Establishment and characterization of androgen-independent human prostate cancer cell lines, PcBra1, PcBra2, and PcBra3

One of the main obstacles for understanding biological events involved in cancer is the lack of experimental models for in vitro studies especially for prostate cancer (PC).There are a limited number of PC cell lines being the majority originated from metastatic tumors mostly acquired from American Tissue Cell Culture which demands importation an expensive and bureaucratic process. Also it is well known that there are ethnic differences between populations concerning the behavior of tumors and the research based on cell lines derived from Brazilians should be interesting. Our aim was to develop tumor cell lines from primary PC.

Mitochondrial DNA depletion and its correlation with TFAM, TFB1M, TFB2M and POLG in human diffusely infiltrating astrocytomas

Mitochondrial DNA (mtDNA) alterations and their clinical and pathological implications have been analyzed in several human malignancies. A marked decrease in mtDNA copy number along with the increase in malignancy was observed in diffusely infiltrating astrocytomas (24 WHO grade II, 18 grade III, and 78 grade IV or GBM) compared to non-neoplastic brain tissues, being mostly depleted in GBM. Although high relative gene expression levels of mtDNA replication regulators (mitochondrial polymerase catalytic subunit (POLG), transcription factors A (TFAM), B1 (TFB1M) and B2 (TFB2M)) were detected, it cannot successfully revert the mtDNA depletion observed in our samples. On the other hand, a strong correlation among the expression levels of mitochondrial transcription factors corroborates with the TFAM role in the direct control of TFB1M and TFB2M during initiation of mtDNA replication. POLG expression was related to decreased mtDNA copy number, and its overexpression associated with TFAM expression levels also have an impact on long-term survival among GBM patients, interpreted as a potential predictive factor for better prognosis. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Human T-cell lymphotropic virus type 1 infective dermatitis emerging in adulthood

Background Infective dermatitis (ID) is a rare dermatologic condition of childhood that has been linked to human T-cell lymphotropic virus type 1 (HTLV-1). Objective To analyze the clinical and laboratory features associated with adult-onset ID linked to HTLV-1. Methods From December 1995 to December 2007, four patients with ID were followed in the dermatology outpatient clinic of the ""Hospital das Clinicas"" of the University of Sao Paulo Medical School, Sao Paulo, Brazil. Epidemiologic data were collected and dermatologic examination was performed. Patients were submitted to histopathologic, hematologic, virologic, and immunologic investigations. Results All patients had a diagnosis of ID according to previously established criteria, despite being adults. HTLV-1 infection was demonstrated by enzyme-linked immunosorbent assay, Western blotting assays, and polymerase chain reaction. The male to female ratio was 1 : 3 and the median age at diagnosis was 42 years. The cutaneous manifestations were erythematous, scaly, and crusted lesions in all patients, and ichthyosis in three of the four cases. Histopathologic study showed lymphocytic epidermotropism in two cases. The median proviral load was 281 copies/10,000 peripheral blood mononuclear cells. Immunodeficiency was not observed in any case. The therapies used were antimicrobials, corticosteroids, and phototherapy. Conclusions Although many authors have considered ID to be a form of childhood dermatitis, we have described four cases that fulfilled the major criteria for ID, except for onset in adulthood.

Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients

Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe One was Bug, which amplified a sequence from the B1 gene The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were positive results were observed in 33.6% (50) patients The sensitivities were 98% for cnPCR (B22/B23), and 86 and 98% for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66 4% patients. The specificities were 97% for cnPCR and qrtPCR (B1Tg). and 88.8% for RETg These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers However, B1Tg PCR had good specificity, but lower sensitivity Among the patients, 20 had blood and CSF collected simultaneously Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive), (iii) positive molecular diagnosis with CSF and negative with blood, and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

The frequency of human papillomavirus findings in normal oral mucosa of healthy people by PCR

The human papillomavirus (HPV) is a DNA virus, which belongs to papillomaviridae family, being of low and high risk, which infect the skin and mucous membranes and can induce benign and malign tumor formation. In the oral mucosa they have been associated with oral papilloma, focal epithelial hyperplasia, leucoplakia and oral neoplasia. Aim: to study the frequency of HPV finding in oral mucosa of normal people. Materials and methods: Prospective study, cross-sectional cohort. One hundred volunteers, young adults, healthy, aged between 20 and 31 years, university students with no history, no complains, without oral or oropharyngeal lesions. They were submitted to a questionnaire with questions regarding HPV infection epidemiology. The samples were harvested by brushing and analyzed by PCR. Results: The results were negative for HPV in all samples. Conclusion: It seems we had high social and economical class individuals, with nutrition rich in carotenoyds and vitamin C, low smoking and alcohol consumption and heterosexual habits with predominant monogamy and regular use of condoms.

Human directed aggression in Brazilian domestic cats: owner reported prevalence, contexts and risk factors

Aggression by cats towards humans is a serious behavioural, welfare and public health problem, although owners may believe it is an inevitable part of cat ownership. There has been little scientific investigation of the risk factors associated with this problem. One hundred and seven owners in the Sao Paulo region of Brazil, took part in a survey aimed at investigating the perceived prevalence of the problem, defining the most common contexts of human directed aggression and identifying associated potential risk factors. Human directed aggression occurred in 49.5%, of cats and was most commonly associated with situations involving petting and play, followed by protection of a resource, when startled, when observing an unfamiliar animal and least commonly when unfamiliar people were present. Pedigree status, neuter status, a history of early trauma, sensitivity to being stroked, the absence of other cats in the home, relationship with other animals, level of background activity at home, access to the outside and tendency to be alone (meaning tendency to staying far from the family members) were all associated with an increased risk in one or more context. However, sex, age, age when acquired, source of pet, attachment to a specific household member, type of domestic accommodation, relationship with another cat if present and contact with other animals did not appear to increase the risk. The results suggest sensitivity to being stroked and background levels of stress in the home are the most pervasive risk factors, and future research should aim to investigate these factors further. These data are of relevance when advising owners about the risk and development of this problem. (C) 2009 ESFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Developing human minor salivary glands: morphological parallel relation between the expression of TGF-beta isoforms and cytoskeletal markers of glandular maturation

Morphogenesis of salivary glands involves complex coordinated events. Synchronisation between cell proliferation, polarisation and differentiation, which are dependent on epithelial-mesenchymal interactions and on the microenvironment, is a requirement. Growth factors mediate many of these orchestrated biological processes and transforming growth factor-beta (TGF-beta) appear to be relevant. Using immunohistochemistry and immunofluorescence, we have mapped the distribution of TGF-beta 1, 2 and 3 and compared it with the expression of maturation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. TGF-beta 1 first appeared during canalisation stage in the surrounding mesenchyme and, in the more differentiated stages, was expressed in the cytoplasm of acinar cells throughout the adult gland. TGF-beta 2 was detected since the bud stage of the salivary gland. Its expression was observed in ductal cells and increased along gland differentiation, TGF-beta 3 was detected from the canalisation stage of the salivary gland, being weakly expressed on ductal cells, and it was the only factor detected on myoepithelial cells. The data suggest that TGF-beta have a role to play in salivary gland development and differentiation.

Development of human minor salivary glands: expression of mucins according to stage of morphogenesis

The formation of salivary glands entails the proliferation of epithelial cells from the stomatodeum into the underlying ectomesenchyme, culminating in a complex network of ducts and acinar bulbs. The extent to which mucins regulate this process is unknown, but they appear to mediate luminal space formation and maturation. Our aim was to examine mucin expression patterns during the morphogenesis of human salivary glands. Mucin expression - MUC1, 2, 3, 4, 5AC, 5B, 6, and 16 - was analyzed in specimens of developing human salivary glands, obtained from fetuses at 4-24 weeks` gestation, and fully developed salivary glands by immunohistochemistry. Expression patterns were analyzed qualitatively according to the development stage of the salivary glands. Mucins 1, 3, 4, 5B, and 16 were expressed during salivary gland development - being stronger in all ductal segments by the final phases of branching morphogenesis and in mature glands. Acinar cells were negative for most mucins, including MUC1 in mature salivary glands. Mucins 2, 5AC, and 6 were not expressed. Mucins MUC1, 3, 4, 5B, and 16 are expressed in developing human salivary glands and in mature glands, suggesting important roles in the maturation and maintenance of the ductal network.

Oral manifestations of human T-cell lymphotropic virus infection in adult patients from Brazil

Objective: Human T-cell lymphotropic virus type 1 (HTLV-1) was the first human retrovirus discovered and its pathogenesis is related to T cells infection. This study aimed to verify the presence of oral manifestations in a Brazilian population of patients who was seropositive for HTLV, and identify risk factors for oral manifestations. Subjects and methods: An assessment was made of 139 patients at the Emilio Ribas Institute of Infectious Diseases. Results: A total of 112 (80.5%) patients were HTLV-1, 26 (18.7%) were HTLV-2+. About 35.2% of patients had myelopathy/tropical spastic paraparesis (HAM/TSP), with 48 of them being HTLV-1+ and one patient was seropositive for HTLV-1 and -2. The most common oral manifestations were: xerostomia (26.8%), candidiasis (20.8%), fissured tongue (17.9%), and loss of tongue papillae (10.0%). A multivariate logistic regression analysis showed that HAM/TSP is an independent risk factor for xerostomia (P = 0.02). The patients who were HAM/TSP+ were three times more likely to develop xerostomia when compared with patients without HAM/TSP (odds ratio = 2.69, 95% confidence interval = 1.17-6.17). Conclusion: Despite the fact that the findings of this study suggest a relationship between xerostomia and HAM/TSP, more studies should be developed to show what the association would be between xerostomia presented by HTLV patients and pathogenesis of the virus.

Differential patterns of receptor activator of nuclear factor kappa B ligand/osteoprotegerin expression in human periapical granulomas: Possible association with progressive or stable nature of the lesions

Receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) are expressed in apical periodontitis, suggesting a role for these molecules during lesion development. However, the profiles of RANKL/OPG expression in periapical lesions remain unknown. In this study we investigated the patterns of RANKL and OPG mRNA expression by real-time polymerase chain reaction in human periapical granulomas (N = 44) and compared them with sites presenting characteristic bone resorbing activity: healthy (n = 14) and orthodontically stretched and compressed periodontal ligament (n = 26), healthy gingiva (n = 24), chronic gingivitis (n = 32), and chronic periodontitis (n = 34) samples. Both RANKL and OPG mRNA expression was higher in periapical granulomas when compared with healthy periodontal ligament. Distinct patterns of RANKL and OPG expression ratio were found in the granulomas and in different physiologic and pathologic conditions, with characteristic bone resorption activity potentially being indicative of the stable or progressive nature of the lesions. Lesions with radiographic image smaller than 5 mm showed higher RANKL/OPG expression than images greater than 5 mm. Periapical granulomas presented heterogeneous patterns of RANKL and OPG expression, ranging from samples with RANKL/OPG ratio similar to that seen in sites with minimal or absent bone resorption to samples with RANKL/OPG expression pattern comparable with active bone resorption sites.

Effect of growth hormone on in vitro osteogenesis and gene expression of human osteoblastic cells is donor-age-dependent

it has been demonstrated that the effect of GH on bone tissue is reduced with aging. In this study we tested the hypothesis that the action of GH on osteoblastic cells is donor-age-dependent by investigating the effect of GH on the development of osteoblastic phenotype in cultures of cells from adolescents (13-16 years old), young adults (18-35 years old), and adults (36-49 years old). Osteoblastic cells derived from human alveolar bone were cultured with or without GH for periods of up to 21 days, and parameters of in vitro osteogenesis and gene expression of osteoblastic markers were evaluated. GH increased culture growth, collagen content and alkaline phosphatase (ALP) activity in cultures from adolescents and young adults, whereas non-significant effect was observed in cultures from adults. While GH significantly increased the bone-like formation in cultures from adolescents, a slightly effect was observed in cultures from young adults and no alteration was detected in cultures from adults. Results from real-time PCR demonstrated that GH upregulated ALP, osteocalcin, type I collagen, and Cbfa1 mRNA levels in cultures from adolescents. In addition, cultures from young adults showed higher ALP mRNA expression and the expression of all evaluated genes was not affected by GH in cultures from adults. These results indicate that the GH effect on both in vitro osteogenesis and gene expression of osteoblastic markers is donor-age-dependent, being more pronounced on cultures from adolescents.