Trends and challenges in tuberculosis in a medium-sized southern European setting.

Trends in tuberculosis (TB) admissions over 40 years at the Infectious Diseases Clinic of Perugia University Hospital, Perugia, Italy, show that in the last decade non-Italian TB case admissions outweighed those of Italians, with a large number of cases from Eastern Europe (25.2%) and Africa (23.4%). Non-Italians tended to be younger and were generally new pulmonary TB cases, and drug resistance was also more common. Overall, the number of multidrug-resistant cases increased. Only one case occurred in a native-born Italian, and five of seven cases had newly diagnosed TB. In low TB incidence settings such as Perugia, Italy, TB prevention and control programmes for the foreign-born need to be reinforced.

Retroperitoneal abscess: an uncommon localization of tubercular infection.

We describe a rare case of a 29-year-old immunocompetent Nigerian male affected by an abdominal abscess due to Mycobacterium tuberculosis infection. Diagnosis was achieved with cultures from surgical drainage. No pulmonary, renal, or gastrointestinal involvement was identified. The patient was successfully treated with standard four-drug antitubercular therapy.

Staphylococcus aureus: study of genomic similarity of strains isolated in veterinary pathology using amplified fragment length polymorphism (AFLP).

Staphylococcus aureus is a widespread pathogen causing infections in different animal species. The extensive use of antibiotics, particularly methicillin, causes the rise of antibiotic-resistant strains (MRSA). In order to verify the epidemiology and genetic relatedness among MRSA and sensible strains (MSSA), an accurate fingerprinting technique, the amplified fragment length polymorphism (AFLP), was carried out. The isolates were cultured, subdivided on MRSA and MSSA and submitted for the genomic DNA extraction that was utilized for AFLP. The data were analysed for genetic similarity using the Dice coefficient. The results of genomic analysis among MRSA and MSSA and within them revealed that the major component of variation was due to variation within strains (82.12%), while variance among strains was lower (17.88%). The low level of genomic similarity found among S. aureus strains implies high level of genetic diversity. Different similarity was found as well in all strains independently of the source.

Differential effector and secretory functions of microglial cell lines derived from BCG-resistant and -susceptible congenic mouse strains.

Using congenic strains of mice susceptible (bcg(s)) or resistant (bcg(r)) to BCG, murine microglial cell lines, RR4.R (BCG-resistant) and RR8.S (BCG-susceptible), were established in vitro. Comparative studies revealed that, although phagocytic to a similar extent, RR4.R cells were more active than RR8.S cells in terms of antimycobacterial activity. Interestingly, cells of resistant genotype secreted more nitric oxide, TNF-alpha and IL-12, but less IL-6, than susceptible cells, when stimulated with IFN-gamma alone or in combination with lipopolysaccharide. Nevertheless, no significant differences were observed between the two cell lines in terms of IL-1 beta or IL-10 secretion, or on assessment of cytokine production following exposure to a massive dose of lipopolysaccharide. Overall, these data provide the first evidence that resistant/susceptible genotype influences antimycobacterial activity, NO and cytokine production in microglial cells, the prototype of cerebral macrophages.

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.

A new assay for the determination of low-molecular-weight nitrosothiols (nitrosoglutathione), NO, and nitrites by using a specific and sensitive solid-state amperometric gas sensor.

Nitric oxide (NO) is generated in biological systems and plays an important role as a bioregulatory molecule. Its ability to bind hemoglobin and myoglobin is well known. Moreover, it may lose an electron forming the nitrosyl group involved in the formation of S-nitrosothiols. The main problem in analyzing NO is its extreme reactivity. We have tackled this task by using an amperometric sensor to determine free NO, S-nitrosothiols (such as S-nitrosoglutathione), and nitrite in cell-free systems and murine microglial cell cultures. The determination of nitrosothiols is of biochemical relevance and a difficult task particularly at low concentration values. In this article we describe a new method based on the reductive cleavage of the S-NO bond by cuprous ions followed by a solid-state amperometric determination. The system described by us is sensitive, rapid, does not require previous purification steps, is easy to perform, and is inexpensive. For this reason, we think that it may represent an important analytical improvement. It has been suggested that nitrosothiols may exert biological activity by acting as a reservoir of NO. We tested the production of nitrite and of RSNO in stimulated, cultured murine microglial cells and we have shown that nitrite accumulates in these conditions. GSNO also accumulates, provided that GSH is present in the medium.

A low virulent strain of Candida albicans enhances brain anticryptococcal defenses: characterization of the local immune reaction by RT-PCR and histochemical analysis.

Here we studied the involvement of PCA-2, a low-virulent strain of Candida albicans known to act as a potent stimulating agent in the development of cryptococcal meningoencephalitis. To this purpose, mice received saline or PCA-2 intracerebrally 7 days before lethal local challenge with Cryptococcus neoformans. We found that, following C. neoformans challenge, PCA-2-treated but not saline-treated mice exhibited (a) delayed brain colonization, (b) enhanced median survival times, (c) massive local immune reaction consisting of abundant astrocytes, microglial and inflammatory cells, and (d) a peculiar trend of cytokine gene expression, including high steady-state levels of interleukin (IL)-1 beta and tumor necrosis factor alpha transcripts, fluctuating levels of interferon gamma and inducible nitric oxide synthase mRNA and lately detectable IL-6 gene expression. PCA-2-mediated immunostimulating properties were partially impaired by aminoguanidine or pentoxifylline treatment, further strengthening the conclusion that soluble mediators, including proinflammatory cytokines and nitric oxide, are important defense elements against cryptococcal meningoencephalitis.

Enhanced resistance to Cryptococcus neoformans infection induced by chloroquine in a murine model of meningoencephalitis.

Although the pathogenesis of cerebral cryptococcosis is poorly understood, local immune cells, such as microglia and astrocytes, likely play a critical role in containing infection. Chloroquine (CQ) is a weak base that accumulates within acidic vacuoles and increases their pH. Consequently, proteolytic activity of lysosomal enzymes and intracellular iron release/availability are impaired, resulting in decreased availability of nutrients crucial to microorganism survival and growth in the host. We found that CQ enhances BV2 microglial-cell-mediated anticryptococcal activity in vitro. The phenomenon is (i) evident when both unopsonized and opsonized microorganisms are used and (ii) mimicked by NH4Cl, another weak base, and by bafilomycin A1, an inhibitor of vacuolar-type H+-ATPases. In vivo, intracerebral administration of CQ before lethal local challenge with Cryptococcus neoformans results in a significant augmentation of median survival time and a marked reduction of yeast growth in the brain and is associated with the enhancement of local interleukin 1beta (IL-1beta) and IL-6 mRNA transcripts. Overall, these results provide the first evidence that CQ enhances anticryptococcal host defenses.

Potent antifungal effects of a new derivative of partricin A in a murine model of cerebral cryptococcosis.

A new member of the polyene family, N-dimethylaminoacetyl-partricin A 2-dimethylaminoethylamide diaspartate (SPA), was investigated and was found to be more effective than amphotericin B (i) in vivo by enhancing mouse resistance to cryptococcal meningoencephalitis and (ii) in vitro by potentiating the anticryptococcal activity of murine microglial cells.

Iron regulates microglial cell-mediated secretory and effector functions.

Iron homeostasis and macrophage physiology are tightly intertwined. In the present study, we evaluated the influence of iron loading on the constitutive and interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-induced functional and secretory properties of microglial cells, using the in vitro established murine cell line BV-2. We demonstrate that iron augments the basal and IFN-gamma plus LPS-enhanced anti-Candida albicans activity exerted by BV-2 cells and that the phenomenon occurs with no enhancement of phagocytic activity. Furthermore, when the secretory properties of IFN-gamma plus LPS-treated BV-2 cells were assessed, we found that tumor necrosis factor remains unchanged while nitric oxide production is significantly reduced in iron-loaded cells. The addition of the iron chelator deferiprone (L1) reverts the effects of iron on BV-2 functional and secretory properties. These data suggest that iron differently affects secretory and effector functions of BV-2 microglial cells, thus implying that iron interferes with murine microglial cell physiology.

Biomolecular events involved in the establishment of brain anticandidal resistance.

Using a murine model, we have demonstrated the establishment of cerebral resistance to local lethal challenge with Candida albicans strain CA-6, by previous intracerebral (i.c.) infection with the low-virulent strain PCA-2. Here we show that i.c. infection with PCA-2 is effective in drastically reducing brain colonization following secondary infection with CA-6. As assessed by colony forming unit assay and histopathological analysis, microbial counts are impaired, granuloma formation and hyphal growth are also reduced in brains of PCA-2- and CA-6-infected mice with respect to CA-6-challenged mice. Furthermore, using PCR studies, we found that, while PCA-2 (i.e. healing infection) induces transient cytokine gene expression in the mouse brain, CA-6 lethal challenge results in long-lasting (until mouse death) high levels of all cytokine gene transcripts assessed. Finally brains from mice that will resist CA-6 challenge, because of previous infection with PCA-2, also exhibit a transient induction of all cytokine genes. Only IL-1 beta remains highly expressed at all time- points tested. Overall, these results provide evidence that healing and non-healing C. albicans i.c. infections differ in the immune reaction(s) locally evoked, at least in terms of cytokine gene expression, strongly suggesting cytokine involvement in the establishment of brain anticandidal resistance.

Influence of the Bcg locus on macrophage response to the dimorphic fungus Candida albicans.

The Bcg/Ity/Lsh gene (candidate Nramp) controls natural resistance to several parasites, such as Mycobacterium bovis, Leishmania donovani, and Salmonella typhimurium. Using two macrophage (M phi) cell lines (B10R and B10S) derived from mouse strains congenic at Bcg, we found that M phi s from resistant mice (B10R M phi s) act more effectively against the two morphogenetic forms of the dimorphic fungus Candida albicans compared with M phi s from susceptible mice (B10S M phi s). Moreover, when assessed for tumor necrosis factor secretion in response to the hyphal form of C. albicans, B10R M phi s are significantly more effective at expressing this secretory function than are B10S M phi s, closely resembling the trend of response to lipopolysaccharide. Overall, these results provide insight into the influence of the Bcg locus on the M phi response to C. albicans.

Differential susceptibility of yeast and hyphal forms of Candida albicans to macrophage-derived nitrogen-containing compounds.

Candida albicans is a dimorphic fungus capable of transition from the yeast form (Y-Candida) to the hyphal form (H-Candida). Both Y-Candida and H-Candida are known to be growth inhibited by murine macrophages (M phi) in vitro. In the present report, we demonstrate that M phi exposed to interferon gamma (IFN-gamma) plus lipopolysaccharide (LPS) show enhanced anti-Y-Candida and anti-H-Candida activities. To further investigate the phenomenon, Y-Candida and H-Candida were assessed for susceptibilities to M phi-derived supernatants. Only the growth of H-Candida, and not that of Y-Candida, is impaired by cell-free supernatants from M phi treated with IFN-gamma plus LPS. In contrast, no H-Candida growth inhibition occurs when supernatants from M phi exposed to IFN-gamma plus LPS in the presence of NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthesis, are employed. Finally, supernatants from M phi incubated with sodium nitroprusside, an NO-generating agent, also show anti-H-Candida activity. In conclusion, these results indicate that H-Candida but not Y-Candida is susceptible to extracellular antifungal mechanisms employed by M phi, which likely involve stable nitrogen-containing compounds.

Role of nitric oxide and melanogenesis in the accomplishment of anticryptococcal activity by the BV-2 microglial cell line.

In the present paper, we investigated the involvement of cryptococcal melanogenesis and macrophage nitric oxide (NO) production in the accomplishment of anticryptococcal activity by microglial effector cells, using the murine cell line BV-2. We demonstrate that the constitutive levels of anticryptococcal activity exerted by BV-2 cells is significantly enhanced upon interferon gamma plus lipopolysaccharide treatment. The phenomenon, which occurs with no enhancement of phagocytic activity, is associated with the production of high levels of NO and is abolished by addition of NG-monomethyl-L-arginine. Comparable patterns of results are observed employing either unopsonized or opsonized microbial targets, the latter microorganisms being markedly more susceptible to BV-2 cell antimicrobial activity. Furthermore, melanization of Cryptococcus neoformans significantly reduces its susceptibility to BV-2 antimicrobial activity, regardless of the fact that activated macrophages or opsonized microorganisms have been employed. In conclusion, our results provide evidence that NO-dependent events are involved in the fulfillment of anticryptococcal activity by activated microglial cells and that fungal melanization is a precious escamotage through which C. neoformans overcomes host defenses.

Biomolecular events involved in anticryptococcal resistance in the brain.

We have recently shown that intracerebral (i.c.) administration of heat-killed Cryptococcus neoformans (HCN) enhances mouse resistance to a subsequent local challenge with lethal doses of viable yeast cells. Here we show that i.c. administration of HCN is also effective in significantly delaying brain colonization of mice intravenously infected with viable C. neoformans. PCR analysis revealed that interleukin 6 (IL-6) and IL-1 beta gene expression occurs in brain of HCN-treated mice but not in brains of saline-treated controls. In contrast, no differences are observed in terms of tumor necrosis factor alpha and IL-1 alpha gene transcripts, which are slightly and highly detectable, respectively, in saline-treated mice and which remain such also following HCN treatment. Furthermore, i.c. administration of exogenous IL-6 or IL-1 beta, but not tumor necrosis factor alpha, before local challenge with viable C. neoformans results in significantly reduced microbial counts in the brain and blood and in increased mouse survival. Taken together, these observations provide initial evidence that brain anticryptococcal resistance involves elicitation of a local cytokine response, involving primarily IL-6 and IL-1 beta.

Differential susceptibility of yeast and hyphal forms of Candida albicans to proteolytic activity of macrophages.

The dimorphic transition of Candida albicans from the yeast (Y-Candida) to the hyphal (H-Candida) form is a complex event whose relevance in fungal pathogenicity is still poorly understood. Using a cloned macrophage (M phi) cell line (ANA-1), we have previously shown that a M phi can discriminate between the two fungal forms, eliciting different secretory responses. In the present study, we investigated the susceptibility of Y-Candida and H-Candida to M phi proteolytic activity. In particular, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) techniques were employed to analyze the patterns of lyticase proteinaceous extracts from cell walls of Y-Candida and H-Candida which had been unexposed or exposed to ANA-1 M phis for 3 h. Silver staining allowed detection of a complex protein pattern in both forms of C. albicans, qualitatively and quantitatively differing from each other, mainly at molecular masses below 106 kDa. Western blot staining with anti-C. albicans mannan antibodies and convalescent-phase sera of mice previously infected systemically or intracerebrally with C. albicans showed that, after contact with M phis, Y-Candida but not H-Candida proteinaceous cell wall components are profoundly modified, with substantial reduction and/or disappearance of many bands. Our experimental approach provides initial insights into the differential susceptibility of Y-Candida and H-Candida to the proteolytic activity of M phis.

Different events involved in the induction of macrophage tumor necrosis factor by Candida albicans and lipopolysaccharide.

Using an in vitro experimental model, we have recently demonstrated that Candida albicans in its hyphal form (H-Candida), similarly to lipopolysaccharide (LPS), enhances tumor necrosis factor (TNF) secretory response in the cloned macrophage (M phi) population ANA-1. Here we show that H-Candida and LPS each differ in their requirements for intact protein kinase functions, susceptibility to 0.4-microns micropore-size membranes, and sensitivity to polymyxin B. These results, together with the synergistic effect occurring between H-Candida and LPS in inducing TNF response, indicate the existence of different receptor(s) and/or signal-transduction pathway(s) through which the two stimuli act.

Comparative studies on functional and secretory properties of macrophage cell lines derived from different anatomical sites.

In the present study, we compared four macrophage (M phi) cell lines from different anatomical origins for functional and secretory activities against the two morphogenetic forms of the fungus Candida albicans. We show that all the cell lines actively phagocytize the yeast and exert antimicrobial activity against both forms of Candida, although M phi of microglial origin are the most effective. When assessed for secretory properties, microglial M phi exhibit a peculiar pattern with respect to other M phi populations under either basal or stimulated conditions. In particular, only microglial M phi fail to respond to the hyphal form of the fungus (H-Candida), which instead acts as a potent tumor necrosis factor inducer in the other M phi cell lines. When exposed to H-Candida, microglial M phi are indistinguishable from other M phi in their ability to modulate specific surface adhesion molecules. In addition to strengthening the knowledge on functional heterogeneity among M phi, our data provide evidence on the peculiar behavior of microglial M phi. To what extent M phi heterogeneity may be related to tissue homeostasis is discussed.

Anticryptococcal resistance in the mouse brain: beneficial effects of local administration of heat-inactivated yeast cells.

Using a murine model, we have previously shown that brain resistance to local infection with opportunistic fungi is affected by manipulation of the host myelomonocytic compartment. Here, we demonstrate that intracerebral administration of heat-inactivated Cryptococcus neoformans (H-CN) yeast cells results in a consistent enhancement of mouse survival to subsequent local challenge with lethal doses of C. neoformans. The phenomenon, more pronounced upon double H-CN treatment, is associated with (i) massive local inflammatory response, (ii) reduced growth of the fungus within the brain, and (iii) induction of delayed-type hypersensitivity. Moreover, H-CN treatment confers protection against local heterologous challenges. Our data provide initial evidence that intracerebral administration of H-CN results in the establishment of aspecific and specific immune responses; the mechanisms of elicitation and relative contributions to host antimicrobial resistance remains to be elucidated.