Passage of uranium through human cerebral microvascular endothelial cells: influence of time exposure in mono- and co-culture in vitro models.

PURPOSE: Depleted uranium (DU) has several civilian and military applications. The effects of this emerging environmental pollutant on human health raise some concerns. Previous experimental studies have shown that uranium (U) exposure can disturb the central nervous system. A small quantity of U reaches the brain via the blood, but the effects on the blood-brain barrier (BBB) remain unclear. MATERIALS AND METHODS: In the present work, two cell culture models were exposed to DU for different times to study its cytotoxicity, paracellular permeability and extracellular concentration of U. The well-known immortalized human cerebral microvascular endothelial cells, hCMEC/D3, were cultured on the filter in the first model. In the second model, human primary cells of pericytes were cultured under the filter to understand the influence of cell environment after U exposure. RESULTS: The results show that U is not cytotoxic to hCMEC/D3 cells or pericytes until 500 µM (1.6 Bq.L-1). In addition, acute or chronic low-dose exposure of U did not disturb permeability and was conserved in both cell culture models. However, U is able to reach the brain compartment. During the first hours of exposure, the passage of U to the abluminal compartment was significantly reduced in the presence of pericytes. Electronic microscopy studies evidenced the formation of needlelike structures, like urchin-shaped precipitates, from 1 h of exposure. Analytical microscopy confirmed the U composition of these precipitates. Interestingly, precipitated U was detected only in endothelial cells and not in pericytes. U was localized in multilamellar or multivesicular bodies along the endo-lysosomal pathway, suggesting the involvement of these traffic vesicles in U sequestration and/or elimination. CONCLUSIONS: We show for the first time the in vitro passage of U across a human cerebral microvascular endothelial cells, and the intracellular localization of U precipitates without any cytotoxicity or modification of paracellular permeability. The difference between the results obtained with monolayers and co-culture models with pericytes illustrates the need to use complex in vitro models in order to mimic the neurovascular unit. Further in vivo studies should be performed to better understand the passage of U across the blood-brain barrier potentially involved in behavioral consequences.

Cardiac performance assessment during cardiopulmonary exercise test can improve the management of children with repaired congenital heart disease.

BACKGROUND: Children with repaired congenital heart disease (CHD) have impaired maximal aerobic capacity (VO2max). Determining the causes of their VO2max alteration remains challenging. Cardiac output measure using thoracic impedancemetry during cardiopulmonary exercise tests (CPET) can help to understand the determinants of VO2max in children with open-heart repaired CHD. METHOD: We analyzed CPET in 77 children with repaired CHD. Among them, 55 patients had residual lesions. Patients with repaired CHD were compared with 44 age-matched healthy individuals. Maximal oxygen content brought to capillaries (QO2max) and oxygen muscle diffusion capacity (DO2) were assessed using cardiac output measure, Fick principle and simplified Fick law. RESULTS: In the 55 patients with residual lesion, VO2max, QO2max and DO2 were lower than those of controls (76.1 vs 86% of theoretical value, p < 0.01; 2.15 vs 2.81 L/mn, p < 0.001; 24.7 vs 28.8 ml/min/mmHg, p < 0.05). Decrease in QO2max was due to both impaired stroke volume and chronotropic insufficiency (48 vs 53 ml/m2 and p < 0.05; 171 vs 185/min p < 0.001). Patients without residual lesion (22/77) had normal VO2max with lower maximal heart rate compensated by higher SV (p < 0.05). CONCLUSION: Aerobic capacity was normal in children without residual lesions after CHD repair. Patients with residual lesion have impaired VO2max due to both lower central and peripheral determinants. Measuring cardiac performance during CPET allowed a better selection of patients with altered cardiac reserve that can benefit from residual lesion treatment and find the good timing for intervention. Detection of peripheral deconditioning can lead to a rehabilitation program.

[Cardiac rehabilitation in adults with congenital heart diseases]. / Réadaptation cardiaque des adultes avec cardiopathies congénitales.

The great advances in the medical and surgical management of congenital heart diseases have allowed many children to reach adulthood with often a good hemodynamic result. Nevertheless most of these adults have a limitation of their functional capacity. This limitation is more or less important, penalizes them in their daily life and alters their quality of life. The origin of this limitation is generally multifactorial. It is linked, of course, to the severity of the heart disease and the quality of the operative result. But there is very often a physical deconditioning. It can be secondary to the heart disease but is often secondary to a lack of physical activity. It is the parents, sometimes overprotective, but frequently the doctors who imposed, often wrongly, this restriction. It is essential to take this dimension into account in view of the important benefits expected for health and quality of life. Cardiac rehabilitation is a privileged tool for providing advice in a suitable environment. This requires close collaboration between cardiac rehabilitators and congenital cardiologists to offer appropriate care. We bring here some reflections and the basic elements to guide the re-training of these patients.

Cut-off values for normal sperm morphology and toxicology for automated analysis of rat sperm morphology and morphometry.

We used automated sperm morphology analysis to investigate rat sperm morphometry and morphology in Sprague-Dawley and Wistar rats in three research centers to develop normal baseline values for sperm morphometry and to quantify the percentage of morphologically normal sperm in healthy rats. The participating centers were IRSN in Paris, France (Sprague-Dawley rats), University of the Western Cape, South Africa (Wistar rats) and Stellenbosch University (Wistar rats), South Africa. All three centers used identical sperm isolation techniques from the cauda epididymis, the same staining protocols, identical computer-aided sperm morphometry analysis (CASMA) software and microscopes with similar optics. With CASMA, fully automated analysis of the different parts of stained sperm, e.g., head, acrosome, mid-piece, can be performed, many sperm morphometric features can be measured accurately and eventually normal sperm morphology can be defined. We found that it is possible to distinguish sperm morphometric characteristics of Sprague-Dawley and Wistar rats. We also developed cut-off values for evaluating the percentage normal sperm in these two rat strains using the automatic analysis mode. Normal sperm morphology varied between 67 and 74% by contrast with previous findings of > 90%.

DNA methylation and potential multigenerational epigenetic effects linked to uranium chronic low-dose exposure in gonads of males and females rats.

INTRODUCTION: An increased health problem in industrialised countries is the contemporary concern of public and scientific community as well. This has been attributed in part to accumulated environmental pollutants especially radioactive substances and the use of nuclear power plants worldwide. However, the outcome of chronic exposure to low doses of a radionuclide such as uranium remains unknown. Recently, a paradigm shift in the perception of risk of radiotoxicology has emerged through investigating the possibility of transmission of biological effects over generations, in particular by epigenetic pathways. These processes are known for their crucial roles associated with the development of several diseases. OBJECTIVE: The current work investigates the epigenetic effect of chronic exposure to low doses of uranium and its inheritance across generations. Materials and Methods To test this proposition, a rodent multigenerational model, males and females, were exposed to a non-toxic concentration of uranium (40mgL-1 drinking water) for nine months. The uranium effects on were evaluated over three generations (F0, F1 and F2) by analysing the DNA methylation profile and DNMT genes expression in ovaries and testes tissues. RESULTS: Here we report a significant hypermethylation of testes DNA (p <0.005) whereas ovaries showed hypomethylated DNA (p <0.005). Interestingly, this DNA methylation profile was significantly maintained across generations F0, F1 and F2. Furthermore, qPCR results of both tissues imply a significant change in the expression of DNA methyltransferase genes (DNMT 1 and DNMT3a/b) as well. CONCLUSION: Altogether, our work demonstrates for the first time a sex-dependance and inheritance of epigenetic marks, DNA methylation, as a biological response to the exposure to low doses of uranium. However, it is not clear which type of reproductive cell type is more responsive in this context.

Hepatocytes cocultured with Sertoli cells in bioreactor favors Sertoli barrier tightness in rat.

The lack of a reliable in vitro system to assess reprotoxicity is an emerging problem in the context of European law for Registration, Evaluation, Authorization and Restriction of Chemicals (REACH, 2007), as it requires a reduction in animal utilization for testing. Furthermore, in vitro reprotoxicological tests would be more relevant and greatly improved by integrating both hepatic metabolism and the blood-testis barrier. Here, we took advantage of an integrated insert in a dynamic microfluidic platform (IIDMP) to co-cultivate hepatocytes in biochip and Sertoli cells in the bicameral chamber. This microfluidic tool has been previously demonstrated to be helpful in cell function and/or quality improvement. We demonstrate that permeability of the Sertoli barrier is reduced by dynamic coculture in our system. Exometabolomics analysis reveals that interactions between hepatocytes and Sertoli cells may have been mediated by the polyamines increase and/or mid-chain fatty acid decrease in the circulating medium. These metabolic changes may be involved in permeability reduction by contributing to modifying junction protein quantity and localization. The present study gives an example of IIDMP as an in vitro partitioning/transport model for cell culture and toxicological testing. Further, based on both our previous results using an intestinal-hepatic cell coculture and the present study, IIDMP seems to be well-suited for (i) assessing the dose-response effect of chemicals within the rodent or human male reproductive tract, and (ii) improving the quality of reprotoxicological assays by including hepatic metabolism. Copyright © 2016 John Wiley & Sons, Ltd.

Treatment of Disseminated Aspergillosis with Posaconazole in 10 Dogs.

BACKGROUND: Few effective treatments for disseminated Aspergillus infections in dogs are available. Posaconazole has potent and broad-spectrum activity against Aspergillus spp., but its use has not yet been sufficiently evaluated in dogs. HYPOTHESIS/OBJECTIVES: The aim of this study was to determine the safety and efficacy of posaconazole for the treatment of naturally occurring disseminated Aspergillus infections in dogs. ANIMALS: Ten client-owned dogs with disseminated aspergillosis. METHODS: Prospective, nonrandomized, noncontrolled study with posaconazole administered to dogs at dosage of 5 mg/kg p.o. q12h. The primary veterinarian or the veterinary specialist caring for the dogs provided patient data. RESULTS: The treatment response for dogs with disseminated disease while receiving posaconazole was defined as clinical remission (n = 4) and clinical improvement (n = 6). There was a high rate of relapse during treatment or after cessation of treatment in both groups, and most dogs died or were euthanized due to progressive disease. Excluding 1 dog concurrently treated with terbinafine that remains alive 5 years after diagnosis, the mean survival time for dogs was 241 days (range 44-516 days). Three other dogs lived >1 year after starting treatment. No clinically relevant adverse events or increases in serum liver enzyme activity occurred during treatment with posaconazole. CONCLUSIONS AND CLINICAL IMPORTANCE: Posaconazole appears to be safe and well-tolerated for treatment of disseminated Aspergillus infections in dogs. Long-term survival >1 year is possible with prolonged treatment, but relapse is common.

Masitinib mesylate for metastatic and non-resectable canine cutaneous mast cell tumours.

Masitinib mesylate is a tyrosine kinase inhibitor approved for the treatment of gross, non-metastatic grade II and III canine mast cell tumours (MCTs). This study evaluated the use of masitinib as a frontline and rescue agent for metastatic and non-metastatic canine MCTs. Identification of toxicities and prognostic factors in these dogs was of secondary interest. Twenty-six dogs were included in this study. The overall response rate to masitinib was 50%. The median survival time for dogs that responded to masitinib was 630 days versus 137 days for dogs that did not respond (P = 0.0033). Toxicity was recorded in 61.5% of treated dogs, but the majority of adverse events were mild and self-limiting. Response to masitinib, not tumour grade, stage or location, was the most significant prognostic factor for survival in dogs with MCTs.

The induction of a secondary corpus luteum on day 12 post-ovulation can delay the time of luteolysis in high-producing Holstein cows.

Luteolysis before the time of maternal recognition of pregnancy is one cause of low fertility in high-producing dairy cows. The objective of this study was to assess whether induction of a secondary corpus luteum (CL) late in the luteal phase would delay the time of luteolysis. Twenty high-producing Holstein cows were synchronized to ovulation (Day 0) with the Ovsynch protocol and received hCG (1500 IU im) on Day 12. Corpora lutea formation (as evaluated by ultrasonography) and plasma P4 concentrations were monitored from Days 4 to 36. hCG treatment induced the formation of one secondary CL (CL2) in 11 of 20 cows (55%) from the dominant follicle (mean diameter: 14.2 ± 0.9 mm) of two-wave (3/11) and three-wave (8/11) cycles. The maximal diameter of the CL2 (23.3 ± 1.9 mm) was reached approximately 6 days after hCG treatment and was correlated with its structural lifespan (p < 0.01). Cows that formed a CL2 after hCG had higher mean plasma P4 concentrations on Day 14 (+4.5 ng/ml) and Day 18 (+3.0 ng/ml) compared with cows without CL2 (p < 0.05). The structural regression of CL2 begun approximately 8 days after that of the CL1, and the median time at which the first drop in circulating P4 levels occurred was later in cows that formed a CL2 than in those that did not (Day 26 vs Day 18; p < 0.01). Thus, the induction of a CL2 by hCG on Day 12 might reduce the risk of premature luteolysis in high-producing dairy cows after insemination.

In situ reconstruction in native and prosthetic aortic infections using cryopreserved arterial allografts.

OBJECTIVES: To evaluate overall survival and complications of cryopreserved arterial allografts in aortic graft infections and infected aortic aneurysms. METHODS: A retrospective review of consecutive patients was conducted with native or prosthetic aortic infections, who underwent local debridement and in situ implantation of a cryopreserved aortic allograft from September 2004 to June 2012 at the Henri Mondor University Hospital. Patient characteristics, indications for allograft implantation, perioperative events, bacteriological data, and events related to follow-up were identified. The primary outcome was overall survival. Overall survival was estimated using the Kaplan-Meier method. Predictors of postoperative mortality were identified using uni- and multivariate analysis with a Cox proportional hazard regression. RESULTS: During the study period, 54 patients (45 [83%] men, mean age 66.2 ± 10.2 years) underwent aortic reconstruction using cryopreserved allografts. Indications were native aortic infection in 17 patients and prosthetic graft infection in 37 patients, including seven aortoenteric fistulae. Twelve aortic reconstructions (22%) were performed as emergency procedures. The median duration of follow-up was 12.1 months (range 0.4-83.6). The 30-day mortality rate was 28%. The overall mortality rate was 39% at a median follow-up of 12.1 months. Early significant postoperative complications occurred in 52% of patients. The graft-related mortality rate was 7%. The graft-related complication rate was 19%. During follow-up, there were two recurrences of aortic infection and two recurrences of allograft limb occlusion. Multivariate survival analysis identified age, chronic renal disease, prosthetic infection, emergent procedure, and coronary disease as independent predictors for postoperative mortality. CONCLUSION: This experience with cryopreserved aortic allografts in aortic reconstructions shows an unsatisfactory 30-day survival rate, as well as a substantial early graft-related complication rate. Longer follow-up is needed in order to support the preferential use of cryopreserved allografts based on their long-term behavior.

(±)-2,2-Dimethyl-5-oxotetrahydrofuran-3-carboxylic acid (terebic acid): a racemic layered structure.

A racemic crystalline form of terebic acid, C(7)H(10)O(4), which is an important industrial chemical compound, is reported for the first time. The crystal structure is stabilized by O-H···O and C-H···O hydrogen bonds which form racemic double layers parallel to (001).

[Use of platelet concentrates: a regional experience]. / Utilisation des concentrés plaquettaires : expérience d'une région.

PURPOSE OF THE STUDY: The Établissement français du sang (EFS) distributes two types of platelet concentrates: using a single donor in aphaeresis platelet concentrate (SDAP), versus pooled platelet concentrates (PPC). A retrospective study performed by the Blood Derivatives Group at Observatory for Drugs, Medical Devices and Therapeutic Innovations (OMEDIT), in collaboration with EFS and haemovigilance correspondents from eight regional health care establishments, has analyzed platelet concentrates prescriptions and the position of the prescribers concerning PPC supply. MATERIAL AND METHODS: Between the 2nd and 6th June 2008, 151 platelet concentrates were supplied by ESF. Data were collected for 144 platelet concentrates and in 83 transfused patients with an average age of 50years. During this study, 33 PPC (23%) and 111 SDAP (77%) were supplied. RESULTS: With regards to the 111 SDAP, the supply of PPC was refused in 47 cases (42%), accepted in 18 cases (16%) and unknown for 46 cases (42%). A total of 51 PPC could be supplied during this period, which represented 35% of platelet concentrates prescriptions. The rate of platelets before transfusion was known for 121 platelet concentrates, the median was 32G·L(-1) for SDAP and 44G·L(-1) for PPC. CONCLUSION: More frequent PPC use, with comparable therapeutic efficacy, could be interesting in a context of increasing platelet concentrates consumption in health care establishments. Moreover, prescribers did not seem to be against the idea. An information pamphlet on platelet concentrates was drafted and distributed to prescribers in order to promote the prescription of PPC. A second assessment is planned to measure the impact of this communication.

Novel mutations in scavenger receptor BI associated with high HDL cholesterol in humans.

The scavenger receptor class B, member 1 (SR-BI), is a key cellular receptor for high-density lipoprotein (HDL) in mice, but its relevance to human physiology has not been well established. Recently a family was reported with a mutation in the gene encoding SR-BI and high HDL cholesterol (HDL-C). Here we report two additional individuals with extremely high HDL-C (greater than the 90th percentile for age and gender) with rare mutations in the gene encoding SR-BI. These mutations segregate with high HDL-C in family members of each proband and are associated with a 37% increase in plasma HDL-C in heterozygous individuals carrying them. Both mutations occur at highly conserved positions in the large extracellular loop region of SR-BI and are predicted to impair the function of the SR-BI protein. Our findings, combined with the prior report of a single mutation in the gene encoding SR-BI, further validate that mutations in SR-BI are a rare but recurring cause of elevated HDL-C in humans.

An engineered 3D blood-testis barrier model for the assessment of reproductive toxicity potential.

We have developed an in vitro model that replicates the composition, organization, and barrier and spermatogenesis functions of the in vivo rat blood-testis barrier. This engineered blood-testis barrier (eBTB) is based on a three-dimensional (3-D) culture in a bicameral chamber of testicular cells isolated from 18-day-old rats. Peritubular cells were cultured on the bottom of the insert. On the top of the insert, a mixture of Sertoli and germ cells were coated within an artificial extracellular matrix, thereby mimicking the basement membrane. The matrix composition was defined to obtain a cord-like organization. This structure was revealed depending on morphogenetic gradients, and was made of polarized Sertoli cells and germ cells in the center of the structure. The in vivo functionality of the BTB was characterized by tight junctions between Sertoli cells. Claudin-11 protein immunodetection suggests that these junctions were also implicated in vitro in the cord-like structure, suggesting the presence of a physical compartment with apical and basal spaces. Measurement of the trans-epithelial electrical resistance characterized the relationship between the Sertoli cells, peritubular cells, and matrix/cells that influenced the tightness of their junctions during the course of the culture. In vitro germ cell differentiation was confirmed with the detection of haploid cells. The development of the eBTB under optimum conditions addresses the involvement of new models, testing the barrier and spermatogenesis functions that are sensitive to chemical compounds from the environment. In this way, the eBTB could be used as an alternative method to animal reprotoxicity studies, and would be of high interest in the scope of regulatory requests for chemical risk assessment.

Review paper: Cancer chemopreventive compounds and canine cancer.

Canine cancer has become more prevalent in recent years because of increased life expectancy and greater attention to the health of pets. The range of cancers seen in dogs is as diverse as that in human patients, and despite more intensive therapeutic interventions, fatality rates remain unacceptably high in both species. Chemoprevention is therefore an important means of confronting this disease. Because domestic pets share our environment, greater cross-application and study of the protumorigenic and antitumorigenic factors in our shared environment will benefit all species, leading to the development of new families of less toxic antitumorigenic compounds based on novel and established molecular targets. Currently, the most interesting cancer preventive agents are nonsteroidal anti-inflammatory drugs, peroxisome proliferator-activated receptor-gamma ligands, and dietary compounds. This article provides an overview of what is known about how these agents affect molecular signaling in neoplastic disease, with reference to reported application and/or study in dogs where available.

Masitinib is safe and effective for the treatment of canine mast cell tumors.

BACKGROUND: Activation of the KIT receptor tyrosine kinase is associated with the development of canine mast cell tumors (MCT). HYPOTHESIS/OBJECTIVE: To evaluate the efficacy of masitinib, a potent and selective inhibitor of KIT, in the treatment of canine MCT. ANIMALS: Two hundred and two client-owned dogs with nonmetastatic recurrent or nonresectable grade II or III MCT. METHODS: Double-blind, randomized, placebo-controlled phase III clinical trial. Dogs were administered masitinib (12.5 mg/kg/d PO) or a placebo. Time-to-tumor progression (TTP), overall survival, objective response at 6 months, and toxicity were assessed. RESULTS: Masitinib increased overall TTP compared with placebo from 75 to 118 days (P = .038). This effect was more pronounced when masitinib was used as first-line therapy, with an increase in the median TTP from 75 to 253 days (P = .001) and regardless of whether the tumors expressed mutant (83 versus not reached [P = .009]) or wild-type KIT (66 versus 253 [P = .008]). Masitinib was generally well tolerated, with mild (grade I) or moderate (grade II) diarrhea or vomiting as the most common adverse events. CONCLUSIONS AND CLINICAL IMPORTANCE: Masitinib is safe and effective at delaying tumor progression in dogs presenting with recurrent or nonresectable grade II or III nonmetastatic MCT.

Antigen and antibody testing for the diagnosis of blastomycosis in dogs.

BACKGROUND: Early diagnosis and treatment are associated with an improved prognosis in blastomycosis. The diagnosis of blastomycosis may be missed by cytology, histopathology, culture, or serology. An enzyme immunoassay (EIA) for detection of Blastomyces dermatitidis galactomannan antigen in body fluids has been used for rapid diagnosis of blastomycosis in humans. HYPOTHESIS: Measurement of Blastomyces antigen in urine or serum by the MVista Blastomyces antigen EIA is more sensitive than measurement of anti-Blastomyces antibodies for diagnosis of blastomycosis in dogs. METHODS: Serum and urine samples from 46 dogs with confirmed blastomycosis were tested for Blastomyces antigen and serum was tested for anti-Blastomyces antibodies. RESULTS: The sensitivity for the detection of antigen in urine was 93.5% and it was 87.0% in serum. The sensitivity of antibody detection by agar gel immunodiffusion (AGID) was 17.4% and it was 76.1% by EIA. Antigen and antibody decreased during itraconazole treatment. CONCLUSIONS AND CLINICAL IMPORTANCE: Antigen detection is a more sensitive test for diagnosis of blastomycosis than antibody testing by AGID, the only commercially available method. Antigen concentrations decreased with treatment.

Successful surgical treatment for a single coronary artery arising from the pulmonary artery.

The case of a 17-day-old infant who underwent correction of an aortic coarctation with pulmonary artery banding for ventricular septal defect is reported. Ischemic cardiac failure 3 weeks later led the authors to diagnose a single coronary artery originating from the pulmonary artery. At the age of 2 months, the infant underwent successful reimplantation of the coronary artery onto the ascending aorta and surgical closure of the septal defect. Normalization of the left ventricular function was observed at the 2-year follow-up assessment.

Effects of L-asparaginase on plasma amino acid profiles and tumor burden in cats with lymphoma.

BACKGROUND: L-Asparaginase (Elspar(a)), is an Escherichia coli-derived enzyme that depletes lymphoma cells of asparagine, inhibiting protein synthesis and resulting in cell death. The single agent response rate in cats with lymphoma and impact of L-asparaginase on plasma amino acid concentrations is unknown. HYPOTHESES: L-Asparaginase significantly reduces plasma asparagine concentrations and has demonstrable efficacy against untreated lymphoma in cats. ANIMALS: Thirteen cats with confirmed lymphoma (LSA) of any anatomic site were given 1 dose 400 IU/kg IM) of L-asparaginase for initial LSA treatment. METHODS: Plasma collected at 0, 2, and 7 days after L-asparaginase therapy was assayed for ammonia, asparagine, aspartic acid, glutamine, and glutamic acid concentrations. Cats were restaged 7 days later to assess tumor response. RESULTS: Eight cats had T-cell LSA, 4 cats had B-cell LSA, and 1 cat's immunophenotype was unknown. Two complete and 2 partial responses to L-asparaginase were seen. Four cats had stable disease, and 5 cats had progressive disease. Ammonia and aspartic acid concentrations were increased from baseline at 2 and 7 days posttreatment. Asparagine concentrations were decreased from baseline at 2 days but not 7 days posttreatment. Glutamic acid concentrations were increased at day 2 compared to day 7 posttreatment but not compared to baseline. Glutamine concentrations were unchanged. CONCLUSIONS AND CLINICAL IMPORTANCE: L-asparaginase significantly reduced asparagine concentrations within 2 days of treatment, but this effect was lost within 7 days. The apparent overall response rate of feline LSA to L-asparaginase in this study was 30%.