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Biomedical subjects

K Nagaraju

Publications and source records attributed to K Nagaraju.

At least 19 recordsLinked to original sources
PubMed2005

Early onset of inflammation and later involvement of TGFbeta in Duchenne muscular dystrophy.

OBJECTIVE: To identify stage-specific induction of molecular pathology pathways in Duchenne muscular dystrophy (DMD). METHODS: We performed mRNA profiling using muscles from fetopsies, infants (aged 8 to 10 months), and symptomatic patients (aged 5 to 12 years) with DMD, and age- and sex-matched controls. We performed immunohistochemistry to determine changes at the protein level and protein localization. RESULTS: Activated tissue dendritic cells, expression of toll-like receptor 7, and strong induction of nuclear factor-kappaB pathways occurred soon after birth in DMD muscle. Two muscle wasting pathways, atrogin-1 and myostatin, were not induced at any stage of the disease. Normal muscle showed accumulation of glycolytic and oxidative metabolism capacity with increased age, but this accumulation failed in DMD. The transforming growth factor (TGF)-beta pathway was strongly induced in symptomatic patients, with expression of TGFbeta type II receptor and apoptosis signal-regulating kinase 1 proteins on subsets of mature DMD myofibers. CONCLUSIONS: Our data show stage-specific remodeling of human dystrophin-deficient muscle, with inflammatory pathways predominating in the presymptomatic stages and acute activation of TGFbeta and failure of metabolic pathways later in the disease.

Child↗
PubMed2001

Conditional tissue-specific expression of the acid alpha-glucosidase (GAA) gene in the GAA knockout mice: implications for therapy.

Both enzyme replacement and gene therapy of lysosomal storage disorders rely on the receptor-mediated uptake of lysosomal enzymes secreted by cells, and for each lysosomal disorder it is necessary to select the correct cell type for recombinant enzyme production or for targeting gene therapy. For example, for the therapy of Pompe disease, a severe metabolic myopathy and cardiomyopathy caused by deficiency of acid alpha-glucosidase (GAA), skeletal muscle seems an obvious choice as a depot organ for local therapy and for the delivery of the recombinant enzyme into the systemic circulation. Using knockout mice with this disease and transgenes containing cDNA for the human enzyme under muscle or liver specific promoters controlled by tetracycline, we have demonstrated that the liver provided enzyme far more efficiently. The achievement of therapeutic levels with skeletal muscle transduction required the entire muscle mass to produce high levels of enzyme of which little found its way to the plasma, whereas liver, comprising <5% of body weight, secreted 100-fold more enzyme, all of which was in the active 110 kDa precursor form. Furthermore, using tetracycline regulation, we somatically induced human GAA in the knockout mice, and demonstrated that the skeletal and cardiac muscle pathology was completely reversible if the treatment was begun early.

Animals↗
PubMed2001

Novel fragments of the Sjögren's syndrome autoantigens alpha-fodrin and type 3 muscarinic acetylcholine receptor generated during cytotoxic lymphocyte granule-induced cell death.

OBJECTIVE: To determine whether the Sjögren's syndrome autoantigens alpha-fodrin and the type 3 muscarinic acetylcholine receptor (M3R) are cleaved during cytotoxic lymphocyte granule-induced death, to yield novel fragments. METHODS: Primary salivary gland epithelial cells, human salivary gland cells, and HeLa cells were incubated with granule contents. The susceptibility to cleavage and the generation of novel fragments of Sjögren's syndrome autoantigens in this form of apoptosis was assessed by immunoblotting. Cleavage of M3R was further characterized by assays performed on the M3R molecule generated by in vitro translation. RESULTS: This study demonstrated that alpha-fodrin was uniquely cleaved during cytotoxic lymphocyte granule-induced cell death, generating a 155-kd fragment distinct from those generated by caspase 3 in other forms of apoptosis. The study also demonstrates that M3R (which is restricted in expression to the peripheral autonomic organs) was efficiently cleaved by granzyme B (but not by caspases) at several sites, both in vitro and in intact cells. This is the first description of cleavage of a transmembrane autoantigen by granzyme B. CONCLUSION: The observation that both ubiquitously expressed autoantigens (e.g., alpha-fodrin, La, and nuclear mitotic apparatus protein) and tissue-restricted autoantigens (e.g., M3R) targeted in Sjögren's syndrome are specifically cleaved by granzyme B, generating unique fragments, strongly suggests that a common biochemical event (novel autoantigen cleavage during granule-induced epithelial cell death) is responsible for selecting this apparently unconnected group of molecules for a high-titer autoantibody response. The data focus attention on the role of cytotoxic lymphocytes in the initiation and propagation of Sjögren's syndrome.

Animals↗
PubMed2001

Immunological capabilities of skeletal muscle cells.

Muscle is the target of immunological injury in several muscle diseases. It is important therefore to understand the immunological capabilities of muscle cells themselves. Although it is conventional to discuss the effects of the immune system on other cells, tissues or organs, the system's boundaries cannot be sharply drawn, and in an increasing number of ways, the immunological capabilities of non-immune tissues are recognized as determining the course of immune-inflammatory processes. Muscle cells have an inherent ability to express and respond to a variety of immunologically relevant surface molecules, cytokines, and chemokines under inflammatory conditions. The ability of muscle cells to process and present antigens to the immune cells is currently debated; thus, this review is aimed at examining the immunological capabilities of skeletal muscle cells in vitro and in vivo.

Antigen Presentation↗
PubMed2001

Update on immunopathogenesis in inflammatory myopathies.

Previous studies on myositis have suggested that these disorders are autoimmune in nature, and have provided evidence that myositis-specific autoantibodies and autoreactive T cells are present in affected patients. Recent studies provide evidence for the upregulation of various immunologically relevant cell surface molecules, cytokines, and chemokines, suggesting active cell-cell interactions. Understanding these interactions may provide novel therapeutic targets in these diseases. The role of skeletal muscle cells and their contribution to the immune response has become more important as a result of the advent of therapeutic strategies such as myoblast implantation, DNA vaccination, and gene therapy for various disease conditions. Understanding the immunologic capabilities of skeletal muscle cells may provide important clues not only to the mechanisms of the autoimmune response, but also to the use of skeletal muscle as the site of transgene expression to correct genetic defects.

Antibody Formation↗
PubMed2000

Adeno-associated virus in normal and myositis human skeletal muscle.

The normal tissue tropism of adeno-associated virus (AAV) is poorly defined, although the majority of humans test seropositive for this virus. Eighty-five muscle biopsy specimens were tested for AAV genomes; AAV DNA was identified in 17% of normal and 10% of Duchenne muscular dystrophy muscle biopsy specimens, but in only 3% of peripheral blood samples. AAV genomes were absent from all 37 muscle biopsy specimens from patients with myositis tested. Muscle is a major target organ for AAV, and infection is associated with autoimmune disease of muscle.

Adolescent↗
PubMed2000

Conditional up-regulation of MHC class I in skeletal muscle leads to self-sustaining autoimmune myositis and myositis-specific autoantibodies.

In the human inflammatory myopathies (polymyositis and dermatomyositis), the early, widespread appearance of MHC class I on the surface of muscle cells and the occurrence of certain myositis-specific autoantibodies are striking features. We have used a controllable muscle-specific promoter system to up-regulate MHC class I in the skeletal muscles of young mice. These mice develop clinical, biochemical, histological, and immunological features very similar to human myositis. The disease is inflammatory, limited to skeletal muscles, self-sustaining, more severe in females, and often accompanied by autoantibodies, including, in some mice, autoantibodies to histidyl-tRNA synthetase, the most common specificity found in the spontaneous human disease, anti-Jo-1. This model suggests that an autoimmune disease may unfold in a highly specific pattern as the consequence of an apparently nonspecific event-the sustained up-regulation of MHC class I in a tissue-and that the specificity of the autoantibodies derives not from the specificity of the stimulus, but from the context, location, and probably the duration of the stimulus. This model further suggests that the presumed order of events as an autoimmune disease develops needs to be reconsidered.

Animals↗
PubMed2000

The inhibition of apoptosis in myositis and in normal muscle cells.

The mechanism of injury and death of muscle cells in the inflammatory myopathies (dermatomyositis, polymyositis, and inclusion body myositis) remains obscure. We and others have not detected apoptosis in the muscle biopsies from patients with myositis despite clear evidence of cell damage and loss. We provide evidence in this study that Fas ligand (FasL) as well as Fas is present on muscle cells and inflammatory cells in myositis biopsies: Fas is present on most muscle cells and lymphocytes, and FasL is present on degenerating muscle cells and many infiltrating mononuclear cells. The expression of both Fas and FasL in the inflamed tissue makes the absence of apoptosis more striking. To address the mechanisms of this resistance to classical apoptosis in muscle cells, we have investigated the expression of the antiapoptotic molecule FLICE (Fas-associated death domain-like IL-1-converting enzyme)-inhibitory protein (FLIP) in muscle biopsies of myositis patients and in cultured human skeletal muscle cells. Using laser capture microscopy, we have shown that FLIP is expressed in the muscle fibers and on infiltrating lymphocytes of myositis biopsies. Furthermore, we have shown that FLIP, but not Bcl-2, is expressed in cultured human skeletal muscle cells stimulated with proinflammatory cytokines, and inhibition of FLIP with antisense oligonucleotides promotes significant cleavage of poly(ADP-ribose) polymerase autoantigen, a sensitive indicator of apoptosis. These studies strongly suggest that the resistance of muscle to Fas-mediated apoptosis is due to the expression of FLIP in muscle cells in the inflammatory environment in myositis.

Apoptosis↗
PubMed2000

Isolation, genomic organization, and expression analysis of the mouse and rat homologs of MEFV, the gene for familial mediterranean fever.

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever with serositis or synovitis. Recently the FMF gene (MEFV) was cloned; the protein product, pyrin/marenostrin, is thought to regulate inflammation in myeloid cells. In this manuscript we report the mouse and rat homologs of MEFV. The murine gene contains ten exons with a coding sequence of 2304 bp, while the rat homolog has nine exons with a coding sequence of 2253 bp. A considerable amino acid sequence homology was observed between the mouse and human (47.6% identity and 65.5% similarity) and between the mouse and rat genes (73.5% identity and 82.1% similarity). The predicted rodent proteins have several important domains and signals found in human pyrin, including a B-box zinc finger domain, Robbins-Dingwall nuclear localization signal, and coiled-coil domain. However, perhaps because of an ancient frame-shift mutation, neither the mouse nor the rat protein has an intact C-terminal B30.2 domain, in which most FMF-associated mutations have been found in human MEFV. Nevertheless, like the human gene, mouse Mefv is expressed in peripheral blood granulocytes but not lymphocytes. Consistent with its expression in granulocytes, Mefv was detected at high levels in the primary follicles and marginal zones of the splenic white pulp. Mefv is localized on mouse Chromosome (Chr) 16, region A3-B1, extending a region of synteny with human Chr 16p13.3. Development of knockout and knockin mouse models may provide further insights into the functional evolution of this gene.

Amino Acid Sequence↗
PubMed2000

Modulation of disease severity in mice with targeted disruption of the acid alpha-glucosidase gene.

Glycogen storage disease type II (GSDII) is a recessively inherited disorder caused by defects in lysosomal acid alpha-glucosidase. In an attempt to reproduce the range of clinical manifestations of the human illness we have created null alleles at the acid alpha-glucosidase locus (GAA) with several gene targeting strategies. In each knockout strain, enzyme activity was completely abolished and glycogen accumulated at indistinguishable rates. The phenotypes, however, differed strikingly. Acid alpha-glucosidase deficiency on a 129xC57BL/6 background resulted in a severe phenotype with progressive cardiomyopathy and profound muscle wasting similar to that in patients with glycogen storage disease type II. On a 129/C57BL/6xFVB background, homozygous mutants developed a milder phenotype with a later age of onset. Females were more affected than males irrespective of genetic background. As in humans with glycogen storage disease type II, therefore, other genetic loci affect the phenotypic expression of a single gene mutation.

Age Factors↗
PubMed1999

Costimulatory markers in muscle of patients with idiopathic inflammatory myopathies and in cultured muscle cells.

In an attempt to understand the mechanisms of cell injury in the inflammatory myopathies, we analyzed the expression of costimulatory molecules, CTLA4, CD28, CD86, CD40, and CD154 as well as HLA class I, HLA class II, and ICAM-I in normal muscle and in muscle biopsies from patients with polymyositis (PM) or dermatomyositis (DM). By immunohistochemical staining, DM and PM biopsies showed the presence of CTLA4, CD28, CD86, and CD40 on inflammatory cells. More strikingly, however, low levels of CTLA4 and CD28 were observed on muscle cells. The expression of CTLA4 and CD28 on nonlymphoid cells has not been previously reported. These unexpected findings were confirmed in cultured normal human myoblasts: various proinflammatory cytokines induced the expression of CTLA4 and CD28 on normal human muscle cells. The sequences of the cDNAs were found to be identical to the sequences for these molecules in T cells. The data suggest a novel complexity in the network of cellular interactions between the infiltrated immune cells and the muscle cells in which the normal relationship between infiltrating inflammatory cells and target tissue is under a previously unrecognized set of controls.

Abatacept↗
PubMed1998

Targeted disruption of the acid alpha-glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II.

We have used gene targeting to create a mouse model of glycogen storage disease type II, a disease in which distinct clinical phenotypes present at different ages. As in the severe human infantile disease (Pompe Syndrome), mice homozygous for disruption of the acid alpha-glucosidase gene (6(neo)/6(neo)) lack enzyme activity and begin to accumulate glycogen in cardiac and skeletal muscle lysosomes by 3 weeks of age, with a progressive increase thereafter. By 3.5 weeks of age, these mice have markedly reduced mobility and strength. They grow normally, however, reach adulthood, remain fertile, and, as in the human adult disease, older mice accumulate glycogen in the diaphragm. By 8-9 months of age animals develop obvious muscle wasting and a weak, waddling gait. This model, therefore, recapitulates critical features of both the infantile and the adult forms of the disease at a pace suitable for the evaluation of enzyme or gene replacement. In contrast, in a second model, mutant mice with deletion of exon 6 (Delta6/Delta6), like the recently published acid alpha-glucosidase knockout with disruption of exon 13 (Bijvoet, A. G., van de Kamp, E. H., Kroos, M., Ding, J. H., Yang, B. Z., Visser, P., Bakker, C. E., Verbeet, M. P., Oostra, B. A., Reuser, A. J. J., and van der Ploeg, A. T. (1998) Hum. Mol. Genet. 7, 53-62), have unimpaired strength and mobility (up to 6.5 months of age) despite indistinguishable biochemical and pathological changes. The genetic background of the mouse strains appears to contribute to the differences among the three models.

Alleles↗
PubMed1998

A variety of cytokines and immunologically relevant surface molecules are expressed by normal human skeletal muscle cells under proinflammatory stimuli.

Muscle is an attractive target for gene therapy and for immunization with DNA vaccines and is also the target of immunological injury in myositis. It is important therefore to understand the immunologic capabilities of muscle cells themselves. In this study, we show that proinflammatory stimuli induce the expression of other cytokines such as IL-6, transforming growth factor-beta (TGF-beta), and granulocyte-macrophage colony-stimulating factor (GM-CSF) by muscle cells themselves, as well as the up-regulation of human leucocyte antigen (HLA) class I, class II and intercellular adhesion molecule-1 (ICAM-1). Thus, muscle cells have an inherent ability to express and respond to a variety of cytokines and chemokines. The levels of HLA class I, class II and ICAM-1 in inflamed muscle may be affected by the secreted products of the stimulation.

Animals↗
PubMed1998

Lack of in vitro lymphoproliferative response to hepatitis B surface antigen in healthy vaccine recipients.

A majority of HBsAg vaccine recipients show good anti-HBs antibody responses but poor antigen specific lymphoproliferative responses. We investigated the basis for this poor in vitro antigen specific proliferative responsiveness in vaccinees who had received the standard three dose schedule (0, 1 and 6 months) of plasma derived HBsAg vaccine. Peripheral blood mononuclear cells (PBMC) from 26 of 29 (89.7%) vaccinees failed to show lymphoproliferative responses to HBsAg in spite of having a very good anti-HBs antibody response (geometric mean titre 3154 IU/1). The mitogen (phytohaemagglutinin, PHA) and antigen (purified protein derivative, PPD) driven lymphoproliferative responses in these individuals were normal. Addition of exogenous recombinant interleukin-2 (rIL-2) along with HBsAg had no effect in the response to HBsAg in six of nine vaccinees, who were tested six months after the third vaccine dose or in four unvaccinated controls. However, in three vaccinees who did not have lymphoproliferative response to HBsAg alone, addition of exogenous rIL-2 resulted in a synergistic response. These data suggest that HBsAg reactive cells are few in the peripheral circulation of a majority of individuals following the standard three dose schedule of vaccination and addition of exogenous rIL-2 induces a response only in a subgroup of individuals. The inability of HBsAg to induce a T cell proliferative response may have implications for the maintenance of protective immunity and immunological memory following vaccination.

Adult↗
PubMed1997

Functional implications of hepatitis B surface antigen (HBsAg) in the T cells of chronic HBV carriers.

This study was undertaken to investigate the role of T-lymphocyte-derived soluble factors in the maintenance of the hepatitis B virus (HBV) chronic carrier state. Cell-free supernatants from the peripheral blood T lymphocytes of chronic HBV carriers were produced by incubating them for 48 h in tissue culture medium. These supernatants were added to in vitro hepatitis B s antibody (HBsAb)-producing cultures of peripheral blood mononuclear cells from hepatitis B surface antigen (HBsAg) vaccinees stimulated with HBsAg or pokeweed mitogen. T-cell supernatants from chronic carriers suppressed in vitro HBsAb antibody synthesis, whereas those from control subjects did not. This suppression was antigen specific as the supernatants did not suppress synthesis of total IgG or IgM. HBV viral sequences were demonstrable, by Southern and dot-blot hybridization, in the T cells secreting this factor. We also demonstrated the presence of HBsAg in T-cell supernatants derived from these cells. These results show that HBsAg of T-cell origin may have a role in suppressing HBsAb production. Our observations point to the role of HBsAg-specific cellular and humoral responses in favouring persistence of the chronic HBV carrier state.

Adult↗
PubMed1996

Experimental autoimmune uveitis: molecular mimicry and oral tolerance.

Intraocular inflammatory disease or uveitis, which affects the uveal tract and the retina of the eyes in human, is the major cause of visual impairment. Experimental autoimmune uveitis (EAU) is a T-cell-mediated autoimmune disease directed against retinal proteins and has been studied in several mammalian species including subhuman primates as a model for human posterior uveitis. Autoimmune responses provoked by molecular mimicry occur when the nonself and host determinants are similar enough to cross-react yet different enough to break immunological tolerance, and is one of the proposed mechanisms for induction of autoimmune diseases. Therapeutic immunomodulatory strategies have been used to induce antigen-specific peripheral immune tolerance in animal models of T-cell-mediated autoimmune diseases by oral administration of autoantigens. Oral tolerance leads to unique mechanisms of tissue and disease-specific immunosuppression, which would circumvent the immunotherapeutic problem of multiple target tissue autoreactivity. Several groups have investigated the effects of delivering autoantigens across gastric mucosal surfaces. This review briefly discusses molecular mimicry and the mechanism of induction of oral tolerance with respect to immunopathogenesis of T-cell-mediated autoimmune disease in general and EAU in particular.

Animals↗