Comprehensive Profiling of an Aging Immune System Reveals Clonal GZMK+ CD8+ T Cells as Conserved Hallmark of Inflammaging

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Human genetic approaches to diseases of lymphocyte activation

Our laboratory focuses on the study of the molecular regulation of T lymphocyte homeostasis, particularly as it relates to immunological tolerance, apoptosis, and autoimmune diseases. Through intense molecular research on the regulation of lymphocyte fate, the Fas receptor and other tumor necrosis factor receptors as well as their ligands have emerged as key regulators of T lymphocyte apoptosis. We are studying genetic abnormalities of this death pathway, particularly in the context of autoimmune lymphoproliferative syndrome (ALPS) and other non-ALPS conditions affecting lymphocyte homeostasis. These studies have led to further investigations of the regulation of the NF-kappaB signaling pathway, the molecular basis for programed cell death and viral cytopathicity, mechanisms of autoimmunity, and the regulation of mature T-cell tolerance. Our investigations promise to provide insight into the molecular mechanisms behind the regulation of immune response and contribute to the development of novel diagnostic and treatment methods for autoimmune diseases.     

Modulation of dendritic cells using granulocyte-macrophage colony-stimulating factor (GM-CSF) delays type 1 diabetes by enhancing CD4+CD25+ regulatory T cell function

Abnormalities in DC function are implicated in defective immune regulation that leads to type-1 diabetes (T1D) in NOD mice and humans. In this study, we used GM-CSF and Flt3-L to modulate DC function in NOD mice and observed the effects on T1D development. Treatment with either ligand at earlier stages of insulitis suppressed the development of T1D. Unlike Flt3-L, GM-CSF was more effective in suppressing T1D, even when administered at later stages of insulitis. In vitro studies and in vivo adoptive transfer experiments revealed that CD4+CD25+ T cells from GM-CSF-treated mice could suppress effector T cell response and T1D. This suppression is likely mediated through enhanced IL-10 and TGF-β1 production. Adoptive transfer of GM-CSF exposed DCs to naive mice resulted in an expansion of Foxp3+ T cells and a significant delay in T1D onset. Our results indicate that GM-CSF acted primarily on DCs and caused an expansion of Foxp3+ Tregs which delayed the onset of T1D in NOD mice.     

CO(2) and pH independently modulate L-type Ca(2+) current in rabbit carotid body glomus cells

The carotid bodies respond to changes in arterial O(2), CO(2), and pH, and Ca(2+) influx via voltage-gated Ca(2+) channels is an important step in the chemoreception process. The objectives of the present study were as follows: 1) to determine whether hypercapnia modulates Ca(2+) current in glomus cells, and if so, to determine if this modulation is secondary to changes in pH; 2) to examine the mechanism of CO(2) modulation of the Ca(2+) current; and 3) to determine whether the effects of hypercapnia and hypoxia on Ca(2+) channel activity in glomus cells are synergistic. The effects of CO(2) on Ca(2+) current were monitored in glomus cells isolated from rabbit carotid bodies using both perforated and conventional patch-clamp techniques. Raising CO(2) in the extracellular solution from 5 to 10% (hypercapnia) reversibly augmented the whole-cell Ca(2+) current. This augmentation was rapid and increased the whole-cell Ca(2+) current similarly in both the perforated and the conventional patch configurations by 16 +/- 2% (n = 5) and 15 +/- 1% (n = 32), respectively. The following observations suggest that the effects of CO(2) are not secondary to changes in pH: 1) isohydric hypercapnia (pH maintained at 7.4) augmented the Ca(2+) current by 24 +/- 2% (n = 6); 2) decreasing the pH of the extra- or intracellular solutions decreased the Ca(2+) current by 43 +/- 4% (n = 8) and 13 +/- 1% (n = 5), respectively; and 3) hypercapnia did not shift the half-maximal activation voltage (V(1/2)), whereas intracellular and extracellular acidosis alone caused shifts in V(1/2). Furthermore, 100 nM of a membrane-permeable protein kinase A inhibitor prevented the augmentation by CO(2), and 500 microM 8-Br-cAMP mimicked the effect of CO(2) by augmenting the Ca(2+) current by 10 +/- 2% (n = 6). Also, cyclic AMP levels in carotid bodies increased from 1.98 +/- 0.6 to 9.0 +/- 2 pmol/microg protein in response to hypercapnia. In contrast, decreasing pH in the nominal absence of CO(2) did not affect cAMP levels in rabbit carotid bodies. Further, nisoldipine, but not omega-conotoxin MVIIC, prevented augmentation of the Ca(2+) current by CO(2). In addition, when combined, hypercapnia and hypoxia augmented the Ca(2+) current by 26 +/- 4% (n = 7), which is greater than either stimulus alone, suggesting the effects are additive. Taken together, these results indicate that L-type Ca(2+) current is augmented by hypercapnia. The effect of CO(2) is not secondary to changes in pH and seems to be mediated by a protein kinase A-dependent mechanism. Furthermore, hypercapnia and hypoxia act additively in stimulating Ca(2+) current in glomus cells.     

Congenital methemoglobinemia due to NADH-methemoglobin reductase deficiency in three Indian families

Congenital methemoglobinemia is a relatively rare clinical disorder characterized by life-long cyanosis, caused by either an inherited mutant hemoglobin (Hb-M) or deficiency of physiologically active NADH-dependent methemoglobin reductase (NADH-MR). NADH-MR deficiency leads to two different types of recessive congenital methemoglobinemia. In type I, cyanosis is the only major symptom and NADH-MR deficiency is restricted only to the red blood cells. In type II, cyanosis is associated with severe mental retardation and neurological impairment. The objective of this study is to establish the cause of cyanosis in our cases of congenital methaemoglobinemia. Erythrocyte NADH-MR activity was assayed spectrophotometrically. Spectral analysis of the hemolysate treated with potassium ferricyanide was recorded between 400-700 nm and Hb electrophoresis on starch gel at pH 7.0 was done to rule out the presence of Hb-M. NADH-MR deficiency was detected in 3 families. There was a history of consanguinity in one of these cases. The three propositi presented with breathlessness, fever and peripheral cyanosis. There was no history of cardiac illness or exposure to drugs and chemicals. There were no signs and symptoms of mental retardation. The presence of Hb-M was ruled out. Hb-A2, Hb-F, G6PD activity and reduced glutathione levels were normal. NADH-MR activity in all the cases ranged from 4.1 to 9.2 IU/g Hb (normal range 7.0-24.0 IU/g Hb). We describe NADH-MR deficiency in three unrelated cases (age 4 months to 6 years) where the activity of the enzyme was 30-40% of normal. These three cases of congenital methemoglobinemia are due to type-I NADH-MR deficiency without mental retardation.     

Serum concentrations of oestradiol-17beta, progesterone, relaxin and chorionic gonadotrophin during blastocyst implantation in natural pregnancy cycle and in embryo transfer cycle in the rhesus monkey

The present study was undertaken to assess the temporal association between the profiles of serum concentrations of oestradiol-17beta, progesterone, chorionic gonadotrophin (CG) and relaxin in pregnancies established naturally, and after embryo transfer, as well as in failed pregnancies in rhesus monkeys. In naturally mated cycles (group 1) a conception rate of 75% was obtained. In group 1, the mean day of CG detection in serum was 11.5 +/- 1.9 day post-ovulation, and for relaxin, 9.0 +/- 2.5 day post-ovulation. In group 2, embryo transfer to synchronous, non-mated surrogate recipients was performed; seven embryo transfer cycles yielded three pregnancies which were allowed to continue to term and normal infants were delivered. In embryo transfer cycles the mean day of CG detection was 14.8 +/- 1.8 day post- ovulation, and for relaxin, 11.4 +/- 2.6 day post-ovulation. A delay of about 3 days was observed in the appearance in circulation of CG (P < 0.05) and also of relaxin (P < 0.05) between natural mated and embryo transfer conception cycles. Significant differences (P < 0.05 for progesterone and P < 0.03 for oestradiol) were obtained for the areas under the curves for progesterone and oestradiol between days 12 and 16 in conception cycles compared with failed pregnancies. These data provide the first observation of the normal hormonal signals associated with maternal recognition of transferred embryos during the peri- implantation period, and suggest that the use of such an experimental primate embryo transfer model may help to elucidate components of maternal and embryonic signal-response mechanisms during embryo implantation.      

The role of size, sequence and haplotype in the stability of FRAXA and FRAXE alleles during transmission

Factors involved in the stability of trinucleotide repeats during transmission were studied in 139 families in which a full mutation, premutation or intermediate allele at either FRAXA or FRAXE was segregating. The transmission of alleles at FRAXA, FRAXE and four microsatellite loci were recorded for all individuals. Instability within the minimal and common ranges (0-40 repeats for FRAXA, 0-30 repeats for FRAXE) was extremely rare; only one example was observed, an increased in size at FRAXA from 29 to 39 repeats. Four FRAXA and three FRAXE alleles in the intermediate range (41-60) repeats for FRAXA, 31-60 for FRAXE) were unstably transmitted. Instability was more frequent for FRAXA intermediate alleles that had a tract of pure CGG greater than 37 although instability only occurred in two of 13 such transmissions: the changes observed were limited to only one or two repeats. Premutation FRAXA alleles over 100 repeats expanded to a full mutation during female transmission in 100% of cases, in agreement with other published series. There was no clear correlation between haplotype and probability of expansion of FRAXA premutations. Instability at FRAXA or FRAXE was more often observed in conjunction with a second instability at an independent locus suggesting genomic instability as a possible mechanism by which at least some FRAXA and FRAXE mutations arise.      

Increased secretory capacity of mouse adrenal chromaffin cells by chronic intermittent hypoxia: involvement of protein kinase C

Previous studies have shown that catecholamine secretion from the adrenal medulla plays a critical role in chronic intermittent hypoxia (CIH)-induced alterations in cardiovascular function. In the present study we examined the cellular mechanisms associated with the effects of CIH on adrenal chromaffin cell catecholamine secretion. Experiments were performed on adult male mice (C57/BL6) that were exposed to 1–4 days of CIH or to normoxia. Perforated patch electrical capacitance recordings were performed on freshly prepared adrenal medullary slices that permit separating the chromaffin cell secretion from sympathetic input. CIH resulted in a significant increase in the readily releasable pool (RRP) of secretory granules, and decreased stimulus-evoked Ca²⁺ influx. Continuous hypoxia (CH) either for 2.5 h (equivalent to hypoxic duration accumulated over 4 days of CIH) or for 4 days were ineffective in evoking changes in the RRP and Ca²⁺ influx. CIH activated PKC in adrenal medullae as evidenced by increased phosphorylation of PKC at Thr⁵¹⁴ and PKC inhibitors prevented CIH-induced increases in the RRP and restored stimulus-evoked attenuation of Ca²⁺ influx. CIH resulted in elevated thio-barbituric acid reactive substances (TBARSs, an index of oxidized proteins) and an antioxidant prevented CIH-induced changes in the RRP, suggesting the involvement of reactive oxygen species (ROS). These results demonstrate that CIH increases the RRP in adrenal chromaffin cells via ROS-mediated activation of PKC and suggest that CIH can directly affect the secretory capacity of chromaffin cells and contribute, in part, to elevated catecholamine levels.     

Segment-2 sequence analysis and cross-neutralization studies on some Indian bluetongue viruses suggest isolates are VP2-variants of serotype 23

Sequence analysis of segment 2 (seg-2) of three Indian bluetongue virus (BTV) isolates, Dehradun, Rahuri and Bangalore revealed 99% nucleotide identity amongst them and 96% with the reference BTV 23. Phylogenetic analysis grouped the isolates in ‘nucleotype D’. The deduced amino acid (aa) sequence of the Bangalore isolate showed a high variability in a few places compared to other isolates. B-cell epitope analyses predicted an epitope that is present exclusively in the Bangalore isolate. Two-way cross serum neutralization confirmed that Bangalore isolate is antigenically different from the other two isolates. The results of this study suggest that these three isolates are VP2 variants of BTV 23. This signifies that non-cross-neutralizing variants of the same BTV serotype should be included in vaccine preparation.