Biomedical subjects

J M Sharma

Publications and source records attributed to J M Sharma.

At least 37 records · Page 2Linked to original sources

PubMed1996

Nitric oxide inducing factor as a measure of antigen and mitogen-specific T cell responses in chickens.

We describe here an assay to measure responses of T cells to in vitro stimulation with antigens and a T cell mitogen (ConA). Spleen cells from chickens immunized with live viruses and an inactivated antigen produced macrophage activating factors (MAF) in response to in vitro stimulation with homologous antigens. The production of MAF, quantitated by the induction of NO in a retrovirus transformed macrophage cell line, HD11 (Beug et al., 1979, Cell 18, 375) was antigen-specific and correlated well with T cell proliferation. Further studies showed that production of MAF was abrogated by cyclosporin A, anti-CD4 and anti-CD8 monoclonal antibodies. These data suggested that production of MAF required T cell activation and can be used as measure of antigen and mitogen-specific T cell responses in chickens.

Animals↗

PubMed1996

Suppressor macrophages mediate depressed lymphoproliferation in chickens infected with avian reovirus.

A previous study indicated that spleens from reovirus-infected chickens contained macrophages that were primed to produce nitric oxide (NO). The presence of these primed macrophages correlated with depressed in vitro T cell mitogenesis. The current studies indicated that splenic adherent macrophages from virus-exposed chickens inhibited concanavalin A (ConA) induced proliferation of normal spleen cells. ConA-stimulated spleen cells from uninfected chickens, but not virus-exposed chickens, produced large quantities of interleukin-2 (IL-2) and a factor that induced NO production. This factor was tentatively named NO inducing factor (NOIF). The removal of macrophages from the spleens of virus-exposed chickens by plastic adherence resulted in partial recovery of ConA-induced proliferation and the production of normal levels of IL-2 and increased levels of NOIF, although these remained below normal. However, nonadherent spleen cells produced substantial quantities of NO, which indicated an incomplete removal of macrophages. Because removal by plastic adherence did not result in the depletion of all macrophages, spleen cells were panned with anti-CD3 antibody to obtain an almost pure population of T cells. Fractionated T cells from virus-exposed chickens proliferated vigorously to ConA and produced normal levels of IL-2 and NOIF. When splenic adherent cells from virus-exposed chickens were added to purified T cells, the T cells failed to respond to ConA. Addition of splenic adherent cells from virus-free chickens did not induce mitogenic inhibition. Further, the addition of purified T cells from the spleens of reovirus-infected chickens to T cells from virus-free birds did not adversely affect T cell mitogenesis. These data indicated that reovirus infection in chickens does not compromise the functional capabilities of T cells but induces suppressor macrophages that inhibit T cell functions.

Animals↗

PubMed1996

Protective efficacy of a recombinant herpesvirus of turkeys as an in ovo vaccine against Newcastle and Marek's diseases in specific-pathogen-free chickens.

We investigated the potential of a herpesvirus of turkey (HVT)-based recombinant virus (rHVT) as an in ovo vaccine to protect specific-pathogen-free chickens against Newcastle disease (ND) and Marek's disease (MD). The rHVT, designed to express fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of the lentogenic Hitchner B1 strain of ND virus (NDV), as well as glycoproteins A and B of the GA strain of serotype 1 MD virus (MDV) was efficacious in protecting chickens against ND and MD. No adverse effects on hatchability or the survival of chickens were observed following in ovo vaccination with rHVT. A single administration at embryonation day 18 (ED18) or at hatch protected chickens against challenge-exposures with virulent MDV strain RB-1B and velogenic NDV strain GB-Texas (NDV-GB-TX). Vaccinated chickens developed antibodies against both viruses as detected by serological tests, namely, hemagglutination inhibition, virus neutralization and western immunoblotting for NDV, and immunofluorescence and radioimmunoprecipitation assays for MDV. PCR analysis showed that in ovo vaccination with rHVT resulted in a persistent infection leading to systemic immunity against ND for up to 8 weeks of age, the longest period of time tested in this study. However, virus isolation tests indicated that rHVT-vaccinated chickens were only partially protected from the replication of NDV-GB-TX in the trachea. The results of the study indicate that rHVT is safe for both ED18 and posthatch vaccination for ND and MD, and because the vaccine persists, it may induce longer lasting immunity than conventional live NDV vaccines.

Animals↗

PubMed1996

Antigen-specific lymphoproliferative responses to tetanus toxoid: a means for the evaluation of Marek's disease virus-induced immunosuppression in chickens.

Antigen-specific lymphoproliferative responses were examined in chickens following immunization with tetanus toxoid (Ttx). The immune competence of chickens was assessed by mitogen assay utilizing phytohemagglutinin (PHA)-stimulation and Ttx-specific antigen proliferation assay (Ttx-APA). Immune spleen cells but not peripheral blood leucocytes demonstrated specific proliferation following stimulation in vitro in a Ttx-APA. In this study, we examined firstly the effects of Marek's disease (MD)-associated immunosuppression on specific immune responses. The humoral and cell-mediated immune responses were monitored by enzyme-linked immunosorbent assay (ELISA) and Ttx-APA, respectively. Secondly, we examined if vaccination against MD using a conventional herpesvirus of turkeys (HVT) vaccine and two recombinant HVT (rHVT) vaccines would affect the development of Ttx-specific immune responses. The rHVT vaccines used in this study included two constructs: one expressing both Newcastle disease virus (NDV) and MD virus (MDV) genes (HVT/NDV/MDV), and another expressing only MDV genes (HVT/MDV). The mitogenic responses of spleen cells of the vaccinated chickens were inconsistent allowing no definitive conclusions about vaccinal immunosuppression. The results of the Ttx-APA indicated that Ttx-specific lymphoproliferative responses provide a meaningful measure of immunosuppression. The MDV-induced immunosuppression resulted in the inhibition of Ttx-specific lymphoproliferation in vitro. Both HVT and rHVT vaccines were not immunosuppressive as indicated by the development of normal Ttx-specific lymphoproliferative responses in chickens. These results indicate that vaccination against MD results not only in the prevention of tumor formation but also protection from possible virus-induced immunosuppression.

Animals↗

PubMed1996

In vivo and In vitro interferon induction in chickens by S -28828, an imidazoquinolinamine immunoenhancer.

Imiquimod and its analogs belonging to a class of imidazoquinolinamines, activate immune system via cytokine induction, and have antitumor and antiviral effects in mammals. In this study, we showed that a related analog, designated S-28828, induced interferon (IFN) and macrophage activating cytokine(s) (macrophage activating factor, MAF) in chickens in vivo, ex vivo, and in vitro. IFN and MAF were detectable in the serum of chickens following oral administration. Serum IFN levels were the highest at 2 h after treatment. Although there was no detectable IFN in sera of chickens at 8, 24, and 48 h after treatment, high levels of interferon inducible enzyme, 2'-5' oligoadenylate synthase (2'5'OAS) were present at these time points. In vitro and ex vivo studies showed that spleen cells, bone marrow (BM) cells, and peripheral blood leukocytes (PBL) were capable of producing IFN and MAF, although spleen cells produced the highest levels. Our results suggest that S-28828 administered orally may be a useful immunoenhancing and antiviral agent for chickens.

2',5'-Oligoadenylate Synthetase↗

PubMed1996

Pathogenesis of type II avian adenovirus infection in turkeys: in vivo immune cell tropism and tissue distribution of the virus.

Hemorrhagic enteritis virus (HEV), a type II avian adenovirus, causes intestinal hemorrhages and immunosuppression in turkeys. In this study, we exposed turkeys to virulent HEV and examined fractionated spleen cells for the presence of viral DNA by in situ hybridization and amplification of DNA extracted from virus-infected cells by PCR. HEV replication was detected only in the immunoglobulin M-bearing B lymphocytes and macrophage-like cells but not in the CD4+ or CD8+ T lymphocytes. The inability to infect T cells distinguishes type II avian adenoviruses from lymphotropic mammalian adenoviruses which infect and replicate in T cells. Furthermore, these data suggested that HEV-induced immunosuppression in turkeys may be due to the effect of the virus on B lymphocytes and macrophages. We also examined tissue tropism of HEV by in situ hybridization conducted on sections of lymphoid and nonlymphoid tissues. Large numbers of HEV-positive cells were detected in spleen and cecal tonsils. Diminutive viral activity was present in the intestines, the principal site of HEV-induced pathology. Thus, intestinal pathology was not associated with local cytopathic viral replication. This result and our previous observation that cyclosporin A abrogated intestinal hemorrhaging in HEV-infected turkeys strongly suggested that intestinal lesion induced by this virus may be immune system mediated.

Adenoviridae Infections↗

PubMed1996

Immunohistochemical detection of lymphocyte subpopulations in the tarsal joints of chickens with experimental viral arthritis.

We characterized the lymphocytes in the tarsal joint synovium of chickens inoculated with an arthrotropic strain of avian reovirus. Cryostat sections of whole joints taken from 2 days to 35 days postinoculation were analyzed using monoclonal antibodies directed against B lymphocytes, T lymphocytes, and chicken Ia antigen. Plasma cells were morphologically identified using stained sections of whole joints. Time-dependent changes were found in the type and number of positively staining cells. Synoviocytes and cells with a dendritic morphology stained positive for Ia in normal joint sections. T cells, mostly CD8 positive, were present in low numbers in acute phase arthritis (2-6 days postinfection) in the perivascular and superficial regions of the synovium. Subacute arthritis (8-14 days postinfection) was characterized by increased numbers of CD4 and Cd8 T cells in the perivascular and superficial regions. The perivascular T cells began to organize into aggregates, with IgM-positive B cells and plasma cells on the periphery of these aggregates. Some CD8-positive cells were detected on the surface of the articular cartilage. Cells staining positively for Ia were not lymphocytes. Chronic arthritis ( > 14 days postinfection) was characterized by large numbers of T cells in the perivascular and superficial regions, with the CD4-positive T cells found primarily in the lymphoid aggregates of the perivascular regions. IgM-positive B cells were fewer, but more plasma cells, few of which stained positive for IgM, were present. Lymphocytes in chronic arthritis stained positively for Ia. These data suggest that the types, numbers, and activation level of lymphocytes present in the tarsal joints are similar but not identical to those seen in rheumatoid arthritis.

Animals↗

PubMed1995

Reovirus infection in chickens primes splenic adherent macrophages to produce nitric oxide in response to T cell-produced factors.

In this study, we examined the mechanisms by which avian reovirus infection of chickens depresses in vitro proliferative responses of spleen cells to T cell mitogens. We showed an enhanced production of nitric oxide (NO) by phytohemagglutinin (PHA)-stimulated spleen cells from reovirus-infected birds but not from virus-free birds. Since macrophages are a primary source of NO, we compared splenic adherent macrophages from virus-free and virus-exposed chickens. There was a fourfold increase in the number of adherent macrophages from the spleens of virus-exposed chickens. Production of NO by macrophages from virus-exposed chickens required T-cell-produced factors and was not due to direct stimulation of macrophages by PHA. Although T cell products were needed for NO production by macrophages, in an apparent paradox, we found significantly reduced levels of NO-inducing activity in the supernatants of PHA-stimulated spleen cells from virus-exposed chickens than in supernatants from PHA-stimulated normal spleen cells. Cocultures of adherent cells from infected chickens with normal spleen cells indicated that although macrophages secreted NO following PHA stimulation, macrophages ultimately suppressed the continued production of NO-inducing factors by normal spleen cells. We further showed in experiments utilizing NG-monomethyl-L-arginine, an NO synthesis inhibitor, that NO was not responsible for the mitogenic inhibition of spleen cells from virus-exposed chickens. In summary, our results indicated that following reovirus infection, macrophages are primed in vivo and activated in vitro by T-cell-produced factors. Despite the requirement of T cell cytokines for NO production, T cells did not proliferate to mitogenic stimuli, which indicated that the early events (i.e., cytokine secretion) but not the late events (i.e., proliferation) of the T cell activation cascade were functional. Macrophage priming following reovirus infection may have important implications for impaired T cell responsiveness.

Animals↗

PubMed1995

Hemorrhagic enteritis virus induced changes in the lymphocyte subpopulations in turkeys and the effect of experimental immunodeficiency on viral pathogenesis.

We examined the changes in the lymphocyte subpopulations in the spleen and peripheral blood of turkeys and the effects of experimental immunodeficiency in the B and T cell compartments on the pathogenesis of hemorrhagic enteritis (HE) in turkeys. Inoculation of turkeys with hemorrhagic enteritis virus (HEV) induced a drop in the relative proportions of IgM bearing cells on Day 2, 3, and 9 post-infection and an elevation in the relative proportions of CD4+ cells on Day 4 and 6 post-infection. Elevated levels of CD8+ cells were observed in the infected turkeys only on Day 16 after infection. Marked depletion of IgM+ cells may play a role in immunodepression caused by HEV. Cyclophosphamide (CY) treatment induced B cell deficiency in turkeys severely impaired HEV replication in the spleen suggesting that B lymphocytes are important for viral replication. Cyclosporin A (CsA) selectively impaired T cell mitogenesis and protected the turkeys against HEV-induced intestinal hemorrhages. CsA did not prevent viral replication in the spleen or the associated splenomegaly. This result suggested that T cell immunity may be important for intestinal hemorrhaging induced by HEV.

Animals↗

PubMed1995

Molecular cloning and sequence analysis of the penton base genes of type II avian adenoviruses.

We describe here the identification of the penton base gene of hemorrhagic enteritis virus (HEV), a type II avian adenovirus, in a 2477-base pair (bp)-EcoRI fragment of the viral DNA by sequence analysis. Identification is based on an extensive amino acid homology between the HEV-open reading frame and the penton base of a fowl adenovirus (FAV-10) and various human adenoviruses. The 1344 bp-penton base gene of HEV encodes a 448-amino acid polypeptide of molecular weight of 50,843 Da. The nucleotide sequences of penton base genes of HEV and marble spleen disease virus (MSDV) are identical. The HEV penton base lacks the RGD motif, present in most human adenoviruses (Ad2, Ad3, Ad4, and Ad 12) suggesting that HEV may not use alpha v integrins to gain entry into host cells. Further sequence analysis revealed the presence of a Leu-Asp-Val (LDV) motif in the HEV penton base amino acid sequence similar to most of the human adenoviruses. LDV motif on the fibronectin has been shown to interact with the alpha 4 beta 1 integrins on cells, which includes lymphocytes and monocytes. The presence of LDV motif in the penton base of HEV implicates the involvement of alpha 4 beta 1 integrins in the viral internalization into host cells.

Amino Acid Sequence↗

PubMed1995

Nitric oxide production by chicken macrophages activated by Acemannan, a complex carbohydrate extracted from Aloe vera.

Cultures of normal chicken spleen cells and HD11 line cells produce nitric oxide (NO) in response to Acemannan, a complex carbohydrate derived from the Aloe vera plant. Neither cell type produced detectable amounts of NO in response to similar concentrations of yeast mannan, another complex carbohydrate. Nitric oxide production was dose dependent and inhibitable by the nitric oxide synthase inhibitor NG-methyl-L-arginine. In addition, the production of NO was inhibited by preincubation of ACM with concanavalin A in a dose-dependent manner. These results suggest that ACM-induced NO synthesis may be mediated through macrophage mannose receptors, and macrophage activation may be accountable for some of the immunomodulatory effects of ACM in chickens.

Adjuvants, Immunologic↗

PubMed1995

Molecular and functional characterization of turkey interferon.

The turkey interferon (TkIFN) gene encodes a signal peptide and a mature protein of 30 and 162 amino acids, respectively. TkIFN mRNA expression was induced by reoviral double-stranded RNA in fibroblasts. The recombinant TkIFN protein possessed species-specific antiviral activity and in synergy with lipopolysaccharide (LPS) induced bone marrow macrophages to produce nitric oxide (NO). LPS or TkIFN alone did not induce bone marrow macrophages to produce significant amounts of NO, which showed that TkIFN provided one of the two signals necessary to induce NO production in turkey macrophages. Unlike the anti-inflammatory nature of mammalian alpha/beta IFNs, TkIFN augmented the LPS-induced expression of interleukin-8, a proinflammatory cytokine. This finding suggests a role for TkIFN in inflammatory conditions.

Amino Acid Sequence↗

PubMed1994

Virus-induced immunosuppression in chickens.

Reovirus and infectious bursal disease virus are among the naturally occurring viruses that cause immunosuppression in chickens. Both viruses cause necrotic lesions in the bursa of Fabricius and may destroy B cells. This may explain their ability to cause humoral immune suppression. The mechanism(s) of virus-induced suppression of cellular immunity is not well understood. Both viruses inhibit the mitogenic response of T cells in chickens. We have noted that this inhibition may be mediated by inhibitory cytokines such as transforming growth factor-beta and nitric oxide produced by activated macrophages. Preliminary studies have indicated that pretreatment of chickens with an immunomodulator, acemannan, reduced the reovirus-induced inhibition of T cells.

Animals↗

PubMed1992

Evaluation of a modified-live virus vaccine administered in ovo to protect chickens against Newcastle disease.

The B1 strain of Newcastle disease virus (NDV-B1), which is nonpathogenic for newly hatched chickens, killed embryos when it was used to inoculate chicken eggs at embryonation day 18. Treatment of NDV-B1 with an alkylating agent, ethylmethane sulfonate (EMS) markedly reduced the pathogenicity of the virus for 18-day-old chicken embryos. Eggs inoculated with the modified virus (NDV-B1-EMS) hatched, and the virus was isolated from lungs and spleen of 1-day-old chickens. The hatched chickens developed antibody to NDV and were protected against challenge exposure (at 4 weeks of age) with a highly virulent GB-Texas strain of NDV. Presence of maternal antibody to NDV in embryonating eggs did not influence the protective ability of NDV-B1-EMS, which also induced protective immunity when administered to 4-week-old chickens. The 50% protective dose of NDV-B1-EMS in maternal antibody-negative and -positive embryos was calculated to be 10.77 and 17.70 embryo 50% lethal doses, respectively. Results of the study indicated that NDV-B1-EMS may be used as an embryo vaccine to protect chickens against Newcastle disease.

Animals↗

PubMed1991

Overview of the avian immune system.

The avian immune system operates on the same general principles as the mammalian immune system. Antigenic stimulation initiates an immune response that involves cellular cooperation most notably between macrophages, B lymphocytes and T lymphocytes. Macrophages process the antigen and present the antigen to the lymphocytes. B lymphocytes, the principal cells that mediate humoral immunity, transform into plasma cells and produce antibodies. T lymphocytes, most important for cellular immunity, differentiate into functionally diverse subpopulations. The subpopulations of avian T cells have been identified with monoclonal reagents and appear to be similar to those of mammalian T cells. Lymphokines, the soluble products secreted by immune cells, mediate the functions of these cells. Studies on avian lymphokines have lagged behind those on mammalian lymphokines because the genes coding for avian lymphokines have not been cloned. The avian lymphokines studied thus far appear to function along the same lines as the mammalian lymphokines. The immune response in birds is highly regulated and breakdown in regulation often results in immunodepression.

Animals↗

PubMed1991

Hemorrhagic enteritis of turkeys.

Hemorrhagic enteritis (HE), an economically important disease of turkeys is caused by a type II adenovirus. The virus is ubiquitous and is liable to infect most field turkeys. In unprotected turkey flocks, infection with virulent hemorrhagic enteritis virus (HEV) may result in variable mortality and immunodepression. Turkeys younger than 2-4 weeks of age are resistant to clinical HE. This age-related resistance is expressed in the presence or absence of maternal antibodies against HEV. Clinical disease is characterized by HE and splenomegaly. The virus causes intranuclear inclusions in the reticuloendothelial cells. Bursectomy or splenectomy abrogate clinical HE. Field data and laboratory studies indicate that HEV causes immunodepression in the humoral as well as the cellular immune functions of turkeys. The mechanism of immunodepression is not known.

Animals↗

PubMed1989

In situ production of interferon in tissues of chickens exposed as embryos to turkey herpesvirus and Marek's disease virus.

Chicken eggs at embryonation day (ED) 18 or newly hatched chicks were inoculated with turkey herpesvirus (HVT), Marek's disease virus (MDV), or virus-free diluent and, at intervals after inoculation, tissue homogenates of virus-exposed and virus-free chickens or chicken embryos were examined for interferon (IFN) activity. Homogenates of lung, thymus and spleen specimens from chickens given HVT at ED 18 had IFN activity. Activity of IFN in the lungs was studied further. Homogenates of lung specimens from chickens exposed to HVT at hatching also had IFN activity, although the concentration of IFN was lower than that in chickens given HVT at ED 18. The pathogenic isolates of MDV (JM-MDV), but not the attenuated (Md11/75C-MDV) or nonpathogenic (SB1-MDV) isolates, inoculated at ED 18 also induced high lung IFN activity. Exposure to a combination of HVT and SB1-MDV induced IFN activity comparable with that in chickens given HVT alone. The IFN activity in homogenates of lung specimens from virus-exposed chickens was species specific and heat and pH stable, but was destroyed by trypsin treatment. Occasionally, low IFN activity also was detected in homogenates of tissue specimens from virus-free chickens or chicken embryos. This IFN activity could have been produced constitutively or may have been induced by substances (inducers) in the environment.

Animals↗

PubMed1988

Presence of natural suppressor cells in the chicken embryo spleen and the effect of virus infection of the embryo on suppressor cell activity.

Spleen cells but not the thymus or the bursa cells of chicken embryos suppressed the in vitro mitogenesis of spleen cells of adult syngeneic or allogeneic chickens. The natural suppressor cell activity of embryo spleen was present at embryonation day 16, reached peak levels at embryonation day 18 and disappeared at hatch. The embryo spleen cells did not by themselves respond to phytohemagglutinin stimulation in vitro. The suppressive effect of embryonic spleen cells on adult spleen cells was present when the embryonic cells were added at the time of or after initiation of the adult spleen mitogenic cultures. When the embryonic cells were added to the cultures of adult spleen cells after the blastogenic response of the adult cells had peaked, the embryonic cells inhibited the incorporation of the label into adult spleen cell blasts. The suppressive activity of the embryonic spleen cells was mediated by soluble suppressor product(s) secreted by these cells, and direct cell-to-cell contact between embryonic and adult spleen cells was not necessary for suppression to occur. Infection of embryos with turkey herpesvirus and Marek's disease virus reduced the suppressor cell activity of embryonic spleen, although substantial residual suppressor cell activity remained in virus-infected embryos. Several pathogenic or non-pathogenic isolates of infectious bursal disease virus did not appreciably alter the suppressor cell activity of embryonic spleen cells.

Animals↗