Hormone-regulated expression and distribution of versican in mouse uterine tissues

Abstract Background Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination. Methods Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR. Results In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus. Conclusion These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

Estradiol induces transcriptional and posttranscriptional modifications in versican expression in the mouse uterus

We have previously shown the differential expression of versican in the mouse uterus under ovarian hormone influence. We also demonstrated there is not a direct correlation between mRNA levels and protein expression, suggesting posttranscriptional events, such as alteration in mRNA stability. This posttranscriptional effect may result in the elongation and stabilization of transcripts poly(A) tail. Thus, the aim of this study was to analyze whether estradiol (E2) regulates versican mRNA stability and expression in a dose-related and time-dependent manner. For this purpose female mice were ovariectomized and treated with a single injection of 0.1 or 10 μg E2. To block transcription a group of females received a single injection of alpha-amanitin before hormone administration. Uterine tissues were collected 30 min, 1, 3, 6, 12 and 24 h after treatments and processed for quantitative real time PCR (qPCR), RACE-PAT Assay and immunohistochemistry. qPCR showed that versican mRNA levels are higher than control from 3 to 24 h after E2 administration, whereas after transcription inhibition versican mRNA unexpectedly increases within 3 h, which can be explained when transcriptional blockers alter the degradation rate of the transcript, resulting in the superinduction of this mRNA. Accordingly, analysis of versican transcript poly(A) tail evidenced a longer product 3 h after treatment, but not after 12 h. Versican immunoreaction becomes conspicuous in the superficial stroma only 3 h after E2 injection, whereas the whole stroma is immunoreactive from 6 h onward. These results demonstrate that E2 modulates versican at the transcriptional and posttranscriptional levels in a time-dependent manner.

Amino acids in squamous cell carcinomas and adjacent normal tissues from patients with larynx and oral cavity lesions

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)

Study of electron densities of normal and neoplastic human breast tissues by Compton scattering using synchrotron radiation

Electron densities of 33 samples of normal (adipose and fibroglangular) and neoplastic (benign and malignant) human breast tissues were determined through Compton scattering data using a mono-chromatic synchrotron radiation source and an energy dispersive detector. The area of Compton peaks was used to determine the electron densities of the samples. Adipose tissue exhibits the lowest values of electron density whereas malignant tissue the highest. The relationship with their histology was discussed. Comparison with previous results showed differences smaller than 4%. (C) 2012 Elsevier Ltd. All rights reserved.

Epiphany root canal sealer prepared with resinous solvent is irritating to rat subcutaneous tissues

Objective: This study assessed the biocompatibility of the Epiphany endodontic sealer prepared with resinous solvent of Epiphany system (Thinning resin) in rat subcutaneous tissues. Study Design: Polyethylene tubes were filled with the sealer and 4 groups were established: GI, Epiphany prepared with 1 drop of resinous solvent (RS); GII, Epiphany prepared with 1 drop of RS and photoactivated; GIII, Epiphany associated with self-etch primer and prepared with 1 drop of RS; GIV, Epiphany associated with self-etch primer, prepared with 1 drop of RS and photoactivated. The filled tubes were implanted into 4 different regions of the dorsum of 20 adult male rats. Results: After 7, 14 and 21 days, all groups presented a moderate to severe chronic inflammation, necrosis and foreign-body giant cells. At 42 days, although the intensity of chronic inflammatory reaction decreased, the other features still were observed. Conclusion: The Epiphany sealer prepared with the RS was irritating to rat subcutaneous tissues.

Compton scattering spectrum as a source of information of normal and neoplastic breast tissues' composition

In this work we measured X-ray scatter spectra from normal and neoplastic breast tissues using photon energy of 17.44 key and a scattering angle of 90 degrees, in order to study the shape (FWHM) of the Compton peaks. The obtained results for FWHM were discussed in terms of composition and histological characteristics of each tissue type. The statistical analysis shows that the distribution of FWHM of normal adipose breast tissue clearly differs from all other investigated tissues. Comparison between experimental values of FWHM and effective atomic number revealed a strong correlation between them, showing that the FWHM values can be used to provide information about elemental composition of the tissues. (C) 2012 Elsevier Ltd. All rights reserved.

High Rates of Detection of Respiratory Viruses in Tonsillar Tissues from Children with Chronic Adenotonsillar Disease

Chronic tonsillar diseases are an important health problem, leading to large numbers of surgical procedures worldwide. Little is known about pathogenesis of these diseases. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases, we developed a cross-sectional study to determine the rates of viral detections of common respiratory viruses detected by TaqMan real time PCR (qPCR) in nasopharyngeal secretions, tonsillar tissues and peripheral blood from 121 children with chronic tonsillar diseases, without symptoms of acute respiratory infections. At least one respiratory virus was detected in 97.5% of patients. The viral co-infection rate was 69.5%. The most frequently detected viruses were human adenovirus in 47.1%, human enterovirus in 40.5%, human rhinovirus in 38%, human bocavirus in 29.8%, human metapneumovirus in 17.4% and human respiratory syncytial virus in 15.7%. Results of qPCR varied widely between sample sites: human adenovirus, human bocavirus and human enterovirus were predominantly detected in tissues, while human rhinovirus was more frequently detected in secretions. Rates of virus detection were remarkably high in tonsil tissues: over 85% in adenoids and close to 70% in palatine tonsils. In addition, overall virus detection rates were higher in more hypertrophic than in smaller adenoids (p = 0.05), and in the particular case of human enteroviruses, they were detected more frequently (p = 0.05) in larger palatine tonsils than in smaller ones. While persistence/latency of DNA viruses in tonsillar tissues has been documented, such is not the case of RNA viruses. Respiratory viruses are highly prevalent in adenoids and palatine tonsils of patients with chronic tonsillar diseases, and persistence of these viruses in tonsils may stimulate chronic inflammation and play a role in the pathogenesis of these diseases.

Molecular Analysis of the Differentiation Potential of Murine Mesenchymal Stem Cells from Tissues of Endodermal or Mesodermal Origin

Mesenchymal stem cells (MSCs) have received great attention due to their remarkable regenerative, angiogenic, antiapoptotic, and immunosuppressive properties. Although conventionally isolated from the bone marrow, they are known to exist in all tissues and organs, raising the question on whether they are identical cell populations or have important differences at the molecular level. To better understand the relationship between MSCs residing in different tissues, we analyzed the expression of genes related to pluripotency (SOX2 and OCT-4) and to adipogenic (C/EBP and ADIPOR1), osteogenic (OMD and ALP), and chondrogenic (COL10A1 and TRPV4) differentiation in cultures derived from murine endodermal (lung) and mesodermal (adipose) tissue maintained in different conditions. MSCs were isolated from lungs (L-MSCs) and inguinal adipose tissue (A-MSCs) and cultured in normal conditions, in overconfluence or in inductive medium for osteogenic, adipogenic, or chondrogenic differentiation. Cultures were characterized for morphology, immunophenotype, and by quantitative real-time reverse transcription-polymerase chain reaction for expression of pluripotency genes or markers of differentiation. Bone marrow-derived MSCs were also analyzed for comparison of these parameters. L-MSCs and A-MSCs exhibited the typical morphology, immunophenotype, and proliferation and differentiation pattern of MSCs. The analysis of gene expression showed a higher potential of adipose tissue-derived MSCs toward the osteogenic pathway and of lung-derived MSCs to chondrogenic differentiation, representing an important contribution for the definition of the type of cell to be used in clinical trials of cell therapy and tissue engineering.

Tributyltin in Crustacean Tissues: Analytical Performance and Validation of Method

The hermit crab Clibanarius vittatus is a typical organism from intertidal regions being considered as a good bioindicator of tributyltin presence at these environments. Thus this study presents the analytical performance and validation method for TBT quantification in tissues of C. vittatus by gas chromatography with pulsed flame photometric detector (GC-PFPD) after extraction with an apolar solvent (toluene) and Grignard derivatization. The limits of detection of the method (LOD) were 2.0 and 2.8 ng g(-1) for TBT and DBT (dibutyltin), respectively, and its limits of quantification (LOQ) were 6.6 and 8.9 ng g(-1) for TBT and DBT, respectively. The method was applied to samples from Santos Estuary, Sao Paulo State, Brazil. TBT and DBT concentrations ranged from 26.7 to 175.0 ng g(-1) and from 46.2 to 156.0 ng g(-1), respectively. These concentrations are worrisome since toxic effects (such as endocrine disruption) have been reported for other organisms even under lower levels of registred at this study.

Histomorphometrical and proliferative aspects of placenta and uterus of the collared peccary (Tayassu tajacu)

The histomorphometric and proliferative characteristics of the collared peccary (Tayassu tajacu) placenta and uterus were analyzed. The material was examined by standard histological techniques and histochemistry (PAS, Perls and Alcian Blue pH 0.5 and 2.5%) and the cellular proliferation by AgNORs and flow cytometry. All the analyzed morphometric variables differed between pregnant and non-pregnant uteri in the luteal phase using the Dunnet test. Height and gland diameter of uterine glands increased linearly during pregnancy, with an intense positive PAS and Perls reaction in all stages. The cells with more than seven AgNORs per nuclei and the cells in the G2M cell cycle phase in the maternal tissue also increased after 70 days of pregnancy. The uteroplacental ridges had a linear increase in size with two distinct areas, base and top, with uterine epithelium and trophoblastic cells changing their morphology following the placental ridge development. Flow cytometry analysis showed the percentage of cells in each cell cycle phase with a quadratic behavior for stages G2/M in the maternal tissue, suggesting an increase in proliferative capacity of maternal tissue after 65 days of pregnancy. The same quadratic effect was observed in the G0/G1 phase in both maternal and fetal tissues. Cells in apoptosis showed cubic behavior in both tissues. The morphometric and cellular dynamic aspects observed in this study have not been previously described and they extend our knowledge of functions relating to maternal-fetal dynamics in this species.

Expression of Clock Genes in Human Subcutaneous and Visceral Adipose Tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41 +/- 11 yrs of age) presenting a wide range of BMI (21.4 to 48.6 kg/m(2)) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p < .0001) in SAT in comparison to VAT was PER1 of female (372%) and male (326%) subjects. Different patterns of expression between the AM and PM periods were observed, in particular REV-ERBa, which was reduced (p < .05) at the PM period in SAT and VAT of both women and men (women: similar to 53% lower; men: similar to 78% lower), whereas CLOCK expression was not altered. Relationships between clock genes were different in SAT vs. VAT. BMI was negatively correlated with SATPER1 (r = -.549; p = .001) and SATPER2 (r = -.613; p = .0001) and positively with VATCLOCK (r = .541; p = .001) and VATBMAL1 (r = .468; p = .007) only in women. These data suggest that the circadian clock machinery of adipose tissue depots differs between female and male subjects, with a sex-specific effect observed for some genes. BMI correlated with clock genes, but at this moment it is not possible to establish the cause-effect relationship. (Author correspondence: mzanquetta@gmail.com)

HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/ RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.

Expression of tlr4, md2 and cd14 in equine blood leukocytes during endotoxin infusion and in intestinal tissues from healthy horses

The expression of tlr4, md2 and cd14 was studied in equine blood leukocytes and in intestinal samples using real time PCR. The stability of three commonly used reference genes, glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxantine ribosyltransferase (HPRT) and succinate dehydrogenase complex subunit A (SDHA), was evaluated using qbase(PLUS). The equine peripheral blood mononuclear cells (eqPBMC) examined were either stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the CpG oligodeoxynuclotide 2216 (CpG-ODN 2216) or obtained from horses before, during and after infusion of endotoxin. Intestinal tissue from healthy horses was sampled at ileum, right dorsal colon and rectum. Ranking of the three reference genes used for normalisation identified the combination HPRT/SDHA as most suitable both when determined ex vivo in leukocytes obtained from experimentally induced endotoxaemia and in eqPBMC activated in vitro while HPRT/GAPDH were most appropriate for the intestinal samples. The relative amounts of mRNA for TLR4 and MD-2 increased threefold during in vitro activation of the cells with CpG-ODN 2216 but was decreased in cultures stimulated with PMA/ionomycin. A transient elevation in the transcription of tlr4 and md2 was also evident for equine blood leukocytes following endotoxaemia. The levels of mRNA for CD14 on the other hard remained unaffected both during the induction of endotoxaemia and in the in vitro stimulated PBMCs. A low steady expression of TLR4, MD-2 and CD14 mRNA was demonstrated for the intestinal samples with no variation between the intestinal segments analysed. Thus, the foundation for real time PCR based levels of analysis of mRNA for all three components in the equine LPS receptor complex in different intestinal segments was set, making it possible to carry out future expression studies on clinical material. (C) 2012 Elsevier B.V. All rights reserved.

Systemically alendronate was incorporated into dental tissues but did not cause morphological or mechanical changes in rats teeth

This study evaluated the effect of the systemic use of sodium alendronate in rats in vivo. Forty-five Wistar rats aged 36 to 42 days and weighing 200 to 230 g were randomly assigned to a control group (n = 20), which received distilled water, and an experimental group (n = 25), which received 2 weekly doses of 1 mg/kg of chemically pure sodium alendronate. The animals were killed after 60 days of treatment. The tibias were removed for analysis of bone mineral density by dual-energy X-ray absorptiometry (DXA). Then, the maxillary incisors were extracted for analysis of the mineralized dental tissues using fluorescence spectroscopy (FS), scanning electron microscopy (SEM), bright field microscopy (BFM), and cross-sectional microhardness (CSMH) testing. DXA and CSMH data were subjected to statistical analysis by Kruskal-Wallis test (5% significance level). The experimental group presented higher bone mineral density than the control group by DXA. FS analysis revealed presence of alendronate in the mineralized dental tissues of the specimens of the experimental group. Significant morphological differences were not found by SEM and BFM. Enamel and dentin (100 and 300 mu m from the dentinoenamel junction) CSMH data did not show significant difference between the control and experimental groups. Based on the obtained results, we conclude that while alendronate increased the bone mineral density and was incorporated into the mineralized dental tissues it did not cause significant alterations in the morphology and microhardness of rat incisor enamel and dentin. Microsc. Res. Tech. 75:12651271, 2012. (C) 2012 Wiley Periodicals, Inc.

IL-5 and IL-17A are critical for the chronic IgE response and differentiation of long-lived antibody-secreting cells in inflamed tissues

Prolonged survival of long-lived antibody-secreting cells in the BM has been implicated as a key component of long-term humoral immunity. The current study was designed to uncover the extrinsic signals required for the generation and maintenance of ASC in several niches (peritoneum, spleen and bone-marrow). Our results show that protein mixture of the Thalassophryne nattereri venom induced a chronic Th2 humoral response that is characterized by splenic hyperplasia with GC formation and venom retention by follicular DCs. Retention of B1a in the BM were observed. In the late phase (120 d) of chronic venom-response the largest pool of ASC into the peritoneal cavity consisted of B220(neg)CD43(high) phenotype; the largest pool of ASC into spleen was constituted by B220 positive cells (B220(high) and B220(low)), whereas the largest pool of ASC into in the BM was constituted by the B220(high)CD43(low) phenotype; and finally, terminally differentiated cells (B220(neg)CD43(high)) were only maintained in the inflamed peritoneal cavity in late phase. After 120 d a sustained production of cytokines (KC, IL-5, TNF-alpha, IL-6, IL-17A and IL-23) and leukocytes recruitment (eosinophils, mast cells, and neutrophils) were induced. IL-5- and IL-17A-producing CD4+ CD44+ CD40L+ Ly6C+ effector memory T cells were also observed in peritoneal cavity. Finally, treatment of venom-mice with anti-IL-5- and anti-IL17A-neutralizing mAbs abolished the synthesis of specific IgE, without modifying the splenic hyperplasia or GC formation. In addition, IL-5 and IL-17A negatively regulated the expansion of B1a in peritoneal cavity and BM, and promoted the differentiation of these cells in spleen. And more, IL-5 and IL-17A are sufficient for the generation of ASC B220(neg) in the peritoneal cavity and negatively regulate the number of ASC B220(Pos), confirming that the hierarchical process of ASC differentiation triggered by venom needs the signal derived from IL-5 and IL-17A. (C) 2012 Elsevier Ltd. All rights reserved.