Márcia Pereira de Oliveira

Evaluation of the stability and immunogenicity of autoclaved and nonautoclaved preparations of a vaccine against American tegumentary leishmaniasis

This study was designed to evaluate the immunogenicity of autoclaved and nonautoclaved preparations of a vaccine composed of whole antigens from killed promastigotes of Leishmania amazonensis. Leishmanin skin-test (LST)-negative volunteers were immunized with either autoclaved or nonautoclaved vaccine preparations (32 and 36 subjects, respectively) that had been maintained at 4 degrees C for one year before the onset of this trial. Immunological tests were performed two days before and 40 days after vaccination. The LST conversion rates induced by the autoclaved and nonautoclaved vaccines were significantly different: 59% and 83%, respectively. Leishmania antigen-stimulated proliferative responses of peripheral blood mononuclear cells (PBMC) were significantly higher after vaccination than before vaccination in both groups. The CD8+ subset was predominant over the CD4+ subset among the leishmania-reactive cells after vaccination in both groups. The production of IFN-gamma by the leishmania antigen-stimulated PBMC was significantly higher after vaccination than before vaccination in the group receiving the nonautoclaved vaccine but not in the autoclaved vaccine group. IL-2 was found both before and after vaccination with no differences between its levels in these time points in either group. IL-4 was not detected for either group during the study period.

TUMOR NECROSIS FACTOR-A IN HUMAN AMERICAN TEGUMENTARY LEISHMANIASIS

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine produced by activated macrophages and other cells. In order to verify whether the serum levels of TNF-alpha in American tegumentary leishmaniasis patients are associated with the process of cure or aggravation of the disease, 41 patients were studied: 26 cases of cutaneous leishmaniasis (CL) and 15 of mucocutaneous leishmaniasis (MCL). During active disease the serum levels of TNF-alpha of MCL patients were significantly higher than those of CL patients and control subjects (healthy individuals and cutaneous lesions from other etiologies). The MCL patients had serum titers of TNF-alpha significantly lower at the end of antimonial therapy than before therapy. After a six-month follow-up, the MCL patients had serum levels of TNF-alpha similar to those observed at the end of the therapy as well as to those of CL patients and control subjects. No significant variation in the serum levels of TNF-alpha was observed in CL patients throughout the study period (before, at the end of therapy and after a six-month follow-up). The possible relationship between the high TNF-alpha serum levels and severity of the disease is discussed.

SYBR Green-based Real-Time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases

BackgroundLeishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between Leishmania isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk.ResultsA SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between Leishmania subgenera. A panel of reference strains representing subgenera Leishmania and Viannia was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95°C ± 0.01 and 77.36°C ± 0.02 for Leishmania and Viannia, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together L. (V.) braziliensis and L. (V.) shawii with a bootstrap value of 100%; while for L. (L.) infantum and L. (L.) amazonensis, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus Viannia amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus Leishmania (average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected Lutzomyia spp. captured in two Brazilian localities.ConclusionsFor all tested samples, a characteristic amplicon melting profile was evidenced for each Leishmania subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil.

Immune factors involved in the cervical immune response in the HIV/HPV co-infection.

Aims: Immune factors influencing the progression of cervical intraepithelial neoplasia (CIN) to cancer remain poorly defined. This study investigates the expression of RANTES, MIP1α, COX1, COX2, STAT3, TGFβRI, IL10R, TNFαRII and TLR4 in the cervical immune response in HIV/HPV (human papillomavirus) co-infected women. Methods: Cervical biopsies of 36 patients were assayed by immunohistochemistry, and the Ventana Benchmark System was used for HIV-nef detection. Results: Cervices from HIV-positive patients exhibited nef in cells mainly around blood vessels, and showed a decreased expression of all the immune factors tested except IL10R and STAT3, while RANTES (5.54 cells/mm2) was highly expressed in comparison with controls (1.41 cells/mm2, p = 0.028). COX1 was decreased in the HIV/HPV- (0.32 cells/mm2, p = 0.017) and HPV-infected patients (0.21 cells/mm2, p = 0.015) compared with controls (3.28 cells/mm2). Conclusions: It is suggested that RANTES in HIV/HPV co-infection may influence the development of CIN leading to progression to cervical cancer.

Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil

Leishmania RNA virus (LRV) has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ), no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V.) guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V.) brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

Clinical features of cutaneous and disseminated cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Paraty, Rio de Janeiro

Background  American tegumentary leishmaniasis (ATL) caused by Leishmania (Viannia) braziliensis is endemic in Rio de Janeiro State (RJ), where the disease shows epidemiologic and clinical characteristics distinct from those of ATL in other Brazilian regions. Paraty is the second most important endemic area in RJ; however, reports on leishmaniasis in this region refer to the occurrence of the disease without describing its characteristics.

An evaluation of p16INK4a expression in cervical intraepithelial neoplasia specimens, including women with HIV-1

Although several studies have evaluated the role of p16(INK4a) as a diagnostic marker of cervical intraepithelial neoplasia (CIN) and its association with disease progression, studies regarding the role of p16(INK4a) in human immunodeficiency virus (HIV)-infected patients remain scarce. The present study was designed to determine the potential utility of p16(INK4a) as a diagnostic marker for CIN and invasive cervical cancer in HIV-positive and negative cervical specimens. An immunohistochemical analysis of p16(INK4a) was performed in 326 cervical tissue microarray specimens. Performance indicators were calculated and compared using receiving operating characteristics curve (ROC)/area under the curve. In HIV-1-negative women, the percentage of cells that was positive for p16(INK4a) expression was significantly correlated with the severity of CIN (p < 0.0001). A ROC curve with a cut-off value of 55.28% resulted in a sensitivity of 89%, a specificity of 81%, a positive predictive value of 91% and a negative predictive value of 78%. HIV-seropositive women exhibited decreased expression of p16(INK4a) in CIN2-3 specimens compared with HIV-negative specimens (p = 0.031). The ROC data underscore the potential utility of p16(INK4a) under defined conditions as a diagnostic marker for CIN 2-3 staging and invasive cervical cancer. HIV-1 infection, however, is associated with relatively reduced p16(INK4a) expression in CIN 2-3.

Cytochemical analysis of the content of chicken thrombocytes vacuoles

The content of the large vacuoles present in chicken thrombocytes was analyzed by the use of cytochemical techniques which indicated the presence of basic proteins, unsaturated fatty acids, sugars rich in viccinal hydroxyl groups, linked to proteins or lipids and acid phosphatase. These substances perform one of the most conspicuous functions of these cells, which is phagocytosis. In addition, thrombocytes are committed to hemostasis. Both functions make these cells similar to human platelets, even though having different origins and morphologic characteristics. The large vacuoles described are connected to the open canalicular system (OCS) and together with other cellular structures they contribute to the endocytic system.

Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

Is Leishmania (Viannia) braziliensis parasite load associated with disease pathogenesis

BACKGROUND Leishmania (Viannia) braziliensis is the main etiological agent of tegumentary leishmaniasis in the Americas. Parasite molecular diversity and host immune status contribute to extensive variations in its clinical presentation within endemic areas of Brazil. Pentavalent antimonials have been used for more than 60 years as the first-line drug for all cases, despite the potential for severe side effects and refractoriness. In Rio de Janeiro, Brazil, most L. (V.) braziliensis infections are benign with a scarcity of parasites, although metastasis and refractory infections can arise. In this scenario, the use of novel molecular tools can be useful for diagnosis and to assess tissue parasitism, and is of benefit to clinical and therapeutic management. METHODS In this study, parasite load was assessed by real-time PCR based on the leishmanial small subunit ribosomal RNA gene. RESULTS AND CONCLUSION The data revealed a tendency to higher tissue parasitism in the skin compared to mucous lesion sites and a reduction with disease progression. Parasite load was lower in poor compared to good responders to antimonials, and was also reduced in recurrent lesions compared to primary ones. However, parasite load became higher with sequential relapses, pointing to an immune system inability to control the infection. Therefore the parasite burden does not seem to be a good predictor of disease progression.