Heterozygosis and pathogenicity of Cryptococcus neoformans AD-hybrid isolates.

Nineteen Cryptococcus neoformans AD-hybrid isolates were investigated to assess whether hybrid genomic background could affect virulence in a mouse model. The level of heterozygosity of each strain was analyzed using primers specific for allele A and D of 15 polymorphic genes. Virulence was tested in a mouse model of systemic infection by measuring time of survival. In addition, the putative virulence attributes, melanin, phospholipase, and capsule production, as well as growth at 39°C and UV sensitivity were investigated. Eight strains showed to be heterozygous in up to 70% of loci, other eight strains were heterozygous in less than 60% of loci, while the remaining three strains were homozygous at all tested loci. Mice infected with hybrids with a high percentage of heterozygosis showed significantly (P < 0.01) shorter survival than mice infected with the other hybrids. Mortality was not correlated with the mating-type locus pattern, as well as it was not correlated with the level of expression of the different virulence attributes investigated. The present study confirms that hybridization in C. neoformans could represent an important evolutionary driving force in increasing the fitness of this yeast in the environment and in the host.

Fluconazole susceptibility of Candida spp. isolates collected over nine years in a teaching hospital of Ancona, Italy.

The in vitro activity of fluconazole was investigated against 476 yeast isolates collected during a 9-year period (1997-2005) from patients hospitalised in a teaching hospital of Ancona. They included 373 isolates of Candida albicans, 53 of Candida glabrata and 50 of Candida parapsilosis. Minimum inhibitory concentrations (MICs) determined in accordance with the Clinical Laboratory Standards Institute methodology showed that 96% of the isolates were susceptible (MIC < or =8.0 microg/ml). The uncommon, resistant isolates (MIC > or =64 microg/ml) were randomly distributed over time. Our data show that resistance to fluconazole in this geographical area is a rare event and suggest that this triazole can still represent first-line therapy in our institution.

Comparative analysis of pathogenicity of Cryptococcus neoformans serotypes A, D and AD in murine cryptococcosis.

OBJECTIVES: To characterize the pathogenicity of 15 strains of Cryptococcus neoformans belonging to several serotype/mating type allele patterns (Dalpha, Da, A(alpha), A(a), A(alpha)/D(a) and D(alpha)/A(a)) in experimental models of murine cryptococcosis. METHODS: CD1-infected mice were examined for survival and fungal loads in either brain or lung during the course of infection. RESULTS: All strains, with the exception of one Da strain, produced melanin in vitro. Similarly, all strains were encapsulated and produced phospholipase. When CD1 mice were challenged intravenously (i.v.) with 5x10(5)CFU/mouse and observed for 60 days post-infection, a significant variation of mortality rate was observed among mice infected with different strains. A(alpha) and A(alpha)/D(a) strains all produced 100% mortality within the study period with mean survivals significantly shorter than those of mice infected with strains belonging to any other allele type (P<0.0001). A wide range of pathogenicity was shown by haploid and diploid strains presenting D(alpha) allele. This finding was confirmed by an intranasal model of challenge. To investigate the progression of infection, the mice were challenged i.v. with 5x10(4)CFU/mouse and tissue burden experiments (brain and lung) were performed on days 6 and 12 post-infection. Only the mice infected with A(alpha) and A(alpha)/D(a) strains showed a >1 log(10) increase of CFU/g in both tissues throughout the study period. CONCLUSIONS: Our results suggest that the presence of the A(alpha) mating type allele in either haploid or diploid strains is correlated with virulence, while the presence of the A(a) or D(a) allele in haploid strains is associated with moderate or no virulence. Finally, either haploid or diploid strains presenting D(alpha) allele vary in virulence.

Genotypic variation and antifungal susceptibilities of Candida pelliculosa clinical isolates.

At the Istituto Ricovero Cura Carattere Scientifico, Ospedale Maggiore di Milano, Italy, Candida pelliculosa accounted for 3.3 and 4.4 % of all Candida species other than Candida albicans collected during 1996 and 1998, respectively. Genetic variability was investigated by electrophoretic karyotyping and inter-repeat PCR, and the susceptibility to five antifungal agents of 46 strains isolated from 37 patients during these 2 years was determined. Combination of the two typing methods yielded 14 different DNA types. Although the majority of DNA types were randomly distributed among different units, one DNA type was significantly more common in patients hospitalized in a given unit compared with those from other wards (P=0.034), whereas another DNA type was more frequently isolated in patients hospitalized during 1996 than in those hospitalized during 1998 (P=0.002). Fluconazole, itraconazole and posaconazole MIC90 values were 16, 1 and 4 microg ml-1, respectively. All isolates but three were susceptible in vitro to flucytosine. All isolates were susceptible in vitro to amphotericin B. These data suggest that there are possible relationships among strains of C. pelliculosa, wards and time of isolation. Amphotericin B seems to be the optimal drug therapy in infections due to this yeast species.

Involvement of alpha(v)beta3 integrin-like receptor and glycosaminoglycans in Candida albicans germ tube adhesion to vitronectin and to a human endothelial cell line.

The present study was undertaken to investigate the expression of alpha(v)beta3 and alpha(v)beta5 integrin-like vitronectin receptors (VNRs) on Candida albicans germ tube and their involvement in its adhesion to vitronectin (VN) and human endothelial cells. By immunofluorescence and FACS analysis, several monoclonal antibodies directed against human alpha(v) or beta3 integrin subunit or alpha(v)beta3 and alpha(v)beta5 heterodimers, positively stained C. albicans germ tubes. C. albicans germ tubes specifically adhered (45-50%) to VN and this adhesion was markedly inhibited by RGD-, but not RGE-containing peptides. Adhesion of C. albicans germ tubes to VN was strongly inhibited by anti-alphav, anti-beta3 or anti-alpha(v)beta3, but not by alpha(v)beta5 monoclonal antibody. C. albicans germ tube adhesion to VN was also inhibited by glycosaminoglycans (GAGs) such as heparin or chondroitin sulphate. Finally, we show that C. albicans germ tubes adhere to the human EA.hy 926 endothelial cell line. This adhesion is markedly blocked by anti-beta3 monoclonal antibody, GRGDSP peptide or heparin, and is completely abolished by their combination. Overall these results indicate that C. albicans germ tube adherence to VN and to a human endothelial cell line is mediated by alpha(v)beta3, but not by alpha(v)beta5-like integrin, and depends on GAGs which may act by regulating alpha(v)beta3 integrin-like/VN adhesive interaction.

Local anticandidal immune responses in a rat model of vaginal infection by and protection against Candida albicans.

Humoral (antibody [Ab]) and cellular Candida-specific immune responses in the vaginas of pseudoestrus rats were investigated during three successive infections by Candida albicans. After the first, protective infection, Abs against mannan and aspartyl proteinase antigens were present in the vaginal fluid, and their titers clearly increased during the two subsequent, rapidly healing infections. In all animals, about 65 and 10% of vaginal lymphocytes (VL) were CD3(+) (T cells) and CD3(-) CD5(+) (B cells), respectively. Two-thirds of the CD3(+) T cells expressed the alpha/beta and one-third expressed the gamma/delta T-cell receptor (TCR). This proportion slightly fluctuated during the three rounds of C. albicans infection, but no significant differences between infected and noninfected rats were found. More relevant were the changes in the CD4(+)/CD8(+) T-cell ratio, particularly for cells bearing the CD25 (interleukin-2 receptor alpha) marker. In fact, a progressively increased number of both CD4(+) alpha/beta TCR and CD4(+) CD25(+) VL was observed after the second and third Candida challenges, reversing the high initial CD8(+) cell number of controls (estrogenized but uninfected rats). The CD3(-) CD5(+) cells also almost doubled from the first to the third infection. Analysis of the cytokines secreted in the vaginal fluid of Candida-infected rats showed high levels of interleukin 12 (IL-12) during the first infection, followed by progressively increasing amounts of IL-2 and gamma interferon during the subsequent infections. No IL-4 or IL-5 was ever detected. During the third infection, VL with in vitro proliferative activity in response to an immunodominant mannoprotein antigen of C. albicans were present in the vaginal tissue. No response to this antigen by mitogen-responsive blood, lymph node, and spleen cells was found. In summary, the presence of protective Ab and T helper type 1 cytokines in the vaginal fluids, the in vitro proliferation of vaginal lymphocytes in response to Candida antigenic stimulation, and the increased number of activated CD4(+) cells and some special B lymphocytes after C. albicans challenge constitute good evidence for induction of locally expressed Candida-specific Ab and cellular responses which are potentially involved in anticandidal protection at the vaginal level.

Impairment of rat thymocyte differentiation and functions by neonatal capsaicin treatment is associated with induction of apoptosis.

The direct and indirect interaction between the nervous system and its transmitters with the immune system was evaluated in the rat by using the neurotoxin capsaicin (Caps). In the present study we investigated the effect of Caps administration to neonatal rats on thymocyte subpopulation distribution and functions at different times after treatment. Caps treatment results in a marked reduction of thymus weight and cellularity. As shown by immunofluorescence and FACS analysis, profound depletion of double negative (DN), double positive (DP), and single positive (SP) CD4(+) cells was already evident at day 7 after treatment and persisted until day 28. Reduced numbers of SP CD8(+) cells were observed only at later time points. Analysis of TCR phenotype indicates that CD5(+) TCR gamma/delta(+) are particularly sensitive to neonatal Caps treatment. Caps-induced thymocyte depletion was associated with reduced proliferation in response to T cell mitogens. Moreover, in situ TUNEL reaction and agarose gel electrophoresis indicate that neonatal Caps treatment induces apoptosis of thymus cells. Morphological analysis reveals the presence of apoptotic cells in the subcapsular thymus cortical region. Overall our results suggest that Caps when administered at birth, profoundly affects T cell differentiation, likely through its ability to activate apoptotic cell death program.

Alterations of T cell distribution and functions in prenatally cypermethrin-exposed rats: possible involvement of catecholamines.

The synthetic pyrethroid insecticide, cypermethrin (50 mg/Kg) was given during gestation to pregnant rats by gavage in corn oil. Prenatal cypermethrin-exposure induces a marked and long-lasting increase of adrenaline (A) and noradrenaline (NA) plasma concentrations. The enhancement of plasma catecholamine levels was accompanied by a marked increase of CD5+, CD4+, and CD8+ total T cell numbers in the peripheral blood, while in the spleen a reduction of all T cell subsets was observed. In addition, peripheral blood lymphocytes (PBL) from rats prenatally exposed to cypermethrin showed an enhanced capability to proliferate in response to different doses of Concanavalin A (ConA), or human recombinant interleukin-2 (hrIL-2), whereas an impaired proliferative response was observed in the spleen. The percent increase of NA, but not A plasma concentrations paralleles the immunomodulatory effects induced by cypermethrin neonatal exposure on T cell distribution and mitogen-induced proliferation in the peripheral blood and spleen. Collectively, our results suggest that the changes in mitogen-induced proliferative responses in the peripheral blood and spleen of prenatally cypermethrin-exposed rats may be attributable to pesticide-induced catecholamine release, which causes an increased output of CD5+, CD4+, and CD8+ T cells from the spleen to the peripheral blood, and a consequent lymphocytosis.

Evidence for alphavbeta3 and alphavbeta5 integrin-like vitronectin (VN) receptors in Candida albicans and their involvement in yeast cell adhesion to VN.

The expression of integrin vitronectin (VN) receptors on Candida albicans yeasts and their involvement in the adhesion to VN were investigated. By immunofluorescence and cytofluorimetric analysis, several antibodies directed against human alphav, beta3, beta5, alphavbeta3, or alphavbeta5 integrin positively stained C. albicans yeasts. Biochemical analysis on yeast lysates with anti-human alphav, beta3, or beta5 antibody revealed molecular species of 130, 110, 100, and 84 kDa. The 130-kDa band was identified as alphav, whereas the doublet of 110/100 kDa and the 84-kDa band likely correspond to the beta3 and beta5 subunits, respectively. Some 48%-54% of Candida yeasts specifically adhered to VN, and this binding was strongly inhibited by anti-human alphav, beta3, alphavbeta3, and alphavbeta5 antibodies and by RGD- but not RGE-containing peptides. In addition, VN inhibited C. albicans adherence to a human endothelial cell line. Thus, C. albicans in the yeast phase expresses VN receptors antigenically related to the vertebrate alphavbeta3 and alphavbeta5 integrins, which mediate its adhesion to VN.

Neurokinin type-1 receptor antagonist inhibits enhancement of T cell functions by substance P in normal and neuromanipulated capsaicin-treated rats.

Substance P (SP) plays a major role in the regulation of the interaction between immune and nervous systems. SP administration stimulates Con A-induced proliferation of spleen and peripheral blood lymphocytes from normal and neonatally capsaicin treated rats, which correlated with enhanced IL-2 production and expression of activation antigens such as IL-2 receptor alpha chain (CD25) and RT1B MHC class II molecule. Moreover, SP markedly increased the percentage of CD5+ and CD4+ T lymphocytes in the peripheral blood of capsaicin-treated rats. Concomitant administration of SP with the non-peptide Neurokinin-1 receptor (NK1R) antagonist SR140333 completely inhibited the SP-mediated augmentation of Con A-induced PBL proliferation and IL-2 production as well as of CD4+ CD25+ and CD4+ RT1B+ T cell numbers in normal and capsaicin-treated rats. SR 140333 also blocked the increased percentage of peripheral blood CD4+ T cells induced by SP in capsaicin-treated rats.

Cypermethrin-induced alteration of thymocyte distribution and functions in prenatally-exposed rats.

The synthetic pyrethroid insecticide, cypermethrin (50 mg/kg) was given during gestation to pregnant rats by gavage in corn oil. Prenatal cypermethrin exposure induced a significant decrease in the absolute number of all thymocyte subsets during the first 30 days after birth, being the double negative CD4-CD8-, single positive CD4 and CD8 T cells preferentially affected. Later on day 60 and 90 double positive CD4+CD8+ and single positive thymocytes gradually recovered, while the total number of CD4 CD8 cells was increased. Moreover, thymocytes from rats prenatally exposed to cypermethrin showed an impaired ability to proliferate in response to different doses of Concanavalin A (ConA) and human recombinant interleukin-2 (hrIL-2) and to produce and/or release IL-2. Overall, our results indicate that cypermethrin administered during prenatal period can affect multiple steps in thymocyte differentiation pathways resulting in an altered cell subset distribution and an impairment of thymocyte functions.

Protective role of antimannan and anti-aspartyl proteinase antibodies in an experimental model of Candida albicans vaginitis in rats.

The role of antibodies (Abs) in the resistance to vaginal infection by Candida albicans was investigated by using a rat vaginitis model. Animals receiving antimannoprotein (anti-MP) and anti-aspartyl proteinase (Sap) Ab-containing vaginal fluids from rats clearing a primary C. albicans infection showed a highly significant level of protection against vaginitis compared to animals given Ab-free vaginal fluid from noninfected rats. Preabsorption of the Ab-containing fluids with either one or both proteins MP and Sap sequentially reduced or abolished, respectively, the level of protection. A degree of protection against vaginitis was also conferred by postinfectious administration of anti-Sap and anti-MP monoclonal antibodies (provided the latter were directed against mannan rather than protein epitopes of MP) and by intravaginal immunization with a highly purified, polysaccharide-free Sap preparation. Postinfectious administration of pepstatin A, a potent Sap inhibitor, greatly accelerated the clearance of C. albicans from rat vagina. No anti-MP or anti-Sap Abs were elicited during a C. albicans vaginal infection of congenitally athymic nude rats. Although they were as able as their euthymic counterparts to clear the primary infection, these animals did not show increased resistance to a rechallenge, demonstrating that induction of anticandidal protection in normal rats was a thymus-dependent Ab response. Overall, our data strengthen the concept that Abs against some defined Candida antigens are relevant in the mechanism of acquired anticandidal protection in vaginitis. The T-cell dependence of this protection may also provide a link between cell-mediated and humoral immunity in vaginal infection.