[Stress and periodontitis]. / Stress en parodontitis.

Gingivitis and periodontitis can worsen with reduced immune fitness. Various causes can reduce immune fitness in a host, as a result of which the balance between the host and the microbiome is disturbed. Among others, lifestyle factors, such as stress and smoking, can have a negative influence on immune fitness. An association has been demonstrated between stress and periodontitis and also acute necrotising ulcerative gingivitis or periodontitis. There are indications that neurons are able to secrete pro-inflammatory cytokines and chemokines that worsen chronic inflammatory reactions in the periodontium and compromise immune fitness. In vitro studies show high cortisol levels may contribute to the increased growth of P. gingivalis. Stress as a risk factor for periodontitis and the role of stress as a negative influence on the results of periodontal treatment are difficult to estimate clinically. Nevertheless, attention to and awareness of stress as an aspect of the comprehensive set of risk factors for periodontitis can diminish its negative impact on immune fitness.

Prospective multicentre study on azole resistance in Aspergillus isolates from surveillance cultures in haematological patients in Italy.

OBJECTIVES: This study was conducted to assess the prevalence of azole resistance in Aspergillus isolates from patients with haematological malignancies or who were undergoing haematopoietic stem cell transplantation and to identify the molecular mechanism of resistance. METHODS: In this 28-month prospective study involving 18 Italian centres, Aspergillus isolates from surveillance cultures were collected and screened for azole resistance, and mutations in the cyp51A gene were identified. Resistant isolates were genotyped by microsatellite analysis, and the allelic profiles were compared with those of resistant environmental and clinical isolates from the same geographical area that had been previously genotyped. RESULTS: There were 292 Aspergillus isolates collected from 228 patients. The isolates belonged mainly to the section Fumigati (45.9%), Nigri (20.9%), Flavi (16.8%) and Terrei (4.8%). Three isolates showed itraconazole resistance: Aspergillus fumigatus sensu stricto, Aspergillus lentulus (section Fumigati) and Aspergillus awamori (section Nigri). The itraconazole resistance rates were 1% and 1.48% considering all Aspergillus spp. isolates and the Aspergillus section Fumigati, respectively. The prevalence of azole resistance among all the patients was 1.3%. Among patients harbouring A. fumigatus sensu stricto isolates, the resistance rate was 0.79%. The A. fumigatus isolate, with the TR34/L98H mutation, was genotypically distant from the environmental and clinical strains previously genotyped. CONCLUSIONS: In this study, the Aspergillus azole resistance rate was 1% (3/292). In addition to A. fumigatus sensu stricto, A. lentulus and A. awamori azole-resistant isolates were identified. Therefore, it is important have a correct identification at the species level to address a rapid therapy better, quickly understand the shift towards cryptic species and have an updated knowledge of the local epidemiology.

Microbiological and parasitological survey of zoonotic agents in apparently healthy feral pigeons.

Microbiological and parasitological investigation was carried out on a colony of feral pigeons, located in a green area near the main hospital of a Central Italy city. One hundred pigeons were submitted to clinical examination. Cloacal swabs, grouped in pool of 4 samples, were analyzed to detect the presence of Coxiella burnetii, Chlamydia psittaci, Chlamydophila spp. using a biomolecular procedure, while individual cloacal samples were examined for Salmonella spp., Campylobacter spp., and yeasts by means of a specific culture media. An ELISA test was used to determine the presence of Giardia spp., and Cryptosporidium spp. coproantigens. Individual serological samples were also tested with the modified agglutination test (MAT) in order to detect antibodies against Toxoplasma gondii. The pigeons did not show any clinical signs. The cloacal pools proved to be negative for C. burnetii DNA while three pools were positive for C. psittaci or Chlamydophila spp. DNAs. Salmonella spp. was not detected. C. jejuni and C. coli were found in 13% and 4% of the samples, respectively. No Giardia spp. and Cryptosporidium spp. were detected. Thirty-three out of 100 samples (33%) were positive for yeast colonies. The seroprevalence for T. gondii was 8%. Although with moderate incidence, potentially zoonotic agents were present thus highlighting the need for sanitary surveillance on feral pigeon colonies.

SIMIFF study: Italian fungal registry of mold infections in hematological and non-hematological patients.

PURPOSE: We compared the risk factors, the diagnostic tools and the outcome of filamentous fungal infections (FFIs) in hematological patients (HAEs) and non-hematological patients (non-HAEs). METHODS: Prospective surveillance (2009-2011) of proven and probable FFIs was implemented in 23 Italian hospitals. RESULTS: Out of 232 FFIs, 113 occurred in HAEs and 119 in non-HAEs. The most frequent infection was invasive aspergillosis (76.1 % for HAEs, 56.3 % for non-HAEs), and the localization was principally pulmonary (83.2 % for HAEs, 74.8 % for non-HAEs). Neutropenia was a risk factor for 89.4 % HAEs; the main underlying condition was corticosteroid treatment (52.9 %) for non-HAEs. The distribution of proven and probable FFIs was different in the two groups: proven FFIs occurred more frequently in non-HAEs, whereas probable FFIs were correlated with the HAEs. The sensitivity of the galactomannan assay was higher for HAEs than for non-HAEs (95.3 vs. 48.1 %). The overall mortality rate was 44.2 % among the HAEs and 35.3 % among the non-HAEs. The etiology influenced the patient outcomes: mucormycosis was associated with a high mortality rate (57.1 % for HAEs, 77.8 % for non-HAEs). CONCLUSIONS: The epidemiological and clinical data for FFIs were not identical in the HAEs and non-HAEs. The differences should be considered to improve the management of FFIs according to the patients' setting.

Identification of animal glue and hen-egg yolk in paintings by use of enzyme-linked immunosorbent assay (ELISA).

We report the development of an indirect ELISA procedure for specific identification of chicken-egg yolk and animal glues in painting micro-samples. The results presented integrate previously published work on ELISA recognition of bovine ß-casein and chicken ovalbumin in painting materials. The integrated final ELISA procedure-optimised for protein extraction, immuno-reagent concentrations, blocking solution, incubation time, and temperature-enables multiplex identification, in single samples, of proteinaceous materials, i.e. chicken-egg yolk and albumen, animal glues, and bovine milk and/or casein, mainly used by painters in the past. The procedure has been systematically tested on laboratory models of mural and easel paintings, both naturally and artificially aged, to assess possible inhibitory effects on the immuno-reaction caused by inorganic painting materials (pigments and substrates) and by protein degradation resulting from aging processes. Real samples from case studies, which had previously been investigated and characterised by spectroscopy and chromatography, were successfully studied by use of the developed ELISA procedure. The commercial availability of all the immuno-reagents used, the affordable analytical equipment, and the specificity, sensitivity, and rapidity of ELISA make this method very attractive to diagnostic laboratories in the field of cultural heritage science. Possible further developments to the analytical potential of this technique include improvement of antibody performance and inclusion of other classes of bio-molecules as analytical targets.

The rs5743836 polymorphism in TLR9 confers a population-based increased risk of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case-control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P=7.3E-9) and Italian (OR, 1.84; P=6.0E-5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.

Development of an analytical protocol for a fast, sensitive and specific protein recognition in paintings by enzyme-linked immunosorbent assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) analysis of proteins offers a particularly promising approach for investigations in cultural heritage on account of its appreciated properties of being highly specific, sensitive, relatively fast, and cost-affordable with respect to other conventional techniques. In spite of that, it has never been fully exploited for routine analyses of painting materials in consideration of several analytical issues that inhibited its diffusion in conservation science: limited sample dimensions, decrease of binder solubility and reduced availability of antibody bonding sites occurring with protein degradation. In this study, an ELISA analytical protocol suited for the identification of aged denatured proteins in ancient painting micro-samples has been developed. We focused on the detection of bovine ß-casein and chicken ovalbumin as markers of bovine milk (or casein) and chicken albumen, respectively. A systematic experimentation of the ELISA protocol has been carried out on mock-ups of mural and easel painting prepared with 13 different pigments to assess limits and strengths of the method when applied for the identification of proteins in presence of a predominant inorganic matrix. The analytical procedure has been optimized with respect to protein extraction, antibodies' concentrations, incubation time and temperature; it allows the detection of the investigated proteins with sensitivity down to nanograms. The optimized protocol was then tested on artificially aged painting models. Analytical results were very encouraging and demonstrated that ELISA allows for protein analysis also in degraded painting samples. To address the feasibility of the developed ELISA methodology, we positively investigated real painting samples and results have been cross-validated by gas chromatography-mass spectrometry.

Prognostic significance of genetic variants in the IL-23/Th17 pathway for the outcome of T cell-depleted allogeneic stem cell transplantation.

T helper (Th) 17 cells have emerged as important mediators in infectious and inflammatory diseases and, recently, in transplant rejection. We analyzed the associations between five common genetic variants in the IL-23/Th17 signaling pathway, namely in IL17A, IL17F and IL23R genes, and clinical outcome in T cell-depleted allogeneic SCT (allo-SCT). In the multivariate analysis, variants in IL23R and IL17A genes were the most important prognostic factors. Thus, patient GA genotype at rs11209026 in IL23R was associated with improved overall survival (hazard ratio (HR)=0.48; P=0.028) and, in donor, with decreased risk of fungal infections (P=0.05). In contrast, patient TC and CC genotypes at rs8193036 in IL17A gene were associated with increased risk of CMV infection (HR=3.68; P=0.011) and patient acute GVHD (HR=7.08; P=0.008), respectively. These results suggest that genetic variants in the IL-23/Th17 inflammatory pathway are important prognostic factors for the clinical outcome of allo-SCT. Although validation studies are ultimately required, our results would suggest the potential usefulness of IL-23/Th17 genotyping in donor selection and patient evaluation.

Identification of proteins in painting cross-sections by immunofluorescence microscopy.

Immunofluorescence microscopy offers a highly specific analytical tool for unambiguous recognition and mapping of proteins in complex matrices. In the present work, the analytical potentials of immunofluorescence microscopy have been exploited to provide recognition of proteinaceous binders in painting cross-sections. An optimised analytical protocol is proposed for the identification of ovalbumin and of bovine serum albumin as markers of egg white and casein, respectively. The study has been carried out on laboratory model samples simulating both easel and mural paintings. The obtained results demonstrated the effectiveness of the method, suggesting the potential future use of immunofluorescence microscopy as a routine diagnostic tool in conservation science. Possible developments of the proposed methodology in order to improve the specificity of the method and its detection sensitivity are presented and discussed.

Polymorphisms in toll-like receptor genes and susceptibility to pulmonary aspergillosis.

Toll-like receptors (TLRs) are important components of innate immunity. We investigated the association between polymorphisms in the TLR2, TLR4, and TLR9 genes and susceptibility to noninvasive forms of pulmonary aspergillosis. A significant association was observed between allele G on Asp299Gly (TLR4) and chronic cavitary pulmonary aspergillosis (odds ratio [OR], 3.46; P =.003). Susceptibility to allergic bronchopulmonary aspergillosis was associated with allele C on T-1237C (TLR9) (OR, 2.49; P =. 043). No particular polymorphism was associated with severe asthma with fungal sensitization. These findings reinforce the importance of innate immunity in the pathogenesis of different forms of aspergillosis.

[Presence of the microbiological risk in Umbrian sawmills]. / Presenza del rischio microbiologico nel settore delle falegnamerie umbre.

The purpose of this study was to quantify and identify the airborne microbial contamination in Umbria Sawmills. In this paper we reported the preliminary results of our analysis. Microbial contaminants (fungi and bacteria) were assessed with passive (IMA Standard) and active (SAS microbial sampler) methods. There were significant differences of bacterial and/or fungal CFU/m3 values between the outdoor and indoor environments during the normal sawmills activity. Staphylococcus, Sphingomonas, Pasteurella, were the most predominant bacteria. The most predominant isolated fungi belong to Cladosporium, Penicillium, Alternaria and Aspergillus genus.

Immunity to Aspergillus fumigatus: the basis for immunotherapy and vaccination.

Efficient responses to fungi require different mechanisms of immunity. Dendritic cells (DCs) are uniquely able to decode the fungus-associated information and translate it into qualitatively different T helper (Th) immune responses. Murine and human DCs phagocytose conidia and hyphae of Aspergillus fumigatus through distinct recognition receptors. The engagement of distinct receptors translates into disparate downstream signaling events, ultimately affecting cytokine production and co-stimulation. Adoptive transfer of different types of DCs activates protective and non-protective Th cells as well as regulatory T cells, ultimately affecting the outcome of the infection in mice with invasive aspergillosis. The infusion of fungus-pulsed or RNA-transfected DCs also accelerates recovery of functional antifungal Th 1 responses in mice with allogeneic hematopoietic stem cell transplantation. Patients receiving T cell-depleted allogeneic hematopoietic stem cell transplantation are unable to develop antigen-specific T cell responses soon after transplant due to defective DC functions. Our results suggest that the adoptive transfer of DCs may restore immunocompetence in hematopoietic stem cell transplantation by contributing to the educational program of T cells. Thus, the remarkable furictional plasticity of DCs can be exploited for the deliberate targeting of cells and pathways of cell-mediated immunity in response to the fungus.

Humoral response against Cryptococcus neoformans mannoprotein antigens in HIV-infected patients.

Twenty-four sera from healthy donors, 18 from HIV-positive patients (< 200 CD4+/mm3) and 18 sera collected before and during cryptococcosis from HIV-positive patients were analysed for the presence of humoral response to C. neoformans mannoproteins. Our results show that samples from healthy subjects and from HIV-positive patients had one of three antibody response profiles: (i) presence of reactive antibodies against both 105 and 80 kilodalton mannoproteins; (ii) presence of reactive antibodies against one of the two mannoproteins; or (iii) absence of reactive antibodies. Importantly the percentage of unreactive sera increased 6-fold in HIV-positive patients and more than 10-fold in patients with cryptococcosis. In addition, in the latter patients no variation of humoral response before and during cryptococcosis was observed. These results suggest that HIV-positive patients show a marked difficulty in mounting or maintaining antibody response to mannoprotein and this could contribute to predisposition to cryptococcosis.

Multicenter comparative evaluation of six commercial systems and the national committee for clinical laboratory standards m27-a broth microdilution method for fluconazole susceptibility testing of Candida species.

Fluconazole susceptibility among 800 clinical Candida isolates (60% C. albicans) and two control strains (C. krusei ATCC 6258 and C. parapsilosis ATCC 22019) was tested with the NCCLS M27-A method (gold standard) and six commercial products (Candifast, disk, Etest, Fungitest, Integral System Yeasts, and Sensititre YeastOne). Results were classified as susceptible, susceptible-dose dependent, or resistant using M27-A breakpoints or, for Fungitest, Integral System Yeasts, and Candifast, as susceptible, intermediate, or resistant, according to the manufacturers' instructions. Concordance with NCCLS M27-A results was analyzed with the chi(2) test. Intra- and interlaboratory reproducibility was also evaluated. NCCLS M27-A (90.1%), Etest (93.1%), Sensititre YeastOne (93.1%), disk (96.7%), Fungitest (92.6%), Integral System Yeasts (40.6%), and Candifast (6.0%) classified the indicated percentages of C. albicans isolates as susceptible. Among non-C. albicans strains, the percentages of susceptible isolates were as follows: NCCLS M27-A, 74.0%; Etest, 83.8%; Sensititre YeastOne, 64.1%; disk, 60.6%; Fungitest, 76.6%; Integral System Yeasts, 28.3%; and Candifast, 27.4%. All methods except Candifast and Integral System Yeasts showed good agreement with NCCLS M27-A results for both C albicans and non-C. albicans isolates. Intralaboratory reproducibility was excellent for NCCLS M27-A, Etest, Sensititre YeastOne, disk, and Fungitest (88 to 91%). Similar results emerged from the interlaboratory reproducibility evaluation. Our findings indicate that some commercial methods can be useful for fluconazole susceptibility testing of clinical Candida isolates. Those characterized by a lack of medium standardization and/or objective interpretative criteria should be avoided. Particular caution is necessary when testing is being done for clinical and epidemiological purposes.

Microbial growth and air pollution in carbonate rock weathering. Preliminary results of a in situ experimental study.

Preliminary results on limestone weathering caused by air pollution and microbial colonization are presented in this study. Outdoor exposure experimental assays were performed on Scaglia limestone samples. Samples were exposed in two areas in Perugia (Italy) that differ for degree of urban air pollution. At different times of exposure, ranging from 1 to 12 months, microbial contamination and textural modifications of sampled surfaces were evaluated by microbiological procedures, X-ray diffraction, and scanning electron microscopy. After one year of exposure a significant fungal colonization and the presence of weathering products (i.e. gypsum) were detected on sampled surfaces.

Early induction of interleukin-12 by human monocytes exposed to Cryptococcus neoformans mannoproteins.

Interleukin-12 (IL-12) production by human monocytes stimulated with mannoproteins (MPs) of Cryptococcus neoformans was investigated. The results reported show that secreted or cell-associated MPs induce an early and significant production of IL-12. MPs show different capabilities to quantitatively affect IL-12 production; MP2, an 8. 2-kDa MP purified from the culture supernatant of C. neoformans, appears to be the most potent stimulator. Cytochalasin B inhibits both internalization and IL-12 induction by MP. In addition, a drastic reduction of IL-12 was observed when monocytes were cultured in the absence of normal human serum or treated with soluble mannan. Early production of IL-12 promotes early secretion of gamma interferon by T cells but does not influence the magnitude of the MP-induced lymphoproliferative response. Overall our results identify cryptococcal antigens responsible for rapid and potent induction of IL-12 in monocytes. MPs appear to regulate IL-12 secretion by internalization via the endocytic pathway and by interaction with monocyte receptors or serum factors.

A new azole derivative of 1,4-benzothiazine increases the antifungal mechanisms of natural effector cells.

The most widely used drug for treatment of candidiasis is fluconazole (FCZ). Recently, a new derivative of 1,4-benzothiazine, compound FS5, was developed. FS5 had an appreciable protective effect against murine candidiasis. The present study was designed to dissect the antifungal mechanisms triggered by FS5 and to establish whether this compound could enhance the antimicrobial abilities of natural effector cells. The results show that intraperitoneal injection of FS5 in mice (i) induced an increase in circulating neutrophil levels comparable to that observed in FCZ-treated mice; (ii) enhanced phagocytosis and the killing activities of macrophages (Mphis) isolated from the spleen or peritoneal cavity, with the latter effect correlating with induction of nitric oxide synthesis and production by Mphis; and (iii) increased the levels of expression and synthesis of tumor necrosis factor alpha. These results suggest that the compound-induced synthesis of antimicrobial and proinflammatory molecules by heterogeneous Mphi populations is part of the beneficial effect of FS5 exerted against murine candidiasis.

Tetanus toxin impairs accessory and secretory functions in interferon-gamma-treated murine macrophages.

Tetanus neurotoxin (TT), a product of microbial origin, acts as a zinc endopeptidase on vesicle-associated membrane proteins (VAMP). We have demonstrated that TT displays inhibitory effects on secretory and accessory functions in the murine macrophage (Mphi) cell line GG2EE. Nitric oxide (NO) secretion was decreased when interferon (IFN)-gamma-pretreated GG2EE Mphis were coincubated with a fungal costimulus (SMP200) and TT. When heat-inactivated TT was used this effect was not evident. The TT-mediated phenomenon was dose-dependent and specific since, under the same experimental conditions, it did not affect interleukin-6 or tumor necrosis factor-alpha secretion. Furthermore, IFN-gamma-induced major histocompatibility complex class II molecule expression and GG2EE accessory function, assessed by SMP200-stimulated lymphoproliferation, were also inhibited by TT. Such inhibition was incomplete, in line with our previous results showing that TT partially cleaves VAMP proteins in murine Mφ.

Cryptococcus neoformans differently regulates B7-1 (CD80) and B7-2 (CD86) expression on human monocytes.

To induce a specific response in primary resting T cells, two signals must be provided by antigen-presenting cells (APC). The first antigen-specific signal is mediated by formation of the T cell receptor major histocompatibility complex molecule ternary complexes. The second signal is delivered by interaction of either B7-1 or B7-2 expressed by APC with CD28 or CTLA-4 on T cells. In this study, we examined the modulation of B7-1 and B7-2 molecules on human monocytes exposed to encapsulated or acapsular Cryptococcus neoformans or Candida albicans. In our experimental system, C. albicans or acapsular C. neoformans are able to induce B7-1 expression while the encapsulated yeast is a poor stimulator. A modest increase of B7-2 expression was also observed after monocyte treatment with acapsular C. neoformans or C. albicans, while the encapsulated yeast was ineffective in inducing B7-2 molecules. Kinetic analysis showed the maximum expression of B7-1 after 24 to 48 h. Addition of the opsonic IgG1 mAb 2H1 to monocytes and C. neoformans significantly increased B7-1, but not B7-2, expression. The contribution of B7-1 and B7-2 co-stimulatory (CS) molecules to cryptococcal-specific T cell activation was analyzed and a substantial inhibition of T cell proliferation was observed. In this study we provide the first demonstration of fungal interference in the regulation of CS molecules. Our results suggest a potential mechanism for poor inflammatory responses observed in C. neoformans infections.

Azole derivatives of 1,4-benzothiazine as antifungal agents.

A series of azole derivatives of 1,4-benzothiazine 7-14 was synthesized and evaluated for the in vitro and in vivo activity against Candida albicans. Secondary alcohol 10 and its ether derivative 13 showed very good efficacy against systemic candidiasis in a murine experimental model.