Moiz Bakhiet

CD8 is critically involved in lymphocyte activation by a T. brucei brucei-released molecule

T. brucei brucei released a lymphocyte triggering factor (TLTF), which triggered purified CD8+, but not CD4+, T cells to interferon gamma (IFN-gamma) mRNA expression and secretion and to [3H]thymidine incorporation. TLTF also induced mRNA for transforming growth factor beta, but not for interleukin-4. The action of this TLTF on mononuclear cell (MNC) cultures was blocked by anti-CD8 antibodies and by soluble CD8. MNCs from a mutant mouse strain lacking CD8 expression were not triggered by TLTF. IFN-gamma provides a growth stimulus for T. brucei brucei, and infected CD8- mice had much lower parasitemia and survived longer than CD8+ mice. The host-parasite interaction in experimental African trypanosomiasis thus involves parasite release of TLTF, which by binding to CD8 triggers CD8+ cells to produce the parasite growth-promoting cytokine IFN-gamma.

Depletion of CD8+ T cells suppresses growth of Trypanosoma brucei brucei and interferon-gamma) production in infected rats.

Sprague‐Dawley rats infected with Trypanosoma brucei brucei showed a strong and rapid induction of splenocyte IFN‐γ (within 12 h post‐infection) as measured by a single cell assay for IFN‐γ secretion. Depiction of CD8+ cells in infected rats abrogated the IFN‐γ production, suppressed parasite growth and increased survival of the animals. Induction of MHC class I antigens in the paraventricular and supra‐optic hypothalamic nuclei caused by the trypanosome infection was also inhibited by the CD8+ cell depletion. It is suggested that the CD8+ cells are involved directly or indirectly in growth regulation of the parasite and that IFN‐γ induced by the parasite may be one of the factors that trigger MHC expression and immunosuppression.

RANTES promotes growth and survival of human first-trimester forebrain astrocytes

We have examined the role of α and β chemokines in the promotion of the ontogenetic development of the brain. RANTES was expressed preferentially in human fetal astrocytes in an age-dependent manner. Astrocytes from 5-week-old brains showed high proliferation and reduced survival, whereas 10-week-old astrocytes exhibited opposite effects. These effects were suppressed by anti-RANTES or anti-RANTES receptor antibodies and were enhanced by recombinant RANTES. RANTES induced tyrosine phosphorylation of several cellular proteins and nuclear translocation of STAT-1 in astrocytes. Interferon-γ (IFN-γ) was required for RANTES effects because RANTES induced IFN-γ and only 10-week-old astrocytes expressed the IFN-γ receptor. Blocking of IFN-γ with antibody reversed the effects of RANTES, indicating that cytokine/chemokine networks are critically involved in brain development.

A Trypanosoma brucei brucei-Derived Factor that Triggers CD8+ Lymphocytes to Interferon-γ Secretion: Purification, Characterization and Protective Effects In Vivo by Treatment with a Monoclonal Antibody against the Factor

A protein factor that stimulates CD8+ lymphocytes to produce and secrete IFN‐γ has been purified from Trypanosoma brucei brucei (T.b. brucei). This was accomplished by raising monoclonal antibodies (MoAbs) against a fraction of T.h. brucei obtained by gel filtration, whieh contained high levels of material inducing rat mononuelear cells (MNC) to IEN‐y production. MoAbs from four hybridomas strongly inhibited trypanosome‐induced IFN‐γ production. One of them (MO1) was used for purification of the trypanosome‐derived lymphocyte triggering factor (TLTF) by affinity chromatography, SDS electrophoresis of the purified TLTF displayed a band of 42‐45 kDa MW. Gel filtration of homogenates of whole parasites yielded several peaks of IFN‐γ‐inducing activity with a lowest MW of 41 46 kDa. Bioactivity of all peaks was blocked by MO1. suggesting that a single molecule, or a single epitope of additional molecules, is responsible for the different peaks with IFN‐γ‐indueing activity. IFN‐γ released from MNC stimulates T.b. brucei growth. Blocking of TLTF in vitro with MO1 inhibited MNC‐supported growth of the parasites. To study the in vivo relevance of TLTF in the course of experimental African trypanosomiasis, MO1 was used to treat rats and mice at different times after infection. Treatments instituted at different time‐points after infection suppressed parasite growth, abrogated the IFN‐γ production by splenocytes indueed by the infection and prolonged survival of the animals. The data support the hypothesis that TLTF and IFN‐γ have a erueial regulatory function in the parasite host interactions and that these moleeules influence the disease eourse during experimental African trypanosomiasis.

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‐1β, IL‐4, IL‐6, IL‐10, IL‐12, tumour necrosis factor‐alpha (TNF‐α), TNF‐β, interferon‐gamma (IFN‐γ), and transforming growth factor‐beta (TGF‐β) 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‐1β, IL‐4, IL‐6 and IFN‐γ was also evaluated by immunohistochemistry. In the brain of Hib‐inoculated rats, there was marked mRNA expression of IL‐1β, IL‐6, TNF‐α, IL‐12 and IFN‐γ. IL‐1β, IL‐6 and TNF‐α 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‐γ and TNF‐β were up‐regulated within 8 h p.i. IL‐10 and TGF‐β 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‐1β, IL‐6 and TNF‐α, but higher levels of TNF‐β and detectable mRNA expression of IL‐4 when compared with Hib‐inoculated rats. IL‐12, IFN‐γ, IL‐10 and TGF‐β 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‐1β, IL‐4, IL‐6 and IFN‐γ 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.

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.

TNF-α and IL-8 in Acute Stroke and the Modulation of these Cytokines by Antiplatelet Agents

Stroke is associated with elevation of several proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-8 that are correlated with central nervous system (CNS) injury. Anti-platelet therapies are important agents in stroke management. The role of antiplatelets as anti-inflammatory agents is not known in acute stroke patients. Furthermore, their effect on induction of potential cytokines as TNF-α and IL-8 in those patients is still not clear. Thus, we herein examined the induction of TNF-α and IL-8 in acute stroke patients and examined the effects of the antiplatelets drugs aspirin, clopidogrel and dipyridamole, and piracetam in their induction. Cytokines were detected intracellularly in leukocytes from patients who had first acute ischemic stroke and from matched controls by immunocytochemistry. The results showed significant increase of spontaneously produced TNF-α and IL-8 in patients compared to control. This induction was significantly inhibited differently by each drug and dual drug agents. The data of this work suggest that antiplatelets agents may have a role in inhibition of stroke mediated proinflammatory cytokine effects, which may initiate a new aspect of the role of antiplatelets in the treatment of acute ischemic stroke.

Chemokines Are Upregulated During Orthodontic Tooth Movement

In the early stage of orthodontic tooth movement, an acute inflammatory response characterized by the migration of leukocytes occurs. This response suggests the presence of specific chemotactic signals that may play a role in the mechanism of bone remodeling, in particular in resorption. The aim of the present study was to explore the induction of potential chemokines at the resorption side during orthodontic tooth movement. Monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-2 (MIP-2) were examined by in situ hybridization using radioactive synthetic oligoneucleotide probes. Mesial movement of the upper first molars was performed with a fixed appliance for 3, 7, and 10 days. The results demonstrated that MCP-1, RANTES, and MIP-2 were highly expressed during orthodontic movement. On day 3, MCP-1 showed maximum induction in the pressure zone, followed in intensity by RANTES and MIP-2, although not in the contralateral control side. The induction of these chemokines had declined on day 7 and reached low levels on day 10. Our data suggest that chemokines are induced early in the application of force, and such induction may contribute to the early inflammatory response that may be responsible in part for the ensuing bone remodeling.

Chemokines are produced in the brain early during the course of experimental African trypanosomiasis.

African trypanosomiasis is characterized by progressive central nervous system (CNS) involvement. Using single and double immunohistochemistry, we evaluated the induction of alpha- and beta-chemokines in brains of Sprague-Dawley rats infected with Trypanosoma brucei brucei (T. b. brucei) and identified their cellular source. The results showed high production of MIP-2, RANTES and MIP-1alpha and to a lower extend MCP-1 in infected animals compared to controls. MIP-2, RANTES and MIP-1alpha were produced early by astrocytes and microglia and later by macrophages and T-cells. These findings suggest that chemokines may contribute to the immunopathogenesis that occurs in the CNS early during infections.

Modulation of Early Immune Responses and Suppression of Trypanosoma brucei brucei Infections by Surgical Denervation of the Spleen

Objective: To examine critical interactions between the nervous system and the immune system during experimental African trypanosomiasis. Methods and Results: Inoculation of Trypanosoma brucei brucei resulted in early interferon (IFN)-γ production, elevated corticosterone and prostaglandin E2 (PGE2) levels and increased splenocyte proliferation, as measured by enzyme-linked immunospot assay, radioimmunoassay and thymidine incorporation assay, respectively. Splenic denervation suppressed IFN-γ, corticosterone and PGE2 production, enhanced splenocyte proliferation, and significantly reduced parasitemia and prolonged rat survival. Conclusions: Our data show substantial effects of the nervous system on early immune responses that may influence the outcome of this disease. These effects were not dependent on cytokine inhibitory mediators such as prostaglandins or stress hormones. More investigations are required to understand the evident neural control over the immune system during infectious challenges, which may assist in novel therapeutic approaches.