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.

Combined effects of intermittent fasting with swimming-based high intensity intermittent exercise training in Wistar rats.

High caloric intake and physical inactivity are known precursors to the development of several chronic metabolic diseases. For obesity and sedentarism, High Intensity Intermittent Exercise (HIIE) and Intermittent Fasting (IF) have emerged as individual strategies to attenuate their negative effects by improving metabolism. To study their combined effects, Wistar male rats (n = 74, 60 days old) were divided into four groups: Sedentary Control (C), swimming-based HIIE only (HIIE), Intermittent Fasting only (IF), and swimming-based HIIE associated with Intermittent Fasting (HIIE/IF). Over an eight-week period swimming performance, body composition, weight and feeding behavior were analyzed. The final morphology of white adipose tissue showed a significant reduction in adipocyte size consistent with a higher number of cells per area in exercised animals (vs C and IF, p < 0.05), which also displayed characteristics of browning through UCP-1 levels and CD31 staining. These results suggest that the increased performance in the HIIE/IF group is, in part, by modifications of WAT metabolism through the browning process.

Randomized prospective study comparing conventional In Vitro Fertilization technique to Intravaginal Culture with the INVOCELL™ device for 3 and 5 days.

OBJECTIVE: The objective was to analyse and compare the formation and quality of the embryos developed using conventional in vitro fertilization (IVF) and IVC techniques with an INVOCell™ device. METHODS: Two groups were formed, with eight couples in each, one in culture for three days (D3) and another in culture for five days (D5), using intravaginal culture technique with an INVOcell device and a conventional in vitro fertilization technique. RESULTS: Embryo formation in Group D5 showed 46.7% (IVC) and 40% (IVF) of recovered blastocysts. In the group D5, the conventional IVF, better embryo development dynamics was observed, with 66% of expanded blastocysts, against 28% in the IVC. Group D3 showed 75% (IVC) and 53% (IVF) of embryo formation. Embryonic quality in Group D3 demonstrated that IVF embryos had a better synchrony in the number and quality of blastomeres. All embryos recovered in Group D3, in both techniques, did not show fragmentation. The pH of the medium contained in the INVOCell™ device in both Groups D5 and D3 showed no differences. The means were 7.26 and 7.25, respectively. The pH of the medium used in IVF was 7.29 in both groups. Microbiological analyzes of the culture media contained in the INVOCell™ devices used in Group D5 were negative. CONCLUSIONS: The results showed that the IVC technique, using the INVOCell™ device, provided a healthy and balanced environment for the development and obtaining of quality embryos with three and five days of culture.

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.

Low Inflammatory Stimulus Increases D2 Activity and Modulates Thyroid Hormone Metabolism during Myogenesis In Vitro.

Thyroid hormone (TH) signaling controls muscle progenitor cells differentiation. However, inflammation can alter muscle TH signaling by modulating the expression of TH transporters (Slc16a2), receptors (Thra1), and deiodinase enzymes (Dio2 and Dio3). Thus, a proinflammatory environment could affect myogenesis. The role of a low-grade inflammatory milieu in TH signaling during myogenesis needs further investigation. Herein, we aimed to study the impact of the bacterial lipopolysaccharide (LPS)-induced inflammatory stimulus on the TH signaling during myogenesis. C2C12 myoblasts differentiation was induced without (CTR) or with 10 ng/mL LPS presence. The myoblasts under LPS stimulus release the proinflammatory cytokines (IL-6 and IL-1ß) and chemokines (CCL2 and CXCL-1). LPS decreases Myod1 expression by 28% during the initial myogenesis, thus reducing the myogenic stimulus. At the same time, LPS reduced the expression of Dio2 by 41% but doubled the D2 enzymatic activity. The late differentiation was not affected by inflammatory milieu, which only increased the Slc16a2 gene expression by 38%. LPS altered the intracellular metabolism of TH and reduced the initial myogenic stimulus. However, it did not affect late differentiation. Increased intracellular TH activation may be the compensatory pathway involved in the recovery of myogenic differentiation under a low-grade inflammatory milieu.

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.

Gaps in the knowledge of thyroid hormones and placental biology†.

Thyroid hormones (THs) are required for the growth and development of the fetus, stimulating anabolism, and oxygen consumption from the early stages of pregnancy to the period of fetal differentiation close to delivery. Maternal changes in the hypothalamic-pituitary-thyroid axis are also well known. In contrast, several open questions remain regarding the relationships between the placenta and the maternal and fetal TH systems. The exact mechanism by which the placenta participates in regulating the TH concentration in the fetus and mother and the role of TH in the placenta are still poorly studied. In this review, we aim to summarize the available data in the area and highlight significant gaps in our understanding of the ontogeny and cell-specific localization of TH transporters, TH receptors, and TH metabolic enzymes in the placenta in both human and rodent models. Significant deficiencies also exist in the knowledge of the contribution of genomic and nongenomic effects of TH on the placenta and finally, how the placenta reacts during pregnancy when the mother has thyroid disease. By addressing these key knowledge gaps, improved pregnancy outcomes and management of women with thyroid alterations may be possible.

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.

Estradiol and Progesterone Levels are Related to Redox Status in the Follicular Fluid During in vitro Fertilization.

Studies have reported a possible association between the levels of oxidative stress biomarkers in follicular fluid (FF) and infertility treatment outcomes. FF analysis can provide important information about oocyte quality. This study aimed to evaluate the possible correlation between oxidative stress biomarker and intrafollicular hormone levels and clinical and laboratory parameters in women during controlled ovarian stimulation. These women were undergoing in vitro fertilization with intracytoplasmic sperm injection (ICSI).The FF samples were acquired from September 2012 to February 2014 from women undergoing private fertility treatment in Rio de Janeiro, Brazil. A total of 196 women who were undergoing ICSI and had different infertility diagnoses were recruited. The FF from each patient (average patient age of 36.3 ± 4.3 years) was collected following puncture of just one follicle with the largest diameter. After ruling out blood contamination by spectrophotometry, 163 patient samples were utilized in the study. In the FF, the progesterone levels were negatively correlated with (a) hydrogen peroxide scavenging capacity (HPSC) (r = -0.294, P < 0.0001), (b) total number of follicles (r = -0.246, P < 0.001) and (c) total number of oocytes punctured (r = -0.268, P = 0.0001). The concentration of serum estradiol exhibited a positive correlation with intrafollicular HPSC (r = 0.165, P = 0.037). Our data indicate that the FF levels of estradiol and progesterone are related to the FF redox status, which is closely associated with the number of oocytes obtained during ICSI procedures.

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.

Comparative study of calcium and calcium-related enzymes with differentiation markers in different ages and muscle types in mdx mice.

Sarcolemma instability and increased calcium influx in muscle fibers are characteristics of the Duchenne muscular dystrophy. Excessive calcium activates calcium-dependent enzymes, such as calpains (CAPN) and matrix metalloproteases (MMP). Here, we analyzed calcium deposits, the activity of CAPN and MMP and the expression of Myh, SERCA and myogenic regulatory factors in different skeletal muscles during myonecrosis (4-weeks) and regeneration (12-weeks) phases of the mdx muscular pathology. Alizarin red staining was used to assess calcium deposits, casein and gelatin zymography were performed to evaluate CAPN and MMP activity, and qPCR was used to evaluate the expression of Myh, Capn, Atp2a1 and Atp2a2, Myod1 and Myog. We observed the following characteristics in mdx muscles: (i) calcium deposits almost exclusively in mdx muscles, (ii) lower CAPN1 activity in mdx muscles, (iii) higher CAPN2 activity in mdx muscles (only at 12 wks), (iv) autolyzed CAPN activity exclusively in mdx muscles, (v) lower expression of Capn1 and higher expression of Capn2 in mdx muscles; (vi) lower expression of Atp2a1 and Atp2a2 in mdx muscles, (vii) higher MMP (pre pro MMP2, pro MMP2, MMP2 and MMP9) activity in mdx muscles, (viii) MMP2 activity exclusively in mdx muscles at 12 wks, (ix) MMP9 activity exclusively in mdx muscles, (x) higher expression of Myog in mdx muscles at 12 wks, and (xi) lower expression of Myh (Myh7, Myh2, Myh1, Myh4) in mdx muscles, particularly Myh7 and Myh2. The collection of our results provides valuable information for a better characterization of mdx pathology phenotype.

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.

Thyroid Function Disruptors: from nature to chemicals.

The modern concept of thyroid disruptors includes man-made chemicals and bioactive compounds from food that interfere with any aspect of the hypothalamus-pituitary-thyroid axis, thyroid hormone biosynthesis and secretion, blood and transmembrane transport, metabolism and local action of thyroid hormones. This review highlights relevant disruptors that effect populations through their diet: directly from food itself (fish oil and polyunsaturated fatty acids, pepper, coffee, cinnamon and resveratrol/grapes), through vegetable cultivation (pesticides) and from containers for food storage and cooking (bisphenol A, phthalates and polybrominated diphenyl ethers). Due to the vital role of thyroid hormones during every stage of life, we review effects from the gestational period through to adulthood, including evidence from in vitro studies, rodent models, human trials and epidemiological studies.

Role of thyroid hormone in skeletal muscle physiology.

Thyroid hormones (TH) are crucial for development, growth, differentiation, metabolism and thermogenesis. Skeletal muscle (SM) contractile function, myogenesis and bioenergetic metabolism are influenced by TH. These effects depend on the presence of the TH transporters MCT8 and MCT10 in the plasma membrane, the expression of TH receptors (THRA or THRB) and hormone availability, which is determined either by the activation of thyroxine (T4) into triiodothyronine (T3) by type 2 iodothyronine deiodinases (D2) or by the inactivation of T4 into reverse T3 by deiodinases type 3 (D3). SM relaxation and contraction rates depend on T3 regulation of myosin expression and energy supplied by substrate oxidation in the mitochondria. The balance between D2 and D3 expression determines TH intracellular levels and thus influences the proliferation and differentiation of satellite cells, indicating an important role of TH in muscle repair and myogenesis. During critical illness, changes in TH levels and in THR and deiodinase expression negatively affect SM function and repair. This review will discuss the influence of TH action on SM contraction, bioenergetics metabolism, myogenesis and repair in health and illness conditions.

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.

Clinical aspects of pancreatogenic diabetes secondary to hereditary pancreatitis.

BACKGROUND: Hereditary pancreatitis is a rare inherited form of pancreatitis, characterized by recurrent episodes of acute pancreatitis with early onset and/or chronic pancreatitis, and presenting brittle diabetes, composed of episodes of nonketotic hyperglycemia and severe hypoglycemia. The existing literature regarding this form of diabetes is scarce. In this report, clinical features of pancreatogenic diabetes secondary to hereditary pancreatitis are presented along with recommendations for appropriate medical treatment. RESULTS: Clinical data from five patients of a family with pancreatogenic diabetes secondary to hereditary pancreatitis were analyzed. The average time between hereditary pancreatitis and diabetes diagnosis was 80 ± 24 months (range: 60-180 months) with a mean age of 25.6 ± 14.7 years (range: 8-42 years), four patients used antidiabetic agents for 46 ± 45 months and all progressed to insulin therapy with a mean dose of 0.71 ± 0.63 IU/kg (range: 0.3-1.76 IU/kg). The glycemic control had a high variability with average capillary blood glucose of 217.00 ± 69.44 mg/dl (range: 145-306 mg/dl) and the average HbA1c was 9.9 ± 1.9% (range: 7.6-11.6%). No ketoacidosis episodes occurred and there were several episodes of hospitalization for severe hypoglycemia. CONCLUSIONS: Diabetes mellitus secondary to hereditary pancreatitis presents with early onset, diverse clinical presentation and with extremely labile glycemic control. Diabetes treatment varies according to the presentation and insulin is frequently necessary for glycemic control.

Perinatal exposure to glyphosate-based herbicide alters the thyrotrophic axis and causes thyroid hormone homeostasis imbalance in male rats.

Glyphosate-based herbicides (GBHs) are widely used in agriculture. Recently, several animal and epidemiological studies have been conducted to understand the effects of these chemicals as an endocrine disruptor for the gonadal system. The aim of the present study was to determine whether GBHs could also disrupt the hypothalamic-pituitary-thyroid (HPT) axis. Female pregnant Wistar rats were exposed to a solution containing GBH Roundup®Transorb (Monsanto). The animals were divided into three groups (control, 5mg/kg/day or 50mg/kg/day) and exposed from gestation day 18 (GD18) to post-natal day 5 (PND5). Male offspring were euthanized at PND 90, and blood and tissues samples from the hypothalamus, pituitary, liver and heart were collected for hormonal evaluation (TSH-Thyroid stimulating hormone, T3-triiodothyronine and T4-thyroxine), metabolomic and mRNA analyses of genes related to thyroid hormone metabolism and function. The hormonal profiles showed decreased concentrations of TSH in the exposed groups, with no variation in the levels of the thyroid hormones (THs) T3 and T4 between the groups. Hypothalamus gene expression analysis of the exposed groups revealed a reduction in the expression of genes encoding deiodinases 2 (Dio2) and 3 (Dio3) and TH transporters Slco1c1 (former Oatp1c1) and Slc16a2 (former Mct8). In the pituitary, Dio2, thyroid hormone receptor genes (Thra1 and Thrb1), and Slc16a2 showed higher expression levels in the exposed groups than in the control group. Interestingly, Tshb gene expression did not show any difference in expression profile between the control and exposed groups. Liver Thra1 and Thrb1 showed increased mRNA expression in both GBH-exposed groups, and in the heart, Dio2, Mb, Myh6 (former Mhca) and Slc2a4 (former Glut4) showed higher mRNA expression in the exposed groups. Additionally, correlation analysis between gene expression and metabolomic data showed similar alterations as detected in hypothyroid rats. Perinatal exposure to GBH in male rats modified the HPT set point, with lower levels of TSH likely reflecting post-translational events. Several genes regulated by TH or involved in TH metabolism and transport presented varying degrees of gene expression alteration that were probably programmed during intrauterine exposure to GBHs and reflects in peripheral metabolism. In conclusion, the role of GBH exposure in HPT axis disruption should be considered in populations exposed to this herbicide.

Differential Effects of Sepsis and Chronic Inflammation on Diaphragm Muscle Fiber Type, Thyroid Hormone Metabolism, and Mitochondrial Function.

BACKGROUND: The diaphragm is the main respiratory muscle, and its function is compromised during severe illness. Altered local thyroid hormone (TH) metabolism may be a determinant of impaired muscle function during illness. METHODS: This study investigates the effects of bacterial sepsis and chronic inflammation on muscle fiber type, local TH metabolism, and mitochondrial function in the diaphragm. Two mouse models were used: sepsis induced by S. pneumoniae infection or chronic inflammation induced by subcutaneous turpentine injection. In vitro, the effect of bacterial endotoxin (LPS) on mitochondrial function in C2C12 myotubes was studied. RESULTS: Sepsis induced a transient increase in the fiber type I profile and increased Dio3 expression while decreasing Dio2, Thra1, and Slc16a2 expression. Triiodothyronine positively regulated genes Tnni2 and Myog were decreased, indicating reduced TH signaling in the diaphragm. In contrast, chronic inflammation increased the fiber type II profile in the diaphragm as well as Thra1, Thrb1, and Myog expression while decreasing Dio3 expression, suggesting increased TH responsiveness during chronic inflammation. LPS-stimulated C2C12 myotubes showed decreased Dio2 expression and reduced basal oxygen consumption as well as non-mitochondrial respiration. The same respiratory profile was induced by Dio2 knockdown in myotubes. CONCLUSIONS: The in vivo results show differential effects of sepsis and chronic inflammation on diaphragm muscle fiber type, TH metabolism, and mitochondrial function, while the in vitro results point to a causal role for altered TH metabolism in functional muscle impairment. These findings may be relevant for the pathogenesis of impaired respiratory function in critical illness.