Effect of estrogens on apoptosis in the pituitary of viscachas (Lagostomus maximus maximus).

Estrogens, acting through their receptors (ERα and ERß), regulate cell turnover in the pituitary gland, influencing cell proliferation and apoptosis across various species. However, their role in pituitary processes in seasonally reproducing animals remains poorly understood. This study aims to investigate the influence of estrogens, through the expression of their specific receptors, on the apoptosis of PD cells in relation to sexual maturity, the reproductive cycle, and pregnancy in a seasonal reproductive rodent (Lagostomus maximus maximus). ERα and caspase-3-cleaved (CASP3c) immunoreactive (-ir) cells were identified through immunohistochemistry. Apoptotic cells were detected using the TUNEL technique, with quantitative analysis facilitated by image analysis software, alongside measurement of serum estradiol levels using radioimmunoassay The immunostaining pattern for ERα included nuclear (ERαn) and cytoplasmic (ERαc) staining. In male viscachas, ERα expression significantly increases from immature to adult animals, correlating with the rise in serum estradiol levels and a decrease in the percentage of apoptotic cells. During the gonadal regression period in adult males, a decrease in the number of ER-ir cells and serum levels of estradiol corresponds with an increase in the number of apoptotic cells. In females, serum levels of estradiol peaked during mid-pregnancy, coinciding with a significant decrease in the number of apoptotic cells in the PD. Simultaneously, the percentage of ERαn-ir cells reaches its maximum value during late pregnancy, indicating the need to maintain the protective action of this gonadal hormone throughout the extensive pregnancy in these rodents. Regional ERα receptor expression and apoptotic cells appear to be associated with distinct PD cell populations and their hormonal responses. Finally, elevated estradiol levels coincide with diminished apoptotic cells in the male reproductive cycle and during pregnancy, suggesting an antiapoptotic role of estradiol in this species.

Variations in the adenohypophysis of the expression of proliferating cellular nuclear antigen, oestrogen and androgen receptors in relation to gonadal steroids during pregnancy of viscachas (Lagostomus maximus maximus).

Viscachas are native rodents of South America that present a long pregnancy of ~154 days. In this work, we analysed variations in the expression of proliferating cellular nuclear antigen, oestrogen and androgen receptors (ERα and AR) in pituitary pars distalis (PD) and pars tuberalis (PT) in relation to oestradiol and testosterone serum levels in non-pregnant and pregnant viscachas. In PD, cell proliferation increased with pregnancy and lactotrophs proliferated during mid-pregnancy (MP). ERα nuclear-immunoreactive cells (ERαn-ir) were maximal in late pregnancy and AR expression did not vary during pregnancy. In PT, cell proliferation and AR expression increased during pregnancy, but ERα expression was very scarce. The immunostaining pattern of receptors was different in PD and PT. The peak of serum oestradiol and testosterone occurred during MP. Our results suggest that cell proliferation and gonadal receptors might be differentially regulated in the pituitary by oestradiol and testosterone during viscacha pregnancy.

A Seasonal and Age-Related Study of Interstitial Cells in the Pineal Gland of Male Viscacha (Lagostomus maximus maximus).

The pineal gland of viscacha exhibits histophysiological variations throughout the year, with periods of maximal activity in winter and minimal activity in summer. The aim of this work is to analyze the interstitial cells (IC) in the pineal gland of male viscachas in relation to season and age. The S-100 protein, glio-fibrillary acidic protein (GFAP), and vimentin were detected in adult and immature animals by immunohistochemistry (IHC). Double-IHC was also performed. The S-100 protein was localized within both, IC nucleus and cytoplasm. GFAP was present only in the cytoplasm. Vimentin was expressed in some IC, besides endothelial cells, and perivascular spaces. In the adult males, the morphometric parameters analyzed for the S-100 protein and GFAP exhibited seasonal variations with higher values of immunopositive area percentage in winter and lower values in summer, whereas the immature ones showed the lowest values for all the adult animals studied. Colocalization of S-100 protein and GFAP was observed. The IC exhibited differential expression for the proteins studied, supporting the hypothesis of the neuroectodermal origin. The IC generate an intraglandular communication network, suggesting its participation in the glandular activity regulation processes. The results of double-IHC might indicate the presence of IC in different functional stages, probably related to the needs of the cellular microenvironment. The morphometric variations in the proteins analyzed between immature and adult viscachas probed to be more salient in the latter, suggesting a direct relationship between the expression of the S-100 protein and GFAP, and animal age. Anat Rec, 2017. © 2017 Wiley Periodicals Inc. Anat Rec, 300:1847-1857, 2017. © 2017 Wiley Periodicals, Inc.

Androgen Receptors Expression in Pituitary of Male Viscacha in relation to Growth and Reproductive Cycle.

The aim of this work was to study the androgen receptors (AR) expression in pituitary pars distalis (PD) of male viscachas in relation to growth and reproductive cycle. AR were detected by immunocytochemistry and quantified by image analysis. Pituitary glands from fetus, immature, prepubertal, and adult viscachas during their reproductive cycle were used. In the fetal PD, the immunoreactivity (ir) was mainly cytoplasmic. In immature and prepubertal animals, AR-ir was cytoplasmic (ARc-ir) and nuclear (ARn-ir) in medial region. In adult animals, ARn-ir cells were numerous at caudal end. AR regionalization varied between the PD zones in relation to growth. In immature animals, the ARn-ir increased whereas the cytoplasmic expression decreased in relation to the fetal glands. The percentage of ARc-ir cells increased in prepubertal animals whereas the nuclear AR expression was predominant in adult viscachas. The AR expression changed in adults, showing minimum percentage in the gonadal regression period. The variation of nuclear AR expression was directly related with testosterone concentration. These results demonstrated variations in the immunostaining pattern, regionalization, and number of AR-ir cells throughout development, growth, and reproductive cycle, suggesting the involvement of AR in the regulation of the pituitary activity of male viscacha.

Effectiveness of bisphosphonate therapy in a community setting.

BACKGROUND: Osteoporosis is a major cause of morbidity and mortality. Clinical trials have shown the effectiveness of bisphosphonates, the most commonly prescribed treatments, in reducing fracture risk. The population-based effectiveness of bisphosphonates in clinical practice is uncertain. METHODS: This retrospective cohort study used a matched design that compared time to clinical fracture in at-risk community women who initiated a bisphosphonate medication between 7/1/1996 and 6/30/2006 to those who did not. The study was conducted in an HMO in Oregon and Washington. Clinical electronic databases provided data. Eligible members were newly treated women aged > or = 55 years with either a BMD T-score of < or = -2.0 or a prior qualifying clinical fracture. They did not have contraindications for bisphosphonate therapy or a diagnosis associated with secondary osteoporosis (n=1829). They were matched to a similar comparison group (n=1829; total N=3658). The primary outcome was the first new incident fracture validated through chart review (closed clinical fracture of any bone except face, skull, finger, or toe or pathological fracture secondary to malignancy) during follow-up. An intention-to-treat analysis used Cox proportional hazards models to estimate the hazard ratio of fracture for treated relative to comparison patients, adjusting for differences in potential confounders. RESULTS: Treated and comparison patients were similar in mean age (72.0 years) and history of fracture (about 45%). The treated group had more women with T-scores of < or = -2.5 (67.3% vs. 54.7%) and a lower mean weight (146.6 lb vs. 151.8 lb). Only about 45% of treated patients had a bisphosphonate medication possession ratio (MPR) of > or = 0.80. During follow-up, 198 (10.8%) of patients in the treated group had incident fractures, vs. 179 (9.8%) of patients in the comparison group. After adjustments, patients in the treated group were 0.91 (95% CI 0.74-1.13) as likely to have an incident fracture as the comparison patients (p=0.388). The treatment effect remained non-significant after accounting for MPR. CONCLUSIONS: In this analysis of a community cohort of post-menopausal women at risk, the fracture risk of patients who received bisphosphonates did not differ significantly from those who did not. An enhanced understanding of this lack of treatment effect is urgently needed.

Prognostic value of ankle-brachial index and dobutamine stress echocardiography for cardiovascular morbidity and all-cause mortality in patients with peripheral arterial disease.

BACKGROUND: Peripheral arterial disease (PAD) is associated with an excessive risk for cardiovascular events and mortality. To determine measures prognostic of adverse events, ankle-brachial index (ABI) was compared with dobutamine stress echocardiography (DSE) in patients referred to our vascular center for the evaluation of PAD. METHODS: The medical records of consecutive patients referred for the concurrent evaluation of PAD and coronary artery disease (CAD) between 1992 and 1995 were reviewed for subsequent cardiovascular events and death. RESULTS: Among 395 patients (mean age, 69.7 +/- 9.6 years; 40% women), 341 had abnormal ABI and 268 had abnormal DSE (95 fixed and 173 stress-induced wall motion abnormalities). During a mean follow-up of 4.7 years, 27.3% of patients experienced a cardiovascular event, and 39.4% died. By multivariate analysis, ABI provided the strongest prediction of all-cause mortality (hazard ratio [HR], 2.34; 95% confidence interval [CI], 1.36 to 4.05; P = .002). Conversely, DSE with inducible or fixed wall motion abnormalities showed no association with cardiovascular events or increased mortality in multivariate analysis. The only DSE variable independently predictive of mortality was decreased left ventricular ejection fraction (<50%) at peak stress (HR, 1.70; 95% CI, 1.22 to 2.36; P = .002). Statin and aspirin therapy, but not beta-blockers, were protective. There was no relation between ABI and wall motion index score at rest or after stress. CONCLUSIONS: In high-risk patients referred to our vascular center for the evaluation of PAD, the assessment of ABI provided a strong independent prediction of all-cause mortality. Therefore, proper interpretation of this simple, affordable, and reproducible measure extends beyond the assessment of PAD severity. Although a poor left ventricular response to dobutamine was also predictive, other echo variables were not.

A measles virus vaccine strain derivative as a novel oncolytic agent against breast cancer.

Breast cancer is the most common malignancy and the second leading cause of female cancer mortality in the United States. There is an urgent need for development of novel therapeutic approaches. In this study, we investigated the antitumor potential of a novel viral agent, an attenuated strain of measles virus deriving from the Edmonston vaccine lineage, genetically engineered to produce carcinoembryonic antigen (CEA) against breast cancer. CEA production as the virus replicates can serve as a marker of viral gene expression. Infection of a variety of breast cancer cell lines including MDA-MB-231, MCF7 and SkBr3 at different multiplicities of infection (MOIs) from 0.1 to 10 resulted in significant cytopathic effect consisting of extensive syncytia formation and massive cell death at 72-96 h from infection. All breast cancer lines overexpressed the measles virus receptor CD46 and supported robust viral replication, which correlated with CEA production. TUNEL assays indicated an apoptotic mechanism of syncytial death. The efficacy of this approach in vivo was examined in a subcutaneous Balb C/nude mouse model of MDA-MB-231 cells. Intravenous administration of MV-CEA at a total dose of 1.2 x 10(7) TCID50 resulted in statistically significant tumor growth delay ( p=0.005) and prolongation of survival ( p=0.001). In summary, MV-CEA has potent antitumor activity against breast cancer lines and xenografts. Monitoring marker peptide levels in the serum could serve as a low-risk method of detecting viral gene expression during treatment and could allow dose optimization and individualization of treatment. Trackable measles virus derivatives merit further exploration in breast cancer treatment.

Adenoviral vectors expressing fusogenic membrane glycoproteins activated via matrix metalloproteinase cleavable linkers have significant antitumor potential in the gene therapy of gliomas.

BACKGROUND: Fusogenic membrane glycoproteins (FMG) such as the gibbon ape leukemia virus envelope (GALV) glycoprotein are potent therapeutic transgenes with potential utility in the gene therapy of gliomas. Transfection of glioma cell lines with FMG expression constructs results in fusion with massive syncytia formation followed by cytotoxic cell death. Nevertheless, ubiquitous expression of the GALV receptor, Pit-1, makes targeting desirable in order to increase the specificity of the observed cytopathic effect. Here we report on use of matrix metalloproteinase (MMP)-cleavable linkers to target the cytotoxicity of FMG-expressing adenoviral vectors against gliomas. METHODS: Replication-defective adenoviruses (Ad) were constructed expressing the hyperfusogenic version of the GALV glycoprotein linked to a blocking ligand (C-terminal extracellular domain of CD40 ligand) through either an MMP-cleavable linker (AdM40) or a non-cleavable linker (AdN40). Both viruses also co-expressed the green fluorescent protein (GFP) via an internal ribosomal entry site. RESULTS: The glioma cell lines U87, U118, and U251 characterized by zymography and MMP-2 activity assay as high, medium and low MMP expressors, respectively, the MMP-poor cell lines TE671 and normal human astrocytes were infected with AdM40 and AdN40 at different multiplicities of infection (MOIs) from 1-30. Fusion was quantitated by counting both number and size of syncytia. Infection of these cell lines with AdN40 did not result in fusion or cytotoxic cell death, despite the presence of infection, as demonstrated by GFP positivity, therefore indicating that the displayed CD40 ligand blocked GALV-induced fusion. Fusion was restored after infection of glioma cells with AdM40 at an MOI as low as 1 to an extent dependent on MMP expression and coxsackie adenovirus receptor (CAR) expression in the specific cell line. Western immunoblotting demonstrated the presence of the cleaved CD40 ligand in the supernatant of fused glioma cells. Use of the MMP inhibitors 1,10 phenanthroline and N-hydroxy-5,5-dimethylpiperazine-2-carboxamide completely abolished AdM40-induced fusion, while the non-specific serine protease inhibitor soybean trypsin inhibitor did not affect it, thus demonstrating specificity of the observed effect. Intratumoral treatment of BalbC/nude mice bearing subcutaneous U87 glioma xenografts with AdM40 at a total dose of 1.2 x 10(10) plaque-forming units (pfu) resulted in statistically significant tumor regression as compared with control animals either treated with AdN40 (p = 0.01) or untreated animals (p = 0.01). Treatment with AdM40 also resulted in survival improvement as compared with AdN40-treated animals (p = 0.006) or untreated animals (p = 0.001). Histopathologic examination of treated tumors demonstrated extensive syncytia formation. CONCLUSIONS: Our data indicate that AdM40, a replication-defective adenovirus expressing the GALV fusogenic glycoprotein, attached to a blocking ligand via an MMP-cleavable linker, can target the cytotoxicity of GALV in MMP-overexpressing glioma lines and xenografts, and maintain significant antitumor activity both in vitro and in vivo. Given the high frequency of MMP overexpression in gliomas, AdM40 represents a potentially promising agent in the gene therapy of these tumors.