Le paludisme, une maladie inflammatoire, est caractérisé par une production du facteur de nécrose tumoral (TNF)-α, du facteur d'inhibition de migration des macrophages (MlF) et d'interféron (lFN)-y, qui inhibent les précurseurs érythropoïétiques de la moelle osseuse. Dans ce contexte, le MlF est sécrété durant les infections par Plasmodium, et sa synergie avec l'lFN-y et le TNF-α contribue à inhiber la différenciation érythropoïétiques et la production d'hémoglobine (Hb). Cet travail à démontrer que la neutralisation in vivo du MlF avec un anticorps monoclonal durant l'infection par Plasmodium chabaudi adami DK amène à une diminution du pic de la parasitémie et de façon inattendue à une production accrue de TNF-α en début d'infection et au pic d'infection. La neutralisation du MlF altère la production d'IFN-y et l'IL-10 durant l'infection. Au moment où la parasitémie est faible, une diminution significative de la production d'lFN-y, accompagnée par une chute importante de l'IL-10, est observée dans la rate de souris infectées et traitées avec l'anti-MIF. Par contre, au pic de l'infection, la production de l'IFN-y et l'IL-10 augmente chez les souris traitées avec l'anti-MIF. En plus de ces effets inhibiteurs au pic de l'infection, la neutralisation du MlF entraîne une diminution du pourcentage de réticulocytes en circulation en début d'infection et cet effet est accompagné par une légère diminution des érythrocytes basophiliques (EryA) dans la rate. La concentration d'hémoglobine sanguine était plus élevée chez les souris traitées avec l'anticorps anti-MIF, et ce, au pic de l'infection, ce qui suggère que la neutralisation du MlF, par son effet sur la parasitémie, diminue la destruction des érythrocytes par le parasite. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Paludisme, Hémoglobine, Cytokine inflammatoire, MlF, Parasitémie.

Previous work has shown that the spinal cord is capable of supporting a simple form of instrumental learning. Subjects that receive controllable shock to an extended hind limb will increase the duration of limb flexion over time in order to reduce net shock exposure. Exposure to as little as 6 minutes of uncontrollable stimulation prior to instrumental testing can elicit a long-lasting learning deficit. Prior work has suggested that this deficit may reflect an overexcitation of spinal neurons akin to central sensitization, and that learning is inhibited by the saturation of plasticity. The experiments in this dissertation were designed to test the role of the cytokine tumor necrosis factor alpha (TNFa) in the induction and expression of the deficit. It is believed that the inflammatory properties of TNFa may mediate the excitatory processes that lead to maladaptive spinal functioning. Experiments 1 and 2 tested the necessity of endogenous TNFa in the deficit produced by uncontrollable shock. These experiments showed that the inhibition of endogenous TNFa blocks both the induction and expression of the shock-induced deficit, suggesting a necessary role for TNFa in mediating the inhibition of spinal learning. Conversely, Experiment 3 was designed to test the sufficiency for TNFa in producing a learning deficit. I found that treatment with exogenous TNFa undermined spinal learning in a dose-dependent fashion, whether given immediately, or 24 hours prior to testing. Experiment 4 demonstrated that the long-term TNFa-induced deficit is mediated by TNFa receptor activity, as a TNF inhibitor given prior to testing blocked the expression of this deficit. As TNFa has been shown to be predominantly of glial origin, I next assessed the role that glia play in the TNFa-induced deficit. Experiment 5 showed that inhibiting glial metabolism prior to TNFa treatment blocked the capacity for TNFa to produce a long-term deficit. Experiment 6 assessed the potential for TNFa inhibition to block the deficit induced by lipopolysaccharide (LPS), an agent known to induce TNFa. TNFa has also been shown to drive neural excitation by increasing the trafficking of calciumpermeable AMPA receptors to the active zone of the post-synaptic bouton. Experiment 7 showed that selectively antagonizing these receptors prior to testing blocked the TNFa- induced deficit, suggesting a possible post-synaptic mechanism by which TNFa exerts its effects. Finally, histological evidence was sought to reinforce the previous behavioral findings. Experiment 8 used quantitative RT-PCR to assess the differential expression of TNFa mRNA in uncontrollably shocked subjects as compared to those receiving controllable shock and no shock. To determine concentrations of TNFa protein, an ELISA was run in Experiment 9 comparing uncontrollably shocked subjects to unshocked controls.

Burn induces the inflammation response, and causes the intestinal barrier failure. The failure of intestinal barrier may cause organ damage. Pervious studies have shown that the increase of iNOS activity is closely related to the organ damage after burn. The expression of iNOS is regulated by the activation of NF-£eB, and that is regulated by MAPKs. The pro-inflammatory cytokines play important roles to promote the inflammation through activating a series of signal transduction cascade, via binding to their receptors on cell membrane. The signal transduction cascades are turn on, MAPKs and NF-£eB are activated and the expression of iNOS is promoted. In this study, the role of pro-inflammatory cytokine interleukin-1 receptor (IL-1R) in burn induced intestinal damage was focused on. In experiments, the animals (C57BL/6 mice) were undergone 30~35 % total body surface area (TBSA) burn. The change of intestinal permeability was examined, and intestinal mucosa was assayed for the activation of iNOS and MAPKs by immunoblotting, and the activation of NF-£eB was detected by EMSA. The results reveal that activation of NF-£eB, intestinal permeability and expression of iNOS were increased after burn in wild type mice (WT). ERK MAPK plays an important role to regulate the activation of NF-£eB and expression of iNOS. Surprisingly, the permeability had no change after burn in IL-1R knock out mice (KO). The activation of ERK, NF-£eB and the expression of iNOS were also measured in KO. The levels of p-ERK, NF-£eB activation and iNOS expression were low in KO. When WT mice were treated with U0126 (5 mg/kg i.p.) right after burn to block the activation of ERK, the activation of ERK and NF-£eB, the expression of iNOS, and the intestinal permeability were all decreased significantly. To sum up, the changes in iNOS expression, NF-£eB activation, and intestinal permeability increase are mostly related to the activation of ERK after burn. IL-1 R plays a promotion role in ERK, NF-£eB activation, and iNOS expression that lead to the increase in intestinal permeability and promote damage in intestine.

Equine infectious anemia virus (EIAV) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia and viremia, termed chronic EIA. Most infected horses control the virus by immune mechanisms and become inapparent carriers. To further our understanding of the equine immune response to EIAV, a multi-probe ribonuclease protection assay (RPA) was developed to quantitate equine-specific cytokine mRNAs. Eleven template plasmids specific to ten equine cytokine genes and the ?-actin gene were generated, from which radiolabeled anti-sense RNA probes were produced. The RPA simultaneously quantitated mRNA levels of interleukin (IL)-1, IL-1, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, interferon (IFN)-, transforming growth factor (TGF)-1 and tumor necrosis factor (TNF)- in equine peripheral blood mononuclear cells and equine monocyte-derived macrophages (EMDM). The assay detected as few as 5105 RNA molecules and displayed coefficients of variation of 0.03-0.08 when normalized to -actin expression. Using this RPA, cytokine expression in EMDM infected with 2 molecularly cloned viruses (EIAV17 and EIAV19) was determined. EIAV17 varies from EIAV19 only in env, rev and LTR and causes fatal disease in Shetland ponies. When added to EMDM cultures, virulent EIAV17 stimulated expression of IL-1, IL-1, IL-6, IL-10 and TNF-. These cytokine mRNAs were significantly elevated by 0.5 to 1 hr post infection (hpi) and returned to basal levels by 12 to 24 hpi, indicating modulation by early event(s), such as receptor binding. In contrast to EIAV17, EIAV19 is avirulent in vivo and failed to induce any of the tested cytokines in EMDM. These data show a direct correlation between the virulence of the EIAV clone and the induction of cytokines. The cytokines stimulated by EIAV17 may contribute to EIA-associated symptoms, enhance viral replication in the host, and regulate the host immune response. To determine whether cytokine induction requires EIAV17 replication, EMDM cultures were exposed to UV-inactivated EIAV17 and cytokine induction was monitored. UV-inactivation did not block cytokine induction by EIAV17, suggesting dispensability of viral replication. Given that EIAV17 induces cytokines in a rapid and replication-independent manner, the activation of cytokine expression is likely mediated by binding of EIAV17 to equine macrophage receptor(s).

Clinical and epidemiological studies support a strong association between chronic inflammation and cancer. Inflammatory related cytokines, such as IL-1£\, IL-1RN, IL-1£], IL-4, IL-6, IL-8, IL-10, TNF-£\ and TGF£]-1, might play important role in carcinogenesis of oral squamous cell carcinoma (OSCC). Two case-control studies were carried out to evaluate the association of 16 various polymorphisms of 9 inflammatory-related genes with the risk for OSCC and the risk for betel quid (BQ)-related oral precancer lesions (OPL) and BQ-related OSCC. Then the association between various IL-1B C-511T/T-31C haplotypes with plasma levels of IL-1£] was evaluated. One case-contol study included 363 OSCC case patients and 487 healthy controls as well as the other case-control study included 227 BQ-related OSCC cases, 116 BQ-related OPL patients and 209 BQ-related controls. All subjects were recruited and genotyped by use of the PCR-RFLP techniques or TaqMan real-time PCR method from November 2003 and May 2007 at Kaohsiung Veterans General Hospital. Then, 9 OSCC case patients, 9 OPL patients, and 9 controls were selected and matched on sex, age as well as the quantity of BQ-chewing, alcohol drinking, and cigarette smoking for evaluation of plasma levels of IL-1£] by use of ELISA. In the single locus analysis, the variant genotype of RP1RP2 or RP2RP2 (VS. RP1RP1) of IL-4 intron 3 VNTR (AOR = 0.67, 95% CI = 0.45-0.99; AOR = 0.65, 95% CI = 0.45-0.95), TA or AA (VS. TT) genotype of IL-8 T-251A (AOR = 1.55, 95% CI = 1.05-2.30; AOR = 2.50, 95% CI = 1.46-4.27), TT (VS. CC) genotype of IL-8 C+781T (AOR = 2.01; 95% CI = 1.11-3.63), and GA combined with AA (VS. GG) genotype of TNFA G-308A (AOR = 0.40; 95% CI = 0.25-0.66) were associated with risk of OSCC, as compare with those genotypes of healthy controls. However, CC (VS. TT) genotype of IL-10 T-819C (AOR = 0.24, 95% CI = 0.08-0.74) and CC (VS. AA) genotype of IL-10 A-592C (AOR = 0.25, 95% CI = 0.08-0.79) were significantly associated with reduced risk of BQ-related OPL, as compared with those genotypes of BQ controls. In addition, the variant genotype of 2/2 or 1/2 (VS. 1/1) of IL-1RN intron2 VNTR (AOR = 0.11, 95% CI = 0.01-0.97; AOR = 0.48, 95% CI = 0.27-0.87), TC (VS TT) genotype of IL-1B T-31C (AOR = 1.82, 95% CI = 1.14-2.92), AA (VS. TT) genotype of IL-8 T-251A (AOR = 1.92, 95% CI = 1.01-3.66), GG (VS. TT) genotype of IL-8 T+396G (AOR = 2.18; 95% CI = 1.12-4.21), and GA combined with AA (VS. GG) genotype of TNFA G-308A (AOR = 0.46, 95% CI = 0.27-0.79) were significantly related with risk of BQ-related OSCC, as compared with BQ controls. Moreover, CC (VS. TT) genotype of IL-10 T-819C (AOR = 3.33, 95% CI = 1.07-10.42) was associated with increased risk of BQ-related OSCC, as compared with those genotypes of BQ-related OPL. In the haplotype analysis, -590C/RP2 (VS. -590T/RP1) haplotype of IL-4 (AOR = 0.69; 95% CI = 0.49-0.98) and -251A/+781T (VS. -251T/+781C) haplotype of IL-8 (AOR = 1.57, 95% CI = 1.19-2.06) were related with risk of OSCC, as compared with those haplotypes of healthy controls. However, -511C/-31C (VS. -511C/-31T) haplotype of IL-1B (AOR = 0.00, 95% CI = 0.00-0.01) and -1082A/-819C/-592C (VS. -1082A/-819T/-592A) haplotype of IL-10 (AOR = 0.66, 95% CI = 0.44-0.98) were strongly associated with reduced risk of BQ-related OPL, as compared with those genotypes BQ controls. In addition, -889C/2/-511C/-31T or -889T/1/-511C/-31T or -889T/1/-511T/-31C (VS. -889C/1/-511C/-31T) haplotypes of IL-1 family genes (AOR = 0.47, 95% CI = 0.26-0.87; AOR = 0.31, 95% CI = 0.13-0.73; AOR = 3.26; 95% CI = 1.34-7.93) were associated with risk of BQ-related OSCC, as compared with those genotypes of BQ controls. On the contrary, -511T/-31T (VS. -511C/-31T) haplotype of IL-1B (AOR = 0.34, 95% CI = 0.12-0.97) and -889T/1/-511C/-31T (VS. -889C/1/-511C/-31T) haplotype of IL-1 family genes (AOR = 0.27, 95% CI = 0.10-0.71) were associated with reduced risk of BQ-related OSCC, as compared with those haplotypes of BQ-related OPL. Finally, in the stratification analysis, the combined effects of three genes (IL-8 T-251A, IL-4 intron 3 VNTR and TNFA G-308A) had a significantly increased risk of OSCC among male, older group (¡Ö50 years old), Fukienece combined with Aborigine population, never-BQ chewers, never as well as heavy smokers, or light and heavy drinkers, compared with healthy controls. The results suggested that gene-environment combined effect were associated with the risk of OSCC. In ELISA assay, plasma IL-1£] levels in BQ-related OSCC and OPL were found significantly higher than those in haplotypes for IL-1B -511T/-31C and IL-1B -511T/-31T compared with the combined effects of IL-1B -511C/-31C and IL-1B -511C/-31T. In conclusion, polymorphisms of IL-1RN, IL-4 and TNFA were associated with the decreased risk of OSCC or BQ-related OPL, but IL-8 was with increased risk of OSCC or BQ-related OPL. Furthermore, polymorphisms of IL-1B and IL-10 had contrary effect between BQ-related OSCC and OPL. Additionally, plasma levels of IL-1£] was correlated with various IL-1B C-511T/T-31C haplotypes but not with correlated with the status of disease.

Universiẗat, Diss.--Berlin, 2005.

Thesis (doctoral)--Universität, Düsseldorf, 2000.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Leishmaniasis is a spectrum of diseases caused by several species of protozoan parasites belonging to the genus, Leishmania. The disease, which is transmitted by Sandflies, ranges from self-healing cutaneous lesions to the life-threatening visceral leishmaniasis. Cutaneous leishmaniasis, caused by L. major, is the most common form of the disease. With no vaccine available for use in humans, cutaneous leishmaniasis remains a global public health problem. Since understanding the factors that regulate effective immunity to cutaneous leishmaniasis is critical for the development of an affective vaccine and treatment strategies, the overall aim of my thesis was to decipher the host and pathogen factors that regulate immunity in cutaneous leishmaniasis. Firstly, I show that parasite dose affects the expansion of different T cell subsets following L. major infection; with low dose infection inducing more CD8+ T cells while high dose infection induced more CD4+ T cells. However, although CD8+ T cells were important for optimal primary immunity following low dose infection, they where dispensable during secondary immunity. Secondly, I found that blockade of LIGHT, (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) significantly impaired DC maturation, expression of co-stimulatory molecules, and early cytokine production (IL-12 and IFN-γ) following L. major infection. Interestingly, LIGHT was completely dispensable during secondary immunity in wild type mice but was critical for effective secondary immunity in CD40 deficient mice. Thirdly,I compared disease progression and immune response in CD40 and CD40L deficient mice infected with L. major under identical experimental conditions. I found significant differences in disease progression and immune response between CD40KO and CD40L KO mice infected with virulent L. major and treated with recombinant IL-12. My data revealed a novel pathway (CD40L-Mac-1 interaction) for IL-12 production and resistance to Leishmania major. Collectively, this thesis provides novel insights into the mechanisms involved in the development and maintenance of protective immunity against cutaneous leishmaniasis, which could lead to the development of a more efficient and effective immunotherapeutic and/or vaccination strategies against the disease. / October 2015

The eis gene of M. tuberculosis is believed to play a role in the intracellular survival of this pathogen. Significantly higher levels of antibodies to Eis were detected by ELISA in the sera of patients with tuberculosis as compared to healthy controls. PBMCs from recovered TB donors were also found to demonstrate significantly higher levels of proliferation in response to stimulation with the Eis protein than PBMCs from either active TB cases or healthy controls. Neither the active TB population nor the healthy controls showed significant levels of IFN- or IL-4 secretion in response to stimulation of PBMC with Eis or ESAT-6. Far Western analysis determined that Eis interacts with a ~65 kDa protein that localizes to the cytoplasmic fraction of M. tuberculosis lysate. Real-time PCR analysis of M. tuberculosis infected U-937 macrophages showed that the eis gene is constitutively expressed during infection. Using immunofluorescence microscopy (IF), the Eis protein was detected within the cytoplasm of M. tuberculosis infected macrophages indicating that the protein was being released/secreted from the mycobacterium containing phagosomes. Western blot analysis of the cytoplasm of macrophages infected with M. tuberculosis expressing green fluorescent protein confirmed these results. Western blot analysis also detected the presence of native Eis both in the culture supernatant of infected macrophages and vesicles released from the macrophages. IF also detected the presence of Eis in uninfected macrophages. The Eis protein in the cytoplasm of M. tuberculosis infected macrophages was also found to colocalize with EEA1, an endosomal marker, indicating a possible association of the protein with early endosomes. Eis was also shown to elicit higher levels of IL-10 secretion than PPD in human monocytes. Infection of monocytes from healthy tuberculin reactors with M. tuberculosis wild type and eis mutant demonstrated that eis plays a role in modulation of IL-10/TNF- secretion in response to infection. Bioinformatic analysis of the amino acid sequence of Eis indicates that Eis is an acetyltransferase of the GCN5 related family of N-acetyltransferases. Further work is required to determine the role Eis plays in the survival of M. tuberculosis within the macrophage.