Claudia Maria Leite Maffei

Chemokine Production and Leukocyte Recruitment to the Lungs of Paracoccidioides brasiliensis-Infected Mice Is Modulated by Interferon-γ

Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.

Leukotrienes are involved in leukocyte recruitment induced by live Histoplasma capsulatum or by the β‐glucan present in their cell wall

The inflammatory cell influx towards the peritoneal cavity in mice inoculated i.p. with live or dead Histoplasma capsulatum or with its subcellular preparations was studied. We also evaluated the effects of dexamethasone (Dexa) or MK886, an inhibitor of leukotriene (LT) biosynthesis, on the recruitment of leukocytes. Live yeast form of fungus (LYH) induced an increase in neutrophils (NE) which was highest 4 to 24 h after inoculation. Mononuclear cell (MN) migration beginning at 24 h with a gradual increase over 48 and 168 h, and an eosinophil (EO) recruitment occurs between 24 and 48 h. NE and EO recruitment induced by dead mycelial form of fungus (DMH) was greater than that observed for dead yeast form of fungus (DYH). A similar leukocyte migration pattern was seen after i.p. injection of the alkali‐insoluble fraction (F1) from DYH (F1Y) and F1 from DMH (F1M) this being more active than former. The difference in concentration of β‐glucan in DYH and DMH could explain the different inflammatory capacity exhibited by the two forms of H. capsulatum. LT seems to be the principal mediator of leukocyte migration in response to LYH, DYH or DMH or to β‐glucan. However, other mediators appear to contribute to NE and EO migration since the treatment with Dexa was more effective in inhibiting cell migration than MK886. Complement dependent leukocyte migration may participate in this recruitment. Treatment with MK886 completely abolished MN cell migration, indicating its dependence on the presence of LT.

In vitro antifungal drug susceptibilities of dermatophytes microconidia and arthroconidia

OBJECTIVES Arthroconidia have been considered as the primary cause of infection by dermatophytes. However, the in vitro antifungal testing evaluates the responses mainly of microconidia or hyphae, and dermatophytes in vivo often produce arthroconidia, a cellular structure presumably more resistant to antifungals. The aim of this study was to compare the in vitro susceptibility of microconidia and arthroconidia of Trichophyton rubrum, Trichophyton tonsurans and Trichophyton equinum to griseofulvin, itraconazole, terbinafine, fluconazole, amphotericin B and hygromycin B. METHODS Microconidia and arthroconidia were produced in vitro, and their susceptibility to each drug was evaluated by assessing the CLSI M38-A broth microdilution method. RESULTS Arthroconidia of all strains analysed appeared to be more resistant to fluconazole, griseofulvin and itraconazole than microconidia. The MIC of terbinafine was the same for microconidia and arthroconidia for all strains, and the MIC of amphotericin B for microconidia and arthroconidia was the same for isolates of T. equinum and T. tonsurans, but differed for T. rubrum. Finally, the level of resistance of microconidia for all strains towards the antibiotic hygromycin B was from 25 to 400 mg/L. CONCLUSIONS The difference in the susceptibility between microconidia and arthroconidia depends on the drug and on the strain, and may be one of the causes of therapeutic failure. Also, the level of resistance to the antibiotic hygromycin B presented by microconidia of these isolates will allow the use of hygromycin resistance as a dominant marker in fungal transformation procedures in future studies of gene function.

Deficiency of IL-12p40 subunit determines severe paracoccidioidomycosis in mice

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis. To investigate the role of interleukin (IL)-12 in this disease, IL-12p40-/- deficient mice (IL-12p40-/-) and wild type mice (WT) were infected intravenously with viable yeast cells of P. brasiliensis 18 isolate. We found that, unlike WT mice, IL-12p40-/- mice did not control fungal proliferation and dissemination and succumbed to infection by day 21 after inoculation. Additionally, IL-12p40-/- mice presented a higher number of granulomas/mm2 in lung tissue than WT mice, and showed unorganized granulomas containing high numbers of yeast cells. Moreover, IL-12p40-/- mice did not release detectable levels of IFN-gamma, but they produced high levels of IL-10, as well as IgG1 antibody. Additionally, splenocytes from both infected IL-12p40-/- and WT mice exhibited a suppressed Con-A-induced T cell proliferative response. Our findings suggest that the IL-12p40 subunit mediates resistance in paracoccidioidomycosis by inducting IFN-gamma production and a Th1 immune response.

In vitro susceptibility ofTrichophyton rubrum isolates to griseofulvin and tioconazole. Induction and isolation of a resistant mutant to both antimycotic drugs

The in vitro susceptibility of three clinicalTrichophyton rubrum isolates to griseofulvin and tioconazole, determined by the minimal inhibitory concentration (MIC), was 2 and 0.5 to 1.0μg/ml, respectively. One mutant (gril) obtained after mutagenic treatment of one of these isolates was selected and showed simultaneous resistance to griseofulvin (MIC > 2000μg/ml) and tioconazole (MIC=1.0μg/ml). The clinical importance and the possibility of a multidrug resistance (MDR)-type mechanism being involved in this event is discussed.The in vitro susceptibility of three clinical Trichophyton rubrum isolates to griseofulvin and tioconazole, determined by the minimal inhibitory concentration (MIC), was 2 and 0.5 to 1.0 micrograms/ml, respectively. One mutant (gril) obtained after mutagenic treatment of one of these isolates was selected and showed simultaneous resistance to griseofulvin (MIC > 2000 micrograms/ml) and tioconazole (MIC = 1.0 microgram/ml). The clinical importance and the possibility of a multidrug resistance (MDR)-type mechanism being involved in this event is discussed.

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS(-/-)) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS(-/-) mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS(-/-) mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS(-/-) mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS(-/-) mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-gamma and TNF-alpha) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.

Experimental systemic infection with Cryptococcus neoformans var. grubii and Cryptococcus gattii in normal and immunodeficient mice

Cryptococcus neoformans (Cn) var. grubii or Cryptococcus neoformans var. neoformans infection is usually associated with immunocompromised hosts, whereas Cryptococcusgattii more frequently causes disease in immunocompetent hosts. We examined the effects of immunodeficiency and glucocorticoid-induced immunosuppression on systemic murine infection induced by i.v. inoculation with these pathogens. SCID and immunocompetent BALB/c and C57BL/6 mice were infected with <or=107 yeast of Cn var. grubii or C. gattii; immunosuppressed BALB/c mice were infected with <or=106 yeast. Mortality was inoculum size-dependent in each model system, for both organisms. Following infection with 106 CFU of either Cn var. grubii or C. gattii immunocompetent BALB/c mice survived longer than immunosuppressed mice (P<0.0001 in both cases); no differences were found using lower inocula. SCID mice infected with Cn var. grubii or C. gattii died sooner than BALB/c mice (P<0.0013, all comparisons). Unexpectedly, BALB/c mice infected with C. gattii developed external lesions. Immunocompetent mice developed rectal prolapse more frequently whereas immunosuppressed mice developed more frequent skin lesions, predominantly on the tail. The fungal burden was especially high in rectum, skin and lung tissues. Histologic examination showed extensive infection of the rectum and skin and pneumonitis. Determination of CFU from various organs of immunocompetent BALB/c mice infected i.v. with 105 CFU of C. gattii or Cn var. grubii showed significant temporal increase of burdens of Cn var. grubii in brain and liver (P<0.003); other organs showed decreasing fungal burden. C.gattii was recovered only from liver and lungs, no CFU were detected in the other organs. As opposed to epidemiologic observations, our results demonstrate no predilection by C. gattii for infection of immunocompetent over immunosuppressed hosts; immunosuppression increased the risk of severe cryptococcosis by both varieties, especially at high inocula. This is the first report of C. gattii inducing experimental cutaneous and intestinal mucosal infection; Cn var. grubii did not affect these tissues, indicating differences in tissue tropism of these pathogens.

Ocorrência de dermatófitos em amostras de unhas, pés e mãos coletadas de estudantes universitários

The objective of this paper was to evaluate the occurrence of dermatophytes, specifically in the nails, feet and hands of university students with and without lesions. Two hundred and eighty samples were collected; 31 (11.1%) were positive by direct examination, while only 20 (7.1%) showed dermatophyte growth in culture, as well as direct positive examination. Trichophyton rubrum was the most frequently isolated (80%) dermatophyte followed by T. mentagrophytes (20%). Considering the sites analyzed, dermatophyte occurrence was: 10.4% in toenails, 5% in foot skin, 2.5% in fingernails and 0.4% in hand skin.

CLINICAL‐EPIDEMIOLOGIC STUDY OF ALOPECIA AREATA

Background. Alopecia areata is a common disease and may be associated with autoimmune disease, atopy, Down syndrome, emotional stress, and foci of sepsis.

An outbreak of scalp white piedra in a Brazilian children day care

White piedra is a superficial mycosis caused by Trichosporon spp. that affects the hair shaft of any part of the body. It is presented an outbreak of scalp white piedra seen in 5.8% of the children frequenting a day care in Northeastern of São Paulo State, Brazil. Mycological exam and culture identified T. cutaneum in all five cases, and scanning electron microscopy of nodules around hair shaft infected by Trichosporon spp. is demonstrated comparing them with those of black piedra and with nits of Pediculous capitis.