Johan R. Boelaert

Differential cytokine pattern in the spleens and livers of BALB/c mice infected with Penicillium marneffei: protective role of gamma interferon.

ABSTRACT Penicillium marneffei is an intracellular opportunistic fungus causing invasive mycosis in AIDS patients. T cells and macrophages are important for protection in vivo. However, the role of T-cell cytokines in the immune response against P. marneffei is still unknown. We studied by semiquantitative reverse transcription-PCR and biological assays the patterns of expression of Th1 and Th2 cytokines in the organs of wild-type (wt) and gamma interferon (IFN-γ) knockout (GKO) mice infected intravenously with P. marneffei conidia. At 3 × 105 conidia/mouse, a self-limiting infection developed in wt BALB/c mice, whereas all GKO mice died at day 18 postinoculation. Splenic and hepatic granulomas were present in wt mice, whereas disorganized masses of macrophages and yeast cells were detected in GKO mice. The infection resolved faster in the spleens than in the livers of wt mice and was associated with the local expression of type 1 cytokines (high levels of interleukin-12 [IL-12] and IFN-γ) but not type 2 cytokines (low levels of IL-4 and IL-10). Conversely, both type 1 and type 2 cytokines were detected in the livers of wt animals. Disregulation of the cytokine profile was seen in the spleens but not in the livers of GKO mice. The inducible nitric oxide synthase mRNA level was low and the TNF-α level was high in both spleens and livers of GKO mice compared to wt mice. These data suggest that the polarization of a protective type 1 immune response against P. marneffei is regulated at the level of individual organs and that the absence of IFN-γ is crucial for the activation of fungicidal macrophages and the development of granulomas.

Anti-HIV effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors

ObjectiveWe tested the effects of chloroquine (CQ) on glycosylation of HIV particles and in combination with protease inhibitors (PIs) on HIV replication and on P-glycoprotein (P-gp)/multidrug resistance protein-1 (MRP1). DesignCD4+ cell lines were infected with laboratory strains and peripheral blood mononuclear cells were infected with primary isolates for evaluation of the anti-HIV effects. Peripheral blood lymphocytes were evaluated for of P-gp and MRP1 functions. MethodsHIV replication was assessed by enzyme-linked immunosorbent assay. HIV glycosylation was measured by metabolic labeling of viral particles with [3H] glucosamine. Synergism was tested using isobolograms. P-gp and MRP1 functions were assayed using rhodamine 123 (Rh123) and carboxyfluorescein (CF) efflux assays, respectively. ResultsCQ alone inhibited HIV replication and glycosylation in a dose-dependent manner. In combination with indinavir (IDV), ritonavir, or saquinavir (SQV), CQ had a synergistic effect at concentrations found in plasma of subjects receiving malaria prophylaxis. CQ decreased the 50% effective concentration of IDV in primary isolates from Africa and restored the response to IDV or SQV in 3 PI-resistant isolates. CQ increased the block of Rh123 and CF efflux activity exerted by PIs. ConclusionThe inhibitory effects of CQ on HIV glycosylation are associated with synergistic effects in combination with PIs. The CQ/PI combination exerts combined inhibitory effects on P-gp and MRP1 function.

Iron metabolism and HIV infection: reciprocal interactions with potentially harmful consequences?

Humans with advanced human immunodeficiency virus (HIV) infection present some evidence suggestive of iron accumulation. Ferritin concentrations increase with HIV disease progression, and iron accumulates in several tissues. Iron excess may exert negative effects in individuals with HIV. Indeed, iron accumulation seems to be associated with shorter survival, and a number of investigations point to an iron‐mediated oxidative stress in subjects with HIV infection. The observations on humans infected with HIV are in part supported by in‐vitro findings. Indeed, in‐vitro HIV infection is associated with changes in iron metabolism, and an iron‐mediated oxidative stress is likely to contribute to viral cytopathogenicity. Furthermore, it is interesting to point out that the interaction between iron and HIV may be reciprocal, since viruses with a life‐cycle involving a DNA phase require chelatable iron for optimum replication. This combined evidence suggests that iron metabolism is an important area for virus/host interaction. These observations may be relevant to both laboratory monitoring and clinical treatment of individuals with HIV. Copyright

Iron overload exacerbates experimental meningoencephalitis by Cryptococcus neoformans

This study was aimed at investigating the effects of iron overload on the onset and outcome of cerebral cryptococcosis. To this purpose, iron dextran-administered mice were intracerebrally challenged with virulent melanogenic and avirulent non-melanogenic strains of Cryptococcus neoformans. The results shown here provide the first evidence that iron overload exacerbates the outcome of cryptococcal meningoencephalitis, irrespective of the fungal strain employed; pathogen colonization of the brain is facilitated, local cytokine response is delayed and/or prevented.

The outcome of Leishmania major experimental infection in BALB/c mice can be modulated by exogenously delivered iron.

We previously established that Leishmania promastigotes express a transferrin receptor and that iron chelators inhibit promastigote growth in vitro. Thus, we were interested in modulating the vertebrate host iron pool and to monitor whether such changes will affect the outcome of L.u2009major infection in BALBu2009/u2009c mice, inoculated in the footpad with 106 stationary phase promastigotes. Treatment of mice with desferrioxamine resulted in a slight delay of the development of cutaneous lesions. In contrast and unexpectedly, systemic iron delivery, at early time points of parasite delivery, significantly limited footpad pathology. Accordingly, parasite loads at the site of parasite delivery, the draining lymph node, liver and spleen were significantly reduced in iron‐loaded mice. Importantly, the protective effect of iron delivery correlated with the presence, at the site of inoculation, of lower levels of IL‐4 and IL‐10 transcripts while both IFN‐γ and inducible nitric oxide synthase transcripts were at higher levels. The presence of more type 1 cytokine transcripts was further supported by the increased levels of IgG2a in their sera. These data strongly suggest that susceptibility to L.u2009major as assessed in the footpad model is modifiable by interventions that alter the iron status of the host at early time points of parasite delivery.

Differential effects of iron load on basal and interferon-gamma plus lipopolysaccharide enhance anticryptococcal activity by the murine microglial cell line BV-2.

Here we evaluated the influence of intracellular iron levels on the constitutive and interferon (IFN)-gamma plus lipopolysaccharide (LPS) enhanced anticryptococcal activity by the murine microglial cell line BV-2. We demonstrated that iron loading via ferric nitrilotriacetate (FeNTA) resulted in a significant increase in the constitutive levels of anticryptococcal activity, while the enhancing effects by IFN-gamma plus LPS were prevented. Accordingly, a major increase was observed in the levels of thiobarbituric reactive substance (TBARS) produced upon iron loading under basal conditions, whereas IFN-gamma plus LPS treatment, that per se did not affect TBARS production, prevented by about 50% the enhancement otherwise occurring in response to iron loading. The potential involvement of multiple effector system and their relation to intracellular iron will be discussed.

Nramp1 gene affects selective early steps in macrophage-mediated anti-cryptococcal defense

Abstract Cryptococcus neoformans is an opportunistic fungus responsible for severe and often recurrent meningoencephalitis in immunodepressed patients. Initial evidence suggests that C. neoformans is a facultative intracellular pathogen; however, the strategies by which C. neoformans undergoes survival and eventually proliferation have not been elucidated. We investigated the role of Nramp1 gene in macrophage-mediated anti-cryptococcal defense. Using cell lines expressing the functional, mutated or knockout gene, it was established that Nramp1 (1) is not involved in the phagocytic event, (2) influences anti-cryptococcal activity in the early steps but not at later times, and (3) is unrelated to the biomolecular pathways through which C. neoformans impairs macrophage secretory response. Although the functional role of Nramp1 is still far from being elucidated, the present data add insight into its involvement in macrophage-mediated antimicrobial defense, particularly in the initial steps allowing C. neoformans growth inhibition.