Rats were randomly classified into AMI group, sham operation group and recombinant human tumor necrosis factor receptor:Fc fusion protein (rhTNFR:Fc) group. Spontaneous and induced VFs were recorded. Monophasic action potentials (MAPs) among different zones of myocardium were recorded at eight time points before and after ligation and MAP duration dispersions (MAPDds) were calculated. Then expression of TNF-α among different myocardial zones was detected. After ligation of the left anterior descending coronary artery, total TNF-α expression in AMI group began to markedly increase at 10 min, reached a climax at 20–30min, and then gradually decreased. The time-windows of VFs and MAPDds in the border zone performed in a similar way. At the same time-point, the expression of TNF-α in the ischemia zone was greater than that in the border zone, and little in the non-ischemia zone. Although the time windows of TNF-α expression, the MAPDds in the border zone and the occurrence of VFs in the rhTNFR:Fc group were similar to those in the AMI group, they all decreased in the rhTNFR:Fc group. Our findings demonstrate that TNF-α could enlarge the MAPDds in the border zone, and promote the onset of VFs. The purpose of this study was to explore the effects of tumor necrosis factor- It has been demonstrated that ischemic myocardium could greatly express TNF-α [α release early in the course of AMI contributes to myocardial injury [α) improves cardiac function and reduces infarct size. The effect of TNF-α on the arrhythmias remains unclear. Shimoda found that TNF-α lever was higher in AMI patients with malignant ventricular arrhythmias than control [α rose in AMI patients and was related to the occurrence of ventricular arrhythmias [α in AMI rats was closely related to the occurrence of ventricular arrhythmias [α plays an important role in the occurrence of VFs remains unknown. Tumor necrosis factor-so forth –3. Recenso forth , 5. Acutso forth . During so forth –9. A varss TNF-α , 11 duril injury . Sugano l injury , 14 foun control . Our prehythmias . Then, ohythmias , 18. Venhythmias . Whetherα in different regions of ischemic myocardium by immunohistochemistry and real-time fluorescence quantitative PCR, recorded the monophasic action potentials (MAPs) and occurrence of VFs, and then explored the effect of TNF-α expression on the occurrence of VFs and the potential mechanisms.When AMI occurs, in accordance with different degrees of ischemia, myocardium could be divided into the ischemic zone, the nonischemic zone, and the border zone . This stAll experimental procedures were approved by the Institutional Authority for Laboratory Animal Care and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health . Acute myocardial infarction (AMI) was induced in male Wistar rats weighing 250~300 g. All rats were anesthetized with pentobarbital sodium (30~35 mL/kg) by intraperitoneal injection. Under controlled ventilation, a thoracotomy through a left parasternal 3, 4 intercostal spaces was performed, the pericardium was incised, and the anterior wall of the left ventricle was exposed. Left anterior descending coronary artery (LAD) proximal end was ligated with 6-0 line at the junction of the pulmonary conus and the left atrial appendage, which could induce extensive infarction of left ventricular anterior wall. When the ventricular anterior wall turned to be pale or cyanosed and ECG showed ST-segment elevated, myocardial infarction model succeeded. n = 80), sham-operation group (n = 80), and recombinant human tumor necrosis factor receptor:Fc fusion protein (rhTNFR:Fc) group (n = 80). Anterior wall myocardial infarction was produced in AMI group by ligating the left anterior descending coronary artery (LAD); there was no ligation but operation in sham-operation group. rhTNFR:Fc group was treated with rhTNFR:Fc (10 mg/kg), a TNF-α antagonist, 24 hours before LAD ligation. ECG and spontaneous VFs were observed during the whole experiment. Monophasic action potentials (MAPs) among the ischemic zone, the border zone, and the non-ischemia zone were observed at baseline, 10 min, 20 min, 30 min, 60 min, 3 h, 6 h, and 12 h after ligation, and monophasic action potential duration disperses (MAPDds) were calculated, while VFs were induced by S1S2 programmed electrical stimulation and recorded. Subsequently, expression of TNF-α among different zones was detected by histochemistry and real-time fluorescent quantitative PCR.Two hundred and forty Wistar rats were randomized into AMI group . The signals were inputted into the computer by the A/D converter. Pacing with 6 Hz at high right atrium, MAPs were recorded at baseline, 10 min, 20 min, 30 min, 60 min, 3 h, 6 h, and 12 h after ligation. After measuring MAPD90 of five points of each zone, Spontaneous and induced VFs were recorded with BL-420F biological signal acquisition and processing system during the whole experiment. VFs were induced by the S1S2 programmed electrical stimulation with the Y-2 electric stimulator  at different time points before and after LAD ligation .μm thick slices. After peroxidase was inactivated by hydrogen peroxide at room temperature for 10 min, the slices were repaired by microwave for 10 min, incubated with BSA at 37°C for 40 min, with a 1 : 200 dilution of goat antirat TNF-α/TNFSF1A antibody  TNF-α antibody at 4°C overnight, with a 1 : 100 dilution of biotinylated rabbit antigoat secondary antibody  at 37°C for 40 min, with avidin-biotin-horseradish peroxidase complexes at 37°C for 60 min, then colored with diaminobenzidine, stained with haematoxylin, and, finally, observed. Selecting five sights at each slice, TNF-α expression in every secition was measured and analyzed by HMIAS Series Color Medical Image Analyze System .Fresh myocardial tissues of rats were fixed with formalin, dehydrated, transparent, embedded in paraffin, and then cut into 5–8 α-kit (TOYOBO Japan), amplifying with SYBR Green mix (TOYOBO Japan), and detecting fluorescence by SLAN Real-time Quantitative PCR Detection System . The following oligonucleotides were used as rat sense and antisense primers; upper primer: 5′-CAGCCGATTTGCCATTTCAT-3′, lower primer: 5′-ACGCCAGTCGCTTCACAGAG-3′ . Thermal cycling conditions were 95°C, 15 s, 56°C, 15 s, 72°C, 45 s, 40 cycles. With housekeeping gene β-actin (249 bp) as internal control, the relative quantity of TNF-α mRNA was analyzed.Myocardial cell total RNA was extracted with TRIzol reagent . Then, reverse transcription with ReverTra Ace-α and VFs. Statistical significance was assumed at P < .05.All values were expressed as mean ± SD. Results were analyzed by analysis of variance followed by a two-sided Dunnett's test or Student-Newman-Keuls test when appropriate. Linear correlation analysis was used for the relationship between TNF-α in acute ischemic myocardium began to increase at 10 min after infarction, reached a climax at 20–30 min, and recovered gradually then. At the same time point, TNF-α in the ischemic zone was higher than the others, and the second was in the border zone. Compared to the AMI group, TNF-α detected by immunohistochemistry in rhTNFR:Fc group was significantly less than the AMI group (P < .05), and the expression of TNF-α in the sham group was extremely low  was not the same too. In the ischemic zone, the ischemic extent of different myocardial cells was similar, their response to the external stimulations was identical, and, thus, there were no significant MAPDds.Expression of TNF-α expression in the occurrence of VFs. Our findings suggested that TNF-α promotes acute ischemic VFs through enlarging MAPDds of the border zone. The underlying mechanisms need to be further studied. Besides, whether there are some other pathways in the process that TNF-α causes VFs remains unknown.The study is only a preliminary research into the possible role of TNF-α increased greatly after acute myocardial infarction. TNF-α could enlarge the MADds in the border zone and promote the onset of VFs while rhTNFR:Fc could diminish the MADds in the border zone and lessen the onset of VFs in AMI rats. Our results demonstrated that TNF-α expressed by ischemic myocardium may play an important role in the occurrence of VFs in AMI rats.The expression of TNF-


Neurons express two families of transporter-like proteins − Synaptic Vesicle protein 2  and SV2-related proteins (SVOP and SVOPL). Both families share structural similarity with the Major Facilitator (MF) family of transporters. SV2 is present in all neurons and endocrine cells, consistent with it playing a key role in regulated exocytosis. Like SV2, SVOP is expressed in all brain regions, with highest levels in cerebellum, hindbrain and pineal gland. Furthermore, SVOP is expressed earlier in development than SV2 and is one of the neuronal proteins whose expression declines most during aging. Although SV2 is essential for survival, it is not required for development. Because significant levels of neurotransmission remain in the absence of SV2 it has been proposed that SVOP performs a function similar to that of SV2 that mitigates the phenotype of SV2 knockout mice. To test this, we generated SVOP knockout mice and SVOP/SV2A/SV2B triple knockout mice. Mice lacking SVOP are viable, fertile and phenotypically normal. Measures of neurotransmission and behaviors dependent on the cerebellum and pineal gland revealed no measurable phenotype. SVOP/SV2A/SV2B triple knockout mice did not display a phenotype more severe than mice harboring the SV2A/SV2B gene deletions. These findings support the interpretation that SVOP performs a unique, though subtle, function that is not necessary for survival under normal conditions. Synaptic vesicles are a specialized class of recycling endosome that mediate the release of neurotransmitters. They contain the proteins required to fill with neurotransmitter, target to active zones at the presynaptic membrane and fuse in response to elevations in cytoplasmic calcium. Among the proteins specific to synaptic vesicles are two transporter-like proteins termed synaptic vesicle protein 2 (SV2) -3 and SVThere are three isoforms of SV2 in mammals  and two SVOPs (SVOP and SVOPL). All contain structural features of the Major Facilitator (MF) transporter superfamily . PhylogeTM) [SV2 is essential for survival ,11. MiceTM) . BecauseReverse genetic studies indicate that the SV2s are positive modulators of evoked vesicle fusion. Loss of SV2A or SV2A and SV2B reduces synaptic release probability ,11,13,14in silico screen for SV2 homologs and it shares 20–22% sequence identity with SV2 [SVOP was identified in an with SV2 . SVOP iswith SV2 ,17. Thiswith SV2 . Likewiswith SV2 .In an effort to elucidate the function of SVOP, we generated mice lacking SVOP and examined their phenotypes. We also created SVOP/SV2A/SV2B triple knockout mice to determine if the absence of all three proteins would reveal a function essential to development or neurotransmission.Animal studies were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Washington.Flox/Flox) and heterozygous (SVOPWT/Flox) as well as wild-type (SVOPWT/WT) mice.A BAC clone containing the entire SVOP gene from a C57BL/6 mouse genomic library was obtained from BACPAC Resources Center. An 11.4 kb HindIII –KpnI fragment containing exons 2–5 was isolated. This fragment was subcloned into pBluescript II KS(-) to construct a conditional gene targeting vector for SVOP as depicted in Flox/Flox) were crossed to Mox2Cre/+ transgenic females, which express Cre recombinase under the control of the ubiquitous Mox2 promoter, including in germline cells [Flox/Flox mice with Mox2Cre/+ transgenic mice resulted in deletion of exons 2 and 3 in the SVOP gene. The resulting mRNA encodes a protein that is truncated at a.a. 18. Offspring carrying the Cre gene were heterozygous for the SVOP gene disruption (SVOP +/-Mox2 Cre/+). These mice were bred with wild type mice to remove Mox2-Cre gene. SVOP +/- mice are bred to produce homozygous (SVOP-/-), heterozygous (SVOP+/-) as well as wild-type (SVOP+/+) mouse colonies.Male mice homozygous for the targeted SVOP gene . Samples were prepared for standard SDS-PAGE and subjected to Western blot analyses. The efficiency of SVOP knockdown by Cre was measured using a polyclonal antibody against SVOP. Expression levels of SV2 and synaptotagmin were detected with a monoclonal anti-SV2  respecti°C in a temperature-controlled chamber. The extracellular solution consisted of 119 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.5 mM MgCl2, 20 mM HEPES, 30 mM glucose, and 0.01 mM glycine, pH 7.3 (310 mOsm). The pipette solution contained the following: 131 mM K-gluconate, 17.5 mM KCl, 9 mM NaCl, 1 mM MgCl2, 10 mM HEPES, and 0.2 mM EGTA, pH 7.2 (328 mOsm). Cells were held at −60 mV and stimulated with a 1 ms depolarization to +20 mV to evoke neurotransmitter release. Excitatory post synaptic currents (EPSCs) were acquired with Pulse software (HEKA Elektronik) and reported as the mean ± SEM.Isolated neurons were subjected to whole-cell voltage-clamp recording. Data were acquired using an EPic-9 amplifier . Recording electrodes were typically 2.5–3.5 MΩ, and series resistance was compensated up to 70% with 10 µs feedback compensation. Cells with series resistance >20 MΩ (before series resistance compensation) or leak currents >250 pA were excluded from analyses. All experiments were performed at 21To measure spontaneous miniature excitatory postsynaptic current (mEPSC), neurons were held at −60 mV and spontaneous events were monitored during a 5 min recording period. The amplitude and frequency of mEPSCs were analyzed with Mini Analysis software .+/-crosses were quickly removed and homogenized in buffered sucrose solution supplemented with a protease inhibitor cocktail . After centrifugation at 1300×g for 10min, postnuclear supernatants (PNS) were collected and used for Western blot analysis. Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Blots were probed for the indicated proteins. Antibody binding was detected by chemiluminescence and imaged using a Kodak Image Station 440. Actin signals in each lane were used to normalize signals to total protein loading. Antibodies used include: a monoclonal antibody against synaptophysin (Millipore), a monoclonal antibody against VGlut1 (Millipore), a monoclonal antibody against Vesicle Associated Membrane Protein 2 (Vamp2) (Synaptic Systems), a polyclonal antibody against the vesicular proton ATPase (116 kDa subunit) (Synaptic Systems), a monoclonal antibody against α –adaptin (Sigma), a polyclonal antibody against clathrin heavy chain (Abcam), a monoclonal antibody against actin (Sigma), a monoclonal antibody against SV2 [Brains from littermate pups of SVOPinst SV2 , and a p-/-) and 12 wild type (SVOP+/+) littermates at 8-10 weeks of age were used in this analysis of motor coordination. Within each genotype, 6 males and 6 females were included. The mice were housed in a temperature and humidity-controlled facility on a 12h: 12h light: dark cycle and tested near the end of the light phase. All mice were transported to the testing room 15 min prior to the beginning of experiment to allow them to habituate to the environment.Twelve SVOP knockout mice  was used for testing. Mice were placed on a rod that accelerated smoothly from 4 rpm to 40 rpm over a 5 min period. The latency to fall or the latency to cling to the rod and passively rotate was recorded. Animals were given three trials per day for 4 consecutive days. Animals were allowed to rest at least 10 min between trials to avoid fatigue.-/- mice and six SVOP+/+ littermates at 9 weeks of age were included in circadian behavioral studies. Within each genotype, 3 males and 3 females were included. Mice were individually housed in cages, and food and water were available ad lib. Cages were equipped with a running wheel and kept in a ventilated, light-tight chamber. Wheel-running activity was monitored continuously using Clocklab software (Actimetrics) and data were collected in 10-min bins. Mice were housed under a 12:12 LD (12 h light : 12 h dark) cycle (lights on 7: 00 AM PST) with 200-lux intensity for 12 d before release into constant darkness (DD) for 14 d. Free-running periods during DD cycles were determined using periodogram analysis with the El Temps software .Six SVOPloxP sites in the introns preceding exon 2 and after exon 3 in the SVOP gene. Excision of this portion of the gene by Cre recombinase produces a frame shift and, in turn, a truncated protein. Breeding mice heterozygous for a floxed SVOP gene produced homozygous (SVOPFlox/Flox) and heterozygous (SVOPWT/Flox) as well as wild-type (SVOPWT/WT) mice at near Mendelian frequencies (71:146:64), indicating that insertion of loxP sites did not affect viability or development. Western blotting of brain samples from the three genotypes of mice detected similar levels of SVOP, indicating insertion of loxP sites did not affect SVOP gene expression (not shown).Because SVOP is expressed early in development ,17, disrFlox/Flox mice in which SVOP was knocked out by expression of Cre recombinase. Cultures from wild type and SVOPFlox/Flox mice were infected with lenti virions expressing either Cre-eGFP fused to a nuclear localization sequence (NLS) or eGFP alone. The lentiviral expression system achieved more than 95% infection efficiency, as judged by the presence of green fluorescence in neurons. As shown in Flox/Flox mice but not SVOPWT/WT mice. Expression of eGFP did not alter SVOP expression in either type of neuron. Expression of the synaptic vesicle proteins SV2 and synaptotagmin did not differ between cultures, suggesting that the effect was specific to expression of SVOP.To assess the role of SVOP in synaptic transmission, we compared spontaneous and evoked release in isolated autaptic neurons from wild-type mice and from SVOPWhole-cell voltage-clamp recordings were performed to assess neurotransmission in the presence and absence of SVOP. Neurons were selected for recording based on the intensity of eGFP fluorescence with preference given to brighter cells. To assess quantal size and spontaneous neurotransmission, we measured the amplitude and frequency of miniature excitatory post-synaptic currents (mEPSC).p values <0.001 suggesting a significant difference between neurons lacking SVOP and controls. However, the D values  were small (ranging from 0.0483 to 0.0921) and visual inspection did not reveal a consistent difference in the distributions  between the four groups revealed no significant difference in average mEPSC amplitude  . As with mEPSC amplitude, however the D values were low (ranging from 0.0295 to 0.098), and visual inspection of the cumulative frequency plots revealed no consistent shift . Western blot analysis revealed loss of SVOP protein expression  (p=0.90). These results indicate that loss of SVOP does not impair cerebellar functioning or motor learning, consistent with SVOP being non-essential to neurotransmission in the cerebellum.The cerebellum plays an important role in motor control, and cerebellar deficits result in decreased motor coordination . We asse-/-mutant mice was indistinguishable from that of wild types (23.90 ± 0.20 h)  . The triple knockout mice demonstrated a phenotype no more severe than that of SV2A/B knockouts. Like SV2A/B knockout mice, they appeared normal at birth, but failed to grow, developed spontaneous seizures at approximately one week of age, and died within 3 weeks of birth. The lack of a more severe phenotype than observed with loss of SV2 alone suggests that SVOP does not perform a function redundant to that of SV2.SVOP has been proposed to perform a function redundant to that of SV2. If true, loss of both gene families should result in a more severe phenotype than the loss of either one alone. To test this, we generated SV2A/SV2B/SVOP triple knockout mice. SV2B knockout and SVOP knockout mice are phenotypically normal. SV2B/SVOP double knockout mice also had no gross abnormalities. Crossing SV2A We report here that mice lacking SVOP are phenotypically normal. They demonstrate no differences in development, viability, fertility, seizure activity, major synaptic protein levels, synaptic transmission, motor coordination or circadian rhythms when compared to wild-type mice. Furthermore, mice lacking SVOP and the two major isoforms of SV2 (A and B) appear phenotypically identical to mice lacking SV2A and SV2B. Thus it appears that SVOP does not perform an essential function, or one that is redundant with SV2. Instead SVOP performs a function not apparent under the conditions assayed in our studies, suggesting that its role in nervous system functioning is subtle or specialized.SVOP joins the ranks of apparently non-essential synaptic vesicle proteins like synaptophysin, another protein unique to synaptic vesicles . We noteWhile our results suggest that SVOP does not perform a function redundant to that of SV2, it remains possible that loss of SVOP function is compensated by another protein. The most likely candidate is SVOP-like protein (SVOPL) . SVOPL sIn studies of rodent and human mRNA expression during aging, SVOP mRNA decreased significantly with aging ,19. To dIn conclusion, our findings support the interpretation that SVOP performs a function that can be compensated for under normal conditions. Future studies of synaptic vesicle protein networks will likely elucidate how each protein contributes to synaptic functioning.


Tumor necrosis factor-α (TNF-α), an important proinflammatory cytokine, exerts a variety of physiologic and pathogenic effects that lead to tissue destruction. Recent laboratory evidence indicates that TNF-α have either protective or adverse effects on primary open angle glaucoma (POAG). Inheritance of the TNF-α (−863) C allele has been associated with an elevated risk of Alzheimer disease. The neuronal injuries associated with Alzheimer disease have several similarities with the optic nerve changes often seen with POAG. In this study we investigated the possible association between the TNF-α (−863) polymorphism and the development of POAG.A total of 234 patients with POAG were recruited and compared with 230 healthy controls in a Chinese population. Sequence-specific primers with 3′ end mismatches were used to identify the presence of specific allelic variants by polymerase chain reaction (PCR) amplification. Patients and controls were genotyped for the A/C polymorphism at position −863 of the TNF-α gene promoter region.The frequency of the TNF-α (−863)A allele  and the carriers of the TNF-α (−863)A allele  were lower in POAG patients compared with those in controls. There is a reduced risk of POAG associated with homozygosity for the TNF-α (−863)A allele (AA genotype) compared with that in the control population .The TNF-α (−863)A allele polymorphism may be a protective factor in the development of POAG. Glaucoma is a progressive optic neuropathy characterized by degeneration of retinal ganglion cells, cupping of the optic nerve heads and visual field defects often related to elevated intraocular pressure. Glaucoma affects 70 million people worldwide, and constitutes the second largest cause of bilateral blindness in the world . PrimaryMYOC), optineurin (OPTN) and WD repeat domain 36 (WDR36) [CYP1B1) and latent-transforming growth factor beta-binding protein 2 (LTBP2) mutations are more frequent in congenital and developmental glaucoma [OPA1), apolipoprotein E, E-cadherin, neurotrophin-4 (NTF-4) and opticin (OPTC), have been reported to elevate the risk of retinal degeneration, a characteristic of glaucoma [Glaucoma is a complex clinical trait and its inheritance has been shown to follow both Mendelian and non-Mendelian models . At leas (WDR36) . Cytochrglaucoma -9. Otherglaucoma -14. HoweThere is evidence to support the hypothesis that the immune system plays a potential pathogenic role in glaucomatous optic nerve degeneration ,16. BalaTNF-α promoter region has been reported to be associated with Alzheimer disease [-α protein may act to promote the development of β-amyloid deposits [A C/A polymorphism at position −863 of the  disease ,26. Therdeposits . Similardeposits ,29. Tamudeposits  have shoRecent studies have shown that ischemic or pressure-loaded glial cells produce TNF-α, which results in oligodendrocytes death and the subsequent apoptosis of retinal ganglion cells . Tezel eTNF-α promoter polymorphisms and glaucoma risk [TNF-α (−863) C allele has been associated with an elevated risk of Alzheimer disease. A C/A polymorphism at position −863 of TNF-α promoter region is considered to be an important enhancer of transcriptional activation associated with elevated levels of TNF-α. Given the potential similarities in cellular events leading to neurodegeneration between Alzheimer disease and glaucoma, we hypothesized that TNF-α (−863) polymorphism may be a genetic factor predisposing affected individuals to glaucoma due to its effect on TNF-α protein expression. Thus, we investigated the distribution of the TNF-α (−863) polymorphism in patients with POAG and compared them with that in a healthy control population.To date, several publications have evaluated the association between oma risk -39. HoweSubjects were recruited from the outpatient clinic at the Department of Ophthalmology of Taichung Veterans General Hospital, Taiwan from January 2009 to July 2011. POAG patients were invited to participate in the study when they came to the clinic for a previously scheduled visit, and were enrolled after informed consent was obtained. Normal control subjects were recruited during their visits to the outpatient clinic for various other reasons. In total, 234 patients with POAG and 230 normal controls were enrolled . The meaAll participants received comprehensive ophthalmologic examinations, including visual acuity testing with refraction, corneal thickness, IOP measurement, Humphrey 30–2, slit lamp examination, Stratus optical coherence tomography , and dilated slit lamp stereo biomicroscopy.Comprehensive ophthalmologic history and longitudinal follow-up data were also obtained for each individual. The definition of POAG included characteristic arcuate, Bjerrum, Seidel and/or paracentral scotoma and/or nasal step on Humphrey 30–2 with reference to Anderson’s criteria for minimal abnormality in glaucoma , and corUnrelated control subjects were recruited from those attending the clinic for senile cataract, floaters, refractive errors, or itchy eye. All normal control subjects had no systemic disease and no family history of glaucoma. Glaucoma in these patients was excluded using the same diagnostic criteria used for the POAG patients after the same ophthalmic examination procedure. If there was any doubt whether the subject had NTG or POAG, the subject was excluded. The study was performed with the approval of the Human Study Committee of Taichung Veterans General Hospital. Written informed consent was obtained from all study subjects before enrollment.TNF-α (−863), we used the primer sequences and primer mixtures previously described by Fanning et al. [Blood samples were collected from each subject (5 ml) and genomic DNA was isolated using the Quiagen QiaAmp Blood mini kit . Polymorphisms were determined using sequence-specific primers (SSPs). PCR-SSP uses SSPs with 3′ end mismatches and identifies the presence of specific allelic variants through PCR amplification. For the polymorphisms in the g et al.  and Grutg et al. . The proTNF-α in selected individuals with each genotype  to confirm the validity of PCR assay. Complete matching results in PCR assay and DNA sequence were obtained. To ensure accuracy, each test was performed three times for each sample.PCR products were run on ethidium bromide-stained 2% agarose gels. We sequenced the 5′ regulatory region (containing the promoter) of 2 test, and Fisher’s exact test was used if any expected frequency was lower than 5. Age and gender were compared between the control and POAG groups using the Mann–Whitney U test and χ2 test, respectively. Odds ratios were computed to assess the strength of associations between the presence of each genotype and the clinical diagnosis of POAG. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 13.0 .Genotype and allele frequencies between the control and POAG groups were compared using the χTNF-α (−863) genotypes and alleles are shown in TNF-α (−863)A/A was less frequent than in controls A allele was significantly decreased in POAG group . In comparison with controls there were significantly fewer carriers of the TNF-α (−863) A allele among patients with POAG , with an odds ratio of 0.63 AA was less frequent in POAG patients than in controls . The frequency of the TNF-α (−863)A allele was significantly decreased in POAG group . There were significantly fewer carriers (AA or CA) of the TNF-α (−863) A allele among patients with POAG than control (37% versus 48%). Our results indicate that TNF-α (−863) A allele polymorphism may be a protective factor in the development of POAG. Inheritance of the TNF-α (−863) C allele has been associated with elevated risk of developing Alzheimer disease [In this study, the genotype  disease ,26. Ther disease , and McK disease ,29 have TNF-α, a pleoprotic proinflammatory cytokine, is upregulated in several neurodegenerative disorders including multiple sclerosis, Parkinson disease, Alzheimer disease ,44, multTNF gene lies within the human leukocyte antigen (HLA) class III region, located 250 kb centromeric to the HLA-B locus and 850 kb telomeric to the class II HLA-DR locus on chromosome 6p21.3. Recent studies have reported new promoter polymorphisms in the TNF-α gene, located at position −863, which may be responsible for transcriptional regulation of TNF-α protein production [TNF-α, the TNF-α (−863)A allele has also been linked with 31% lower transcriptional activity in reporter gene studies owing to reduced binding affinity of the transcription nuclear factor (NF-κB), especially the form p50-p50 [The oduction . In TNF- p50-p50 ,57. The  p50-p50 ,58.TNF promoter polymorphisms and glaucoma risk [TNF-α (−308)G/A polymorphism has been reported to be associated with POAG in a Chinese population; Razeghinejad et al. [TNF-α (−308)G/A polymorphism in the development of POAG and PEXG in Iranian patients. The TNF-α (−308)G/A polymorphism has been reported to be associated with PEXG in the Pakistani and Turkish population [OPTN, the expression of which is induced by TNF-α, and their association with polymorphisms in the promoter region of TNF-α at position −308,-857, and −863 in Japanese patients with POAG. However, no significant difference in genotype or allelic frequency was noted between patients and Japanese control for the three single-nucleotide polymorphisms of TNF-α. Our study showed that the TNF-α (−863)A allele is associated with POAG protection in a Chinese population. The genotype and allele frequency in our control population was different from that observed in Japanese populations [TNF-α (−863)A allele were lower in Japanese populations compared with those in Chinese populations . In the Japanese study, the frequency of the AA genotype was as rare as 0.5%. In contrast, the frequency of the AA genotype at −863 was reported to be more than 10% in Chinese populations. These findings indicate Chinese and Japanese have a different genetic background at this site. This phenomenon may be due to ethnic differences, which may lead to controversial results.Several publications have evaluated the association between the oma risk -39. Lin oma risk  found thd et al.  revealedpulation ,39. Funapulation  investigulations . The freTNF-α (−863)A allele polymorphism may be a protective factor in the development of POAG. Besides the TNF-α gene, previous studies have noted other cytokine genes  and growth factor gene (insulin-like growth factor-II) polymorphisms are associated with an elevated risk of POAG [IL-1β (+3953)T allele were significantly more common in POAG patients than in controls. In future studies, proteomic analyses may be useful for determining the exact roles of cytokines genes in the development of POAG.In our study, we noted the  of POAG ,35,59-61Controversy exists regarding whether or not there is an absolute increased level of TNF protein in the vitreous or plasma that coincides with an increased level in retinal ganglion tissue among patients with POAG. The genotyping data obtained in the present study may now enable researchers to identify potential subpopulations within POAG patients, who we predict are likely to have increased circulating cytokines in the vitreous fluid. These data also further strengthen the current hypothesis that neuro-inflammation is a contributing component in the pathogenesis of POAG.TNF-α (−863) allele frequency and different level of IOP or stage of POAG. The association still needs further study.Elevated IOP is an important risk factor for glaucoma. Thus, an understanding of the genes that are involved in apoptosis, oxidative stress, mitochondrial dysfunction and regulating IOP should assist in the elucidation of the mechanisms of glaucoma. In our study, glaucoma features such as IOP and vertical cup-disk ratio were included. There were no significance between the TNF-α (−863) A allele is associated with POAG protection in a Chinese population. Further studies on POAG patients with different ethnicities are required to further define this association. The possibility that linkage disequilibrium between TNF-α gene polymorphisms and other genes on chromosome 6 is the cause of increased risk of POAG can not be excluded.The results of our genetic analysis showed that the TNF-α (−863) A allele polymorphism may be a protective factor in the development of POAG. The data indicate that therapeutic strategies designed to reduce TNF-protein production or activity might be a valuable treatment for POAG. Further insight into the mechanism underlying this relationship may suggest new strategies for reducing the intensity of the inflammatory response and attenuating the progression of POAG.In conclusion, our results indicate that the


The literature highlights several actions of IL-10 and TNF-α during pregnancy. The actions of these cytokines seem to be antagonistic and dependent on the balance between them, which is orchestrated by the specific immunosuppressive action of IL-10. TNF-α has a characteristic inflammatory action, and it is an additional diabetogenic factor in pregnancy. The loss of the control of the production of these cytokines, with increase of TNF-α, is related to the risk for developing obstetric complications, particularly recurrent fetal loss, gestational diabetes mellitus, hypertensive syndromes, and fetal growth restriction. However, study results are controversial and are not clearly defined. These issues are attributed to the heterogeneity of the studies, particularly regarding their sample sizes and sources, the evaluation methods, and the multiplicity of factors and conditions that influence cytokine production. These questions are fundamental and should be addressed in future investigations to obtain more consistent results that can be applied to obstetric practice.The purpose of this study was to review the literature regarding the action of the cytokines interleukin 10 (IL-10) and tumor necrosis factor-alpha (TNF- Cytokines belong to a diverse family of small, soluble proteins that are expressed by several cell types and tissues and act as immunologic mediators. The subpopulations of these cytokine-producing cells, which classically include T helper 1 (Th1) and T helper 2 (Th2) cells, play fundamental roles in differentiating the nature of the immunologic response. If Th1 cytokines predominate, the immune system will generate a cell-mediated response (cytotoxic type) that is targeted at intracellular pathogens or cancer cells [γ) and by interleukin 12 (IL-12). The Th2 subpopulation is characterized by IL-4, IL-5, and IL-13 [α) and the interleukins 1-beta (IL-1β) and IL-6 have clear proinflammatory actions and are classified in the subpopulation of Th1 lymphocytes [To date, at least 22 interleukin types have been identified . The Th1nd IL-13 . Tumor nphocytes . (IL-10) is a special type of cytokine that, in humans, plays a dual immunologic role that is either stimulatory and counterregulatory or immunosuppressive. This role excludes it from the Th1 and Th2 classes. However, IL-10 was originally described as a Th2 cytokine because of its anti-inflammatory action in rodents [Interleukin 10 rodents , 8, 9.α is essential in the orchestration of the cytokine cascade, and it is a therapeutic target in many inflammatory diseases. The increased production of TNF-α has been related to the pathogenesis of various diseases, including rheumatoid arthritis, Crohn's disease, atherosclerosis psoriasis, sepsis, diabetes mellitus, and obesity [TNF- obesity .α, IL-1β, and IL-6 are some of the fundamental cytokines in early pregnancy. It participates in blastocyst implantation and, adversely, in first-trimester losses. As pregnancy develops, high TNF-α concentrations have been related to the development of preeclampsia and gestational diabetes mellitus (GDM), reduced IL-10 levels, and preterm birth [In pregnancy, the immunologic system plays an important role that ensures normal pregnancy development and to promote the development of complications . Pregnanrm birth , 13, 14.rm birth . Trophobrm birth . Accordirm birth , 17.Because of the association between immunologic responses and obstetric outcomes, the early identification of immunologic alterations would facilitate the prevention of adverse pregnancy outcomes. However, the immunologic profile of human pregnancy needs to be more clearly defined .+and CD8+ cells, immunoglobulin production by B cells, the cytotoxic activity of natural-killer cells, and the maturation of dendritic cells, which results in tolerance induction [From the 1980s to the 1990s, maternal tolerance to alloantigens was explained by the predominance of Th2 immunity over Th1 immunity, which protected the fetus from maternal “attack”. Th1 predominance was observed in cases of recurrent , 19 and nduction , 25, 26.Several paradigms have been proposed to explain the modulation of the immunologic system in pregnancy . More reThaxton and Sharma  proposedα, IL-1β, and IL-6, and an increase of counterregulatory cytokines, such as IL-10. According to these studies, these alterations reinforce the role of immune modulation in the maintenance and development of normal pregnancy [From implantation until the end of a term pregnancy, the interaction between pro- and anti-inflammatory cytokines, of which IL-10 seems to be the key-cytokine, is fundamental to promote normal pregnancy outcomes. Recent findings of healthy pregnancy development show a global reduction of proinflammatory cytokines, such as TNF-α in pregnancy; we emphasized factors that are of interest to obstetrics.We sought to clarify the true role of cytokines in human pregnancy by conducting a review of the literature regarding the action of the cytokines IL-10 and TNF-Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS), Scientific Electronic Library Online (Scielo), and PubMed were consulted for this review. National and international articles were located from the following key words: “cytokines,” “pregnancy,” “placenta,” “interleukin 10” and “tumor necrosis factor-alpha.” In some searches, these words were used in English. Initially, the articles were not limited to a specific time period; however, those that were published in the last 15 years were preferred. All authors evaluated the studies that were included in the review.The National Library of Medicine (Medline), IL-10 is a particularly intriguing cytokine. In humans, it is characterized as pleiotropic and has dual immunologic functions. This dual function means that it is both stimulating and counterregulatory (immunosuppressive), which excludes it from the Th1/Th2 paradigm; however, it was originally described as a cytokine that displayed Th2 activity in rodents , 8, 9.IL-10 is encoded on chromosome 1, and polymorphisms in the gene's promoter region can interfere with its regulation , 33. Itsα, and IFN-γ [IL-10 is the key cytokine at the beginning of pregnancy because it is involved in various important events, which include placental formation. IL-10 has a protective effect on the fetal-placental unit because it inhibits the secretion of inflammatory cytokines, such as IL-6, TNF-nd IFN-γ ; jointlynd IFN-γ . Accordind IFN-γ , IL-10 ind IFN-γ , this chOther results have described decreased placental IL-10 production in the decidual region and the trophoblast in pregnancies that are complicated by intrauterine growth restriction , and thiIn summary, IL-10 is an important cytokine for pregnancy maintenance and development. During this specific period, its immunosuppressive action plays a key role in (i) regulating the balance of pro- and anti-inflammatory signs that orchestrate the adequate development of pregnancy and (ii) in placental growth and remodeling, which are also important for a favorable pregnancy outcome.α is an inflammatory cytokine that belongs to the subpopulation of Th1 lymphocytes, and it is encoded on chromosome 6. This cytokine regulates a number of cell functions, including cell proliferation, differentiation, and apoptosis. Macrophages are the primary producers of TNF-α and are also highly responsive to it [TNF-ve to it .TNF receptors are members of a large protein family, and they exist in almost every type of cell that is involved in immunologic and inflammatory responses. These receptors appear as trimers in the cytoplasmic membrane even after binding with TNF. When a cytokine binds to a type-I TNF receptor (TNF-RI), either gene expression or apoptosis may occur. The binding of the TRAAD adaptor protein (a death domain that is associated with the TNF receptor) occurs in the receptor's cytoplasmic domain, and this interaction is followed by the binding of two intermediate signaling factors, the TNF receptor-associated factor (TRAF) and the receptor interaction protein; this process promotes the gene expression of inflammatory mediators and antiapoptotic proteins . To promote caspase activation and apoptosis induction, the TRAAD adaptor protein binds to the Fas-associated death domain (FAAD). Another action mechanism of such cytokines includes the direct binding of the cytoplasmic tails of other TNF receptor family members to FAAD, which leads to apoptosis or to TRAF and thus induces gene expression .α increases with pregnancy development, and the primary production source of this cytokine appears to be the placenta [α can exacerbate insulin resistance, which is normal in pregnancy; this favors the development of GDM [α regulation during pregnancy can prevent the deleterious effects of insulin resistance. According to some authors, such verification contradicts the classical concept that insulin resistance in pregnancy is exclusively induced by placental hormones such as progesterone, human chorionic gonadotropin, prolactin, and estradiol [TNF-placenta . Increast of GDM . This cht of GDM . Therefostradiol .α in pregnant women with GDM; however, the results remain controversial. Some studies observed increased TNF-α in the blood of mothers who developed GDM [Various studies have evaluated the concentrations of circulating TNF-oped GDM –49. Howeoped GDM .α production was observed in the placenta and the subcutaneous adipose tissue in women with GDM and noncontrolled hyperglycemia when compared with those with well-controlled GDM . According to the authors, these observations suggest that the tissues of women with GDM increase the release of TNF-α in response to hyperglycemia. Because TNF-α is involved in the metabolic regulation of glucose, lipids, and insulin resistance, these data are consistent with the hypothesis that TNF-α is involved in GDM development [Significantly increased TNF-elopment , 51.α have been described in the newborns of diabetic and nondiabetic mothers and in other high-risk pregnancies. TNF-α decreases in the macrosomic offspring of mothers with GDM [α concentrations were observed in pregnant women with preeclampsia (hypertension with proteinuria) when compared with those with gestational hypertension (hypertension without proteinuria). This result suggests that TNF-α can be used as a marker of severity in pregnancy hypertensive syndromes [Other effects of TNF-with GDM , and conwith GDM . Increasyndromes .α is related to obesity, glucose intolerance, type-2 diabetes mellitus, and GDM, and it is positively correlated with body mass index (BMI) [α production in maternal adipose tissue is enhanced by the placental production of TNF-α, which makes it an important factor in the pathogenesis of insulin resistance and GDM. This challenges the traditional theory that reproductive hormones alone reduce insulin sensitivity during pregnancy [α seems to be the marker of “demetabolism” and maternal glycemic control in pregnancies that are complicated by type-2 diabetes mellitus and GDM.In summary, TNF-ex (BMI) , 48, 53.regnancy , 49, 54.α in normal pregnancy. The actions of these cytokines seem to be antagonistic and dependent on the balance between them, which is orchestrated by the specific immunosuppressive action of IL-10. TNF-α has a characteristic inflammatory action, and it is an additional diabetogenic factor in pregnancy. The loss of the control of the production of these cytokines, with increase of TNF-α, is related to the risk for developing obstetric complications, particularly recurrent fetal loss, GDM, hypertensive syndromes, and fetal growth restriction. However, these results remain controversial and are not completely reproducible. These issues are attributed to the heterogeneity of the studies, which are especially related to their source (in vivo compared with in vitro), sample size , evaluation methods (immunohistochemistry or molecular biology tools), and the multiplicity of factors and conditions that influence cytokine production. These issues are fundamental and must be considered in future investigations to produce more consistent results that can benefit the field of obstetrics.The literature describes various functions of IL-10 and of TNF-


We used L5 spinal nerve transection (SNT) mice as a neuropathic pain model. These animals were found to display up-regulated mRNA expression of activating transcription factor 3 (ATF3) and neuropeptide Y (NPY), injury markers, and interleukin (IL)-6, an inflammatory cytokine in the ipsilateral L5 DRG. Injection of LV-TNF-shRNA3 onto the proximal transected site suppressed significantly the mRNA levels of ATF3, NPY and IL-6, reduced mechanical allodynia and neuronal cell death of DRG neurons. These results suggest that lentiviral-mediated silencing of TNF-α in DRG relieves neuropathic pain and reduces neuronal cell death, and may constitute a novel therapeutic option for neuropathic pain.Neuropathic pain can be a debilitating condition. Many types of drugs that have been used to treat neuropathic pain have only limited efficacy. Recent studies indicate that pro-inflammatory mediators including tumor necrosis factor α (TNF-α) are involved in the pathogenesis of neuropathic pain. In the present study, we engineered a gene therapy strategy to relieve neuropathic pain by silencing TNF-α expression in the dorsal root ganglion (DRG) using lentiviral vectors expressing TNF short hairpin RNA1-4 (LV-TNF-shRNA1-4) in mice. First, based on its efficacy in silencing TNF-α  Neuropathic pain is defined as pain caused by a lesion or disease in the somatosensory nervous system A number of animal models have been developed that can be used to study the mechanism of neuropathic pain and to examine the efficacy of new therapies Recently, peripheral nerve and spinal nerve injury models have also been used for research into the pathogenesis of neuropathic pain. Studies in these models revealed that pro-inflammatory mediators, such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6, are up-regulated in the dorsal root ganglion (DRG) and may be important mediators of neuropathic pain in rodents Here, we present a gene therapy strategy that relieves SNT-induced neuropathic pain by silencing TNF-α expression in DRG using RNA interference technology with lentiviral vectors. This strategy was effective in suppressing neuropathic pain and protecting DRG neurons from cell death. These results constitute a proof of principle that the therapeutic strategy can potentially be used to treat neuropathic pain and sensory neuronopathy in people.All animal protocols were approved by the Institutional Animal Care and Use Committee of Shiga University of Medical Science (Approved Number: 2012-8-5). All procedures were performed in accordance with the guidelines of the Research Center for Animal Life Science of Shiga University of Medical Science.http://www.broadinstitute.org/rnai/trc. Accessed on 2014 Feb 20.).The full-length mouse TNF-α cDNA was inserted into pLL3.7 plasmid including elongation factor 1 (EF-1) promoter and DsRed as a reporter gene (TNF-α over-expression plasmid: LV-TNF-DsRed). Four short hairpin RNAs (shRNA) were individually inserted into pLL3.7 plasmid including U6 promoter and enhanced green fluorescent protein (GFP), as a reporter gene, driven by cytomegalovirus (CMV) promoter (TNF-α silencing plasmid: LV-TNF-shRNA1-4). The full-length TNF-α coding sequence (NM_013693) was isolated from RAW cells with lipopolysaccharide stimulation. shRNA sequences against mouse TNF-α were designed using a web site from The RNAi Consortium (TRC) algorithm (Available: 8 infectious units (IFU)/ml at −80°C after the determination of IFU using 293T cells. Finally, we produced LV-TNF-DsRed (mouse TNF-α and DsRed expression vector), LV-GFP (control vector expressing GFP without shRNA sequence) and LV-TNF-shRNA1-4 (TNF-α silencing vector with GFP expression).To produce recombinant lentiviral particles, psPAX2 (a packaging plasmid), pMD2.G , a gift of Dr. Didier Trono , and TNF-α over-expression plasmid (or silencing plasmid) were co-transfected into 293T cells using the calcium phosphate method as described previously 5 cells per well. The confluency of 293T cells was approximately 70% at the time of infection. At 6 h after infection of LV-TNF-DsRed (3.6×105 IFU), 3.6×105 IFU LV-GFP or LV-TNF-shRNA1-4 was separately added into each well of LV-TNF-DsRed-infected 293T cells. At 72 h later, DsRed and GFP expression were observed under fluorescence microscopy and TNF-α mRNA expression was determined by quantitative RT-PCR analysis.One day before infection, 293T cells were split to 6 well-culture dishes at a density of 1.0×10Male 9–10 week-old C57BL6  mice weighing 19.0–22.0 g were used in this study. The mice were housed in separated cages and the room kept under 12:12 light-dark cycle with free access to food and water.5 IFU/μl) of lentiviral vector was injected onto proximal transected site of left L5 spinal nerve using a Hamilton syringe  with a 30G needle. The right spinal nerve, as a control side, was clearly exposed without transection or administration of lentiviral vector.The neuropathic pain model by L5 spinal nerve transection (SNT) was generated by the method described previously  and at 1, 3, 5, 7, 10 and 14 days after SNT, using a dynamic planter aesthesiometer  as described previously Animal tissues were removed under deep anesthesia and immediately frozen in liquid nitrogen. Total RNA was extracted from cultured cells and frozen tissues using RNeasy mini kit  with DNase I  treatment. Reverse transcription was performed from 100 ng of total RNA in each tube using Prime Script perfect Real time (Takara Bio Inc.).5′-CACGTCGTAGCAAACCACCAAGTGG-3′ and 5′-GATAGCAAATCGGCTGACGGTGTGG-3′ for mouse TNF-α, 5′-AGGACGACGGCAACTACAAGAC-3′ and 5′-GAAGTTCACCTTGATGCCGTTC-3′ for GFP, 5′-AAATCCCATCACCATCTTCCA-3′ and 5′-AATGAGCCCCAGCCTTCTC-3′ for human GAPDH (for 293T cell), 5′-AACGACCCCTTCATTGAC-3′ and 5′-TCCACGACATACTCAGCAC-3′ for mouse GAPDH (for mouse tissues). For RT-PCR by TaqMan method, the following primers were used: Mn_99999915_g1  for mouse GAPDH and 5′-CATCTTCTCAAAATTCGAGTGACAA-3′, 5′-TCGGAGTAGACAAGGTAGAACCC-3′ and 5′-FAM-GCACGTCGTAGCAAACCACCACCAAGTGGA-TAMRA-3′ (probe) for mouse TNF-α. The PCR reaction consisted of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds. The PCR products were analyzed with FAS-IV .RT-PCR was performed with the following primers: −ΔΔCt method with GAPDH as a control, which expression levels were confirmed not to be changed among day -2, 3, 7 and 14.For quantification of each gene, real time PCR assay was performed using a LightCycler 480  with SYBR Green method (for activating transcription factor 3 (ATF3), calcitonin gene related peptide (CGRP), IL-6, neuropeptide Y (NPY), human GAPDH, mouse GAPDH and mouse TNF-α) and TaqMan method (for mouse GAPDH and mouse TNF-α). The PCR parameters were one cycle of 95°C for three min, followed by 50 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec. The emitted fluorescence for each reaction was measured three times during the annealing extension phase, and amplification plots were analyzed using LightCycler 480 software, version 1.5 . Potential genomic DNA contamination was controlled by the use of intron-encompassing primers and DNase digestion. GAPDH and MAPK6 have been reported to be the most stable reference genes for use in normalizing transcript level of interest genes although the expression of other many house-keeping genes reduced on the early phase in DRG after nerve injury Animals were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg) and intracardially perfused with 30 ml of PBS followed by a fixative containing 4% paraformaldehyde in 0.1 M PB. After perfusion fixation, animal tissues were kept in the same fixative at 4°C for 18 h and permeated with 15% (w/v) sucrose buffer with agitation. Each tissue was embedded in Tissue-Tek O.C.T compound , frozen with liquid nitrogen and cut on a cryostat at 8 μm sections collected on MAS (Matsunami aggressive silane)-coated glass slides. Sections were blocked with 3% normal goat serum in PBS at room temperature for 15 min and processed for immunohistochemistry. The following antibodies were used: anti-TNF-α , anti-GFP , anti-cleaved caspase3 , and AlexaFluor 488, 594 and 633  as primary and secondary antibodies. For marker stains, we used 4′,6-diamidino-2-phenylindole (DAPI) for nuclei and fluorescent Nissl  for neurons. Fluorescence images were captured and analyzed using Nikon C1si and the software, EZ-C1 version 3.90 .To evaluate the neuronal cell survival in ipsilateral L5 DRG after SNT, we perfused mice transcardially with 4% paraformaldehyde in 0.1 M PBS, and isolated DRG tissues and embedded them in paraffin. L5 DRG paraffin blocks were sectioned at 10 μm thickness and stained with 0.1% Cresyl violet . After Nissl staining (Cresyl violet), all visible neurons in each left L5 DRG were counted in consecutive sections . For the evaluation of apoptosis after SNT with or without gene therapy, we counted the number of cleaved caspase3 positive neurons. Approximately 300 neurons were analyzed in consecutive sections  for each left L5 DRG.in vivo were evaluated by counting the number of GFP or TNF-α positive neurons with the neuronal marker stain of fluorescent Nissl (NeuroTrace 435/455 for GFP or NeuroTrace 530/615 for TNF-α) in DRG after SNT with or without gene therapy. We analyzed approximately 300 neurons in consecutive sections  for each L5 DRG.Transduction efficacy and treatment effects of gene therapy by lentiviral vector p<0.05.Statistical analysis was performed with SPSS 17.0 software . All data are shown as mean ± standard error of the mean (S.E.). The one-way ANOVA followed by Tukey's test was used to calculate statistical significance for multiple data sets. For behavioral test, two-way ANOVA was used. Data were considered significant difference at We induced neuropathic pain in mice by SNT on the left side of the L5 spinal nerve at a location close to the spine   in bilateral DRG tissues after infection of LV-TNF-shRNA3 targeting the transected left L5 spinal nerve. Infection-induced GFP mRNA expression in ipsilateral L5 DRG was confirmed between day 3 to day 14 after local injection onto the left L5 spinal nerve (We quantified the relative degree of TNF-α silencing after LV-TNF-shRNA3 gene therapy by quantitative RT-PCR of TNF-α mRNA and immunohistochemistry of TNF-α protein in L5 DRG tissues. In SNT with LV-GFP group (control group), TNF-α mRNA expression increased approximately 4-fold compared with pretreatment expression levels in ipsilateral L5 DRG from day 3 to day 14  treatment by measuring the mechanical threshold every other day till day 7 and again on day 10 and 14 after treatment Figure 8This study demonstrated that silencing of TNF-α expression with lentiviral vectors suppressed neuropathic pain induced by SNT. Lentiviral vectors targeted to DRG tissues decreased TNF-α expression in the affected  DRG neurons. In addition, silencing of TNF-α expression in DRG resulted in the inhibition of neuronal cell death and apoptosis caused by SNT. Furthermore, injury markers and inflammatory cytokines that were up-regulated by SNT were greatly attenuated by lentiviral gene therapy. Therefore, gene therapy-mediated TNF-α silencing may be an efficacious treatment strategy for protecting DRG neurons and ameliorate neuropathic pain induced by nerve injury.+ channels and increases membrane K+ ion conductance by a non-voltage-gated mechanism in DRG neurons Neuropathic pain is caused by neuronal damage or dysfunction Spinal nerve injury leads to activated caspase3 and increased apoptosis IL-6 is up-regulated in DRG in response to peripheral nerve injury DRG-targeted gene delivery is a promising therapeutic option for the treatment of neuropathic pain. A number of viral gene vectors are available for gene delivery, including adenoviral (Ad) vectors, adeno-associated viral (AAV) vectors, and lentiviral vectors In general, Ad vectors have high transduction efficiency in most cells with the coxsackie virus and adenovirus receptor (CAR) compared to AAV and lentiviral vectors after attachment to the target cells in vivo gene therapy in vivo in rodents It is also important to consider the toxicity of various viral vectors. Ad vectors have been shown to induce a potent immune response against the capsid, double-stranded DNA genome, viral proteins expressed from the vector backbone, or incorporated transgenes, a limitation for in vivo, it was reported mainly by three different approaches, intrathecally, direct injection into sciatic nerve or into the DRG itself For delivering transgenes by viral vectors to the DRG Unexpectedly, we found that spinal nerve injection led to high level transduction of the DRG. It was much higher than that reported in a previous study that utilized direct injection of lentiviral vectors into DRG in rats Clinical reports suggest that anti-TNF-α drugs, such as etanercept Considering the gene therapy of sensory neuronopathy, we previously reported the intrathecal administration of DRG-targeting fiber-modified helper-dependent adenovirus (HDAd) vectors In conclusion, we presented a strategy to relieve neuropathic pain using lentiviral shRNA vectors. Our results support previous evidence for TNF-α in the pathogenesis of neuropathic pain. In addition, we showed that TNF-α silencing by lentiviral shRNA vectors protects injured DRG neurons from apoptosis. Silencing TNF-α by lentiviral vector-mediated gene transfer targeted to DRGs with injection onto injury site of spinal nerve may provide a novel therapeutic alternative for the treatment of neuropathic pain.


Nipah virus (NiV) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile encephalitis in humans and a predominantly respiratory disease in pigs. Periodic deadly outbreaks, documentation of person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective means of therapy and prevention. In this report, we describe the vaccine potential of NiV virus-like particles (NiV VLPs) composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and importantly, they retained their antigenic potential. Finally, these particles, formulated without adjuvant, were able to induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease. Since it was first recognized in 1998, Nipah virus (NiV) has caused several outbreaks in humans of encephalitic disease associated with high lethality. In the first outbreak, which was in Malaysia and Singapore, 265 humans became sick and some ∼40% of them died. Epidemiological links pointed to human contact with sick pigs in commercial piggeries, and the outbreak was brought under control through culling of approximately ∼1.1 million pigs Henipavirus in the subfamily Paramyxovirinae, family Paramyxoviridae. It has several distinctive genetic and biologic features NiV is a member of the genus Two vaccination strategies for NiV disease prevention have already been explored experimentally: A canarypox virus-based vaccine vector approach was effective as veterinary vaccine In this study we have explored the potential of NiV virus-like particles (VLPs) as a vaccine. Plasmid-mediated expression of selected viral proteins results in the spontaneous assembly and release of VLPs. These particles make highly effective immunogens because they possess several features of the authentic virus such as their surface structure and dimensions 0 form. Recently, the vaccine potential of native VLPs of NDV The budding capacity of virus proteins as VLPs, the protein-protein interactions that facilitate this process, and the central role of M protein in VLP assembly and release has been described for several paramyxoviruses such as Sendai virus (SeV), Newcastle disease virus (NDV), respiratory syncytial virus (RSV), paramyxovirus simian virus 5 (PIV-5) and human parainfluenza virus type 1 (hPIV1) in vitro. It has also allowed us to test for immunogenicity in vivo in Balb/c mice; note that although NiV does not cause disease in these animals, NiV proteins injected in them are known to induce robust neutralizing antibody response Here we describe NiV VLPs composed of the two surface glycoproteins G, and F, and the matrix protein M. The G and F proteins were included because they mediate attachment and entry into the host cell In this study, careful assessment of immunogenicity has shown for the first time, that these NiV VLPs are able to induce neutralizing antibody response. We have also provided a detailed methodology to optimize production of the VLPs for research purposes. Beyond this, we have provided the first CryoEM study of NiV VLPs and thus provide a careful assessment of their morphology. We further demonstrate that NiV VLPs can trigger “fusion from without” upon addition to cells. To our knowledge this is a first for an enveloped VLP. Finally, we have shown that NiV VLPs activate innate immune signaling in “infected” cells and provide a transcriptional profile of this response. Based on all these attributes, NiV M, F and G-protein-containing VLPs show promise as vaccine and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease.NiV expression plasmids pCAGGS- G, F, and M are all under the control of chicken beta actin promoter 293T cells were grown in Dulbecco's complete medium to achieve semi-confluent (80–90% density) cell monolayers. The cells were transiently transfected with the plasmids constructs using the lipid reagent Lipofectamine 2000 according to the general guidelines provided by the manufacturers' instructions (Invitrogen Inc). At 48 hrs post-transfection, the VLP-containing cell supernatants (SUP) were harvested for concentration and purification of the VLPs. Because of the fusogenic property of our VLPs, there was widespread syncytia formation at this time point although the cells were still adherent.VLPs released in the transfected-cell SUP were harvested and clarified by centrifugation at 3,500 rpm for 30 minutes at 4°C and concentrated by sucrose density gradient centrifugation based on previous descriptions VLP infectivity assay, similar to those described previously was used to determine the relative concentrations of the constituent plasmids, and to determine the kinetics of VLP formation for optimal production. This assay provides only a comparative assessment of VLP formation since it only accounts for VLPs that are able to incorporate and passage minigenomes. However, based on the assumption that the ratio of empty and minigenome-containing VLPs will be equivalent in each reaction, the method provides an indirect means to determine the optimal set of conditions for VLP production as determined by VLP-incorporated minigenome-encoded CAT enzyme activity. Briefly, the steps involved in the VLP infectivity assay were 1) transfection of NiV minigenome construct and co-transfection with full complement of the NiV protein expression plasmids, N, P, L, M, F and G, using Lipofectamine 2000. 2) following replication (48 hours post-transfection), passage of equal volume of VLP-containing transfected cell SUP on to fresh cells previously transfected with N, P and L plasmids and 3), determination of CAT activity in the VLP infected cells 48 hours later. Replication of the VLP-incorporated incoming mingenomes based on reporter gene activity indicates the level of particle formation and release, VLP infectivity, and successful minigenome packaging.Since the ratio of the protein expression plasmids used at transfection and the time of harvest may have a bearing on the level of VLP formation, a minigenome-based FAST CAT Assay kit (Molecular Probes) was used according the manufacturer's instructions and allowed accurate quantification of CAT enzyme levels over a wide linear range.VLPs were purified as described. The particles were adsorbed on Formvar carbon coated copper grid by floating it on a drop of VLP suspension for 15 minutes, the grids were blotted, and then negatively stained with 2% aqueous uranyl acetate for viewing by transmission electron microscopy.The VLPs were vitrified as reported previously Unfixed VLPs were used for immunogold labeling to limit antibody reactivity to the cell surface proteins. The particles were adsorbed on formvar coated nickel grids, stained with NiV specific primary antibody  diluted in buffer (1% BSA in 0.05 M tris buffer) rinsed in wash buffer (0.1% BSA in 0.05 M tris buffer), stained with colloidal gold labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories), washed, and then negatively stained with 2% uranyl acetate for viewing by EM.The total protein concentration of the purified VLP preparations was measured by the BCA (Bicinchoninic Acid) method (Thermo Scientific Laboratories).VLP composition was determined by western blot analysis. Briefly, purified VLPs resuspended in endotoxin free PBS were lysed by resuspending them in equal amount of 2x SDS protein-loading buffer and loaded into a 12% SDS-polyacrylamide gel with a 4% stacking gel. 293T cell lysates processed similarly were run in parallel as negative cell control. Following electrophoresis to resolve the protein bands, and transfer to membrane, the blot was incubated with NiV-specific HMAF primary antibody at a dilution of 1∶1000 dilution, overnight at 4°C, and HRP-conjugated anti-mouse secondary antibody  at a 1: 20,000 dilution for one hour at room temperature. The proteins were revealed using western blot detection reagents according to instructions provided by the manufacturer .These studies were undertaken with the approval of the Institutional Biosafety Committee (Protocol# #01/08-2010-1) and the Institutional Animal Care and Use (IACUC) Committee (Protocol # 0904028). Five to six week old female Balb/c mice (Harlan Laboratories) were housed in microisolater cage for 4 days in the Animal Resource Center at the University of Texas Medical Branch before beginning the immunization protocol. Mice in groups of five were immunized by subcutaneous inoculation of four different concentrations of VLPs  prepared just prior to use in sterile endotoxin free PBS. No adjuvant was used. A group of five mice inoculated with sterile endotoxin free PBS served as negative control group. Mice in the four treatment groups (A through D) were boosted (6 µg/mouse) on days 15 and 29; the negative control group received PBS. Blood was collected from the submandibular vein of the animals on days −1, 14, 21, 28 and 35; they were euthanized on day 35.50).Two-fold dilutions of test sera were made in 50 µl cell culture medium. Under biohazard level 4 conditions, each of the diluted sera were mixed with 50 µl of NiV diluted to generate ∼30 plaque forming units and incubated for 30 min at 37°C. The pre-incubated virus-antibody mix was added to Vero cell monolayers grown in 96 well plates and incubated for 30 min at 37°C when the inoculum was removed and replaced 150 µl of cell media. After incubation at 37°C for 24 h, the cells fixed in 100% ice-cold methanol and staining by indirect immunofluorescence assay as follows: The wells in the plate were blocked with BSA/PBS and stained with rabbit sera raised against the G protein of HeV, and goat anti-rabbit Alexa Fluor 488 conjugate (Invitrogen) diluted 1∶1000 in blocking buffer. Viral plaques were visualized and counted, and neutralizing antibody titers were reported based on reduction in plaque count by 50% relative to the untreated control  on glass slides to obtain monolayered preparations and then stored at 4°C, and used as antigen within three weeks of preparation. On the day of use, the slides were washed in PBS, permeabilized with Triton-X-100 and blocked with BSA/PBS. After incubation with two fold dilutions of the test sera, the cell monolayers were washed and stained with Alexa fluor 488-conjugated goat anti-mouse antibody according the manufacturer's (Molecular Probes) instructions. Negative and positive controls were run in parallel with each batch.2 Profiler PCR array (SABiosciences). The 96 well array format included mediators of TLR signaling including adaptors and proteins that interact with the TLRs, and members of NFKB, JNKp38, NF/IL6 and IRF signaling pathways downstream of TLR signaling. Briefly, 293 cells grown overnight in 60 mm dishes were exposed to 10 µg of purified VLPs suspended in 1 ml of OPTI-MEM (Invitrogen). “Mock particles” -mediated signal transduction using RTIn preliminary studies it was found that co-expression of NiV G, F and M proteins in 293T cells resulted in the formation of VLPs that bud out into the transfected cell SUP and that they can be harvested, concentrated and purified as described under The importance of protein ratios for VLP formation was alluded to in a previous NDV study where the expression plasmids were co-transfected at “pre-determined concentrations” to produce VLP-incorporated protein ratios analogous to those in virus infected cells or F or M expression plasmids were, in turn, co-transfected with fixed concentrations of the other two plasmids. The minigenome and N, P and L plasmids were transfected using a predetermined ratio VLP infected cells were harvested 48 hours later and tested for optimal particle production based on incoming minigenome-encoded CAT activity. This time point was chosen because maximal VLP formation was also found to be time dependent and optimal at 48 hours post-passage (data not shown). The reproducibility of the results was verified in an independent repeat experiment. Results presented in We have chosen a minigenome-based functional assay, the VLP infectivity assay . The particles were viewed by EM after negative staining. The use of unfixed particles assured that only the surface-exposed antigens would be reactive. Numerous VLPs with the gold particles decorating their surface were seen; Syncytium formation is a classical feature of NiV and other paramyxovirus-induced cytopathology that can be blocked by virus-specific neutralizing antibody. A similar observation was made when 293 cells were “infected” with the NiV VLPs. Briefly, the VLPs were pre-incubated with NiV-specific antibody, Junin virus (JV)-specific antibody, and with OPTI-MEM I (Invitrogen Inc) medium only for one hour at 37°C before inoculating onto near confluent 293 cell monolayers grown overnight in 60 mm dishes. The inoculum was removed after incubation for 3 hours at 37°C, replaced with OPTI-MEM I, and the plates were further incubated overnight at 37°C overnight. The monolayers were then viewed for the formation of syncytia after staining with crystal violet. The results in Mice in groups of five were inoculated subcutaneously with four different concentrations of purified VLPs and boosted as described under All sera were tested individually by plaque reduction neutralization method by doubling dilution of each sample (1∶5 to 1∶80) as described under A PCR array format (SABiosciences) was used to investigate modulation in transcription profile of 84 genes involved in innate immune responses to include TLR signaling family and members of the downstream signaling pathways, NFKB, NF/IL6, IRF and JNKp38. These genes represent key sensors of non-self that signal, and ultimately shape the nature of innate immune response that modulates the type and duration of adaptive immune responses Using a minigenome-based functional assay, we have established conditions (described under Results and shown in No vaccine for NiV disease has been developed so far that would be both safe and protective for humans. The two vaccination strategies that have already been explored are the canary pox-based vector approach Native VLPs like the ones we have produced allow the viral proteins to be presented to the immune system in the same conformation as in the virion for effective B and T cell response NiV M, F, G and N protein-containing VLPs consistent in size and morphology to the parental virus have also been reported in a previous study which evaluated protein-protein interaction that facilitate VLP formation Immunogold labeling of our unfixed NiV VLPs confirmed that the surface proteins in our VLPs were functionally assembled and they were biologically active . We coulformulated without adjuvant, compares favorably with the levels of such response induced at an equivalent time point by some replication competent pseudotype viruses Neutralizing antibody response is the critical correlate of protection mediated by prophylactic vaccines Immunogenicity to native VLPs has been reported previously for one other paramyxovirus namely NDV The nature of innate immune response dictates the type and duration of adaptive immune response Finally, as described above, our VLPs were highly effective as immunogens, able to induce neutralizing antibody response in all animals with primary inoculation of as little as 7 µg VLP protein each. Fusogenic property of our VLPs may be critically relevant in this regard in the light of recent findings, and would need to be experimentally verified by comparing the potency of fusion-competent and fusion-defective VLPs as vaccine In conclusion, we have been successful in producing substantial quantities of NiV VLPs needed to characterize NiV VLPs, we have demonstrated their many virus-like properties, and their effectiveness as immunogens in Balb/c mice. These findings are the basis on which we will be undertaking future challenge studies in the hamster model of NiV disease


Calanus finmarchicus. A siphon flow was used to generate a consistent fluid signal and the behavioral threshold and magnitude of the escape response was quantified in the dark and in the light. The results show that C. finmarchicus initiated their escape reaction further from the siphon and traveled with greater speed in the light than in the dark. However, no difference was found in the escape distance. These results suggest that copepods use information derived from multiple sensory inputs to modulate the sensitivity and strength of the escape in response to an increase risk of predation. Population and IBM models that predict optimal vertical distributions of copepods in response to visual predators need to consider changes in the copepod's behavioral thresholds when predicting predation risk within the water column.The timing and magnitude of an escape reaction is often the determining factor governing a copepod’s success at avoiding predation. Copepods initiate rapid and directed escapes in response to fluid signals created by predators; however little is known about how copepods modulate their behavior in response to additional sensory input. This study investigates the effect of light level on the escape behavior of  For a predator, an unsuccessful predation event results only in its continued hunger Copepods are heavily preyed upon by visual predators, mainly fish The biological and physical environment of pelagic copepods requires highly discriminate and yet rapid behavioral responses. Living at low Reynolds numbers, chemical stimuli are transported to the copepod's sensors largely through the slow process of laminar fluid displacement and Fickian diffusion Mechanoreception is a primary mechanism for the remote detection and discrimination of predators In addition to mechanical signals, copepods also detect and respond to light Calanus finmarchicus are prey to visual predators including fish Calanus finmarchicus CV and adult stages to fluid mechanical signals in the light and dark. We hypothesized that, in response to the higher predation risk from visual predators in the light, C. finmarchicus will initiate an escape reaction at a lower threshold (further from the source) in the light than in the dark. In addition to the lower behavioral threshold, the magnitude of the escape response was hypothesized to be greater when the perceived predation threat was higher.Calanus finmarchicus) were cultured in large 5000 L flow-through silos at the Institute of Marine Research's Austevoll Research Station, Norway. Animals were maintained at 12.5°C on a mixed diet of Rhodomonas baltica, and Isochrisis sp. at a food level of 2×104 cells mL−1. Individual adult stage copepods were collected in a large beaker and held for ∼2 hours in 20 L buckets at 12.5°C in the dark prior to testing. Copepods (100–150 per treatment) were placed within the filming vessel  and allowed to acclimate to the test condition for 10 minutes. Animals were filmed for 30 minutes. To test if light levels modulate copepod escape characteristics, a constant fluid mechanical signal was maintained and tested the response of copepods in the dark and at light levels found at 20 m during an average Bergen, Norway summer (see below).Copepods  °C. Each experimental condition was run in triplicate with 100–150 animals per replicate . Each replicate was filmed for 30 minutes. Animals were not used more than once.A siphon flow was used to create a stable fluid mechanical disturbance. The resulting flow fields are well-characterized and have been used to analyze copepod escape behavior  surface . The exp−4 W cm−2) and darkness at a depth of ∼20 meters Calanus finmarchicus in the fiords outside of Bergen Norway during mid Spring.Copepod escape responses were observed under two light settings . The light beam was positioned above the tank to produce a homogeneous light field at the top of the filming vessel. Light levels used in these experiments simulated dusk −1where Q is the volume exiting the siphon −1. This is well below the escape threshold for most species V = Q  at which the copepod initiated an escape reaction. Once initiated, the magnitude of the escape reaction was assessed by the measuring the average speed of the entire escape reaction and the total distance traveled during the escape.The threshold and magnitude of escape response of CV and adult stage Cyclops sp. Oithona spThe appendages (and their motion) involved in an escape reaction have been described for Calanus finmarchicus initiated the escape reaction (threshold) and the total distance traveled during the escape reaction were not normally distributed. Therefore, differences in the threshold distance and travel distance were analyzed using the non-parametric Mann-Whitney ranked sum test. The escape speeds, which were normally distributed, were analyzed using a 2 tailed t-test.The distances from the siphon at which The escape characteristics of a total of 116 copepods were examined at the two light levels. Each replicate tank produced between 15–26 escape reactions in both the light and dark treatments. No significant difference was found between replicates within the same treatment and they were, therefore, pooled for further analyses. All animals entrained by the siphon initiated an escape reaction. Two copepods initiated an escape reaction but were captured by the siphon. The escape characteristics of these animals were not analyzed as part of this study.C. finmarchicus to the fluid mechanical signal was quantified as the distance from the siphon at which the animals initiated their escape reaction  from the siphon mouth in the dark and 9.4 mm (4.2 BL) in the light; an increase of 36%.The behavioral threshold for reaction . The disreaction . Within reaction  and 2. Hreaction ; Fig. 3.C. finmarchicus was significantly faster in the light than in the dark (−1 (±53.6 BL s−1). In the light, the average escape speed was 140 mm s−1 (±63.6 BL s−1), an increase of 18% compared to the escape speeds in the dark. The higher escape speeds, however, did not result in a greater distance traveled during the escape (2<0.01) or the total distance traveled (r2<0.06) during the escape.Once the escape reaction was initiated, the strength of the response was quantified by the speed of the escape and total distance traveled. Escape speed of the dark ; Fig. 4.e escape . Median The distance from the predator at which an organism initiates an escape response and the strength of the escape (speed and distance traveled) can be the determining factors governing an organism's ability to avoid predation Calanus finmarchicus initiated the escape reaction showed no significant difference with respect to the angle from the siphon  would transport the copepod well outside this capture volume providing the copepod a temporary reprieve from the threat of predation. The risk of a second attack would depend in part on the visual acuity of the predator, light level and the optical quality of the water , escape reactions would occur at 125 mm (56BL) from the siphon. Assuming that the neurophysiological thresholds are similar to those reported above, C. finmarchicus do not escape at their neurophysiological threshold but rather initiate escapes only in response to much higher signal strength. The intermediate value of the escape threshold suggests two opposing forces which determine the upper and lower limits of the magnitude of the stimulus needed to cause an escape reaction Fluid mechanical disturbances become neurological signals through the motion of one or several of the numerous mechanoreceptive setae that adorn the antennules of copepods Less obvious, and more difficult to assess, are the conditions that give rise to an inhibition of the escape reaction despite being within the neurophysiological detection limit of the copepod It has long been hypothesized that copepods attempt to decrease predation risk from visual predators by vertically migrating out of the photic zone during the day


Silver-impregnated fabrics are widely used for their antibacterial and antifungal effects, including for clinical clothing such as surgical scrub suits (scrubs). This study investigated whether silver impregnation reduces surface bacterial contamination of surgical scrubs during use in a veterinary hospital. Using agar contact plates, abdominal and lumbar areas of silver-impregnated nylon or polyester/cotton scrubs were sampled for surface bacterial contamination before (0 h) and after 4 and 8 h of use. The number of bacterial colonies on each contact plate was counted after 24 and 48 h incubation at 37 °C. Standard basic descriptive statistics and mixed-effects linear regression were used to investigate the association of possible predictors of the level of bacterial contamination of the scrubs with surface bacterial counts.Silver-impregnated scrubs had significantly lowered bacterial colony counts (BCC) at 0 h compared with polyester/cotton scrubs. However, after 4 and 8 h of wear, silver impregnation had no effect on BCC. Scrub tops with higher BCC at 0 h had significantly higher BCC at 4 and 8 h, suggesting that contamination present at 0 h persisted during wear. Sampling from the lumbar area was associated with lower BCC at all three time points. Other factors  also affected BCC at some time points. Silver impregnation appeared to be ineffective in reducing bacterial contamination of scrubs during use in a veterinary hospital. Enterococcus and Staphylococcus spp. can survive for more than 90 days on clothing worn by health care workers , University of Liverpool, were randomly assigned to wear either silver-impregnated nylon scrubs (Buckley Lamb) or standard polyester/cotton scrubs (Urban Scrubs Ltd and Lundau Scrubs) on each day of the study. These staff groups were chosen because they were typically working inside the theatre suite for the whole day, allowing the mid-abdominal and mid-lumbar surface of each scrub top to be sampled for surface bacterial contamination prior to use (0 h), after 4 h use and after 8 h use.Four centimetre diameter agar contact plates  were placed directly onto the fabric for 1–3 s. A different, adjacent section of fabric was sampled at each of the three time points to avoid sampling areas contaminated with agar from previous plates. Before and after each sampling, the researcher’s hands were cleaned with chlorhexidine surgical scrub .To allow statistical analysis of other possible influences on the level of bacterial contamination, participants each day completed a short questionnaire about factors that might affect how contaminated their scrubs had become . If a paContact plates were incubated at 37 °C and the number of bacterial colonies on each plate was counted after 24 and 48 h incubation, allowing counting of both faster-growing and slower-growing colonies. Each day, two randomly-selected unused contact plates were incubated alongside the sample plates as negative controls. Both types of scrubs were laundered daily in a standard commercial washing machine  at 40 °C using a standard commercial biological washing powder , then tumble dried in a commercial drier (JLA 92830ELG). Both types of scrubs were folded and returned to the theatre changing area immediately after laundry and stored in open plastic baskets overnight.To satisfy requirements for ethical approval, at the end of the study participants completed a short anonymous debriefing questionnaire about their participation. Each set of scrubs, participant, contact plate and questionnaire was assigned a unique identification number and all collected data were recorded against these numbers, ensuring that the researcher performing the bacterial colony counting was blinded to the type of scrub suit and participant’s identity and job type.Bacterial colony counts were recorded on a spreadsheet (Microsoft Excel) and transferred into STATA11 (StataCorp) for statistical analysis.10(count + 1). After performing standard basic descriptive statistics, the association of the log10 transformed cell count with possible predictors of the level of bacterial contamination of the scrubs (questionnaire data as shown in P ⩽ 0.2. These variables were then included in a mixed-effects linear regression model with staff identity and/or scrub suit identity as a random variable: the type of scrub suit (silver or not) was also forced into the model.Data from contact plates where bacterial overgrowth had occurred and individual colonies could not be counted were coded as missing. If a contact plate contained 100 or more colonies, an accurate count could not be determined due to merging of adjacent colonies, so counts from these plates were recorded as ‘100+’ and recoded to a value of 150 before statistical analysis. All counts were transformed to logP < 0.05 on a two-sided null hypothesis.The second method included all possible predictors in a mixed-effects linear regression. In both cases, the only random effect needed by the model was staff identity. The Wald statistic was used to determine the significance of predictors in the final model. Graphical methods were used to examine the residuals for normality and equality of variance, revealing that the required assumptions were met. Since the two approaches gave very similar results, only the second is reported here. Predicted means were obtained using the STATA ‘margins’ command using its default settings. For a given level of a factor (e.g. type of scrub suit), the predicted mean provided values estimated for the model that would have been obtained if all individuals had this level of the factor. Statistical significance was defined as All participants gave fully informed consent to take part in the study and were informed that they could withdraw from the study at any time for any reason. The study was approved by the University of Liverpool Research Ethics Committee.Mixed-effects linear regression revealed that, at 0 h, samples taken from silver-impregnated scrubs had significantly lower BCC after both 24 and 48 h incubation . In sampRestraint of patients during the day was associated with increased BCC in samples taken at 4 h and incubated for 24 or 48 h . Two of Although the antibacterial and antifungal properties of silver-impregnated fabrics are well known , our stuIn our study, sampling from the abdominal area of the scrubs was consistently associated with higher BCC than sampling from the lumbar area and patient restraint was associated with increased BCC. This suggested that patients and the wearer’s hands were the main source of contamination of the scrub tops, since the abdominal area is most likely to come into contact with the patient during restraint or lifting and is more likely than the lumbar area to be touched by the wearer. Identification of the bacteria contaminating the scrubs might have allowed confirmation of the source of contamination but the large number of colonies grown on the contact plates precluded their identification in this study.2 species and damage to the cell envelope  medications in our theatre suite are usually only handled for a short period around the time that they are administered to the patient. Thus, contact with medications implies close patient contact and thus possibly increased scrub suit contamination. This hypothesis is supported by the association between handling of patients and increased BCC in the samples taken after 4 h wear.Previous data have suggested that one of the other predictors associated with patient contact  should be associated with increased BCC, since contamination with organic materials reduces the effectiveness of silver as an antibacterial agent . In our Contamination of the surgical scrubs with urine was associated with lower BCC in samples taken at 8 h and incubated for 48 h. We believe this association to be due to chance and not causal because it only occurred at this one time point and visible contamination of scrubs with urine should have prompted the scrub top to be changed, thus excluding data from it on that day from the study. Also, there may have been errors in reporting (see below).There are several possible limitations to this study. First, the number of samples in each group was lower than that assumed in the power calculation, possibly resulting in type II errors; this may account for restraint of patients being associated with higher BCC in samples taken at 4 h, but not at 8 h, for example. Unfortunately, more samples could not be taken in the time available for the study. Second, although the questionnaires were completed anonymously (to encourage full disclosure) and as soon as was practical (at the end of the working day), there may have been errors or omissions in the responses, leading to over or under-reporting of some types of contamination. However, any such errors should not alter any effects based on scrub type, sampling location, participant identification or date, since the accuracy of these data was not dependent on reporting by the participants. Third, the difficulty in counting large numbers of bacterial colonies due to colony merging and overgrowth may have resulted in underestimation of the number of colonies on some sample plates, which may have affected the results.This study suggests that silver impregnation of fabric is ineffective in reducing surface bacterial contamination of surgical scrub tops during use in a veterinary surgical suite. Furthermore, bacterial contamination of scrub suits present when they are first put on may persist through the working day, although silver impregnation does appear to be effective in reducing bacterial contamination on the scrubs during storage. The apparent lack of efficacy of silver in the face of ongoing bacterial contamination may be clinically significant, since in a busy theatre suite bacteria could be transferred between patients via the scrubs. Precautions such as wearing disposable plastic aprons during patient contact, regularly changing surgical scrubs during the working day and strict hand hygiene may be more effective in reducing bacterial contamination than buying specialist ‘antibacterial’ scrubs.None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.


To discern altered control of myocardial remodeling by PKG, HFPEF was compared with aortic stenosis and HF with reduced EF (HFREF).Prominent features of myocardial remodeling in heart failure with preserved ejection fraction (HFPEF) are high cardiomyocyte resting tension (Fpassive) -4 and capassive) . In expepassive) . The prepassive was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Cardiomyocyte Fpassive was higher in HFPEF (7.6±0.4 kN/m2) than in AS  and in HFREF . In-vitro administration of PKG acutely lowered cardiomyocyte stiffness in all groups with the largest decrement in Fpassive in DHF patients. PKG activity in myocardial tissue homogenates was significantly lower in HFPEF (5.11±0.62 pmol/min/mg) than in both AS  and HFREF . Immunohistochemical determination of myocardial PKG activity by pVASP/VASP ratio provided confirmatory evidence as it was also significantly lower in HFPEF (0.70±0.03) than in both AS  and HFREF . Lower PKG activity in HFPEF than in aortic stenosis or HFREF was associated with higher cardiomyocyte Fpassive (P<0.001) and related to lower cGMP concentration (P<0.001) and higher nitrosative/ oxidative stress (P<0.05). Reduced PKG activity and lower myocardial cGMP concentration in HFPEF did not result from altered myocardial sGC or PDE5A expression, which was similar in all groups nor from unequal BNP expression, which was comparable in HFPEF and AS. The downregulated cGMP-PKG signaling in HFPEF was therefore related to low myocardial nitric oxide bioavailability because of high nitrosative/oxidative stress.Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese (P<0.05) or had diabetes mellitus (P<0.05). Left ventricular myocardial biopsies were procured transvascularly in HFPEF and HFREF and perioperatively in aortic stenosis. Fpassive and was related to increased myocardial nitrosative/oxidative stress. The latter was probably induced by the high prevalence in HFPEF of metabolic comorbidities. Correction of myocardial PKG activity could be a target for specific HFPEF treatment.Low myocardial PKG activity in HFPEF was associated with raised cardiomyocyte F