Increased peripheral blood TCD4+ counts and serum SP-D levels in patients with chronic paracoccidioidomycosis, during and after antifungal therapy.

BACKGROUND: The main clinical forms of paracoccidioidomycosis (PCM) are the acute/subacute form (AF) and the chronic form (CF), and they both display considerable clinical variability. The immune responses of PCM patients, during and after treatment, remain neglected, mainly in the case of CF patients, due to the high prevalence of pulmonary sequelae. OBJECTIVE: To evaluate the distribution of whole blood T cell subsets, serum cytokines, and biomarkers of pulmonary fibrosis in PCM patients, according to the clinical form and at different time points, during the antifungal therapy. METHODS: Eighty-seven PCM patients, from an endemic area in Brazil, were categorised into groups, according to the clinical form (AF or CF) and the moment of treatment. The peripheral blood T lymphocyte subsets of these patients were analysed using fluorescence-activated cell sorting. The serum levels of cytokines, basic fibroblast growth factor and surfactant protein-D (SP-D) were also analysed. FINDINGS: In the CF patients, an expansion of the peripheral blood TCD4+ cells was observed during the treatment, and this persisted even after two years of antifungal treatment. In addition, these patients showed high serum levels of SP-D. CONCLUSION: Our findings highlight the immunological changes CF patients undergo, during and after treatment, possibly due to the hypoxia triggered by pulmonary fibrosis and emphysema.

Phenotypic and functional evaluations of peripheral blood monocytes from chronic-form paracoccidioidomycosis patients before and after treatment.

BACKGROUND: Paracoccidioidomycosis (PCM) is systemic mycosis caused by the thermal dimorphic fungus of genus Paracoccidioides, leading to either acute/subacute (AF) or chronic (CF) clinical forms. Numerous CF patients after treatment exhibit sequels, such as pulmonary and adrenal fibrosis. Monocytes are cells that are involved in the inflammatory response during active infection as well as in the fibrogenesis. These cells comprise a heterogeneous population with distinct phenotypic and functional activities. The scope of this study was to identify changes regarding functional and phenotypical aspects in monocytes comparing CF PCM patients on antifungal treatment versus non-treated patients (PMC-p). METHODS: Twenty-three CF PCM composed of 11 non-treated patients (NTG) and 12 patients in apparent cure (ACG) were studied. Sixteen healthy individuals were used as control group (CG). Monocyte subsets were determined by immunophenotyping based on CD14 and CD16 expression. Cellular function was measured in vitro with and without stimulation with lipopolysaccharide (LPS) and P. brasiliensis exoantigen (PbAg) for 24 hours. Independent samples were compared using unpaired t tests, dependent samples were analyzed by paired t-test. Groups of more than two independent samples were analyzed using an ANOVA, with Tukey's post-test. Significance was set up at p <0.05. RESULTS: Our results showed high counts of peripheral blood CD14+CD16+ and CD14+CD16++ monocytes in untreated PCM-p accompanied by intense production of pro-inflammatory cytokines (IL-1ß and TNF-α) and profibrotic growth factors (TGF-ß1 and bFGF) by monocytes challenged with P. brasiliensis antigens. After the introduction of antifungal therapy, the counts of CD14+CD16+ cells returned to baseline while CD14+CD16++ counts remained high. Interestingly, counts of CD14+CD16++ monocytes remained elevated even 52 ± 7 months after successful antifungal treatment. Furthermore, the ACG-patients showed preserved pro-inflammatory activity in the presence of specific antigen stimuli and high spontaneous production of TNF-α by monocytes. CONCLUSIONS: Infection with Paracoccidioides leads to initiation of a specific proinflammatory response by monocytes of PCM-p during active disease and in the apparent cure. A profibrotic profile by monocytes was observed only at admission. Furthermore, PCM-p with apparent cure showed high spontaneous production of TNF-α and high counts of CD14+CD16++ monocytes, probably induced by hypoxia duo to fibrotic sequelae.

Is the production of reactive oxygen and nitrogen species by macrophages associated with better infectious control in female mice with experimentally disseminated and pulmonary mucormycosis?

Different levels of resistance against Rhizopus oryzae infection have been observed between inbred (BALB/c) and outbred (Swiss) mice and are associated with the genetic background of each mouse strain. Considering that macrophages play an important role in host resistance to Rhizopus species, we used different infectious outcomes observed in experimental mucormycosis to identify the most efficient macrophage response pattern against R. oryzae in vitro and in vivo. For this, we compared BALB/c and Swiss macrophage activity before and after intravenous or intratracheal R. oryzae infections. The production of hydrogen peroxide (H2O2) and nitric oxide (NO) was determined in cultures of peritoneal (PMΦ) or alveolar macrophages (AMΦ) challenged with heat-killed spores of R. oryzae. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) were measured to confirm our findings. Naïve PMΦ from female BALB/c mice showed increased production of H2O2, TNF-α, and IL-10 in the presence of heat-killed spores of R. oryzae. Naïve PMΦ from female Swiss mice were less responsive. Naïve AMΦ from the two strains of female mice were less reactive to heat-killed spores of R. oryzae than PMΦ. After 30 days of R. oryzae intravenous infection, lower fungal load in spleen from BALB/c mice was accompanied by higher production of H2O2 by PMΦ compared with Swiss mice. In contrast, AMΦ from BALB/c mice showed higher production of NO, TNF-α, and IL-10 after 7 days of intratracheal infection. The collective findings reveal that, independent of the female mouse strain, PMΦ is more reactive against R. oryzae upon first contact than AMΦ. In addition, increased PMΦ production of H2O2 at the end of disseminated infection is accompanied by better fungal clearance in resistant (BALB/c) mice. Our findings further the understanding of the parasite-host relationship in mucormycosis.

Enhanced expression of NLRP3 inflammasome components by monocytes of patients with pulmonary paracoccidioidomycosis is associated with smoking and intracellular hypoxemia.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermally dimorphic fungi of the genus Paracoccidioides that affects predominantly 30-60-year-old male rural workers. The main clinical forms of the disease are acute/subacute, chronic (CF); almost all CF patients develop pulmonary fibrosis, and they also exhibit emphysema due to smoke. An important cytokine in this context, IL-1ß, different from the others, is produced by an intracellular multimolecular complex called inflammasome that is activated by pathogens and/or host signs of damage. Inflammasome has been recognized for its contribution to chronic inflammatory diseases, from that, we hypothesized that this activation could be involved in paracoccidioidomycosis, contributing to chronic inflammation. While inflammasome activation has been demonstrated in experimental models of Paracoccidioides brasiliensis infection, no information is available in patients, leading us to investigate the participation of NLRP3-inflammasome machinery in CF/PCM patients from a Brazilian endemic area. Our findings showed increased priming in mRNA levels of NLRP3 inflammasome genes by monocytes of PCM patients in vitro than healthy controls. Similar intracellular protein expression of NLRP3, CASP-1, ASC, and IL-1ß were also observed in freshly isolated monocytes of PCM patients and smoker controls. Increased expression of NLRP3 and ASC was observed in monocytes from PCM patients under hypoxia in comparison with smoker controls. For the first time, we showed that primed monocytes of CF-PCM patients were associated with enhanced expression of components of NLRP3-inflammasome due to smoke. Also, hypoxemia boosted this machinery. These findings reinforce the systemic low-grade inflammation activation observed in PCM during and after treatment.

Gliotoxin Aggravates Experimental Autoimmune Encephalomyelitis by Triggering Neuroinflammation.

Gliotoxin (GTX) is the major and the most potent mycotoxin that is secreted by Aspergillus fumigatus, which is capable of injuring and killing microglial cells, astrocytes, and oligodendrocytes. During the last years, studies with patients and experimental models of multiple sclerosis (MS), which is an autoimmune disease of the central nervous system (CNS), suggested that fungal infections are among the possible initiators or aggravators of this pathology. The deleterious effect can occur through a direct interaction of the fungus with the CNS or by the toxin release from a non-neurological site. In the present work, we investigated the effect of GTX on experimental autoimmune encephalomyelitis (EAE) development. Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein and then intraperitoneally injected with three doses of GTX (1 mg/kg b.w., each) on days 4, 7, and 10. GTX aggravated clinical symptoms of the disease in a dose-dependent way and this outcome was concomitant with an increased neuroinflammation. CNS analyses revealed that GTX locally increased the relative expression of inflammatory genes and the cytokine production. Our results indicate that GTX administered in a non-neuronal site was able to increase neuroinflammation in EAE. Other mycotoxins could also be deleterious to many neurological diseases by similar mechanisms.

Altered distribution of peripheral blood dendritic cell subsets in patients with pulmonary paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi from the genus Paracoccidioides in Latin America. PCM-patients (PCM-p) are classified as having acute/subacute or chronic (CF) clinical forms. CF is responsible for 75%-90% of all cases, affects mainly adults over 30 years old and the clinical manifestation are associated mainly with lungs and mucosa of upper airdigestive tract. In addition, the CF patients exhibit fibrosis of the lungs, oral mucous membranes and adrenals, and pulmonary emphysema. Consequently, CF PCM-p with active disease, as well as those that have been apparently cured, seem to be an interesting model for studies aiming to understand the long-term host-fungi relationship and hypoxia. Dendritic cells (DCs) constitute a system that serve as a major link between innate and adaptive immunity composed of several subpopulations of cells including two main subsets: myeloid (mDCs) and plasmacytoid (pDCs). The present study aimed to access the distribution of PBDC subsets of CF PCM-p who were not treated (NT) or treated (apparently cured - AC). CF PCM-p were categorized into two groups, consisting of 9 NTs and 9 ACs. Twenty-one healthy individuals were used as the control group. The determination of the PBDC subsets was performed by FACS (fluorescence-activated cell sorting) and the dosage of serum TNF-α, IL1ß, IL-18, CCL3, IL-10 and basic fibroblast growth factor (bFGF) by ELISA (enzyme-linked immunosorbent assay). A high count and percentage of mDCs was observed before treatment, along with a low count of pDCs in treated patients. Furthermore, the mDC:pDC ratio and serum levels of TNF-α was higher in both of the PCM-p groups than in the control group. In conclusion, our findings demonstrated that active PCM influences the distribution of mDCs and pDCs, and after treatment, PCM-p retained a lower count of pDCs associated with pro-inflammatory profile. Therefore, we identified new evidences of persistent immunological abnormalities in PCM-p after treatment. Even these patients showing fungal clearance after successful antifungal treatment; the hypoxia, triggered by the persistent pulmonary sequelae, possibly continues to interfere in the immune response.

Dermatophyte-host relationship of a murine model of experimental invasive dermatophytosis.

Recognizing the invasive potential of the dermatophytes and understanding the mechanisms involved in this process will help with disease diagnosis and with developing an appropriate treatment plan. In this report, we present the histopathological, microbiological and immunological features of a model of invasive dermatophytosis that is induced by subcutaneous infection of Trichophyton mentagrophytes in healthy adult Swiss mice. Using this model, we observed that the fungus rapidly spreads to the popliteal lymph nodes, spleen, liver and kidneys. Similar to the human disease, the lymph nodes were the most severely affected sites. The fungal infection evoked acute inflammation followed by a granulomatous reaction in the mice, which is similar to what is observed in patients. The mice were able to mount a Th1-polarized immune response and displayed IL-10-mediated immune regulation. We believe that the model described here will provide valuable information regarding the dermatophyte-host relationship and will yield new perspective for a better understanding of the immunological and pathological aspects of invasive dermatophytosis.

Increased peripheral blood TCD4+ counts and serum SP-D levels in patients with chronic paracoccidioidomycosis, during and after antifungal therapy

BACKGROUND The main clinical forms of paracoccidioidomycosis (PCM) are the acute/subacute form (AF) and the chronic form (CF), and they both display considerable clinical variability. The immune responses of PCM patients, during and after treatment, remain neglected, mainly in the case of CF patients, due to the high prevalence of pulmonary sequelae. OBJECTIVE To evaluate the distribution of whole blood T cell subsets, serum cytokines, and biomarkers of pulmonary fibrosis in PCM patients, according to the clinical form and at different time points, during the antifungal therapy. METHODS Eighty-seven PCM patients, from an endemic area in Brazil, were categorised into groups, according to the clinical form (AF or CF) and the moment of treatment. The peripheral blood T lymphocyte subsets of these patients were analysed using fluorescence-activated cell sorting. The serum levels of cytokines, basic fibroblast growth factor and surfactant protein-D (SP-D) were also analysed. FINDINGS In the CF patients, an expansion of the peripheral blood TCD4+ cells was observed during the treatment, and this persisted even after two years of antifungal treatment. In addition, these patients showed high serum levels of SP-D. CONCLUSION Our findings highlight the immunological changes CF patients undergo, during and after treatment, possibly due to the hypoxia triggered by pulmonary fibrosis and emphysema.

The effect of phosphoethanolamine intake on mortality and macrophage activity in mice with solid ehrlich tumors

The aim of the present study was to examine the effect of a diet rich in synthetic PEtn on the metabolism macrophages of tumor-bearing mice. The results demonstrated that PEtn increased the animals' survival time. In addition, the treated animals released smaller amounts of hydrogen peroxide (H2O2) and nitric oxide (NO) than the non-treated animals, particularly after day 14. From the results it could be concluded that H2O2 and NO were important in the modulation of neoplastic growth, and pointed to a promising role of PEtn in the control of human neoplasms.

Histological evaluation of the lesion induced by inoculation of Leishmania mexicana in the cheek pouch of the hamster

We have studied the role of the immune response in the morphology of the leishmaniotic granuloma induced in the cheek pouch of hamsters, an immunologically privileged site, after inoculation of 3 x 10(5) Leishmania mexicana. Animals were histologically and immunologically evaluated until 120 days after inoculation. Independent of the time of sacrifice, the animals were always non-reactors to the footpad test (FPT). At histology, the introduction of L. mexicana in the cheek pouch leads to an abscess that evolves to a granulomatous reaction rich in amastigote forms, and later it leads to resolution, even in the absence of immune response detectable by FPT. Our results demonstrate that the development of immune response is not preponderant for the control of infection induced by L. mexicana inoculated subcutaneously in the cheek pouch of the hamster. It also suggests that the macrophages present in the leishmaniotic granuloma are capable of eliminating this parasite, even in the absence of immune response evaluated by FPT.

Experimental dermatophytosis in hamsters inoculated with Trichophyton mentagrophytes in the cheek pouch

This study presents the results of T. mentagrophytes inoculation in the cheek pouch of the hamster, an immunologically privileged site. Forty two animals were used: 21 inoculated with 10(6) fungi in the cheek pouch (group 1) and 21 inoculated initially with 10(6) fungi in the foot pad and 15 days later in the cheek pouch, with the same amount of fungi (group 2). Animals were sacrificed at 20 hours, 3, 7, 14, 30, 60, and 120 days; samples from inoculated cheek pouch, and foot pads submitted to the foot pad test (FPT), were collected. Independent of group and time of evolution of infection, animals did not develop delayed hypersensitivity evaluated through the FPT. The pre-inoculation of fungi in the foot pad did not change the morphology of lesions induced in the cheek pouch. Therefore, in animals of group 1 and 2, the introduction of the fungus in the cheek pouch resulted in focal lesion composed of a sterile acute inflammatory infiltrate, with abscess formation that evolved to a macrophagic reaction, and later to resolution even in the absence of immune response detectable by FPT. Our results indicate that in spite of the important role of the immune response in the spontaneous regression of dermatophytosis, other factors are also an integral part in the defense against this fungal infection

Dermatophytosis: association between ABO blood groups and reactivity to the trichophytin

The authors investigated the relationship between dermatophytosis and ABO blood groups through blood typing, identification of isolated dermatophytes and specific cellular immune response of 40 individuals carriers of this mycosis. They verified that the fungus Trichophyton rubrum, isolated from 54.5 percent of the patients, was more frequent in individuals belonging to blood group A. The cellular immune response, evaluated through the trichophytin antigen, was positive in 25 percent of the studied patients; the presence of immediate reactions (30 minutes) was verified in 35 percent. The blood group distribution among patients with dermatophytosis and control groups was, respectively: 47.5 percent X 36 percent in group A, 40 percent X 50 percent in group O, 12.5 percent X 11 percent in group B. Even though the authors have found a higher number of patients belonging to blood group A infected by T. rubrum, these results suggest that there is no statistical evidence that these individuals are more susceptible to dermatophytosis

Algumas consideraçöes a respeito da reaçäo de Rubino / Rubino's reaction

Säo comentados alguns aspectos relativos a reaçäo de Rubino, visando tanto uma melhor identificaçäo dos elementos envolvidos como despertar o interesse dos pesquisadores para esta prova. Os seguintes aspectos säo discutidos: 1) influência da temperatura sobre os elementos da reaçäo; 2) correlaçäo entre a reaçäo de Rubino e: a) alteraçöes humorais observadas nas hanseníase, b) imunidade mediada por células; 3) variaçöes na reatividade sorológica; 4) valor prognóstico e crério de cura

Efeito da talidomida no desenvolvimento da lesäo induzida pelo paracoccidioides brasiliensis na bolsa jugal do hamster / Effect of thalidomide on the development of paracoccidioides brasiliensis induced lesion in the hamste's cheek pouch

Considerando que a talidomida, droga antinflamatória, inibe a produçäo local de TNF-a em granulomas epitelóides e que a inoculaçäo de Paracocciodioides brasiliensis(P. brasiliensis) na bolsa jugal do hamster resulta na formaçäo de granulomas epitelióides bem estruturados, utilizamos no presente estudo este modelo experimental para investigar o seu efeto no desenvolvimento da lesäo paracoccidioidomocótica, na presença e na ausência de resposta imune. Para isso, 48 hamsters, 24 dos quais foram previamente sensiblizados constituíram o grupo A e os previamente sensibilizados, o grupo B. De cada grupo , 12 animais receberam injeçöes diárias de talidomida e 12 constituíram os subgrupos-controle. Os animais foram sacrificados aos 7,14,28 e 60 dias, sendo avaliados quanto à morfologia das lesöes de inoculaçäo e á presença de lesöes de disseminaçäo. Histologicamente, as lesöes na bolsa jugal eram constituídas por granulomas epitelióides, melhor definidos a partir do 14§ dia. Nos animais do grupo B, as lesöes sempre exibiam halo de células mononucleares, enquanto que naqueles do grupo A, este tornou-se mais evidente ao final da experimentaçäo. Os resultados revelaram que, independente de pré-sensibilizaçäo, a administraçäo de talidomida resultou na ausência de área necrótica no centro da lesäo, reforçando a idéia de seu uso em eventos inflamatórios com destruiçäo tecidual

Inoculaçäo de mycobacterium leprae na bolsa jugal do hamster / Mycobacterium leprae seeding at hamster cheek pouch

A utilizaçäo da bolsa jugal do hamster, foi avaliada pela inoculaçäao de 6,0x10.8 M. leprae/ml no tecido subepitelial de 60 animais, empregando como grupo controle, 12 hamsters inoculados no coxim plantar. Os animais foram sacrificados em 20 e 48 horas e aos 7, 14, 21 e 28 dias pi. A evoluçäo da lesäo de inoculaçäo foi analisada pelo exame histológico em cortes corados pela hematoxilina-eosina e faraco-fite. A avaliaçäo da viabilidade bacilar na bolsa jugal do hamster foi realizada 7, 14, 21 e 28 dias pi pelo teste de recuperaçäo bacilar em camundongos. Os resultados nos permitiram concluir que a resposta inflamatória ao M. leprae na bolsa jugal evoluiu para formaçäo de granulomas macrofágicos semelhantes aos da hanseníase virchoviana humana porém, o teste de recupraçäo bacilar sugeriu que näo houve multiplicaçäo dos bacilos. As lesões do coxim plantar evoluiram para granulomas epiteliódes semelhantes aos da hanseníase dimorfa

Avaliaçäo do levamizole como imuno-estimulador na hanseníase / Levamizole assessment as immuno-stimulator in leprosy

Pacientes hansenianos classificados como portadores da forma clínica virchoviana foram inicialmente submetidos a avaliaçäo imulógica específica (Mitsudina) e inespecífica (PPD, tricofitina, DNCB e determinaçäo do percentual de linfócitos T no sangue periférico). A seguir, foram estimulados com levamizole (150 mg/dia, 2 vezes por semana, durante 2 meses) e novamente submetidos a avaliaçäo imunológica, visando determinar o efeito deste modulador sobre o defeito imunológico descrito nestes pacientes

Aspectos biológicos do tatu Dasypus novemcinctus / Biologycal aspects of the armadillo Dasypus novemcinctus

Nesta revisäo, säo apresentados dados de literatura sobre a biologia do tatu D. novemcinctus, com ênfase aos estudos experimentais em laboratório. Os trabalhos descrevem as características morfológicas do tatu, a regular produçäo de gêmeos univitelinos e os aspectos genéticos. Além disso, säo ressaltados ainda a hipotermia corporal, estudos teratogênicos com talidomida em fêmeas prenhas e os casos, já superiores a uma centena, de infecçäo natural pelo M. leprae