Suitability of DNA extracted from archival specimens of fruit-eating bats of the genus Artibeus (Chiroptera, Phyllostomidae) for polymerase chain reaction and sequencing analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Epstein-Barr virus-associated gastric carcinoma in Brazil: comparison between in situ hybridization and polymerase chain reaction detection

Epstein-Barr virus (EBV) has been associated with 10% of gastric carcinomas. The aim of this study was to determine the frequency of EBV in gastric carcinomas in Brazil assessed by in situ hybridization (ISH) and PCR, which would contribute to the characterization of the clinical and pathological aspects of EBV-associated gastric carcinomas. One hundred and ninety-two gastric carcinoma cases were collected at hospitals in two Brazilian states. Seventy-three out of 151 cases were PCR(+), while 11/160 cases were ISH(+). Nine out of eleven ISH(+) cases displayed a diffuse staining pattern and 2 out of 11 a focal pattern. Both techniques showed that the EBV(+) cases were characterized by their association with males, older patients, lower gastric region, intestinal type, advanced stage and poorly to moderately differentiated tumors. The concordance between the two techniques was 55.8% (Cohen's kappa index = 0.034). Four cases were ISH(+)/PCR(-), while 49 cases were PCR(+)/ISH(-). Only two cases showed stained lymphocytes by ISH and one of them was PCR(-). The observed discrepancy between the two techniques could not be explained just by the elevated accuracy of PCR. ISH(+)/PCR(-) carcinomas may be encountered if EBV is not present in the whole tumor tissue or if there are polymorphisms in the sequences of the viral genome amplified. on the other hand, the high frequency of PCR(+) results associated with the absence of ISH staining in lymphocytes and/or tumors cells suggests that the virus may be present in tumor cells or other cell types without expressing EBER1, the target of the ISH technique.

Correlation among immune response, morphogenesis of the granulomatous reaction and spleen lymphoid structure in murine experimental paracoccidioidomycosis

We studied the correlation among cellular immune response, the pattern of lung granulomatous lesions and alterations in spleen lymphoid structure in Swiss mice inoculated intravenously with Paracoccidioides brasiliensis strain 18. The animals were evaluated at 24, 48 and 96 h after infection and further studied weekly for 18 weeks by: (i) the macrophage migration inhibition test with phytohemagglutinin (PHA) and P. brasiliensis antigen (PbAg); and (ii) histopathology of the lung and spleen lesions. One group of animals was gamma -irradiated (8 Gy), infected under the same conditions and evaluated for the pattern of lung granulomatous lesions and spleen lymphoid structure at 24, 48 and 96 h after infection. During the first week of infection, the non-irradiated animals presented a positive response to PHA and PbAg, compact granulomas in the lungs and a typical hyperplasia of the spleen white pulp. However, from weeks 2 to 5, a depression of the cell-mediated immunity (CMI) response to PHA and PbAg was observed in association with granulomas presenting only large mononuclear cells and lacking both giant cells and a peripheral halo of small mononuclear cells. This pattern of granuloma formation was similar to that seen in gamma -irradiated animals, whose cells involved in CMI were absent. After week 7, the non-irradiated animals showed granulomas characterized by the presence of giant cells and a peripheral halo of small mononuclear cells. This type of granuloma was formed concomitantly with recovery of the CMI and of the lymphoid structure of the spleen. The results showed a correlation among granulomas composed of large mononuclear cells, hypoplasia of the splenic tissue and impaired CMI. This correlation indicated that although granuloma morphogenesis per se does not depend on the activation of CMI, this response is important at later stages during modulation of the cellular composition of the granulomas.

Deletion of Epstein-Barr virus latent membrane protein 1 gene in United States and Brazilian Hodgkin's disease and reactive lymphoid tissue: High frequency of a 30-bp deletion

A 30-basepair (bp) deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene has been reported in nasopharyngeal carcinoma and EBV-associated malignant lymphomas. Prior studies have found the deletion in about 10% to 28% of cases of Hodgkin's disease (HD), particularly in cases with aggressive histology. We studied the prevalence of 30-bp LMP1 gene deletion in EBV-positive HD in the United States (US) (12 cases) and Brazil (26 cases) with comparison to reactive lymphoid tissues (21 cases) and HD without EBV-positive Reed-Sternberg cells (15 cases). We studied the status of the LMP1 gene by Southern blot hybridization of polymerase chain reaction (PCR) products obtained after amplification with primers spanning the site of the deletion. We also performed EBV typing, EBER1 in situ hybridization, and LMP1 protein immunohistochemistry. EBV was detected in 12/26 (46%) cases of HD from the US and 26/27 (96%) cases of Brazilian HD. The 30-bp LMP1 gene deletion was observed in 4/12 (33%) cases of EBV-positive HD from US, and 12/26 (46%) cases of Brazilian EBV-positive HD, including 3 cases of type B EBV, as compared with 12/21 (57%) reactive lymphoid tissues and 9/15 (60%) cases of EBV-negative HD. US and Brazilian HD showed a higher prevalence of the 30-bp LMP1 gene deletion, compared with studies of others. The unexpected finding of high incidence of 30-bp deletion in LMP1 gene in reactive lymphoid tissue and HD without EBV-positive Reed-Sternberg cells suggests that this deletion may not be relevant to HD pathogenesis in most cases. Copyright (C) 1997 by W.B. Saunders Company.

Histopathological changes induced by extracts from the tissue covering the stingers of Potamotrygon falkneri freshwater stingrays

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

Reaction of dogs' teeth to root canal filling with mineral trioxide aggregate or a glass ionomer sealer

This study was conducted to observe the reaction of apical tissues of dogs' teeth after root canal filling with gutta-percha and mineral trioxide aggregate (MTA) or a glass ionomer (Ketac-Endo) as a sealer. The root canals were instrumented and filled by the lateral condensation technique with the sealers studied. Animals were killed 6 months later, and the specimens were removed and prepared for histological analysis. Results showed no inflammatory reaction of apical tissue and total closure of the apical foramen of all the teeth sealed with MTA. The teeth sealed with Ketac-Endo showed two cases of partial closure and different degrees of chronic inflammatory reaction. In conclusion, MTA exhibited better biological properties than Ketac-Endo. Copyright © 1999 by The American Association of Endodontists.

The detection of Toxoplasma gondii by comparing cytology, histopathology, bioassay in mice, and the polymerase chain reaction (PCR)

The objective of this study was to compare the different methods of detecting Toxoplasma gondii in sheep tissue, tested serologically positive by the indirect immunofluorescent antibody test (IFAT). Brain, diaphragm, and blood samples were collected from 522 sheep slaughtered at the São Manuel abattoir, São Paulo State, Brazil. Brain and diaphragm samples from IFAT seropositive animals were digested by both trypsin and pepsin and then injected into mice. Part of the digested samples was used to prepare slides for Giemsa staining and in the polymerase chain reaction (PCR). Tissue fragments were fixed in formalin and examined using hematoxilin-eosin (HE). Forty of the sheep (7.7%) were IFAT positive. T. gondii was isolated in 23 (59.0%) of the 39 mice with pepsin-digested brain samples and in 27 (69.0%) of the 39 with trypsin-digested brain samples. Injection of diaphragm samples led to T. gondii isolation in 26 (66.7%) of the 39 pepsin-digested samples and 21 (53.8%) of the 39 trypsin-digested samples. Cytological and hystopathological examination of both brains and diaphragms was negative in all examined sheep. PCR was positive in 7 (17.9%) of the trypsin and 2 (5.1%) of the pepsin-digested samples, while 9 (23.1%) of the trypsin and 3 (7.7%) of the pepsin-digested samples showed T. gondii DNA. T. gondii isolation rate in mice (n = 34; 85.0%) was significantly higher than detection by PCR (n = 15; 37.5%). © 2001 Elsevier Science B.V.

Comparative analysis of tissue reactions to anesthetic solutions: histological analysis in subcutaneous tissue of rats.

Postanesthetic pain is a relatively common complication after local anesthesia. This complication may be caused by the anesthetic technique or by the anesthetic solution used. Tissue reactions induced by the anesthetic solutions may be one of the factors resulting in pain after anesthesia. The objective of this study was to comparatively analyze tissue reactions induced by different anesthetic solutions in the subcutaneous tissue of rats. The following solutions were utilized: 2% lidocaine without vasoconstrictor; a 0.5% bupivacaine solution with 1:200,000 adrenaline; a 4% articaine solution and 2% mepivacaine, both with 1:100,000 adrenaline; and a 0.9% sodium chloride solution as a control. Sterilized absorbent paper cones packed inside polyethylene tubes were soaked in the solutions and implanted in the subcutaneous region. The sacrifice periods were 1, 2, 5, and 10 days after surgery. The specimens were prepared and stained with hematoxylin and eosin for histological analysis. The results showed that there is a difference in tissue irritability produced by the local anesthetic solutions. The results also showed that there is no relation between the concentration of the drug and the inflammatory intensity, that the mepivacaine and articaine solutions promoted less inflammatory reaction than the bupivacaine, and that the lidocaine solution produced the least intense inflammation.

Tamoxifen down-regulates CaMKII messenger RNA levels in normal human breast tissue

Tamoxifen was proven to reduce the incidence of breast cancer by 49% in women at increased risk of the disease in the Breast Cancer Prevention Trial. In order to identify potential candidates to explain the preventive effect induced by tamoxifen on breast cancer, normal breast tissue obtained from 42 fibroadenoma patients, randomly assigned to receive placebo or tamoxifen, was analyzed by the reverse Northern blot and RT-PCR techniques. The cDNA fragments used on Northern blot membranes were generated by the Human Cancer Genome Project funded by the Ludwig Institute for Cancer Research and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil). Total RNA was obtained from normal breast tissue from patients with clinical, cytological and ultrasound diagnosis of fibroadenoma. After a 50-day treatment with tamoxifen (10 or 20 mg/day) or placebo, normal breast tissue adjacent to the tumor was collected during lumpectomy with local anesthesia. One differentially expressed gene, Calcium/calmodulin-dependent protein kinase II (CaMKII), was found to be down-regulated during TAM treatment. CaMKII is an ubiquitous serine/threonine protein kinase that has been implicated in the diverse effects of hormones utilizing Ca2+ as a second messenger as well as in c-fos activation. These results indicate that the down-regulation of CaMKII induced by TAM might represent alternative or additional mechanisms of the action of this drug on cell cycle control and response to hormones in normal human breast tissue.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system. © 2006 Coelho-Castelo et al; licensee BioMed Central Ltd.

Toxoplasma gondii: Detection by mouse bioassay, histopathology, and polymerase chain reaction in tissues from experimentally infected pigs

In the present study, we evaluated three techniques, mouse bioassay, histopathology, and polymerase chain reaction (PCR) to detect Toxoplasma gondii infection in tissues from experimentally infected pigs. Twelve mixed breed pigs, seronegative for T. gondii using an indirect immunofluorescent antibody test (IFAT), were used. Ten pigs were infected with 4 × 104 VEG strain oocysts, and two were maintained as uninfected controls. Animals were killed 60 days pos infection. Muscle (heart, tongue, diaphragm, and masseter) and brain samples were collected to investigate the presence of T. gondii tissue cysts by the different assay methods. For the bioassay, samples of brain (50 g) and pool of muscle samples (12.5 g of tongue, masseter, diaphragm, and heart) were used. PCR was performed using Tox4 and Tox5 primers which amplified a 529 bp fragment. The DNA extraction and PCR were performed three times, and all tissue samples were tested individually (brain, tongue, masseter, diaphragm, and heart). For histopathology, fragments of tissues were fixed in 10% of buffered formal saline and stained with HE. Histopathological results were all negative. PCR showed 25/150 (16.6%) positive samples, being 17/120 (14.1%) and 8/30 (26.6%) from muscle, and brain tissues, respectively. Tissue cysts of T. gondii were identified by mouse bioassay in 54/98 (55.1%) samples, being 31/48 (64.6%) from muscle samples, and 23/50 (46.0%) from brain samples. Toxoplasma gondii isolation in muscle samples by mouse bioassay was higher than in PCR (P < 0.01). Results indicate that DNA from pig tissues interfered with 529-bp-PCR sensitivity, and mouse bioassay was better than PCR in detecting T. gondii in tissues from pigs. © 2006 Elsevier Inc. All rights reserved.

Female sex hormones mediate the allergic lung reaction by regulating the release of inflammatory mediators and the expression of lung E-selectin in rats

Background: Fluctuations of estradiol and progesterone levels caused by the menstrual cycle worsen asthma symptoms. Conflicting data are reported in literature regarding pro and anti-inflammatory properties of estradiol and progesterone.Methods: Female Wistar rats were ovalbumin (OVA) sensitized 1 day after resection of the ovaries (OVx). Control group consisted of sensitized-rats with intact ovaries (Sham-OVx). Allergic challenge was performed by aerosol (OVA 1%, 15 min) two weeks later. Twenty four hours after challenge, BAL, bone marrow and total blood cells were counted. Lung tissues were used as explants, for expontaneous cytokine secretion in vitro or for immunostaining of E-selectin.Results: We observed an exacerbated cell recruitment into the lungs of OVx rats, reduced blood leukocytes counting and increased the number of bone marrow cells. Estradiol-treated OVx allergic rats reduced, and those treated with progesterone increased, respectively, the number of cells in the BAL and bone marrow. Lungs of OVx allergic rats significantly increased the E-selectin expression, an effect prevented by estradiol but not by progesterone treatment. Systemically, estradiol treatment increased the number of peripheral blood leukocytes in OVx allergic rats when compared to non treated-OVx allergic rats. Cultured-BAL cells of OVx allergic rats released elevated amounts of LTB4 and nitrites while bone marrow cells increased the release of TNF-α and nitrites. Estradiol treatment of OVx allergic rats was associated with a decreased release of TNF-α, IL-10, LTB4 and nitrites by bone marrow cells incubates. In contrast, estradiol caused an increase in IL-10 and NO release by cultured-BAL cells. Progesterone significantly increased TNF- α by cultured BAL cells and bone marrow cells.Conclusions: Data presented here suggest that upon hormonal oscillations the immune sensitization might trigger an allergic lung inflammation whose phenotype is under control of estradiol. Our data could contribute to the understanding of the protective role of estradiol in some cases of asthma symptoms in fertile ans post-menopausal women clinically observed. © 2010 de Oliveira et al; licensee BioMed Central Ltd.

A single multiplex SNaPshot reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. DNA was extracted from blood spotted on filter paper using Chelex protocol. Forty SNPs were selected targeting haplogroup-specific mutations in Europeans, Africans and Asians (only precursors of Native Americans haplogroups A2, B2, C1, and D1) and two non-coding SNPs were chosen to increase the power of discrimination between individuals (SNPs positions 16,519 and 16,362). It was done using a modified version of a previously published multiplex SNaPshot minisequencing reaction established to resolve European haplogroups, adding SNPs targeting Africans (L0, L1, L2, L3, and L*) and Asians (A, B, C, and D) haplogroups based on SNPs described at PhyloTree.org build 2. PCR primers were designed using PerlPrimer software and checked with the Autodimer program. Thirty-three primer-pairs were used to amplify 42 SNPs. Using this panel, we were able to successfully classify 160 individuals into their correct haplogroups. Complete SNP profiles were obtained from 10. pg of total DNA. We conclude that it is possible to build and genotype more than 40 mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations. © 2011.

The Number of Bleaching Sessions Influences Pulp Tissue Damage in Rat Teeth

Introduction: Hydrogen peroxide tooth bleaching is claimed to cause alterations in dental tissue structures. This study investigated the influence of the number of bleaching sessions on pulp tissue in rats. Methods: Male Wistar rats were studied in 5 groups (groups 1S-5S) of 10 each, which differed by the number (1-5) of bleaching sessions. In each session, the animals were anesthetized, and 35% hydrogen peroxide gel was applied to 3 upper right molars. Two days after the experimental period, the animals were killed, and their jaws were processed for light microscope evaluation. Pulp tissue reactions were scored as follows: 1, no or few inflammatory cells and no reaction; 2, <25 cells and a mild reaction; 3, between 25 and 125 cells and a moderate reaction; and 4, 125 or more cells and a severe reaction. Results from each experimental group were compared between groups and within groups to the corresponding unbleached upper left molars and analyzed for significant differences using the Kruskal-Wallis test (P < .05). Results: All tissue sections showed significant bleaching-induced changes in the dental pulp. After 1 bleaching session, necrotic tissue in the pulp horns and underlying inflammatory changes were observed. The extent and intensity of these changes increased with the number of bleaching sessions. After 5 sessions, the changes included necrotic areas in the pulp tissue involving the second third of the radicular pulp and intense inflammation in the apical third. Conclusions: The number of bleaching sessions directly influenced the extent of pulp damage. © 2013 American Association of Endodontists.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.