SummaryAdvanced glycosylation endproducts (AGEs), the glucose-derived adducts that form nonenzymatically and accumulate on tissue proteins, are implicated in many chronic complications associated with diabetes and aging . We have previously described a monocyte/macrophage surface receptor system thought to coordinate AGE protein removal and tissue remodeling, and purified a corresponding 90-kD AGE-binding protein from the murine RAW 264 .7 cell line. To identify AGEbinding proteins in normal animals, the tissue distribution of 125I-AGE rat serum albumin taken up from the blood was determined in rats in vivo. These uptake studies demonstrated that the liver was a major site of AGE protein sequestration . Using a solid-phase assay system involving the immobilization of solubilized membrane proteins onto nitrocellulose to monitor binding activity, and several purification steps including affinity chromatography over an AGE bovine serum albumin matrix, two rat liver membrane proteins were isolated that specifically bound AGES, one migrating at 60 kD (p60) and the other at 90 kD (p90) on SDS-PAGE . NH2-terminal sequence analysis revealed no significant homology between these two proteins nor to any molecules available in sequence databases. Flow cytometric analyses using avian antibodies to purified rat p60 and p90 demonstrated that both proteins are present on rat monocytes and macrophages. Competition studies revealed no crossreactivity between the two antisera; anti-p60 and anti-p90 antisera prevented AGE-protein binding to rat macrophages when added alone or in combination . These results indicate that rat liver contains at least two novel and distinct proteins that recognize AGE-modified macromolecules, although p90 may be related to the previously described 90-kD AGE receptor isolated from RAW 264.7 cells . The constitutive expression of AGE-binding proteins on rat monocytes and macrophages, and the sequestration of circulating AGE-modified proteins by the liver, provides further evidence in support of a role for these molecules in the normal removal of proteins marked as senescent by accumulated glucose-derived covalent addition products, or AGES .G lucose and other reducing sugars attach nonenzymatical ly to the amino groups of proteins in a concentrationdependent manner. Over time, these initial Amadori adducts undergo further rearrangements, dehydrations, and crosslinking with other proteins to accumulate as a family of complex structures that are referred to as advanced glycosylation endproducts (AGEs)t . Although this chemistry has been studied by food chemists for many years, it was only in the past decade that the presence of AGES in living tissues has t Abbreviations used in this paper. AGE, advanced glycosylation endproduct ; FFI, 2-furoyl-4(5)-(2-fumnyl)-1H-imidazole; NC, nitrocellulose ; RSA, rat serum albumin; TBIR, tissue-to-blood isotope ratio. 515 been established . The excessive deposition of these products on structural body proteins as a function of age and elevated glucose concentration, (1)...

During normal aging and in chronic diabetes the excessive accumulation of reactive glucose-protein or glucose-lipid adducts known as advanced glycosylation endproducts (AGEs) has been shown to induce tissue dysfunction, in part through interaction with AGE-specific receptors on monocyte/macrophages and other cells. Recognizing that circulating lymphocytes trafficking through tissues interact with tissue AGEs, we searched for the expression of AGE-binding sites on peripheral blood T lymphocytes. Resting rat and human T cells bound 125I-AGE-albumin with an affinity of 7.8 x 10(7) M-1, whereas, after stimulation with phytohemagglutinin (PHA) for 48 h, binding affinity increased to 5.8 x 10(8) M-1. Flow cytometric analysis of resting rat T cells using polyclonal antibodies raised against rat liver AGE-binding proteins (p60 and p90) revealed the constitutive expression of both immunoreactivities. The number of resting CD4+ and CD8+ T cells positive for anti-p60 antibody binding (34.2 and 58.5%, respectively) increased to 92 and 90% of cells after 48-h stimulation with PHA. Exposure of PHA-activated T lymphocytes to AGE-albumin enhanced expression of interferon gamma (IFN-gamma) mRNA 10-fold and induced greater elaboration of the mature protein than did exposure to unmodified protein or PHA treatment alone. These data indicate that T cells contain an inducible system of surface receptors for AGE-modified proteins, and that receptor occupancy is linked to lymphokine production. This T cell AGE-receptor system might serve to target lymphocytes to AGE-rich tissues and involve them in the regulation of tissue homeostasis either by assisting in macrophage-dependent clearance of AGE-proteins, or by exerting direct antiproliferative action on mesenchymal cells. Under conditions of excessive AGE-protein and AGE lipid accumulation (e.g., aging and diabetes), enhanced production of AGE-induced IFN-gamma may accelerate immune responses that contribute to tissue injury.

SummaryThe regulation of mKNA encoding transforming growth factor 3 (TGF-/3) and interleukin 2 (I1,2) in normal human T cells was explored using novel competitor DNA constructs in the quantitative polymerase chain reaction and accessory cell-independent T cell activation models. Our experimental design revealed the following: (a) TGF-3 mRNA and IL-2 mRNA are regulated differentially in normal human T cells, quiescent or signaled with the synergistic combinations of: sn-l,2-dioctanoylglycerol and ionomycin or anti-CD3 monoclonal antibody (mAb) and anti-CD2 mAb; (b) the steady-state level of TGF-B mRNA in the stimulated T cells, in contrast to that of II.2 mRNA, is increased by the immunosuppressant cyclosporine (CsA); and (c) the paradoxical effect of CsA on TGF-3 mRNA levels is also appreciable at the level of production of functionally active TGF-3 protein. Our findings, in addition to demonstrating the utility of the competitor DNA constructs for the precise quantification of immunoregulatory cytokines, suggest a novel and unifying mechanistic basis for the immunosuppression and some of the complications (e.g., renal fibrosis) associated with CsA usage.T ransforming growth factor 3 (TGF-3), a 25-kD homodimeric multifunctional cytokine and a secretory product of many cell types including T cells, is a potent inhibitor of T cell growth and differentiation (1-3). I1,2, on the other hand, promotes T cell growth and their acquisition of specialized effector functions (4). TGF-B and I1,2, thus, have the potential to exert diametrically opposite effects on the expression of antigen-specific T cell immunity. It was of interest, therefore, to quantify, simultaneously, the regulation of mRNA encoding TGF-3 and Ib2 in normal human T cells. Also, in view of the ability of TGF-3 to inhibit the antiallograft response (5), it was considered important to quantify the effect of cyclosporine (CsA) on the induction of TGF-fl mKNA in normal human T cells, and compare it to its well-characterized inhibitory effect on the induction of II.-2 mRNA (6). Materials and Methods Isolation and Activation of T Cells. T cells were isolated fromnormal human PBMC with a sequential muhi-step procedure that yields >98% CD2 antigen-positive cells and <1% cells that are positive for the DR, CD14, CD25, or CD56 antigens (7). Accessory cell-independent T cell activation was accomplished with either sn-l,2-dioctanoylglycerol (DAG; 10.0/zg/ml) and ionomycin (1.0 #M) or crosslinked anti-CD2 (OKT11; 0.5 #g/ml) and anti-CD3 (OKT3; 0.5 #g/ml) mAbs (7).Design and Synthesis of Competitor DNA Constructs. Fig. 1 illus-trates the design, synthesis, and authentication of the 290-bp TGF-/~ competitor DNA construct created for the quantification of TGF-B mRNA by PCR. As shown, the oligonucleotide primer pair was designed to amplify a region in the TGF-3 gene that contains a MseI restriction site. The MseI digestion of the 246-bp TGF-3 PCK product yielded 210-and 36-bp subfragments that were annealed with a 44-bp DNA insert synthesized in vitro to have cohesive en...

Tumor necrosis factor/cachectin (TNF), initially perceived as a secretory product of activated macrophages, is a potent proinflammatory polypeptide (1, 2). A wide range of,biological outcomes have been ascribed to this hormone that is now recognized to be secreted by additional cells of the immune system, including T cells (3) . TNF, on the one hand, is thought to contribute to the host armamentarium in the arena of defence against infectious agents and in the control of tumor growth . On the other hand, TNF has also been implicated as a major mediator of septic shock and ofcachexia associated with chronic disease states . A possible reason for the differential outcomes might reside in the quantitative rather than qualitative aspects ofTNF physiology ; "appropriate" amounts of TNF being protective to the host and an excess being deleterious. A qualitative explanation might be that the cell-bound rather than secreted version of TNF is advantageous to the host. In this line of reasoning, we have examined whether activated normal human T cells display TNF on their cell surface. Our line of enquiry was prompted also by an earlier report ofinduced human monocytes expressing TNF on their cell surface (4).Our first demonstration that normal human T cells, signaled with a synergistic combination of 12-o-tetradecanoyl phorbol-13-acetate (TPA) and ionomycin, display TNF on their cell surface forms the basis ofthis report . Furthermore, we show that the cell surface TNF is -26 kD, and that cyclosporine (CsA) and methylprednisolone (MP), block the expression ofTNF in T cells at a pretranslational site (by Northern analysis for TNRspecific transcripts) . Our observations confirm and extend the observation that induced human monocytes express a membrane version of TNF (4), and are in broad agreement with an elegant study (published while this manuscript was in preparation) that murine cytotoxic cell lines express a membrane-bound cytotoxin that is immunologically related to TNF (5).

In searching for a candidate mechanism for the immunosuppressive as well as fibrogenic consequences of cyclosporine usage, we have explored the hypothesis that cyclosporine stimulates transcription of transforming growth factor-Ol (TGF-/30, a multifunctional cytokine endowed with immunosuppressive and fibrogenic properties. Our results demonstrate that cyclosporine (i) stimulates TGF-I~I promoter-dependent transcription of chloramphenicol acetyl transferase gene in transiently transfected human A-549 cells, (ii) stimulates the synthesis of TGF-O1 RNA transcripts in human T cells, and (iii) permits the expression/emergence of DNA regulatory proteins (retinoblastoma control factor-1 (RCF-1) and RCF-2) that bind and regulate TGF-~I promoter activity. Our studies demonstrate for the first time that cyclosporine stimulates TGF-~I gene transcription and suggest a novel mechanism of action of cyclosporine.

Tuberculosis (TB) is one of the prevalent causes of death worldwide, with 95% of these deaths occurring in developing countries, like India. The causative agent, Mycobacterium tuberculosis (MTb) has the tenacious ability to circumvent the host’s immune system for its own advantage. Macrophages are one of the phagocytic cells that are central to immunity against MTb. These are highly plastic cells dependent on the milieu and can showcase M1/M2 polarization. M1 macrophages are bactericidal in action, but M2 macrophages are anti-inflammatory in their immune response. This computational study is an effort to elucidate the role of miRNAs that influences the survival of MTb in the macrophage. To identify the miRNAs against critical transcription factors, we selected only conserved hits from TargetScan database. Further, validation of these miRNAs was achieved using four databases viz. DIANA-microT, miRDB, miRanda-mirSVR, and miRNAMap. All miRNAs were identified through a conserved seed sequence against the 3′-UTR of transcription factors. This bioinformatics study found that miR-27a and miR-27b has a putative binding site at 3′-UTR of IRF4, and miR-302c against IRF5. miR-155, miR-132, and miR-455-5p are predicted microRNAs against suppressor of cytokine signaling transcription factors. Several other microRNAs, which have an affinity for critical transcription factors, are also predicted in this study. This MTb-associated modulation of microRNAs to modify the expression of the target gene(s) plays a critical role in TB pathogenesis. Other than M1/M2 plasticity, MTb has the ability to convert macrophage into foam cells that are rich in lipids and cholesterol. We have highlighted few microRNAs which overlap between M2/foam cell continuums. miR-155, miR-33, miR-27a, and miR-27b plays a dual role in deciding macrophage polarity and its conversion to foam cells. This study shows a glimpse of microRNAs which can be modulated by MTb not only to prevent its elimination but also to promote its survival.

Type 2 diabetes (T2D) is a syndrome of multiple metabolic disorders and is genetically heterogeneous. India comprises one of the largest global populations with highest number of reported type 2 diabetes cases. However, limited information about T2D associated loci is available for Indian populations. It is, therefore, pertinent to evaluate the previously associated candidates as well as identify novel genetic variations in Indian populations to understand the extent of genetic heterogeneity. We chose to do a cost effective high-throughput mass-array genotyping and studied the candidate gene variations associated with T2D in literature. In this case-control candidate genes association study, 91 SNPs from 55 candidate genes have been analyzed in three geographically independent population groups from India. We report the genetic variants in five candidate genes: TCF7L2, HHEX, ENPP1, IDE and FTO, are significantly associated (after Bonferroni correction, p<5.5E−04) with T2D susceptibility in combined population. Interestingly, SNP rs7903146 of the TCF7L2 gene passed the genome wide significance threshold (combined P value = 2.05E−08) in the studied populations. We also observed the association of rs7903146 with blood glucose (fasting and postprandial) levels, supporting the role of TCF7L2 gene in blood glucose homeostasis. Further, we noted that the moderate risk provided by the independently associated loci in combined population with Odds Ratio (OR)<1.38 increased to OR = 2.44, (95%CI = 1.67–3.59) when the risk providing genotypes of TCF7L2, HHEX, ENPP1 and FTO genes were combined, suggesting the importance of gene-gene interactions evaluation in complex disorders like T2D.

Nitric oxide (*NO) is a short-lived free radical with many functions including vasoregulation, synaptic plasticity, and immune modulation and has recently been associated with AIDS pathology. Various pathophysiological conditions, such as viral infection, trigger inducible nitric oxide synthase (iNOS) to synthesize NO in the cell. NO-derived species can react with thiols of proteins and form nitrosothiol adducts. HIV-1 protease (HIV-PR) contains two cysteine residues, Cys67 and Cys95, which are believed to serve a regulatory function. We have found that HIV-PR is inactivated by nitric oxide produced in vitro by NO donors and by iNOS. Sodium nitroprusside inhibited HIV-PR by 70%, and S-nitroso-N-acetylpenicillamine completely inhibited the enzyme. Furthermore, iNOS generated sufficient NO to inhibit HIV-PR activity by almost 90%. This inactivation was reversed by the addition of reducing agents. Treatment of HIV-PR with NO donors and ritonavir (a competitive peptide inhibitor) indicates that NO exerts its effect through a site independent of the active site of HIV-PR. Using electrospray ionization mass spectrometry, we found that NO forms S-nitrosothiols on Cys67 and Cys95 of HIV-PR which directly correlate with a loss of activity. These data indicate that NO may suppress HIV-1 replication by directly inhibiting HIV-PR.