Isolation of human monoclonal autoantibodies derived from pancreatic lymph node and peripheral blood B cells of islet autoantibody-positive patients

Aims/hypothesis Autoantibodies against pancreatic islets and infections by enteroviruses are associated with type 1 diabetes, but the specificity of immune responses within the type 1 diabetic pancreas is poorly characterised. We investigated whether pancreatic lymph nodes could provide a source of antigen-specific B cells for analysis of immune responses within the (pre)diabetic pancreas. Methods Human IgG antibodies were cloned from single B lymphocytes sorted from pancreatic lymph node cells of three organ donors positive for islet autoantibodies, and from the peripheral blood of a patient with type 1 diabetes. Antibodies to insulinoma-associated antigen 2 (IA-2), GAD65, zinc trans- porter 8 (ZnT8) and Coxsackie B virus proteins were assayed by immunoprecipitation and by immunofluorescence on pan- creatic sections. Results Human IgG antibodies (863) were successfully cloned and produced from 4,092 single B cells from lymph nodes and peripheral blood. Reactivity to the protein tyrosine phosphatase domain of the IA-2 autoantigen was detected in two cloned antibodies: one derived from a pancreatic lymph node and one from peripheral blood. Epitopes for these two antibodies were similar to each other and to those for circulat- ing antibodies in type 1 diabetes. The remaining 861 antibod- ies were negative for reactivity to IA-2, GAD65 or ZnT8 by both assays tested. Reactivity to a Coxsackie viral protein 2 was detected in one antibody derived from a peripheral blood B cell, but not from lymph nodes. Conclusions/interpretation We show evidence for the infre- quent presence of autoantigen-specific IgG+ B lymphocytes in the pancreatic-draining lymph nodes of islet autoantibody- positive individuals

Cerebrospinal Fluid B Cells Correlate with Early Brain Inflammation in Multiple Sclerosis

Background: There is accumulating evidence from immunological, pathological and therapeutic studies that B cells are key components in the pathophysiology of multiple sclerosis (MS). Methodology/Principal Findings: In this prospective study we have for the first time investigated the differences in the inflammatory response between relapsing and progressive MS by comparing cerebrospinal fluid (CSF) cell profiles from patients at the onset of the disease (clinically isolated syndrome, CIS), relapsing-remitting (RR) and chronic progressive (CP) MS by flow cytometry. As controls we have used patients with other neurological diseases. We have found a statistically significant accumulation of CSF mature B cells (CD19+CD1382) and plasma blasts (CD19+CD138+) in CIS and RRMS. Both B cell populations were, however, not significantly increased in CPMS. Further, this accumulation of B cells correlated with acute brain inflammation measured by magnetic resonance imaging and with inflammatory CSF parameters such as the number of CSF leukocytes, intrathecal immunoglobulin M and G synthesis and intrathecal production of matri

Neuromyelitis optica MOG-IgG causes reversible lesions in mouse brain.

INTRODUCTION: Antibodies against myelin oligodendrocyte glycoprotein (MOG-IgG) are present in some neuromyelitis optica patients who lack antibodies against aquaporin-4 (AQP4-IgG). The effects of neuromyelitis optica MOG-IgG in the central nervous system have not been investigated in vivo. We microinjected MOG-IgG, obtained from patients with neuromyelitis optica, into mouse brains and compared the results with AQP4-IgG. RESULTS: MOG-IgG caused myelin changes and altered the expression of axonal proteins that are essential for action potential firing, but did not produce inflammation, axonal loss, neuronal or astrocyte death. These changes were independent of complement and recovered within two weeks. By contrast, AQP4-IgG produced complement-mediated myelin loss, neuronal and astrocyte death with limited recovery at two weeks. CONCLUSIONS: These differences mirror the better outcomes for MOG-IgG compared with AQP4-IgG patients and raise the possibility that MOG-IgG contributes to pathology in some neuromyelitis optica patients

Neuromyelitis optica MOG-IgG causes reversible lesions in mouse brain.

INTRODUCTION: Antibodies against myelin oligodendrocyte glycoprotein (MOG-IgG) are present in some neuromyelitis optica patients who lack antibodies against aquaporin-4 (AQP4-IgG). The effects of neuromyelitis optica MOG-IgG in the central nervous system have not been investigated in vivo. We microinjected MOG-IgG, obtained from patients with neuromyelitis optica, into mouse brains and compared the results with AQP4-IgG. RESULTS: MOG-IgG caused myelin changes and altered the expression of axonal proteins that are essential for action potential firing, but did not produce inflammation, axonal loss, neuronal or astrocyte death. These changes were independent of complement and recovered within two weeks. By contrast, AQP4-IgG produced complement-mediated myelin loss, neuronal and astrocyte death with limited recovery at two weeks. CONCLUSIONS: These differences mirror the better outcomes for MOG-IgG compared with AQP4-IgG patients and raise the possibility that MOG-IgG contributes to pathology in some neuromyelitis optica patients

Pregnant women become insensitive to cold stress

BACKGROUND: The function of the hypothalamic-pituitary-adrenal (HPA) axis is known to be altered during pregnancy, but it has not been tested with a natural stressor. METHODS: A group of pregnant women (n = 10) were tested towards the end of pregnancy (mean 36.8 ± 2.5 weeks gestation) and about 8 weeks postpartum (mean 7.8 ± 1.5 weeks), together with a matched control group, with a one minute cold hand stressor test. Saliva samples were collected before and 10 and 20 minutes after the test, and stored for later radioimmunoassay of cortisol. RESULTS: The control group showed a highly significant response to the test. The pregnant group showed no response, and the postpartum group a variable but non significant one CONCLUSIONS: This shows that the HPA axis becomes hypofunctional to a natural stressor at the end of pregnancy. It is suggested that one possible evolutionary function for this is to protect the fetus from the stress responses of the mother

Subcellular distribution of glutathione and cysteine in cyanobacteria

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria

Heart Rate-Corrected QT Interval Helps Predict Mortality after Intentional Organophosphate Poisoning

INTRODUCTION: In this study, we investigated the outcomes for patients with intentional organophosphate poisoning. Previous reports indicate that in contrast to normal heart rate-corrected QT intervals (QTc), QTc prolongation might be indicative of a poor prognosis for patients exposed to organophosphates. METHODS: We analyzed the records of 118 patients who were referred to Chang Gung Memorial Hospital for management of organophosphate poisoning between 2000 and 2011. Patients were grouped according to their initial QTc interval, i.e., normal (<0.44 s) or prolonged (>0.44 s). Demographic, clinical, laboratory, and mortality data were obtained for analysis. RESULTS: The incidence of hypotension in patients with prolonged QTc intervals was higher than that in the patients with normal QTc intervals (P = 0.019). By the end of the study, 18 of 118 (15.2%) patients had died, including 3 of 75 (4.0%) patients with normal QTc intervals and 15 of 43 (34.9%) patients with prolonged QTc intervals. Using multivariate-Cox-regression analysis, we found that hypotension (OR = 10.930, 95% CI = 2.961-40.345, P = 0.000), respiratory failure (OR = 4.867, 95% CI = 1.062-22.301, P = 0.042), coma (OR = 3.482, 95% CI = 1.184-10.238, P = 0.023), and QTc prolongation (OR = 7.459, 95% CI = 2.053-27.099, P = 0.002) were significant risk factors for mortality. Furthermore, it was revealed that non-survivors not only had longer QTc interval (503.00±41.56 versus 432.71±51.21 ms, P = 0.002), but also suffered higher incidences of hypotension (83.3 versus 12.0%, P = 0.000), shortness of breath (64 versus 94.4%, P = 0.010), bronchorrhea (55 versus 94.4%, P = 0.002), bronchospasm (50.0 versus 94.4%, P = 0.000), respiratory failure (94.4 versus 43.0%, P = 0.000) and coma (66.7 versus 11.0%, P = 0.000) than survivors. Finally, Kaplan-Meier analysis demonstrated that cumulative mortality was higher among patients with prolonged QTc intervals than among those with normal QTc intervals (Log-rank test, Chi-square test = 20.36, P<0.001). CONCLUSIONS: QTc interval helps predict mortality after intentional organophosphate poisoning

Rheotaxis in Larval Zebrafish Is Mediated by Lateral Line Mechanosensory Hair Cells

The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. This system includes cell clusters, called superficial neuromasts, located on the surface of head and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond to disturbances in the water and convey information to the brain via the lateral line ganglia. The convenient location of mechanosensory hair cells on the body surface has made the lateral line a valuable system in which to study hair cell damage and regeneration. One way to measure hair cell survival and recovery is to assay behaviors that depend on their function. We built a system in which orientation against constant water flow, positive rheotaxis, can be quantitatively assessed. We found that zebrafish larvae perform positive rheotaxis and that, similar to adult fish, larvae use both visual and lateral line input to perform this behavior. Disruption or damage of hair cells in the absence of vision leads to a marked decrease in rheotaxis that recovers upon hair cell repair or regeneration