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Biomedical subjects

A Sher

Publications and source records attributed to A Sher.

At least 109 records · Page 6Linked to original sources
PubMed1996

In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha.

To examine the function of IL-10 synthesis during early infection with the intracellular protozoan Toxoplasma gondii, IL-10 knockout (KO) mice were inoculated with an avirulent parasite strain (ME-49). In contrast to control littermates that displayed 100% survival, the IL-10-deficient animals succumbed within the first 2 wk of the infection, with no evidence of enhanced parasite proliferation. The mortality in the IL-10 KO mice was associated with enhanced liver pathology characterized by increased cellular infiltration and intense necrosis. Levels of IL-12 and IFN-gamma in sera of infected IL-10-deficient animals were four- to sixfold higher than those in sera from control mice, as were mRNA levels for IFN-gamma, IL-1 beta, TNF-alpha, and IL-12 in lung tissue. Similarly, macrophages from IL-10 KO mice activated in vitro or in vivo with T. gondii produced higher levels of TNF-alpha and IL-12 than macrophages from control animals. Moreover, spleen cells from IL-10 KO mice infected with T. gondii secreted more IFN-gamma than splenocytes from nondeficient animals. In vitro depletion experiments indicated that CD4+ lymphocytes are the major source of the latter cytokine in the spleen cell populations, and in vivo depletion with anti-CD4 Abs protected the IL-10 KO mice from parasite-induced mortality. Together the data suggest that endogenous IL-10 synthesis plays an important role in vivo in down-regulating monokine and IFN-gamma responses to acute intracellular infection, thereby preventing host immunopathology.

Acute Disease↗
PubMed1996

Calpain is the target antigen of a Th1 clone that transfers protective immunity against Schistosoma mansoni.

A CD4+ clone (clone B), characterized as Th1 based on its selective production of IFN-gamma and IL-2, was established from C57Bl/6 mice protectively immunized against Schistosoma mansoni by intradermal vaccination with soluble worm Ags, plus bacillus Calmette Guerin. In agreement with previous results demonstrating an IFN-gamma-dependent cell-mediated protective mechanism in this vaccination model, Ag-elicited peritoneal macrophages from syngeneic recipients of this clone were activated to kill schistosome larvae (schistosomula) in vitro. Moreover, recipients of clone B displayed significant resistance against cercarial challenge. By screening a battery of lambda(gt11) clones from an adult worm cDNA library, one recombinant (25B) was identified that stimulated clone B specifically. Analysis of the 25B cDNA insert revealed a nucleotide sequence identical with that of the large subunit of schistosome calpain, a Ca2+-activated neutral proteinase. By expressing the products of PCR subcloning, we identified a 146-amino acid region of the 25B gene containing immunologic activity equivalent to the whole polypeptide. Overlapping peptides spanning this region were synthesized, and a core epitope was identified with the sequence EWKGAWCDGS. Since clone B responds to supernatants from cultured schistosomula, we postulate that the recognition of calpain released by invading larvae and resulting induction of Th1 cytokines accounts for the protection mediated by the adoptively transferred clone. Our findings thus implicate calpain as a target of protective immunity in schistosomes and provide the first example of a candidate vaccine Ag for this parasite identified on the basis of T cell reactivity.

Adoptive Transfer↗
PubMed1996

A role for CD4+ NK1.1+ T lymphocytes as major histocompatibility complex class II independent helper cells in the generation of CD8+ effector function against intracellular infection.

Major histocompatibility complex (MHC) class II (A beta) knockout mice were vaccinated with ts-4, an attenuated mutant strain of Toxoplasma gondii, which in normal animals induces strong T cell immunity mediated by interferon gamma (IFN-gamma). After challenge with the lethal parasite strain RH, the knockout mice displayed decreased resistance consistent with absence of CD4+ effectors. Nevertheless, these animals generated CD8+ lymphocyte effectors capable of mediating partial protection through IFN-gamma secretion. Moreover, in vivo neutralization experiments indicated that the development of resistance in knockout mice depends on CD4+ cells as well as interleukin 2 (IL-2). The identity of the IL-2-producing protective cell population was further characterized as CD4+, NK1.1+ by in vitro depletion studies and reverse transcriptase-PCR analysis of fluorescence-activated cell sorter (FACS)-purified CD4+ NK1.1+ T lymphocytes. These results demonstrate that in the absence of conventional MHC class II-restricted CD4+ T lymphocytes, CD8 priming persists and mediates partial protective immunity to T. gondii. Moreover, the data argue that CD4+, NK1.1+ cells, previously implicated in the initiation of T helper cell 2 (Th2) responses through their production of IL-4, can also play a role as alternative IL-2-secreting helper cells in Th1-mediated host resistance to infection.

Animals↗
PubMed1996

Infection of human immunodeficiency virus 1 transgenic mice with Toxoplasma gondii stimulates proviral transcription in macrophages in vivo.

Human immunodeficiency virus (HIV) 1 transgenic mice expressing low or undetectable levels of viral mRNA in lymphoid tissue were infected with the intracellular protozoan Toxoplasma gondii. Exposure to this parasite resulted in an increase in HIV-1 transcript in lymph nodes, spleens, and lungs during the acute phase of infection and in the central nervous system during the chronic stage of disease. In vivo and ex vivo experiments identified macrophages as a major source of the induced HIV-1 transcripts. In contrast, T. gondii infection failed to stimulate HIV-1 transcription in tissues of two HIV-1 transgenic mouse strains harboring a HIV-1 proviral DNA in which the nuclear factor (NF) kappa B binding motifs from the viral long terminal repeats had been replaced with a duplicated Moloney murine leukemia virus core enhancer. A role for NF-kappaB in the activation of the HIV-1 by T. gondii was also suggested by the simultaneous induction of NF-kappaB binding activity and tumor necrosis factor alpha synthesis in transgenic mouse macrophages stimulated by exposure to parasite extracts. These results demonstrate the potential of an opportunistic pathogen to induce HIV-1 transcription in vivo and suggest a mechanism for the in vivo dissemination of HIV-1 by macrophages.

AIDS-Related Opportunistic Infections↗
PubMed1996

T cell-derived IL-3 induces the production of IL-4 by non-B, non-T cells to amplify the Th2-cytokine response to a non-parasite antigen in Schistosoma mansoni-infected mice.

We describe a novel amplification mechanism underlying the increased early IL-4 production observed in Schistosoma mansoni-infected mice in response to a non-parasite Ag, sperm whale myoglobin (SwMb). Earlier studies have shown that splenic Fc epsilon R+ non-B, non-T (NBNT) cells from schistosome-infected mice secrete IL-4 after stimulation with parasite Ag. We now demonstrate that purified NBNT cells from SwMb-immunized S. mansoni-infected mice do not respond directly to SwMb, but produce IL-4 in response to IL-3. Accordingly, we show that the early SwMb-specific IL-4 response of spleen cells (SC) from immunized infected mice is dependent on IL-3 and on CD4+ T cells. Thus, most of the early SwMb-induced IL-4 from SC of infected mice appears to be produced by NBNT cells triggered by IL-3 synthesized by SwMb-specific CD4+ T cells. IL-3-induced IL-4 production was also observed in purified NBNT cells from immunized uninfected mice, but the frequency and/or IL-4-producing capacity of splenic IL-3-responsive cells was found to be 8 to 16 times higher in immunized infected animals. IL-4 production by purified CD4+ cells from immunized infected mice was also seen after SwMb stimulation, but this response showed slower kinetics than those of total SC, was IL-3-independent, and on average threefold greater than that by CD4+ cells from immunized uninfected controls. Thus, increased SwMb-induced IL-4 production in immunized S. mansoni-infected mice results from direct synthesis by CD4+ T cells, as well as their stimulation via IL-3 of an expanded population of NBNT cells. The latter pathway may serve as an amplification loop for Th2-cytokine responses.

Animals↗
PubMed1996

Toxoplasma gondii infection induces specific nonresponsiveness in lymphocytes bearing the V beta 5 chain of the mouse T cell receptor.

We recently reported a superantigen activity associated with Toxoplasma gondii tachyzoites that in vitro induces preferential expansion of V beta 5+ T lymphocytes following parasite stimulation of nonimmune cells. In the experiments presented in this work, V beta 5+ lymphocyte function was examined ex vivo using mice undergoing chronic and acute infection with the avirulent parasite strain ME49 or acutely infected with the attenuated mutant ts-4. Cells bearing the TCR V beta 5 chain were found to be increased by 1.5- to twofold during acute infection, whereas during the chronic phase, modest decreases (approximately 20%) in cells of the latter subset were observed. When splenocytes from chronically infected animals were stimulated in vitro with tachyzoites, the preferential expansion of V beta 5+ lymphocytes seen using cells from normal mice was not observed. Furthermore, when purified T lymphocytes were cultured with plate-bound V beta 5-specific mAb, we found that in contrast to normal and acutely infected animals, cells from chronically infected and ts-4-vaccinated mice were nonresponsive to TCR-induced stimulation (70 to 90% reduction relative to normal cells). In control experiments, mAb to CD3 and V beta 8 elicited normal responses in the same animals. Similarly, in contrast to normal splenocytes, cells from chronically infected mice failed to produce IFN-gamma in response to anti-V beta 5 mAb. These data indicate that V beta 5+ cells are rendered nonresponsive as a result of in vivo encounter with T. gondii, and as such they provide the first demonstration of V beta-specific anergy induced by a protozoan parasite.

Animals↗
PubMed1996

Induction and regulation of IL-15 expression in murine macrophages.

IL-15 is a recently described cytokine which resembles IL-2 in its biologic activities, stimulating T cell and NK cell proliferation and activation as well as enhancing B cell expansion and Ab production. Unlike IL-2, IL-15 is not produced by lymphocytes, but instead (at least among cells of the immune system) appears to be synthesized primarily by monocyte/macrophages. We have examined the induction of IL-15 in murine macrophages (by semiquantitative reverse transcriptase-PCR and bioassay) in response to a variety of different macrophage-activating stimuli and compared the regulation of IL-15 production to that of IL-12 and TNF-alpha. Optimal induction of IL-15, in each of the macrophages populations tested, was found to require both priming (IFN-gamma) and triggering (LPS, mycobacteria, or Toxoplasma gondii) stimuli. When compared with IL-12 mRNA synthesis by the same macrophages, IL-15.mRNA production was more resistant to inhibition by the down-regulatory cytokines IL-14, IL-13, and TGF-beta. Moreover, IL-10, which is inhibitory for most other monokines, increased levels of IL-15 mRNA found after stimulation. These data establish IL-15 as a product of the macrophage/monocyte lineage, which is up-regulated on activation. IL-15 could thus play an important role in the initiation of immune responses by microbial agents.

Animals↗
PubMed1996

Toxoplasma gondii: evidence for interleukin-12-dependent and-independent pathways of interferon-gamma production induced by an attenuated parasite strain.

Immunity in mice infected with Toxoplasma gondii is dependent upon the ability to generate protective levels of the cytokine IFN-gamma. In this report, we present evidence that the attenuated vaccine strain, ts-4, induces the latter cytokine by both IL-12-dependent and -independent pathways. In contrast, strain ME49 appears to induce IFN-gamma wholly in dependence upon IL-12. Thus, 88% of wildtype C57BL/6 mice treated with anti-IL-12 mAb survive ts-4 infection, unlike similarly treated ME49-infected animals. Moreover, while anti-IL-12 treatment reduced early IFN-gamma and nitric oxide production to background levels in ts-4-infected scid animals, the same treatment in infected C57BL/6 mice had no effect on production of the latter mediators. In addition, we found that anti-IL-12 treatment induces 100% mortality in CD(4+)-deficient MHC class II knockout mice infected with ts-4. Finally, production of nitric oxide (a molecule implicated in parasite control) was abrogated in ts-4-infected scid mice following depletion of IFN-gamma producing NK cells. Together, our results suggest that ts-4 induces IL-12-dependent and -independent IFN-gamma production in normal mice, but ME49 induces the latter cytokine only in dependence upon IL-12. Our data, furthermore, implicate involvement of T cells in the IL-12-independent component of the IFN-gamma response.

Animals↗
PubMed1996

Fluid conductance of cancellous bone graft as a predictor for graft-host interface healing.

The aim of this study is to test the hypothesis that the fluid conductance of cancellous bone graft is a deciding factor in graft-host union. Cylindrical cancellous bone specimens were trephined from fresh porcine femoral heads in a direction either parallel or perpendicular to the femoral neck axes. The graft after a defatting and freeze-drying process was placed in a perfusion apparatus. The pressure drop across the bone graft and the induced flow were measured and the conductance to fluid flow was calculated as the slope of the flow-pressure relation. Grafts of different flow conductance were transplanted into 35 rabbits to replace segments of tibiae. Nine weeks after grafting, 29 rabbits which had completed the follow-up without incidents were sacrificed. The decalcified sections of the grafts and the adjacent tibiae were examined microscopically for histological events during graft-host interface healing. Perfusion data indicated that fluid conductance ranged from 0.05 x 10(-10) to 13.4 x 10(-10) m3 s-1 Pa-1 and was higher in the direction parallel to the femoral neck axis, inversely proportional to the length of the graft and directly proportional to the square of porosity. Data from rabbit model supported the hypothesis stated above. A threshold conductance was found to be 1.5 x 10(-10) m3 s-1 Pa-1, below which revascularisation and the formations of osteoblasts and fibrous tissues could not be attained. How the low conductance led to non-union at the graft-host interface was briefly discussed.

Animals↗
PubMed1996

Molecular analysis of decreased interleukin-12 production in persons infected with human immunodeficiency virus.

Human immunodeficiency virus (HIV) disease is associated with loss of type 1 responses, including interleukin (IL)-12 production. The dramatic drop in p70 production seen at early stages of disease was found not to be associated with a similarly decreased p40 mRNA expression. p35 mRNA expression was more extensively reduced than p40 mRNA expression at these early stages. Monocytes infected in vitro with HIV displayed decreased p35 expression and p70 production, suggesting that such decreased IL-12 expression may contribute to reduced IL-12 production in HIV-positive patients' cells. In addition, treatment of cells with IL-10 increased IL-10 mRNA expression and decreased p40 expression in both HIV-positive and -negative cells, while neutralization of IL-10 increased p40 mRNA levels. These observations, together with the observed hyperproduction of IL-10 in HIV-positive patients, may explain the dysregulation of IL-12 production seen in HIV disease.

Base Sequence↗
PubMed1996

Biochemical characterization and protein kinase C dependency of monokine-inducing activities of Toxoplasma gondii.

Previous reports have indicated that the early induction of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), IL-1beta, and IL-10 is crucial for the establishment and regulation of host cell-mediated immunity to the intracellular protozoan parasite Toxoplasma gondii. In this study, we demonstrate that a soluble tachyzoite extract (soluble tachyzoite antigen) can trigger the expression of these four monokines by murine inflammatory macrophages. Further characterization revealed that the parasite molecules in soluble tachyzoite antigen responsible for monokine induction are heat stable at 100 degree C but differ in sensitivity to protease digestion. Thus, the tachyzoite factors that stimulate TNF-alpha and IL-to expression were found to be more resistant to treatment with proteinase K than those responsible for IL-12 and IL-10 induction. Similarly, while the factors responsible for the induction of all four monokines were found to be sensitive to periodate oxidation, the TNF-alpha-stimulating activity was partially resistant to treatment with the compound at a low concentration (1 mM). A further dichotomy in monokine induction signals was inferred from experiments with isoquinoline sulfonamide protein kinase inhibitors. The latter work suggested that the pathways for TNF-alpha and IL-1beta are protein kinase C dependent, while expression of IL-12 and expression of IL-10 share distinct signal transduction mechanisms involving other kinases. Together, these data argue that monokine induction by T. gondii is mediated by glycoproteins that may belong to distinct groups in terms of their biochemical properties and intracellular signaling pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Successful T cell priming in B cell-deficient mice.

B cells are an abundant population of lymphocytes that can efficiently capture, process, and present antigen for recognition by activated or memory T cells. Controversial experiments and arguments exist, however, as to whether B cells are or should be involved in the priming of virgin T cells in vivo. Using B cell-deficient mice, we have studied the role of B cells as antigen-presenting cells in a wide variety of tests, including assays of T cell proliferation and cytokine production in responses to protein antigens, T cell killing to minor and major histocompatibility antigens, skin graft rejection, and the in vitro and in vivo responses to shistosome eggs. We found that B cells are not critical for either CD4 or CD8 T cell priming in any of these systems. This finding lends support to the notion that the priming of T cells is reserved for specialized cells such as dendritic cells and that antigen presentation by B cells serves distinct immunological functions.

Animals↗
PubMed1995

IL-12-induced protection against blood-stage Plasmodium chabaudi AS requires IFN-gamma and TNF-alpha and occurs via a nitric oxide-dependent mechanism.

The effects of IL-12 administration on the development of protective immunity to blood-stage Plasmodium chabaudi AS were analyzed. Treatment of susceptible A/J mice on the day of infection and for 5 days postinfection with various doses 0.025-0.3 microgram) of rIL-12 significantly decreased the peak parasitemia level, but only treatment with 0.1 microgram resulted in increased survival. Treatment of resistant B6 mice with 0.1 microgram of rIL-12 using the same regimen also significantly decreased the peak parasitemia level, but 40% of the animals died. Treatment of these mice with anti-IL-12 mAb resulted in a more severe course of infection, but survival was not significantly altered. The mechanism of IL-12-induced resistance was examined in A/J mice during infection. Compared with spleen cells from untreated mice, cells from IL-12-treated mice produced significantly higher levels of IFN-gamma spontaneously as well as in response to Con A or Ag stimulation on day 7 postinfection. Significantly higher levels of INF-gamma and TNF-alpha were found in the sera of IL-12-treated mice, which correlated with high levels of the nitric oxide (NO) metabolite, NO3-. Furthermore, CD4+T cell depletion was found to abrogate IL-12-induced resistance. Administration of neutralizing mAb against IFN-gamma or TNF-alpha to IL-12-treated mice showed that simultaneous depletion of both cytokines resulted in 100% mortality. The role of NO was investigated by administration of aminoguanidine, a selective inhibitor of cytokine-inducible nitric oxide synthase, to IL-12-treated mice. Significantly increased mortality was observed following treatment twice daily with 9 mg of aminoguanidine, but there was no effect on parasitemia. In conclusion, these results demonstrate that IL-12 regulates the development of resistance to P. chabaudi AS via a CD4+ Th1 response, which involves the cytokines IFN-gamma and TNF-alpha, and is in part NO dependent. Therefore, IL-12, given in the appropriate dose, may be useful in the induction of protective immunity to blood-state malaria.

Animals↗
PubMed1995

An IL-12-based vaccination method for preventing fibrosis induced by schistosome infection.

The harmful fibrosis which often occurs in the context of infectious disease involves the excessive deposition of connective tissue matrix, particularly collagen, and is mostly resistant to pharmacological and immunological intervention. In schistosomiasis, fibrosis is associated with the granulomatous response to parasite eggs trapped in the liver. We have previously shown that interleukin (IL)-12 administered peritoneally with eggs prevents subsequent pulmonary granuloma formation on intravenous challenge with eggs. Here we show that sensitization with eggs plus IL-12 partly inhibits granuloma formation and dramatically reduces the tissue fibrosis induced by natural infection with Schistosoma mansoni worms. These results are an example of a vaccine against parasites which acts by preventing pathology rather than infection. IL-12 is known to favour the priming of TH1 rather than Th2 cells, and the effects on fibrosis are accompanied by replacement of the Th2-dominated pattern of cytokine expression characteristic of S. mansoni infection with one dominated by Th1 cytokines. Elevated Th2 cytokine expression and fibrosis are common manifestations of a wide variety of infectious diseases and atopic disorders which might be ameliorated by vaccination with antigen and IL-12.

Animals↗
PubMed1995

HIV infection suppresses type 1 lymphokine and IL-12 responses to Toxoplasma gondii but fails to inhibit the synthesis of other parasite-induced monokines.

Individuals infected with Toxoplasma gondii normally develop resistance to the parasite, resulting in an asymptomatic chronic infection. In AIDS patients, this resistance is lost leading to reactivation of infection and development of encephalitis. To characterize the cytokine response of T. gondii-infected individuals, PBMC were cultured in vitro in the presence or absence of crude tachyzoite Ags (STAg). When stimulated with STAg, PBMC from T. gondii-infected donors, but not controls, produced high levels of Type 1 lymphokines (IL-2 and IFN-gamma) as well as the monokine IL-12, in the absence of detectable Type 2 lymphokines (IL-4 and IL-5). In contrast, cells of individuals from both groups produced high levels of IL-1, IL-6, and TNF-alpha when exposed to the same Ag preparation. By using highly purified elutriated cells, we demonstrated that monocytes are a major source of these monokines. The above findings were further expanded by analyzing the cytokine responses induced by STAg in PBMC from patients co-infected with T. gondii and HIV. Our results demonstrate that parasite-specific IL-2 and IFN-gamma responses are greatly impaired even before AIDS development, as is IL-12 synthesis by PBMC from HIV-infected individuals stimulated with STAg. In contrast, the release of IL-6 and TNF-alpha triggered by STAg is either not affected or augmented during HIV infection.

AIDS-Related Opportunistic Infections↗
PubMed1995

IL-12 prevents mortality in mice infected with Histoplasma capsulatum through induction of IFN-gamma.

Histoplasma capsulatum is a pathogenic fungus found in discrete geographic locations throughout the world. The fungus invades the reticuloendothelial organs such as the spleen and liver of immunocompetent hosts where it is usually controlled. However, in individuals with immune deficiency, histoplasmosis is a severe and potentially fatal disease. Resistance to this infection is due primarily to a cellular immune response mediated by T cells and macrophages. Moreover, IFN-gamma is critical in activating macrophages to kill the organism. Herein we study the regulation of cytokine induction in mice infected with H. capsulatum and the effects of IL-12 in the course of infection. Mice infected with H. capsulatum and treated with neutralizing Abs to IFN-gamma, TNF-alpha, or IL-12 experienced accelerated mortality, indicating that endogenous production of these cytokines plays an important role in response to infection. In contrast, mice treated with IL-12 or a neutralizing Ab to IL-4 at the initiation of infection had substantially diminished mortality. Moreover, mice infected and treated with IL-12 show a two- to threefold increase in the amount of IFN-gamma following in vitro stimulation with specific H. capsulatum Ag compared with the control infected mice. The protective effect of IL-12 could be abrogated if a neutralizing Ab to IFN-gamma was given at the same time, demonstrating that the role of IL-12 in protection was mediated by IFN-gamma. Additionally, infected mice treated with IL-12 had a severalfold decrease in the colony counts of H. capsulatum in spleen cells after 5 days of infection as compared with control animals. Lastly, spleen cells from infected animals treated with IL-12 showed a striking decrease in their proliferative response to mitogen or H. capsulatum Ag. Responses could be restored by adding inhibitors of IFN-gamma or of nitric oxide to the in vitro cultures. The above observations suggest that IL-12 may be useful in immunologic intervention against this opportunistic pathogen.

Animals↗