Posttranslational modification of gluten shapes TCR usage in celiac disease

Posttranslational modification of Ag is implicated in several autoimmune diseases. In celiac disease, a cereal gluten-induced enteropathy with several autoimmune features, T cell recognition of the gluten Ag is heavily dependent on the posttranslational conversion of Gln to Glu residues. Evidence suggests that the enhanced recognition of deamidated gluten peptides results from improved peptide binding to the MHC and TCR interaction with the peptide-MHC complex. In this study, we report that there is a biased usage of TCR Vβ6.7 chain among TCRs reactive to the immunodominant DQ2-α-II gliadin epitope. We isolated Vβ6.7 and DQ2-αII tetramer-positive CD4(+) T cells from peripheral blood of gluten-challenged celiac patients and sequenced the TCRs of a large number of single T cells. TCR sequence analysis revealed in vivo clonal expansion, convergent recombination, semipublic response, and the notable conservation of a non-germline-encoded Arg residue in the CDR3β loop. Functional testing of a prototype DQ2-α-II-reactive TCR by analysis of TCR transfectants and soluble single-chain TCRs indicate that the deamidated residue in the DQ2-α-II peptide poses constraints on the TCR structure in which the conserved Arg residue is a critical element. The findings have implications for understanding T cell responses to posttranslationally modified Ags.     

Intragastric generation of antimicrobial nitrogen oxides from saliva--physiological and therapeutic considerations

Salivary nitrite is suggested to enhance the antimicrobial properties of gastric juice by conversion to nitric oxide (NO) and other reactive nitrogen intermediates in the stomach. Intubated patients exhibit extremely low gastric levels of NO, because they do not swallow their saliva. The present investigation was designed to examine the antibacterial effects of human saliva and gastric juice. Furthermore, we studied a new mode of NO delivery, involving formation from acidified nitrite, which could prevent bacterial growth in the gastric juice of intubated patients in intensive care units. The growth of Escherichia coli ATCC 25922 and the formation of NO and nitroso/nitrosyl species were determined after incubation of gastric juice with saliva from healthy volunteers that was rich (nitrate ingestion) or poor (overnight fasting) in nitrite. In a stomach model containing gastric juice from intubated patients, we inserted a catheter with a silicone retention cuff filled with ascorbic acid and nitrite and determined the resulting antibacterial effects on E. coli and Candida albicans. Saliva enhanced the bactericidal effect of gastric juice, especially saliva rich in nitrite. Formation of NO and nitroso/nitrosyl species by nitrite-rich saliva was 10-fold greater than that by saliva poor in nitrite. In our stomach model, E. coli and C. albicans were killed after exposure to ascorbic acid and nitrite. In conclusion, saliva rich in nitrite enhances the bactericidal effects of gastric juice, possibly through the generation of reactive nitrogen intermediates, including NO. Acidified nitrite inside a gas-permeable retention cuff may be useful for restoring gastric NO levels and host defense in critically ill patients.     

Rapid generation of rotavirus-specific human monoclonal antibodies from small-intestinal mucosa

The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03-0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with K(d) < 1 × 10(-10)). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.     

Strong genetic evidence of DCDC2 as a susceptibility gene for dyslexia

We searched for linkage disequilibrium (LD) in 137 triads with dyslexia, using markers that span the most-replicated dyslexia susceptibility region on 6p21-p22, and found association between the disease and markers within the VMP/DCDC2/KAAG1 locus. Detailed refinement of the LD region, involving sequencing and genotyping of additional markers, showed significant association within DCDC2 in single-marker and haplotype analyses. The association appeared to be strongest in severely affected patients. In a second step, the study was extended to include an independent sample of 239 triads with dyslexia, in which the association--in particular, with the severe phenotype of dyslexia--was confirmed. Our expression data showed that DCDC2, which contains a doublecortin homology domain that is possibly involved in cortical neuron migration, is expressed in the fetal and adult CNS, which--together with the hypothesized protein function--is in accordance with findings in dyslexic patients with abnormal neuronal migration and maturation.     

Morphometric studies of prepyramidal and pyramidal neurons of various vertebrates]

Praepyramidal and pyramidal neurons were demonstrated in homologous telencephalic areas of Salmo irideus (Gibbons 1855), Rana temporaria L., Rattus norvegicus, forma alba, by means of the Golgi-technique. A comparative morphometrical analysis was made concerning the following parameters: main dendritic length, length of the pericaryon, number of dendritic spines in the first 50-micrometer-dendritic segment and length of the spines-free zone. From these data a quotient was calculated and expressing the theoretical dendritic length per ten spines. There is an increase of length of the pericaryon, length of the spines-free zone, main dendritic length and a pseudo-decrease of the number of dendritic spines in the first 50 micrometer-dendritic segment in rats, as compared with Rana and Salmo. The results are discussed with respect to following problems: homology, phylogenesis and corticalisation.     

Comparative psychology and the grand challenge of drug discovery in psychiatry and neurodegeneration

Drug discovery for brain disorders is undergoing a period of upheaval. Faced with an empty drug pipeline and numerous failures of potential new drugs in clinical trials, many large pharmaceutical companies have been shrinking or even closing down their research divisions that focus on central nervous system (CNS) disorders. In this paper, we argue that many of the difficulties facing CNS drug discovery stem from a lack of robustness in pre-clinical (i.e., non-human animal) testing. There are two main sources for this lack of robustness. First, there is the lack of replicability of many results from the pre-clinical stage, which we argue is driven by a combination of publication bias and inappropriate selection of statistical and experimental designs. Second, there is the frequent failure to translate results in non-human animals to parallel results in humans in the clinic. This limitation can only be overcome by developing new behavioral tests for non-human animals that have predictive, construct, and etiological validity. Here, we present these translational difficulties as a “grand challenge” to researchers from comparative cognition, who are well positioned to provide new methods for testing behavior and cognition in non-human animals. These new experimental protocols will need to be both statistically robust and target behavioral and cognitive processes that allow for better connection with human CNS disorders. Our hope is that this downturn in industrial research may represent an opportunity to develop new protocols that will re-kindle the search for more effective and safer drugs for CNS disorders.     

Post-translational modifications in MeHg-induced neurotoxicity

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.