Anatomical features of the urethra and urinary bladder catheterization in female mice and rats. An essential translational tool

PURPOSE: To present fundamental anatomical aspects and technical skills necessary to urethra and urinary bladder catheterization in female mice and rats. METHODS: Urethral and bladder catheterization has been widely utilized for carcinogenesis and cancer research and still remains very useful in several applications: from toxicological purposes as well as inflammatory and infectious conditions to functional aspects as bladder dynamics and vesicoureteral reflux, among many others. RESULTS: Animal models are in the center of translational research and those involving rodents are the most important nowadays due to several advantages including human reproducibility, easy handling and low cost. CONCLUSIONS: Although technical and anatomical pearls for rodent urethral and bladder access are presented as tackles to the advancement of lower urinary tract preclinical investigation in a broaden sight, restriction to female animals hampers the male microenvironment, demanding future advances.

Inorganic UV filters

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of the expression of p53, MDM2, and SUMO-1 in oral lichen planus

Objective: The objective of this study was to compare the expression of proteins p53, MDM2, and SUMO-1 in oral lichen planus (OLP) lesions, epithelial dysplasia, and squamous cell carcinoma. Materials and Methods: The sample consisted of the following five groups of cheek mucosa lesions: normal mucosa (NM), inflammatory fibrous hyperplasia (IFH), lichen planus, epithelial dysplasia, and squamous cell carcinoma. The tissue samples were stained with hematoxylin-eosin and submitted to immunohistochemistry using anti-p53, anti-MDM2, and anti-SUMO-1 antibodies. Results: The results of this study demonstrated similar expression of p53 and MDM2 between OLP, oral epithelial dysplasia and, to a lesser extent, between OLP and oral squamous cell carcinoma (OSCC). However, for SUMO-1 a similar expression was observed in OLP, NM, and IFH. Conclusions: The results demonstrated overexpression of important proteins (p53 and MDM2) related to regulatory mechanisms of apoptosis in OLP, suggesting that there is a favorable environment for malignant transformation. The expression of SUMO-1 in OLP was similar to NM and IFH, suggesting that alterations of this protein occur at later stages of carcinogenesis, because important overexpression occurred in oral epithelial dysplasia and OSCC. © 2013 John Wiley & Sons A/S.

Expression of annexin-A1 and galectin-1 anti-inflammatory proteins and mRNA in chronic gastritis and gastric cancer

Objective. The anti-inflammatory proteins annexin-A1 and galectin-1 have been associated with tumor progression. This scenario prompted us to investigate the relationship between the gene and protein expression of annexin-A1 (ANXA1/AnxA1) and galectin-1 (LGALS1/Gal-1) in an inflammatory gastric lesion as chronic gastritis (CG) and gastric adenocarcinoma (GA) and its association with H. pylori infection. Methods. We analyzed 40 samples of CG, 20 of GA, and 10 of normal mucosa (C) by the quantitative real-time PCR (qPCR) technique and the immunohistochemistry assay. Results. High ANXA1 mRNA expression levels were observed in 90% (36/40) of CG cases (mean relative quantification RQ = 4.26 ± 2.03) and in 80% (16/20) of GA cases (mean RQ = 4.38 ± 4.77). However, LGALS1 mRNA levels were high (mean RQ = 2.44 ± 3.26) in 60% (12/20) of the GA cases, while low expression was found in CG (mean RQ = 0.43 ± 3.13; P < 0.01). Normal mucosa showed modest immunoreactivity in stroma but not in epithelium, while stroma and epithelium displayed an intense immunostaining in CG and GA for both proteins. Conclusion. These results have provided evidence that galectin-1 and mainly annexin-A1 are overexpressed in both gastritis and gastric cancer, suggesting a strong association of these proteins with chronic gastric inflammation and carcinogenesis. © 2013 Yvana Cristina Jorge et al.

Profiles of gene polymorphisms in cytokines and toll-like receptors with higher risk for gastric cancer

Background: Chronic inflammation and gastric carcinogenesis show a close association, so gene polymorphisms that modify the intensity of the inflammatory response may contribute to variations in gastric cancer risk. Aims: The purpose of this study was to investigate the combined effect of the pro- and anti-inflammatory cytokines and toll-like receptors polymorphisms on the chronic gastritis and gastric cancer risk in a Brazilian population sample. Methods: We evaluated 669 DNA samples (200 of gastric cancer [GC], 229 of chronic gastritis [CG], and 240 of healthy individuals [C]). Ten polymorphisms were genotyped: IL-1RN and TLR2 -196 to -174 del using the allele-specific PCR method and TNF-A (rs1800629; rs1799724), TNF-B (rs909253), IL-8 (rs4073; rs2227532), IL-10 (rs1800872) and TLR4 (rs4986790; rs4986791) using PCR-RFLP. Results: Polymorphisms TNF-A-308G/A, IL-8-251A/T, TNF-B + 252A/G and TLR4 + 1196C/T were not associated with risk of any gastric lesion. However, an association with increased risk for GC was observed for polymorphisms IL-1RNL/2 (p < 0.001), TNF-A-857C/T (p = 0.022), IL-8-845T/C (p < 0.001), IL-10-592C/A (p < 0.001), TLR2ins/del (p < 0.001), and TLR4 + 896A/G (p = 0.033). In CG, an association was observed only with polymorphisms IL-1RNL/2 and IL-10-592A/C (p < 0.001 for both). A combined analysis of these six polymorphisms associated with GC revealed a profile with two to four combined genotypes which confer a higher risk of gastric carcinogenesis, with an OR increased 2.95-fold to 50.4-fold, highlighting the combinations IL-1RN2/TNF-A-857T/IL-8-845C, IL-1RN2/IL-8-845C/TLR2del, IL-1RN2/IL-10-592A/TLR4 + 896G, IL-10-592A/TLR2del/ TLR4 + 896G, and IL-1RN2/TNFA-857T/IL8-845C/TLR2del. Conclusions: Our findings evidenced that the combined effect of polymorphisms in genes involved in the inflammatory process may potentiate the risk of gastric cancer, thus emphasizing the importance of evaluating multiple polymorphisms together. © 2012 Springer Science+Business Media New York.

Digit ratio (2D:4D) is associated with gastric cancer

Background: Sex steroid exposure during early human development may influence disease susceptibility. Digit ratio (2D:4D) is a putative marker for prenatal hormone exposure and sensitivity, as well as the action of genes closely related to carcinogenesis. Digit ratio could act as a possible marker for cancer predisposition. Aims: The aim of this study is to investigate the possible correlations between right hand, left hand and right minus left (R. - L) 2D:4D and gastric cancer (GCA) in men and women and assess the correlations with tumor staging and histological diagnosis. Methods: Digital images of the right and left hand palms of patients diagnosed with GCA (n = 57, 42 males, 15 females) and age and sex-matched controls (n = 59, 41 males, 18 females) were obtained. Means for 2D:4D were compared. Data were analyzed by repeated-measures one-way ANOVA and Student's t-test for finger measurements and group comparisons and Pearson's and Spearman's tests for correlations with tumor staging (α = 0.05). Results: GCA group presented significantly higher left 2D:4D, but significantly lower R. - L in comparison to healthy controls, particularly so for males. Digit ratio did not correlate to clinical staging or TNM staging. However, low R. - L was significantly related to adenocarcinomas. Conclusions: Early developmental conditions, including prenatal testosterone seem to play a role on the malignant transformation of gastric lesions. The 2D:4D pattern found for gastric cancer parallels that earlier described for breast cancer. The findings suggest that 2D:4D could add to the list of etiological factors and be a putative marker for the screening of patients' susceptibility to develop gastric cancer. © 2012 Elsevier Ltd.

Effects of Ginkgo biloba on chemically-induced mammary tumors in rats receiving tamoxifen

Background: Ginkgo biloba extract (GbE) is used extensively by breast cancer patients undergoing treatment with Tamoxifen (TAM). Thus, the present study investigated the effects of GbE in female Sprague-Dawley (SD) rats bearing chemically-induced mammary tumors and receiving TAM.Methods: Animals bearing mammary tumors (≥1 cm in diameter) were divided into four groups: TAM [10 mg/kg, intragastrically (i.g.)], TAM plus GbE [50 and 100 mg/kg, intraperitoneally (i.p.)] or an untreated control group. After 4 weeks, the therapeutic efficacy of the different treatments was evaluated by measuring the tumor volume (cm3) and the proportions of each tumor that were alive, necrotic or degenerative (mm2). In addition, labeling indexes (LI%) were calculated for cell proliferation (PCNA LI%) and apoptosis (cleaved caspase-3 LI%), expression of estrogen receptor-alpha (ER-α) and p63 biomarkers.Results: Overall, the tumor volume and the PCNA LI% within live tumor areas were reduced by 83% and 99%, respectively, in all TAM-treated groups when compared to the untreated control group. GbE treatment (100 mg/kg) reduced the proportions of live (24.8%) and necrotic areas (2.9%) (p = 0.046 and p = 0.038, respectively) and significantly increased the proportion of degenerative areas (72.9%) (p = 0.004) in mammary tumors when compared to the group treated only with TAM. The expression of ER-α, p63 and cleaved caspase-3 in live tumor tissues was not modified by GbE treatment.Conclusions: Co-treatment with 100 mg/kg GbE presented a slightly beneficial effect on the therapeutic efficacy of TAM in female SD rats bearing mammary tumors. © 2013 Dias et al.; licensee BioMed Central Ltd.

Dragon's blood Croton palanostigma induces genotoxic effects in mice

Ethnopharmacological relevance Dragon's blood is a dark-red sap produced by species from the genus Croton (Euphorbiaceae), which has been used as a famous traditional medicine since ancient times in many countries, with scarce data about its safe use in humans. In this research, we studied genotoxicity and clastogenicity of Croton palanostigma sap using the comet assay and micronucleus test in cells of mice submitted to acute treatment. Material and methods HPLC analysis was performed to identify the main components of the sap. The sap was administered by oral gavage at doses of 300 mg/kg, 1000 mg/kg and 2000 mg/kg. For the analysis, the comet assay was performed on the leukocytes and liver cells collected 24 h after treatment, and the micronucleus test (MN) on bone marrow cells. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Results and conclusion The alkaloid taspine was the main compound indentified in the crude sap of Croton palanostigma. The results of the genotoxicity assessment show that all sap doses tested produced genotoxic effects in leukocytes and liver cells and also produced clastogenic/aneugenic effects in bone marrow cells of mice at the two higher doses tested. The PCE/NCE ratio indicated no cytotoxicity. The data obtained suggest caution in the use of Croton palanostigma sap by humans considering its risk of carcinogenesis. © 2013 Elsevier B.V. All rights reserved.

Efeitos da polpa do fruto de annona crassiflora mart., nos parâmetros bioquímicos e na carcinogênese de colon de ratos

A. crassiflora Mart. a Brazilian savannah fruit, is a source of phytochemical compounds that possess a wide array of biological activities, including free radical scavenging. This native fruit proved to potentialize the mutagenic process in previous in vivo investigations. The aim of the present study was to investigate the effects of A. crassiflora Mart. pulp intake on colonic cell proliferation and on the development of Aberrant Crypt Foci (ACF) in male Wistar rats. The animals were fed with either a commercial diet or a diet supplemented with A. crassiflora Mart. pulp mixed in 1%, 10% or 20% (w/w) for 4 weeks or 20 weeks. The carcinogen 1,2-dimethylhydrazine dihydrochloride (4 doses, 40 mg kg-1 each) was used to induce colonic ACF. After euthanasia, the blood, liver and colon samples were collected for biochemical determinations, oxidative stress or ACF development analysis, respectively. Immunohistochemical analyses of the colonic mucosa were performed using antibodies against proliferating cell nuclear antigen (PCNA) in normal-appearing colonic crypt and β-catenin in ACF. There was no ACF development in the colon from groups treated with A. crassiflora Mart. pulp. Also, the biochemical and oxidative stress analysis, PCNA labeling and ACF development (number, multiplicity or cellular localization of β-catenin) were unchanged as a result of marolo pulp intake. Thus, the present results suggest that A. crassiflora Mart. pulp intake did not exert any protective effect in the colon carcinogenesis induced by DMH in rats.

Chromosomal imbalances in successive moments of human bladder urothelial carcinoma

Objective: To understand developmental characteristics of urinary bladder carcinomas (UBC) by evaluating genomic alterations and p53 protein expression in primary tumors, their recurrences, and in the morphologically normal urothelium of UBC patients. Methods: Tumors and their respective recurrences, six low-grade and five high-grade cases, provided 19 samples that were submitted to laser microdissection capture followed by high resolution comparative genomic hybridization (HR-CGH). HR-CGH profiles went through two different analyses-all tumors combined or classified according to their respective histologic grades. In a supplementary analysis, 124 primary urothelial tumors, their recurrences, and normal urothelium biopsied during the period between tumor surgical resection and recurrence, were submitted to immunohistochemical analyses of the p53 protein. During the follow-up of at least 21 patients, urinary bladder washes citologically negative for neoplastic cells were submitted to fluorescence in situ hybridization (FISH) to detect copy number alterations in centromeres 7, 17, and 9p21 region. Results and Conclusions: HR-CGH indicated high frequencies (80%) of gains in 11p12 and losses in 16p12, in line with suggestions that these chromosome regions contain genes critical for urinary bladder carcinogenesis. Within a same patient, tumors and their respective recurrences showed common genomic losses and gains, which implies that the genomic profile acquired by primary tumors was relatively stable. There were exclusive genomic alterations in low and in high grade tumors. Genes mapped in these regions should be investigated on their involvement in the urinary bladder carcinogenesis. Successive tumors from same patient did not present similar levels of protein p53 expression; however, when cases were grouped according to tumor histologic grades, p53 expression was directly proportional to tumor grades. Biopsies taken during the follow-up of patients with history of previously resected UBC revealed that 5/15 patients with no histologic alterations had more than 25% of urothelial cells expressing the p53 protein, suggesting that the apparently normal urothelium was genomically unstable. No numerical alterations of the chromosomes 7, 17, and 9p21 region were found by FISH during the periods free-of-neoplasia. Our data are informative for further studies to better understand urinary bladder urothelial carcinogenesis. © 2013 Elsevier Inc.

Expression, purification and molecular analysis of the human ZNF706 protein

Background: The ZNF706 gene encodes a protein that belongs to the zinc finger family of proteins and was found to be highly expressed in laryngeal cancer, making the structure and function of ZNF706 worthy of investigation. In this study, we expressed and purified recombinant human ZNF706 that was suitable for structural analysis in Escherichia coli BL21(DH3). Findings. ZNF706 mRNA was extracted from a larynx tissue sample, and cDNA was ligated into a cloning vector using the TOPO method. ZNF706 protein was expressed according to the E. coli expression system procedures and was purified using a nickel-affinity column. The structural qualities of recombinant ZNF706 and quantification alpha, beta sheet, and other structures were obtained by spectroscopy of circular dichroism. ZNF706's structural modeling showed that it is composed of α-helices (28.3%), β-strands (19.4%), and turns (20.9%), in agreement with the spectral data from the dichroism analysis. Conclusions: We used circular dichroism and molecular modeling to examine the structure of ZNF706. The results suggest that human recombinant ZNF706 keeps its secondary structures and is appropriate for functional and structural studies. The method of expressing ZNF706 protein used in this study can be used to direct various functional and structural studies that will contribute to the understanding of its function as well as its relationship with other biological molecules and its putative role in carcinogenesis. © 2013 Colombo et al.; licensee BioMed Central Ltd.

An approach to local immunotoxicity induced by adjuvanted vaccines

The occurrence of injection site reactions following immunization is the most frequently reported toxicity manifestation of vaccines; however, the different types of local reactions and the different mechanisms involved are still unclear. Here, the current advances in adjuvants and the role that adjuvants play in local reactions are reviewed. The role of adjuvants in the formation of the loco-regional complex (LRC), which consists of the injection site, draining lymphatic vessels and regional lymph nodes, is also discussed. Finally, strategies and recommendations for the rational design of adjuvanted vaccines are discussed, with a particular interest in the reduction of local inflammation. © 2013 Elsevier B.V.

Methylation pattern of THBS1, GATA-4, and HIC1 in pediatric and adult patients infected with helicobacter pylori

Background: Helicobacter pylori infection is usually acquired in childhood and persists into adulthood if untreated. The bacterium induces a chronic inflammatory response, which is associated with epigenetic alterations in oncogenes, tumor-suppressor genes, cell-cycle regulators, and cell-adhesion molecules. Aim: The aim of this study was to analyze the effect of H. pylori infection on the methylation status of Thrombospondin-1 (THBS1), Hypermethylated in cancer 1 (HIC1) and Gata binding protein-4 (GATA-4) in gastric biopsy samples from children and adults infected or uninfected with the bacterium and in samples obtained from gastric cancer patients. Methods: The methylation pattern was analyzed with methylation-specific PCR. Results: Our results showed that H. pylori infection was associated with methylation of the promoter regions of the THBS1 and GATA-4 genes in pediatric and adult samples (p < 0.01). HIC1 showed the lowest level of methylation, which was not an early event during gastric carcinogenesis. Conclusions: The results from this study indicate that methylation of THBS1 and GATA-4 occurs in the early stages of chronic gastritis and gastric cancer in association with H. pylori infection; however, in gastric cancer samples, other mechanisms cooperate with the down-regulation of these genes. Methylation of HIC1 may not be the principal mechanism implicated in its down-regulation in gastric cancer samples. © 2013 Springer Science+Business Media New York.

Impact of Epstein-Barr virus load, virus genotype, and frequency of the 30bp deletion in the viral BNLF-1 gene in patients harboring the human immunodeficiency virus

Patients infected with the human immunodeficiency virus (HIV) are at higher risk of developing Epstein-Barr Virus (EBV)-associated lymphomas. The usefulness of monitoring EBV in peripheral blood mononuclear cells (PBMCs) of patients infected with HIV has not been established. The aim of this study was to evaluate the EBV viral load in PBMCs, the frequency of viral genotypes, and the presence of the 30-bp deletion in the BNLF-1 gene. DNA samples from 156 patients attending the HIV/AIDS Day Clinic at Botucatu School of Medicine, Sao Paulo State University were evaluated. The EBV viral load was detectable by real time PCR in 123/156 (78.8%) cases and was higher in patients not receiving antiretroviral treatment or under therapeutic failure than in patients under successful highly active antiretroviral therapy (HAART) (P=0.0076). Overall, the profile of patients with high EBV viral load included elevated HIV viremia (P=0.0005), longer time of HIV diagnosis (P=0.0026), and increased levels of T CD8 + lymphocytes (P=0.0159). The successful amplification of the EBNA-2 gene by nested-PCR was achieved in 95 of 123 (77.2%) cases, of which 75.8% were EBV-1, 9.5% EBV-2, and 14.7% were co-infected with both EBV-1 and -2. The analysis of the BNLF-1 gene was possible in 99 of 123 (80.5%) cases, of which 50.5% had the 30-bp deletion. EBV-1 was more common than EBV-2, which may reflect the fact that the cohort was predominantly Caucasian and heterosexual. J. Med. Virol. 85:2110-2118, 2013. © 2013 Wiley Periodicals, Inc.

Efeitos de ultradiluições de drogas carcinogênicas iniciadoras e da dexametasona na carcinogênese hepática de ratos

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC