The aim of this study was to assess the role of TLR2, TLR4 and MyD88 accessory molecule in the effector and secretory response of macrophages to viable microbial agents. Using TLR-deleted macrophage cell lines generated from the bone marrow of genetically engineered mice (TLR4 gene-deficient, MyD88- and TLR2-knockout mice) and wild-type control mice, we found that TLR2-deleted macrophages exhibit increased ability to contain Candida albicans infection compared to TLR2+/+ counterpart. In contrast, both MyD88-/- and TLR4-/- macrophages retain levels of functional activity comparable to that of the respective wild-type MyD88+/+ and TLR4+/+ controls. The difference in anticandidal effector functions observed between TLR2-/- and TLR2+/+ macrophages is abrogated upon opsonization of the fungal target and interestingly is not observed when using other microbial targets, such as Streptococcus pneumoniae and Helicobacter pylori. When tested for secretory response to C. albicans, TLR2-deleted macrophages show a pattern of cytokine production similar to that of TLR2+/+ controls. Finally, flow cytometry analysis reveals that TLR2-deleted macrophages express only TLR4, while, as expected, TLR2+/+ macrophages are both TLR2 and TLR4 positive; in no cases, modulation of such markers occurs in macrophages exposed to C. albicans infection. In conclusion, these data indicate that TLR2 and TLR4 have different biological relevance, in which TLR2 but not TLR4, is involved in the accomplishment of macrophage-mediated anticandidal activity, while the secretory response to C. albicans appears to be TLR4 but not TLR2-dependent.

Macrophages are important antimicrobial effectors, whose efficacy is greatly enhanced by interferon-gamma (IFNgamma). We recently engineered a mouse macrophage cell line to express the IFNgamma gene in a inducible manner. Such macrophages, Mphi10, include a construct containing the IFNgamma gene under the control of the synthetic promoter HRE3x-Tk. Picolinic acid (PA) is a catabolite of tryptophan, known to exert costimulatory activities on macrophages and expected to act on transcriptional elements within HRE3x-Tk promoter. Since evidence exists on the efficacy of engineered macrophages as carriers of therapeutic genes against tumors, we tested Mphi10, under basal conditions and following exposure to PA, as IFNgamma-producing cells in in vitro models of fungal infection. We found that Mphi10 constitutively exhibited anticryptococcal and anticandidal activity, low but detectable levels of IFNgamma mRNA and undetectable levels of nitric oxide synthase (iNOS) transcripts. Treatment with PA caused time-dependent enhancement of antifungal activity. The phenomenon was associated with the induction of both IFNgamma and iNOS gene expression and was followed by IFNgamma and NO production. The effect of the Mphi10-produced IFNgamma on other cells was also investigated by a transwell co-culture system. A major enhancement of phagocytosis and antifungal activity was observed in BV2 microglial cells that had been co-cultured with Mphi10. Such an increase was only evident when Mphi10 had been pretreated with PA and was abrogated by concomitant addition of anti-IFNgamma antibodies. In conclusion, we show that Mphi10 respond to PA with the production of IFNgamma, which retains the ability to induce antifungal activity in the producing macrophages as well as in other macrophage populations. The potential use of Mphi10 as vectors for therapeutic genes in infectious diseases is discussed.

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 Nrampl 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.

No abstract

INTRODUZIONEGranulicatella adiacens è un batterio del gruppo dei NVS (nutritionally variant streptococci) e può essere responsabile di infezioni gravi quali infezioni protesiche ed endocarditi (3). L'interazione del microrganismo con la fibronectina pare giocare un ruolo importante nell'instaurarsi dell'endocardite infettiva (4). Un precoce intervento chirurgico appare opportuno in pazienti con insufficienza cardiaca causata da distruzione valvolare. Le endocarditi da NVS non sono molto frequenti, sono tuttavia associate ad elevati tassi di mortalità, complicanze ed insuccesso terapeutico. Tale quadro è in parte conseguenza della difficile identificazione microbiologica di questi microrganismi (1, 2). Il caso clinico pervenuto alla nostra osservazione riguarda una donna di 81 anni, portatrice di protesi valvolare biologica ed affetta da insufficienza mitralica, ricoverata in un reparto internistico a causa di febbre recidivante. L'ecografia trans-esofagea mostrava la presenza di vegetazioni sui lembi mitralici della paziente. Sono stati raccolti, da accessi venosi distinti e in rapida successione, due set di emocolture per la ricerca di microrganismi aerobi ed anaerobi. MATERIALI E METODII set di emocolture erano entrambi composti dal flacone per ricerca di aerobi associato al flacone per la ricerca di anaerobi (Bact/Alert FA e Bact/Alert FN, bioMérieux). I sistemi utilizzati per l´identificazione sono Vitek2 e Api Rapid 32 Strep. (bioMérieux). Isolamento colturale di Granulicatella adiacens da emocolture in paziente portatore di protesi valvolare SUMMARYThe clinical case studied concerns a woman 81 years old, with a history of prosthetic valve and mitral insufficiency, admitted to internal medicine ward of NOCSAE hospital as a result of a recurrent fever. Due to the suspicion of endocarditis and with the aim to identify the presence of aerobic/anaerobic microorganisms, two set of blood cultures collected within 24 hours were sent to the Laboratory of microbiology. All the bottles were incubated into the Bact-Alert 3D System (bioMérieux). After an 19 hours incubation time, the samples were identified as positive by the automated system; consequently they cultured on a blood agar and selective media, according to our laboratory operational protocol.