Biomedical subjects

William M Baldwin

Publications and source records attributed to William M Baldwin.

At least 19 recordsLinked to original sources

PubMed2007

Antibody to human leukocyte antigen triggers endothelial exocytosis.

Although antibodies to HLA play a role in the pathogenesis of diseases processes such as rejection of transplanted organs, the precise mechanisms by which antibodies cause tissue injury are not completely understood. We hypothesized that antibodies to host tissues cause inflammation in part by activating endothelial exocytosis of granules that contain prothrombotic mediators such as von Willebrand Factor (VWF) and proinflammatory mediators such as P-selectin. To test this hypothesis, we treated human endothelial cells with murine monoclonal antibody W6/32 to HLA class I and then measured exocytosis by the release of VWF and the externalization of P-selectin. Antibody to HLA activates endothelial exocytosis in a dose-dependent manner over time. The biologically active complement split product, C5a, adds a slight but significant increase to antibody induction of exocytosis. Antibody to HLA alone or with C5a did not damage the cells. Cross-linking of HLA appears to play a role in the ability of antibody to activate exocytosis, because the W6/32 monovalent Fab fragment did not activate VWF release, but the bivalent Fab'2 was effective in triggering exocytosis. To explore the in vivo effects of antibody upon graft injury, we infused W6/32 Fab'2 antibody to human HLA into severe combined immunodeficient/beige mice that had been transplanted with human skin grafts. Antibody to HLA activated exocytosis and inflammation in human skin grafts. Our data show that antibody to host antigens can activate human endothelial cell exocytosis and leukocyte trafficking. By triggering vascular inflammation, antibody activation of exocytosis may play a role in transplant rejection.

Autoantibodies↗

PubMed2006

Antibody and complement mediated injury in transplants following sensitization by allogeneic blood transfusion.

BACKGROUND: Many patients on the waiting list for transplants are sensitized from previous blood transfusions, pregnancy, or transplants. We investigated the role of complement in acute and chronic pathology in hearts transplanted to sensitized rats. METHODS: Blood was transfused from allogeneic PVG.R8 rats or control isogeneic PVG.1U rats to C6-sufficient and -deficient PVG.1U rats. Three weeks later hearts were transplanted from PVG.R8 donors and low-dose cyclosporin A was initiated. RESULTS: Allogeneic but not isogeneic blood transfusion elicited strong immunoglobulin (Ig) M, IgG1 and IgG2b alloantibody responses. Sensitization caused accelerated acute rejection of cardiac allografts by C6-sufficient recipients (4 days). In contrast, allografts functioned over 40 days in all C6-deficient recipients, but sensitization caused increased interstitial fibrosis and chronic vasculopathy. Circulating alloantibodies were associated with deposits of C4d on the vascular endothelium together with pericapillary accumulation of neutrophils and macrophages in the grafts. In contrast, T cells accumulated in periarterial lymphatics that did not have C4d deposits. CONCLUSIONS: Presensitization by allogeneic blood transfusion causes accelerated acute graft rejection in the presence of the complete complement cascade. In the absence of C6, macrophages colocalized with deposits of C4d and T cells accumulated in the periarterial lymphatics.

Animals↗

PubMed2006

Decreased capacity of immune cells to cause tissue injury mediates kidney ischemic preconditioning.

Ischemic preconditioning (IP) is a well-established phenomenon, and the underlying mechanisms of IP are thought to involve adaptive changes within the injured tissue. Because one of the main functions of immune cells is to harbor memory, we hypothesized that circulating immune cells could mediate IP by responding to an initial ischemia reperfusion injury (IRI) and then mediate decreased injury after a second IRI event. C57BL/6 mice underwent 30 min of bilateral renal clamping or sham operation. At 5 days after ischemia, purified leukocytes from spleen were adoptively transferred into T cell-deficient (nu/nu) mice. After 1 wk, these mice underwent 30 min of renal IRI. The nu/nu mice receiving leukocytes from ischemic wild-type mice had significantly reduced renal injury compared with nu/nu mice receiving leukocytes from sham-operated, wild-type mice. Infiltration of neutrophil and macrophage in postischemic kidney did not correlate with the protection. No difference in kidney C3d or IgG deposition was detected between groups. Given that inducible NO synthase (iNOS) has been implicated in IP, leukocytes from ischemic or sham-operated, iNOS-deficient mice were transferred into nu/nu mice. Effects similar to those of wild-type transfer of ischemic leukocytes were demonstrated; thus, iNOS was not mediating the IP effect of leukocytes. This is the first evidence that immune cells are primed after renal IRI and thereby lose the capacity to cause kidney injury during a second episode of IRI. This finding may also be relevant for elucidating the mechanisms underlying cross-talk between injured kidney and distant organs.

Adoptive Transfer↗

PubMed2006

The production of red blood cell alloantibodies in mice transfused with blood from transgenic Fyb-expressing mice.

BACKGROUND: A murine model would be useful to identify which immune mechanisms could be manipulated to treat or prevent red blood cell (RBC) alloimmunization in patients who become sensitized to multiple or widely expressed antigens. STUDY DESIGN AND METHODS: Transgenic mice (B6CBAF1/J-Tg-Fy(b)) expressing the human Fy(b) antigen of the Duffy (Fy) blood group were donors. Recipient B6CBA-F1 mice received four weekly intravenous (IV) transfusions: either 0.3 mL of washed buffy coat-depleted RBCs or 0.3 mL of RBCs with spleen cells. Titers of immunoglobulin M (IgM) and immunoglobulin G (IgG) were measured in recipient serum samples by flow cytometry with RBCs from donor mice as target cells. Recipient serum samples were also tested against human RBCs of various Fy phenotypes. Additionally, RBC survival studies were performed in alloimmunized mice utilizing biotin-labeled Fy(b) transgenic mouse RBCs. RESULTS: B6CBA-F1 mice receiving washed buffy coat-depleted RBCs first made IgM, followed by IgG alloantibodies to transgenic mouse Fy(b)-positive RBCs. Recipients of Fy(b)-positive RBCs mixed with spleen cells also produced IgM and IgG alloantibodies, but at a slower rate than recipients of washed buffy coat-depleted RBCs. Serum samples showed specificity for Fy3, Fy(b), and Fy6. Decreased survival of transfused RBCs was evident at 24 hours after transfusion. CONCLUSIONS: It is possible to elicit the formation of anti-Fy alloantibodies by IV transfusion in mice that lack Fy antigens. The transfusion of RBCs alone was adequate to stimulate alloantibody production in B6CBA-F1 recipient mice. The survival of transfused Fy(b)-positive RBCs is diminished in sensitized mice. This model will be useful in further studies of RBC alloimmunization.

Animals↗

PubMed2005

HMG-CoA reductase inhibitors inhibit endothelial exocytosis and decrease myocardial infarct size.

Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors protect the vasculature from inflammation and atherosclerosis by cholesterol dependent and cholesterol independent mechanisms. We hypothesized that HMG-CoA reductase inhibitors decrease exocytosis of Weibel-Palade bodies, endothelial cell granules whose contents promote thrombosis and vascular inflammation. We pretreated human aortic endothelial cells with simvastatin for 24 hours, then stimulated the cells with thrombin, and measured the amount of vWF released into the media. We then measured the effect of simvastatin on myocardial infarction in mice. Simvastatin decreased thrombin-stimulated Weibel-Palade body exocytosis by 89%. Simvastatin inhibited exocytosis in part by increasing synthesis of nitric oxide (NO), which S-nitrosylated N-ethylmaleimide sensitive factor (NSF), a critical regulator of exocytosis. Simvastatin treatment attenuated myocardial infarct size by 58% in wild-type but not eNOS knockout mice. Furthermore, simvastatin decreased endothelial exocytosis and neutrophil infiltration into ischemic-reperfused myocardium, which was mediated in part by P-selectin contained in Weibel-Palade bodies. However, simvastatin did not affect exocytosis and inflammation in myocardial infarcts of eNOS knockout mice. Inhibition of endothelial exocytosis is a novel mechanism by which HMG-CoA reductase inhibitors may reduce vascular inflammation, inhibit thrombosis, and protect the ischemic myocardium. These findings may explain part of the pleiotropic effects of statin therapy for patients with cardiovascular disease.

Animals↗

PubMed2005

Signal transducer and activator of transcription 3alpha and specificity protein 1 interact to upregulate intercellular adhesion molecule-1 in ischemic-reperfused myocardium and vascular endothelium.

OBJECTIVE: Intercellular adhesion molecule-1 (ICAM-1) is upregulated rapidly on endothelial cells during ischemia-reperfusion (I-R) and mediates tissue leukocyte accumulation. The ICAM-1 proximal promoter contains a signal transducer and activator of transcription (Stat) binding motif (gamma-interferon activation site [GAS] sequence), which flanks a specificity protein 1 (Sp1) binding site. We examined the roles of Stat and Sp1 in the regulation of ICAM-1 after myocardial I-R. METHODS AND RESULTS: Open-chest anesthetized rats underwent coronary artery occlusion for 35 minutes and reperfusion for 0 to 240 minutes. Stat became activated within 15 minutes after reperfusion, primarily in vascular endothelial cells; the activated Stat protein was identified as Stat3 (alpha-isoform). After phosphorylation on serine 727 (p-S727), Stat3alpha was found in association with the transcriptional regulator Sp1, and the complex bound to an ICAM-1-GAS probe. ICAM-1 expression increased after I-R and lagged shortly behind Stat3alpha activation. In cultured human umbilical vein endothelial (HUVE) cells, activation of Stat3alpha after hypoxia-reoxygenation (H-R) was dependent on the small GTPase Rac1. Transfection of a dominant-negative Stat3 (Y705F) adenovirus or a GAS decoy oligonucleotide reduced ICAM-1 mRNA expression after H-R. Using a reporter gene transfected into HUVE cells, mutation of the GAS element in the ICAM-1 promoter resulted in reduced transcriptional activity after H-R. Sp1 coimmunoprecipitated with p-S727 Stat3 during H-R, and Sp1 or Stat3alpha interfering RNA markedly reduced ICAM-1 mRNA expression. CONCLUSIONS: The Sp1-Stat3 complex appears to play an important role in the upregulation of ICAM-1 transcription after reoxygenation or reperfusion.

Animals↗

PubMed2005

Regulation of platelet granule exocytosis by S-nitrosylation.

Nitric oxide (NO) regulates platelet activation by cGMP-dependent mechanisms and by mechanisms that are not completely defined. Platelet activation includes exocytosis of platelet granules, releasing mediators that regulate interactions between platelets, leukocytes, and endothelial cells. Exocytosis is mediated in part by N-ethylmaleimide-sensitive factor (NSF), an ATPase that disassembles complexes of soluble NSF attachment protein receptors. We now demonstrate that NO inhibits exocytosis of dense granules, lysosomal granules, and alpha-granules from human platelets by S-nitrosylation of NSF. Platelets lacking endothelial NO synthase show increased rolling on venules, increased thrombosis in arterioles, and increased exocytosis in vivo. Regulation of exocytosis is thus a mechanism by which NO regulates thrombosis.

Blood Platelets↗

PubMed2005

Terminal complement components mediate release of von Willebrand factor and adhesion of platelets in arteries of allografts.

BACKGROUND: Both humoral and cellular immune responses can cause arterial injury in organ transplants, but the manifestations of these different inflammatory mechanisms have not been dissected fully. The present study was designed to define the effects of the terminal complement components on arterial injury in vivo. METHODS: The authors have developed congenic rat strains with a C6 deficiency. The absence of C6 terminates the cascade of complement after C5 cleavage and prevents the assembly of the membrane attack complex. Hearts were transplanted from PVG.1A (RT1) rats to major histocompatibility complex-incompatible C6-deficient (C6-) or C6-sufficient (C6+) PVG.1U (RT1) rats. RESULTS: PVG.1A (C6-) cardiac grafts were rejected acutely (6-7 days) by untreated PVG.1U (C6+) recipients but survived significantly longer in PVG.1U (C6-) recipients (8 to >30 days). Arteries of cardiac allografts in C6+ recipients demonstrated extensive endothelial injury evidenced by release of von Willebrand factor (vWF) and accompanied by platelet aggregation. In contrast, vWF was retained in Weibel-Palade storage granules of arterial endothelial cells in cardiac allografts that were rejected by C6- recipients. In the absence of C6, intimal alterations were limited to lifting of endothelial cells from supporting stroma by infiltrating mononuclear cells, duplicating the clinical lesion described as endotheliitis or intimal arteritis. Delaying graft rejection with a short course of cyclosporine did not decrease vWF release and platelet aggregation in PVG.1U (C6+) recipients. CONCLUSIONS: Mononuclear cell infiltration of the arterial intima occurs in the absence of C6, but C6 deficiency limits the release of vWF from arterial endothelial cells.

Animals↗

PubMed2005

Clearance of complement by human vascular endothelial cells: effects of hypoxia/reoxygenation and IL-1beta activation.

Antibody-mediated rejection is characterized by deposits of complement (C) C4 and C3 split products on endothelial cells (ECs). C3 split products are critical mechanistically and diagnostically because they are deposited in amplified quantities, bind covalently to ECs and act as ligands for leukocytes. This study was designed to determine whether cultured vascular human ECs could clear covalently bound C3 split products from their surface. An immunoglobulin M (IgM) antibody against beta(2)-microglobulin of major histocompatibility complex class I antigens was used to activate C in human serum. Some cells were exposed to hypoxia/reoxygenation and/or interleukin 1beta (IL-1beta) prior to incubation with antibody. C3b/iC3b and C3d deposition on the cell surface was measured by flow cytometry. Incubation with antibody followed by human serum caused a dose-dependent deposition of C3b/iC3b and C3d. Over half of deposited C3b/iC3b and one-third of C3d were cleared from the cell surface during a 3-7-h incubation period with human serum. Neither hypoxia/reoxygenation nor IL-1beta further increased the deposition of C3b/iC3b and C3d, and only slightly modulated their rates of clearance. In summary, human ECs rapidly clear iC3b and C3d from their surface. This finding may have important diagnostic and mechanistic implications to transplantation because C3d is used as a marker of antibody-mediated rejection.

Antibodies, Monoclonal↗

PubMed2005

Impact of MHC class II incompatibility on localization of mononuclear cell infiltrates to the bronchiolar compartment of orthotopic lung allografts.

Chronic pathological changes in transplanted lungs are unique because they center on the airways. We examined the relative role of MHC class I and II antigens in causing bronchial pathology in orthotopic lung transplants to rats maintained on cyclosporin A (CsA). Transplants mismatched for MHC class II antigens had significantly more peri-bronchiolar infiltrates than MHC class I incompatible transplants. No significant increase in infiltrates was found in lung transplants incompatible for MHC class I plus II antigens compared to MHC class II antigens alone. Immunohistochemistry demonstrated that MHC class II antigen expression was confined to macrophages in MHC class I incompatible transplants, but was upregulated on bronchial epithelium in transplants with MHC class II incompatibilities. Vascular endothelium was notably devoid of MHC class II antigen expression in all transplants. However, both peri-bronchial and peri-vascular infiltrates were frequently cuffed by alveolar macrophages and type II pneumocytes that expressed MHC class II antigens. PCR analysis demonstrated that IFN-gamma and regulated on activation, normal T cells expressed and secreted (RANTES) were upregulated in MHC class II incompatible transplants. Thus, MHC class II incompatible orthotopic lung transplants in rats maintained on CsA immunosuppression undergo a bronchiolcentric upregulation of alloantigens.

Animals↗

PubMed2005

Antibody-mediated rejection in human cardiac allografts: evaluation of immunoglobulins and complement activation products C4d and C3d as markers.

Antibody-mediated rejection (AMR) in human heart transplantation is an immunopathologic process in which injury to the graft is in part the result of activation of complement and it is poorly responsive to conventional therapy. We evaluated by immunofluorescence (IF), 665 consecutive endomyocardial biopsies from 165 patients for deposits of immunoglobulins and complement. Diffuse IF deposits in a linear capillary pattern greater than 2+ were considered significant. Clinical evidence of graft dysfunction was correlated with complement deposits. IF 2+ or higher was positive for IgG, 66%; IgM, 12%; IgA, 0.6%; C1q, 1.8%; C4d, 9% and C3d, 10%. In 3% of patients, concomitant C4d and C3d correlated with graft dysfunction or heart failure. In these 5 patients AMR occurred 56-163 months after transplantation, and they responded well to therapy for AMR but not to treatment with steroids. Systematic evaluation of endomyocardial biopsies is not improved by the use of antibodies for immunoglobulins or C1q. Concomitant use of C4d and C3d is very useful to diagnose AMR, when correlated with clinical parameters of graft function. AMR in heart transplant patients can occur many months or years after transplant.

Biomarkers↗

PubMed2004

Peroxynitrite inhibition of Coxsackievirus infection by prevention of viral RNA entry.

Although peroxynitrite is harmful to the host, the beneficial effects of peroxynitrite are less well understood. We explored the role of peroxynitrite in the host immune response to Coxsackievirus infection. Peroxynitrite inhibits viral replication in vitro, in part by inhibiting viral RNA entry into the host cell. Nitrotyrosine, a marker for peroxynitrite production, is colocalized with viral antigens in the hearts of infected mice but not control mice. Nitrotyrosine coprecipitates with the viral polypeptide VP1 as well. Guanidinoethyl disulfide, a scavenger of peroxynitrite, blocks peroxynitrite inhibition of viral replication in vitro and permits an increase in viral replication in vivo. These data suggest that peroxynitrite is an endogenous effector of the immune response to viruses.

Animals↗

PubMed2004

Antibody, complement and accommodation in ABO-incompatible transplants.

Many facets of accommodation have been explored since this process was first observed in ABO-incompatible renal allografts over 17 years ago. Intriguing new pieces of the puzzle have emerged to be fitted into the picture in several places. For example, vascular endothelial cells can be stimulated to secrete substantial amounts of blood group A and B antigens linked to von Willebrand factor; the antibody response to A and B antigens stimulated by ABO-incompatible renal allografts can show epitope spreading; complement can inhibit inflammation through actions of some complement split products, particularly iC3b and C3a; endothelial cells can upregulate various cytoprotective mechanisms; and clinically, new protocols for achieving accommodation have been implemented with improved results.

ABO Blood-Group System↗

PubMed2004

Antithymocyte globulin is associated with complement deposition in cardiac transplant biopsies.

Polyclonal antithymocyte globulin preparations contain antibodies with reactivity to endothelial cells. Therefore, we investigated whether treatment with this reagent caused complement deposition in human cardiac transplants. Frozen tissue was available from endomyocardial biopsies of 75 patients, who were transplanted between April 1995 and April 2000. Nine of these patients were converted from cyclosporin A (CsA) to horse antithymocyte globulin (ATGAM) in the first month after transplantation. All of the biopsies were stained by immunofluorescence for C4d as evidence of activation of the classical pathway of complement. In addition, biopsies from patients treated with ATGAM and control patients were stained for deposition of horse immunoglobulin (Ig)G. All nine patients who received ATGAM had deposition of horse IgG and C4d. Two color stains demonstrated that the horse IgG colocalized with the C4d staining. No staining for horse IgG or C4d was evident in biopsies obtained before ATGAM treatment. Likewise, no staining for horse IgG was detected in seven control patients who had C4d staining. Most patients treated with ATGAM had no histologic evidence of rejection, but did have myocyte damage and macrophage infiltration. Thus prophylactic treatment with ATGAM is associated with the deposition of horse IgG and activation of complement in the transplant.

Animals↗

PubMed2004

Non-complement- and complement-activating antibodies synergize to cause rejection of cardiac allografts.

Alloantibodies (AlloAbs) are a clinically significant component of the immune response to organ transplants. In our experimental model, B10.A (H-2a) cardiac transplants survived significantly longer in C57BL/6 (H-2b) immunoglobulin knock-out (IgKO) recipients than in their wild-type (WT) counterparts. Passive transfer of a single 50-200-microg dose of complement-activating IgG2b AlloAbs to IgKO recipients reconstituted acute rejection of cardiac allografts. Although passive transfer of a subthreshold dose of 25 microg of IgG2b or a single 100-200-microg dose of non-complement-activating IgG1 AlloAbs did not restore acute rejection to IgKO recipients, a combination of these AlloAbs did cause acute graft rejection. Histologically, rejection was accompanied by augmented release of von Willebrand factor from endothelial cells. IgG1 AlloAbs did not activate complement on their own and did not augment complement activation by IgG2b AlloAbs. However, IgG1 AlloAbs stimulated cultured mouse endothelial cells to produce monocyte chemotactic protein 1 (MCP-1) and neutrophil chemoattractant growth-related oncogene alpha (KC). TNF-alpha augmented IgG1 induced secretion of MCP-1 and KC. These findings indicate that non-complement-activating AlloAbs can augment injury to allografts by complement-activating AlloAbs. Non-complement-activating AlloAbs stimulate endothelial cells to produce chemokines and this effect is augmented in the milieu of proinflammatory cytokines.

Animals↗

PubMed2004

Beyond C4d: other complement-related diagnostic approaches to antibody-mediated rejection.

Complement is a multifunctional system of receptors and regulators as well as effector molecules. Both the pathogenic and diagnostic power of complement is based on the capacity of the complement system to amplify innate and adaptive immunity. This amplification is accomplished through two strategies: (1) enzymatic reactions in the complement cascade, and (2) stimulation of leukocytes, platelets and parenchymal cells through specific receptors or receptor-independent pore formation. The mechanisms by which complement mediates and modifies nonspecific inflammation, antibody-mediated injury and T-cell responses are of particular significance to the pathogenesis of transplant rejection. Understanding the mechanisms by which complement integrates the interactions of leukocytes, platelets and parenchymal cells offers opportunities to further refine the diagnosis of rejection.

Animals↗

PubMed2003

Complement in transplant rejection: diagnostic and mechanistic considerations.

After decades of neglect, complement has been rediscovered as a potent mediator and diagnostic indicator of inflammation and rejection in organ transplants. In part, this reflects a better understanding of the biology of complement, but it also reflects changes in clinical practice. The relevance of complement to clinical transplantation has increased as access to transplantation continues to be extended. Extended criteria for organ donors include older donors and non-heart beating donors. Simultaneously, the criteria for recipients have been extended to include more presensitized and blood group incompatible recipients. All of these variables can increase complement activation. As a result, several components of complement have received attention as potential diagnostic tools, and, with more sophisticated reagents, evidence of complement activation has been found in larger numbers of biopsy samples. Understanding the biology of complement is important to appreciate fully the diagnostic and mechanistic implications of complement activation in organ transplants. Mechanistically, a series of effector molecules in the complement cascade mediate proinflammatory functions that can account for chemotaxis and activation of cells of the innate immune system, such as granulocytes and monocytes. Simultaneously, many of these same complement mediators activate and disrupt the endothelial cell interface between the recipient and the transplant. In addition, there is growing appreciation that complement can stimulate B and T lymphocytes of the adaptive immune system. More recent evidence indicates that complement participates in the non-inflammatory clearance of apoptotic cells. Therefore, the complement cascade can be activated by multiple mechanisms and various components of complement can modulate the response to transplants in different directions.

Animals↗

PubMed2002

Membrane attack complex contributes to destruction of vascular integrity in acute lung allograft rejection.

The lung is known to be particularly susceptible to complement-mediated injury. Both C5a and the membrane attack complex (MAC), which is formed by the terminal components of complement (C5b-C9), can cause acute pulmonary distress in nontransplanted lungs. We used C6-deficient rats to investigate whether MAC causes injury to lung allografts. PVG.R8 lungs were transplanted orthotopically to MHC class I-incompatible PVG.1U recipients. Allografts from C6-sufficient (C6(+)) donors to C6(+) recipients were rejected with an intense vascular infiltration and diffuse alveolar hemorrhage 7 days after transplantation (n = 5). Ab and complement (C3d) deposition was accompanied by extensive vascular endothelial injury and intravascular release of von Willebrand factor. In contrast, lung allografts from C6-deficient (C6(-)) donors to C6(-) recipients survived 13-17 days (n = 5). In the absence of C6, perivascular mononuclear infiltrates of ED1(+) macrophages and CD8(+) T lymphocytes were present 7 days after transplantation, but vascular endothelial cells were quiescent, with minimal von Willebrand factor release and no evidence of alveolar hemorrhage or edema. Lung allografts were performed from C6(-) donors to C6(+) recipients (n = 5) and from C6(+) donors to C6(-) recipients (n = 5) to separate the effects of systemic and local C6 production. Lungs transplanted from C6(+) donors to C6(-) recipients had increased alveolar macrophages and capillary injury. C6 production by lung allografts was demonstrated at the mRNA and protein levels. These results demonstrate that MAC causes vascular injury in lung allografts and that the location of injury is dependent on the source of C6.

Acute Disease↗