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Girish J Kotwal

Publications and source records attributed to Girish J Kotwal.

At least 19 recordsLinked to original sources
PubMed2006

Candidate inhibitors of porcine complement.

Therapeutic complement inhibition is a promising strategy for treatment of a number of diseases as judged from rodent studies. The species distance from rodents to humans may limit the clinical relevance of these studies. The pig is an alternative animal for studies of human diseases like sepsis and ischemia/reperfusion injury. However, available complement inhibitors for use in pigs are scarce. The aim of the present study was to investigate and compare the efficacy of selected candidate inhibitors of porcine complement in vitro for possible future application in vivo. Sera from three different pigs were each incubated with three different activators of the complement system (zymosan, heat-aggregated immunoglobulin G (HAIGG) and Escherichia coli). Three groups of complement inhibitor candidates were tested: serine protease inhibitors (FUT-175 and C1-inhibitor), monoclonal antibodies (anti-factor B (fB) and anti-factor D (fD)) and a recombinant regulatory protein (vaccinia virus complement control protein (VCP)). Read-out was the terminal C5b-9 complement complex (TCC). The serine protease inhibitors FUT-175 and C1-inhibitor dose-dependently inhibited TCC formation in zymosan-, HAIGG- and E. coli-activated porcine sera, but with different efficacy. Complete inhibition of TCC was obtained using 0.2 mg/mL FUT-175, but required 16 mg/mL of C1-inhibitor. The monoclonal anti-fB and -fD antibodies both inhibited TCC formation dose-dependently, but in different ways. Anti-fB at high dose (1 mg/mL) completely inhibited TCC formation in sera activated with zymosan and virtually completely in sera activated with HAIGG, but not in sera activated with E. coli. Anti-fD inhibited all three activators at low dose (0.05 mg/mL), and approximately 50% TCC reduction was obtained. The recombinant complement regulatory protein VCP efficiently and dose-dependently inhibited TCC formation with a complete inhibition found at 0.05 mg/mL for all three activators. All candidates tested inhibited porcine complement activation, but in different ways and to different degrees. Of the complement-specific candidates, VCP inhibited all activators completely at low doses.

Animals↗
PubMed2006

The structure of complement C3b provides insights into complement activation and regulation.

The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus complement-control protein. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the complement response. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1-MG6; nomenclature follows ref. 5) arranged in a ring, termed the beta-ring. However, because neither C3 nor C3c is active in complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for 'complement C1r/C1s, Uegf, Bmp1') domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.

Animals↗
PubMed2006

Structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation.

The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5-30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were approximately 100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.

Amino Acid Sequence↗
PubMed2005

Structural basis for antagonism by suramin of heparin binding to vaccinia complement protein.

Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.

Binding Sites↗
PubMed2005

Vaccinia virus complement control protein diminishes formation of atherosclerotic lesions: complement is centrally involved in atherosclerotic disease.

Complement is known to be activated in atherosclerotic lesions, but the importance of this event in disease pathology is a matter of debate. Studies of rabbits fed a high-fat diet have indicated complement activation as a rate-limiting step, whereas results from genetically modified mouse strains (ApoE-/- or LDLR-/-) have failed to support this finding. To resolve whether this reflects differences between species or between genetically driven and diet-induced disease, we studied the effect of a complement inhibitor, vaccinia virus complement control protein (VCP), on C57BL/6 mice, the background strain of ApoE-/- and LDLR-/- mice. Atherosclerosis was induced by a high-fat diet, and VCP (20 mg/kg) was injected once per week after the eighth week. Fatty streak development was monitored at 15 weeks by microscopic examination of oil red-O-stained sections from the root of the aorta. VCP injections led to significant (50%) reduction of lesion size (P = 0.004). Lesions were marked by gradual accumulation of lipids and macrophages but did not develop beyond the fatty streak stage. VCP activity disappeared from serum in 4 days, and the possibility therefore exists that a higher level of protection may be achieved by more frequent injections. We conclude that the development of fatty streaks in diet-induced atherosclerotic disease can be significantly retarded by prophylactic treatment with a complement inhibitor. These results support previous findings from complement-deficient rabbits and suggest that the pathogenesis of atherosclerosis in diet-induced disease differs from that induced by major defects in lipid metabolism.

Animals↗
PubMed2005

Vaccinia virus complement control protein ameliorates collagen-induced arthritic mice.

The main objective of this study was to investigate the therapeutic efficiency of recombinant vaccinia virus complement control protein (rVCP) on collagen-induced arthritis (CIA) in DBA-1/J mice. Arthritis was induced in DBA-1J mice by injecting bovine collagen emulsified in complete Freunds adjuvant. We used rVCP to block complement activation and investigated its effect on different aspects of CIA including osteoclast formation and bone destruction. The osteoclast-like cells were detected using immunohistochemistry. Joint destruction was studied using X-ray of the intact knee joints. Cartilage destruction was monitored by staining the paraffin sections with toluidine blue. ELISA was used to measure the cytokine levels in the serum. Blocking complement activation in DBA/1J arthritic mice with rVCP resulted in significant inhibition of the clinical progression of the disease and reduction in joint destruction as revealed by X-ray analysis and toluidine blue staining of the joint sections. Inhibition of complement reduced the production of proinflammatory cytokines and the number of osteoclast-like cells in arthritic joints. In conclusion, blocking of complement in CIA by rVCP inhibits the inflammation and the formation of osteoclast-like cells and reduces cartilage destruction.

Animals↗
PubMed2005

The vaccinia virus N1L protein influences cytokine secretion in vitro after infection.

The vaccinia virus N1L ORF encodes a protein that enhances virulence and replication of the virus by an unknown mechanism. It has been studied for its ability to enhance viral replication and dissemination in the brain and more recently has been linked to an immunomodulatory role in which it inhibits the activation of cytokine transcription activators in Toll-like receptor signaling pathways after pathogen recognition. The effect of N1L on the release of cytokines from human primary monocytes was investigated. Secretion of the proinflammatory, antiviral cytokines TNF-alpha, IL-1beta, IFN-alpha, IFN-beta, and the anti-inflammatory cytokine IL-10 was found to be inhibited by the presence of the N1L protein.

Animals↗
PubMed2005

The vaccinia virus N1L ORF may encode a multifunctional protein possibly targeting different kinases, one of which influences ATP levels in vivo.

As the single-most potent virulence factor of the vaccinia virus, the 13.8-kDa protein enhances viral replication in the brain by an unknown mechanism. Due to the high energy demands of the brain and the at times inadequate energy supply and small energy reserves to support physiologic activity, the ability of this organ to support energy requirements for replication of a virus is unlikely. We investigated the possible role of the 13.8-kDa protein in the enhancement of adenosine triphosphate (ATP) utilization in the brain to sustain viral replication. In vitro and in vivo monitoring and comparison of ATP levels in mouse brain tissue infected with a wild-type vaccinia virus or a 13.8-kDa deletion strain (vGK5) revealed differences in ATP utilization and a significant difference in ATP levels in vivo after 5 days of infection. Because of poor replication of the wild-type Lister vaccinia virus in the brain, a role for the 13.8-kDa protein in the modulation of ATP levels to support viral replication in the brain could not be conclusively implicated. Evaluation of the amino acid sequence and predicted secondary structure of the 13.8-kDa protein and sequence and structural homologs thereof provided evidence of putative dimerization and adenine binding sites and a possible kinase-related function for this protein.

Adenine↗
PubMed2005

Curcumin inhibits the classical and the alternate pathways of complement activation.

Curcumin (Cur), the golden yellow phenolic compound in turmeric, is well studied for its medicinal properties. In the current investigation, Cur dissolved using sodium hydroxide solution (CurNa) was tested for in vitro complement inhibitory activity and compared with rosmarinic acid (RA) and quercetin (Qur) dissolved using sodium hydroxide (RANa and QurNa, respectively) and the vaccinia virus complement control protein (VCP). The comparative study indicated that CurNa inhibited the classical complement pathway dose dependently (IC50 = 404 microM). CurNa was more active than RANa, but less active than QurNa. VCP was about 2,212, 2,786, and 4,520 times more active than QurNa, CurNa, and RANa, respectively. Further study revealed that CurNa dose dependently inhibited zymosan-induced activation of the alternate pathway of complement activation.

Analysis of Variance↗
PubMed2005

Anti-HIV, anti-poxvirus, and anti-SARS activity of a nontoxic, acidic plant extract from the Trifollium species Secomet-V/anti-vac suggests that it contains a novel broad-spectrum antiviral.

Enveloped animal viruses such as human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus, human papillomavirus, Marburg, and influenza are major public health concerns around the world. The prohibitive cost of antiretroviral (ARV) drugs for most HIV-infected patients in sub-Saharan Africa and the serious side effects in those who have access to ARV drugs make a compelling case for the study of complementary and alternative therapies. Such therapies should have scientifically proved antiviral activity and minimal toxic effects. A plant extract, Secomet-V, with an anecdotal indication in humans for promise as an anti-HIV treatment, was investigated. Using a previously described attenuated vaccinia virus vGK5, we established the antiviral activity of Secomet-V. Chemical analysis showed that it has an acidic pH, nontoxic traces of iron (<10 ppm), and almost undetectable levels of arsenic (<1.0 ppm). The color varies from colorless to pale yellow to dark brown. The active agent is heat stable at least up to sterilizing temperature of 121 degrees C. The crude plant extract is a mixture of several small molecules separable by high-pressure liquid chromatography. The HIV viral loads were significantly reduced over several months in a few patients monitored after treatment with Secomet-V. Secomet-V was also found to have antiviral activity against the SARS virus but not against the West Nile virus. Secomet-V, therefore, is a broad-spectrum antiviral, which possibly works by neutralizing viral infectivity, resulting in the prevention of viral attachment.

Anti-HIV Agents↗
PubMed2005

Cloning and expression of a functionally active truncated N-glycosylated KSHV ORF4/KCP/kaposica in the methylotrophic yeast Pichia pastoris.

Kaposi's sarcoma herpesvirus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement control protein, KCP) that binds complement components and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Based on primary sequence analysis and comparison with other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by polymerase chain reaction (PCR) three regions of the predicted ORF 4 from human herpes virus-8 (HHV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris, to produce separately the four N-terminal sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane-binding domain (KCP-M, medium), and the full-length protein (KCP-F, full). Expression of the viral proteins was confirmed by SDS-PAGE, immunologic detection, and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. KCP-S directly from expression media could inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice as efficient in inhibiting the classic pathway-mediated lysis of erythrocytes than is the vaccinia virus complement control protein, which also contains four sushi domains.

Base Sequence↗
PubMed2005

Herbal complement inhibitors in the treatment of neuroinflammation: future strategy for neuroprotection.

The upregulated complement system plays a damaging role in disorders of the central nervous system (CNS). The classical and alternate pathways are two major pathways activated in neuroinflammatory disorders such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, spinal cord injury, HIV-associated dementia, Parkinson's disease, and mad cow disease. Failure of currently available anti-inflammatory agents, especially cyclooxygenase inhibitors, in offering significant neuroprotection in large epidemiologic clinical trials of CNS disorders suggests an urgent need for the development of new neuroprotective agents. The positive preclinical outcomes in treating CNS disorders by complement regulatory molecules, such as vaccinia virus complement control protein, suggest the possibility of using complement-inhibitory molecules as neuroprotective agents. Several active ingredients of herbal origin are found to have complement-inhibitory activity. These herbal ingredients along with other anti-inflammatory roles might be useful in treating neuroinflammation associated with CNS disorders. Active ingredients of herbal origin with complement inhibitory ingredients are summarized and classified according to their chemical nature and specificity towards the major pathways activating the complement system. The structure activity relationship of some specific examples is also discussed in this report. This information might be helpful in formulating a natural panacea against complement-mediated neuroinflammation.

Complement Inactivating Agents↗
PubMed2005

Administration of vaccinia virus complement control protein shows significant cognitive improvement in a mild injury model.

Previous studies have shown that traumatic mild brain injury in a rat model is accompanied by breakdown of the blood brain barrier and the accumulation of inflammatory cells. A therapeutic agent, vaccinia virus complement control protein (VCP), inhibits both the classic and the alternative pathways of the complement system and, in so doing, prevents cell death and inflammation. With the use of a rat mild injury model, the effects of VCP on spatial learning and memory were tested. Training in a Morris water maze consisted of a total of 16 trials over a 2-day period before rats were anesthetized and subjected to mild (1.0-1.1 atm) lateral fluid percussion injury (FPI) 3.0 mm lateral to the sagittal suture and 4.5 mm posterior to bregma. Ten microl of VCP (1.7 mg/ml) was injected into the injury site immediately after FPI. Two weeks post-FPI the rats were assessed in the Morris water maze for spatial learning and memory. Neurologic motor function tests were carried out after FPI for 14 consecutive days and again after 28 days. The Morris water maze data show that FPI plus saline-injected rats spent a significantly (P <0.05) larger amount of time in one of the incorrect quadrants than did the FPI plus VCP-injected group. Neurologic evaluations 24 hours postinjury revealed differences in sensorimotor function between groups. The results suggest that in a mild injury model, VCP influences neurologic outcome and offers some enhancement in spatial memory and learning.

Animals↗
PubMed2005

Humanized recombinant vaccinia virus complement control protein (hrVCP) with three amino acid changes, H98Y, E102K, and E120K creating an additional putative heparin binding site, is 100-fold more active than rVCP in blocking both classical and alternative complement pathways.

Vaccinia virus complement control protein (VCP) is able to modulate the host complement system by regulating both pathways of complement activation. Efficient downregulation of complement activation depends on the ability of the regulatory protein to effectively bind the activated third (C3b) and fourth (C4b) complement components. Based on native crystallographic structure, molecular modeling, and sequence alignment with other Orthopoxviral complement control proteins (CCPs) and their host homologs, putative sites have been found on VCP as contact points for C3b/C4b. Here, we report that using site-directed mutagenesis, modified proteins have been generated. In addition, we report that the generated modified proteins with postulated contact point substitutions have shown greater ability to regulate both the classical and the alternative pathways of complement activation than the recombinant Western Reserve VCP, with one modified protein showing nearly 100-fold more potency in regulating both complement activation pathways independently. The augmented in vitro inhibitory activity of the modified protein together with the newly created putative heparin binding site suggests its promising potential as a competent therapeutic agent in modulating various complement-mediated ailments, for example, traumatic brain injury, Alzheimer's disease, rheumatoid arthritis, multiple organ dysfunction syndrome, reperfusion injury, and xenorejection.

Amino Acid Sequence↗
PubMed2005

Intervention strategies and agents mediating the prevention of xenorejection.

Xenotransplantation, the transplantation of cells, tissues, and/or organs across species, has proven to be an enormous challenge, resulting in only limited achievements over the last century. Unlike allotransplantation, the immunologic barriers involved in xenotransplant rejection are aggressive and usually occur within minutes in a hyperacute fashion. The use of organs from phylogenetically related concordant species may not be practical. Discordant xenotransplantation is characterized by hyperacute graft rejection, and to use nonprimate discordant organs for human benefit will require manipulation of the taxonomic differences. The hyperacute rejection process is primarily due to the attachment of preformed xenoreactive antibodies to the donor vascular endothelium, which results in hyperactivation of the complement system beyond the control of the natural complement regulatory proteins. Understanding the complex and diverse immune components involved in hyperacute, acute, and accelerated rejections has resulted in the development of different hematologic and molecular strategies. Plasmapheresis has been used to remove xenoantibodies, and xenoperfusion techniques are used to create a suitable and familiar environment for the xenograft. Various molecular approaches, such as the development of transgenic animals expressing human complement regulatory proteins such as CD59 or decay accelerating factor (DAF), to downregulate complement activation or the production of pigs lacking the xenoreactive antigen by knockout of the Gal alpha-1,3-galactosyl transferase gene have also been attempted. A combination of these techniques together with the administration of soluble complement inhibitors such as the vaccinia virus complement control protein (VCP) may well contribute to prolong graft survival. However, various issues including the possible emergence of new viral infections have confounded the topic of xenotransplantation. Here the different modulatory approaches and agents mediating interventions in xenorejection are discussed.

Acute Disease↗
PubMed2005

In vitro mutagenicity studies of the antiretrovirals AZT, Didanosine, and 3TC and a plant antiviral extract Secomet-V derived from the Trifollium species.

The plant extract Secomet-V has previously been shown by Kotwal et al. to have potent antiviral activity. It was tested for mutagenicity with the Ames gene mutation test in Salmonella and the chromosome damage (clastogenic) micronucleolus (MN) test in human lymphocytes. These tests predict long-term carcinogenesis activity of the agents tested. Secomet-V (with charcoal added) demonstrated weak clastogenic activity, but powerful mutagenic activity in the Ames test with the addition of exogenous metabolic activation. The mutagenic activity of the conventional antiretroviral drugs AZT, Didanosine (DID), and 3TC alone and in dual combinations was also assessed for the first time for Salmonella mutagenicity without any mutagenic effects. AZT, DID, and 3TC have also been tested for MN induction; DID and 3TC resulted negatively, whereas AZT was positive in a dose-related manner. The dual combinations of AZT and DID, 3TC and DID plus 3TC did not result in any additive or synergistic effect. Purification in the absence of charcoal results in a drastic reduction in extract mutagenicity, which is almost reduced completely by further ultrafiltration (cutoff <3,000 Da). This fraction, which is a mixture of molecules of less than 3,000 Da, still possesses the capability to induce sister chromatid exchanges in human lymphocytes. This could be due to residual mutagenicity or, more likely, to the slowdown of the DNA replication process. These findings open new possibilities for HIV therapy, because this antiviral activity of Secomet-V purified in the absence of charcoal and further filtered through a 3,000-Da filter is devoid of mutagenic activity and therefore safe for long-term use.

Anti-HIV Agents↗
PubMed2004

Structure of vaccinia complement protein in complex with heparin and potential implications for complement regulation.

Vaccinia virus complement control protein (VCP), a homolog of the regulators of the complement activation family of proteins, inhibits complement activation through mechanisms similar to human fluid-phase complement regulators factor H and C4b-binding protein. VCP has a heparin-binding activity that assists vaccinia in host interactions. Interaction with cell-surface polyanions like heparin is centrally important in the functioning of fluid-phase complement regulators and is the basis of host-target discrimination by the alternative pathway. We report the structure of VCP in complex with a heparin decasaccharide, which reveals changes in VCP that might be pertinent to complement regulation. Properties that VCP shares with fluid-phase complement regulators suggest that such conformational changes may be of relevance in the functioning of other complement regulators. Additionally, comparison of VCP-heparin interactions with potentially similar interactions in factor H might enable understanding of the structural basis of familial hemolytic uremic syndrome, attributed to mutational disruption of heparin and C3b binding by factor H.

Amino Acid Sequence↗