Immunostaining of ßA-Activin and Follistatin Is Decreased in HPV(+) Cervical Pre-Neoplastic and Neoplastic Lesions.

The activin-follistatin system regulates several cellular processes, including differentiation and tumorigenesis. We hypothesized that the immunostaining of ßA-activin and follistatin varies in neoplastic cervical lesions. Cervical paraffin-embedded tissues from 162 patients sorted in control (n = 15), cervical intraepithelial neoplasia (CIN) grade 1 (n = 38), CIN2 (n = 37), CIN3 (n = 39), and squamous cell carcinoma (SCC; n = 33) groups were examined for ßA-activin and follistatin immunostaining. Human papillomavirus (HPV) detection and genotyping were performed by PCR and immunohistochemistry. Sixteen samples were inconclusive for HPV detection. In total, 93% of the specimens exhibited HPV positivity, which increased with patient age. The most detected high-risk (HR)-HPV type was HPV16 (41.2%) followed by HPV18 (16%). The immunostaining of cytoplasmatic ßA-activin and follistatin was higher than nuclear immunostaining in all cervical epithelium layers of the CIN1, CIN2, CIN3, and SCC groups. A significant decrease (p < 0.05) in the cytoplasmic and nuclear immunostaining of ßA-activin was detected in all cervical epithelial layers from the control to the CIN1, CIN2, CIN3, and SCC groups. Only nuclear follistatin immunostaining exhibited a significant reduction (p < 0.05) in specific epithelial layers of cervical tissues from CIN1, CIN2, CIN3, and SCC compared to the control. Decreased immunostaining of cervical ßA-activin and follistatin at specific stages of CIN progression suggests that the activin-follistatin system participates in the loss of the differentiation control of pre-neoplastic and neoplastic cervical specimens predominantly positive for HPV.

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.

BACKGROUND: 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. METHODS: The objective of the current study was to compare the results of liquid-based cytology and HPV DNA testing through polymerase chain reaction in 99 penile samples collected from men presenting with condyloma acuminate or male partners of HPV-infected women who had attended a public health service in the city of Belo Horizonte, Minas Gerais, Brazil. Classic and nonclassic cytomorphologic signs were adopted to evaluate the presence of HPV infections in penile smears. RESULTS: HPV DNA was detected in 93 (93.9%) of the 99 samples analyzed. Koilocytosis was detected in 1 smear and nonclassic signs were detected in 23 smears, 22 of which were found to be positive for HPV DNA. CONCLUSIONS: The cytopathologic detection of HPV infection in penile samples collected for liquid-based cytology is low, even when cytologic nonclassic signs are applied, and does not appear to improve the diagnosis of HPV infection in men.

Molecular detection of Chlamydia trachomatis and HPV infections in cervical samples with normal and abnormal cytopathological findings.

It has been suggested that Chlamydia trachomatis (CT) and human papillomaviruses (HPV) co-infection could contribute to development of intraepithelial lesions. In this study, HPV and CT-DNA were investigated in 250 cervicovaginal samples of patients from Minas Gerais, Brazil. The cytological analysis revealed that 70% of samples (175) were negative, 5.2% (13) presented atypical squamous or glandular cells of undetermined significance (ASCUS/AGUS), 12.4% (31) presented low-grade squamous intraepithelial lesion (LSIL), 10.8% (27) high-grade squamous intraepithelial lesion (HSIL), and 1.6% (4) invasive carcinoma. HPV-DNA and HPV/CT co-infection was observed in 40% (100/250) and in 5.2% (13/250) of samples, respectively. Among the positive cytological samples, HPV-DNA was detected in 73.3% and CT-DNA in 9.33% and in 13%, if only the HPV positive samples were considered. The highest co-infection rate (15.4%) was observed among ASCUS/AGUS samples. Although a significant association was found for HPV infection and the precursor lesions of cervical cancer, it was not possible to establish a significant association between these lesions and CT or HPV/CT co-infection.

Evaluation of MAT, IgM ELISA and PCR methods for the diagnosis of human leptospirosis.

Serum samples from 125 patients with clinical suspicion of leptospirosis were tested by the microscopic agglutination test (MAT), IgM ELISA and PCR for the diagnosis of the disease. Most patients were adult males and 74.1% (p<0.001) of the patients exposed to water and 77.6% (p<0.001) of those exposed to animals, were respectively considered confirmed or probable cases by MAT. The clinical symptoms mainly observed among the patients considered confirmed or probable cases were fever (95.6%), jaundice and headache (79.4%), myalgia (77.9%), nausea and vomiting (64.7%). About 63% of the confirmed or probable cases were patients that lived in Belo Horizonte, a big city of the Minas Gerais state, Brazil, showing the occurrence of urban leptospirosis. Among the 47 confirmed cases of leptospirosis diagnosed by MAT, 44 (94%) serum samples were positive by IgM ELISA and 17 (36%) were PCR positive. Among the 33 probable cases, 10 (30%) samples showed positive amplification by PCR. By considering MAT as the standard test, the sensitivity and specificity of IgM ELISA was 96.6% and 93.3%, respectively. A relevant finding in our study was the number of positive cases verified by PCR (13-29%) and IgM ELISA (3-7%) among the 45 unconfirmed cases by MAT, demonstrating the value of PCR in the early diagnosis of human leptospirosis.

Potential application of low-stringency single specific primer-PCR in the identification of Leptospira in the serum of patients with suspected leptospirosis.

In this study we tested the potential use of low-stringency single specific primer-PCR (LSSP-PCR) for genetically typing Leptospira directly from biological samples. Serum samples obtained from 29 patients with clinically suspected leptospirosis were amplified by specific PCR, using the previously selected G1 and G2 primers. The PCR products of approximately 300 bp were subsequently used as a template for LSSP-PCR analysis. We were able to produce genetic signatures from the leptospires present in the human samples, which permitted us to make a preliminary identification of the infective serovar by comparing the LSSP-PCR profiles obtained directly from serum samples with those from reference leptospires. Thus, LSSP-PCR has the potential to become a useful diagnostic tool for identifying leptospires in biological samples without the need for bacteria isolation and culture.

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.