Hair phenotype in non-syndromic deafness.

The GJB2 gene is located on chromosome 13q12 and it encodes the connexin 26, a transmembrane protein involved in cell-cell attachment of almost all tissues. GJB2 mutations cause autosomal recessive (DFNB1) and sometimes dominant (DFNA3) non-syndromic sensorineural hearing loss. Moreover, it has been demonstrated that connexins are involved in regulation of growth and differentiation of epidermal tissues. Hence, mutations in GJB2 gene, which is responsible for non-syndromic deafness, may be associated with an abnormal skin and hair phenotype. We analyzed hair samples from 96 subjects: a study group of 42 patients with hearing impairments of genetic origin (38 with a non-syndromic form, 4 with a syndromic form), and a control group including 54 people, i.e. 43 patients with other, non-genetic hearing impairments and 11 healthy volunteers aged up to 10 years old. The surface structure of 49 hair samples was normal, whereas in 45 cases it was altered, with a damaged appearance. Two hair samples were considered unclassifiable: one from the patient heterozygotic for the pendrin mutation (Fig. 2C), the other from a patient from Ghana with a R134W mutation (Fig. 2D). Among the 43 altered hair samples, 31 belonged to patients with connexin mutations and the other 12 came from patients without connexin mutations.

Response of bovine gammadelta T cells to activation through CD3.

Since the T cell receptor of gammadelta T cells is associated with CD3 molecules, it is a reasonable postulate that signal transduction through CD3 would occur in gammadelta T cells as it does in alphabeta T cells. However, while a small percentage of bovine gammadelta T cells divided in cultures of peripheral blood mononuclear cells (PBMCs) in response to stimulation by anti-CD3 monoclonal antibody (mAb) the majority of viable gammadelta T cells at the end of the culture period had not. This was assessed by carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis here and previously [Res. Vet. Sci. 69 (2000) 275]. When intracytoplasmic staining for interferon-gamma (IFN-gamma) was also used here to assess activation through CD3, a small proportion of gammadelta T cells (approximately 14%) produced IFN-gamma during the first 4 h of culture and by 72 h of culture that number had doubled. By comparison, a much larger proportion of CD4 and CD8 T cells stimulated with anti-CD3 mAb divided and although the percentage of CD4 and CD8 T cells that produced IFN-gamma at 4 h was similar to that of gammadelta T cells, by 72 h the majority of CD4 and CD8 T cells were IFN-gamma(+). Addition of IL-2 did not increase the proportion of gammadelta T cells that responded to anti-CD3 stimulation by cell division. To test the hypothesis that gammadelta T cells were inhibited from responding by other mononuclear cell populations within PBMC, monocytes were removed from the PBMC or gammadelta T cells were purified by magnetic-bead sorting. Only a small distinct population of the sorted cells underwent multiple cell divisions in response to anti-CD3 mAb and removal of monocytes resulted in only a moderate increase in gammadelta T cell replication. The anti-CD3 mAb stimulation system may provide a useful system to evaluate the difference in the requirements for activation and clonal expansion for gammadelta T cells versus alphabeta T cells.

Activation of bovine peripheral blood gammadelta T cells for cell division and IFN-gamma production.

Bovine peripheral blood gammadelta T cells have been evaluated for effector function (IFN-gamma production) and clonal expansion in a variety of systems including following activation by mitogens, IL-12, and stimulation, through the T cell receptor (TCR) with anti-CD3 monoclonal antibody (mAb), a cell-bound molecule and a soluble antigenic extract. To evaluate cell division, carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis were used, while IFN-gamma production was evaluated by intracytoplasmic staining. It was found that bovine gammadelta T cells produced IFN-gamma and clonally expanded when stimulated through the TCR/CD3 complex by a cell-associated autologous molecule on monocyte, by bacterial components following in vivo sensitization of gammadelta T cells with a leptospira vaccine or by anti-CD3 mAb. In addition, gammadelta T cells were activated efficiently for effector function but not clonal expansion by culturing with IL-12. In contrast, stimulation by Con A or PMA/ionomycin induced efficient replication but only low level IFN-gamma production which was not enhanced by the presence of IL-12. In several systems the amount of IFN-gamma produced per cell by gammadelta T cells was less than that produced by CD4 T cells in the same cultures.

Evaluation of cell replication by bovine T cells in polyclonally activated cultures using carboxyfluorescein succinimidyl ester (CFSE) loading and flow cytometric analysis.

Studies reported here demonstrated that carboxyfluorescein succinimidyl ester (CFSE) loading of lymphocytes and flow cytometric analysis is a powerful assay to assess the kinetics and extent of cellular replication by bovine T-cell subpopulations in heterogeneous cultures of peripheral blood mononuclear cells (PBMC) where subpopulation interactions can occur. As CFSE analysis allows determination of the proportion of lymphocytes that divided, as well as the number of cell divisions each cell underwent, distinctions in responses among mitogen-stimulated cultures could be made even when(3)H-thymidine incorporation was equivalent. When combined with surface staining for detection of differentiation antigens, differences among T-cell subpopulations with regard to the number of divisions their members had undergone, were found. Anti-CD3 mAb stimulated both CD8(+)and CD4(+)T cells to undergo several cell divisions in 72 hours, while there was essentially no division by gamma delta T cells. In contrast, in concanavalin A-stimulated cultures, all T-cell subpopulations had divided.

Rapid changes occur in the percentage of circulating bovine WC1(+)gamma delta Th1 cells.

gamma delta T cells found in the peripheral blood of cattle include a major subpopulation distinguished by expression of WC1. These cells are distinct from the WC1(-)gamma delta T cell population based on T cell receptor gene usage. We documented that a group of 6-month-old calves allowed free-range grazing and access to their mothers had a significantly greater proportion of total gamma delta T cells in their blood, attributable to the WC1(+)gamma delta T cell subpopulation, compared to age and breed-matched calves held in conventional housing. When the animals with the greater proportion of gamma delta T cells were transferred to conventional housing there was a decrease in the WC1(+)population so that by 3 weeks after transfer there was no longer a significant difference between the two groups. To investigate the biological activities of WC1(+)gamma delta T cells, the cells were purified by flow cytometric sorting. In vitro, they responded to stimulation by irradiated monocytes in autologous mixed leukocyte reaction (AMLR) cultures but not to direct stimulation through the T cell receptor (T c R) by anti-delta monoclonal antibody. After stimulation in the AMLR, WC1(+)gamma delta T cells had a Th1 cytokine profile characterised by production of IFN -gamma and lack of IL -4. Thus we propose that higher levels of the WC1(+)gamma delta T cells may provide calves with a mechanism to produce Th1 cytokines and that the level of these cells may be modulated according to environment or stress since both groups of calves were apparently disease-free.