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Hyaluronic acid (HA) has several clinical applications (aesthetic surgery, dermatology, orthopaedics and ophtalmology). Following recent evidence, suggesting antimicrobial and antiviral properties for HA, we investigated its effects on 15 ATCC strains, representative of clinically relevant bacterial and fungal species. The in vitro system employed allowed to assess optical density of broth cultures as a measure of microbial load in a time-dependent manner. The results showed that different microbial species and, sometimes, different strains belonging to the same species, are differently affected by HA. In particular, staphylococci, enterococci, Streptococcus mutans, two Escherichia coli strains, Pseudomonas aeruginosa, Candida glabrata and C. parapsilosis displayed a HA dose-dependent growth inhibition; no HA effects were detected in E. coli ATCC 13768 and C. albicans; S. sanguinis was favoured by the highest HA dose. Therefore, the influence of HA on bacteria and fungi warrants further studies aimed at better establishing its relevance in clinical applications.

Recently, we showed that herpes simplex virus 1 (HSV-1)-infected monocytes have altered antifungal defences, in particular they show augmented phagocytosis of Candida albicans followed by a failure of the intracellular killing of the ingested fungi. On the basis of these functional data, comparative studies were carried out on the gene expression profile of cells infected with HSV-1 and/or C. albicans in order to investigate the molecular mechanisms underlying such virus-induced dysfunction. Affymetrix GeneChip technology was used to evaluate the cell transcription pattern, focusing on genes involved in phagocytosis, fungal adhesion, antimicrobial activity and apoptosis. The results indicated there was: (a) prevalent inhibition of opsonin-mediated phagocytosis, (b) upregulation of several pathways of antibody- and complement-independent phagocytosis, (c) inhibition of macrophage activation, (d) marked dysregulation of oxidative burst, (e) induction of apoptosis.

It has been recently demonstrated that Candida albicans isolates with distinct genomic backgrounds (namely, b and c genotypes) express different susceptibility to antifungal activity by human monocytes in vitro. We show here that, although comparable in their ability to undergo dimorphic transition and in susceptibility to phagocytosis by microglial cells, the b and c isolates show striking differences in terms of intracellular survival. Only the c genotype resists indeed to intracellular killing and eventually replicates inside microglial cells, that in turn respond to fungal infection, preferentially towards the c genotype, with nuclear factor-κB (NF-κB) activation and increased Mip1 α production. These data indicate that C. albicans -microglial cell interaction is strictly dependent upon fungal genotype, strengthening the potential significance of genotyping as prognostic parameter in clinical infections by C. albicans .

The aim of this study was to examine three serial isolates of Cryptococcus neoformans from a patient with AIDS for genotypical and phenotypical characteristics. The isolates were obtained during a first episode of cryptococcosis (simultaneous sampling of blood and cerebrospinal fluid) and after a relapse 3 years later (sampling of cerebrospinal fluid). Pulsed-field gel electrophoresis and random amplification of polymorphic DNA revealed that the blood isolate 1525 (first episode) was different from the two cerebrospinal fluid isolates (1526, first episode; 1782, relapse), yet the cerebrospinal fluid isolates were indistinguishable from each other regardless of the analysis performed. Phenotypical studies showed that isolate 1782 had significantly improved resistance to phagocytosis and killing by monocytes and polymorphonuclear cells and an altered efficacy in evoking cytokine response (augmentation of tumour necrosis factor-alpha, interleukin [IL]-1beta, IL-10, and interferon-gamma, decrease of IL-12). Interestingly, capsule size and antifungal drug resistance remained unchanged, while production of phospholipase and protease was consistently enhanced in the 1782 isolate with respect to the 1525 and 1526 isolates. In conclusion, in serial Cryptococcus neoformans isolates from a patient with AIDS, phenotypical changes but not molecular changes were documented, thus supporting the role of microevolution as a pathogenetic mechanism(s) for persistence/reactivation of fungal organisms.

The hyphal wall protein 1 (HWP1) gene of Candida albicans encodes for a fungal cell wall protein, required for hyphal development and yeast adhesion to epithelial cells; yet, its role in pathogenesis remains largely unknown. In the present study, we analyzed two C. albicans laboratory strains, the DAY286 (HWP1/HWP1) and the null mutant FJS24 (hwp1/hwp1) and six clinical isolates [3 harbouring the homozygous HWP1 gene (HWP1/HWP1) and 3 the heterologous gene (HWP1/hwp1)]. Biofilm production, fungal HWP1 mRNA levels and ultrastructural morphology were investigated; also, the susceptibility of these strains to microglial cells was evaluated, in terms of fungal damage and immune cell-mediated secretory response. When comparing the two laboratory strains, biofilm was produced to a similar extent independently on the genetic background, while the susceptibility to microglial cell-mediated damage was higher in the hwp1/hwp1 mutant than in the HWP1/HWP1 counterpart. Also, transmission electron microscopy revealed differences between the two in terms of abundance in surface adhesin-like structures, fungal cell wall shape and intracellular granules. When comparing the clinical isolates grouped according to their HWP1 genotype, reduced biofilm production and increased susceptibility to microglial cell-mediated damage occurred in the HWP1/hwp1 isolates with respect to the HWP1/HWP1 counterparts; furthermore, upon exposure to microglial cells, the HWP1/HWP1 isolates, but not the HWP1/hwp1 counterpart, showed enhanced HWP1 mRNA levels. Finally, both laboratory and clinical isolates exhibited reduced ability to stimulate TNFα and nitric oxide production by microglial cells in the case of heterozygous or null mutant HWP1 genotype. Overall, these data indicate that C. albicans HWP1 genotype influences pathogen morphological structure as well as its interaction with microglial cells, while fungal biofilm production results unaffected, thus arguing on its role as virulence factor that directly affects host mediated defences.

Candida albicans is the most prevalent human fungal pathogen associated with biofilm formation on indwelling medical devices. Under this form, Candida represents an infectious reservoir difficult to eradicate and possibly responsible for systemic, often lethal infections. Currently, no information is available on the occurrence and persistence of pathogenic viruses within C. albicans biofilm. Therefore, the aim of this study was to investigate whether Herpes Simplex Virus type 1 (HSV-1) and Coxsackievirus type B5 (CVB5) can be encompassed in Candida biofilm, retain their infectivity and then be released. Thus, cell-free virus inocula or HSV-1-infected cells were added to 24h-old fungal biofilm in tissue culture plates; 48 h later, the biofilm was detached by washing and energetic scratching and the presence of virus in the rescued material was end-point titrated on VERO cells. Planktonic Candida cultures and samples containing only medium were run in parallel as controls. We found that both HSV-1 and CVB5 free virus particles, as well as HSV-1 infected cells remain embedded in the biofilm retaining their infectivity. As a second step, the influence of biofilm on virus sensitivity to sodium hypochlorite and to specific neutralizing antibodies was investigated. The results showed that virus encompassment in fungal biofilm reduces virus sensitivity to chemical inactivation but does not affect antibody neutralization. Overall, these data provide the first in vitro evidence that viruses can be encompassed within Candida biofilm and then be released. Thus, it may be speculated that Candida biofilm can be a reservoir of viruses too, posing a further health risk.

By means of a double-chamber model, different root canal filling materials and procedures were compared. Briefly, the root canals of single-rooted human teeth, extracted for periodontal reasons, were instrumented and obturated by gutta-percha/Pulp Canal Sealer EWT (PCS) or by Resilon, in association with different sealers (Real Seal, RelyX Unicem or Meta). Obturation was achieved by traditional continuous wave of condensation technique (TCWCT), a modified version of it (MCWCT), or single cone technique (SCT). The obturated roots, inserted in a double-chamber model, were sterilized by gamma irradiation. Next, Enterococcus faecalis was added to the upper chamber and the specimens were incubated at 37 °C for up to 120 days; the development of turbidity in the lower chambers’ broths indicated bacterial leakage through the obturated root canals. The kinetics of leakage were analyzed in different groups by means of Kaplan–Meier statistics and compared by log-rank test. The results showed that root canals obturated with either gutta-percha/PCS using the MCWCT, Resilon/Real Seal SCT or Resilon/RelyX Unicem using the TCWCT displayed significantly better performance than the remaining groups (p

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 (AFR1) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1-overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1Delta mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1-overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans-microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.

Recently, we have shown a differential susceptibility of non-pathogenic vs. pathogenic leptospires to phagocytosis and killing by microglial cells. Although all ingested to some extent, only the pathogenic strains survived intracellularly while the non-pathogenic ones were killed in a time-dependent manner. By the same infection model, here we demonstrate that microglial cells respond to Leptospira infection with a time- and dose-dependent induction of molecular signals (p38 phosphorilation and NF-kB activation) and the production of soluble factors (cytokines and nitric oxide). Such bio-molecular response is predominantly observed against the pathogenic Leptospira; the phenomenon is reproduced by leptospiral lipoproteins and, to a lower extent, by leptospiral-derived LPS. These data provide initial evidence that Leptospira affects microglial cell response in a different manner depending upon the virulence of the infecting strain; specific bacterial components happen to be involved in the induction of such pathogen-induced immune response.

Microglial cells, the resident phagocytes in the brain, share many phenotypical and functional characteristics with peripheral macrophages, suggesting that they may participate in an innate immune response against microorganisms invading the central nervous system (CNS). In this study, we demonstrate that the microglial cells constitutively exhibit antibacterial activity in vitro against Streptococcus pneumoniae. By using a Pneumococcal surface protein C (PspC)-deleted strain and its wild-type counterpart, we found that the extent of such an activity is significantly influenced by the presence of a PspC molecule on the bacterial surface. The PspC- mutant FP20 is indeed more susceptible than the PspC+ strain HB565 to microglial killing. Interestingly, this phenomenon is observed when using a medium supplemented with heat-inactivated foetal bovine serum (FBS). Electron microscopy studies indicate that the microglial cells interact more efficiently with PspC- than with PspC+ pneumococci. Moreover, upon infection with the PspC- mutant, microglial cells produce levels of TNF-alpha, MIP-2, IL-10 and nitric oxide, significantly higher than those observed with PspC+ bacteria. These findings indicate that the lack of PspC significantly enhances the susceptibility of S. pneumoniae to both bactericidal activity and secretory response by the microglial cells, suggesting that this molecule may play an important role in the invasion of CNS by pneumococcus.

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.

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.

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.

In mice, the gene encoding Nramp1 (natural resistance-associated protein 1) exists in two allelic forms, differing for a point mutation. According to Nramp1 genotype, extensive literature documents a clear-cut distinction of inbred strains in two non-overlapping groups that phenotypically express resistance (Nramp1r) and susceptibility (Nramp1s) to systemic infections. Here, we provide evidence that Nramp1r (DBA/2) and Nramp1s (Balb/c) mice differently handle intracerebral infection with Mycobacterium bovis BCG. Distinct trends of microbial clearance from the brain and also different patterns of local immune responses occur, thus arguing on the involvement of Nramp1 gene product on the accomplishment of cerebral anti-mycobacterial defenses.

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.

The opportunistic fungal pathogen, Cryptococcus neoformans, shows a marked predilection for the central nervous system (CNS). This can be partially explained by its ability to synthesize melanin starting from the catecholamines, highly concentrated at the CNS level. Two cryptococcal strains, the avirulent non-melanogenic strain Sb26 and the virulent melanogenic revertant strain Sb26Rev, were used in a murine model of intracerebral (i.c.) infection, in order to evaluate their virulence and immunomodulating properties at the cerebral level. We found that, unlike Sb26Rev, Sb26 i.c. infection was never lethal regardless of the challenging dose. Sb26Rev infection resulted in massive CNS tissue damage, associated with little or no cytokine response, as established by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Differently, Sb26 infection failed to alter CNS structure, while inducing IL-12 p40, TNF-alpha, IL-1beta, IFN-gamma and iNOS specific-gene expression as well as IL-12, TNF-alpha and IL-1beta cytokine production. Interestingly, all Sb26 infected mice survived a subsequent lethal challenge with Sb26Rev. The phenomenon was associated with enhanced IL-12, TNF-alpha and IL-1beta production and was strictly specific, as shown by heterologous challenges and delayed type of hypersensitivity assay. Overall, we provide evidence that protective immunity against cerebral cryptococcosis is established by means of an avirulent strain of C. neoformans.

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.

We investigated Legionella and Pseudomonas contamination of hot water in a cross-sectional multicentric survey in Italy. Chemical parameters (hardness, free chlorine, and trace elements) were determined. Legionella spp. were detected in 33 (22.6%) and Pseudomonas spp. in 56 (38.4%) of 146 samples. Some factors associated with Legionella contamination were heater type, tank distance and capacity, water plant age, and mineral content. Pseudomonas presence was influenced by water source, hardness, free chlorine, and temperature. Legionella contamination was associated with a centralized heater, distance from the heater point >10 m, and a water plant >10 years old. Furthermore, zinc levels of 50 microg/L appeared to be protective against Legionella colonization [corrected]. Legionella species and serogroups were differently distributed according to heater type, water temperature, and free chlorine, suggesting that Legionella strains may have a different sensibility and resistance to environmental factors and different ecologic niches.