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G J Kotwal

Publications and source records attributed to G J Kotwal.

At least 37 records · Page 2Linked to original sources
PubMed1999

Virokines: mediators of virus-host interaction and future immunomodulators in medicine.

A decade ago, after the discovery of the major secretory protein of vaccinia virus with structural similarity to complement control, the term virokine was coined. The term virokine, simply refers to a virally encoded secretory protein. During the past decade several virokines were discovered and most of them have been found to have immunomodulatory effects. A subset of virokines which resemble cytokine/chemokine receptors have been termed viroceptors. Examples of viroceptors include the TNF receptor homolog and the IL-1 beta receptor homolog. During the past several years animal models have been developed to try to understand the in vivo role of the virokines. It has become evident from at least two studies that quite a few of the virokines down-regulate the inflammatory response elicited during infection. This down-regulation at least in one model system seems to indicate that it ensures the preservation of the viral habitat (site of infection). One obvious spinoff of the research on virokines is a new class of immunomodulators has become available as therapeutics in alleviating the symptoms of inflammatory disease conditions.

Adjuvants, Immunologic↗
PubMed1998

The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins.

Sequencing and computer analysis of the left (52,283 bp) and right (49,649 bp) variable DNA regions of the cowpox virus strain GRI-90 (CPV-GRI) has revealed 51 and 37 potential open reading frames (ORFs), respectively. Comparison of the structure-function organization of these DNA regions of CPV-GRI with those previously published for corresponding regions of genomes of vaccinia virus, strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR); and variola major virus, strains India-1967 (VAR-IND), Bangladesh-1975 (VAR-BSH); and alastrim variola minor virus, strain Garcia-1966 (VAR-GAR), was performed. Within the left terminal region under study, an extended DNA sequence (14,171 bp), unique to CPV, has been found. Within the right region of the CPV-GRI genome two segments, which are unique to CPV DNA (1579 and 3585 bp) have been found. Numerous differences have been revealed in the genetic structure of CPV-GRI DNA regions, homologous to fragments of the genomes of the above-mentioned orthopoxvirus strains. A cluster of ORFs with structural similarity ot immunomodulatory and host range function of other poxviruses have also been detected. A comparison of the sequences of ORF B, crmA, crmB, crmC, IMP, and CHO hr genes of CPV Brighton strain (CPV-BRI) with the corresponding genes in strain GRI-90 have revealed an identity at the amino acid level ranging from 82 to 96% between the two strains. The findings are significant in light of the recent demonstration of CPV as an important poxvirus model system to probe the precise in vivo role(s) of the unique virally encoded immunomodulatory proteins. Also, the presence of a complete and intact repertoire of immunomodulatory proteins, ring canal proteins family, and host range genes indicates that CPV may have been the most ancient of all studied orthopoxviruses.

Amino Acid Sequence↗
PubMed1998

Molecular mimicry of the inflammation modulatory proteins (IMPs) of poxviruses: evasion of the inflammatory response to preserve viral habitat.

Microorganisms encode numerous immunomodulators that resemble, in structure and function, molecules captured over the millennia from their hosts [G. J. Kotwal J. Leukoc. Biol. 62, 415-429]. The vaccinia virus complement control protein (VCP) was the first soluble microbial protein to have a postulated role in the immunomodulation and evasion of host defense [G. J. Kotwal and B. Moss Nature 355, 176-179]. Purified bioactive VCP has been shown to bind to C3 and C4, block the complement cascade at multiple sites [G. J. Kotwal et al. Science 250, 827-830; R. Mckenzie, G. J. Kotwal et al. J. Infect. Dis. 166, 1245-1250] and exhibit a greater potency than the human complement 4b binding protein, C4b-BP [G. J. Kotwal, Am. Biotech. Lab. 9, 76]. The importance of this protein to poxviruses was further demonstrated in rabbits and guinea pigs through the use of recombinant virus lacking an intact DNA coding for VCP [Isaacs, G. J. Kotwal, and B. Moss Proc. Natl. Acad. Sci. 89, 628-672]. Studies in mice have shown that the homolog of VCP in cowpox virus (CPV), referred to as the inflammation modulatory protein (IMP) can, in a mouse model, significantly diminish the specific footpad swelling response [C. G. Miller, S. N. Shchelkunov, and G. J. Kotwal Virol. 229, 126-133]. To determine the precise cellular changes at the site of infection, BALB/c mice were subcutaneously injected (in the backs) with CPV or a recombinant virus lacking IMP, CPV-IMP. Differences in histology were observed by staining the adjoining skin tissue sections with hematoxylin & eosin or by removal of the connective tissue and staining with May-Grunwald-Geimsa. All mice that were injected with the CPV-IMP experienced severe tissue destruction and formation of nodular lesions compared with the mice injected with CPV. Microscopic examination indicated significantly greater cellular infiltration and destruction of skeletal muscle cells in the sections of connective tissue and adjoining skin tissue, respectively, of the mice injected with the CPV-IMP [G. J. Kotwal et al. Mol. Cell. Biochem. in press]. Thus IMP preserves the tissue at the site of infection (viral habitat). In this review, we present evidence for molecular mimicry and evolutionary relationship to other homologs of IMP and discuss their relationships with other IMPs such as the poxviral chemokine and cytokine receptor-like proteins.

Amino Acid Sequence↗
PubMed1998

Detection of the membrane-retained carboxy-terminal tail containing polypeptides of the amyloid precursor protein in tissue from Alzheimer's disease brain.

A major hallmark of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques consisting primarily of amyloid beta peptide (A beta) which is derived from a larger beta-amyloid precursor protein (APP). APP is processed via secretory and endosomal/lysosomal pathways by a group of proteases called secretases. During the processing of APP, the carboxy-terminal tail fragment has been suggested to remain within the cell. To investigate the fate of this fragment, we generated an antibody specific for a nine amino acid residue, the sequence of which was derived from the carboxy-terminal putative cytoplasmic tail of APP. Computer analysis of the entire APP gene, searching for regions of greatest antigenicity, surface probability, hydrophilicity, and presence of beta turns, indicated that the cytoplasmic tail region is an immunodominant region of APP. The peptide coupled to keyhole limpet hemocyanin protein, produced a very high titer antibody (1:1 x 10(6)). To evaluate the specificity of the antibody, immunoprecipitation of in vitro transcribed and translated DNA encoding the carboxy-terminal amino acids of APP in wheat germ extract was carried out. A single immunoprecipitated band of the correct size was seen by SDS-PAGE. The antibody was also able to specifically detect the accumulation of the stable C-terminal tail containing fragments of APP in neurites of the amygdala and hippocampus regions of the human brain tissue from AD subjects, but did not react with age-matched control normal brain tissue. The localization of the C-terminal tail of APP within the brain tissue of AD patients underscores the likely importance of the C-terminus in the pathogenesis of AD.

Alzheimer Disease↗
PubMed1998

The inflammation modulatory protein (IMP) of cowpox virus drastically diminishes the tissue damage by down-regulating cellular infiltration resulting from complement activation.

Vaccinia virus (VV) and other pathogenic poxviruses encode for a complement control protein. The VV complement control protein or VCP, was one of the first soluble microbial proteins postulated to have an active role in the immunomodulation of the host defense. Since then, 2 other poxviruses, including variola virus and cowpox virus (CPV), were found to have corresponding proteins. Based upon earlier studies which demonstrated the role of the CPV complement control protein in modulating the specific tissue responses in BALB/c and congenic-matched C5-sufficient and C5-deficient mice, the CPV equivalent has been renamed the inflammation modulatory protein (IMP), so as to specifically reflect its function. In this study, the in vivo cellular response of mice injected with CPV or a recombinant virus lacking the IMP sequence (CPV-IMP) was examined using a connective tissue air pouch model. Microscopic examination revealed that CPV-IMP caused a significant mononuclear cell infiltration into the connective tissue and adjacent dermal tissue of the skin. To characterize IMP's ability to regulate the observed cellular infiltration through both complement derived and non-complement derived chemotactic factors, footpad and skin connective tissue of C3 knockout mice and footpad of MIP-1alpha knockout mice received injections of CPV and CPV-IMP. In comparison to the matched control, significantly greater footpad specific swelling response was seen in C3 -/- mice injected with CPV. This indicates an important role for C3 in poxvirus pathogenesis. However, MIP-1 alpha -/- mice injected with CPV-IMP recovered earlier than mice injected with CPV alone. This indicates that the function of IMP in vivo in mice with a complete repertoire of immune components is to limit cellular infiltration by down regulating the complement derived chemotactic analphylotoxins, thereby modulating the inflammatory response contributing to a diminished tissue pathology and preservation of viral habitat.

Animals↗
PubMed1997

The cowpox virus-encoded homolog of the vaccinia virus complement control protein is an inflammation modulatory protein.

Vaccinia virus complement control protein (VCP) is encoded by vaccinia virus, with its homolog encoded by other pathogenic poxviruses including variola virus. Since rodents are the primary reservoir hosts of cowpox virus (CPV) and since CPV encodes a highly conserved functional homolog of VCP, termed here the inflammation modulatory protein (IMP), the effects of injection of CPV into the footpads of mice was determined in order to study the precise in vivo effects of IMP. Macroscopic examination of the site of injection with a recombinant virus lacking IMP (CPV-IMP) showed greater tissue damage, with more hemorrhage and induration, than sites injected with the wild-type cowpox virus. In addition, the measurement of the specific swelling response carried out for several weeks revealed significantly greater swelling in mice injected with CPV-IMP. Thus, IMP modulates the complement-activated inflammatory response in vivo. Furthermore, the diminished destruction of host tissue observed in the presence of IMP indicates symbiosis in which the virus ensures the preservation of surrounding host tissue, possibly to support the growth of its progeny.

Amino Acid Sequence↗
PubMed1997

Microorganisms and their interaction with the immune system.

Microorganisms interact with the immune system in multiple ways. In an interaction between a microorganism and its host, the defense of the host does not go unchallenged. Microorganisms have for decades been suspected of possessing the capabilities of hiding from and escaping the consequences of immune surveillance. Escape mechanisms like antigenic variation, latency, and genomic integration can best be described as passive mechanisms for avoiding interaction with the host immune system, to differentiate them from the more engaging and host-directed active mechanisms of interaction. Studies of the mechanism of direct entry of viruses (HIV, measles, and enteroviruses), bacteria (streptococci and staphylococci), and parasites (Leishmania and plasmodium) into immune cells like CD4+ T cells or macrophages, as reported very recently, indicate an even more aggressive mode of interaction. This aggressive mechanism of interaction with the components of the host immune system allows the microbe not only to block the normal function of immune components on the surface of immune cells from functioning, but also to obliterate a vital immune function, cellular immunity, causing immunosuppression, e.g. the depletion of CD4+ T cells due to the entry and replication of HIV. Collectively, microorganisms have evolved various mechanisms by which they can actively block almost any cellular, humoral, or systemic immune response. One general feature of the proteins that assist microorganism to immunomodulate and actively evade host defense is their structural and therefore functional similarity to the host proteins, which they effectively mimic. Understanding the different mechanisms by which microorganisms interact with the immune system can impact the design of live vaccines as well as the development of novel therapeutic immunomodulators that can provide medicine with powerful new tools to manage immune disorders, allograft rejection, remote multiple organ failure resulting from trauma, autoimmune diseases, etc.

Animals↗
PubMed1996

IFN-gamma-induced MHC class II gene expression is suppressed in endothelial cells by dextran sulfate.

IFN-gamma-activated endothelial cells actively participate in initiating immune responses by interacting with immunocompetent cells via class II MHC proteins. In this study, dextran sulfate, a synthetic heparin analogue, was shown to selectively inhibit IFN-gamma-induced surface expression of HLA-DR molecules by human umbilical cord vascular endothelial cells, but not other cytokine-induced molecules such as ELAM-1 or ICAM-1. Inhibition occurred regardless of whether dextran sulfate was added 24 h before, at the same time as, or 24 h after IFN-gamma stimulation of cells. In addition, both high (500 kDa) and low (5 kDa) molecular mass dextran sulfate molecules were able to block class II expression, whereas treating cells with naturally occurring polysulfated glycosaminoglycans such as heparin, heparan, and chondroitin sulfate did not produce any suppressive effects. Radiolabeling of cells with [35S]methionine followed by radioimmunoprecipitation using anti-HLA-DR alpha mAb demonstrated that biosynthesis of class II proteins was specifically blocked. RT-PCR and Southern blotting were utilized to examine transcription of the HLA-DR alpha gene and demonstrated an absence of HLA-DR alpha mRNA from dextran sulfate-treated and IFN-gamma-induced cells. Dextran sulfate also prevented transcription of the gene encoding CIITA, a transactivator protein required for IFN-gamma-inducible expression of class II genes. Thus, dextran sulfate apparently inhibited this step or an earlier one in the intracellular signaling pathway for IFN-gamma in human endothelial cells, subsequent to IFN-gamma binding to its cell surface receptor.

Base Sequence↗
PubMed1996

Two chemotactic factors, C5a and MIP-1alpha, dramatically alter the mortality from zymosan-induced multiple organ dysfunction syndrome (MODS): C5a contributes to MODS while MIP-1alpha has a protective role.

Multiple organ dysfunction syndrome (MODS) is a major cause of morbidity and mortality in surgical intensive care units. It is characterized by progressive failure of two or more organs remote from the origin of injury. Since MODS results from a severe generalized inflammatory response, both chemokines and complement have had a proposed role in its pathophysiology. The availability of macrophage inflammatory protein 1 alpha (MIP-1alpha) knockout mice and congenic C5-deficient and C5-sufficient mice allowed us to investigate the individual contribution of these immune modulators in MODS. It has been demonstrated in this assay that MIP-1alpha has a protective role against MODS mortality, while C5a contributes to MODS mortality. Using a zymosan-induced MODS murine model, the absence of MIP-1alpha increased mortality four-fold, whereas the absence of C5 decreased mortality four-fold. Therefore, MIP-1alpha-dependent mediators are essential in the prevention of MODS related deaths, while C5-dependent mediators of inflammation can be considered to be contributing to the development of MODS related deaths.

Animals↗
PubMed1995

Severe and prolonged inflammatory response to localized cowpox virus infection in footpads of C5-deficient mice: investigation of the role of host complement in poxvirus pathogenesis.

Poxviruses are a large, complex group of highly successful pathogens that cause disease in humans and other animals. They encode several proteins postulated to be involved in the evasion of host immunity and therefore serve as excellent models for understanding virus-host interaction during the early stages of viral infection. Vaccinia virus, the best characterized member of the poxviridae family, encodes a 35-kDa major secretory polypeptide termed vaccinia virus complement control protein (VCP), which is structurally related to the family of human and mouse complement control proteins. Members of the family of complement control proteins have been shown to inhibit complement-mediated opsonization of bacteria and induction of inflammatory and phagocytic responses in vitro. Insertional inactivation of the VCP gene results in attenuation of viral virulence in vivo. The role of host complement in the inflammatory response to poxvirus infection has not been systematically investigated. Prior to determining the role of VCP on inflammatory responses in vivo, we decided to investigate the role of host complement in the progression of viral infection. We have compared the effects of injection of cowpox virus, primarily a rodent virus, into footpads of congenic mice strains B10.D2/nSnJ (C5-sufficient) and B10.D2/oSnJ (C5-deficient). The effects of the injection were monitored macroscopically by measuring the specific swelling response immediately following primary injection and subsequently after reinfection and by histological examination of the stained sections of the footpads. Our results indicate that there is a significant variation in the primary response in the two different mouse strains to cowpox virus infection. The specific swelling response observed in measurements from the footpads of the B10.D2/oSnJ mice was significantly greater, persisted for a longer duration, and was accompanied by severe ulceration, edema, induration, and hemorrhaging. Reinjection of the footpads after a 3-month period, during which time the swelling had subsided and the footpad had fully recovered to its original size and appearance, showed no significant differences between the two strains. This strongly suggests that the host complement plays a significant role during the initial response to poxvirus infection.

Animals↗
PubMed1993

Routine laboratory diagnosis of hepatitis C virus infection.

Hepatitis C virus (HCV) is the major cause of parenterally transmitted non-A, non-B hepatitis. The analysis of the genomic sequence of HCV has facilitated the development of a number of diagnostic assays for testing circulating antibodies in serum from patients with HCV infection. Besides the first-generation ELISA and RIBA, which employed the C100-3 non-structural polypeptide, second-generation tests employing both structural and non-structural polypeptides are being rapidly introduced. Several coded panels were employed in a comparative study of HCV-SP ELISA (utilizing a new synthetic peptide whose sequence was derived from the structural region) along with first- and second-generation tests. On the basis of the results, evidently antigens corresponding to the structural components of the virus are more sensitive and specific for the early detection of HCV antibodies than tests using non-structural epitopes. Additionally epitopes of the structural region elicit a very strong antibody response in laboratory animals. An example of one such application is the detection of HCV specific antigens in semen from patients diagnosed with non-A, non-B (NANB) hepatitis. Semen samples from 9 patients clinically diagnosed as having NANB hepatitis were tested by an ELISA using antibodies against HCV-specific structural antigens. The semen from all 9 patients had HCV-specific structural antigens in comparison to semen from 5 healthy donors. Semen from 5 of the 9 patients had significant levels of the HCV-specific antigen. This approach to detecting HCV antigens could, if rigorously tested, evolve into promising new assays for detecting HCV.

Amino Acid Sequence↗
PubMed1992

Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope.

An enzyme-linked immunosorbent assay (ELISA) was developed by using a synthetic polypeptide (SP) whose sequence was derived from the structural region of hepatitis C virus (HCV). Results of several coded panels of sera obtained from volunteer blood donors and patients with apparent non-A, non-B hepatitis and/or hepatitis B virus used in this ELISA were compared with those of a commercially available first-generation C-100 ELISA (using nonstructural HCV antigens), an experimental second-generation C-200/C-22 ELISA (using both structural and nonstructural HCV antigens), and recombinant immunoblot assays RIBA-I and RIBA-II. In the majority of cases, the results obtained with the HCV-SP ELISA correlated well with those obtained by RIBA-II and C-200/C-22 ELISA. In contrast, many samples that were repeatedly reactive in the C-100 ELISA results were nonreactive with RIBA and HCV-SP ELISA. In addition, HCV-SP detected HCV-specific antibody that appeared within a month of infection and coincided with the earliest increase in alanine aminotransferase. In summary, we have developed an ELISA based on a structural HCV synthetic polypeptide, HCV-SP, that has high specificity and sensitivity and is capable of detecting specific antibodies in the acute phase of HCV infection.

Acute Disease↗
PubMed1992

Vaccinia virus complement-control protein prevents antibody-dependent complement-enhanced neutralization of infectivity and contributes to virulence.

The role of a viral gene product in evasion of the host immune response was investigated. The antibody-dependent complement-enhanced neutralization of vaccinia virus infectivity was prevented by the culture medium from vaccinia virus-infected cells. The vaccinia virus complement-control protein (VCP) was identified as the secreted product of vaccinia virus gene C21L and has homology to a group of eukaryotic genes encoding regulators of complement activation. Thus, the culture medium from cells infected with a C21L deletion mutant was VCP deficient and had little or no effect on antibody-dependent complement-enhanced neutralization. In addition, the anticomplement effect was associated with the C21L-encoded protein partially purified from the medium of cells infected with wild-type virus. Antibody-dependent, complement-enhanced neutralization of vaccinia virus occurred with a complement source that was deficient in the classical pathway complement component C4 and required the alternative pathway complement factor B. Furthermore, the presence of VCP abrogated the complement-enhanced neutralization in C4-deficient serum. Together with previous hemolysis data, the present result suggests that VCP can inhibit both the classical and alternative pathways of complement activation. Skin lesions caused by the C21L deletion mutant were smaller than those caused by wild-type virus, demonstrating an important role for VCP in virulence. The C21L deletion mutant also was attenuated in C4-deficient guinea pigs, consistent with in vitro studies. Vaccinia virus appears to have acquired the ability to regulate the complement cascade for the purpose of evading the host immune response.

Animals↗
PubMed1992

Detection of hepatitis C virus-specific antigens in semen from non-A, non-B hepatitis patients.

Semen samples from nine patients clinically diagnosed as having non-A, non-B hepatitis (NANBH) were tested by an ELISA using antibodies raised in rabbits against HCV-specific antigens. The semen from all nine patients had elevated levels of HCV-specific antigen in comparison to semen from five healthy donors. Semen from five of the nine patients had significant levels of the HCV-specific antigen. Seven of the eight serum samples from these patients were reactive with the standard C-100 HCV ELISA. Eight of these nine patients had serum reactive for HCV-specific antibodies in our ELISA using HCV-specific antigens. This more direct evidence for viral presence supports the earlier epidemiological data suggesting that HCV could be transmitted sexually.

Antibody Specificity↗