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Recently described forkhead box protein 3 (FoxP3) transcription factor is a key molecule in CD4+ CD25hi+ T-cell characterization. Invariant NK T (iNKT) cells are also characterized as regulatory cells modulating the immune response by rapidly producing Th1 and Th2 cytokines. We aimed to analyze cellular markers important in regulatory features of human iNKT cells and to study their role in functional assays. iNKT cells were single cell sorted from peripheral mononuclear cells of healthy individuals after immunostaining of invariant TCR α-chain. We found FoxP3 expression in human iNKT clones. Randomly selected iNKT cell clones (CD4+, double negative, CD8+) expressed FoxP3 mRNA and protein at different levels upon stimulation as supported by various approaches. FoxP3 mRNA and protein expression was detected in unstimulated iNKT cells as well. Furthermore, different stimulations changed the FoxP3 expression in iNKT cells over time and the most dramatic changes were observed upon anti-CD3 stimulation. Both the supernatant of iNKT cells and iNKT cells themselves exerted similar stimulation effects on PBMC proliferation in functional assays and these stimulations showed a negative correlation with FoxP3 expression. Our data indicate that the FoxP3 expression in iNKT cells may be a key transcriptional factor in controlling the regulatory function of the iNKT cells.

Invariant NKT (iNKT) cells are considered to be important in some autoimmune diseases including Type 1 diabetes mellitus (T1DM). So far, the published data are contradictory in regard to the role of iNKT cells in T1DM. We aimed to study iNKT cell frequency and the function of different iNKT cell subgroups in T1DM. We compared the results of four subject groups: healthy (H), long-term T2DM (ltT2DM; more than 1 year), newly diagnosed T1DM (ndT1DM; less than 3 months), and ltT1DM (more than 1 year) individuals. We measured the iNKT cell frequencies by costaining for the invariant TCR α-chain with 6B11-FITC and Vα24-PE. After sorting the Vα24+6B11+ cells, the generated iNKT clones were characterized. We tested CD4, CD8, and CD161 expression and IL-4 and IFN-γ production on TCR stimulation. The CD4+ population among the iNKT cells was decreased significantly in ltT1DM versus ndT1DM, ltT2DM, or H individuals. The T1DM iNKT cell cytokine profile markedly shifted to the Th1 direction. There was no difference in the frequency of iNKT cells in PBMC among the different patient groups. The decrease in the CD4+ population among the iNKT cells and their Th1 shift indicates dysfunction of these potentially important regulatory cells in T1DM.

The development of adipose tissue in the chick embryo was investigated using two groups of fertile eggs which differed by 1.7-fold in their initial yolk lipid levels. The triacylglycerol content of the subcutaneous adipose depot in both groups increased dramatically from day 12 of the 21-day embryonic period, attaining a maximal value just prior to hatching. During this period, the amount of triacylglycerol deposited in the adipose tissue was very highly correlated with the amount of lipid transferred from the yolk. The triacylglycerol content of the depot was also dependent on the initial yolk lipid content. During the hatching period, the amount of adipose triacylglycerol remained approximately constant in the group with the higher initial yolk lipid content but, in the case of the group with the lower initial yolk lipid levels, decreased by approximately 25%. The size distribution of adipocytes isolated from the tissue was determined by computerized image analysis microscopy. The mean adipocyte diameter increased from approximately 6 to 35 microns between days 12 and 19, irrespective of the initial yolk content, although development within the eggs with the lower initial yolk content resulted in a decrease in cell size over the hatching period. Both the triacylglycerol and phospholipid fractions of the isolated adipocytes contained substantial proportions (approximately 6%, w/w) of docosahexaenoic acid (DHA) at days 12 and 14, and lower levels of this fatty acid at the later stages. The amount (mg/depot) of DHA in adipose triacylglycerol decreased dramatically over the hatching period. The amount (mg/brain) of DHA in brain phospholipid increased by more than 5-fold between day 12 of development and hatching. A possible explanation for the data may be that DHA is preferentially mobilized from adipose tissue in order to deliver the fatty acid to the developing neural tissues in a form suitable for uptake.

There is a growing body of evidence to suggest that the autoimmunity observed in type 1 diabetes mellitus (T1DM) is the result of an imbalance between autoaggressive and regulatory cell subsets. Therapeutics that supplement or enhance the existing regulatory subset are therefore a much sought after goal in this indication. Here, we report the results of a double blind, placebo controlled, phase I clinical trial of a novel antigen-specific therapeutic in 12 subjects with recently diagnosed T1DM. Our primary objective was to test its safety. The study drug, human insulin B-chain in incomplete Freund's adjuvant (IFA) was administered as a single intramuscular injection, with subjects followed for 2 years. All subjects completed therapy and all follow-up visits. The therapy was generally safe and well-tolerated. Mixed meal stimulated C-peptide responses, measured every 6 months, showed no statistical differences between arms. All patients vaccinated with the autoantigen, but none who received placebo, developed robust insulin-specific humoral and T cell responses. Up to two years following the single injection, in peripheral blood from subjects in the experimental arm, but not the control arm, insulin B-chain-specific CD4+ T cells could be isolated and cloned that showed phenotypic and functional characteristics of regulatory T cells. The induction of a lasting, robust immune response generating autoantigen-specific regulatory T cells provides strong justification for further testing of this therapy in type 1 diabetes. (clinicaltrials.gov identifier NCT00057499).

Type 1 diabetes mellitus (T1DM) in humans is characterized by the T-cell-dependent destruction of the insulin producing pancreatic beta cells; however, the precise pathogenesis of the disease, especially the initiation of pathologic immune response, is still largely unknown. We hypothesized that the function of human CD4+ T cells is altered in T1DM and analyzed unstimulated human peripheral blood CD4+ T-cell gene expression. We used a novel three-way comparison of DNA microarray data of CD4+ T cells isolated from patients with new onset T1DM, patients with long-term Type 2 diabetes (T2DM), and from healthy control subjects in order to eliminate any possible influence of glucose homeostasis on our findings. We analyzed the T1DM specific gene-expression changes and their functional relevance to T1DM autoimmunity. Our genetic and functional data show that T1DM CD4+ T cells are down-regulated specifically affecting key immune functions and cell cycle. Histone deacetylase gene expression, a key regulator of epigenetic modification is also reduced. The CD4+ T cells showed impaired function, including an abnormal immune response, which may be a key element that leads to the breakdown of self-tolerance.

Triacylglycerols from the initial yolk and from the adipose tissue of the chick embryo were subjected high-performance liquid chromatography in the silver ion mode to resolve molecular species of triacylglycerol on the basis of the degree of unsaturation. A total of 12 such species was resolved from the yolk samples, with only one of these species containing docosahexaenoic acid. In contrast, 17 species were resolved from adipose tissue at day 12 of embryo development, of which 8 contained docosahexaenoic acid. The major species containing this fatty acid consisted of docosahexaenoic acid in combination with palmitic and oleic acids. The amounts of the species consisting of C16 and C18 fatty acids in the adipose tissue increased continuously from day 12 until just before hatching at day 19 and then decreased slightly over the hatching period. In contrast, the amounts within the adipose depot of the species containing docosahexaenoic acid reached a maximal level by day 16 and then decreased dramatically to almost undetectable levels during the hatching period. The possibility that adipose tissue triacylglycerol in the chick embryo may function as a temporary store of docosahexaenoic acid for subsequent delivery to the developing neural tissues is suggested.

Changes in the amounts of the major fatty acids present in the lipids of the yolk complex and the embryo were delineated during embryogenesis of the chicken. The rates of transfer of palmitic, oleic, linoleic, linolenic and arachidonic acids from the lipids of the yolk complex were essentially identical. In contrast, docosahexaenoic acid (DHA) was preferentially transferred from the yolk complex at a rate which was significantly higher than that exhibited by the other major fatty acids. The rates of accumulation of both arachidonic acid and DHA in the lipids of the whole embryo were significantly greater than the rates observed for the C16 and C18 fatty acids, particularly between days 12 and 16 of the 21-day embryonic period. Analysis of the fatty acid composition of plasma lipid throughout development indicated that the triacylglycerol fraction contained relatively high proportions (up to approx. 14% w/w of total fatty acids) of DHA, but much lower proportions (approx. 3%) of arachidonic acid. In contrast, plasma phospholipid was enriched in arachidonic acid (up to approx. 18%), but contained much lower proportions (generally less than 3%) of DHA. A considerable amount of DHA was incorporated into adipose tissue triacylglycerol, so that by the time of hatching, the tissue represented a major store of this fatty acid. Over the hatching period, the amount of DHA in adipose triacylglycerol decreased dramatically, by up to 85%, whereas there was little or no change in the amounts of the other major fatty acyl components in this tissue. The amount of DHA as a component of brain phospholipid increased continuously throughout the developmental period studied. However, by the time of hatching, the amount of DHA in brain phospholipid represented less than 10% of the amount of this fatty acid originally present in the lipids of the yolk.