PI3K inhibition by BKM120 results in anti-proliferative effects on corticotroph tumor cells.

PURPOSE: Cushing's disease is associated with significant morbidity; thus, additional tumor-directed drugs with the potential to exert antineoplastic effects on corticotroph adenoma cells are desired. The phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway, which plays regulatory role in cell survival and proliferation, is activated in pituitary adenomas. The present study evaluated the effects of BKM120 (Buparlisib), an oral PI3K inhibitor, on cell viability, apoptosis, cell cycle phase distribution, and ACTH production in mouse corticotroph tumor cells. METHODS: AtT-20/D16v-F2 mouse pituitary corticotroph tumor cells were treated with increasing concentrations of BKM120 or vehicle. Cell viability was measured using an MTS-based assay. Apoptosis was evaluated by Annexin V staining. Cell cycle analysis was performed by propidium iodide DNA staining and flow cytometry. Gene expression of cell cycle regulators (Cdkn1b, Ccnd1, Ccne1, Cdk2, Cdk4, Myc, and Rb1) was assessed by qPCR. Protein expression of p27, total and phosphorylated Akt was assessed by Western blot. ACTH levels were measured in the culture supernatants by chemiluminescent immunometric assay. RESULTS: Treatment with BKM120 decreased AtT-20/D16v-F2 cell viability, induced a G0/G1 cell cycle arrest, reduced the phosphorylation of Akt at Serine 473, and increased p27 expression. Furthermore, BKM120 treatment diminished ACTH levels in the cell culture supernatants. CONCLUSION: In vitro inhibition of PI3K/AKT pathway by BKM120 resulted in anti-proliferative effects on corticotroph tumor cells, decreasing cell viability and ACTH production. These encouraging findings shape the path for further experiments with the inhibition of PI3K/AKT pathway in Cushing's disease.

Polymorphisms of the adiponectin gene in gestational hypertension and pre-eclampsia.

Adiponectin is a hormone involved in energy homeostasis by regulating glucose and lipid metabolism. In addition, the adiponectin gene (ADIPOQ) has polymorphisms that can modulate the circulating concentration of adiponectin. Abnormal adiponectin levels have been associated with pre-eclampsia (PE); however, the influence of genetic polymorphisms on the development of hypertensive disorders of pregnancy is unclear. The aim of this study was to examine whether ADIPOQ polymorphisms are associated with gestational hypertension (GH) and/or PE. We studied 401 pregnant women: 161 healthy pregnant (HP), 113 pregnant with GH and 127 pregnant with PE. ADIPOQ polymorphisms -11391G>A (rs17300539), -11377C>G (rs266729), 45T>G (rs2241766) and 276G>T (rs1501299) were genotyped by allelic discrimination assays using real-time PCR. Haplotypes were inferred using the PHASE 2.1 program. We observed that the genotypic frequencies of the -11377C>G polymorphism were different in PE compared with HP (P<0.0125), with the CT genotype being more commonly found in PE patients than in HP women (P<0.0125). However, allelic frequencies of this single-nucleotide polymorphism were similar between PE and HP (P>0.0125). No difference was observed when GH and HP groups were compared (both P>0.0125). In addition, we found no difference in genotype or allele distributions for the -11391G>A, 45T>G and 276G>T polymorphisms when we compared GH or PE with HP (all P>0.0125). In conclusion, we found a modest association between the CG genotype of the -11377C>G polymorphism and PE.

The effect of light-curing access and different resin cements on apical bond strength of fiber posts.

PURPOSE: This study evaluated the effect of light-curing access on the bond strength of fiber glass posts to the apical area of bovine roots using self-adhesive cement or dual-cured cement with an etch-and-rinse adhesive system. MATERIALS AND METHODS: The root canals of 60 bovine teeth were endodontically treated and filled. A 15-mm-length post space was prepared and roots were randomly divided into three groups, simulating the levels of light-curing access: coronal (C), with 15-mm post space; middle (M), in which the coronal thirds of roots were cut out, leaving a 10-mm post space; and apical (A), in which the coronal and middle thirds of roots were cut out, leaving a 5-mm post space. Fiber glass posts (Reforpost # 3, Angelus) were cemented with RelyX U100 (3M ESPE) or RelyX ARC/Scotchbond Multi Purpose Plus (SBMP) (3M ESPE) (n=10) and light-cured. After 24 hours, the apical thirds of roots were sectioned perpendicularly to the long axis and submitted to a push-out test (0.5 mm/min, 200 N). The Kruskal-Wallis test compared the three levels of light-curing access, and the Mann-Whitney test compared the cements. RESULTS: The bond strength was significantly higher in the groups C (p=0.028) and M (p=0.016) when U100 was used, whereas it was similar for both cements in group A. The bond strengths of posts cemented with ARC/SBMP were significantly higher in group A compared to group C (p=0.031). CONCLUSIONS: The type of cement used and the light-curing access level influenced the bond strength between glass fiber posts and root canals. The bond strength of the RelyX ARC/SBMP cement proved to be more dependent on photoactivation than was the RelyX U100 cement. The light-curing access level did not influence the apical bond strength of RelyX U100.

PP002. Study of polymorphisms of the adiponectin gene in women with gestational hypertension and preeclampsia.

INTRODUCTION: Adiponectin is involved in energy homeostasis by regulating glucose and lipid metabolism. Additionally, it presents anti-inflammatory and anti-atherosclerotic functions. Polymorphisms in adiponectin gene (ADIPOQ) can modulate the concentrations of adiponectin. The influence of these polymorphisms on the development of gestational hypertension (GH) and preeclampsia (PE) is unknown. OBJECTIVES: The aim of this work was to examine the influence of polymorphisms in the gene ADIPOQ on the development of gestational hypertension and preeclampsia. PATIENTS AND METHODS: We studied 401 pregnant women: 161 healthy pregnant (HP), 113 pregnant with gestational hypertension (GH) and 127 pregnant with preeclampsia (PE). Polymorphisms ADIPOQ -11391G>A (rs17300539), -11377C>G (rs266729), 45T>G (rs2241766) and 276G>T (rs1501299) were genotyped by allelic discrimination by PCR in real time. Haplotypes were inferred using the PHASE 2.1 program. RESULTS: There were no statistically significant differences in allele and genotype frequencies of the polymorphisms studied. In the analysis of haplotypes, we observed small differences in haplotype frequencies between groups, however, none of these differences was statistically significant (P>0.05). CONCLUSION: We found no association between the genotypic and allelic variants of the ADIPOQ gene polymorphisms with the development of gestational hypertension and preeclampsia.

Efeitos da fumaça de cigarro sobre a osteogênese e a resistência mecânica óssea em ratos / Effects of cigarette smoke on osteogenesis and bone mechanical resistance in rats

This paper reports a study of the effects of exposure to cigarette smoke on osteogenesis and the mechanical strength of bone in rats. Twelve male rats were separated into two groups (n=6): CT (control) and CI (cigarette). All the animals had free access to rat chow and water throughout the experiment. Group CI was exposed to the smoke of 6 cigarettes/day for 12 weeks. After 6 weeks of exposure to cigarette smoke, a defect was produced on the parietal bone and dense hydroxyapatite (DHA) ceramic bodies were implanted into cavities made surgically in the tibia of the animals, in each group. After surgery, the CT and CI groups returned to normal experimental conditions and, at the end of 12 weeks, they were euthanized, and their tibiae and parietal bones removed for histological processing, while the femurs were subjected to biomechanical tests in a MTS TestStar II three-point flexion module. Consumption of solid and liquid diet was satisfactory in both groups, all animals gaining weight throughout the experiment. CI animals showed a smaller volume of newly formed bone in the parietal defect (8.l9±0.2) and around the DHA implant in the tibia (33±0.5) than the rats in the CT group (14.4±0.5 and 39±1 respectively). The maximum force needed to break the femur was smaller in CI (119±3.2) than in CT (140±6.5). The results of this study led to the conclusion that exposure to cigarette smoke interfered with osteogenesis in the bone defect and around the DHA implant and reduced the maximum force required to completely break the femur, revealing that bone fragility can be caused by tobacco smoke.

O presente estudo teve como objetivo avaliar os efeitos da exposição à fumaça de cigarro sobre a osteogênese e a resistência mecânica do osso em ratos. Foram utilizados doze ratos machos, divididos em dois grupos (n=6): grupo CT (controle) e grupo CI (cigarro). Durante 12 semanas, os animais do grupo CI foram expostos à fumaça de seis cigarros/dia. Após seis semanas de exposição à fumaça de cigarro, uma falha óssea de 5mm foi produzida no osso parietal e corpos cerâmicos de hidroxiapatita densa (HAD) foram implantados em cavidade produzida cirurgicamente na tíbia dos animais do grupo CI e CT. Após as cirurgias, os animais retornaram aos protocolos experimentais e, ao término de doze semanas de experimentação, foram eutanasiados, as tíbias e os ossos parietais foram coletados para processamento histológico e os fêmures encaminhados para ensaio biomecânico em um módulo MTS TestStar II®. A exposição à fumaça do cigarro não interferiu no ganho de peso dos animais e os consumos de dieta líquida e sólida foram satisfatórios entre os grupos. Os animais do grupo CI apresentaram menor volume de osso neoformado na falha óssea (8,9±0,2) produzida no osso parietal e ao redor do implante de HAD na tíbia (33±0,5). A força máxima necessária para romper o fêmur dos animais foi menor no grupo CI (119±3,2) do que no grupo CT (140±6,5). Com bases nos resultados obtidos no presente trabalho, pôde-se concluir que a exposição à fumaça do cigarro interferiu na osteogênese da falha óssea e ao redor do implante de HAD, diminuiu a força máxima necessária para a ruptura completa dos fêmures e demonstrando a fragilidade óssea causada pelo hábito tabagista.

Mineralocorticoid receptor p.I180V polymorphism: association with body mass index and LDL-cholesterol levels.

BACKGROUND/AIMS: Aldosterone and the mineralocorticoid receptor (MR) play a major role in sodium balance and blood pressure control. They are also involved in adipocyte metabolism. The aim of this study was to analyze the association between the MR p.I180V polymorphism with hypertension and markers of cardiovascular risk. DESIGN AND METHODS: Case-control study nested within a cohort of 2063 subjects followed since birth to date. All subjects (age 23-25 yr old) from the entire cohort with systolic and diastolic hypertension (no.=126) were paired with 398 normotensive controls. MR p.I180V genotype association with anthropometric and biochemical markers of cardiometabolic risk was tested. RESULTS: There was a significant association of the MR p.I180V genotype with body mass index (BMI) and LDL-cholesterol level (p<0.01). Hypertensive subjects carrying the polymorphic G allele (AG or GG genotypes) presented significantly higher BMI (30.0+/-6.0 vs 28.7+/-5.6 kg/m(2); p<0.01) and higher LDL-cholesterol (139.9+/-60.3 vs 109.9+/-35.5 mg/dl; p<0.01). The frequency of the polymorphism MR p.I180V was similar between hypertensive subjects and controls (p=0.15). CONCLUSIONS: The MR p.I180V polymorphism seems to be associated with cardiovascular risk factors including BMI and LDL-cholesterol levels. This original in vivo finding reinforces the role of MR in adipocyte biology and in cardiovascular disease.

Ultrasonographic diagnosis: cranial vena cava thrombosis in a cow.

A 5-year-old Hereford cow was donated to Oregon State University, College of Veterinary Medicine. Two weeks before presentation the cow was treated by a left-flank laparotomy to correct a uterine torsion that revealed an excessive amount of abdominal fluid and a full term dead fetus. Ultrasonographic evaluation of the right cranioventral thoracic cavity was performed owing to physical examination (muffled heart sounds, areas of silence) and thoracocentesis findings suggestive of pleuritis. Ultrasonography revealed hypoechoic pleural effusion and a mass of mixed echogenicity visible within the right atrial lumen extending into the cranial vena cava. A diagnosis of cranial vena cava thrombosis was demonstrated at necropsy. The clinical, ultrasonographic, and pathological features of this less common condition (cranial vena cava thrombosis) in cattle are discussed.

Cardiopulmonary and sedative effects of intravenous administration of low doses of medetomidine and xylazine to adult horses.

OBJECTIVE: To determine the cardiopulmonary and sedative effects of medetomidine hydrochloride in adult horses and to compare those effects with effects of an equipotent dose of xylazine hydrochloride. ANIMALS: 10 healthy adult female horses. PROCEDURE: 5 horses were given medetomidine (4 microg/kg of body weight, i.v.), and the other 5 were given xylazine (0.4 mg/kg, i.v.). Heart rate, respiratory rate, arterial blood pressures, pulmonary arterial blood pressures, and cardiac output were recorded, and sedation and ataxia scores were assigned before and every 5 minutes after drug administration for 60 minutes. Rectal temperature and blood gas partial pressures were measured every 15 minutes after drug administration. RESULTS: Arterial blood pressure was significantly decreased throughout the study among horses given medetomidine and was significantly decreased for 40 minutes among horses given xylazine. Compared with baseline values, cardiac output was significantly decreased 10, 20, and 40 minutes after administration of medetomidine and significantly increased 40 and 60 minutes after administration of xylazine. Despite the significant decrease in respiratory rate in both groups, results of blood gas analyses were not significantly changed over time. Ataxia and sedation scores were of similar magnitude for the 2 groups, but ataxia persisted slightly longer among horses given medetomidine. Horses resumed eating hay 10 to 55 minutes after drug administration. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that equipotent low doses of medetomidine and xylazine induce comparable levels of ataxia and sedation and similar cardiopulmonary changes in adult horses.

Hemodynamic and metabolic alterations associated with intravenous infusion of a combination of adenosine triphosphate and magnesium chloride in conscious horses.

OBJECTIVE: To determine hemodynamic and metabolic effects of IV infusion of ATP-MgCl2 combination and maximal safe IV infusion rate in conscious horses. ANIMALS: 6 adult female horses. PROCEDURE: All horses received an IV infusion of ATP-MgCl2 combination, beginning at a rate of 0.05 mg of ATP/kg of body weight/min, which was increased by 0.05 mg/kg/min increments at 10-minute intervals until a rate of 1.0 mg/kg/min was achieved. Data were collected prior to the start of the infusion, at the end of each infusion rate, and at 15-minute intervals for the next hour after discontinuation of the infusion. Measured or calculated hemodynamic variables included cardiac output, cardiac index, heart rate, stroke volume, systemic and pulmonary arterial pressures, and systemic and pulmonary vascular resistances. Arterial blood gas tensions, CBC, plasma biochemical profiles, urine volume and specific gravity, and selected clinical signs of disease also were evaluated. RESULTS: Intravenous infusion of ATP-MgCl2 significantly increased cardiac output, decreased systemic vascular resistance, and caused mild pulmonary hypertension. Magnitude of the hemodynamic alterations was dependent on rate of infusion. Maximal safe infusion rate for these horses was 0.3 mg/kg/min. All horses became lethargic, and their appetites diminished during the infusion; 5 horses had mild signs of abdominal discomfort. Flank sweating was observed in all horses as infusion rate increased. Urine volume and specific gravity and hematologic, biochemical, and arterial blood gas alterations were detected during and after infusion. CONCLUSIONS AND CLINICAL RELEVANCE: Intravenous administration of ATP-MgCl2 in healthy, conscious, adult horses caused various metabolic and hemodynamic alterations that were without appreciable detrimental effects.

Plasma and urine nitric oxide concentrations in horses given below a low dose of endotoxin.

OBJECTIVE: To quantify plasma and urine nitric oxide (NO) concentrations before and after low-dose endotoxin infusion in horses. ANIMALS: 11 healthy adult female horses. Procedure-Eight horses were given endotoxin (35 ng/kg of body weight,i.v.) over 30 minutes. Three sentinel horses received an equivalent volume of saline (0.9% NaCl) solution over the same time. Clinical signs of disease and hemodynamic variables were recorded, and urine and plasma samples were obtained to measure NO concentrations prior to endotoxin infusion (t = 0) and every hour until postinfusion hour (PIH) 6, then every 2 hours until PIH 24. Blood for hematologic and metabolic analyses and for serum cytokine bioassays were collected at 0 hour, every hour until PIH 6, every 2 hours through PIH 12, and finally, every 6 hours until PIH 24. RESULTS: Differences in plasma NO concentrations across time were not apparent, but urine NO concentrations significantly decreased at 4 and 20 to 24 hours in endotoxin-treated horses. Also in endotoxin-treated horses, alterations in clinical signs of disease, and hemodynamic, metabolic, and hematologic variables were significant and characteristic of endotoxemia. Serum interleukin-6 (IL-6) activity and tumor necrosis factor (TNF) concentrations were increased above baseline values from 1 to 8 hours and 1 to 2 hours, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Plasma and urine NO concentrations did not increase in horses after administration of a low dose of endotoxin, despite induction of an inflammatory response, which was confirmed by increased TNF and IL-6 values characteristic alterations in clinical signs of disease, and hematologic, hemodynamic and metabolic variables.

Duration of effects of phenylbutazone on serum total thyroxine and free thyroxine concentrations in horses.

The objectives of this study were to determine if phenylbutazone decreased serum thyroxine (TT4) and free thyroxine (FT4) concentrations using radioimmunoassay and equilibrium dialysis techniques in horses, and, if so, an additional objective was to determine the duration of this decreased concentration once phenylbutazone administration was discontinued. Serum TT4 and FT4 concentrations were determined before and after administration of 4.4 mg/kg of phenylbutazone i.v. bid for 5 days. Treatment with phenylbutazone caused a significant decrease in TT4 and FT4 concentrations (P < .05). Serum TT4 concentration significantly decreased after day 4 of treatment and remained significantly below baseline value for 10 days after discontinuing phenylbutazone administration; it returned to a value not different from the baseline value by the 11th day. Serum FT4 concentration significantly decreased after day 4 of treatment and remained significantly below the baseline value for only 1 day after phenylbutazone administration was discontinued; it returned to a value not different from the baseline value by the 3rd day after discontinuation of phenylbutazone. These results indicate that serum TT4 and FT4 should not be used to evaluate thyroid function in horses receiving phenylbutazone. In addition, results should be interpreted cautiously when phenylbutazone has been administered within 2 days (for FT4) or within 10 days (for TT4) of sample collection.