Many caregivers with chronically ill relatives suffer from depression. However, the relationship of depression to other outcomes of chronic caregiving remains unclear. This study tested a hypothesized model which proposed that hours of care, stressful life events, social support, age and gender would predict caregivers' outcomes through perceived caregiver stress. Depression was expected to mediate the relationship between perceived stress and outcomes of chronic caregiving .The sample for this secondary data analysis consisted of 236 and 271 subjects from the Americans' Changing Lives, Wave 1, 1986, and Wave 2, 1989, data sets. Measures were constructed from the original study. Structural equation modeling was used to test the hypothesized model, and an exploratory structural modeling method, specification search, was used to develop a data-derived model. Cross-validation was used to verify the paths among variables.Hours of care, age, and gender predicted caregivers' outcomes directly or through perceived caregiver stress (p < .01). Depression mediated the relationship between perceived stress and psychological outcomes and explained 40% and 11% of the variance in self-esteem and marital satisfaction, respectively.Depression predicted psychological outcomes. Whether depression predicts physical health outcomes needs to be further explored. It is estimated that 31–55% of caregivers of chronically ill elderly relatives experience depression  and mostFactors that may be related to stress and depression in caregivers include hours of care, stressful life events, social support, age, and gender. Studies have found that hours of care were significantly related to caregivers' anxiety/depression and somatic symptoms ,11, and Social support may enhance the ability of the individual to cope with events or change the individual's cognitive appraisal of events . QuayhagAge has been shown to have indirect effects on depression through its influence on perceived stress, the coping process, and perceived efficacy . YoungerTsai et al. have suggested that stress and depression are emotional aspects of coping mechanisms and depression is the outcome of perceived caregiver stress . Stress Although the associations between depression and self-esteem and marital satisfaction have been examined extensively, only a few studies have been conducted in the context of caregiving. Caregivers have been shown to have lower self-esteem , and thiA recently developed Theory of Caregiver Stress  based onData for the study were obtained from the Americans' Changing Lives (ACL) Survey: Wave 1, 1986 (N = 3,617), and Wave 2, 1989 (N = 2,867) . The ACLOnly individuals with experience in caregiving to a chronically ill aged relative were included in the analyses reported here. This reduced the number of cases available for study to 335 from Wave 1 and 271 from Wave 2. The two samples were not completely independent because 99 cases were included in both waves; therefore, to ensure the independence of samples, these 99 cases were dropped from Wave 1. The final samples from Wave 1 and Wave 2 were thus 236 and 271, respectively.Since the study was a secondary data analysis, indicators of the study variables  were selected from the Americans' Changing Lives Survey questionnaires, based on face validity. That is, the questions selected gave the appearance of measuring the content of interest. Exploratory factor analysis, confirmatory factor analysis, and internal consistency tests were then conducted to confirm the underlying structures of established scales and develop outcome measures for the current study.Hours of care were the total hours estimated by the caregiver in the past year, categorized as less than 20 hours, 20 to 39 hours, 40–79 hours, 80–159 hours, and 160 hours or more. Providing more hours of care was expected to indicate more burden of caregiving.Stressful life events were measured by a 12-item checklist of negative or undesirable events, such as being robbed or burglarized, losing a job, being physically attacked, or experiencing the death of spouse, death of a parent, death of a close friend/relative, serious illness, life-threatening illness/accident, divorce/separation, serious financial problem, death of children, and other such events. Respondents were asked to report whether they had experienced any of these events within the past 2 years. A simple score, the stressful life events index, was created by summing the number of events reported by each respondent. A high score reflected more stressful life events.Social support was measured by two items: friends/relatives' love and care, and their willingness to listen. Alphas reliabilities were .73 and .74 for Wave 1 and Wave 2, respectively. Higher scores indicated greater support from friends/relatives. Demographic data included age, defined as the chronological age of the caregiver, and gender, coded as biological sex identity.Perceived caregiver stress was measured by one item asking how much stress the caregiver felt about caring for or arranging care for the elderly relative. Responses were on a 5-point scale ranging from not stressful to very stressful; a higher score reflected more perceived stress. Other studies have shown that stress was associated psychosocial well-being, such as depression ,43; in tDepression was measured by the 11-item Center for Epidemiological Studies Depression (CES-D) scale , which aPhysical function was defined as consisting of functional health, number of chronic illnesses, and self-rated health. Functional health was measured by asking the caregiver whether the caregiver was bedbound, and whether the caregiver had difficulty bathing, climbing stairs, walking, or doing heavy housework, and the degree of difficulty of these tasks. Higher scores reflected a higher level of physical function. The number of chronic illnesses was the sum of the following: arthritis or rheumatism, lung disease, hypertension, heart disease, diabetes, cancer, circulation problems, stroke, fracture, and urinary incontinence. A low score on this measure indicated high physical function. Self-rated health was measured by a single item that asked caregivers to rate their own health on a 4-point scale ranging from poor to excellent. A high score reflected high physical function.The caregiver's self-esteem was measured by five items: "I take a positive attitude toward self," "I am no good at all," "I see myself as a failure," "I have the feeling of being pushed around in life," and "I perceive myself able to solve problems." These items were measured on a 4-point scale ranging from strongly agree to strongly disagree. A higher score indicated higher self-esteem.Marital satisfaction was also measured by five items: "Overall satisfaction with relationship," "love and affection expressed from spouse or significant other," "spouse treats me well," "thinking about divorce or separation," and "things happened that I can never forget." Higher scores indicated more marital satisfaction.Cronbach's alphas for all measures were above the acceptable criterion of .70 in both waves except for self-esteem in Wave 1. However, that measure was on the margin of acceptance, at .68. Since Cronbach's alpha is a conservative estimate of internal consistency , the selUnivariate and bivariate analyses were used to examine the descriptive findings. To test the appropriateness of the indicators for each latent variable in both waves, the following procedures were used. First, a single indicator was extracted when applicable , and summary scale scores were used as single indicators. This strategy was used to reduce the number of parameter estimations in a complex model; it is considered appropriate when individual factor item loadings in a specific scale are high . Second,2], the goodness-of-fit index [GFI], and the adjusted goodness-of-fit [AGFI]) and comparative fit indices . Values of GFI, AGFI, RNI, and RNFI between 0.90 and 1.00 were considered to indicate a good fit between the model and the data [A covariance matrix derived from data in the Wave 2 sample was analyzed as input data in the process of model testing. Hypothesized models were tested using the maximal likelihood procedure in the LISREL statistics program. The model tests used absolute goodness-of-fit indices . Depression also differed significantly (p < 0.05). Respondents in Wave 1 reported more depression than those in Wave 2, with mean scores on the 11-item CES-D of 16.00 (SD = 4.14) and 15.15 (SD = 3.87) for Waves 1 and 2, respectively.2 = 237.22; d.f. = 73; GFI = .89; AGFI = .84; RNFI = .85).When the hypothesized model was tested to determine whether depression mediated the relationship between perceived caregiver stress and caregiving outcomes , the statistics showed a moderate fit between the model and the data , close to that of the measurement model, with an insignificant difference .Since the hypothesized model fit the data only moderately well, a data-derived model was built to compare with the hypothesized model. After all the insignificant paths in the hypothesized model had been dropped, paths were added at each step to improve the goodness-of-fit statistics based on the modification index and pre-set assumptions. The resulting data-derived model had a good fit with the data  declare that they have no competing interests.Author 1, PT, developed the research proposal, carried out the data analysis, interpreted the data and participated in the sequence alignment of the manuscript. Author 2, MMJ, participated in developing the research proposal and interpreting the data.The pre-publication history for this paper can be accessed here:


In mammals, males usually have one X and one Y chromosome and females have two X chromosomes. This crucial difference sets the sexes apart, but also creates a problem—female cells have the potential to turn out twice as much X-based gene product as necessary. Males with multiple–X chromosome syndromes face a similar problem. As if to avoid an overdose of X-related proteins, cells in the early embryo inactivate all but one X chromosome. The choice of which X (or Xs) to inactivate is apparently random, but once made, it persists across cell divisions and the specializations that determine a cell's ultimate fate, or type.Xist—which resides on the X chromosome—has a central role in X chromosome inactivation. It creates a special RNA molecule that spreads from its point of production down the length of the X chromosome, repressing its genes and inactivating the chromosome. After about one cell cycle, this gene silencing no longer requires Xist RNA. Daughter cells somehow remember which X to keep mute. In this month's PLoS Biology, geneticist Anton Wutz and colleagues at the Research Institute of Molecular Pathology in Vienna show that Xist expression during a critical period very early in embryonic development creates a chromosomal memory, independent of X silencing, that might help maintain X inactivation across cell generations.A gene called Xist gene mediates alterations to histones  along the X chromosome, which result in heterochromatin formation. As this structure is passed on to daughter cells, X silencing is perpetuated.The molecular underpinnings of X inactivation seem to center on histones, the protein spools around which DNA coils its length. DNA and histones form complexes called chromatin, which undergoes many structural modifications that have important effects on gene expression. For example, tightly packed chromatin inhibits gene expression in its closely curled segments. Not surprisingly, the inactivated X chromosome is coiled into this dense form, called heterochromatin. In the standing model of X inactivation, the Xist gene into the X chromosome of male mouse embryonic stem cells, so they could turn Xist expression “on” and “off” at will. The stem cells represent the earliest, unspecialized cells of a mouse embryo. Since the cells can be induced to differentiate in culture, they provide the opportunity to study the relationship between differentiation and X chromosome inactivation . Using this system, the authors have shown previously that Xist must act during a critical window very early during stem cell differentiation—within the first 24 hours. Wutz and colleagues now show that after that window the X chromosome inactivation can still be reversed, but after an additional 24 hours, it cannot. There appears to be a “memory” of Xist action, which leads to the permanent shutting down of the chromosome.To explore the molecular changes that mediate X chromosome inactivation, Wutz and colleagues inserted a special Xist and the establishment of irreversible silencing. By looking at the kinetics of histone modification, gene silencing, and Xist action, Wutz and colleagues further show that although certain histones are methylated at specific locations during this period in response to Xist, these modifications do not themselves constitute the chromosomal memory. The nature of the memory remains mysterious. Further experiments, perhaps looking at different histone modifications, will be required. Clarification of the events that lead to X inactivation will also improve our knowledge of how changes in the organization and structure of chromosomes can influence the activity of genes.The importance of this observation is that it establishes a new step in the process of X chromosome inactivation—between the action of


It is possible that the insulin secreting liver cells will be destroyed by the immune system because hepatocytes express major histocompatibility complex (MHC) class I molecules and exhibit constitutive Fas expression; moreover the liver has antigen presenting activity. Together with previous reports that proinsulin is a possible autoantigen in the development of Type 1 diabetes, the autoimmune destruction of insulin producing liver cells is a distinct possibility.The liver has been suggested as a suitable target organ for gene therapy of Type 1 diabetes. However, the fundamental issue whether insulin-secreting hepatocytes To address this question, transgenic Non-Obese Diabetic (NOD) mice which express insulin in the liver were made using the Phosphoenolpyruvate Carboxykinase (PEPCK) promoter to drive the mouse insulin I gene (Ins).in vivo and release it when exposed to 100 nmol/l glucagon in vitro. The amount of insulin produced was however significantly lower than that produced by the pancreas. The transgenic PEPCK-Ins NOD mice became diabetic at 20–25 weeks of age, with blood glucose levels of 24.1 ± 1.7 mmol/l. Haematoxylin and eosin staining of liver sections from these transgenic NOD PEPCK-Ins mice revealed the absence of an infiltrate of immune cells, a feature that characterised the pancreatic islets of these mice.The liver cells were found to possess preproinsulin mRNA, translate (pro)insulin These data show that hepatocytes induced to produce (pro)insulin in NOD mice are not destroyed by an ongoing autoimmune response; furthermore the expression of (pro)insulin in hepatocytes is insufficient to prevent development of diabetes in NOD mice. These results support the use of liver cells as a potential therapy for type 1 diabetes. However it is possible that a certain threshold level of (pro)insulin production might have to be reached to trigger the autoimmune response. Ferber also showed that this population of trans-differentiated liver cells was induced to produce the prohormone convertases PC1/3 and PC2 leading to complete processing of proinsulin. The amount of insulin produced was sufficient to ameliorate streptozotocin-induced hyperglycaemia in the mice.Genetic alteration of non-pancreatic cells in a diabetic person to synthesise, store and secrete insulin in the same manner as a pancreatic β cell is a potential therapy of type 1 diabetes. The hepatocyte has been suggested as a suitable target cell for such gene therapy . Such ce glucose . Moreove glucose . Further glucose  has receFor this gene therapy approach to be viable, the insulin producing liver cells must not be destroyed by the immune system or else this could lead to liver damage. This fundamental question has not been addressed by previous studies with (pro)insulin producing hepatocytes as diabetes in these models was induced chemically rather than by autoimmune means -9. The ain vivo, transgenic Non-Obese Diabetic (NOD) mice that express insulin in the liver were created using the Phosphoenolpyruvate Carboxykinase (PEPCK) promoter [To address this question promoter  to direcpromoter . HoweverThe liver of the transgenic PEPCK-Ins NOD mice were characterised with respect to insulin mRNA transcription, (pro)insulin content and (pro)insulin release. The blood sugar levels of the animals were monitored and the livers of the animals were analysed for any evidence of immune cell infiltration.The PEPCK promoter was generously donated by Dr R. W. Hanson (Case Western Reserve University, Cleveland, Ohio, USA). DNA modifying enzymes and competent bacteria for transformation were purchased from Promega (Madison, Wisconsin, USA). RPMI 1640, α-MEM and fetal bovine serum (FBS) were purchased from Trace Biosciences . Hybond-N+ nylon membrane for the Southern Blot and ribonuclease protection assay (RPA) was from Amersham International (Bucks, UK). Human insulin and rat insulin standards for the in-house radioimmunoassay (RIA) were obtained from Novo Nordisk (Bagsvaerd, Denmark).The transfection of the HEP G2 cells with the PEPCK-Ins transgene was carried out using Lipofectamine (GibcoBRL, Madison, Wisconsin, USA). Cells were trypsin harvested and seeded 24 hr prior to transfection at a density of 200,000 cells/well in 24 well tissue culture plates (Becton Dickinson, New Jersey, USA) to achieve 60–80% confluence at the time of transfection. 10μl of Lipofectamine was added to 300μl of serum free media and incubated at room temperature for 30 minutes. After the incubation, 300μl of DNA solution (3–4μg) was added and the lipid:DNA complex solution incubated at room temperature for 15 minutes. The cells were washed once with 1 ml of serum free medium prior to the transfer of 300μl of the lipid:DNA complex solutions to the cells. The cells were then incubated for 5 h at 37°C in an air incubator. The DNA/lipid complex media was removed and replaced with 2 mL of α-MEM 10% FBS which contains Antimycotic and Antibiotic solution (10μL/mL) (Sigma, St Louis, Missouri, USA). In addition, for the PEPCK-Ins transgene, a final concentration of 100 nM glucagon was added to the media to upregulate the PEPCK promoter. The cells were incubated at 37°C in an air incubator for 48–60 hr. After the incubation, the conditioned culture medium was collected and assayed for mouse (pro)insulin.(Pro)insulin was measured by an in-house RIA using rat insulin standard (Novo Nordisk Laboratories, Bagsvaerd, Denmark). The lowest value on the rat insulin standard curve used in the assay was 0.25 ng/ml. The intra and interassay coefficients of variance were <5% and 10% respectively.To direct the expression of the mouse insulin I gene to the liver of the transgenic mice, the 5' flanking sequences (-460 bp to +73 bp) of the rat PEPCK gene  were useThe mice were fed ad libitum with a standard diet and kept under a light-dark cycle of 12 h in compliance with the animal ethics of our institution. The facilities used to breed and maintain the mice were specific pathogen free, with air passaged through a HEPA filter. The transgenic PEPCK-Ins NOD mice and the wild type littermates were monitored for natural development of diabetes. The diabetic PEPCK-Ins transgenic NOD mice were maintained by daily insulin injections and sacrificed 4 weeks after they became diabetic.Blood glucose levels of the wild type as well as the transgenic NOD mice were performed using the Medisense Precision QID Blood Glucose Sensor System (Bedford, Massachusetts, USA). The mice were bled by pricking the tail vein and 5μl of the blood placed on to the Precision Plus blood glucose electrode. A mouse was classified as being diabetic if a reading of ≥ 15 mmol/l was obtained on more than one occasion.- Reverse Transcriptase from GibcoBRL (Grand Island, New York, USA) to synthesize the first strand cDNA as recommended by the manufacturer. The cDNA sample was then used as a template in PCR amplification (30 cycles) using primers specific for mouse insulin I mRNA. The sequence of primers used were:The total RNA from liver and pancreas was isolated using TRIzol (Life Technologies, Grand Island, New York, USA) . The det(Forward) TAA CCC CCA GCC CTT AGT GAC CAG CTA TAA(Reverse) AAA GTT TTA TTC ATT GCA GAG GGG TGG GGCThe PCR products were run on a 1.2% Tris Acetate EDTA agarose gel, photographed and analysed using the Gel Doc 1000 system .in vitro transcription method using the DIG RNA labeling kit (SP6/T7) from Boehringer Mannheim (Mannheim, Germany), which utilises DIG-dUTP, according to the manufacturer's recommendations.Insulin mRNA in the samples was also visualised using the ribonuclease protection assay (RPA) method. The plasmid used as the template to transcribe the riboprobe for mouse insulin I was a generous gift from Chirgwin J. M. (San Antonio, Texas, USA). The Digoxigenin (DIG) labelled insulin riboprobe was synthesized by the Hybridisation and RNase treatment were performed using the RPA II kit from Ambion according to the manufacturer's recommendations. Next the samples were loaded and run on a 6% polyacrylamide/8 M urea denaturing mini-gel in Tris borate EDTA buffer. The RNA bands on the gel were transferred onto a nylon membrane by using an electro-gel transfer apparatus and the membrane was fixed by using an UV cross linker (Hybaid, UK).For the visualisation of the RNA bands on the nylon membrane, the DIG Wash and Block Buffer Set and the DIG Chemiluminescent Detection Kit, both of which were purchased from Boehringer Mannheim, were used. The membrane was then exposed to a standard Kodak X-ray film for 30 minutes. The film was subsequently analysed using the Gel Doc 1000 system.3 explants and spread on a filter paper placed on a sterile wire grid in the inner well of the organ culture dish such that the tissue was exposed to air above and the RPMI 1640 10% FBS medium below. Sterile saline was placed in the outer well to maintain a humidified environment. The tissue in the organ culture dishes were incubated for 24 hr in a humidified 5% CO2 and air incubator at 37°C. Culture supernatant from all media changes of respective wells were pooled separately and kept at -20°C until the RIA was performed. The results were expressed per mg of tissue.Pancreatic and hepatic tissue organ cultures were performed using organ culture dishes from Becton Dickinson (New Jersey, USA) . Briefly(Pro)insulin was extracted by homogenising a weighed sample of liver or pancreas in acid-ethanol (solution of 0.18 mol/l HCl in 70% ethanol) and incubating overnight at 4°C. The next day, the samples were spun down and the supernatant removed. The (pro)insulin content was determined by RIA and the results expressed per mg tissue.To detect the transcription expression of (pro)insulin, in situ hybridisation (ISH) was performed on pancreatic and liver tissues of diabetic transgenic PEPCK-Ins NOD mice and diabetic wild type NOD mice using a modification of a previously described method (20). Briefly, 4μm thick paraffin sections were deparaffinized with xylene, ethanol and air dried. The sections in 0.01 mol/l citric buffer, pH 6.0 were then treated with microwave irradiation for 10 min. Following this procedure the sections were treated with proteinase K (Invitrogen, Carlsbad, CA, USA) 1μg/ml for 15 min at 37°C. They were washed 3 times with phosphate buffered saline for 5 min and dried with ethanol. The sections were then hybridized with [35]-S-labelled riboprobes (1μg/ml) overnight at 57°C. The [35]-S-labelled riboprobes were synthesized by in vitro transcription (SP6/T7) incorporation of [35]-S-dUTP using RNA polymerase (Roche, Mannheim, Germany). The template used was mouse insulin I cDNA that was generously donated by Chirgwin JM (San Antonio, Texas, USA). The hybridisation buffer consisted of a 25μl hybridisation cocktail . After hybridisation, the slides were soaked in 2X standard saline citrate (SSC), rinsed with deionised water and then treated with RNase A (20μg/ml) for 30 min at room temperature. High stringency washes were then performed on the slides with 2XSSC followed by 0.2XSSC. The slides were then dried by ethanol, air dried for 20 min and the results viewed by NBT2 emulsion autoradiography (Kodak, Rochester, New York). For controls, sense-probe instead of anti-sense was used for hybridisation. The photos were taken using the Olympus IX70 microscope (Melville, New York) with a dark field condenser.2O2, and then incubated in PBS containing 10% goat serum or 1% BSA for 20 minutes at room temperature to block nonspecific binding.Sections of the liver and pancreas from the transgenic mice were washed twice in Phosphate Buffered Saline (PBS), fixed in 10% formalin overnight and embedded into paraffin. Consecutive 5μm sections were cut and placed on poly-L-lysine-coated slides. After dewaxing and serial alcohol rehydration, the tissue sections were treated with HFor insulin staining, primary insulin antibody  at 1:1250 was added and the sections were incubated overnight. The next morning the sections were washed in PBS and incubated with anti-guinea pig IgG (1:400) at room temperature for 20 minutes. Thereafter the sections were washed again with PBS, and biotinylated anti-rabbit / mouse antibody added to the sections for 15 minutes followed by streptavidin-peroxidase conjugate for a further 15 minutes. Finally, the sections were treated with substrate-chromogen AEC. A standard concentration (0.1%) of haematoxylin was added as a counterstain. The primary antibody was omitted for the negative control. The haematoxylin and eosin staining of sections was performed automatically by the Jung Autostainer XL machine (Leica, New Jersey, USA).The log rank-test was used to determine whether the occurrence of diabetes in the transgenic mice was significantly different from the wild type NOD mice. The non-parametric t-test (Mann-Whitney U test) was used to determine whether the blood glucose levels of the diabetic transgenic and wild type NOD mice were significantly different.To confirm that the PEPCK-Ins transgene Figure  was funcFrom a series of two microinjections, 43 mice were obtained of which 4 were positive for the PEPCK-Ins transgene when analysed by PCR Figure  and SoutOf the two lines only one was found to transcribe insulin mRNA in the liver, as determined by mouse insulin I RT-PCR Figure  and mousin vivo. This was not possible since any (pro)insulin released from the liver would be indistinguishable from that secreted from pancreatic β cells. Two methods were used to analyse if (pro)insulin was produced from liver tissue removed from the mice. The liver was both extracted for its hormonal content, and placed in organ culture with analysis of the conditioned culture medium for (pro)insulin. In both cases, (pro)insulin was measurable. The (pro)insulin content was 0.5 ± 1 ng/mg (n = 5), which is equivalent to 8% of the (pro)insulin content of the pancreas (Table The ability of the liver cells from PEPCK-Ins positive transgenic mice to produce (pro)insulin was examined on liver removed from mice that had been sacrificed. Ideally production of (pro)insulin from the liver would be sought In situ hybridisation confirmed the presence of (pro)insulin mRNA in the pancreatic and liver sections of diabetic PEPCK-Ins transgenic mice Figure  and 3C bOne out of 14 males (7%) and four out of 25 females (16%) of the F1 and F2 progeny of the PEPCK-Ins transgenic NOD mice became diabetic insulin content of the liver of the diabetic mice was determined. This was similar to that from the liver of normoglycaemic transgenic mice Table . (Pro)inAt the end of the study, 5 heterozygous transgenic PEPCK-Ins NOD mice had become diabetic. Nevertheless, preproinsulin mRNA was localised to the hepatocytes by in situ hybridisation and was detected in total RNA extracts of the livers of these transgenic mice by both RT-PCR and RPA. (Pro)insulin was detected in acid ethanol extracts of the livers from the transgenic mice by RIA. Furthermore, (pro)insulin was released from explants of transgenic liver placed in organ culture. These results indicate that the liver cells from these transgenic PEPCK-Ins NOD mice did synthesise and produce (pro)insulin.Histological examination of the livers of the diabetic transgenic mice showed no infiltration of the liver by immune cells even though insulitis was observed in the pancreatic sections. This indicates that hepatic insulin production did not cause the development of tolerance to insulin in these PEPCK-Ins transgenic NOD mice. The lack of infiltration of immune cells in the livers of the diabetic transgenic mice also suggests that the mouse insulin I mRNA transcribing hepatocytes were not targeted or destroyed by the immune system. In addition, as the transgenic mice were sacrificed 4 weeks after the onset of diabetes, this reduces the possibility of delayed destruction of the (pro)insulin expressing hepatocytes. During this time, hepatic insulin expression was at least equal to that found in the pancreas. At the apparently low levels of transgene expression, these results suggest that (pro)insulin is not targeted by the immune system in this transgenic PEPCK-Ins mouse model superimposed on the autoimmune diabetes model of the NOD mouse. Likewise, Lipes et al reported that when pituitary cells in transgenic NOD mice were made to produce insulin, the cells were not targeted by the immune system even though the pancreatic β cells were. No cellular infiltrate also was shown when the pituitary cells that produced (pro)insulin were taken outside the blood brain barrier and transplanted beneath the renal capsule of NOD mice . Our datThe blood glucose levels of the diabetic transgenic NOD PEPCK-Ins mice (24.8 ± 1.9 mmol/l) were significantly lower (P = 0.004) than those of the diabetic wild type NOD mice (>33 ± 2.1 mmol/l). This is consistent with some (pro)insulin being released from the liver. These results are in agreement with those of Valera et al in which the majority of their high copy number PEPCK human insulin C57BL/6 mice also became diabetic when injected with streptozotocin despite the constitutive release of human (pro)insulin from the livers of the mice (7). Similarly, the transgenic mice which Valera et al made diabetic by streptozotocin injections also had lower blood glucose levels compared to the diabetic wild type NOD mice suggesting that the constitutive hepatic insulin release lowered the blood glucose levels of the diabetic mice but not to normal. This lack of therapeutic effect could be due to the fact that the PEPCK promoter is induced by glucagon and cyclic AMP. The high glucose levels in the diabetic transgenic mice might have resulted in the inhibition of endogenous glucagon release and thus shutting down (pro)insulin production in the liver.However it is possible that in some of our transgenic mice, enough (pro)insulin was produced to prevent diabetes. This would explain the significantly lower incidence of diabetes in the transgenic PEPCK-Ins (13%) compared to the wild type (28%) NOD mice (P < 0.001). One possibility is a protective effect of insulin, whether by allowing exhausted beta cells to rest or by altering the makeup of the T cells in the pancreas destroying the β cells there. The first reason was the basis for the large North American trial in pre-diabetic people in the late 90's with small doses of parenteral insulin . The secIt is possible that a critical amount of (pro)insulin needs to be produced for an autoimmune effect to be observed. The (pro)insulin content of the transgenic liver cells in normoglycaemic mice was much lower than that of a pancreatic β cell and was also lower than that in the liver of PEPCK-human insulin C57BL/6 transgenic mice produced by Valera et al , resultiAnother possible explanation for the lack of an autoimmune effect is that presentation of insulin peptides by hepatocytes might be limited by the ability of the liver cells to cleave proinsulin. The enzymes responsible for this in the β cell, prohormone convertases (PC) 1/3 and PC2, are absent from normal liver cells, but the endopeptidase furin is present . CleavagIn summary, we have shown that transgenic NOD mice that produce (pro)insulin in their liver do not develop cellular infiltration of their liver when autoimmune destruction of pancreatic β cells occur. Furthermore the expression of (pro)insulin in hepatocytes is insufficient to prevent development of diabetes in NOD mice. These results offer hope that eventually liver cells, or a subpopulation of them, may be of value as a therapy for type 1 diabetes.


The formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites.2) binding region of gelsolin (an uncapper of actin filaments) blocks granule mobilization as well as secretion of oxygen radicals. The inhibitory effect of PBP10 is, however, receptor specific and affects the FPRL1-, but not the FPR-, induced cellular response. The transient rise in intracellular calcium induced by the active receptors is not affected by PBP10, suggesting that the blockage occurs in a parallel, novel signaling pathway used by FPRL1 to induce oxygen radical production and secretion. Also the FPR can activate neutrophils through a PBP10-sensitive signaling pathway, but this signal is normally blocked by the cytoskeleton.We report that a cell permeable ten amino acid peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate (PIP2-binding peptide PBP10 selectively inhibits FPRL1-mediated superoxide production and granule mobilization. Furthermore, the activity of this novel PBP10 sensitive pathway in neutrophils is modulated by the actin cytoskeleton network.This study demonstrates that the two very closely related chemoattractant receptors, FPR and FPRL1, use distinct signaling pathways in activation of human neutrophils. The PIP Despiteubstrate . The dis calcium . It has  calcium . Further calcium -15. Howe calcium ) derived50 values .Despite the indistinguishable activation by FPR and FPRL1 shown in Fig. 2) binding region segment 2 of the cytoskeletal protein gelsolin [2 as substrate to generate the signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP3) [The amino acid sequence of PBP 10 peptide corresponds to the phosphatidylinositol 4,5-bisphosphate (PIPgelsolin . Followigelsolin , an enzye (PIP3) . The oxi9–25 (Table The inhibition induced by PBP10 on neutrophil oxidase activity was linked to the receptor FPRL1 rather than to an FPRL1-specific agonist, since the same inhibition pattern was obtained when WKYMVM was replaced by serum amyloid A (SAA) (Table 2 depends on the peptide sequence, as truncation of the peptide either at the N terminus or C terminus reduces the PIP2 binding affinity [50 = 1 μM compared to 0.05 μM for PBP10) suggesting that PIP2 binding is of importance for inhibition of the neutrophil oxidase response induced by FPRL1.Interaction of PBP10 with PIPaffinity . Accordi2 binding capacity but is also membrane permeable. This feature has been shown to be coupled to the rhodamine B part of the molecule, i.e., the unconjugated peptide QRLFQVKGRR exhibits the same PIP2-binding characteristics as PBP10 but lacks the ability to cross the plasma membrane [The PBP10 peptide possesses not only PIPmembrane . The unc2 is utilized for generation of IP3 that induces a transient increase in intracellular calcium [2+ with EGTA (data not shown), suggesting that the rise in intracellular free Ca2+ relates to a mobilization from calcium storing organelles rather then from an influx through ion channels in the plasma membrane. Treatment of the cells with PBP10 prior to stimulation did not alter the calcium responses, neither induced by fMLF nor by WKYMVM . Interestingly, the receptor selectivity of PBP10's inhibitory effect was lost when the receptors were first uncoupled from the cytoskeleton by cytochalasin B Fig. . In accoThe two formyl peptide receptor family members FPR and FPRL1 possess a high degree of amino acid identity in the signaling cytoplasmic domains , and the2 binding region segment 2 of the cytoskeletal protein gelsolin [2 would be the phosphoinositide remodeling enzyme PI3K that converts PIP2 into phosphatidylinositol 3,4,5-trisphosphate (PIP3) which is of importance for cell locomotion and the associated dynamic reorganization of cytoskeletal components. The precise target for PIP3 has however not yet been defined [Despite the indistinguishable activation by FPR and FPRL1, functional differences between these two highly homologous receptors emerge when they are challenged by the membrane permeant polyphosphoinositide-binding peptide PBP10 (rhodamine B-QRLFQVKGRR) prior to activation. The FPRL1-mediated neutrophil activity was totally inhibited by the peptide PBP10 whereas there was no effect on FPR-mediated responses. As mentioned, the amino acids in the PBP 10 peptide correspond to the PIPgelsolin . One of  defined . The inh2 binding activity but also crosses the cell membrane of neutrophils and other cells. Interaction of rhodamine labeled peptides with PIP2 depends on the peptide sequence, and truncation of the peptide reduces the PIP2 binding affinity [2-binding characteristics as PBP10 but lacking the ability to cross the plasma membrane [2 binding is of importance for peptide-induced inhibition.The basic properties of PBP 10 have been described earlier ,21,34, amembrane  had no e2 is utilized for generation of IP3 which in turn is responsible for inducing transients in intracellular calcium [2+ elevation has been claimed to be required but not sufficient for the generation of an NADPH-oxidase activating signal from FPR [2+ [2+ levels from a requirement for a Ca2+ transient, and we have earlier shown that receptor mobilization can occur and the oxidase can be activated without any transient rise in cytosolic Ca2+ [2+ induced by WKYMVM, the secretory response was blocked and PBP10 selectively inhibited the FPRL1 induced granule mobilization.Plasma membrane localized PIP calcium . Triggerfrom FPR ,24. Likefrom FPR , has bee FPR [2+ ,27. Theslic Ca2+ ,35,36. DThe GPCR family is very diverse and the transmission of signals by such receptors is a critical function in many cell/organ systems. Signaling through GPCRs is highly complex, evidently not only with respect to the wide variety of mechanisms that regulate different functional responses , but als322FPR336) has a fairly high (45–50%) identity with the actin-binding cytoskeletal proteins vinculin and coronin, and this region also participates in FPR interaction with the G-protein [iα2 complex of the pertussis toxin sensitive G-proteins and, to a lesser extent Giα3 [Regulation of FPR signaling has been suggested to involve mechanisms that depend on direct receptor interaction with the membrane cytoskeleton . It is w-protein . It is oent Giα3 . The molent Giα3 . It is hThe relation of the biochemical/biophysical activities of the PBP10 peptide to its effect on the FPRL1-triggered cell function is not obvious and is likely to be complex. The sensitivity to PBP10 seems to be unique to FPRL1, but this receptor is expressed also in other cells such as astrocytes, neuroblastoma, and microglia cells . FPR is The neutrophil formyl peptide receptor family members FPR and FPRL1 share 69% of amino acid identity and mediate almost indistinguishable cellular responses. Thus, the assumption that FPR and FPRL1 use the same signaling pathways has been generally accepted. However, in this study we clearly demonstrate a fundamental difference in intracellular signaling between these two very closely related neutrophil formyl peptide receptor members, one being PBP10 sensitive and the other not. This novel PBP10 sensitive signaling pathway utilized by FPRL1 is also used by FPR but only when the cytoskeleton network is disrupted.7 /ml) in Krebs-Ringer phosphate buffer containing glucose (10 mM), Ca2+ (1 mM), and Mg2+ (1.5 mM) . The cells were stored on melting ice and used within 120 min of preparation.Neutrophil granulocytes were isolated from buffy coats obtained from healthy adults. After dextran sedimentation at 1 × g, hypotonic lysis of the remaining erythrocytes, and centrifugation in a Ficoll-Paque gradient , the neu2 (WKYMVM) and the annexin I peptide (annexin9–25 Ac-QAWFIENEEQEYVQTVK) were synthesized and HPLC-purified by Alta Bioscience (University of Birmingham, Birmingham, United Kingdom), and Ross-Petersen ApS (Holte, Denmark). The formylated peptide N-formyl-Met-Leu-Phe (fMLF), C5a, and, phorbol myristate acetate (PMA) were from Sigma Chemical Co. (St. Louis, Missouri). IL-8 was from R&D systems (Minneapolis, MN) and dissolved in KRG containing 0.5% (w/v) bovine serum albumin. Serum amyloid A (SAA) was from Pepro Tech Inc. (UK). The C5a and SAA were dissolved in water while the other peptide agonists were dissolved in dimethyl sulfoxide to 10-2 M and stored at -70°C until use. Further dilutions were made in KRG.The hexapeptide Trp-Lys-Tyr-Met-Val-Met-NHThe peptide QRLFQVKGRR (gelsolin residues 160–169) and related peptides were prepared by solid phase peptide synthesis and coupled to rhodamine as described earlier [5–106 neutrophils, horseradish peroxidase (4 U) and isoluminol (20 μM) with and without cytochalasin B or PBP10. The measuring tubes were equilibrated for 5 to 10 minutes at 37°C and the cells were activated by addition of a receptor specific peptide agonist, fMLF for FPR and WKYMVM for FPRL1. The light emission was recorded continuously. Details about the CL technique are given in [Neutrophil production and release of superoxide anions was measured by means of an isoluminol-enhanced chemiluminescence (CL) assay . The CL given in .7/ml in Ca2+-free KRG supplemented with bovine serum albumin (BSA, 0.1%) were incubated with the acetoxymethylated derivative fura-2/AM (2 μM) at room temperature for 30 minutes. The cells were washed twice and resuspended in KRG, adjusted to 2 × 107/ml and kept protected from light on ice until use. Cells with or without PBP10 were equilibrated for 5 minutes at 37°C, after which the peptide agonist was added. The fura-2 fluorescence was followed with a luminescence spectrometer  using excitation wavelengths of 340 nm and 380 nm, and an emission wavelength of 510 nm and the [Ca2+]i was calculated as described earlier [Neutrophils at a density of 2 × 10 earlier .3), after which the cells were incubated at 4°C with phycoerythrin-conjugated anti-CR3 antibodies  before analysis using a FACScan (Becton Dickinson, Mountain View, CA).The exposure of CR3 (CD11b/CD18) on the neutrophil cell surface was assessed by immunostaining and FACS-analysis. Cells challenged with peptide agonists with or without PBP10 were fixed with ice-cold paraformaldehyde and washed with FACSwash (PBS, 0.02% NaNHorseradish peroxidase (HRP) was from Boehringer-Mannheim (Mannheim, Germany). Isoluminol, cytochalasin B, and pertussis toxin were purchased from Sigma. Dextran and Ficoll-Paque were from Pharmacia , and Wortmannin was from Calbiochem (La Jolla, CA). Fura-2AM was from Molecular Probes Inc. (Eugene, OR).Two-tailed, paired Students's t-tests were performed to determine statistical significance, and a P-value of < 0.05 was regarded as significant.GPCR – G-protein coupled receptorPBP – Phosphoinositide binding peptideFPR – formyl peptide receptorFPRL1 – formyl peptide receptor-like 1PIP2 – phosphatidylinositol 4,5-bisphosphatePI3K – phosphatidylinositol 3-kinasefMLF – formyl-methionly-leucyl-phenylalaninThe scientific question raised in the paper was formulated during discussions between HF and CD, about the mechanisms behind receptor activation/deactivation/reactivation (see ref ). HF was


How does your brain pass the time while you're sleeping? If you've ever wrestled the demons of insomnia, you know what sleepless nights can do to your mental agility. Sleep cycles in mammals are characterized by two distinct, successive sleep stages: slow wave and rapid eye movement (REM). Both stages of sleep have uniquely associated electrical activity in the brain, which neuroscientists can measure by placing elec-trodes on the brain during sleeping and waking states. What neuroscientists can't easily measure is the purpose of these two sequential sleep stages.The notion that sleep helps to improve memory was introduced over 80 years ago. Since then, several studies have demonstrated that sleep deprivation following the acqui-sition of a new memory strongly impairs its consolidation. Insight into the mechanisms underlying this effect came from the observation that neuronal activity patterns detected during waking reappear during ensuing sleep, suggesting that newly acquired “memory traces” may be replayed in the brain to solidify neural connections and thus “consolidate” memory. These reverberating patterns of activity have been observed in both mammals and birds, pointing to a very general biological phenomenon.Still, the relationship between brain reverberation and memory consolidation remains unclear for a number of reasons. First, studies to date have observed only subtle, short-lived reverberations lasting less than an hour and can't explain the memory-disrupting effects of sleep deprivation applied several hours and even days after initial memory encoding. And since brain reverberation in mammals has only been investigated in the hippocampus and cerebral cortex, it is unclear whether the phenomenon is specific to this neural circuit or is a more general property of the brain. Furthermore, reverberation studies have so far relied on neural activity measured in animals that were highly trained on specific laboratory tasks and therefore may simply not be representative of the acquisition of new memories. Finally, experience-dependent neural reverberation has been detected in both phases of sleep as well as waking, but no quantitative comparison of the different contributions of each state has been established.In a study designed to address these concerns, Sidarta Ribeiro and his colleagues at Duke University in Durham, North Carolina, recorded over a hundred neurons continuously over the course of the normal sleep--wake cycle in rats, focusing on four major forebrain areas that are essential for rodent-specific behaviors. Halfway through the recording time, animals were transiently allowed to explore four strictly novel objects, each of them designed to provide different spatial and sensory cues. The researchers found that in all the forebrain areas examined the neuronal firing patterns recorded when the rats initially explored the new objects reverberated for up to 48 hours after these objects were removed. The reverberation of neuronal activity sampled when rats explored familiar environs was insignificant. Reverberation was most significant during slow-wave sleep (a state that accounts for nearly 40% of a rat's life), decreased during waking periods, and was highly variable during REM sleep.In this study, Ribeiro et al. demonstrate that long-lasting neuronal reverberation following novel waking experiences can occur in several forebrain sites and is strongly enhanced during slow-wave sleep. Because neuronal reverberations are sustained for long periods, this may provide a mechanism to recall and amplify memories until they are effectively stored. On the basis of differences observed between REM and slow-wave sleep in this and previous studies, the authors propose that the two phases of sleep play separate and complementary roles in memory consolidation. Thus, the two stages of sleep give the brain a chance to process the novel events of the day in peace.


A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Δ30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate.A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Δ30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys.Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Δ30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Δ30, designated rDEN2Δ30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Δ30 in SCID-HuH-7 mice.The rDEN2Δ30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for The rDEN2Δ30 and rDEN2Δ30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine. The increased prevalence of disease caused by the mosquito-borne dengue (DEN) viruses  has intensified the effort to generate a vaccine that would both confer protection and be economically feasible for use in countries with limited resources for healthcare . Risk faImmunity to the DEN viruses is primarily mediated by neutralizing antibodies directed against the envelope (E) glycoprotein, and most vaccine strategies aim to induce antibody against this major protective antigen. Live attenuated tetravalent vaccines appear to be the best vaccine candidates since they are economical to manufacture and they induce long-term immunity with the live attenuated yellow fever virus vaccine serving as a successful model flavivirus vaccine . SeveralWe have previously described attenuated and immunogenic monovalent vaccine candidates for DEN-1, DEN-2, DEN-3, and DEN-4 that were generated by two distinct recombinant methodologies. Using the first methodology, nucleotides 10478–10507 were deleted from the 3' UTR (Δ30) of a wild type cDNA clone for DEN-4 to generate a vaccine candidate, rDEN4Δ30, which is safe, attenuated, and immunogenic in rhesus monkeys and humans . IncorpoWe have found it prudent to pursue several strategies to develop a live attenuated virus vaccine for each dengue serotype recognizing that it has been a challenge to achieve a satisfactory balance between attenuation and immunogenicity ,18-20. TVero cells (African green monkey kidney) were propagated in OptiPro SFM (Invitrogen, Grand Island, NY) supplemented with 4 mM L-glutamine (Invitrogen). HuH-7 cells (human hepatoma) were maintained in D-MEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1 mM L-glutamine and 0.05 mg/ml gentamicin (Invitrogen). C6/36 cells  were maintained at 32°C in Minimal Essential Medium (MEM) containing Earle's salts and 25 mM HEPES buffer (Invitrogen) and supplemented with 10% FBS, 2 mM L-glutamine, and 0.1 mM non-essential amino acids (Invitrogen).Aedes albopictus mosquitoes, and subsequent passage in C6/36 cells before determination of genomic sequence.A dengue virus type 2 isolate, Tonga/74, was provided by Dr. Duane Gubler (CDC, Fort Collins, CO). The virus was isolated during a 1974 dengue outbreak in the South Pacific island of Tonga . The virViral RNA was isolated from DEN-2 Tonga/74 wild type virus using the QIAamp Viral RNA mini kit . Reverse transcription was performed using random hexamer primers and the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Overlapping PCR fragments of approximately 2000 base pairs were generated using DEN-2 specific primers and Advantage cDNA polymerase . Both strands of the resulting PCR fragments were sequenced directly on a 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) using DEN-2 specific primers in BigDye terminator cycle sequencing reactions (Applied Biosystems) and the results were assembled into a consensus sequence. To determine the nucleotide sequence of the genomic 5' and 3' regions, the 5' cap nucleoside of the viral genome was removed with tobacco acid pyrophosphatase (Epicentre Technologies, Madison, WI), followed by circularization of the genome using RNA ligase (Epicentre Technologies). An RT-PCR fragment spanning the ligation junction was generated and sequenced using DEN-2 primers. For the DEN-2 Tonga/74 consensus sequence, GenBank accession number AY744147 was assigned.SpeI restriction site at genomic nucleotide 2353; Fragment R was modified to contain a SpeI restriction site also at genomic nucleotide 2353, and, to stabilize the eventual full-length clone, two additional mutations at nucleotides 2362 – 2364 and 2397 were created to ensure that translation stop codons were present in all reading frames other than that used to synthesize the virus polyprotein; Fragment A was modified at nucleotide 3582 to ablate a naturally occurring SpeI restriction site and at nucleotide 4497 to ablate a naturally occurring KpnI restriction site; Fragment C was modified at nucleotide 9374 to ablate a naturally occurring KpnI restriction site; and Fragment Y, representing the 3' end of the genome was abutted to a KpnI restriction site. All mutations introduced into the cloned cDNA fragments were translationally-silent, thereby preserving the wild-type polyprotein sequence. Each fragment was added incrementally between the AscI and KpnI restriction sites of DEN-4 cDNA clone p4 (GenBank accession number: AY648301) to generate a full-length DEN-2 cDNA clone (p2) with the same vector background successfully used to generate rDEN-4 and rDEN4Δ30 virus [cDNA fragments of DEN-2 Tonga/74 were generated by reverse-transcription of the genome as indicated in Figure 30 virus . cDNA clUsing site-directed mutagenesis, an attenuating amino acid change characterized in the NS3 gene of DEN-4  was introduced into the p2Δ30 cDNA clone . A mutagAcc65I (isoschizomer of KpnI which cleaves leaving only a single 3' nucleotide) and were transcribed in vitro using the AmpliCap SP6 Message Maker kit (Epicentre Technologies, Madison, WI). Purified transcripts were then transfected into Vero or C6/36 cells. Viruses recovered in C6/36 cells were passaged 3 times in Vero cells, and all viruses were biologically cloned by terminal dilution in Vero cells. The genomes of recombinant viruses used to infect rhesus monkeys were completely sequenced as described above to identify adventitious mutations that had accumulated during transfection and biological cloning.cDNA clones were linearized with Prkdcscid) (Taconic, Germantown, NY) were injected intraperitoneally with 107 HuH-7 cells suspended in 0.2 ml phosphate-buffered saline. Tumors were detected in the peritoneum, and mice were infected by direct inoculation of the tumor with 104 PFU of virus in 0.05 ml Opti-MEM (Invitrogen). On day 7 post-infection, serum was obtained from cardiac blood and stored at -70°C. Virus titer in serum samples was determined by plaque assay in Vero cells.Four to six week-old SCID mice (Tac:Icr:Ha(ICR)-5 PFU virus diluted in L-15 medium (Invitrogen) or with a mock inoculum. Serum was collected on days 0–6, 8, 10, 12 and 28 after inoculation and stored at -70°C. Virus titer was determined for each serum sample by plaque assay in Vero cells and serum neutralizing antibody titer was determined for serum from days 0 and 28 by plaque reduction neutralization test. On day 28, monkeys were challenged with 105 PFU of DEN-2 Tonga/74, and serum was collected on days 29–34, 36, and 56. Virus titer was determined for serum from days 28–34 and 36 and serum neutralizing antibody titer was determined for serum from day 56.The DEN-2 viruses were evaluated in rhesus macaques using established methods . DEN virAedes aegypti and Toxorynchites amboinensis mosquitoes was evaluated as previously described [A. aegypti were fed blood meals containing serial 10-fold dilutions of virus. After 21 days, viral antigen was detected in head and midgut preparations by immunoflourescence assay using DEN-2-specific hyperimmune mouse ascitic fluid and fluorescein isothyocyanate conjugated goat anti-mouse IgG (KPL, Gaithersburg, MD), and the mosquito infectious dose-50% (MID50) was determined. T. amboinensis were inoculated intrathoracically with a 0.2 ul dose containing serial ten-fold dilutions of virus and incubated for 14 days. Head preparations were made and antigen visualized as described above.Replication in escribed . Briefly10PFU/ml in Vero cells.A full-length cDNA clone, p2, was constructed that matched the genomic consensus sequence of the American genotype DEN-2 isolate, Tonga/74, with the exception of translationally-silent modifications made to facilitate cloning Figure . The pre10PFU/ml serum in SCID-HuH-7 mice, and an att phenotype in SCID-HuH-7 mice has been shown to be a predictor of reduced replication in rhesus monkeys [10PFU/ml (Table P < 0.05), and was similar to that observed for the well-characterized rDEN4Δ30 virus [As an initial evaluation of replication of the DEN-2 Tonga/74 virus and the rDEN-2 viruses, replication in SCID mice transplanted with HuH-7 human hepatoma cells (SCID-HuH-7 mice) was tested. Wild-type viruses from each DEN serotype have been shown to replicate to approximately 6.0 log monkeys ,14,15,17Aedes aegypti fed on an infectious bloodmeal  and for Toxorynchites amboinensis inoculated intrathoracically  were lower than DEN-2 and rDEN-2, the differences were not as dramatic as had been observed for rDEN1Δ30 and rDEN4Δ30 when compared to their parent viruses [The replication (viremia), immunogenicity, and protective efficacy of the DEN-2 viruses in monkeys were studied. Monkeys inoculated with the DEN-2 Tonga/74 wild-type isolate were viremic for an average of 4.5 days with a mean peak titer of 2.1 log viruses ,14. The att mutation that has been characterized in another DEN serotype was imported into a homologous region in DEN-2. One such mutation, the 4995 mutation in DEN-4 NS3 at amino acid 158 (Ser→Leu), was previously incorporated into the DEN-4 vaccine candidate, rDEN4Δ30, and found to further attenuate the virus for SCID-HuH-7 mice and rhesus monkeys [10PFU/ml . The virus titer of rDEN2Δ30-4995 virus in SCID-HuH-7 mice was over 60-fold reduced from that of the rDEN-2 parent virus.Replication in the SCID-HuH-7 mouse model was used as an initial assessment of the rDEN2Δ30-4995 virus phenotype. Table Development of a live-attenuated tetravalent dengue vaccine has been complicated by two major factors. First, monovalent vaccine candidates that exhibit a satisfactory balance between attenuation and immunogenicity have been difficult to identify ,18-20. S10PFU/ml [10PFU/ml. However, the 10-fold reduction of replication of rDEN4Δ30 and rDEN2Δ30 in SCID-HuH-7 mice, compared to that of their respective wild type parents, was similar. To date, rDEN4Δ30 is the only Δ30 vaccine candidate that has been tested in humans, and it was found to be both safe and immunogenic [It was hoped that each of the four components of a tetravalent vaccine, consisting of DEN-1, -2, -3, and -4 wild type viruses, each with the common 30 nucleotide deletion mutation in the 3' UTR, would exhibit a similar level of attenuation in animal models -15. Unfo10PFU/ml ,14, the unogenic . If the To construct a further attenuated derivative of rDEN2Δ30, the 4995 mutation present in the NS3 gene of DEN-4 at amino acid 158 (Ser→Leu) was introduced into the homologous region of the NS3 protein of rDEN2Δ30 . AlthougEpidemiologic and molecular pathogenesis studies of DEN-2 strains support the concept that the DEN-2 Tonga/74 virus, from which the vaccine candidates were derived, may naturally have a lower level of virulence than other DEN-2 viruses. If the DEN-2 Tonga/74 parent virus is naturally attenuated to some degree, only a small incremental increase in attenuation might be required to satisfactorily attenuate it for humans. Gubler et al. investigated the 1974 outbreak of DEN-2 infection in the Pacific island of Tonga . In compSince the Tonga/74 outbreak, additional evidence has emerged that supports the suggestion that there are at least two circulating lineages of DEN-2 viruses that differ in virulence -29. The Aedes mosquitoes in comparison to Asian DEN-2 viruses [Aedes mosquitoes in our laboratory [Aedes aegypti mosquitoes fed an infectious blood meal. Since rDEN-2 and rDEN2Δ30 viruses were infectious by intrathoracic inoculation of Toxorynchites mosquitoes, the lack of infectivity for A. aegypti was likely caused solely by the inability of the DEN-2 Tonga/74 viruses to establish a midgut infection. Decreased infectivity for Aedes mosquitoes could serve to help limit transmission of the vaccine virus.The American genotype DEN-2 viruses exhibit decreased infectivity for  viruses ,25. Consboratory  whereas boratory . Howeverboratory ,27. At t10PFU/ml in Vero cells that would permit economical manufacture. Second, the viruses are derived from the DEN-2 Tonga/74 strain, a member of the American genotype, which has been associated with decreased virulence. Third, rDEN2Δ30 was attenuated for replication in SCID-HuH-7 mice and slightly attenuated for rhesus monkeys while inducing a protective neutralizing antibody response. Fourth, rDEN2Δ30-4995 was more attenuated in SCID-HuH-7 mice than rDEN2Δ30. Fifth, the DEN-2 Tonga/74 strain, like other members of the American genotype, is poorly infectious for Aedes aegypti mosquitoes which would help to limit uncontrolled transmission of the vaccine virus.The live-attenuated DEN-2 virus candidates described here, rDEN2Δ30 and rDEN2Δ30-4995, have several properties desired in a live attenuated virus vaccine for humans. First, both viruses reached a titer over 6.0 logThe authors declare that they have no competing interests.J.B. recovered viruses, conducted animal studies, and drafted the manuscript. C.H. and S.W. constructed the DEN-2 cDNA clone and C.H. performed sequencing. K.H. performed mosquito studies. B.M. and S.W. supervised the study and participated in planning and design. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


While the nucleus of a cell may be its command headquarters, mitochondria are equally vital—they are the power plants of the cell, and without them all cellular activity would quickly and irrevocably come to a halt. Testifying to their origins as once free-living bacteria, mitochondria have their own DNA, comprising 37 genes in humans on a single circular chromosome. Whether they invaded their ancestral hosts as parasites or were captured as subcellular collaborators, they have long since left their independent ways behind. Their meager complement of genes is far fewer than is needed to produce these complex organelles; it is clear from analyzing the nuclear genome that most of the mitochondria's presumed ancestral genes have been taken into the cell's nucleus, where they are under the strict control of their host.The transplanted mitochondrial genes have been faithfully doing their job under new management since they were first appropriated, probably hundreds of millions of years ago. However, not all of their DNA descendants have continued to make themselves so useful. For, in addition to many of the mitochondria's original genes, the human genome houses over 200 mitochondrial genetic fragments, useless pieces of code whose only remaining function is to be replicated generation after generation.Detritus from other sources is even more common within the genome, and most of it seems to be harmless. But in this issue, Ricchetti and colleagues show that mitochondrial fragments may not be quite so benign. They have continued to invade the human genome, even into the present day, and a large proportion of them take up residence within nuclear genes, possibly disrupting them and causing human diseases.Scanning the entire human genome, Ricchetti and colleagues found a total of 211 nuclear sequences of mitochondrial origin (NUMTs). Of these, they selected 42, which appeared to be the most recent integrations, for detailed study. Only 14 of them were also found in DNA from our closest relatives, chimpanzees, indicating that the rest arose after the human–chimp split approximately 5 million years ago. While 35 of the 42 were found in all humans tested, the rest were not, suggesting a still more recent origin for these among human populations.The authors also made two surprising discoveries about the location of these human-specific NUMTs. They were not evenly distributed across the entire genome; instead, for reasons that are unclear, there were a disproportionate number of them on two chromosomes—the Y chromosome, present only in males, and number 18. Furthermore, NUMTs were not randomly scattered among all the DNA of the chromosomes. Rather, they were much less likely to be found in non-coding “junk” DNA and much more likely to have inserted themselves within highly active genes. This phenomenon is likely to be related to the mechanism by which a NUMT enters the chromosome—it relies on the machinery that repairs breaks in the DNA, and these breaks are more common in genes that are frequently transcribed. Such insertions can cause disease, as shown by the recent discovery of a hemophilia patient with a NUMT interrupting his clotting factor gene.Much remains to be learned about the functional and temporal dynamics of NUMT insertions, but their potential for harm suggests that many NUMTS, unlike much of the rest of the flotsam that litters our genome, may be selected against quickly. Combined with their differential distribution among human ethnic groups, this may make them valuable markers for tracking both long- and short-term trends in human evolution and migration.


Most people threatened by AIDS, tuberculosis, and unsafe drinking water are poor and have little or no influence over the global politics of public health The rancour surrounding the Bangkok AIDS summit of July 2004 has exposed a series of fundamental disagreements surrounding the global politics of public health. These tensions range from access to cheaper lifesaving drugs to disputes over the role of poverty and gender equality in the promotion of sexual health.The world is now experiencing the most profound public health challenge of the last forty years: we have witnessed the appearance of new diseases such as Ebola, SARS, and in particular, AIDS, combined with the alarming resurgence of diseases previously thought to have been under control, such as malaria and tuberculosis ,2. The AYet the very idea of “public health” sits uncomfortably alongside the current emphasis of bio-medical science on the molecular realm of DNA coding and the development of lucrative Western markets for new pharmaceutical products such as sildenafil (Viagra). The needs of the majority—the global poor—scarcely feature within this tactical alliance between the biomedical sciences and corporate power. Since most people threatened by AIDS, tuberculosis, unsafe drinking water, and other health threats are poor, they have little or no influence over the global politics of public health.If we are to make sense of the current public health crisis, we need to explore interconnections between political, economic, and social developments that are ignored by the fragmentary emphasis of the biomedical sciences. The current impetus toward economic globalization is causing widespread social and economic disruption, ranging from wild currency fluctuations to the systematic collapse of viable agricultural systems An examination of the social impact of the global public health crisis shows that it is women and children who have been most badly affected. One of the least addressed dimensions to the externally imposed structural adjustment programmes on developing countries is the harmful impact on the sexual health of women caused by their increased economic dependence on men. Sexual health programmes based on abstinence, for example, ignore the difficulties women face in negotiating safer or non-penetrative forms of sex. It is married women in southern Africa and south Asia who make up the largest and most vulnerable group of women, since they are at risk of being infected by their husbands.A new generation of HIV-positive women activists, such as the Nigerian journalist and AIDS campaigner Rolake Odetoyinbo Nwagwu, are now engaged in a vital struggle to challenge the social attitudes and economic inequalities that have driven the devastating impact of AIDS on women and children in developing countries. The spread of AIDS in more traditional societies is closely linked with patriarchal power structures.. The sustenance of these structures is assured by the rise of poverty-fuelled ethnic and religious chauvinism, which undermines the prospects for developing more progressive approaches to social policy. And in regions where war or civil strife prevail, the vulnerability of women and children to sexual violence, economic exploitation, and disease is even greater so that we cannot consider public health questions separately from issues surrounding political stability and social justice.In order to understand better the political dynamics behind public health we need to recognize that the development of modern forms of governance has emerged in tandem with new approaches toward the administration of human populations. The rise of the 19th-century industrial city, for example, necessitated the development of much more sophisticated forms of urban governance in order to tackle the threat of epidemic disease and enable these new cities to function effectively as centres of economic activity. But these new spaces of public health control, which scholars such as Michel Foucault have so vividly described, had a shadowy “other”. This “other” was represented by the increasingly squalid conditions endured by the mass of the population in European colonies, exemplified by the devastating modern outbreaks of bubonic plague in cities such as Baghdad, Lagos, and Bombay The Italian political philosopher Giorgio Agamben has elaborated on this distinction between “inside” and “outside” under modern systems of governance to reveal the persistence of what he terms conditions of “bare life” within even the most sophisticated legal and political systems A striking manifestation of this systematic exclusion of the poor from medical care is provided by the Bush administration's efforts to stymie access to affordable antiretroviral drugs. A dramatic standoff in 2001 between the global pharmaceutical industry and public health activists in South Africa, following the import of cheap generic antiretroviral drugs manufactured in Brazil, led to the historic Doha Declaration on intellectual property rights and public health In order to widen access to affordable drugs, the current production of generic medicines will have to be massively expanded, but the Bush administration and its corporate allies in the pharmaceutical industry has worked assiduously to undermine the potential impact of the Doha agreement Of the $15 billion pledged by the Bush administration in the fight against AIDS most of this money will be focused on 15 selected countries. The countries were chosen by their willingness to abide by bilateral trade deals to prevent the production of cheaper generic drugs and their willingness to stress the centrality of abstinence as a strategy for AIDS prevention (a strategy that the religious Right has pushed for) ,17. It sThe US government has sought to highlight the inadequacies of health care infrastructure in developing countries as a further justification for the diversion of attention from the cost of drugs. But primary health care services have themselves been undermined by the structural adjustment and trade policies promoted by Western financial institutions and their corporate backers. The global politics of AIDS is therefore caught in a neo-liberal vicious spiral from which it is impossible to disentangle the needs for social and institutional reform in the worst affected regions from the challenge of widening access to available treatments.Public health pioneers of the past, such as the German bacteriologist Robert Koch, were not only scientists but also political advocates for social change. Improvements in health care were perceived as part of a nexus of reforms that ranged from better housing and nutrition to an extension of voting rights to ensure that the poor had adequate political representation.The contemporary politics of public health needs to be considered, however, in relation to wider discourses on security and human welfare that are quite different from those of the 19th century. The current Western preoccupation with the threat of international terrorism, for example, has distracted attention from the much more real threats to human well-being created by poverty and ill health. The vast resources—both financial and logistical—now being poured into “security” contrast starkly with the inadequate funding provided for the Global Fund for HIV/AIDS, Tuberculosis, and Malaria. The increasingly unilateral and self-interested stance of the US illustrates the tensions between attempts to build international forms of governance for health care and the continuing centrality of national interests or global institutions that explicitly represent the economic power of a relatively small group of nations The paradox is that widening global disparities in wealth and poverty play a role in fostering the social and political conditions in which religious fanaticism and hatred for the West can flourish. So any “war on terror” that fails to address the causes of poverty, despair, and insecurity in the lives of the world's poor will ultimately create a more dangerous world for everyone.


A recent paper suggests that genes can interact in networks to limit variation of phenotype. Similar principles might apply to the regulation of ion channels in nerve cells Individuals within a wild population show remarkably little morphological variation, given the amount of environmental variation they encounter during development and the amount of genetic variation within the population. This phenotypic constancy led to the proposal that individuals were somehow buffered, or canalized, against genetic and environmental variation . ClearlyDrosophila and Arabidopsis have suggested that Hsp90 (heat shock protein 90), a member of a family of proteins expressed at high temperatures (heat shock), may be an excellent candidate for bringing about canalization (Hsp83 (the gene encoding Hsp90) show increased levels of morphological abnormalities; (2) individuals treated with a pharmacological inhibitor of Hsp90 show severe morphological abnormalities; (3) the normal function of Hsp90 is to stabilise the tertiary structure of signal transduction molecules involved in developmental pathways; and (4) this function may be compromised by environmental factors, e.g., heat shock.Experiments in both lization . SeveralHsp90 may not, however, be uniquely placed to act as an evolutionary buffer producing canalization. Recent theoretical work has suggested that canalization may be an emergent property of complex gene networks and may not require specific mechanisms of protein stabilisation and environmental coupling such as those provided by Hsp90 . Siegal Thus, populations derived from ‘knockouts’ express phenotypic variation that was not expressed by the wild-type network, suggesting that any of the genes within the network may buffer genetic variation. This suggests that at least one aspect of generating evolutionary buffering is not unique to Hsp90. But can genes that, unlike Hsp90, are not conditional upon the environment act as evolutionary buffers? To test this, Sacchromyces cerevisiae, in which each gene may be ‘knocked out’ in turn and the expression of the remaining genes determined, to demonstrate that their simulations also had application to biological gene networks. Using these data, they showed that ‘knockouts’ show greater variability in gene expression than wild-type yeast, suggesting that buffering has been disrupted.The simulations described by + and K+ channels as well as numerous Cl−, Ca2+, and voltage-independent leak channels. The electrical properties of a single neuron are dependent, though not exclusively, upon the suite of ion channels expressed within that neuron. The properties of a neural network, which generates behaviour, are determined both by the intrinsic expression patterns of ion channels within neurons and the connectivity between neurons. The nervous system develops as an interaction between experience and genetically programmed events. One mechanism by which this interaction is achieved is ion channel compensation  may be compensated for by regulating the expression of other ion channels. ‘Knockout’ of one of these compensating ion channels may reveal the change in voltage-dependent Na+ channel properties, resulting in a shift in the output of the neuron. This hypothesis has several testable predictions. For example, ‘knocking out’ an ion channel should increase the variation in the activity of particular neurons among individuals in a population. This variation in neural activity may produce an effect on the behaviour of the whole organism.It is possible, therefore, that the networks of genes regulating ion channel expression may act as evolutionary buffers. The relationship between neural activity and the network of ion channel encoding genes may stabilise the neural activity in relation to both the genetic and environmental variation. The stabilisation of neural activity may have consequences for the generation of adaptive behaviour, which is constructed from neural activity. It is possible that ion channels could canalize the evolution of the nervous system by reducing behavioural variation and therefore removing the substrate on which natural selection may act. For example, changes in voltage-dependent NaDrosophila photoreceptors (Studying canalization in ion channel gene networks may have significant advantages over studying developmental gene networks because it is relatively straightforward to measure the amounts of ion channels expressed in single identified neurons, to alter the expression of individual ion channels, and to relate these alterations to behaviour. I am currently pursuing the impact of ion channel compensation in eceptors , 2003c.


Neck dissection has traditionally played an important role in the management of patients with regionally advanced head and neck squamous cell carcinoma (HNSCC) treated with radical radiotherapy alone. However, with the incorporation of chemotherapy in the therapeutic strategy for advanced HNSCC and resultant improvement in outcome the routine use of post chemo-radiotherapy neck dissection is being questioned.Published data for this review was identified by systematically searching MEDLINE, CANCERLIT & EMBASE databases from 1995 until date with restriction to the English language.There is lack of high quality evidence on the role of planned neck dissection in advanced HNSCC treated with chemo-radiotherapy. A systematic literature search could identify only one small randomized controlled trial (Level I evidence) addressing this issue, albeit with major limitations. Upfront neck dissection followed by chemo-radiotherapy resulted in better disease-specific survival as compared to chemoradiation only. Several single arm prospective and retrospective reports were also identified with significant heterogeneity and often-contradictory conclusions.Planned neck dissection after radical chemo-radiotherapy achieves a high level of regional control, but its ultimate benefit is limited to a small subset of patients only. Unless there are better non-invasive ways to identify residual viable disease, the role of such neck dissection shall remain debatable. A large randomized controlled trial addressing this issue is needed to clarify its role and provide evidence-based answers. The optimal management of the neck in loco-regionally advanced head & neck squamous cell carcinomas (HNSCC) following primary chemo-radiotherapy remains controversial ,2. TradiPublished data for this review was identified by systematically searching the MEDLINE, CANCERLIT & EMBASE databases from 1995 until date with restriction to the English language. "Head & Neck cancer" OR "HNSCC" was combined with "chemo-radiotherapy" OR "chemo-radiation" as Medical Subject Heading (MeSH) terms and each of the following phrase used as text words: "adjuvant neck dissection"; "planned neck dissection"; and "neck management". Relevant cross-references were also considered.There is only one small randomized control trial (American Society of Clinical Oncology  Level I In absence of high quality evidence, the best available evidence tempered with clinical judgment often guides decision-making. Two of the recently published reports ,12 somewArgiris  et al evBrizel  et al idApart from the afore-mentioned two reports, there are a few reasonably large studies (involving >50 patients: Table The potential benefit of planned neck dissection after a course of intensive chemo-radiotherapy in terms of improved regional control with or without an impact on survival needs to be weighed against the expected morbidity associated with the surgical procedure ,14,25. OPlanned neck dissection after radical chemo-radiotherapy achieves a high level of regional control, but its ultimate benefit is limited to a small subset of patients only. The morbidity of such dissection is small, but significant. Its impact on survival is yet to be completely realized. In the majority of patients it is either unnecessary because there is no residual disease in the neck or futile because of unsalvageable primary recurrence or distant metastases. Nevertheless, it is recommended that planned neck dissection be performed for patients with less than a complete response in the neck after combined modality therapy to optimize regional control, provided the primary is controlled and there is no evidence of distant metastases. It should also be performed as part of salvage surgery for locally persistent or residual disease at primary site. The criterion for planned neck dissection for patients with advanced nodal disease with a CCR in the neck following chemo-radiotherapy should incorporate not only the nodal staging but also the actual size of the involved lymph nodes. Unless there are better non-invasive ways to identify residual viable disease, which could include functional imaging like Positron Emission Tomography and biological assays like hypoxia markers, the role of such neck dissection shall remain debatable. A large randomized controlled trial across several institutions addressing these issues is needed to clarify the role of planned neck dissection in advanced HNSCC treated with primary chemo-radiotherapy and provide evidence-based answers.No source of funding involved in this reviewNone declared.Dr JP proposed the idea of systematic review on the issueDr TG did the literature search & prepared the manuscriptDr JP critically reviewed and revised the manuscript


There have been contradictory reports regarding the B cell helping function of CD57+ GC-Th cells in production of immunoglobulin (Ig). Furthermore, the cytokine and co-stimulation requirement for their helper activity remains largely unknown. To clarify and gain more insight into their function in helping B cells, we systematically investigated the capacity of human tonsil CD57+ GC-Th cells in inducing B cell Ig synthesis.The function of CD57+ GC-Th cells are highly efficient in helping B cell production of all four subsets of Ig (IgM, IgG, IgA and IgE) compared to other T-helper cells located in germinal centers or interfollicular areas. CD57+ GC-Th cells were particularly more efficient than other T cells in helping GC-B cells but not naïve B cells. CD57+ GC-Th cells induced the expression of activation-induced cytosine deaminase (AID) and class switch recombination in developing B cells. IgG1-3 and IgA1 were the major Ig isotypes induced by CD57+ GC-Th cells. CD40L, but not IL-4, IL-10 and IFN-γ, was critical in CD57+ GC-Th cell-driven B cell production of Ig. However, IL-10, when added exogenously, significantly enhanced the helper activity of CD57+ GC-Th cells, while TGF-β1 completely and IFN-γ partially suppressed the CD57+ GC-Th cell-driven Ig production.We demonstrated that CD57+CD4+ T cells in the germinal centers of human lymphoid tissues are the major T helper cell subset for GC-B cells in Ig synthesis. Their helper activity is consistent with their capacity to induce AID and class switch recombination, and can be regulated by CD40L, IL-4, IL-10 and TGF-β.CD57 CD57+ G of IL-2 ,18. CD57on in GC . Based uh2 cells . Using apression . The mic5+ cells ,23 and d5+ cells . Becauseientists ,25-27. T+ GC-Th cells in inducing B cell Ig synthesis in naïve vs. germinal center B (GC-B) cells in comparison with other T cell subsets in human tonsils. We show that CD57+ GC-Th cells are more efficient than other germinal center or interfollicular T cells in supporting B cell production of Ig. CD57+ GC-Th cells, when compared to other T cells, have better helper activity for GC-B cells than for naïve B cells. CD57+ GC-Th cells induced the expression of activation-induced cytosine deaminase (AID) and CSR in developing B cells. CD40L, but not other major cytokines, is critical for the helper activity of CD57+ GC-Th cells. IL-10 positively and TGF-β1 negatively regulate the helper activity of CD57+ GC-Th cells.To clarify and gain more insight into their function in helping B cells, we systematically investigated the capacity of human tonsil CD57+ CD4+ T cells are located in germinal centers surrounded by IgD+ naïve B cells  surrounding GC. Although some are found in IFA, CD69+ CD4+ T cells were also preferentially found in GC , CD4+CD57-CD69+ cells (mainly in GC and a minor proportion in IFA), and CD4+CD57-CD69- cells (mainly in IFA).We examined the distribution of T helper cell subsets in human tonsils based upon the expression of CD4, CD57 and CD69. As reported previously ,19,28,29C Figure . In cont+ T cell population was fractionated into CD57+ GC-Th cells , total CD57- T cells, CD57-CD69+ T cells and CD57-CD69- T cells  in the composition of Ig that they induced.The relative composition of IgM, IgG, IgA and IgE produced in response to CD57r Figure . Class-s+ GC-Th cells have the capacity to induce AID in B cells. Naïve B cells were co-cultured with CD57+ GC-Th cells, and AID expression was examined  were compared, AID expression preceded the expression of IgG3 transcript by 1–2 days in culture , whereas CD4+CD57-CD69- interfollicular T cells did not express CD40L at significant levels. There was no significant difference in the expression of surface CD40L between CD57+ and CD57- CD69+ cells.Cytokines and CD40L regulate B cell maturation and Ig production. We examined whether CD40L, IL-4, IL-10 and IFN-γ play any roles in the CD57E Figure . In this+ GC-Th cells  employed in our study appear to favor the detection of the suppressive function of TGF-β. Taken together, these results imply that Th1, Th2 and regulatory T cells, if present in germinal centers, could positively or negatively control the function of CD57+ GC-Th cells in regulation of humoral immune responses. Indeed, there are regulatory T cells in GCs that express surface TGF-β and can effectively suppress the function of CD57+ GC-Th cells [CD40L appears to be essential for the helper activity of CD57tivation . Anotherynthesis . We did Th cells .+ GC-Th cells in supporting CSR and Ig synthesis in B cells, and revealed the factors that regulate their activity, thereby substantiating the so-far inconclusive function of CD57+ GC-Th cells. The fact that these T cells have preferential and efficient helper activity for GC-B cells and are specifically localized in GCs in large numbers suggests that CD57+ GC-Th cells are probably the major T helper subset responsible for supporting B cell differentiation for Ig production in germinal centers.Our results demonstrated the capacity of CD57+ T cells (purity >97%) were isolated by depleting non-CD4+ T cells using a magnetic bead depletion method (Miltenyi Biotec, Auburn, CA). After staining of the isolated CD4+ T cells with appropriate antibodies, CD57+ GC-Th cells (purity >95%) were isolated by a positive magnetic selection method (Miltenyi Biotec). CD4+CD57-CD69+ and CD4+CD57-CD69- T cell subsets (purity >95%) were further isolated from the CD57- T cell fraction by magnetically selecting CD69+ T cells (Miltenyi Biotec). Total B cells were isolated by rosetting with 2-amino-ethylisothiouronium bromide (AET)-treated sheep red blood cells followed by CD4+ T cell depletion (CD19+ cells > 99.5%). Naïve B cells (CD19+IgD+ cells >99%) were isolated from the total B cell fraction by depleting CD38+ T cells followed by positive magnetic selection of IgD+ B cells. CD19+CD38+IgD+/- GC-B cells (purity >95%) were isolated from the tonsil CD19+ B cells as described before [Mononuclear cells were prepared by density gradient centrifuge on histopaque 1077 (Sigma-Aldrich, St. Louis) from human tonsil pathological specimens obtained from young patients (3–10 yr) undergoing tonsillectomy to relieve obstruction of respiratory passages and improve drainage of the middle ear at Sagamore Surgical Center (Lafayette, IN). The use of human pathological specimens in this study was approved by the institutional review board at Purdue University. CD45 T and 105 B cells were co-cultured, unless indicated otherwise, in each well of 48-well plates in the presence of Staphylococcal enterotoxin B . Cells were incubated in 5% CO2 incubators at 37°C for 3–8 days. Recombinant IL-4, IL-10, and TGF-β1 were purchased from R&D systems (Minneapolis, MN). Recombinant IFN-γ was obtained from BD Pharmingen (San Diego, CA). Purified CD154-blocking antibody (24–31) was obtained from Ancell Corporation (Bayport, MN). IL-4-blocking antibody (MP4-25D2) was purchased from BD Pharmingen. Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems. All antibodies and reagents added to culture were azide-free. Cytokines were added at saturating concentrations: IL-4 (40 ng/ml), IL-10 (40 ng/ml), IFN-γ (200 ng/ml) and TGF-β1 (10 ng/ml). Neutralizing antibodies were added at following concentrations: anti-CD40L (20 μg/ml), anti-IL-4 (5 μg/ml), anti-IL-10 (5 μg/ml), anti-IFN-γ (2.5 μg/ml) and isotype antibody (5 μg/ml).All cell cultures were performed in RPMI1640 medium supplemented with 10% FBS, gentamycin, streptomycin, and penicillin. To cross-link the B cell receptors, isolated B cells were incubated for 2 h at 4°C with Sepharose-conjugated rabbit Ab to human Ig μ chain and human Ig (H + L) chain , and then washed with cold PBS. 10T cells were stained with anti-human CD57 , anti-human CD69 , anti-human CD4 , and anti-human CD3 . B cells were stained with anti-CD19 , anti-human IgD (IAb-2, FITC, BD Pharmingen), anti-human CD38 , and anti-human CD3 . Stained cells were analyzed using a 4-color FACSCalibur™ (BD Biosciences).Frozen sections of tonsils were acetone-fixed and stained using antibodies to CD57 , CD69 , IgD  and/or CD4 . Stained sections were analyzed using a confocal microscopy system (Bio-Rad MRC 1024UV and Nikon Diaphot 300 microscope) at Purdue Cytometry Lab.Culture supernatants were assayed by ELISA as previously described . The senTotal RNA was extracted from cultured cells with Trizol reagent (Invitrogen, Carlsbad, CA), and was reverse-transcribed into cDNAs with SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's protocol. The primer pairs used in this study were designed by Cerutti et al. : IgM, FRTotal RNA was extracted from freshly isolated or cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA), and was reverse-transcribed into cDNAs with SuperScript™ II Reverse Transcriptase. RT-PCR amplification of AID was performed using the two primers: AID-forward (5'-GAT GAA CCG GAG GAA GTT TC-3') and AID-reverse (5'-TCA GCC TTG CGG TCC TCA CAG-3'), which generated a specific 351 bp PCR product after 30 cycles of PCR reaction (30 s at 94°C, 30 s at 60°C, and 60 s at 72°C). β-actin was also amplified as a control.Student's paired t-test was used. P values smaller than 0.05 were considered significant.GC, germinal center; GC-Th cells, germinal center T helper cells; GC-B cells, germinal center B cells; AID, activation-induced cytosine deaminase; CSR, class switch recombination; SEB, staphylococcal enterotoxin B.CHK conceived, coordinated the study, analyzed the results and wrote the text. JRK, HWL and SGK participated in experiments, data analysis, making figures and proofreading the manuscript. PH provided specimens and helped perform the study. All authors read and approved the manuscript.


New York Times crossword puzzle, you probably have a gift for insight. The puzzles always have an underlying hint to solving them, but on Saturdays that clue is insanely obtuse. If you had all day, you could try a zillion different combinations and eventually figure it out. But with insight, you'd experience the usual clueless confusion, until—voilà—the fog clears and you get the clue, which suddenly seems obvious. The sudden flash of insight that precedes such “Aha!” moments is characteristic of many types of cognitive processes besides problem-solving, including memory retrieval, language comprehension, and various forms of creativity. Although different problem-solving strategies share many common attributes, insight-derived solutions appear to be unique in several ways. In this issue, researchers from Northwestern and Drexel Universities report on studies revealing a unique neural signature of such insight solutions.If you're one of those insufferable people who can finish the Saturday Eureka!, which Archimedes reportedly shouted upon realizing that water displacement could be used to compute density. Illustrating the strong emotional response elicited by such a sudden insight, Archimedes is said to have run home from the baths in euphoric glee—without his clothes.Mark Jung-Beeman, John Kounios, Ed Bowden, and their colleagues recount the storied origin of the term Among other characteristics that typically distinguish insight from “noninsight” solutions, people feel stuck before insight strikes; they can't explain how they solved the problem and might say they were not even thinking about it; the solution appears suddenly and is immediately seen as correct. But are the neural processes involved in arriving at a solution through insight actually distinct from those related to more mundane problem-solving?Recent findings suggest that people think about solutions, at an unconscious level, prior to solving insight problems, and that the right cerebral hemisphere (RH) appears to be preferentially involved. Jung-Beeman et al. predicted that a particular region of the RH, called the anterior superior temporal gyrus (aSTG), is likely involved in insight because it seems critical for tasks that require recognizing broad associative semantic relationships—exactly the type of process that could facilitate reinterpretation of problems and lead to insight.pine, crab, sauce) and asked to think of one word that would form a compound word or phrase for each of the words (can you figure it out?). Neural activity was mapped with fMRI while the participants were given 124 similar word problems—which can be solved quickly with or without insight, and evoke a distinct Aha! moment about half the time they're solved. Subjects pressed a button to indicate whether they had solved the problem using insight, which they had been told leads to an Aha! experience characterized by suddenness and obviousness.To test this hypothesis, Jung-Beeman et al. mapped both the location and electrical signature of neural activity using functional magnetic resonance imaging (fMRI) and the electroencephalogram (EEG). In the first experiment, thirteen people were given three words , the authors conclude that the increase does not simply reflect the emotional jolt associated with insight.In a second experiment, 19 new participants engaged in the same type of problem-solving tasks as the first group while their brain waves were measured with an EEG. The researchers then analyzed the EEG recordings to look for differences between insight and noninsight solutions in brain wave activity. The researchers found that 0.3 seconds before the subjects indicated solutions achieved through insight, there was a burst of neural activity of one particular type: high-frequency (gamma band) activity that is often thought to reflect complex cognitive processing. This activity was also mapped to the aSTG of the RH, providing compelling convergence across experiments and methods.at least one additional component. Further, the fact that the effect occurred in RH aSTG suggests what that process may be: integration of distantly related information. Distinct neural processes, the authors conclude, underlie the sudden flash of insight that allows people to “see connections that previously eluded them.”Problem-solving involves a complex cortical network to encode, retrieve, and evaluate information, but these results show that solving verbal problems with insight requires