IgVH gene analysis suggests that peritoneal B cells do not contribute to the gut immune system in man

The contribution of peritoneal B cells to the intestinal lamina propria plasma cell population is well documented in mice, but unknown in humans. We have analyzed immunoglobulin (Ig) genes of human peritoneal B cells, because such genes show distinctive characteristics in mucosal B cells, particularly highly mutated variable regions. Here, we report the characteristics of variable region genes used by IgM, IgA and IgG in peritoneal cells. We focused on the properties of IgV(H)4-34 to allow comparisons of like-with-like between different isotypes and cells from different immune compartments. We observed that the IgM genes were mostly unmutated, and that the mutated subset had less mutations than would be expected in a mucosal B cell population. Likewise, the IgV(H)4-34 genes used by IgA and IgG from peritoneal B cells had significantly lower numbers of mutations than observed in the mucosal counterparts. Other trends observed, while not reaching statistical significance, followed the trend of peripheral B cells. The peritoneal B cell population had more IgA1 than IgA2 sequences, and there was no dominance of J(H)4 in the IgA from peritoneum or spleen, in contrast to the mucosal sequences. Overall, this study suggested that human peritoneal B cell are either peripheral or mixed in origin; they are unlikely to represent an inductive compartment for the mucosal B cell system.     

TNF-alpha impairs peripheral tolerance towards beta-cells,and local costimulation by B7.1 enhances the effector function of diabetogenic T cells

Maintenance of peripheral tolerance and inactivation of autoreactive T cells is based on a delicate balance between pro-inflammatory and protective cytokines that is poorly understood. We have here addressed how the local expression of the inflammatory cytokine TNF-alpha can impair peripheral tolerance and lead to autoreactivity. After transplantation of pancreata that are immunogenic due to beta-cell expression of B7.1 and TNF-alpha, into thymectomized and euthymic syngeneic mice, we found that only euthymic mice rejected the grafts. This result suggests that under normal circumstances autoreactive T cells are functionally inactivated, and initiation of an autoreactive response requires de-novo generation of T cells. By contrast, thymectomized mice expressing TNF-alpha on the endogenous islets rejected the grafts, showing that expression of TNF-alpha prevents functional silencing of the autoreactive T cells. Thus, this study provides a mechanism by which TNF-alpha and possibly chronic inflammatory responses may promote autoimmune diseases. Furthermore, we have investigated whether B7.1 can enhance T cell responses of already activated T cells leading to islet rejection. By transplantation of wild-type and B7.1-expressing islets into overtly diabetic mice we found that only the wild-type islets could restore normoglycemia, suggesting that costimulation by B7.1 is required in the expansion or effector phase of the response.     

Isolation of exfoliated colonocytes from human stool as a new technique for colonic cytology

Cell exfoliation in the gut is an important cell renewal mechanism. To approach its investigation we applied a novel immunomagnetic technique for isolation of exfoliated cells from human stool. Exfoliated colonocytes were isolated from 168 stool samples. The cells were assessed microscopically using conventional stains and immunohistochemistry. The technique allowed us to obtain well-preserved colonocytes displaying characteristic features of well-differentiated colonic epithelium and positive immunostaining for cytokeratin 5/8. No mucin-producing cells were found. Exfoliated cells did not produce inducible nitric oxide synthase, albeit cultured colon carcinoma cells HT-29 analysed in parallel showed strong immunostaining. Analysis of exfoliated cell numbers in consecutive stool samples from the same subjects revealed considerable interindividual variation. Overall exfoliated colonocyte numbers were relatively low, isolation being unaffected by addition during the procedure of excessive amounts of HT-29 cells. Apoptosis was extremely rare among exfoliated colonocytes. Well-preserved exfoliated colonocytes can be consistently isolated from human faeces using a simple procedure. Our findings suggest that the actual process of cell exfoliation in the human colon may be much less intense than is generally accepted. Exfoliated cell isolation from human stool constitutes a convenient non-invasive approach that can be used for diagnostic and research purposes.     

Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride

Summary The effects of various modifications of rabbit skeletal myosin subfragment 1 on thermal denaturation of subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (V_i) or beryllium fluoride (BeF_x) have been studied by differential scanning calorimetry. It has been shown that specific modifications of SH₁ group of Cys-707 by different sulfhydryl reagents, trinitrophenylation of Lys-83, and reductive methylation of lysine residues promote the decomposition of the S1·ADP·V_i complex and change the character of structural transitions of the subfragment 1 molecule induced by the formation of this complex, but they have much less or no influence on subfragment 1 thermal stability in the S1·ADP·BeF_x complex. Thus, the differential scanning calorimetric studies on modified subfragment 1 preparations reveal a significant difference between S1·ADP·V_i and S1·ADP·BeF_x complexes. It is suggested that S1·ADP·V_i and S1·ADP·BeF_x complexes represent structural analogues of different transition states of the ATPase cycle, namely the intermediate states S1^**·ADP·P_i and S1^*·ATP, respectively. It is also proposed that during formation of the S1·ADP·V_i complex the region containing both Cys-707 and Lys-83 plays an important role in the spread of conformational changes from the active site of subfragment 1 ATPase throughout the structure of the entire subfragment 1 molecule. In such a case, the effects of reductive methylation of lysine residues on the subfragment 1 structure in the S1·ADP·V_i complex are related to the modification of Lys-83.     

Demonstration of immunoglobulin on the surface of thymus lymphocytes

Immunoglobulin molecules on the surface of mouse thymus cells have been shown by immunofluorescence. Ninety-five to 100 per cent of thymus lymphocytes were found to bind polyvalent rabbit anti-mouse immunoglobulin, although the density of the fluorescence was much less than that on the surface of B lymphocytes derived from the spleen.

Two purified antisera, an anti-IgM and an anti-kappa chain serum, also stained the cells.

The resynthesis of these immunoglobulin molecules in vitro was demonstrated after they had been removed with pronase.

     

Expression and characterization of a soluble rubella virus E1 envelope protein

Individual specific antigenic rubella virus (RV) structural proteins are required for accurate serological diagnosis of acute and congenital rubella infections as well as rubella immune status. The RV envelope glycoprotein E1 is the major target antigen and plays an important role in viral-specific immune responses. The native virion is difficult to produce in large quantities and the protein subunits are also difficult to isolate without loss of antigenicity. The production of a soluble RV E1 (designated E1ΔTm) using the baculovirus-insect cell expression system is described. In contrast to wild-type RV E1, the genetically engineered E1ΔTm protein lacks a transmembrane anchor. It behaved as a secretory protein and was secreted abundantly from insect cells. Pulse-chase studies were used to examine the synthesis, glycosylation, and secretion of E1ΔTm by the insect cells. The secreted E1ΔTm protein was purified from serum-free medium by onestep immunochromatography. The purified E1ΔTm protein retained full antigenicity and may be a convenient source of E1 protein for use in diagnostic assay and rubella vaccine development.