Immunostaining of ßA-Activin and Follistatin Is Decreased in HPV(+) Cervical Pre-Neoplastic and Neoplastic Lesions.

The activin-follistatin system regulates several cellular processes, including differentiation and tumorigenesis. We hypothesized that the immunostaining of ßA-activin and follistatin varies in neoplastic cervical lesions. Cervical paraffin-embedded tissues from 162 patients sorted in control (n = 15), cervical intraepithelial neoplasia (CIN) grade 1 (n = 38), CIN2 (n = 37), CIN3 (n = 39), and squamous cell carcinoma (SCC; n = 33) groups were examined for ßA-activin and follistatin immunostaining. Human papillomavirus (HPV) detection and genotyping were performed by PCR and immunohistochemistry. Sixteen samples were inconclusive for HPV detection. In total, 93% of the specimens exhibited HPV positivity, which increased with patient age. The most detected high-risk (HR)-HPV type was HPV16 (41.2%) followed by HPV18 (16%). The immunostaining of cytoplasmatic ßA-activin and follistatin was higher than nuclear immunostaining in all cervical epithelium layers of the CIN1, CIN2, CIN3, and SCC groups. A significant decrease (p < 0.05) in the cytoplasmic and nuclear immunostaining of ßA-activin was detected in all cervical epithelial layers from the control to the CIN1, CIN2, CIN3, and SCC groups. Only nuclear follistatin immunostaining exhibited a significant reduction (p < 0.05) in specific epithelial layers of cervical tissues from CIN1, CIN2, CIN3, and SCC compared to the control. Decreased immunostaining of cervical ßA-activin and follistatin at specific stages of CIN progression suggests that the activin-follistatin system participates in the loss of the differentiation control of pre-neoplastic and neoplastic cervical specimens predominantly positive for HPV.

Yerba mate consumption by ovariectomized rats alters white adipose tissue.

INTRODUCTION: Menopause and post-menopause are characterized by low levels of estrogen that can be associated with the emergence of metabolic diseases. While hormone replacement therapy can alleviate many symptoms, it can also exacerbate other diseases such as breast cancer. In the search for natural alternatives, Ilex paraguariensis (Yerba Mate) has been identified as a potential therapy for the onset of obesity. Here, the effect of MATE consumption on white adipose tissue (WAT) was studied in ovariectomized rats, an animal model for post-menopause hormone loss. METHODS: Four groups of animals were used: ovariectomy with MATE (OVX MATE) and without MATE (OVX), as well as sham surgery with MATE (Sham MATE) and without MATE (Sham). MATE was provided by gavage at 1 g/kg of body weight for eight weeks before measuring biochemical parameters in plasma and characterizing WAT morphology. RESULTS: The consumption of Yerba MATE significantly decreased weight gain in ovariectomized rats and presented near control levels of triglycerides, total cholesterol, and LDL. A morphometric analysis of WAT showed a significant decrease in the area occupied by adipocytes in the group that consumed MATE. Finally, MATE consumption increased the UCP1 content in the WAT of the ovariectomized group. Yerba MATE treatment was also associated with higher levels of SIRT1 protein. CONCLUSION: MATE consumption has a preventive effect on the weight gain observed in ovariectomized rats and potential benefits in naturally avoiding the onset of obesity post menopause.

Cylindrospermopsin Disrupts Estrous Cycle and Increases Spermatogenesis in Mice.

Cylindrospermopsin (CYN) is a water-soluble cyanotoxin that has been linked to several cases of poisoning in the world. In vitro studies have shown that CYN acts as an endocrine disruptor by inhibiting progesterone synthesis in primary cell cultures of women, showing estrogenic activity. However, in vivo assessment of CYN in the female and male reproductive systems remains unknown. We thus aimed to evaluate the in vivo effects of CYN in both the female and male reproductive systems of mice. A single intraperitoneal exposure to 64 µg of CYN/kg body weight was performed in females. Estrous cycle was evaluated daily by vaginal cytology, and serum progesterone and estradiol levels were measured after 50 days. We showed an impairment in the estrous cycle as well as a decrease in circulating plasma progesterone levels. In males, weekly intraperitoneal doses of 20 µg of CYN/kg body weight were given and groups were killed after one, two, or four doses. CYN increased the testosterone levels in the groups that received one or two doses of CYN. Additionally, CYN induced a transient increase in spermatozoa in males after four doses. Our results highlight that CYN interferes with both male and female reproductive systems and may lead to infertility. As far as we know, this is the first report showing the impacts of CYN on the mammalian reproductive system, suggesting a threat from this cyanotoxin to human and environmental health.

Sepsis Disrupts Mitochondrial Function and Diaphragm Morphology.

BACKGROUND: The diaphragm is the primary muscle of inspiration, and its dysfunction is frequent during sepsis. However, the mechanisms associated with sepsis and diaphragm dysfunction are not well understood. In this study, we evaluated the morphophysiological changes of the mitochondrial diaphragm 5 days after sepsis induction. METHODS: Male C57Bl/6 mice were divided into two groups, namely, cecal ligation and puncture (CLP, n = 26) and sham-operated (n = 19). Mice received antibiotic treatment 8 h after surgery and then every 24 h until 5 days after surgery when mice were euthanized and the diaphragms were collected. Also, diaphragm function was evaluated in vivo by ultrasound 120 h after CLP. The tissue fiber profile was evaluated by the expression of myosin heavy chain and SERCA gene by qPCR and myosin protein by using Western blot. The Myod1 and Myog expressions were evaluated by using qPCR. Diaphragm ultrastructure was assessed by electron microscopy, and mitochondrial physiology was investigated by high-resolution respirometry, Western blot, and qPCR. RESULTS: Cecal ligation and puncture mice developed moderated sepsis, with a 74% survivor rate at 120 h. The diaphragm mass did not change in CLP mice compared with control, but we observed sarcomeric disorganization and increased muscle thickness (38%) during inspiration and expiration (21%). Septic diaphragm showed a reduction in fiber myosin type I and IIb mRNA expression by 50% but an increase in MyHC I and IIb protein levels compared with the sham mice. Total and healthy mitochondria were reduced by 30% in septic mice, which may be associated with a 50% decrease in Ppargc1a (encoding PGC1a) and Opa1 (mitochondria fusion marker) expressions in the septic diaphragm. The small and non-functional OPA1 isoform also increased 70% in the septic diaphragm. These data suggest an imbalance in mitochondrial function. In fact, we observed downregulation of all respiratory chain complexes mRNA expression, decreased complex III and IV protein levels, and reduced oxygen consumption associated with ADP phosphorylation (36%) in CLP mice. Additionally, the septic diaphragm increased proton leak and downregulated Sod2 by 70%. CONCLUSION: The current model of sepsis induced diaphragm morphological changes, increased mitochondrial damage, and induced functional impairment. Thus, diaphragm damage during sepsis seems to be associated with mitochondrial dysfunction.

ZIKV Disrupts Placental Ultrastructure and Drug Transporter Expression in Mice.

Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome.

Sepsis Impairs Thyroid Hormone Signaling and Mitochondrial Function in the Mouse Diaphragm.

Background: Sepsis can cause the nonthyroidal illness syndrome (NTIS), resulting in perturbed thyroid hormone (TH) signaling and reduced thyroxine (T4) levels. TH is a major regulator of muscle function, via its influence on mitochondria. This study aimed at evaluating the relationship between TH signaling, mitochondrial function, and the antioxidant defense system in the diaphragms of septic mice. Methods: Male C57Bl/6 mice were divided into two groups: cecal ligation and puncture (CLP) and sham. Twenty-four hours after surgery, plasma, diaphragms, and livers were collected. TH metabolism and responses were analyzed by measuring messenger RNA (mRNA) expression of Dio1 in the liver, and Thra, Thrb, Dio2, Slc16a10, and Slc16a2 (encodes MCT 10 and 8), in the diaphragm. T4 plasma levels were measured by radioimmunoassay. Damage to diaphragm mitochondria was assessed by electron microscopy and real-time polymerase chain reaction (qPCR), and function with oxygraphy. The diaphragm antioxidative defense system was examined by qPCR, analyzing superoxide dismutase (SOD) 1 (Sod1), mitochondrial superoxide dismutase (SOD 2; Sod2), extracellular superoxide dismutase (SOD 3; Sod3), glutathione peroxidase 1 (Gpx1), and catalase (Cat) expression. The effect of TH replacement was tested by treating the mice with T4 and triiodothyronine (T3) (CLP+TH) after surgery. Results: CLP mice presented reduced total plasma T4 concentrations, downregulated Dio1, and upregulated Il1b mRNA expression in the liver. CLP mice also displayed downregulated Thra, Thrb, Slc16a10, and Slc16a2 expression in the diaphragm, suggesting that TH signaling was compromised. The expression of Ppargc1a (encoding PGC1a) was downregulated, which correlated with the decrease in the number of total mitochondria, increase in the percentage of injured mitochondria, downregulation of respiratory chain complex 2 and 3 mRNA expression, and reduced maximal respiration. In addition, septic animals presented a three-fold increase in Ucp3 and G6pdh expression; downregulated Sod3, Gpx1, and Cat expression; and upregulated Sod2 expression, potentially due to elevated reactive oxygen species levels. The mitochondrial number and the percentage of injured mitochondrial were similar between sham and CLP+TH mice. Conclusions: Sepsis induced responses consistent with NTIS, resulted in mitochondrial damage and functional impairment, and modulated the expression of key antioxidant enzymes in the diaphragm. Thus, impaired diaphragm function during sepsis seems to involve altered local TH signaling, mitochondrial dysfunction, and oxidative stress defense.

Reduced mitochondrial respiration and increased calcium deposits in the EDL muscle, but not in soleus, from 12-week-old dystrophic mdx mice.

Mitochondria play an important role in providing ATP for muscle contraction. Muscle physiology is compromised in Duchenne muscular dystrophy (DMD) and several studies have shown the involvement of bioenergetics. In this work we investigated the mitochondrial physiology in fibers from fast-twitch muscle (EDL) and slow-twitch muscle (soleus) in the mdx mouse model for DMD and in control C57BL/10J mice. In our study, multiple mitochondrial respiratory parameters were investigated in permeabilized muscle fibers from 12-week-old animals, a critical age where muscle regeneration is observed in the mdx mouse. Using substrates of complex I and complex II from the electron transport chain, ADP and mitochondrial inhibitors, we found in the mdx EDL, but not in the mdx soleus, a reduction in coupled respiration suggesting that ATP synthesis is affected. In addition, the oxygen consumption after addition of complex II substrate is reduced in mdx EDL; the maximal consumption rate (measured in the presence of uncoupler) also seems to be reduced. Mitochondria are involved in calcium regulation and we observed, using alizarin stain, calcium deposits in mdx muscles but not in control muscles. Interestingly, more calcium deposits were found in mdx EDL than in mdx soleus. These data provide evidence that in 12-week-old mdx mice, calcium is accumulated and mitochondrial function is disturbed in the fast-twitch muscle EDL, but not in the slow-twitch muscle soleus.

Gestational age-dependent gene expression profiling of ATP-binding cassette transporters in the healthy human placenta.

The ATP-binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal-fetal interface. We and others have demonstrated a gestational age-dependent expression pattern of two ABC transporters, P-glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational-age dependent manner in normal human pregnancy. Using the TaqMan® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.

Acute Effects of Viral Exposure on P-Glycoprotein Function in the Mouse Fetal Blood-Brain Barrier.

BACKGROUND/AIMS: Viral infection during pregnancy is known to affect the fetal brain. The toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viruses known to elicit adverse fetal neurological outcomes. The P-glycoprotein (P-gp) efflux transporter protects the developing fetus by limiting the transfer of substrates across both the placenta and the fetal blood-brain barrier (BBB). As such, inhibition of P-gp at these blood-barrier sites may result in increased exposure of the developing fetus to environmental toxins and xenobiotics present in the maternal circulation. We hypothesized that viral exposure during pregnancy would impair P-gp function in the placenta and in the developing BBB. Here we investigated whether the TLR-3 ligand, polyinosinic:polycytidylic acid (PolyI:C), increased accumulation of one P-gp substrate in the fetus and in the developing fetal brain. METHODS: Pregnant C57BL/6 mice (GD15.5) were injected (i.p.) with PolyI:C (5 mg/kg or 10 mg/kg) or vehicle (saline). [3H]digoxin (P-gp substrate) was injected (i.v.) 3 or 23h post-treatment and animals were euthanized 1h later. Maternal plasma, 'fetal-units' (fetal membranes, amniotic fluid and whole fetus), and fetal brains were collected. RESULTS: PolyI:C exposure (4h) significantly elevated maternal plasma IL-6 (P<0.001) and increased [3H]digoxin accumulation in the fetal brain (P<0.05). In contrast, 24h after PolyI:C exposure, no effect on IL-6 or fetal brain accumulation of P-gp substrate was observed. CONCLUSION: Viral infection modeled by PolyI:C causes acute increases in fetal brain accumulation of P-gp substrates and by doing so, may increase fetal brain exposure to xenobiotics and environmental toxins present in the maternal circulation.

Hereditary Pancreatitis Associated With the N29T Mutation of the PRSS1 Gene in a Brazilian Family: A Case-Control Study.

Hereditary pancreatitis (HP) is an autosomal-dominant disease with incomplete penetrance manifesting as early-onset chronic relapsing pancreatitis. A mutation in the PRSS1 gene is present in greater than 70% of HP kindreds and leads to a gain-of-function characterized by the increased autocatalytic conversion of trypsinogen to active trypsin, promoting autodigestion and damage to acinar cells. Other genetic defects observed in the pathogenic mechanism of pancreatitis include mutations in the genes encoding SPINK1, CTRC, and CPA1. There are few reports of HP in Latin America, and no families have been investigated in Brazil. A case-control observational study was conducted at Clementino Fraga Filho University Hospital in Brazil. Patients with suspected HP and healthy controls were enrolled in this study, and a detailed questionnaire was administered to patients with HP. PRSS1 and SPINK1 genes were analyzed by DNA sequencing, and a family that fit the HP diagnostic criteria was identified. The neutral polymorphism c.88-352A > G in the SPINK1 gene was found to be prevalent in the individuals studied, but no important alterations were found in this gene. Ten out of 16 individuals in this family carried the N29T mutation in the PRSS1 gene, with 2 clinically unaffected mutation carriers. The median age of HP onset was 6 years. Pancreatic exocrine failure occurred in 6 patients, 5 of whom also had diabetes mellitus. Surgical procedures were performed on 3 affected members, and no cases of pancreatic cancer have been reported thus far. This study identified the first PRSS1 gene mutation in a Brazilian family with HP.

Thyroid hormone contributes to the hypolipidemic effect of polyunsaturated fatty acids from fish oil: in vivo evidence for cross talking mechanisms.

n-3 polyunsaturated fatty acids (n-3 PUFA) from fish oil (FO) exert important lipid-lowering effects, an effect also ascribed to thyroid hormones (TH) and TH receptor ß1 (TRß1)-specific agonists. n-3 PUFA effects are mediated by nuclear receptors, such as peroxisome proliferator-activated receptors (PPAR) and others. In this study, we investigated a role for TH signaling in n-3 PUFA effects. Euthyroid and hypothyroid adult rats (methimazole-treated for 5 weeks) received FO or soybean oil (control) by oral administration for 3 weeks. In euthyroid rats, FO treatment reduced serum triglycerides and cholesterol, diminished body fat, and increased protein content of the animals. In addition, FO-treated rats exhibited higher liver expression of TRß1 and mitochondrial α-glycerophosphate dehydrogenase (mGPD), at protein and mRNA levels, but no alteration of glutathione S-transferase or type 1 deiodinase. In hypothyroid condition, FO induced reduction in serum cholesterol and increase in body protein content, but lost the ability to reduce triglycerides and body fat, and to induce TRß1 and mGDP expression. FO did not change PPARα liver abundance regardless of thyroid state; however, hypothyroidism led to a marked increase in PPARα liver content but did not alter TRß1 or TRα expression. The data suggest that part of the effect of n-3 PUFA from FO on lipid metabolism is dependent on TH signaling in specific steps and together with the marked upregulation of PPARα in liver of hypothyroid rats suggest important in vivo consequences of the cross-talking between those fatty acids and TH pathways in liver metabolism.

Thyroid hormone receptor beta mutation causes severe impairment of cerebellar development.

Cerebellar development on the postnatal period is mainly characterized by cellular proliferation in the external granular layer (EGL) followed by migration of granular cells in the molecular layer through the Bergmann glia (BG) fibers in order to form the granular layer in the adult. All these events are drastically affected by thyroid hormones (TH), which actions are mainly mediated by alpha (TRalpha) and beta (TRbeta) nuclear receptor isoforms. Here, we analyzed the effects of a natural human mutation (337T) in the TRbeta locus, which impairs T3 binding to its receptor, on the mouse cerebellum ontogenesis. We report that target inactivation of TRbeta-TH binding leads to a smaller cerebellum area characterized by impaired lamination and foliation. Further, TRbeta mutant mice presented severe deficits in proliferation of granular precursors, arborization of Purkinje cells and organization of BG fibers. Together, our data suggest that the action of TH via TRbeta regulates important events of cerebellar ontogenesis contributing to a better understanding of some neuroendocrine disorders. Further, our data correlate TRbeta with cerebellar foliation, and provide, for the first time, evidence of a receptor-mediated mechanism underlying TH actions on this event.

Estrogen modulates neuromedin B effects on thyrotropin and prolactin release in vitro.

Neuromedin B(NB), a bombesin-like peptide, has been shown to inhibit thyrotropin (TSH) release in pituitary explants of male rats and to stimulate Prolactin (PRL) release in male pituitary cell cultures. We investigated the effect of estrogen status of female rats on the response of thyrotrophs and lactotrophs to neuromedin B (NB) in vitro. Ovariectomized rats were treated with near-physiological or high doses of 17beta estradiol benzoate (0.7 or 14 EB microg/100 gBW/daily, 10 days) or with vehicle (OVX). EB treatment induced a dose-dependent increase in serum prolactin and an increase in pituitary NB content, measured by specific RIA, that was similar in both EB groups (P < 0.05). TSH release from isolated hemipituitaries of OVX rats was significantly reduced (P < 0.05) in the presence of 10(-7) M NB. OVX + EB0.7 glands responded to NB with a not statistically significant dose-dependent decrease in TSH release. However, glands from hyperestrogenized rats (OVX + EB14) required a higher dose (10(-5) M) of NB to inhibit TSH release (P < 0.05). PRL release was highly increased (p < 0.001) by the presence of 10(-5) M NB only in glands of hyperestrogenized rats, while no effect of NB was observed in the other groups. In conclusion, estrogen status of female rats modulates the inhibitory effect of NB on TSH release in vitro and hyperestrogenism is required for stimulatory effect of NB on PRL release in vitro. It is suggested that the induction of PRL release by neuromedin B is a pharmacological rather than a physiological effect, but neuromedin B may contribute to the increased release of PRL associated with hyperestrogenism.