Agglutinating activity of gliadin-derived peptides from bread wheat: implications for coeliac disease pathogenesis

The PT-digest of bread wheat gliadin was very active in agglutinating undifferentiated human K562(S) cells. This activity was quantitatively, but not qualitatively, similar to that of Con A or WGA. Moreover, Con A-induced cell agglutination was inhibited by mannan and mannose, WGA-induced agglutination by NAG only, and cell agglutination induced by bread wheat gliadin peptides was inhibited by each of these three saccharides. Not only was mannan the most active saccharide in preventing cell agglutination induced by bread wheat gliadin peptides, but it was also able to dissociate agglutinated cells. As compared to the PT- digest of whole bread wheat gliadin, the digest obtained from purified A-gliadin was tenfold more active. The PT-digest of durum wheat gliadin did not show any agglutinating activity.     

Recombinant Vectors Based on Porcine Adeno-Associated Viral Serotypes Transduce the Murine and Pig Retina

Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.     

Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer

Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.     

Celiac disease association with CD8+ T cell responses: identification of a novel gliadin-derived HLA-A2-restricted epitope

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8(+) T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123-132) by CD8(+) T lymphocytes from celiac patients. A-gliadin 123-132-specific IFN-gamma production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123-132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123-132 epitope recognized human APC pulsed with either whole recombinant alpha-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.     

Celiac anti-tissue transglutaminase antibodies interfere with the uptake of alpha gliadin peptide 31–43 but not of peptide 57–68 by epithelial cells

Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31–43 (but not peptide 57–68) uptake by cells, thereby impairing the ability of p31–43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31–43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.