Novel predictive assay for contact allergens using human skin explant cultures.

Contact allergens sensitize the immune system by the binding to and subsequent activation of Langerhans cells (LCs), the antigen-presenting cells of the skin. At present, new chemicals are usually tested for their contact allergenicity in animal models. To develop an animal-replacing predictive in vivo assay for the identification of potential contact allergens, we compared the effects of epicutaneous application of six known contact allergens, five known irritants and two dermatologically inactive chemicals on LCs in skin biopsy cultures from seven healthy donors. Immunohistochemical analysis of cryostat sections of all the biopsies treated with contact allergens showed 1) a large reduction in the number of LCs in epidermis, as evaluated by a decrease in human leukocyte antigens (HLA)-DR-expressing cells, and CD1a-expressing cells and 2) accumulation of the remaining LCs at the epidermal-dermal junction. In contrast, the irritants, inactive chemicals, and solvents did not induce these changes. Morphometrical analysis indicated that the contact allergen-induced reduction in the number of HLA-DR+ and CD1a+ LCs per millimeter of epidermis was significant and was dependent on the concentration of the contact allergens. Flow cytometric analysis of isolated epidermal cells confirmed the immunohistochemical findings. In combination, these results suggest that the culture of ex vivo human skin explants provides a promising model to predict potential allergenicity of newly produced chemical compounds and can therefore replace current animal models.     

Activation-induced polarized recycling targets T cell antigen receptors to the immunological synapse; involvement of SNARE complexes

The mechanism by which T cell antigen receptors (TCR) accumulate at the immunological synapse has not been fully elucidated. Since TCRs are continuously internalized and recycled back to the cell surface, we investigated the role of polarized recycling in TCR targeting to the immunological synapse. We show here that the recycling endosomal compartment of T cells encountering activatory antigen-presenting cells (APCs) polarizes towards the T cell-APC contact site. Moreover, TCRs in transit through recycling endosomes are targeted to the immunological synapse. Inhibition of T cell polarity, constitutive TCR endocytosis, or recycling reduces TCR accumulation at the immunological synapse. Conversely, increasing the amount of TCRs in recycling endosomes before synapse formation enhanced their accumulation. Finally, we show that exocytic t-SNAREs from T cells cluster at the APC contact site and that tetanus toxin inhibits TCR accumulation at the immunological synapse, indicating that vesicle fusion mediated by SNARE complexes is involved in TCR targeting to the immunological synapse.     

Polymerisation by the chromate/arsenite system

The aqueous polymerisation of methyl methacrylate initiated by the chromate/arsenite system has been studied. It is observed that the polymerisation by the above redox system is catalysed by OH^? though the parent reaction between chromate and arsenite is catalysed by H^⊕. It is also observed that traces of Cu^2⊕ inhibit both the polymerization reaction as also the parent reaction. From these observations it is concluded (1) intermediate valency states of chromium has no initiating power in alkali solution and (2) the redox reaction between chromate and arsenite is a chain reaction involving single electron transfer and the intermediate As^4⊕ thus produced is the initiating species.     

Increased levels of inflammatory mediators in children and adults infected with Vibrio cholerae O1 and O139

Investigations were carried out to study the production of factors associated with the innate immune response in the systemic and mucosal compartments in adults and children infected with Vibrio cholerae O1 and V. cholerae O139. The levels of nonspecific mediators of the innate defense system, i.e., prostaglandin E(2) (PGE(2)), leukotriene B(4) (LTB(4)), and lactoferrin (Lf), as well as myeloperoxidase (MPO), were elevated at the acute stage of the disease in stools obtained from both O1- and O139-infected adults and children. In the systemic compartment, the levels of Lf were increased after onset of disease, which in children remained elevated up to convalescence compared to the healthy controls. Increased concentrations of C-reactive protein were seen in the sera of adult cholera patients at the acute stage of infection. Elevated levels of the nitric oxide (NO*) metabolites (nitrite and nitrate [NO(2)(-) and NO(3)(-)]) were detected in plasma but not in urine. The activity of the scavenger of reactive oxygen species, superoxide dismutase, was higher in the plasma of adults immediately after the onset of disease, suggesting that an active scavenging of reactive oxygen species was taking place. The concentration of 8-iso-prostaglandin F(2 alpha) remained unchanged in the systemic and mucosal compartments in the study subjects. After the recovery of patients from cholera, the concentration of the majority of the metabolites decreased to baseline levels by day 30 after the onset of infection. Immunohistochemical staining showed increased tissue expression of MPO, Lf, and inducible nitric oxide synthase at the acute stage in the duodenal biopsies of adults and rectal biopsies obtained from children with cholera. Very little difference was seen in the levels of the different inflammatory mediators in patients infected with V. cholerae O1 or the encapsulated V. cholerae O139. In summary, these results suggest that elevated concentrations of Lf, MPO, PGE(2), LTB(4), and NO*, as well as other metabolites, during the acute stage of the disease indicate that the innate defense system, as well as the inflammatory process, is activated in both adults and pediatric patients infected with V. cholerae O1 and O139.     

Regulation of NK cell function in vivo by the dose of tumour transplanted in the peritoneum

Antigen dose is known to regulate T cell activation and anergy. Similarly, dose of antigen also regulates NK cell lytic potential and phenotype development. Resident peritoneal cells of rat contain a small population of NK and NKT cells. Inoculation of AK-5 tumour cells intraperitoneally modulate the cytotoxic function of NK and NKT cells present in the peritoneal exudate cells (PEC) in a dose dependent manner. Low dose of tumour causes activation of NK and NKT cell cytotoxic function and enhanced NK and NKT cell population in PEC, whereas, high doses of tumour cause inactivation of NK and NKT cell cytotoxic function and depletion of the two sub-populations in the peritoneum. Different doses of tumour inoculation in the peritoneal cavity did not suppress the cytotoxic function of NK cells from spleen suggesting that a direct interaction between NK cells and tumour cells is required for the suppression of NK cell cytotoxic function. Tumour inoculation induced secretion of IL-2, IL-12, IFN-gamma and TNF-alpha by tumour infiltrating mononuclear cells (TIM) in ascitic fluid as well as in serum. The levels of IL-2, IL-12, IFN-gamma and TNF-alpha secretion were higher in animals, which rejected tumours as compared with the animals that failed to reject the tumours. Injection of anti IL-12 and anti IFN-gamma antibody reduced the survival rate of tumour injected animals, however, anti IL-2 antibody had no effect on the survival of animals. Following incubation with AK-5 tumour cells, activated NK cells upregulated perform expression, whereas, there was upregulation of CD95 expression in inactivated NK cells.