Analysis of pancreas-infiltrating T cells in diabetic NOD mice: fusion with BW5147 yields a high frequency of islet-reactive hybridomas.

Diabetes in NOD mice is an autoimmune disease which is characterized by the infiltration of islets of Langerhans by large numbers of T cells. Some of these infiltrating T cells are clearly islet-cells-specific; however, many or most of these T cells could be attracted nonspecificity into these lesions. To study NOD pancreas-infiltrating T cells, we fused these cells with BW5147 to make T cell hybridomas. Ninety-four pancreas-derived T hybrids were analyzed of which 12 responded specifically to islet cells by secreting IL-2. Only CD3+, CD4+ hybrids responded to islet cells in our assay, and a large proportion of these hybrids were islet-cell reactive. T cell receptor (TCR) V beta element usage was heterogeneous in islet-reactive hybridomas.     

CD4+ and CD8+ T cells mediated direct cytotoxic effect against Trichophyton rubrum and Trichophyton mentagrophytes

Background The cellular immune system is the most dominant factor in curing acute dermatophytosis. However, the exact immune mechanisms involved in generating this defense are complex and still obscure. The aim of this study was to investigate the fungicidal mechanism of T cells in the normal population versus patients with chronic fungal infections. Methods Thirty patients were included in the study: 15 patients with chronic dermatophytosis and 15 normal healthy patients with a history of acute dermatophytosis. The procedures were performed as follows. 1) Proliferation and cytotoxic activity of lymphocytes cultured with various dermatophytes homogenate such as, Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum gypseum. 2) CD4⁺ and CD8⁺ T cells were separated by magnetic beads before culture with fresh spores of either T. mentagrophytes or T. rubrum. 3) Routine histology and ultrastructural study were performed to illustrate the mode of activity of the T cells against the dermatophytes. Results The study showed that both CD4 and CD8 possess cytotoxic activity against dermatophytes. However, the results demonstrated a suppression of lymphocyte proliferation response and a significant lower cytotoxic effect in chronic patients. Ultra structure and histological evaluation of the culture of hyphae with CD4⁺ or CD8⁺ T cells showed more prominently destructive effects in the culture of cells that had been obtained from normal population than those of patients with long‐lasting fungal infections. Conclusion The study suggests a selective impairment of lymphocyte function against dermatophytes, in patients with chronic dermatophytoses.     

T-cell receptor alpha-chain variable-region haplotypes of normal and autoimmune laboratory mouse strains.

We used Southern blotting and mRNA analysis to characterize allelic polymorphisms among genes of the T-cell antigen receptor (TCR) alpha-chain variable-region (V alpha) locus in a large panel of normal and autoimmune-susceptible or autoimmune-contributing strains of laboratory mice. Four major V alpha haplotypes were defined on the basis of multiple restriction fragment length polymorphisms for each of nine V alpha subfamily probes used. Southern blotting also revealed haplotype-specific loss of bands within some V alpha subfamilies, consistent with the deletion of particular V alpha genes or sets of genes from haplotype to haplotype. In contrast to the situation in the V beta locus, however, deletion of entire V alpha subfamilies was not observed. The nature of V alpha allelic variability was further explored by using an RNase protection assay to analyze expressed V alpha mRNA sequences in thymocyte RNA. Such analysis revealed both shared and unique patterns of V alpha mRNA expression among the different haplotypes and supported the conclusion that haplotype differences sometimes involve V alpha gene deletions. Interestingly, a disproportionate number of, but not all, autoimmune-susceptible strains, including NZB, SJL, SWR, PL/J, and NOD, share a common V alpha haplotype. The identification of murine TCR V alpha haplotypes should provide a basis for understanding the role of TCR diversity in normal immunoregulatory and immune-response phenomena, as well as autoimmune-disease predisposition.     

Immunoglobulin kappa light chain variable region gene complex organization and immunoglobulin genes encoding anti-DNA autoantibodies in lupus mice.

We have investigated the genetic origin of autoantibody production in several strains of mice that spontaneously develop a systemic lupus erythematosus-like disease. Restriction fragment length polymorphism analyses of gene loci encoding kappa light chain variable regions (Igk-V) demonstrated, as shown previously for the Ig heavy chain locus, that autoantibody production and disease occur in different Igk-V haplotypes. Moreover, autoimmune mice with known genetic derivation inherited their Igk-V loci essentially unaltered from their nonautoimmune ancestors. New Zealand black lupus mice, with unknown genetic derivation, had a possibly recombinant Igk-V haplotype, composed of V kappa loci that were primarily indistinguishable from those of nonautoimmune strains from either of the two potential donor haplotypes. The heavy and light chain gene segments (variable, diversity, joining) encoding anti-DNA antibodies were diverse and often closely related, or even identical, to those found in antibodies to foreign antigens in normal mice. Only 1 of 11 sequenced variable region genes could not be assigned to existing variable region gene families; however, corresponding germline genes were present in the genome of normal mice as well. These data argue against abnormalities in the genes and mechanisms generating antibody diversity in lupus mice and suggest a remarkable genetic and structural diversity in the generation of anti-DNA binding sites.     

Microbiological and Toxicological Effects of Perla Black Bean (Phaseolus vulgaris L.) Extracts: In Vitro and In Vivo Studies

Abstract: We investigated the microbiological and toxicological effects of three Perla black bean extracts on the growth and culture of selected pathogenic microorganisms, the toxicity over Vero cell lines and an in vivo rat model. Three different solvents were used to obtain Perla black bean extracts. All three Perla black bean extracts were tested for antibacterial and antiparasitic activity and further analysed for intrinsic cytotoxicity (IC₅₀). Methanol Perla black bean extract was used for acute toxicity test in rats, with the up‐and‐down doping method. All Perla black bean extracts inhibited bacterial growth. Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella oxytoca, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis and Listeria monocytogenes showed inhibition, while Escherichia coli and Enterobacter aerogenes did not. Acidified water and acetic acid Perla black bean extract were tested in parasites. The best IC₅₀ was observed for Giardia lamblia, while higher concentrations were active against Entamoeba histolytica and Trichomonas vaginalis. The Vero cells toxicity levels (IC₅₀) for methanol, acidified water and acetic acid Perla black bean extract were [mean ± S.D. (95% CI)]: 275 ± 6.2 (267.9–282.0), 390 ± 4.6 (384.8–395.2) and 209 ± 3.39 (205.6–212.4) µg/ml, respectively. In vivo acute toxicity assays did not show changes in absolute organ weights, gross and histological examinations of selected tissues or functional tests. The acetic acid and methanol Perla black bean extract proved to exhibit strong antibacterial activity and the acidified water Perla black bean extract exerted parasiticidal effects against Giardia lamblia, Entamoeba hystolitica and Trichomonas vaginalis. The three Perla black bean extracts assayed over Vero cells showed very low toxicity and the methanol Perla black bean extract in vivo did not cause toxicity.     

Reversal by Calcium Ions of the Growth Inhibition of Debaryomyces nicotianae Caused by Antifungal Polyene Antibiotics

Only Debaryomyces nicotianae strain 77, of seven different yeast strains tested, was found to be resistant to heptamycin and other antifungal heptaenes when grown in a rich medium. This strain, however, like the other six, was completely susceptible to these antibiotics in a minimal medium. Addition of yeast extract to the minimal medium abolished the heptamycin effect; calcium ions fully duplicated the effect of yeast extract; Mg(2+) and Mn(2+) were also effective but less so than Ca(2+). Ca(2+) also counteracted the activity of the heptaenes ascosin and trichomycin. Complete reversal of the polyene inhibition by Ca(2+) was obtained if the cation was added simultaneously with the antibiotic; addition of Ca(2+) 2 hr after the polyene was without effect. Addition of Ca(2+) in the absence of the polyene caused a slight, if any, growth stimulation of D. nicotianae 77. Cholesterol also counteracted polyene activity; this was due to the formation of a complex with the antibiotic which prevented the polyene from reaching the site of action-the cytoplasmic membrane. No evidence for complex formation between heptamycin and calcium was found. The importance of Ca(2+) in membrane structure, as evidenced from heptaene studies, is discussed.     

The heart of children with steroid-resistant nephrotic syndrome: is it all podocin?

Mutations in the gene NPHS2 encoding podocin are responsible for a recessive form of steroid-resistant nephrotic syndrome (SRNS). The common phenotype is of massive proteinuria in early childhood that tends to progress to end-stage renal failure. Extrarenal manifestations have not been described. Twenty-two children with SRNS from six unrelated Arab families were found to be homozygous for the R138X mutation in NPHS2. Eighteen patients underwent cardiac evaluation at diagnosis of SRNS while they had normal BP and preserved renal function. Cardiac anomalies were detected in 16 (89%) children: Left ventricular hypertrophy in eight, pulmonary stenosis in six, discrete subaortic stenosis in two, and Ebstein anomaly and ventricular septal defect in one each. The remaining four affected individuals were assessed only once they had end-stage renal failure. They had severe left ventricular hypertrophy and experienced repeated episodes of heart failure. Two control groups were equally evaluated. The first consisted of 37 siblings without nephrotic syndrome, of whom only one carrier had a cardiac defect (P < 0.001). None of the second group, which included 22 children with persistent nephrotic syndrome as a result of other causes, had a cardiac anomaly (P < 0.001). Cardiac disorders in homozygotes for mutations in NPHS2 cannot be attributed to an association by chance or to a state of persistent nephrotic syndrome. Because human podocin mRNA is expressed in fetal heart, it is speculated that it may have a role in normal cardiac development. Cardiac evaluation is recommended at the time of diagnosis of SRNS due to mutations in podocin.     

Paternal isodisomy of chromosome 7 with cystic fibrosis and overgrowth

We have diagnosed a boy with cystic fibrosis (CF) due to paternal UPD presenting with overweight and developmental delay, not typical features to CF patients. Two previously reported patients with paternal UPD(7) did not present overgrowth. The discrepancy between the phenotype of this boy and the other two patients raises the question of imprinted genes or homozygotization of a disease-causing gene in paternal UPD7.     

V beta repertoire in rats and implications for endogenous superantigens.

Endogenous superantigens of mice, encoded by mammary tumor virus proviral integrants, induce intrathymic deletion of entire T cell populations that express specific V beta gene products, a phenomenon proposed to be important in self-tolerance and prevention of toxic responses to exogenous microbial superantigens. Evidence for the presence of V beta selecting/deleting endogenous superantigens in other species is lacking. We report here that rats do not exhibit endogenous superantigen-induced V beta clonal deletions despite their strong response to bacterial superantigens. These findings indicate that endogenous superantigens are not obligatory in V beta repertoire shaping.