Biomedical subjects

Gregory L Stahl

Publications and source records attributed to Gregory L Stahl.

At least 19 recordsLinked to original sources

PubMed2006

C5-blocking antibody reduces fluid requirements and improves responsiveness to fluid infusion in hemorrhagic shock managed with hypotensive resuscitation.

Hypotensive resuscitation strategies and inhibition of complement may both be of benefit in hemorrhagic shock. We asked if C5-blocking antibody (anti-C5) could diminish the amount of fluid required and improve responsiveness to resuscitation from hemorrhage. Awake, male Sprague-Dawley rats underwent controlled hemorrhage followed by prolonged (3 h) hypotensive resuscitation with lactated Ringer's or Hextend, with or without anti-C5. Anti-C5 treatment led to an estimated 62.3 and 58.5% reduction in the volume of Hextend and lactated Ringer's, respectively. In the subgroup of animals with a positive mean arterial pressure (MAP) response to fluid infusion following prolonged hypotension, anti-C5 treatment led to an estimated 4.7- and 4.1-fold increase in mean arterial pressure response per unit Hextend and lactated Ringer's infused, respectively. We observed no significant postresuscitation metabolic differences between the anti-C5 groups and controls. Whether anti-C5 could serve as a volume-sparing adjunct that improves responsiveness to fluid administration in humans deserves further study.

Animals↗

PubMed2006

Mannose-binding lectin binds IgM to activate the lectin complement pathway in vitro and in vivo.

Recent evidence has implicated a role for the MBL-dependent lectin pathway in gastrointestinal and myocardial ischemia/reperfusion (I/R)-induced injury. However, previous studies have implicated IgM and the classical pathway as initiators of complement activation following I/R. Thus, we investigated the potential interaction between MBL and IgM leading to complement activation. Using surface plasmon resonance, we demonstrate that MBL does bind human IgM. Subsequently, functional complement activation was demonstrated in vitro following sensitization of human RBCs with mouse anti-human CD59 IgM and more lysis was observed with MBL sufficient sera compared to MBL deficient (KO) sera. Similarly, treatment of human endothelial cells with mouse anti-human CD59 IgM, MBL and MASP-2 activated and deposited C4. These data suggest that the presence of both IgM and MBL can activate the lectin pathway in vitro. Serum ALT levels increased significantly in sIgM/MBL-A/C KO mice reconstituted with WT plasma compared to sIgM/MBL-A/C KO mice reconstituted with MBL-A/C KO plasma following gastrointestinal (G) I/R. Similarly, intestinal C3 deposition was greater in sIgM/MBL-A/C KO mice reconstituted with WT plasma compared to sIgM/MBL-A/C KO mice treated with MBL-A/C KO plasma. These data indicate for the first time that both IgM and MBL-A/C are required for GI/R-induced complement activation and subsequent injury.

Animals↗

PubMed2006

Role of complement component C5 in cerebral ischemia/reperfusion injury.

We evaluated the role of complement component C5 during the course of cerebral ischemic reperfusion injury in a rat model of middle cerebral artery occlusion (MCAO). Systemic C5 inhibition was achieved with an anti-C5 monoclonal antibody, which significantly prevented the deterioration of the motor functions by reducing cerebral lesion and edema. Our results show that activated C5 complement components played an important role in cerebral tissue inflammation resulting from ischemia/reperfusion injury.

Animals↗

PubMed2006

Glomerular activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease.

IgA nephropathy (IgAN) is characterized by glomerular co-deposition of IgA and complement components. Earlier studies showed that IgA activates the alternative pathway of complement, whereas more recent data also indicate activation of the lectin pathway. The lectin pathway can be activated by binding of mannose-binding lectin (MBL) and ficolins to carbohydrate ligands, followed by activation of MBL-associated serine proteases and C4. This study examined the potential role of the lectin pathway in IgAN. Renal biopsies of patients with IgAN (n=60) showed mesangial deposition of IgA1 but not IgA2. Glomerular deposition of MBL was observed in 15 (25%) of 60 cases with IgAN and showed a mesangial pattern. All MBL-positive case, but none of the MBL-negative cases showed glomerular co-deposition of L-ficolin, MBL-associated serine proteases, and C4d. Glomerular deposition of MBL and L-ficolin was associated with more pronounced histologic damage, as evidenced by increased mesangial proliferation, extracapillary proliferation, glomerular sclerosis, and interstitial infiltration, as well as with significantly more proteinuria. Patients who had IgAN with or without glomerular MBL deposition did not show significant differences in serum levels of MBL, L-ficolin, or IgA or in the size distribution of circulating IgA. Furthermore, in vitro experiments showed clear binding of MBL to polymeric but not monomeric patient IgA, without a significant difference between both groups. Together, these findings strongly point to a role for the lectin pathway of complement in glomerular complement activation in IgAN and suggest a contribution for both MBL and L-ficolin in the progression of the disease.

Adult↗

PubMed2006

Polyglycolic acid-induced inflammation: role of hydrolysis and resulting complement activation.

Tissue and organ replacement have quickly outpaced available supply. Tissue bioengineering holds the promise for additional tissue availability. Various scaffolds are currently used, whereas polyglycolic acid (PGA), which is currently used in absorbable sutures and orthopedic pins, provides an excellent support for tissue development. Unfortunately, PGA can induce a local inflammatory response following implantation. Therefore, we investigated the molecular mechanism of inflammation in vitro and in vivo. Degraded PGA induced an acute peritonitis, characterized by neutrophil (PMN) infiltration following intraperitoneal injection in mice. Similar observations were observed using the metabolite of PGA, glycolide. Dissolved PGA or glycolide, but not native PGA, activated the classical complement pathway in human sera, as determined by classical complement pathway hemolytic assays, C3a and C5a production, and C3 and immunoglobulin deposition. To investigate whether these in vitro observations translated to in vivo findings, we used genetically engineered mice. Intraperitoneal administration of glycolide or dissolved PGA in mice deficient in C1q, factor D, C1q and factor D, or C2 and factor B demonstrated significantly reduced PMN infiltration compared to congenic controls (WT). Mice deficient in C6 also demonstrated acute peritonitis. However, treatment of WT or C6 deficient mice with a monoclonal antibody against C5 prevented the inflammatory response. These data suggest that the hydrolysis of PGA to glycolide activates the classical complement pathway. Furthermore, complement is amplified via the alternative pathway and inflammation is induced by C5a generation. Inhibition of C5a may provide a potential therapeutic approach to limit the inflammation associated with PGA-derived materials following implantation.

Animals↗

PubMed2005

Mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury.

The mannose-binding lectin (MBL), a circulating pattern recognition molecule, recognizes a wide range of infectious agents with resultant initiation of the complement cascade in an Ab-independent manner. MBL recognizes infectious non-self and altered self in the guise of apoptotic and necrotic cells. In this study, we demonstrate that mice lacking MBL, and hence are devoid of MBL-dependent lectin pathway activation but have fully active alternative and classical complement pathways, are protected from cardiac reperfusion injury with resultant preservation of cardiac function. Significantly, mice that lack a major component of the classical complement pathway initiation complex (C1q) but have an intact MBL complement pathway, are not protected from injury. These results suggest that the MBL-dependent pathway of complement activation is a key regulator of myocardial reperfusion ischemic injury. MBL is an example of a pattern recognition molecule that plays a dual role in modifying inflammatory responses to sterile and infectious injury.

Animals↗

PubMed2005

Human peptidoglycan recognition protein S is an effector of neutrophil-mediated innate immunity.

Innate immune responses to bacteria require cooperative interactions between host recognition molecules and phagocytes. The peptidoglycan recognition proteins (PGRPs) are a large group of proteins found in insects and mammals that bind to bacterial peptidoglycan (PGN). PGRP-S is located with other antimicrobial proteins, such as lysozyme, in the granules of human neutrophils. Whereas both PGRP-S and lysozyme recognize PGN, the exact binding specificity of human PGRP-S, its functional activity, and its potential synergy with other neutrophil-derived bactericidal proteins such as lysozyme have not been determined. Here we show that human PGRP-S binds to and inhibits the growth of Staphylococcus aureus (containing lysine-type PGN) and Escherichia coli (containing mesodiaminopimelic acid-type PGN). The binding affinity and thus antimicrobial activity of PGRP-S is determined by the third amino acid in the PGN stem peptide. Furthermore, the antimicrobial effect of PGRP-S against E coli is synergistic with lysozyme, and lysozyme and PGRP-S colocalize in neutrophil extracellular traps (NETs), suggesting that these granule-derived proteins act together to kill bacteria trapped in the NETs. Taken together, these results indicate that human PGRP-S plays a role in innate immunity in the context of neutrophils by contributing to the killing of intracellular and extracellular bacteria.

Anti-Infective Agents↗

PubMed2005

Gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving C1q.

Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal ischemia-reperfusion (GI/R). The role of the classical and lectin complement pathways in GI/R injury was evaluated using C1q-deficient (C1q KO), MBL-A/C-deficient (MBL-null), complement factor 2- and factor B-deficient (C2/fB KO), and wild-type (WT) mice. Gastrointestinal ischemia (20 min), followed by 3-h reperfusion, induced intestinal and lung injury in C1q KO and WT mice, but not in C2/fB KO mice. Addition of human C2 to C2/fB KO mice significantly restored GI/R injury, demonstrating that GI/R injury is mediated via the lectin and/or classical pathway. Tissue C3 deposition in C1q KO and WT, but not C2/fB KO, mice after GI/R demonstrated that complement was activated in C1q KO mice. GI/R significantly increased serum alanine aminotransferase, gastrointestinal barrier dysfunction, and neutrophil infiltration into the lung and gut in C1q KO and WT, but not C2/fB KO, mice. MBL-null mice displayed little gut injury after GI/R, but lung injury was present. Addition of recombinant human MBL (rhuMBL) to MBL-null mice significantly increased injury compared with MBL-null mice after GI/R and was reversed by anti-MBL mAb treatment. However, MBL-null mice were not protected from secondary lung injury after GI/R. These data demonstrate that C2 and MBL, but not C1q, are necessary for gut injury after GI/R. Lung injury in mice after GI/R is MBL and C1q independent, but C2 dependent, suggesting a potential role for ficolins in this model.

Alanine Transaminase↗

PubMed2005

Role of C5 in the development of airway inflammation, airway hyperresponsiveness, and ongoing airway response.

The role of complement component C5 in asthma remains controversial. Here we examined the contribution of C5 at 3 critical checkpoints during the course of disease. Using an mAb specific for C5, we were able to evaluate the contribution of C5 during (a) the initiation of airway inflammation, (b) the maintenance of airway hyperresponsiveness (AHR), and (c) sustainment of an ongoing airway response to allergen provocation. Our results indicate that C5 is probably activated intrapulmonarily after infections or exposures to allergen and C5 inhibition has profound effects at all 3 critical checkpoints. In contrast to an earlier report, C5-deficient mice with established airway inflammation did not have elevated AHR to nonspecific stimuli. In the presence of airway inflammation, C5a serves as a direct link between the innate immune system and the development of AHR by engaging directly with its receptors expressed in airways. Through their powerful chemotactic and cell activation properties, both C5a and C5b-9 regulate the downstream inflammatory cascade, which results in a massive migration of inflammatory cells into the bronchial airway lumen and triggers the release of multiple harmful inflammatory mediators. This study suggests that targeting C5 is a potential clinical approach for treating patients with asthma.

Animals↗

PubMed2005

CpG oligodeoxynucleotide protection in polymicrobial sepsis is dependent on interleukin-17.

CpG oligodeoxynucleotides (ODNs) may prevent mortality from infection. We have identified a therapeutic benefit in treating sepsis with phosphorothioate ODN sequences containing the CpG motif. Sepsis was induced in rats by cecal ligation and puncture (CLP), and treatment with CpG ODNs reduced sepsis mortality from 80% to 15% during a 108-h period. Protection from mortality was dose dependent. Bacterial load in peritoneal fluid was reduced in CpG ODN-treated versus non-CpG ODN-treated rats after CLP. Lung injury, as determined by total myeloperoxidase activity, was also reduced in CpG ODN-treated versus non-CpG ODN-treated rats after CLP. Indirect evidence suggests that CpG-induced expression of interleukin (IL)-23 as levels of p40--but not p35--were significantly increased in both plasma and peritoneal lavage fluid in CpG ODN-treated versus non-CpG ODN-treated rats 24 h after CLP. Anti-IL-17 antibody inhibited the CpG-mediated prevention of mortality. These data suggest that IL-17 may mediate CpG-inducible host defenses during intraabdominal sepsis.

Animals↗

PubMed2004

Hypoxia-regulated therapeutic gene as a preemptive treatment strategy against ischemia/reperfusion tissue injury.

Ischemia and reperfusion represent major mechanisms of tissue injury and organ failure. The timing of administration and the duration of action limit current treatment approaches using pharmacological agents. In this study, we have successfully developed a preemptive strategy for tissue protection using an adenoassociated vector system containing erythropoietin hypoxia response elements for ischemia-regulated expression of the therapeutic gene human heme-oxygenase-1 (hHO-1). We demonstrate that a single administration of this vector several weeks in advance of ischemia/reperfusion injury to multiple tissues such as heart, liver, and skeletal muscle yields rapid and timely induction of hHO-1 during ischemia that resulted in dramatic reduction in tissue damage. In addition, overexpression of therapeutic transgene prevented long-term pathological tissue remodeling and normalized tissue function. Application of this regulatable system using an endogenous physiological stimulus for expression of a therapeutic gene may be a feasible strategy for protecting tissues at risk of ischemia/reperfusion injury.

Animals↗

PubMed2004

A stable aspirin-triggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils.

Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A(4) methyl ester, an aspirin-triggered lipoxin A(4)-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10-50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.

Amino Acid Sequence↗

PubMed2004

Molecular basis for complement component 6 (C6) deficiency in rats and mice.

Complement component C6 is a part of the lytic membrane attack complex formed during complement activation. Animal modeling to define the role of C5a vs. C5b-9 in human disease has used rodents deficient in C6, yet the molecular basis for the deficiencies has not been ascertained. Oligonucleotides derived from a 493 bp EST sequence of the rat C6 gene were used to isolate full-length transcripts of rat C6 mRNA. Sequence analysis confirmed that the derived amino acid sequence for rat C6 is highly homologous to human and mouse. We identified a 31 bp deletion in exon 10 of the C6 gene that leads to C6 deficiency in a strain of PVG rats (PVG/c-) and developed a PCR-based genotyping test. In addition, we identified four point mutations in the mouse C6 gene that may result in C6 deficiency observed in the Peru-Coppock mouse strain. A serendipitous finding from this study was a coagulation defect in the C6 deficient mice and rats. C6 deficient mice or rats demonstrated prolonged tail bleeding times that was reversed by treatment with purified rat C6 protein. Further, adenosine diphosphate induced platelet aggregation were markedly reduced in C6 deficient rats. The molecular basis for these coagulations defects is unknown at present.

Amino Acid Sequence↗

PubMed2004

Inhibition of C5 or absence of C6 protects from sepsis mortality.

Inhibiting complement anaphlytoxin C5a during sepsis may prevent sepsis mortality. Although human anti-C5 antibodies exist, their therapeutic use in microbial sepsis has been avoided because of the hypothesis that inhibiting C5b will prevent formation of the bactericidal membrane attack complex (MAC) and worsen clinical outcome. We wished to test the hypothesis that inhibition of C5 would improve outcomes in sepsis. Sepsis was induced in rats by laparotomy and cecal ligation and puncture (CLP) by an IACUC-approved protocol. Sham animals underwent laparotomy without CLP. Following CLP rats were randomized to receive a single IV dose of purified IgG ant-C5 antibody (Ab) or control IgG Ab. Anti-C5 Ab treated rats (n = 20) had significantly lower mortality vs. controls (n = 21), 20% vs. 52% (P = 0.019, log-rank). Analysis of bacterial load by culture of spleen and liver homogenates showed a reduction in colony forming units in anti-C5 Ab treated rats vs. control IgG (P = 0.003 and 0.009, respectively). Anti-C5 treatment reduced lung injury as measured by total MPO content of lung tissue (P = 0.024). Finally, rats genetically deficient in C6 production, unable to form MAC but capable of producing C5a and C5b, were protected from CLP-induced sepsis mortality. Our results show that in anti-C5 antibody therapy prevents CLP sepsis-induced mortality and improves lung injury. Inhibition of the complement MAC does not increase bacterial load or mortality, therefore, the use of anti-C5 therapy may be beneficial rather than detrimental in sepsis.

Animals↗

PubMed2004

Initiation of complement activation following oxidative stress. In vitro and in vivo observations.

Ischemia and reperfusion of organs/tissues induce a state of inflammation that can lead to tissue injury. Focus on development of effective therapeutics based on sound pre-clinical work and the role of leukocytes in models of human disease has not lead to a successful clinical trial for anti-leukocyte technologies. For the past >30 years, it has been known that complement activation plays a role in the inflammation and tissue injury associated with ischemia/reperfusion (I/R) injury. In the last 10 years, several complement inhibitors have made their way from the bench to bedside. Will a complement inhibitor eventually be approved for clinical treatment of I/R type diseases? What pathway(s) are involved in I/R injury, and what role do they play? What specific complement components are needed for resolution of inflammation and what components need to be inhibited to decrease tissue injury? This short review will focus on the current state of the art knowledge about complement, complement pathways, complement components and several promising clinical biologics that inhibit complement activation. This review is not a complete review of complement in ischemia/reperfusion injury, but it raises important questions about the role of complement, its pathways and the current knowledge in the area of ischemia/reperfusion injury.

Animals↗

PubMed2003

Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators.

PGs and leukotrienes (LTs) mediate cardinal signs of inflammation; hence, their enzymes are targets of current anti-inflammatory therapies. Products of arachidonate 15-lipoxygenases (LO) types I and II display both beneficial roles, such as lipoxins (LXs) that stereoselectively signal counterregulation, as well as potential deleterious actions (i.e., nonspecific phospholipid degradation). In this study, we examined transgenic (TG) rabbits overexpressing 15-LO type I and their response to inflammatory challenge. Skin challenges with either LTB(4) or IL-8 showed that 15-LO TG rabbits give markedly reduced neutrophil (PMN) recruitment and plasma leakage at dermal sites with LTB(4). PMN from TG rabbits also exhibited a dramatic reduction in LTB(4)-stimulated granular mobilization that was not evident with peptide chemoattractants. Leukocytes from 15-LO TG rabbits gave enhanced LX production, underscoring differences in lipid mediator profiles compared with non-TG rabbits. Microbe-associated inflammation and leukocyte-mediated bone destruction were assessed by initiating acute periodontitis. 15-LO TG rabbits exhibited markedly reduced bone loss and local inflammation. Because enhanced LX production was associated with an increased anti-inflammatory status of 15-LO TG rabbits, a stable analog of 5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid (LXA(4)) was applied to the gingival crevice subject to periodontitis. Topical application with the 15-epi-16-phenoxy-para-fluoro-LXA(4) stable analog (ATLa) dramatically reduced leukocyte infiltration, ensuing bone loss as well as inflammation. These results indicate that overexpression of 15-LO type I and LXA(4) is associated with dampened PMN-mediated tissue degradation and bone loss, suggesting that enhanced anti-inflammation status is an active process. Moreover, they suggest that LXs can be targets for novel approaches to diseases, e.g., periodontitis and arthritis, where inflammation and bone destruction are features.

Administration, Topical↗

PubMed2003

Antiadhesive role of apical decay-accelerating factor (CD55) in human neutrophil transmigration across mucosal epithelia.

Neutrophil migration across mucosal epithelium during inflammatory episodes involves the precise orchestration of a number a cell surface molecules and signaling pathways. After successful migration to the apical epithelial surface, apically localized epithelial proteins may serve to retain PMN at the lumenal surface. At present, identification of apical epithelial ligands and their PMN counter-receptors remain elusive. Therefore, to define the existence of apical epithelial cell surface proteins involved in PMN-epithelial interactions, we screened a panel of antibodies directed against epithelial plasma membranes. This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers. Microsequence analysis revealed that OE-1 recognized human decay-accelerating factor (DAF, CD55). DAF is a highly glycosylated, 70-80-kD, glycosyl-phosphatidyinositol-linked protein that functions predominantly as an inhibitor of autologous complement lysis. DAF suppression experiments using antisense oligonucleotides or RNA interference revealed that DAF may function as an antiadhesive molecule promoting the release of PMN from the lumenal surface after transmigration. Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface. The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes.

Amino Acid Sequence↗

PubMed2003

Role for complement in mediating intestinal nitric oxide synthase-2 and superoxide dismutase expression.

Inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD) play an important role in the pathology of ischemia-reperfusion. This study sought to determine if the proinflammatory effects of complement modulate iNOS and SOD in the rat after gastrointestinal ischemia and reperfusion (GI/R). An inhibitory or noninhibitory anti-complement component 5 (C5) monoclonal antibody (18A or 16C, respectively) was administered before GI/R. RT-PCR revealed a significant increase in intestinal iNOS mRNA compared with sham after GI/R that was attenuated significantly by 18A. Immunohistochemistry demonstrated increased iNOS protein expression within the intestinal crypts after GI/R. Cu/Zn SOD (mRNA and protein) was unaffected by GI/R, whereas Cu/Zn SOD activity was reduced significantly. Mn SOD protein expression was decreased significantly by GI/R. Anti-C5 preserved Cu/Zn SOD activity and Mn SOD protein expression. Staining for nitrotyrosine showed that anti-C5 treatment reduced protein nitration in the reperfused intestine. Immunohistochemistry demonstrated prominent phosphorylated (p) inhibitory factor-kappaB (IkappaB)-alpha staining of intestinal tissue after GI/R, whereas anti-C5 reduced p-IkappaB-alpha expression. These data indicate that complement may mediate tissue damage during GI/R by increasing intestinal iNOS and decreasing the activity and protein levels of Cu/Zn SOD and Mn SOD, respectively.

Animals↗