Concentrations Of Fetal Serum Research Articles

Integration of MUTZ-Langerhans cells into a 3D full-thickness skin equivalent and influences of serum reduction and undefined medium supplements on differentiation

The MUTZ-3 cell line is a surrogate for Langerhans cells (LCs) employed in New Approach Methodologies for assessing the skin sensitizing potential of chemicals. However, MUTZ-3 cells must first be differentiated to achieve the LC-typical phenotype. As all protocols use high fetal calf serum (FCS) concentrations, we aimed at reducing, or even replacing FCS, while maintaining MUTZ-LC characteristics. Additionally, we assessed the impact of the poorly defined 5637-conditioned medium (5637CM) on MUTZ-LC differentiation.With reducing the FCS content by 75 %, the desired differentiation status was achieved after 7 instead of 14 days, identified by elevated CD207 and CD1a expression. Culture with Ultroser G, a synthetic surrogate for FCS, resulted in an insufficient number of MUTZ-LCs. 5 % FCS-differentiated MUTZ-LCs could be activated with DNCB, an extreme sensitizer, as demonstrated by increased CD83 expression. 5637CM did not affect MUTZ-LC differentiation and is therefore not needed as a supplement.For their intended role in an immunocompetent skin model to assess the sensitizing potential of chemicals, MUTZ-LCs were successfully integrated into the Phenion® Full-Thickness skin model, as demonstrated by CD1a expression. These results are important steps towards medium standardization and the generation of an immunocompetent skin model according to the 3R principles.

Construction of a 12-metal Zn(II)-Nd(III) nanocluster for ratiometric fluorescence detection of inflammatory marker neopterin

A 12-metal Zn(II)-Nd(III) cluster 1 (sizes: 1.8 nm × 2.0 nm × 2.0 nm) was synthesized from a long-chain type Schiff base ligand. It displays ratiometric fluorescence response to neopterin (Neo) with high selectivity and sensitivity, which can be expressed by the equation I545 nm /I1060 nm = A·[Neo]2 + B·[Neo] + C. 1 is used to quantitatively test Neo concentrations in fetal calf serum (FCS) and urine, and the recovery ranges are 98.57%–103.82% and 99.25%–103.50%, respectively, while the relative standard deviations (RSDs) are 7.89%–9.46% and 1.85%–4.16%, respectively. The limits of detection of 1 to Neo in FCS and urine are 0.034 and 0.021 μmol/L, respectively.

Optimal isolation, culture, and invitro propagation of spermatogonial stem cells in Huaixiang chicken.

Spermatogonial stem cells (SSCs) comprise the foundation of spermatogenesis and hence have great potential for fertility preservation of rare or endangered species and the development of transgenic animals and birds. Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs invitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8-79 days of age) of Huaixiang chicken. We found that the testes of 21-day-old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02 g/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic-activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30 ng/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20 ng/mL), bFGF (30 ng/mL), or LIF (5 ng/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21-day-old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs.

Ratiometric Fluorescence Detection of Levofloxacin Based on a Cube-like Zn(II)-Eu(III) Nanocluster: Functionalized Sodium Alginate Film for the Detection in Serum and Medicine.

A cube-like Zn(II)-Eu(III) nanocluster 1 (molecular sizes: 1.8 × 2.0 × 2.0 nm) was constructed by the use of a new long-chain Schiff base ligand. It shows a ratiometric fluorescence response to levofloxacin (LFX) with high sensitivity and selectivity, which can be expressed as I615nm/I550nm = A*[LFX]2 + B*[LFX] + C. It is used to quantitatively detect the LFX concentrations in fetal calf serum (FCS) and tablets sold in pharmacy. Filter paper strips bearing 1 can be used to qualitatively detect LFX by a color change to red under a UV lamp. 1 and its hybrid with sodium alginate (SA), 1@SA, display potential applications in the qualitative detection of LFX in FCS and the medicine. The limit of detection of 1 to LFX is as low as 2.1 × 10-2 nM.

Metabolic Profiling of SH-SY5Y and Neuro2A Cells in Relation to Fetal Calf Serum (FCS) Concentration in Culture Media.

The neuroblastoma cell lines SH-SY5Y and Neuro2A are commonly utilized models in neurobiological research. DMEM supplemented with different nutrients and 5-10% Fetal Calf Serum (FCS) is typically used for culturing these cell lines. During special treatments, a reduced FCS content is often deployed to reduce cellular proliferation or the content of bioactive compounds. The impact of the reduction of FCS in culture media on the metabolic profile of SH-SY5Y and Neuro2A cells is currently unknown. Using an Amplex Red Assay, this study showed that the consumption of L-glutamine decreased after FCS reduction. Glucose and pyruvate consumption increased in both cell lines after the reduction of FCS. Thus, lactate production also increased with reduced FCS concentration. The reduction of FCS in the cell culture medium resulted in a reduced aerobic ATP production for SH-SY5Y cells and a complete shut down of aerobic ATP production for Neuro2A cells, measured using the Seahorse XF Real-Time ATP Rate Assay. Utilizing the Seahorse XF Glutamine Oxidation Stress Test, Neuro2A cells showed an increased utilization of L-glutamine oxidation after reduction of FCS. These results indicate that changes in FCS concentration in culture media have an impact on the different energy production strategies of SH-SY5Y and Neuro2A cells which must be considered when planning special treatments.

Effect of Genetically Reduced Maternal Myostatin on Late Gestation Maternal, Fetal, and Placental Metabolomes in Mice.

Myostatin (gene symbol: Mstn) is an autocrine and paracrine inhibitor of muscle growth. Pregnant mice with genetically reduced levels of myostatin give birth to offspring with greater adult muscle mass and bone biomechanical strength. However, maternal myostatin is not detectable in fetal circulations. Fetal growth is dependent on the maternal environment, and the provisioning of nutrients and growth factors by the placenta. Thus, this study examined the effect of reduced maternal myostatin on maternal and fetal serum metabolomes, as well as the placental metabolome. Fetal and maternal serum metabolomes were highly distinct, which is consistent with the role of the placenta in creating a specific fetal nutrient environment. There was no effect from myostatin on maternal glucose tolerance or fasting insulin. In comparisons between pregnant control and Mstn+/- mice, there were more significantly different metabolite concentrations in fetal serum, at 50, than in the mother's serum at 33, confirming the effect of maternal myostatin reduction on the fetal metabolic milieu. Polyamines, lysophospholipids, fatty acid oxidation, and vitamin C, in fetal serum, were all affected by maternal myostatin reduction.

Soluble factors from TLR4- or TCR-activated cells contribute to stability of the resting phenotype and increase the expression of CXCR4 of human memory CD4 T cells.

It has been proposed that cytokines can induce activation of resting T cells in an antigen-independent manner. However, experimental conditions have included the use of fetal serum and nanogram concentrations of added cytokines. To evaluate the effect of cytokines and chemokines generated by activated immune cells on the phenotypic profile of human memory CD4 T cells, the cells were cultured in FBS-free conditions in the presence of IL-15 and 5% of hAB serum and incubated with conditioned medium (CM) obtained from PBMC activated through the TCR using anti-CD3/CD28/CD2 antibodies (TCR-CM) or through TLR4 using bacterial LPS (TLR4-CM). Cytokines and chemokines present in the CMs were evaluated by ProcartaPlex immunoassay. Cell viability, proliferation, and surface markers were determined by flow cytometry on day 2, 5, and 8 of culture. Cell viability was maintained by TLR4-CM plus IL-15 for 8days but decreased in the presence of the TCR-CM plus IL-15. In combination with IL-15, the TLR4-CM, but not the TCR-CM, maintained the expression of CD3 and CD4 stable. Both conditions stabilized the expression of CD45RO and CCR5. Thus, the TLR4-CM better supported the viability and stability of the memory phenotype. None of the CMs induced proliferation or expression of activation markers; however, they induced an increased expression of CXCR4. This study indicates that resting memory CD4 T cells are not activated by, but may be sensitive to soluble factors produced by antigen or PAMP-stimulated cells, which may contribute to their homeostasis and favor the CXCR4 expression.

Utilization of Aminoguanidine Prevents Cytotoxic Effects of Semen.

Studies of human semen in cell or tissue culture are hampered by the high cytotoxic activity of this body fluid. The components responsible for the cell damaging activity of semen are amine oxidases, which convert abundant polyamines, such as spermine or spermidine in seminal plasma into toxic intermediates. Amine oxidases are naturally present at low concentrations in seminal plasma and at high concentrations in fetal calf serum, a commonly used cell culture supplement. Here, we show that, in the presence of fetal calf serum, seminal plasma, as well as the polyamines spermine and spermidine, are highly cytotoxic to immortalized cells, primary blood mononuclear cells, and vaginal tissue. Thus, experiments investigating the effect of polyamines and seminal plasma on cellular functions should be performed with great caution, considering the confounding cytotoxic effects. The addition of the amine oxidase inhibitor aminoguanidine to fetal calf serum and/or the utilization of serum-free medium greatly reduced this serum-induced cytotoxicity of polyamines and seminal plasma in cell lines, primary cells, and tissues and, thus, should be implemented in all future studies analyzing the role of polyamines and semen on cellular functions.

MO396: Development, Establishment and Validation of in Vitro and EX Vivo Assays of Vascular Calcification

Abstract BACKGROUND AND AIMS Vascular calcification is one major complication in patients with chronic kidney disease (CKD), with a misbalance in calcium and phosphate metabolism playing a crucial role. The mechanisms underlying vascular calcification have not been entirely revealed to date. As studies aiming at the identification and characterization of the involved mediators are highly relevant, standardized operating protocols (SOP) for reliable assays to make different studies comparable, are needed. METHOD We are dealing with the following in vitro and ex vivo experimental conditions to study vascular calcifications: induction of calcification by phosphate and CaCl2, incubation time, as well as foetal calve serum concentration and ageing. Furthermore, the location and inhibition of vascular calcifications are studied. Human aortic smooth muscle cells are used for in vitro experiments and rat aorta for ex vivo experiments. The degree of calcification is estimated by quantification of calcium concentrations and by von Kossa staining. RESULTS As a result, a step-by-step protocol for performing experiments on vascular calcification was created. CONCLUSION Through the established protocol and examination of decent modifications nuances, we aim to promote standardized research on vascular calcification.

Maternal and fetal serum concentrations of magnesium after administration of a 6-g bolus dose of magnesium sulfate (MgSO4 ) to women with imminent preterm delivery.

Magnesium sulfate is used world-wide to treat pregnant women at imminent risk of preterm delivery in order to protect the brain of the premature infant. Previous research has shown that magnesium sulfate decreases the risk of cerebral palsy by ~30% in infants born preterm. Despite this, the dosage required for optimal neuroprotection remains unknown. We aimed to investigate whether 6 g magnesium sulfate given as a single bolus dose was tolerable for the women and infants and whether the desired target concentration in the mother's blood was reached and non-toxic level in the infant could be ensured. In total, 49 women who were at risk of delivery prior to 32 weeks of gestation were recruited. They received a bolus dose of 6 g magnesium sulfate intravenously between 1 and 24h prior to giving birth and were closely monitored during and after infusion. Blood samples from the patients were analyzed at different time-points (20-30min after start of infusion, 1, 2, 6 and 24h) post-administration. Blood samples from the umbilical cord were also taken directly after birth to assess the concentration of magnesium in the infant. None of the women who received magnesium sulfate reached serum magnesium concentrations >3.3 mmol/L. In all, 72% of the women showed serum magnesium levels within the therapeutic interval (2.0-3.5 mmol/L) and no adverse events were observed during the infusion. The serum magnesium levels in the mothers declined to pre-bolus-levels within 24h after delivery. Serum magnesium levels in the umbilical cord samples ranged from 0.87 to 1.4 mmol/L, which means that all but two were within the normal expected range for a newborn premature infant. A bolus dose of 6 g magnesium sulfate was well tolerated and without any serious side effects in either mother or infant. Most of our women reached the targeted concentration range of serum magnesium levels after infusion was completed. Their infants had magnesium levels within acceptable levels, regardless of gestational week or mother's body mass index.

Developmental toxicity of Nafion byproduct 2 (NBP2) in the Sprague-Dawley rat with comparisons to hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) and perfluorooctane sulfonate (PFOS).

Nafion byproduct 2 (NBP2) is a polyfluoroalkyl ether sulfonic acid that was recently detected in surface water, drinking water, and human serum samples from monitoring studies in North Carolina, USA. We orally exposed pregnant Sprague-Dawley rats to NBP2 from gestation day (GD) 14-18 (0.1-30mg/kg/d), GD17-21, and GD8 to postnatal day (PND) 2 (0.3-30mg/kg/d) to characterize maternal, fetal, and postnatal effects. GD14-18 exposures were also conducted with perfluorooctane sulfonate (PFOS) for comparison to NBP2, as well as data previously published for hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX). NBP2 produced stillbirth (30mg/kg), reduced pup survival shortly after birth (10mg/kg), and reduced pup body weight (10mg/kg). Histopathological evaluation identified reduced glycogen stores in newborn pup livers and hepatocyte hypertrophy in maternal livers at≥10mg/kg. Exposure to NBP2 from GD14-18 reduced maternal serum total T3 and cholesterol concentrations (30mg/kg). Maternal, fetal, and neonatal liver gene expression was investigated using RT-qPCR pathway arrays, while maternal and fetal livers were also analyzed using TempO-Seq transcriptomic profiling. Overall, there was limited alteration of genes in maternal or F1 livers from NBP2 exposure with significant changes mostly occurring in the top dose group (30mg/kg) associated with lipid and carbohydrate metabolism. Metabolomic profiling indicated elevated maternal bile acids for NBP2, but not HFPO-DA or PFOS, while all three reduced 3-indolepropionic acid. Maternal and fetal serum and liver NBP2 concentrations were similar to PFOS, but ∼10-30-fold greater than HFPO-DA concentrations at a given maternal oral dose. NBP2 is a developmental toxicant in the rat, producing neonatal mortality, reduced pup body weight, reduced pup liver glycogen, reduced maternal thyroid hormones, and altered maternal and offspring lipid and carbohydrate metabolism similar to other studied PFAS, with oral toxicity for pup loss that is slightly less potent than PFOS but more potent than HFPO-DA.

MO583IN VITRO AND EX VIVO EXPERIMENTS OF VASCULAR CALCIFICATION

Abstract Background and Aims Vascular calcification (VC) is one major complication in patients with chronic kidney disease whereas a misbalance in calcium and phosphate metabolism plays a crucial role. The mechanisms underlying VC have not been entirely revealed to date. Therefore are the studies aiming at the identification and characterization of the mediators/uremic toxins involved in VC ongoing and highly relevant. However, currently many different protocols being used in the studies of vascular calcification processes. This complicates the comparison of study outcomes, composing systematic reviews, and meta-analyses. Moreover, the reproducibility of data is hampered, and the efficiency in calcification research through the lack of a standardized protocol is reduced. In this study, we developed a standardized operating protocol for in vitro and ex vivo approaches to aiming at the comparability of these studies. Method We analysed in vitro and ex vivo experimental conditions to study VC. Vascular smooth muscle cells (HAoSMCs) were used for in vitro experiments and aortas from Wistar rats were used for ex vivo experiments. The influence of the following conditions was studied in detail: • Phosphate and calcium concentrations in calcifying media. • Incubation time. • Fetal calf serum (FCS) concentration. The degree of calcification was estimated by quantification of calcium concentrations that were normalized to protein content (in vitro) or to the dry weight of the aortic ring (ex vivo). Additionally, the aortic rings were stained using the von Kossa method. Optimal conditions for investigating medial vascular calcification were detected and summarized in the step-by-step protocol. Results We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings were highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium. An optimized protocol for studying vascular calcification in vitro and ex vivo was developed and validated. The final protocol (Figure 1) presented will help to standardize in vitro and ex vivo approaches to investigate the processes of vascular calcification. Conclusion In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies.

Development and Verification of a Linked Δ 9-THC/11-OH-THC Physiologically Based Pharmacokinetic Model in Healthy, Nonpregnant Population and Extrapolation to Pregnant Women.

Conducting clinical trials to understand the exposure risk/benefit relationship of cannabis use is not always feasible. Alternatively, physiologically based pharmacokinetic (PBPK) models can be used to predict exposure of the psychoactive cannabinoid (-)-Δ9-tetrahydrocannabinol (THC) and its active metabolite 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC). Here, we first extrapolated in vitro mechanistic pharmacokinetic information previously quantified to build a linked THC/11-OH-THC PBPK model and verified the model with observed data after intravenous and inhalation administration of THC in a healthy, nonpregnant population. The in vitro to in vivo extrapolation of both THC and 11-OH-THC disposition was successful. The inhalation bioavailability (Finh) of THC after inhalation was higher in chronic versus casual cannabis users (Finh = 0.35 and 0.19, respectively). Sensitivity analysis demonstrated that 11-OH-THC but not THC exposure was sensitive to alterations in hepatic intrinsic clearance of the respective compound. Next, we extrapolated the linked THC/11-OH-THC PBPK model to pregnant women. Simulations showed that THC plasma area under the curve (AUC) does not change during pregnancy, but 11-OH-THC plasma AUC decreases by up to 41%. Using a maternal-fetal PBPK model, maternal and fetal THC serum concentrations were simulated and compared with the observed THC serum concentrations in pregnant women at term. To recapitulate the observed THC fetal serum concentrations, active placental efflux of THC needed to be invoked. In conclusion, we built and verified a linked THC/11-OH-THC PBPK model in healthy nonpregnant population and demonstrated how this mechanistic physiologic and pharmacokinetic platform can be extrapolated to a special population, such as pregnant women. SIGNIFICANCE STATEMENT: Although the pharmacokinetics of cannabinoids have been extensively studied clinically, limited mechanistic pharmacokinetic models exist. Here, we developed and verified a physiologically based pharmacokinetic (PBPK) model for (-)-Δ9-tetrahydrocannabinol (THC) and its active metabolite, 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC). The PBPK model was verified in healthy, nonpregnant population after intravenous and inhalation administration of THC, and then extrapolated to pregnant women. The THC/11-OH-THC PBPK model can be used to predict exposure in special populations, predict drug-drug interactions, or impact of genetic polymorphism.

Design of a structure-based fluorescent biosensor from bioengineered arginine deiminase for rapid determination of L-arginine

Rationally designed mutations on recombinant arginine deiminase (ADI) could act as a ‘turn-off’ L-arginine (L-Arg) fluorescent biosensor and provide an alternative method for rapid determination of L-Arg. Double mutations were introduced on the Cys251➔Ser251 and Thr265➔Cys265 of recombinant ADI, rendering a single cysteine present on the protein surface for the site-specific attachment of a fluorophore, fluorescein-5-maleimide. The double mutations on ADI (265C) and its fluorescein-labelled form (265Cf) conserved the catalytic efficiency of wild-type ADI. Upon binding to L-Arg, 265Cf induced structural conformational changes and rendered the fluorescein moiety to move closer to Trp264, resulting in fluorescence quenching. The duration of fluorescence quenching was dependant on the L-Arg concentration. A linear relationship between the time at the maximum rate of fluorescence change and L-Arg concentrations, which ranged from 2.5 to 100 μM, was found with R2 = 0.9988. The measurement time was within 0.15–4 min. Determination of L-Arg concentration in fetal bovine serum could be achieved by the standard addition method and without sample pre-treatment. The result showed a good agreement with the one determined by mass spectrometry, suggesting our biosensor as a promising tool for the detection of L-Arg in biological samples.

Development, establishment and validation of in vitro and ex vivo assays of vascular calcification

ObjectiveVascular calcification (VC) is one major complication in patients with chronic kidney disease, with a misbalance in calcium and phosphate metabolism playing crucial role. The mechanisms underlying VC have not been entirely revealed to date. As studies aiming at the identification and characterization of the involved mediators are highly relevant, we developed a standardized operating protocol for in vitro and ex vivo approaches in this study to aiming at the comparability of these studies. Approach and resultsWe analyzed in vitro and ex vivo experimental conditions to study VC. Therefore, vascular smooth muscle cells were used for in vitro experiments and rat aorta for ex vivo experiments. The degree of calcification was estimated by quantification of calcium concentrations and by von Kossa staining. As a result, a step-by-step protocol for performing experiments on VC was established. We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings was highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium. ConclusionIn the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies.

Foetal development of endocrine organs in dog.

The canine adenohypophysis starts to be identifiable from 25day of pregnancy. ACTH-immunoreactive cells migrate until day 38 after which the number of ACTH-producing cells increases but their distribution does not change. The STH- and LH-producing cells first appear on day 38 of pregnancy. The primordium of the adrenal glands appears as a slender structure on day 27 and forms the definitive cortical structure on day 35. The histological pattern of the foetal adrenal cortex differs from the post-natal structure in so far as the three cortical zones (definitive zone, transitional zone and foetal zone) extend from the outside towards the inside of gland. The mass of foetal and neonatal adrenals is more than 10 times larger than the adult adrenals relative to body weight. The cortisol concentration in the amnion is slightly lower than in the allantois but the foetal serum cortisol concentration is significantly higher than in the maternal and foetal fluid compartments. The thyroxine concentrations in the allantois and amnion fluids exceed the foetal serum concentrations except in the ninth week of pregnancy, but thyroxine levels in foetal fluids and serum are below the physiological levels of adult animals. The exocrine and endocrine functions of the pancreas develop and act in parallel. Pancreatic cells are first detected at 30days when the branched structure is clearly detectable immunohistochemically, and at that time, insulin-positive β-cells and α-cells are visible as well. The foetal serum glucose concentration exceeds the healthy adult range, but the glucose concentration in the allantois and amnion fluid remains below the physiological blood glucose concentration of mature dogs. The insulin concentration in the allantois fluid greatly exceeds the foetal serum and amnion insulin concentrations.

Aspects of a Methodological Approach to Determination of Interferon Alpha Specific Activity

Specific antiviral activity is one of the key indicators characterising pharmaceutical quality and pharmacological efficacy of interferon alpha products (IFN-α). Specific activity is determined using a bioassay measuring antiviral activity in cell culture. The aim of the study was to select the most appropriate conditions for in vitro determination of IFN-α product specific activity. Materials and methods: Vero, MDBK, Hep-2, and A-549 homologous and heterologous cell cultures, as well as vesicular stomatitis Indiana virus (VSV) and murine encephalomyocarditis (EMC) virus at a dose of 100 TCD50 /0.1 mL were used for determination of specific antiviral activity. The international reference standard of recombinant interferon alpha-2b activity (Interferon alpha 2b, human, rDNA, E. coli-derived, 2nd WHO International Standard, 1999, NIBSC Code No. 95/566) and human recombinant interferon alpha 2b in the form of solution (batch No. 040214, Pharmapark LLC, Russia) were used as IFN-α samples. Results: the analysis of the obtained data helped to determine: the combinations of cell lines and the indicator virus most sensitive to IFN-α; the optimal concentration of fetal serum in the medium, and the optimal time parameters; the preferred method of reporting test results. Conclusions: the following test conditions were found to be optimal: the MDBK/VSV and Hep-2/EMC combinations proved to be the most sensitive to IFN-α; the optimal period of interferon and cell culture incubation—24 hours; the optimal concentration of fetal bovine serum in the culture medium used for diluting interferon products—2–5%. The instrumental procedure is preferred for reporting the results of interferon antiviral activity determination, because it is up-to-date, reliable, accurate and time-efficient.

Optimisation of cell and ex vivo culture conditions to study vascular calcification.

Medial vascular calcification (MVC) is a highly prevalent disease associated with a high risk of severe, potentially lethal, complications. While animal studies may not systematically be circumvented, in vitro systems have been proven useful to study disease physiopathology. In the context of MVC, the absence of a clinically relevant standardized in vitro method prevents the appropriate comparison and overall interpretation of results originating from different experiments. The aim of our study is to establish in vitro models mimicking in vivo vascular calcification and to select the best methods to unravel the mechanisms involved in MVC. Human aortic smooth muscle cells and rat aortic rings were cultured in different conditions. The influence of fetal calf serum (FCS), alkaline phosphatase, phosphate and calcium concentrations in the medium were evaluated. We identified culture conditions, including the herein reported Aorta Calcifying Medium (ACM), which allowed a reproducible and specific medial calcification of aortic explants. Studying cells and aortic explants cultured, the involvement of bone morphogenetic protein 2 (BMP2) pathway, fibrosis and apoptosis processes in in vitro MVC were demonstrated. Expression of osteoblastic markers was also observed suggesting the occurrence of transdifferentiation of smooth muscle cells to osteoblasts in our models. The use of these models will help researchers in the field of vascular calcification to achieve reproducible results and allow result comparison in a more consistent way.

Insulin-Like Peptide 3 (INSL3) Serum Concentration During Human Male Fetal Life.

Context: Insulin-like peptide 3 (INSL3), a protein hormone produced by Leydig cells, may play a crucial role in testicular descent as male INSL3 knockout mice have bilateral cryptorchidism. Previous studies have measured human fetal INSL3 levels in amniotic fluid only.Objective: To measure INSL3 serum levels and mRNA in fetal umbilical cord blood and fetal testes, respectively.Design: INSL3 concentrations were assayed on 50 μl of serum from male human fetal umbilical cord blood by a non-commercial highly sensitive and specific radioimmunoassay. For secondary confirmation, quantitative real-time PCR was used to measure INSL3 relative mRNA expression in 7 age-matched human fetal testes.Setting: UT Southwestern Medical Center, Dallas, TX and Medical University of South Carolina, Charleston, SC.Patients or other Participants: Twelve human male umbilical cord blood samples and 7 human male testes were obtained from fetuses 14–21 weeks gestation. Male sex was verified by leukocyte genomic DNA SRY PCR.Interventions: None.Main Outcome Measures: Human male fetal INSL3 cord blood serum concentrations and testicular relative mRNA expression.Results: INSL3 serum concentrations during human male gestational weeks 15–20 were 2–4 times higher than published prepubertal male levels and were 5–100 times higher than previous reports of INSL3 concentrations obtained from amniotic fluid. Testicular fetal INSL3 mRNA relative expression was low from weeks 14–16, rose significantly weeks 17 and 18, and returned to low levels at week 21.Conclusions: These findings further support the role of INSL3 in human testicular descent and could prove relevant in uncovering the pathophysiology of cryptorchidism.

The feto-maternal immune response to equine placentitis.

Ascending placentitis is one of the leading causes of abortion in the horse. Minimal work has focused on its effect on fetal fluids or the antenatal immune response of the fetus. Placentitis was induced via transcervical inoculation of Streptococcus equi ssp Zooepidemicus, and fluids/serum/tissues were collected 4-6days later following euthanasia. Cytokine concentrations were detected using a multiplex immunoassay within fetal fluids (amniotic and allantoic) and serum (maternal and fetal) in inoculated and control mares. In addition, tissues from fetal (spleen, liver, lung, umbilicus, amnioallantois) and maternal (spleen, liver, lung, chorioallantois, endometrium) origin were analyzed in inoculated and control mares utilizing qPCR for expression of cytokines. No difference in cytokine concentrations in maternal or fetal serum was noted between inoculated and control mares. Concentrations of IL-1β, IL-6, IL-10, and GRO were upregulated in the amniotic fluid following inoculation, with a trend toward higher IL-6 concentration in allantoic fluid. The amnioallantoic tissue separating the two fluids had higher expression of IL-1β and IL-6 following inoculation, while chorioallantois and endometrium upregulated IL-1β and IL-8 expression. IL-1β was upregulated in the maternal spleen following inoculation. Fetal spleens were upregulated in expression of IL-1β, GRO, and IL-6, while IL-6 was higher in fetal liver after inoculation than in controls. The maternal response to placentitis is primarily pro-inflammatory while the fetus appears to play a regulatory role in this inflammation. Additionally, amniotic fluid sampling may be more diagnostic of ascending placentitis than circulating cytokines.