Annamaria Ravara Vago

Genetic Characterization of Trypanosoma cruzi Directly from Tissues of Patients with Chronic Chagas Disease : Differential Distribution of Genetic Types into Diverse Organs

We have previously shown that a low-stringency single-specific primer-polymerase chain reaction (LSSP- PCR) is a highly sensitive and reproducible technique for the genetic profiling of Trypanosoma cruzi parasites directly in tissues from infected animals and humans. By applying LSSP-PCR to the study of the variable region of kinetoplast minicircle from T. cruzi, the intraspecific polymorphism of the kinetoplast-deoxyribonucleic acid (kDNA) sequence can be translated into individual kDNA signatures. In the present article, we report on our success using the LSSP-PCR technique in profiling the T. cruzi parasites present in the hearts of 13 patients with chagasic cardiopathy and in the esophagi of four patients (three of them with chagasic megaesophagus). In two patients, one with the cardiodigestive clinical form of Chagas disease and the other with cardiopathy and an esophageal inflammatory process, we could study both heart and esophagus and we detected distinct kDNA signatures in the two organs. This provides evidence of a differential tissue distribution of genetically diverse T. cruzi populations in chronic Chagas disease, suggesting that the genetic variability of the parasite is one of the determining factors of the clinical form of the disease.

Comparative study of the presence of Trypanosoma cruzi kDNA, inflammation and denervation in chagasic patients with and without megaesophagus.

Neuronal lesions have been considered the hallmark of chagasic megaesophagus, but the role of Trypanosoma cruzi and the participation of the inflammatory cells in this process are still debated. In the present study we counted neurons in the oesophagus from patients with and without megaesophagus and further examined these samples for the presence of parasite kDNA and cells with cytolytic potential (Natural Killer cells, cytotoxic lymphocytes and macrophages). The presence of parasite kDNA was demonstrated in 100% of cases with megaesophagus and in 60% of patients without megaesophagus. When analysed for the number of neurons, the patients without megaesophagus could be classified into 2 groups, as having normal or a decreased number of neurons. The former group did not show any inflammatory process, but interestingly, all patients without megaesophagus presenting decreased number of neurons also presented both parasite kDNA and inflammatory process in the organ. We further observed that the numbers of cytotoxic cells in the myenteric plexus region inversely correlate with the number of neurons. These data together strongly suggest that chronic lesions in chagasic megaesophagus might be a consequence of immune-mediated mechanisms, that last until the chronic phase of infection, and are dependent on the persistence of parasite in the hosts tissue.

KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

Background Visceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region. Principal Findings KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World. Conclusions LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains.

Minichromosome maintenance 7 protein is a reliable biological marker for human cervical progressive disease.

Objective This study focused on comparing the expression levels of p16, Ki-67, and minichromosome maintenance 7 (MCM7) protein in normal and affected cervical epithelium to ascertain the biological significance of these markers in detecting progressive cervical disease. Methods A quantitative and based on-scanning-microscopy analysis of the three markers expression was performed in normal and cervical intraepithelial neoplasia (CIN) I, II, and III tissues. p16 area as well as p16, Ki-67, and MCM7 positive cells or nuclei were evaluated according to their distribution and extent through the cervical epithelium. Results A clear p16 over-expression was observed in all the dysplastic epithelium tissue samples. The quantitative analysis of p16 area as well as the number of p16 positive cells was able to better discriminate the CIN lesions grades than the usual semi-quantitative analysis. The average Ki-67 labeling indexes for the normal epithelium, CIN I, CIN II, and CIN III groups were 19.8%, 27.3%, 32.8%, and 37.1%, respectively, whereas the mean MCM7 labeling indexes for the correspondent grades were 27.0%, 30.4%, 50.5%, and 67.2%. The Ki-67 and MCM7 labeling indexes were closely correlated with the CIN histological grade, with higher labeling indexe values obtained from the more severe lesions (p<0.05), being the MCM7 labeling indexes the highest values in all the CIN categories (p<0.05). Conclusion We observed a good correlation among the p16, Ki-67, and MCM7 data. In addition, MCM7 demonstrated to be a more efficient and sensitive marker to assess disease progression in the uterine cervix.

Detection of human papillomavirus infection in penile samples through liquid-based cytology and polymerase chain reaction

The human papillomavirus (HPV) is strongly related to cervical cancer and its precursor lesions. However, unlike in the case of women, there are limited data regarding HPV infection in men. Analysis of male HPV infection is frequently hindered by the lack of consistency in collection methods, sample adequacy, and low sensitivity of cytologic analysis.

Chronic Chagas disease: presence of parasite DNA in the oesophagus of patients without megaoesophagus

We have previously amplified Trypanosoma cruzi DNA by polymerase chain reaction (PCR) from the oesophagus of chagasic patients with megaoesophagus, whilst immunohistochemical analysis failed to detect T. cruzi antigen in the oesophagus of chagasic patients without megaoesophagus. During 2000-01, we tested for the presence of T. cruzi DNA in oesophageal tissue from 9 chronic chagasic patients without megaoesophagus and 5 were positive by PCR, which suggests that other factors, besides simply the presence of the parasite, should be considered in the understanding of the pathogenesis of megaoesophagus.

Langerhans Cells Ascertaining in Cervical Tissues Obtained from Women withCervical Intraepithelial Neoplasia

Objectives: Impairment of cell-mediated immunity in cervical cancer (CC) and intraepithelial neoplasia (CIN) has been reported. In this study, Langerhans cells (LC) subpopulations were quantitatively evaluated in cervical tissuesamples obtained from Brazilian patients exhibiting progressive grades of CIN, (CIN)I (n=3), CINII (n=3) and CINIII (n=3), in addition to three normal controls. Methods: The precise number of cervical LC was determined in the entire area of lesioned epithelium from CIN samples, by performing a morphometric analysis of Langerhans cells positive for two distinct markers, S100 and Langerin (Lang) detected either by Immunofluorescence or Immunohistochemistry analysis. Results: In normal cervix, a higher density of Langerin+ cells was observed whereas S100+ LC were predominant in pre-neoplastic lesions samples. Increased numbers of intraepithelial S100+ and Lang+ cells were observed in CIN samples, with an important predominance of Langerhans cell in CINII samples, despite the LC marker or the histological technique employed for the analysis. Curiously, a severe decrease in S100+, but particularly in Lang+ Langerhans cells was observed in cervical tissues exhibiting CINIII. Conclusions: Our results suggest that the morphometric evaluation of Langerhans cells number is an effective approach to determine LC number in cervical tissues, and that those immune-cells are possibly involved in the surveillance against the cervical lesion development.