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1.  Transfection of Trypanosoma cruzi with Host CD40 Ligand Results in Improved Control of Parasite Infection  
Infection and Immunity  2005;73(10):6552-6561.
We have previously shown that infection by Trypanosoma cruzi, a parasitic protozoan, is reduced by injection of CD40 ligand (CD40L)-transfected 3T3 fibroblasts (D. Chaussabel, F. Jacobs, J. de Jonge, M. de Veerman, Y. Carlier, K. Thielemans, M. Goldman, and B. Vray, Infect. Immun. 67:1929-1934, 1999). This prompted us to transfect T. cruzi with the murine CD40L gene and to study the consequences of this transfection on the course of infection. For this, epimastigotes (Y strain) were electroporated with the pTEX vector alone or the pTEX-CD40L construct, and transfected cells were selected for their resistance to Geneticin G418. Then strain Y-, pTEX-, and pTEX-CD40L-transfected epimastigotes were transformed by metacyclogenesis into mammalian infective forms called Y, YpTEX, and YpTEX-CD40L trypomastigotes. Transfection of the CD40L gene and expression of the CD40L protein were assessed by reverse transcription-PCR and Western blot analysis. The three strains of parasites were infective in vitro for mouse peritoneal macrophages. When organisms were inoculated into mice, a very low level of parasitemia and no mortality were seen with the YpTEX-CD40L strain compared to the Y and YpTEX strains. Furthermore, the proliferative capacity and the secretion of gamma interferon were both preserved in spleen cells (SCs) from YpTEX-CD40L-infected mice but not with SCs from Y- and YpTEX-infected mice. These results suggest that the CD40L produced by transfected T. cruzi is involved in the modulation of an antiparasite immune response. Moreover, mice surviving YpTEX-CD40L infection resisted a challenge infection with the wild-type strain. Taken together, our data demonstrate the feasibility of generating a T. cruzi strain expressing a bioactive host costimulatory molecule that counteracts the immunodeficiency induced by the parasite during infection and enhances protective immunity against a challenge infection.
PMCID: PMC1230987  PMID: 16177330
2.  Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity 
Journal of Clinical Microbiology  2001;39(4):1396-1401.
Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.
PMCID: PMC87945  PMID: 11283062
3.  Biochemical and Genetic Evidence for Phospholipase C Activity in Mycobacterium ulcerans 
Infection and Immunity  2000;68(5):2995-2997.
This study reports the existence of phospholipase C and D enzymatic activities in Mycobacterium ulcerans cultures as determined by use of thin-layer chromatography to detect diglycerides in hydrolysates of radiolabeled phosphatidylcholine. M. ulcerans DNA sequences homologous to the genes encoding phospholipase C in Mycobacterium tuberculosis and Pseudomonas aeruginosa were identified by sequence analysis and DNA-DNA hybridization. Whether or not the phospholipase C and D enzymes of M. ulcerans plays a role in the pathogenesis of the disease needs further investigation.
PMCID: PMC97516  PMID: 10769001
4.  Trypanosoma cruzi Infects Human Dendritic Cells and Prevents Their Maturation: Inhibition of Cytokines, HLA-DR, And Costimulatory Molecules 
Infection and Immunity  1999;67(8):4033-4040.
Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas’ disease. Despite the many immune system disorders recognized in this infection and the crucial role played by dendritic cells (DC) in acquired immune responses, it was not known whether these cells could be infected by T. cruzi trypomastigotes and the consequences of such an infection on their immune functions. We now provide evidence that human monocyte-derived DC can be infected by T. cruzi and can support its intracellular multiplication. Interestingly, this infection has functional consequences on immature DC and on their maturation induced by lipopolysaccharide (LPS). First, after T. cruzi infection, the basal synthesis of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) was impaired. Furthermore, the process of maturation of DC induced by LPS was drastically affected by T. cruzi infection. Indeed, secretion of cytokines such as IL-12, TNF-α, and IL-6, which are released normally at high levels by LPS-activated DC, as well as the up-regulation of HLA-DR and CD40 molecules, was significantly reduced after this infection. The same effects could be induced by T. cruzi-conditioned medium, indicating that at least these inhibitory effects were mediated by soluble factors released by T. cruzi. Taken together, these results provide new insights into a novel efficient mechanism, directly involving the alteration of DC function, which might be used by T. cruzi to escape the host immune responses in Chagas’ disease and thus might favor persistent infection.
PMCID: PMC96695  PMID: 10417171
5.  CD40 Ligation Prevents Trypanosoma cruzi Infection through Interleukin-12 Upregulation 
Infection and Immunity  1999;67(4):1929-1934.
Because of the critical role of the CD40-CD40 ligand (CD40L) pathway in the induction and effector phases of immune responses, we investigated the effects of CD40 ligation on the control of Trypanosoma cruzi infection. First, we observed that supernatants of murine spleen cells stimulated by CD40L-transfected 3T3 fibroblasts (3T3-CD40L transfectants) prevent the infection of mouse peritoneal macrophages (MPM) by T. cruzi. This phenomenon depends on de novo production of nitric oxide (NO) as it is prevented by the addition of N-nitro-l-arginine methyl ester, a NO synthase inhibitor. NO production requires interleukin (IL)-12-mediated gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) synthesis as demonstrated by inhibition experiments using neutralizing anti-IL-12, anti-IFN-γ, and anti-TNF-α monoclonal antibodies (MAb). We found that an activating anti-CD40 MAb also directly stimulates IFN-γ-activated MPM to produce NO and thereby to control T. cruzi infection. To determine the in vivo relevance of these in vitro findings, mice were injected with 3T3-CD40L transfectants or 3T3 control fibroblasts at the time of T. cruzi inoculation. We observed that in vivo CD40 ligation dramatically reduced both parasitemia and the mortality rate of T. cruzi-infected mice. A reduced parasitemia was still observed when the injection of 3T3-CD40L transfectants was delayed 8 days postinfection. It was abolished by injection of anti-IL-12 MAb. Taken together, these data establish that CD40 ligation facilitates the control of T. cruzi infection through a cascade involving IL-12, IFN-γ, and NO.
PMCID: PMC96548  PMID: 10085038
6.  Effects of Granulocyte-Macrophage Colony-Stimulating Factor and Tumor Necrosis Factor Alpha on Trypanosoma cruzi Trypomastigotes 
Infection and Immunity  1998;66(6):2722-2727.
We have previously shown that the addition of exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) to nonactivated mouse peritoneal macrophages (MPM) limits Trypanosoma cruzi infections in vitro (E. Olivares Fontt and B. Vray, Parasite Immunol. 17:135–141, 1995). Lower levels of infection were correlated with a higher level of production of tumor necrosis factor alpha (TNF-α) in the absence of nitric oxide (NO) release. These data suggested that GM-CSF and/or TNF-α might have a direct parasitocidal effect on T. cruzi trypomastigotes, independently of NO release. To address this question, T. cruzi trypomastigotes were treated with recombinant murine GM-CSF (rmGM-CSF), recombinant murine TNF-α (rmTNF-α), or both cytokines in a cell-free system. Treatment with rmGM-CSF but not rmTNF-α caused morphological changes in the parasites, and most became spherical after 7 h of incubation. Both cytokines exerted a cytolytic activity on the trypomastigotes, yet the trypanolytic activity of rmTNF-α was more effective than that of rmGM-CSF. Viable rmGM-CSF- and rmTNF-α-treated parasites were less able to infect MPM than untreated parasites, and this reduction in infectivity was greatest for rmGM-CSF. Treatments with both cytokines resulted in more lysis and almost complete inhibition of infection. The direct parasitocidal activity of rmTNF-α was inhibited by carbohydrates and monoclonal antibodies specific for the lectin-like domain of TNF-α. Collectively, these results suggest that cytokines such as GM-CSF and TNF-α may directly control the level of T. cruzi trypomastigotes at least in vitro and so could determine the outcome of infection in vivo.
PMCID: PMC108261  PMID: 9596739

Results 1-6 (6)