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Biomedical subjects

J Padmanabhan

Publications and source records attributed to J Padmanabhan.

29 records · Page 2Linked to original sources
PubMed1994

Lymphoid organ production of immunomodulatory eicosanoids in mice resistant to neonatal tolerance induction.

Neonatally induced tolerance of class I and class II alloantigens is difficult to achieve in certain I-E non-expressing hosts that received semiallogeneic cells at birth from strains of mice that express I-E molecules. Although clonal deletion occurs ubiquitously after infusion of the tolerogen-bearing inoculum, the majority of these mice ultimately regain the capacity to reject donor-specific skin graft challenges in adulthood and this is associated with a reacquisition of I-E recognizing and alloreactive T cells as well as a loss of donor chimeric cells. In this study, we determined whether production levels of the eicosanoids prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), both potent modifiers of lymphocyte function, were altered in lymphoid organs concomitant with a breakdown of tolerance in these mice. The levels of TxB2 and PGE2 produced by lymphoid organs were measured in the early/late post-partum periods and immediately before and after skin grafting in B10.S mice (H-2s/I-E-) that had been injected at birth with (B10.S x B10.A)F1 (H-2k/d, I-E+) lymphohematopoietic cells. Phenotypic (e.g., %V beta 11+ T cells) and functional parameters of host donor-reactive effector cell populations along with chimerism were determined simultaneously. We found that TxB2 and PGE2 production fluctuated in the early postnatal periods in naive mice and that the neonatally injected counterparts showed a significant alteration from this pattern, particularly with PGE2. As adults, injected hosts maintained an altered pattern of eicosanoid metabolism and this was accentuated after the rejection or acceptance of a donor-specific skin allograft. Specific patterns emerged after transplant challenge such that neonatally injected mice deleted of V beta 11+ T cells before grafting differed in their eicosanoid secretory profiles; moreover, injected mice that accepted (i.e., tolerant) the donor-specific allograft had a markedly different TxB2 and PGE2 profile than injected/rejecting hosts. In naive mice, the application of 2 subsequent grafts elicited a release of splenic TxB2 and PGE2 that mimicked the pattern seen in the neonatally injected hosts after 1 graft--these latter results give preliminary indication that the generation of memory T cells and the re-exposure to specific alloantigen coincides with a derangement in eicosanoid metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Differential patterns of T cell clonal deletion in neonatal H-2 tolerance and I-E/Mls induced self-tolerance.

The pattern of clonal deletion of putative I-E-reactive (V beta 11) and Mls-reactive (V beta 3) T cells was evaluated and compared by cytofluorographic and immunohistochemical methods in a model of neonatal H-2 tolerance and in I-E- or Mls-bearing strains of mice which normally delete these cell populations (self-tolerance). The ontogeny of deletion of V beta 11+ cells was studied by evaluating thymic changes from birth until maturity in B10.S (H-2s/I-E-), B10.A (H-2k/d/I-E+) and B10.S mice intravenously infused at birth with (B10.SxB10.A)F1 lymphohaematopoietic cells. The reduction in V beta 11+ cells was most prevalent in the medullary region of the naive B10.A and neonatally injected B10.S animals and was corroborated by flow cytometry which demonstrated a marked reduction in single CD4 and CD8 positive B beta 11 T cells when compared to naive B10.S mice. However, immunohistochemistry illustrated that 'deletion' was never complete since V beta 11+ cells remained in the thymic cortex and splenic lymphoid follicles. By comparison, DBA/2 mice (Mlsc+ and previously documented to have decreased levels of V beta 3+ cells) showed a different pattern of deletion of V beta 3+ T cells than what was found for T cells bearing V beta 11 in animals deleting this population. DBA/2 thymi contained fewer thymic V beta 3- cells and there was more complete elimination of these cells, particularly in the periphery, by flow cytometry and immunohistology. The mice which do not delete V beta 3 cells (Mlsc-) showed that the majority of V beta 3- cells were located in the medulla with a few cells distributed in the cortical region. This pattern was notably different than the distribution of V beta 11 cells in thymi. Despite their location by histology, the majority of remaining V beta 3+ cells were dual CD4/CD8 positive (CD4+CD8+) by flow cytometric analysis. Our data illustrate that V beta 11 and V beta 3 T cells appear to be eliminated (i.e. 'deleted') at similar stages of maturation (single positive) during self-tolerance as well as in a neonatal H-2 tolerance model. However, the degree of elimination and the location of the cells remaining in these mice is dramatically different, depending on which T cell population is being evaluated and which deleting ligand is presented intrathymically. Thus, the accepted tenet of dual CD4+CD8+ cells localizing to the thymic cortex appears to have exceptions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Cytochalasins induce actin polymerization in human leukocytes.

We studied the effect of cytochalasins (B, D, and E) on the F-actin content in human neutrophils and lymphocytes using NBD-phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration- and time-dependent increase in the F-actin content in both cell types. The order of potency was cytochalasin D greater than E greater than B. The increase in F-actin content was accompanied by a decrease in the G-actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin-binding or sequestering proteins. 2-deoxyglucose (20 mM) caused a decrease in the basal F-actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than current models suggest.

Actin Cytoskeleton↗
PubMed1992

Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescein diacetate in the presence of a calmodulin inhibitor.

Dichlorofluorescein (DCFH) oxidation assay measures hydrogen peroxide (H2O2), which is a derivative of superoxide anion. We found that a calmodulin antagonist, W-13, which is known to inhibit superoxide anion generation enhanced the capacity of human neutrophils to oxidize DCFH. To investigate this discrepancy we studied the role of nitric oxide (NO) in DCFH oxidation. Pure NO was capable of oxidizing DCFH, and the product formed had spectral properties identical to oxidized DCFH produced by H2O2. The arginine analog, NG-monomethyl-L-arginine (NMMA), which inhibits NO production, in combination with W-13 completely inhibited the stimulus-induced increase in DCFH oxidation. We conclude that the oxidation of DCFH in human neutrophils can occur by either H2O2 or NO.

Arginine↗
PubMed1992

Age-related alterations in actin cytoskeleton and receptor expression in human leukocytes.

We studied a number of parameters, which may all depend upon cytoskeletal function, comparing lymphocytes and granulocytes (PMN) from young and old healthy donors. F-actin content was measured by NBD-phallacidin staining, followed by flow cytometry and was expressed as mean channel fluorescence (MCF). There were no differences in the basal F-actin content of PMN obtained from young (under 35 years old) and old donors (above 65 years). In contrast, the basal F-actin content was higher in lymphocytes obtained from the old donors (MCF, 56.8 +/- 2.9 vs 48.1 +/- 2.6 in the young; mean +/- SEM, n = 20, p less than .03). Stimulus-induced actin polymerization was slightly lower in the older age-group both in PMN and lymphocytes, but a significant difference was found only in PMN stimulated with the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (MCF 97.9 +/- 4.7 vs 88.6 +/- 3.4; young vs old, mean +/- SEM, n = 20, p less than .05). Interleukin-2 receptor expression was measured by staining with FITC-conjugated anti-CD25 antibodies and flow cytometry, following stimulation with phytohemagglutinin (PHA) or pokeweed mitogen (PWM). Perturbation of the cytoskeletal system with pentoxifylline, which has been shown to decrease F-actin content and inhibit the expression of several cell surface receptors, had similar effects on leukocytes from young and old donors.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1990

Stimulus-specific effects of pentoxifylline on neutrophil CR3 expression, degranulation, and superoxide production.

The effects of pentoxifylline (Trental) on human neutrophil CR3 up-modulation, degranulation, and superoxide production were studied. We used the chemotactic peptide fMLP and the phorbol ester PMA as soluble stimuli, and beta-glucan particles as a CR3-specific solid phase stimulus of neutrophil superoxide production. Since neutrophils have adenosine A2 receptors, we compared effects of pentoxifylline to effects of adenosine, and we also looked at the effect of cytochalasin B, which breaks up actin filaments. Pentoxifylline inhibited both CR3 up-modulation and degranulation of myeloperoxidase and lysozyme. Pentoxifylline is a more potent inhibitor of fMLP- compared to PMA-induced degranulation, and is especially potent against superoxide production. While pentoxifylline is less potent than adenosine in its inhibition of fMLP-induced superoxide production, it is more potent in its inhibition of PMA- and beta-glucan particle-stimulated superoxide production. Cytochalasin B, which enhances degranulation and fMLP-stimulated superoxide production, was found to inhibit beta-glucan particle-stimulated superoxide production. These findings are consistent with the hypothesis that pentoxifylline can affect both the cytoskeletal architecture of unstimulated neutrophils and the activation and responses of neutrophils which involve actin polymerization and receptor-cytoskeletal interactions.

Adenosine↗