Biomedical subjects

M Bakhiet

Publications and source records attributed to M Bakhiet.

At least 55 records · Page 3Linked to original sources

PubMed1998

Mutations in the promoter of adenylyl cyclase (AC)-III gene, overexpression of AC-III mRNA, and enhanced cAMP generation in islets from the spontaneously diabetic GK rat model of type 2 diabetes.

Glucose-induced insulin release is decreased in the spontaneously diabetic GK rat, a nonobese rodent model of type 2 diabetes. Forskolin restores the impaired insulin release in both the isolated perfused pancreas and isolated islets from these rats (Abdel-Halim et al., Diabetes 45:934-940, 1996). We demonstrate here that the insulinotropic effect of forskolin in the GK rat is due to increased generation of cAMP and that it is associated with overexpression of adenylyl cyclase (AC)-III mRNA and gene mutations. The AC-III mRNA overexpression was demonstrated by in situ hybridization using oligonucleotide probes binding to different regions of the rat AC-III mRNA. It was associated with the presence of two point mutations identified at positions -28 bp (A --> G) and -358 bp (A --> C) of the promoter region of the AC-III gene and was demonstrable in both GK rat islets and peripheral blood cells. Transfection of COS cells with a luciferase reporter gene system revealed up to 25-fold increased promoter activity of GK AC-III promoter when compared with normal rat promoter (P < 0.0001). In conclusion, forskolin restores the impaired insulin release in islets of the GK rat through enhanced cAMP generation. This is linked to overexpression of AC-III mRNA in GK islets due to two functional point mutations in the promoter region of the AC-III gene.

Adenylyl Cyclases↗

PubMed1998

Interactions between human embryonic forebrain cells and the cytokines interferon-gamma and interleukin-4.

In the present study we investigated the capacity of human first trimester forebrain cells at different gestational ages to produce IFN-gamma and IL-4. We also studied the effects of IFN-gamma on their proliferation, survival and expression of MHC antigens. IFN-gamma but not IL-4 was spontaneously produced after 24 h cultures. Furthermore, IFN-gamma exhibited anti-proliferative effects and induced MHC expression on these cells. However, the IFN-gamma exposed cultures showed significantly higher cell survival compared to un-exposed cultures. Co-culture with IL-4 blocked the IFN-gamma production and reversed its anti-proliferative effects. These interactions suggest important roles for cytokines in the survival, proliferation and differentiation of human embryonic and fetal forebrain cells.

Cell Division↗

PubMed1998

Cytokines and anti-cytokine autoantibodies during experimental african trypanosomiasis in mice with disrupted interferon-gamma and interferon-gamma receptor genes.

We studied cytokines and anti-cytokine autoantibodies (Aabs) during T.b.brucei infections in IFN-gamma-/-, IFN-gammaR-/- and wild-type mice. Increased serum levels of IFN-gamma, TNF-gamma and IL-4 with decreased Aabs to these cytokines were recorded early during infections in all mice (except IFN-gamma in IFN-gamma-/- mice). Later, these responses were reversed, and surprisingly Aabs reacting to IFN-gamma in the IFN-gamma -/- mice were detected. To examine the possibility that an IFN-ç immunoreactive molecule might be expressed due to infections and upon gene deletion, anti-IFN-gamma antibody was inoculated and resulted in abrogation of such Aabs. The scenario was different for IL-10 and TGF- since IFN-gammaR-/- and wild-type mice showed low cytokines and high Aabs early during infections, but later high cytokines and low Aabs were registered. Interestingly, IFN-gamma-/- mice exhibited reversed levels of both IL-10 and TGF-beta, and also of their Aabs. Fab fragments of purified serum immunoglobulins showed binding and neutralizing effects in biological assays. Pre-absorption of the Fab fragments with a cytokine inhibited the binding and neutralization effects of this cytokine, but not of other cytokines. These results highlight an important role for autoimmunity in cytokine regulation, and that genomic deletion of IFN-gamma modulates cytokines and their Aab responses in experimental African trypanosomiasis.

Animals↗

PubMed1997

The gene for a T lymphocyte triggering factor from African trypanosomes.

An early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-gamma (IFN-gamma). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-gamma do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8(+) T lymphocytes to proliferate and to secrete IFN-gamma. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8(+), but not CD4(+), T cells to secrete IFN-gamma. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

Amino Acid Sequence↗

PubMed1997

Cytokine mRNA profiles during the course of experimental Haemophilus influenzae bacterial meningitis.

Intraperitoneal inoculation of Haemophilus influenzae type b (Hib) to 3-week-old Sprague-Dawley rats resulted in nonlethal meningitis with high levels of leukocytes in the cerebrospinal fluid (CSF) and positive bacterial culture. Using in situ hybridization, levels of cytokine mRNA-expressing cells were determined in the brain, CSF, and spleen from Hib-inoculated and uninfected control rats. IFN-gamma, IL-1 beta, IL-4, IL-6, IL-10, IL-12, and TNF-alpha mRNA levels were elevated at 12 hr postinoculation (pi) in spleen and CSF. At this time point, strong expression of IL-6 and TGF-beta was detected in the brain, and also of IL-10 at 48 hr while IFN-gamma and IL-12 were expressed at very low levels throughout the observation time. Delayed cytokine induction occurred in CSF compared to spleen and brain. TGF-beta was high in CSF at 48 hr, and some elevation of IL-1 beta, IL-6, IL-10, TNF-alpha, IFN-gamma, and IL-12 was evident at 72 hr pi. This may suggest measures that promote production of TGF-beta and/or IL-10 should be evaluated in treatment of bacterial meningitis.

Animals↗

PubMed1997

Haemophilus influenzae and Streptococcus pneumoniae induce different intracerebral mRNA cytokine patterns during the course of experimental bacterial meningitis.

Using in situ hybridization with radiolabelled oligonucleotide probes, we studied the mRNA expression of IL-1beta, IL-4, IL-6, IL-10, IL-12, tumour necrosis factor-alpha (TNF-alpha), TNF-beta, interferon-gamma (IFN-gamma), and transforming growth factor-beta (TGF-beta) in the brain during the lethal course of experimental meningitis in a rat model inoculated intracisternally with Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae and in uninfected control rats inoculated with the same volume of PBS. The production of IL-1beta, IL-4, IL-6 and IFN-gamma was also evaluated by immunohistochemistry. In the brain of Hib-inoculated rats, there was marked mRNA expression of IL-1beta, IL-6, TNF-alpha, IL-12 and IFN-gamma. IL-1beta, IL-6 and TNF-alpha were up-regulated throughout the observation period at 2, 8 and 18 h post-inoculation (p.i.), with similar patterns of induction. The Th1 cytokines IFN-gamma and TNF-beta were up-regulated within 8 h p.i. IL-10 and TGF-beta were down-regulated at 18 h p.i., while IL-4 was not detected. In contrast, the brain of S. pneumoniae-inoculated rats showed lower levels of IL-1beta, IL-6 and TNF-alpha, but higher levels of TNF-beta and detectable mRNA expression of IL-4 when compared with Hib-inoculated rats. IL-12, IFN-gamma, IL-10 and TGF-beta exhibited similar patterns of induction in the brains of Hib- and S. pneumoniae-inoculated rats. At 18 h p.i., immunohistochemistry showed similar patterns of IL-1beta, IL-4, IL-6 and IFN-gamma as mRNA expression in the brains of Hib- and S. pneumoniae-inoculated rats. The differences of cytokine profiles induced by the two bacterial strains may imply that different immunomodulating approaches should be considered, depending on etiology.

Animals↗

PubMed1997

Tyrosine kinases are required for interferon-gamma-stimulated proliferation of Trypanosoma brucei brucei.

The tyrosine kinase activity of Trypanosoma brucei brucei upon stimulation with interferon-gamma (IFN-gamma) was investigated. IFN-gamma induced a rapid and strong increase of tyrosine phosphorylation of several cellular proteins that reached maximum after 5 min and was followed by a decrease to control levels after 120 min. The tyrosine kinase-specific inhibitor tyrphostin A47 at a concentration of 10(-6) M reduced IFN-gamma-induced protein phosphorylation. In vitro application of 10(-6) M tyrphostin A47 to the trypanosome cultures caused a significant reduction of [3H]thymidine uptake by IFN-gamma-stimulated trypanosomes. In animals, 2 x 0.5 mg of tyrphostin A47 (injected intraperitoneally) caused a significant reduction of parasite growth compared with the vehicle dimethyl sulfoxide or the inactive compound tyrphostin A1. In conclusion, tyrosine kinases are strongly up-regulated in IFN-gamma-stimulated T. b. brucei, and specific tyrosine kinase inhibitors can prevent trypanosome growth in vitro and in vivo.

Animals↗

PubMed1997

Leishmania aethiopica derived from diffuse leishmaniasis patients preferentially induce mRNA for interleukin-10 while those from localized leishmaniasis patients induce interferon-gamma.

Exposure of peripheral blood mononuclear cells to promastigotes of Leishmania aethiopica derived from patients with the self-limiting, localized form of the disease (local cutaneous leishmaniasis; LCL) preferentially induced mRNA for interferon (IFN)-gamma but little for interleukin (IL)-10. In contrast, stimulation of the same cells with promastigotes derived from patients with the persistent, disseminated form of the disease (diffuse cutaneous leishmaniasis; DCL) stimulated the expression of IL-10 rather than IFN-gamma. In general, parasites derived from LCL patients induced more expression of other cytokines tested, including IL-4, IL-6, and transforming growth factor-beta, although tumor necrosis factor-alpha was equivalent in cultures stimulated with LCL or DCL promastigotes. The results suggest that the antigen-specific immunosuppression observed in DCL patients and the resulting clinical picture could in part be due to the properties of the infecting parasite to induce more IL-10 than IFN-gamma.

Animals↗

PubMed1997

A systemic downregulation of gamma interferon production is associated with acute shigellosis.

Production of cytokines by peripheral blood mononuclear cells from Shigella-infected patients was assessed. The frequencies of tumor necrosis factor alpha (TNF-alpha), TNF-beta, and transforming growth factor beta mRNA-expressing cells were persistently upregulated during the course of shigellosis in comparison to those of healthy controls. In contrast, the frequency of gamma interferon (IFN-gamma) mRNA-expressing cells was significantly reduced during the acute stage compared to that during the convalescent stage and to that of healthy Bangladeshi controls (P < 0.01). Constitutive IFN-gamma production in Bangladeshi controls was significantly upregulated compared to that in Swedish controls.

Adult↗

PubMed1997

Potential role of autoantibodies in the regulation of cytokine responses during bacterial infections.

An immunoregulatory mechanism involving release of neutralizing autoantibodies (Aabs) to self cytokines during bacterial infections is presented herein. Intraperitoneal inoculation of Haemophilus influenzae type b into Sprague-Dawley rats resulted in a self-limiting meningitis. High levels of cells expressing mRNA for gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) were detected 12 to 48 h postinoculation (p.i.) in splenocytes, and large numbers of IFN-gamma-secreting cells were present in the spleen on day 3 p.i. These levels were undetectable at days 9 and 14 p.i. Increased titers of Aabs of immunoglobulin G (IgG) isotypes to both cytokines were observed, with a peak at day 7 p.i. and with very low levels at day 30. Upon reinoculation with H. influenzae type b at day 30, regeneration of Aabs was recorded 7 days later (i.e., at day 37). To elucidate their regulatory importance, Aabs dose-dependently inhibited IFN-gamma production by splenocytes, IFN-gamma-induced major histocompatibility complex expression by peritoneal macrophages, and TNF-alpha-induced thymocyte proliferation. To control the specificity of these Aabs, Fab fragments of purified serum Igs from day p.i. exhibited binding and neutralizing effects. Furthermore, preincubation of the sera with a cytokine inhibited the binding and neutralization effects of that particular cytokine, but not those of any other cytokine. Aab-producing B cells were cloned, and their supernatants had similar effects. Our data suggest a role for autoimmunity in cytokine regulation and suggest that a maintained balance of this mechanism may protect from sequelae.

Animals↗

PubMed1997

Growth hormone stimulates production of interferon-gamma by human peripheral mononuclear cells.

There is substantial evidence for interactions between the immune and endocrine systems at different levels. In the present study we investigated whether human growth hormone (hGH) could stimulate proliferation of interferon-gamma-secreting cells (IFN-gamma-SC), and production of IFN-gamma. Human peripheral blood mononuclear cells (PBMC) taken from 15 normal subjects were incubated with varying doses (200,400,600 and 800 ng/ml) of recombinant hGH. Samples of cells were also incubated with PBS buffer (without hGH) to serve as controls. Effects of hGH were studied by enumerating IFN-gamma-SC and by measuring the concentration of IFN-gamma using an Immunospot assay and an enzyme-linked immunosorbent assay, respectively. The results showed that hGH significantly increased both the number of IFN-gamma-SC and the concentration of IFN-gamma in a dose-dependent manner. The maximum effects were obtained in the presence of (400 ng/ml) hGH (15 +/- .5 IFN-gamma-SC/10(6) PBMC and 300 +/- 55 U/ml IFN-gamma) compared to controls (4 +/- 2 IFN-gamma-SC/10(6) PBMC and 50 +/- 10 U/ml IFN-gamma). The results of the present study suggest that hGH might influence the immune system by stimulating the proliferation of IFN-gamma-SC and the production of IFN-gamma.

Human Growth Hormone↗

PubMed1997

Cytokine gene expression during experimental Escherichia coli pyelonephritis in mice.

PURPOSE: We studied nine inflammatory and immunoregulatory cytokines in acute pyelonephritis and urethral obstruction in mice to better understand the processes underlying kidney inflammation and scarring. MATERIALS AND METHODS: Experimental acute pyelonephritis was established in Bki NMRI outbred mice by bladder inoculation of Escherichia coli, followed by 6 h urethral obstruction. The numbers of cytokine mRNA expressing cells for interleukin-1 (IL-1), IL-4, IL-6, IL-10, IL-12, tumor necrosis factor alpha (TNF-alpha), TNF-beta, transforming growth factor beta (TGF-beta) and interferon gamma (IFN-gamma) were determined in the kidneys and spleens from the infected, non-infected but obstructed and untouched mice using in situ hybridization with radio-labelled oligonucleotide probes at 12 h, 48 h and 6 d after release of the urethral obstruction. RESULTS: Kidney cell expression of IL-1, IL-6 and TNF-alpha mRNA was observed already at 12 h and persisted on day 6 in the infected animals. A significant proinflammatory cytokine response occurred also in the non-infected obstructed animals, albeit later and at lower levels. A marked increase of IL-4, IL-10, TGF-beta and IFN-gamma mRNA producing cells was also found in the kidneys of these two groups again with higher levels in the infected animals. Very high numbers of splenocytes expressing mRNA for IL-1 were observed especially in the infected animals. A high proportion of splenocytes further expressed mRNA for IL-6, TNF-alpha, IL-4, IL-10, IFN-gamma and TGF-beta, again with highest numbers in the infected group of animals. CONCLUSIONS: The present study extends previous knowledge about the local and systemic cytokine expression profiles during acute pyelonephritis and after urethral obstruction. Of particular interest was the marked kidney cell expression of mRNA for TGF-beta, presumed to be important both for obstructive and post-infectious renal scarring.

Acute Disease↗

PubMed1996

Control of parasitemia and survival during Trypanosoma brucei brucei infection is related to strain-dependent ability to produce IL-4.

We studied non-MHC gene-dependent expression of a number of cytokines in relation to host defense and survival during Trypanosoma brucei brucei (Tbb) infection in mice. In particular, the role of IL-4 was explored with use of genomically IL-4-disrupted mice and in vivo Ab blocking. Splenocytes from MHC-identical B10.Q (relatively resistant) mice showed day 5 postinfection higher numbers of IL-4 mRNA expressing cells than C3H.Q (highly susceptible). A trypanosome-derived lymphocyte triggering factor, which is released by Tbb to polyclonally activate CD8+ T cells, stimulated naive splenocytes in vitro to a higher IL-4 response in B10.Q than in C3H.Q mice. The C3H.Q mice developed an extremely high parasitemia, showed a low Ab response against the variant surface glycoprotein (VSG), and had a mean survival time of 42 days. Conversely, B10.Q mice had lower parasitemia, mounted higher anti-VSG response, and had a mean survival time of 56 days. Deletion of the IL-4 gene had no influence on the infection in C3H.Q mice, while in B10.Q mice the deletion was associated with lower anti-VSG Ab levels and higher parasitemia. Paradoxically, B10.Q mice with disrupted IL-4 gene survived longer than the wild type. Anti-IL-4 Ab-blocking experiments in vivo displayed an enhanced parasitemia and prolonged survival in infected B10.Q mice. We conclude that 1) a non-MHC gene-related and CD8+-dependent ability to produce IL-4 partly determines the susceptibility to Tbb infection; and 2) IL-4, although involved in controlling the levels of parasitemia by its effects on immunoglobulin synthesis, also can have toxic effects on the animals.

Animals↗

PubMed1996

Both CD4+ and CD8+ T cells are essential to induce experimental autoimmune myasthenia gravis.

CD4+ T cells have been shown to be crucial in the development of experimental autoimmune myasthenia gravis (EAMG). The role of CD8+ T cells in EAMG is less well established. We previously showed that antibody depletion of CD8+ T cells in rats effectively suppresses EAMG. To further study the role and relationship of CD4+ versus CD8+ T cells in induction of EAMG, CD4-/-, CD8-/-, and CD4-8- mutant C57BL/6 mice and the parent CD4+8- wild-type mice were immunized with Torpedo acetylcholine receptor (AChR) plus complete Freund's adjuvant. Clinical EAMG was nearly completely prevented in CD4-8-, CD4-/-, and CD8-/- mice. This was associated with strongly reduced AChR-specific T and B cell responses, and with reduced levels of AChR-reactive interferon gamma (IFN-gamma) and interleukin 4 (IL-4) mRNA-expressing cells in lymphoid organs when compared with CD4+8+ wild-type mice. We conclude that (a) both CD4+ and CD8+ T cells are essential for development of EAMG, and a collaboration between these cell types may be necessary; (b) CD4+ as well as CD8+ T cells secrete IFN-gamma and IL-4, and both cytokines are involved in the development of EAMG; and (c), besides T cells, other immune cells might also be responsible for help of anti-AChR antibody production.

Animals↗

PubMed1996

Human and rodent interferon-gamma as a growth factor for Trypanosoma brucei.

An example for the bidirectional exchange of activating signals between a pathogen and immunocompetent cells in the host is presented. Trypanosoma brucei, which include subspecies that cause African sleeping sickness, secrete a molecule that triggers lymphocytes to produce interferon (IFN)-gamma. We now report that proliferation of T. brucei is stimulated in axenic cultures by IFN-gamma. The growth-enhancing effect on the pathogen is inhibited by anti-IFN-gamma receptor (R) antibodies and does not occur after exposure to other cytokines, i.e. IFN-alpha, IFN-beta and tumor necrosis factor (TNF)-alpha. While rodent-pathogenic T. brucei strains are stimulated by rat IFN-gamma, human pathogenic strains are more potently stimulated by human IFN-gamma. Rat and human IFN-gamma can partially block each others effects. Mice with disrupted IFN-gamma genes have reduced parasitemia and prolonged survival, while the outcome is reversed in mice that lack the IFN-gamma R gene.

Animals↗

PubMed1996

Different trypanozoan species possess CD8 dependent lymphocyte triggering factor-like activity.

Trypanosoma brucei brucei (T. b. brucei) release a molecule, trypanosome derived lymphocyte triggering factor (TLTF), which stimulates CD8+ cells to produce cytokines and to proliferate. We now report that T. evansi, T.b. gambiense and T.b. rhodesiense also contain factor(s) with similar activity. Thus, homogenates from these parasite taxa triggered mouse or rat lymphoid tissue of mononuclear cells (MNC) to produce interferon gamma (IFN-gamma) and to proliferate. These responses were dependent on CD8 since the activity was blocked by (a) anti CD8 antibodies, (b) occurred in CD8+ but not in CD8- mice and (c) was recorded in human CD8+ but not in CD4+ enriched peripheral blood mononuclear cells (PBL). The presence of TLTF or TLTF-like molecules in the trypanozoan species was also examined by T.b. brucei directed anti-TLTF Mabs using two Mabs with inhibitory activity and one with stimulatory activity. The lymphocyte triggering activity of T.b. gambiense and T.b. rhodesiense, but not T. evansi, was affected by the anti-TLTF Mabs. We conclude that T. evansi, T.b. rhodesiense and T.b. gambiense similar to T.b. brucei, all possess molecule(s) which CD8 dependently trigger lymphocytes. The latter three, related parasite taxa, share TLTF antibody binding epitopes.

Animals↗

PubMed1996

Induction of interferon-gamma, transforming growth factor-beta, and interleukin-4 in mouse strains with different susceptibilities to Trypanosoma brucei brucei.

A Trypanosoma brucei brucei-derived lymphocyte triggering factor (TLTF) induced CD8+ T cells to produce IFN-gamma, which in turn stimulates parasite growth. This parasite-host interaction was studied in mouse strains that are either relatively susceptible (C3H/He) or resistant (C57Bl/6J) to infection, as well as in athymic nude mice. In all mouse strains, T. b. brucei infection caused a strong induction of IFN-gamma production by spleen mononuclear cells (MNC). In vivo blocking of IFN-gamma by intraperitoneal injection of mouse monoclonal anti-IFN-gamma antibody suppressed parasite growth and increased survival of both C3H/H3 and C57Bl/6J animals, suggesting that, irrespective of strain-related disease susceptibility, IFN-gamma is a growth-promoting stimulus for T. b. brucei. Spleen MNC from noninfected mice of all strains were in vitro like-wise strongly induced to IFN-gamma production when exposed to TLTF. This suggests that CD8+ expressing T cell receptor (TCR) alpha/beta, gamma/delta-bearing T cells and NK cells may all be triggered to IFN-gamma production by TLTF. In all mouse strains, TLTF also caused an increase in the number of cells expressing mRNA for TGF-beta in vitro. However, significant triggering to IL-4 mRNA expression only occurred in the relatively disease-resistant C57Bl/6J strain. As IL-4 is required for the synthesis and class switches of immunoglobulins, which are essential host immune defenses against T. b. brucei, the degree of resistance may be related to inherent strain ability to produce IL-4 in response to TLTF.

Animals↗

PubMed1996

Lovastatin inhibits interferon-gamma-induced Trypanosoma brucei brucei proliferation: evidence for mevalonate pathway involvement.

Interferon-gamma (IFN-gamma) is an essential immunoregulating molecule that has recently been shown to have a growth stimulatory effect on Trypanosoma brucei brucei (T. b. brucei). The signalling pathway(s) involved during this triggering are unknown. Since the different products from the biosynthesis pathway utilizing mevalonate have several important cellular functions, ranging from cholesterol synthesis to growth control, we here investigate the possible role for the mevalonate pathway in IFN-gamma-driven parasite proliferation. Thus, lovastatin, a hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase-inhibiting drug, was incubated at different concentrations in vitro with T. b. brucei. The parasites were then stimulated with a broad concentration range of rIFN-gamma. The effect on proliferation or growth was measured either by the tritium-labeled thymidine incorporation assay or by direct counting of parasites from the cultures using light microscopy. The maximum proliferative response was obtained with IFN-gamma at a concentration of 10(3) U/ml added to 10(6) parasites. This response was markedly decreased with lovastatin, even at a low concentration (0.1 mM). The effect of lovastatin was reversed by the addition of 10 mM mevalonate. IFN-gamma at a concentration of 10(4) U/ml showed no proliferative effect. Addition of mevalonate to this concentration of IFN-gamma gave a threefold increase in parasite proliferation. Our data suggest that a low concentration of IFN-gamma induces parasite growth, a high concentration has the opposite effect, and both these events are regulated by activity or inactivity of the mevalonate pathway.

Animals↗