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Biomedical subjects

V Natarajan

Publications and source records attributed to V Natarajan.

At least 19 recordsLinked to original sources
PubMed2000

Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection.

To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the presence of modified cells in proportions equivalent to or below those in the circulation. The cell infusions were well tolerated and were not associated with substantive immunologic or virologic changes. Thus, adoptive transfer of genetically modified HIV-antigen-specific T cells was safe. Sustained survival in the circulation was achieved when modified CD4(+ )and CD8(+) T cells were infused together after ex vivo costimulation, indicating the important role played by antigen-specific CD4(+) T cells in providing "help" to cytotoxic effectors. (Blood. 2000;96:467-474)

Adult↗
PubMed2000

Optimization of ion-exchange displacement separations. I. Validation of an iterative scheme and its use as a methods development tool.

Displacement chromatography has been demonstrated to be a powerful, high-resolution preparative tool. The performance of displacement systems can be affected by a variety of factors such as the feed load, flow-rate, initial salt concentration and the displacer partition ratio. Thus, the optimization of displacement separations is a uniquely challenging problem. In this manuscript, an iterative optimization scheme has been presented whereby one can identify the optimum operating conditions for displacement separations at a given level of loading on a given resin material. The solid film linear driving force model has been employed in concert with the Steric Mass Action formalism of ion-exchange chromatography to describe the chromatographic behavior in these systems. Simple pulse techniques have been employed to estimate the transport parameters. The iterative scheme has been validated using a rigorous Feasible Sequential Quadratic Programming algorithm. Finally, the utility of the iterative optimization scheme as a methods development tool for displacement separations has been demonstrated for a difficult separation. The results indicate that the use of the optimization scheme leads to significantly better performance than standard rules of thumb.

Algorithms↗
PubMed2000

Optimization of ion-exchange displacement separations. II. Comparison of displacement separations on various ion-exchange resins.

A variety of stationary-phase materials are currently available for the chromatographic purification of biomolecules. However, the effect of various resin characteristics on the performance of displacement chromatography has not been studied in depth. In Part I, a novel iterative scheme was presented for the rapid optimization of displacement separations in ion-exchange systems. In this article, the optimization scheme is employed to identify the optimum operating conditions for displacement separations on various ion-exchange resin materials. In addition, the effect of different classes of separation problems (e.g., diverging, converging or parallel affinity lines) on the performance of displacement separations is also presented. The solid film linear driving force model is employed in concert with the Steric Mass Action isotherm to describe the chromatographic behavior in these systems. The results presented in this article provide insight into the effects of resin capacity and efficiency as well as the type of separation problem on the performance of various ion-exchange displacement systems.

Algorithms↗
PubMed2000

Involvement of c-Src in diperoxovanadate-induced endothelial cell barrier dysfunction.

Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.

Animals↗
PubMed1999

HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression.

Identifying the immunologic and virologic consequences of discontinuing antiretroviral therapy in HIV-infected patients is of major importance in developing long-term treatment strategies for patients with HIV-1 infection. We designed a trial to characterize these parameters after interruption of highly active antiretroviral therapy (HAART) in patients who had maintained prolonged viral suppression on antiretroviral drugs. Eighteen patients with CD4(+) T cell counts >/= 350 cells/microliter and viral load below the limits of detection for >/=1 year while on HAART were enrolled prospectively in a trial in which HAART was discontinued. Twelve of these patients had received prior IL-2 therapy and had low frequencies of resting, latently infected CD4 cells. Viral load relapse to >50 copies/ml occurred in all 18 patients independent of prior IL-2 treatment, beginning most commonly during weeks 2-3 after cessation of HAART. The mean relapse rate constant was 0.45 (0.20 log(10) copies) day(-1), which was very similar to the mean viral clearance rate constant after drug resumption of 0.35 (0.15 log(10) copies) day(-1) (P = 0.28). One patient experienced a relapse delay to week 7. All patients except one experienced a relapse burden to >5,000 RNA copies/ml. Ex vivo labeling with BrdUrd showed that CD4 and CD8 cell turnover increased after withdrawal of HAART and correlated with viral load whereas lymphocyte turnover decreased after reinitiation of drug treatment. Virologic relapse occurs rapidly in patients who discontinue suppressive drug therapy, even in patients with a markedly diminished pool of resting, latently infected CD4(+) T cells.

Adult↗
PubMed1999

Induction of endothelial monolayer permeability by phosphatidate.

Released into the vasculature from disrupted cells or transported to the surface of adjacent effectors, phosphatidate and related lipids may potentiate endothelial cell activation. However, the effect of these lipids on endothelial monolayer barrier integrity has not been reported. The present study documents the induction of endothelial monolayer permeability by phosphatidate. Both long (di-C18:1) and medium (di-C10; di-C8) chain length phosphatidates increased permeability of bovine pulmonary artery endothelial cell monolayers assessed using a well characterized assay system in vitro. Barrier disruption effected by dioctanoyl (di-C8) phosphatidate was markedly potentiated by the addition of propranolol, an inhibitor of endothelial cell "ecto"-phosphatidate phosphohydrolase (PAP), a lipid phosphate phosphohydrolase (LPP) that efficiently hydrolyzes extracellular substrate. Disruption of barrier function by phosphatidate did not result from its non-specific detergent characteristics, since a non-hydrolyzable but biologically inactive phosphonate analog of dioctanoyl phosphatidate, which retains the detergent characteristics of phosphatidate, did not induce permeability changes. Furthermore, neither diacylglycerol nor lyso-PA effected significant increases in monolayer permeability, indicating the observed response was due to phosphatidate rather than one of its metabolites. Phosphatidate-induced permeability was attenuated by preincubation of endothelial cells with the tyrosine kinase inhibitor, herbimycin A (10 microg/ml), and enhanced by the tyrosine phosphatase inhibitor, vanadate (100 microM), implicating a role for activation of intracellular tyrosine kinases in the response. In addition, phosphatidate increased the levels of intracellular free Ca(2+) in endothelial cells and ligated specific binding sites on endothelial cell plasma membranes, consistent with the presence of a phosphatidate receptor. Since phosphatidate generated within the plasma membrane of adherent effectors potentially interacts with endothelial membranes, we evaluated the influence of phosphatidate-enriched neutrophil plasma membranes on endothelial monolayer integrity. The effects of ectopic phosphatidate on endothelial monolayer permeability were mimicked by phosphatidate confined to neutrophil plasma membranes. We conclude that phosphatidate may be a physiologic modulator of endothelial monolayer permeability that exerts its effects by activating a receptor-linked, tyrosine kinase-dependent process which results in mobilization of intracellular stored Ca(2+)and consequent metabolic activation.

Animals↗
PubMed1999

[Phosphodiesterase 5--the enzyme inhibited by sildenafil (Viagra)].

Phosphodiesterases are enzymes that catalyze the degradation of the cyclic nucleotides, cyclic AMP and cyclic GMP, to the corresponding 5' nucleotide monophosphates. Ten different phosphodiesterase families have been described to date. These enzymes exist as homodimers and there is structural similarity between the different families. However, they differ in several respects like selectivity for cyclic nucleotides, sensitivity for inhibitors and activators, physiological roles and tissue distribution. Interest in these enzymes has increased tremendously, both within the medical community and in the general public as a consequence of sildenafil (Viagra), the medication recently introduced for the treatment of erectile dysfunction. Sildenafil mediates its effects by inhibiting phosphodiesterase 5. Some biochemical and molecular biological aspects of this enzyme are presented here. To achieve satisfactory erection, normal penile innervation is required. Nitrogen monoxide, the transmitter substance in these nerves, activates guanylyl cyclase, thereby increasing cyclic GMP production. The increased levels of cyclic GMP cause relaxation of smooth muscles in penile vessels and this leads to an erection. Erection is dependent on elevated levels of cyclic GMP and sildenafil mediates its effects by inhibiting the degradation of cyclic GMP. Other functions that are mediated by the phosphodiesterases explain visual disturbances, flushing and decreased blood pressure that are some of the side effects seen with sildenafil treatment. Furthermore, the potentially fatal consequence of combining sildenafil and nitrovasodilators is discussed.

Cyclic GMP↗
PubMed1999

Tumor necrosis factor-alpha stimulates attachment of small cell lung carcinoma to endothelial cells.

Tumor cell attachment to endothelial cells (ECs) is an important step in the metastasis of small cell lung carcinoma (SCLC). Tumor necrosis factor-alpha (TNF-alpha) stimulation of ECs increases the attachment of some malignant cell types to ECs by affecting the expression of cell adhesion molecules (CAMs). Similarly, the inhibition of EC protein kinase C (PKC) and tyrosine kinase (TK) pathways modulates TNF-alpha-mediated effects on CAM expression. We hypothesized that TNF-alpha would increase SCLC attachment to ECs by affecting CAM expression through activation of PKC and TK pathways. To test this hypothesis, human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-alpha (0 to 500 U/mL) for variable time periods (1 to 24 hours), and the attachment of H82 cells (an SCLC cell line) to the HUVECs was quantified. TNF-alpha stimulation of the HUVECs increased H82 attachment from 28.1% +/- 1.6% to 48.8% +/- 1.7% (P < .05). Preincubation of HUVECs with the PKC inhibitors bis-indolylmaleimide (BIN) or calphostin C or the TK inhibitors genistein or herbimycin A (HMA) blocked the TNF-alpha-induced increase in H82 cell attachment. The addition of antibodies to vitronectin (Vn) or beta1-integrin to TNF-alpha-activated HUVECs before the addition of the H82 cells also significantly decreased H82 attachment, whereas the addition of antibodies to E-selectin, P-selectin, vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), neural cell adhesion molecule (NCAM), sialyl-Lewis(x), fibronectin (Fn), alpha(v)-integrin, alpha3-integrin, alpha4-integrin, or alpha5-integrin had no effect on SCLC attachment. In summary, the TNF-alpha-mediated increase in SCLC attachment to ECs appears to be mediated by the activation of EC PKC and TK pathways as well as through effects on the function or expression of EC Vn and beta1 integrin.

Carcinoma, Small Cell↗
PubMed1999

Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60(src).

Inflammatory diseases of the lung are characterized by increases in vascular permeability and enhanced leukocyte infiltration, reflecting compromise of the endothelial cell (EC) barrier. We examined potential molecular mechanisms that underlie these alterations and assessed the effects of diperoxovanadate (DPV), a potent tyrosine kinase activator and phosphatase inhibitor, on EC contractile events. Confocal immunofluorescent microscopy confirmed dramatic increases in stress-fiber formation and colocalization of EC myosin light chain (MLC) kinase (MLCK) with the actin cytoskeleton, findings consistent with activation of the endothelial contractile apparatus. DPV produced significant time-dependent increases in MLC phosphorylation that were significantly attenuated but not abolished by EC MLCK inhibition with KT-5926. Pretreatment with the Rho GTPase-inhibitory C3 exotoxin completely abolished DPV-induced MLC phosphorylation, consistent with Rho-mediated MLC phosphatase inhibition and novel regulation of EC MLCK activity. Immunoprecipitation of EC MLCK after DPV challenge revealed dramatic time-dependent tyrosine phosphorylation of the kinase in association with increased MLCK activity and a stable association of MLCK with the p85 actin-binding protein cortactin and p60(src). Translocation of immunoreactive cortactin from the cytosol to the cytoskeleton was noted after DPV in concert with cortactin tyrosine phosphorylation. These studies indicate that DPV activates the endothelial contractile apparatus in a Rho GTPase-dependent fashion and suggests that p60(src)-induced tyrosine phosphorylation of MLCK and cortactin may be important features of contractile complex assembly.

Actins↗
PubMed1999

Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells.

Reactive oxygen species (ROS) are implicated in the pathophysiology of several vascular disorders including atherosclerosis. Although the mechanism(s) of ROS-induced vascular damage remains unclear, there is increasing evidence for ROS-mediated modulation of signal transduction pathways. Exposure of bovine pulmonary artery endothelial cells to hydrogen peroxide (H(2)O(2)) enhanced tyrosine phosphorylation of 60- to 80- and 110- to 130-kDa cellular proteins, which were determined by immunoprecipitation with specific antibodies focal adhesion kinase (p125(FAK)) and paxillin (p68). Brief exposure of cells to a relatively high concentration of H(2)O(2) (1 mM) resulted in a time- and dose-dependent tyrosine phosphorylation of FAK, which reached maximum levels within 10 min (290% of basal levels). Cytoskeletal reorganization as evidenced by the appearance of actin stress fibers preceded H(2)O(2)-induced tyrosine phosphorylation of FAK, and the microfilament disruptor cytochalasin D also attenuated the tyrosine phosphorylation of FAK. Treatment of BPAECs with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-AM attenuated H(2)O(2)-induced increases in intracellular Ca(2+) but did not show any consistent effect on H(2)O(2)-induced tyrosine phosphorylation of FAK. Several tyrosine kinase inhibitors, including genistein, herbimycin, and tyrphostin, had no detectable effect on tyrosine phosphorylation of FAK but attenuated the H(2)O(2)-induction of mitogen-activated protein kinase activity. We conclude that H(2)O(2)-induced increases in FAK tyrosine phosphorylation may be important in H(2)O(2)-mediated endothelial cell activation.

Actins↗
PubMed1998

Activation-induced down-regulation of retinoid receptor RXRalpha expression in human T lymphocytes. Role of cell cycle regulation.

A 5.4-kilobase mRNA, the expression of which is down-regulated after treatment of human peripheral blood mononuclear cells (PBMCs) with various T cell-activating agents, was isolated using an mRNA differential display method. Nucleotide sequence analysis identified the 5' end of this RNA as human retinoid receptor RXRalpha mRNA. Here, we report the nucleotide sequence of 3.6 kilobases of this RNA, which represents the 3' end of RXRalpha mRNA, the sequence of which has not been previously described. Activated PBMCs also expressed lower levels of RXRalpha protein, and a DNA binding assay showed that the activation-induced loss of RXRalpha mRNA and protein expression correlated with the loss of DNA binding activity of this protein. We present evidence that the transition from G0/G1 to S phase of the cell cycle results in the down-regulation of RXRalpha expression and that cell cycle inhibitors, which block the cells in G1 phase, prevent this down-regulation. The decrease in the levels of RXRalpha mRNA was found to be regulated at the post-transcriptional level and involved new protein synthesis. These observations indicate that the levels of RXRalpha expression in T lymphocytes are coupled to cell cycle progression, and there is tight regulatory control of RXRalpha expression during the transition from G0/G1 to S phase of the cell cycle.

Base Sequence↗
PubMed1998

Characterization of the gene encoding the human type II cGMP-dependent protein kinase.

The type II cGMP-dependent protein kinase (cGK) plays a pivotal role in the regulation of intestinal fluid balance in man. Furthermore, mice carrying a null mutation for the gene encoding the type II cGK develop as dwarfs indicating that this enzyme has other less characterized roles. The present report describes the isolation and characterization of bacterial artificial chromosome (BAC)- and P1-derived artificial chromosome (PAC)-clones containing the gene encoding the human type II cGK. The gene was estimated to cover at least 125 kb and consisted of 19 exons separated by introns of various lengths. The splice junctions of the type II cGK gene corresponded well with the structure of the gene encoding human type I cGK and with the splice junctions observed in the Drosophila melanogaster DG2 gene. 5'-rapid amplification of cDNA-ends established the presence of a non-translated exon.

Amino Acid Sequence↗
PubMed1998

RAR-, not RXR, ligands inhibit cell activation and prevent apoptosis in B-lymphocytes.

We have previously shown that retinoids inhibit activation of human peripheral blood B-lymphocytes. In the present paper, we wished to explore the involvement of nuclear retinoid-specific receptors in this process by using ligands specific for the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We found that the RAR-specific ligand TTAB reduced anti-IgM-induced B-cell activation in a dose-dependent manner. Thus, at 100 nM of TTAB, DNA synthesis was reduced by approximately 60%. In contrast, the RXR-selective ligand SR11217 had no effect on DNA synthesis. Similar findings were obtained when the expression of the activation antigen CD71 (appears late in G1) was examined. The role of retinoids in apoptosis of resting peripheral blood B-lymphocytes was examined using the same receptor-selective ligands. Again, we found that the RAR-selective ligands were more potent effectors than were the RXR-selective ligands. In spite of the inhibitory effects of retinoids on B-cell proliferation, the same retinoids significantly promoted the survival of the cells. Thus, 10 nM TTAB significantly reduced spontaneous apoptosis of in vitro cultured B-cells at day 3 from 45% to 30%, as determined by vital dye staining and DNA end-labeling. Again, the RXR-specific ligand SR11217 had no effect. Interestingly, we found that CD40 ligand was able to potentiate the retinoid-mediated inhibition of apoptosis. By reverse transcriptase polymerase chain reaction (PCR), we found that peripheral blood B-lymphocytes expressed RARalpha, RARgamma, and RXRalpha, but not RARbeta, RXRbeta, or RXRgamma. Hence, the lack of effect of the RXR-specific ligand SR11217 on growth and apoptosis was not due to absence of RXRs. In conclusion, the ability of retinoids to inhibit growth and prevent apoptosis of normal human B-lymphocytes indicates a dual role of retinoids in this cell compartment, and it appears that both effects of retinoids are mediated via RARs and not RXRs.

Alitretinoin↗
PubMed1998

Expression of retinoic acid receptor and retinoid X receptor subtypes in rat liver cells: implications for retinoid signalling in parenchymal, endothelial, Kupffer and stellate cells.

In the present study, a systematic examination of the relative expression pattern of the nuclear retinoid receptors (RAR and RXR) in various liver cells was performed. Our data demonstrate that RXRalpha is the dominant receptor in all liver cells, and that RARbeta is also expressed at a high level in most cells. More specifically, RARbeta and RARalpha were the most predominant of the RAR subtypes in parenchymal cells, while all three RAR subtypes were equally expressed in endothelial and Kupffer cells. The total level of expression of all the RXR subtypes were larger than the total level of expression of all the RAR subtypes in all liver cells. This is in agreement with the observation that RXR is a heterodimer partner not only for RAR, but also for other members in the steroid/thyroid receptor superfamily of ligand-dependent transcription factors.

Animals↗
PubMed1998

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate.

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [32P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [32P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.

Animals↗
PubMed1998

Peripheral expansion of pre-existing mature T cells is an important means of CD4+ T-cell regeneration HIV-infected adults.

The CD4+ T-cell pool in HIV-infected patients is in a constant state of flux as CD4+ T cells are infected and destroyed by HIV and new cells take their place. To study T-cell survival, we adoptively transferred peripheral blood lymphocytes transduced with the neomycin phosphotransferase gene between syngeneic twin pairs discordant for HIV infection. A stable fraction of marked CD4+ T cells persisted in the circulation for four to eighteen weeks after transfer in all patients. After this time there was a precipitous decline in marked cells in three of the patients. At approximately six months, marked cells were in lymphoid tissues in proportions comparable to those found in peripheral blood. In two patients, the proportion of total signal for the transgene (found by PCR analysis) in the CD4/CD45RA+ T-cell population relative to the CD4/CD45RO+ population increased in the weeks after cell infusion. These findings indicate that genetically-marked CD4+ T cells persist in vivo for weeks to months and that the CD4+ T-cell pool in adults is maintained mostly by the division of mature T cells rather than by differentiation of prethymic stem cells. Thus, after elements of the T-cell repertoire are lost through HIV infection, they may be difficult to replace.

Adult↗