Biomedical subjects

J Vidya Sarma

Publications and source records attributed to J Vidya Sarma.

27 records · Page 2Linked to original sources

PubMed2003

Anti-complement strategies in experimental sepsis.

Using the cecal ligation/puncture (CLP) model of sepsis in rodents, evidence was obtained for excessive activation of the complement system, which leads to nearly total loss of innate immune protective functions of blood neutrophils. These defects are associated with profound defects in chemotaxis, respiratory burst (H2O2 production) and phagocytosis. The molecular mechanisms of these defects are linked to the complement activation product C5a. In CLP rats and mice, the C5a receptor (C5aR) is widely up-regulated in organs, in part owing to the production of interleukin-6 (IL-6). The up-regulation of C5aR in the thymus is linked to C5a-dependent induction of apoptosis in thymocytes, as revealed by caspase activation, increased binding of C5a and DNA laddering. Such events in thymocytes are prevented if rats first are treated with anti-C5a or with anti-C5aR at the time of CLP. Treatment of CLP rats and mice with anti-C5a, anti-IL-6 or anti-C5aR dramatically improves survival rates after CLP, indicating a linkage between C5a and C5aR in the harmful outcomes of sepsis in rodents. Studies are underway in humans with sepsis to determine whether similar mechanisms are in play.

Animals↗

PubMed2003

Murine complement interactions with Pseudomonas aeruginosa and their consequences during pneumonia.

Complement is necessary for defense against lung infection with Pseudomonas aeruginosa in mice. We studied in vitro interactions between complement and P. aeruginosa and in vivo effects of complement depletion to better understand this relationship. In vitro, P. aeruginosa strain UI-18 was resistant to killing by mouse serum. However, C3 opsonized the organism (via the alternative and mannose binding lectin [MBL] pathways), and C5 convertase activity on the bacterial surface was demonstrated. In vivo, compared with normal mice, complement-deficient mice experienced higher mortality and failed to sterilize their bronchoalveolar space within 24 h of inoculation. These changes did not seem to be a result of decreased inflammation because complement-deficient mice had normal neutrophil recruitment, greater lung myeloperoxidase content, and, by 24 h, a 35-fold higher level of the CXC chemokine KC. Lung static pressure-volume curves were abnormal in infected animals but were significantly more so in complement deficient mice. These data indicate that although P. aeruginosa is resistant to serum killing, C3 opsonization and C5 convertase assembly occur on its surface. This interaction in vivo plays a central role in host survival beyond just recruitment and activation of phagocytes and may serve to limit the inflammatory response to and tissue injury resulting from bacterial infection.

Animals↗

PubMed2002

Expression and function of C5a receptor in mouse microvascular endothelial cells.

The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a K(d50) of 3.6 nM and to approximately 15,000-20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [(125)I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-gamma, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-alpha and MIP-1alpha). Although LPS or IFN-gamma alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-gamma, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.

Adjuvants, Immunologic↗

PubMed2002

Complement-induced impairment of innate immunity during sepsis.

This study defines the molecular basis for defects in innate immunity involving neutrophils during cecal ligation/puncture (CLP)-induced sepsis in rats. Blood neutrophils from CLP rats demonstrated defective phagocytosis and defective assembly of NADPH oxidase, the latter being due to the inability of p47(phox) to translocate from the cytosol to the cell membrane of neutrophils after cell stimulation by phorbol ester (PMA). The appearance of these defects was prevented by in vivo blockade of C5a in CLP rats. In vitro exposure of neutrophils to C5a led to reduced surface expression of C5aR and defective assembly of NADPH oxidase, as defined by failure in phosphorylation of p47(phox) and its translocation to the cell membrane, together with failure in phosphorylation of p42/p44 mitogen-activated protein kinases. These data identify a molecular basis for defective innate immunity involving neutrophils during sepsis.

Animals↗

PubMed2002

Anti-c5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis.

Sepsis and trauma are the two most common causes of disseminated intravascular coagulation and multiple organ dysfunction syndrome. Both disseminated intravascular coagulation and the systemic inflammatory response syndrome often lead to multiple organ dysfunction syndrome. The current studies have evaluated the relationship between the anaphylatoxin, C5a, and changes in the coagulation/fibrinolytic systems during the cecal ligation and puncture (CLP) model of sepsis in rats. CLP animals treated with anti-C5a had a much improved number of survivors (63%) compared to rats treated with pre-immune IgG (31%). In CLP rats treated with pre-immune IgG there was clearly increased procoagulant activity with prolongation of the activated partial thromboplastin time and prothrombin time, reduced platelet counts, and increased levels of plasma fibrinogen. Evidence for thrombin formation was indicated by early consumption of factor VII:C, subsequent consumption of factors XI:C and IX:C and anti-thrombin and increased levels of the thrombin-anti-thrombin complex and D-dimer. Limited activation of fibrinolysis was indicated by reduced plasma levels of plasminogen and increased levels of tissue plasminogen activator and plasminogen activator inhibitor. Most of these parameters were reversed in CLP rats that had been treated with anti-C5a. Production of C5a during sepsis may directly or indirectly cause hemostatic defects that can be reduced by blockade of C5a.

Animals↗

PubMed2002

Activator protein-1 activation in acute lung injury.

The role of activator protein-1 (AP-1) in inflammation is primarily unknown. AP-1 was evaluated in nuclear extracts from alveolar macrophages and whole lung nuclear extracts during acute lung injury after deposition of IgG immune complexes. AP-1 activation occurred in macrophages and in whole lung extracts, but with distinctly different time courses. Low levels of constitutive AP-1 were observed in normal rat lung as determined by the electrophoretic mobility shift assay. Increased AP-1 was detected 2 hours after initiation of the inflammatory response in lung with a further increase by 4 hours, while AP-1 activation was found in alveolar macrophages 0.5 hour after onset of the inflammatory response. mRNAs and proteins for c-fos, c-jun, jun-B, and jun-D were all up-regulated in whole lung tissues and in alveolar macrophages during acute lung injury induced by IgG immune complex deposition. Depletion of lung macrophages sharply reduced AP-1 activation, as did anti-tumor necrosis factor-alpha, whereas complement depletion showed no effect on lung AP-1 activation. The data suggest that activation of AP-1 occurs in both alveolar macrophages and in the lung, and this activation process is macrophage- and tumor necrosis factor-alpha-dependent.

Acute Disease↗

PubMed2002

Generation of C5a by phagocytic cells.

The complement activation product, C5a, is a powerful phlogistic factor. Using antibodies to detect human or rat C5a, incubation at pH 7.4 of human blood neutrophils or rat alveolar macrophages (AMs) with C5 in the presence of phorbol 12-myristate 13-acetate (PMA) led to generation of C5a. Rat AMs activated with lipopolysaccharide also generated C5a from C5. With activated neutrophils, extensive cleavage of C5 occurred, whereas activated macrophages had much more selective proteolytic activity for C5. Peripheral blood human or rat mononuclear cells and rat alveolar epithelial cells when stimulated with phorbol ester all failed to demonstrate an ability to cleave C5, suggesting a specificity of C5 cleavage by phagocytic cells. With rat AMs, C5a generation was time-dependent and was blocked if AMs were pretreated with inhibitors of transcription or protein synthesis (actinomycin D or cycloheximide). Similar treatment of activated human polymorphonuclear leukocytes only partially reduced C5a generation after addition of C5. C5a generated by activated AMs was biologically (chemotactically) active. This generation was sensitive to serine protease inhibitors but not to other classes of inhibitors. These data indicate that phagocytic cells, especially lung macrophages, can generate C5a from C5. In the context of the lung, this may represent an important C5a-generating pathway that is independent of the plasma complement system.

Animals↗

PubMed2002

Protection of innate immunity by C5aR antagonist in septic mice.

Innate immune functions are known to be compromised during sepsis, often with lethal consequences. There is also evidence in rats that sepsis is associated with excessive complement activation and generation of the potent anaphylatoxin C5a. In the presence of a cyclic peptide antagonist (C5aRa) to the C5a receptor (C5aR), the binding of murine 125I-C5a to murine neutrophils was reduced, the in vitro chemotactic responses of mouse neutrophils to mouse C5a were markedly diminished, the acquired defect in hydrogen peroxide (H2O2) production of C5a-exposed neutrophils was reversed, and the lung permeability index (extravascular leakage of albumin) in mice after intrapulmonary deposition of IgG immune complexes was markedly diminished. Mice that developed sepsis after cecal ligation/puncture (CLP) and were treated with C5aRa had greatly improved survival rates. These data suggest that C5aRa interferes with neutrophil responses to C5a, preventing C5a-induced compromise of innate immunity during sepsis, with greatly improved survival rates after CLP.

Animals↗

PubMed2000

Role of RANTES in experimental cardiac allograft rejection.

The host response to alloantigen results in upregulation of Class II MHC antigens and associated cytokine production (especially IL-2 and interferon-gamma (IFN-gamma)) as well as lymphocytic infiltration and cellular activation which leads to graft damage/destruction. RANTES (Regulated upon Activation, Normal T-cell Expressed and presumably Secreted) is a member of the beta subfamily (CC) of chemokines and has been shown to function as a lymphocyte chemoattractant. We now describe the requirement for RANTES in cardiac heterotopic allograft (brown Norway into Lewis rats) rejection in rats. By Northern blot analysis, mRNA could be detected in allografts at 6 and 8 days post-transplantation but not in isogenic (Lewis) grafts. RANTES protein could be demonstrated by Western blot analysis in homogenates from allografts at day 8 but not at day 0 and could not be identified in isogenic cardiac transplants. By immunostaining, RANTES protein was present in mononuclear cells of allografts at day 6 but was absent in the isogenic transplants. When rats were treated with anti-RANTES serum, there was a significant delay in rejection time (cessation of beating) of the allografts. These data demonstrate that expression of RANTES in rat cardiac allografts is linked to the rejection phenomenon.

Amino Acid Sequence↗