [i.e., housing manager] and having the client lead those interventions [] as opposed to, we [the service providers] meet[ing] with management, then meet[ing] with the client we really include the client in those interventions, so that the client really is aware of everyone that's involved, what's being discussed and then they are really empowered to be part of the action plan. when individuals with hoarding behaviour are more involved in directing their own support, they may experience greater control in such decision making which can lead to empowerment. as observed by another community collaborative member, [empowerment provides] a sense of control in a situation that they may feel a lack of control. the most successful reported treatment for those with compulsive hoarding behaviour is the use of behavioural treatment, in other words, a cognitive behavioural model. to be motivated to discard their possessions, insight into their hoarding behaviour members of the community collaborative said that individuals with hoarding behaviour seemed to gain insight into their behaviour as a result of the support provided by this collaborative. this observation was expressed during the focus group interview by the social worker: the insights that come out as a part of the intervention, as you go along, then they [clients with hoarding behaviour] start to reveal some insight as to how did i ever get to this place? and, i ca n't believe this happened to me and i ca n't believe that i'm actually, i'm making some decisions now that i was not able to before. during an individual interview, one individual with hoarding behaviour reflected on the changes in her own behaviour of accumulating things: i would still be walking down one little path between the bathroom, bedroom and one side of the kitchen and that would have been it. another interviewee with hoarding behavior expresses insight into her hoarding behavior: they [the social worker] finally were able to get me to accept this condition that it probably was not my fault and that i could probably face it she also said i need to continue both thinking about and maybe following up [] with counseling [] is there an answer why i have become a person who allows clutter around myself [] ?. in the one-on-one interviews, individuals reported an improved feeling of independence and a sense of empowerment. this occurred because the collaborative team joined forces, they were united by a common goal of supporting, to the best of their ability, the needs of this population who they noticed to be struggling more and more and who they were being called upon more frequently to try to assist. on individual and collective levels, the members of the community collaborative experienced three significant benefits as a consequence of participating in this group. for example, members of the collaborative were able to access the expertise of other professionals, they maximized the use of their own skills and knowledge and significantly enhanced their understanding of hoarding behaviour. working on the collaborative team allowed the individual members an opportunity to access a broader range of skills and knowledge than those who were found solely in their own area of expertise or their own organization. in one instance, a professional social worker described how she could now present the risks associated with hoarding behaviour more objectively to a client with greater confidence as she could make reference to and more easily call on the authoritative role of the local fire department. because the firefighter and social worker were both members of the collaborative when i mention to her [the client with compulsive hoarding behavior] the possibility of having someone from [the fire department] come and just do an assessment to let her know what her risk level is [i.e., of eviction from her home], she was suddenly open to that. as a result of working collaboratively, members also got to know more about the professional resources available to them in the community through their ongoing communication together. as one health professional of the community collaborative said: it is a professional benefit to see and use the expertise around this table for the benefit of the individual clients. evaluating collaborative planning practices must consider not just the purpose of the collaboration but the value of its relational interactions. for example, asking how social relations are changed can reveal how certain conditions are impacted for the group. access to new areas of expertise is one descriptor of the quality of social relations. the sharing of expertise between and amongst the members was said to directly benefit the older people with hoarding behaviour using the services of this full house. in the words of one member of the community collaborative, a benefit of the collaborative process was working with everybody, to partner, to ensure that we're getting our clients the best support. participating in this group allowed team members to maximize the use of their own expertise. one member, a public health inspector, recounted a time when such a collaborative approach was not used to support older adults with hoarding behaviour illustrating the tremendous limitations of working in isolation: before [the community collaborative existed] it was [up to] the health inspector to try and play all roles and just sort of go and visit once a week and try and nag people into cleaning up. which was mostly unsuccessful and was n't really our job. i mean we are not social workers, we're not mental health workers, we are public health inspectors. functioning alone, the health inspector had to operate as the only contact for this client group. working in isolation took away time from the job he/she was actually trained and hired to do forcing them to work beyond their professional scope of practice. as further evidence of the value of being able to maximize one's own expertise, the professional organizer, who provides hands-on assistance with the cleanup of client homes, can now maximize her cleaning and organizational skills while directing clients ' emotional issues to a trained professional. as described by the social worker during the focus group interview: a big portion of her [the professional organizer's] time was addressing the [clients] emotional issues. so we've now learned that when those issues come up, it's a direct link back to me. members of the community collaborative described how their partnering with one another as professionals helped to enhance their knowledge and understanding of compulsive hoarding behaviour. gaining new knowledge and a more enlightened understanding of compulsive hoarding behaviour was said to be the result of participating in this ongoing process. one member of the collaborative described the value of the increase of his knowledge stating another professional benefit [of being a member of the community collaborative] is deepening my personal understanding of what hoarding is and what the dynamics are. certainly, it's helped me in recognizing that it's multi-faceted. group members said they were then able to take their learning back to their representative organizations: i think working with [the collaborative] has really helped to educate me and hopefully the rest of [name of organization]. successful aging in place requires that support by community-based organizations exists that it is available and accessible and responsive to a variety of their needs; therefore, knowledge about aging and its long list of associated issues, such as compulsive hoarding behaviour, is imperative. the aim of our discussion is to explain several matters that underpin the major themes of our results. explaining why such themes evolved and their relationship to the broader phenomenon being studied is what strauss and corbin refer to as the process of theorizing. at the heart of this study is community-based planning as a phenomenon of which a number of related concepts are embedded: aging in place, social support, collaboration, vulnerable populations, and community services. overall, our research attempted to discover how a collaborative approach to planning for and addressing the needs of older adults with hoarding behaviour, living in the community, provided value. for these vulnerable adults living in edmonton, alberta, canada, a collaborative planning approach that involved multiple agencies (representing varying sectors) that worked continuously to improve their quality of life made a difference. and, the members of this community collaborative also benefitted. from this approach they were able to access the professional expertise of the other group members, maximize the use of their own skills and knowledge, overall, giving them an opportunity to generate new insights into hoarding behaviour which they described as helping them provide the best possible care and support to this population. it is feasible; therefore, to use such results to inform the many ways to age in place more successfully in late life. currently there is no systemic, long-term process to support older people living in the community that have compulsive hoarding behavior. nor in canada, is there an overall strategy to plan for our aging population, therefore aging in place, at a national, political scale, is not yet a priority. but, because people with this behavior will increase in numbers, and the complexities associated with their need to live safely in their own communities, a national strategy must also address their specific and unique needs. as emphasized by the canadian health services research foundation, our study also finds a need for enhanced integration, cooperation, and coordination at the system and at the service delivery levels. that is, integration and collaboration between health and social services, between ranges of sectors, between disciplines of front line workers, and between government ministries. collaboration and integration need to be part of the foundation upon which aging in one's own home and community can be realized. in addition, our study supports the findings of keonig et al. who found that when having to facilitate ethical dilemmas for this population, older adults with hoarding behavior benefit from the use of teams whose members have a variety of disciplines. well evidenced in the health services planning literature, applied to an aging population, is the need for improved collaboration, both at a principle and a practice-based level. not only do our findings align with the literature but it provides further insight into the challenges associated with older individuals that want and deserve to remain in their own homes. our particular case is specific to older individuals that have hoarding behaviour that were supported through the efforts of a community collaborative planning approach. the current literature supports collaboration and integration at varying levels of the health and social support system. this population will increase in numbers over time and they deserve to remain in their own communities with the support of surrounding agencies and organizations that work togather to best support older individuals with hoarding behaviour to age well in their own homes. at the heart of several concepts and themes arising from interviews with older people with compulsive hoarding behavior and members of a community collaborative working to support this population is an approach founded on collaboration between and amongst service providers. results demonstrated that when a highly collaborative approach to planning is used, there were quite direct benefits for older adults with hoarding behavior and, at the same time, there were benefits for the members of the community collaborative. this approach to planning for the health and social needs of this population resulted in people with hoarding behavior being able to remain in their own homes when eviction was a potential, enhancing their safety, helping to minimize their isolation, and creating opportunities to increase control in their own decision making. the members of the community collaborative could now access the expertise of other professionals, maximize their own expertise, and they generated new insight and understanding of the experience of older adults living with hoarding behaviour in edmonton. although our data conveys that this approach to planning has quite positive outcomes, our data is short term and situational. our use of a single, one-time only interview method only allows us to draw insights and observations about that moment in time and not over an extended trajectory. as well, our study is grounded in five interviews with older adults with compulsive hoarding behavior. and although a collaborative approach to addressing the needs of older people with hoarding behaviour conveyed comprehensive benefits, collaboration as a planning approach is rarely the complete answer or solution to people's social and health needs. health integrated delivery systems, for example, are far more comprehensive but do view collaborative planning as a core principle. viewed in this light, aging in place may not always be possible, but it must be realized that community-level social and health related supports maximize the quality of later life while aging at home. and further building on that is the need for a well-coordinated model of care [5, 35] where supports are comprehensive, easily accessible, and well connected.

this paper reports on and synthesizes new research that examines how a collaborative community response can promote successful aging in place for older adults with hoarding behaviour. through interviews with older adults with hoarding behaviour, who used a particular community support and a focus group interview with members of the community collaborative that directed supports for this population, our findings suggest that there were valuable outcomes for both groups. these older adults with hoarding behaviour were able to remain in their own homes, their safety was enhanced, their sense of isolation was minimized, empowerment was fostered, and they gained valuable insight into their behaviour. the members of the community collaborative were able to access the expertise of other professionals, maximize their own expertise, and they generated an enhanced understanding of the experience of older adults living with hoarding behaviour in edmonton. this study is a significant addition to the much too sparse literature about the community planning needs of older adults with hoarding behaviour. it offers knowledge that is integral to theories and principles of better aging in place but attempts to translate this into practice.

PMC3195538

pubmed-759

hand-assisted laparoscopic donor nephrectomy (haldn) has become the method of choice for removing living donor kidneys. similar to open live donor nephrectomy cases, however, most laparoscopic donor nephrectomy cases have been limited to the left side because of the longer renal vein than on the right side, the greater technical ease of transplantation, and reasonable positioning of the transplanted kidney in a recipient. in addition, although there have been debates about this issue, some authors have emphasized the left-side preference by arguing that left-sided haldns are successful even in cases with multiple left renal arteries [1,3-5]. obviously, several indications, including significantly lower function in the right kidney than in the left one [6-10] or a woman of child-bearing age, should prompt consideration of the right rather than the left kidney. however, fewer reports have been made on right laparoscopic donor nephrectomy because most right donor nephrectomies have been performed in open surgery owing to a lack of experience and the technical difficulties in performing laparoscopic procedures. moreover, the surgical outcomes of right laparoscopic donor nephrectomies (especially haldns) have not yet been reported in korea. the aim of this study, therefore, was to report the safety and feasibility of right-sided haldn. between may 2006 and may 2009, 16 patients underwent right-sided haldn in our institution, by the same operator, who had experienced more than 500 cases of open donor nephrectomies and more than 300 cases of left-sided haldns. we retrospectively analyzed these cases after collecting demographic information on donors including age, sex, relation to a recipient, body mass index (bmi), and indications for the right-sided approach. preoperative donor evaluation included history taking, physical examination, laboratory tests, renal ultrasonography, intravenous pyelography (ivp), and renal function testing by radionuclide renal scan (99mtc-diethylenetriamine penta-acetic acid; dtpa). three-dimensional spiral computerized tomography was used to define the renal parenchyma and vasculature. surgical demographics included intraoperative and postoperative parameters such as operative time, delivery time, warm ischemic time (wit), estimated blood loss (ebl), intraoperative and postoperative complication rates, length of hospital stay (los), and serum creatinine levels of donors (at the time of discharge) and recipients (4 weeks postoperatively). operative time was defined as the time interval from the initial skin incision to closure of the skin. delivery time was calculated as the time interval from renal artery stapling to being placed in ice slush, and wit was calculated as the time interval from renal artery stapling to back-table flushing. under general endotracheal anesthesia, the patient was placed on a flexed table in a left-down partial flank position. an axillary roll was placed beneath the donor's arm, and the right arm was maintained on an armrest. a 6 to 8 cm incision was made for the hand (a) below the umbilicus along the border of the right rectus muscle, and an 11 mm trocar was placed 5 cm above the hand port for the camera (b). a 12 mm trocar (c) was placed 5 cm above the camera port for the hem-o-lok (weck closure systems, research triangle park, usa) or endogia (conmed, new york, usa), and we performed most of our procedures through this port. additionally, a 5 mm trocar (d) was placed below the xiphoid process for liver retraction. when needed, we placed an additional 10 mm trocar at the right subcostal margin in the right mid-clavicular line (fig., we incised the gerota's fascia and entered the perirenal space after incising the lateral line of toldt and medially reflecting the ascending colon and duodenum. for complete mobilization of the kidney, the perirenal fat and adjacent tissues were sufficiently dissected. the ureter was dissected to the level of the external iliac vessels and divided, leaving enough margins to ensure blood supplies around it. then, the ascending colon, right transverse colon, and duodenum were widely mobilized to provide a maximal exposure of the right renal hilum and inferior vena cava (ivc). the renal hilum was skeletonized by meticulous dissection of its adjacent structures with great care to avoid any injuries to the hilar vessels (fig. a harmonic scalpel (ultracision; ethicon endo-surgery, flower mound, usa) was used in all of these procedures for the dissection and coagulation of tiny vessels and other peritoneal structures. after 25% mannitol (250 ml) and diuretics were intravenously administered 20 minutes before arterial clamping, the renal artery was clamped with 2 or 3 hem-o-loks and divided (fig. 2b), and a 30 mm endogia stapler was used to transect the renal vein. to gain a maximal length of the right renal vein, the kidney must be gently retracted laterally with the help of the surgeon's left hand to extend the right renal vein, and the endogia stapler must be positioned at the junction of the ivc and right renal vein (fig. thereafter, the right kidney was removed by the surgeon's left hand through the hand-port device. the staple lines were excised, and the artery was flushed with cold kidney preservation solution. after the abdomen was carefully reinspected at a reduced intraperitoneal pressure, bleeding was controlled, and a jp drain was inserted. the procedures were successfully performed on all 16 patients, and none of the patients experienced intraoperative complications or required conversion to laparotomy. the mean age of the donors was 38.310.4 years, the ratio of males to females was 1.71:1, and the mean bmi was 238.0. the reasons for right donor nephrectomy were as follows: 10% difference in split renal function as determined by radionuclide renal scan dtpa (n=15) and right renal stone (n=1). the numbers of right renal arteries of the 16 patients were as follows: single renal artery (n=13), single renal artery with an early branching artery that supplied the upper pole of kidney (n=2), and duplicated renal artery (n=1). the numbers of right renal veins of the patients were as follows: single renal vein (n=15), duplicated renal vein (n=1). the mean operative time in our series was 192.131.6 minutes, with a mean delivery time of 122.222.4 seconds, a mean wit of 191.842.5 seconds, a mean ebl of 199.071.4 ml, and a mean los of 4.180.39 days. the mean serum creatinine level in the donors was 1.170.19 mg/dl at discharge. concerning graft function, the mean serum creatinine level on day 28 was satisfactory in the recipients (1.160.18 mg/dl). no major or minor complications occurred in any of the 16 patients who underwent our right-sided haldn during the step of clipping and dividing the renal vessels of the donors. as for the recipients, there were no technical problems in the anastomosis of the renal vein, and ureteral anastomoses were also successful without any postoperative sequelae such as ischemic ureteral stricture or leakage of urine. in the case of the donor who had a right renal stone, the stone-bearing kidney was transplanted and the recipient underwent subsequent successful elective shock wave lithotripsy in the third posttransplantation week, and no recurrence of calculi had occurred at the 2-year follow-up. haldn, which was initially reported by wolf et al, may be the safest option for removing living donor kidneys because the surgeon can use the hand in the surgical field by making an abdominal incision at the start of operation. this digital palpation is a valuable tool that provides surgeons with a tactile sensation that permits them to trace the vascular structures and retract the adjacent structures. therefore, this technique can minimize intraoperative injury, give immediate management to emergent situations such as bleeding, and especially minimize wit as compared with pure laparoscopic donor nephrectomy. for these reasons, haldn has spread quickly and widely, and it has become the method of choice for living donor nephrectomy. similar to open live donor nephrectomy, most laparoscopic donor nephrectomy procedures have been limited to the left side because of the longer renal vein than on the right side, the greater technical ease of transplantation, and reasonable positioning of the transplanted kidney in the iliac fossa of a recipient. however, despite all these advantages of left nephrectomy, not all potential donors have the same situation conducive to left nephrectomy., donors who have significantly lower function in the right kidney than in the left one as determined by dtpa scanning must undergo right nephrectomy to preserve their future renal function [6-10]. smaller right kidneys and undiagnosed lesions within the right donor kidney are also indications for right donor nephrectomy. in addition, some previous studies reported that women at a fertile age who want a future pregnancy must undergo right kidney donation because there is a higher chance of pyelonephritis and hydronephrosis on the right side during the gestational period. some authors recommended that the right kidney should be chosen in such cases, whereas others report that the presence of renal artery multiplicity does not have a significant impact on the outcomes of the renal donors or recipients when performing ldn [1,3-5]. in our institution, multiplicity of the renal artery is not included in the criteria by which we select the side of the kidney for donor nephrectomy. until recently, most transplantation centers have hesitated to perform right-sided haldn and have continued to perform right donor nephrectomy by the open technique. the reasons for this choice include a lack of experience, concerns that the shorter length of the right renal vein poses technical challenges for transplant surgeons in transplanting the kidney and thereby increases the risk of intraoperative and postoperative vascular complications in the recipient, and concerns about an increase in delivery time and wit. unlike the general method of left-sided haldn in which a hand port is positioned by making a midline incision just above the umbilicus, our method allows surgeons to stand in the most comfortable position so that they can use both hands most freely by making an abdominal incision for the hand below the umbilicus along the border of the right rectus muscle. we widely mobilized the ascending colon, right transverse colon, and duodenum, and thereby provided a maximal exposure of the right renal hilum and ivc. furthermore, we gently retracted the right kidney laterally with the help of the surgeon's left hand to extend the right renal vein, and positioned the endogia stapler at the junction of the ivc and right renal vein so that we could achieve a maximal length of the right renal vein. we transected the renal vein by use of the endogia stapler instead of by using satinsky clamps to reduce the operative time by eliminating the need for intracorporeal suturing. also, because we believed that the use of the endogia stapler would waste a similar length of vein compared with the use of a hem-o-lok clip, we used the endogia stapler instead of a hem-o-lok clip for the renal vein. ko et al reported an opinion similar to ours regarding this matter. compared with the surgical outcome of left-sided haldns published previously (we used the results of ' the single renal artery group ' from choi et al), the 16 right-sided haldns in our study showed satisfactory results in not only fundamental surgical parameters such as operative time, ebl, and intraoperative and postoperative complication rates, but also the postoperative serum creatinine level of the recipients, delivery time, and wit, which are thought to be the most important parameters in kidney transplantation (table 2). based on the result that there were no technical problems in the anastomosis of the renal vein in the recipients, we suggest that our technique enabled us to harvest renal veins with appropriate lengths. there have been several reports on the surgical outcomes of right laparoscopic donor nephrectomy in international journals. liu et al reported similar surgical outcomes in 19 left-sided haldns and 6 right-sided haldns. keller et al reported that 36 right-sided procedures out of 230 ldns showed similar results in ebl, amount of blood transfusion, operative time, los, and delayed graft function compared with left-sided haldn. there have been more reports about the excellent surgical outcomes of right laparoscopic donor nephrectomy compared with left-sided operations [21-23]. when we reviewed the surgical parameters, mean delivery time and wit were slightly higher in the right-sided haldn group than in the left-sided haldn group. these results seem to be caused by the differences in the operative procedures between the groups. in left-sided haldn, 2 different ports are simultaneously prepared, 1 for hem-o-lok (for arterial clamping) and 1 for endogia (for vein stapling). by use of these 2 ports, the renal artery is divided with the hem-o-lok after the endogia is positioned at the accurate site for renal vein stapling. this technique enables renal vein stapling immediately after renal artery dividing, thereby reducing delivery time and wit. on the other hand, in right-sided haldn, only 1 port (c) in addition to a port for liver traction is actually available. thus, after the renal artery is divided by using a hem-o-lock instrument and the instrument is removed, we then insert the endogia into the same port, place it in the appropriate position for renal vein stapling, and fire it. it is conceivable that the anatomically short length of the right renal vein will not always make right-sided haldn difficult. it is possible for surgeons to obtain an additional length of the right renal vein by performing an ex vivo microvascular reconstruction technique (bench surgery) by using the intrarenal vein in right donor nephrectomy. right-sided haldn is safe and technically feasible in donors and shows a favorable graft outcome. the results of this study suggest that right-sided haldn may be preferable for living donor nephrectomy in patients with significantly lower function in the right kidney than in the left one.

purposewe aimed to prove the safety and feasibility of right-sided hand-assisted laparoscopic donor nephrectomy (haldn). materials and methodsbetween may 2006 and may 2009, 16 patients underwent right-sided haldn at our institution. of these patients, 15 showed significantly lower renal function in the right kidney than in the left one and 1 had a stone in the right kidney. when the right renal vein was divided, an endogia stapling device was placed on the wall of the inferior vena cava to gain a maximal length of the vein. we evaluated intraoperative and postoperative parameters such as operative time, delivery time, warm ischemic time, estimated blood loss, intraoperative and postoperative complication rates, length of hospital stay, and serum creatinine levels of donors (at the time of discharge) and recipients (4 weeks postoperatively), comparing the right-sided haldn group (our study) with a left-sided haldn group (from a previously reported study). resultsa total of 16 right-sided haldns were successfully performed without any complications or open conversion. all of the intraoperative and postoperative parameters were similar between the right-sided haldn and left-sided haldn groups. there were no technical problems in the recipients in the anastomosis of the renal vein, and the ureteral anastomoses were also successful. conclusionsright-sided haldn is safe and technically feasible in a donor, showing favorable graft outcomes. the results of our study suggest that right-sided haldn may be preferable in patients with significantly lower renal function in the right kidney than in the left one.

PMC2855464

pubmed-760

pyomyositis is a primary pyogenic infection of skeletal muscle, an uncommon cause of musculoskeletal infection. although relatively common in tropical areas, it is very rare in temperate areas1. in contrast, pyomyositis is observed in immunodeficient hosts, particularly in the human immunodeficiency virus (hiv) infected condition2. in nonhiv infected cases, skeletal muscle tissue is resistant to bacterial infection, even in the severe bacterial septic condition3. in approximately 80% of cases, the location of the infection is unifocal, and the lower extremities, especially the thigh, are the most common site2. in the early stages of pyomyositis, there are no typical features of this disease. because of its rarity, it is often misdiagnosed as muscle hematoma, cellulitis or neoplasm4. therefore, prompt imaging procedures, aggressive surgical interventions and appropriate antibiotic therapy are very important to cure this disease without any complications. here, we report a case of a japanese woman who developed multiple abscesses, which were, surprisingly, disseminated over 30 parts throughout the body, under poorly controlled diabetic conditions accompanied by ketoacidosis, but was successfully treated with prompt and appropriate therapy. a 26yearold woman was referred to kawasaki medical school hospital, kurashiki, japan, because of a high fever and right thigh pain. she had mild pain of the right thigh, a sense of thirst and general fatigue. furthermore, her bodyweight reduced by 6 kg during 2 weeks, and severe thigh pain, and highgrade fever developed and progressively worsened. she had untreated type 2 diabetes, but did not have any history of trauma or illicit drug use. her grandmother had adultonset type 2 diabetes, but except for this there was no family history of diabetes including maturityonset diabetes of the young and mitochondrial diabetes. on admission, her body mass index was 23.0 kg/m (height 156 cm and body weight 56 kg). emergent computed tomography (ct) showed a giant mass in the right adductor muscle (figure 1a, b). the drastic elevation of plasma glucose (470 mg/dl) and glycated hemoglobin levels (15.2%) were shown with severe ketoacidosis. despite prehospital administration of insulin (total 50 u) and bicarbonate, blood gas analysis still showed metabolic acidosis (ph 7.339, hco3 10.9 meq/l, paco2 20.8 mmhg, pao2 107.5 mmhg, base excess 12.8 meq/l, lactate 1.22 meq/l under oxygen inhalation 2 l/min by nasal cannula). leukocytosis and an increased creactive protein (28.21 mg/dl) were also observed. renal and liver function, serum creatinine kinase, and myoglobin levels were not elevated. (a, b) computed tomography with contrast enhancement in the right thigh on admission. (a) frontal slice. (c) disseminated abscesses throughout the patient's body including the kidney and muscle (on day 7). based on these findings, we diagnosed this patient as primary pyomyositis, and immediately started broadspectrum antibiotics therapy (doripenem 3.0 g/day, clindamycin 1200 mg/day). aggressive hydration with saline (4.5 l/day) and intravenous continuous insulin infusion was immediately started. furthermore, we carried out surgical drainage and removed 700 ml of yellowish pus. surprisingly, the followup ct showed multiple pyomyositis disseminated over 30 parts throughout her body, as well as a kidney abscess (figure 1c). we immediately restarted clindamycin, and changed fulconazole to micafungin with additional percutaneous renal drainage. we used broadspectrum antibiotics and an antifungal drug intravenously for a total of 66 days. there was no clear evidence of osteomyelitis, which is closely associated with pyomyositis, but in consideration of the possibility of osteomyelitis, we decided to carry out longterm antibiotics and antifungal therapy. indeed, despite a good clinical course, lowgrade fever and persistent mild elevation of crp (approximately 1.5 mg/dl) were observed. therefore, we continued antibacterial and antifungal agents until the normalization of these infectious markers. in the findings of followup ct with the enhancement, the origin bulky abscess in the adductor muscle and systemic intramuscular abscess completely disappeared (figure 2). on day 66 of the hospitalization, when the value of creactive protein remained within the normal range for 7 days, we stopped all intravenous agents. complete disappearance of origin and disseminated abscesses. left panel, computed tomography on admission or day 7; right panel, computed tomography on day 96. it is known that skeletal muscle is resistant to bacterial infection, but under various immunodeficient conditions, such as severe lymphopenia with hiv infection or diabetic ketoacidosis, the host's defense system against microbiota is destroyed, which easily leads to opportunistic infection. indeed, there is a close relationship between diabetes itself and the incidence of serious infections5, 6, especially sepsis7. in addition, because of its rarity, the accurate and timely diagnosis of pyomyositis is challenging. in the present patient, severe hyperglycemic state with diabetic ketoacidosis and severe infectious signs led to the diagnosis of pyomyositis. furthermore, typical ct imaging of the bulky abscess in the right adductor muscle was helpful for the diagnosis. to the best of our knowledge, there have been no reports showing as many as 30 disseminated abscesses. because there seemed to be no other additional immunodeficiency cause, we assume that the delay of the visit to the medical institution was the main cause of pyomyositis observed in this patient. stage 1 is the invasive stage where local symptoms and lowgrade fever are observed, but other physical findings are typically absent. however, only a minority of patients (< 2%) visits a medical institution at this stage1. stage 2 is the suppurative stage with abscess formation. around 23 weeks after the initial onset, patients complain about progressive fever and severe pain of the affected sites. stage 3 is the late stage, where sepsis and dissemination of the infection are observed if the abscess remains untreated in the previous stages1. this progressive stage can be occasionally associated with lethal conditions, especially septic shock, multiple organ failure and rhabdomyolysis6, 8. our patient was very afraid of undergoing all treatment, and hesitated to visit a medical institution. we believe it is likely that such a delay progressed the pyomyositis in this patient to the most severe and lifethreatening condition. therefore, it would be very difficult to save her life, if the only one piece of the following situation is missing: precise and timely diagnosis, intensive surgical approach, longstanding antibiotics therapy, and general supportive care. as observed in the present patient, staphylococcus aureus is the most common (approximately 70%) causative bacteria in both tropical and temperate conditions4. treatment of pyomyositis depends on the stage of the disease. in the invasive stage, a key feature of prognosis of pyomyositis is recurrence, but it is uncommon (< 30% of all cases). the mortality rate is<1.5% in the early stage, but it becomes as high as 15% in the late stage10. therefore, we have to continue antibiotics and antifungal agent for a long enough period. in fact, the present patient was successfully treated with surgical interventions, in combination with longstanding broadspectrum antibiotics and antifungal therapy. fortunately, during the longstanding broadspectrum antibiotics and antifungal therapy, there were no adverse events including the appearance of resistant bacteria, pseudomembranous colitis, liver dysfunction and electrolytes abnormalities. pyomyositis is a rare infectious disease, but physicians should be aware of its possibility in poorly controlled diabetic patients. in addition, the present case strongly suggests that we should continue appropriate therapies until the complete disappearance of abscesses.

abstractprimary pyomyositis is a pyogenic and uncommon infection of skeletal muscle, which is mainly observed in tropical areas and/or human immunodeficiency virus patients. in nonhuman immunodeficiency virus infected patients, the most common cause is diabetes mellitus. because of its rarity, the accurate diagnosis is often challenging. staphylococcus aureus is the most common causative bacteria. according to the severity, pyomyositis is divided into three stages, and the late stage is occasionally lethal. the present case was compatible with the most advanced stage. therefore, it was very difficult to save her life without precise and timely diagnosis. furthermore, in the invasive stage, surgical drainage and broadspectrum antibiotics should be given for a long enough period. here, we report a case of a japanese woman who developed disseminated abscesses under poorly controlled diabetic conditions accompanied by ketoacidosis, but was successfully treated without any sequelae.

PMC4931217

pubmed-761

in recent years, core training has been widely studied since it has been considered a pivotal issue in health, rehabilitation and sports performance (hibbs et al., 2008). however, the definition of the core varies with the interpretation of the literature (hibbs and thompson, 2008). anatomically, the core region has been described as the area bounded by the abdominal muscles in the front, by paraspinal and gluteal muscles in the back, by diaphragm on the top and by pelvic floor and girdle musculature at the bottom (richardson et al., 1999). the core represents the connection between lower and upper limbs and should be considered as a functional unit in which different muscles interact, even if not located in the thoraco-lumbar region (such as shoulders and pelvic muscles). however, literature concerning core training sometimes fails to distinguish between concepts of core stability and core strength. faries and greenwood (in hibbs and thompson, 2008) formulated the following clear definitions: core stability refers to the ability to stabilize the spine as a result of muscle activity, while core strength refers to the ability of muscles contractions to produce and transfer force as a result of muscle activity. since strength and motor control are complementary qualities, the core training programmes can target mainly, but not exclusively, at muscle strengthening and/or motor control of core musculature. motor control training seems to require low intensity stabilization exercises focused on efficient integration of low threshold recruitment of local and global muscle systems. conversely, core strength training seems to require high intensity and overload training of the global muscle system. vezina and hubley-kozey (2000) suggested that core stability programmes should include muscle activation below 25% of maximum voluntary contraction (mvc), while core strength training should include activation higher than 60% of mvc to result in strength benefits. the available evidence suggests that to adequately train the core muscles in athletes, strength and conditioning specialists should focus on implementing multi-joint full body exercises, rather than core-specific exercises (martuscello et al., 2013). exercises involving the full body linkage such as plank exercises, have been advocated to the capacity of transmitting force through the body linkage (schoenfeld et al., 2014). training with labile systems has been documented to offer unique opportunities for linkage training challenges (mcgill et al., 2015). several studies examined core muscle activation during the execution of various exercises on stable and unstable surfaces (for a review see: behm et al., 2010). the use of unstable surfaces contacting the subject s feet or hands is becoming popular in strength training. instability can be obtained through the use of many devices and techniques including, but not limited to, unstable platforms such as bosu or swiss balls. more recently, suspension training systems have been added to the list of instability training devices. in suspension training, lower or upper limbs many core directed exercises are designed with such a device, creating a wide variety of challenges. these exercises consist of multi-planar and multijoint movements, and are executed with complex techniques. it is important to quantify the muscle contraction intensity since it is a key factor in establishing training effects induced by this sort of exercises. although considerable research has examined more traditional means of instability training (behm and drinkwater, 2010), little previous research has evaluated the effects of suspension training on muscle activation. in particular, some studies focused on core-directed exercises (atkins, 2014; byrne et al., 2014; czaprowski et al., 2014; mok et al., 2014; snarr and esco, 2014), whereas others investigated the effect of the application of suspension system on core muscle activity in push exercises (calatayud et al., 2014 further investigation of these exercise approaches is needed to understand their influence on muscle activation and joint load levels. the primary purpose of this study therefore, was to examine differences in core muscle activation across four full-body linkage exercises using a suspension training system. these exercises were chosen from a spectrum of whole body linkage exercises focused on the anterior core musculature executed in instable conditions, including a roll-out, bodysaw, pike, and knee-up. although the selected exercises were mainly focused on anterior slings, we wanted to provide a comprehensive view of core muscle activation by monitoring rectus abdominis, internal and external oblique, and paraspinal muscles. it was hypothesized that significant differences would be found in core muscles among exercises. the second aim of the study was to determine which of these exercises would reach the threshold of 60% of mvc, expected to be high enough to increase muscle strength. it was hypothesized that the four exercises would elicit muscle activity in excess of 60% of mvc in the rectus abdominis, i.e. the muscle on which the main focus was put considering the selected exercises. seventeen healthy participants were recruited (age 27.32.4 years, body height 1725 cm, body mass 69.29.3 kg). all participants were physically active, declaring three practice sessions per week of resistance training. inclusion criteria for study participation were as follows: no past or present neurological or musculoskeletal trunk or limb pathology, no cardiorespiratory disease, no history of abdominal, shoulder or back surgery, and no psychological problems. participants were instructed to refrain from performing strenuous physical activity in the 24 hours preceding all experimental sessions. the study was previously approved by the research ethics committee of the department of medical sciences, university of turin. the surface electromyographic (emg) signals were obtained from six trunk muscles with concentric bipolar electrodes (code, spes medica, battipaglia, italy). before the placement of the electrodes, the skin was slightly abraded with adhesive paste and cleaned with water in accordance to seniam recommendation for skin preparation (hermens et al., 2000). the electrodes were placed according to the instructions described in previous methodological works (beretta piccoli et al., 2014; boccia and rainoldi, 2014) lower rectus abdominis: on the lower part of the rectus abdominis, 3 cm lateral to the midline; upper rectus abdominis: on the upper part of the rectus abdominis, 3 cm lateral to the midline; external oblique: 14 cm lateral to the umbilicus, above the anterior superior iliac spine (asis); internal oblique: 2 cm lower with respect to the most prominent point of the asis, just medial and superior to the inguinal ligament; lower erector spinae: 2 cm lateral to the l5-s1; upper erector spinae: 6 cm lateral to the l1-l2. the electrodes were placed only on the left (randomly chosen) side of the body; the reference electrode was positioned on the wrist. the signal of a biaxial electrogoniometer (sg 150, biometrics ltd, gwent, uk) positioned at the level of the shoulders (for the roll-out and bodysaw) or the hips (for the pike and knee-tuck), depending on which joint was more involved during the exercise, was used as a trigger to highlight exercise repetitions. the emg signals were synchronized with the electrogoniometer signal, amplified (emg-usb, ot bioelettronica, torino, italy), sampled at 2048 hz, bandpass filtered (3-db bandwidth, 10-450 hz, 12 db/oct slope on each side), and converted to digital data by a 12-bit a/d converter. samples were visualized during acquisition and then stored in a personal computer using ot biolab software (version 1.8, ot bioelettronica, torino, italy) for further analysis. the participants recruited were instructed with regard to the correct technique of suspension exercise and the mvc procedure during the first experimental session conducted one week before the measurement session. the participants were asked to refrain from physical activity 24 hours before the measurements. during the measurement session, participants performed 4 exercises with the use of suspension straps (trx suspension trainer; fitness anywhere lcc, san francisco, ca, usa) in random order. the exercises were selected based on a previous study (behm and drinkwater, 2010) that indicated them as important in developing core strength. at the beginning of the measurement session, three mvc exercises were performed twice for 5 s, with 2 min rest between them. the following standardized exercises (ng et al., 2002) were used to activate maximally the trunk muscles (figure 1): upper rectus abdominis (ura) and lower rectus abdominis (lra): body supine with hips and knees flexed 90, with feet locked. participants flexed the trunk (i.e. crunch execution) against resistance at the level of the shoulders;external oblique (eo) and internal oblique (io): side-lying with the hip at the edge of the bench and feet locked by a second operator. participants performed side-bend exercise against resistance at the level of the shoulder;lower erector spinae (les) and upper erector spinae (ues): prone position with asis at the edge of the bench and feet locked by a second operator. upper rectus abdominis (ura) and lower rectus abdominis (lra): body supine with hips and knees flexed 90, with feet locked. participants flexed the trunk (i.e. crunch execution) against resistance at the level of the shoulders; external oblique (eo) and internal oblique (io): side-lying with the hip at the edge of the bench and feet locked by a second operator. participants performed side-bend exercise against resistance at the level of the shoulder; lower erector spinae (les) and upper erector spinae (ues): prone position with asis at the edge of the bench and feet locked by a second operator. standardized exercises used to maximally activate trunk muscles: lower rectus abdominis and upper rectus abdominis (left); internal oblique and external oblique (middle); lower erector spinae and upper erector spinae (right). participants were required to achieve a range of motion with the correct technique execution and to maintain a neutral position of the spine and pelvis in each exercise. a certified strength and conditioning coach monitored the exercise performance to ensure that the exercise was properly executed considering its technique. each exercise was repeated three times and lasted 6 s. a metronome set at 30 beats per minute was used to ensure proper timing (with 4 beats for each repetition): 2 s from the initial position to the final position (concentric phase); 2 s of maintenance (isometric phase); and 2 s returning to the starting position (eccentric phase). the exercises were performed with 3 min of rest in-between to allow complete recovery. the random order of the exercises allowed to mitigate the effects of cumulative fatigue on emg estimates. the following exercises were used (figure 2): roll-out: participants assumed an inclined standing position while placing each hand on the strap handles, with elbows and wrists placed below the shoulders, arms perpendicular to the floor and shoulders flexed approximately 45; they then performed a shoulder flexion moving the hands forward;bodysaw: participants assumed a prone position, they placed elbows below the shoulders, both forearms touching the floor, while placing each foot on the strap handle; participants then flexed the shoulders and extended the elbows pushing the body backwards;pike: participants assumed a push-up position with the feet in strap handles, then they flexed hips to approximately 90, while keeping the knees fully extended;knee-tuck: participants assumed a push-up position while placing each foot in the strap handle, then they flexed both hips and knees to approximately 90, bringing the knees forward. roll-out: participants assumed an inclined standing position while placing each hand on the strap handles, with elbows and wrists placed below the shoulders, arms perpendicular to the floor and shoulders flexed approximately 45; they then performed a shoulder flexion moving the hands forward; bodysaw: participants assumed a prone position, they placed elbows below the shoulders, both forearms touching the floor, while placing each foot on the strap handle; participants then flexed the shoulders and extended the elbows pushing the body backwards; pike: participants assumed a push-up position with the feet in strap handles, then they flexed hips to approximately 90, while keeping the knees fully extended; knee-tuck: participants assumed a push-up position while placing each foot in the strap handle, then they flexed both hips and knees to approximately 90, bringing the knees forward. initial and final positions of each exercise: 1) roll-out; 2) bodysaw; 3) pike; 4) knee-tuck. the average rectified value (arv) of emg signals was computed off-line with numerical algorithms using non-overlapping signal epochs of 0.5 s (hibbs et al., 2011). the mean value of arv over the two repetitions was calculated for each muscle and normalized with respect to the maximum arv obtained during the correspondent mvc. the normality assumption of the data was evaluated with the shapiro-wilk test; homoscedasticity and autocorrelation of the variables were assessed using the breusch-pagan and durbin-watson tests. the differences between exercises (pike bodysaw knee-tuck roll-out) and between muscles (lra ura eo io les ues) were compared with the 2-way analysis of variance (anova). for the purpose of this report statistical analyses were conducted using the r statistical package (version 3.0.3, r core team, foundation for statistical computing, vienna, austria). all participants managed to complete each exercise trial and thus, were included in the data analysis. figure 3 shows the box plots of the activation values (% of mvc) of each muscle during the four exercises. muscle activation (median, ir) figure 3each box plot shows the muscle activation (as percentage of maximum voluntary contraction) during exercise. table 1muscle activation (median, ir) expressed as percentage values of electromyographic amplitude normalized to maximum voluntary contraction. results of the two-way anova after tukey multiple comparisons are reported as symbols; p<0.01.lower rectus abdominisupper rectus abdominisexternal obliqueinternal obliquelower erector spinaeupper erector spinaepike57 (36) 41 (48) 55 (21)23 (20)12 (7)9 (4)bodysaw100 (42) 57 (52)59 (33)32 (20)4 (3)8 (6)knee-tuck54 (50) 44 (41) 42 (7) 18 (26)8 (5)6 (5)roll-out140 (89) 67 (78) 71 (44) 40 (31)9 (5)11 (6)indicates statistically significant difference between the indicated exercise (explained in row) with respect to the pikeindicates statistically significant difference between the indicated exercise (explained in row) with respect to the bodysawindicates statistically significant difference between the indicated exercise (explained in row) with respect to the knee-tuckindicates statistically significant difference betweenthe indicated exercise (explained in row) with respect to the roll-out each box plot shows the muscle activation (as percentage of maximum voluntary contraction) during exercise. muscle activation (median, ir) expressed as percentage values of electromyographic amplitude normalized to maximum voluntary contraction. results of the two-way anova after tukey multiple comparisons are reported as symbols; p<0.01. indicates statistically significant difference between the indicated exercise (explained in row) with respect to the pike indicates statistically significant difference between the indicated exercise (explained in row) with respect to the bodysaw indicates statistically significant difference between the indicated exercise (explained in row) with respect to the knee-tuck indicates statistically significant difference between the indicated exercise (explained in row) with respect to the roll-out the normalized lra activity was 140% (ir, 89%) of mvc during the roll-out, 100% (ir, 42%) of mvc during the bodysaw, 57% (ir, 36%) of mvc during the pike and 54% (ir, 50%) of mvc during the knee-tuck. the normalized lra values were significantly higher (p<0.01) during the roll-out and bodysaw compared to the pike and knee-tuck. the roll-out exercise showed significantly greater activation (p<0.01) than the bodysaw. the normalized ura activity was 67% (ir, 78%) of mvc during the roll-out, 57% (ir, 52%) of mvc during the bodysaw, 41% (ir, 48%) of mvc during the pike and 44% (ir, 41%) of mvc during the knee-tuck. the normalized ura values were significantly higher (p <0.01) during the roll-out compared to the pike and knee-tuck. the normalized eo activity was 71% (ir, 44%) of mvc during the roll-out, 59% (ir, 33%) of mvc during the bodysaw, 55% (ir, 21%) of mvc during the pike and 42% (ir, 7%) of mvc during the knee-tuck. the normalized eo values were significantly higher (p<0.01) during the roll-out compared to the knee-tuck. the normalized io activity was 40% (ir, 31%) of mvc during the roll-out, 32% (ir, 20%) of mvc during the bodysaw, 23% (ir, 20%) of mvc during the pike and 18% (ir, 26%) of mvc during the knee-tuck. during all exercises the normalized io values were not significantly higher (p<0.01). the normalized les activity was 9% (ir, 5%) of mvc during the roll-out, 4% (ir, 3%) of mvc during the bodysaw, 12% (ir, 7%) of mvc during the pike and 8% (ir, 5%) of mvc during the knee-tuck. during all exercises the normalized les values were not significantly higher (p<0.01). the normalized ues activity was 11% (ir, 6%) of mvc during the roll-out, 8% (ir, 6%) of mvc during the bodysaw, 9% (ir, 4%) of mvc during the pike and 6% (ir, 5%) of mvc during the knee-tuck. during all exercises the normalized ues values were not significantly higher (p<0.01). table 2 shows the estimate (difference of means) at 95% of the confidence interval after tukey multiple comparisons; in this case only exercise factor was considered. estimate at 95% of the confidence interval after tukey multiple comparisons with the exercise factor considered. the estimate shows the difference of means (% of maximum voluntary contraction). indicates the statistical significance of the adjusted p-value. the roll-out exercise showed significantly (p<0.01) higher activation compared to the bodysaw (16%, ci 8-23%), pike (26%, ci 18-33%) and knee-tuck (29%, ci 21-37%). pike and knee-tuck exercises showed significantly higher activation compared to the bodysaw of 10% (28%) and 13% (6-21%). suspension training has become increasingly popular as a training tool. despite this popularity, relatively little research exists on the effects of such training on muscle activation magnitude. the first objective of the study was to investigate the activation differences of four exercises (roll-out, bodysaw, pike and knee-tuck) to better characterize suspension training. our findings indicate that suspension exercises could be an effective strategy to reach high to very high activation of abdominal muscles such as the rectus abdominis and external oblique. to facilitate comparisons between exercises and previous studies, we categorized muscle activation into four levels according to previous studies, with<21% as low, 2140% as moderate, 4160% as high, and>60% as very high (escamilla et al., 2010). exercises used in the present study provide a range of medium to high intensity exercises through which participants or athletes can progress during a training or rehabilitation programme (blanchard and glasgow, 2014) (figure 2). roll-out exercise was the most challenging for core musculature, followed by bodysaw, pike and knee-tuck exercises (table 2). the roll-out showed the highest activation of rectus abdominis and oblique muscles compared to other exercises. although lra showed much greater activation in roll-out and bodysaw compared to pike and knee-tuck exercises, the other muscles showed smaller differences. these findings could suggest that in the exercises characterized by shoulder flexion (such as roll-out and bodysaw), the increased requirement of core stability was reflected more by the lower rectus abdominis. according to vezina and hubley-kozey (2000), the exercises that generate muscle activity greater than 60% of mvc might be more conducive to developing muscular strength. the rectus abdominis (both parts) and eo reached activation higher than 60% of mvc (or very close to that threshold, 55%) in the roll-out and bodysaw; consequently these exercises can be considered suitable for strength training of these muscles. although in the knee-tuck and pike, the rectus abdominis and eo did not reach the threshold of 60%, they presented high activation levels (41-60% mvc). while strengthening of the core is important, an activation level below 60% might be beneficial in increasing muscle endurance within the core. since the core muscles are primarily composed of type i fibres (haggmark and thorstensson, 1979), muscular endurance should also be a major concern when designing strength and conditioning programmes (vezina and hubley-kozey, 2000). due to large demand for muscle activation, all the proposed exercises might be appropriate for extremely fit individuals in the latter stages of a progressive abdominal strengthening or rehabilitation programme. this is an expected result as all exercises focused on anterior abdominal wall muscles. this finding confirms that in the herein selected whole-body linkage exercises, the activation of core muscles can be mainly focused on abdominal muscles while keeping the paraspinal muscles involved with low intensity. although no direct comparison can be made between the selected suspension exercises compared to previously reported similar exercises, it is possible to highlight the following differences. we can compare only the activation of the rectus abdominis, since for oblique muscles we used a different normalization exercise than the other three studies. plank exercises are frequently included in spine stabilization programmes as a means of improving motor control for spine stabilization. when plank exercises are performed on stable or unstable support surfaces, the reported activation level of the rectus abdominis and eo ranges from low to moderate (garcia-vaquero et al., 2012). when executed in suspension condition, rectus abdominis muscles also showed moderate activation (byrne and bishop, 2014). only when the planks were performed with a similar technique (instability on lower limb and shoulder flexion) was the activation similar to that reported here, which was very high for the rectus abdominis (mcgill and andersen, 2015). therefore, we can assume that our exercises were more challenging than an isometric plank in a stable condition. in the roll-out, we found very high activation of lra (140%) and ura (67%). these levels were higher than previously reported values obtained during the execution of the roll-out with the swiss-ball (about 50-60% for rectus abdominis) (escamilla and lewis, 2010; marshall and desai, 2010) and similar to the values reported with the use of the power wheel, being very high for ura (76%) and lra (81%) (escamilla et al., 2006). in the pike, we found high activation of lra (57%) and ura (41%). the values reported for the pike executed with the swiss ball (escamilla and lewis, 2010) and power wheel (escamilla and babb, 2006) were similar for ura (swiss ball 47%; power wheel 41%) and lra (swiss ball 55%; power wheel 53%). in the knee-tuck, we observed high activation of lra (54%) and ura (44%). otherwise, the values reported for the knee-tuck executed with the swiss ball (escamilla and lewis, 2010) and power wheel (escamilla and babb, 2006) were lower for both ura (swiss ball 32%; power wheel 41%) and lra (swiss ball 35%; power wheel 45%). our findings suggest that the two parts of the rectus abdominis can be activated differently according to the needs of the motor task (kibler et al., 2006). this finding could be explained by the possibility to (voluntary or involuntary) modulate the activation ratio between rectus abdominis parts in order to achieve the best control of the core region. this could be justified by the metameric innervation of rectus abdominis muscles (duchateau et al., 1988), although this issue is still controversial (monfort-panego et al., 2009). however, lra muscles were generally more active than ura because of confounding methodological factors. mvcs of the lra and ura in fact were estimated by a standardized exercise to activate maximally the trunk muscles: it could be argued that the same exercise fully activated ura whereas it failed to fully activate lra. hence, the emg amplitude recorded during mvc was not the maximum achievable. consequently, throughout experimental exercises, lra seemed relatively more active than ura because its reference value of mvc was underestimated., arv estimates of emg signals exceeded the mvc reference values (arv higher than 100%). this inconsistency might be due to incomplete activation during mvc (as in the case of the lower rectus abdominis) and other confounding factors related to emg technique (relative shift of muscle belly with respect to electrodes occurring in dynamic tasks and different activation between isometric and dynamic tasks, among others). as widely reported, variability of muscular activation between participants was high. this suggests that performing these exercises, some individuals might produce more or less activation than the average activity indicated here. although 17 individuals participated in this research, the differences in their fitness level and exercise experience could have affected the performance of the exercises and the resulting activation levels. crosstalk between muscles was minimized by using an innovative detection system based on concentric-ring electrodes which had been reported as having higher spatial selectivity compared to the traditional detection systems and reducing the problem of crosstalk from nearby muscles (farina and cescon, 2001). findings from this study, based on electromyographic analysis, showed that roll-out exercise was the most challenging. moreover, roll-out and bodysaw exercises executed in suspension activated the rectus abdominis and external oblique muscles at intensities higher than, or very close to, 60% of the maximum voluntary contraction. based on these findings, we can assume that roll-out and bodysaw exercises can be used to adequately strengthen the antero-lateral, superficial aspect of the core region, and thus they can be considered core strength exercises. these findings appear to have particular relevance for well-trained individuals given the high demand imposed by these exercises.

abstracta quantitative observational laboratory study was conducted to characterize and classify core training exercises executed in a suspension modality on the base of muscle activation. in a prospective single-group repeated measures design, seventeen active male participants performed four suspension exercises typically associated with core training (roll-out, bodysaw, pike and knee-tuck). surface electromyographic signals were recorded from lower and upper parts of rectus abdominis, external oblique, internal oblique, lower and upper parts of erector spinae muscles using concentric bipolar electrodes. the average rectified values of electromyographic signals were normalized with respect to individual maximum voluntary isometric contraction of each muscle. roll-out exercise showed the highest activation of rectus abdominis and oblique muscles compared to the other exercises. the rectus abdominis and external oblique reached an activation higher than 60% of the maximal voluntary contraction (or very close to that threshold, 55%) in roll-out and bodysaw exercises. findings from this study allow the selection of suspension core training exercises on the basis of quantitative information about the activation of muscles of interest. roll-out and bodysaw exercises can be considered as suitable for strength training of rectus abdominis and external oblique muscles.

PMC5384053

pubmed-762

spasticity is a frequent consequence of stroke1, 2. in the lower leg, this malposition or malalignment causes mechanical stress to foot joints in the lateral forefoot area, and joints under this stress are likely to be inflamed and painful4. foot pain causes difficulty in standing and walking, and thus limits daily living activities. the malposition or malalignment of the foot can be managed by the control of spasticity. it was proposed that correcting the malposition or malalignment can reduce abnormal mechanical pressure in specific areas of the foot, which leads to a reduction in foot pain. of the several options available for the management of spasticity, the clinical usefulness of nerve and motor point blocks with alcohol has been well demonstrated5,6,7. in the current study, whether a reduction of ankle spasticity using nerve and motor point blocks with 20% ethyl alcohol can manage foot pain was examined. a 58-year-old woman visited the rehabilitation department of a university hospital due to piercing pain (numeric rating scale [nrs]: 8) in the left fifth metatarsal head for two years. erythema and edema were present on the lateral aspect of the head of the fifth metatarsal bone (fig. 1.(a) an image of the patient s left foot shows erythema and edema in the fifth metatarsal head area. (b) the left foot x-ray showed that the fourth and fifth intermetatarsal angle was 7.7). the patient provided informed signed consent for participation in the study. the study was approved by the research ethics committee of yeungnam university hospital (yuh-16-0425-d7). on the foot x-ray, the fourth and fifth intermetatarsal angle of the left foot was 7.7 (fig. she had a history of intracerebral hemorrhage on the right basal ganglia about five years previously. the degree of her spasticity was a modified ashworth scale (mas)8 of 1 +. additionally, slight motor weakness (medical research council9: 4 +) in the left upper and lower extremities was shown. other neurological symptoms, including sensory and cognitive deficits, were not present. at first, 0.5 ml of 2% lidocaine with 10 mg triamcinolone acetonide was injected into the tender point of the left fifth metatarsal head area. (a) an image of the patient s left foot shows erythema and edema in the fifth metatarsal head area. (b) the left foot x-ray showed that the fourth and fifth intermetatarsal angle was 7.7 it was proposed that the ankle spasticity in the patient caused repetitive pressure and shearing between the soft tissue and head of the fifth metatarsal bone. thus, we considered the possibility that controlling spasticity might be helpful in the management of pain from the tailor s bunion, and decided to control spasticity in this patient using a nerve or motor point block with 20% ethyl alcohol. to reduce the spasticity of the left ankle plantar flexor, the medial and lateral motor branches to the gastrocnemius muscle of the left tibial nerve the block of nerve branches was performed based on the method described by jang et al5. to manage spasticity in the left ankle supinator a motor point block was performed in the left tibialis posterior muscle with 5 ml of 20% ethyl alcohol. both nerve and motor points were located using the nerve stimulator at the popliteal area and at the mid-calf level, respectively. the cathode stimulating needle (teflon-coated, 23-gauge needle) was slowly advanced in the direction of the nerve and posterior tibialis muscle. the needle hub was connected to injection tubing that bore the syringe containing the 20% ethyl alcohol. contractions were seen and palpated as the tip came close to the nerve or motor point of the tibialis posterior. at this stage, the intensity of the current was between 3 ma and 5 ma. after the needle was placed close to the targeted nerve or motor point, the tip is assumed to be in contact with the nerve or motor point when maximum contraction is obtained with minimum current. the needle was finally positioned at a current of 1 ma with nerve block and 3.5 ma with motor point block. additionally, the patient underwent the stretching exercises for the left ankle plantar flexors and ankle supinators for 2 weeks after the alcohol block (monday through friday: 15 mins 1 time/day). to evaluate dynamic foot pressure, the foot pressure measurement system (fpms) (tpscan; biomechanics, goyang, korea) was used10. dynamic foot contact pressure data during gait were recorded on the fpms floor mat (40.5 40.5 cm) at the middle of the gait test. the foot pressure was presented as a color: red is the highest pressure followed by orange, yellow, green, and blue. foot contact pressure was evaluated twice before and one- month after the nerve and motor point block procedure. before the procedure, contact pressure was highly distributed to the lateral forefoot, however, reduced distribution was observed in the medial forefoot and hindfoot (fig. 2.foot pressure measurement imagethe foot pressure image of a normal foot and the images of the patient s foot pressure showing alterations pre-alcohol block and post-alcohol block.). after the procedure, the distribution of foot contract pressure was found to be similar to that of a normal control (56-year-old female individual). foot pressure measurement image the foot pressure image of a normal foot and the images of the patient s foot pressure showing alterations pre-alcohol block and post-alcohol block. at the follow-up evaluation, two weeks after the alcohol block, the spasticity in the left ankle plantar flexor and supinator had disappeared (mas: 0). in addition, ankle clonus was not present. moreover, the pain in the left fifth metatarsal head was significantly reduced from nrs 8 to nrs 1. at one, two, and three months after the alcohol block the effects on ankle spasticity and foot pain were sustained. in the present study, foot pain related to tailor s bunion was reduced after spasticity treatment using nerve and motor point block with 20% ethyl alcohol. although the incidence of foot pain in patients with stroke has not been investigated, patients with stroke are prone to have foot pain due to deteriorated musculoskeletal position or alignment induced by spasticity or motor weakness. the tailor s bunion or bunionette, which was first described by davies in 194911, is a painful bony prominence on the lateral, dorsolateral, or plantar aspect of the head of the fifth metatarsal bone12. the conflict between the fifth metatarsal head and footwear has been reported to be the cause of tailor s bunions12. in the patient, the spasticity in the ankle plantar flexor and supinator appeared to cause repeated mechanical stress and chronic irritation in the fifth metatarsal head area, which is thought to induce a tailor s bunion. for the management of foot pain due to tailor s bunion, corticosteroids were injected into the tender point of the fifth metatarsal head area; however, the patient s pain was unresponsive to this injection. it was considered that non-controlled spasticity can lead to continuous mechanical stress or irritation and repeated occurrence of inflammation in the fifth metatarsal head area. therefore, the spasticity was controlled using a block with 20% ethyl alcohol on the motor branches to the gastrocnemius muscle of the tibial nerve and the motor points of the posterior tibialis muscle. after the block with ethyl alcohol, spasticity in this patient had almost disappeared (from mas 1+to mas 0). also, the patient received the stretching exercises for the ankle plantar flexors and the ankle supinators. the stretching exercise is known to effectively reduce spasticity13, 14 by increasing tissue extensibility15. it was thought that the reduced spasticity in the patient was attributed, at least in part, to the stretching exercises. it is thought that the mechanical stress and irritation in the fifth metatarsal head area during walking were significantly reduced with the disappearance of spasticity in this patient. when the stress and irritation were reduced after the management of spasticity, the pain due to tailor s bunion appeared to be significantly reduced. in addition, the fpms was used for detailed evaluation of the effect of the 20% ethyl alcohol block. before the alcohol block, after the control of spasticity with the alcohol block, increased contract foot pressure in the hindfoot and medial forefoot were observed. this change in pressure distribution is concurrent with the results of several previous studies10, 16, 17. using fpms, it was confirmed that mechanical stress abnormally concentrated on the fifth metatarsal head area was corrected to near normal following the alcohol blocks on the tibial nerve branches and the motor point of the tibialis posterior muscle. the correction of foot contact pressure distribution appears to have contributed to the pain reduction in the fifth metatarsal head area. in conclusion, the case of a patient with chronic hemiparetic stroke whose pain due to tailor s bunion showed relief following the control of spasticity using nerve and motor point blocks with 20% ethyl alcohol was reported. this study showed that deteriorated foot position or alignment induced by spasticity and malposition/malalignment-related foot pain can be successfully managed by nerve and motor point blocks with 20% ethyl alcohol. on the basis of the clinical experience in this study, when clinicians treat foot pain in patients with stroke, they should consider the possibility that spasticity can contribute to the development of foot pain. this is first study to show that management of spasticity can reduce foot pain in a patient with hemiparetic stroke. however, since this study reports a single case, further studies involving a larger number of patients are necessary.

[ purpose] this study report a case of a patient with hemiparetic stroke who showed significantly reduced foot pain when ankle spasticity was reduced using nerve and motor point blocks with 20% ethyl alcohol. [subject and methods] a 58-year-old woman with left hemiparesis following intracranial hemorrhage five years previously presented with pain in the left fifth metatarsal head for two years (numeric rating scale[nrs]: 8). erythema and edema were observed on the lateral aspect of the head of the fifth metatarsal bone. she was diagnosed with a tailor s bunion. spasticity was observed in the left ankle plantar flexor and ankle supinator (modified ashworth scale: 1 +). using 20% ethyl alcohol, a block in the medial and lateral motor branches to the gastrocnemius muscle of the left tibial nerve and the motor point of the left posterior tibialis muscle was performed. [results] after the alcohol block, spasticity had almost disappeared and foot pain was significantly reduced (nrs: 1). results from the foot pressure measurement system test showed foot contact pressure was highly distributed to the lateral forefoot pre-block. after the block, the distribution of foot contract pressure was similar to normal distribution. [conclusion] clinicians should consider the possibility that spasticity can contribute to foot pain.

PMC5430290

pubmed-763

alzheimer's disease (ad), the most common form of dementia affecting populations aged over 65 years worldwide, is sporadic, genetically non-obvious, and rarely inherited (1). the main well-known pathological features of the disease include the abnormal extracellular deposition of misfolded amyloid- (a) senile plaques in brain parenchyma and cerebral vessels, the intracellular accumulation of hyperphosphorylated tau () in neurofibrillary tangles (nfts), chronic neuroinflammation, neuronal loss and severe brain atrophy as well as progressive loss of memory (2,3). consequently, in the last 30 years, the deregulation of a metabolism (oligomerization, aggregation and plaque formation) has been the major target for therapeutic intervention (4), and only marginal effects have been registered (57), suggesting the need for preventive/curative treatments or alternative solutions. therefore, although the causes of ad remain unknown and cures or universally effective treatments, are not available, most experts have highlighted a broad constellation of contributing risk factors. among these risk factors, alcohol consumption, associated with extensive cognitive problems (8), including alcoholic dementia (9), has been targeted. similar features have been denoted between the effects of alcohol on cognition, brain disorder and brain biochemistry with the biological effects of ad, suggesting that the use of alcohol may constitute a risk for aggravating or developing ad (10). in line with this view, ad patients with a habitual drinking history have shown cognitive improvement during the clinical course of abstinence (11). by contrast, this effect was not observed in patients consuming high amounts of alcohol prior to diagnosis of ad (11). in line with these data, there is another emerging body of literature that contends alcohol consumption, particularly red wine, may rather serve as a protective factor for cognitive decline (12,13). several epidemiological studies have shown that low or moderate wine consumption can be effective in retarding age-related cognitive decline (14), possibly linked to polyphenols present in beverages. however, the studies promoting the benefits of alcohol on ad exclusively focus on moderate alcohol consumption (12), the restriction of alcohol to only an elderly population (13), and broad classification of cognitive decline (15). relevant issues remained regarding the protection, the aggravating or detrimental effects of alcohol consumption or whether protective effects are simply influenced by the quantity and/or frequency of drinking. despite the limiting factors identified regarding the beneficial effects of alcohol consumption (16), a single-target therapeutic strategy appears to produce only suboptimal results and a broader neuroprotective approach, at least theoretically, appears more appealing (17). thus, the aim of the present review was to discuss the association between alcohol consumption and ad. heavy alcohol consumption impairs cognitive performance with immediate and long-term effects on the brain anatomy and neuropsychological functioning (1821). cognitive impairment is related to clinical dementia as it accelerates shrinkage and atrophy of the brain, leading to critical determinant of neurodegenerative changes and cognitive decline in aging (22). however, these morphological changes induced by alcohol consumption may be reversible unlike ad or aging (23), as atrophy decreases, with cognitive improvement after abstinence from alcohol (24,25). other data showed that morphological changes in the brain are associated with the loss of a number of nerve cells occurring in the white matter, which largely comprises nerve fibers that connect neurons (26) and/or cortical cholinergic neurons (24), known to be affected in ad. this link renders plausible that alcohol use may be linked to ad as the cholinergic system plays an important role in memory. this role is confirmed and its deficits are well established in ad (27). chronic alcohol use causes degeneration of cholinergic neurons (28), or decreases receptors of cholinergic system in ad. these may aggravate the reduction of cholinergic neurons already present in ad patients however, the improvement of cognitive function in alcoholics after abstention from alcohol suggests that cognitive deficits may reflect neurochemical alterations rather than neuronal loss (24,28). therefore, without appearing as an accelerator of ad process, alcohol may induce its effects on the cholinergic system, independently from the cholinergic deficits caused by ad (29,30). however, alcohol-related brain damage appears to differ in young and older alcohol consumers (24), although data suggest that alcohol abuse may accelerate aging-related changes in the brain at any age and that older adults may be more vulnerable to the effects of alcohol (8). apart from cholinergic deficit, another negative effect of heavy alcohol consumption on cognitive function may be attributed to nutritional deficiency or vascular change (31,32), which consist of damage that may be relatively irreversible even after abstinence from heavy alcohol consumption (11). therefore, despite the above evidence showing cognitive detrimental effects of heavy alcohol consumption, to the best of our knowledge, no study has established a clear association between alcohol consumption and ad (33). more consistently, recent genetical study on a japanese population provided evidence that the mitochondrial aldehyde dehydrogenase 2 (aldh2*2, metabolizes acetaldehyde into acetate, protecting against oxidative stress and playing an important role in the development of ad), and two functional single-nucleotide polymorphisms (snps) of the dopamine--hydroxylase (dbh) gene, involved in the pathophysiology of alcoholism and whose activity is reduced in the neocortex of ad), did not modify the risk for developing ad, suggesting that the polymorphism of the aldh2 and dbh genes were not associated with ad (34). nevertheless, future studies must be undertaken on other populations worldwide. low-to-moderate alcohol intake is considered to protect against neurodegeneration pathology (13,15), dementia (3539) and cognitive deterioration (4043). of the biologic mechanisms suggested to explain such potential beneficial effects on the brain, there are mainly the antioxidant properties of wine flavonoids (44), the effects against a (45) and the prevention of ischemia or stroke by alcohol (46). specifically, polyphenols, members of a large family of plant-derived compounds, are molecules containing one or more phenolic group. there are thousands of polyphenols that have been identified thus far including, bioflavonoids (anthocyanins, flavanols, favanols, favones, flavanones, isofavones and proanthocyanins), coumestans, ligans and stilbenoids (47). polyphenolics are in general antioxidant molecules that reduce the in vitro process aggregation of a, reducing the neuronal death of cortical neurons preventing neurodegeneration (48). the morin for example, a specific flavonoid described in red wine exhibited significant effects in preventing aggregation of a (49). in agreement with these data, red wine cabernet sauvignon significantly reduced the number of a plaque-induced neuropathology and attenuated a spatial memory decrease in an adult tg2576 mouse model of ad (50). resveratrol is known to protect against cardiovascular diseases (which are risk factors for developing ad) and various types of cancer, together with the promotion of the antiaging effect, the modulation of pathomechanisms of debilitating neurological disorder such as strokes, ischemia and huntington's disease, as well as protection against neuronal degeneration (14,51). other natural molecules including fulvic acid, altered the aggregation mechanism of proteins, a critical protein involved in the stabilization of microtubule and axonal transport, found to be involved in ad pathogenesis (52). however, whatever the facts around the benefits that may follow the potential benefits of low-to-moderate consumption of alcohol, the importance of drinking patterns and specific beverages consumed remain elusive. the operational definition of low or moderate drinking which may vary greatly across studies (53) and the concept of a moderate drinker, which may also be imprecise, comprising a wide range measure that may include those consuming less than one drink a day (54). red wine consumption appears to promote far more protective effects than the consumption of other ethanol containing beverages (55). this is in keeping with variations that may be introduced by consumers who sometimes associate the consumption of tobacco, or the possible toxicity effects of chronic exposure of the liver to alcohol (56), which can also contribute to brain alteration in regions involved in memory (57). taken together, the data provide insufficient evidence to suggest abstainers should initiate alcohol consumption to protect against dementia or ad. based on the abovementioned research data, no relationship between alcohol consumption and ad exists. in addition, although low-to-moderate consumption of alcohol may protect against ad, leading to benefits on neurodegeneration, a, oxidative stress and neurofibrilary tangle formation suggest bias regarding the definition of low-to-moderate consumption as well as the variability of beverages containing alcohol. in addition, the absence of studies on the possible side effects of chronic exposure to alcohol on peripheral organs such as liver, and kidney lead to the necessity to delineate global and standard protocols for advanced studies. these studies render the benefits associated with low-to-moderate alcohol consumption against ad. however, the results should be considered as controversial and insufficient to suggest abstainers that initiate alcohol consumption in a preventive manner against ad.

alzheimer's disease (ad) is a neurodegenerative disease characterized by dense deposition of amyloid- (a) protein in the brain, failure of the memory and dementia. at present, there is no cure for ad and current treatments only provide a temporary reduction of symptoms. thus, there is a need for effective preventive/curative strategic approaches. accordingly, epidemiological studies have reported a reduction in the prevalence of ad in individuals ingesting low amounts of alcohol, while a moderate consumption of ethanol may protect against a. these data are conflicting with other observations that assigned detrimental effects of heavy alcohol use on brain function, which are apparently similar to those observed in ad. these discrepancies questioned whether or not alcohol is a protective agent against the development of ad, whether the probable protective effects are influenced by the quantity and/or frequency of drinking. these issues are addressed in this review with the aim to suggest the real risk of alcohol for developing or preventing ad.

PMC4998119

pubmed-764

furthermore, the most frequent indication for surgery is spinal stenosis.1 after decompression (laminectomy or foraminotomy), the structural integrity of the lumbar spine can be weakened, and requires additional support. this is achieved by vertebral fusion, which is the current standard for outcome and the most commonly used procedure. interbody arthrodesis can be performed through anterior, lateral, posterior, or transforaminal approaches. the vertebral disks are replaced with bone to promote arthrodesis, the sagittal height is restored with cages, and supplemental stabilization is completed by internal fixation using transpedicular instrumentation. the result is a bloc of vertebral segments that are permanently fixed and rigid. this resolves the immediate situation, but in the long term the proximal segment is overloaded, which might aggravate the degeneration, lead to hypermobility and osteophyte formation, become symptomatic, and require further surgery.2 this phenomenon is inherent to the principle of fusion and can not be avoided, with an annual incidence of 2.5% and an estimated 10-year prevalence of 22.2%.3 the increased stresses on the adjacent disk and facets might be dependent on the rigidity of the stabilization.4 in such circumstances, the need for an implant that will allow for dynamic stabilization is paramount. these were the premises that led to the introduction of the total facet arthroplasty system, aimed at restoring normal segmental kinematics. dynamic stabilization was not a new idea,5,6 but facet arthroplasty was designed to mimic natural movement of the vertebral segments without overstraining the adjacent vertebral disks, which was not possible before.7,8 we performed a prospective observational study on 14 cases operated on in our department between 2005 and 2008. the patients were diagnosed with lumbar stenosis due to hypertrophy of the articular facets on one to three levels. dynamic posterior stabilization was performed using the total facet arthroplasty system (archus orthopedics, redmond, wa, usa) (figure 1). we therefore implanted nine at l4l5 and four at l3l4 (see figure 2 and tables 13). necessary criteria to consider the patient suitable for this implant were: degenerative spinal stenosis, lateral or central, at l3l4 and/or l4l5 levels, with imaging confirmation through at least one of computed tomography, magnetic resonance imaging, x-ray, and myelography, which can show: compression on cauda equina, dural sac, or roots impingement on nerve roots due to bony or soft-tissue elements hypertrophic facets with lateral or central compression in cases of spondylolisthesis, at the level proposed for this new implant, the grade of severity should be no greater than grade i neurogenic claudication thigh or leg discomfort, pain, paresthesia, muscular weakness, fatigue, sensation of heaviness around the leg, pain in the medial thigh and lumbar region, tingling sensation that can be worsened by walking or by orthostatism and relieved with rest or lying down on a bed age between 40 and 80 years patients who have been referred for laminectomy due to spinal stenosis up to a maximum l3. exclusion criteria (patients not suitable for this implant) were: patients under 40 years or over 80 years of age spondylolisthesis of grade ii or more and retrolisthesis at the proposed level for the implant more than three levels proposed for the laminectomy levels other than l3l5. the implant is manufactured using the most recent technology with a variable geometry, which allows an exact choice of angles and dimensions in order to reproduce the intervertebral movements perfectly. biomechanical tests performed during our study demonstrated that this implant allows relatively normal flexion and extension of the involved vertebrae, as shown in figures 1 and 2. the new implant is made up of eleven modular components: two superior curved rods two inferior rods to which the connectors are adapted a transverse rod with two cups at the ends to control tracking tests indicated that the best way to fix the implants was with acrylic cement (figure 3). from biomechanical studies, the technical specifications indicated that this system resists for more than 10 million cycles, which roughly translates to flexion and extension movements for average human performance for over 10 years. the adhesion of the implant to the cement can take two to three times its maximum duty loads without debonding. this new implant can withstand more than twice the load it takes to detach a pedicle-screw fusion system (the current standard of care) from bone without loosening from the spine. a standard posterior median approach is used, with associated laminectomy and foraminectomy for decompression. in nine cases, we performed a laminectomy at two levels, and in four cases laminectomy was performed at one and three levels. this was followed by foraminotomy and inferior-facet resection at l3 or l4 accordingly. the next step was drilling of the 24 mm transpedicular tunnels for application of the probes at the two levels. because this is a cemented procedure, it is very important to keep the walls intact in order to prevent extravasations of the cement into the medullary canal or along the roots. the device is made up of eleven modular components: two superior curved rods, two inferior rods to which the connectors are attached, a transverse rod with two cups at the ends to control tracking, and two connectors. it is fixed through the pedicles to the vertebral bodies, using poly(methyl methacrylate) cement with increased barium content (same as in vertebroplasty). connections between all four elements are made with bolts, the same as used for transpedicular instrumentation. it is very important that the two bolts are positioned parallel to the sliding surfaces of the distal stems. the medullary canal and the holes for conjugation were verified again, and the wound was closed with a vacuum drain. the most frequent level of decompression was l4l5 (nine cases), followed by l3l4 (seven cases). in nine cases, laminectomy was performed on two levels, in two cases on three levels and in two cases on only one level (table 4). the average time necessary to perform the surgery was about 60 minutes, but with experience this might be reduced. without complications, the established protocol for the follow-up period was clinical and radiological exams at 3 and 6 months and then yearly, corroborated with function and pain according to a visual analog scale and oswestry score. the preliminary results of the initial 20 patients have been previously presented.911 of the 13 patients available for long-term follow-up, four showed progression of the degeneration, both at the operated level, as well as the proximal segment, with limited range of motion but no clinical deterioration. this is important, because it allows for immediate recovery without a long period of rest waiting for consolidation, as is the case with fusion. functional scores, together with dynamic radiographic imaging, confirmed the functional efficacy of this new implant (table 5, figures 4, 5, 6 and 7). the most important aspect of our case series is that it is one of only very few long-term reports of clinical outcomes using total facet arthroplasty. since the procedure s conception and clinical use, the limited articles studying facet joint replacement focused on kinematic7 motion of the adjacent level,8 disk pressures, and load sharing.12 these experimental cadaver analyses proved conceptual validity, but long-term outcomes were uncertain. we have presented proof of the potential validity of this system, though based on a small number of patients. nevertheless, all of the 13 patients available for follow-up showed the minimum clinically significant improvement in oswestry and visual analog scales, which persisted throughout the follow-up period.13 adjacent-segment disease does not yet have a precisely determined etiology. it can be attributed to decompression, fusion, and pedicle screw misplacement, as well as preexisting degeneration.1417 therefore, there is still effort being made toward improving sagittal and postural balance with better solutions for lumbar arthrodesis.18 currently, there are several implants on the market boasting dynamic posterior stabilization.5 the dynesys device (zimmer, warsaw, in, usa) has received the most attention. longitudinal imaging studies have found potential protective effects on adjacent-disk disease, especially for seriously degenerated disks.18 biomechanical research has shown that dynamic stabilization has less overall range of motion than the intact spine.19 in addition, intradiscal pressures were altered compared to the normal disk.20 even though dynamic stabilization distributes mobility more physiologically compared to fusion, over the cranial and caudal adjacent segments21 adjacent-disk degeneration seems to continue.22 with long-term follow-up, almost half of the patients showed some degree of progression of degeneration, but satisfaction remained very high.23 as with our cohort, residual range of motion is reduced, as well as the need for secondary surgery.24 yet with all favorable results, there are also reports that show inferior long-term functional and clinical outcomes compared to fusion, except for older patients, who appear to be more satisfied.25 the cemented fixation of the pedicle screws provides great mechanical strength, especially in osteoporotic bone, and might reduce some of the complications and cyst osteolysis encountered with osteointegration.26,27 in addition, cemented fixation allows immediate movement, support, and rehabilitation, and has not yet demonstrated any adverse events. we can conclude that total facet arthroplasty represents a feasible long-term solution for dynamic stabilization of the lumbar spine after decompression. it also has the potential advantage of preserving normal biomechanics of the lumbar segments, which might reduce the impact of adjacent-disk degeneration.

facet degeneration can lead to spinal stenosis and instability, and often requires stabilization. interbody fusion is commonly performed, but it can lead to adjacent-segment disease. dynamic posterior stabilization was performed using a total facet arthroplasty system. the total facet arthroplasty system was originally intended to restore the natural motion of the posterior stabilizers, but follow-up studies are lacking due to limited clinical use. we studied the first 14 cases (long-term follow-up) treated with this new device in our clinic. all patients were diagnosed with lumbar stenosis due to hypertrophy of the articular facets on one to three levels (maximum). disk space was of normal height. the design of this implant allows its use only at levels l3l4 and l4l5. we implanted nine patients at the l4l5 level and four patients at level l3l4. postoperative follow-up of the patients was obtained for an average of 3.7 years. all patients reported persistent improvement of symptoms, visual analog scale score, and oswestry disability index score. functional scores and dynamic radiographic imaging demonstrated the functional efficacy of this new implant, which represents an alternative technique and a new approach to dynamic stabilization of the vertebral column after interventions for spine decompression. the total facet arthroplasty system represents a viable option for dynamic posterior stabilization after spinal decompression. for the observed follow-up, it preserved motion without significant complications or apparent intradisk or adjacent-disk degeneration.

PMC4049884

pubmed-765

innate immunity mediated through pamp recognition by prrs is the earliest stage of immunity against viral infection. the subsequent modulation of the adaptive immune response by prr signaling has been studied using cells and mice deficient in specific tlrs, rlrs, or their associated signaling adaptor proteins. recent studies demonstrated, for example, that the absence of specific tlr pathways impairs adaptive immune responses against a variety of viruses (18, 19). rlr signaling also seems to be critical for the outcome of japanese encephalitis virus (jev), vesicular stomatitis virus (vsv), influenza virus, and encephalomyocarditis virus (emcv) infection (11, 20, 21). in mice lacking rig-i, stimulator 1, jev, vsv, influenza virus, and ecmv were more virulent and replicated to higher levels than in wild-type mice, suggesting that rlr pathways are essential for controlling infection by these viruses (11, 21, 22). for example, irf3 target genes induced by rlr signaling directly control viral replication in infected tissues (23, 24). thus, depending on the nature of virus infection, tlrs and rlrs may work together or independently to mount an efficient immune response. it is thought that the innate immune system protects the host from infection in a nonspecific way. this, along with differences in cellular location likely serve to distinguish self-rna from nonself pamp rna, thus avoiding type i ifn induction in response to components of host nucleic acids. for example, tlr7 recognizes a specific motif within uridine-rich ribonucleotide sequences (25, 26), which are hypothesized to be unique to rna viruses. tlr9 recognizes dna pamp ligands and triggers signaling through the myd88 adaptor protein to induce type i ifn production (27). the recognition of nonself dna ligands by tlr9 might also be sequence dependent, and some studies have implicated the sugar-base-backbone sequence of pamp dna as a recognition factor (2830). unlike tlrs, which are found either on the cell surface or within membrane-bound vesicles, rlrs are found in the cytoplasm where cellular rna is also present (1). rig-i preferentially recognizes single-stranded rna (ssrna) over dsrna (15, 31). ssrnas containing a terminal 5 triphosphate (ppp), but not 5oh or a 5-methylguanosine cap, bind to the rig-i repressor domain and promote a conformational change that activates rig-i signaling (10, 1316). rna ligands of rig-i are longer than 23 nucleotides, have a linear structure, and contain a uridine- or adenosine-rich ribonucleotide sequence (3133). host mrna, trna, mirna, snrna, and rrna are not ideal rig-i ligands because their length, structure, 5 end modifications, and interactions with ribonucleoproteins limit their recognition. self-rnas therefore do not typically trigger innate immune programs or type i ifn expression (34). mda5 triggers innate immune signaling in response to emcv infection and synthetic dsrna, such as poly i: c (11, 20). but ssrna viruses such as dengue virus and west nile virus, as well as reovirus, a dsrna virus, have also been shown to trigger signaling partially via mda5 (12, 35). however, in these studies, mda5 appeared only to amplify type i ifn production as compared with rig-i, whose actions were essential to initiate innate immunity (12). (17) in this issue describes a plausible mechanism of rna ligand recognition by mda5, providing new insights into the differential roles of rlrs. the study reveals that mda5 binding to dsrna does not depend on its 5 end modification, but rather on its length. dsrnas with lengths of 2, 3, and 4 kb were shown to increasingly activate mda5 signaling, whereas rig-i recognized ssrna and, to a lesser extent, short dsrna motifs. these results help explain how reovirus can trigger both rig-i and mda5 signaling. the genome of reovirus comprises at least 10 segments of dsrna that vary in length, including long (3.9 kbp), medium (2.2 kbp), and short (1.2 kbp) rna segments. show that rig-i and mda5 recognize the short and long dsrna segments of the reovirus genome, respectively. in the case of emcv infection, the authors show that the viral genome forms long dsrna via its antisense replication intermediate, and this dsrna is recognized by mda5 but not by rig-i. on the other hand, vsv, which triggers rig-i dependent immunity, forms medium-length dsrna replication intermediates that are not recognized by mda5. propose a model for pamp recognition by mda5 in which only long dsrna viral genomes or stable long duplex viral rnas formed during virus replication serve as mda ligands. this model might explain why mda5 ignores self-dsrna, which is typically not present as long duplex rnas. the current observations advance our understanding of how mda5 discriminates between potential rna ligands, but several questions about recognition of rna by rig-i and mda5 still remain. it is widely accepted that ssrna viruses generate dsrna during their replication process (36, 37). however, only mda5 recognizes emcv (8 kb), whereas rig-i recognizes other viruses with lengthy rna genomes, such as vsv (11 kb), jev (11 kb), and hepatitis c virus (9.6 kb). mda5 's failure to recognize these latter viruses may be due to their inability to form long or perhaps stable dsrna. it is also possible that recognition by mda5 depends on specific compartmentalization of the pamp rna with mda5, as differences in the subcellular site of viral genome replication may influence the pamp the mechanism by which mda5 discriminates between pamp ligands in terms of their length is also unclear. demonstrate that mda5 binds long, capped, di- or mono-5 phosphate dsrna, whereas rig-i binds to short dsrna or 5ppp uncapped ssrna. when rig-i binds to 5ppp ssrna, it changes conformation, which disrupts the inhibitory interaction between the rig-i repressor domain and the cards. this alteration results in the initiation of downstream signaling by the cards and exposes a 17- or 30-kd trypsin-resistant fragment corresponding to the rig-i repressor domain, thereby marking the activation of rig-i (10, 15, 16). however, poly i: c binding to rig-i promotes a distinct conformational change that results in a 66-kd trypsin-resistant fragment, indicating that pamp-bound rig-i undergoes a distinct conformational change depending on the nature of its ligand. structural studies validate that rig-i prefers ssrna ligands, whereas dsrna binds only inefficiently to rig-i, possibly due to constrains imposed by the location of basic residues within the rna binding groove of its repressor domain. the charged residues are proposed to anchor 5ppp ssrna within the pocket, which would be less amenable to binding dsrna ligands (15, 16). these observations suggest that rig-i may have two ways to distinguish different rna species: efficient recognition of 5ppp ssrna through its repressor domain (10, 15, 16) and comparably inefficient recognition and binding of dsrna (10, 15). by extension, these conformational alterations of rig-i might provide clues about how dsrna ligands interact with mda5 to stimulate its activation. thus, mda5 might become active upon binding long dsrna through a ligand-induced conformation change that places its card into a signaling-active conformation. this might involve dsrna interactions with its helicase domain and c-terminal region in a fashion similar to the binding of poly i: c to rig-i. others have demonstrated that although very short dsrna (2030 bp) fail to trigger rig-i dependent signaling (15, 17), longer dsrnas ranging from 70 kb to 2 kb can at least weakly engage rig-i. these data suggest that the length of dsrnas may dictate the interaction with the rig-i helicase domain and/or repressor domain in a manner that supports a rig-i dsrna complex. however, the mechanism of length discrimination of dsrna pamps by these rlrs has not yet been revealed. the third rlr family member, lgp2, has been defined as a regulator of rig-i signaling based on its suppression of rig-i function when overexpressed in cultured cells (10, 38, 39). however, recent studies imply that lgp2 may also function as either a positive or negative regulator of rlr signaling by forming a heterodimer with rig-i or mda5 when bound to rna ligand (40, 41). such interactions between the rlrs may also broaden their pamp recognition profiles to accommodate other ligands, including ssrna or dsrna of varied length and composition, or even dna rna duplexes. structure and function analyses of rna interactions with rlrs or heteromeric rlr complexes will further define the molecular basis of self- versus nonself discrimination. the rlrs are adenosine triphosphate (atp) binding proteins that hydrolyze atp as a result of binding rna ligand (15). however, the role of the atpase activity in the function of rlrs is not fully understood. rlrs contain multiple domains, including two domains known as the walker a motif and the walker b (also known as the dexd/h-box) motif, each located within the helicase domain (42). walker a and b motifs comprise an atp binding region wherein specific amino acid residues contact the -phosphate of the nucleotide and mediate atp hydrolysis upon substrate binding (4345). mutant rig-i with an inactive walker a motif retains ssrna binding function but fails to trigger downstream signaling (15, 46). thus, atp hydrolysis is not essential for pamp binding but is required for downstream signaling. recent studies revealed that the rig-i repressor domain, in addition to binding to the rig-i cards, forms a complex with the helicase domain linker region that likely stabilizes rna ligand binding (10, 15, 16). moreover, it seems that motifs in the rlr helicase domain could also be involved in rna substrate binding according to structure and function studies of other dexd/h-box rna helicases (42). such studies indicate that the rna binding sites and the walker a and b motifs mediate specific interactions (47). these observations present a model in which a repressor domain helicase domain internal complex forms an rna binding pocket whose association with pamp rna may serve to initiate the atpase activity of rig-i, which then triggers a conformation change that allows innate immune signaling. the repressor helicase domain interaction of rlrs may thus be the key determinant of pamp discrimination by rig-i and mda5 (15, 17). it also seems that rig-i atpase activity is involved in the unwinding of dsrna (15). the importance of this activity for innate immune function is unclear, but it might support rig-i interaction with dsrna ligands by catalyzing their conversion to ssrnas. the breadth of prrs expressed in different cells and tissues and their distinct intracellular distribution provide the means for pamp detection and immune activation against a variety of microbial pathogens at the local site of infection or in the microbial niche. these include the membrane-associated cytosolic replication sites of rna viruses, extracellular sites of microbe interaction, and endosomal sites of microbial trafficking and metabolism (fig. the recent identification of the dai (dlm-1/zbp1) protein as a cytoplasmic sensor of microbial dna (48) suggests the existence of other prrs that detect cytosolic pamp dna. it is reasonable to speculate that, similar to tlrs and rlrs, the cytoplasmic dna sensor molecules also exhibit ligand specificity to cover a variety of dna pamps in association with their specific microbial niche, thus providing a further basis for self- versus nonself discrimination. understanding the processes of pamp ligand recognition within each microbial niche will provide a foundation for the design of appropriate vaccines, adjuvants, and immunotherapies.

retinoic acid inducible gene (rig)-i like receptors (rlrs) are cytosolic rna helicases that sense viral rna and trigger signaling pathways that induce the production of type i interferons (ifns) and proinflammatory cytokines. rlrs recognize distinct and overlapping sets of viruses, but the mechanisms that dictate this specificity were unknown. a new study now provides evidence for size-based discrimination of double-stranded rna (dsrna) by rlrs and suggests how host cells recognize a variety of rna viruses.

PMC2442628

pubmed-766

the parainfluenza virus 5 (piv5) belongs to the paramyxoviridae family, which contains significant pathogens to mammals such as measles, mumps, and hendra viruses. two glycoproteins in the lipid envelope, a receptor-binding protein (hn, h, or g) and a fusion protein (f), are required for membrane fusion. the f protein, similar to the influenza hemagglutinin (ha) and the hiv env protein, is synthesized as a homotrimer and is activated by proteolytic cleavage, which creates a highly hydrophobic n terminus called the fusion peptide (fp) that is essential for membrane fusion. the cleaved protein is anchored to the virus envelope by a hydrophobic c-terminal transmembrane (tm) domain. two heptad repeats, hra and hrb, lie next to the fp and tm domains, respectively. crystal structures of the water-soluble portions of a number of viral fusion proteins have been determined and have provided much of the current understanding of the mechanism of protein-mediated virus cell membrane fusion. it is known that fusion proteins undergo multiple conformational changes to provide the necessary energy for membrane fusion. the conformations that have been observed correspond to the prefusion states before and after cleavage, an extended prehairpin state and the postfusion hairpin state. the hairpins are formed between two heptad-repeat domains common in class i fusion proteins and give rise to a six-helix bundle (6hb) that is characteristic of the postfusion state of these trimeric proteins. a consequence of this 6hb is that it enforces close proximity of the neighboring fp and tm domains in the merged membrane, but no direct structural evidence of this close packing in the membrane has yet been reported. for the parainfluenza f protein, the crystal structures of the uncleaved prefusion state, the cleaved prefusion state, and the postfusion state have been determined, and an extended prehairpin structure was observed by electron microscopy. in comparison, structural information about the membrane-bound fp and tm domains is still scarce. solution and solid-state nmr studies of the influenza and hiv fusion peptides in detergent micelles and lipid bilayers have provided insights into the mechanisms of virus cell fusion. the ha fusion peptide is predominantly -helical, but the exact tertiary structure depends on the peptide length and the membrane-mimetic environment. a 20-residue construct adopts an obliquely inserted boomerang conformation in detergent micelles, but in lipid bilayers at fusogenic ph, it also samples a small population of a helical hairpin conformation. a 23-residue construct that includes the conserved gxxxg and gxxg motifs adopts a helical hairpin conformation already in detergent micelles, with the hairpin stabilized by gly gly to ala mutation at residue 8 results in a mixture of hairpin and boomerang structures. it is -helical in detergent micelles but a -strand in lipid bilayers containing more than 20% cholesterol. solid-state nmr data indicate that both helical and strand conformations of the hiv fp insert into the lipid membrane but cross-linked trimers insert more deeply than monomers and are also more fusogenic. the conformational polymorphism of these viral fusion peptides indicates the importance of the lipid environment in regulating membrane fusion. however, the lipid environment is important not only for modulating the fp structure but also for directly influencing the membrane curvature and hydration during fusion. a large number of computational analyses and experimental studies have probed the structures of membrane intermediates during fusion; however, few studies have combined or correlated the fp structure with the membrane-intermediate structure. we recently reported the first solid-state nmr structural study of the piv5 fusion peptide in lipid bilayers. we found that the peptide adopted an -helical conformation in the negatively charged popc/popg membrane but a -strand conformation on the surface of neutral popc and dmpc bilayers. in the current study, we have determined the complete backbone conformation of the popc/popg-bound piv5 fp using chemical shift constraints. the dopc/dopg membrane retains the same negative surface charge as the popc/popg membrane but increases the unsaturation and disorder of the lipid chains. surprisingly, this change did not increase the fp mobility but converted the peptide from an -helical structure to a partial -strand structure. in the dope membrane, the piv5 fusion peptide mainly adopts a -strand conformation, similar to its structure in neutral pc membranes, but the -strand is inserted into the dope membrane rather than surface bound. moreover, the peptide changes the phase behavior and hydration of the dope membrane. these results suggest the structural roles of the piv5 fusion peptide during membrane fusion. the fusion peptide used in this study corresponds to residues 103129 of the piv5 f protein, with the amino acid sequence of fagvviglaalgvataaqvtaavalvk. to increase the peptide solubility, a lys tag kkkk was appended to the c terminus through a flexible dioxa linker (nh(ch2ch2o)2ch2co). five c-, n-labeled peptides were synthesized by primm biotech (cambridge, ma): gval-fpk4, igalv-fpk4, gvtaa-fpk4, vlaat-fpk4, and aaqv-fpk4 (table 1). the labeled residues cover all except for four residues at the n and c termini (f103, a104, v128, and k129) of the peptide. fpk4 was reconstituted into popc/popg (4:1), dopc/dopg (4:1), and dope membranes at a peptide/lipid molar ratio of 1:20. briefly, the peptide was dissolved in trifluoroethanol (tfe) and mixed with lipids in chloroform. hcl, 1 mm edta, 1 mm nan3, ph 7.5) or phosphate buffer (10 mm na2hpo4nah2po4, 1 mm edta, 1 mm nan3, ph 7.5) and dialyzed for a day. the proteoliposomes were centrifuged at 55 000 rpm at 4 c to obtain membrane pellets, which were equilibrated to 3040 wt% water before being transferred to 4 mm magic-angle-spinning (mas) rotors. mas nmr experiments were carried out on bruker avance-600 (14.1 t) and dsx-400 mhz (9.4 t) spectrometers. c chemical shifts were referenced to the adamantane ch2 signal at 38.48 ppm on the tms scale, and the n chemical shifts were referenced to the n-acetylvaline signal at 122.0 ppm on the liquid ammonia scale. p chemical shifts were referenced to the hydroxyapatite signal at 2.73 ppm on the phosphoric acid scale. two-dimensional (2d) c c correlation spectra were measured using a h-driven c spin diffusion experiment with h irradiation (darr) during a mixing time of 2060 ms. experimental temperatures ranged from 233 to 303 k to investigate fpk4 conformation in both the gel and liquid-crystalline (lc) phases of the membrane. n c correlation spectra were measured using a redor-based pulse sequence with a coherence transfer time of 857 s. one-dimensional (1d) static and mas p spectra were measured between 273 and 313 k to probe the membrane morphology and structure. the size of the p chemical shift anisotropy (csa) is characterized by its span, defined as the difference between the 0 edge and the 90 edge of the uniaxial powder pattern, =0 90. a 2d p the depth of insertion of the fusion peptide was measured using 2d h spin diffusion experiments in either the lc phase or the gel phase. the lc-phase experiment was applied to dopc/dopg-bound peptide with igalv and aaqv labels using a h t2 filter of 0.81.0 ms and a spin diffusion mixing time of 9625 ms. spin diffusion buildup curves were quantified after correcting for h t1 relaxation and were simulated using diffusion coefficients of 0.012 and 0.30 nm/ms for the lipid and peptide, respectively. the water peptide interfacial diffusion coefficient (dwp) was 0.0020.003 nm/ms while the lipid peptide coefficient (dlp) was 0.00250.005 nm/ms. the gel-phase spin diffusion experiment was carried out on popc/popg- and dopc/dopg-bound fpk4. the intensity ratios between the water and lipid ch2 cross peaks of each residue were measured to compare residue-specific depths. we recently reported that popc/popg-bound fpk4 exhibited only -helical chemical shifts for nine labeled residues, suggesting that this membrane promotes a single conformation of the peptide. to obtain the complete backbone conformation in this anionic membrane, we labeled additional residues (table 1). figures 1 and 2 show the 2d c c and n c correlation spectra of fpk4 in gel-phase popc/popg bilayers. consistent with the previous study, most residues exhibited -helical chemical shifts and a single set of signals. modest conformational disorder was manifested at residues a118v121 as reduced intensities and peak multiplicity. for example, the q120 cc cross peak is 4-fold weaker than the a118 and a119 peak (figure 1c), suggesting dynamic disorder at q120. a recent hexamer model of piv5 fusion peptide placed q120 in the interior of the hexamer and postulated that this residue may be involved in intermolecular h-bonding. since oligomeric assembly and h-bonding should order and immobilize the peptide, our data does not support this model for fpk4 in the popc/popg membrane. 2d c c correlation spectra of piv5 fpk4 in gel-phase popc/popg (4:1) bilayers. shown at the top is the amino acid sequence with labeled residues color-coded according to samples. (a) gvtaa-fpk4 spectrum, measured at 253 k with 20 ms mixing. (b) vlaat-fpk4 spectrum, coadded from two spectra measured at 243 k with 20 ms mixing and 253 k with 60 ms mixing. (c) aaqv-fpk4 spectrum, measured at 253 k with 20 ms mixing. c correlation spectra of piv5 fpk4 in gel-phase popc/popg (magenta), dopc/dopg (black), and popc (blue) membranes. the peptide shows predominantly -strand chemical shifts in the popc membrane, -helical chemical shifts in the popc/popg membrane, and mixed strand and helix chemical shifts in the dopc/dopg membrane. most residues in the gvtaa and vlaat samples show two sets of chemical shifts in the dopc/dopg bilayer. the aaqv sample shows nearly identical -helical chemical shifts in the popc/popg and dopc/dopg membranes. the assigned c and n chemical shifts of popc/popg-bound fpk4 (table 2) allow us to obtain a backbone conformational model of the peptide. all 23 residues (g105l127) exhibit -helical chemical shifts as the dominant signals, with positive c and co secondary shifts and negative c secondary shifts (figure 3a). using talos+, we obtained backbone (,) torsion angles (table 3), which indicate a nearly ideal -helical conformation in the popc/popg membrane. chemical shifts were measured from 2d spectra at 233253 k. italics indicate the second conformation. c chemical shifts are referenced to tms, and n chemical shifts are referenced to liquid ammonia. from the gvtaa sample (g105, v106, t122, a123, and a124). from the vlaat sample (v107, l110, a111, a116, and t117). from the igalv sample (i108, g109, a112, l113, and v125). from the gval sample (g114, v115, a126, and l127). from the aaqv sample (a118, a119, q120, and v121). c and n secondary chemical shifts of fpk4 in (a) popc/popg and (b) dopc/dopg membranes. fpk4 shows clear -helical chemical shifts (red) in popc/popg bilayers and mixed helical and strand chemical shifts (blue) in dopc/dopg bilayers. the random coil values of zhang et al. were used to calculate the secondary shifts. the dopc/dopg values were predicted from the main set of chemical shifts. fpk4 undergoes intermediate-time scale motion in the lc phase of the popc/popg membrane. the resulting line broadening precludes the lc-phase h spin diffusion experiment for measuring the insertion depth of the peptide. therefore, we carried out the gel-phase spin diffusion experiment, which resolves the water, lipid, and peptide h signals in the indirect dimension by h homonuclear decoupling. strong cross peaks between lipid protons and peptide c signals indicate deep insertion of the peptide into the membrane. in addition, well-inserted peptides exhibit similar h intensity patterns as the lipid chain carbons, while surface-bound peptides exhibit different h cross sections, with much higher water cross peaks than lipid cross peaks. figure 4a shows representative gel-phase h spin diffusion spectra of fpk4 in the popc/popg membrane. by 25 ms, the peptide shows strong cross peaks with both lipids and water, and the peptide c and lipid ch2 cross sections have similar h chemical shifts, linewidths, and intensity distributions (figure 4b), indicating that fpk4 is well inserted into the hydrophobic region of the membrane. with a shorter mixing time of 4 ms, more residue-specific depth information is obtained, since different residues give different relative intensities between the water and lipid cross peaks (figure 4c): terminal residues such as g105 and a126 have higher water/lipid intensity ratios than middle residues such as a112 and l113, indicating that the two termini are in closer contact with water. the water/lipid intensity ratios (figure 4d) are the lowest between a111 and t117 (0.100.13) and higher for both the n and c termini (0.170.35), consistent with a membrane-spanning topology of the peptide. the intensity profile is asymmetric, with the n terminus having higher values than the c terminus, indicating that the n-terminal half of the peptide is more exposed to the membrane surface. depth of insertion of fpk4 in the popc/popg membrane from gel-phase spin diffusion. (a) representative 2d spectra with 0 and 25 ms spin diffusion mixing at 258 k. (b) h cross sections for the peptide c peaks (red) and the lipid ch2 peak (black). already at 4 ms, the peptide and lipid h cross sections have similar intensity patterns, indicating that the peptide is well inserted into the membrane. (c) c cross sections extracted from the water (blue) and lipid ch2 (black) h chemical shifts from the 4 ms 2d spectra. the n- and c-terminal residues have higher water/lipid intensity ratios than the middle residues. (d) water/lipid intensity ratios for all labeled sites. since fpk4 undergoes intermediate-time scale motion in the popc/popg membrane at ambient temperature, we searched for a different lipid membrane that may speed up the helix motion. fast motion not only gives higher-resolution nmr spectra but may also allow helix orientation to be determined from motional order parameters without requiring macroscopically aligned samples. the most obvious choice is the dopc/dopg (4:1) membrane, since it has the same membrane surface charge as the popc/popg bilayer while having a 18 c lower gel-to-lc phase-transition temperature due to the presence of a double bond in both acyl chains of each lipid. surprisingly, the increased disorder and dynamics of the dopc/dopg membrane did not speed up motion of the fusion peptide but changed the peptide conformation. 1d c cp-mas spectra (figure 5) show high-intensity -strand signals for various residues at ambient temperature and few -helical signals. when the membrane is cooled to the gel phase, the -helix signals become detectable and comparable in intensity as the -strand signals. thus, the remaining -helical conformation has similar intermediate-time scale motion between the dopc/dopg and the popc/popg membranes, but the new -strand structure is immobilized in the lc phase. the increased disorder of the lipid chains shifted the conformational equilibrium of the fusion peptide toward -strand, without changing the mobility of the -helical segment. representative 1d c cp mas spectra of dopc/dopg-bound fpk4 as a function of temperature. the vlaat-fpk4 spectra are shown. at high temperature, mainly -strand chemical shifts (blue dotted lines) are observed, while at low temperature, both -helical (red dashed lines) and -strand chemical shifts are detected. figure 6 shows 2d c c darr spectra of four labeled peptides in the gel and lc phases of the dopc/dopg membrane. in the gel phase, the exceptions are g105, v106, i108, and g109, which exhibit only -strand signals, and q120 and v125, which display only -helical chemical shifts. increasing the temperature decreased the intensities of the helix signals while retaining the strand signals. the position of the peptide at which the helix and strand have comparable intensities is t117. the signals of several ala residues partially overlap in the short-mixing-time spectra but become resolved by inter-residue cross peaks at long mixing times. for example, the 300 ms 2d spectrum (figure s1a, supporting information) shows -strand l110a111 cross peaks and -helical a116t117 cross peaks, indicating that a111 is primarily in the strand conformation while a116 is mostly helical. a118 and a119 show chemical shifts for all three conformations, but the -helix intensity dominates the strand and coil intensities (figure 6 g, h). finally, the n-terminal half of the peptide underwent a slow conformational change from -helical to -strand in the dopc/dopg membrane: v107, l110, and a111 initially showed -helical chemical shifts, which converted to -strand chemical shifts at equilibrium (figure s1b, supporting information). however, the more c-terminal a116 and t117 in the same vlaat sample remained stably -helical. 2d c c correlation spectra of dopc/dopg-bound fpk4 in the gel phase (233 or 243 k, left column) and the lc phase (303 k, right column). c correlation spectra (figure 2) confirmed the mixed strand/helix conformation of the n- and c-terminal halves of the dopc/dopg-bound peptide. two sets of chemical shifts were observed for many residues, but residues g105l113 show dominant -strand peaks while residues a118v125 have dominant -helical peaks. comparison of the peptide spectra for three lipid membranes, popc/popg, dopc/dopg, and popc, highlights the membrane-induced conformational polymorphism of fpk4. the -helical chemical shifts of the c-terminal half of the peptide are the same between the popc/popg and dopc/dopg membranes, whereas the -strand chemical shifts of the n-terminal residues differ between the popc and dopc/dopg membranes. for example, the chemical shifts of i108, a112, and l113 in the dopc/dopg membrane are intermediate between the corresponding chemical shifts in the popc and popc/popg membranes (figure 2c). on the basis of the cross-peak intensities in the low-temperature 2d c c spectra, we quantified the -helical content of each residue (table s1, supporting information). residues up to l113 are less than 35% -helical, whereas residues a116v125 are greater than 50% helical. the increasing helicity toward the c terminus was consistently observed for all labeled peptides, independent of minor variations in the hydration and salt content of the samples. the talos+ predicted backbone (,) torsion angles of the major conformer of dopc/dopg-bound fpk4 (table 3) confirm the n-terminal -strand and c-terminal -helical structures of the peptide. for this mixed conformation, oligomerization, if present, is expected to be parallel rather than antiparallel. this is consistent with the cross-peak pattern detected at long mixing times. the labeled residues within gvtaa- and igalv-fpk4 lie at the two ends of the peptide. thus, if antiparallel packing or a hairpinlike structure were present, we would observe inter-residue cross peaks between the n- and c-terminal residues. the 500 ms 2d spectra (figure s2, supporting information) of these samples show only sequential inter-residue cross peaks such as g105v106, t122a123, i108g109, and a112l113 but no long-range cross peaks, thus ruling out antiparallel packing and the helical hairpin conformation. since fpk4 adopts a surface-bound -strand structure in neutral pc membranes but an inserted -helical structure in the anionic popc/popg membrane, the topology of the partial -strand peptide in the anionic dopc/dopg membrane is not immediately obvious. we thus measured the depth of the peptide in the dopc/dopg membrane, using both the lc-phase h spin diffusion experiment and the gel-phase experiment. by 100 ms, the 2d h c correlation spectra at 293 k (figure s3, supporting information) showed clear cross peaks between lipid-chain protons and peptide c for both the -strand and -helical residues, indicating that the entire peptide is inserted into the dopc/dopg membrane. this is confirmed by the fast lipid-to-peptide spin diffusion buildup rates for both conformations (figure s3c, f, supporting information). more residue-specific depth information is obtained from the gel-phase spin diffusion spectra obtained at 243 k. by 4 ms, the peptide h cross section is already similar to the lipid h cross section (figure 7a), indicating equilibration of the h magnetization among the peptide, lipid, and water. similar to the popc/popg case, fpk4 has higher water/lipid cross-peak intensity ratios for the terminal residues than the central residues (figure 7b, c), indicating that the peptide spans the bilayer thickness. but in contrast to the popc/popg-bound fpk4, the c-terminal -helical residues are significantly more exposed to water than the n-terminal -strand residues (figure 7c). the lc-phase spin diffusion spectra (figure s3c, f, supporting information) also exhibit slightly faster lipid peptide spin diffusion buildup rates for the n-terminal residues than the c-terminal residues. it is not fully clear whether it is the backbone conformation (helix versus strand) or the residue position (n or c termini) that causes the different insertion asymmetry between the popc/popg and dopc/dopg membranes. however, the minor -strand conformation of the c-terminal a123 and a124 has lower water/lipid intensity ratios than the -helical counterpart, while the minor -helical conformation of the n-terminal l110 has higher water exposure than -strand l110 (figure 7c), suggesting that conformation may be the more important determinant of depth: the -strand conformation is more deeply inserted than the -helical conformation into the dopc/dopg membrane. depth of insertion of fpk4 in the dopc/dopg membrane from gel-phase spin diffusion spectra measured at 243 k. (a) h cross sections of the peptide c peaks (red) and lipid ch2 peak (black). by 4 ms, the peptide and lipid signals have equilibrated, indicating that the peptide is well inserted into the membrane. (b) c cross sections from the water (blue) and lipid ch2 (black) h chemical shifts of the 4 ms 2d spectra. the c-terminal -helical residues have higher water cross peaks than the n-terminal -strand residues, and the -helical a123/a124 have higher water cross peaks than the -strand a123/a124. (c) water/lipid intensity ratios of all labeled residues in the dopc/dopg membrane (blue and red symbols). open symbols indicate the minor conformation. for comparison, the popc/popg-bound fpk4 data are also shown (black open symbols). to investigate whether fpk4 causes curvature and dehydration to the dopc/dopg membrane, we measured the static and mas p spectra (figure s4a, b, supporting information). fpk4 displayed little perturbation of the structure of the dopc/dopg membrane: the lamellar-bilayer powder pattern is retained, and the isotropic chemical shift is unchanged. however, the mas isotropic line width is significantly broadened by the peptide (from 30 to 130 hz), and the p transverse relaxation times of dopc and dopg decreased from 18.3 and 19.4 ms, respectively, for the peptide-free membrane to 2.4 and 2.0 ms for the peptide-bound membrane (figure s4c, supporting information). thus, the apparent p linewidths are largely homogeneous, and the fusion peptide slows down the lipid headgroup motion without changing its average conformation. finally, the 2d p h correlation spectrum shows clear water lipid cross peaks (figure s4d, supporting information) for both dopc and dopg, indicating that fpk4 retains the hydration of the membrane surface. the p mas spectrum (figure s4b, supporting information) exhibits a small isotropic peak at 2.2 ppm. this peak can be assigned to the phosphate buffer, since samples prepared in tris or hepes buffer did not show this peak (data not shown). we previously observed the same isotropic peak in static and mas p spectra of fpk4-containing popc and dmpc membranes, and the peak intensity increased with the peptide concentration. the latter led to the erroneous conclusion that this peak resulted from a peptide-induced high-curvature isotropic phase. we now attribute the concentration dependence of this p peak to electrostatic attraction between the cationic lys tag and the phosphate ions. similar cases of phosphate buffer interactions with membrane peptides have been reported in the literature. thus, the -strand fpk4 that binds to the surface of the popc membrane does not cause curvature on the sub-10 nm scale. however, this does not exclude the possibility that the peptide may cause curvature on larger length scales of 50100 nm, which would not manifest as a narrow peak in the static p spectra. to further investigate whether fpk4 induces membrane curvature, we studied the structure and lipid interactions of dope-bound fpk4. the small headgroup of dope and its disordered acyl chains create spontaneous negative curvature to the membrane, causing an inverse hexagonal phase (hii) in a wide temperature range. the dope phase diagram has been measured using nmr and x-ray diffraction, and the lamellar (l)hii transition temperature (th) is known to depend on the hydration: above 16 water molecules per lipid, the membrane converts to the hii phase by 283 k. if fpk4 causes membrane curvature, then th will be affected: positive curvature generation by the peptide increases th while negative curvature generation lowers the transition temperature. figure 8 shows the static p spectra of dope membranes without and with fpk4 from 273 to 313 k. at 273 k, pure dope membrane shows an l-phase powder pattern with a chemical shift anisotropy span of+44.5 ppm. above 273 k, the p spectrum shows increasing intensities of a narrower line shape with a span of 21.5 ppm, which is inverted from the l line shape around the isotropic p chemical shift. the pure dope membrane fully converted to the hii phase by 283 k (figure 8a), consistent with the reported th value. upon fpk4 binding, the lhii transition shifted to higher temperatures and was complete only by 293 k, indicating that fpk4 exerted positive membrane curvature. in addition, an isotropic peak appeared in the spectra (figure 8b). in principle moreover, small-angle x-ray diffraction data of dope containing the ha fusion peptide indicated the presence of inverted bicontinuous cubic phases as well as an increase of the lhii transition temperature, and independent md simulations also predicted the same effect. thus, the isotropic p peak seen here is most likely due to cubic-phase formation in the dope membrane, which suggests that the piv5 fusion peptide causes both positive and negative curvatures; that is, the peptide generates negative gaussian curvature. also known as saddle-splay curvature, negative gaussian curvature results from the product of positive and negative principal curvatures and is present at membrane pores and protrusions formed during membrane budding and scission. (a, b) static p spectra of the membrane without (a) and with (b) fpk4 from 273 to 313 k. fpk4 increased the l-to-hii phase transition temperature and caused a small isotropic peak. h correlation spectrum of fpk4-bound dope membrane with a spin diffusion mixing time of 225 ms. (d) h cross sections from the 2d p h spectra of peptide-free and peptide-bound dope membranes, compared with the 1d h single-pulse spectrum (top). the equilibrium fpk4 conformation in the dope membrane is predominantly -strand, after the transient existence of a mixed strand/helix conformation (figure 9a, b). the -strand shows clear cross peaks with lipid ch2 protons in the 2d h c correlation spectra (figure 9c), indicating that the peptide is embedded in the hydrophobic region of the hexagonal-phase cylinders (figure 10e). the 2d p h correlation spectrum of the fpk4-bound dope membrane shows a much weaker water p cross peak than the peptide-free membrane (figure 8c, d), indicating that the -strand fpk4 dehydrates the dope membrane in addition to causing curvature to this membrane. conformation and depth of fpk4 in the dope membrane. (a) 2d c c correlation spectrum of a fresh gvtaa-fpk4 sample at 243 k. the peptide exhibits both helix and strand signals. (b) c cp-mas spectra of the initial and equilibrated gvtaa-fpk4 at 246 k. at equilibrium, most residues exhibit -strand chemical shifts. (c) 100 ms 2d c h correlation spectrum at 293 k, in the hii phase membrane. lipid peptide cross peaks are observed, indicating that the -strand peptide is inserted into the hydrophobic region of the dope membrane. piv5 fusion peptide conformations in lipid membranes from solid-state nmr and outside the membrane from crystal structures. (a) fusion peptide is fully -helical in popc/popg bilayers but adopts a mixed strand/helix conformation in dopc/dopg bilayers. the peptide is inserted into both membranes, but the depicted tilt angle is hypothetical. the structures were built using (,) torsion angles predicted by talos+. (b) prefusion crystal structures of the piv5 f protein in the uncleaved (green) and cleaved (red) states. the fusion peptide domain has similar conformations before and after cleavage and has a bend near t117. (c) prefusion crystal structures of the influenza ha in the uncleaved (green) and cleaved (red) states. the n-terminal half of the fusion peptide is rotated around n12 before and after cleavage. seven residues (t122v128) of the fusion peptide are detected and show -helical structure extended from hra. (e) schematic of the piv5 fusion peptide conformation in the dope membrane. the lipid cylinders and water radius are drawn to scale using 1518 water molecules per lipid based on the dope phase diagram. (f) the hemifusion stalk intermediate showing both negative and positive membrane curvatures and dehydration between two opposing bilayers. the present solid-state nmr data indicate at least four distinct conformations and membrane topologies of the piv5 fusion peptide. in the popc/popg membrane, the peptide adopts a membrane-spanning -helical conformation (figure 10a). the popc/popg membrane has a hydrophobic thickness of 27 at 30 c based on x-ray scattering data. the full -helix has a length of 34 from g105 to l127 c based on the talos+ structural model. thus, the helix may be tilted by 3540 to achieve optimal hydrophobic match between the popc/popg bilayer thickness and the peptide length. in the dopc/dopg membrane, fpk4 adopts a mixed conformation with an n-terminal -strand (residues 105113) and a c-terminal -helix (residues 116125). no chemical shift constraints were measured for residues g114, a115, a126, and l127. the current structural model assumed a126l127 to be similarly helical as in the popc/popg-bound fpk4 and g114 and a115 to be random coil due to its position near a likely bend (see below). the overall dimension of the mixed helix/strand conformation is not known without long-range distance constraints. however, since the -strand is much more extended than the -helix, the peptide is likely to be significantly tilted to match the hydrophobic thickness of the dopc/dopg bilayer, which is similar to that of the popc/popg bilayer. in the dope membrane (figure 10e), the chemical shift constraints suggest a predominantly -strand peptide, which is inserted into the hydrophobic region between the lipid cylinders. finally, fpk4 adopts a surface-bound -strand structure in neutral popc and dmpc membranes, as we showed previously. the four conformations and topologies of fpk4 suggest several principles for the influence of the lipid membrane on the fusion peptide structure. first, while the entire sequence of the fusion peptide is capable of conformational polymorphism, the c terminus has a higher propensity for the -helical structure. second, anionic membranes promote the -helical conformation, as shown by the difference between the popc/popg membrane and the popc membrane and by the difference between dopc/dopg and dope membranes. third, more disordered membranes shift the peptide conformational equilibrium toward -strand, as shown by the difference between the popc/popg and dopc/dopg membranes. the third observation, while initially unexpected, can in fact be understood by the fact that lipid unsaturation not only changes membrane dynamics but also membrane curvature. cone-shaped lipids (with negative intrinsic curvature) such as oleic acids, cis-unsaturated lipids, and phosphatidylethanolamine promote stalk formation, whereas inverted-cone shaped lipids (with positive curvature) such as lysophosphocholine inhibit fusion by preventing stalk formation. thus, the more unsaturated dopc/dopg lipids change the membrane curvature in addition to membrane dynamics compared to the popc/popg lipids. the higher -strand content of the fusion peptide in the dopc/dopg membrane thus suggests that the -strand structure may be the active form in hemifusion intermediates. our chemical shift analysis indicates that the piv5 fusion peptide has a higher conformational disorder in the middle of the sequence, near g114t117. this region is not only the transition point between the strand and helix segments in the dopc/dopg-bound peptide but also has multiple conformations and residual dynamics in the popc/popg membrane. the prefusion crystal structures of several viral fusion proteins and the nmr structures of other fusion peptides suggest that conformational disorder in the middle of fusion peptide domains may be general. for example, in the uncleaved piv5 f protein, the c-terminal part (a118v128) of the buried fusion peptide shows an -helical structure extended from hra, whereas the n-terminal part (f103t117) has a mixed conformation of random coil (f103i108), -helix (g109l113), and -strand (v115a116) (figure 10b). after cleavage, the first four residues of the fp undergo an orientational change while the other residues are mostly unaffected. in the prefusion ha crystal structures, the fp is unstructured in both uncleaved and cleaved states, but the n-terminal segment undergoes a large-amplitude rotation with respect to the rest of the protein after cleavage (figure 10c). isolated ha fusion peptides bound to dpc micelles exhibit -helical conformations, but the middle of the peptide is disordered and forms the bend of the helical hairpin in the 23-residue construct and the bend of the boomerang structure in the 20-residue construct. the exact significance of this mid-domain disorder for membrane fusion is not clear, but we speculate that the disorder may be useful for controlling oligomerization, the degree of peptide insertion into the membrane, and membrane hydration. for example, if the -sheet conformation is indeed more effective in dehydrating the lipid membrane than the -helical conformation, as suggested by the current dope data and the previous popc and dmpc data, then a mixed strand/helix conformation may be useful for dehydrating one of the two surfaces of the lipid bilayer. mutagenesis data of the fusion peptide domain of the piv5 f protein indicated a competition between protein transport, surface expression, and membrane fusion, but the n-terminal residues f103v115 appear to be more important for membrane fusion than other functions. mutations of g105, g109, and g114 to ala reduced protein expression but increased membrane fusion. g109a and g114a mutants showed 25% lower expression levels but 10-fold higher membrane fusion than the wild-type protein. while fusion peptides are generally rich in gly and ala residues, in piv5 fp, all three gly residues are located in the n-terminal region while over half of the ala residues are located in the c-terminal region. in contrast, ha and hiv fusion peptides have a more uniform distribution of gly residues. the high gly content of the n-terminal half of the piv5 fusion peptide may be one of the reasons for the stronger -strand propensity of the n-terminal half. meanwhile, the helix propensity of the c-terminal segment may be related to the neighboring -helical hra domain, as seen in the postfusion crystal structure of piv5 f, which shows -helical t122v128 in the fp domain. since the n-terminal domain is more important for membrane fusion and has a stronger propensity for the -strand conformation, the -strand conformation may be more critical for membrane fusion. this is also consistent with the ability of the -strand conformation in causing membrane dehydration, as discussed below. static p nmr spectra indicate that the piv5 fusion peptide neither causes curvature nor dehydration to the popc/popg and dopc/dopg membranes but causes curvature to the dope membrane. the peptide increased the lhii transition temperature by about 10 k and generated a small amount of an isotropic phase. we attribute this signal to a cubic phase, which would suggest that the fp promotes negative gaussian curvature. increasing experimental evidence and simulations indicate that generation of negative gaussian curvature may be a common property of viral fusion peptides. recent small-angle x-ray diffraction data of the ha fusion peptide in methylated dope showed that the peptide shifted the lhii phase transition to higher temperatures and additionally promoted the formation of inverted bicontinuous cubic phases, lm3 m and pn3 m, which possess negative gaussian curvature. this result revises earlier literature that concluded that the ha fusion peptide promoted only negative curvature. these earlier studies were based on differential scanning calorimetry experiments, which may not be able to resolve the cubic phases from the l and hii phases, and on p nmr spectra that showed clear isotropic peaks but poor-sensitivity powder patterns that can not be definitively assigned to either the l phase or the hii phase. therefore, these earlier data can not be interpreted as stabilization of the hii phase by the ha fusion peptide, but they do indicate the generation of an isotropic phase, which is consistent with cubic-phase formation. molecular dynamics simulations of membranes containing the ha fusion peptide indicate that the peptide systematically shifted the lipid phase diagram toward more positive mean curvature and bicontinuous cubic phases. for the hiv gp41 fusion peptide, p nmr spectra and cryo-tem micrographs of dope-containing lipid membranes showed the presence of an isotropic phase. thus, all reliable evidence converges to indicate that influenza, hiv, and piv5 fusion peptides cause negative gaussian curvature to pe-rich membranes. on the basis of the intensity of the p isotropic peak, the piv5 fpk4 construct used here has weaker curvature-generating ability than the influenza and hiv fusion peptides. in addition to generating membrane curvature, fpk4 also partially dehydrated the dope membrane (figure 8c, d). for the hii-phase dope, this means a reduction of the water-core diameter of the cylinders. figure 10e depicts the hexagonal cylinders, where the relative dimensions of the water pore and the hydrophobic chains match the values reported from x-ray diffraction data of dope at 1518 water molecules per lipid, which is the hydration level of the fpk4-containing dope membrane. at this hydration, the water channel radius is 19, while the distance between the centers of two cylinders in adjacent layers (dhex) is 70, whose hydrophobic portion (32) is traversed by the -strand fpk4. this peptide location is consistent with x-ray scattering data that showed that the hiv fusion peptide increased the dhex value of dope at peptide concentrations above 2 mol %. the increased hexagonal spacing results from a compensatory effect of an increased hydrocarbon volume, which implicates the hiv fusion peptide to be embedded in the hydrocarbon region, and a decreased water volume, which agrees with the dehydration seen in the current 2d p h correlation spectra of the dope membrane. thus, the piv5 and hiv fusion peptides exert similar changes to the dope membrane. the cross section of the inverse-hexagonal phase dope, in which the opposed lipid chains of different cylinders experience negative curvature (figure 10e), is similar but not identical to the cross section of the hemifusion stalk intermediate (figure 10f), since the latter also contains lipids experiencing positive curvature. the hemifusion stalk is topologically more similar to inverted bicontinuous cubic phases, which are the likely cause of the isotropic peak in the p spectra. the lipids that experience positive curvature in the cubic phase should correspond to lipids in the distal leaflet of the opposing membranes. on the basis of the conformation, topology, and lipid and water interactions of piv5 fpk4 in the four lipid membranes obtained from solid-state nmr, we propose the following relations between the fp structure and viral membrane fusion. when the fp is released from the globular head of the f protein, it inserts into the target cell membrane in an -helical structure. as the protein rearranges its structure and hra forms a coiled-coil trimer in the prehairpin intermediate, the fusion peptides of the three proteins interact in the cell membrane to form a homotrimer. when several trimers cluster in regions of the membrane containing high concentrations of unsaturated lipids with their ensuing negative intrinsic curvature, the gly-rich n-terminal half of the fusion peptide converts to a -strand conformation, which dehydrates the membrane surface and exerts negative gaussian curvature to the membrane. at this point, depending on the local lipid composition, the fusion peptide may be partially -strand (in pc-rich membranes) or fully -strand (in pe-rich membranes), and the peptide is well inserted unless the local membrane composition is predominantly neutral pc. when the water-soluble ectodomain completes its conformational change to a six-helix bundle, the fp and tm domains are forced into close proximity, which may revert the fusion peptide to the -helical conformation, which may in turn reduce membrane curvature and increase membrane hydration. multiple lines of evidence obtained here suggest the -strand conformation of the fusion peptide to be the most relevant structure in hemifusion intermediates, responsible for remodeling the membrane to acquire the curvature and low hydration necessary for progression to complete fusion.

viral fusion proteins catalyze the merger of the virus envelope and the target cell membrane through multiple steps of protein conformational changes. the fusion peptide domain of these proteins is important for membrane fusion, but how it causes membrane curvature and dehydration is still poorly understood. we now use solid-state nmr spectroscopy to investigate the conformation, topology, and lipid and water interactions of the fusion peptide of the piv5 virus f protein in three lipid membranes, popc/popg, dopc/dopg, and dope. these membranes allow us to investigate the effects of lipid chain disorder, membrane surface charge, and intrinsic negative curvature on the fusion peptide structure. chemical shifts and spin diffusion data indicate that the piv5 fusion peptide is inserted into all three membranes but adopts distinct conformations: it is fully -helical in the popc/popg membrane, adopts a mixed strand/helix conformation in the dopc/dopg membrane, and is primarily a -strand in the dope membrane. 31p nmr spectra show that the peptide retains the lamellar structure and hydration of the two anionic membranes. however, it dehydrates the dope membrane, destabilizes its inverted hexagonal phase, and creates an isotropic phase that is most likely a cubic phase. the ability of the -strand conformation of the fusion peptide to generate negative gaussian curvature and to dehydrate the membrane may be important for the formation of hemifusion intermediates in the membrane fusion pathway.

PMC3985871

pubmed-767

continued improvements in therapeutic medicine have resulted in prolonged survival of patients with cancer, and consequently, physicians must manage the long-term complications of cancer and its treatment. bone metastases are a common complication in patients with a variety of solid tumors,1 and the skeleton is 1 of the 3 most frequent sites of metastasis (including lung, liver, and bone).2 all cancers have the potential to metastasize to bone; however, tumors of the breast, prostate, lung, kidney, and thyroid do so most frequently.1 these tumors show an intense osteotropism and represent approximately 80% of cases of bone metastases. a devastating complication for patients, the development of bone metastases signals that their disease has become incurable. furthermore, bone metastases can result in potentially debilitating skeletal-related events (sres) including pathologic fracture, the need for orthopedic surgery to treat or prevent a pathologic fracture, spinal cord compression, severe bone pain requiring radiotherapy, and potentially life-threatening hypercalcemia of malignancy. treatment of patients with bone metastases should involve a multidisciplinary team of experts, including oncologists, radiation oncologists, and orthopedic surgeons, with the primary goals of relieving pain, improving quality of life, preventing sres, and restoring functional independence, to minimize the impact on patients lives. management options for patients with bone metastases include pharmaceutical agents (eg, bisphosphonates [bps] and analgesics), radiotherapy, and surgery. these treatments are normally used in combination, depending on the severity of bone destruction and the life expectancy of the patient. analogues of pyrophosphate, bps have a high affinity for the mineralized surface of bone and were initially recognized for their ability to reduce bone resorption by inhibiting osteoclasts. however, preclinical studies have demonstrated direct and indirect anticancer activities for some bps, such as reducing cancer cell proliferation, inducing cancer cell apoptosis, antiangiogenic effects, and inhibiting cancer cell adhesion and invasion of the extracellular matrix.3 additionally, bps have been shown to reduce bone tumor area in multiple animal models.3 moreover, zoledronic acid (zol) has demonstrated anticancer benefits in some early breast cancer trials and in other settings, including metastatic disease.4 two different types of bps are currently utilized for treating patients with bone metastases from breast cancer those that contain nitrogen and those that do not. those without nitrogen are known as first-generation bps (eg, clodronate) and inhibit osteoclast- mediated bone resorption mostly via inhibition of mitochondrial atp. nitrogen-containing bps (eg, pamidronate, risedronate, ibandronate, and zol) prevent bone resorption by inhibiting farnesyl diphosphate synthase. on the basis of systematic review and meta-analyses of published data from clinical trials of bps,5,6 it is clear that bps reduce sre risks in patients with metastatic bone disease from breast cancer. furthermore, zol has been shown to significantly delay the onset of sres and reduce the ongoing risk of sres, supporting initiation of therapy as soon as bone metastases are diagnosed and continuing until performance status significantly declines.6,7 to date, the majority of evidence supports the use of the intravenous (iv) nitrogen-containing bp zol for preventing sres in patients with multiple myeloma or bone metastases secondary to any solid tumor.6,8 when administered intravenously, bps have been associated with a transient acute-phase reaction including fever, arthralgia, and bone pain (described as flu-like symptoms). these reactions generally occur with the first infusion only, are usually self-limiting, resolve within 1 to 2 days of administration, and can typically be managed with nonprescription analgesics.9 in the majority of patients, reactions are infrequent with subsequent infusions.9 the underlying cause of the characteristic acute-phase reaction with iv zol is believed to be through transient release of cytokines such as tumor necrosis factor alpha and interferon from immune cells and activation of the immune system (eg, v9v2 t cells) against cancer cells.10 we report here a case study documenting a dramatic difference between the safety profiles of a generic zol and the brand-name zol formulations. a 50-year-old hispanic woman presented in january 2007 with cancer in her right breast (stage iiia, t3, n2, m0). a tru-cut biopsy was performed, revealing poorly differentiated, infiltrating, her2/ neu- negative ductal carcinoma with vascular permeation and estrogen- and progesterone-receptor strong positive staining in 10% of cells. the patient received 4 cycles of neoadjuvant 5-fluorouracil, epirubicin, and cyclophosphamide (fec) chemotherapy with good clinical response, followed by right modified radical mastectomy in may 2007. postmastectomy histopathology revealed multicentric, poorly differentiated, infiltrating ductal carcinoma, with 5 of 10 nodes positive. she received adjuvant therapy with 4 cycles of docetaxel and radiotherapy, followed by sequential endocrine therapy with tamoxifen for approximately 6 months followed by letrozole, beginning in november 2007. in march 2009, after 22 months of adjuvant endocrine therapy and surveillance, the patient reported lower back pain. a bone metastasis was detected in the lumbar spine, for which she received external beam radiotherapy to the lumbar spine and began therapy with the brand-name zol (zometa; 4 mg every 34 weeks) plus second-line adjuvant letrozole. she received zometa for the first 3 cycles with good tolerance, and reported no acute adverse events. as of june 2009, our institution s policies dictated that she receive generic zol for continued monthly bp therapy. on infusion of generic zol, the patient experienced extreme weakness, nausea, vomiting (all grade 2, as defined by common terminology criteria for adverse events [ctcae]), and incapacitating bone pain (ctcae grade 3). as a result, the use of weak opioids such as tramadol (50 mg iv every 8 hours) in conjunction with paracetamol (500 mg every 8 hours) was necessary for pain control, and the patient had to be hospitalized for 2 to 3 days of evaluation and monitoring after each of the 2 generic zol infusions received. the severity of adverse events experienced by the patient was the same after both the first and second infusions of generic zol. because of the noticeable differences in toxicity profiles between generic zol and zometa, the patient (under her physician s care) decided to purchase zometa and assess tolerability. thereafter, the patient continued therapy with zometa without complications, and experienced a meaningful reduction in bone pain and improved mobility until eventually succumbing to her disease in february 2011. written informed consent was obtained from the patient s family for publication of this case report. infusion of iv bps, including zol, is known to be associated with a transient acute-phase reaction (flu-like symptoms) that is generally mild and manageable with nonprescription analgesics. here we have reported a case in which generic zol resulted in hospitalization of the patient because of nausea, vomiting, severe bone pain, and weakness. these debilitating symptoms resulted in increased use of medical resources including nursing care, laboratory tests, and pharmaceuticals including opioids, in addition to standard hospitalization fees. because the patient had already received zometa without experiencing these acute complications, it was very likely that the new generic zol was responsible for the differences in tolerability. this suggests that the safety profile of zometa was better than that of the generic zol used in this patient. in the case reported herein, we observed a clear increase in the severity of adverse events associated with the use of generic zol. the same phenomenon of increased toxicity with generic zol has been observed in numerous other patients at our institution; however, until now it was not possible to make direct comparisons between generic zol and zometa in the same patient. in this case study experience, the generally mild adverse events were amplified so much that the patient required hospitalization after generic zol infusion, which is of great concern. furthermore, although not observed in this case report, we have observed increases in the frequencies of more severe adverse events such as kidney damage and osteonecrosis of the jaw (onj) in patients receiving generic zol. interestingly, according to the package inserts, both the active substance (4 mg zol) and inactive ingredients (mannitol, sodium citrate, and sterile water for injection) were the same for the generic zol and zometa formulations. therefore, the precise reason for the new, acute adverse events experienced after the generic zol infusions remains unknown; however, we can not rule out that patient awareness of the change from zometa to generic zol may have at least contributed to the perceived severity of her symptoms. we believe strongly that before institutions can ethically require the substitution of generic zol for zometa, it will be important to closely monitor and re-evaluate both the efficacy and safety of generic zol formulations in patients with bone metastases. our institutional experience suggests that the generic zol used in our patient presents a potential danger to patient safety, perhaps resulting from insufficient manufacturing and/or testing processes. clearly, more information is needed to validate the safety and efficacy of generic zol formulations, and this will be of direct interest to oncologists, but will also have relevance to all healthcare professionals who are confronted by decisions regarding the increasing number of generic drug choices. those of us who work in healthcare are entrusted with our patients best interests, and should be highly concerned about the quality and regulation of generic pharmaceuticals such as the emerging formulations of zol. ideally, in the future it will be possible to have international regulatory bodies with the resources and power to monitor and regulate the day-to-day quality of generic medicines, particularly in emerging or developing countries. however, until that time, it is important that treating physicians monitor patients under their care to ensure their safety when new drug formulations are introduced into clinical practice. although these may be silent substitutions enacted by pharmacists, unexpected toxicities, such as those observed in our patient treated with generic zol, can alert us to emerging safety concerns. by reporting this case study, we hope to increase vigilance and allow more rapid identification and management of toxicities with substandard zol formulations and allow other patients to benefit from our experience.

intravenous zoledronic acid (zol) is an integral component for the management of patients with bone metastases, but can be associated with transient flu-like symptoms, which generally occur only with the first infusion and are typically manageable with nonprescription analgesics. a 50-year-old woman with a bone metastasis secondary to breast cancer received radiation therapy, brand-name zol (zometa), and letrozole. during the first 3 cycles of zometa (4 mg every 34 weeks), no acute adverse events were reported. for the next 2 cycles she was switched to generic zol and experienced severe toxicity (nausea, vomiting, extreme weakness, and incapacitating bone pain) that required hospitalization. toxicity differences between generic zol and zometa led the patient to pay additional costs for zometa, and subsequent zometa infusions were without incident. this is the first case report documenting a clinically significant difference between the safety profiles of a generic formulation of zol and brand-name zometa.

PMC3480876

pubmed-768

anterior cruciate ligament (acl) has a primary role in the limitation of anterior translation of the tibia as regards to the femur. they are also important for the compensation of stability in the acl in the knee lesions and patients with acl. the hamstring muscle weakness and hamstrings weak relationship with the quadriceps are risk factors for acl injuries. one of the most commonly used treatments is the method in which knee flexor tendons are used as autograft. as shown in previous studies, after using this method reconstructed knee recovered up to the 90% of flexion muscle strength of counter knee (harter et al., 1990; lipscomb et al., 1982; simonian et al., 1997). it is also shown in previous studies that semitendinosus which is also used as acl graft has regeneration potential from more proximal and with similar morphology (eriksson et al., 2001; ferretti et al. although there is a development of regeneration close to normal morphology; knee flexors abnormalities in neurological function can be a barrier to motor units function by preventing their healing. however, morphological factors such as muscle atrophy in these patients is not the only factor that determines the maximum power; in previous studies, it is shown that despite the absence of morphological abnormalities the afferent feedback deficiency from acl may interfere with the activation of gamma motor unit of the muscles around the knee (konishi et al. even if the knee flexor tendon regeneration is morphologically normal and that motor unit complex may prevent the function of the knee flexors by causing neurologic abnormalities. therefore, while comparing knee flexor muscle strength of patients who underwent acl reconstruction with normal uninjured knee, muscle weakness due to the possibility of neurologic abnormalities can not be ignored. after acl reconstruction, which muscle groups are more affected from frequently developing thigh muscle atrophy is a matter of debate. muscle strength tests are used to evaluate recovery of acl after treatment and the effectiveness of treatment. according to the study carried out, after acl reconstruction using hamstring tendon grafts, weakness in the knee flexor muscle strength can be observed up to 24 months. in addition, previous studies showed that in patients who underwent acl reconstruction quadriceps muscle torque per unit volume is significantly lower compared the intact knee (konishi et al., 2007b). in our study, we aimed to evaluate the effect of thigh circumference difference between patients knees who were administered the acl reconstruction with hamstring tendon autograft and intact knees, on torque between the hamstring and quadriceps muscles. as is known, development of muscle weakness in quadriceps after acl lesions caused by neurological dysfunction was shown to be a natural consequence. in our study, we planned to evaluate the effect of thigh circumference difference between operated extremity and intact extremity on knee flexorsmuscle strength. fifty-five patients (54 males, 1 female, mean age 28.156.47) who underwent acl reconstruction in our clinic and with at least 6 months follow-up period available were included in our study. while choosing the patients, not having any disease nor interventions previously on intact knees has taken into account. patients who have symptoms and signs such as discharge, inflammation, instability, locking, limitation of motion, anxiety while exercising were removed from the study. all operations were applied in teaching and research hospital by the same surgeon. the condition that autogenous hamstring tendon graft usage in acl reconstruction was searched for the patients. in all patients, same postoperative rehabilitation program which is indicated below for the first week walking with a full load, 090 of passive knee extension, bringing the active flexion, quadriceps and hamstring muscle training, heel shift, straight leg rise; between 12 weeks, hamstring training in the prone position, asideleg lift, walkingin the water if a swimming pool exist; between 23 weeks, terminal extension and hamstring stretch studies with weight; 34 weeks, if knee flexion has reached to 90 the pedals ergonometric work, walking back in the pool (if possible), doing full daily activities; 612 weeks, rising at finger tips and starting to closed kinetic chain exercises, preparations for the transition to the sport aiming to increase strength, durability and propsiosepsiyo; in the 3rd month, cycling, running and scissoring in water; in the 4th month, in addition to weight training activities beginning to proprioceptive and on the stairs activities, doing straight running; in 68 months beginning to sport-specific movements, ensuring to return to contact sports were taken into account. each patient was laid in the supine position, knees at full extension and relaxed position prior to measurement of the circumference of the thigh muscles. both thigh circumference were measured and recorded from 15 cm proximal to the upper limit of the patella for measuring. the determined length difference between there constructed knee and the intact knee power measurements of quadriceps and hamstring muscle groups in patients extremities who underwent operation and who did not in were done by using cybex ii dynamometer (humac). during the measurements the maximum torque (peak torque) values at 60/sec, 240/sec infrequency, the application of standard equipment, data collection and heating procedures were performed before the measurement. patients were told to continue their usual daily activities not to do tiring activities in the day before the test. on the test day before the beginning of measurements patients patients pelvis were stabilized with the help of a belt, thighs were supported with pillows, ankle cuff was placed directly on top of malleolar. patients forearms were positioned so that the rotational axis of the forearms were aligned with rotational axis of knees. during the test the range of motion was adjusted to be 0 extension and 90 flexion. in order to get support patients were allowed to hold seats on the sidebar during the test. before starting the recording of the data patients did three times sample repetition at both angle rates. concentric exercise were carried out for 5 times in maximal flexion and extension to patients after one minute rest. first, 60/sec speed after a minute rest the test was continued with 240/sec speed. several patients developed thigh pain during measurements and measurements were repeated after the test was terminated. number cruncher statistical system (ncss) 2007 and power analysis and sample size (pass) 2008 statistical software (utah, usa) were used for statistical analysis. besides descriptive statistical methods (mean, standard deviation, median, frequency, rate, minimum, maximum) for qualitative comparison of data pearson s chi-square test, fisher-freeman-halton test, fisher s exact test, and yates continuity correction test (yates adjusted chi-square) were used for the evaluation of data. age identifier values and follow-up periods of patients are shown in table 1. thigh circumference of intact extremity and operated extremity of patients are shown in table 2. peak torque of the mean extensor and flexor muscle strength and percentage values relative to each other, are shown in tables 3, 4, and 5. statistically significant relationship was observed at the level of 66.0% percent between the thigh diameter difference and cybex extension 60 of patients in negative direction (while the thigh diameter difference increasing the cybex extension 60 percent decreasing) (r: 0,660; p=0.001; p<0.01). statistically significant relationship was observed at the level of 55.0% percent between the thigh diameter difference and cybex extension 240 of patients in negative direction (while the thigh diameter difference increasing the cybex extension 240 percent decreasing) (r: 0,550; p=0.005; p<0.01) (fig. statistically significant relationship was observed at the level of 55.0% percent between the thigh diameter difference and cybex flexion 60 of patients in negative direction (while the thigh diameter difference increasing the cybex flexion 60 percent decreasing) (r: 0,555; p=0.002; p<0.01). it was observed a relation between the thigh diameter difference and cybex flexion 240 of patients in negative direction (while the thigh diameter difference increasing the cybex flexion 240 percent decreasing) at the level of 28.1% percent and this situation was found to be statistically significant (r: 0,281; p=0.079; p>0.05) (fig. after our study, in accordance with our findings it is still possible to encounter the thigh atrophy in average 28 months after acl reconstruction surgery even under physical rehabilitation programs and appropriate follow-up. in line with earlier studies, quadriceps muscle mass is often seen as responsible for the present thigh atrophy, however negative effects of the hamstring muscle group on muscle atrophy which is determined with manual thigh circumference measurements are undeniable. as shown in previous studies, in patients with acl lesions the development of neurological dysfunction is not available in the knee flexors comparing with quadriceps muscle group. comparative studies which was done in patients with acl lesion and in patients without any knee problems showed that there was no significant difference between the speed of flexion and torque per unit volume of the knee flexor muscle. despite this, in the same study it was found that isokinetic torque that occurred in the knee with acl lesions at 60/sec was less than robust knee (konishi et al., 2012). in addition, it is shown that the acl reconstruction with hamstring tendon is not effective on muscle torque power of knee flexors and flexion speed of these muscles by showing that detection of changes in the robust knee is the same. in the light of these data, it might lead to think that acl lesions does not cause neurological dysfunction in the knee flexors. in another study there was a comparison between patients who were administered to the acl reconstruction and patients who did not have any knee problem, it has been identified that muscle torque power on per unit volume of the quadriceps was significantly lower in the patients who were administered to reconstruction. in addition, quadriceps weakness is associated with the neurologic dysfunction that is developed after acl reconstruction has been shown by other researchers (hart et al., 2010; rice et al., 2009; snyder-mackler et al. it has been shown that muscle weakness in the quadriceps that is induced by the acl lesions is a natural result linked to the development of neurological dysfunction. in our study we have showed the loss of torque power of thigh atrophy on quadriceps at the 60/sec, 240/sec frequency even in the patients who have an appropriate rehabilitation program after acl reconstruction and under going follow-up process. although, it has been shown in previous studies that acl lesion development in the thigh did not lead to neurological dysfunction on hamstring; in our measurements, it has been determined statistically that atrophy developed in the thigh is effective on the hamstrings muscle torque power at least as it is on quadriceps, especially 60/sec frequency. it could not be shown that this weakness is correlated to thigh atrophy as well as quadriceps at 240/sec flexion frequency. these difficulties are likely to be related to neurological dysfunction occurred in the quadriceps muscles. due to different muscle weakness mechanisms which are developing according to the morphological structure and muscle properties, it is inevitable for the clinician to consider these changes in diagnosis and rehabilitation stages. the point that we want to emphasize in our study is; it ca nt be ignored that muscle weakness mechanisms developing in the thigh circumference vary according to the thigh muscle group and knee flexors play an important role in thigh atrophy when determining an appropriate rehabilitation program after anterior cruciate ligament reconstruction application.

after anterior cruciate ligament (acl) reconstruction, which muscle groups are more affected from frequently developing thigh muscle atrophy is a matter of debate. we evaluate the effect of thigh circumference difference between patients knees who were administered the acl reconstruction with hamstring tendon autograft and intact knees, on torque between the hamstring and quadriceps muscles. fifty-five patients at least 6 months follow-up period available were included in our study. power measurements of quadriceps and hamstring muscle groups in patients extremities were done by using isokinetic dynamometer. the maximum torque values at 60/sec, 240/sec in frequency, positions of flexion and extension were determined. in accordance with our findings it is still possible to encounter the thigh atrophy in average 28 months after acl reconstruction surgery even under physical rehabilitation programs and appropriate follow-up. it is inevitable for the clinician to consider these changes in diagnosis and rehabilitation stages. it ca nt be ignored that muscle weakness mechanisms developing in the thigh circumference vary according to the thigh muscle group and knee flexors play an important role in thigh atrophy when determining an appropriate rehabilitation program after reconstruction application.

PMC4415756

pubmed-769

every year, it is estimated that tens of thousands of pregnant women in malaria-endemic areas are infected with plasmodium falciparum. frequently, placental infection occurs, owing to the accumulation of p. falciparum-infected erythrocytes in the intervillous space, despite the absence of parasites in peripheral blood. the complications of malaria during pregnancy are maternal anaemia, preterm delivery, and low birth weight of newborns, which increase perinatal morbidity [1, 3, 4]. the world health organization (who) recommends intermittent preventive treatment with sulfadoxine-pyrimethamine (iptsp) during pregnancy, with at least two doses after quickening (1820 weeks) not more frequently than monthly, use of insecticide-treated bed nets (itns) and prompt treatment of clinical malaria. intermittent preventive treatment consists of delivering a curative treatment dose of an antimalarial at predefined intervals, regardless of the parasitological status of the woman, and the efficacy of this protocol has been demonstrated in a number of malaria-endemic countries [58]. placental plasmodium screening in the central african republic (car) in 1990 showed a rate of 37.1% in women who had been given chemoprophylaxis with chloroquine. in 2006, the ministry of health of the car adopted and implemented the new who recommendations for malaria prevention during pregnancy. the aim of the study reported here was to estimate the prevalence of malaria in thick peripheral blood smears and placental blood from a sample of women who gave birth during september 2009 at two main maternities of bangui, the capital of car. secondly, we assessed the women's coverage rates with the three components of the who package for malaria management during the current pregnancy and identify pregnant women characteristics associated with iptsp and itns. we conducted a cross-sectional study in the two main maternity clinics of bangui, the castors health centre and the communautaire hospital, in september 2009. the climate is tropical, and rainfall peaks are observed from april to november, and temperature ranges from 19c to 32c. the main parasite is plasmodium falciparum, and malaria transmission is perennial with peaks during the rainy season, but no data on the intensity of malaria transmission (entomological inoculation rates) is available in car. malaria represents more than 40% of morbidity in bangui, as well as in other car areas. the castors health centre and the communautaire hospital provide antenatal and delivery services and have established programmes for the prevention of malaria and other infectious diseases, such as mother-to-child transmission of hiv infection. each year, it is estimated that 12,000 women deliver at those centres, representing 70% of all women who deliver in bangui. all women are screened for malaria by microscopic analysis of 4% giemsa-stained thick blood smears during visits to the antenatal clinics, performed either at the same health centre or at one of the national reference biomedical laboratories (the national laboratory for clinical biology and public health and the institut pasteur de bangui), depending on each woman's choice. antimalarial treatment is prescribed on the basis of clinical symptoms if the woman can not afford the laboratory fees immediately. for asymptomatic women, iptsp is given free of charge as directly observed therapy during the antenatal visit. administration of two doses is recommended for hiv-negative women and three doses for hiv-positive women. all women from whom we obtained written informed consent were eligible for the study immediately after delivery. a study midwife administered a standardized questionnaire to record sociodemographic data (age, residence area, literacy, number of gravidities, and monthly income), hiv serological status, intake of antimalarial medications (iptsp and other antimalarial prescriptions), and bed net use. malaria prevalence of 30% at the time of childbirth was used as a proxy to calculate the sample size in this study. thus, a number of 323 women was necessary assuming 90% power at 5% significance level. because of lack of an ethical committee in the car, this project was reviewed and approved by an ad hoc scientific committee of the university of bangui in charge of validating scientific study protocols in the car. the study was conducted under a collaborative agreement with the university of marseille, france. peripheral venous and placental blood from each woman was used to prepare thick blood films. placental blood was obtained as follows: immediately after delivery, the paracentric side of the maternal placenta was cleaned with sterile water and incised, and thick blood films were prepared from a droplet collected by aspiration through a 21-gauge needle attached to a 5-ml syringe, as described previously [12, 13]. the thick smears were air-dried and stained with 4% giemsa. at each of the two study sites, an experienced microscopist immediately examined the stained smears by light microscopy (100 oil immersion) to detect asexual forms of p. falciparum malaria parasites. on peripheral blood slides, malaria parasites were counted in 200 leukocytes, and the parasite density per microlitre of blood was estimated as the number of parasites counted multiplied by 40 under the assumption of a leukocyte count of 8000/l of blood. for both types of blood film, a result was considered to be negative if no parasites were detected per 200 leukocytes. women with a positive peripheral blood result were given antimalarial treatment (either artemether-lumefantrine or quinine, depending on a clinical evaluation and individual tolerance). all the slides were analysed twice at the biomedical laboratory of the institut pasteur de bangui. data were double-entered into epiinfo software version 3.5.1, and the database was checked and data entry errors corrected with the epiinfo software the association between sociodemographic criteria and malaria, use of iptsp and itns was examined using the chi-squared test, and association between those variables was tested by calculating the odds ratios (ors). overall, 328 pregnant women delivering at the two health centres were included in the study: 168 (51.2%) at the castors and 160 (48.8%) at the communautaire hospital. the mean age was 23 years (range, 1439 years), and 33.8% were aged less than twenty years. most of those women do not have any personal monthly income (57.9%), but the majority of them have at least secondary educational status. hiv infection had been screened for 58.2% (191/328) of the population, resulting an infection prevalence of 9.1% (15/164; 27 hiv results could not be cross-checked on the antenatal clinic cards). sleeping daily under bed net was reported by 81.5% (95% ci, [77.385.7 ]) of the women, and 42.4% (95% ci, [36.848.0 ]) had itns. at the two study sites, checking of antenatal clinic cards showed that 93.3% (95% ci, [90.895.8 ]) of the women had presented at least one antenatal visit. less than one fourth of the women (24.1%; 95% ci, [19.328.9 ]) had attended an antenatal clinic during the first trimester of pregnancy, while the majority (55.6%; 95% ci, [50.061.2 ]) had attended a clinic during the second trimester. overall, 35.6% (95% ci, [31.140.1 ]) completed four antenatal visits. the distribution of first antenatal attendance according to gestational age at each study site is shown in figure 1. of the women who received iptsp, 75.4% (95% ci, [69.181.8 ]) were given curative prescriptions of other antimalarial drugs, independently of the timing of iptsp doses (figure 2). the antenatal clinic cards of 182 women (55.5%; 95% ci, [50.160.1 ]) showed a history of at least one curative treatment for malaria. of these women, 27.0% had been prescribed an antimalarial drug two or three times. although 228 antimalarial prescriptions were recorded on antenatal clinic cards during the current pregnancy, only 56 laboratory results were positive out of the total 73 blood smears analysed. the antimalarial drugs prescribed were quinine (66.7% or 152/228; 95% ci, [60.672.8]), artemisinin-based combinations (15.5%; 95% ci, [10.720.1 ]) and artemisinin monotherapy (18.0%; 95% ci, [13.023.0]). at least one dose of iptsp was recorded for 54.6% (95% ci, [49.260.0 ]) of our study population; only 30.5% (95% ci, [23.837.2 ]) had received at least two doses. most of these women (78.2%; 95% ci, [72.284.3 ]) had received the first iptsp dose between the fourth and seventh months of pregnancy; however, 11.7% (95% ci, [7.016.4 ]) had received the first dose during the first trimester. multigravid women, were less likely to use two doses of iptsp (or=0.14; 95% ci, [0.080.24], p<0.0001) and itns (or=0.16; 95% ci, [0.100.28], p<0.001) compared to primigravid women. use of iptsp (two doses) was associated with lucrative activities (or=4.20; [2.556.92], p<0.0001) and secondary or university educational status (or=2.22; 95% ci, [1.333.72], p=0.002). women with secondary or university educational status were also likely to use ints (or=1.90; 95% ci, [1.203.01], p=0.01). details on association analysis between sociodemographic characteristics and those preventive tools use are shown in table 1. overall, peripheral blood p. falciparum infection at delivery was found in 2.8% (95% ci, [1.04.6 ]) of peripheral blood and in 4.0% (95% ci, [2.06.0 ]) of placental blood. of the women with placental malaria, 77.0% (10/13) declared not using any bed net and 53.8% (7/13) had not taken any antimalarial drug during pregnancy. iptsp and itns use was found not to be associated with the women sociodemographical characteristics. moreover, there is no statistically significant association between these laboratory findings and those characteristics. the prevalence of malaria among pregnant women in the car (2.8% of thick peripheral blood smears and 4.0% of placental slides) was lower than in other areas of intense malaria transmission, such as gabon, where the rates were 34.4% in maternal blood and 53.6% in placental blood films. a recent review of randomized clinical trials and surveys on the efficacy of ipt showed overall placenta-positive rates 10%. falade and coauthors in nigeria reported that the prevalence of placental parasitaemia was 10.5% in women given iptsp and 17% in those with no chemoprophylaxis. three years after initiation of iptsp in car, coverage with at least one dose of iptsp was slightly more than 50%. although who recommends two doses iptsp for 80% of pregnant women, our estimate in this study was 30.5%. the low prevalence of placental malaria in our study is therefore probably due to the combination of iptsp, other antimalarial drug, and use of itns. hence, the relatively low prevalence of malaria at delivery is not surprising in bangui. indeed, a similar finding of malaria prevalence at the time of delivery is observed in cte d'ivoire, where vanga-bosson and coauthors report a prevalence of 4.8% of placental malaria, in an area where iptsp coverage rate (2 doses) does not exceed 50% and, in thailand, where proportion of positive results for p. falciparum in maternal blood and in placental blood were estimated at 3.0% and 3.8%, respectively. in thailand, the authors report that this relative lower prevalence was due to antimalarial treatment with artemisinin derivatives. in our study, otherwise, our findings show that more than 80% of the women slept under a bed net. indeed, use of itns was found to reduce the incidence of uncomplicated malarial episodes in areas of stable malaria by 50% in comparison with no nets use and by 39% in comparison with untreated nets use. in our study, the lower proportion of use of iptsp and itns in multigravid women could be due to less health conscious of those women of their pregnancy. inversely, the relative high proportion of use of iptsp and itns in women with high level of education is due to the fact that higher educational status implies more health consciousness and is a factor influencing assimilation of health education programmes. women with salary or other personal income are also likely to be compliant with iptsp and itns, possibly because they are able to afford health care fees. the main limitation of our study is the only use of microscopic examination of blood smears. even if, the microscopic analysis remains the standard detection of plasmodium, submicroscopic infections are common during pregnancy [21, 22]. hence, molecular methods (polymerase chain reaction or pcr) and rapid diagnostic tests (rdts) provide finding approximately twice as many infections as microscopy [2325]. however, determination of the possible impact of these submicroscopic infections to poor birth outcomes and maternal health is critical, and pcr is not feasible routinely. to this end, microscopic examination to detect these infections is still essential, and implementation of rdts use is challenging [21, 28]. our results indicate that, although the recommended coverage rates of pregnant women with iptsp and itns are not reached in bangui, the prevalence of the main indicator of infection, placental malaria, is relatively low. the widespread presumptive prescription and consumption of antimalarial agents could indisputably be the cause of clearance of the existing peripheral and placental plasmodium infection and decreased the risk of new infections over the pregnancy period. indeed, symptoms suggestive of malaria are very frequent among pregnant women attending antenatal clinics, thus implying frequently unnecessary large use of antimalarial drugs. for this purpose, strengthening national malaria control activities, taking into account prompt laboratory diagnosis and sociodemographic particularities, should contribute to the achievement of high coverage rate with the who preventive package components. otherwise, cohort studies are needed to assess the real efficacy of iptsp in preventing malaria during pregnancy. blood samples analysis were achieved by a. manirakiza, m. moyen, d. djalle, n. madji and r. laganier. data analysis and interpretation were achieved by a. manirakiza, j.delmont, e. serdouma, g. soula, and alain le faou.

introduction. the aim of this study was to estimate the prevalence of malaria among women giving birth in bangui. association between sociodemographic characteristics of those women and malaria, as well as prevention compliance (use of intermittent preventive treatment with sulfadoxine-pyrimethamine (iptsp) and insecticide-treated bed nets (itns)), was analyzed. methods. during september 2009, a survey was conducted on 328 women who gave birth at two main maternities of bangui. information was obtained by standardized questionnaire about sociodemographic criteria, iptsp, other antimalarial treatment, and use of bet nets. smears prepared from peripheral and placental blood were analysed for malaria parasites. findings and discussion. positive results were found in 2.8% of thick peripheral blood smears and in 4.0% of placental slides. a proportion of 30.5% of the women had received at least two doses of iptsp during the current pregnancy. only a proportion of 42.4% of this study population had itns. multigravid women were less likely to use iptsp and itns. however, use of iptsp was associated with personal income and secondary or university educational status. hence, although this relatively prevalence was observed, more efforts are needed to implement iptsp and itns, taking into account sociodemographic criteria.

PMC3253579

pubmed-770

at prince of wales hospital, hong kong (7), from january 2007 through august 2008, influenza infection was diagnosed for>460 hospitalized adult patients for whom acute febrile respiratory illnesses had been diagnosed. nasopharyngeal aspiration and immunofluorescence assays (ifa) were used for rapid diagnosis of influenza a and b infection, confirmed by virus isolation. thirteen (2.8%) patients had signs of confusion or altered consciousness, together with fever and respiratory symptoms (mean sd age 77.7 8.8 years). we studied 3 patients from whom csf was obtained for analysis, and who fulfilled the definition of influenza-associated acute encephalopathy (altered mental status>24 hours within 5 days of influenza onset and without alternative explanation) (1,2,46). nasopharyngeal aspirates were subjected to ifa, virus isolation, and subsequent subtyping (7). csf specimens were subjected to virus isolation using mdck cells, and reverse transcription pcr to detect influenza virus rna by using h1/h3 subtype-specific primers. herpes simplex virus, herpes zoster virus, and enterovirus dna/rna was detected using pcrs (technical appendix). csf and plasma samples collected on the same day were analyzed simultaneously for the concentrations of 11 cytokines/chemokines by bead-based multiplex flow cytometry. their assay methods and plasma reference ranges (established from>100 healthy persons) have been described (technical appendix) (7). in csf, in patients without central nervous system (cns) disease/infection, cytokines/chemokines are either undetectable (e.g., interleukin-6 [il-6], cxcl8/il-8, cxcl10/ip-10, cxcl9/mig) or present at low levels (e.g., ccl2/mcp-1) (810). concentrations of oseltamivir phosphate (op) and its biologically active metabolite oseltamivir carboxylate (oc) were measured in csf and plasma taken simultaneously from 1 patient who received concurrent treatment, using tandem mass spectrometry (11). the clinical and virologic findings are summarized in table 1. all case-patients were elderly (7286 years of age), but none were known to have neuropsychiatric illness, dementia, or to be taking psychotropic medication. none had received updated influenza vaccination (6). confusion and altered consciousness developed in patients 1 and 2 one to 2 days after the onset of fever and cough. these patients had no meningismus, focal neurologic deficit, hypotension, respiratory distress, or metabolic disturbances. oseltamivir was given to patient 2 only when influenza a was later confirmed by nasopharnygeal aspirate/ifa; patient 1 did not receive antiviral treatment. patient 3 had fever, severe chronic obstructive pulmonary disease exacerbation requiring noninvasive ventilatory support, complicated by acute coronary syndrome. he was given oseltamivir, 75 mg 2/day, after influenza a infection was confirmed. agitation and confusion developed in the patient on day 34 of illness (onset after the third dose of oseltamivir), despite resolution of the patient s respiratory failure. these symptoms were followed by involuntary, tremulous movements involving all 4 limbs, while at rest and during movement.*copd, chronic obstructive pulmonary disease; ct, computed tomographic scan; npa, nasopharyngeal aspirate; csf, cerebrospinal fluid; rt-pcr, reverse transcription pcr; hsv, herpes simples virus; hzv, herpes zoster virus. in all cases, there was no hypoglycemia, and liver and renal function test results were normal. an electroencephalogram was performed and showed generalized slowing of background consistent with moderate encephalopathic change (similar to that observed in septic encephalopathy) (1,6). findings are consistent with previous reports on adult cases of influenza-associated encephalopathy: patients are all unvaccinated, pleocytosis and cerebral imaging abnormalities (even with magnetic resonance imaging) are usually absent, and symptoms are generally self-limiting (1,6). most reports have mentioned influenza a as a cause of encephalopathy, and more commonly subtype h3n2 (16). despite apparently normal csf findings, high concentrations of cytokines/chemokines were detected in the csf and plasma specimens of all patients (table 2). plasma concentrations of il-6, cxcl8/il-8, cxcl10/ip-10, ccl2/mcp-1, and cxcl9/mig were elevated at median values of 2.0, 2.8, 11.9, 3.7, and 2.1 the upper limits of their respective reference ranges (comparable to or higher than that observed in other hospitalized influenza patients) (table 2) (7). il-6, cxcl8/il-8, cxcl10/ip-10, and ccl2/mcp-1 were consistently detected, and were elevated at median values of 2.6, 15.0, 3.4, and 20.0 the upper limits of their respective plasma reference ranges. the csf/plasma concentration ratios of cxcl8/il-8 and ccl2/mcp-1 were>3 (median csf/plasma ratio 5.4 and 8.0, respectively).*csf, cerebrospinal fluid ;, test not done due to inadequate sample; ud, undetectable (i.e., below the detection limit of the cytokine/chemokine assay). cytokines: interleukin (il)1, il-6, il-10, il-12p70, tumor necrosis factor (tnf-). chemokines: cxcl8/il-8, monokine induced by interferon- (ifn-) (cxcl9/mig), ifn-inducible protein-10 (cxcl10/ip-10), monocyte chemoattractant protein1 (ccl2/mcp-1), and regulated upon activation normal t cell expressed and secreted (ccl5/rantes). the assay sensitivities of il-1, il-6, il-10, il-12p70, tnf-, il8, mig, ip-10, mcp-1, rantes, and ifn- are 2.5, 3.3, 3.7, 1.9, 7.2, 0.2, 2.5, 2.8, 2.7, 1.0, and 7.1 pg/ml, respectively. 39 adult influenza patients hospitalized with cardio-respiratory complications (8), the median (interquartile range) plasma concentrations of il-6, il-8, ip-10, mcp-1, and mig were 10.6 (4.218.4), 5.4 (2.58.7), 7,043.0 (4,025.11,2381.1), 76.5 (49.5-97.0), and 992.1 (499.11,992.3) pg/ml, respectively. in csf, in subjects without neurologic disease/infection, these cytokines/chemokines are either undetectable or present at low levels (911). in a pediatrics influenza cohort, csf cytokine levels were substantially higher in encephalopathy cases when compared to those with febrile seizure; csf/plasma concentration was<1 (9). csf/plasma cytokine concentration ratio consistently>3 (3.512.1), in addition to csf cytokine concentrations being above the plasma reference ranges. for ifn-, il-12p70, tnf-, il-10, il-1 and rantes, because of their low/undetectable levels, the csf/plasma ratios were not calculated. csf specimens from patients 1 and 2 were collected at the peak of symptoms, and before antiviral treatment (if given); csf from patient 3 was collected when persistent tremor developed 18 hours after the ninth dose of oseltamivir; the drug was stopped afterward. simultaneous csf and plasma oc and op concentrations were determined for patient 3, as symptoms progressed at 18 h after oseltamivir. the concentrations (mean sd) of oc in duplicate csf and plasma samples were 18.3 0.9 ng/ml and 143.8 3.3 ng/ml, respectively; the csf/plasma concentration ratio was 12%13%. the op plasma concentration was 1.05 0.03 ng/ml; it was not detectable in the csf. we report 3 adults with acute encephalopathy (altered consciousness, confusion) associated with influenza. high csf and blood cytokine/chemokine (cxcl8/il-8, ccl2/mcp-1, il-6, cxcl10/ip-10) levels were detected. influenza virus is rarely detected in the csf, and pleocytosis is often absent (1,2,46). high levels of cytokines (e.g., il-6, soluble tumor necrosis factor receptor 1) can be consistently found in csf/blood specimens, correlating with disease severity and outcomes (hyperactivated cytokine response is absent in febrile seizure associated with influenza) (24,8). we found a broader range of cytokines/chemokines being activated (7); for certain cytokines (cxcl8/il-8, ccl2/mcp-1), the csf concentrations were 3 those in plasma. il-6, cxcl8/il-8, ccl2/mcp-1 and cxcl10/ip-10 have been shown to play pathogenic roles in cns viral infections, cerebral injury, and acute brain syndrome in susceptible patients (7,9,12). the high csf/plasma ratios suggest that for some cytokines, activation within the cns might have occurred along with respiratory-tract and systemic productions (cytokines are not detected in csf normally; (table 2) (4,710,12). resident macrophages/monocytes, astrocytes, microglial and endothelial cells in the cns are shown to release cytokines/chemokines when stimulated by viral/influenza infection; activation mechanisms without involving overt cns invasion have been suggested (1,4,9,1214). cytokines may cause direct neurotoxic effects, cerebral metabolism changes, or breakdown of the blood-brain-barrier (endothelial injury) to produce symptoms (14,8,1214). whether early viral suppression by antivirals can lead to attenuation of these cytokine responses and better outcomes warrants further study (7). we measured oseltamivir concentrations because of the concerns over its neuropsychiatric side-effects in children and adolescents. however, only the active metabolite (oc) was detected in the csf of patient 3; the csf/plasma concentration ratio was 12%13% (18.3/143.8 ng/ml) at 18-hours postdose. this degree of csf penetration is similar to that observed among healthy patients, with a cmax csf/plasma concentration ratio of 3.5% (at 8 hours), and a ratio of 10% at 18 hours (concentration-time profiles for plasma/csf differ). assuming a similar ratio, the csf op concentration would have fallen below the assay s detection limit (0.25 ng/ml) by 18 hours (11,15). the low csf drug-penetration, together with high cytokines in csf and symptom progression despite drug withdrawal suggest that the manifestations of patient 3 may have been disease-related. further investigations on the cns effects of oseltamivir in the clinical setting are needed .. our study is limited by the small patient number and the lack of feasibility in obtaining csf for study/comparison in influenza patients without neurologic symptoms. further studies on the clinical spectrum of influenza encephalopathy and encephalitis in adults (1,6) and their pathogenesis are indicated. in conclusion,

we report acute encephalopathy associated with influenza a infection in 3 adults. we detected high cerebrospinal fluid (csf) and plasma concentrations of cxcl8/il-8 and ccl2/mcp-1 (csf/plasma ratios>3), and interleukin-6, cxcl10/ip-10, but no evidence of viral neuroinvasion. patients recovered without sequelae. hyperactivated cytokine response may play a role in pathogenesis.

PMC2874350

pubmed-771

a maximum mouth opening that is smaller than the size of a complete denture can make prosthetic treatment challenging. this article describes a simple technique used to fabricate maxillary and mandibular custom sectional impression trays for making definitive impressions in patients with microstomia. microstomia is defined as an abnormally small oral orifice.1 this disorder is described as a reduction in the oral aperture size associated with facial burns, diffuse scleroderma, traumatic injuries, and surgical reconstruction involving the orbicularis oris muscle. microstomia can result in multiple debilitating sequelae such as inability to masticate, droolin g, speech problems due to poor articulation, impaired delivery of oral hygiene and dental care, and psychological problems secondary to facial disfigurement.28 several methods of prosthodontic treatment for microstomia patients have been presented, and numerous devices to expand the oral commissure have been described.3,912 the prosthetic rehabilitation of microstomia patients presents difficulties at all stages, from preliminary impressions to prosthesis fabrication.13 because such patients have small oral openings, using conventional methods for making definitive dental impressions and fabricating dentures may be extremely difficult. making the accurate impressions represents the initial difficulty in the prosthetic rehabilitation of such patients. the recommended techniques for obtaining preliminary impressions for microstomia patients include the use of modeling plastic impression compound, the use of stock impression trays with heavy and light body silicone impression materials, and flexible impression trays with silicone putty. the casts obtained from these preliminary impressions are then used for making custom sectional impression trays. these trays typically consist of 2 locking devices or assemblies, 1 situated anteriorly and the other posteriorly, which join and provide stability to both the sections of the trays. after the definitive impressions are made, these trays are reassembled extraorally and are poured in dental stone to obtain definitive, working casts. several studies have described various techniques that are used for making custom sectional impression trays.1418 different devices used for connecting the custom sectional trays include hinges,14 plastic building blocks (lego; lego systems inc, enfield, conn., usa),15,16 orthodontic expansion screws,17 or locking levers.18 this article describes a simple, cost-effective, and time-saving method for fabricating custom sectional impression trays using easily available dual die-pins and sleeves as potential devices for interlocking the sectional trays. the locking mechanism design includes an anterior locking assembly for the maxillary and mandibular custom trays and a posterior locking assembly for only the maxillary custom tray. by using the conventional method, fabricate the maxillary and mandibular custom sectional impression trays by using autopolymerizing acrylic resin (dpi-rr, dental products of india, mumbai, india) on the preliminary casts. make the handles of the trays (minimum dimensions with 13 mm height, 10 mm length, and 10 mm width) such that they incorporate the metal sleeves of the dual die-pins (m.r. section both the custom impression trays at the midline by using a diamond disk (dfs, germany). steps in the fabrication of the anterior lock assembly in the maxillary and mandibular custom sectional impression trays:-the assembly basically consists of 2 dual die-pins and 2 sleeves. closely juxtapose the 2 sleeves such that the smaller keyway of 1 sleeve faces the larger keyway of the other sleeve. join these sleeves by inserting the 2 dual die-pins and making the assembly a rigid joint (figure 1a d).-make a slot on the inside portion of the handles on each half of the sectioned custom trays to incorporate the sleeve (figure 2a&b).-attach the sleeves in the slots by using the autopolymerizing acrylic resin as mentioned previously (figure 3).-verify the position of the attached sleeves by inserting die-pins such that the halves juxtapose precisely in both the sectioned trays.-fabricate an anterior assembly for the mandibular sectional tray in a similar manner (figure 7). steps in the fabrication of the posterior lock assembly in the maxillary custom sectional tray:-cut the 2 dual die-pins that are attached to sleeves halfway through their heights (figure 4) by using a carborundum disk (dentorium, new york, usa). only the broader upper halves are used for fabricating the posterior assembly and the lower halves are discarded.-attach the half-cut sleeves on the posterior parts of the sectioned custom tray halves using acrylic resin such that the greatest dimension of each sleeve is oriented in the anteroposterior direction. check for parallelism between the sleeves using a dental surveyor and verify the fit of the die-pins in the respective sleeves (figure 5).-fabricate an acrylic resin block (6 mm height, 10 mm width, and 4 mm longer than the distance between the 2 attached sleeves) using the autopolymerizing acrylic resin (figure 4).-transfer the respective points of the half-cut die-pin heads on the acrylic bar by using a pressure spot indicator (coltene psi, switzerland) and drill slightly oversized holes in the acrylic bar on the marked points by using a bur (261-ef023, brasseler, usa)-with the closely juxtaposed sectioned trays and the die-pins of the anterior assembly placed in position, secure the heads of the half-cut die-pins into the holes by using the autopolymerizing acrylic resin (figure 5&figure 6). the 2-piece custom-made tray described above allows for a functional impression to be made despite the difficulties associated with microstomia. the anterior locking assembly described in this article does not require any special alteration in the conventional custom tray design. the die-pins with metal sleeves used in this technique provide a greater degree of stability and a precise union of the 2 sections of the tray. the joint can be made more rigid by activating the 2 prongs of each dual die-pin away from each other. this technique used for fabricating custom sectional impression trays does not require any special devices or complex locking joints. the only additional materials used are the dual die-pins which are commercially available at a minimum cost. it is often difficult to use conventional methods for fabricating dentures for patients with limited mouth opening. this article described a simple, time-saving, and cost-effective method used to fabricate custom sectional impression trays for making definitive impressions in patients with microstomia.

objective: a maximum mouth opening that is smaller than the size of a complete denture can make prosthetic treatment challenging. this article describes a simple technique used to fabricate maxillary and mandibular custom sectional impression trays for making definitive impressions in patients with microstomia.

PMC3420830

pubmed-772

heterogenous vancomycin intermediate staphylococcus aureus (hvisa) strains have been reported as indicators for reduced vancomycin susceptibility in s. aureus, and various studies associated its presence with vancomycin treatment failure. it has been shown that methicillin resistant s. aureus (mrsa) has the propensity to evolve into hvisa phenotype during in vitro exposure to subinhibitory concentrations of vancomycin. during the last decade, hvisas had been isolated in many countries including those in south east asia; nevertheless, in our knowledge, its emergence has not been reported in malaysia. as a pilot study, we investigated the prevalence of hvisa among mrsa strains isolated at hospital kuala lumpur (hkl) in a 3-month period and determined factors associated with its infections. hospital kuala lumpur is the largest hospital in malaysia with the highest mrsa burden in the country. in this hospital, vancomycin is used as the standard first line treatment for mrsa infection; however, recently, its efficacy has been a subject of discussion due to several anecdotal vancomycin treatment failure cases in hospital kuala lumpur. we also wondered if some of the mrsas isolated in the hospital were actually hvisas with reduced susceptibilities to vancomycin that could not be detected by routine microbiological tests used in our hospital diagnostic laboratory. to investigate this, from 25 february to 25 may 2009, we collected a total of 320 index mrsa isolates (first mrsa isolated from the corresponding patients) and established them as strains for vancomycin resistance testing. as it is cost, time and labor consuming to perform vancomycin population analysis on all 320 strains to test for heterogenous vancomycin resistance, strains were first screened for the phenotype using glycopeptide resistance detection (grd) etest antibiotic strips (ab biodisk, sweden). after grd screening, a total of 8 strains were defined as presumptive hvisa, no visa strain was detected. following that, to confirm the results of the grd screening interestingly, area under the curve (auc) analyses of the strains ' population analysis profiles confirmed that 7 out of the 8 tested strains were hvisa (table 1), giving a prevalence rate of 2.19%. table 1heterogenous vancomycin intermediate staphylococcus aureus strains and their corresponding patients in this study.specimen noagegenderprimary diagnosisspecimendiabetes mellitusrenal failuremalignancyadmission to icudays of hospital staydays of iv vcmon beta-lactamarea under curve ratio68214maleright hip osteomyelitisnasal swabnononoyes44 days0yes0.9058253femaleleft diabetic foot ulcerpus swabyesyesnoyes52 days14yes0.9318258maleacute ventriculitiscsfyesnonoyes98 days14yes1.0125220malegluteal sarcoma with hapsputumnonoyesyes24 days10yes0.9097871malepemphigus folliaceouspus swabyesnonono34 days7yes1.012154femaleacute encephalitis with haptracheal aspiratenononoyes32 days14yes0.9646029femalemeningo-encephalitis with hapsputumnononoyes38 days10yes0.98icu, intensive care unit; iv, intravenous; vcm, vancomycin; csf, cerebrospinal fluid; hap, hospital acquired pneumonia. icu, intensive care unit; iv, intravenous; vcm, vancomycin; csf, cerebrospinal fluid; hap, hospital acquired pneumonia. all hvisa strains isolated in this study were hospital acquired as they were isolated from their corresponding patients after 48 hours of hospital admission. to determine factors associated with the 7 hvisa infections, demographic data of all corresponding patients of each index mrsa isolate were retrieved from medical records. medical history of each patient such as diabetes mellitus, renal failure, malignancy, together with prescription history of vancomycin and beta-lactam antibiotics (as these were the only classes of antibiotics prescribed to the corresponding patients of the study isolates during this investigation), length of hospitalization and intensive care unit (icu) admission were recorded. continuous variables were then assessed by independent samples t-test, while categorical variables were analyzed using pearson's chisquare. calculations were performed using statistical package for social science (spss) 12.0 (spss inc., chicago, usa) where a p-value of<0.05 was considered as significant. after performing multivariate linear regression, we found that icu admission (p<0.004), hospitalization of more than 14 days (p<0.014) and vancomycin administration of more than 7 days (p<0.016) were independent factors associated with hvisa infections in our group of patients. our findings were in line with those of charles et al. in 2004, where hvisa/visa infections were found to be associated with longer antibiotic treatment periods and longer hospitalization. in a separate report it seems that patients who are severely ill, hospitalized for long durations with icu admissions might have a higher chance of developing hvisa infections. as many patients in hkl fulfill some or all of the above criteria, taking it together, we suspect that the prevalence of hvisa in hkl might be high; however, these strains are not being actively detected by the hospital diagnostics laboratory. as hvisa and mrsa with reduced vancomycin susceptibility has been reported to cause treatment failure, given the hvisa prevalence rate detected in this study, it is not surprising that vancomycin treatment failure cases among mrsa infected patients are increasing in hkl. in our study, we employed the grd test as a screening tool for hvisa before confirming the resistance with population analysis, and found that the grd etest was fairly specific with only one false positive result. in a review, howden and colleagues have reported the test's sensitivity as 9394% with a 8295% specificity for hvisa detection. therefore, the grd might be considered a good screening tool for hvisa in hospitals where most hospitalized patients are severely ill with long hospitalization durations. once identified as hvisa infected, optimal treatment could be prescribed to the corresponding patient to prevent vancomycin treatment failure, thereby increasing the chance of a good clinical outcome for the patient. as the strains used in this study were collected in a short span of 3 months, and that vancomycin treatment failure is on the rise in hkl, we suspect that the actual prevalence of hvisa in this hospital might be even higher. we found the grd test useful for hvisa screening, nevertheless pap-auc analysis still remains the gold standard for hvisa confirmation. a more comprehensive, case control study involving major hospitals in the country would be important to better understand the significance and distribution of hvisa in malaysian hospitals.

in a 3-month study done in hospital kuala lumpur (hkl), 7 out of 320 methicillin resistant staphylococcus aureus isolates were confirmed as heterogeneous vancomycin intermediate s. aureus (hvisa) using the glycopeptide resistance detection e-test and population analysis, giving a prevalence rate of 2.19%. this is the first report of hvisa in malaysia.

PMC3892648

pubmed-773

it exhibits broad bioactivity including inducing apoptosis of scar cells and anti-inflammatory and anticancer [3, 4] properties, which have been used in the traditional chinese medicine for treatment of hypertrophic scar, inflammation, cancer, and other diseases without obvious toxicity or side effect in clinic. several preparions, hydrogel, lotion, injection, and liposome, for example, have been reported in the literature. we are further interested in the form of liquid crystalline nanoparticles (cubosome), for cubosome consists of a curved bicontinuous lipid bilayer extending in three dimensions and separating two congruent networks of water channels [911], which can enclose hydrophilic, amphiphilic, and hydrophobic substances ranging from low-molecular-weight drugs to proteins, peptides, amino acids, and nucleic acids. compared to liposomes, cubosomes showed better storing stability at room temperature and could endure heat treatment [1315]. cubosomes could exist at almost any dilution level in water and drug leakage was less concerned compared with liposome. reported that cubosomes had a higher permeability coefficient (4.5-fold) compared to eye drops when dexamethasone was used as a model drug. therefore, we consider that the cubosome might represent a promising vehicle containing matrine for effective ocular drug delivery. hplc, lc/ms/ms, and esi-qtof-ms/ms methods have been used to determine matrine in samples at present [6, 17, 18]. the aim of this study is to establish and validate a simple, sensitive, and accurate hplc method to determine matrine combined in liquid crystalline nanoparticles. glycerol monooleate (dimodan mo/d kosher, material number 116703) was kindly provided by danisco cultor (brabrand, denmark) and used as received. poloxamer 407 (peo98pop67peo98) was a gift from basf (ludwigshafen, germany). milli-q-grade water purified through a millipore system (elga labwater, sartorius, uk) was used throughout this study. pbs (ph 6.8) was made according to the chinese pharmacopoeia (2010). liquid crystal nanoparticles were prepared through the fragmentation of glycerol monooleate/poloxamer 407 bulk cubic gels. glycerol monooleate (3 g) and poloxamer 407 (300 mg) were first melted at 60c in a hot water bath until they were homogeneous, after which matrine was added to dissolve/blend under continuous stirring. water (6.7 ml) was then added gradually and the mixture was vortex-mixed to achieve a homogeneous state. after equilibration for 48 hours at room temperature, the cubic phase gel was formed. by adding 20 ml of water, the cubic gel was disrupted by mechanical stirring. subsequently, the crude dispersion was fragmented for 10 min by intermittent probe sonication (jy-96 iin, ningbo scientz biotechology co., ltd, china) at 200 w energy input using a pulse mode (9-second pulses interrupted by 18-second breaks) under cooling in a 20c water bath. the resulting milky coarse dispersion was homogenized using a high-pressure homogenizer (avestin em-c3, ottawa, canada) at certain high pressures and cycles to obtain an opalescent dispersion of the cubic nanoparticles. the final dispersion of liquid crystal nanoparticles was stored at room temperature for further studies. the hplc analysis was carried out using a shimadzu system that is equipped with an lc-20at pump, spd-20a uv/vis detector connected to shimadzu spin chrome software. the chromatographic assay was performed on a reversed-phase ods-bp c18 column (5 m, 4.6 mm 250 mm) at ambient temperature 25c. the mobile phase under isocratic mode was a mixture of methanol-pbs (ph 6.8)-triethylamine (50: 50: 0.1%, v/v). the mobile phase was degassed by an ultrasonic bath and filtered with 0.45 m membrane under vacuum. all the calculations concerning the quantitative analysis were carried out by an external standard method based on peak areas. to prepare the stock solution, matrine (10 mg) was accurately weighed into 50 ml volumetric flask, made up to volume with methanol, and then the volume was adjusted to 50 ml. this solution was further diluted with methanol to yield solutions containing 100.0, 50.0, 25.0, 12.5, 6.3, 3.1, and 1.6 g/ml. the chromatogram peak area of each known concentration was calculated. results from each analysis were subjected to regression analysis. 0.2 ml (or 200.0 mg) of the nanoparticles was accurately transferred into a 10 ml volumetric flask, dissolved, and made up to volume with methanol. then, the sample solutions were filtered using a 0.45 m filter membrane and injected (10 l) into the hplc system three times under optimized chromatographic conditions. the method was validated in terms of parameters of specificity, linearity, sensitivity, accuracy, precision, and reproducibility according to the international conference on harmonisation. the specificity of the method was assessed by comparing chromatograms of matrine working solution, blank excipients sample without matrine, and equal concentrations samples of compound liquid crystalline nanoparticles made as the previous procedure. the linearity of the method was studied by injecting seven known concentrations of the standard in the range of 1.6200 g/ml. the sensitivity of the method was evaluated with limit of detection (lod) and limit of quantification (loq). lod and loq were established at a signal-to-noise ratio (s/n) of 3 and 10, respectively. the accuracy of the method was tested by comparing the percent analyte recovered by the optimum method at three concentration levels (80.0, 100, and 120.0 g/ml). intraday precision was determined by injection of standard solutions of matrine at 3 concentration levels (50, 25, and 12.5 g/ml), on the same day. the specificity was evaluated by analyzing blank excipients sample, matrine standard solution, and liquid crystalline nanoparticles samples. the retention times of matrine at a flow rate of 1.0 ml/min was 16.3 min. the calibration curves for matrine were found to be linear within the range of 1.6 to 200.0 g/ml. the regression equation was y=10706x 2959 (r=1.0), where y is peak area and x is the concentration (g/ml) of matrine standard solution. the correlation coefficient indicated a good linear relationship between peak area and concentration over a wide range. the lod (signal/noise ratio of 3: 1) was calculated as 1.3 10 g/ml and the loq (signal/noise ratio of 10: 1) was determined as 3.9 10 g/ml. mean recovery for matrine at three concentration levels (80.0, 100, and 120.0 g/ml) was found to be 102.1 1.9% (rsd=1.96%, n=3), 102.6 1.9% (rsd=2.94%, n=3), 100.5 2.1% (rsd=2.12%, n=3), respectively. the mean concentration was 9.5 mg/ml (rsd=1.4%, n=3). several mobile phase systems including methanol-water, ethanol-wate-kh2po4, and acetonitrile-ethanol-h3po4 systems have been tested in this study. however, the chromatogram of standard matrine might disappear or appear in a wide range with inaccurate calculation area (see figure 2). matrine crystalline has 4 forms, namely,, ,, and matrine. the incorrect mobile phase may change the matrine solution to nanocrystalline because of the solubility. the chromatographic method was eventually carried out using an isocratic system with a mobile phase of methanol-pbs (ph 6.8)-triethylamine (50: 50: 0.1%) applied at a flow rate of 1 ml/min with detection wavelength at 220 nm. under these optimum mobile phase conditions, elution of analyte was completed in less than 20.0 min and retention time of matrine was 16.3 min. the method was validated according to ich guidelines with the parameters of specificity, linearity, sensitivity, accuracy, precision, and reproducibility. a simple, rapid, selective, and sensitive hplc method has been developed and validated for the determination of matrine when formulated in cubosome particles. the present study is the first report on the matrine determination combined with particle dispersion system. the method can be used for controlling the quality of the cubosome and helpful for further investigation.

a reversed-phase high-performance liquid chromatographic method has been developed to quantitatively determine matrine in liquid crystal nanoparticles. the chromatographic method is carried out using an isocratic system. the mobile phase was composed of methanol-pbs(ph6.8)-triethylamine (50: 50: 0.1%) with a flow rate of 1 ml/min with spd-20a uv/vis detector and the detection wavelength was at 220 nm. the linearity of matrine is in the range of 1.6 to 200.0 g/ml. the regression equation is y=10706x 2959 (r2=1.0). the average recovery is 101.7%; rsd=2.22% (n=9). this method provides a simple and accurate strategy to determine matrine in liquid crystalline nanoparticle.

PMC4009329

pubmed-774

however, outcomes of anatomic arthroplasty with an osteoarthritic rotator-cuff-deficient shoulder have been limited. the reverse shoulder arthroplasty (rsa) is a potential solution for shoulder osteoarthritis with deficient rotator cuff. theoretical advantages of rsa are an increased lever arm of the deltoid muscle through a medialized center of rotation of the prosthesis (increasing deltoid efficiency), increased prosthetic stabilization through humeral lengthening (increasing deltoid tension), and decreased mechanical torque at the glenoid component (decreasing glenoid loosening) these factors are related to the indication for surgery, surgeon's experience, characteristics of the implant, characteristics of the surgical technique, type of approach, or postoperative rehabilitation, among others. unfortunately, the analysis of outcomes of rsa depending on the type of prosthesis, type of approach, and indication for surgery has not been well reported to date. the purpose of this study was to compare the clinical and functional outcomes of rsa depending on the surgical approach, type of prosthesis, and indication for surgery through a comprehensive, systematic review of the literature. the methodology for this study was reported following the prisma statement for systematic review and meta-analysis. all human studies reporting clinical and/or functional outcomes in patients treated with primary or revision rsa were assessed for eligibility. studies were included if they had a level of evidence between i and iv, were written in english, had a minimum of 2 years of follow-up and had a minimum sample size of 10 patients. studies reporting complications only, nonoriginal articles, or studies with insufficient outcome data the authors are not aware of any relevant publication related to rsa before 1985, so the search was limited to this period. the keywords and search strategy employed in this study included the following: (reverse or inverse) and shoulder and (arthroplasty or replacement or prosthesis), limited to human studies published in the above-mentioned period. thus, cinahl, ebsco-sportdiscus, and the cochrane central register of controlled trials were also used to search for relevant publications in the same period. articles of potential interest were reviewed in detail (full text) by two authors and a decision was made regarding inclusion or exclusion. clinical and functional outcomes were extracted from all included studies in a systematic way using a table template by one author, which was then verified by another author. in cases of disagreement between both authors with regard to study inclusion or data extraction, a reference list of all included articles was reviewed to search for potential studies not previously identified. mean, standard deviation (sd), and range were extracted (whenever provided) in the preoperative and postoperative periods for the following variables: constant score, american shoulder and elbow society (ases) score, simple shoulder test, range of motion (rom), and satisfaction. furthermore, relevant information regarding level of evidence, type of prosthesis (either with medialized or lateralized center of rotation), type of approach (either deltopectoral or superolateral), indication for rsa, sample size, percentage of females in the sample, follow-up, and age of patients was extracted from all studies. the pubmed search yielded 329 citations, from which 174 were clinical studies in humans that were reviewed in further detail. about 32 met inclusion criteria and additional database searches and review of the reference list from included articles yielded a final number of 35 articles included in the descriptive analysis of clinical outcomes [figure 1]. from all 174 articles assessed for eligibility, the senior author had to review four of them because of disagreement between the two authors conducting the systematic review. literature search flow chart the 35 included studies were grouped depending on the approach and type of prosthesis: deltopectoral approach associated with prosthesis with a medialized center of rotation (dm group; n=18 studies), lateralized (dl group; n=8 studies), and a combination of approaches associated with a medialized prosthesis (cm group; n=9 studies). the latter group was created because the authors employed different approaches, but clinical outcomes were not specified depending on the type of approach (all these studies employed a prosthesis with a medialized center of rotation). prostheses with a medialized center of rotation included in this study were the following: delta iii (depuy, france), delta xtend (depuy, warsaw, in, usa), aequalis (tornier, france), smr modular shoulder system (systema multiplana randelli, lima-lto, san daniele de friuli, italy), and exactech (gainesville, fl, usa). prostheses with a more lateralized center of rotation included in this study were the following: reverse shoulder prosthesis (djo surgical, austin, texas), and arrow anatomical shoulder system (mulhouse, france). the 35 studies included a total sample of 2049 patients with a mean (sd) percentage of females of 71.6% (13.4), age of 71.5 years (3.7), and follow-up of 43.1 months (18.8); the respective data separately in groups was, dm, 1085 patients, 73.4% (10.2), 72.4 years (3.1), and 38.1 months (8.2). dl, 241 patients, 69.7% (12.5), 70.1 years (1.7), and 40 months (7.6) and cm group, 723 patients, 73.1% (7.5), 73 years (2.8), and 50.6 months (33.9). clinical outcomes depending on the type of approach-type of prosthesis in the analysis of clinical outcomes depending on the indication for rsa, not all 35 studies could be included because results were not always specified by indication. the number of studies included (total subjects involved) by indications for rsa were cuff tear arthropathy 12 (581); revision of anatomic prosthesis 10 (263); failed rotator cuff repair 5 (150); fracture sequelae 4 (82); rheumatoid arthritis 3 (52); massive cuff tear 2 (68); primary osteoarthritis with degenerative cuff tear 2 (51); posttraumatic osteoarthritis 2 (59); and revision of reverse prosthesis 1 (14). indications of rsa for tumors and acute fractures were not included due to limited data. mean (sd) for percentage of females, age, and follow-up depending on indications was the following: cuff tear arthropathy 74% (12), 72.5 years (3.4), and 34.6 months (8), respectively; revision of anatomic prosthesis 66.5% (11.7), 68.2 years (2.7), and 38.2 months (6.6); failed rotator cuff repair 69% (19.8), 69.8 years (3.6), and 39.6 months (11.8); fracture sequelae 70% (8.1), 73.2 years (5.3), and 37.6 months (8.2); and rheumatoid arthritis 87.6% (9.8), 68.2 years (2.9), and 56 months (22.2), respectively. for massive cuff tear, primary osteoarthritis with degenerative rotator cuff, and posttraumatic osteoarthritis only the mean (range) follow-up was provided: 34 months (range 24-118), 38 months (range 24-81), and 42 months (range 24-97), respectively. for revision of reverse prosthesis, percentage of females, and mean (sd) of age and follow-up were 28%, 70.6 years (8.7), and 33 months (11.2), respectively. table 2 summarizes the clinical outcomes depending on the most common indication for rsa in the included studies. the purpose of this study was to compare the clinical and functional outcomes of rsa depending on the type of prosthesis (with either medialized or lateralized center of rotation), type of approach, and indication for surgery. the principal finding of this study was that both types of prostheses clearly improved the outcomes, but lateralized prostheses had more pre-to-postoperative differences (improvement) for ases total and pain scores and external rotation compared with medialized prostheses. in addition, outcomes depending on each indication considerably improved, but those corresponding to revision of anatomic prosthesis, failed rotator cuff repair, and fracture sequelae demonstrated lower improvements compared to cuff tear arthropathy. the postoperative patient's satisfaction with surgery was very high (overall mean of 90%) in both types of prostheses and for all indications for surgery. to the best of our knowledge, this is the first systematic review aimed to investigate the clinical and functional outcomes of rsa depending on the type of prosthesis, type of approach, and an indication for surgery. khan et al. conducted a comprehensive, systematic review aimed to investigate the outcomes of rsa depending for cuff tear arthropathy, massive cuff tear, and rheumatoid arthritis. however, the authors only included delta ii prostheses and the review included studies up to 2010. the present investigation found many references in the last 2 years and in addition, different type of prostheses, more indications, and a higher number of studies were analyzed. based on the present study and on the existing literature, rsa is an excellent surgical solution with great improvements in clinical outcomes for cuff tear arthropathy, massive cuff tear, 42 failed rotator cuff repair, rheumatoid arthritis, fracture sequelae, revision of anatomic prosthesis, and revision of reverse prosthesis. in addition, both types of prostheses demonstrated excellent improvements in the postoperative period with regard to all outcomes. the fact that prostheses with lateralized center of rotation had greater improvement in ases and external rotation have to be interpreted with caution, as this study had some limitations. first and foremost, a pooled analysis of the results (meta-analysis with inferential statistics) was not possible for methodological reasons, as nearly all studies did not report the sd in the outcomes and an accurate meta-analysis could not be, therefore, conducted. in addition, most of the studies did not disclose the outcomes depending on the indications for surgery, so this parameter had to be considered separately to avoid a significant decrease in the number of studies included in the comparisons of outcomes. thus, only the type of prosthesis and type of approach could be analyzed altogether. second, as almost all included studies were case series, the comparison of outcomes depending on the type of prosthesis, approach, and an indication was indirect in nature with a greater potential influence of uncontrolled variables. third, the influence of several factors potentially influencing the outcomes could not be assessed because of limited information, heterogeneity of studies, and small number of studies included for some comparisons (which would decrease even more the available data if more subgroups were done). finally, it must be mentioned that the ases score in medialized prosthesis was only reported by one study, which may decrease the value of the comparison of this parameter between medialized and lateralized prostheses. it must be first recognized that there are a considerable number of potential factors not controlled in this analysis that may have a potential influence on the outcomes: different surgeon's experience, different rehabilitation protocols (given the multicentric nature of this study), type (eccentric or concentric) and size of glenosphere, location and orientation of the glenosphere and humeral components (inferiorly placed glenosphere, anteversion/retroversion, of the humeral component) degree of fatty infiltration of the teres minor muscle, degree of bone stock, soft tissue tensioning, status of the subscapularis muscle, humeral osteotomy angle, or previous surgery. in addition, no attempt was made to analyze data based on differences in humeral components, medialized versus lateralized, high neck angle versus low neck angle, sit-on-top versus sit inside, and cemented versus uncemented. in the present study, the influence of the type of approach on the outcomes of rsa could not be well determined. some studies used a combination of approaches, and the outcomes were not specified depending on whether the approach was deltopectoral or superolateral. therefore, some studies were grouped as cm to refer to studies using a combination of approaches (and a medialized prosthesis). in some ways, differences between groups dm and cm may be explained by differences in the type of approach, as a type of prosthesis in both groups has a medialized center of rotation. however, considering that the cm has a combination of approaches rather than a unique superolateral approach, no clear conclusions can be drawn regarding its influence on the outcomes of rsa. there are some studies that have found that the surgical approach does not have an influence on the outcomes of rsa. clearly, further research is needed in this aspect to better elucidate the influence of the type of approach on the outcomes of rsa. well-designed level i- or ii-evidence comparative studies are needed before clear conclusions can be established. nonetheless, the clinical relevance of this research question (influence of the type of approach) may be questioned, as some surgeons may be forced to adopt a certain approach based on the surgical history of the patient or the characteristics of the patient's disorder itself. the type of prosthesis (with a medialized or lateralized center of rotation) seems to have a much more relevant influence on the outcomes of rsa. given that many studies only employed the deltopectoral approach, the type of prosthesis was more easily isolated. thus, the comparison between dm and dl may show the influence of the center of rotation on the outcomes. essentially, both groups demonstrated great improvements in outcomes in the postoperative period. unfortunately, no studies employing a lateralized center of rotation reported the constant score, so no comparisons were possible between dm and dl for this parameter. in addition, the ases score was only reported by one study in the dm group so no accurate conclusions can be drawn for this parameter. specifically, the dl group demonstrated greater improvements in external rotation compared to the dm group. the reasons for lower improvement in external rotation in medialized prostheses have been suggested by boileau et al., and grammont and baulot. a medialized center of rotation may imply that the humeral cup impinges the posterior neck of the scapula when the arm is at the side. in addition, as the posterior deltoid theoretically provides some external rotation when coupled with some abduction, the medialization of the center of rotation may decrease the efficacy of the posterior deltoid to assist in the external rotation. also, the status of the teres minor may influence the degree of external rotation, but this variable was not controlled in the vast majority of studies. conducted an interesting study in which the center of rotation of a medialized prosthesis was lateralized by placing a bone autograft from the humeral head between the base plate and the scapula. this bony lateralization of the center of rotation demonstrated good integration and the authors found 53 of external rotation and a constant score of 66. unfortunately, this was a case series and comparisons with medialized prostheses were only conducted based on the existing literature. the disadvantage of metallic, as opposed to bony, lateralization may be the higher torque or shear force applied to the glenoid component, which may lead to a higher rate of glenoid loosening and screw breakage witnessed. therefore, bony lateralization was suggested to provide a benefit to external rotation without the potentially disastrous consequences of metallic lateralization. in a similar way, valenti et al. reported the outcomes of a lateralized prosthesis and concluded that less medialization of rsa improves external rotation, thus facilitating activities of daily living of older patients. however, the authors did not compare the outcomes with a sample of patients undergoing rsa with a medialized prosthesis, so their conclusion was again based on a comparison with the existing literature. no level i- or ii-evidence studies aimed to compare the outcomes of rsa depending on the type of prosthesis were found in the literature. most common indications for rsa were cuff tear arthropathy, revision of anatomic prosthesis, failed rotator cuff repair and fracture sequelae. this study shows that the indication for surgery may have an impact on the outcomes of rsa, which is in accordance with the existing literature. some authors found that patients with no previous surgery undergoing rsa had higher postoperative scores in ases (total, pain, and function) and constant (total and pain) compared to patients with previous surgery. however, other authors observed no significant differences in the improvement or postoperative values of constant score, ases, simple shoulder test, visual analogue scale for pain and function, oxford shoulder score, university of california in los angeles (ucla) shoulder scale, and rom between patients with and without previous surgery. for specific indications, the present study demonstrated that cuff tear arthropathy had higher improvements in constant score (total, pain, and activity), ases score (total, pain, and function), simple shoulder test, forward flexion, and abduction compared to revision of anatomic prosthesis. although there were no inferential statistics in the present study, these results are both in agreement and disagreement with previous studies. found that patients with cuff tear arthropathy had significantly higher improvements in constant score compared with patients undergoing revision of the prosthesis. the authors found a higher improvement (no p value provided) in ases score and external rotation in cuff tear arthropathy compared with revision of prosthesis, but no differences (no p value provided) in forward flexion. regarding the constant score, wall et al. found that cuff tear arthropathy and primary osteoarthritis with degenerative rotator cuff had a higher postoperative constant score (no p value provided) compared to revision of anatomic prosthesis, massive cuff tear, and posttraumatic osteoarthritis in fact, revision of anatomic prosthesis and posttraumatic osteoarthritis had significantly worse postoperative constant score compared to the other indications. found that patients with cuff tear arthropathy and posttraumatic osteoarthritis had higher improvements (no p value provided) in external rotation compared to revision of prosthesis, massive cuff tear, and primary osteoarthritis with degenerative rotator cuff. for forward flexion, cuff tear arthropathy and revision of anatomic prosthesis had the highest improvement (no p value provided) compared with massive cuff tear, posttraumatic osteoarthritis, and primary osteoarthritis with degenerative rotator cuff. reported a case series in which patients underwent hemiarthroplasty, anatomic total shoulder arthroplasty, or rsa and outcomes were analyzed depending on the indication for surgery. the authors found that the primary osteoarthritis with degenerative rotator cuff and cuff tear arthropathy had a significantly higher improvement of the constant score compared to rheumatoid arthritis and avascular necrosis. unfortunately, the number of rsa in the groups of primary osteoarthritis with degenerative rotator cuff, rheumatoid arthritis, and avascular necrosis was 2, 6, and 0, respectively. therefore, the significant differences are likely explained by anatomic prostheses instead of rsa. similarly, walch et al. found that cuff tear arthropathy, primary osteoarthritis with degenerative rotator cuff, and massive cuff tear had significantly higher improvement of the constant score compared with the revision of the prosthesis and posttraumatic osteoarthritis. there is a clear need for future studies specifically comparing the use of medialized and lateralized rsa, as the present comparison was indirect in nature given that no comparative studies of this parameter have been published to date. further clarification is needed to know to which extent there are significant differences in functional outcomes as well as in external rotation between both models. it is probable that differences on the implanted humeral side may have profound outcome implications, yet there has been no focus on this side of the joint in rsa outcome studies. in addition, the exact impact of the type of approach on the outcomes needs to be better delineated. in any further study utilizing more than one approach and type of prosthesis, furthermore, as the results of rsa may differ depending on the indications for surgery, disclosure of outcomes for indications is also warranted. finally, there are two methodological recommendations regarding the presentation of studies to facilitate further meta-analyses. first, it is important from a methodological and statistical point of view to report the sd in all parameters collected. most studies employing prostheses with medialized center of rotation used the constant score, whereas studies utilizing lateralized prostheses used the ases score. other investigations employed the simple shoulder test, the oxford shoulder score, or the ucla shoulder scale. only rom is systematically provided in the published studies, but more homogeneity is required to facilitate further meta-analyses. both types of prostheses (with medialized and lateralized center of rotation) clearly improved all the reported outcomes, but lateralized prostheses had more improvement in external rotation compared to medialized prostheses. all outcomes of rsa implanted for all types of indications significantly improved in the postoperative period, but those corresponding to revision of anatomic prosthesis, failed rotator cuff repair, and fracture sequelae demonstrated lower improvements compared with cuff tear arthropaty. the rsa is a surgical procedure with high patient satisfaction regardless of the type of prosthesis or the indication for surgery. there is no conflict of interest or financial aid from any organization regarding the material discussed in the manuscript. thomas w. wright, m.d. is a consultant for exactech, inc., gainesville, florida, and receives royalties and institutional research support on products cited to this article.

many factors influence the outcomes of reverse shoulder arthroplasty (rsa). the purpose of this study was to compare the clinical and functional outcomes of rsa depending on the surgical approach, type of prosthesis, and indication for surgery through a comprehensive, systematic review.a literature search was conducted (1985 to june 2012) using pubmed, cinahl, ebsco sportdiscus, and cochrane central register of controlled trials. levels i iv evidence, in-vivo human studies (written in english with minimum of 2 years of follow-up and sample size of 10 patients) reporting clinical and/or functional outcomes after rsa were included. the outcomes were analyzed depending on the surgical approach, type of prosthesis (with medialized or lateralized center of rotation), or indication for surgery.a total of 35 studies were included involving 2049 patients (mean [sd] percentage of females, age, and follow-up of 71.6% [13.4], 71.5 years [3.7], and 43.1 months [18.8], respectively). studies using deltopectoral approach with lateralized prostheses demonstrated greater improvement in external rotation compared with medialized prostheses with the same approach (mean 22.9 and 5, respectively). in general, rsa for cuff tear arthropathy demonstrated higher improvements in constant and american shoulder and elbow society scores, and range of motion compared with revision of anatomic prosthesis, failed rotator cuff repair, and fracture sequelae.lateralized prostheses provided more improvement in external rotation compared to medialized prostheses. indications of rsa for cuff tear arthropathy demonstrated higher improvements in the outcomes compared with other indications. rsa demonstrated high patient's satisfaction regardless of the type of prosthesis or indication for surgery.level of evidence: level iv.

PMC4325387

pubmed-775

non-small cell lung cancer (nsclc) can exhibit rearranged driver oncogenes, which are possible targets for therapy.1 the most frequently identified driver oncogenes in nsclc leading to targeted therapy are epidermal growth factor receptor (egfr) and anaplastic lymphoma kinase (alk).1,2 the prevalence of these driver oncogenes is significantly influenced by race, smoking habits, and gender.3 the frequency of egfr-mutated nsclc in asian patients is known to be higher than in caucasian patients.3 in contrast, prevalence of alk rearrangements in nsclc is reported to be similar in caucasian and asian patients.3 in recent cohort studies with afro-americans, there is no significant difference in the prevalence of egfr mutations or alk rearrangements with caucasian americans.4,5 about other races, however, less or no data are available.6,7 for obvious reasons, little is known about africa. cancer, however, is a significant health problem in this continent, as it is estimated that there were 715,000 new cancer cases in 2008, and additional data from southern africa and northern africa already confirm that lung cancer is the leading cause of death related to cancer.8 to our knowledge, there is no literature available about the prevalence of lung cancer and rearranged driver oncogenes in sub-saharan africans. as the latter is influenced by race and smoking habits that may differ from afro-americans; we have studied the prevalence of egfr mutations and alk translocations in a case series of patients of sub-saharan african ethnicity with nsclc. we retrospectively studied all patients of sub saharan african ethnicity who have been treated for nsclc stage iv in our hospital (university hospital saint pierre) in brussels, belgium. the ethics committee of saint-pierre hospital deemed ethical approval not necessary as it was a retrospective study. investigations were performed on biopsies obtained during flexible bronchoscopy and these samplings were immediately fixed in neutral buffered formalin and embedded in paraffin.9 driver oncogenes on biopsy specimens were researched by using the truseq amplicon cancer panel (illumina, illumina inc., san diego, ca, usa). not only were egfr/alk mutations investigated, but also other mutations included in the panel, such as tumor protein p53 (tp53), proto-oncogene b-raf (braf) and kirsten rat sarcoma viral oncogene homolog (kras). from july 2012 to november 2015, 6 patients of sub-saharan origin with nsclc stage iv have been treated in our hospital. three patients originated from congo, an ex-belgian colony, 1 from djibouti, lfrom cameroon, and 1 from guinea. egfr mutation was present in 3/6 patients and alk rearrangement was present in 1/6 patients. two egfr mutations were common, i.e., l858r (exon 21 point mutation) and deletion exon 19.10 the significance of the third egfr mutation (t710i in exon 18) is unknown. all egfr mutations and both patients without egfr mutation and alk rearrangement were considered as light smokers (< 10 pack-years). in our small case series, 4 out of 6 sub-saharan patients with nsclc had a driver oncogene, either egfr mutation or alk rearrangement. the prevalence of rearranged driver oncogenes in nsclc is known to be influenced by race, gender, and smoking status.3 differences in prevalence of egfr mutation status in nsclc between asian and caucasian patients have already been extensively studied since the early 2000s.2,3 egfr mutations (exons 1822) are present in ~30% east asian patients with lung cancer, and more specifically in 35%47% of east asian patients with lung adenocarcinoma, whereas egfr mutations are present in ~7% of caucasian patients with lung cancer, and in 13%18% of caucasian patients with lung adenocarci-noma.3,4 even within the asian population, egfr mutation status in nsclc varies.6 the east asian population is the largest asian group studied (japanese, korean, and chinese patients).3,4,6 in a large cohort of 907 indian patients, an egfr mutation was found in 23% of patients with nsclc and in 26% of lung adenocarcinomas.6 recently, egfr mutations in nsclc in afro-american patients have been investigated.4,5,11 there seems to be no difference in prevalence of egfr mutations in ncslc between caucasians and afro-americans. yamaguchi et al described a prevalence of egfr mutations in nsclc of 18.4% in white patients and in 18.2% of black patients in their patient cohort.4 bollig-fischer et al found a prevalence of 8% of egfr mutations in both black and white patients with nsclc.11 it is not clear whether these results for afro-american patients can be extrapolated to sub-saharan african patients with nsclc. errihani et al performed the only study, to our knowledge, on the prevalence of egfr mutation status in lung cancer patients in the african continent.7 they examined in a moroccan patient cohort the prevalence of egfr mutations in advanced lung adenocarcinoma. a prevalence of 21% egfr mutations in lung adenocarcinoma was found (29 out of 137 patients), which lies between the prevalence of egfr mutations in caucasian and asian patients with nsclc.7 it is unclear whether the results of the moroccan (north african) cohort also apply to sub saharan african patients with nsclc. besides race, the presence of driver oncogenes is influenced by gender and smoking history.3 in this limited series, the role of gender was impossible to assess. finally, smoking status independently influences egfr mutation status in nsclc.2,3,4 the prevalence is higher in never smokers or light smokers. in our study, 3 out of 6 patients had an egfr mutation. this incidence seems high, but interestingly, all our 6 patients were non-smokers or light smokers. in europe, the prevalence of tobacco use among adults is approximately 19% for women and 38% for men.12 in the african continent, at first sight, smoking prevalence does not seem to be different, as among males it ranges from 14% in swaziland to 40% in niger.13 however, according to a recent analysis of the american cancer society, most african countries remain in the early stages of the tobacco epidemic, with tobacco use relatively low compared to the rest of the world.14 on this basis, it is likely that the proportion of lung adenocarcinoma in non-smokers is still high and this may contribute to explaining the frequent egfr mutations in our case series. indeed, egfr mutations in nsclc have been found in 35%40% of caucasian never smokers, and in 55%70% of asian never smokers.3,4 we believe this explanation also applies for the finding of one alk translocation in our small series. the overall prevalence of alk translocation in nsclc is thought to be 3%5%, without ethnic difference, but with a higher prevalence in non-smokers.3,4 even if these results can not be extrapolated due to the small number of patients, the early stages of the tobacco epidemic in africa are probably associated with a high frequency of egfr mutation and alk rearrangement in nsclc. this will probably change with time, as africa is becoming a future epicenter of tobacco epidemic.13 in this small series, 4/6 patients of sub-saharan origin with nsclc presented driver oncogene rearrangements. this may be related to the fact that africa is still in the early stages of the tobacco epidemic, leading to suggest that the prevalence of driver oncogenes is high in this population. as the burden of lung cancer in sub-saharan countries is expected to rise in the next decennia,8 further investigations of nsclc subgroups in this racial group are required.

non-small cell lung cancer can exhibit driver oncogenes, including epidermal growth factor receptor (egfr) and anaplastic lymphoma kinase (alk), that are possible targets for therapy. the prevalence of these rearranged driver oncogenes is influenced by race, smoking habits, and gender. most data come from caucasian and asian populations. to our knowledge, there is no literature available about the prevalence of driver oncogenes in sub-saharan africa, where the tobacco epidemic is still in the early stage. in this small case series, 6 patients of sub-saharan african ethnicity with stage iv lung adenocarcinoma are described. egfr mutation was present in 3/6 patients and alk rearrangement in 1/6 patients. this incidence seems high but interestingly, all patients were non-smokers or light smokers. in this series, the high prevalence of driver oncogene was probably related to low smoking habits and these initial data in sub-saharan africans suggest high prevalence of driver mutations for this reason.

PMC5310716

pubmed-776

according to the results of a nationwide survey, direct-vision internal urethrotomy (dviu) is used for most urethral strictures in the united states and the situation should be similar in korea. according to the high recurrence rate of strictures after dviu, the most cost-effective strategy for the management of short, bulbar urethral strictures is to reserve urethroplasty for patients in whom a single dviu procedure fails. repeated dviu can increase the length and density of spongiofibrosis, thus making definitive surgical intervention more difficult. thus, it might be more cost-effective to go straight to primary urethroplasty because most patients want a cure. for bulbar urethral strictures of 2 cm or less, excision and end-to-end anastomosis remains the ideal procedure with excellent long-term results reported [5-7]. unfortunately, there have been few studies on the surgical outcomes of end-to-end anastomosis for bulbar urethral stricture in korean patients. we therefore performed a retrospective evaluation of patients who underwent bulbar end-to-end anastomosis to report our experience with the surgery and to assess the factors affecting surgical outcome. we reviewed the medical charts of 33 patients who underwent excision and end-to-end anastomosis for bulbar urethral strictures by a single surgeon and who completed at least 6 months of follow-up. the patients ' records were reviewed with respect to etiology of stricture, previous treatment, preoperative evaluation, surgical findings, follow-up results, and early and late complications. preoperative evaluation included history, physical exam, urinalysis, urine culture, uroflowmetry, and retrograde and voiding cystourethrography. the most common cause of stricture was blunt perineal trauma (straddle injury) in 18 patients (54.6%), followed by iatrogenic causes in 8 patients, idiopathic causes in 4 patients, and infection in 3 patients (table 1). about two-thirds of the patients (63.6%) underwent dviu, dilation, or multiple treatments before referral to our center (table 2). at presentation, 20 patients (60.6%) had a suprapubic cystostomy. in the remaining 13 patients with slow stream, the mean maximal flow rate (mfr) was 5.4 ml/s (range, 2.4 to 8 ml/s) the standard surgical technique of anastomotic urethroplasty was applied while the patient was positioned in a slightly hyperextended lithotomy position. after mobilization of the bulbar urethra, the area of fibrosis was completely excised and the healthy ends of the urethra were spatulated. urethral mobilization was required, extending in some cases to the penoscrotal junction distally and perineal body proximally. dorsal anastomosis was performed with interrupted 4-0 or 5-0 polyglactin sutures and ventral anastomosis was performed in two layers with the urethral mucosa first and then the corpus spongiosum. at the end of the procedure, a 14-fr silastic foley urethral catheter was exclusively placed and a small drain was left under the bulbospongiosus muscle for 2 to 3 days. patients were discharged with oral antibiotics until the catheter was removed, usually after 14 days. the urethral catheter was removed when there was no extravasation on urethrography of the pericatheter. the catheter was left in place an additional 1 to 2 weeks when extravasation was present. uroflowmetry was performed 3, 6, and 12 months after surgery in the first year and annually thereafter. patients underwent retrograde urethrography or urethroscopy if they developed voiding symptoms, such as slow or splayed stream. chi-square test or fisher exact test was used to assess the significance of categorical risk factors for surgical failure, and student t-test or wilcoxon rank sum test was used to assess significance in continuous factors, e.g., age or operation time. six patients (18.2%) required corporal separation to achieve a tension-free anastomosis. mean excised stricture length was 1.5 cm (range, 0.8 to 2.3 cm). stricture length was less than 1 cm in 3 patients (9.1%), 1 to 2 cm in 26 patients (78.8%), and more than 2 cm in 4 patients (12.1%). the urethral catheter was removed a mean of 16.5 days (range, 13 to 24 days) postoperatively. at a mean follow-up of 42.6 months (range, 8 to 96 months), 29 of the 33 patients (87.9%) had no evidence of recurrent stricture. in one case in the success group, meatal stenosis was successfully treated with a single urethral dilation. because there was no evidence of recurrence of bulbar stricture by retrograde urethrography or urethroscopy, the surgical outcome of this patient was classified as successful. in the success group, the mean mfr after surgery was 21.65 ml/s. patients aged less than 50 years (n=11) showed better mfr with mean of 27.4 ml/s (range, 18 to 48 ml/s) than did those aged 50 years or more (n=18), who had a mean mfr of 18.14 ml/s (range, 12 to 47 ml/s). six patients who had benign prostatic hyperplasia preoperatively had an mfr less than 15 ml/s. two of these patients were treated by laser prostate surgery and the other two patients were well controlled by medication. strictures recurred in four patients (12.1%) at a mean follow-up of 3.5 months (range, 2.5 to 4.7 months). of the four recurrences, one patient was managed successfully by dviu, whereas the remaining three patients did not respond to dviu or dilation. these three patients underwent ventral onlay graft urethroplasty using buccal mucosa at 6, 13, and 14 months after end-to-end anastomosis, respectively. although all patients had excellent outcomes, with good urinary stream and not requiring any intervention after the reoperation, further follow-up is needed because of the short follow-up time (range, 4 to 7 months). the recurrence rate was significantly higher in the patients with nontraumatic causes than in the patients with traumatic etiology. the stricture etiology of the four recurrent cases was iatrogenic in three patients and infection in one patient. other variables did not affect the surgical outcome of end-to-end urethroplasty. early complications were minor, including catheter-related infection and epididymitis that was easily treated with antibiotics in one patient each. with respect to late complications, intermittent perineal or scrotal pain bothered eight patients (24.2%) and was relieved by analgesics. two patients complained of a decrease in ejaculatory force and volume. in the seven patients who had erectile dysfunction preoperatively all these cases had a traumatic etiology. however, no patient had new onset of erectile dysfunction postoperatively. in the bulbar urethra, many variables, such as length, severity, and location of stricture, can influence surgical outcome. the surgical technique should be selected mainly according to stricture length, but the stricture etiology and density of the spongiofibrosis tissue should also be taken into account. for the treatment of a short segmental bulbar urethral stricture (< 2 cm), dviu or end-to-end urethroplasty is commonly accepted as standard therapy. when the stricture is limited in focal area, if the stricture is more than 1 cm in length, single dviu followed by end-to-end urethroplasty is commonly used as a cost-effective strategy. dorsal or ventral onlay substitution urethroplasty using a buccal mucosa graft is currently suggested for a longer (> 2 cm) strictures, where the urethral lumen is relatively well preserved and the spongiofibrosis around the lumen is limited to 1 mm. augmented anastomotic urethroplasty, with complete excision of the worst stricture segment, is currently recommended for strictures that cover a particularly dense and narrow area of 1 to 2 cm in length [14-16]. both ventral and dorsal onlay free grafts survive well with equal success rates [14-16]. short bulbar strictures are generally amenable to complete excision with primary anastomosis via a perineal incision, affording a high success rate of 95%, as reported by santucci et al.. published their series of 260 patients with bulbar stricture who underwent end-to-end anastomosis with a mean follow-up of 50.2 months. the stricture length ranged from 0.5 to 4.5 cm (mean, 1.9 cm) and the authors reported a success rate of 98.8%. recently, barbagli et al. described a success rate of 90.8% in 153 patients who underwent bulbar end-to-end anastomosis with a mean follow-up of 68 months. in 2002, jezior and schlossberg summarized the surgical outcomes of excision and primary anastomosis for bulbar stricture on the basis of major series reported in the literature. these series showed a success rate of 93% in 443 patients with a range of 65% to 100% between series. in our series of 33 patients with bulbar stricture, end-to-end anastomosis had a success rate of 87.9% with a mean follow-up of 42.6 months. many variables such as age, operation time, stricture length, previous operation history, preoperative voiding status, and etiology of stricture were evaluated as potential risk factors of recurrence. no clear consensus exists on stricture etiology and the success rate with respect to excision and end-to-end anastomosis. it is believed, however, that inflammatory strictures are more extensive, generally involving more of the urethra and corpus spongiosum, and are less likely to yield a successful result. reported the highest failure rate in patients with strictures, which was related to prolonged indwelling catheter drainage. in our series, the stricture etiology of the four failure cases was iatrogenic in three patients and infection in one patient. therefore, the most common cause of stricture in the surgical failure group was iatrogenic, arising after previous endoscopic surgery (n=1) or following prolonged indwelling catheter placement (n=2). although initial postoperative retrograde urethrography findings were normal, stricture recurred in four patients at a mean follow-up of 3.5 months (range, 2.5 to 4.7 months). a typical case of recurrent stricture after excision and end-to-end anastomosis is illustrated in fig. 1. to get the best results for end-to-end anastomosis, complete excision of unhealthy urethra and accompanying spongiofibrosis and tension-free anastomosis are essential. failure to remove all abnormal urethra is thought to be the primary cause of surgical failure and stricture recurrence. the main cause of surgical failure in our series was also assumed to be inadequate excision of the urethral stricture. retrograde urethrography often combined with voiding cystourethrography is a conventional preoperative tool for evaluation of the extent of urethral involvement. however, the static retrograde urethrography image can both underestimate (by as much as 50%) and overestimate the length of the stricture. intraoperative urethrocystoscopy can be used as an adjunct to retrograde urethrography to estimate the extent of stricture. some advocate urethral ultrasonography to accurately determine stricture length. in one study, intraoperative ultrasonography of the anterior urethra recurrence might have been prevented in our series by a wider excision of suspicious spongiofibrosis or augmented anastomotic urethroplasty. we have no clear explanation for why the patients with traumatic etiology had better surgical outcomes than did those with nontraumatic etiology. the most likely reason for the better results in the traumatic group is that spongiofibrosis developed from outside to inside, which makes it easier to identify the extent of stricture. on the contrary, spongiofibrosis propagated from inside to outside in urethral strictures of nontraumatic causes, especially those with iatrogenic or infectious causes. in addition to complete excision of abnormal urethral mucosa and spongiofibrosis, tension-free anastomosis is important for achieving the best results. the ideal stricture length for excision and end-to-end anastomosis has been a contentious issue. guralnick and webster insisted that this operation should be limited to strictures of 1 cm or less, because excision of a 1-cm urethral segment with opposing 1-cm proximal and distal spatulations results in a 2-cm urethral shortening. in general, the best stricture length manageable by excision and primary anastomosis is 2 cm or less. however, strictures longer than 2 cm can be managed successfully in selected patients with end-to-end anastomosis [5-7]. morey and kizer reported on a selected cohort of 22 patients with proximal bulbar urethral strictures longer than 2.5 cm that were treated with an extended anastomotic approach and suggested that the ability of the urethra to be reconstructed is proportional to the length and elasticity of the distal urethral segment. they reported a 91% success rate, concluding that defects up to 5 cm can be successfully excised and primarily reconstructed in select young men with proximal bulbar strictures. in our series, the majority of patients (87.9%) had an excised urethral length of 2 cm or less; no cases had a stricture length more than 2.5 cm. the literature suggests that the influence of previous treatment on surgical outcome is controversial [2,5-7,22]. in the recent series reported by santucci et al. and eltahawy et al., 55% and 69.2% of the patients had failed attempts of urethroplasty or dviu, respectively. despite this fact, furthermore, previously failed urethrotomy did not influence the long-term outcome of urethroplasty., the only group of patients who had a lower success rate (78.6%) had undergone undergone multiple treatments (dilation, dviu, or urethroplasty), whereas the other groups (prior single or no treatment) showed similar success rates ranging from 92.1% to 100% without any statistical significance. it was also suggested that endoscopic or open urethral manipulation before anastomotic urethroplasty for posttraumatic urethral stricture has a significant impact on the outcome of urethral reconstruction. in our study, 21 of 33 patients (63.6%) underwent prior single or multiple treatments, whereas 12 patients (36.4%) had no previous treatment. although previous treatment did not affect surgical outcome, all recurrent cases had a history of one or more dvius. most patients feel satisfied with the surgical outcome despite some minor postoperative complications. in our series, the most frequent postoperative ejaculation disorder was decreased force of ejaculation (20%) or semen sequestration in the urethral bulb (3.3%). yucel and baskin suggested that most likely surgical damage to the branches of the perineal nerves or bulbospongiosus muscles may have a role in determining the loss of efficient bulbar urethral contraction, thus causing difficulties in expelling semen and urine. although the success rate of bulbar urethroplasty is high, some argued that this is the urologist's view and not necessarily the patient's view. whereas the urologist concentrates on voiding efficiency, the patient is much more concerned with cosmetic effects and adverse effects, especially on sexual performance. recently, a patient-reported outcome measure for urethral stricture surgery was devised and validated. a major limitation of this study was that the number of involved patients was not enough to obtain statistical significance in the multivariate analysis. for example, etiology of stricture was a significant factor that influenced recurrence after surgery, but an exact odds ratio could not be calculated because the recurrence rate of the traumatic group was 0%. instead, we could calculate the estimated odd ratio, but the range of the confidence interval was so wide that the interpretation of the results was limited. excision and end-to-end anastomosis for short, bulbar urethral stricture has an acceptable success rate of 87.9% with minor complications. however, strictures recurred early (less than 5 months) in four patients (12.1%). all recurrences occurred in the patients with nontraumatic causes (iatrogenic in three, infection in one patient). therefore, careful consideration is needed when choosing a surgical procedure if the stricture etiology is nontraumatic.

purposealthough direct-vision internal urethrotomy can be performed for the management of short, bulbar urethral strictures, excision and end-to-end anastomosis remains the best procedure to guarantee a high success rate. we performed a retrospective evaluation of patients who underwent bulbar end-to-end anastomosis to assess the factors affecting surgical outcome. materials and methodswe reviewed 33 patients with an average age of 55 years who underwent bulbar end-to-end anastomosis. stricture etiology was blunt perineal trauma (54.6%), iatrogenic (24.2%), idiopathic (12.1%), and infection (9.1%). a total of 21 patients (63.6%) underwent urethrotomy, dilation, or multiple treatments before referral to our center. clinical outcome was considered a treatment failure when any postoperative instrumentation was needed. resultsmean operation time was 151 minutes (range, 100 to 215 minutes) and mean excised stricture length was 1.5 cm (range, 0.8 to 2.3 cm). at a mean follow-up of 42.6 months (range, 8 to 96 months), 29 patients (87.9%) were symptom-free and required no further procedure. strictures recurred in 4 patients (12.1%) within 5 months after surgery. of four recurrences, one patient was managed successfully by urethrotomy, whereas the remaining three did not respond to urethrotomy or dilation and required additional urethroplasty. the recurrence rate was significantly higher in the patients with nontraumatic causes (iatrogenic in three, infection in one patient) than in the patients with traumatic etiology. conclusionsexcision and end-to-end anastomosis for short, bulbar urethral stricture has an acceptable success rate of 87.9%. however, careful consideration is needed to decide on the surgical procedure if the stricture etiology is nontraumatic.

PMC3715707

pubmed-777

it is known as the fast enemy that should be treated and destroyed very fast as well. most women do not like to hear the word cancer, and feel worried and stressed over it. it can be the beginning of learning how to fight, getting the facts, and finding hope. this is followed by lack of patient's personal control over the current treatment method and uncertainty of its outcome. therefore, anxiety is associated with cancer; it is the most prevalent psychological symptoms perceived by cancer patients as a response to a threat, and so many patients are anxious. in one study done by ashbury et al, 77% of 913 patients within 2 years of treatment recalled experiencing anxiety. however, anxiety after cancer diagnosis is not necessarily abnormal, may not present a problem, or may even be a constructive part of dealing with problems. the most common cancer and the number one cause of cancer death amongst women in malaysia is breast cancer. if not detected and treated promptly, breast cancer can metastasize, spreading to the lymph glands and other parts of the body including the lungs, bones, and liver. usually, cancer is named after the body part in which it originated; thus, breast cancer refers to the erratic growth and proliferation of cells that originate in the breast tissue. the term breast cancer refers to a malignant tumor that has developed from cells in the breast. the breast is composed of two main types of tissues: glandular tissues and stromal (supporting) tissues. glandular tissues house the milk-producing glands (lobules) and the ducts (the milk passages), while stromal tissues include fatty and fibrous connective tissues of the breast. the breast is also made up of lymphatic tissue-immune system tissue that removes cellular fluids and waste. breast cancer is characterized by the uncontrolled growth of abnormal cells in the milk-producing glands of the breast or in the passages (ducts) that deliver milk to the nipples. the early stage of breast cancer usually refers to the cancer that is confined to the fatty tissue of the breast. it may then spread to underlying tissues of the chest wall and then to other parts of the body. furthermore, worldwide, breast cancer is the leading cause of cancer death in women, and more than one million women are diagnosed each year. in addition to that, more than 500,000 women every year die from the disease worldwide. anxiety, tension, worry, stress, and strain are all common feelings and it is a part of our life today. simple worry or stress will not drive us to look for specialist, but when these feelings become a chronics and interfere with our lives we need to do something and look for ways to manage it in order to function well. anxiety can be defined as an unpleasant subjective experience associated with the perception of real threat; therefore, it is a common symptom in connection with cancer. furthermore, it can be described as an emotional state characterized by feelings of unpleasant expectation and a sense of imminent danger. according to stark, et al. autonomic hyper-arousal with acceleration of heart rate and respiration, tremor, sweating, muscle tension, and gastrointestinal changes are common physiological experiences. apprehension, feeling powerless, and fearing loss of control are psychological aspects. according to kazdin, anxiety is an emotion that characterized by feelings of tension, worry, and stress as well as physiological changes such as increased blood pressure. furthermore, medical news today defines anxiety as a general term for several disorders that cause nervousness, fear, apprehension, and worrying. these disorders affect how we feel and behave, and they can manifest real physical symptoms. mild anxiety is vague and unsettling, while severe anxiety can be extremely debilitating, having a serious impact on daily life.. we may worry about things that might happen or have a restless night of sleep. but, people with an anxiety problem worry so much that it affects their lives in negative ways. as stated above, anxiety is one of the most dominant psychological challenges associated with cancer. in another word, patients anxiety increases once they discover that they suffer from breast cancer, they may also become more anxious as cancer spreads or treatment becomes more intense. consequently, the level of anxiety experienced by one person with cancer may differ from the anxiety experienced by another. many anxiety cases associated with cancer were treated from this sickness, but others were not. therefore, psychologists need to give support and hope to breast cancer's patients; they need to help them cope with their feeling and pain. moreover, cancer's patients may experience anxiety at different situation as while undergoing a screening test, waiting for the results, receiving a diagnosis, undergoing treatment, or anticipating a recurrence of their cancer. the anxiety associated with cancer may increase feelings of pain, interfere their ability to sleep, causes nausea and vomiting, and interfere with their quality of life. and for most patients, cancer requires facing uncertainty, worries about cancer treatment effects, fear of cancer progression and death, guilt, and spiritual questioning. a study by ashbury et al., indicated that 77% of patients within 2 years of treatment recalled experiencing anxiety. on the other hand, anxiety after cancer diagnosis is not necessarily to be normal, understanding the nature of the anxiety in cancer patient populations is important because abnormal anxiety is troublesome the psychological wellbeing of the patients sherbourne and sheard. interviewing some breast cancer patients reported that their anxiety is characterized by a number of typical symptoms and signs such as shivering or tremor. the level of anxiety experienced by one person may differ from the level of anxiety experienced by another. anxiety in breast cancer patients is associated with death anxiety, fear of death as a result of their symptoms. according to pollak this type of anxiety is lower for people who have a positive sense of well-being and sense of meaning in life. in addition, evidence indicates that religious beliefs influence their level of anxiety [figure 1]. many researchers have investigated the differences in anxiety level among women receiving different breast cancer treatments. recent study done by lim, indicated that anxiety presents in all treatment types for breast cancer. moreover, the anxiety level in women who underwent chemotherapy was highest before the first chemotherapy infusion, mediated by age and trait anxiety. this result confirms the needs for more research and studies on anxiety among breast cancer patients. in addition, the villadeguadarrama free article reported that the cancer-related anxiety may manifest as physical symptoms, such as rapid heartbeat, tightness in the chest or shortness of breath. the patient may also experience digestive symptoms, such as nausea, vomiting, or diarrhea. thinking about anxiety that can lead to physical symptoms, the overall symptoms of anxiety among cancer patients include: excessive, ongoing worry and tension, an unrealistic view of problems, restlessness or a feeling of being edgy, irritability, muscle tension, headaches, sweating, difficulty concentrating, nausea, the need to go to the bathroom frequently, tiredness, trouble falling or staying asleep, trembling, and being easily startled. symptoms of anxiety and depression have been found to be common in patients with cancer, frequently occurring around the time of diagnosis and during the period of chemotherapy. high-depression burden has been found at the time when patients experience adverse effects of chemotherapy. significant difference in psychological distress has been found depending on age, gender, and living situation, with those living alone experiencing higher levels of distress in a sample of icelandic cancer patients during the treatment period. a study of women at high risk for breast cancer showed significantly higher levels of depressive symptoms and feelings of emotional alienation than did a standardized test group, with 27% of this population defined as having a level of psychological distress justifying psychological counseling. a second study also documented an increase in distress among first-degree relatives of breast cancer patients. an israeli study found that first-degree relatives who have physical symptoms of breast pathology respond with more emotional distress than do women of normal risk in the same situation. patients with cancer face most of the stressors associated with diagnosis, illness, and treatment. cancer diagnosis and treatment brings changes in patients personal paths of life, in their daily activities, work, relationships, and family roles, and it is associated with a high level of patient psychological stress. if you are really caring for someone who is having the symptoms of anxiety, encourage him or her to get help. people are differing in the way they perceive their sickness and they are also differ in the way they cope with their anxiety. some are easily disturbed by their feeling of being anxious and other may take it as a challenge and they try to look for ways to overcome their feeling. there are many treatments for clinical depression including medicines, counseling, or a combination of both. therefore, the diagnosis of anxiety in cancer is usually complicated by the overlap of anxiety and sickness symptoms such as cancer-related fatigue and pain. many common symptoms of major depression were observed in cancer patients who do not endorse full depression symptoms. different studies showed that various kinds of coping strategies are used to overcome the anxiety in different types and stages of cancer. for instance, reuter et al., stated that patients using ineffective coping strategies have higher levels of anxiety and depression and that benefiting from social support results in a marked reduction in the levels of anxiety and depression. moreover, the importance of social support to good mental health outcomes is well established. as well as the positive effect of good social support, the detrimental effect of negative interactions with significant others in the social environment has also become apparent in psychiatric and other conditions. eventually, people are unique in the way they perceive and cope with the anxiety, also the way one patient uses is different from the way other uses. undoubtedly, it is related to the personality, strength, faith, and hope patients is having toward their sickness. worry, tension, fear, and stress are interrelated to the anxiety and depression among them. patients are differing in the way they perceive their problem as well as the way they cope with the anxiety associate with it. the huge literatures on anxiety, its effects, coping, counseling, and mental health are evidence of the extensive belief that the way people cope is somehow linked to their belief and faith. in conclusion, anxiety has a great effect on the feeling of breast cancer patients and it leads to high level of coping mechanisms.

cancer is a disease wherein abnormal cells divide without control and are able to attack other tissues. most of the patients and their families face some degree of depression, anxiety, and fear when cancer becomes a part of their lives. they feel helpless and eager to find ways on how to get rid of it. the study focuses on anxiety among breast cancer patients. it aims at investigating cancer, its symptoms, and effects the disease has on the anxiety level of patients.

PMC3498772

pubmed-778

neurological soft signs (nss) have long been considered one of the functional features (tsuang and faraone, 1999; tsuang et al., 1991) and endophenotypes (chan and gottesman, 2008; chan et al., 2010a) of schizophrenia spectrum disorders. however, most of the studies of nss have been limited to the use of clinical ratings. instead, recent findings from both structural and functional imaging studies have shown that nss may be associated with specific brain alterations. specifically, in individuals with psychosis, more nss have been associated with smaller volumes of the inferior frontal lobes (thomann et al., 2009a), the pre-central gyrus (heuser et al., 2011; mouchet-mages et al., 2011), the global cortical sulci (dazzan et al., 2004; gay et al., 2013) as well as the cerebellum (ho et al., 2004; thomann et al., nss may be responsible for some of the observed clinical manifestations in schizophrenia (keshavan et al., 2003; mouchet-mages et al., 2011; schroder et al., 1999). a recent meta-analysis of structural and functional imaging findings suggests that nss are associated with volume reductions of the pre-central gyrus, the cerebellum, the inferior frontal gyrus and the thalamus. furthermore, the same meta-analysis also suggests that functional imaging studies support an association between nss and altered neural activation in the inferior frontal gyrus, the bilateral putamen, the cerebellum and the superior temporal gyrus in patients with schizophrenia (zhao et al.,, these findings support the presence of a dysfunction of neural circuitry that underlies the presence of these minor neurological abnormalities, which are already present at illness onset. on the other hand, empirical findings have also shown that non-psychotic first-degree relatives of schizophrenia patients exhibit a higher prevalence of nss compared to healthy controls (egan et al. a meta-analysis comparing the prevalence of nss between patients with schizophrenia, non-psychotic first-degree relatives and healthy controls indicated a mean effect size of 0.8 and 0.97 for patients and their non-psychotic first-degree relatives and for non-psychotic first-degree relatives and controls respectively (chan et al., 2010b). these results are consistent with the argument that nss are familial in nature and segregate with the illness. however, no studies had examined the brain correlates of nss in healthy relatives of patients with schizophrenia. furthermore, all the aforementioned neuroimaging studies in schizophrenia patients used conventional subtraction analysis and did not examine the underlying connectivity between the brain regions identified. given that some nss, such as the fist palm (fep) task (first described by luria, see heuser et al., 2011), involve the functional integration or regulation of areas involved in motor sequencing rather than being directly activated, a connectivity approach is more appropriate to identify the specific network involved. only one study (rao et al., 2008) adopted a psychophysiological interaction (ppi) (friston et al., 1997) method to re-analyze their previous findings on the fep task in healthy volunteers (chan et al., 2006). this new analysis showed enhanced functional connectivities between the left- and right sensorimotor cortices (smc) and the right inferior and middle prefrontal cortices during fep task performance compared to a simple palming task. these findings suggest a regulatory role of the prefrontal cortex on fep task execution. in this study, we examined whether prefrontal regions are involved in the integration or regulation of neural activity underlying motor sequencing in patients with first-episode schizophrenia and their non-psychotic first degree relatives (fdr). more specifically, we aimed to identify any frontal regions where coupling in these areas and the sensorimotor cortex (smc) significantly differed between the complex fep task and a simple control motor task using ppi analysis. we hypothesized that patients with first-episode schizophrenia and their non-psychotic fdrs would show a reduced dysfunction of the prefrontal cortex and smc while performing the fep task. thirteen right-handed first-episode schizophrenia patients were recruited from the mental health center, peking university, beijing, for the study. participants were recruited to the study if they met the following inclusion criteria: a) diagnosis of schizophrenia ascertained by experienced psychiatrists according to the dsm-iv (apa, 1994); b) aged 1840 years; and c) illness duration within 2 years. the exclusion criteria were: a) a history of neurological disorders; b) a lifetime history of substance abuse and c) an estimated iq lower than 70. current symptom severity was assessed with the positive and negative syndrome scale (kay et al., 1987). fourteen non-psychotic fdrs of the patients taking part in the study were also invited to participate. potential participants were excluded if they a) met the dsm-iv criteria for substance abuse; b) were suffering from any clinically unstable medical disorder; c) had a history of head injury (past or present); and d) had an estimated iq lower than 70. fourteen healthy controls matched with the patients in age, gender and handedness were recruited from local universities through advertisements. the institutional review board of the institute of psychology, the chinese academy of sciences, approved the study protocol. behavioral neurological soft signs were examined with the abridged version of the cambridge neurological inventory (cni) (chan et al., 2009). this abridged version offers instructions for eliciting and rating a comprehensive range of nss in motor coordination, sensory integration and disinhibition. rao et al., 2008) (for the earliest description, please see luria, 1966; in heuser et al., 2011). in brief, the task consisted of three right-hand motor tasks which varied in complexity. in the simple palm tapping (pt) task, participants were required to repeat only one right palm tapping in the prone position. in the intermediate complex pronation/supination (ps) task, participants were required to perform right palm tapping in the prone and supine positions alternatively. in the complex fep task, participants were required to successivley place their right hand in a fist resting position vertically (fist), a palm resting position vertically (edge), and a palm resting position horizontally (palm). a resting condition (a) without any hand movement when participants were asked to focus on the screen was used as the control baseline of the pt task, and the pt task was in turn used as the control baseline of the ps and fep tasks. participants were asked to practice the motor actions correctly at a constant rate before entering the scanner. during scanning, their performance was monitored by the experimenter through the window of the scanner room. in the formal imaging task, participants were instructed to perform the three tasks at a similar pace throughout the entire experiment. pt and ps tasks were executed in 1 s and the fep task was executed in 1.5 s. the experimenter monitored the task performance outside the scanner room to ensure that the participants performed the correct hand movements. first, a resting condition lasted for 20 s and then 6 s of counting backward reminded the participants to get ready for their hand movement. after backward counting, the pt or ps or fep task lasted for 40 s. one of these three hand movements would be presented on the screen. participants were required to conduct the hand movements according to the demonstration on the screen. the sequence of the three hand movement tasks was optimized and counterbalanced within the three runs. the functional imaging data was originally acquired in a ge 3 t sigma scanner (general electric, waukesha, wi, usa) with a standard ge birdcage-type rf coil using a standard t2*-weighted epi sequence. the epi parameters were: tr=2 s te=60 ms, fov=24 24 cm, matrix=64 64, flip angle=60, 22 axial slices (5 mm thick/1.2 mm sp, from superior to inferior). the spatial resolution for the functional images was 3.75 3.75 6.2 mm. high-resolution anatomical images were also obtained using the standard t1-weighted sequence (66 axial slices, 2.0 mm thick/interleaved, fov=24 24 cm, matrix=256 256). images were analyzed with statistical parametric mapping software (spm8, wellcome department of imaging neuroscience, london, uk) implemented in matlab 2009b (mathworks inc., sherborn, ma, usa). three dummy scans in the beginning of the experiment were removed automatically from the dataset. images were registered to the first icbm 152, which was based on 152 brains and was created by the montreal neurological institute (mni) with a 2 2 2 mm resolution. in the final step of pre-processing, the images were spatially smoothed by an isotropic gaussian kernel with fwhm of 8 mm. conventional analyses were first conducted at the individual-level using voxel-wise general linear modeling (glm) and four t-contrasts were defined between the tasks and the corresponding baselines, i.e., pt vs. rest, ps vs. rest, fep vs. rest and fep vs. pt. data from the contrasts of the first level model in the healthy control group, the schizophrenia group and the fdr group were entered into this model. a threshold of alphasim corrected p<0.01 and cluster size larger than 15 voxels were used to identify the activations associated with each contrast. furthermore, we used a regression model to find the regions with group difference. we set up an f contrast to find the regions with main effect of group difference. signal change percentage was retrieved from the rois with group difference for post-hoc analysis. ppi analysis was used to estimate functional integration during task execution under different motor complexity conditions. the left smc was determined a-priori as the reference region for the ppi analysis because the motor tasks in the present study were only completed with the right hand. this region was defined by using a sphere with a radius of 8 mm and a center at the peak activation in the left smc activation (mni coordinates=[36 28 52], from the conventional analysis of the contrast of pt vs. rest). we performed voxel-wise ppi analysis at individual-level for the left smc to see if any other brain areas connected to the smc showed a significant increase in functional coupling (the slope of regression) during the fep task compared with a control task (e.g., the pt or ps task). for each participant, the activation time course signal in the reference region (i.e., the first eigenvariate time series, adjusted by effect of interest) was extracted from the conventional glm and entered into the ppi analysis as the first regressor representing the physiological variable. a second regressor representing the motor tasks with different complexity was entered into the ppi analysis as the psychological variable. the psychophysiological interaction between task complexity and activation signal in the reference region was designated as the regressor of interest in the ppi analysis. thereafter we performed group-level random effect analysis on the individual results using one sample t-tests for the contrast fep vs. pt. group-level paired t-tests were conducted for the contrast fep vs. ps. areas of significant activation were identified at a threshold of uncorrected p<0.001 and cluster size larger than 15 voxels. there was no significant difference between patients with schizophrenia, their fdrs and healthy controls in the three subscales and total score of cni. conventional activation of the pt, ps and fep tasks in the three groups, after co-varying for age, is shown in table 3. the left frontal parietal region was significantly activated when participants performed the pt, ps and fep tasks with their right hand. moreover, in the pt rest contrast, activation in the left frontal parietal region, together with the left medial frontal region, were lowest in patients with schizophrenia, intermediate in the fdrs and highest in healthy controls (scz<rel<hc). we further used a regression model and conducted a post-hoc analysis to identify the regions that were different across groups both linearly and non-linearly. in the pt rest contrast, we confirmed that the left frontal precentral gyrus (30 34 64, k=100, f(2,37)=20.23) was linearly activated in all three groups (scz<rel<hc). the left medial frontal region (hc=rel>scz, 8 18 64, k=136, f(2,37)=15.59) and the left middle temporal gyrus (hc>scz=rel, 48 68 10, k=124, f(2,37)=11.7) were non-linearly activated in all three groups. the activations associated with task complexity were examined in the fep ps and the fep pt contrasts. in the fep pt contrast, the bilateral middle frontal regions were activated in the healthy control group. the left middle frontal region was activated in the fdr group, but there was no frontal region activation in first episode schizophrenia patients. 2 illustrates the frontal activation in both healthy controls and fdrs. in the fep ps contrast, the left middle frontal region was also activated in the fdr group, but not in the healthy control group or the schizophrenia group. the ppi analysis identified right frontal regions in which the activity showed significant activation coupling to activity in the left smc during performance of the more complex fep task relative to the simple pt or ps tasks in the healthy control group, but not in the schizophrenia group. in this study, we report for the first time the neural activation and connectivity elicited by execution of the fep task in patients with first episode schizophrenia and their healthy first degree relatives. our main finding is that patients with first episode schizophrenia do not show, in comparison with healthy controls and their fdrs, activation of the left middle frontal gyrus in the execution of the fep versus a simpler motor task like the pt. this provides evidence of a frontal dysfunction in these patients when performing a complex motor task. the right inferior frontal gyrus was found to modulate the activation of the sensorimotor cortex with the increase in motor complexity in healthy controls. a frontal dysfunction was also demonstrated by our second main finding, suggesting that with increase in task difficulty, patients with first episode schizophrenia do not show functional connectivity between the sensorimotor cortex and the right frontal gyrus, in contrast to healthy controls. the presence of a prefrontal dysfunction in schizophrenia is supported by extensive evidence from both structural and functional neuroimaging studies. prefrontal areas have been consistently reported as reduced in volume in patients with schizophrenia (chan et al., 2011; dazzan et al., furthermore, fmri studies have shown altered activation of frontal areas during executive and working memory tasks, particularly with increasing capacity demand (barch et al., 2012; callicott et al., 2000; perry et al., 2001). at a functional level, patients with schizophrenia also show an excess of signs reflecting frontal release, such as abnormalities in eye movements and short-term memory deficits, which again point to a frontal cortical alteration (hyde et al., 2007). in this study, we tested the activation of frontal areas during a complex motor task, the fep task, which has long been considered in neurological and neurocognitive studies as indicative of frontal lobe lesions (luria, 1966). two previous fmri studies have reported that in healthy individuals, the execution of this task did not induce activation of prefrontal areas, but induced activation of other parts of the cortex, including the sensorimotor areas (chan et al., 2006; umetsu et al., 2002). a subsequent study, using the same ppi approach that we used in this study, showed that this finding could reflect an indirect involvement of the prefrontal cortex, which could exert an integrative and regulatory function on neural circuitry involved in complex motor sequences. we found here that in healthy individuals, the execution of the fep task, compared to a simpler motor act, was indeed associated with both direct activation of the prefrontal cortex, and greater coupling of prefrontal areas and sensorimotor cortex. interestingly, a recent meta-analysis of the brain connectome suggests that the frontal lobe represents a hub (a region highly interconnected with other brain regions and valuable for integrative information processing and adaptive behavior) particularly affected in patients with schizophrenia (crossley et al., 2014). there is a paucity of studies on brain activation during complex motor tasks in these patients. however, neuroimaging studies that examined gross motor coordination signs such as finger-to-thumb operation tests in these patients have reported reduced activation in the smc and the supplementary motor area (sma) in comparison to healthy controls (schrder et al., a later similar study used pronation/supination tests and found significant reduced activation in the smc in patients with schizophrenia compared to healthy controls (schroder et al., 1999). the lack of activation we found with a task like the fep, which requires fine motor coordination and requires executive processes like inhibition, planning and updating, could be interpreted as part of the hypofrontality hypothesis of schizophrenia reflecting a reduced function of the prefrontal cortex (callicott et al., 2000). response relationship of the left frontal parietal activation across patients, their fdrs and healthy controls. here, in the pt vs. rest contrast, activation was lowest in the schizophrenia group, intermediate in the fdr group and highest in the healthy control group. although these differences were highlighted with a motor sequence less complex than the fep, the finding suggests that at least part of the pathophysiological substrate that underlies motor difficulties in psychosis may be linked to a genetic susceptibility to schizophrenia. indeed, deficits in motor skills have been reported extensively in individuals at risk of schizophrenia because of either genetic loading or prodromal features (blanchard et al., 2010; first, the clinical sample size was relatively small. however, the merit of the present sample is that it included both first-episode schizophrenia patients and their non-psychotic siblings. secondly, the use of ppi analysis may not fully detect the connectivity changes related to the performance of the fep task. however, ppi is an appropriate method to test the relationship between two simple mental activities (friston et al., 1997). other more sophisticated approaches, such as dynamic casual modeling, may help identify further subtle connectivity changes in the performance of the fep task. thirdly, the use of the screen to synchronize the motor actions might induce the activation of mirror neurons. however, the contrasts between any two of the three motor actions (fep, pt, and ps) should minimize this effect by subtraction methods. finally, we should also consider the validity of our task, since previous subtraction analyses of the fep task did not elicit activation of the frontal cortex. however, the fact that the left frontal parietal region was significantly activated when participants performed the pt, ps and fep tasks with their right hand suggests that this hand movement imaging task was valid. notwithstanding these limitations, our findings suggest that the fep task may be a useful endophenotype of schizophrenia, as it fulfills the criteria of being familial and shows a dose previous study had demonstrated that patients with schizophrenia showed aberrant brain activation in the premotor area after a week of motor training (kodama et al., 2001). given this temporal stability of nss, the fact that this is an imaging endophenotype rather than a simple behavioral rating arguably enhances its precision. this task is also simpler to carry out when compared to a full nss scale. we believe that our findings add valuable information to the understanding of the origin and underlying neural mechanism of motor coordination signs. future studies should recruit a larger sample of first-episode, preferably medication-nave schizophrenia patients for further validation of our findings.

neurological soft signs have been considered one of the promising neurological endophenotypes for schizophrenia. however, most previous studies have employed clinical rating data only. the present study aimed to examine the neurobiological basis of one of the typical motor coordination signs, the fist edge palm (fep) task, in patients with first-episode schizophrenia and their non-psychotic first degree relatives. thirteen patients with first-episode schizophrenia, 14 non-psychotic first-degree relatives and 14 healthy controls were recruited. all of them were instructed to perform the fep task in a 3 t ge machine. psychophysiological interaction (ppi) analysis was used to evaluate the functional connectivity between the sensorimotor cortex and frontal regions when participants performed the fep task compared to simple motor tasks. in the contrast of palm-tapping (pt) vs. rest, activation of the left frontal parietal region was lowest in the schizophrenia group, intermediate in the relative group and highest in the healthy control group. in the contrast of fep vs. pt, patients with schizophrenia did not show areas of significant activation, while relatives and healthy controls showed significant activation of the left middle frontal gyrus. moreover, with the increase in task complexity, significant functional connectivity was observed between the sensorimotor cortex and the right frontal gyrus in healthy controls but not in patients with first episode schizophrenia. these findings suggest that activity of the left frontal parietal and frontal regions may be neurofunctional correlates of neurological soft signs, which in turn may be a potential endophenotype of schizophrenia. moreover, the right frontal gyrus may play a specific role in the execution of the fep task in schizophrenia spectrum disorders.

PMC4596919

pubmed-779

metastatic melanoma in the brain is a serious event in patients with melanoma because of the poor prognosis and potential impact on quality of life. symptomatic metastases represent the initial site of metastatic spread in 20% but may occur at any time during the course of the disease. autopsy data have shown that up to 75% of patients who died from metastatic melanoma had brain metastases [2, 3]. two large institutional series of 686 and 702 patients [3, 4] indicate a generally poor outcome, with the majority (up to 95%) dying directly from brain metastases. there were some differences in survival according to treatment received, being 8.9 months for surgery plus whole brain radiotherapy (wbrt), 8.7 months for surgery alone, 3.4 months for wbrt alone, and 2.1 months for supportive care. these differences are a probable reflection of patient selection based on the number of cerebral metastasis, performance status, and extent of extracranial metastasis. the radiation therapy oncology group recursive partitioning analysis (rpa) classes have been validated in melanoma. age (> 65 year old) and the number of neurological symptoms (weakness and fatigue) are associated with poorer survival. ulceration and location on the head and neck region are two main primary tumour characteristics that are associated with poorer survival. the number of cerebral metastases is a significant prognostic factor with better prognosis seen in single or oligometastatic disease (2-3 cerebral metastases). patients with more than 3 metastases had a median survival of 3.5 months compared with 5.9 months for those with 3 or less metastases (p=0.005). more recently, there is debate on whether it is the number of metastases or the overall intracranial tumour volume that is the relevant factor. the worst outcome is seen in patients with leptomeningeal disease. in all large cohorts of patients with melanoma brain metastases, md anderson cancer center analysed the outcomes of 743 patients with metastatic melanoma in the brain treated between 1986 and 2004. the median survival for patients diagnosed before 1996 was 4.14 months compared with 5.92 months for patients diagnosed in 1996 or later (hr 0.75 95% ci 0.590.95, p=0.02). the increased use of mri as a screening tool for brain metastases over time may have contributed to this improvement. in addition, earlier diagnosis of patients with lower burden, asymptomatic brain metastasis might allow for more frequent use of locally directed treatment such as stereotactic radiosurgery or surgical excision. a similar study of patients from the memorial sloan kettering cancer center noted that age>65, presence of extracranial metastases, presence of neurologic symptoms and four or more metastases are predictors for poorer survival, although some of these features are self-predicting in that more aggressive treatment options are less likely to be offered. the management of metastatic melanoma in the brain depends on the combination of patient, tumor, and treatment factors. the dominant factor determining management has been the number of cerebral metastases. with the wider availability of stereotactic radiosurgery facilities enabling the effective treatment of multiple metastases in a single treatment session, reports increasingly suggest that the use of stereotactic radiosurgery to treat multiple metastases may have merit, particularly if there are less than 10 lesions, all under 3 cm in size and with limited oedema or mass effect [1113]. recent data suggested that the total volume of the metastatic lesions rather than the number of metastases was the limiting factor for radiosurgery technique. our general approach in the management of melanoma metastases in the brain is shown in figure 1. for patients with a single or oligometastases, the management depends on the performance status, neurological status, the characteristic of the metastases (number, size, and location), and the extent of the extracranial disease. those with more favourable characteristics should be considered for more aggressive local treatment of the individual metastasis. surgery has a role in confirming the diagnosis especially as there is no clear relationship between the primary melanoma and the development of brain metastasis. in addition, surgical resection can also provide quick relief in symptoms associated with disease such as shunting in hydrocephalus. the prognosis is poor, with the majority succumbing to progressive intracranial metastases within months, irrespective of treatment. application of the rtog recursive partitioning analysis to 74 patients with cerebral metastases from melanoma produced median survival of 10, 6, and 2 months, respectively, for rpa classes i iii, respectively, with a median survival of 5.5 months for the entire group. initial management includes the use of steroids, typically 416 mg of dexamethasone per day. this usually results in rapid symptomatic, but often short-term, improvement in approximately 50% of patients. whole brain radiation therapy may produce a small survival advantage compared with steroids alone and may allow reduction in the steroid dose. in addition to whole brain radiation therapy, surgical removal, or stereotactic radiosurgery of a dominant and symptomatic lesion should be considered. conversely, patients with a poor performance status who have not responded to steroids may be better treated with supportive care. the technique of whole brain radiation therapy is a pair of parallel opposed lateral 6 mv photon fields. commonly used regimens are 20 gy in 5 fractions and 30 gy in 10 fractions. for good performance patients with minimal extracranial disease, there might be an advantages for higher dose of whole brain radiation therapy based on a retrospective study. rades et al. compared the outcomes of 33 patients treated with 30 gy in 10 fractions with 18 patients treated with higher doses (40 gy in 20 fractions or 45 gy in 15 fractions). in the multivariate analysis, higher doses (p=0.010), less than four brain metastases (p=0.012), no extracranial metastases (p=0.006), and rpa class 1 (p=0.005) were associated with improved overall survival. in an attempt to improve survival of patients with multiple brain metastases, radiation has been combined with a variety of chemotherapy agents, including temozolomide, thalidomide, and fotemustine without much success [1518]. the most recent study is a phase 2 study combining whole brain radiation therapy (30 gy in 10 fractions) with temozolomide and thalidomide in 39 patients. the term radiosurgery was originally coined by lars leksell to describe the use of a multisource cobalt system (gamma knife) to deliver radiation to a defined target using stereotactic principle. it aims to deliver an ablative dose to the target while limiting the dose to surrounding normal tissue. the latest version of the gamma knife (perfexion) uses 192 cobalt sources arranged circumferentially in a noncoplanar fashion, permitting smaller doses to the surrounding normal brain tissue and a lower integral dose. the associated improvements in planning software and design modifications have resulted in the ability to treat multiple targets in one session. historically, this has relied on frame-based stereotactic approaches that can accurately localise the tumour and target the beam in three-dimensional space. options now include frameless image-guided approaches such as fixed beam intensity-modulated radiotherapy, helically delivered intensity-modulated radiotherapy (tomotherapy), and image-guided robotic radiosurgery (cyberknife). arc-based intensity-modulated radiotherapy techniques (vmat and rapidarc) can also achieve highly conformal image-guided treatment in very short treatment times. linear accelerator-based stereotactic radiosurgery will use a limited number of fields, usually (but not always) in a coplanar fashion. the dose of radiosurgery depends on the size of the target lesion and the location. the rtog 90-05 study was designed to determine the maximum tolerable dose of radiosurgery in patients with recurrent previously irradiated brain metastases (excluding lesions in the brain stem). the maximum tolerable doses of single fraction radiosurgery for patients with recurrent previously irradiated brain metastases were 24 gy, 18 gy, and 15 gy for tumors 20 mm, 2130 mm, and 3140 mm in maximum diameter, respectively. in the multivariate analysis, those who were treated on a linear accelerator (versus the gamma knife) had a 2.84 greater risk of local progression. however there are no randomised data showing clear superiority of any one stereotactic radiosurgery system. mathieu et al. from the university of pittsburgh reviewed the experience of 244 patients with 754 melanoma metastases treated with gamma knife radiosurgery without adjuvant whole brain radiation therapy. overall, 54 patients (30.9%) had progression of at least one metastasis after radiosurgery. fifty-one patients (24.8%) underwent whole brain radiation therapy after radiosurgery because of the development of multiple new brain lesions. multiple lesions and failure to provide systemic immunotherapy were predictors for the occurrence of new brain metastases, which developed in 41.7% of the patients. on multivariate analysis, the use of whole brain radiation therapy was not a factor that influenced local control or distant intracranial control (p=0.061). a more recent update of the university of pittsburgh's experience on 333 consecutive patients with 1570 metastatic melanoma lesions treated with gamma knife radiosurgery showed the long-term local control rate was 73% and the actuarial survival rates were 70% at 3 months, 47% at 6 months, 25% at 12 months, and 10% at 24 months. about 25% of 259 patients who had followup imaging after stereotactic radiosurgery had evidence of delayed intratumoral haemorrhage. factors associated with longer survival included controlled extracranial disease, better performance status, fewer number of brain metastases, no prior use of whole brain radiation therapy or chemotherapy, treatment with immunotherapy, and no intratumoral hemorrhage before radiosurgery. the potential morbidity of stereotactic radiosurgery includes progression or worsening of cerebral oedema symptomatic in 46% of patients within 1-2 weeks of treatment, seizures within 1-2 days in 26%, and delayed radiation necrosis in 211% [21, 24, 25]. this risk increases with prior treatment, larger volumes treated (both larger lesions and larger numbers of lesions), and larger doses delivered. the role for whole brain radiation therapy after surgery or stereotactic radiosurgery of the single or oligometastasis is controversial and there is no level 1 evidence in this scenario. the rationale of whole brain radiation therapy is to treat microscopic disease at the site of initial metastasis and elsewhere in the brain to maintain long-term cerebral control. adjuvant systemic therapy is generally not used as the brain is considered a sanctuary site for chemotherapy although this assumption has been recently challenged by responses in the patients with b-raf mutant melanoma treated with b-raf inhibitor. however opponents of whole brain radiation therapy argue that melanoma is radioresistant and that whole brain radiation therapy can potentially cause late neurocognitive deficits. the australia and new zealand melanoma trials group (anzmtg) and trans-tasman radiation oncology group (trog) are conducting a phase 3 randomised trial to address the role of whole brain radiation therapy after local treatment of 1 to 3 melanoma metastases. eligible patients are randomised to whole brain radiation therapy or observation after their local treatment of the brain metastasis. is reported, clinicians will have to rely on data from other randomised trials included patients with metastatic disease from all histologies. several randomised studies including metastasis from all histologies have provided good evidence for the use of whole brain radiation therapy after local treatment of oligometastases in terms of an improvement in intracranial control. aoyama et al. compared whole brain radiation therapy and stereotactic radiosurgery (65 patients) to stereotactic radiosurgery alone in patients with 14 brain metastases of any histology. over 65% of the patients had metastatic lung cancer, while the number of melanoma patients per arm was not mentioned. the whole brain radiation therapy dose was 30 gy in 10 fractions over 2 weeks. there was no difference between the two groups with respect to overall survival, neurological toxicity, neurological functional preservation, and neurological death. the median survival time was 7.5 months with whole brain radiation therapy plus stereotactic radiosurgery compared to 8 months with stereotactic radiosurgery alone. the 12-month actuarial brain tumour local recurrence rate was 46.8% in the whole brain radiation therapy plus stereotactic radiosurgery group and 76.4% in the stereotactic radiosurgery alone group (p=0.001). fifty-five patients had new brain metastases at distant sites (21 in the whole brain radiation therapy plus stereotactic radiosurgery group and 34 in the stereotactic radiosurgery-alone group). the 12-month actuarial rate of developing distant brain metastases was 41.5% in the whole brain radiation therapy plus stereotactic radiosurgery group and 63.7% in the stereotactic radiosurgery-alone group (p=0.003). the univariate analysis showed that patients with 24 metastases had a higher risk for developing distant intracranial disease than those with single metastasis (p=0.03), but this did not reach significance on multivariate analysis (p=0.06). more recently, eortc reported a randomized trial of 359 patients with 1 to 3 brain metastases from all solid tumours randomised to either observation or whole brain radiation therapy of 30 gy in 10 fractions after local treatment (surgery or stereotactic radiosurgery). the majority (53%) of patients had primary lung cancer and only 5% had metastatic melanoma. after surgery, at 2 years, whole brain radiation therapy significantly reduced the probability of relapse at initial sites from 59% to 27% (p=0.001) and at distant intracranial sites from 42% to 23% (p=0.008). after stereotactic radiosurgery, whole brain radiation therapy reduced the probability of relapse at initial sites from 31% to 19% (p=0.04) and at distant intracranial sites from 48% to 33% (p=0.023) at 2 years. the median progression-free survival was slightly longer in the whole brain radiation therapy arm compared with the observation arm (4.6 months versus 3.4 months; p=0.02) but there was no difference in overall survival between the two arms. eighty-one percent of the patients had single metastases but there was no analysis of one versus more than one metastases. this trial included neurocognitive and the quality of life assessments which have not yet been reported. there are also a number of single institution retrospective series in melanoma patients with their inherent selection biases. reviewed the outcomes of 686 patients at the sydney melanoma unit (now the melanoma institute australia). there was no significant difference in the median survival between the 158 patients treated with surgery and whole brain radiation therapy and the 47 patients treated with surgery alone (8.9 months versus 8.7 months, p=0.21). sampson et al. also reported no difference in median survival in patients treated with whole brain radiation therapy after surgery or surgery-alone patients (median survival of 9 months, p=0.99). however, patients treated with whole brain radiation therapy were more likely to remain without neurological deficits or experience an improvement (81.7%) after completion of therapy than those who did not (57.7%, p=0.01). treatment was stereotactic radiosurgery alone (61 patients), stereotactic radiosurgery with whole brain radiation therapy (12 patients), and salvage stereotactic radiosurgery after whole brain radiation therapy (30 patients). the overall incidence of distant brain metastasis-free survival did not differ significantly between the group that received initial stereotactic radiosurgery alone and the group that received stereotactic radiosurgery and whole brain radiation therapy (17.6% versus 0%, p=0.27). however this study did not have the statistical power to detect a difference in distant brain metastasis free survival. the initial number of brain lesions (single versus multiple) was the only factor with a significant effect on distant brain metastasis-free survival at 1 year: 23.5% for single metastases and 0% for multiple lesions (p<0.05). samlowski et al. performed a retrospective analysis of 44 melanoma patients with five or less brain metastases treated with stereotactic radiosurgery and showed that the addition of whole brain radiation therapy did not improve survival. survival analysis showed that combined treatment of local and whole brain radiation therapy offered significantly better survival (p<0.0001). the median survival was 8.8 months for the combined therapy group, 4.8 months for the local-therapy-alone group. md anderson reported a series of patients with solitary melanoma brain metastasis and no extracranial disease. twenty-two patients received surgical excision and whole brain radiation therapy whole brain radiation therapy and 12 patients were treated with surgery alone. despite the small sample size, intracranial recurrence rates favoured the combination (5/22 versus 9/12 surgery alone, p=0.01). median overall survival was 18 months in the combination therapy group versus 6 months for surgery alone (p=0.002). these data argue that whole brain radiation therapy can decrease intracranial progression and may even convey a survival benefit in patients without active extracranial disease as a competing cause of death. one concern of delivering whole brain radiation therapy after local treatment of oligometastases is the potential neurological deficit. preclinical and early clinical evidence suggests that a neural stem cell compartment in the hippocampus is central to the pathogenesis of neurocognitive deficits observed after cranial irradiation. modern intensity-modulated radiotherapy technologies, such as helical tomotherapy and volumetric modulated arc therapy can conformally avoid the hippocampus during whole brain radiation therapy and therefore potentially reduce the risk of neurocognitive deficit. a rtog review of 371 patients with less than 10 brain metastasis from all histologies showed that only 3% of the metastatic deposit were within the 5 mm region around the hippocampus and none within the hippocampus itself. there is an ongoing clinical trial (rtog 0933) examining the effect of hippocampal avoidance whole brain radiation therapy technique on the neurocognitive function in patients with brain metastases from all histologies. the role of radiation therapy in the management is highly variable due to the natural history of the disease. to provide optimal management of the patient with melanoma although there have been no randomized trials especially in patients with melanoma brain metastasis, treatment can be guided by the application of evidence for the treatment of brain metastasis in general. a promising new approach to deliver radiation therapy while sparing the hippocampus

brain metastasis is common in patients with melanoma and represents a significant cause of morbidity and mortality. there have been no specific randomized trials for patients with melanoma brain metastasis, so treatment is based on management of brain metastasis in general and requires multidisciplinary expertise including radiation oncology, neurosurgery, medical oncology, and palliative care. in this paper, we summarize the prognosis, general management, and the role of radiation therapy in the management of metastatic melanoma in the brain.

PMC3332202

pubmed-780

growing recognition that resources for health care are scarce has led to broad acceptance that the evidence base should include economic as well as clinical evidence. in the uk, this is reflected in the work of the national institute for health and clinical excellence (nice) whose national guidance on health care for england and wales is explicitly informed by evidence of cost effectiveness as well as clinical effectiveness. the principal benefits from most clinical treatments are in the form of health gains to patients. while there can also be nonhealth benefits, for example, from earlier return to work or in terms of reduced burden on informal carers, these tend to be relatively small. since an objective of all health care systems is to maximise the amount of health produced (allowing for other objectives such as equity), it is not surprising that the economic questions most commonly addressed when evaluating clinical treatments are concerned with identifying the most cost-effective ways of producing health. there is now a growing movement toward incorporating the principles of evidence-based medicine in evidence-based public health. at the end of 2005, nice announced that it was extending its remit to include guidance on the promotion of good health and the prevention of ill health thus explicitly recognising the contribution of public health in improving health. of these, 1235 (73%) were classed as cost effectiveness (cea) or cost utility (cua) studies which assess how 68 cost effectively a public health programme produces health. only 43 studies (2.5%) were classed as cost benefit analyses (cba)the technique of economic evaluation which addresses the broader issue of whether or not a public health programme is worthwhile. this paper considers the fundamental differences between cea/cua and cba in the context of providing an evidence base to inform public health policy. using selected studies from the database produced by the team that undertook the review, this paper examines how information provided by cea/cua studies might lead to inefficient recommendations for public health policy. it does this by briefly reviewing the principles of the different techniques of economic evaluation, considers the fundamental differences between public health and clinical treatments in terms of their objectives, and then, on the basis of information from a selection of studies, suggests a simple method for assessing where cea/cua of public health programmes might make a misleading contribution to the economic evidence base for public health. within health care, a cost effectiveness analysis will assess an intervention against a comparator in terms of cost per unit of health effect achieved. these units can be specific, for example, true positive cases of presymptomatic disease detected in a screening programme, or more generic, for example, life years saved. in cost utility analysis, the unit of effectiveness is the quality adjusted life year (qaly) or other single index measures which capture both life length of life and quality of life. the technique's name refers to the fact that the quality of life element is determined by the utilities, or values, attached to different health states. thus while cua outcomes take account of preferences they can still be regarded as a form of cea since they seek to find the least cost way of producing a health-related unit of effect. nice determined early on that, where possible, cua was the preferred form of analysis to provide the economic evidence for health care interventions. cea/cua, however, only compare alternatives ways of pursuing a single objective in this case to maximise health gain. an unambiguous result, however, is produced only if the intervention in question is both more effective and less costly than the comparator (or vice versa). dominant and there are no economic arguments for not adopting it over the comparator. whether or not the higher costs are worth incurring, that is, whether additional resources should be allocated to the treatment of these patients is an allocative efficiency question which can not be answered by cea/cua. in order to assist decision makers, nondominant results are commonly presented in the form of incremental cost effectiveness ratios (icers) which show the extra cost of achieving the extra health effects. cost effectiveness acceptability curves (ceacs) deal with the uncertainty surrounding the estimates by showing the probability that a nondominant intervention will have an icer below a range of thresholds which represent the maximum amounts that a payer would be willing to pay for an extra unit of effect. for example, nice currently regards an icer of 30 000 per qaly as being at the upper limit for the interventions to be recommended for use in the british national health service. while the question of how much society is willing to pay for extra health benefits remains a live issue, the focus on assessing the cost effectiveness of clinical treatments through cea/cua remains the norm. results of these analyses clearly depend on which costs, including cost savings, are included, that is, on the perspective adopted. in the uk costs (positive and negative) to other sectors are not considered at least in the primary analysis. this remains a contentious issue as exemplified in a recent nice appraisal of drugs for alzheimer's disease where a major issue was whether or not to include cost borne by informal carers and a recent editorial in the british medical journal has called for a rethink on continued use of a narrow perspective even for health service interventions. the uk faculty of public health has adopted sir donald acheson's definition of public health as the science and art of preventing disease, prolonging life and promoting health through organised efforts of society. the faculty regards the key elements of public health as being population based, emphasising collective responsibility for health, recognising the key role of the state, and emphasising partnerships with all those who contribute to the health of the population. these principles make it clear that public health programmes can produce health in ways which do not necessarily involve health professionals or involve the use of health services. while these principles are captured in most definitions of public health, the definition by allin et al. ends with and involves mobilising local, regional, national and international resources to create conditions in which people can be healthy in contrast to producing health by treating illness, creating the conditions in which people can be healthy will often achieve important nonhealth benefits in addition to health. evidence that such nonhealth benefits are positively valued was demonstrated in a study by cropper which examined people's preferences for different life saving programmes. when asked to choose between programmes, a belief that a programme would produce benefits in addition to life saving was shown to significantly increase the probability of that programme being preferred over another with the same life saving benefits. cost benefit analysis (cba) is the technique of economic evaluation which addresses allocative efficiency. it explicitly addresses the question how much more or how much less of society's resources should be allocated to achieving this goal or to this type of healthcare ?. its foundations are rooted within welfare economics where the aim is to assess how social welfare is affected by a particular project. cba does this by identifying and measuring all costs and all benefits; defined as everything of value that results (positive or negative) and regardless of who gains or loses, that is, using a societal perspective. when all gains and losses are measured in commensurate terms (i.e., money) healthcare objectives can be compared with each other or with those in other sectors of the economy. if the total value of the benefits (gains) exceeds the total value of the costs (losses), then the proposal passes the cost benefit test and total social welfare is increased by implementing the programme. although in principle cea/cua are capable of capturing avoided costs in sectors other than health care, these analyses would still not capture the full range of benefits that would be picked up in a cba. for example, a cua of a proposed policy to reduce air pollution would be based on the narrow premise that the objective of the policy is solely to produce health. if the analysis showed an incremental cost/qaly above a predetermined threshold, even if that analysis included cost savings to sectors other than health such as reduced cleaning costs due to reduced pollution, the implication would be that resources should not be allocated to this intervention. a cba, however, which included nonhealth as well as health benefits say the value attached to breathing clean air independent of any health implications might show the same proposal to pass the cost benefit test. in this example, a policy decision taken on the basis of the cua would mean forgoing an opportunity to improve social welfare. it is, of course, possible that nonhealth benefits could be negative which reinforces the importance of not excluding them, particularly where they are significant. although economists have developed numerous methods to assign money values to costs and benefits which do not have associated market prices, this inevitably is not an easy task which might explain why comprehensive cbas remain rare. it is well recognised that many studies which include the word cost benefit analysis in their title are, in reality, not cba studies at all. such mistitled studies frequently use that term because they regard cost savings from avoided future illness as benefits and thus feel that their analysis has covered both sides of the cost benefit calculus. some economic studies avoid valuation problems by simply listing the costs and consequences of any activity without aggregation. such cost-consequences analyses (ccas) are, strictly, not economic evaluations as they can not provide answers to either cost effectiveness or allocative efficiency questions. however, by identifying costs and consequence they can be an important aid to decision making beyond cost effectiveness ratios and a recent report from the public health research consortium in the uk has called for the intersectoral impacts of public health interventions to be presented in the form of a cost-consequences analysis. in terms of an emerging public health evidence base, there thus appears to be a problem. if the benefits from public health interventions frequently include more than just health gains then, arguably, its evidence base ought to include a smaller proportion of cost effectiveness and cost utility analyses than does the evidence base for clinical treatments. nevertheless, as shown in the review, nearly three quarters of the economic evaluation undertaken in the area of public health have to date been cost effectiveness or cost utility studies. in order to illustrate the partial nature of the information provided from cea/cua evaluations in public health, hence the potential for inefficient health policy, the present study examined the nature and relative importance of benefits which were included in some of the economic studies which went beyond cost effectiveness or cost utility analyses in the recent review. studies from the review were identified for possible selection if they were not classed as cost effectiveness or cost utility studies. it was evident from examination of abstracts, however, that despite their classification many of these studies had limited their analyses to health benefits alone. omission of nonhealth benefits, even where they might be significant, is not necessarily a weakness in these studies. if the value of the health benefits alone can be shown to exceed the value of all of the costs, then the intervention passes the cost benefit test without the need to consider any nonhealth benefits. including them would only reinforce the conclusion already reached although clearly, incomplete assessment would make comparisons of the benefit: cost ratios between programmes problematic. of these two had been classed within the review as cba; the remainder being cca or multimethod apart from one case where no classification was given. the work by aunan et al. evaluated the costs and benefits of implementing to reduce air pollution programme. reduced damage to public health, building materials, and agricultural crops from reduced emissions of air pollutants the possible benefits from implementing the measures described by the national energy efficiency improvement and energy conservation program were evaluated using saved energy from various sectors: households, transportation, industry, service, energy, and agriculture. health benefits included those from acute respiratory symptoms, chronic respiratory symptoms, infant deaths, and lung cancer. crop loss due to so2 was seen to be a great concern for crop production. the analysis indicated that the annual benefit of improved health alone is likely to exceed the investment needed to implement the programme. thus, the policy would pass the cost benefit even without inclusion of the significant benefits due to reduced damage to materials and crops. the work by miller et al. modelled the potential health and economic impacts of implementing a medically prescribed heroin programme among canadian injecting drug users over 5 years. the potential impact of the programme was estimated by comparing hospitalisation and emergency use costs. reductions in criminal activity costs accounted for fully 63% of the total reduction in costs. other costs avoided since the implementation of the programme such as the costs of social housing, use of social services, counseling, and employment programs were identified but not included in the model. although this study did not claim to be a cba it provides an example of an intervention whose nonhealth benefits were significantly larger than the health benefits. had the researchers attempted a cba, the nonhealth benefits could have made the difference between the programme passing or failing the cost benefit test. the work by zeng-sui et al. assessed the impact on enteric infectious disease by providing deep-well tap water across six villages in china. health benefits included reductions in diarrhoea, dysentery, viral hepatitis, cholera, and reduce mortality form liver cell cancer. nonhealth benefits included reductions in lost wages or earnings of patients and of their relatives who looked after them during the illness and the avoided costs of transportation, supplemental nutrition, and the value of gifts sent by relatives to assist towards their recuperation (but interestingly not the value of the gift to the recipient which illustrates how the gifts should have been regarded as a financial transfer rather than an economic cost avoided). for example, water-related conditions such as skin and eye infections, dermatosis, gynaecological conditions, parasitic enteric diseases, and vector-borne diseases were mentioned but not included. other intangible benefits such as the improved service that will benefit future generations were also mentioned but not included in the analysis. overall assessed benefits were more than double the costs. the work by guria et al. evaluated the incremental outcomes of road safety programmes and driving campaigns enforced in new zealand and compared them with their resource costs. in addition to loss of life and reduced quality of life resulting from injury, the study also included the social costs of injuries and property damage avoided. other benefits such as the development of a safety culture, improvement of road user behaviour, and the safety quality of vehicles were mentioned but not included. the study showed that road safety programmes aimed at reducing high-risk behaviours produced high returns. if 90% of road safety expenditure is attributed to the period of investment, then the benefit to cost ratio would be 12.3: 1 for 19931995 and 7.9: 1 for 19941996. the work by aehyung et al. presented a cba of the onchocerciasis (river blindness) control programme. nonhealth benefits included additional agricultural output as a result of a more productive labour force and additional agricultural land made available through the control of onchocerciasis. other nonhealth benefits such as the reduction of lost production time by family members when providing care and improved parenting were mentioned but not included in the study. the net present value (npv) ranged from us$485 million to us$3,792 million (1987 dollars) depending on the assumptions used. a positive npv is another way of saying that the programme passed the cost benefit test. the work by fleming et al. estimated the costs and benefits of brief physician advice with problem drinkers in primary care settings. health care benefits included avoided cost from the perspective of the managed care organisation, the use of equipment, personnel, emergency medical care, hospitalisations, treatments, and clinic visits. the study indicated that physician-delivered advice can reduce not only medical costs but also social costs associated with alcohol consumption. the total economic cost of the intervention was $80,210, or $205 (1993 dollars) per study patient. the study by cohen et al. estimated the potential benefits from saving a high-risk youth by estimating the lifetime costs associated with the career criminal drug abuser and high school drop out. antisocial behaviour of career criminals was included as an externality and thus seen as imposing as a social cost. assessed nonhealth benefits included the avoided social costs from stolen property and lost wages. as this study did not examine the costs of interventions aimed at reducing antisocial behaviour, it was not a cba. however, the range and magnitude of the nonhealth benefits which were included in the valuation exercise illustrates what would be missed if a cea/cua study focussing solely on the health benefits had been undertaken. the work by caulkins et al. on school-based drug prevention programmes focused on reducing drug consumption, particularly cocaine, as an objective of the nation's drug control efforts. the study reported the quantity of cocaine consumed, the cost of drug use, and the social value of cocaine control. benefits from the prevention program included reductions in the use of other drugs including marijuana, alcohol, and cigarettes. nonhealth benefits included lower crime rates, higher productivity, and an increase in the number of pupils graduating from high school graduation. the programme was deemed to be affordable and social benefits were shown to exceed the total costs which justified implementation of the programme. the work ginsberg et al. estimated the costs of making the wearing of bicycle helmets compulsory in israel. benefits included resource saving from fewer head injuries in terms of hospitalisation, emergency room visits, ambulatory care, rehabilitation, long-term care, and special education. the authors called their analysis conservative as it did not consider reduced pain, worry, grief, work losses for ambulatory visits or even time off from housework as a result of bicycle injuries. nor did we consider the intangible benefits of the lessening of anxiety concerning crashes by cyclists or by their friends and relatives. inclusion of these additional benefits was unnecessary as the partial benefits (total=us$60.7 million) clearly exceeded total costs (us$20.1 million) without their inclusion (dollar base year not stated). in this example, the health service savings alone (us$44.2 million) were sufficient for the proposed policy to pass the cost benefit test. if, however, the results showed that the productivity gains (us$7.5 million) were needed to tip the balance, then their omission would have meant a lost opportunity to increase social welfare. benefits included savings from hospital admissions, professional services, rehabilitation, prescriptions, home health care, and medical equipment. the study also included benefits from avoided productivity losses due to children not being able to work when they became adults if they were killed or permanently disabled and included a value for avoided pain, grief, and suffering. for example, it mentioned but did not include productivity losses from parents dealing with injured children. nevertheless, it still showed a benefit to cost ratio of nearly 13: 1. in this case, however, annual productivity benefits ($ 660 million) were nearly 3 times those of health service benefits ($ 230 million) (1992 dollars) which in a higher-cost programme might have made the difference between passing or failing the cost benefit test. it is evident that many public health interventions produce benefits in addition to health and in many cases these can be substantial. some of these involve resource savings which, if regarded as negative costs could in principle be included in a cea/cue provided that a societal perspective were adopted. others, however, for example, reductions in criminal activity, are clearly of value independent of any cost savings and these would not feature in a cost effectiveness or cost utility study. the extent to which the noninclusion of nonhealth benefits in evaluation by cea/cua represents a problem in the sense that it could potentially lead to foregone opportunities to increase social welfare might be predicted by considering where the intervention in question would sit along a continuum of intent. at one extreme of such a continuum would be public health measures whose intent was solely to produce health. for example, a policy to add folic acid to flour has been advocated with the specific intent of reducing the incidence of neural tube defects (ntd) in newborns. where health gains are the sole objective of a public health programme then, on the same principles used within health care, they can be assessed in terms of cost effectiveness. if the addition of folic acid to flour were shown to have a low incremental cost/qaly, then this public health measure would be a cost-effective way of producing health relative to interventions within health care. even in this example, however, there could still be a case for directly addressing allocative efficiency through cba, for example, if women of childbearing age receive immediate reassurance from the knowledge that eating fortified food reduces their risk of conceiving a baby with an ntd. equally, making consumption of folic acid compulsory removes freedom of choice which to many could be a highly negatively valued nonhealth outcome. moreover, although there are no rules within the methodology of economic evaluation to prevent a cua being undertaken from a broad perspectiveand there have been recent calls to do just that such evaluations remain uncommon. in the folic acid example, the cost/qaly derived from a study which adopted a health service perspective would not include savings to other agencies such as special education which in the case of children with ntd could be substantial. further along the continuum of intent would be public health interventions where health is the primary concern but other objectives will clearly also be achieved. thus, while a road safety intervention may be advocated primarily to reduce injury and death on the roads, it is evident that a reduction in accidents will also produce savings in terms of property damage. the study by guria et al. on road safety in new zealand included property damage in deriving its cost benefit ratios. further still along the continuum would be public health interventions which clearly address multiple objectives. for example, illegal drug use is known to cause many social problems as well as health problems. thus the study by caulkins et al. included reduced crime and increased productivity among the benefits of schools-based drug prevention programmes, even taking account of intangible benefits such as an increase in the proportion of pupils graduating from high school. another example is the onchocerciasis prevention programme examined by aehyung et al. which, although driven by a desire to reduce the incidence of river blindness, would also free previously oncho-ridden tracts of land for settlement and cultivation. in both these examples, cuas would have given misleading information for policy. further still along the continuum would be programmes which could be perceived as being only incidentally preventive in the sense that the effect of pursuing another policy objective would incidentally have a positive effect on health. an example here could be improvements in housing which are undertaken to provide people with more pleasant places to live but which can at the same time affect respiratory illnesses or reduce injuries. the health effects of such programme can be assessed via a health impact assessment. ultimately almost any public policy can be seen as containing an element of public health. for example, macroeconomic policies to stimulate economic growth are clearly driven by concerns other than health, yet economic growth reduces unemployment and the relationship between unemployment and ill health is long established. public health programmes can have nonhealth benefits which may not be captured when a cost effectiveness/cost utility approach to economic evaluation is undertaken. a preanalysis examination of where any public health intervention would be located on a continuum of intent (relative importance of health versus nonhealth benefits) could identify where evaluation by cost effectiveness or cost utility analysis might produce inappropriate conclusions for policy. many of the public health programmes which to date have been assessed by cea/cua, in particular, those addressing smoking cessation have shown incremental cost effectiveness ratios which are far below current thresholds. in such cases, equally, where a cba is undertaken and the value of the health benefits alone is anticipated to clearly outweigh the costs, addition of nonhealth benefits will not affect the decision on whether to implement the programme and hence their inclusion is again unnecessary. most public health programmes, however, are unlikely to allow such obvious a priori conclusions to be drawn and it is here that consideration of where the programme sits along the suggested continuum of intent will increase the likelihood that the most appropriate technique of economic evaluation will be used.

economic evaluations of clinical treatments most commonly take the form of cost effectiveness or cost utility analyses. this is appropriate since the main sometimes the only benefit of such interventions is increased health. the majority of economic evaluations in public health, however, have also been assessed using these techniques when arguably cost benefit analyses would in many cases have been more appropriate, given its ability to take account of nonhealth benefits as well. an examination of the nonhealth benefits from a sample of studies featured in a recent review of economic evaluations in public health illustrates how overfocusing on cost effectiveness/cost utility analyses may lead to forgoing potential social welfare gains from programmes in public health. prior to evaluation, programmes should be considered in terms of the potential importance of nonhealth benefits and where these are considerable would be better evaluated by more inclusive economic evaluation techniques.

PMC2798564

pubmed-781

prostate cancer is the second most common cause of cancer death in men in the united states. in the united states, the age-adjusted number of new cases of prostate cancer was 147.8 per 100,000 men per year based on 2007~2011 data and the age-adjusted number of deaths was 23.0 per 100,000 men per year based on 2006~2010 data. according to 2008~2010 data, approximately 15.3% of men will be diagnosed with prostate cancer at some point during their lifetime, and in 2011, an estimated 2,707,821 men were living with prostate cancer in the united states.1 the most common sites of prostate cancer metastasis include the bone, lymph nodes, lungs, liver, pleura, and adrenal glands.2 rarely has metastatic prostate cancer been reported to involve the gastrointestinal tract.3,4,5,6,7,8,9,10,11 here, we report the case of a 64-year-old man with prostate cancer that had metastasized to the rectum and stomach. a 64-year-old african-american man presented with anemia (hemoglobin level of 6.8 g/dl) in september 2012. he had a medical history of prostate cancer (gleason score of 5+4=9) diagnosed in june 2011, hypertension, coronary artery disease with stent placement, and insertion of bilateral percutaneous nephrostomy tubes due to hydronephrosis. the patient was being treated with flutamide and leuprolide acetate for prostate cancer since june 2011. in september 2012, he presented with the complaint of dark-colored stools for a week. at that time, he did not experience any chest pain, shortness of breath, palpitations, dizziness, or headache, and no history of hemoptysis or hematemesis was reported. physical examination of the patient was unremarkable except for the findings of a cachectic elderly man with pale conjunctiva and bilateral nephrostomy tubes. laboratory data on admission revealed a hemoglobin level of 6.8 g/dl, hematocrit of 21%, platelet count of 281,000/mm, white cell count of 4,500/mm, and normal prothrombin time and activated partial thromboplastin time. iron studies revealed a total iron level of 80 mg/dl, total iron binding capacity of 227 mg/dl, ferritin level of 357 ng/ml, and reticulocyte count of 0.9%. a complete metabolic profile showed a blood urea nitrogen level of 54 mg/dl and a creatinine level of 3.3 mg/dl. the patient had a computed tomography (ct) scan performed without contrast on his abdomen and pelvis. the ct scan revealed multiple cystic lesions in the liver, marked thickening of the wall of the stomach (fig. the patient also underwent a colonoscopy, which revealed circumferential nodularity and poor distensibility of the rectal lumen (fig. biopsy of the rectum indicated diffuse infiltration of high-grade neoplastic cells that were positive for human prostatic acid phosphatase and prostate-specific membrane antigen. these findings were consistent with a diagnosis of metastatic, poorly differentiated carcinoma of the prostate. since it was known that the patient had hormone-refractory prostate cancer, he was started on docetaxel and received 3 cycles until february 2013, at which point he was lost to follow-up. in may 2013, microscopic examination revealed sheets of atypical cells with prominent nucleoli and no glandular pattern within the gastric lamina propria (fig. immunohistochemical studies demonstrated that the tumor cells were positive for prostate specific antigen (psa) and alpha-methylacyl-coenzyme a racemase (amacr, p504s) (fig. the patient's serum psa level was more than 1,000 mg/dl at that time. the bone scan, as well as the ct scan of the abdomen and pelvis, showed no metastases but showed retroperitoneal lymphadenopathy. at that time, the patient had an eastern cooperative oncology group performance status of 3. the patient and family refused further chemotherapy for metastatic prostate cancer and opted for palliative care instead. the patient was admitted multiple times for similar complaints and was managed with supportive care. prostate cancer is the most common noncutaneous cancer in men in the united states and is among the most commonly diagnosed cancers in many developed countries. classic risk factors for this cancer include older age, african-american race/ethnicity, and a family history of prostate cancer. the increase in prostate cancer incidence rates in groups that have migrated from countries with low rates to countries with high rates strongly suggests the importance of environmental factors in its etiology. kolonel12 reported that the the incidence rates of prostate cancer steadily increased in the japanese group with migration from mainland japan to hawaii, and the united states mainland in that order based on the ethnic studies. the majority of men are diagnosed with prostate cancer at an age older than 65 years, and the vast majority of prostate cancer deaths occur in this older age group. the median age at prostate cancer diagnosis is 71 years in caucasians and 69 years in african-americans in the united states. metastatic prostate cancer has a poor prognosis and median survival time ranges from 1 to 3 years.3 prostate cancer preferentially spreads to the skeleton. more than 80% of men who die from prostate cancer are identified with bone metastases at autopsy.13 in contrast to most other cancers, prostate cancer predominantly forms osteoblastic metastases. the vertebral column, pelvis, ribs, and proximal long bones are the most common sites of skeletal metastases. hematogenous, lymphatic, and direct infiltrations are the typical routes of spread.14 patients with prostate cancer can be anemic due to bone marrow involvement. metastasis of prostatic carcinoma to the gastrointestinal tract is a very rare occurrence and presents a diagnostic challenge.6,7,8,9,10,11,13,14 because the prostate is richly supplied with lymphatic channels, metastasis to the gastrointestinal tract may occur via the lymphatic route. this should be taken into consideration during the work up for anemia in a patient with advanced prostate cancer, as supportive management will depend on the etiology of the anemia. in patients with suspected gastrointestinal bleeding or signs of iron deficiency anemia, upper and lower endoscopy two postmortem studies reported gastric metastasis from primary prostate cancer in 1% to 4% of cases.15,16 histologically, primary adenocarcinoma of the stomach is composed of atypical glands with cribriform (back to back) formation and a mucin-producing infiltrating growth pattern. carcinoma in situ or high-grade dysplasia is usually found adjacent to the carcinoma. in contrast, tumor cells of metastatic carcinoma from prostate cancer usually have prominent nucleoli and no glandular formation. the prostate biomarker amacr has been used in conjunction with morphology with very high sensitivity and specificity in diagnostically challenging cases. amacr, also known as racemase or p504s, is an enzyme identified by cdna subtraction and microarray technology. it is a sensitive and specific immunohistochemical marker that has been found to be consistently up-regulated in prostate carcinoma.17,18 treatment for metastatic prostate cancer is palliative. several new agents have been introduced for the treatment of metastatic prostate cancer in the past two decades, with excellent disease control and good patient tolerability. the management of advanced prostate cancer with metastases to the gastrointestinal tract includes local control measures, supportive care, and treatment of the underlying cancer. if the disease progresses and hormone-refractory metastatic prostate cancer is diagnosed, alternative treatments include chemotherapy, immunotherapy with sipuleucel-t, androgen receptor antagonist drugs such as enzalutamide, and androgen synthesis inhibitors such as abiraterone. although rare, it is important to consider the possibility of prostate carcinoma metastasizing to the gastrointestinal tract in patients presenting with gastrointestinal bleeding and a history of prostatic adenocarcinoma. it is crucial to distinguish primary gastrointestinal cancer from metastatic lesions, especially in cases of a previous history of cancer at another site, for appropriate management. this can be achieved by determining the histopathologic classification of the tumor and by immunohistochemical staining for psa.

prostate cancer is the second most common cause of cancer death in men in the united states. the most common sites of metastasis include the bone, lymph nodes, lung, liver, pleura, and adrenal glands, whereas metastatic prostate cancer involving the gastrointestinal tract has been rarely reported. a 64-year-old african-american man with a history of prostate cancer presented with anemia. he reported the passing of dark colored stools but denied hematemesis or hematochezia. colonoscopy revealed circumferential nodularity, and histology demonstrated metastatic carcinoma of the prostate. esophagogastroduodenoscopy showed hypertrophic folds in the gastric fundus, and microscopic examination revealed tumor cells positive for prostate-specific antigen. bone scanning and computed tomography of the abdomen and pelvis did not show metastasis. it is crucial to distinguish primary gastrointestinal cancer from metastatic lesions, especially in patients with a history of cancer at another site, for appropriate management.

PMC4286907

pubmed-782

while the country relies heavily on its natural resources and agricultural land to provide food and livelihoods for its rapidly growing population, the inland fisheries sector is perhaps the most valuable, which officially accounts for about 12% of gross domestic product (gdp) and provides most cambodians with their key source of animal protein, calcium, and vitamin a. the pollution of the aquatic environment with both essential and nonessential elements has attracted serious concern in the recent years because they are indestructible and most of them have toxic effects on organisms. heavy metals, particularly cadmium, lead, and arsenic constitute a significant potential health threat to human. fish is one of the sources of protein, vitamin, and mineral, and it contains essential nutrients required in human diet. fish has been widely accepted as a very important source of animal protein for supplementing both infants and adults diet [5, 6]. fish drying is an age long practice of processing fish across the world to prolong its shelf-life and to conserve the quality. it is a slow process and when drying of these animal products took a long time, bacterial spoilage during the slow operation occurred. besides, osd will not lower moisture content below about 15%, which is still too high for storage stability of food products. it also exposes products to birds, insects, and rodents and makes the products susceptible to contamination with foreign materials, such as dust and litter. the main contaminants that are likely to arise from bird droppings include fungus such as histoplasma capsulatum and cryptococcus neoformans and bacteria chlamydophila psittaci. in addition, insects and fungi that thrive in moist conditions render the products unusable. in recent times, smoking kiln and solar drying system (sds) are used to obtain product of high quality. sds is a solar energy process that is well matched for drying of agricultural and fishery products in the tropical and subtropical countries. according to, phnom penh receives an average of 5.3 hours of full sun every day with an average of about 2,490 hours of sunshine per year. the maximum fluctuation in solar radiation volume throughout the year is relatively low and has been estimated at 17%. many studies have reported on various methods of fish drying in tropical and subtropical countries. r. m. davies and o. a. davies reported six different types of traditional fish processing techniques in nigeria whereas an experimental study was carried out on solar tunnel dryer to dry fish. a hybrid solar drying system with diesel burner was employed to dry salted silver jewfish in johor, malaysia [13, 14], and its drying characteristic was compared under open and solar drying. the effect of traditional fish processing was investigated in terms of its nutritional value, proximate composition of raw and cooked thai freshwater and marine fish, mineral composition, and proximate analysis of dried salted molva molva l. and merluccius merluccius l.. changes in nutritional and chemical composition of fried sardine (clupea pilchardus) were reported to be produced by microwave reheating and frozen storage. it has been observed that different drying methods and processing have different effect on nutritional composition of fish. the skin is compared to the flesh because the skin of channa striatus has been known to possess toxic and lethal components despite being edible among the local people. bearing in mind that the quality of dried fish using different drying methods can not be the same, as such, the objective of this study is to investigate the toxic and essential elements of striped snakehead fish (channa striata) dried using osd and sds in cambodia. samples of the striped snakehead fish (channa striata) as shown in figure 1 were obtained from cambodia. the mean length and weight of fresh fish were about 35 cm and 600 g, respectively. the striped snakehead fish was gutted and washed before cutting them prior to be subjected to two different methods of drying (osd and sds). the striped snakehead fish was soaked in separate containers that contained a 25% (w/v) brine solution of nacl for 4 hr. a fish-to-brine ratio of 1: 4 l was used [22, 23]. solar drying system is shown in figure 2(a), which comprised the forced-convection indirect type. the system consisted of a v-groove solar air collector, fans, electric heater, and drying chamber. the solar collector was of the back-pass v-groove which was connected in series. experiment was done between 9:00 a.m. and 5:00 p.m. corresponding to the sunshine duration in cambodia. drying experiment has been done on 62 kg striped snakehead. it was divided equally and then placed on 12 trays, as shown in figure 2(b). during this process the drying temperature setting in drying chamber was fixed at 50c and the flow rate was fixed at 0.07 kg/s. the data measured were air temperature (ambient temperature, air temperature inlet, and outlet of the collector), solar radiation, and air velocity, as well as the air temperature before it entered the dryer chamber, the temperature inside the dryer chamber, and the temperature of the air out of the dryer chamber. air temperature was measured by t-type thermocouple, and the intensity of solar radiation was measured by pyranometer. the striped snakehead fish was placed on the bamboo tray in osd from 9:00 a.m. to 5:00 p.m. at night, fishes were piled in plastic bins which were kept inside until the next morning and then continued to dry in the osd. the striped snakehead fish was dried until the final moisture content reached about 40% w.b. the trace and minor elements content in samples were determined by using inductively coupled plasma mass spectrometry (icp-ms elan 9000) (perkinelmer, sciex usa). the icp-ms was set with the condition as stated in table 1. for samples preparation, all the samples were processed using acid digestion method based on modified standard procedure. samples were rinsed using deionized water to get rid of all the contaminant, 5 g (wt/wt) of the samples was placed in the 50 ml beaker, and 10 ml of concentrated nitric acid was added. after the mixture was evaporated to the desired volume, 2 ml of hydrogen peroxide solution was added. the process of adding hydrogen peroxide solution, heating, and cooling were repeated until the sample solution turned into clear sight. sample solution was diluted with deionized water until the volume eventually reached 100 ml. for standard solution, multielement stock solution calibration number 3 from perkin elmer was used in this study. the aim of the present study is merely to compare the level of the toxic and essential elements in the striped snakehead fish using osd and sds methods of drying instead of analyzing the quality of the fried fish product; hence, analysis of microbiology was not yet considered. performance of sds for striped snakehead fish is shown in table 2. in this study, the inductive coupled plasma mass spectrometry (icp-ms) assay has been used to measure the elemental content in striped snakehead fish sample. recoveries of trace metal contents in the present study are shown in tables 35. a total number of 12 trace and minor elements (pb, cd, as, zn, mn, cu, cr, mg, mo, fe, ni, and se) in the dried striped snakehead fish from cambodia have been determined using icp-ms after acid digestion. the concentration of 12 elements was determined based on their classification of toxic metals (as, pb, and cd) in table 3 and nutritional trace elements (fe, mn, mg, se, mo, cu, ni, zn, and cr) in tables 4 and 5. the test was done in triplicates with the number of samples n=3 for each analysis. table 3 shows the concentration of three heavy metals content in the skin and the flesh of the fish using two different drying methods. out of these three toxic elements, as was detected at the highest concentration followed by pb and cd. as far as the skin samples are concerned, the fish subjected to osd showed higher level of as, pb, and cd (8.72 ppb, 5.72 ppb, and 0.54 ppb) compared to the samples dried by sds (7.32 ppb, 3.97 ppb, and 0.30 ppb). on the other hand, the flesh of the fish also accumulated high amount of as, pb, and cd (11.53 ppb, 3.87 ppb, and 0.31 ppb) during the process of osd compared to when sds technique (10.17 ppb, 1.43 ppb, and 0.18 ppb) was used. however, the levels of arsenic in both skin and flesh samples using both drying methods were well below the acceptable limit of 130 ppb for arsenic. the acceptable limits for pb and cd are 240 ppb and 60 ppb, respectively. as far as the skin samples are concerned, the nutritional trace elements were higher in the samples subjected to sds with concentration of between 3.41 ppb and 2,019.69 ppb compared to osd skin sample of within the range of 2.55 ppb to 1,550.55 ppb recorded for se, mn, ni, cu, cr, fe, mo, and mg as presented in table 4. this means that the beneficial trace metals were conserved in the skin of the fried fish during the process of drying by sds technique. generally, it was seen in table 5 that sds method of drying produced lower concentration of the beneficial trace elements (mn, se, cr, fe, mo, and mg) in the flesh of the fish samples compared to using conventional osd method of drying the fish. the levels of mn under sds and osd method were, respectively, 4.68 ppb and 4.73 ppb compared to the tolerable upper intake level (ul) for manganese in 70 kg adult at 11,000 g per day which corresponds to 157 ppb. although low level of manganese intake is necessary for human health, exposure to high manganese level has the potential to cause neurotoxicity. as far as selenium is concerned, the sds and osd method displayed its concentration of 5.52 ppb and 5.62 ppb. according to the institute of medicine, the recommended dietary intake of selenium is 55 g per day for 70 kg adult equivalent to 0.80 ppb. the se levels in both samples are still considered within acceptable limit for consumption because the tolerable ul limit for selenium in 70 kg adult was set at 400 g per day which corresponds to 5.71 ppb. in one study, selenium is reported to reduce vulnerability to mercury toxicity in humans and has protective effect for neonates against neurotoxicity from prenatal mn exposure. chromium was detected at, respectively, 9.16 ppb and 10.67 ppb in sds and osd dried fish which is 20 times greater than the recommended average daily intake level (adequate intake) in 70 kg adult of 35 g per day for 70 kg adult equivalent to 0.50 ppb. however, no adverse effects have been convincingly associated with excess intake of chromium from food or dietary supplements. besides, it has been found that some fish are capable of bioaccumulating cr level nearly 100 times the concentration of cr in the water. the levels of fe under sds and osd method were, respectively, 98.80 ppb and 130.71 ppb compared with the tolerable ul for iron in 70 kg adult was set at 45,000 g per day which corresponds to 642.86 ppb. the levels of mg under sds and osd method were, respectively, 3000.58 ppb and 3231.07 ppb compared with the tolerable ul value for magnesium in 70 kg adult was set at 350,000 g per day which corresponds to 5,000 ppb. it can be deduced that the levels of mn, se, cr, fe, and mg in the muscle of striped snakehead fish using both methods of drying were generally low when compared with the ul limit values. this is not the case for the level of molybdenum which recorded 239.04 ppb and 272.04 ppb in sds and osd samples. logically, this is not considered safe for human consumption because it is almost 100 times the tolerable ul value for mo in 70 kg adult as was set at 2,000 g per day or 28.57 ppb. nevertheless, the extremely high level of molybdenum in fishery products will not cause any harmful effects associated with high molybdenum level in human such as gout, anemia, and symptoms of copper deficiency. this is because of the rapid renal clearance of the majority of ingested molybdenum, which will likely prevent deleterious effects in the event of high intake. it is interesting to note that when sds method was employed, the nutritious elements (mn, cr, fe, and mo) which were detected in high quantity in the skin of the fish samples (table 4) were found to be in lower amount in the flesh of the same sample of the fish (table 5). this is expected because when these important trace elements were greatly concentrated in the skin of the fish, they would not be accumulated in high concentration in the flesh of the same sample of dried fish. ni and cu however demonstrated higher level in both the skin and flesh of the fish samples under the technique of solar-assisted drying compared to osd method. therefore, this finding actually supported using of sds method of drying the fish because the reddish coloration of the fishes dried under this condition is due to the presence of higher content of cu in both the skin and flesh samples of solar-powered drying compared to both the skin and flesh samples of dried fishes subjected to osd. in addition, the quality of fish was also preserved in the sds method whereby no formation mold was observed after 5 days of packaging (figure 4) compared to the presence of mold formation in osd dried striped snakehead fish (figure 5). the finding from this study is important because it demonstrated that the different method of fish drying can influence their elemental contents and it is recommended that the drying method using sds has proven to contain higher content of the nutritious trace elements compared to using the conventional open drying system. however, the level of toxic heavy metals only showed slight difference between the two systems. as conclusion, solar drying system is recommended for healthier eating and longer shelf-life of dried striped snakehead fish.

the content of 12 elements in cambodian dried striped snakehead fish was determined using inductively coupled plasma mass spectrometry. the present study compares the level of the trace toxic metals and nutritional trace elements in the fish processed using solar drying system (sds) and open sun drying (osd). the skin of sds fish has lower level of as, pb, and cd compared to the osd sample. as such, the flesh of the fish accumulated higher amount of toxic metals during osd compared to sds. however, arsenic was detected in both samples within the safe limit. the nutritional elements (fe, mn, mg, se, mo, cu, ni, and cr) were higher in the skin sample sds fish compared to osd fish. these beneficial metals were not accumulated in the flesh sample sds fish demonstrating lower level compared to drying under conventional system. the reddish coloration of the sds fish was due to the presence of high cu content in both the skin and flesh samples which possibly account for no mold formation 5 days after packaging. as conclusion, drying of cambodian c. striata using solar-assisted system has proven higher content of the nutritious elements compared to using the conventional system despite only slight difference in the toxic metals level between the two systems.

PMC4320906

pubmed-783

multiple sequence alignments (msas) are an essential first step for a number of computational approaches such as protein secondary structure/function prediction, phylogeny inference, and many other common tasks in sequence analysis.15 several software tools for generating multiple sequence alignments are available,6 but none of them is suitable for all types of data sets.7 consequently, in order to generate a true alignment, a need rises for inspection and adjusting alignments by hand, which is a very laborious job.5 furthermore, handling hundreds of thousands of alignments is another problem in the domain of bioinformatics. many popular alignment editors such as jalview,5 strap,8 seaview,9 mega,10 cinema,11 and base-by-base12 are available. all these tools either do not support tens of thousands of sequences (where length of each sequence is more than 2,500 base pairs) or they do not have user-friendly editing features. jalview and strap do not work when the size of an alignment exceeds 30.01 mb. seaview and mega can load big alignments, but the editing features provided by them involve multiple steps. many software tools provide graphical user interfaces to reconstruct msas, but they do not allow loading multiple sequence files at the same time. msa comparison tools such as suitemsa6 and sinicview13 permit to compare msas, but they support alignments comprising less than 1,000 sequences. multiple formats of msas exist, but fasta is the most popular format. presently, there is no tool that can convert the format of an alignment of unlimited size into fasta format. many tools, such as matgat14 and sequence identity and similarity (sias),15 are available, to calculate the identity matrix but they do not support more than 1,000 sequences. this article describes ivistmsa, which is a software package of seven graphical tools for multiple sequence alignments. all tools allow the user to load tens of thousands of sequences to edit/analyze and compare them. ivistmsa implemented a divide-and-conquer (dnc) approach to calculate efficiently consensus/conserved sequences, identity matrix, sum-of-pairs score (sps), colum score (cs), and conserved regions of two alignments. sps of a column is calculated as the ratio of the sum of scores of all pairs of residues in every column of test alignment by sum of scores of all pairs of residues in the similar column of reference alignment. to calculate sps of the entire alignment, sum of sum of scores of each column of the test alignment cs is computed as the ratio of matched columns (between test and reference alignments) by the number of considered columns in the test alignment. the sequence file with 807 sequences named as bba0039.tfa available in rv100 folder enclosed in a zipped file named as this data set was replicated to generate an alignment with 1,614 sequences, which was replicated to generate 3,228 sequences, and so on. replication was done in order to save time and avoid from constructing big alignments from an msa method. all tools of ivistmsa (fig. 1) are written in java programming language. xml was used for saving the state of the work performed in msapad and msa comparator. a computing machine having core i7 3.34-ghz processor and 8-gb ram was used to write and analyze ivistmsa. most of the tools of ivistmsa use dnc approach for performing efficient computations on msas. the dnc approach has been implemented using a power feature of multithreading provided by java programming language. dnc approach divides an alignment horizontally into subalignments, and java threads are generated for each fragment. all java threads return results to the main thread, which computes the final value. msapad uses dnc approach to compute consensus, conserved sequence(s), and distance matrix to construct a phylogenetic tree. step 1 calculates number of subalignments for a given msa. by default, each alignment is divided into two subalignments. step 2 creates a list/array for holding size of each sub-msa. step 3 calculates the size of the first subalignment and stores it into the first position of the list. step 5 subtracts one from the submsas variable because up to now, the size of the first sub-msa has been calculated. variable is used to store the size of subalignments at correct positions in the list. figure 2b shows the overall process of dividing msa into sub-msas, computations performed by each thread, and the final computation by the main thread. the alignment-rendering model has played a very important role to display, edit, and analyze very big alignments efficiently. the model sits between the alignment and its viewer and manages efficiently various types of manipulations on the alignments. these manipulations include finding a residue, all types of editing features provided by ivistmsa, changing color schemes, and loading the alignment itself. abstracttablemodel class provided as part of the swing library of the java programming language. results showed that, in contrast to all other alignment editors written in java programming language such as strap, jalview, and base-by-base, it can load more than 400% big alignments. msapad allows the user to save the alignment, find a single residue or sequence name, add the sequence at the start or end of an alignment, add a sequence before or after a selected sequence, and move a sequence up or down (fig. 4b). it can find consensus and conserved sequence/regions of an alignment of 120-mb size in just 27 seconds. it allows the user to calculate a phylogenetic tree using neighbor joining% identity and blosum 62 matrix. the tree is drawn using archaeopteryx, which is an open-source software tool for displaying and analyzing a phylogenetic tree. archaeopteryx and jmol are embedded to view and analyze phylogenetic trees and protein 3d chemical structures, respectively (fig. its version 1.0.2 is already published in life science journal.16 data structures used in mqatv1.0.2 were improved, and it is now more efficient than qscore program (http://www.drive5.com/qscore/) and fastsp as well. the new version of mqat allows the user to visualize the conserved and nonconserved regions of the selected alignments from the main window of mqat. figure 5a displays the main window of mqat, which allows the user to select an alignment. results showed that the msa comparator can display conserved and nonconserved regions of two alignments comprising more than 8,000 sequences with a sequence length of 2,696 base pairs in less than 12 seconds. msa reconstruction tool provides graphical interfaces for clustal omega, clustaw2, mafft, muscle, and biojava implementation for msas. its unique feature is that the user can load multiple sequence files at the same time. now the user does not need to sit before the system and wait for the completion of the process so that the next sequence file may be uploaded. using msa reconstruction tool of ivistmsa interfaces for clustal omega, clustalw2, mafft, muscle, and biojava can be loaded by clicking the msa reconstruction drop-down list (fig. fatsa generator can convert clustalw, msf, phylip, pir, gde, mega, and nexus formats of alignments of unlimited size into fasta format. we converted successfully an alignment of msf format comprising 102,102 sequences into fasta format in less than 1 second. the other important feature of this tool is that the user can load alignments of different formats. fasta generator recognizes the format of an alignment automatically and converts it into fasta format. msa i d calculator allows the user to calculate the identity matrix of an alignment. results showed that it can calculate the identity matrix of more than 11,000 sequences with a sequence length of 2,696 base pairs in less than 100 seconds. tree calculation tool calculates a phylogenetic tree using neighbor joining% identity and blosum 62 matrix and permits the user to draw the tree using archaeopteryx, which is an open-source software tool for drawing and analyzing a phylogenetic tree. ivistmsa is a suite of seven interactive visual tools to generate, view, edit, and analyze msas. presently, a lot of msa editing and analyzing tools are available. the popular and widely used tools include jalview,5 seaview,9 mega,10 strap,8 pfaat,17 base-by-base,12 and cinema.11 jalview, strap, pfaat, and base-by-base are java programs, whereas seaview, mega, and cinema are written in c++ language. results showed that msapad loaded more than 50,000 sequences with a sequence length of 2,696 base pairs, whereas other alignment editors written in java programming language could load less than 12,000 sequences only. alignment editors written in c++ language loaded more sequences than msapad, but their editing features are not user friendly. msapad allows the user to edit a residue at its own position without opening a new interface, whereas all other tools allow editing of an alignment in a new interface. msapad provides the feature to insert a sequence at any location of an alignment, whereas most of the available editing tools allow the user to insert a sequence at the end of an alignment. similarly moving a column and sorting an alignment by column are also the unique features of msa-pad. some of the alignment editors such as homed18 and maligned19 are no longer maintained by their authors. several tools such as suite msa,6 sinicview,13 altavist,7 and balibase c program20 are available to compute sps and cs but they can not process more than 1,000 sequences. results showed that the msa comparator was 5,200% efficient as compared to balibase c program. it calculated conserved regions of two alignments comprising more than 8,000 sequences with a sequence length of 2,696 base pairs in less than 12 seconds, whereas suitemsa allowed the user to compute conserved regions of two alignments consisting of less than 1,000 sequences. many software tools such as strap, suitemsa, pfaat, and seaview provide graphical user interfaces for generating msas, but they do not allow the user to load several sequence files simultaneously. msa reconstruction tool allows the user to load several sequence files at a time to align them one by one using clustal omega, clustalw2, mafft, muscle, or biojava. lot of tools and web servers such as alter21 and readseq22 are available for converting formats of msas, but they process alignments comprising a few thousand sequences. fasta generator allows the user to convert seven msa formats (clustalw, msf, phylip, pir, gde, and nexus) into fasta format. it can generate an identity matrix of more than 500 sequences with sequence length of 2,696 base pairs, which was very time consuming. since, sias is a web application, it was also not a good tool for calculating identity matrix of more than 1,000 sequences with a sequence length of 2,696 base pairs. msa i d calculator is an efficient tool, which calculated the identity matrix of more than 11,000 sequences with a sequence length of 2,696 base pairs in less than 100 seconds. comparison indicates that msapad, msa comparator, fasta generator, and msa i d calculator are more efficient than other similar tools available in the market for multiple sequence alignments. msapad allows the user to edit and analyze 409% more data than jalview, strap, cinema, and base-by-base. msa comparator (mqat version 2.0.1) allows the user to visualize consistent and inconsistent regions of reference and test alignments of more than 21-mb size in less than 12 seconds. msa reconstruction tool allows a user to upload several sequence files through the graphical user interfaces of clustal omega, clustalw2, mafft, and muscle, and then align them one by one. fasta generator converts the other seven formats of alignments of unlimited size into fasta format in a few seconds. msa i d calculator is a tool that allows a user to calculate the identity matrix of more than 11,000 sequences with a sequence length of 2,696 base pairs in less than 100 seconds. tree and distance matrix calculation tools generate phylogenetic tree and distance matrix, respectively, using the neighbor joining% identity and blosum 62 matrix. ivistmsa allows scientists to view, edit, interpret, and analyze very big alignments. project name: ivistmsa project home page: http://ivistmsa.com/ operating system(s): tested on windows but it should run on other platforms as well programming language: java 1.7 execution requirements: jdk1.7 or higher any restrictions to use by nonacademics: none

ivistmsa is a software package of seven graphical tools for multiple sequence alignments. msapad is an editing and analysis tool. it can load 409% more data than jalview, strap, cinema, and base-by-base. msa comparator allows the user to visualize consistent and inconsistent regions of reference and test alignments of more than 21-mb size in less than 12 seconds. msa comparator is 5,200% efficient and more than 40% efficient as compared to balibase c program and fastsp, respectively. msa reconstruction tool provides graphical user interfaces for four popular aligners and allows the user to load several sequence files at a time. fasta generator converts seven formats of alignments of unlimited size into fasta format in a few seconds. msa i d calculator calculates identity matrix of more than 11,000 sequences with a sequence length of 2,696 base pairs in less than 100 seconds. tree and distance matrix calculation tools generate phylogenetic tree and distance matrix, respectively, using neighbor joining% identity and blosum 62 matrix.

PMC4362671

pubmed-784

dn is one of the most serious diabetic microvascular complications and the leading cause of end-stage renal diseases (esrd); it brings about heavy social and economic burden worldwide, particularly in the developed countries. both type 1 and type 2 diabetic patients presented indistinguishable and variable pathological changes and clinical course; the prognosis is difficult to predict because of diverse pathogenesis. clinically, dn is characterised by different degrees of proteinuria, albuminuria, increased serum creatinine (scr), decreased glomerular filtration rate (gfr), and esrd [1, 2]. importantly, dn also increases the risks for the development of diabetic macrovascular complications including heart attacks and strokes [3, 4]. pathologically, dn associated histological structural changes include glomerular mesangial expansion, glomerular basement membrane (gbm) thickening, glomerular sclerosis known as kimmelstiel-wilson lesions caused by excessive extracellular matrix (ecm) proteins accumulations, and tubulointerstitial fibrosis in the advanced stages [1, 5]. arterial hyalinosis of the afferent and efferent arterioles is often prevalently caused by endothelial dysfunction and inflammation [2, 6, 7], which will lead to glomerular hyperfiltration. in the development and progression of dn, resident kidney cells are affected by hyperglycemia: including mesangial cells, podocytes, endothelial cells, smooth muscle cells, inflammatory cells, myofibroblasts, and cells of tubular and collecting duct system. multiple contributors including environmental and genetic factors are associated with the pathogenesis of dn, which cause metabolic, hemodynamic, and biochemical changes in the diabetic kidneys. main pathways leading to dn include intracellular pkc activation and increased polyol pathway flux, production of reactive oxygen species (ros) and advanced glycation end products (ages), and hypertension and glomerular hyperfiltration leading to shear stress and mechanical stretch [8, 9]. increased blood glucose activates the renin-angiotensin system (ras), tgf--smad-mapk pathway, jak-stat pathway, and g-protein signaling; aberrant expression of ecm proteins and deregulated expression of cyclin kinases and their inhibitors; transcription factor such as nf-b, proinflammatory cytokines like tnf and il-1, and toll-like receptors 4 (tlr4), which are considered to exert hemodynamic, proinflammatory, and profibrotic effects on kidney cells [8, 13]. there is cross-talk among the above-mentioned signaling pathways, which can amplify aberrant pathogenetic genes expression and lead to the progression of dn. in addition, the phenomenon of metabolic memory regulated by epigenetic mechanisms can promote these genes expressions [14, 15]. although a lot of biochemical and molecular mechanisms and pathways have been broadly studied in the pathogenesis of dn, the undeniable fact is that the progressive incidence and prevalence of dn worldwide still exist, suggesting that more investigations will be needed in the future. emerging evidences suggest that multiple signaling pathways activations and key transcription factors (tfs) are associated with the pathophysiology of dn, which could be influenced by epigenetically regulated mechanisms in chromatin (histones form a complex structure with dna), including dna methylation, posttranslational modifications (ptms), and noncoding rnas (ncrna), which can modulate gene expression in the cell-type-specific pattern. core histones are subject to diverse ptms including histone lysine acetylation (hkac), histone lysine methylation (hkme), phosphorylation, ubiquitination, and sumoylation. we have implicated the roles of hkme in the pathogenesis of dn, especially in the metabolic memory phenomenon pertinent to dn [2, 16, 17]. global acetylation alterations have been seen in a lot of human diseases including cancer and nervous system diseases, whereas the roles of hac in the pathogenesis of dn are rarely mentioned. recently, some studies showed that hac level is linked to dn, hats and hdacs also participate in the pathogenesis of dn, and the research regarding hac and the covalent enzymes is not enough to yield a clear picture about dn so far. in this review, we describe some progress associated with the molecular mechanism underlying dn, with specific emphasis on hac and acetylation on nonhistone proteins as important regulators of gene expression in renal cell under diabetic conditions; the regulators of hac such as hats as well as hdacs in the development and progression of dn; the inhibitors of hats/hdacs in the dn pathogenesis and their therapeutic potentials for dn. dynamic balance of histone acetylation and deacetylation can regulate gene expression, chromosome assembly, mitosis, and ptms, by altering the chromatin structure and the accessibility to tfs without affecting the sequence of dna. hac is highly reversible and dynamic, which can be catalyzed by hats or hdacs, respectively. hkac at n-terminal tails can facilitate gene transcription through neutralizing the positive charge of histone residues and weakening the binding of histone to negatively charged dna [22, 23]. hkac, such as h3k9ac, h3k14ac, and h4kac, is generally linked to permissive gene expression, while histone deacetylation is often associated with chromatin condensation and gene transcriptional repression [25, 26]. several previous studies have shown that hkac at the insulin gene promoter was specific to cells and islet-derived precursor cells, which was highly correlated with the recruitment of p300 [27, 28]. in vitro studies with hdac inhibitors (hdaci) suggested that hkac was essential in the development of pancreas. these findings can not fully demonstrate the underlying mechanism of dn; in this review, we will discuss the current opinions of hac and nonhistone acetylation on inflammation, fibrosis, and oxidative stress in the development and progression of dn (table 1). diabetic patients showed that levels of h3 acetylation at lysine 9 and 14 and h4 acetylation at lysine 5, 8, and 12 were increased at tnf- and cox-2 inflammatory genes promoters in human blood monocytes. another study showed that oxidized lipids could increase h3k9/14ac at mcp-1 and il-6 gene promoters in a creb/p300-dependent manner, along with the inflammatory genes expression. advanced dn in db/db mice underwent by uninephrectomy is specifically associated with increased acetylation of h3k9 and h3k23. a recent study revealed that acetylation of h3k9, h3k18, and h3k23 were significantly increased in the renal cortex of akita mice, hg and nab-induced h3k9 and h3k18 acetylation was elevated in the mesangial cells also, which were associated with inflammatory factors such as mcp-1, icam-1, vcam-1, and inos expression linked to the development of dn. thioredoxin-interacting protein (txnip) has been demonstrated to play an important role in the pathogenesis of dn. hg-induced txnip expression was associated with the stimulation of activating h3k9ac in mcs of diverse species, which could drive the expression of proinflammatory genes predisposing to dn. tgf-1 is established to be involved in the pathogenesis of dn, the underlying mechanism of which is still unclear. tgf-1 treatment could increase acetylation of histone (h3k9, h3k14, and h3k27) as well as ets-1 in mouse renal glomerular mesangial cells; furthermore, acetylation of ets-1 and histone h3 was increased in glomeruli from diabetic db/db mice also, both of which can increase mir-192 expression contributing to dn. tgf-1 treatment increased h3k9/14ac at the pai-1 and p21 promoters near smad and sp1 binding sites in rmcs, acetylation of smads was also increased [36, 37], and hg-treated rmcs exhibited increased levels of h3k9/14ac that can be blocked by tgf-1 antibodies, which played an important role in tgf-1 and hg-induced deregulated gene expression associated with hypertrophy and fibrosis linked to dn. hg stimulation can also increase h3k9/14ac at the rage, pai-1, and mcp-1 promoters, which can be further augmented by hg+ang ii (hg/a), suggesting the key roles of h3k9/14ac in the key dn-related genes expression. excessive h3k9/14ac levels were reported at the ctgf, pai-1, and fn-1 promoters in diabetic kidneys, which were associated with p300/cbp activation. although there is a conflicting result in an animal study that the level of h3k9/14ac was decreased in the stz-induced type 1 diabetic rat kidney [40, 41], the majority of hac is involved in the development and progression of dn. for the past few years, the phenomenon metabolic memory has been implicated in the pathogenesis of diabetes and its complications such as dn. a study of patients from dcct conventional treatment groups showed that there was association between hba1c level and h3k9ac; hyperacetylated promoters included more than 15 genes related to the nf-b pathway and could be enriched in genes associated with diabetic complications, which may be a possible epigenetic explanation along with hkme [16, 17, 43] for metabolic memory phenomenon in humans. endoplasmic reticulum stress (ers) is an important mechanism responsible for the pathogenesis of dn. histone h4 acetylation levels are increased at glucose-regulated protein (grp78) promoters and decreased at c/ebp-homologous protein (chop) promoters, which are associated with renal cell apoptosis, proteinuria, and increases of scr; these results provide initial experimental evidences for understanding the mechanism of dn. apart from hac, nonhistone proteins acetylation can also take part in the pathogenesis of dn. fork box o4 (foxo4) transcription factor can be activated to promote podocyte apoptosis by ages through bcl2111 expression, at the same time, age-bsa can also increase foxo4 acetylation; a recent study showed that alteration of foxo4 acetylation and downregulation of sirt1 expression in dm promote podocyte apoptosis; foxo4 acetylation reduction could be a therapeutic potential for preventing diabetic podocyte loss. enhanced nf-b acetylation level was present in both diabetic rats and hg-treated rmc leading to dn in another study, which can be dampened by 3,5-diiodothyronine (t2) involved regulation of sirt1; acetylation of nf-b p65 and stat3 was increased in both mice and human diabetic kidneys and ages induced human podocytes, suggesting their critical roles in dn. p65 acetylation was also increased by hg in rmcs, pns could protect diabetic kidney through decreasing induction of inflammatory cytokines and tgf-1. smad 3 acetylation has been implicated in the pathogenesis of dn recently [49, 50], overexpression of transcription factor srebp-1 induces glomerulosclerosis of dn; srebp-1a k333 acetylation by cbp is required for smad3 association and srebp-1 transcriptional activity; both smad3 and srebp-1a activation regulates tgf-1 transcriptional responses associated with dn, srebp-1 inhibition could be a novel therapeutic strategy for dn. nephrin acetylation in diabetic podocytopathy has seldom been addressed before, a recent study showed that nephrin acetylation was reduced in stz-induced diabetic mice kidney; increasing mir-29a may protect diabetic podocytopathy by modulating nephrin acetylation. type a hats (nuclear) exist in nucleus, including (1) gnat (gcn5) family such as gcn5, p/caf, and elp3, (2) myst (hmof/myst1, hbo1/myst2, moz/myst3, morf/myst4, and tip60) family, (3) p300/cbp, (4) basal tf family (tfiiic and taf1), and (5) nrcf family, src, and actr/ncoa3, which can acetylate nucleosomal histones and other chromatin-associated proteins, while type b hats are cytoplasmic and acetylate newly synthesized histones. hkac is generally mediated by hats including p300, cbp, p/caf, and tip60, which is associated with gene activation via adding acetyl groups. in addition, hats can also regulate gene expression through acetylation of nonhistone proteins such as smads, p53, sp1, and nf-b. among the studies of hats and their links with dn development, in vitro and in vivo studies showed that hats cbp and p/caf recruitment was increased under diabetic conditions, which led to upregulated hkac at inflammatory genes promoters continent with the gene expression [30, 53]. it was implicated that p300 played important roles in oxidative stress-induced parp and nf-b signaling in hg-treated endothelial cells and diabetic kidneys [5355]; further study showed that hg upregulated p300, which increased hac at promoters of key ecm protein fn, as well as vasoactive factors such as et-1 and vegf in endothelial cells. another study showed that tgf-1 increased h3k9/14ac by recruiting the hats p300 and cbp; tgf-1 treatment also increased association of p300 with smad2/3 and sp1, cotransfection experiments showed that p300 and cbp, but not p/caf, upregulated transcriptional activity of pai-1 and p21 promoters and increased tgf-1-induced gene expression. on the contrary, inhibition of cbp and p300 by overexpressing dominant-negative mutants p/caf was found sharply increased in the renal cortex of akita mice, while gcn5 was significantly decreased in the hg group, suggesting that the inflammatory genes expressions were related to dn. in vivo and in vitro results of another report showed that p/caf was closely related to h3k18ac levels at inflammatory molecules icam-1 and mcp-1 promoters, which could be a potential therapeutic agent for inflammation-related renal diseases including dn. all the data implied that hats have critical roles in acetylating both histones and nonhistone proteins in the pathogenesis of dn; these results point to the necessity of further studies on the hats activity in the development of dn, which may be therapeutic targets in the future. in preclinical trials, small-molecule hats inhibitors have been shown to sensitize cancer cells to ionizing irradiation. curcumin, the p300/cbp inhibitor, extracted from rhizomes of turmeric curcuma longa, which was supposed to be a new target molecule for treating cns disorders and cancer [61, 62], was firstly reported to prevent the development of dn involved in the changes of ptms of histone h3 including acetylation and phosphorylation and the changes in hsp-27 and p38 expression in diabetic rats. curcumin could also prevent hg-induced key ecm genes and vasoactive factors (enos and et-1) expression levels associated with dn in endothelial cells; it was able to reverse the upregulation of vasoactive factors, tgf-1 and ecm protein fn in stz-induced diabetic kidneys, which was associated with p300 and nf-b activity changes. curcumin was also found to reverse hg-induced cytokines (il-6, tnf-, and mcp-1) production in human monocytes via epigenetic changes involving nf-b, but dietary curcumin failed to decrease albuminuria either before or after diabetes induction. curcumin analogue, c66, has been demonstrated to significantly and persistently prevent renal injury and dysfunction in diabetic mice via downregulation of jnk activation and consequent suppression of diabetes-related increases in p300/cbp expression and histone acetylation (h3k9/14ac). in a recent study, c646, a novel p300/cbp specific inhibitor, has been declared to specifically suppress the growth of cbp-deficient hematopoietic and lung cancer cells in vivo and in vitro. in another in vitro study, histone h3ac activated tgf-1/smad3 pathway during emt of human peritoneal mesothelial cells; c646 could reverse the mesenchymal phenotype transition. c646 was also reported reversing acetylation involved in hg-induced txnip expression leading to dn. to date, 18 hdacs have been identified in humans and divided into 4 distinct classes based on their homology to yeast hdac, in which class i (hdac1, 2, 3, and 8), class ii including iia (hdac4, 5, 7, and 9) and iib (hdac6 and 10), and class iv (hdac11) have structurally similar zinc-dependent active sites, whereas class iii, sirtuins (sirts1-7), are zinc-independent but require cofactor nicotinamide adenine dinucleotide (nad). hdacs can remove acetyl groups from conserved lysine residues and nonhistone proteins and generally act as corepressors with some exceptions. most research related to the epigenetics of dn has focused on hac; different classes of hdacs are involved in distinct pathways that engaged in the pathogenesis of dn. overexpression of hdac1 and hdac5 blocked tgf-1-induced gene expression, whereas inhibition of hdacs upregulated h3k9/14ac and gene expression, further supporting the key inhibitory roles of hdacs in tgf-1-induced gene expression. a recent study showed that hdac1 was significantly decreased in the renal cortex of akita mice, while the levels of hdac2 in akita and wt mice were unchanged, and hdac1 was significantly decreased in hg-cultured hbzy-1 cell, which can upregulate diabetes-, hg-, and nab-induced histone hyperacetylation leading to inflammatory factors elevation associated with dn. glomerular sclerosis is also a core characteristic of dn resulting from excessive ecm deposition in the glomerular mesangium and the loss of glomerular epithelial cells, followed by aberrant fibrosis in the glomerular structure. hdac2 activity was markedly increased in the kidneys of type 1 and type 2 murine models and tgf-1 treated nrk52-e cells, which played an important role in the development of dn. knockdown of hdac2 in cell culture reduced ecm components accumulation, further implicating the role of hdac2 in the fibrosis. oxidative stress is also of the view to play an important role in regulating fibrosis in dn; a potent oxidative stress inducer h2o2 can increase hdac2 levels, which may be an underlying mechanism in the pathogenesis of dn. hdac4 is regarded as a contributor to podocyte injury in type 1 and type 2 diabetic models and diabetic patients and could suppress autophagy related with podocyte injury in dn by deacetylating stat1, suggesting that hdac4 is important to accelerate dn in epigenetic and nonepigenetic mechanisms [70, 71]. sirts have been shown to be involved in diverse cellular processes such as insulin secretion, cell cycle, and apoptosis. dysfunction of sirt1 may contribute to abnormal cancer metabolism, cancer stemness, neurological disorders, obesity, and diabetes. a previous study showed that decreased sirt1 level in diabetic kidney and intermittent fasting (if) prevents this decrease; sirt1-dependent deacetylation is thought to mediate p53 expression and activation, which could play a renoprotective effect of if in diabetes. another report showed that resveratrol could prevent decreased sirt1 and increased p53 expression in diabetic kidney, which could be responsible for preventing apoptosis in type 1 diabetic kidney. resveratrol has also been demonstrated to reduce oxidative stress and maintain mitochondrial function related with sirt1 activation in hg-treated mcs and db/db diabetic mice [75, 76]. sirt1 in proximal tubules (pt) has been reported to attenuate diabetic albuminuria by suppressing the overexpression of tight junction protein claudin-1 via hypermethylation of the claudin-1 gene in podocytes [77, 78]. another previous report showed that sirt1 could inhibit tgf-1-induced glomerular mesangial cell apoptosis via smad7 deacetylation, and overexpression of sirt1 attenuated ros-induced apoptosis in mesangial cells through p53 deacetylation and provided a new therapeutic strategy for kidney glomerular diseases; tsg has been proven to protect dn through inhibiting tgf-1 expression partially mediated by sirt1 activation. conditional sirt1 deletion in podocytes of diabetic db/db mice developed more acetylation of nf-b p65 and stat3, proteinuria, and kidney injury compared with db/db mice without sirt1 deletion, suggesting the protective roles of sirt1 in tfs acetylation on dn. dietary restriction was reported to ameliorate dn through regulation of the autophagy via restoration of sirt1 in diabetic fa/fa rats. the beneficial effects of sirt1 on age-associated dn correlate with the activation of nrf2/are antioxidative pathway [83, 84]. all the findings suggested the possibility of sirt1 as the target of treatment in dn [8587]. taken together, these studies highlight important and different roles of hdacs in the pathways, and most of them are beneficial, suggesting hdacs will be the targets for the prevention of dn despite the fact that further studies are needed. the present hdacis include both natural and synthetic compounds and are subdivided into 5 categories: short-chain fatty acids, cyclic peptides, benzamides, electrophilic ketones, and small-molecule hydroxamic-acid-derived compounds [52, 88]. hdacis are regarded as potential anticancer agents and are promising for the treatment of a lot of diseases such as inflammation and neurological diseases. recently, hdacis have been identified as a novel class of potential therapeutic agents for dn. here we list some progress of hdacis applied in the treatment of dn regarding antifibrotic, anti-inflammatory, and antioxidative effects. nevertheless, most of the hdacis are nonselective and target both nuclear histones and cytoplasmic nonhistone proteins. it was found that millimolar concentrations of n-butyrate induce accumulations of acetylated histones in cells in the 1970s and inhibited deacetylation [72, 90, 91]. sodium butyrate (nab, a nonselective inhibitor of hdacs), a short-chain fatty acid, can upregulate hac levels, promote tumor cell senescence and apoptosis, and inhibit tumor cell proliferation. nab was used as animal feed additive and played a major role in the treatment of neurodegenerative conditions. in vivo, it was reported that nab could not only decrease blood glucose, creatinine, and urea but also ameliorate histological changes, fibrosis, apoptosis, and dna damage in the kidneys of juvenile diabetic rats. saha (suberoylanilide hydroxamic acid, vorinostat), a nonselective hdaci, designed and synthesized as a hybrid polar compound that can strongly induce erythroid differentiation [72, 93], is orally bioavailable and clinically applicable. saha can reduce albuminuria, glomerular hypertrophy, and glomerular type iv collagen deposition through an enos-dependent mechanism, without affecting blood pressure or blood glucose concentration. indeed, another study showed that saha attenuated early renal enlargement in stz-induced diabetic rats, which is supposed to be mediated partly through downregulating egfr. these results indicated the key role of saha in attenuating fibrosis and oxidative damage in dn. trichostatin a (tsa), the natural product isolated from a streptomyces strain, originally identified as an antifungal antibiotic, was discovered to have potent hdac inhibition activity in 1990. tsa was reported to act as an agent in preventing dn in diabetic rats, by blocking tgf-1-induced ecm accumulation and emt in diabetic kidneys as well as in renal epithelial cells; knockdown of hdac2 had similar effect of tsa treatment mediated by ros. valproic acid (vpa), a broad-spectrum hdaci, is a first-line drug used for the treatment of epilepsy and migraine. vpa treatment alleviated renal injury and fibrosis in stz-induced diabetic kidney by preventing myofibroblast activation and fibrogenesis through hdac4/5/7 inhibition in a dose-dependent manner, vpa has also been proven to ameliorate the podocyte and renal injuries by facilitating autophagy and inactivation of nf-b/inos pathway. a recent study showed that vpa can attenuate renal injury in a rat model of dn, by upregulating the histone h4 acetylation levels at the promoter of grp78 and downregulating the histone h4 acetylation at the promoter of chop. to our knowledge, at the time of the present review, the molecular implications of hdacis were identified in the treatment of dn, and the development of selective hdacis in preventing dn may be part of the most prevalent areas in the drug discovery. recent research has concentrated on histone modifications to provide a reliable theoretical basis for clinical treatment. a comprehensive understanding of hac mechanisms can give rise of novel therapeutic options for dn. increasing in vitro and in vivo evidences implicated that reversible histone and nonhistone acetylation play important roles in the pathogenesis of dn, suggesting that hac regulation could be promising therapeutic targets for dn. hats and a small number of hdacs provide a central mechanism for regulating gene expression and cellular signaling events in dn (table 2). experimental evidences suggest that hats/hdacs inhibitors and a large number of hdacs can delay the development and progression of dn (tables 2 and 3). hats inhibitor curcumin and its analogue c66 could protect renal injuries in diabetic patients and diabetic animal models; apelin-13 and esculetin treatment could be innovative therapeutic agents for dn via regulation of hac also [33, 40, 41]. continued research is needed to better understand the roles of hac in the process of dn, the modifiers and the mechanism that regulate them, and address the curative potential of more selective hats inhibitors and hdaci in treating dn.

diabetic nephropathy (dn) remains a leading cause of mortality worldwide despite advances in its prevention and management. a comprehensive understanding of factors contributing to dn is required to develop more effective therapeutic options. it is becoming more evident that histone acetylation (hac), as one of the epigenetic mechanisms, is thought to be associated with the etiology of diabetic vascular complications such as diabetic retinopathy (dr), diabetic cardiomyopathy (dcm), and dn. histone acetylases (hats) and histone deacetylases (hdacs) are the well-known regulators of reversible acetylation in the amino-terminal domains of histone and nonhistone proteins. in dn, however, the roles of histone acetylation (hac) and these enzymes are still controversial. some new evidence has revealed that hats and hdacs inhibitors are renoprotective in cellular and animal models of dn, while, on the other hand, upregulation of hac has been implicated in the pathogenesis of dn. in this review, we focus on the recent advances on the roles of hac and their covalent enzymes in the development and progression of dn in certain cellular processes including fibrosis, inflammation, hypertrophy, and oxidative stress and discuss how targeting these enzymes and their inhibitors can ultimately lead to the therapeutic approaches for treating dn.

PMC4917685

pubmed-785

the chia (salvia hispanica) seed was used as an offering to the aztec gods, and, because of its religious use, it essentially disappeared for 500 years. this is an annual herbaceous plant belonging to the lamiaceae or labiatae family. in pre-columbian times, it was one of the basic foods of several central american civilizations, less important than corn and beans, but more important than amaranth. the chemical composition reports contents of protein (1525%), fats (3033%), carbohydrates (2641%), dietary fiber (1830%), and ash (4-5%). chia seeds have been investigated and recommended due to their high levels of proteins, antioxidants, dietary fiber, vitamins, and minerals but particularly due to their oil content with the highest proportion of -linolenic acid (-3) compared to other natural sources known to date. chia seeds contain up to 39% of oil, which has the highest known content of -linolenic acid, up to 68%. chia seed gum has the potential for industrial use because of its slimy properties, evident even at very low concentration, and because the plant, native to america, grows well in semiarid regions that have few practical plants. chia gum begins to emerge from seeds as soon as they are placed in water. the exudate is either partially cross-linked or is bound to the seed surface, since it is not easily separated from the seed. separation can be accomplished by strong stirring, preferably in the presence of sand to aid in dislodgment or cleavage of insolubilizing bonds. for research purposes, gum has been removed by extraction of seeds with a 6 m urea solution. chia gum is composed of -d-xylopyranosyl, -d-glucopyranosyl, and 4-o-methyl--d-glucopyranosyluronic acid unit in the ratio 2: 1: 1. the polysaccharide seems to consist of a repeating unit. extracted gum has a slimy, mucilaginous character at very low concentrations, giving it wide potential use in a variety of industrial applications, especially in certain foods and food preparations. the objective of the present study was to determine the physicochemical properties of fatted and defatted gums from chia (salvia hispanica) seeds. chia (s. hispanica l.) seeds were obtained in the yucatan state of mexico. reagents were of analytical grade and purchased from j. t. baker (phillipsburg, nj, usa), sigma (sigma chemical co., st. louis, mo, usa), merck (darmstadt, germany), and bio-rad (bio-rad laboratories, inc. seeds of chia were submitted to gum extraction with water at a 1: 20 ratio (w/v) for 30 min and at a 50c temperature. after that, the suspension was milled in a mixer and then it was boiled again at 50c under stirring for 15 min. the crude mixture, containing water, gum, and seeds, was centrifuged at 9460 g at 15c for 3 h. the recovered gum (fcg) was dried at 40c for 24 hours and milled. one portion of the recovered gum was partly defatted (pdcg) in a soxhlet. standard aoac procedures were used to determine nitrogen (method 954.01), fat (method 920.39), ash (method 925.09), crude fiber (method 962.09), and moisture (method 925.09) contents in the fatted and defatted chia gums. approximate water absorption capacity was first determined by weighing out 0.1 g (d.b.) of sample, adding water until saturation (approximately 5 ml), and centrifuging at 2000 g for 10 min in a beckman gs-15r centrifuge. approximate water absorption capacity was calculated by dividing the increase in sample weight (g) by the quantity of water needed to complete original sample weight to 15 g. water absorption capacity (wabc) was then determined by placing samples in four tubes, adding different quantities of water to bracket the measurement (1.5 and 0.5 ml water above original weight and 1.5 and 0.5 ml water below; one in each tube), agitating vigorously in a vortex for 2 min, and centrifuging at 2000 g for 10 min in a beckman gs-15r centrifuge. average water absorbed was calculated and the wabc was calculated, expressed as g water absorbed per g of sample. briefly, 0.1 g (d.b.) of sample was placed in an equilibrium microenvironment at 98% relative humidity, generated by placing 20 ml of saturated potassium sulfate saline solution in tightly sealed glass flasks and placing these in desiccators at 25c. the sample was left in the microenvironment until reaching constant weight (72 h). briefly, 0.1 g (d.b.) of sample was weighed and then stirred into 20 ml of distilled water or corn oil (mazola, cpi international) for one minute. these fibrous suspensions were then centrifuged at 2200 g for 30 min and the supernatant volume was measured. water-holding capacity was expressed as g of water held per g of sample, and oil-holding capacity was expressed as g of oil held per g of gum. corn oil density was 0.92 g/ml. apparent viscosity was evaluated using an adaptation of the li and chang method, using a brookfield viscometer model dv-ii (brookfield engineering lab., stoughton, ma) with spindle 27 (for small samples) and share rate range from 2.5 to 100 rpm at 25c. the samples were dispersed in water to 0.5, 1.0, 1.5, 2.0, and 2.5% (w/v, db). the results were expressed in pa.s and data was fixed to an ostwald-de waele model to determine the consistency index (k) and flow behavior index (n). this percentage was lower than reported by sciarini et al. in gleditsia triacanthos seeds (11.934.16%). however, the chia gum's yield was higher than reported by oomah et al. in flaxseed (3.68%). the proximal composition (table 1) showed that pdcg registered a higher content of protein, ash, and nfe than fcg. the fiber content of fcg (28.96%) was similar to that reported by vazquez-ovando et al. in a fiber-rich fraction of chia seeds (29.56%). the moisture content of both gums was similar to that reported by kader et al. in acacia glomerosa (9.09%) but lower than reported in guar gum (10.36%), xanthan gum (11.08%), and gleditsia triacanthos (14.08%). with respect to the nfe content, both gums registered lower values than reported by vazquez-ovando et al. in a fiber-rich fraction of chia seeds (34.52%). however, the ash content of fcg and pdcg was higher than that reported by kader et al. in arabic gum (acacia senegal, 3.6%) although lower than reported by sciarini et al. in xanthan gum (9.35%). the protein content of both gums was higher than registered in corn gum (5.1%) and mesquite gum (5.8%), this last one with important emulsifying properties attributed to its protein content according to bosquez. in this respect, establish that hydrocolloids rich in protein, such as gelatin, arabic gum, and mesquite, are good stabilizers because they have sufficient hydrophobic groups to act as bonding points as well as hydrophilic groups that reduce surface tension in a liquid-liquid or liquid-gas interface. on the other hand, yadav et al. establish that the lipid content in the gums may also play an important role in stabilization of oil-water emulsions. however, bosquez established that carbohydrates avoid flocculation and coalescence of oil droplets to extend in the aqueous solution. these findings suggest that fcg and pdcg could act as good emulsifiers and stabilizers in the food industry. water absorption capacity is indicative of a structure's aptitude to spontaneously absorb water when placed in contact with a constantly moist surface or when immersed in water. water adsorption capacity is the ability of a structure to spontaneously adsorb water when exposed to an atmosphere of constant relative humidity. wabc was higher in fcg (44.08 g/g of sample) than pdcg (36.2 g/g of sample). the high values of wabc obtained here could be due to the proteins present in the gums, which would have a large number of exposed hydrophilic sites interacting with water. the wabc of fcg and pdcg was higher than reported by vzquez-ovando et al. in a fiber-rich fraction of chia seeds (11.73 g/g of sample), who establish that fiber content is an important factor in the increment of this property for its capacity to form gels and to hold water; this justifies the higher value of wabc in fcg. on the other hand, pdcg (0.84 g/g of sample) registered a higher value of wadc than fcg (0.27 g/g of sample). the wadc of dcg was also higher than registered by vzquez-ovando et al. in a fiber-rich fraction of chia seeds (0.3 g/g of sample), similar to that reported in carrots (0.82 g/g of sample) but lower than the value registered in beet bagasse (1.58 g/g of sample). however, whc of both gums was higher than that reported by vzquez-ovando et al. and baquero and bermdez in a fiber-rich fraction of chia seeds (15.41 g/g of fiber) and passion fruit peel (8.7 g/g of fiber), respectively. a similar behavior was observed with orange waste (7.658.23 g/g of fiber). soluble fiber and the denaturalized proteins may have increased the whc of both gums, thus enhancing the swelling ability, an important function of proteins in preparation of viscous foods such as soups, gravies, dough, and baked products. on the other hand, fcg showed a higher ohc than pdcg, which might be related to its higher value of fat. however, both gums registered higher ohc values than those registered in guar and xanthan gum (46 g oil/g fiber) although similar to that reported in arabic gum (8-9 g oil/g fiber). this functional property has been attributed to the physical entrapment of oil for molecules such as lipids and proteins. for the above mentioned, the ohc registered in chia gums could be due to protein and fat contents as well as factors as particle size and the absence of hemicellulose. chia gum seems to possess an adequate fat absorption capacity, allowing it to play an important role in food processing, since fat acts on flavor retainers and increases the mouth feel of foods. both gums showed a non-newtonian behavior where viscosity presented a relation directly proportional to the concentration and inversely proportional to the shear rate. the maximum viscosity reached between both gums was registered by pdcg (55.4 pas) at 2.5%. in general, pdcg registered a higher viscosity profile than fcg suggesting that the fat content was the principal factor that generated this behavior. at this respect, report that gums with higher oil absorption as arabic gum show less viscosity (2.34 pas at 3.8%). according to table 2, the rheological behavior of the fcg and pdcg dispersions was a shear thinning or pseudoplastic type due to registered values of n<1. the results suggest the use at low concentrations of dcg in products as yoghurts, sauces, toppings, and pastries among others that require high viscosity, whilst fcg could be used in sauces, mayonnaises, and meat products as emulsifying and stabilizer. the results obtained here show that chia gums present interesting physicochemical properties for the food industry. the partly defatted chia gum showed a very good ability to water holding (110.5 g/g); however, their ability of oil holding (11.67 g/g) and water absorption (36.26 g/g) was minor compared to the fatted chia gum, which provided a greater retention of oil holding (25.79 g/g) and water absorption (44.08 g/g). rheological behavior of gums was shear thinning or pseudoplastic type. from a functional point of view, chia gum also is an important food ingredient due its emulsifier and stabilizer potentials.

chia (salvia hispanica l.) constitutes a potential alternative raw material and ingredient in food industry applications due to its dietary fiber content. gum can be extracted from its dietary fiber fractions for use as an additive to control viscosity, stability, texture, and consistency in food systems. the gum extracted from chia seeds was characterized to determine their quality and potential as functional food additives. the extracted chia gum contained 26.2% fat and a portion was submitted to fat extraction, producing two fractions: gum with fat (fcg) and gum partly defatted (pdcg). proximal composition and physicochemical characterization showed these fractions to be different (p<0.05). the pdcg had higher protein, ash, and carbohydrates content than the fcg, in addition to higher water-holding (110.5 g water/g fiber) and water-binding capacities (0.84 g water/g fiber). the fcg had greater oil-holding capacity (25.7 g oil/g fiber) and water absorption capacity (44 g water/g fiber). in dispersion trials, the gums exhibited a non-newtonian fluid behavior, specifically shear thinning or pseudoplastic type. pdcg had more viscosity than fcg. chia seed is an excellent natural source of gum with good physicochemical and functional qualities, and is very promising for use in food industry.

PMC4745557

pubmed-786

bacterial pathogens can develop antibiotic resistance either by mutations, or by the acquisition of antibiotic resistance genes from other microorganisms through horizontal gene transfer (hgt). since bacterial pathogens were (presumably) susceptible to antibiotics at the time of the development of these compounds, it is reasonable to think that resistance genes have been acquired from non-pathogenic microorganisms. indeed, the analysis of escherichia coli plasmids from bacterial strains isolated before and after the use of antibiotics for therapy demonstrated that the plasmid families were similar, but incorporated resistance genes after the antibiotic era (datta and hughes, 1983). since the resistance genes did not originate in bacterial pathogens, the sources for these genes would be environmental microorganisms (martinez et al., 2009a; davies and davies, 2010). indeed, the fact that most antibiotics currently used in clinics originated in environmental microorganisms (waksman and woodruff, 1940) led to the proposal that the origin of resistance genes are the antibiotic-producing organisms, where resistance genes may play an auto-protective role (benveniste and davies, 1973; davies, 1997). recent work indicates that indeed antibiotic-producing environmental microorganisms harbor a large number of resistance genes that could be potentially transferred to human pathogens (dcosta et al., 2006). nevertheless, the presence of resistance genes in the environment is not confined to antibiotic producers (aminov, 2009). for instance, the quinolone resistance gene qnra originated in the water-borne bacteria shewanella algae, which is not known to produce an antibiotic (poirel et al., 2005). given that quinolones are synthetic drugs, the existence of these determinants indicate the antibiotic resistance genes can have disparate functions in their original hosts, in such a way that the universe of potential resistance genes that can be incorporated into mobile genetic elements is even larger than predicted from the analysis of antibiotic producers. support for this statement is the finding of genes that contribute to intrinsic resistance in different bacterial species (fajardo et al., 2008 ;, 2009; alvarez-ortega et al., 2010; liu et al., 2010) and functional metagenomic analyses indicating that the wide dissemination of a large number of resistance genes (dcosta et al., 2006; sommer et al., 2009) in all analyzed ecosystems (including the human gut) whether or not contaminated by human activities. it would be expected that this diversity of resistance genes in microorganisms, that can confer an antibiotic resistant phenotype on their transfer to a new host might be mirrored by a large variability of resistance genes, acquired by hgt, in human pathogens. however, the number of different resistance determinants found among human bacterial pathogens is low in comparison to those present in the different metagenomes. this indicates that the transfer of a resistance gene from its original host to a human pathogen might be constrained by different bottlenecks, as discussed in this review. the existence of resistance genes in natural ecosystems, even those without any record of pollution by antibiotics was reported more than four decades ago (gardner et al., 1969). however, detailed studies on this topic are more recent. in the last years an increasing number of studies of the presence of resistance genes in non-clinical ecosystems have been published. briefly, two different methodologies are applied; one is the search for any potential gene that confers resistance on expression in a heterologous host by using functional genomic techniques (dcosta et al., 2006, 2011; the other is the search for resistance genes already present in human pathogens, usually by pcr, in metagenomic dna (koike et al., 2007). whereas, in the first analysis the purpose is to characterize any gene that can cause resistance if transferred and hence study the potential natural resistome of the studied ecosystem, the second type of study analyses contamination by resistance genes already acquired by human pathogen. functional metagenomics serves to define novel mechanisms of resistance (potentiality, see martinez et al., 2007), but predicting whether such mechanisms will be transferred to human pathogens is not obvious (see below). indeed, the fact that the origin of the antibiotic resistance genes currently present in human pathogens is known in only a few cases indicates that defining the environmental resistome is a needed but not sufficient condition for predicting the emergence of resistance. it is important to note however that the finding of novel mechanisms of resistance can be a valuable tool for the design of antibiotic modifications before resistance arises (wright, 2007; martinez et al., 2011). the analysis of the presence in different ecosystems (contaminated and pristine) of genes that have been already acquired by human pathogens would provide information on the stability of these elements, the reservoirs and the factors that enrich their presence in nature. these studies can be used to evaluate the risks for human health from pollution of natural ecosystems by antibiotic resistance determinants, together with antibiotics that serve as selectors of resistance themselves (martinez, 2008, 2009). this knowledge might serve for the identification of intervention strategies to reduce the impact of anthropogenic activities on the enrichment of resistance elements, already present in mobile genetic elements (mges), in natural (non-clinical) ecosystems (baquero et al., 2008). the relevance that farming and transport of food-borne animals or pets (guardabassi et al., 2004; aarestrup, 2005; moreno et al., 2008), as well as the transport of goods (ruiz et al., 2000), or human migration (kumarasamy et al., 2010) may have for the dissemination or resistance is well known. for these processes, procedures for tracking the presence and dissemination of resistance genes more difficult will be the implementation of such studies for analyzing the role of wild animals in the spread of resistance (gilliver et al., 1999; livermore et al., 2001; allen et al., 2010). important in this respect is the finding of resistance in migratory birds that can disseminate both antibiotic resistance determinants and infective resistant bacteria all over the world (middleton and ambrose, 2005; steele et al., 2005; simoes et al., 2010). between these types of studies are functional analyses on the resistance mobilome, those resistance genes that are already present on mobile elements, irrespective of whether or not they have been acquired by human pathogens. the transfer of a potential resistance gene from the chromosome of an environmental bacterium to a human pathogen requires it to be mobilizable after its capture by a translocative element and its integration in an mge. this means that once the resistance element has been incorporated in a mobile element, the possibility of its acquisition by a human pathogen can be high, especially if this element is present in the human bacterial population. unfortunately, studies on the environmental resistance mobilome are difficult and still rare (szczepanowski et al., 2008, 2009; the first requirement for the transfer of a resistance gene is that both the donor and the receptor share the same habitat. in the case of pathogenic bacteria, the pathogens need not co-exist with the donor, because a chain of microorganisms may link the donor and the recipient. however, since acquiring resistance genes might confer a fitness cost (see below), the establishment of a successful gene-transfer chain is possible only with positive selection for the resistance determinant. in other words, unless resistance is selected (mainly by antibiotics), it is unlikely that mges containing resistance genes will be fixed in the populations of environmental microorganisms en route to human pathogens. since the natural concentrations of antibiotics in non-clinical ecosystems are much lower than at hospitals (davies, 2006), only in the case of pollution by antibiotics (aquaculture, waste disposal from cities, farms, or industries) a positive selection for mges containing resistance genes can be envisaged. following this reasoning, it has been proposed that the possibility of a given resistance gene being transferred to a human pathogen will largely depend on whether the habitat where the donor micro-organism is present close to human-linked ecosystems (baquero et al., 2009). for instance, it would be rare for resistance genes found in deep soil allocations (brown and balkwill, 2009) or at a glacial ice core (miteva et al., 2004) would be transferred to human pathogens. in contrast, it has been suggested that ecosystems such as waste-water treatment plants or farms, where human pathogens and environmental bacteria co-exist in the presence of contaminating antibiotic residues, might be hot-spots for the acquisition of resistance genes by bacterial pathogens (baquero et al., 2008; aminov, 2011). for those microorganisms sharing the same ecosystem, some of them are more prone to exchange genetic material than others. the organisms that can share genes, have been named as genetic exchange communities (jain et al., 2003). as stated in (skippington and ragan, 2011), gene exchange communities can vary widely in spatial extent, taxonomic diversity, density of internal connectivity, and involvement of vector types. these communities usually share some plasmid (or transposon) types and do not possess strong restriction/modification systems that would impede the interchange of dna. as a consequence, the entrance of a resistance gene, located in a proficient mge into a well established gene exchange community might allow its spread among different organisms and consequently fixation in populations of bacterial pathogens. this spread will be modulated by specific fitness costs that preclude the stability of the gene in some bacterial species. by founder effect, we refer to the situation in which the first gene to arrive is the one to win (baquero et al., 2009). when there are several resistance determinants with a similar substrate profile, usually one prevails once transferred to human bacterial pathogens. as we will discuss later, this situation can be the consequence of differential fitness costs, nevertheless, a certain degree of serendipity might be the basis of the successful transfer, spread and fixation of a given resistance determinant. one example of this situation is the tem-1 beta-lactamase, which, followed by shv-1 and oxa enzymes, has been the predominant plasmid-encoded beta-lactamase in enterobacteriaceae for many years (simpson et al., 1980; medeiros, 1997). the tem-1 beta-lactamase was acquired soon after the introduction of the first generation of beta-lactams for therapy, and plasmids coding this beta-lactamase spread rapidly among bacterial pathogens. the study of several different ecosystems has shown that there exist a large number of beta-lactamases nearly everywhere, which can confer resistance to the same antibiotics as tem-1. antibiotic resistance genes are acquired and maintained because of the strong selective pressure of antibiotics. once bacteria have acquired a determinant that allow them to resist antibiotics, there is not a selection pressure for replacement of the determinant already present in bacterial populations. this situation can change if the selective pressure is altered, for example when new antibiotics are launched into clinical use (livermore, 2009; salverda et al., the introduction of beta-lactamase inhibitors and novel beta-lactams for which tem-1 presented low activity generated two different processes: (i) evolution of the tem-enzyme that most likely occurred in clinical settings when bacterial pathogens were exposed to the novel selective pressure (ii) acquisition of novel beta-lactamase coding genes by human pathogens with novel substrate profiles. it is generally assumed that the acquisition of an antibiotic resistance determinant confers a fitness cost (andersson and levin, 1999), meaning that in the absence of selection, resistant bacteria will be outcompeted by the susceptible ones. in the case of genes acquired by hgt, these costs might be the consequence of the metabolic load imposed by the replication, transcription, and translation of the novel genetic elements. if this was the unique cause of fitness costs, the disadvantage of carrying one or another resistance gene will be similar and the fitness cost would not constitute a relevant bottleneck in selecting one resistance determinant over another. however, different studies have shown that, at least on occasion, the introduction of a given resistance gene does not impose a non-specific metabolic burden but leads to specific changes in bacterial physiology. this may be the case for ampc beta-lactamase genes, which are infrequently found on salmonella plasmids unless the plasmid also harbors the repressor of their expression (verdet et al., 2000) or elements that compensate the biological costs associated to ampc expression (hossain et al., 2004). it has been found that ampc alters the physiology of salmonella, decreasing its virulence and hence a differential fitness cost that decreases the probability of dissemination of specific gene among salmonella strains (morosini et al., 2000). this example indicates that the fitness costs can be gene-specific and do not necessarily derive from a general metabolic burden. in this context, those resistance determinants conferring high fitness costs are unlikely to be fixed in bacterial populations because they would be outcompeted by other resistance determinants which lower fitness costs (martinez et al., 2011). this reasoning must be however modified by the chances of acquiring compensatory mutations (andersson and hughes, 2011; martinez et al., 2011). if a resistance determinant confers high fitness costs, but compensatory mutations are easily selected, the probability of being maintained in bacterial pathogens is high. in such cases, if the compensatory mutations occur in the chromosome, not in the mge, the acquisition of the resistance gene by a new host implies a new fitness cost, and as a consequence the spread of the resistance determinant will be compromised. however, it the mutation occurs in the mge, the chances for spread will be enhanced. if the acquisition of resistance confers fitness costs it is logical to suppose that resistant organisms will be outcompeted by their susceptible, fitter counterparts in the absence of selection. however, some resistant strains present no-cost (rozen et al., 2007; balsalobre and de la campa, 2008) and even some resistance determinants can be beneficial under certain conditions (alonso et al., 2004; maughan et al., 2004; luo et al., 2005; perkins and nicholson, 2008; michon et al., finally, some fitness costs can be compensated by mutations that do not impede to keep resistance (bjorkman et al., 1998 ;, 2002; paulander et al., 2007; lind et al., 2010; shcherbakov et al., this indicates that reversing resistance once established can be a difficult task (andersson and hughes, 2010). maintenance of resistance genes in habitats without a strong antibiotic pressure is favored as well by second-order selection processes. this means that the selection for one antibiotic will select for the whole array of resistance genes present in this specific mge. furthermore, mges besides resistance genes may carry other elements such as heavy-metal resistance determinants (baker-austin et al., 2006), or genes coding for production of siderophores, toxins, or bacteriocins (de lorenzo and martinez, 1988; clewell, 1990; herrero et al., 2008); these can confer an ecological advantage in some ecosystems and thus co-select resistance in the absence of antibiotics. cross-selection might also be a relevant second-order process that allows maintenance of resistance in the absence of selection. certain resistance determinants, such as multidrug (mdr) efflux pumps confer resistance to different compounds (antibiotics, biocides, or heavy metals; martinez et al., 2009b). this means that selection with the biocide or the heavy metal might result in cross-resistance to the antibiotic (hernandez et al. a final mechanism for the maintenance of resistance is based on the inherent systems for plasmid stability. plasmids frequently encode toxin/antitoxin systems, which provoke death of bacteria that lose the plasmid (hayes, 2003; hayes and van melderen, 2011). if one such plasmid incorporates an antibiotic resistance determinant, the probabilities for its maintenance will be high. taking these considerations into account, resistance genes might evade elimination (andersson and hughes, 2011) in the absence of antibiotics; indeed, resistance determinants present in human pathogens have been found on identical mges, in antibiotic-pristine habitats (pallecchi et al. livermore et al., 2001) and primitive human populations without any known exposure to antibiotics (grenet et al., 2004; bartoloni et al., these observations indicate that anthropogenic activity has enriched for a small number of resistance genes in natural ecosystems and that this type of pollution will be difficult to eradicate (salyers and amabile-cuevas, 1997; martinez, 2009). supporting this notion, analyses of soils sampled in the netherlands from 1940 to 2008, this, in spite of the fact that restrictions on non-therapeutic use of antibiotics in agriculture and in waste management procedures have been strongly enforced (knapp et al., 2010). research on antibiotic resistance has been mainly focused on bacterial pathogens isolated from infections or in clinical settings. however, the fact that hgt-acquired genes originated in natural, non-clinically relevant microorganisms and that the first step in the transfer of resistance likely occurs in natural ecosystems emphasizes the need to analyze resistance in non-clinical ecosystems. furthermore, the constant release of antibiotic resistance determinants already present in mges located in human pathogens, and in some circumstances associated with selective concentrations of antibiotics, may disrupt natural microbiota, which then serve as reservoirs for resistance genes. non-culture based methods have demonstrated their value for the analysis of resistance in natural ecosystems. among them, functional metagenomics provides the means to identify novel mechanisms of resistance independently of whether they will be acquired by bacterial pathogens. on the other hand, pcr analyses for specific genes serve to define reservoirs and to study elements like pollution in the dissemination and maintenance of resistance. functional metagenomic studies indicate that very few among the resistance genes present in nature have been transferred to human pathogens. whereas the founder effect can provide stochasticity to these acquisitions, other factors such as fitness costs, ecological connectivity, which includes the formation of gene exchange communities, are relevant bottlenecks that serve to modulate the acquisition of resistance genes by animal or human pathogens. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

it is generally accepted that resistance genes acquired by human pathogens through horizontal gene transfer originated in environmental, non-pathogenic bacteria. as a consequence, there is increasing concern on the roles that natural, non-clinical ecosystems, may play in the evolution of resistance. recent studies have shown that the variability of determinants that can provide antibiotic resistance on their expression in a heterologous host is much larger than what is actually found in human pathogens, which implies the existence of bottlenecks modulating the transfer, spread, and stability of antibiotic resistance genes. in this review, the role that different factors such as founder effects, ecological connectivity, fitness costs, or second-order selection may have on the establishment of a specific resistance determinant in a population of bacterial pathogens is analyzed.

PMC3249888

pubmed-787

spinal cord stimulation (scs) has been used since the 1960s to treat chronic refractory pain conditions. in particular, scs has been extensively used to manage the painful symptoms related to chronic lumbosacral radiculopathy following failed back surgery syndrome and complex regional pain syndromes. randomized controlled trials of scs for failed back surgery syndrome have reported favorable long-term results.1,2 concurrently, there has been significant technologic advancement in the equipment for scs. specifically, implanting physicians can offer patients rechargeable implantable pulse generators, improved anchoring systems, and more stimulating lead contacts to ensure coverage for paresthesia in painful somatic regions. thus, there is more widespread use of scs therapy and acceptance among treating physicians. despite the positive findings and increasing experience of implanting physicians using scs therapy, complications remain a common occurrence. an analysis of the available literature by turner et al found that just over one third of patients have a complication.3 prospective data from a multicenter evaluation of scs for failed back surgery syndrome showed a similar rate of complications, even in the hands of experienced implanters.4 these adverse events are usually lead migration, infection, lead breakage, and unwanted stimulation.4,5 an allergic or immunologic/ inflammatory reaction to the system components is thought to be rare, and has been estimated by cameron to be 0.1%.5 however, the true incidence may be underestimated because clinicians may fail to include it in the differential diagnosis, attributing an inflammatory reaction of the soft tissue surrounding scs components to infection. in addition, only two articles detail the cutaneous reaction to the components of scs systems.6,7 the purpose of this paper is to detail the presentation, clinical course, and histologic findings in three patients with cutaneous reactions to spinal cord stimulator equipment components. the patient was a 61-year-old male who, despite two previous laminectomies, epidural injections, and multimodal analgesia (oxycodone/acetaminophen, cyclobenzaprine, ibuprofen), complained of severe pain secondary to left-sided chronic lumbosacral radiculopathy. the pain was 9/10 in intensity, located at his lower back on the left side with radiation down his posterior thigh, behind his knee to his lateral leg, and to the bottom of the sole of his foot. he had a past medical history of irritable bowel syndrome, squamous cell carcinoma of the skin, gastroesophageal reflux disease, and depression. magnetic resonance imaging revealed no evidence of spinal stenosis, foraminal narrowing, or nerve root displacement, so he was implanted with an scs consisting of two eight-contact leads, two lead extensions, and a rechargeable implantable pulse generator (eon, st jude medical neuromodulation division, plano, tx, usa) following a successful trial of 7 days. he was seen one week after the operation and his wounds, which included the abdomen for the generator, flank for the extension, and back for lead insertion and anchoring, were noted to be dry with intact incisions, and no erythema, drainage, or tenderness. at one month post operation, the patient developed a left-sided rash on his flank and abdomen, with no fevers or chills (figure 1). however, he had an area of erythema/dermatitis that seemed to be localized to the area over the length of the lead extensions connecting the implantable pulse generator to the leads. it was noted that this rash appeared to be an area of reactive inflammation, as opposed to infection, and he was diagnosed with a hypersensitivity reaction to the implanted wires. he was given topical 1% hydrocortisone cream and seen 7 days later, with resolution of the rash. forty-three days after implantation, the patient presented with complaints concerning wound drainage. the abdominal and flank incisions had dehisced, with device exposure and were draining pink, clear fluid. his white blood cell count was 6.8 and the differential was abnormal for eosinophils at 5.4%, with no bandemia or elevation in fraction of neutrophils. because of concerns for potential early infection, urgent explantation of the entire scs device was performed. exploration of the wounds did not reveal any purulent material, but the subcutaneous tissue directly involving the wound was frail, bled easily with manipulation, and was difficult to coagulate. wound cultures were positive for rare coagulase negative staphylococci as well as rare staphylococcus aureus, and the patient was treated for 2 weeks with intravenous vancomycin. clinically, however, he did not manifest with the usual clinical findings suggestive of device infection. one month later, all incisions were well healed. because the initial reaction to the scs equipment involved the flank to the abdomen but not the back, the buttock was targeted as a potential site for the implantable pulse generator. in an attempt to rule out a hypersensitivity reaction, an in vivo allergy test using a portion of the extension lead was done (figure 3). a 4 cm lead extension piece was placed in the left buttock via a 12 gauge angiocatheter upon removal of the introducer. the extension fragment was placed into the catheter which was removed leaving the lead in the subcutaneous tissue. the puncture site was closed with 4.0 monocryl suture (ethicon inc, somerville, nj, usa). after a 2-month follow-up period one month later, the system was reimplanted, with the pulse generator pocket site now in the left buttock. now three years following reimplantation, his pain is well managed with a combination of scs and medications. the patient was a 44-year-old gentleman with a past surgical history of decompression laminotomy, discectomy, and fusion at l5/s1. one year following the surgery, the patient continued to have worsening low back pain, with radiation down his lateral calf into the lateral aspect of his dorsal foot. the patient described the pain as being 8/10 in severity, with an achy quality that was partially alleviated with oxycodone/ acetaminophen. he had a past history of depression and colitis, and had no known drug allergies. magnetic resonance imaging had revealed prior posterior decompression of posterior fusion at the l5/s1 level with metallic hardware artifacts with grade 1 anterolisthesis with posterior uncovering of the disc and superimposed left paracentral protrusion. there was also enhancing tissue encasing the thecal sac and the s1 nerve roots at the l5s1 level. the patient had no sustained improvement with transforaminal epidural injections. following a one-week trial of scs, he reported excellent relief and was subsequently implanted with dual eight-contact leads and the implantable eon pulse generator was placed in the left buttock region. after two weeks, the patient complained of increased pain from the mid back incision. on physical examination the patient was treated for a presumptive superficial wound infection with antibiotics for ten days with resolution of the pain and white count. however, despite resolution of the infection, the patient still had a skin inflammatory change that persisted for 5 weeks following implantation (figure 4). because of poor wound healing, the decision was made to remove the dual leads, silastic anchors with associated 0 silk sutures, and the implantable pulse generator. intraoperative frozen sections revealed acute and chronic inflammation, with a foreign body giant cell reaction (figure 5a and b). the removal of the soft inflammatory tissue in figure 4 revealed the underlying leads, and the pocket floor of the implantable pulse generator that had excess lead coils was notable for soft tissue inflammation. interestingly, there was no fibrotic tissue deposition surrounding the leads, anchors, or the floor of the implantable pulse generator pocket. the patient was a 40-year-old right-handed female, who presented to the pain center with a diagnosis of complex regional pain syndrome affecting the right upper extremity. this condition was believed to be the result of two right shoulder operations for supraspinatus tendon tears. the pain was burning diffusely across her right shoulder, extending over her scapula and down her arm into her hand, with an intensity of 7/10. she was allergic to nickel, oxycodone/acetaminophen, metronidazole, scopolamine, and tramadol. after multiple interventions including stellate and interscalene blocks, transcutaneous electrical nerve stimulation, physical therapy, intravenous lidocaine, and multimodal pharmacotherapy failed to bring appreciable relief, she underwent a one-week trial of scs. after reporting a greater than 50% pain intensity reduction, she had a fully implanted dual 8 contact system placed via an upper back incision at the t2/3 level, with the implantable eon pulse generator placed in the right buttock region. a small pocket for lead extensions was made on the right flank just below the bra line at the t9 level. she had reported some clear drainage from the upper back wound, but there was none noted on examination. five weeks later she called to say that she was developing a partially opened wound at the lead insertion site as well as the lead extension site, with copious amounts of clear drainage. examination of the wound revealed minimal erythema with no hardware exposure but it had grossly evident soft tissue inflammation. her white blood cell count was 8.7 but she had an erythrocyte sedimentation rate of 53 and a c-reactive protein of 11.8. because of persistent wound inflammation and intermittent clear drainage, the leads, silastic anchors (associated 0 ethibond sutures, ethicon inc), and lead extensions were removed 7 weeks post implantation. the clinical plan was to reinsert the leads at a lower level if it was proven to be less immunoreactive using the methods described in case 1. figure 6a and b displays the histology of the specimen results sent from the upper back wound. the wounds healed rapidly with removal of the leads and anchors. one month after removal of the leads, the patient called to report a minor amount of bleeding from the buttock wound where the implantable pulse generator had been implanted. she was afebrile and her white blood cell count was 8.1, erythrocyte sedimentation rate was 45, and c-reactive protein was 13.8. an inflammatory reaction of the pocket floor was noted with very friable tissue (figure 7). the fibrous floor of the pocket that is usually well formed at 3 months post-implantation was disrupted by inflammation., all her wounds were healing well and the drainage had ceased from the buttock. allergic or other inflammatory reactions to the components of spinal cord stimulation systems have only rarely been reported.68 a search of the literature revealed only one recent detailed case series to our knowledge, ie, a report from france describing two cases of cutaneous eruption related to spinal cord stimulators.6 the first of these cases was a foreign body type reaction to the silicone component of the neurostimulatory electrodes, with histology of the affected tissue showing a foreign body granuloma formation. a similar foreign body granuloma reaction occurred after device removal at the surgical scar, in response to silicone particles. the second case was that of a contact dermatitis reaction thought to be due to the silicone part of the stimulator, with histology of affected tissue showing a contact dermatitis pattern. delayed hypersensitivity patch testing confirmed a specific sensitivity to silicone. while reports of inflammatory reactions to spinal cord stimulator devices are exceedingly rare, reactions to other devices, such as pacemakers and cardiac defibrillators, have been reported less rarely.9 the apparent higher rate of inflammatory reaction to cardiac rhythm management devices compared with scs may in part relate to the fact that the former has a higher implantation rate. two types of delayed inflammatory responses might occur in response to implanted devices, ie, delayed hypersensitivity responses to a specific antigen (eg, the metal polyurethane or silicone rubber of a device) or foreign body giant cell granuloma reactions to device material(s). delayed hypersensitiv-ity responses to an allergen are mediated by t cells and monocytes/macrophages, rather than antibodies. contact dermatitis is a form of delayed hypersensitivity reaction to antigen at the surface of the skin, but a similar process can occur to substances inoculated intradermally or subdermally. the patient in case 1 developed such a reaction likely to the polyurethane lead extensions because the rash was along the entire length of their course. this rash was treated successfully with topical hydrocortisone, but the patient presented within 2 weeks with wound dehiscence predisposing to early infection as suggested by the cultures and inflammatory histology. we suspect that the proximity of hydrocortisone treatment to the healing wounds coupled with an inflammatory reaction likely reduced the tensile strength, leading to dehiscence. while contact dermatitis presented approximately one month after implantation, it may present as soon as 8 days, as recently reported after implantation of a peripheral nerve stimulator.10 for contact dermatitis (delayed hypersensitivity reactions to items on the surface of the skin), patch testing is a diagnostic technique that can be utilized. this involves placing a suspected substance on the surface of the skin for 48 hours, then looking for an inflammatory response of the skin at 4896 hours. while this technique can be helpful in some cases, even with contact dermatitis, there can be false negatives and false positives. there is little known about whether patch testing can predict responses to a device that is implanted subcutaneously. the immune system response that occurs with absorption of an allergen from the surface of the skin through the epidermis can be different from that which occurs when the same substance is implanted. the literature provides little insight on the reliability of patch testing to predict an inflammatory response to implanted devices. because of the authors prior experience of false negatives on skin patch testing and the aforementioned complications of case 1, it was thought necessary to try an in vivo test of a piece of lead extension in another site for placement of the implantable pulse generator (figure 3). interestingly, this 2-month test of implantation of the lead extension fragment in the buttock region did not result in any reaction, and the device was successfully implanted. we are unable to explain why an inflammatory dermatitis occurred in the flank/abdominal region but was absent in the back and buttock area. in addition to contact dermatitis, a delayed hypersensitivity reaction may manifest as a granuloma, as reported in the french case series. with delayed hypersensitivity reactions, an antigen is taken up by macrophages or monocytes and is then presented to t cells which can specifically recognize that antigen (ie, there is memory from previous exposure to the antigen). this leads to recruitment of further inflammatory cells to the area, including macrophages that can in turn form giant cells. overall, this inflammatory pattern involving t cells and macrophages is referred to as a granuloma. a foreign body reaction can invoke a similar inflammatory pattern, but it is not in response to a particular immunologically recognized antigen. the inflammatory reaction in response to a foreign body starts as the body tries to respond to the foreign substance by attempting to clean the substance out from the body. macrophages will remain at the foreign body site for an extended period of time, and recruit other inflammatory cells to the area via secretion of chemokines. granulation tissue can form at the site of the foreign body reaction (similar to granulation tissue seen with wound healing, but in a foreign body reaction, the healing process is unable to complete itself). therefore, the foreign body reaction consists of persistent inflammation, characterized by foreign body giant cells and granulomas seen on histology. foreign body giant cells form when macrophages encounter a large foreign body (such as the components of an scs implant). because the macrophage cells can not phagocytose the foreign body, the cells fuse together to form a giant cell composed of many fused macrophages. in this case series, the histology of the affected tissue from cases 2 and 3 was that of a foreign body giant cell reaction (figure 5a and b, and figure 6a and b). in case 2, the clinical impression is that the polyurethane leads are the likely allergens, given that the inflammatory response was seen in the implantable pulse generator pocket floor where the excess lead coils are placed. the polyurethane-coated leads and extensions are even more likely to be the offending stimulus, given that the reaction was in all three wounds, including lead insertion, lead extension, and implantable pulse generator. component materials, which include silicone rubber (eg, anchors), polyurethane (leads, extensions), titanium (implantable pulse generator), and platinum/iridium (electrodes), are part of an allergy test kit (st jude medical neuromodulation) and can be applied topically as a skin patch test.11 among the current three vendors, materials used for the components of scs devices are similar on account of regulation by the us food and drug administration. testing was pursued because, given the location of the inflammatory reaction, it was not felt that allergy testing would alter future clinical decision-making in cases 2 and 3. all patients who receive an implanted device develop some degree of foreign body reaction around the device. why some patients but not others develop a substantial, pathologic, and clinically detrimental level of reaction is not fully known.12,13 interestingly, none of the three patients in this case series had a history of prior allergies to metal, rubber, or autonomic dysfunction to suggest predisposition to cutaneous reactions. in summary, delayed inflammatory responses to the components of scs devices can manifest via t cell/ monocyte-mediated delayed hypersensitivity reactions or foreign body giant cell reactions. contact dermatitis, granuloma formation, and foreign body reactions with giant cell formation are possible in response to scs devices. the role of skin patch testing remains uncertain when testing for foreign body reactions because these occurred one month after implantation. while the lead/extension polyurethane component is suspected as the most immunogenic source in this case series, other materials of the scs device can not be excluded. excision of the inflamed tissue and histologic evaluation is the key for diagnosis and distinguishing between infection and inflammation. infection may occur as a complication of poor wound healing because of an underlying inflammatory response to the component(s) of the scs device.

the use of spinal cord stimulation (scs) devices to treat chronic, refractory neuropathic pain continues to expand in application. while device-related complications have been well described, inflammatory reactions to the components of these devices remain underreported. in contrast, hypersensitivity reactions associated with other implanted therapies, such as endovascular and cardiac rhythm devices, have been detailed. the purpose of this case series is to describe the clinical presentation and course of inflammatory reactions as well as the histology of these reactions. all patients required removal of the entire device after developing inflammatory reactions over a time course of 13 months. two patients developed a foreign body reaction in the lead insertion wound as well as at the implantable pulse generator site, with histology positive for giant cells. one patient developed an inflammatory dermatitis on the flank and abdomen that resolved with topical hydrocortisone. in vivo testing with a lead extension fragment placed in the buttock resulted in a negative reaction followed by successful reimplantation of an scs device. inflammatory reactions to scs devices can manifest as contact dermatitis, granuloma formation, or foreign body reactions with giant cell formation. tissue diagnosis is essential, and is helpful to differentiate an inflammatory reaction from infection. the role of skin patch testing for 96 hours may not be suited to detect inflammatory giant cell reactions that manifest several weeks post implantation.

PMC3738259

pubmed-788

non-specific odontogenic infections are among the most frequent disorders of bacterial origin that affect individuals during the course of their life. before antibiotic treatment was available, the mortality rate of these disorders was 1040%, but the discovery of antibiotics led to a significant improvement of this rate. however, the last 1015 years have witnessed a rebound of severe odontogenic infections caused by highly aggressive antibiotic-resistant bacteria. the main cause indicated by the literature for the development of bacteria resistant to antibiotic therapy is the incorrect or inefficient administration of antibiotics. currently, a number of antibiotics for inflammatory dental pulp disorders or established abscesses are administered. the efficiency of these antibiotics is extremely limited because of the circulation disorders present in the inflammatory focus and, in addition, there is a high risk for inducing adverse reactions and antibiotic resistance of the bacteria involved in the development of the septic process. for this reason, we believe that it is extremely important to evaluate the way in which antibiotics are administered and their efficiency on the bacterial flora involved in the development of odontogenic suppurations, in the absence of surgery. the aim of this study is to prospectively determine the type of antibiotics used by patients with odontogenic head and neck soft tissue infections, from the point of view of their efficiency on the bacterial flora involved in the development of the septic process. the study included 10 randomly selected patients with suppurations of odontogenic origin who presented to the clinic of oral and maxillofacial surgery cluj-napoca in the period january 2014 july 2014. the patient selection criteria were: perimaxillary soft tissue infections of odontogenic origin, disease duration of at least 5 days, antibiotic treatment duration of at least 4 days, known type and mode of administration of antibiotic medication, antibiotic treatment prescribed by the family doctor or the dentist, patient treated under continuous hospitalization, patient from which biological samples were taken for bacteriological examination and antibiogram determination, no other systemic pathology types with a possible influence on the immune response, adult patient having signed an informed consent for participation in the study. the variables monitored for each patient were: general data (age, sex), location of infection, time from onset to presentation, type of antibiotic treatment and route of administration used, bacteriological examination and antibiogram result, postoperative evolution. after the emergency admission of the patients, surgery was performed under locoregional anesthesia. after the asepsis and antisepsis of the operative field, biological samples were collected in a closed environment and were subsequently sent for bacteriological examination and antibiogram determination. the results obtained were centralized in the study data base. for the development of contingency tables, microsoft excel software was used, and the statistical interpretation of the results was performed using microsoft excel. of the patients included in the study, 6 were males and 4 were females. the mean age of the patients was 35.4 years, with a minimum age of 22 years and a maximum age of 49 years. the mean age of female patients was 28.75 years and that of male patients 39.83 years. regarding the location of the septic process in perimaxillary soft tissues, the majority of the infections were located in the submandibular gland area, followed by those located in the genial region (figs. 1, 2). in the case of the patients included in the study, a time period between 5 and 12 days, with a mean of 7.4 days, lapsed from the onset of the septic process to the presentation for specialized treatment. during this time period, all patients used antibiotic treatment, with a mean of 1.2 antibiotic types; 8 of the 10 patients had one antibiotic type, and 2 patients had 2 antibiotic types. 3), and the most frequent route of administration was the oral route, only in two cases the antibiotics being administered by intravenous route. the bacteriological result obtained from the purulent secretion samples collected from the septic focus evidenced microbial polymorphism (tab. i). in half of the patients included in the study, the identified bacterial flora had no sensitivity to the antibiotics 4), and in one patient in whom several bacterial strains were detected, some of these were sensitive to the antibiotic administered to the patient. after the incision and the drainage of the suppuration were performed, along with the change of the antibiotic scheme according to the antibiogram, the patients postoperative evolution was favorable. the objectives of the study were reached and the main types of antibiotics administered to patients with odontogenic septic processes complicated by perimaxillary suppurations were determined. it can be seen that the majority of the patients included in the study were young adults, which is confirmed by other literature studies. authors analyzing extensive groups of patients evidence the fact that odontogenic infections mainly affect patients in the third decade of life, which is confirmed in this study only by female patients, male patients having a more advanced age, but without a significant difference. of the patients included in the study, however, it can not be concluded based on the presented data that the male sex is more frequently affected by cervical inflammatory disorders of odontogenic origin, because the patient inclusion criteria were very restrictive and the number of patients included in the study was limited. most authors opine that there is a higher incidence of odontogenic infections among male patients, but the differences between the two sexes are extremely varied. regarding the antibiotics prescribed to the patients included in the present study, it can be seen that more than half of the patients received amoxicillin treatment with or without beta-lactamase inhibitors. the majority of the patients took the antibiotic without beta-lactamase inhibitors, which is contrary to literature studies, which show that the main antibiotic administered for odontogenic infections is amoxicillin with beta-lactamase inhibitors. the administration of an effective antibiotic in odontogenic infections is particularly important in the attempt to limit the septic process. when the antibiotic has no effect on the main bacterial strains involved in the development of the infection and only eliminates less aggressive pathogens, the premises for extremely severe and very difficult to control infections are created. bacteriological examination evidenced the presence of a varied bacterial flora, but in the majority of the cases, a single bacterial strain in each patient was obvious. the presence of a single bacterial strain in each patient is surprising, given the fact that at the level of the infected dental pulp or periodontal space, the main sources of bacterial flora for odontogenic suppurations, an increased number of bacterial species are concomitantly identified. thus, it is possible that the early administration of bacterial therapy may select the majority of the bacterial species sensitive to the administered antibiotic and a single bacterial species may remain in the septic focus. this hypothesis is also supported by the antibiogram result, which evidenced no sensitivity of the identified bacteria to the administered antibiotic. another possible cause of the identification of a single bacterial strain might be the technical limitations of microbiology laboratories or the way of collecting biological samples, which pose difficulties in identifying some bacterial strains, particularly anaerobic ones. the fact that most of the administered antibiotics were not active on the identified bacterial flora is an alarm signal. the lack of efficiency of the antibiotic on the main bacterial strains involved in the development of the septic process implicitly leads to an increase of the difficulty of treatment of these infections. some authors indicate the use of antibiotics as a single treatment, in the absence of a preliminary bacteriological examination, as the main factor favoring the development of severe odontogenic infections such as necrotizing fasciitis. the exhaustion of the action of common antibiotics on the bacteria involved in the development of common cervical infections leads the practitioner to use niche antibiotics that should not be used under normal conditions. a limitation of this study is the fact that it does not take into account possible cases of patients with odontogenic infections who received antibiotic treatment alone and who had a favorable evolution. these cases can not be monitored in such studies because these patients do not ask for specialized help. most common antibiotics used as a single therapy for the treatment of cervical infections of odontogenic origin have a limited action, and the association of antibiotic treatment with surgery is recommended. the administration of antibiotics according to the bacteriological examination and antibiogram, associated with surgery, led to a favorable evolution of the patients included in this study.

background and aims.odontogenic infections are among the main types of disorders located in the cephalic extremity. the aim of this study was to determine the efficiency of empirically administered antibiotics on the bacterial strains identified at the infection sites. patients and method.the study included 10 randomly selected patients with odontogenic cervical soft tissue infections, who received antibiotic treatment prescribed by the family doctor or the dentist. the bacterial flora involved in the development of the septic process, the type of antibiotic administered to the patient and the sensitivity of the identified bacterial flora to the administered antibiotic were determined. results.in the 10 selected patients, 14 bacterial strains were detected; 7 patients had a single bacterial strain, and 3 patients had two or three types of bacteria. of the administered antibiotics, amoxicillin was the most widely used (33.3% of the cases), followed by amoxicillin with beta-lactamase inhibitors (25% of the cases). in half of the patients, there was no sensitivity of the bacteria detected in the septic focus to the empirically administered antibiotic, and in 10% of the cases, partial sensitivity was evidenced. conclusions.empirical administration of antibiotics without the association of surgery did not prove to be effective in the treatment of cervical infections of odontogenic origin.

PMC4508612

pubmed-789

in particular, musculoskeletal pain and lower back muscle injuries in nurses working in the geriatric setting are very high because of the significant number of patient transfers to wheelchairs that involve lifting [14]. it has been reported that physical therapists working in rehabilitation settings who perform 610 patients transfers per day are 2.4 times more likely to develop lower back injuries than therapists who do not perform transfers. a previous study on occupational and physical therapists showed that transferring or lifting patients was associated with 26.6% of all injuries during work-related activities. moreover, a study on various nursing work activities showed that during the transfer of patients, the nurses ' heart rate (hr) increased to approximately 125 beats/min (bpm) and they had higher levels of neuromuscular fatigue. therefore, the transfer of patients to wheelchairs produces increased burden on the musculoskeletal and cardiovascular systems through changes in joint range of motion (rom), muscle activity, and hr. therefore, there is a higher probability of musculoskeletal and physical strain in healthcare workers who transfer patients. several interventions have been reported to decrease musculoskeletal injuries in healthcare workers [79]. in fact, the implementation of safe patient handling and movement policies by the nursing profession has dramatically decreased work-related injuries and chronic pain. nurses who are more skilled in patient transfer increase the patients ' perceptions of safety and comfort during transfers. some previous literature reported the efforts to decrease musculoskeletal injuries; however, strong evidence for the effectiveness of intervention is lacking. on the other hand, following the adoption of no-lift policies, transfer robotic devices have emerged as tools that have the potential to prevent injuries in healthcare workers. robotic lift and powered devices may decrease both the patients ' effort and the clinicians ' physical burden. one tool emerging from these initiatives is a battery-powered sit-to-stand transfer device that safely lifts and lowers patients between the seated and standing positions [11, 12]. transfer of patients with disabilities who are unable to contribute their own effort depends on powered robotic devices. however, the patients ' physical activity or motivation may increase if the patients can transfer themselves using these assistance devices. recently, a new robotic wheel chair (rwc) has been developed which enables patients to be transferred directly in the sitting position using their own effort and the assistance of a healthcare worker. the manual transfer of disabled patients from the bed to a conventional wheelchair (cwc) is demanding and involves complex movements. patient-handling tasks involved with a cwc can be classified into 3 groups: lifting of the patient, repositioning or turning from the bed towards the direction of the wheelchair, and seating the patient safely in the chair. however, the rwc involves only 1 transfer step, which is that the assistant pushes the patient sitting on a bed forward in the same position to the seat of the rwc. therefore, the rwc may decrease the complexity of transfer and decrease physical load during transfer for healthcare workers. the purpose of this experimental study was to investigate the burden on healthcare workers by measuring rom, muscle activity, and hr during transfer of a simulated patient using either the rwc or a cwc. ten females adults were recruited from an acute hospital and included 6 nurses and 4 rehabilitation therapists who had work experience in transferring patients (mean age: 32.2 9.3 years; range: 2347 years; body weight: 48.8 4.7 kg; height: 157.2 6.7 cm; bmi: 19.7 1.3 kg/m). another female adult (age: 27 years; body weight: 49.0 kg; height: 153.0 cm; bmi: 20.9 kg/m) participated in the study acting as the simulated patient who was transferred from bed to the wheelchairs. the simulation was assumed to be a right hemiplegia patient whose right upper extremity was fixed in a sling. instructions for this procedure were provided by a researcher as, please do not encourage movement of your right lower extremity during transfer to the wheelchair. all the participants provided written, informed consent, and the study was approved by the local ethics committee of the faculty of medicine, tottori university (number 2292). three-dimensional (3d) motion analysis (myomotion analysis system; noraxon usa inc., arizona, usa) consists of combined motion sensors, surface electromyography (emg), and synchronized video recordings. signals of the subjects from these systems were digitally recorded (200 hz and 1500 hz and 30 hz, resp.). in addition, the hr of each subject was measured via a wireless chest-strap electrocardiogram (ecg) monitor (dynascope; fukuda denshi co., ltd., the motion sensor used inertial measurement units, which are widely recognized as a means to overcome the disadvantages of existing optical motion capture systems. the device can measure various kinematic parameters, such as object orientation and velocity, using accelerometers, gyroscopes, and magnetometers. the system has a measurement accuracy of 0.4 degrees for static measurements and 1.2 degrees for dynamic measurements. a standard cwc (matsunaga, co., ltd., the arm and foot supports could swing out upwards (seat width, 40 mm; front height, 42 cm; total length, 95.5 cm; weight, 18 kg). the seat moves back and forth and has an elevating mechanism to adjust the height of the patient (width, 720 mm; length, 750 mm; minimum turning radius, 360 mm; weight, approximately 80 kg; battery, lithium-ion battery) (table 1 and figure 2). the motion sensors used for rom measurements during transfer of the simulated patient were placed on the seventh cervical, seventh thoracic, and fifth lumbar vertebrae and bilaterally on the upper arm, forearm, thigh, shank, and forefoot (figure 3). calibration of the motion sensors was performed before the measurements using the segment model in the standing position. emg electrodes were secured over the muscle bellies of both sides of the biceps, vastus medialis, upper back, and lower back muscles using standard techniques. following practice, an emg signal was recorded during maximum isometric manual testing of each muscle. the signals from the motion sensors, emg, and hr were recorded simultaneously in both experimental conditions (i.e., transfer using either the rwc or the cwc), while the subjects performed the following tasks, once in each situation and in random order: (1) cwc: the subjects supported the trunk of the simulated patient sitting on a bed and lifted the patient to a standing position, converted the patient's position toward the direction of the wheelchair, and seated the patient safely; (2) rwc: the subjects, located on the right side of the simulated patient who was sitting on a bed, supported the pelvis of the patient and pushed the patient directly onto the seat of the rwc. transfer of the simulated patient was assisted until the patient was positioned on the seat (figure 4). the rwc was located at the front of the patient with the height of the seat adjusted to the patient's sitting position using the robotic elevation system. arizona, usa) was used to analyze the signal processing of the motion sensors, emg, and video recordings. in both experimental conditions, the onset and cessation of rom analysis, emg, and ecg were defined as the start of assistance until the end of assistance to transfer the simulated patient and were determined by visual interpretation of the video recordings. rom of the upper extremity, trunk, and lower extremity segments were calculated during both experimental conditions. in both situations, real-time rom during patient transfer was analyzed using data obtained between 2 motion sensors; for example, right elbow-joint motion was analyzed using integrated signals of the accelerometers, gyroscopes, and magnetometers between the right upper arm and right forearm sensors, and the peak rom was identified. for each muscle, the emg data was integrated over 0.01 s intervals during each experimental condition and then normalized for each muscle's emg signal of maximum voluntary contraction (mvc) which was recorded during maximum isometric manual test. mean muscle activity, expressed as% mvc, and mean hr were calculated during both experimental conditions. all statistical analyses were performed using spss for windows version 22 (ibm, co., ltd., table 2 shows the comparison of the motion analyses for cwc and rwc transfer. the peak rom of both shoulder flexion and left ankle abduction during assistive transfer to the rwc were significantly lower than with the cwc. left shoulder abduction, right shoulder rotation, and left knee flexion were significantly higher with the rwc than with the cwc. table 3 shows the comparison of the muscle activation analysis for cwc and rwc transfer. the% mvc of the right biceps, the left upper back muscles, the left lower back muscles, and the right vastus medialis muscle were significantly lower with the rwc than with the cwc. the% mvc of the left biceps was significantly higher with the rwc than with the cwc. there was a significant difference in mean hr during transfer between the 2 conditions (rwc, 87.1 10.9 bpm versus cwc, 99.2 13.2 bpm; p=0.006). we performed power diagnoses for the tests with relevant outcomes and checked that most of them would have sufficient statistical power, for example, shoulder flexion (left 62.4%, right 67.6%), shoulder abduction (left 98.7%, right 8.5%), upper back muscles (left 99.2%, right 10.0%), lower back muscles (left 99.9%, right 20.5%), and hr (88.6%). therefore, our statistician considers that the sample size was valid from a statistical perspective. our study showed that transferring a simulated patient from bed to the rwc decreased the rom on shoulder flexion, back muscle activity, and hr in the subjects compared to using a cwc. these findings suggest that the rwc may have the advantage of decreased muscle activity of the leg or back muscles during transfer compared with the cwc. this is because the rwc enables healthcare workers to push the patient forward in the sitting position; they may not have to lift the patients. in addition, transferring patients using a cwc involves 3 steps: lifting the patient, turning towards the direction of the wheelchair, and seating the patient safely in the chair. this adds complexity and requires the clinician to use more technical methods when transferring the patient. in contrast, the rwc does not involve lifting the patient, who instead can be transferred in 1 step using their own effort to push forward onto the seat of the rwc., the patient does not need to change direction while in the standing position as is the case with the cwc. therefore, transferring to the front using the rwc may decrease the physical load on healthcare workers who often have to perform many patient transfers during a single working day. the peak rom values in the motion analysis of the subjects during transfer to the rwc showed lower shoulder flexion and left ankle abduction compared with transfer to the cwc. transfer of patients from bed to the rwc does not involve lifting and also the foot position while sitting is neutral. in contrast, the technique for transfer to a cwc places higher demand on the shoulder flexion required to lift the patient, and a stride standing position and lower extremity abduction to maintain standing balance. it is not necessary to increase these rom values when transferring a patient to a rwc. on the other hand, rom of left shoulder abduction, right shoulder rotation, and right knee flexion were higher with the rwc. during transfer to the rwc, healthcare workers flex their knees to transfer the patient in the sitting position compared with a standing transfer. shoulder abduction and rotation occur during transfer to the rwc because of the need to hold the pelvis of the patients using both arms. however, pushing the patient forward in the rwc may place a burden on the upper extremities. it is therefore important to recognize the potential of shoulder abduction and rotation muscle overload during transfers using the rwc. with the rwc, lower back muscle activity and lower mean hr during transfers were observed on emg and ecg analyses. in the present study, analysis of the left back muscles during transfer from bed to the cwc showed 48%66% mvc demand in the subjects. in a previous study, emg analysis of the lower back muscles during transfer of patients to we observed similar findings in our study, with the rwc decreasing left back muscle activity to a greater extent (i.e.,% mvc of 29%35%). it is also possible that hr increases because of increased muscle activity with a cwc compared to a rwc. in fact, a previous study showed that nurses rated patient lifting, transfer, and turning as the most physically demanding activities. we suggest that this is an important advantage of the rwc compared to the cwc because of the need to decrease back muscle injury and cardiovascular stress during clinical work [4, 6]. transfer from bed to the rwc requires healthcare workers to bend rather than rotate their trunks, without lifting the patients. this suggests that the rwc provides a simple transfer technique in which it is possible to transfer the load to the upper extremity when transferring patients with higher body weight. the clinical implication of the rwc is that it represents a motorized device for older adults who are physically frail with weakness of the lower extremities and enables them to stand and turn to the wheelchair during transfer. in addition, we hypothesize that transfer using the rwc is advantageous for patients with parkinsonism who can not change their direction when standing. in this situation, we suggest that the rwc needs a robotic function or a sling to carry the patient in the sitting position to a seat. we believe that riding the rwc is suitable for patients with pressure ulcers on the sacral area because the rwc does not have a back support; patients are supported by the breast support and do not have pressure on the sacrum. however, there is no available evidence regarding the pressure of the breast support or the seat. the weakness of the rwc is that this device does not have a back support, although the seat leans forward so that subjects do not fall backwards on the rwc. thus, the use of this device is limited to those who have the ability to sit or stand with assistance. additionally, transferring from the rwc to the cwc or bed is a weak point of this device because subjects who ride on the rwc must turn around and look behind in order to return to the bed. the rwc may need a rearview mirror or an automatic navigation system to correct this problem. therefore, the values obtained in our data analysis may not be applicable to the transfer of patients with paralysis, dysfunction of the lower extremities, or fractures. in addition, the sample size (n=10) might not have been sufficiently large. however, we performed power diagnoses for the tests with relevant outcomes and checked that most of them would have sufficient statistical power. also, as a result of the detailed experimental measurements that we made for all subjects, sufficient objective data for various indices and evidence that transfer of patients in the sitting positon clearly caused less physiological burden were obtained although repetitive load was not studied. the group of patients that would benefit most from transfers in the rwc and the associated implications are not addressed in the present study. lastly, the study did not investigate muscle activity and psychological burden in the simulated patient. shoulder flexion rom, activity of the back muscles, and hr were decreased in the subjects when transferred from bed to the rwc. the rwc enables patients to be transferred in the sitting position directly to the frontal position. it is possible that the rwc will decrease workplace injuries and lower back pain in healthcare workers. using an assisted device to transfer patients without manual lifting has obvious benefits in healthcare and rehabilitation settings.

objectives. the aim of this study was to compare the musculoskeletal and physical strain on healthcare workers, by measuring range of motion (rom), muscle activity, and heart rate (hr), during transfer of a simulated patient using either a robotic wheelchair (rwc) or a conventional wheelchair (cwc). methods. the subjects were 10 females who had work experience in transferring patients and another female adult as the simulated patient to be transferred from bed to a rwc or a cwc. in both experimental conditions, rom, muscle activity, and hr were assessed in the subjects using motion sensors, electromyography, and electrocardiograms. results. peak rom of shoulder flexion during assistive transfer with the rwc was significantly lower than that with the cwc. values for back muscle activity during transfer were lower with the rwc than with the cwc. conclusions. the findings suggest that the rwc may decrease workplace injuries and lower back pain in healthcare workers.

PMC5058567

pubmed-790

divided vascular lesions in the maxillofacial area into two groups: hemangioma and vascular malformations. they are more common in women than men (3:1). about 60% of hemangioma lips, tongue, and buccal mucosa are the most common sites of involvement. however, it is more likely to occur in the gingiva, mandible, palate, floor of the mouth, and parotid gland. this report introduces a rare incidence of hemangioma in the buccal fat pad (bfp) along with phlebolithiasis. the patient was a 28-year-old woman who referred with the chief complaint of a swelling and stiffness in the left cheek. from a clinical perspective on palpation, a moving mass with a stiff area was felt; and in intraoral examination, its position was felt in the anterior ramus. the only point in the patient's history was a course of laser therapy for skin rejuvenation in the left cheek and several other areas in her face. in the medical history of the lesion, there were 3 times of triamcinolone injection in the area during the last 3 years to treat the lesion by another physician. the patient said that reduction in tumor size was seen for a while after these injections. aspiration was performed for the patient through intraoral approach, whose result was negative. magnetic resonance imaging (mri) magnetic resonance imaging revealed a solid heterogeneous mass in the pterygopalatine fossa area with penetration and extension towards both buccal and masticator spaces on the left. the submandibular area and carotid space were normal and no abnormality was seen in the nasopharynx area. mri revealed a solid heterogeneous mass in the pterygopalatine fossa area with penetration and extension toward both buccal and masticator spaces on the left. (b) coronal view of t1 and t2 according to the results of clinical and radiographic examinations of the treatment plan, the excisional biopsy of the studied mass was selected under complete anesthesia. under general anesthesia, the patient underwent a surgery with intraoral access through a cut in the upper area of anterior ramus. after dissection in the upper-side direction, vascular lesion was seen in buccal extension of the bfp [figure 2]. the bfp capsule was intact and the mass had offended no soft/hard adjacent tissue. the possibility of a vascular lesion and second, for the purpose of liposuction for cosmetic goal and remove of the swelling on the patient's cheek, the vascular mass was removed along with anterior lobe, as excisional biopsy through intraoral approach and hence that we did not enter into the vascular lesion [figure 3]. the clinical swelling of the cheek was removed immediately after the surgery. in the macroscopic viewpoint, the lesion was a yellow and dark purple mass measuring 2 cm 3 cm 4 cm along with a hard nodule-like area. microscopic results represented a vascular lesion composed of large amounts of small to large vascular structures covered with endothelial cells. view of the totally excised lesion showing hemangioma with phlebolith histopathologic view of the lesion showing vascular lesion composed of large amounts of small to large vascular structures covered with endothelial cells. hemangiomas usually appear within a few weeks after birth and have a growth rate that exceeds the growth rate of children. in this growth phase, hemangioma will have its own characteristics: endothelial cells getting fatter along with frequent mitotic division, increased number of mast cells, and multilayer basement membrane. following this stage, flat and inactive endothelial cells are located in a context called fibrous fatty tissue with a normal view. sometimes, they can even involve all layers of the skin and offend the muscles. at the cellular level, hemangiomas are characterized by increased birth and death rate of endothelial cells and proliferation of mastocytes during the postnatal proliferative phase in the lesion. derived from young proliferating hemangiomas, capillary endothelium is easily grown in cell culture mediums and forms tubes. in accumulated hemangiomas, hence, a normal hemangioma is an endothelial tumor with a very complex life cycle of cell proliferation and natural regression.. however, their clinical manifestations are not obvious sometimes until late infancy or even childhood. phleboliths consist of a mixture of calcium carbonate and calcium phosphate salts and are thought to form when a fibrous component attaches to a developing phlebolith and becomes calcified. radiologically, they have either a radiolucent or a radiopaque core, and repetition of this calcification causes an onion-like appearance or concentric rings. it should be noted that bfp is also a mass composed of fat tissue covered with a thin capsule membrane and is mainly located in the buccal space. bfp has a rich blood supply and it is a proven fact that bfp has multipotential cells. however, there was no report about the incidence of hemangioma in the bfp until 1956. deighan and barton first pointed to the incidence of a hemangioma case with phlebolithiasis in the bfp mass in 1956. after a review of english literature, only two cases of the incidence of hemangioma in the bfp mass were found, except the above case. ikegami and nishijima reported the incidence of hemangioma in the bfp mass in a 23-year-old patient in 1984. in that case, the tumor was enucleated and the presence of a cavernous hemangioma was confirmed. the last report on the incidence of hemangioma in the bfp was published by tanaka et al. in 2000. the patient was a 3-year-old boy whose tumor was diagnosed 4 months after birth. unlike the report of ikegami and nishijima in which the lesion surface was irregular, in this case, the lesion surface was reported smooth. however, the surface of the removed lesion was also irregular in this report. like the case described in this report, no involvement was reported in the bfp mass adjacent areas in the previously reported cases. when radiographic examinations reveal a radiopaque lesion in the tumor, the differential diagnosis will be easier and there will be two possibilities: hemangioma or sialolithiasis of the parotid gland. however, when phlebolithiasis is not seen, preoperative diagnosis of hemangioma will be very difficult. in the report by tanaka et al., the tumor was removed through extraoral access. however, in the extraoral access, the facial nerve is more likely to be damaged and risk of scar is present. however, tanaka et al. suggested that they used extraoral access because they assumed the probability of large extension of the lesion. this shows the importance of careful radiographic examinations for accurate diagnosis of the lesion limits and the selection of appropriate surgical technique. therefore, if the tumor does not have too large extension according to clinical and radiographic examinations, then intraoral access is preferred. in such cases, the use of only one single imaging modality can not provide enough information with the physician about the diagnosis and treatment of vascular lesions. therefore, the use of mri and computed tomography (ct) is recommended in these cases. the use of ultrasound/color doppler will also be very helpful for validating the mri and ct interpretational results. in general, the incidence of hemangioma in the bfp will be very rare, but in cases where this lesion is suspected, precise preoperative clinical and radiographic examinations are recommended.

hemangiomas are benign vascular neoplasms characterized by an abnormal proliferation of blood vessels. buccal fat pad (bfp) is a rare place for hemangioma. in this report, clinical, radiographic, and histopathological findings are described in a rare case of hemangioma with phleboliths involving the bfp, and a review is made of the international literature on this subject.

PMC4067793

pubmed-791

periodontitis is an inflammatory disease which is initiated and maintained by the gram-negative bacteria of the subgingival biofilm. specific pathogen associated molecular patterns (pamps) and bacterial virulence factors stimulate an inflammatory host response that finally results in destruction of periodontal tissue and tooth loss. chronic periodontitis (cp) and generalized aggressive forms of periodontitis (agp) appear to be associated with certain pathogens, including porphyromonas gingivalis, campylobacter rectus, tannerella forsythia, peptostreptococcus micro, and treponema species [3, 4]. treponema denticola, p. gingivalis, and t. forsythia, characterized as the red complex, were strongly associated with the clinical progression of chronic periodontitis [15]. in contrast, agp was more often diagnosed in patients positive for aggregatibacter actinomycetemcomitans, but there were many individuals with agp who did not harbor this microorganism. in addition to the microbial challenge, other factors, such as genetics, environment, and host factors, play a role in the pathogenesis of these diseases [79]. various compounds, such as cytokines, representing an important pathway of connective tissue destruction in periodontitis, have been detected in gingival crevicular fluid (gcf). il-8, a member of the cxc chemokine family, was originally described by matsushima and oppenheim. it is the most important chemoattractant and activator of human neutrophils and an important mediator for granulocyte accumulation. il-8 is involved in the initiation and amplification of acute inflammatory reactions and chronic inflammatory processes. functions of il-8 are mediated through two receptors (cxcr1 and cxcr2); the expression was detected on numerous cell lineages, including neutrophils and epithelial cells. gingival epithelial cells (gec) are capable of upregulating il8 expression rapidly in response to a. actinomycetemcomitans challenge, facilitating thus the recruitment of neutrophils as a host defense mechanism [13, 14]. il8 expression in gec is induced by p. gingivalis and t. forsythia; il-8 production by gingival fibroblast cultures is also affected by lipopolysaccharides of p. gingivalis and p. intermedia. the il-8 levels in gingival crevicular fluid (gcf) are valuable in detecting the inflammation of periodontal tissue [1820], and periodontal therapy reduces the il-8 levels in gcf. il-8 is encoded by the il8 gene located on chromosome 4q13-21 (genbank accession number m28130.1), consisting of four exons, three introns, and the proximal promoter region. several polymorphisms have been reported in the il8 gene [2326] and some of them can regulate the il-8 production. some of snps in the il8 gene such as 845t/c (rs2227532), 738t/c, 251a/t (rs4073, previously referred to as 353a/t),+396t/g (rs2227307), and+781c/t (rs2227306) have been studied in patients with agp or cp in the brazilian population [2732]. in addition, il8 251 t allele, which was associated with higher production of il-8, increased the risk of developing acute suppurative form of apical periodontitis (ap), whereas il8 251a low-producing allele was associated with chronic nonsuppurative form of ap in the colombian population. in the chinese population, il8 251a allele has been associated with decreased susceptibility to cp. the cross-sectional study in iran has also focused on the study of polymorphisms in the il8 gene but did not specify whether it was for patients with cp or agp. to date, no study analyzing allele, genotype, or haplotype frequencies of il8 gene polymorphisms in patients with periodontitis has been performed in caucasians. the aim of this study was to associate four snps in the il8 gene (rs4073, rs2227307, rs2227306, and rs2227532) and their haplotypes to cp and agp and subgingival bacterial colonization in the czech population. the study was performed with the approval of the committees for ethics of the medical faculty, masaryk university brno and st. written informed consent was obtained from all participants before inclusion in the study, in line with the helsinki declaration. all patients were recruited from the patient pool of the periodontology department, clinic of stomatology, st. exclusion criteria included history of cardiovascular disorders (such as coronary artery diseases or hypertension), diabetes mellitus, malignant diseases, immunodeficiency, current pregnancy, or lactation. controls were selected from subjects referring to the clinic of stomatology for reasons other than periodontal disease (such as dental caries, orthodontic consultations, preventive dental check-ups, etc.) during the same period as patients and matched for age, gender, and smoking status. similarly as patients, all controls had at least 20 remaining teeth and were in good general health. a total of 492 unrelated caucasian subjects of exclusively czech ethnicity from the region of south moravia were included in this case-control association study. diagnosis of nonperiodontitis/periodontitis was based on the detailed clinical examination, medical and dental history, tooth mobility, and radiographic assessment. probing depth (pd) and attachment loss (cal) were collected with a unc-15 periodontal probe from six sites on every tooth present. we used the index of mhlemann to evaluate decreases in alveolar bone levels.generalized cp group (n=278): all patients with chronic periodontitis (cp) fulfilled the diagnostic criteria defined according to cal levels by the international workshop for a classification of periodontal diseases and conditions for chronic periodontitis. inclusion criteria for patients suffering from generalized chronic periodontitis were as follows: 30% of the teeth were affected, pd was 4 mm, and the amount of cal was consistent with the presence of dental plaque.generalized agp group (n=58): patients with aggressive periodontitis with age at disease onset<35 years, attachment loss of 4 mm or more in at least 30% of the teeth (at least three of the affected teeth were not first molars and incisors), and the severity of attachment loss being inconsistent with the amount of dental plaque were included in this study.control group (healthy/nonperiodontitis) (n=156): controls were screened using a who probe and the cpitn (community periodontal index of treatment needs) was assessed; values of the cpitn index in controls were less than 3. generalized cp group (n=278): all patients with chronic periodontitis (cp) fulfilled the diagnostic criteria defined according to cal levels by the international workshop for a classification of periodontal diseases and conditions for chronic periodontitis. inclusion criteria for patients suffering from generalized chronic periodontitis were as follows: 30% of the teeth were affected, pd was 4 mm, and the amount of cal was consistent with the presence of dental plaque. generalized agp group (n=58): patients with aggressive periodontitis with age at disease onset<35 years, attachment loss of 4 mm or more in at least 30% of the teeth (at least three of the affected teeth were not first molars and incisors), and the severity of attachment loss being inconsistent with the amount of dental plaque were included in this study. control group (healthy/nonperiodontitis) (n=156): controls were screened using a who probe and the cpitn (community periodontal index of treatment needs) was assessed; values of the cpitn index in controls were less than 3. in order to adjust for the effect of smoking history on periodontal disease, the subjects (patients and controls) were classified into the following groups: subjects who never smoked (referred to as nonsmokers) and subjects who were former smokers for 5 pack years or current smokers (referred to as smokers). the pack years were calculated by multiplying the number of years of smoking by the average number of cigarette packs smoked per day. dna for genetic analysis was extracted from the peripheral blood leukocytes using standard phenol/chloroform procedures with proteinase k according to sambrook et al.. isolation and storage of dna (working samples at concentrations of 50 ng l at 4c) as well as the genotyping of samples were conducted in the laboratory of the department of pathophysiology, faculty of medicine, masaryk university, brno, czech republic. four snps (845c/t rs2227532, 251a/t rs4073,+396g/t rs2227307, and+781c/t rs2227306) in the il8 gene were genotyped using the 5 nuclease taqman assay for allelic discrimination. individual fluorogenic taqman probes, consisting of an oligonucleotide labelled with both a fluorescent reporter dye, fam, and a quencher dye, vic, were obtained from life technologies (grand island, ny, usa). each reaction mixture was prepared using taqman genotyping master mix (12.5 l), taqman snp genotyping assay (1.25 l), and 50 ng of genomic dna in 17.5 l of dh2o to make a 25.0 l reaction volume. genotyping was carried out simultaneously with 88 samples on 96-well plate (+ 8 negative controls). the pcr thermocycling protocol consisted of 10 min at 95c, followed by 40 cycles of 15 s at 92c and 1 min at 60c. each genotyping plate contained eight wells without any dna template (negative controls) and randomly selected duplicate samples (10% of plate samples). allele genotyping from fluorescence measurements was then obtained using the abi prism 7000 sequence detection system. sds version 1.2.3 software was used to analyze real-time and endpoint fluorescence data. genotyping was performed by one investigator (p. b. l.) unaware of the phenotype. subgingival bacterial colonization (aggregatibacter actinomycetemcomitans, porphyromonas gingivalis, prevotella intermedia, tannerella forsythia, treponema denticola, peptostreptococcus micros, and fusobacterium nucleatum) in subgingival pockets was investigated by the dna microarray based on a periodontal pathogen detection kit (protean ltd., ceske budejovice, cr) in a subgroup of randomly selected subjects (n=151 for cp, n=21 for agp, and n=75 for controls) before subgingival scaling. microbial samples were collected from the deepest pocket in periodontitis patients (and from the deepest sulcus in healthy subjects) of each quadrant by inserting a sterile paper point into a base of the pocket for 20 seconds. this test determined the individual pathogens semiquantitatively as follows: () undetected, which corresponds to the number of bacteria less than 10, (+) slightly positive corresponding to the number of bacteria 10 to 10, (+ +) positive corresponding to the number of bacteria 10 to 10, and (+++) strongly positive, with the number of bacteria higher than 10. comparisons were made between allelic and genotype frequencies in the patients with chronic or aggressive form of periodontitis and control population. the significance of differences in the allele frequencies among groups was determined by fisher's exact test. analysis was used to test for deviation of genotype distribution from hardy-weinberg equilibrium and comparison of differences in genotype combinations among groups. to examine the linkage disequilibrium (ld) between all snps, pairwise ld coefficients (d) and haplotype frequencies variations in the quantity of subgingival bacteria corresponding to the particular genotypes/alleles were tested by and fisher's exact tests. power analysis was performed with respect to the case-control design of the study taking the incidence rate of markers and estimate of the odds ratio (or) as end-point statistical measures. ors with corresponding 95% confidence intervals (ci) were estimated using logistic regression models, adopting age, sex, and smoking as adjusting covariates. 10.0 (statsoft inc., tulsa, ok, usa) and spss software (spss 20.0.1, ibm corporation, 2011) the mean ages for agp patients (37.0 8.2; years sd) and healthy subjects (41.0 12.3) did not differ between the two groups. however, subjects with cp were significantly older (47.9 8.7) than the patients with agp (p<0.05). nearly, 27% of the periodontitis patients (28.0% of cp and 26.0% of agp) and 28.0% of healthy subjects were smokers. there were no significant differences between the subjects with periodontitis and controls regarding the mean percentage of smokers and ratio of males/females (77/79 in controls, 136/142 in patients with cp, and 25/33 in patients with agp). sample size of the study was planned in standard power calculation for case-control design of the study with the null and alternative hypotheses expressed on the basis of or. the design was prospectively optimized assuming the prevalence of examined attribute among controls to be 0.5. the recruited sample (278 cases, 156 controls) allowed a statistically significant detection of or out of the range of 0.551.80 (alpha=0.05, power=0.80). in case of the agp group (58 cases, 156 controls), the statistically significantly detectable ors estimates were out of the range of 0.392.59. allele and genotype frequencies of all investigated il8 polymorphisms were not significantly different between the subjects with cp and/or agp and controls (p>0.05; table 2). considering that in the czech population, snp il8 845tt genotype occurred in 98.4% of cp patients and even 100% of controls and agp patients, we analyzed this polymorphism only in the subgroup of subjects (n=193). based on the previous study the haplotype analysis of these selected snps was performed in the il8 gene (251t/a rs4073,+396t/g rs2227307, and+781c/t rs2227306). all variants in the il8 gene were in tight linkage disequilibrium with each other to various degrees (d=0.7931.000 in controls, d=0.8890.951 in patients with cp, and d=0.8911.000 in patients with agp). the complex analysis revealed differences in il8 haplotype frequencies. specifically, the a(251)/t(+396)/t(+781) and t(251)/g(+396)/c(+781) haplotypes were significantly less frequent in patients with cp (2.0% versus 5.1%, resp., 4.5%, p<0.05) (table 3). the decreased frequency of the tgc haplotype alleles in patients with cp was confirmed by the observation that tgc/ttc haplotype (arranged as genotypes) was less frequent in patients with cp (0.4% versus 2.6%, p<0.05, or=0.09, 95% ci=0.010.96). moreover, an uncommon att/att haplotype (1.15% of the studied population) was found more, but nonsignificantly, in non-periodontitis controls (2.6% versus 0.4%, p=0.07). there was also a nonsignificant trend in the atc/ttc haplotype association with cp (2.2% versus 0.0%, p=0.08, table 4). f. nucleatum occurred less frequently in nonperiodontitis subjects (n=75) positive for t allele of il8+396g/t variant (49.2% versus 77.8%, p<0.02; or=0.28, 95% ci=0.090.89) or tt genotype (21.2% versus 55.6%, p<0.05; or=0.22, 95% ci=0.050.91). in contrast, il8 251 t allele carriers had an increased or for individual presence of a. actinomycetemcomitans in agp (n=21) patients (91.7% versus 40.0%, p<0.01; or=16.5, 95% ci=1.88145.0) and also tt genotype was more often found in a. actinomycetemcomitans presence (83.0% versus 13.3%, p<0.01; or=32.5, 95% ci=2.38443.2). patients with cp (n=151) carrying cc genotype of il8+781t/c variant had less frequent presence of t. forsythia in their subgingival microflora than subjects without this genotype (21.6% versus 35.1%, p<0.05; or=0.51, 95% ci=0.251.05). however, the relationship between periodontal bacteria and il8 gene polymorphisms must be assessed very carefully regarding small numbers of subjects in the respective subgroups. cytokines involved in the inflammatory process, such as il8 and their genes, are important potential modifiers of individual susceptibility to agp or cp. although none of the investigated snps in the il8 gene was individually associated with aggressive or chronic periodontitis, the patients with cp showed lower a(251)/t(+396)/t(+781) and t(251)/g(+396)/c(+781) haplotype frequencies than the controls. the association of tgc haplotype with cp was confirmed by the relationship between tgc/ttc haplotype (arranged as genotypes) and cp. these results confirm the hypothesis that haplotypes are more powerful for detecting susceptibility alleles than individual polymorphisms. our results differ from those obtained by scarel-caminaga et al. who associated atc/ttc and agt/tgc haplotypes with chronic periodontitis in the brazilian population for example, the frequency of the atc haplotype in the czech population was less than 3.4%, compared to 23.7% in the brazilians. in the brazilian population, some haplotypes of il8 845(t/c)/738(t/a)/353(a/t) variants showed significant association to, or protection against, cp. of the three il8 snps, only one polymorphism was the same as in our study (i.e., snp 251 (rs4073) referred to as 353 in brazilian study). polymorphism 845t/c (rs2227532) was also investigated in this study, but regarding a very low frequency of c allele, it was analyzed only in several individuals and therefore it was not included for any further haplotype assessment. to date, eight studies evaluating the association of il8 snps (251,+396, and+781) and cp or agp in different populations (mostly in brazilian but none in the caucasian population) have been performed, with contradictory results [2732, 34, 35]. most of these studies analyzed only the individual snp and were focused on examining of cp; only andia et al. studied the relationship between il8 251 variant and agp; however, no association was found. similarly, kim et al. failed to find any association between cp and allele or genotype distribution of il8 251 snp. in contrast, another study discovered a significant association between il8 251 snp and cp in nonsmokers. the il8 251ta heterozygote genotype was associated with increased levels of il8 mrna transcripts and a allele had an increased risk for developing periodontitis. this is consistent with the observation that the a allele of snp il8 251 tended to be associated with higher il-8 production in lipopolysaccharide- (lps-) stimulated human whole blood. very recently, li et al. found that a allele of il8 251 variant was associated with decreased susceptibility to cp in chinese population.. found a significant difference in the genotype frequencies of il8 251a/t and+396g/t snps between subjects with periodontitis and a control group in hamedan, iran, but did not specify whether it was for patients with cp or agp. conversely, corbi et al. showed that the genetic susceptibility to cp in the il8 gene was not associated with worse periodontal clinical parameters and increased il-8 concentration. with the exception of scarel-caminaga et al, no association between snp at position+396 or+781 and cp in the brazilian population was discovered. several studies have examined polymorphisms in interleukins in connection with a subgingival bacterial colonization in patients with periodontitis. in this study, snps in the gene encoding il-8 were associated with the presence of pathogenic bacteria in subgingival dental plaque. the results showed that il8 251 t allele carriers had an increased or for the individual presence of a. actinomycetemcomitans in agp patients (p<0.01) and an increased or was also found for the presence of t. forsythia for t allele of il8+781 in cp patients (p<0.05). these data are in agreement with the notion that individual genetic susceptibility may influence the host response to infection. nibali et al. found association between il6 snps and a. actinomycetemcomitans, which they confirmed by the haplotype analysis. specifically, il6 174gg genotype was associated with high (above median) counts of a. actinomycetemcomitans (both in all subjects and periodontally healthy subjects only) in indians. but nibali et al. also suggested that only a detection of known periodontopathogenic bacteria could not discriminate different forms of periodontitis. in contrast, schulz et al. found no evidence that snps in il1 gene cluster could be associated with subgingival colonization with a. actinomycetemcomitans and could thus be an independent risk indicator of agp. in addition, finoti et al. observed that periodontal destruction may occur in patients who are considered to be genetically susceptible to cp with a lower microbial challenge because of the presence of the il8 atc/ttc haplotype than in patients without this haplotype. first, the major complicating factor in the study of isolated locus (such as il8) is the nature of periodontitis as a multifactorial disease in which interaction between multiple genes plays a role and each genetic polymorphism has generally only a small effect. in addition, the interaction of gene variants with environmental factors (such as bacterial pathogens), socioeconomic factors, bmi, and others not analyzed in this study, potentially affect the observed phenotype. second, the case-control approach used is generally quite vulnerable to the population stratification, for example, due to different ethnic origin. the present sample, however, is exclusively of the czech caucasian origin, restricted to the limited geographical area populated by quite homogeneous population with low admixture. finally, we did not measure rna expression or protein levels of il-8, therefore, we do not know the functional consequences of these polymorphisms in our subjects. in conclusion, although none of the investigated snps in the il8 gene were individually associated with periodontitis, some haplotypes can be protective against cp in the czech population. clinical significance of these findings is low due to a very low frequency of the protective haplotypes. the individual il8 variants were associated with subgingival colonization with a. actinomycetemcomitans in agp and with t. forsythia in cp in the czech population. however, these relationships must be assessed very carefully regarding small numbers of subjects in the respective subgroups. further studies are needed to clarify the association of these polymorphisms with periodontal diseases in other populations.

objectives. periodontitis is an inflammatory disease characterized by connective tissue loss and alveolar bone destruction. interleukin-8 (il8) is important in the regulation of the immune response. the aim of this study was to analyze four polymorphisms in the il8 gene in relation to chronic (cp) and aggressive (agp) periodontitis. methods. a total of 492 unrelated subjects were included in this case-control association study. genomic dna of 278 patients with cp, 58 patients with agp, and 156 controls were genotyped, using the 5 nuclease taqman assay, for il8 (rs4073, rs2227307, rs2227306, and rs2227532) gene polymorphisms. subgingival bacterial colonization was investigated by the dna-microarray detection kit in a subgroup of subjects (n=247). results. allele and genotype frequencies of all investigated il8 polymorphisms were not significantly different between the subjects with cp and/or agp and controls (p>0.05). nevertheless, the a(251)/t(+396)/t(+781) and t(251)/g(+396)/c(+781) haplotypes were significantly less frequent in patients with cp (2.0% versus 5.1%, p<0.02, or=0.34, 95% ci: 0.150.78, resp., 2.0% versus 4.5%, p<0.05, or=0.41, 95% ci: 0.180.97) than in controls. conclusions. although none of the investigated snps in the il8 gene was individually associated with periodontitis, some haplotypes can be protective against cp in the czech population.

PMC3866791

pubmed-792

obesity has become a major global health challenge. although epidemiological data show that regular physical activity helps prevent obesity, cardiovascular disease, diabetes, and hypertension, the majority of adults fail to maintain physical activity at levels that can promote health. lack of time is the most commonly cited barrier to regular exercise participation, a more time-efficient mode of exercise training has been developed. both high-intensity interval training (hiit; involves near maximal effort at intensities between 80 and 100% of maximal heart rate) [3, 4] and low-volume sprint interval training (sit; involves all-out or supramaximal effort at the intensity of 100% of maximal oxygen uptake) [3, 4] are time-efficient training strategies that can rapidly improve cardiorespiratory fitness [5, 6], muscle metabolic adaptations [7, 8], and insulin sensitivity to resemble the changes elicited by moderate-intensity continuous training (mict). excessive accumulation of body fat, especially intra-abdominal visceral fat, is identified as a potent independent predictor for hyperlipidemia, insulin resistance, and metabolic syndrome, whereas a slight change in the visceral adipose area/volume may significantly alter the risk profile. a growing body of evidence has demonstrated that hiit/sit could reduce body mass [5, 10], total or regional fat mass [6, 1012], and waist circumference [10, 11, 13] and increase fat-free mass [5, 6, 1012]. however, in terms of short-term intervention (e.g., 18 sessions for six weeks), it seems that sit running is effective at improving body composition in active individuals [6, 11], whereas hiit interventions showed inconsistent findings in terms of body composition, including improvement or ineffectiveness [14, 15]. from the view of the influential factors of body composition, confounding factors such as extra physical activity in addition to the training program [5, 10, 11] and/or the dietary intake of the participants [6, 8, 13, 16] given the conflicting results in previous studies [5, 17], whether the hiit intervention induces significant improvements in fat mass, lean mass, and regional fat deposits in comparison with an exercise control remains to be elucidated. although some studies have reported that hiit protocols are enjoyable and adherent for both active male subjects and inactive normal-weight subjects, some researchers have contended that, for the largely inactive and/or obese population, the strenuous nature of sit may produce negative emotions toward exercise adherence and is likely to be a deterrent to participation [18, 20]. given the fact that the power output of the wingate-based protocol with hard resistance (typically 7.5% of body mass) rises quickly and then decreases precipitously over the 30 s, this kind of sit training may not be suitable for the inactive female cohort. as a result little et al. designed a low-demanding hiit model consisting of 10 60 s work bouts at 100% maximal heart rate (hr) interspersed with 60 s of recovery. trapp et al. established a brief hiit protocol of 8 s sprint cycling interspersed with 12 s of rest for 20 min and found that 15 weeks of hiit intervention with this protocol could improve cardiovascular fitness, body composition, and insulin resistance in young women when compared to 40 min of mict with a similar energy expenditure. given that the young women with a higher bmi had a greater fat loss in trapp et al. 's study, we speculated that overweight/obese women and lean women may have different physiological responses to the same hiit training protocol. hormonal abnormalities play a pathogenetic role in the development of excess body fat and are associated with metabolic diseases. a number of hormones, for example, leptin, growth hormone (gh), testosterone, cortisol, and fibroblast growth factor 21 (fgf-21), regulate lipid metabolism and affect muscle protein synthesis and muscle hypertrophy. it has been reported that acute high-intensity interval exercise may result in optimal responses on circulating testosterone, growth hormone, and cortisol in well-trained males as well as in type 1 diabetic individuals. however, it is not clear whether the hormonal responses to an acute bout of high-intensity interval exercise can be sustained after a period of training. collectively, the purpose of this study was mainly to compare the effects of five weeks of hiit intervention or mict on body composition and blood glucose as well as the systemic hormones that may influence body composition and blood glucose in overweight and obese young women. we hypothesized that both training programs would result in similar influences on the improvements of body composition (e.g., reduce fat mass and increase lean mass) and blood glucose, while the improvements are associated with the upregulated gh and testosterone and the downregulated cortisol, leptin, and fgf-21. furthermore, hiit would be more time-efficient and perceived as being easier when compared with mict. the inclusion criteria were being between 18 and 30 years of age, having a classification of inactivity (defined as completion of less than 90 min of moderate-intensity exercise per week over the past six months), and being overweight or obese, defined as having a body mass index (bmi, in kgm) over 23 and a body fat percentage (%) over 30. volunteers who were interested in the study and met the inclusion criteria were required to complete a par-q form and a medical history questionnaire for further eligibility screening. smokers, alcoholics, diabetics, persons with endocrine disorders, and users of oral contraceptive pills or any prescribed medications known to affect body composition or the endocrine system were excluded. then, the subjects underwent a full physical examination to obtain clearance for undertaking vigorous exercise from a doctor. under the assumptions of a within-subject correlation of 0.70 between the pre- and postintervention measures and a power of 0.80 with an effect size of 0.48 based on the primary outcome of vo2peak resulting from high-intensity interval training [3, 29], the sample size for the hiit group after the screening phase, 22 eligible subjects were recruited to participate in this study. they all provided written informed consent before being randomly assigned to either the hiit group (n=11) or the mict group (n=11). one participant in the hiit group and three participants in the mict group quit before completing the training intervention for personal reasons (figure 1). each subject completed a five-week hiit or mict exercise intervention (four sessions per week). before and after the training intervention, subjects underwent a body composition analysis and a vo2peak assessment, and their fasting blood samples were obtained. all pretraining and posttraining measures were conducted in the follicular stage based on the self-reported menstrual cycle survey. because of the different menstrual cycles for the subjects, the starting time for baseline measures and training intervention were different among subjects, with the last subject starting two weeks later than the first one. however, all of the pretraining measures were taken within 48 to 144 h before the training intervention and all of the posttraining measures were taken within 48 to 144 h after the last training session. before baseline measures, each subject visited the laboratory to sign the consent form, become familiar with all testing and training procedures, and provide a three-day diet record. baseline measures were conducted on three different days, separated by at least 24 h, and all measures were completed at least 48 h before the training intervention. subjects were asked to refrain from strenuous activity and caffeine for 48 h and fasted overnight (12 h) prior to the baseline blood sampling. 8 ml blood samples were collected from the cubital veins using serum separation tubes and were left to clot at room temperature for 60 min. then, the blood samples were centrifuged at 3000 rpm for 10 min at 4c, separated for serum, and subsequently frozen at 80c until later analysis. subjects were instructed to come to the laboratory in the morning after a fasting state (12 h). height and weight were determined using standard methods with a stadiometer and an electronic scale (in light clothing and with no footwear) to the nearest 0.1 cm and 0.1 kg, respectively. body mass index was calculated by dividing weight (kg) by height (m) squared. by the same operator, subjects were scanned in a supine position using a dual-energy x-ray absorptiometry scanner (norland xr-36 dxa densitometer, norland corporation, fort atkinson, ws, usa) and were analyzed with a software program (3.7.4/2.1.0; norland corporation). the instrument was calibrated daily using the phantoms provided by the manufacturer, and the value of the intra-assay coefficient of variation (cv) was 0.53%. the abdominal region referred to the area consisting of the line between the two iliac crests, the two edges of the hip, and the lateral sides of the femoral necks. the trunk region was defined as being from the lower edge of the mandibular to the upper edge of the line between the iliac crests, excluding the head and upper limbs. lean mass and fat mass were calculated from the total and regional analysis of the whole body scan. the subjects performed a graded maximal exercise test on a computer-controlled cycle ergometer (monark 839e, sweden) to determine vo2peak and peak power output (ppo). after a two-minute warm-up at 30 w, the subjects pedaled at the initial workload of 50 w and maintained a cycling speed of 60 5 rpm, and the workload was increased by 25 w every three min until volitional exhaustion. respiratory gases were assessed continuously using an automatic gas analyzer (meta-max 3b, cortex biophysik gmbh, leipzig, germany), and the highest oxygen consumption averaged over the final 15 s was identified as the vo2peak. ppo was calculated according to the following formula: ppo=wcom+(t/180) 25, where wcom is the last completed workload, t is the time completed in the final unfinished workload (in seconds), 180 is the increment duration (s) in each workload, and 25 is the workload increment. both the hiit and the mict exercise training were conducted four days per week for five weeks. participants in the hiit group performed 60 repetitions of high-intensity interval exercise (8 s cycling and 12 s passive recovery) on a cycle ergometer (monark 874e, sweden) for 20 min. a prerecorded tape was used to coordinate the hiit intervention, and all subjects worked as hard as they could during the exercise phase. the initial resistance of the exercise phase was 1.0 kg, and once an individual could complete two consecutive sessions at the given workload, resistance would be gradually increased by increments of 0.5 kg until reaching 0.05 body weight. hr (polar f4 m blk, finland) and ratings of perceived exertion (rpe, borg scale) were recorded before and immediately after the completion of the 8 s cycling exercise for every five intervals. during the first and last training sessions the product of all intervention sessions and the mean value of energy expenditure measured during the two mentioned sessions were regarded as the total energy expenditure of hiit. participants in the mict group performed a continuous cycling exercise at 65% of pre-vo2peak on an ergocycle (ergometer 900pc, ergoline, germany) for 40 min; a cycling speed of 60 5 rpm would be maintained throughout each training session. with the increasing fitness indicated by a decreased hr, the energy expenditure for every training session was estimated from an individual's vo2: energy expenditure=5.05 (kcall) vo2 (lmin) exercise time (min), whereas vo2 was determined using an equation for leg cycling ergometry: vo2=7.0+1.8 workload (kgmmin)/body weight (kg). the total energy expenditure of the mict group was calculated as the product of all intervention sessions and the energy expenditure of each session. serum glucose was measured via the glucose oxidase method using a roche/hitachi p800 modular chemistry analyzer (roche diagnostics gmbh, mannheim, germany). serum concentrations of testosterone, cortisol, and gh were analyzed using commercially available electrochemiluminescence immunoassay kits (roche diagnostics gmbh, mannheim, germany), whereas leptin and fgf-21 were measured using a commercial enzyme-linked immunosorbent assays (elisa) kit (abcam, cambridge, uk). the cvs were 1.1% for glucose, 4.5% for testosterone, 4.6% for cortisol, 2.9% for gh, 2.4% for leptin, and 2.6% for fgf-21. subjects in both the hiit and mict groups were instructed to maintain their normal eating habits and normal daily physical activities during the study period. each subject provided a three-day diet inventory one week before and one week after the intervention as well as during the third week of the intervention. energy intake and diet component analyses were conducted by the sports nutrition research center (national institute of sports medicine, china) using the nutrition analysis and management system. daily physical activities were monitored using pedometers (yamax sw-200 digiwalker, japan) for three days per week for a total of seven weeks (on the weeks before and after training and every week during exercise training). posttraining assessments were performed in the same way as described in the pretraining testing protocol and were completed within 48 to 144 h following the last training session. blood samples were taken within 96 to 144 h after the intervention, whereas body composition and vo2peak were determined within 48 to 72 h after the last training session. data were analyzed using pasw software (release 22.0; ibm, ny, usa). independent-sample t-tests were performed to determine the differences in training data (hr and rpe) and energy expenditure between the two groups. a two-way mixed analysis of variance (anova) with repeated measures was used to test for main (time) and interaction effects (time group). significant interactions or main effects were determined using tukey's honestly significant difference post hoc test. as for effect size measure of the main effect and the interaction effect, partial was considered small if <0.06 and large if >0.14. all results were presented as mean standard deviation (sd), and p values of<0.05 were considered significant. there were no significant differences on any measured variables between the two groups on pretraining tests. there was no significant difference in training hr between hiit and mict (164 8 bpm in the hiit group versus 160 12 bpm in the mict group; p=0.435). however, mict is perceived to be significantly harder compared to hiit (13 1 in hiit group versus 15 1 in mict group; p=0.042). in the first training session, the values of energy expenditure were 174 28 kcal in hiit and 301 45 kcal in mict, and the former spent less than the latter (p<0.001). similarly, the total energy expenditure of the intervention in the hiit group (3167 549 kcal) was significantly lower than that in the mict group (6011 505 kcal; p<0.001). the daily calorie intakes (table 3) were not different within group and between groups over time (as evaluated before training, during training, and after training; p>0.05). the proportions of macronutrient intake were approximately 50%, 35%, and 15% for carbohydrates, fat, and protein, respectively, in both groups, with no within-group or interaction differences (p>0.05). physical activities recorded by the pedometers had no within-group or interaction differences before (7673 1145 steps in hiit versus 8062 1367 steps in mict), during (9785 1640 steps in hiit versus 8517 791 steps in mict), and after intervention (7434 1225 steps in hiit versus 7023 849 steps in mict) (p>0.05). after five weeks of exercise training, both hiit and mict resulted in a significant improvement in vo2peak (p=0.006; =0.38) and ppo (p<0.001; =0.61). hiit training increased vo2peak and ppo by 7.9% and 13.8%, respectively, whereas mict training increased vo2peak and ppo by 11.7% and 21.9%, respectively. there were no group differences in the magnitude of improvement in vo2peak and ppo (p>0.05) (table 1). after the intervention, despite no significant changes in weight, bmi, total fat mass (tfm), and total body fatness (tbf) for both groups, the mict group experienced significantly decreased total lean mass (tlm) (1.7 kg or 3.9%; p=0.011) and leg lm (0.6 kg or 3.3%, p=0.018). meanwhile, tlm and leg lm in the hiit group were unchanged (reduced by 0.2% and 0.1%, resp.; p>0.05). in the regions of the trunk and abdomen, no significant changes in lean mass, fat mass, and fatness were observed within group or between groups (table 1). fasting glucose tended to be significantly decreased (p=0.062; =0.213) after the training intervention, but no group difference was found. there were no within-group or group differences in serum levels of testosterone, cortisol, the ratio of testosterone and cortisol (t/c ratio), gh, leptin, or fgf-21 (table 2). for all subjects, no significant correlations were found among the variables of the changes in aerobic capacity, body composition, and the changes in blood parameters. this study showed that five weeks of hiit, despite involving half of the time and exercise energy expenditure when compared to mict, resulted in a similar improvement in aerobic capacity but had no influence on fat mass or lean mass in the trunk or abdomen. hiit subjects seemly lost less lean body mass and lean leg mass than did mict subjects after training. moreover, both short-term training protocols resulted in a trend to decrease fasting serum glucose but had no effects on systemic hormones, including leptin, testosterone, cortisol, gh, and fgf-21, in overweight and obese young women. different forms of hiit have been shown to significantly increase vo2peak [5, 6, 8, 10, 11, 13, 33] and aerobic capacity [10, 11]. the present study found that, after five weeks of this low-volume hiit protocol, the relative vo2peak and ppo were increased by 7.9% and 13.8%, respectively, consistent with an average of 7.3 4.8% increment in vo2peak reported in a meta-analysis after the wingate-based sprint interval intervention in sedentary female cohorts. in accordance with previous studies [5, 17], our study also did not find any additional effect caused by the hiit protocol when compared to mict. moreover, previous studies have demonstrated that, using the same hiit protocol, vo2peak was improved by 15.0% (+ 5.2 mlminkg) in obese men for 12 weeks and was increased by 23.8% (+ 7.6 mlminkg) in sedentary women for 15 weeks, respectively. although the 7.9% (+ 2.5 mlminkg) magnitude of vo2peak was relatively smaller, which might be caused by the shorter duration and differences in exercise intensity, this present study indicates that short-term training with this brief hiit protocol could also result in rapid adaptation in cardiovascular function in inactive obese young women. the possible reasons might be attributed to the upregulated mitochondrial oxidative enzyme activity [7, 8, 21, 34], the enhanced fractional muscle oxygen extraction [6, 35], and/or the increased stroke volume. since the hr monitored during exercises were analogous (164 8 bpm in hiit versus 160 12 bpm in mict) between the two groups, the significantly lower rpe reported in the hiit group may be mainly caused by the interval exercise mode with submaximal exercise intensity, which was 89% of vo2peak during the exercise phases and 76% of vo2peak during the recovery phases according to the data measured in the first hiit session. the game-like nature of hiit, varying between short sprints and recovery intervals, may be helpful in reducing the perception of effort. collectively, compared to mict, the present hiit protocol is a more time-efficient and easier exercise mode for improving cardiorespiratory fitness in the overweight female cohort. to our surprise, the mict group lost 1.7 kg of total lean mass and 0.6 kg of leg lean mass after training. dxa, as a frequently used method to assess body composition, had the smallest detectable differences, at 1.39 kg and 1.30 kg for fat and lean mass in obese children, and the cvs for fat and lean mass were 1.2% and 1.1% in obese females. based on this evidence, we estimate that an approximate 1 kg reduction in total and leg lean mass in the mict group is probably a consequence of measurement error from the dxa. moreover, we did not detect any significant reductions in total and regional fat mass as well as fasting leptin levels following the five-week hiit training intervention. trapp et al. showed that 15 weeks of hiit training with a similar protocol significantly reduced resting leptin levels, and the decreases in leptin levels were positively correlated with the decreases in body weight among normal-weight females. on the contrary, a recent study demonstrated that there were no changes in fasting serum leptin despite improvement in body composition after ten weeks of high-intensity interval training in young women with polycystic ovary syndrome. however, due to the body composition, as assessed using a bioelectrical impedance analysis in their study, we believed the notion that short-term exercise training (12 weeks) does not affect leptin levels and that long-term exercise training that has reduced leptin levels is generally not independent of changes in body fat mass. furthermore, given that fat losses were reported with the same 8 s/12 s protocol but using longer interventions (i.e., 15 weeks and 12 weeks) [5, 10], we speculated that, for a less intense hiit protocol, a longer duration is essential for accumulating measurable alterations in fat loss. there is no definite conclusion regarding whether hiit intervention improves body composition in overweight and obese individuals. several recent studies have shown that hiit interval training reduces total fat mass [5, 10, 12] and abdominal and visceral fat mass [10, 12] and improves lean mass [5, 8, 10, 12] effectively in both obese and nonobese adults, whereas some evidence reported no changes in body composition in overweight individuals [16, 17] or in active men. however, sit, a form of supermaximal exercise intensity of a shorter duration [3, 4], seems to be more effective compared to hiit for improving body composition. a short wingate-based sit, which lasted for two weeks, has been shown to reduce abdominal and subcutaneous fat mass in sedentary overweight/obese men, reflected by decreases in waist (1.4 cm or 1.1%) and hip (1.1 cm or 1.0%) circumferences. consistently, six weeks of running sit interventions led to significant decrement of fat mass and increment of lean mass in recreationally active men and women, and the improvements in body composition were comparable to that of mict. the similar fat losses between hiit and mict may result from the increased excess postexercise oxygen consumption (epoc) [43, 44] and/or the improved muscle oxidative capacity [7, 8, 21], though hiit had a lower total training volume. additionally, in the present study, the training intervention demonstrated a trend toward improved fasting glucose concentrations in the obese female cohort with normal fasting glucose level. previous studies showed that short-term hiit, and even acute hiit, can rapidly improve glucose control in prediabetic [45, 46] and type 2 diabetic patients. on the contrary, some studies reported that, when compared to baseline, short-term wingate-based hiit improved insulin sensitivity but had no substantial advantage for improving fasting blood glucose in healthy sedentary and overweight and obese men. nybo et al. found that 12 weeks of 20-minute high-intensity interval running per week had a similar effect of improving fasting glucose as 60-minute continuous running at 65% vo2peak in sedentary overweight and obese males. taken together, the discrepancy in fasting blood glucose resulting from hiit may be attributable to differences in protocols, intervention durations, and initial fasting glucose levels. we did not find any changes in the basal levels of fgf-21 after the hiit or mict interventions in this population. accumulated evidence derived primarily from animal models indicates that this novel myokine has therapeutic potential for the treatment of type 2 diabetes and has beneficial effects on metabolic disorders. animal studies have demonstrated that both acute exercise and chronic exercise training could increase serum fgf-21 levels in rodents, and the increment is accompanied by increasing serum levels of ketone bodies, glycerol, and free fatty acids. among the few studies examining the effects of exercise/training on fgf-21 levels in humans, it has been shown that a single bout of treadmill running exercise as well as two weeks of daily supervised training increased serum fgf-21 levels in healthy men and women. in the present study, neither exercise regimen had an effect on fasting levels of serum testosterone, cortisol, t/c ratio, and gh, indicating that short-term exercise training, even at a high intensity, can not induce significant effects on the resting hormones in inactive overweight and obese young women. although an acute bout of high-intensity interval exercise or sprint exercise would result in marked increment in cortisol and gh levels, previous studies have shown that the resting levels of cortisol, testosterone, and gh are unlikely to be influenced by exercise training [24, 51]. similar to our study, the fasting levels of gh were unaffected by four to six weeks of hiit/sit in sedentary men or recreationally active males. given that the hormonal changes respond mainly to acute exercise [24, 51], future studies should examine the acute responses of different hormones, as well as body composition, before and after hiit intervention in overweight and obese individuals. first, on the basis of sample estimation from the potential changes of vo2peak, we acknowledged that the small sample size in the present study limits the ability to draw a meaningful conclusion regarding the efficacy of hiit in improving body composition against traditional continuous exercise. second, this study was conducted during a time of the year that people are more likely to gain weight, which started from mid-october and ended in early december. the seasonal factors may also have an influence on body composition since an average net weight gain of up to 0.5 kg in the fall and winter has been reported in a previous study. because of this, for future studies aimed at reducing weight, seasonal factors should be taken into consideration, and a nonexercise control group is needed for interpretation of the relative results. finally, considering the great effect of the combination of a hypoenergetic diet and exercise on weight loss and preserving muscle mass, future studies may consider managing the factors involving demographic characteristics (gender, age, menstrual cycle, etc.), hiit modality (low and high demanding), daily physical activity (intensity and amount), and nutrition status (high- and low-protein diet) thoroughly. this would be helpful to ascertain the impact of high-intensity interval training on metabolic outcomes and the potential roles of hormone meditation during changes in overweight and obese populations. in conclusion, the present study shows that when compared to mict, short-term brief hiit intervention with 8 s of high-intensity interval cycling interspersed with 12 s of rest is a more time-efficient approach and is perceived as being easier for improving aerobic fitness and blood glucose in sedentary overweight and obese young women. neither short-term hiit nor the mict intervention had an effect on body composition or the relevant systemic hormones.

this study was to determine the effects of five-week high-intensity interval training (hiit) on cardiorespiratory fitness, body composition, blood glucose, and relevant systemic hormones when compared to moderate-intensity continuous training (mict) in overweight and obese young women. methods. eighteen subjects completed 20 sessions of hiit or mict for five weeks. hiit involved 60 8 s cycling at ~90% of peak oxygen consumption (vo2peak) interspersed with 12 s recovery, whereas mict involved 40-minute continuous cycling at 65% of vo2peak. vo2peak, body composition, blood glucose, and fasting serum hormones, including leptin, growth hormone, testosterone, cortisol, and fibroblast growth factor 21, were measured before and after training. results. both exercise groups achieved significant improvements in vo2peak (+ 7.9% in hiit versus+11.7% in mict) and peak power output (+ 13.8% in hiit versus+21.9% in mict) despite no training effects on body composition or the relevant systemic hormones. blood glucose tended to be decreased after the intervention (p=0.062). the rating of perceived exertion in mict was higher than that in hiit (p=0.042). conclusion. compared with mict, short-term hiit is more time-efficient and is perceived as being easier for improving cardiorespiratory fitness and fasting blood glucose for overweight and obese young women.

PMC5059579

pubmed-793

insects-resistant population-samples from the el mal field population of t. infestans were collected in november 2010 from infested houses in the chaco province of argentina (s2556.077 w6027.105), where vector control using pyrethroid insecticides is considered ineffective by the authorities responsible for the chagas program of chaco province. field-collected insects were transported to the research center of pest and insecticides (cipein) laboratory and further generations of these insects were bred in the laboratory. the 50% lethal dose (ld50) obtained for deltamethrin for this population is 134 ng/insect, with a high resistance ratio of 1,031 (carvajal et al. a study performed at cipein has shown that eight years after the most recent exposure to deltamethrin, the ld50 did not vary in a population from northern argentina (germano 2013). susceptible population-for comparison, we used a susceptible reference colony, nfs, derived from a domestic field population collected in december 2004 from santiago del estero, argentina. this laboratory colony has been maintained without the introduction of new insects from external sources. a laboratory test with nfs has shown an ld50 of 0.13 (0.11-0.15) ng/i for deltamethrin. this value did not differ statistically from that of the traditional laboratory-susceptible strain maintained at cipein (roca acevedo et al., each population was kept in enclosed boxes (30 x 30 x 30 cm) at 28 1c and 50-60% relative humidity with a photoperiod of 12:12 h (l: d). a pigeon was provided weekly as a blood meal source (who 1994). chemicals-technical grade imidacloprid (98%) provided by dr ehrrenstorfer (augsburg, germany) was used in topical application bioassays. analytical grade acetone was purchased from jt baker (san pedro xalostoc, mexico). in the surface assays and spot-on bioassays, the following formulations of imidacloprid were used: 35% emulsifiable concentrates (ec) (mamboret confi and ec-chemotcnica (argentina), 70% wettable granule (wg) (bayer confidor, argentina) and 10% spot-on (bayer advantage g, argentina). moreover, 2.5% ec deltamethrin (bayer k-othrine, argentina) was used only in surface bioassays. topical application bioassays-t. infestans first instars (5-7 days old) that had been starved since eclosion were selected for toxicity tests according to the world health organization protocol (who 1994). the bioassays consisted of the topical application of 0.2 l of the insecticide diluted in acetone on the dorsal abdomen of the first instar using a 10 l hamilton syringe equipped with an automatic dispenser. in the evaluation of blood feeding, nymphs were previously fed to repletion on a pigeon and the insecticide was applied immediately after feeding. the final concentrations of imidacloprid tested ranged from 0.0025-0.5 mg/ml. all concentrations were replicated at least three times with a minimum of 10 insects per replicate. to calculate the ld values at p<0.05, a minimum of 30 insects per concentration was required and mortality values between 10-90% were observed (robertson et al. mortality was evaluated after 24 h and also after 48 h and 72 h in the delayed toxicity assays, by placing the insects on a circular piece of filter paper (11 cm diameter) and observing their ability to walk. only nymphs that were able to walk from the centre of the filter paper to the border evaluation of formulated insecticides- on glass-insecticides were applied to a square area (96 cm) using a 1 ml pipette and a constant flow to achieve uniform impregnation. the treated surface was dried for 24 h. each replicate consisted of a negative control group (water), a positive control group [ec 2.5% deltamethrin in water at 25 mg active ingredient (ai)/m] and one or more doses of formulated imidacloprid, ranging from 1,000-5,000 mg ai/m. groups of 10 nymphs (3rd-instar nymphs aged 10-20 days, starved since last moult) were confined in glass rings and exposed for 1 h to the treated surfaces. after exposure, the insects were placed in clean flasks with filter paper and were maintained under the laboratory conditions described previously. mortality was recorded after 24 h (germano et al. 2014). on filter paper-circular disks of whatman n 1 filter paper (5.5 cm diameter) the papers (area 23.75 cm) were homogeneously impregnated with 0.1 ml of the formulated insecticide. the control groups were exposed to filter paper homogeneously impregnated with 0.1 ml of pure water. after 1 h, when the solvent had evaporated, the insects (10 1st-instar nymphs per group selected as in topical application bioassays) were held in contact with the treated surface for 1 h. after this period, live insects were placed in clean flasks and were maintained under the laboratory conditions described earlier. mortality was recorded after 24 h (rojas de arias&fournet 2002). on pigeons-selected pigeons were treated with various doses of imidacloprid advantage g. the average weight of the pigeons was 252.7 39.4 g. twenty-one pigeons were used. the experimental design included five groups (4 treated and 1 control) with a minimum of three pigeons per group. prior to the application of the spot-on, we removed several feathers from the pigeon to produce a blind spot or arena where the insects could feed. the insecticide was applied to the base of the neck with a needle-less syringe. the insects were then exposed in the area where the spot-on formulation was applied. we have previously determined that this area was the most feasible site because the insects were removed easily without any additional disturbance to the treated pigeon. the pigeon groups were treated with 1, 5, 20 and 40 mg/ai of the formulation (i.e.,=4, 20, 80 and 160 mg/ai per kg of animal). the control group was manipulated similarly, without the addition of any insecticide, but water. synchronised insects (1st and 3rd-instar), previously starved for 15-20 days, were allowed to feed for 30 min on the treated area of the pigeons. the residual effect of the drug was studied on feeding nymphs at different intervals of time (1, 7, 14 and 21 days post spot-on application) after the administration of the drug. the tests were performed by allowing the insects housed in jars containing 10-30 insects to feed on the pigeon. a total of 1,054 and 717 first and third-instar nymphs were used, respectively. the fed insects were transferred to clean flasks with filter paper and kept under the laboratory conditions described previously. mortality was recorded after 24 h. statistical analysis-in the topical application and surface bioassays, mortality data were analysed using polo plus software v.2.0. dose-mortality data were subjected to a probit analysis to estimate the ld (ng/insect) required to kill 50% of the treated individuals (ld50). in the spot-on bioassays, all mortality data were corrected for control mortality with abbott s equation (abbott 1925). the percentage mortality was determined and transformed to arcsine square-root values for an anova. a 0.05 significance level was chosen as the criterion for biological significance among related treatments. evaluation of delayed toxicity and influence of the blood feeding state -the ld50 values for the susceptible (s) and resistant (r) populations were 5.2 (3.4-7.8) and 9.2 (7.4-11.2) ng/insect, respectively, and did not differ up to 72 h after the initial topical application. we also studied the variation in the toxic effects of imidacloprid relative to the blood feeding condition of the insect because the cuticular distention resulting from blood feeding facilitates the penetration of the insecticide. the blood feeding condition (starvation/feeding) of the insects had no significant influence on the insecticidal activity of the imidacloprid in either population (table). table insecticidal activity of imidacloprid over starved and fed nymphs i of triatoma infestans at 24 hpopulationblood feedingld50 confidencestate(ng/insect)limits (ng/insect)susceptiblestarved5.23.4-7.8fed42.4-7resistantstarved9.27.4-11.2fed10.86.4-19ld: lethal dose. formulation-surfaces-four formulations of imidacloprid were tested against first and third-instar t. infestans on two different surfaces: filter paper and glass. the ecs chemotcnica and mamboret confi and the wg confidor showed no effects, either on glass against third-instar nymphs (at 1,000-5,000 mg ai/m) or on filter paper against first-instar nymphs (at a 100 mg/ml dose). the spot-on advantage g could not be tested on glass because it did not form a film after 24 h of drying. on filter paper, advantage g was effective, with a mortality of 100% with a 100 mg/ml dose in both susceptible and resistant populations. the lc50 obtained for advantage g on filter paper was 22.84 (14.94-36.89)mg/ml. in contrast, a 10 mg/ml dose of deltamethrin on filter paper caused 100% mortality in the susceptible population, whereas no mortality (0%) was found in the resistant population. spot-on on pigeons-a first evaluation of formulations of imidacloprid on glass and filter paper showed that only the spot-on formulation was effective. accordingly, we analysed the effect of the spot-on formulation of imidacloprid by applying different doses of the insecticide to pigeons. the applied dose of 1 mg/ai showed no lethal effects against first and third-instar t. infestans (p>0.05). twenty-four hours after the application of 5 mg/ai to pigeons, nymphs that had fed on the pigeons showed a higher mortality rate (49.8 1% and 40.5 18% for first and third-instar, respectively) than the control group (p<0.01). nymphs fed seven days after a spot-on application did not show significant differences in mortality between the treated and control groups (p>0.05). nymphs fed 14 and 21 days after spot-on application did not show significant differences in mortality between the treated and control groups at any studied dose or nymphal stage (p>0.05). both first and third-instar nymphs fed on pigeons that had been treated with 20 mg or 40 mg of the formulation showed a higher mortality rate than the control group one and seven days post-treatment (p<0.01). the residual effect (7 days after treatment) was higher for 40 mg than for 20 mg (p<0.01) the lethal effect was similar against first and third-instar nymphs at all doses and time intervals (a, b in figure). mortality of triatoma infestans fed at different intervals after the spot-on application. bars with different letters are significantly different (p 0.05) within each day. although the pyrethroids deltamethrin and -cia-lothrin are available, the only insecticides approved by the health service of argentina for use in the field control of t. infestans are the organophosphates fenitrothion and malathion. these insecticides were used to control t. infestans in the 70 s, but because of their high toxicity in mammals, strong odour and tendency to leave stains on the walls after application they were replaced by pyrethroids in the 80 s (schofield&dias 1999). fenitrothion has been shown to be effective against several deltamethrin-resistant populations under laboratory and field conditions (picollo et al., the development of pyrethroid-resistant populations has led to the re-utilisation of either malathion or fenitrothion against triatomines by the health authorities of argentina and bolivia. this is the first study of the efficacy of several formulations of imidacloprid on different surfaces against susceptible and pyrethroid-resistant t. infestans. in an attempt to characterise the toxicology of imidacloprid against t. infestans, we studied the variation in mortality through time after topical application and the influence of the blood feeding condition of the insect on this toxicity. our results showed that the ld50 did not vary significantly up to 72 h after the initial topical application. this result could indicate that the toxicological effects of the imidacloprid remain stable through time. (2001), who found a decrease in the toxicity of imidacloprid through time in the ground beetle h. pennsylvanicus (coleoptera: carabidae) following contact exposure. the blood feeding condition (starvation/feeding) of the insects had no significant influence on the insecticidal activity of the imidacloprid. thus, the rate of penetration associated with physicochemical modifications of the cuticle after blood feeding appears not to alter the toxic effects after topical application. we also analysed various types of commercial formulations of imidacloprid on two different surfaces. thus, the first step, focusing on the field application of the insecticide, is to find a correct formulation of the insecticide. we found that neither the ec nor the wg formulations were effective against t. infestans nymphs. although most surfactants and polymers are biologically inert when applied to insects, these chemicals can profoundly affect the biological activity of the pesticide when used as part of a formulation (scher 1988). traditional spraying is highly effective inside domiciles, but it usually leaves a number of residual individuals of the vector in the peridomestic environment, as reported in the southern part of the chaco region (porcasi et al. these residual populations eventually re-colonise the domestic sites, re-establishing the domestic transmission cycle of t. cruzi (grtler et al. 1995), pyrethroid-impregnated curtains (ferral et al. 2010) and a residual paint formulated as a micro-encapsulate containing an organophosphate and a juvenile hormone analogue (alarico et al. although t. cruzi is transmitted by several species of triatomines, animals such as dogs, cats and chickens are the main domestic reservoirs of t. cruzi in the endemic areas of chagas disease (grtler et al. thus, reithinger et al. (2006) found that deltamethrin-impregnated dog collars reduced the survival and fecundity of exposed kissing bugs on dogs. similarly, a spot-on formulation of fipronil applied on dogs and a pour-on formulation of cypermethrin applied on chickens have been successfully tested against t. infestans (rojas de arias&fournet 2002, amelotti et al. the imidacloprid advantage g spot-on formulation is recommended for treating cat fleas. the recommended dose ranges from 10-40 mg ai/kg. in this study, we tested doses from 4-160 mg ai/kg on pigeons. at a dose of 20 mg ai/kg, 50% of the nymphs were killed 24 h after the application. the doses of 80 and 160 mg ai/kg produced 100% mortality and had a high residual effect until seven days post-treatment. (2009) studied the efficacy in chickens of a pour-on formulation containing cypermethrin. they found that after a week of initial exposure to the insecticide at a dose of 120 mg/chicken, 53% of the treated third-instar nymphs were killed, whereas at day 14, mortality had values similar to the controls (4.9%). this finding is similar to our results because a dose of 160 mg/kg between days 7-14 after initial exposure produced a mortality of 58% and 8%, respectively. rojas de arias and fournet (2002) studied the residual activity of fipronil with a contact test of the insecticide on filter paper against fifth-instar t. infestans. the authors reported a value of lc50 of 106 mg/m. despite the subtle differences in methodology, the lc50 value of 960 mg/m of imidacloprid suggest that this compound has a high contact activity that depends on the type of formulation. thus, an approach involving the use of the spot-on formulation might complement traditional pyrethroid spraying, which has shown a low efficacy in the elimination of t. infestans peridomestic populations. the effectiveness of imidacloprid against t. infestans is reinforced by its lower oral and dermal mammalian toxicity than fenitrothion, the current alternative to pyrethroids. for instance, the oral and dermal ld50 in rats for imidacloprid are 450 mg/kg and>5,000 mg/kg, whereas the values for fenitrothion are 250 mg/kg and 2,500 mg/kg, respectively. additionally, the no-observed effect level in rats is higher for a diet containing 300 mg/kg imidacloprid [based on a unit of 1 kg of body weight than for a diet containing 10 mg/kg fenitrothion (tomlin 1997)]. this concern is highly important if an insecticide must be used indoors and in a domestic environment. because of the increasing number of populations resistant to pyrethroids and the high mammalian toxicity of fenitrothion, it is essential that other insecticidal compounds, especially those with alternative modes of action to pyrethroids and organophosphates, are rapidly made available for t. infestans control programmes in south america. this study has indicated the potential of imidacloprid in the control of chagas disease vectors. however, imidacloprid should be incorporated into an integrated pest management programme because its effectiveness is primarily restricted to domestic and peridomestic animals. moreover, the type of formulation selected is essential in the function of the toxicokinetic and toxicodynamic processes by which the active ingredient (i.e., imidacloprid) affects the insect (t. infestans). a spot-on formulation appears to improve this interaction, resulting in increased mortality of the triatomine vector in the laboratory. further studies are needed to test this type of formulation under semi-field or field conditions and to incorporate this formulation as a complementary strategy for triatomine control.

the prevention of chagas disease is based primarily on the chemical control of triatoma infestans (klug) using pyrethroid insecticides. however, high resistance levels, correlated with control failures, have been detected in argentina and bolivia. a previous study at our laboratory found that imidacloprid could serve as an alternative to pyrethroid insecticides. we studied the delayed toxicity of imidacloprid and the influence of the blood feeding condition of the insect on the toxicity of this insecticide; we also studied the effectiveness of various commercial imidacloprid formulations against a pyrethroid-resistant t. infestans population from the gran chaco ecoregion. variations in the toxic effects of imidacloprid were not observed up to 72 h after exposure and were not found to depend on the blood feeding condition of susceptible and resistant individuals. of the three different studied formulations of imidacloprid on glass and filter paper, only the spot-on formulation was effective. this formulation was applied to pigeons at doses of 1, 5, 20 and 40 mg/bird. the nymphs that fed on pigeons treated with 20 mg or 40 mg of the formulation showed a higher mortality rate than the control group one day and seven days post-treatment (p<0.01). a spot-on formulation of imidacloprid was effective against pyrethroid-resistant t. infestans populations at the laboratory level.

PMC4238768

pubmed-794

the pathology of rheumatoid arthritis (ra) is characterized by the proliferation of synovial cells and angiogenesis, pannus formation. multiple cell types, including lymphocytes, dendritic cells, macrophages, and synovial fibroblasts, contribute to the chronic inflammatory responses of ra, and comprise a major portion of the invasive pannus. in addition, angiogenesis, the process of new blood vessel formation, is highly active in ra, particularly during the earliest stages of the disease [2, 3]. newly formed vessels can maintain the chronic inflammatory state by transporting inflammatory cells to sites of synovitis, and supply nutrients and oxygen to the pannus [2, 3]. angiogenesis is strictly regulated by many inducers and inhibitors, and a number of proangiogenic factors have been suggested to be involved in neovascularization in ra joints. these include acidic and basic fibroblast growth factors, transforming growth factor (tgf)-, angiopoietin, and placenta growth factor (pigf) in addition to vascular endothelial growth factor (vegf) [24]. the final goal of ra treatment is complete disease remission, and not symptomatic relief. at one end of the spectrum of ra treatment outcomes lie a large group of patients who do not respond to single disease-modifying antirheumatic drugs (dmards). recent clinical trials have suggested that several biologic agents, such as tnf- blockers, rituximab, abatacept, and anakinra, are effective at retarding joint destruction and at alleviating ra activity [5, 6]. however, these biologic agents may have serious side effects, such as predispositions to tuberculosis, lymphoma, progressive multifocal leukoencephalopathy, and high cost, which limit their use. it is also a concern that abrupt stoppages or reductions in these treatments may result in a relapse of disease activity. moreover, the pathology of ra suggests that it is unlikely that a single biologic agent that targets a specific subset of immune cells is capable of effecting cure. in this review, we integrate current knowledge concerning how angiogenesis, specifically vegf, contributes to disease exacerbations in ra. in addition, we present a new therapy for ra based on a synthetic anti-vegf hexapeptide that specifically targets the interaction between vegf and its receptor. prospects for the development of pharmacologic regulators of placental growth factor, which is another angiogenic factor implicated in the pathogenesis of ra, also are discussed. vegf is a dimeric glycoprotein that induces the proliferation and migration of endothelial cells to form new blood vessels, and which increases vascular permeability. vegf plays important roles during wound healing, embryonic development, the growths of certain solid tumors, and during ascites formation. several recent reports have demonstrated that vegf is also implicated in the pathogenesis of ra. vegf in synovial fluids is significantly more increased in ra than in osteoarthritis [2, 13, 14], and serum levels of vegf correlate well with ra disease activity, particularly with swollen joint counts. vegf protein and mrna are expressed by synovial macrophages and synovial fibroblasts in the synovial tissues of ra patients, and cultured synovial cells are able to secrete vegf under hypoxic conditions or when stimulated with il-1, il-6, il-17, il-18, -prostaglandin, or tgf-, or by cd40 ligation [24, 1517]. furthermore, vegf knockout mice showed reduced pathology and synovial angiogenesis in antigen-induced models of arthritis. these findings strongly suggest that the inhibition of the angiogenic action of vegf is likely to suppress rheumatoid inflammation. angiogenesis and inflammation are interdependent processes, and inflammatory mediators have significant effects on angiogenesis [2, 3]. furthermore, recent studies have suggested that the reverse is also true [14, 19]. for example, chronic transgenic delivery of placental growth factor (plgf) to murine epidermis resulted in a significant increase in inflammatory response. in addition, in a previous study, we demonstrated that 165-amino acid form of vegf, vegf165, has a direct proinflammatory role in the pathogenesis of ra. in this previous study, recombinant vegf165 was found to increase the productions of tnf- and il-6 by human peripheral blood mononuclear cells (pbmc). moreover, the synovial fluid mononuclear cells of ra patients showed a greater response to vegf165 stimulation than the pbmc of healthy controls (the major cell types that responded to vegf were monocytes). these findings suggest that vegf165 may act as a proinflammatory mediator and as an angiogenic stimulator in ra joints, and thus, they indicate that vegf is an important link between angiogenesis and the inflammatory process. a number of inflammatory cell types participate in maintaining a mutually activating network in ra joints, which leads to the establishment of a self-perpetuating cycle of autoimmunity. it has been documented that vegf165 activates endothelial cells to produce chemokines, such as mcp-1 and il-8 [20, 21], which may recruit monocytes around endothelial cells in synovial membranes, where newly employed macrophages, in addition to resident synoviocytes, can produce tnf- and il-6 when stimulated by vegf165 (as was evidenced by our work) or via cell contact with activated endothelial cells. tnf- and il-6, in turn, further enhance the capacities of macrophages and synoviocytes to secrete vegf165, and stimulate endothelial cells to induce cell-contact-mediated macrophage activation, which generates a positive feedback-loop (figure 1). thus, vegf165 may serve as a functional bridge between endothelial cells and macrophages/synoviocytes. in ra synovium, synovial fibroblasts proliferate abnormally and invade local environments, and in some ways exhibit the characteristics of tumor cells. recently, we demonstrated that vegf is crucially required for the survival of rheumatoid synoviocytes. in this previous study, the ligation of recombinant vegf165 to its receptor prevented synoviocyte apoptosis induced by serum starvation or sodium nitroprusside (snp). vegf165 rapidly triggered pakt and perk activity, and then induced bcl-2 expression in rheumatoid synoviocytes. furthermore, vegf165 completely blocked snp-induced bcl-2 downregulation and snp-induced bax translocation from the cytosol to mitochondria. collectively, these results suggest that vegf functions as an important synoviocyte survival factor in ra. as mentioned above, vegf165 is present at higher levels in sera, synovial fluid, and in the inflamed synovial tissues of ra patients than in those of osteoarthritis patients [13, 14]. therefore, ra synoviocytes are more likely to be stimulated by vegf165 than osteoarthritis synoviocytes, which causes synoviocyte hyperplasia. moreover, hyperplastic synoviocytes in ra joints secrete more vegf165, and thus, generate positive feedback that promotes their survival (figure 1). vegf165 exerts its biological effects by binding with its receptor subtypes, that is, fms-like tyrosine kinase (flt-1), kinase insert domain-containing receptor (kdr) and neuropilin-1 (np-1). flt-1 and kdr exhibit tyrosine kinase activity, and both are expressed in the majority of vascular endothelial cells [8, 24, 25]. kdr is a primary mediator of endothelial cell proliferation in response to vegf165, whereas unlike kdr, flt-1 is present in inflammatory cells, such as, macrophages and monocytes [8, 24, 25]. therefore, in addition to its proangiogenic action, flt-1 is critically involved in monocyte activation, and in addition, it also promotes the mobilization of myeloid progenitors from bone marrow to the blood [8, 24, 25]. on the other hand, np-1 has been demonstrated to function as a nontyrosine kinase receptor for vegf165, and specifically, for the heparin-binding domain of vegf165 [26, 27]. np-1 was initially characterized as a receptor for semaphorin 3a, which mediates the guidance of neuronal cells. in endothelial cells, np-1 is also a coreceptor of vegf, and has been shown to regulate kdr-dependent angiogenesis [26, 27]. furthermore, we previously demonstrated that np-1, rather than flt-1 or kdr, is the major vegf165 receptor in ra synoviocytes. np-1 was found to be highly expressed in the lining layer, and on infiltrating leukocytes and endothelial cells of the rheumatoid synovium. furthermore, the downregulation of np-1 transcripts by short interfering rna caused spontaneous synoviocyte apoptosis, which was associated with both a decrease in bcl-2 expression and an increase in bax translocation to mitochondria. in addition, more recently, we found that flt-1 is highly expressed in the sublining of leukocytes in ra synovium, and in monocytes from the pbmc of active ra patients. furthermore, flt-1 expression levels were found to be well correlated with erythrocyte sedimentation rates, a marker of disease activity, indicating that they reflect inflammatory activity of ra. these findings suggest that chronic inflammatory milieux, such as those generated by high concentrations of proinflammatory cytokines, may upregulate flt-1 expression on ra monocytes. first, vegf165 binding to kdr may lead to an increase in angiogenesis, and thereby, the recruitment of peripheral leukocytes to inflamed synovium, which diminishes the growing burden of synoviocytes by supplying the oxygen and nutrients required for tissue metabolism. second, via interaction with flt-1, vegf165 may directly stimulate the productions of cytochemokines, such as tnf-, il-6, mcp-1, and il-8, which are essential for the perpetuation of chronic inflammation in joints. third, np-1 could hamper synoviocyte apoptosis upon ligation of vegf165, and thus, function as a survival factor, in an autocrine or paracrine fashion. in this manner, vegf165 would simultaneously regulate the developments of synovial inflammation, hyperplasia, and angiogenesis in ra joints (figure 1). the success of anti-vegf antibody (ab) treatment in cancer patients raises the possibility of applying antiangiogenic therapies in other diseases, such as retinopathy, ra, and other inflammatory disorders. given the pleiotropic roles played by vegf and its receptor in ra inflammation [2, 3, 8, 24], it can be postulated that anti-vegf treatment retards chronic synovitis in several ways, as follows: (a) it may decrease nutrient supply to the tumor-like synovium; (b) inhibit leukocyte adhesion and migration by decreasing endothelial cell surface area; (c) decrease chemokine and cytokine productions by activated endothelial cells; (d) reduce the vegf-induced productions of tnf- and il-6 by monocytes/macrophages; (e) abrogate vegf-induced increases in synoviocyte survival. these different mechanisms may occur independently, but it remains to be determined which mechanism plays a dominant role in the quenching of ra inflammation. bevacizumab, a humanized form of anti-vegf ab, was the first antiangiogenic agent approved by the fda in the us for the treatment of metastatic colon cancer, and it was also found to be beneficial for the treatment of lung and renal cell cancer [33, 34]. although bevacizumab is generally well tolerated, it has some serious toxic effects, for example, hypertension, bleeding, and arterial thromboembolism, which occur infrequently [3234]. currently, various different developmental approaches to inhibit vegf and its receptors are in progress. the fda approved pegaptanib, an anti-vegf rna aptamer, for the treatment of neovascular age-related macular degeneration (amd). notably, pegaptanib was found to reduce vision loss in amd patients by about 50% during the first treatment year and to stabilize vision during the second year. other agents which target vegf receptors, such as, chimerized anti-kdr antibody, vegf-trap, and a synthetic ribozyme of flt-1, are also undergoing phase i or ii trials for the treatment of solid tumors and cancer. the effects of vegf and of its receptor antagonists have also been tested in experimental models of ra. neutralizing ab to vegf was found to prevent collagen-induced arthritis and to ameliorate established disease in mice, and treatment with a soluble form of flt-1 receptor significantly attenuated the severity of murine collagen-induced arthritis. interestingly, the failure of anti-kdr ab, but not of anti-flt-1 ab, to block arthritis and atherosclerosis [24, 25, 39], indicates that anti-inflammation, rather than antiangiogenesis, may be primarily responsible for the observed effects of anti-vegf ab. considering that flt-1 tyrosine kinase signaling promotes ra via monocyte/macrophage activation [31, 40], the selective inhibition of flt-1 may be effective at blocking vegf-induced inflammation and angiogenesis with minimal toxicity. however, no clinical trial on anti-vegf inhibitors has been undertaken in ra. indeed, some antirheumatic drugs with well-known clinical efficacy in ra, such as cyclosporin and anti-tnf- ab, have been reported to inhibit vegf production in ra patients [41, 42]. through the screening of positional scanning synthetic peptide libraries, we identified a soluble arginine-rich hexapeptide sequence, rrkrrr (arg-arg-lys-arg-arg-arg), which binds to vegf165, and thereby prevents it from interacting with its receptor. to increase the in vivo stability of this peptide, we changed its peptide structure from the l- to the d-form, and accordingly, were able to increase its half life to more than 24 hours, which makes the peptide more suitable for therapeutic applications. in mice, the hexapeptide rrkrrr significantly inhibited vegf-induced angiogenesis, and also retarded the growth and metastasis of colon carcinoma cells. in addition, it strongly inhibited ongoing paw inflammation in arthritic mice without apparent side effects. when compared with several known vegf antagonists, such as anti-vegf antibody and aptamer, rrkrrr is advantageous from the clinical standpoint because it is a short peptide that is easily synthesized and because it has low immunogenicity. in a similar manner, bae et al., also found that the novel anti-flt-1 hexapeptide, gnqwfi (gly-asn-gln-trp-phe-iie), inhibited angiogenesis and tumor growth without side effects. this peptide selectively binds to flt-1, and thereby, blocks the interaction between flt-1 and vegf or plgf. investigations on the effect of anti-flt-1 peptide gnqwfi on an experimental model of arthritis are under way. plgf is a member of the vegf family, which was first identified in placenta, but is also known to be present in heart, lung, and joints. as a specific ligand for flt-1, plgf has potent angiogenic properties, and it also induces the growth and migration of endothelial cells [24, 25]. in addition, plgf stimulates tissue factor production and chemotaxis in monocytes, and also increases tnf-, il-1, il-6, il-8, and mcp-1 productions by normal and/or rheumatoid monocytes [31, 46], which suggests that it directly modulates the inflammatory process. plgf concentrations were reported to be increased in ra synovial fluids, and to induce vegf production by mononuclear cells. moreover, genetic ablation of plgf prevented the development of antibody-induced arthritis in mice suggesting the critical role of plgf in ra inflammation. plgf exhibits functions that are distinct from those of vegf, in that it regulates the angiogenic switch during the diseased state. it was recently reported that neutralizing ab to plgf inhibits the growth and metastasis of various tumors, including those resistant to vegf inhibitors, and that it enhances the efficacies of chemotherapy and that of anti-kdr ab. unlike anti-kdr ab, anti-plgf ab prevented the infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to anti-kdr ab. furthermore, it did not cause or enhance anti-kdr ab-related side effects, such as, the inhibition of placental vascular development. similar suppressive effects of plgf antagonist on tumor growth were observed in another study, in which an antagonistic plgf peptide (shryrlaiqlhasdsssscv) inhibited the growth and metastasis of human breast cancer xenografts. taken together, plgf antagonists may prevent angiogenesis and tumor growth without affecting normal physiology, and thus, are ideal candidates for ra treatment. we are currently investigating whether synthetic anti-plgf peptides inhibit the severity of arthritis and angiogenesis. we and others have demonstrated that proangiogenic factors, such as vegf and plgf, exert direct proinflammatory [14, 1921, 31, 40, 45, 46] and antiapoptotic effects [23, 29]. in this regards, the developments of synovial inflammation, hyperplasia, and angiogenesis in the joints of ra patients may all be regulated by vegf. given the importance of vegf in the pathology of ra, antiangiogenic therapies, particularly those involving an anti-flt-1 blocking agents, could when administered as a monotherapy or in combination with other biologic agents selectively ameliorate ra symptoms and reverse its fundamental pathology. the antiangiogenic peptides, rrkrrr and gnqwfi, introduced here represent a promising development in the antiangiogenic field.

recent experimental and clinical studies have placed new emphasis on the role of angiogenesis in chronic inflammatory disease. vascular endothelial growth factor (vegf) and its receptors are the best characterized system in the regulation of rheumatoid arthritis (ra) by angiogenesis. furthermore, in addition to its angiogenic role, vegf can act as a direct proinflammatory mediator during the pathogenesis of ra, and protect rheumatoid synoviocytes from apoptosis, which contributes to synovial hyperplasia. therefore, the developments of synovial inflammation, hyperplasia, and angiogenesis in the joints of ra patients seem to be regulated by a common cue, namely, vegf. agents that target vegf, such as anti-vegf antibody and aptamer, have yielded promising clinical data in patients with cancer or macular degeneration, and in ra patients, pharmacologic modulations targeting vegf or its receptor may offer new therapeutic approaches. in this review, the authors integrate current knowledge of vegf signaling and information on vegf antagonists gleaned experimentally and place emphasis on the use of synthetic anti-vegf hexapeptide to prevent vegf interacting with its receptor.

PMC2638142

pubmed-795

the antimetabolite 5-fluorouracil (fu) is widely used in the treatment of solid tumors, including gastrointestinal, breast, head, and neck cancers. cardiotoxicity, a rare adverse effect of 5-fu, has a reported incidence of 1.2% to 18%. the patient presented with typical angina and electrocardiographic changes suggestive of an ischemic coronary event during the continuous infusion of 5-fu. the ischemia recurred when the infusion was stopped, and was relieved by administration of nitroglycerin followed by a sublingual calcium channel blocker. an 83-year-old man who had been diagnosed with stage iiic (t4n2m0) adenocarcinoma of the ascending colon and who had undergone a right hemicolectomy was admitted to inje university seoul paik hospital to begin adjuvant chemotherapy, using the folfox4 regimen (400 mg/m 5-fu bolus infusion followed by the continuous infusion of 600 mg/m 5-fu for 22 hours on days 1 and 2; 200 mg/m leucovorin as a continuous infusion for 2 hours before 5-fu infusion on days 1 and 2; and infusion of 85 mg/m oxaliplatin on day 1). baseline echocardiography performed 1 month before admission showed normal left ventricular systolic function and no regional wall motion abnormality. he was treated with 1,500 mg high-dose 5-fu (1,000 mg/m) per day. on the morning of the 3rd day, he developed a severe, substernal, crushing chest pain during the continuous intravenous infusion of 5-fu (cumulative dose 1,679 mg/m), which was partially relieved by administering sublingual nitroglycerin. the electrocardiogram (ecg) showed st segment elevation with a tall t wave in leads i, avl, and v4-6, and reciprocal st segment depression in leads v1-2 (fig. the troponin-i and ck-mb levels were 0.010 ng/ml (reference range, 0.1) and 3.73 ng/ml (reference range, 4.94), respectively. severe hypokinesia of the lateral wall of the left ventricle was noted on a portable bedside echocardiogram. the 5-fu infusion was stopped, and the chest pain and electrocardiographic changes resolved after intravenous infusion of nitroglycerin at 30 g/min. emergency coronary angiography was then performed, which revealed significant stenosis in the proximal left circumf lex coronary artery (lcx). intracoronary nitroglycerin (200 g) was injected to exclude coronary vasospasm, but no change occurred (fig. 2). intravenous ultrasound (ivus) showed severe luminal narrowing with a heavy concentric plaque in the proximal lcx. percutaneous coronary intervention of the proximal lcx lesion was performed successfully with the implantation of a drug-eluting stent (3.5 16 mm; taxus, national medical center, seoul, korea) (fig. the patient was transferred to the coronary-care unit, where 8 hours later, he reported a recurrence of the anterior chest pain. the ecg also showed st segment elevation and reciprocal st changes similar to those seen in the previous ischemic events (fig. the chest pain and ecg changes persisted despite a 100 g/min nitroglycerin infusion. to rule out acute stent thrombosis, the chest pain and ecg changes were relieved after sublingual administration of 10 mg nifedipine. repeated coronary angiography showed a widely patent stent (fig. 4). the postprocedural troponin-i and ck-mb levels were 0.010 and 4.62 ng/ml, respectively. echocardiography performed the next day also showed the absence of the regional wall motion abnormality and normal left ventricular systolic function. we present here a case of severe cardiotoxicity mimicking acute anterolateral myocardial infarction occurring in a patient receiving 5-fu chemotherapy for adenocarcinoma of the colon. the cessation of 5-fu administration and the subsequent initiation of treatment with a sublingual calcium channel blocker and nitrate resulted in a successful outcome. although the mechanism by which 5-fu exerts its cardiotoxic effects is unknown, the resolution of the patient's chest pain and the normalization of his ecg changes with a vasodilator strongly support the vasospastic hypothesis of 5-fu cardiotoxicity. one study postulated that 5-fu-associated cardiotoxicity is due to the uncoupling of the electromechanical mechanisms that underlie normal myocardial function, which might be mediated at the level of the cell membrane. recently, kuzel et al. suggested that 5-fu promotes a hypercoagulable state (e.g., coronary artery thrombosis) and observed a significant increase in fibrinopeptide a and a decrease in protein c activity during 5-fu administration. the incidence of clinically apparent 5-fu cardiotoxicity is less than 10% in patients receiving the drug. patients with a history of coronary artery disease (cad) have a significantly increased risk of 5-fu-induced cardiotoxicity. although our patient did not have a history of cad, a large atheromatous plaque was found on coronary angiography and ivus. therefore, during 5-fu infusion, close, careful monitoring of patients, especially those with pre-existing cad or cad risk factors, is mandatory. prophylactic calcium channel blockers or nitrates should be administered to patients with cad during 5-fu administration, to prevent vasospasm. one study proposed that impaired renal function is also a risk factor for 5-fu cardiotoxicity. although it is not clear whether the cardiotoxic metabolites undergo renal excretion, the pathophysiological effect of 5-fu on the myocardium is likely to increase with decreased renal function. thus it is necessary to clarify which patients may benefit from optimum anti-angina prophylaxis and careful, close monitoring. the incidence of 5-fu-related cardiotoxicity appears to be dependent on the dosage and delivery system. infusion of 5-fu, which is now being used more frequently and at higher doses instead of bolus therapy, may be a significant factor in the development of 5-fu cardiotoxicity. in one study, nine patients treated with a higher-dose (> 800 mg/m) continuous infusion of 5-fu died suddenly. interestingly, despite stopping the 5-fu, the chest pain and ecg changes recurred in our case. one series reported that 19% of the patients developed reversible angina pectoris during treatment, which lasted for up to 12 hours after cessation of the infusion. the possible mechanisms of delayed angina are the late release of potent vasoactive 5-fu metabolites, which accumulate over time due to degradation of 5-fu. therefore, a calcium channel blocker or nitrates should be administered after stopping the 5-fu when 5-fu-induced cardiotoxicity occurs. the long-term outcome of patients with 5-fu-related cardiotoxicity has not been investigated extensively. as with our case, patel et al. recently reported interval improvements in the left ventricular wall motion abnormalities in echocardiography performed 8 to 15 days following the initial study. when cardiotoxicity occurs, 5-fu treatment is usually discontinued due to its very high recurrence rate (90%). the re-administration of 5-fu is not recommended, and a different chemotherapy regimen should be considered. interestingly, meydan et al. continued 5-fu chemotherapy in one group who experienced 5-fu cardiotoxicity due to the absence of an alternative drug and found that subsequent serious, hemodynamic consequences were easily controlled with nitrate treatment. for the remaining patients, either 5-fu was removed from the combination regimen or an alternative drug was started and no cardiotoxicity developed subsequently. in summary, although 5-fu-associated cardiac toxicity is rare, it may cause angina, myocardial infarction, and even sudden death. physicians should be aware of this potentially lethal side effect and should start the proper treatment when 5-fu cardiotoxicity develops. this case supports the vasospastic hypothesis of 5-fu cardiac toxicity, indicating that a calcium channel blocker may be effective for prevention or treatment of 5-fu cardiotoxicity.

cardiotoxicity associated with 5-fluorouracil (fu) is an uncommon, but potentially lethal, condition. the case of an 83-year-old man with colon cancer who developed chest pain during 5-fu infusion is presented. the electrocardiogram (ecg) showed pronounced st elevation in the lateral leads, and the chest pain was resolved after infusion of nitroglycerin. a coronary angiogram (cag) revealed that the patient had significant atherosclerosis in the proximal left circumflex artery. coronary artery spasm with fixed stenosis was considered, and a drug-eluting stent was implanted. after 8 hours, the patient complained of recurring chest pain, paralleled by st elevation on the ecg. the chest pain subsided after administration of intravenous nitroglycerin followed by sublingual nifedipine. repeated cag showed patency of the previous stent. this case supports the vasospastic hypothesis of 5-fu cardiac toxicity, indicating that a calcium channel blocker may be effective in the prevention or treatment of 5-fu cardiotoxicity.

PMC3443728

pubmed-796

thyroglossal duct cyst (tdc) is a common abnormality of the neck region and can occur at any age, though it is much more common in the pediatric population.1 the most definitive, efficacious management is achieved through the sistrunk procedure, a widely accepted and choice surgical technique, which effectively removes the cystic lesion and reduces recurrence rates.2 3 4 5 common postoperative complications associated with the technique include wound-related infection, pus/abscess, and hematoma/seroma (h-s) formation, with subsequent airway compromise.3 6 surgical drains are placed in patients undergoing the sistrunk procedure to prevent these postoperative complications, but hardly any studies have overtly assessed if drain placement is actually necessary. a recent case series in a pediatric population suggests that routine drain placement may not be necessary.7 as drain placement in the sistrunk procedure may often necessitate postoperative hospitalization, leading to increased cost and patient discomfort, it is important to understand if drain placement offers any substantial advantage. the rationale of our study was to assess if drain placement in the sistrunk procedure makes any significant difference in the prognosis of postoperative complications. the issue assumes significance as a focus of head and neck surgeries (including the sistrunk procedure) now is shifting toward outpatient, same-day, and ambulatory surgeries.5 8 9 studies demonstrate same-day surgery to be a safer, less costly, and reasonable alternative to admission surgery, without increased patient risk.8 9 with a general tendency to reduce hospital stays and with the sistrunk technique being increasingly performed in same-day, outpatient settings, evaluating the impact of drain placement with the technique on postoperative complications and patient morbidity is essential.5 7 with this objective, we conducted a retrospective study at our hospital to explore if same-day, outpatient sistrunk procedure performed without drain placement was a safe alternative versus the same procedure with drain placement. the study focused on postsurgical complications of the sistrunk procedure in both groups (those with or without drain placement) and evaluated whether surgical drain offered any substantial benefit in the sistrunk procedure, and if it was necessary. records of all patients who underwent the sistrunk procedure for tdc surgery in the preceding 10 years (2004 to 2014) were retrieved, and data were searched and extracted. in total, 80 patient records of the sistrunk procedure could be retrieved and were reviewed. certain patients were excluded to standardize the patient population and avoid any bias in the study. all those who had active infection and pus in cyst requiring incision and drainage of the cyst along with sistrunk procedure were also excluded. patients who had incomplete surgery like removal of just the cyst without the tract including the body of the hyoid (which were documented as sistrunk but were not actually sistrunk) were also not included. patients who in addition to sistrunk procedure also underwent other surgical procedures were excluded (e.g., those who underwent papillary carcinoma surgery and proceeded to the sistrunk procedure, or thyroidectomy/lymph node biopsies along with sistrunk procedure, among others). moreover, cases with missing data like no mention of placement or nonplacement of drain were also not included. records missing follow-up information or clues regarding complications after the sistrunk procedure were also not included. based on inclusion-exclusion criteria, of 80 records, only 58 patients were deemed fit for analysis, and the remaining 22 were excluded due to either missing data or inclusion-exclusion criteria. patients who were found suitable for analysis (n=58) were separated into two groups: patients who did not have a drain placed (n=38) during the sistrunk procedure, and those who had a drain placed (n=20). of these 58 patients, 38 were males (65.5%) and 20 (35.5%) were females. the age of the patients varied from 1 to 53 years, with the mean and median age of patients being 18.1 and 13.5 years, respectively. overall mean age (sd) was 18.1 (14.8) y and median age, 13.5 y. all patients had same-day surgery under general anesthesia. the patients came in the morning, were operated upon, and were discharged by evening after their condition stabilized. of the 58 patients, 13 (6 from the drain group and 7 from the no-drain group) were admitted for 1 night and discharged the next day but for reasons unrelated to complications, apparently based on patient or surgeon preference. patient preference included those who had come from distant places and had late evening surgery, those who felt nauseous and had possibly not recovered well from anesthesia, and those who had anticipatory anxiety and reported feeling patients who were admitted due to surgeon preference included one patient who had sickle cell anemia (who did not have a drain) for optimization and another patient (who had a drain) to observe for any anticipatory h-s. both groups of patients had homeostasis secured at time of surgery, and no major complication was noted. none of the patients developed any major complication like damage to great vessels, nerve damage, hypothyroidism, perforation of pharynx/esophagus, or injury to airway. none of the patients required any surgical exploration post sistrunk procedure, except minor wound exploration or incision and drainage of pus. both groups of patients were reviewed, and data were extracted for various postoperative sistrunk-related complications, like presence of h-s (and if the patients required aspiration for h-s or were managed conservatively), wound infection, and pus formation. of the 58 patients, 3 did not return for follow-up and were assumed to be doing fine. the extracted data were stored and analyzed using spss version 16 (armonk, ny, ibm corp). pearson chi-square test was used to see the association of drain or no drain with gender, age, h-s, aspiration, wound infection, pus, and number of follow-up visits. the t test was used to compare if there was any significant difference in mean age of patients in both groups. the parametric nature of the data was accessed statistically, and normality assumption was checked before application of t test. overall, about 10% of patients had h-s, 6.9% of whom needed aspiration for h-s and the rest were managed conservatively; 3.4% had wound infections; and 1.7% had pus collection that required wound exploration or incision and drainage. the comparative overall gross percentages of complications in both groups (drain versus no drain) did not show any significant difference (h-s, 0 versus 15.8%; aspiration needed, 0 versus 10.5%; wound infection, 5.0 versus 2.6%; pus formation, 5 versus 0%; respectively). none of the patients who were admitted overnight (patient or surgeon preference) developed any of the complications discussed. the chi-square test compared both group of patients in terms of any sistrunk procedure-related complications (h-s, aspiration required, wound infection, pus, and number of follow-up visits). the groups did not show any statistical significant difference in sistrunk-related complications in the fisher exact (two-sided) test: h-s (p=0.08), aspiration required (p=0.29), wound infection (p>0.99), and pus formation (p=0.35; table 2). the chi-square (two-sided) test also did not show any statistically significant difference in number of follow-ups for both group of patients (p=0.81; table 2). the t test compared mean age difference between patients with and without drain, and no significant difference in mean age could be seen (p=0.34; table 2). abbreviations: h-s, hematoma/seroma; ns, not significant; sd, standard deviation. note: all percent values are within group percentages. overall mean age (sd) of participants was 18.09 14.84. analysis of data suggests that there is no significant difference in complications post sistrunk procedure (infection, pus/abscess, or h-s) between the drain and no-drain groups. furthermore, no patient admitted overnight developed any of the complications discussed, which supports our methodology because the patients were admitted for reasons not related to complications and admissions were apparently due to patient and surgeon preferences. the findings of our study resonate with that of another recently published case series, which tried to ascertain if drain placement is at all necessary with the sistrunk procedure for treating tdcs in the pediatric population.7 the case series evaluated 30 consecutive pediatric patients (mean age 7.4 years) who underwent the sistrunk procedure without drain placement (study group). importantly, the first 10 patients were hospitalized, despite no drain placement, to observe for any complications in terms of hematomas, seromas, and subsequent airway compromise. however, when no major complications were seen, the next 20 patients undergoing the sistrunk procedure without drain placement were treated with same-day, outpatient surgery. the study also had 21 age-matched controls (mean age 7.5 years) who underwent the sistrunk procedure with drain placement.7 interestingly, the study did not find any statistical difference in the complication rates between the study (no drain) and the control (drain) group (paired t test, p=0.85). conclusively, the author stated that surgical drain placement was not necessary in pediatric patients who underwent the sistrunk procedure.7 our study (which included both children and adults) reinforces the same finding in a pediatric population and extends the generalizability of similar outcome in the sistrunk procedure performed in an adult population. with hardly any studies exploring the need for drain placement in the sistrunk procedure, in fact, use of drains in thyroid/parathyroid surgery has been considered controversial and debatable and may be even contraindicated.10 11 it has been suggested that use of drains is not justified as drains can not substitute for meticulous use of surgical technique and adequate hemostasis, in which noncompliance would eventually still lead to h-s. rather, it has been contended that in noncomplicated surgeries with minimal drainage, placement of a drain could lead to a possible infection.12 13 hence, nonusage of drains in the sistrunk procedure is not only related to reduced hospital stay, less cost, and improved patient comfort, but also has implications in terms of decreased chance of postoperative infections. though not specific to the sistrunk procedure, hurtado-lpez et al tried to analyze the actual value and effectiveness of a drain in thyroid surgery settings and found that presence or absence of drains did not affect the incidence of seroma or hematoma postsurgery.14 it is worth mentioning that although routine use of drains does not seem to be indicated in uncomplicated thyroid surgery cases, in complicated cases or when dead space is large, drains do have a use.15 the same may be applicable while using the sistrunk procedure to surgically excise and manage tdcs, and the surgeon preference for drain placement should depend on merits of its use and not on routine use in the sistrunk procedure. due to the increasing burden of extensive head and neck surgeries requiring mandatory hospitalization, smaller surgeries like the sistrunk procedure be managed as same-day, outpatient treatment. as drain placement in the sistrunk procedure may often require in-patient admission and increased hospital stay, it should not be performed if it does not offer any considerable advantage in decreasing postoperative complications. bratu and laberge evaluated same-day tdc surgery (which included sistrunk procedure) in 100 children in a retrospective review and found that outpatient surgery was a safe alternative to postoperative admission surgery in uncomplicated cases (no comorbidity, congenital defect, or bleeding disorders).5 interestingly, drain placement was one of the factors that prolonged the length of hospital stay in patients. another point worth emphasizing is that 38% patients had drains placed at surgeon discretion with no further details.5 bratu and laberge also stated that outpatient, same-day surgery was safe for routine tdc excision (including the sistrunk procedure), but surgeons who were reluctant to use same-day surgery due to fear of complications needed a shift in behavior to increase outpatient tdc surgery.5 this further emphasizes the rationale of performing the sistrunk procedure as same-day, outpatient surgery and emphasizes that the unnecessary routine use of drain placement with the sistrunk procedure would necessitate admission surgery leading to waste of health resources. the fact that very few complications were observed in our study even after discharge suggests that if homeostasis is adequately achieved, drain placement in sistrunk procedure may not be necessary. however, to take care of any unprecedented postoperative hematoma and/or edema and subsequent airway compromise in patients undergoing same-day, outpatient sistrunk procedure without any drain placement, the surgeon should take all precautions. all patients with tdc having same-day sistrunk procedure should be asked to report any postoperative abnormality like swelling, purulent discharge, or fever postdischarge, as soon as possible and irrespective of the scheduled follow-up. this not only would ensure optimal utilization of health care resources to manage any adverse complication (if any arise) but also would provide cost-benefit information and improved comfort to the patient, ensuring greater patient satisfaction and better overall management of tdcs. smaller sample size is another limitation, as complications of h-s in the sistrunk procedure are relatively rare. due to the retrospective, observational study design, with no randomization possible, there may be a risk of selection bias. although, to the best of our effort, we could not find any clinical or surgical variable to be associated with placement or nonplacement of a drain, its possibility can not be completely ruled out. furthermore, because all surgeries were performed by the consultant and residents under direct supervision of the consultant (and consultant as first assistant), there does not seem to be any performance bias. apparently, drain placement with the sistrunk procedure does not seems to be determined by the person performing the surgery or the surgeon's expertise, and rather appears to be governed by patient and surgeon preference. however, we can not deny it in absolute terms. a study with a larger sample population ideally, however, a randomized trial comparing the sistrunk procedure, with or without drain, should be performed to definitively understand if placing a drain is at all necessary in the sistrunk procedure. this would help create a protocol recommendation and consensus among surgeons managing tdcs, with respect to deciding whether or not a drain should be placed during the sistrunk procedure. to best of our knowledge, ours is the first study that has compared drain or no-drain sistrunk procedure across all age groups; more comparative studies are needed in both pediatric as well as nonpediatric population. surgical placement of a drain did not seem to offer any advantage in patients undergoing the sistrunk procedure at our hospital in terms of reduced postoperative complications. hence, there does not seem to be any apparent need for drain placement when performing the sistrunk procedure in patients with tdcs. moreover, because no major complications were observed in surgically managing uncomplicated cases of tdcs by same-day, outpatient sistrunk procedure without drain placement, it could be considered a safe alternative to the sistrunk procedure with drain placement, which may require overnight hospitalization/admission. this would translate into better patient comfort, greater satisfaction, and reduced surgical costs in patients of tdcs undergoing the sistrunk procedure.

introduction same-day, outpatient sistrunk procedure is commonly performed to manage thyroglossal duct cyst anomalies and may lead to postoperative complications. surgical drains are placed to prevent complications, but recent observations show no advantage and rather increased health care costs and patient discomfort. objective the study evaluated if drain placement in the sistrunk procedure offers any benefit on postoperative complications. methods a retrospective analysis of patient records having undergone same-day, outpatient sistrunk procedure from 2004 to 2014 was done. of 58 (38 male and 20 female) patients included, 38 did not have drains placed and the remaining 20 had drains placed. mean and median age of patients was 18.1 and 13.5 years, respectively. postoperative complications of patients with drains versus those without drains were statistically analyzed. results overall, about 10% of patients had hematoma/seroma (h-s), with 6.9% of patients needing aspiration for h-s; 3.4% had wound infections; and 1.7% had pus formation. no statistically significant differences in sistrunk-related complications between patient groups (with drain or without drain) were seen using fisher exact (two-sided) test: h-s (p=0.08); need for aspiration (p=0.29); wound infection (p>0.05); and pus formation (p=0.35). chi-square test also did not show any significant difference in the groups in terms of number of follow-ups. conclusion surgical placement of a drain in the sistrunk procedure does not seem to offer any advantage in terms of reducing common postoperative complications. same-day sistrunk procedure without any drain placement may be a safer alternative without necessitating hospitalization. more studies with larger sample size are needed for further substantiation.

PMC4593915

pubmed-797

cancer is the second leading cause of death worldwide, accounting for about 600,000 deaths in the united states in 2012. despite significant improvements in treatment, early detection remains the most important prognostic factor predicting of better outcome [24]. current cancer screening methods, including mammography for breast cancer, colonoscopy for colon cancer, computed tomography for lung cancer, prostate-specific antigen for prostate cancer, and papanicolaou stains for cervical cancer, have demonstrated some limitations in terms of sensitivity, specificity, complexity, cost, and compliance. serum tumor-associated antigens (taas) have been extensively studied for early cancer detection because of the simplicity and reliability of the tests used for their determination, such as western blot and enzyme-linked immunosorbent assay (elisa). unfortunately, they are transiently secreted and rapidly eliminated from blood circulation [6, 7] and usually reach a detectable concentration only in advanced stage of the disease. along with taas, autoantibodies are frequently detected in sera from patients affected by different types of neoplasms. this finding has been interpreted as an attempt of the immune system to block invasion and spreading of cancer cells in the organism. circulating autoantibodies have biological and biochemical characteristics that render them particularly suitable to screen subjects at cancer risk. in fact, they may develop early in the process of tumorigenesis, when premalignant or malignant cells begin to express altered molecules as a result of cell transformation [10, 11]. in addition, they can easily be detected in the serum because of the usual high concentration and long-time stability. for these reasons, great efforts have been made in recent years to identify circulating autoantibodies directed against cancer-related proteins in order to build up tests for the early detection of neoplastic disease [1215]. in this study, we investigated the production of autoantibodies against lectin galactoside-binding soluble 3 binding protein (lgals3bp) in patients affected by different types of cancer. lgals3bp, also known as 90k or mac-2 bp, has been largely regarded as a taa, since it is present at elevated concentrations in the blood of cancer patients and is overexpressed in the vast majority of cancer tissues. both high serum and tumor levels of lgals3bp have been associated with a poor outcome in patients with different types of neoplasms [1821]. in fact, it can bind important molecules associated with the membrane of tumor cells, such as galectin-3, galectin-1, and 1-integrins [16, 22, 23]. additionally, lgals3bp can interact with extracellular matrix proteins such as collagen, fibronectin, and laminin [23, 24]. here we show that patients with different types of cancer, but not healthy subjects, develop autoantibodies against lgals3bp. this finding discloses the capability of lgals3bp to trigger a humoral immune response in cancer patients and provides the basis for further investigation on a possible use of anti-lgals3bp antibodies as biomarkers for early diagnosis of cancer. the study population consisted of 71 patients with different types of cancers, 15 gastrointestinal cancers, 13 non-small-cell lung cancers (nsclc), 12 breast cancers, 10 neuroendocrine tumors (net), 10 urogenital tract cancers, 7 melanomas, and 4 others (2 gliomas, 1 tongue cancer, and 1 osteosarcoma). serum was collected from total blood and stored at 20c after adding 0.01% sodium azide. human recombinant lgals3bp was immunoaffinity-purified from serum-free supernatant of human embryonic kidney ebna-293 cells (invitrogen, carlsbad, ca, usa) transfected with lgals3bp cdna. ninety-six well microtiter plates (nalge nunc, denmark) were coated with recombinant purified lgals3bp protein (5 g/ml in pbs) at 4c overnight. the plate was saturated with 1% bsa and 0.05% tween-20 in pbs (saturation buffer) at 37c for 2 h and then incubated with 100 l of serum from healthy donors or patients at 37c for 1 h. the serum was diluted 1: 100 in saturation buffer. after 3 washes with 0.05% tween-20 in pbs (washing buffer), a second incubation was performed with 100 l of biotin-conjugated anti-human igg (sigma, st louis, mo, usa), diluted 1: 2000 in saturation buffer, at 37c for 1 h. after washing, a third incubation was performed with peroxidase-conjugated extravidin (sigma, st louis, mo, usa) diluted 1: 4000 in saturation buffer, at 37c for 45 min. after washing, 100 l of tmb substrate was added to each well and the plate was shaken at room temperature for 15 min. eventually, the reaction was stopped by adding 100 l of 1 m h2so4/well and color revealed by reading absorbance at 450 nm in an automatic elisa reader. to measure serum concentration of lgals3bp a commercially available elisa kit (diesse, siena, italy) purified human recombinant lgals3bp (10 g/well) was separated by 8% sds-page under reducing conditions and transferred to nitrocellulose using standard procedures. membrane was saturated in blocking buffer phosphate buffered saline with 0.05% tween-20 (pbs-t), 5% low-fat milk, 1% bsa and at 4c overnight. after washing in pbs-t, membrane was incubated with serum from controls or patients affected by cancer, diluted in an equal volume with pbs-t, at room temperature for 1 h. after washing in pbs-t, membrane was incubated with biotinylated anti-human igg (sigma, st louis, mo, usa) diluted 1: 1000 in blocking buffer at room temperature for 1 h and then with extravidin peroxidase (sigma, st louis, mo, usa) 1: 500 in blocking buffer for 30 min. to identify the presence of antibodies bound to lgals3bp, the colorimetric substrate dab (3,3-diaminobenzidine) each sample was assayed in triplicate and the mean value was used for statistical analyses. the normal upper cut-off value of anti-lgals3bp antibody in sera was set at the value of the mean+2sd of the absorbance in 54 healthy donors. differences in the proportion over the cut-off limit were evaluated by fisher's exact test. patients affected by different types of cancers showed significant increased levels of lgals3bp autoantibodies (p<0.001) compared to healthy subjects (figure 1(a)). the specific binding of antibodies to lgals3bp was confirmed in western blot, where the presence of anti-lgals3bp igg was detected as two bands at about 97 kda and 66 kda, the exact size of the protein in its full length and cleaved form, respectively (figure 1(b)). among patients, nsclc, gastrointestinal cancer, urogenital tract cancer, and net reached the highest levels of autoantibodies, while there was no significant increase in breast cancer and melanoma (table 2). setting the normal upper cut-off limit of elisa at od 0.99 (the mean+2sd of the absorbance in sera from healthy individuals), the assay showed a sensitivity of 33% (26/71 patients were positive) and a specificity of 98% (only 1 out of 54 controls was positive). all cancer groups, but melanoma, showed autoantibody levels significantly above the cut-off limit (table 2). as expected, the protein was significantly higher in patients with cancer compared to normal subjects (13.19 versus 6.36 g/ml, p<0.001) (table 3), but values did not correlate with the levels of autoantibodies (data not shown). using elisa technique, we show that lgals3bp is able to elicit host immune response with igg autoantibodies production in patients affected by different types of cancer. anti-lgals3bp igg concentrations were higher in patients with nsclc, gastrointestinal cancer, urogenital tract cancer, and net than in those with breast cancer and melanoma, but the number of patients in each subgroup was insufficient to yield a statistically reliable comparison. it is generally accepted that tumor proteins perceived as nonself by the immune system and able to trigger an immune response are often overexpressed, mutated, misfolded, or endowed with posttranslational changes, such as alterations of glycosylation and phosporylation [28, 29]. consistently, lgals3bp may evoke autoantibody production because it is overexpressed in cancer cells, and also because it may carry posttranslational alterations in its glycidic moiety. qualitative and quantitative changes in o- and n-glycosylation of proteins are frequent events in malignancies [30, 31] and differences in the glycosylation pattern of lgals3bp have been reported in some cancer cell lines. finalistically, antitumor directed antibodies are generated in order to halt tumor initiation and progression. as this process initiates early in cancerogenesis, in a preclinical phase of the disease, autoantibodies production has been considered a useful biomarker for early cancer diagnosis [3235]. in this study, the serum levels of anti-lgals3bp igg detected in cancer patients were not correlated with those of lgals3bp, indicating that even small amount of the protein, as expected in the initial phase of cancer growth, may generate high concentrations of autoantibodies. this evidence suggests a possible role for anti-lgals3bp igg in the early detection of cancer. although it is not possible to exclude that the presence of autoantibodies might affect the correct quantification of lgals3bp by elisa, the identification of anti-lgals3bp igg in western blot indicates that the epitopes recognized by these autoantibodies are different from those recognized by the antibody used in elisa. in fact, western blot performed under reducing conditions can detect only autoantibodies directed to epitopes expressed on the primary structure of lgals3bp, while the monoclonal antibody contained in the commercially available elisa kit, known as sp2, recognizes a conformational epitope shaped in the native form of the protein and, for this reason, is not suitable for western blotting. in cancer patients, autoantibodies are frequently directed against cellular proteins that play key roles in tumor progression, including molecules involved in cell cycle, signal transduction, proliferation, and apoptosis [3638]. as a consequence, the identification of the molecular target of autoantibodies might be of relevance in designing new antitumor agents. we can, therefore, speculate that lgals3bp could be a candidate for targeted therapies against cancer. in the past few years, the growing interest in autoantibodies as a possible tool for the early diagnosis of cancer and the identification of new targets for molecular therapy has made the development of high-throughput techniques such as serex (serological analysis of tumor antigens by recombinant cdna expression cloning), phage display, protein microarray, serpa (serological proteome analysis), and mapping (multiple affinity protein profiling) able to detect simultaneously multiple autoantibodies and their cognate taas. with these methods, several new targets have been identified, but collectively single antigens have shown low sensitivity and specificity to be used in clinical screening. to increase sensitivity, autoantibody diagnostic tests combining two or more taas [4143] or evaluating well-known biomarkers in combination with autoantibodies have been developed. for example, a large screening study of high-risk individuals for lung cancer has validated a test measuring autoantibody levels against a panel of six taas (p53, ny-eso-1, cage, gbu4-5, annexin 1, and sox2). in another study, the combination of p53 autoantibodies and ca125 levels increased sensitivity for ovarian cancer from 73.8% (ca125) to 85.7% (ca 125 plus p53 autoantibodies). the determination of anti-lgals3bp igg presented in this study showed a very high specificity (98%), but a low sensitivity (33%), comparable to that reported for autoantibodies against single taa, ranging between 10% and 30%. therefore, our anti-lgals3bp elisa lacks sufficient sensitivity to be used in early cancer diagnosis. nevertheless, the determination of autoantibodies against lgals3bp might be useful to increase the sensitivity of tests combining multiple autoantibodies. preliminary results indicate that using a set of different autoantibodies combined with autoantibodies for lgals3bp will increase the sensitivity for breast cancer patients to 50% and maintain the high specificity (98%). these preliminary results should be tested for other types of cancers as well. in summary, our study demonstrated the presence of autoantibodies against lgals3bp in the serum of patients with different types of cancers. these autoantibodies may be used in developing screening tests for early-stage cancer detection.

purpose. circulating autoantibodies have been extensively investigated as possible markers for early diagnosis of cancer. the present study was carried out to investigate whether anti-lgals3bp igg autoantibodies could be classified as a biomarker for malignant tumors. methods. an in-house developed enzyme-linked immunosorbent assay was used to detect autoantibodies to lgals3bp in sera from 71 patients with various types of cancers and 54 healthy subjects matched by age and gender. results. patients with cancer have significant higher circulating levels of anti-lgals3bp antibodies as compared to control subjects (p<0.001). the test has a sensitivity of 33% and a specificity of 98%. conclusions. anti-lgals3bp igg autoantibodies are a promising biomarker for malignant tumors and could play a role in the development of a multimarker assay for the early detection of cancer.

PMC3850626

pubmed-798

the udder is the milk-producing organ of dairy animals; hence, for optimal production, it should be healthy. mastitis is the inflammatory response of the mammary gland (mg) tissue to physiological and metabolic changes, traumas, and allergies and, most frequently, to injuries caused by various microorganisms. mastitis is considered the utmost threat to the dairy industry from three perspectives: economic, hygienic, and legal (eu directive 46/92, modified by directive 71/94). the intramammary inflammation (imi), accompanied by immunological and pathological changes in the mg tissue, occurs at different degrees of intensities and results in a wide range of consequences regarding physical, chemical, and often microbiological alterations of secreted milk. a wide spectrum of microorganisms, including fungi, yeast, algae, chlamydia, and viruses, have been incriminated in causing mastitis, but bacteria remain the principle causative agents of such complex [1, 2]. the major bacterial mastitis pathogens (staphylococcus aureus, streptococcus agalactiae, s. uberis, s. dysgalactiae, and coliforms) are most often responsible for clinical mastitis (cm). meanwhile, minor pathogens (coagulase-negative staphylococci cns; streptococci other than s. agalactiae, s. uberis, and s. dysgalactiae; corynebacterium spp.;; listeria spp.; leptospira spp.; yersinia spp.; enterobacter spp.; brucella spp.; and mycobacterium spp.) are typically associated with subclinical mastitis (scm) or sometimes associate clinical imis. with the exception of a few pathogens that can invade via the blood stream (e.g., brucella abortus or mycobacterium bovis), infection of the mg occurs by ascension through the ductus papillaris, the only opening of the udder to the outside world, and the pathogens pass to find an environment that is warm, moist, and nutrient-rich and thus suitable for rapid growth and multiplication. to establish a successful infection after traversing the teat end opening mg immunity depends on the complex combination and coordination of nonspecific and specific protective elements, including the anatomical features of the gland as well as cellular and humoral defence components. nevertheless, mg immune defence varies over different stages of lactation in dairy animals and is typically depressed with exposure to stress and around drying-off and parturition, thus increasing susceptibility to mastitis [6, 7]. however, a considerable body of evidence has accumulated suggesting that mastitis is a multifactorial complex, and several management and environmental factors must interact to increase host exposure to mastitis pathogens, reduce the natural resistance of animals to disease, or aid pathogens in gaining entrance to the mg environment to cause infection [2, 4]. the primary defence mechanism of the mg is represented structurally in the teat canal [3, 8, 9], which acts as both a physical barrier and a source of antimicrobial substances. the physical barrier is provided by the smooth muscle sphincter surrounding the teat canal, which prevents escape of milk and constitutes a barricade against the entry of different pathogens by maintaining tight closure [2, 3, 9]. normally, the healthy teat skin is coated with a protective mantle of fatty acids (fas) that slow the growth of bacterial pathogens. additionally, the stratified squamous epithelium of the teat duct produces keratin, a waxy material lining the teat canal, which traps invading bacteria and hinders the migration of microorganisms into the gland cistern. this keratin is composed of (i) bacteriostatic fas of both esterified and nonesterified types, such as lauric, myristic, palmitoleic, and linolenic acids and (ii) fibrous proteins, which bind electrostatically to microorganisms, altering the cell wall and rendering it more susceptible to osmotic pressure changes and, thus, to lysis and death. additionally, these cationic proteins were found to have an inhibitory effect against some pathogens as staph. aureus and s. agalactiae, which was equal to that of proteins isolated from bovine neutrophils. the lipid content and composition of teat duct keratin have been shown to vary throughout the milking process, between lactating and dry dairy animals, and according to the severity of imi. scm was found to not affect the lipid content of teat duct keratin, while cm was shown to be associated either with significantly higher levels of total lipids or with similar lipid composition of uninfected quarters. additionally, the free fas in milk from clinical quarters contained fewer short-chain fas, whereas polyunsaturated fas were significantly higher. recently, sentinel functions for the teat towards invading pathogens have been documented, as the teat canal tissue responded rapidly and intensely, with both expression of several toll-like receptors (tlrs) and production of cytokines and antimicrobial peptides [16, 17]. damage of keratin, perhaps as a result of incorrect intramammary therapy infusion or by faulty machine milking, has been reported to increase susceptibility of the teat canal to bacterial invasion and colonisation. however, the antimicrobial effectiveness of keratin is limited [9, 21] and, despite the potent physical and chemical protection in the teat canal, there are several ways by which bacteria can penetrate the teat canal and cause imi, so much so that a number of pathogens are able to colonize the teat canal for prolonged periods, such as corynebacterium bovis, or cns. aureus deposited a few mm inside the teat canal has also been demonstrated [2224]. also, during milking, it is common for keratin to be flushed out with distention of the teat canal. because the sphincter takes approximately 2 h to regain its contracted position, there is a chance for outside pathogens to enter the teat canal, causing trauma and damage to the keratin or mucous membranes lining the teat sinus [2, 21]. additionally, during mechanical milking, microorganisms present at the teat end may be propelled into or through the teat duct into the cistern. the mg is normally protected by both innate and adaptive immune responses (irs), which coordinate and operate together to provide an optimal defence against infections. the irs also facilitate the constitutive or acute transient presence of a wide range of immune-related components in milk. the adaptive immune system (ais) responds more robustly to threats to which it has previously been exposed; however, it is slow to respond to novel threats. in contrast, the innate immune system (iis) is the first line of defence against pathogens once they have penetrated the physical barrier of the teat canal and before the ais comes into play, and it evolves into a highly effective host defence [33, 34]. this process is mediated via several intracellular signal transduction cascades that trigger an acute upregulation of several innate immune components including different leukocytes, adhesion molecules, and cytokines [3537]. the two most critical components of host innate immunity are pathogen recognition (pr) and the ability to mount a proinflammatory response, a complex interaction of cellular and molecular processes aimed at detecting and subsequently eliminating harmful pathogens [25, 34]. a wide variety of components linked to the innate ir (iir) have been identified in milk, including cellular defence components [e.g., leukocytes], components contributing to humoral defence [e.g., complement system (cs), immune-modulating factors (pro- and anti-inflammatory cytokines), lactoferrin (lf), transferrin (tf), lysozyme (lz), and components of the lactoperoxidase/myeloperoxidase systems], oligosaccharides, gangliosides, reactive oxygen species (ros), acute phase proteins (apps) (e.g., haptoglobin and serum amyloid a), ribonucleases, and a wide range of antimicrobial peptides and proteins. many of these components originate from specialised cells that traffic to the mg [33, 34]. the ability of the iis to recognise and respond to a broad spectrum of pathogens that may or may not have been previously encountered, combined with the speed in mounting a proinflammatory response following initial pr, greatly contributes to the host's ability to control invading pathogens. below, there is a detailed overview of the roles and mechanisms of action of some innate immune factors.. the viable leukocytes inside the mg offer some degree of cellular protection against microbial invasion through their ability to recognise microorganisms and induce a rapid inflammatory response in an attempt to resolve the imi immediately. thus, mg-resident leukocytes likely provide a surveillance function in the uninfected gland. also, these cells may aid in the restructuring of the mg that occurs during involution (i.e., apoptosis). in addition to microbicidal functions of phagocytosis, mg leukocytes secrete a variety of immune-related components into milk including cytokines, chemokines, ros, and antimicrobial proteins and peptides (lf, defensins, and cathelicidins). leukocytes also assist in the repair of damaged tissue caused by shedding and renewal processes. despite the presence of considerable numbers of immune cells in the mg environment, it has been suggested that the mg is immune-compromised when compared to the rest of the body. moreover, the activities of all types of leukocytes in milk have been shown to be reduced compared to those in blood [28, 43, 44]. the migration of immune cells during imi plus desquamation of mg epithelia results in an increase of somatic cell count (scc) accompanied with decreased milk production according to the severity of the process [1, 45] polymorphonuclear neutrophils (pmns) constitute the second line of the iis against imi. even under healthy conditions, pmns are permanently present inside the mg environment, and nursing or milking stimuli accompanied with milk removal were found to induce directed migration of fresh pmn into mammary tissue. bovine neutrophils cross the mg epithelium by diapedesis without causing epithelial cell damage unless the migration is extensive, in which case both mechanical and chemical damage are possible. the neutrophil's multilobulated nucleus allows for easy and rapid migration between endothelial cells, thus arriving as the first recruited immune cell to sites of infection. because only small numbers of mature pmns are stored in the bone marrow, the number of immature neutrophils in circulation increases as a result of mobilisation into circulation during inflammatory conditions. several important functions are not fully developed in immature neutrophils, including those pertaining to phagocytosis, intracellular killing, and chemotaxis. neutrophils are delineated by a plasma membrane that has a number of functionally important receptors. these include l-selectin and 2-integrin adhesion molecules, which promote the binding of pmns to endothelial cells and facilitate their migration to infected foci [39, 49, 51]. membrane receptors for the fc portion of the igg2 and igm classes of igs and for complement components c3b and ic3b are necessary for the phagocytosis of invading bacteria [52, 53]. the activation of c3b regions on bacterial surfaces after binding with abs promotes phagocytosis and binding to cr1 and cr3 receptors on the pmn surface. additionally, lectin-carbohydrate receptors found on neutrophil cell membranes can recognise carbohydrate-rich fimbriae of escherichia coli in the absence of specific opsonins [49, 54], resulting in a process referred to as nonopsonic phagocytosis. the primary function of pmns to engulf, phagocytose, and destroy foreign materials, including invading bacteria, occurs via two parallel systems. the first is an oxygen-dependent (respiratory burst) system that includes the production of hydroxyl and oxygen radicals. the second is an oxygen-independent system that relies on several oxygen-independent reactants such as peroxidases, lzs, hydrolytic enzymes, and lf [1, 41]. in addition to phagocytic activity, pmns also contribute to the modulation of vascular permeability and release several inflammatory mediators that play crucial roles in the coordination of innate and adaptive immune components. furthermore, the intracellular granules of pmns contain several bactericidal peptides including defensins, enzymes (e.g., myeloperoxidase), and neutral and acidic proteases (e.g., elastase; cathepsin types b, d, and g, procathepsins) [5658], which can kill a variety of mastitis pathogens. such proteases as well as plasmin are known to permit the chemotaxis of cells in the site of inflammation and are involved in the limitation in time of the ir (e.g., by the cleavage of some cytokines such as il-2, il-6, and il-8). the exposure of pmns to cytokines and chemoattractants causes rapid mobilization of azurophil granules (containing elastase and cathepsin g mainly) to the cell surface., pmns can wrongly phagocytose milk fat globules, and their proteases can degrade milk casein (caseinolysis), leading to putrefaction of milk, and, together with their released hydroxyl radicals, can damage the mg epithelium which contributes to the decreased synthetic activity of the mg during imi. once pmns perform their tasks, they undergo apoptosis, or programmed cell death, and are removed by macrophages [63, 64]. macrophages. they constitute the predominant cell type found in milk and tissues of both healthy involuted and lactating bmgs [43, 65, 66]. in contrast to neutrophils, macrophages have large horseshoe-shaped nuclei that make their migration between endothelial cells more difficult. macrophages contribute to induction of specific local irs through antigen (ag) processing and presentation to lymphocytes in association with mhc class ii ags [45, 6769]. similar to pmns, macrophages can perform a variety of nonspecific functions including ingestion and phagocytosis of foreign particles, including some invading bacteria (e.g., staph. aureus), and destroying them with proteases and ros [66, 70, 71]. additionally, they can ingest cellular debris and accumulated milk components in involuting mgs. the phagocytic activity of macrophages can be increased in the presence of opsonic abs for specific pathogen. in cattle, mg macrophages bear receptors for igg1 and igg2. unlike neutrophils, macrophages possess fewer fc receptors, which decrease their phagocytic capacity. mg macrophages are considered less effective at phagocytosis compared to blood monocytes because of indiscriminate ingestion of milk components as well as the fact that macrophage proteases can also contribute to damage of mg epithelium [62, 70, 74]. a failure of efficient killing of some mastitis pathogens (e.g., s. uberis) after engulfing and even increased intracellular multiplication of s. uberis as well as lesser stimulatory responses by ifn- to release tnf- and bactericidal products compared to blood monocytes have been also reported. however, it has been demonstrated that the bactericidal activity of mg macrophages can vary according to mg secretion, and dry-off secretion macrophages exerted higher bactericidal activities than lactational macrophages. therefore, the ability of macrophages to secrete substances that augment local inflammatory processes, thereby inducing the migration and bactericidal activities of neutrophils, is believed to be of greater importance to nonspecific defence of the mg than their function as professional phagocytes [7, 67, 70, 75]. lymphocytes recognise a variety of antigenic structures via membrane receptors, which define their specificity, diversity, and memory characteristics. t- and b-lymphocytes and natural killer (nk) cells are distinct lymphocyte subsets that operate in the mg (figure 1), although they differ in function and protein products. during imi, preferential trafficking of certain lymphocyte subpopulations to specific mammary tissue foci occurs [28, 76] and marked changes in milk lymphocyte count and composition during imis have been reported. additionally, the ais response is mainly mediated by memory lymphocytes, which respond quickly to threats to which they have previously been exposed. it must be mentioned that the presence of specific lymphocyte subsets can affect the total lymphocyte function and even the whole ir. for example, the activation of cd8+t-cells during certain bacterial imis, such as staph. aureus, can suppress important host irs and predispose to chronic pattern of imi [78, 79]. unfortunately, the exact roles of lymphocytes during imi and their subsets are complex and are not fully defined. even in healthy mgs, the composition of the lymphocyte population varies during the lactation cycle [28, 76, 80]; the consequences to mg immunity are still not fully understood. additionally, mg lymphocytes exhibit hyporesponsiveness to mitogenic, antigenic, and allogeneic stimuli compared to blood lymphocytes, possibly due to the presence of distinct lymphocyte subsets, high proportion of memory t-lymphocytes present in the mg, and/or less efficient presentation of ags by ag-presenting mg cells. in healthy bmgs, t-cells prevail in both mg secretions and parenchyma and predominantly exhibit the cd8+phenotype, which is in contrast to the blood, where cd4+cells are the predominant t-cell subset. therefore, the ratio of cd4+/cd8+t-cells is lower in milk than in blood. cd4+(t-helper) cells produce a variety of immunoregulatory cytokines following ag-recognition with mhc class ii molecules; and are being memory cells following ag-recognition [4, 7, 27, 81]. on the other hand, it is well established that cd8+cells can exert either cytotoxic or suppressor functions. in coordination with major histocompatibility complex (mhc) class i molecules, cytotoxic t-cells recognise and eliminate altered self-cells via ag presentation, thus being more specific than nk cells. however, it has been suggested that their removal of damaged mammary epithelium could enhance the susceptibility of mg to infection. suppressor t-cells are thought to play roles in control or modulation of the mgir. however, the immunoregulatory roles of cd8+cells are also greatly dependant on lactation stage. cells obtained from midlactation dairy cattle exhibited cytotoxic activity and mainly expressed interferon- (ifn-), whereas cd8+lymphocytes obtained during the postpartum period exhibited no cytotoxic activity and mainly expressed interleukin 4 (il-4). ruminants bear greater levels of t-lymphocytes in secretions and parenchyma of mg relative to blood. there are indications that t-cells can mediate cytotoxicity, similar to nk cells, with variable involvement of mhc molecules; thus, they may be able to destroy altered epithelial cells [83, 84]. t-lymphocytes preferentially migrate to particular epithelial surfaces and do not exhibit extensive recirculation. thus, it has been indicated that t-lymphocytes play a role in antibacterial immunity and may provide a unique barrier function for mucosal microenvironments against bacterial pathogens. the wc1 subpopulation represents a minor portion of t-lymphocytic population in normal mg secretions [28, 86], but they markedly increase following parturition. because of restricted localisation and expression of invariant ag receptors, the exact contribution of these cells to mg immunity is not fully understood. several lines of evidence have been accumulated suggesting that these cells perform specific functions in comparison to circulating and t-cells. recently, it has been addressed that lymphocytes exert some immunoregulatory/suppressive functions, more precisely in the wc1.1 and the wc1.2 cells. on the other hand, it has been reported that wc1 cells are not recruited to the mg during chronic imis caused by staph. one of the main roles of b-lymphocytes is to produce abs against invading pathogens. unlike macrophages and pmns, b-lymphocytes utilise their cell surface receptors to recognise specific pathogens and then internalise, process, and present ags in the context of mhc class ii molecules to t-helper cells. under certain conditions, b-lymphocyte differentiation can be directly stimulated by an ag such as lipopolysaccharides (lps). in contrast to t-lymphocytes, the percentages of b-lymphocytes remain fairly constant regardless of lactation stage [49, 77] or infection. nk cells are large granular nonimmune lymphocytes that differentiate and mature in bone marrow, lymph nodes, spleen, and tonsils before passing to the circulation. nk cells constitute the third type of cell derived from lymphoid progenitors that also generate b- and t-lymphocytes. nk cells utilise their fc receptors to possess a cytotoxic activity critical to the iis in the absence of mhc restriction. nk cells cause lysis of target cells through a diverse repertoire of mechanisms, including ab-dependent cell-mediated cytotoxicity, granule exocytosis, release of cytolytic factors, and receptor-mediated ag-recognition. additionally, they secrete various toxic molecules that may initiate apoptosis in altered cells. nk cells differ from natural killer t-cells in origin, respective effector functions, and lack of specificity for ag-recognition. however, nk cells do not require activation to kill cells that lack self-markers of mhc class i. studies have demonstrated the capability of nk cells to kill both gram-positive (gpb) and gram-negative bacteria (gnb) and, therefore, they may be important in preventing imis [91, 92]. the differences in distribution of cellular components in mg environment between healthy and inflammatory conditions are detailed in table 1. the distribution of leukocytes in healthy mg is somewhat variable during healthy lactating and dry periods. the percentage of pmns tends to increase during early and late lactation, while the percentage of lymphocytes decreases. meanwhile, the proportion of macrophages is highest (68%) in the early postpartum period and lowest (21%) in late lactation. during the dry period the increase at the start of involution is most likely due to an influx of cells resulting from cessation of milk removal, or due to the concentration effect by removal of the liquid phase of the secretion. sccs in milk from uninfected glands at the beginning of the dry period are usually higher than 1 10 cells/ml milk, but by the 7th day of the dry period this count can be as high as 2 10 cells/ml milk. pmn counts are initially high in early involutional secretions, comprising 4080% of scc (similar to colostrum), but are reduced again from the 2nd to 4th week of the dry period and then return to lactational values in the fully involuted udder [43, 66]. unlike in the lactation stage [43, 66] and with exception of the 1st day of the dry period in which they exhibit higher counts, macrophage concentrations are relatively low during the remaining part of early involution and in colostrum, with maximal proportions (30%) peaking by the mid-dry period and remaining constant until calving. lymphocyte concentrations in dry secretions are approximately 30006000 times that in normal milk, and the proportions of b- and t-lymphocytes are approximately 28% and 47%, respectively, approximating proportions in peripheral blood [31, 32]. (3) distribution of cellular components in the mg environment of ovines and caprines. the milk sccs thresholds are higher in milk of small ruminants than in bovine milk. recent studies have indicated an upper scc threshold of 2.5 10 cells/ml milk in healthy ewe's udders or more, up to 6 10 cells/ml milk. similar to bovines, the macrophages are the predominant cell type (4684%) in milk from uninfected ewes [96, 97]. counts of macrophages were higher in early and midlactation milk than in late lactation milk. the rest of the scs population consists of pmns (228%) and lymphocytes (1120%). meanwhile, limited data exist on changes of leukocytes population in infected ewes ' mgs. recorded an increase of pmns percentages to 50% at a scc of 2 10 cells/ml milk and to 90% at a scc over 3 10 cells/ml milk, representing the predominant cell type at inflammatory conditions. likewise, an increase of pmns and macrophages counts within imi of ewe's udder has been reported, whereas lymphocytes decreased. scc of milk from uninfected goats is higher than those of milk of uninfected bovines and sheep. unlike cow and sheep milk where macrophages are the predominant cell type, pmns comprise the major cell type in goat milk from both infected and uninfected mgs [100104]. in healthy status, pmns, macrophages, and lymphocytes comprise 4574%, 1541%, and 920% of scs population, respectively, while epithelial cells are present in low percentage (16%) [98, 103, 105, 106]. with advanced lactation, the pmns increase, manlongat et al. explained this late-lactation rise-up on the presence of higher chemotactic activity in nonmastitic goats udder and concluded that this phenomenon was nonpathological and could play a physiologic regulatory role in mg involution. unfortunately, very little data exist on the distribution of these cells during imi. a study by dulin et al. reported an elevation of pmns to 7186% in infected halves, while macrophages and lymphocytes percentages are being changed to 818% and 511%, respectively. mecs themselves are active contributors to the innate immune and inflammatory responses of mg [108, 109]. they express a range of pr receptors (prrs), most notably the tlrs [35, 36]. additionally, the polymeric-ig receptor (pigr) expressed on the mucosal epithelium facilitates the translocation of igs, particularly iga, across the epithelium into the alveolar lumen. upon bacterial stimulation, mecs secrete a range of innate immune effector molecules and inflammatory mediators, which contribute to attraction and recruitment of circulating leukocytes [38, 111]. it was shown that mecs secrete il-8, a potent neutrophil chemoattractant, in the presence of gpb and their exotoxins, lps from gnb or il-1 [51, 111, 112]. mecs constitute an important source for host defence components as arachidonic acid metabolites [38, 108, 114, 115], apps, lf [111, 116], -defensins [117, 118], cathelicidins and calprotectin, and lps binding protein [bp] (lps-bp), which is involved in host recognition of the bacterial cell wall [17, 119]. supporting results were obtained experimentally on bovine mecs, showing also their ability to express il-1, tumour necrosis factor- (tnf-), il-6, il-8, and growth related oncogene- [gro-] mrna during infection and immune stimulation [111, 114, 120, 121]. mg epithelium may exhibit protective and phagocytic functions via the ingestion and possible digestion of phagocytosed microbes and milk components, including fat globules and casein micelles, through the formation of pseudopodia. experimental studies showed that glutaraldehyde-killed streptococci, staphylococci, and e. coli were phagocytosed by milk secretory cells. moreover, many peptides, proteins, and lipids which are involved in host defence and shown to have antibacterial properties (including xanthine oxidase and sphingolipids) were found in fat globule membranes, which originate from the apical membrane of the mg epithelium [123, 124]. the initiation of rapid and effective iir depends mainly on recognition of the infectious agent [36, 109]. iir of mg is initiated when prrs on the surfaces or within host cells, primarily leukocytes and mecs, bind to particular bacterial motif molecules termed pathogen/microbial-associated molecular patterns (pamps/mamps) [109, 125, 126]. such prrs belong to three different families, namely, the tlr, nucleotide-binding oligomerization domain- (nod-) like receptors (nlr) 1-2, and retinoic acid inducible gene-1- (rig-1-) like receptors, and each of these receptors recognizes a set of bacterial motifs [17, 35, 36, 109]. activation of these prrs initiates a signalling transduction cascade in which nuclear factor-b plays a pivotal role in coordinating multiple signals and directing expression of effector response genes, including cytokines, as well as orchestrating both the local and the systemic immune responses [35, 120, 128130]. in this context, it was not surprising that the expression of prrs increases in infected bovine mgs tissues and epithelia [17, 130135]. till now, they are the best characterized bovine prrs and they recognize a wide range of pamps. thirteen tlrs have been identified among mammals, 10 of which are known to occur in cattle [17, 35, 136]. for example, tlr pairs such as tlr1/2 and tlr2/6 can recognise lipopeptides or lipoproteins, whereas individual tlrs such as tlr2, tlr4, tlr5, and tlr9, respectively, are involved in sensing lipoteichoic acid (lta), lps, flagellin, and 6-base dna motif consisting of an unmethylated cpg-dinucleotide motif (cpg-dna) [35, 36, 109, 137140]. besides recognizing lps motifs, tlr4 also can recognise bacterial-derived elastases and exoenzyme-s [141, 142]. another important prr found on pmns and macrophages in the mg is cd14, which can bind to lps and induces the synthesis and release of tnf-. also, the role of nod1 and nod2 receptors of mecs in sensing peptidoglycans (pgs) of gnb has been addressed [109, 144, 145]. (6) contribution of specific bacterial components to the identification by host iis and induction of irs gram-negative bacteria (gnb). cell wall lps, or endotoxin, is central to the pathogenesis of mastitis caused by gnb. lps is considered the most potent immunostimulant of cell wall components and is the key virulence factor eliciting clinical symptoms [36, 37]. the lps layer of the outer membrane generally contains three regions: o-specific polysaccharide chain, polysaccharide core, and lipid a. lipid a was found to be responsible for most of the pathogenic phenomena associated with gnb imis, including endotoxin shock. recognition of lps is mediated by membrane cd14, lps-lbp, an app present in the bloodstream, and tlr on mecs (primarily tlr4) [35, 37, 64, 146]. as a consequence, initiation of acute ir results in an intense elevation of scc [109, 147], activation of different leukocytes and immune-related genes, and subsequent production of antimicrobial defence proteins and peptides (e.g., lf, lz, and lap), lipid mediators (e.g., cyclooxygenase-2 and 5-lipoxygenase) [149, 150], chemokines (e.g., cxcl5, cxcl8, and rantes) [148, 151, 152], and cytokines, especially il-6, tnf- and insulin-like growth factor-1 [35, 64, 146, 151]. additionally, binding of soluble cd14 to lps stimulates mecs to produce leukocytic chemoattractants such as il-8 [112, 153]. despite the principle role of lps in recognizing gnb by tlrs (tlr1/2 and tlr2/6), it has been illustrated more recently that pgs fragments of e. coli, which are known to activate the cytoplasmic nod1 receptor, could be recognized by bovine mecs and, thus, can induce inflammatory response. although nod1 receptor is cytoplasmic and its activation requires that the agonist is transported into the host cell, it is possible that pgs fragments can reach the cytoplasm of bovine mecs following invasion by e. coli, as proven by some authors. moreover, the expression of membrane transporters under particular circumstances including inflammation could transport pgs fragments, as was shown for muramyl-dipeptide (mdp), a potent nod2 agonist [144, 145]. gram-positive bacteria (gpb). in contrast to gnb, for which lps is the major immunostimulatory molecule, several important compounds have been identified as immune stimulators for gpb species, including cell wall lipoproteins, lta, which is a cell wall component of the murein capsule [36, 119], and pgs in addition to secreted exotoxins. both pg and lta have been shown to induce immune cells, including monocytes and macrophages, to produce inflammatory cytokines and chemokines [159, 160]. pg combined with lta induced the expression of mcp-1 and a slight increase in mcp-3 chemokine expression. in vitro studies have shown that lta alone can induce expression of several cytokines such as il-1 [161, 162], il-6, il-8, and tnf- in mecs, although to a lesser extent than lps [125, 161163]. also, lta proved to induce strongly the secretion of the chemokines cxcl1, cxcl2, cxcl3, and cxcl8, which target mainly neutrophils. the role of lta and other pamps as muramyl-dipeptide in stimulating iis is not only limited to expression of specific cytokines and chemokines, but can potentiate their subsequent effects after production. the staphylococcal lta or muramyl-dipeptide enhances the expression of immune defence genes that are induced by il-17 in mecs in vitro. however, it must also be considered that the virulence of bacterial compounds such as lps and lta may vary somewhat depending on their bacterial origin. more interestingly, lps-bp has been shown to bind lta of gpb cell wall although primarily associated with gnb infection. the induction of the gene encoding lps-bp was observed in all tissues of mg challenged by staph. aureus, and increased concentration of lps-bp has been previously reported in milk and serum after imi with staph. tlr2 plays a major role in the recognition of a variety of components related to gpb including lta and lipoproteins. lta activates cells via the tlr2/tlr6 heterodimer [119, 134, 138, 139, 166], and with physical and functional interactions with tlr1 and tlr6 it allows discriminating the lipid portion of lipoproteins [36, 166]. meanwhile, the roles of tlr1, tlr2, and tlr6 in the recognition of pg remain controversial, and it has been suggested that pg recognition occurs mainly intracellularly rather than from the extracellular compartments. despite the principle role of tlr1 and tlr6 heterodimers with tlr2, significant increases in the expression of tlrs that recognise viral ligands (tlr3 and tlr7) were also observed in bovine mgs challenged with staph. aureus, and a previous study has shown the role of tlr7 in recognition of gpb. aureus and il6 treatment. additionally, expression of intracellular receptors may be important in recognizing staph. aureus which has the potential to invade epithelial cells [170, 171]. lf, an iron-binding glycoprotein, was first isolated from bovine milk in 1939. in the mg environment, it is mainly produced by the secretory epithelium and to lesser extent by pmns. little or no expression of lf occurs in lactating alveoli, and moderate to high expression occurs in the epithelia lining the ducts and cisterns, while lf expression is absent at the proximal end of the teat canal. the regulation of lf expression in mg appears to be reciprocal to that of the other milk proteins. although bovine colostrum contains high levels of lf (up to 5 mg/ml), these levels drop very rapidly as lactation proceeds, so that mature bovine milk normally contains 200485 g/ml lf or less [176, 177], depending on daily milk production and lactation stage. on the other hand, lf increases markedly in dry secretions, with the maximum concentrations attained after 3-4 weeks of involution (2030 mg/ml), nearly 100-fold greater than during lactation. the antibacterial effect of lf is enhanced by increased bicarbonates and low concentrations of the lf inhibitor, citrate, present during the dry period [25, 179, 180]. the increased lf concentration during involution strongly inhibits bacterial growth, and it has been suggested to contribute to the low number of naturally occurring imis during this early dry period. lf contributes to mg immunity, immune modulation, and transcriptional activation of various molecules via several pathways. principally, it exerts its bacteriostatic effect by competing with bacteria for available iron [182184] or by binding to bacterial surfaces [185, 186]. studies have shown the ability of lf to damage the outer membrane of a broad range of gnb by interacting with the lipid a portion of lps and performing proteins in the outer membrane (porins), altering the integrity and permeability of the cell wall [185, 187, 188] and releasing lps, which sensitizes the cell to antibiotics. the binding interactions of lf to gpb are still not fully understood, although it has been shown that lf binds to specific receptors on the cell walls of several gpbspecies associated with imis, including s. uberis, s. agalactiae, and staph. aureus [186, 191], as well as several coagulase-negative staphylococci (cns) (e.g., staph. one study showed that although the antagonistic effect of bovine lf on the adhesion and invasion of cns strains to mecs is weak, it significantly decreased intracellular replication rates. bacteria with high iron requirements are susceptible to the bacteriostatic activities of lf. among mastitis-causing bacteria, aureus, but streptococci are more resistant. for e. coli, it appears that igs are not required for lf to exert a potent bacteriostatic effect. aureus and e. coli, although s. uberis challenged mg shows increased mrna expression of lf-related gene and stimulated the production of lf more than the other two organisms. in this context, some studies showed that bovine lf can enhance adhesion of s. uberis to host cells and increase invasiveness, suggesting that s. uberis has evolved to take advantage of the presence of lf [198, 199]. on the other hand, bovine lf has also been shown to inhibit many pathogenic bacteria, including listeria monocytogenes and enterotoxigenic e. coli [200, 201], and to increase the antibacterial effect of antibiotics synergistically against antibiotic-resistant gpb. as a major component of the specific granules of pmns, lf additionally contributes to both hydrogen peroxide-dependent and hydrogen peroxide-independent bacterial killing and promotes the adhesion and aggregation of pmns to the endothelial surface. another aspect of lf's antibacterial activity is based on activation of the cs via the alternative pathway. lf may also be important in ag-processing by cells of the reticuloendothelial system and in ab production. additionally, lf increases nk cells activities and amplifies the inflammatory response and stimulates the phagocytic and cytotoxic properties of macrophages against invading pathogens [203, 205] such as staph. aureus but still as a potent inhibitor of granulocyte-monocyte colony-stimulating factor [205, 206]. during mastitis, lf levels in lacteal secretions may increase 30-fold, corresponding to the severity of infection [111, 149, 176, 197, 207] and depending on the causative agent, as evidence has accumulated suggesting that different pathogens induce different lf-mediated responses from mecs. the dramatic increase in lf concentrations in milk during acute mastitis is consistent with the role of lf as an acute phase response (apr) protein in the mg, in accordance with the presence of apr elements in the lf gene promoter region. in experimentally induced e. coli mastitis, the mean concentration of bovine lf was 2 mg/ml, whereas in cns mastitis it was<0.2 mg/ml. the expression of lf by mecs in vitro has been shown to be greater upon exposure to s. uberis isolated from acute mastitis compared to s. uberis isolated from chronic mastitis. based on the strong association between lf concentrations and mastitis occurrence, combined with the antibacterial properties of lf, it has been suggested that bovine milk lf plays an important role in defence against e. coli if concentrations exceed 200 g/ml milk [185, 188, 210], while it has little effect against other major pathogens such as staph. tf is another iron-bp in the milk of dairy ruminants, although it is present at low concentrations. the concentration of tf ranges from 1.07 mg/ml in colostrum to 0.020.04 mg/ml in milk of third week postpartum compared to 4-5 mg/ml in serum [213, 214]. in contrast to rodents, pigs, and rabbits, which synthesise tf in the mgs at higher concentrations, tf in the milk of dairy ruminants is not synthesised in the udder and instead comes from blood serum, from transcytosis in the normal gland, and through exudation of plasma during mastitis. like lf, tf can damage the cell membranes of gnb with the release of lps, thereby altering outer membrane permeability. during experimental e. coli imis in dairy cows, tf concentrations were found to rise even before lf elevation, reaching 1 mg/ml in milk and paralleling the concentrations of serum albumin. lz (n-acetylmuramyl hydrolase) is one of the components of antibacterial system in milk [4, 216, 217]. lz has inhibitory or lytic activity mainly against gpb and to lesser extent against gnb by cleaving the 1,4-glycosidic bond between n-acetylmuramic acid and n-acetyl-d-glucosamine residues in pg, thereby disrupting the cell wall [4, 177]. however, milk lz alone is not a significant component of the bmg defence, and only a few mastitis-causing bacteria are killed by lz. nonetheless, lz can synergize with abs, complement, and lf [4, 25]. for example, the binding of cationic lf to the lta of gpb renders staphylococci more susceptible to lz [4, 218]. in healthy conditions, lz concentration of milk shows wide variation among species and is influenced by several factors such as the period of lactation, health, age, and the parity of animals [217, 219]. after parturition, the lz concentration shows successive increase, reaching the peak (0.72 mg/l milk) at the 7th day, and then begins to decrease after the 2nd week postpartum. nevertheless, bovine and buffalo milk contain averages of only 0.0004 and 0.000152 g lz/l milk, respectively, compared to 10 mg lz/100 ml in human milk. a substantial rise (1050-fold) of lysosomal activity of milk has been recorded during mastitis among different dairy species [149, 217, 222, 223]. however, buffalo may exhibit thousandfold greater lz activity and moderately raised sccs in milk without showing signs of mastitis. lz in milk may be derived from blood or locally synthesized, and during imi leucocytes appear to be the source of lz. next to xanthine oxidase, lactoperoxidase is the most abundant enzyme in milk, constituting 0.5% of the total whey proteins (30 mg/l) [225, 226], and nearly similar concentration is present in colostrum [226, 227]. as for many other indigenous enzymes, the level of lactoperoxidase in milk increases with mastitis. locally synthesised lactoperoxidase, in the presence of thiocyanate of hepatic origin and hydrogen peroxide of either bacterial or endogenous origins, can exert antibacterial properties against both gpb and gnb via the generation of activated oxygen products like hypothiocyanate, a reactive metabolite formed from the oxidation of thiocyanate that promotes bactericidal activity of phagocytes [5, 177]. it has been hypothesized that lactoperoxidase may have a synergistic antimicrobial function with lingual antimicrobial peptide (lap), one of the host defence peptides, in mgs of dairy cows. it is mainly located in the primary granules of neutrophils, and together with peroxide and halide it has an important role in the oxygen-dependent antimicrobial system of neutrophils and thus in defence against microorganisms [231, 232]. it catalyses the same peroxidase reaction as lactoperoxidase and additionally catalyses the oxidation of chloride, the product of which provides the bactericidal activity of this system. in vitro, this system has been shown to be potent against major common udder pathogens such as staph. unfortunately, the antibacterial properties attributed to this system are only relevant during the dry period, whereas they were found to be completely inhibited with lactation, mainly due to milk proteins. additionally, the levels of thiocyanate in udder are dependent on the specific dietary composition, and the low oxygen tension of the mg can inhibit the production of hydrogen peroxide, thus limiting the effectiveness of this antimicrobial system against different pathogens incriminated in mastitis. complement system (cs). complement is a collection of proteins that are produced in plasma mainly by liver as well as tissue macrophages and monocytes and for c3 a local synthesis in the mg was suggested. in support of the assumption of a local synthesis, experimental staph. aureus and e. coli imis induced an increase of c3 mrna-expression in mecs. complement components elicit their biological activities through complement receptors located on a variety of cells [7, 134, 233]. the cs is central to iis because it is intimately involved in initiation and control of inflammation, opsonisation of bacterial surfaces, attraction and recruitment of phagocytes (chemoattractants) (e.g., c3a and c5a cleavage fragments), recognition and ingestion of microorganisms by phagocytes (e.g., c3 and c4), and the killing of microorganisms, either directly or through cooperation with phagocytic cells [53, 134, 233235]. nevertheless, it was also gradually appreciated that different proteins of the cs can influence the mgir and constitute an important bridge between iis and ais [53, 235, 236]. the lowest concentrations of complement are observed in the milk of healthy mgs during lactation, and higher levels are observed during late lactation period, in colostrum, and in mammary secretions obtained during involution, presumably due to the mobilisation of complement components by transudation from blood [237240]. the alternative pathway (ap) was found to be the sole complement pathway operating under these healthy conditions, while the classical pathway (cp) is not functional due to lack or lowered presence of c1q component compared to blood [53, 233, 241]. the ap operates with two consequences that are greatly involved in recruitment and activation of phagocytes, mainly pmns: (1) deposition of opsonic c3b and c3bi on bacteria and (2) generation of the proinflammatory fragment c5a [75, 234, 241, 242]. however, the milk from noninflamed mg is generally devoid of significant haemolytic and bactericidal complement-mediated activities, especially during the midlactation period [240, 241, 243, 244], due to strong anticomplement activity of milk on complement mediated hemolysis and the absence of the c1q component required for activation of the cp [5, 177, 244], except for some healthy periods of exerting elevated complement concentrations, where these activities exist in a weak but significant manner [237, 238, 240]. nevertheless, this inhibitory activity does not involve c3b/c3bi deposition on bacteria or the generation of c5a by the ap. unfortunately, the lack of haemolytic activity in bovine normal milk in the absence of inflammation adversely affects a very important function of the cs, opsonisation of bacteria by cs components, mainly c3. however, it has been shown a noteworthy deposition of c3 complement fragments from neat milk of non inflamed mg on some particular udder bacteria, as mastitis-causing staph. aureus, and s. agalactiae even in mid-lactating period by the activation of the ap. in addition, an enhanced chemiluminescence response of pmns against invading pathogens was noticed [53, 245]. on the other hand, the production of extracellular fibrinogen-bp by staph. aureus was found to inhibit complement activation by blocking c3 deposition on the bacterial surface. in contrast, the highest concentrations of complement are observed in mastitic milk, presumably due to the mobilisation of complement components by transudation from blood [233, 238, 239]. relative to the increase in complement concentrations during imi after recruiting plasma components, both bactericidal and haemolytic activities of cs are increased in inflamed mg, and the intensities of these activities correlate with intensity of the ir [233, 247, 248]. gnb (e.g., e. coli) are sensitive to complement lytic action, while some gpb (e.g., staph. aureus) are resistant, although all bacteria show susceptibility to the opsonizing action of c3b and c3bi fragments after activation of the ap [53, 233, 241, 247, 248]. most cytokines have more than one function and often have redundant effects with other cytokines. because of the high affinity of their receptors, cytokines are highly potent and can elicit biological responses even at femtomolar to nanomolar concentrations. numerous cytokines (e.g., tnf-, ifn-, gm-csf, il-8, and il-12) have been detected in normal udders [251, 252], but during imi a complex upregulation of specific cytokines occurs depending on several factors. cytokines act at both local and systemic levels during onset, progression, and resolution of inflammation [253, 254]. they provide relatively short-range communications between cellular immune components, thus linking the innate and adaptive immune branches, and this short communication range is important to limit their effects to the appropriate cells. although cytokines play an essential role in the host response to infection, they can also have deleterious effects. thus, there is a fine balance between the positive and negative effects of cytokines on the host that is dictated by the duration, amount, and location of their expression. a more detailed explanation of the roles of specific cytokines, chemokines, and growth factors in mg during imis is illustrated in table 2. due to their important contributions to the inflammatory process, several studies have illustrated cytokines benefits in immunotherapy of mastitis via enhancing mg immunity (e.g., interferons, mainly ifn-, il-2) [257261], their contributions to control or prevention/immunisation against mastitis pathogens especially e. coli or staph. aureus (e.g., g-gsf, gm-csf, il-2, and ifn-) [262264], and their potentiating effects on response to treatment with antibiotics (e.g., il-1, il-2, and ifn-) [262, 265269]. the efficacy of recombinant cytokines (e.g., recombinant bovine il-2 [rboil-2 ]) in accelerating the involution of mg during dry period, and thus reducing the time in which the mg is particularly susceptible to infection, has been addressed [270, 271]. intramammary infusion of il-2 elicits a considerable increase in scc, which is dominated by macrophages and plasma cells producing igg1, igg2, iga, and igm. on the contrary, the immunotherapeutic properties of rboil-1 are masked by the domination of proinflammatory nature of il-1 [251, 271, 272]. chemokines are important molecules involved in migration and recruiting leukocytes into mg during imi, besides being involved in several immunoregulatory and inflammatory processes [39, 51, 151, 161]. according to arrangement of conserved n-terminal cysteine motifs, chemokines are grouped into 4 families: c, cc, cxc (subdivided into elr and elr), and cx3c. members that contain the motif (elr) are potent chemoattractants for neutrophils and promoters of angiogenesis, whereas those that do not contain the motif (elr) are potent chemoattractants for mononuclear cells [151, 161]. representatives of the elr cxc chemokines are structurally similar, including il-8/cxcl8 and ena-78/cxcl5. chemokines target neutrophils by interacting with one (e.g., cxcl1, cxcl2, and cxcl3) or two (e.g., cxcl8) receptors, cxcr1 and cxcr2, which are expressed by neutrophils of several species including cattle. several molecules which mediate leukocytic trafficking are expressed in the mg tissues and mecs in response to lta from gpb (e.g., cxcl1, cxcl2, cxcl3, and cxcl8) or lps from gnb (e.g., rantes, cxcl5, cxlx8, mcp-1, mcp-2, and mcp-3) and can be also detected in milk [39, 51, 127, 135, 151, 152, 161, 165, 275]. the remarkable induction of chemokine gene expression by the epithelial cell lends strong support to its role in stimulating migration of leukocytes into the mg [39, 63]. host defence peptides (hdps) are a large family of innate immune effector molecules. they are predominantly synthesised in pmns and epithelial cells [5658, 132, 276] and have been shown to be important in the resolution of local infection through both antimicrobial and immune-regulatory properties. defensins are an important family of hdps in cattle owing to variable bactericidal properties [57, 276] and are considered as effector arm of iis as well as representing a putative link between iis and ais [58, 117, 132, 277]. several -defensins, including lap, tracheal antimicrobial peptide (tap), and bovine neutrophil -defensins 1, 4, and 5 (defb1, defb4, and defb5), are expressed in mg tissues in both a constitutive and an inducible manner, or even excreted in milk, in response to bacterial challenge [17, 57, 117, 118, 131, 132, 150, 278]. also, an increase in lap mrna expression in the bovine alveolar tissue at 192 h after milking upon involution has been declared. a broad spectrum of antimicrobial activities has been demonstrated for several bovine -defensins, in particular against several species that cause mastitis as staph. pneumoniae, and ps. aeruginosa [57, 118, 279]. the specific or adaptive immune system [ais] recognises specific determinants of a pathogen mainly via abs molecules, macrophages, and several lymphoid populations, which subsequently facilitate selective elimination [7, 27]. because of the memory function of certain lymphocytes, specific irs can be augmented by repeated exposure to a pathogen. immunoglobulins (igs) are the most important specific soluble humoral factors in adaptive immune defence, linking various parts of the cellular and humoral immune system, and they constitute the main component of the ais present in colostrum and milk [33, 280]. they are able to prevent adhesion of microbes to tissues, inhibit bacterial metabolism, agglutinate bacteria, augment opsonisation and phagocytosis of bacteria, kill bacteria through activation of complement-mediated bacteriolytic reactions, and neutralize toxins and viruses [281, 282]. igs account for up to 7080% of the total protein content in colostrum (20150 g/l) to confer passive immunity to newborns, whereas in milk they account for only 1-2% of total protein (0.51 g/l) [31, 226, 227, 247]. however, ig concentrations in the bmg vary during the lactation cycle, and an increase occurs at the end of lactation igs in milk may be blood-derived or may be produced in situ by ag-activated plasma cells, which traffic to the udder from the blood [77, 284] mediated by chemokines produced locally during imi. the mg plays an active role in regulating the levels of different igs present in colostrum and milk, although the mammary epithelium itself does not synthesise igs. the majority of igs are transported into mammary secretions via specialised receptors (selective receptor-mediated intracellular route). there are four different classes of igs that play dominant roles in mg defence against bacterial pathogens: igg1, igg2, igm, and iga (table 3). functionally, igg1, igg2, and igm act as opsonins and facilitate phagocytosis by pmns and macrophages [49, 247], while iga is thought to play roles in toxin neutralisation and bacterial agglutination, thereby hindering bacterial spread and colonisation [247, 284]. bovine colostrum contains igg1, iga, and igm in concentrations exceeding those of blood. the colostrum/blood ratios for igg1, iga, and igm are approximately 4: 1, 13: 1, and 2: 1, respectively. the most abundant ig class in bovine milk and colostrum is igg1 [287289], while igg2 increases substantially during inflammatory states. in contrast, iga and igm are present at much lower concentrations in healthy bmgs [286, 290]. as mentioned, both innate and adaptive irs are coordinating and operating together in very complicated pathways to provide the optimal defence against infections. pr and ag presentation by innate immune components initiates a proinflammatory response with quantitative and qualitative changes of different immune components in a complex manner. different cytokines and chemokines appear to play essential roles in this process by acting through their variable immunoregulatory roles, thus coordinating mgir. once bacteria contact leukocytes in the milk or the lining mg epithelium accompanied by exerting various virulence mechanisms and liberating toxins, irritation or even damage to mg epithelium and, thereby inflammatory products from damaged epithelium induce locally located leukocytes and healthy mg epithelium to release several chemoattractants for the migration and recruitment of both bone marrow and circulating immune cells into the mg environment, mainly neutrophils [39, 63, 151, 255, 291, 292]. proinflammatory cytokines (il-1, il-6, and il-17) as well as il-8 and tnf- are the main effectors to initiate the inflammatory responses at both local and systemic levels [121, 162, 291, 293, 294]. they act in collaboration with tgf-, gm-csf, and several chemotactic factors (e.g., c3a and c5a complement fragments, leukotriene b4, paf, eicosanoids [as prostaglandin-f2], oxygen radicals, and apps) to potently trigger circulation-into-mg migration of neutrophils via induction of vascular endothelial adhesion molecules expression (mainly for e- and p-selectins), thereby promoting neutrophil transendothelial migration to the infected foci [291, 295, 296]. as a consequence, enhanced expression and adhesiveness of another neutrophil adhesion molecule, mac-1 (known also as cd11b/cd18), occur, which allows neutrophils to bind tightly to activated endothelium in collaboration with another endothelial adhesion molecule, icam-1. this adhesive interaction allows neutrophils to migrate along the endothelial surface and into mg tissues up a concentration gradient of chemoattractants; one of the most potent with long-lasting effect is il-8 [75, 256, 291, 292, 297]. it is thus clear that the migration of immune cells to mg is not a random process and a collaboration of several molecules, chemoattractants, selectins, and integrins is greatly needed to regulate chemotaxis. il-17 has been suggested to enhance leukocytic recruitment into mg via regulating il-8 expression and enhancing expression of several chemokines targeting not only neutrophils (cxcl3 and cxcl8) but also mononuclear leucocytes (ccl2, ccl20) [121, 162, 294]. leukocytes that freshly migrated express greater numbers of cell surface receptors for igs and complement and are more phagocytic than their counterparts in blood. stimulation of microbicidal activities of various leukocytes located inside infected tissues is mainly regulated by certain proinflammatory cytokines (table 2). the activation status and enhancing functions of neutrophils are stimulated mainly by il-1, il-8, ifn-, tnf-, and g-csf; macrophages by il-12, m-csf, and gm-csf; and nk cells by il-2 and il-12. meanwhile, b-lymphocyte differentiation is driven mainly by il-2 and il-6 [27, 256, 298305]. systematically, several physiologic responses occur as a result of imi: (1) generation of febrile response [293, 296, 301, 306, 307], (2) alterations in metabolism and gene regulation in the liver, resulting in elevation of apps levels as well as serum cortisol levels, and (3) changes in vascular permeability, tone, and activation [257, 293, 296, 309]. some cytokines such as tnf-, il-1, and il-6 are responsible for generation of febrile response, and the latter one specifically contributes to the great extent for regulation of the apr through the synthesis of app. il-17 greatly synergizes to generation of inflammatory reactions via enhancing production of il-6 [121, 162], il-8, and gro and the expression of inflammatory cytokines tnf- and il-1 (table 3). likewise, tgf- has been shown to have a potential role in mediating iir and promoting inflammation by upregulating the production of prostaglandins and synergistically enhancing the effects of il-1 and tnf- [311313]. additionally, tgf- has the ability to directly stimulate il-8 and to induce expression of antimicrobial peptides. ags from invading mastitis-causing bacteria are processed mainly within macrophages and b-lymphocytes and appear on the membranes in association with mhc class i or ii; thus they can be recognised by different lymphocytes [27, 45, 6769]. ifn- greatly contributes to upregulating of the mhc-i expression and mhc-ii ag presentation, thus increasing cytotoxic t-cell recognition for foreign peptides, and inducing cd4+t-cell activation [256, 303]. upon recognition of ag-mhc class ii on b-lymphocytes or macrophages, cd4+cells are activated and produce cytokines that have roles in the activation and polarisation of b- and t-lymphocytes, macrophages, and various other cells that participate in the ir [4, 7, 27, 81]. depending on the repertoire of cytokines produced, the t-helper cell response can facilitate either a cell-mediated (th1 type) or a humoral (th2 type) ir. il-2 and ifn- are the major cytokines secreted by th1 cells, and they stimulate cellular responses against intracellular pathogens. in contrast, il-4, il-5, and il-10 are secreted by th2 lymphocytes; these cytokines promote humoral immunity and regulate both macrophage functions and the activity of cytokine production [27, 316]. on the other hand, inflammatory-inducer ifn- and regulatory il-4 are the main cytokines produced by cd8+cytotoxic and cd8+suppressor t-cells, respectively. inflammatory cytokines produced by t-cells in turn induce the proliferation and differentiation of the b-lymphocytes into either ab-producing plasma cells or memory cells [27, 49], and some of them are responsible for increasing fc receptors for igg2. synergistically, activated macrophages release chemotactic signals for neutrophils, thereby amplifying the inflammatory response. macrophages secrete prostaglandins and leukotrienes that augment local inflammatory processes [75, 317] as well as specific cytokines that are known to regulate t-cell differentiation, mainly il-12. regulation of polarising t-helper subsets into either th1 or th2 is the main axis on which some regulatory cytokines (il-4 and il-12) work. il-12 contributes to the ir by favouring the polarising cd4+t-cells towards th1 responses and enhancing the generation of cytotoxic-ifn- producing cd8+cells and also acts as a growth factor for nk cells and an inducer of their cytotoxic activities [254, 318, 320]. thus, it contributes to the production of ifn- from lymphocytes as well as nk cells [254, 318]. in contrast to il-12, il-4 favours the development of th2 subsets and exerts a clear inhibitory effect on ifn- production. compared to the anti-inflammatory il-10 cytokine, the inhibitory effect of il-4 on monokine synthesis is lesser. based on the effects of il-4 and il-12 on polarisation of t-cell subsets, the early preference expressed in the ir is greatly dependent on the balance between il-12 and il-4. resolution of the imi is mediated by upregulation of several inflammatory-antagonist cytokines, including il-10, and tgf-, and in corporation to anti-inflammatory effects elicited by il-6 and il-4. il-10 is the most potent contributor to this process as it downregulates both the generation of all subtypes of t-helper cells and the production of proinflammatory cytokines, chemokines, and eicosanoids by monocytes, macrophages, and neutrophils [85, 253, 291, 324]. il-10 potently inhibits the ability of macrophages to stimulate th1 cells to produce cytokines, principally ifn-, and has an inhibitory effect on lps-induced production of il-1, il-6, and tnf- by macrophage cell lines. in cooperation with il-6, il-10 also upregulates il-1 receptor antagonist and soluble tnf receptors, impairing the ability of the proinflammatory cytokines il-1 and tnf-, respectively, to exert their effects. in contrast, il-10 does not inhibit cytokine production by b-lymphocytes nor does it affect the ability of different phagocytes to stimulate cytokine production by th2 cells. like il-10, the major role of tgf- is to suppress the irs, although some proinflammatory properties have been reported [325, 326]. the anti-inflammatory role of tgf- is exerted through its ability to (1) inhibit macrophage production of chemokines, proinflammatory cytokines, nitric oxide, and ros; (2) limit ifn- production; (3) increase expression of the il-1 receptor antagonist; and (4) enhance macrophage clearance of bacteria and cellular debris [325, 326]. the repair of damaged mg epithelium is mainly mediated by tgf-, which promotes epithelial proliferation and tissue remodelling. tgf-, on the other hand, promotes extracellular matrix deposition, fibrosis, and scarring. thus, restoring healthy structure/homeostasis and scar formation is controlled by the balance between the two tgf types. during the whole process, altered cells are mainly removed by macrophages and cytotoxic t-cells, which recognise and eliminate altered self-cells via ag presentation, with the help of t-cells and nk cells, which mediate cytotoxicity with variable involvement of mhc molecules [27, 83, 84]. in addition to investigating the pathogen virulence mechanisms and the resulting histopathological changes, study of the immunological profile of the mg against a particular pathogen will help provide a better understanding of the nature, rate of development, and severity of mastitis caused by such pathogen and is considered a prerequisite to the development of novel and effective diagnostics and therapeutics. the sensitivity and responsiveness of the mg in terms of specific immune factors varies greatly against different bacteria [37, 131, 134, 165, 196, 275, 329331] and their associated toxins [125, 148, 332, 333]. thus, the high sensitivity of the mg to some mastitis pathogens results in a robust ir, invoking an acute response to infection and likely predisposing to rapid elimination of the invading bacterium with proper host immunity and animal management. aureus and some cns may result in subclinical or chronic imis as a result of poor responsiveness of mg immunity. in attempt to understand the pathogenesis of imis caused by different bacterial species, several studies have assessed the mammary irs towards particular mastitis pathogens, as shown in table 4. unfortunately, most studies regarding mammary irs towards particular pathogens in bovines have focused on staph. most bacterial species causing coliform mastitis elicit a marked acute inflammatory response in comparison to staph. however, the iir varies among different mastitis-causative species. a strong tnf- response to lps was found to be central to the earliest initiation of mgirs and in the development of pyrexia associated with coliform mastitis, endotoxic shock in per acute form [127, 334, 335], leukopenia in peripheral blood, and concurrent increases in milk leukocytes [62, 336, 337]. the powerful chemotaxis and recruitment of leukocytes, mainly pmns, and robust production of a wide variety of cytokines reflect the mg's sensitivity to and response against e. coli compared to staph. aureus [114, 127, 131, 165, 239, 307, 311, 329, 338, 339]. aureus bacteria were used to stimulate isolated mecs, expression of tnf-, il-1, il-6, and il-8 was greater in cells stimulated by e. coli. experimental studies conducted on ovines revealed similar results regarding mgir towards e. coli, and increases in leukocyte recruitment (mainly pmns) and proinflammatory cytokine levels (including il-1, il-8, and tnf- [255, 340 ]) have been reported in response to either e. coli or its endotoxin. occasional increases in gm-csf and ifn- have also been shown [255, 340]. these data explain why e. coli imis follow acute form and why these imis may resolve spontaneously within a short period as declared in previous studies [341, 342]. depending on the levels of chemoattractants and proinflammatory, inflammatory, and regulatory cytokines, the iir is also robust towards kl. aeruginosa, reflecting the strong mgir towards these bacteria. against s. marcescens, however, the mgir is comparatively modest [337, 343]. the number of bacteria isolated from mgs of s. marcescens-infected cows as well as sccs dropped precipitously 24 h and 48 h following infection (pi), respectively, which could reveal elimination of bacterium by mg immune system. though several studies reported strong systemic responses and clinical signs in animals infected with several species of gnb [343345], the accurate investigations focused on the iir towards gnb other than e. coli are considered rare and mostly experimental. further in vivo and in vitro studies are required. aeruginosa infection in humans have revealed that secretion of exotoxin a, exoenzyme s, and elastase by such bacterium inhibits monocyte and neutrophil chemotaxis and respiratory burst, thus altering the ir [346, 347]. unlike the case with e. coli, mgir against staph. aureus was found to be insufficient to eliminate the bacterium, allowing persistence of infection and eventually leading to subclinical or chronic patterns of imi. aureus imi induced strongly il-8 and tnf- gene expression in the mg tissue as well as strong activation of nf-b in mecs and triggered a rapid early expression of -defensin, tlr2, and tlr4 in the inoculated mg and lymph nodes, while impaired proinflammatory activation was paralleled by a complete lack of nf-b activation in mecs challenged by staph. aureus or lta, and only expression of -defensin occurred later than 48 h in inoculated quarters with staph. aureus. in a contradictory study, although all 10 tlrs ' and nod 1-2 expression was upregulated in mg tissues challenged with staph. aureus, with tlr8 having the least expression in comparison to the other prrs, immunohistochemistry analysis of tissues from both staph. this variability in the expression of prrs could be attributed to different strains, but in all conditions how the ir of mg towards staph. aureus is being translated remains as a crucial point. in the last study, expression of proinflammatory cytokines (il6, il17a, and il8) and anti-inflammatory cytokine (il10) meanwhile, the production of these cytokines varied among studies (table 4), which reveal the complexity of mgir towards staph. aureus and illustrate that mgir could be modulated due to pathogen factors suppressing the production of these cytokines. reduced expression and induction of some inflammatory cytokines, including tnf- by lta, the principle immune-stimulator of gram-positive cell wall [17, 125, 126, 163], impaired activation of nf-b and reduced expression and production of chemokines (il-8 and rantes) [134, 165], involved in recruiting leukocytes, which may reflect why the sccs are not elevated in mgs challenged by staph. it has been hypothesized that decreased expression of immune-modulator -1 acid glycoprotein in the alveolar region of mg experimentally challenged with staph. aureus may inhibit the early recruitment of neutrophils to the mg and could be a result of modulation of the host's ir by the pathogen in order to enhance survival. also, since it has been suggested that tgf- was found to block the tlr signalling, the expression of tgf- in imi caused by staph. additionally, various studies have shown that staphylococcal enterotoxins (sea, seb, sec, and toxic shock syndrome toxin-1) act as super ags by activating specific types of t-lymphocytes (mainly cd8+suppressors) and stimulating release of specific cytokines [332, 333, 349]. the presence of high numbers of suppressor cd8+t-cells compared to cd4+t-cells significantly suppresses lymphocyte irs and recruitment [78, 86]; and in addition to unstable expression and release of inflammatory inducers (il-1, il-8, and tnf-) [17, 86, 114, 131, 134, 165, 239, 329], compromised expression and release of inflammatory cytokines (depressed il-2 and c5a levels) [17, 86, 114, 239, 350] and unstable release of anti-inflammatory il-10 could greatly reflect and provide explanation for the suppressive nature of mastitis-causative staph. aureus and why imis caused by such bacterium do not usually undergo resolution and follow subclinical or chronic patterns with persistence of the pathogen. in addition to causing a marked increase in scc, cns can persist similar to staph. unfortunately, few studies have investigated the bovine mgir against cns, and the majority were conducted in ovines or investigated only few aspects of mgirs. in both bovines and ovines [355, 356], the imis caused by staph. simulans were associated with a decline in leukocyte counts for a short period after initiation of the inflammatory process and the absence of a marked systemic cytokine response. however, some proinflammatory cytokines, including il-1, il-8, and tnf-, were elevated in milk [354356]. these observations likely reflect the unsuccessful combat of mg against the invading bacterium and that the sensitivity or responsiveness of mg to inflammatory signals decreased as infection progressed. in experimentally induced ovine imi by staph. epidermidis, counts of leukocyte subsets (including cd4, cd8, wc1, and mhcii) temporarily decreased and then subsequently increased, while the expression of some adhesion molecules (cd11b and cd18) on pmns decreased after 24 h. chromogenes as measured by systemic signs, scc, milk yield, bacterial counts, and some inflammatory indicators (including enzymatic activity and app levels), but cellular and other soluble factors of mg immunity have not been studied. xylosus have been shown to cause cellular responses in both ovines [358, 359] and caprine udders, as indicated by increased scc and leukocyte counts in milk and severe infiltration of mg tissues with mononuclear cells and neutrophils on histopathological investigations. unfortunately, few studies have focused on mgirs against streptococci, despite their substantial contribution to mastitis. to our knowledge dysgalactiae, and few studies were conducted on s. uberis [196, 209, 336, 337, 362364]. although not completely comprehensive, mgir towards s. dysgalactiae subsp. dysgalactiae in one study was represented by increased expression of tlr4 plus release of various cytokines (il-1 and tnf-). most experimental challenge studies showed that mgir against s. uberis was not sufficient to allow successful elimination of the bacterium, although increased expression and production of several inflammatory mediators and antimicrobial components as il-1, il-8, il-10, il-12, ifn-, tnf-, scd14, lps-bp, c5a, and lf have been declared during imis caused by s. uberis. in s. uberis-experimentally infected cows, both numbers of bacteria in milk and sccs remain highly elevated for long time pi, compared to s. marcescens infected cows. neither the influx of pmns into mg infected with s. uberis [336, 337, 365] nor intracellular engulfment by macrophages [70, 366], have resulted in effective reduction in the number of bacteria, and in contrast intracellular replication of s. uberis inside macrophages increased. additionally, it has been accumulated that mgir towards s. uberis is very complex, and different strains of s. uberis can elicit different irs. some studies showed that strain-specific pathogenicity greatly modulates the ir, implying that pathogen factors rather than host factors play an important role in modification of mgir [209, 364]. contradictory results have been obtained in different study when a strain of s. uberis used to induce cm in vivo failed to cause a change in the mrna levels of the immune-related genes by bovine mecs in culture, suggesting that the expression of immune-related genes by mecs may be initiated by host factors and not s. uberis. however, in the same study, challenging bovine mecs with different s. uberis strains resulted in an increase in the mrna expression of a subset of the immune-related genes measured. also, mgirs towards different strains of s. uberis isolated from different imi cases of different intensities varied. expression of il-1 and il-8 from mecs in vitro has been shown to be greater with exposure to living and heat-inactivated s. uberis isolated from acute mastitis than s. uberis isolated from chronic mastitis. more interestingly, a strain of s. uberis that induced acute mastitis in vivo caused twofold and fourfold higher expression of il-8 and il-1, respectively, in isolated mecs in vitro than a strain isolated from a case of chronic mastitis. similar results were obtained in a separate study, indicating that the severity of mastitis induced by different s. uberis strains in vivo can be reflected at the level of the mgir in vitro. in another in vitro study, heat-inactivated s. uberis did not trigger an ir from mecs, although inactivated staph. aureus did, despite the fact that both bacteria are gram-positive and contain lta in their cell walls. continued to particularity of mgir towards s. uberis, an emergence of s. uberis-specific bactericidal t-cells in the mgs of cows after infection or environmental exposure to s. uberis has been documented, suggesting that these specific cells may play a role in control of imi caused by this bacterium. to the best of our knowledge, no studies have been performed to assess the mgir of bovines to the major contagious bacterium s. agalactiae. in a study of s. agalactiae imi in mice, the ir manifested as a massive infiltration of mg by pmns and the release of il-1, il-6, and tnf- in the first 72 h pi; these cytokine levels decreased concurrently with increased levels of il-12 and il-10. results obtained from different studies investigated the mgir towards different mastitis pathogens, demonstrating the complexity of the mgir to an infecting pathogen and indicating that a coordinated response exists between the resident, recruited, and inducible immune factors. in recent years, there has been considerable expansion of our knowledge concerning host mg immune defence against bacterial infections. this defence involves sophisticated mechanisms for detecting various invading bacteria and combating them by the innate and acquired irs. to improve dairy animal resistance against imis, further investigation concerning mg immunology should focus on the following: (1) enhancement of immune functions or at least the maintenance of these functions at normal levels under various lactating and nonlactating conditions, especially during periods of immune suppression; (2) clarifying the roles of specific mammary immune cells, primarily lymphocytes, and in particular the roles of nk cells and cells, which are not fully defined; (3) in vivo and in vitro investigation of mgirs against certain common bacteria in bovines, including s. uberis, s. dysgalactiae, s. agalactiae, coliforms other than e. coli, and cns because most research studies concerning mgirs have focused on staph. aureus and e. coli, as most studies using other pathogens have involved experiments in ovines and focused on cytokine levels only without detailing the cellular responses; (4) clarifying the roles of certain chemokines as rantes and cytokines such as il-17, tgf, and csf in mg, as well as lf effect against gpb because its role is not clearly understood; and (5) changes of leukocytes population in mgs of ovines and caprines during imis.

the health of dairy animals, particularly the milk-producing mammary glands, is essential to the dairy industry because of the crucial hygienic and economic aspects of ensuring production of high quality milk. due to its high prevalence, mastitis is considered the most important threat to dairy industry, due to its impacts on animal health and milk production and thus on economic benefits. the mg is protected by several defence mechanisms that prevent microbial penetration and surveillance. however, several factors can attenuate the host immune response (ir), and the possession of various virulence and resistance factors by different mastitis-causing microorganisms greatly limits immune defences and promotes establishment of intramammary infections (imis). a comprehensive understanding of mg immunity in both healthy and inflammatory conditions will be an important key to understand the nature of imis caused by specific pathogens and greatly contributes to the development of effective control methods and appropriate detection techniques. consequently, this review aims to provide a detailed overview of antimicrobial defences in the mg under healthy and inflammatory conditions. in this sense, we will focus on pathogen-dependent variations in irs mounted by the host during imi and discuss the potential ramifications of these variations.

PMC4590879

pubmed-799

patients with severe stenosis should undergo treatment even if it is well tolerated and is asymptomatic at the beginning. this is due to the possibility of dangerous complications that may emerge if timely treatment is not used.1 balloon valvuloplasty for pulmonary valve stenosis is the treatment of choice for isolated pulmonary stenosis in all childhood age groups.2 the balloon valvuloplasty procedure in treating pulmonary valve stenosis has its origins in the success of surgical valvotomy to relieve the pressure gradient developed across the stenotic pulmonary valve.3 the first catheter attempts to relieve the gradient were described by rubio and limon lason in 19544 and semb et al in 1979.5 the short-term and long-term effects of this therapy are still an area of interest for interventional cardiologists as stronger evidence is needed from studies in different settings and populations. the aim of this study was to investigate characteristics and outcomes of treating pulmonary stenosis with valvuloplasty, and to compare the results among three childhood age groups. all children under 15 years of age who had undergone pulmonary valve balloon valvuloplasty in madani heart center from 20052009 were enrolled in this study. madani heart center is a referral subspecialty center belonging to tabriz university of medical sciences and is located in tabriz, northwest of iran. the diagnosis was based on two-dimensional echocardiography and peak-to-peak pressure gradient difference between the pulmonary artery and right ventricle. the main variables investigated included: age, sex, coincident diseases, baseline right ventricle and pulmonary artery pressure gradient, pulmonary artery-right ventricle pressure gradient at baseline and after 24 hours, remaining residual pressure, annulus size, balloon size, complications, and mortality. percutaneous balloon valvuloplasty was performed on a standard base for any patient with transvalvular gradient>40 mmhg. a catheter, with deflated balloon at a given size, the gradient across the pulmonary outflow was measured and location of the valve was defined using fluoroscopy and a ventriculogram in the anteroposterior and lateral projections. after conducting valvuloplasty and all measurements, the results were compared among three age groups: infants, 15 years, and 515 years. data were entered into the computer and analyzed using ibm spss software (version 16.0; spss inc, chicago, il). distribution, independent t-test or mann whitney u test were used to compare numeric variables. study protocol was approved by regional committee of ethics at tabriz university of medical sciences. noncardiac coincident diseases included two cases of bilateral tonsillar hypertrophy, one congenital cataract, one case of undescended testis, one hemophilia case, and one case of left sided inguinal hernia. mean right ventricular pressure was 95.9 35 mmhg at the first visit and mean pulmonary artery pressure was 26.2 14.7 mmhg. mean difference between the pulmonary artery and right ventricular pressure was 77 35 mmhg. pulmonary valve insufficiency was observed in 52 (59.8%) patients, 36 patients having mild pulmonary valve insufficiency. stenosis remained in 56 patients leading to a mean pressure of 40 26.4 mmhg. two children died under treatment, one of whom was a 3.5-month-old infant who died after 3 days in intensive care unit, suffering from multiple cardiac anomalies. balloon valvuloplasty failed in nearly one-fifth of patients in this study leading to a surgical alternative treatment. although balloon dilatation mortality and morbidity is reported to be greater than after surgery and the recurrence rate is higher following balloon dilatation, the comparison can be problematic due to methodological issues.6 however, since the first report by kan et al,7 many studies have confirmed the safety and efficacy of pulmonary balloon valvuloplasty in infants, children, and adolescents with pulmonary valve stenosis, and it has gained much popularity. indications for intervention in this age group include the prevention of progression of right ventricular outflow tract obstruction, right ventricular hypertrophy, and right ventricular fibrosis. outcome results in this study, as consistent with others, pose the idea of hemodynamic mechanisms being affected after pulmonary balloon valvuloplasty. in a study by alyan et al, it was found that sympathetic overactivity and increased probrain natriuretic peptide levels were associated with the symptomatic status of patients with pulmonary stenosis and associated with a decrease in atrial pressure and probrain natriuretic peptide levels; pulmonary balloon valvuloplasty yielded a decrease in adrenergic overactivity in the patients with pulmonary stenosis.8 although not statistically significant, there were 17 failures and two cases of mortality, descriptively less frequent among children>5 years. failure, mortality, and complication are an inevitable part of cardiothoracic interventions.911 the presence of complications is proportional to age and such complications are mostly found in infants. although younger patients have shown poorer prognosis after valvuloplasty, interestingly the method has been used even for a 700-g neonate with pulmonary stenosis.12 using an appropriate ratio of balloon to valve hinge point diameter is shown to optimize the chance of long-term success. 13 mean balloon diameter/annulus size ratio in the present study varied from 1.2 in older age group to 1.5 among infants. the disruption of the annulus of the pulmonary valve may lead to hemorrhage into the pericardial sac and subsequent tamponade, which is why the choice of the right diameter of balloon is so important. it is best to choose the balloon according to data obtained from echocardiography and angiocardiography.14 werynski et al studied 137 children with isolated pulmonary stenosis who underwent valvuloplasty. the balloon diameter to pulmonary valve annulus ratio was 1.3 in their study and complications were seen in 3.6% of the patients, including one case of a balloon being lodged in the iliac vein. mild pulmonary valve insufficiency was a common finding in the patients of the present study. it is associated with the diameter of balloons used during the intervention. according to literature, the occurrence of this problem ranges 10%50% of patients with pulmonary valve stenosis who underwent surgical treatment or balloon valvuloplasty. for many years the summary data suggest that in long-term observation, serious insufficiency is not well tolerated. in a polish study, none of patients needed reintervention and in the long-term observation there was no insufficiency of the pulmonary valve>ii. but all the patients undergoing balloon valvuloplasty in that study had isolated pulmonary stenosis. new mild pulmonary insufficiency was noted in 28% after pulmonary balloon valvuloplasty in a long-term assessment by fawzy et al.15 like the present study, most studies have used echocardiographic assessments. however, doppler echocardiography tends to overestimate the transvalvular gradient of systolic pressure in mild cases of pulmonary valve stenosis, in comparison to hemodynamic assessment. the study reveals that balloon valvuloplasty can be a useful and effective treatment for pulmonary stenosis in all childhood age groups. it was found that failure may not be uncommon and can lead to a subsequent surgery. also, recurrences of the stenosis should be expected and repeated valvuloplasty may be inevitable.

the aim of this study was to investigate the characteristics and outcomes of treating pulmonary stenosis with percutaneous valvuloplasty, and to compare them among three childhood age groups. all children under 15 years of age who had undergone pulmonary valve balloon valvuloplasty in madani heart center from 20052009 were enrolled in this study. data were analyzed using ibm spss software (spss, inc, chicago, il). mean (standard deviation) age of patients was 55.5 47.4 months. two-thirds of the subjects had moderate pulmonary valve stenosis. balloon valvuloplasty failed in nearly one-fifth of the treated patients. there were 17 failures and two cases of mortality, descriptively less frequent among children>5 years; however, the observed difference was not statistically significant. mild pulmonary valve insufficiency was a common finding.

PMC3373210

pubmed-800

the online version of this article (doi:10.1007/s10654-012-9749-8) contains supplementary material, which is available to authorized users. complications may occur during pregnancy (e.g. hypertension, thromboembolism, diabetes) and during labor (e.g. fetal distress, dystocia and instrumental delivery/cesarean section). it is well known that obesity reduces the likelihood of a successful result of assisted reproduction (art) [3, 4]. miscarriages, particularly early in the pregnancy, are frequent, but the relationship between bmi and the risk of miscarriages among women in the general population is not established as both underweight and obesity have been reported to increase the risk of miscarriages [612]. brewer and balen have recently reviewed how obesity affects adversely both conception and implantation. time to pregnancy is longer and fecundity lower in obese women than in women at optimal weight [1316]. some previous studies has indicated that obesity as young women is associated with ovulatory infertility and menstrual problems later in life also caffeine containing beverages may be a risk factor, although the evidence is weak. such life style factors may confound or modify the relationship between body mass and reproductive health. the aim of this study was to investigate, in a population of 46,000 american women aged 40 and above, how bmi (both low and high) at age 20 influences the frequency of reporting miscarriages, irregular periods or failing to become pregnant even if trying to get pregnant for one straight year or more. the large number of women included facilitates the investigation of effects of underweight (body mass index<18.5 kg/m) as well as obesity (both body mass index 3032.4 kg/m and body mass index 32.5 kg/m). the women were members of the adventist church, thus a large proportion had never (thus also during the childbearing years) smoked or used alcohol. furthermore, the consumption of caffeine containing beverages (coffee or soft drinks) was low with approximately two-thirds never consuming this or using it less frequently than once a month. adventist church members living in the usa and canada, aged 30 years and more, were included in the adventist health study-2 (ahs-2). more than 96,000 participants completed the lifestyle questionnaire which took 13 h to complete. the adventists church encourages a healthy life style with no smoking and alcohol consumption and advises members to follow a vegetarian diet. the comprehensive self-administered questionnaire included sections for medical history, diet, physical activity, supplement use and vegetarian food consumption. information on marital status, ethnic group and lifestyle variables like smoking and the use of alcohol and caffeine containing beverages were available. only 0.3% of the women reported living in a common law marriage, and these women were in the stratified analyses included in the group of ever married women. the female history section included information about, among other topics, menarche, irregular menstruation and difficulties in becoming pregnant (at different points during the life of the women) and the outcome of the pregnancies (including miscarriages/stillbirths, ectopic pregnancies, elective abortion and live births), and the use of oral contraceptives. there were also simple questions about current weight and height as well as weight when aged 20. body mass index (bmi) was computed as weight in kilograms divided by the square of height in meters (kg/m). the three dependent variables considered in our study were ever having experienced a miscarriage, menstrual irregularities and failing to become pregnant even if trying for one straight year. the women were asked to state the number of miscarriages or stillbirths she had experienced. in the main analyses, we dichotomized this information into ever/never having experienced this pregnancy outcome. menstrual irregularities was considered present if the women answered yes to the question have your periods ever had much reduced flow, become irregular or stopped completely for at least 6 months? do not count during or after menopause, or when you were pregnant, or nursing a child. if the women indicated that this happened before the age of 20 only, we did not include her in the group of women with menstrual irregularities. the women answered another question about problems with becoming pregnant: did you ever try for one straight year or more to become pregnant and, during that time, not become pregnant? if the women indicated that this happened before the age of 20 only or that the only reason for the problem was that the husband had fertility problem, we did not include her in the group of women with problems becoming pregnant. bmi was categorized into 6 groups: bmi<18.5, 18.5 bmi<20, 20 bmi<25, 25 bmi<30, 30 bmi<32.5 and bmi 32.5. these groups are in accordance with the main groups recommended by the who for classification of underweight, normal weight, overweight and obesity (< 18.5, 18.524.9, 2529.9, 30), but the present classification is more detailed. the present analyses were limited to 54,369 women who were between the age of 40 and 99 at enrolment. the following missing data led to exclusions: ever having been pregnant was missing for 183 women, information regarding marital status, a key determinant of childbearing, was missing for 1,102 women; an additional 4,727 women had missing information regarding bmi at age 20. we also excluded 808 women with estimated bmi lower than 16.0 kg/m or higher than 60.0 kg/m as these were considered to either reflect incorrect self-reported data concerning weight or height or severe illness. in some situations (24% of the women), the information from the women was missing with regard to the three dependent variables. thus, the number of women included in the analyses varied between 45,701 regarding information concerning irregular periods to 46,582 for information on having tried for one straight year or more to become pregnant but not having become pregnant. in addition to age when completing the questionnaire (5 year age groups) and marital status (7 groups), the following variables were considered as possible confounders of the relationship between bmi at age 20 and the three different indicators of fertility problems: ethnic group (blacks vs. other), level of education, age at menarche, extended use of oral contraceptives (here defined as having used oral contraceptives for 7 or more years both when aged 2029 and when aged 3039), parity, ever smoked and ever regularly used alcohol as well as monthly or more frequently use of caffeine containing beverages. the statistical analyses included simple cross-tabulations, analyses of variance and multiple logistic regression analyses. stratified analyses were conducted in order to control for confounding and evaluating possible effect modification. the p values in the tables test the hypothesis of any difference according to bmi (in 6 categories) rather than a linear trend over bmi categories. in some situations, we also tested for a u-formed relationship, including also a quadratic term in the model. the mean age (standard deviation) of the women at enrollment was 59.9 (14.7). overall, 30.6, 14.1 and 16.5% reported miscarriages, irregular periods, and problems becoming pregnant, respectively. table 1 displays the associations between bmi at age 20 and some relevant variables which may be associated with bmi. women who were obese at age 20 were more likely to be relatively younger when completing the lifestyle questionnaire, never to have been married, to have relatively low education, to be black, and to have early menarche and have relatively low parity. only 1 and 6 %, respectively, of the women were current users of tobacco or alcohol. monthly use of caffeinated beverages as well as ever use of tobacco or alcohol was associated with obesity. table 1unadjusted relationships between body mass at age 20 and demographic, reproductive and life style variables. mean values (sd) or percentagesbody mass index (kg/m) at age 20n<18.518.519.92024.92529.93032.432.5p valuenumber of women47,5496,815 (14.3)10,213 (21.5)25,866 (54.4)3,539 (7.4)506 (1.1)610 (1.3)age at enrolment47,54958.5 (11.9)59.2 (12.3)60.8 (12.9)59.4 (13.3)57.0 (12.8)54.8 (11.2)<0.0001% ever married47,54994.695.694.890.687.782.3<0.0001% with college degree47,12233.134.732.528.523.525.5<0.0001% blacks47,00230.424.223.430.433.835.9<0.0001age at menarche47,22712.9 (1.6)12.7 (1.6)12.5 (1.5)12.2 (1.6)12.1 (1.7)11.7 (1.6)<0.0001live births46,3342.3 (1.7)2.3 (1.6)2.4 (1.7)2.3 (1.8)2.1 (1.8)1.8 (1.9)<0.0001% extended oc use46,9722.12.01.81.72.62.00.33% who consume caffeinated drinks44,91232.735.735.638.544.242.0<0.0001% ever smoked47,25517.016.716.419.527.731.5<0.0001% ever used alcohol47,16136.037.336.040.649.151.5<0.0001used oral contraceptives (oc) for 7 or more years both when aged 2029 and when aged 3039 unadjusted relationships between body mass at age 20 and demographic, reproductive and life style variables. mean values (sd) or percentages used oral contraceptives (oc) for 7 or more years both when aged 2029 and when aged 3039 after adjustments for age and marital status, those reporting ever to have experienced a miscarriage had increased odds of failing to become pregnant even if trying for one straight year; odds ratio (or) 1.51 (95% ci: 1.431.59). increasing number of miscarriages (1, 2 and>2) was linearly related to the odds of reporting failing to become pregnant (or=1.35 (95% ci: 1.271.44), 1.63 (95% ci: 1.491.79) and 2.37 (95% ci: 2.122.64)), respectively compared to the risk in women with no miscarriages). also ever experienced irregular periods was positively related to failing to become pregnant even if trying for one straight year; or=1.72 (95% ci: 1.611.83), but there was very little relationship between the experience of a miscarriage and the likelihood of reporting irregular periods (results not shown in tables) underweight or obesity at age 20 did not have any bearing on the risk of this pregnancy outcome (p=0.16). when adjusted for age when completing the questionnaire and marital status, the odds for a hysterectomy before the age of 40 (15% of the women indicated this) was approximately 35% higher in obese women than in women with normal weight (p=0.003), but after additional adjustments, for education and ethnic group, this relationship was no longer statistically significant (p=0.06) (results not shown in the table). table 2 gives the relationships between bmi at age 20 and the likelihood of reporting at least one miscarriage, irregular periods or failing to become pregnant even if trying for one straight year. the first line represents results of analyses are adjusted for age when completing the questionnaire and marital status, the next two lines when adjusted for an increasing number of possible confounders. table 2relationships between body mass index at age 20 and the likelihood of having experienced a miscarriage, irregular periods or failing to become pregnant even if trying for one straight yearbody mass index (kg/m)n<18.518.519.92024.92529.93032.432.5p valuedependent variableever experienced miscarriage miscarriage46,3341.06 (1.00, 1.12)1.02 (0.97, 1.07)1.001.11 (1.03, 1.20)1.11 (0.92, 1.35)0.97 (0.80, 1.16)0.06 miscarriage44,9751.04 (0.98, 1.10)1.02 (0.97, 1.07)1.001.07 (0.99, 1.16)1.04 (0.85, 1.27)0.92 (0.76, 1.11)0.46 miscarriage42,9791.04 (0.98, 1.11)1.02 (0.97, 1.08)1.001.05 (0.97, 1.14)1.01 (0.82, 1.24)0.87 (0.72, 1.06)0.40menstrual irregularities menstrual irregularities45,7011.03 (0.95, 1.12)0.98 (0.91, 1.04)1.001.18 (1.07, 1.31)1.89 (1.53, 2.33)1.98 (1.64, 2.39)<0.0001 menstrual irregularities44,4291.03 (0.95, 1.12)0.97 (0.90, 1.04)1.001.16 (1.05, 1.28)1.84 (1.49, 2.28)1.91 (1.58, 2.31)<0.0001 menstrual irregularities42,9791.04 (0.96, 1.13)0.97 (0.90, 1.04)1.001.16 (1.04, 1.29)1.79 (1.44, 2.23)1.87 (1.54, 2.27)<0.0001problems with becoming pregnant problems with becoming pregnant46,5821.16 (1.08, 1.25)1.13 (1.06, 1.20)1.001.11 (1.01, 1.22)1.46 (1.17, 1.82)1.55 (1.26, 1.90)<0.0001 problems with becoming pregnant45,2461.14 (1.06, 1.23)1.12 (1.05, 1.20)1.001.11 (1.00, 1.22)1.49 (1.19, 1.87)1.53 (1.25, 1.89)<0.0001 problems with becoming pregnant42,9791.13 (1.04, 1.22)1.13 (1.06, 1.21)1.001.07 (0.97, 1.19)1.36 (1.08, 1.72)1.36 (1.10, 1.69)<0.0001odds ratio (95% ci). adjusted for age when filling in the questionnaire and marital statusalso adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other) and level of educationalso adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as problems with becoming pregnant, menstrual irregularities and age at menarchealso adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as experienced a miscarriage, problems with becoming pregnant and age at menarchealso adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as experienced a miscarriage, menstrual irregularities and age at menarche relationships between body mass index at age 20 and the likelihood of having experienced a miscarriage, irregular periods or failing to become pregnant even if trying for one straight year odds ratio (95% ci). adjusted for age when filling in the questionnaire and marital status also adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other) and level of education also adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as problems with becoming pregnant, menstrual irregularities and age at menarche also adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as experienced a miscarriage, problems with becoming pregnant and age at menarche also adjusted for ever smoking, ever use of alcohol, ethnic background (blacks vs. other), level of education as well as experienced a miscarriage, menstrual irregularities and age at menarche no relationship was found for the risk of any miscarriage. furthermore, we found no linear relationship between body mass index at age 20 and the number of miscarriages (not shown in the table). women with bmi 32.5 kg/m at age 20 had approximately 2.0 and 1.5 higher odds for irregular menstruation or failing to get pregnant, respectively, than women with bmi in the 2024.9 kg/m bracket. underweight (bmi<18.5 kg/m) when aged 20 marginally (approximately 15 %) increased the risk of failing to get pregnant within a year (p value for quadratic term<0.001). also for irregular menstruation, a u-formed relationship was statistically significant (p<0.001), but the increased risk associated with underweight was negligible. adjustments for ever smoking, ever use of alcohol, ethnic background (blacks vs. other) and education in addition to age and marital status had little impact on these relationships (table 2). further adjustments for age at menarche as well ever experienced one of the two other dependent variables included in our analyses did not explain the statistically significantly increased risk associated with obesity. however, the increased risk of failing to become pregnant associated with both underweight and obesity was attenuated with the last set of adjustments (table 2), but even fully adjusted (line 3), there was a statistically significant (p<0.001) u-formed relationship between body mass index at age 20 and problems of becoming pregnant. it may, however, be debatable whether it is correct to adjust for miscarriages and irregular periods when assessing the relationship between body mass index at age 20 and failing to become pregnant, as these variables may be considered intermediary. thus, the impact of these two variables on the relationship with failing to become pregnant was assessed in a separate analysis. when adjusted for irregular periods and miscarriages in addition to smoking, alcohol, ethnic background, education and age at menarche (including 42,979 women in both analyses), the odds ratio associated with obesity (bmi 30 some of the results regarding failing to become pregnant are presented in table 3. as evident, the displayed relationship did not depend on the age when completing the questionnaire (p value for interaction=0.9), or whether the women had ever been married (p value for interaction=0.6). the relationship may be somewhat weaker in blacks than in other ethnic groups (p value for interaction=0.03). table 3stratified analyses of the relationships between body mass index at age 20 and having experienced problems getting pregnantbody mass index (kg/m) at age 20n<18.518.519.92024.92529.93032.432.5p valueall women46,5821.16 (1.08, 1.25)1.13 (1.06, 1.20)1.001.11 (1.01, 1.22)1.46 (1.17, 1.82)1.55 (1.26, 1.90)<0.0001age (years) 405418,5141.21 (1.09, 1.35)1.08 (0.98, 1.19)1.001.23 (1.07, 1.42)1.56 (1.15, 2.12)1.55 (1.18, 2.04)<0.0001 556916,7951.11 (0.98, 1.25)1.10 (1.00, 1.23)1.000.95 (0.79, 1.13)1.42 (0.96, 2.10)1.60 (1.12, 2.28)0.014 70+ 11,2731.15 (0.97, 1.35)1.28 (1.13, 1.46)1.001.10 (0.90, 1.34)1.26 (0.73, 2.17)1.31 (0.72, 2.41)0.008married never2,6071.31 (0.81, 2.12)0.76 (0.45, 1.30)1.001.27 (0.77, 2.09)1.49 (0.58, 3.85)1.64 (0.81, 3.31)0.33 ever43,9751.16 (1.08, 1.25)1.14 (1.07, 1.21)1.001.10 (1.00, 1.21)1.45 (1.16, 1.83)1.53 (1.23, 1.89)<0.0001ethnic group blacks11,5831.12 (0.97, 1.28)0.97 (0.85, 1.10)1.001.22 (1.03, 1.46)1.43 (0.97, 2.11)1.29 (0.90, 1.85)0.03 other34,4891.17 (1.07, 1.27)1.19 (1.11, 1.27)1.001.07 (0.95, 1.20)1.47 (1.12, 1.94)1.69 (1.31, 2.17)<0.0001parity 07,3181.33 (1.13, 1.55)1.11 (0.96, 1.28)1.001.14 (0.93, 1.39)1.52 (0.98, 2.35)1.42 (0.99, 2.04)0.004 1+38,1181.10 (1.01, 1.19)1.13 (1.05, 1.21)1.001.08 (0.96, 1.21)1.31 (1.00, 1.72)1.40 (1.08, 1.81)0.0008smoking never38,3771.18 (1.09, 1.28)1.17 (1.10, 1.26)1.001.14 (1.02, 1.27)1.55 (1.19, 2.01)1.38 (1.07, 1.80)<0.0001 ever7,9441.05 (0.89, 1.25)0.93 (0.80, 1.09)1.000.97 (0.78, 1.21)1.26 (0.83, 1.92)1.77 (1.26, 2.48)0.014alcohol never29,0491.17 (1.07, 1.28)1.17 (1.09, 1.27)1.001.14 (1.01, 1.29)1.44 (1.05, 1.98)1.49 (1.10, 2.01)<0.0001 ever17,1941.14 (1.01, 1.28)1.05 (0.94, 1.16)1.001.05 (0.91, 1.23)1.49 (1.10, 2.03)1.60 (1.21, 2.11)0.002never used tobacco or alcohol27,8911.18 (1.08, 1.30)1.19 (1.10, 1.29)1.001.15 (1.02, 1.31)1.44 (1.04, 2.02)1.35 (0.96, 1.88)<0.0001coffee/caffeine containing soft drinks <monthly28,3751.15 (1.05, 1.26)1.16 (1.07, 1.25)1.001.18 (1.04, 1.34)1.59 (1.18, 2.15)1.55 (1.17, 2.06)<0.0001 monthly or more often15,7871.18 (1.05, 1.34)1.09 (0.98, 1.21)1.000.96 (0.81, 1.13)1.49 (1.06, 2.09)1.66 (1.22, 2.26)0.0005odds ratio (95% ci). adjusted for age when filling in the questionnaire and marital status stratified analyses of the relationships between body mass index at age 20 and having experienced problems getting pregnant odds ratio (95% ci). adjusted for age when filling in the questionnaire and marital status of particulate interest in this population are associations in never smokers and women who have never used alcohol. table 3 demonstrates that the relationships displayed in table 2 were found in women who had abstained from smoking for their entire life and in women who had not, although the relationship may be somewhat stronger in the latter group (p value for interaction=0.04). the association was the same in women who were lifelong abstainers from alcohol and other women (p value for interaction=0.5) and was also found in the 27,891 women who denied ever having used either stimulant (alcohol and tobacco). there was in addition a seemingly interaction for age at menarche, but the relationships for women with early and late menarche were the same (p-value for interaction=0.9) if the two categories of obesity were merged. the same stratified analyses were conducted also for irregular periods and miscarriages, and the relationships displayed in table 2 were found consistently in the different strata of the population. for irregular periods, there were no indications of any significant interactions. for miscarriages, we found a weak u-formed (p=0.003) relationship in parous women which was unaffected by further adjustments for parity. we refer to web appendix (web tables 13) for more detailed presentation of stratified analyses. obesity at age 20 years was in this large study of women associated with increased risk of irregular periods and failing to become pregnant even if trying for one straight year, but not with the risk of experiencing at least one miscarriage. as we are exploring relationships between body mass index at age 20 and reproductive problems, we have restricted the analytical sample to women at an age (aged 40 and above) when they most likely will have experienced reproductive problems if they will ever do so, particularly if these problems should have clinical consequences. as detailed above, we did not include irregular periods or failing be become pregnant before the age of 20 as an outcome in the study, only problems after the age of 20. we do not, however, know when the women experienced her (first) miscarriage; this may have happened as a teenager. due to the mean age of the included women (nearly 60 years), modern treatment for infertility (like in vitro fertilization) has played a minor role for our findings. the relationship between bmi at age 20 and irregular periods and problems of becoming pregnant may be explained by an increased risk of oligo- and anovulation in obese women and a number of other adverse effects of obesity on reproductive physiology in women [13, 25]. our results support previous findings of a u-formed relationship between body mass index at age 23 and problems of becoming pregnant and menstrual problems before age 33. data from a self-administered questionnaire hamper the possibilities for further discussion with regard to etiology. one explanation may be that a relatively high percentage of the obese women may have had polycystic ovary syndrome (pcos). polycystic ovary syndrome affects 510% of women in a general population, and many (at least one out of three) of the patients with pcos are obese [26, 27]. no relationship was found between obesity and the odds of reporting one or more miscarriages. previous population-based studies have not given a consistent picture with regard to body mass index as a risk factor for miscarriages; underweight may be just as important as obesity [612]. traditionally, psos has been thought to play a major role also for the risk of recurrent miscarriages, but this has recently been questioned. the lack of relationship between body mass at age 20 and miscarriages may to some extent be due to misclassification as early miscarriages often are overlooked and any relationship will tend to be attenuated. however, the positive, direct relationship between the number of miscarriages and the odds of reporting failing to become pregnant indicates that information about the miscarriages has some validity and may further suggest that some women have interpreted the question did you ever try for one straight year or more to become pregnant and, during that time, not become pregnant? less precisely than was the intention, answering that they for one straight year or more were not able to get pregnant or give birth to a live born child. given that there is no, or only a very weak, relationship between obesity and the risk of a miscarriage and that some women have interpreted the question as suggested above, this points to a possible stronger relationship between obesity (and possible underweight) and failure to become pregnant than the results presented in table 2 may indicate. whereas irregular periods and failing to become pregnant reflect problems of becoming pregnant, the women who have a miscarriage have conceived and are therefore fertile. thus, according to our findings, weight may be more important for becoming pregnant than for remaining pregnant. when the relationship with failing to become pregnant was adjusted for irregular periods and miscarriages, the odds ratio associated with obesity was attenuated. however, it is debatable whether it is correct to adjust for these variables which most likely are on the causal pathway. if not, obesity (bmi 30 kg/m) increases the odds of having problems of becoming pregnant with approximately 45 %, even after adjustments for other likely confounders. table 1 shows that women who were obese at age 20 were less likely to ever have been married. we adjusted for marital status in all analyses (table 2) and the stratified analyses by ever married status (table 3) clearly demonstrate that the relationship we found is not due to women who never were married and therefore may not have tried to become pregnant. one might assume that experiencing irregular periods, the variable most strongly related to obesity, was independent of marital status, but the risk was found to be higher in never married women. thus, the higher odds for reporting problems of becoming pregnant in obese women may be due to obesity in the male partner. it is however unlikely that the positive (and stronger) association (table 2) between obesity and irregular periods is related to male obesity. the information from the women did not make it possible to differentiate between a miscarriage (a spontaneous loss of a fetus before the 20th week of pregnancy) and a stillbirth (a delivery after 20 completed weeks gestation of a fetus showing no signs of life). the former is much more frequent, and our results will pertain largely to miscarriages.. found that 31% of pregnancies were lost, two out of three before the pregnancy was detected clinically. currently<1% of all pregnancies in the us end as a stillbirth, but the percentage is higher in blacks than in other ethnic groups. the risk of a stillbirth was higher during the childbearing years of the women included in our analysis, though. obesity has in most studies been found to increase the risk of stillbirths [32, 33]. we have used this height when computing the body mass earlier in life, at age 20. however, as the associations we found were basically independent of age at enrollment (aged 4054, 5569, or 70, and thus time since the women were 20 years old), little bias is introduced when applying current height when computing bmi earlier in life. the main weakness of our study is that weight is self-reported and recalled. underweight women tend to overestimate the self-reported weight whereas obese women underestimate it. thus, in women with bmi<18.5 kg/m, the reported bmi is probably higher than the true bmi and the opposite is true for obese women. however, the most important in our context is the ability to rank the women according to bmi and measured and self-reported bmi has been found to be highly correlated (rs=0.94) in this population as in the previous adventist health study (ahs-1). the mean age at enrolment was 59.9 years, and the women were asked to recall their weight nearly 40 years earlier, at age 20. data from the nurses health study indicate that women are able to recall their weight at age 18, the correlation coefficient between recalled and measured weight was 0.87. the women in the nhs cohort (aged 2542) were, however, significantly younger than in our study. data from women who took part in both this adventist health study (ahs-2) and the former one (ahs-1 in 1976) demonstrate strong correlations (r=0.82 for women of all ages) between recalled weight in the 1970s and weight stated in the questionnaires in 1976. thus, misclassification of recalled bmi at least in terms of relative rank appears to be quite small for recall of 2530 years. we do not find it likely that our results can be explained by differential recall of weight at age 20 as this would imply a strong correlation between reporting problems of becoming pregnant or, in particular, irregular periods and falsely recalled overweight and obesity when aged 20 years old. it is probable that a more relevant measure is the percentage of body fat, a measure that was strongly correlated (r=0.84) with bmi in us women aged 2039. information about adipose tissue distribution, like waist circumference or waist/hip-ratio, may have given additional information, although the correlation between bmi and waist circumference in relatively young women is high (r=0.93) according to recent nhanes data. one possible source of bias would be that women who complete the lifestyle questionnaire are survivors. obese, relatively young, women have higher mortality than women with normal weight [40, 41]. however, the mortality in women aged less than 40 is low, particularly in this relatively healthy group of subjects with low smoking prevalence, and the relationships did not depend on the age of the women when completing the questionnaire (table 3 and web tables 13). thus, it is unlikely that survival bias has impacted on our results to any measurable degree. the prevalence of obesity at age 20 (2.4 %) is relatively low, but it is for instance similar to the prevalence of obesity in women included in the swan cohort which was based on women aged 1718 years old in the late 1960s, furthermore, the study population is somewhat selected as all the women were adventists. it could be that obesity is associated with irregular periods and miscarriages differently in this group of women than in the general population. however, we find this unlikely and the stratified analyses did not suggest any interaction with lifestyle. it is large in terms of women included, which has allowed detailed stratified analyses. the main findings were found to be very consistent in the different strata of the population. another related strength is that this study has been conducted in a rather unique us population with a relatively high proportion of women who have abstained from alcohol and smoking for their entire life. additionally, 25% of the analytical population are black adventists of us and caribbean origin; approximately 90% of the remaining 75% are white, non-hispanic women. both underweight and obesity after adjustment for age and marital status and compared to other women, black women were only slightly more likely to report irregular periods (2 %) or failing to become pregnant even if trying for least 1 year (6%). however, blacks were more likely to report at least one miscarriage [or=1.34 (95% ci: 1.271.40)]. however, as detailed in table 3 and the web appendix, stratified analyses demonstrate that there are few indications that ethnicity has influenced our findings significantly. in summary, this large study found that women who were obese when they were 20 years old were at a significantly increased risk of failing to become pregnant even if trying for one straight year. one of the explanations for this seems to be that obese women have difficulties to conceive due to irregular periods, rather than increased risk of miscarriages. supplementary material 1 (docx 36 kb) supplementary material 1 (docx 36 kb )

in a group of 46,000 north-american adventist women aged 40 and above, we investigated the relationships between body mass index (bmi, kg/m2) at age 20 and the proportion of women who reported at least one miscarriage, periods with irregular menstruation or failing to become pregnant even if trying for more than one straight year. approximately 31, 14 and 17 %, respectively, reported the three different problems related to reproduction. positive age- and marital status adjusted relationships were found between bmi at age 20 and periods with irregular menstruation or failing to become pregnant even if trying for more than 1 year, but not with the risk of miscarriages. women with bmi 32.5 kg/m2 when aged 20 had approximately 2.0 (95% ci: 1.6, 2.4) and 1.5 (95% ci: 1.3, 1.9) higher odds for irregular periods or failing to get pregnant, respectively, than women with bmi in the 2024.9 kg/m2 bracket. these relationships were consistently found in a number of strata of the population, including the large proportion of the women who never had smoked or never used alcohol. underweight (bmi<18.5 kg/m2) when aged 20 marginally (approximately 15 %) increased the risk of failing to get pregnant within a year. thus, obesity at age 20 increases the risk of reporting some specific reproductive problems, but not the risk of miscarriages.electronic supplementary materialthe online version of this article (doi:10.1007/s10654-012-9749-8) contains supplementary material, which is available to authorized users.

PMC3539069