The Parasitemia has Contributed to the Severity of Cases of Visceral Leishmaniasis.

Visceral leishmaniasis (VL) occurs due to the evolution, virulence, and adaptation of Leishmania, vector biology, host immune system evasion, and reservoir hosts. Parasitemia can be involved as a warning regarding the clinical severity of VL The present study aims to evaluate the relationship between parasitemia and the prognosis of individuals with VL. Blood and bone marrow samples from individuals with VL were analyzed to identify parasite and quantify or measure parasite burden. Individuals were classified in the clinical score model of risk of death by disease proposed by Coura-Vital et al. (PLoS Negl Trop Dis 8(12): e33742014, 2014). 39/74 individuals presented a better prognosis, and 35/74 individuals presented a worse prognosis. HIV + VL co-infection was present in 32 individuals, of which 12 were considered severe. The group aged 51 to 64 was classified as severe, with a decrease in leukocytes (p-value 0.0295) and neutrophils (p-value 0.0476). L. infantum DNA was identified in blood and bone marrow, in 69 individuals, and not detected in 5 individuals. The quantification of the parasite showed greater parasitemia in bone marrow (P = 0.0003) with an average of 4.70 × 104 Leishmanias/mL about blood, with 0.29 × 104 Leishmanias/mL. Individuals in the age group aged 51 to 64 co-infected with HIV + VL had higher parasitemia (p-value 0.0150) with 2.44 × 104 Leishmanias/mL in blood and bone marrow than in the group aged 20 to 50. Parasitemia, measured by molecular biology in blood and bone marrow, was related to the worst clinical prognosis of VL in the age group aged 51 to 64.

In vitro effects of promethazine on cell morphology and structure and mitochondrial activity of azole-resistant Candida tropicalis.

The aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of Candida tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Three C. tropicalis isolates (two azole-resistant and one azole-susceptible) were evaluated for their planktonic and biofilm susceptibility to promethazine alone and in combination with itraconazole, fluconazole, voriconazole, amphotericin B, and caspofungin. The antifungal activity of promethazine against C. tropicalis was investigated by performing time-kill curve assays and assessing rhodamine 6G efflux, cell size/granularity, membrane integrity, and mitochondrial transmembrane potential, through flow cytometry. Promethazine showed antifungal activity against planktonic cells and biofilms at concentrations of 64 and 128 µg/ml, respectively. The addition of two subinhibitory concentrations of promethazine reduced the antifungal MICs for all tested azole drugs against planktonic growth, reversing the resistance phenotype to all azoles. Promethazine decreased the efflux of rhodamine 6G in an azole-resistant strain. Moreover, promethazine decreased cell size/granularity and caused membrane damage, and mitochondrial membrane depolarization. In conclusion, promethazine presented synergy with azole antifungals against resistant C. tropicalis and exhibited in vitro cytotoxicity against C. tropicalis, altering cell size/granularity, membrane integrity, and mitochondrial function, demonstrating potential mechanisms of cell damage against this yeast species.

Role of regulatory T cells in irinotecan-induced intestinal mucositis.

Intestinal mucositis (IM) is a common side effect of irinotecan-based chemotherapy. The involvement of inflammatory mediators, such as TNF-α, IL1-ß, IL-18 and IL-33, has been demonstrated. However, the role of adaptive immune system cells, whose activation is partially regulated by these cytokines, is yet unknown. Thus, we investigated the role of regulatory T cells (Tregs) in irinotecan-induced IM. C57BL/6 mice were injected with saline or irinotecan (75mgkg-1, i.p.), once a day for 4days, and euthanized at day 1, 3, 5 or 7 following the first dose of irinotecan. For Treg depletion, the mice were pretreated with a low single dose of cyclophosphamide (100mgkg-1, i.p). Intestinal lamina propria lymphocytes were harvested and purified by Percoll gradient. Treg and Th17 cells were identified by flow cytometry. Blood leukocyte count was obtained and ileum samples were collected for histopathological analysis and myeloperoxidase assay. IM caused an accumulation of Tregs and Th17 cells over time. Treg depletion exacerbated intestinal damage, diarrhea, neutrophil infiltration and animal mortality, despite a reduction in Th17 cell number. The frequency of other Th cells increased and was positively correlated with neutrophil infiltration. Tregs showed a negative correlation with neutrophils and the frequency of non-regulatory Th cells. In conclusion, Tregs are important in the control of intestinal damage induced by irinotecan, and their depletion showed a deleterious effect on IM. Activation of these cells appears to be a compensatory mechanism for intestinal inflammation.

Increased frequency of CD4 and CD8 regulatory T cells in individuals under 15 years with multibacillary leprosy.

BACKGROUND: Leprosy is a chronic disease, caused by Mycobacterium leprae, which poses a serious public health problem worldwide. Its high incidence in people under 15 years old in Ceará state, Brazil, reflects the difficulty of its control. The spectrum of clinical manifestations is associated with the immune response developed, with the Th1 and Th2 responses being related to the paucibacillary and multibacillary forms, respectively. Regulatory T cells (Treg), which can suppress Th1 and Th2 response, have received special attention in the literature and have been associated with development of chronic infections. However, their role in leprosy in individuals under 15 years old has not yet been elucidated. We evaluated the frequency of CD4(+)/CD8(+)CD25(high)FOXP3(+) and CD4(+)/CD8(+)CD25(high)FOXP3(high) cells in leprosy patients and household contacts, in both cases under 15 years old. METHODOLOGY/PRINCIPAL FINDINGS: PBMC from 12 patients and 17 contacts were cultured for 72 hours with anti-CD3 and anti-CD28 (activators) or with activators associated with total sonicated fraction of M. leprae. After culture, the frequency of CD4(+)/CD8(+) Treg was identified by flow cytometry. Cells stimulated by activators and antigen from multibacillary patients showed Treg frequencies almost two times that of the contacts: CD4(+)FOXP3(+) (21.93±8.43 vs. 13.79±8.19%, p = 0.0500), CD4(+)FOXP3(high) (10.33±5.69 vs. 5.57±4.03%, p = 0.0362), CD8(+)FOXP3(+) (13.88±9.19 vs. 6.18±5.56%, p = 0.0230) and CD8(+)FOXP3(high) (5.36±4.17 vs. 2.23±2.68%, p = 0.0461). Furthermore, the mean fluorescence intensity of FOXP3 in Treg was higher in multibacillary patients than in the contacts. Interestingly, there was a positive correlation of the bacillary index and number of lesions with the frequency of all Treg evaluated in patients. CONCLUSIONS/SIGNIFICANCE: We have demonstrated for the first time that multibacillary leprosy patients under 15 years old have greater CD4(+) and CD8(+) Treg frequencies and these correlate with clinical and laboratorial aspects of disease. These findings suggest the involvement of these cells in the perpetuation of M. leprae infection.

Efficacy of Plectranthus amboinicus (Lour.) Spreng in a Murine Model of Methicillin-Resistant Staphylococcus aureus Skin Abscesses.

The present work aimed to evaluate the effectiveness of Plectranthus amboinicus (Lour.) Spreng against MRSA clinical isolates. The in vitro antimicrobial activity of the hydroalcoholic extract (HE), the ethyl acetate (EA) fraction and its subfractions were determined by broth microdilution and bioautography against MRSA clinical isolates. The microdilution checkerboard method was used to assess in vitro drug combination studies. To induce abscess formation, bacterial suspensions were added to Citodex and inoculated subcutaneously into male Swiss mice. The treatment protocol consisted of 2 doses of HE, the EA fraction or vancomycin introduced intraperitoneally into mice 3 and 12 h after infection. The EA fraction and its subfractions presented the lowest minimal inhibitory concentrations (MIC, 0.25 to 0.5 mg/mL). The plant samples were bacteriostatic at 2x and 4x MIC and bactericidal at 100 mg/mL. The EA fraction presented synergism with vancomycin and an additive effect with ciprofloxacin. A significant reduction of abscess volume, bacterial cell counts in abscess slurries, and inflammatory scores was observed in the HE and EA fraction-treated groups. The samples were effective in treating the animals in a dose-dependent fashion. The present study proved the effectiveness of P. amboinicus fractions against MRSA using in vitro and in vivo assays.

Anti-PGL1 salivary IgA/IgM, serum IgG/IgM, and nasal Mycobacterium leprae DNA in individuals with household contact with leprosy.

OBJECTIVES: Leprosy household contacts represent a group at high risk of developing the disease. The aim of this study was to detect Mycobacterium leprae subclinical infection in this group through serological and molecular parameters. METHODS: Serum anti-PGL1 IgG/IgM and salivary anti-PGL1 IgA/IgM was investigated using an ELISA, and nasal carriage of M. leprae DNA was detected by PCR, in leprosy household contacts of paucibacillary (PB) and multibacillary (MB) household leprosy patients (n=135), their index cases (n=30), and in persons living in a low endemic city (n=17). RESULTS: Salivary anti-PGL1 IgA and IgM and serum anti-PGL1 IgG showed good correlation comparing contacts and index cases (p<0.01, p<0.005, and p<0.0001, respectively). This was not observed for serum anti-PGL1 IgM (p>0.05). A high frequency of anti-PGL1 IgM positivity was found in IgG-negative samples (p<0.0001). For IgG-positive samples, IgM antibodies were also positive in most of the samples. None of the 17 volunteers living in a low endemic city presented seropositivity for IgG; however, two of them showed positivity for anti-PGL1 IgM. M. leprae DNA was found in the nasal swabs of nine out of the 85 MB household leprosy contacts (10.6%) and in three out of the 50 PB household leprosy contacts (6.0%). CONCLUSION: We strongly suggest that serum IgG/IgM and salivary anti-PGL1 IgA/IgM measurements are used to follow leprosy household contacts.

Estudo sorológico da co-infecção de leishmania infantum e mycobacterium leprae em área de alta endemicidade / Sorological sutdy of coinfection leishmania infantum e mmycobacterium leprae in area of endemicity high

Introdução: A região Nordeste é responsável por 55% dos casos de hanseníase e por quase 50% dos casos de Leishmaniose visceral no Brasil. O Ceará, em especial a capital Fortaleza, é responsável por um grande número de casos novos dessas doenças. Este fato é reforçado pela correlação na distribuição de casos dessas patologias por municípios do estado do Ceará,onde de acordo com os dados da Secretaria de Saúde do Estado (2013), observa-se forte correlação epidemiológica entre os casos de hanseníase e do Leishmaniose visceral nos 184 municípios principalmente em Fortaleza. Objetivos: Nosso objetivo foi analisar a produção de anticorpos IgM anti-PGL1 em pacientes com Calazar sem tratamento. Material e métodos: 28 pacientes com confirmação clínico-laboratorial para Leishmaniose visceral acompanhados no Hospital São José de Doenças Infecciosas. Resultados: Quanto ao gênero, 21 foram do sexo masculino e 7 do sexo feminino, com mediana de idade de 20,5 anos (var. 3 a 76 anos), dos quais 15 pacientes não necessitaram internamento e 13 foram internados por um período médio de 28 dias (var. 5 a 28 dias). A média e desvio-padrão do índice de IgM anti-PGL1 foi de 1,91 + 0,69, sendo 78,6% considerados soropositivos. Conclusão: Não foi observada qualquer diferença entre gênero,idade, necessidade ou não de internamento, ou tempo de tratamento. A alta frequência de IgM anti-PGL1 positiva pode ser secundária à ativiação policlonal que ocorre na Leishmaniose visceral, dificultando a possibilidade de detecção da infecção pelo M. leprae por avaliação sorológica em região de alta endemicidade para Leishmaniose visceral.

Introduction: The Northeast region accounts for 55% of leprosy cases and nearly 50% of cases of visceral leishmaniasis in Brazil. Ceará, in particular the Fortaleza capital is responsible for a large number of new cases of these diseases. This fact is reinforced by the correlation in the distribution of cases of these diseases in the state of Ceará counties where according to the data of the State Health Departament (2013), we observed strong epidemiological correlation between cases of leprosy and visceral leishmaniasis in 184 counties mostly in Fortaleza. Objectives: Our objective was to analyze the production of anti-PGL1 IgM antibodies in patients with visceral leishmaniasis untreated. Materials and Methods: 28 patients with clinical and laboratory confirmation for visceral leishmaniasis followed at SãoJosé Hospital for Infectious Diseases. Results: As togender, 21 were males and 7 females, with a median age of 20,5 years (var 3-76 years.), Of which 15 patients did not require hospitalization and 13 were hospitalized for an average 28 days (var. 5 to 28 days). The mean and standard deviation of the anti-IgM PGL1 index was 1.91 ± 0.69, and 78.6% considered seropositive. Conclusion: It was not observed any difference between gender, age, necessity or not hospitalization, or time treatment. The high frequency of positive IgM anti-PGL1,can be secondary to polyclonal activation occurring in kala-azar, hindering the possibility of detection of M. leprae infection by serologic evaluation in high endemicity area for visceral leishmaniasis.

[The macrophages in the placenta during labor]. / Os macrófagos na placenta durante o trabalho de parto.

PURPOSE: to verify the amount of CD68+ cells in chorionic villosities in placentae from gestations submitted or not to labor. METHODS: transversal study with healthy near-term pregnant women, among whose placentae, 31 have been examined by immunohistochemical technique. Twenty placentae were obtained after vaginal delivery (VAGG) and eleven after elective cesarean sections (CESG). Slides were prepared with chorionic villosities samples and labeled with anti-CD68 antibody, specific for macrophages. Labeled and nonlabeled cells were counted inside the villosities. Non-parametric statistical tests were used for the analysis. RESULTS: among the 6,424 cells counted in the villosities' stroma from the 31 placentae, 1,135 cells (17.6%) were stained by the CD68+. The mean of cells labeled by the anti-CD68 was 22+/-18 for the VAGG group and 20+/-16 for the CESG, in each placentary sample. CONCLUSIONS: there were no significant differences in the percentage of macrophages (CD68+) in association with labor.

Os macrófagos na placenta durante o trabalho de parto / The macrophages in the placenta during labor

OBJETIVO: verificar a quantidade de células CD68+ no estroma das vilosidades coriônicas na placenta de gestações submetidas ou não ao trabalho de parto. MÉTODOS: estudo transversal, com gestantes saudáveis a termo, das quais 31 placentas foram examinadas pela técnica de imunoistoquímica. Vinte placentas foram obtidas após partos vaginais (GVAG) e 11 obtidas em cesarianas eletivas (GCES). Lâminas foram preparadas com amostras de vilosidades coriônicas e submetidas à marcação com anticorpo anti-CD68, específico para macrófagos. Foram contadas as células marcadas e as não marcadas dentro das vilosidades. Testes estatísticos não-paramétricos foram utilizados para a análise. RESULTADOS: entre 6.424 células contadas no estroma das vilosidades das 31 placentas, 1.135 células (17,6%) foram marcadas pelo CD68+. Em cada amostra placentária, a média de células coradas pelo anticorpo anti-CD68 foi de 22±18 para o grupo GVAG e de 20±16 para o grupo GCES. CONCLUSÕES: não houve diferenças significantes no percentual de macrófagos (CD68+) em associação com o trabalho de parto.

PURPOSE: to verify the amount of CD68+ cells in chorionic villosities in placentae from gestations submitted or not to labor. METHODS: transversal study with healthy near-term pregnant women, among whose placentae, 31 have been examined by immunohistochemical technique. Twenty placentae were obtained after vaginal delivery (VAGG) and eleven after elective cesarean sections (CESG). Slides were prepared with chorionic villosities samples and labeled with anti-CD68 antibody, specific for macrophages. Labeled and nonlabeled cells were counted inside the villosities. Non-parametric statistical tests were used for the analysis. RESULTS: among the 6,424 cells counted in the villosities' stroma from the 31 placentae, 1,135 cells (17.6%) were stained by the CD68+. The mean of cells labeled by the anti-CD68 was 22±18 for the VAGG group and 20±16 for the CESG, in each placentary sample. CONCLUSIONS: there were no significant differences in the percentage of macrophages (CD68+) in association with labor.

Cytokine polymorphisms and susceptibility to leprosy / Polimorfismos de citocinas e susceptibilidade à hanseníase

Cytokines play a key role in the regulation of the immune response against infectious diseases. In leprosy, the polymorphisms of pro-inflammatory cytokine genes may contribute to host susceptibility to Mycobacterium leprae. The objective of this study was to investigate the association between cytokine gene polymorphisms and BCG protection against leprosy in Brazilian leprosy cases and controls. DNA samples were obtained from 46 patients with leprosy and 83 healthy controls (not leprosy contacts). All genotyping (TNF alpha, IFN gamma, IL-6, IL-10 and TGF beta) measurements were taken using sequence-specific primers (SSP) - PCR. When compared to the healthy controls, no significant associations were observed between the cytokine gene polymorphisms studied and their susceptibility to leprosy. The most frequent genotypes in this population were TNF alpha ?G? allele and G/G genotype at position -308, ?A? allele of IFN gamma at position +874, G allele in the IL-6 at position -174, G allele (codon 25) and T/C-G/G genotype in TGF beta, and ?A? allele in IL-10 at position -1082. For those individuals that had a BCG scar, the TGF beta and IFN gamma genotype polymorphisms did not show difference among leprosy patients compared to healthy controls. Polymorphisms of the cytokine genes studied were not associated with an increased occurrence of leprosy.

A Invasão trofoblástica e a sua importância clínica / The Invasion of trophoblast cells and its clinical consequence

A Placenta é um órgão importante para o desenvolvimento fetal e para a saúde materna. Durante o início da gravidez humana, os citotrofoblastos extravilosos invadem o útero e transformam as artérias espiraladas em grandes vasos de baixa resistência. A inadequada invasão das artérias espiraladas por estes trofoblastos extravilosos resulta em isquemia placentária e no desenvolvimento de complicações obstétricas como pre-eclâmpsia e a restrição do crescimento fetal intra-útero (RCIU). A alta resistência do fluxo arterial uterino pode ser detectado pela dopplerfluxometria das artérias uterinas. Conseqüentemente, achamos interessante explicar como e quando ocorrem as invasões trofoblásticas na decídua e no miométrio