Equine Placentitis in Mares Induces the Secretion of Pro-Inflammatory Cytokine eIL-1ß and the Active Extracellular Matrix Metalloproteinase (MMP)-9.

Equine placentitis is characterized by infection and inflammation of the placenta. Different biomarkers associated with this inflammatory response have been evaluated in experimentally induced equine placentitis, but not in pregnant mares with spontaneous placentitis. The aim of the current study was to determine the concentration of eIL-1ß and the activity of proMMP-2 and proMMP-9 in the serum of healthy mares and mares with placentitis on days 240 and 320 of gestation to explore whether these biomarkers are associated with equine maternal placentitis and/or with the birth of an infected or inviable foals. Serum samples were collected from sixteen pregnant English Thoroughbred mares, retrospectively classified as follows: (1) healthy mares with full-term gestation; and (2) mares with ultrasonographic signs of placentitis. The health of each foal was examined at birth, and it was decided to classify the cases into four groups: (1) healthy mares delivering a healthy foals (HM-HF, n = 6); (2) mares with USP delivering a healthy foal (USP-HF, n = 3); (3) mares with USP delivering a live septic foal (USP-LSeF, n = 4); and (4) mares with USP delivering a dead foal (USP-DF, n = 3). eIL-1ß was quantified by ELISA, and proMMP-2 and proMMP-9 activity by gelatin zymography electrophoresis. In healthy mares, the serum concentrations of eIL-1ß underwent a significant 16.5-fold increase from day 240 to day 320 of gestation. Although similar results were found in the mares with ultrasonographic signs of placentitis that delivered a healthy foal, those delivering a live septic or nonviable foal exhibited much higher concentrations of eIL-1ß. proMMP-2 and proMMP-9 activity was not associated with maternal placentitis, foal infection, or death. Hence, the presence of placentitis severe enough to affect the health of the foal can be confirmed or discarded by determining the eIL-1ß concentration in mares that have shown ultrasonographic signs of placentitis.

Differential proMMP-2 and proMMP-9 secretion in human pre-implantation embryos at day 5 of development.

Morphological development is the most common non-invasive criterion used to select in vitro human embryos for implantation. With this criterion, however, embryos in cellular arrest go unnoticed. A more accurate criterion is needed to improve the success rate of implantation. Extracellular matrix metalloproteases type 2 (MMP-2) and MMP-9 are key markers of embryonic development and the implantation process, according to various animal studies. The first objective of this study was to examine proMMP-2 and proMMP-9 activity in the culture media of human embryos with good morphological development. Secondly, the results of proMMP-2 and proMMP-9 activity in the culture medium were compared between pregnant and non-pregnant. Forty-two patients were approved by the Ethics and Research Committees of the Instituto Nacional de Perinatología in México City hospital, based on institutional inclusion criteria for in vitro fertilization. On day 5 of development, embryos were transferred to patients, and the culture media secretion profile of proMMP-2 and proMMP-9 activity was determined by substrate gel zymography. After analysis of embryo sac development, each patient was assigned to the pregnant (n=17) or non-pregnant (n=25) group. Our results demonstrate that proMMP-2 was active in the culture media corresponding to all 17 women achieving full-term pregnancy and proMMP-9 in the media corresponding to 11 of these women. Contrarily proMMP-2 and proMMP-9 were active in the culture media corresponding to 3 and 11 of the 25 non-pregnant patients, respectively. The clinical implications of this study suggest the activity evaluation of proMMP-2 and proMMP-9 in embryonic culture media on day 5 of development appears to be a reliable indicator of the quality of embryos and their capacity to establish a pregnancy.

MiR-191-5p is upregulated in culture media of implanted human embryo on day fifth of development.

BACKGROUND: Morphological features are the most common criteria used to select human embryos for transfer to a receptive uterine cavity. However, such characteristics are not valid for embryos in cellular arrest. Even aneuploid embryos can have normal morphology, and some euploid embryos have aberrant morphology. The aim of this study was to quantify the expression profile of hsa-miR-21-3p, -24-1-5p, -191-5p, and -372-5p in culture media on day 5 of in vitro embryo development, and compare the profiles of two groups of media classified by outcome: successful (n = 25) or unsuccessful (n = 25) implantation pregnancy. METHODS: Fifty patients were accepted in the Department of Reproductive Biology of a Hospital in México City, based on the Institutional inclusion criteria for in vitro fertilization. Embryos were transferred to the women on day 5 of cultivation, and the culture media were collected. RNA was isolated from each culture medium with TRIzol reagent, and microRNA (miRNA) expression was detected through RT-PCR with specific primers. Expression bands were quantified by reading optical density. RESULTS: There was a 5.2-fold greater expression of hsa-miR-191-5p in the pregnancy-related culture media (p ≤ 0.001) and a 1.6-fold greater level of hsa-miR-24-1-5p (p = 0.043) in the media corresponding to non-pregnant women. No significant difference existed between the two groups hsa-miR-21-3p (p = 0.38) or hsa-miR-372-5p (p = 0.41). CONCLUSIONS: Regarding adequate in vitro embryo development, hsa-miR-191-5p could possibly represent a positive biomarker, while has-miR-24-1-5p may indicate poor prognosis. This former miRNA modulates IGF2BP-1 and IGF2R, associated with the implantation window. On the other hand, hsa-miR-24-1-5p may be related to a poor prognosis of human embryo development.

Possible Mechanisms Involved in the Vasorelaxant Effect Produced by Anorexigenic Drugs in Rat Aortic Rings.

Anorexigenics are compounds capable of reducing or suppressing appetite. Their three main types act on different neurotransmitters, either norepinephrine, serotonin or a combination of both. Among the drugs that act on norepinephrine are fenproporex, amfepramone and clobenzorex. Derivatives of the thyroid hormone triiodothyronine have also been associated with weight loss and used as a controversial treatment for obesity, despite their known cardiovascular side effects. Recent data suggest a possible vasodilating effect for these four substances that might be beneficial in a subset of patients. Herein we performed a systematic review of the literature (with emphasis on recent reports) to determine the implications and mechanisms of the vasodilating effects of some anorectics, specifically fenproporex, clobenzorex, amfepramone and triiodothyronine. Data analysis showed these four drugs to be vasodilating agents for rat aortic rings. The different mechanisms of action include endothelium-dependent vasodilation via activation of the NO-cGMP-PKG pathway and the opening of calcium-activated potassium channels. The finding of vasodilating activity indicates a potential role for some anorexigenic drugs in the treatment of obesity in hypertensive patients. Further in vivo studies are needed to test the clinical benefits of these four drugs.

Pharmacological characterization of mechanisms involved in the vasorelaxation produced by rosuvastatin in aortic rings from rats with a cafeteria-style diet.

The present study aimed to investigate the possible influence of several inhibitors and blockers on the vascular effect produced by the acute in vitro application of rosuvastatin to phenylephrine-precontracted aortic rings from rats with a semi-solid, cafeteria-style (CAF) diet. It also aimed to examine the effects of rosuvastatin on the expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase in aortic rings from rats with a CAF diet. From comparisons of the effect on phenylephrine-precontracted aortic rings extracted from rats with two different diets (a standard and a CAF diet), it was found that 10(-9) -10(-5) -mol/L rosuvastatin produced lower concentration-dependent vasorelaxation on rings from the CAF diet group. The vasorelaxant effect was unaffected by the vehicle, but it was significantly attenuated by 10(-5) -mol/L N(G) -nitro-l-arginine methyl ester, 10(-2) -mol/L tetraethylammonium, 10(-3) -mol/L 4-aminopyridine, 10(-7) -mol/L apamin plus 10(-7) -mol/L charybdotoxin, 10(-5) -mol/L indomethacin, or 10(-5) -mol/L cycloheximide. Moreover, in aortic rings from rats with a CAF diet, rosuvastatin enhanced the expression of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase. The acute in vitro application of rosuvastatin to phenylephrine-precontracted aortic rings from rats with a CAF diet had a vasorelaxant effect. Overall, the present results suggest that the stimulation of eNOS, the opening of Ca(2+) -activated and voltage-activated K(+) channels, the stimulation of prostaglandin synthesis and enhanced protein levels of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase are involved in this relaxant effect.

Rapid and sensitive determination of levofloxacin in microsamples of human plasma by high-performance liquid chromatography and its application in a pharmacokinetic study.

A rapid, sensitive and simple high-performance liquid chromatographic assay with ultraviolet detection was developed for the quantification of levofloxacin in microsamples (100 µL) of human plasma. The extraction procedure included a protein precipitation technique and a short chromatographic running time (4.5 min). Analyses were carried out on a Symmetry C18 column using a mixture of acetonitrile and 0.01 m potassium dihydrogen aqueous solution (pH 3.4; 14:86 v/v) as mobile phase. The method provided specificity and was linear (r ≥ 0.9992) over the concentration range 0.1-12 µg/mL. The average absolute recovery was 93.59%. The intra- and inter-day coefficients of variation were <6%. Additionally, levofloxacin was stable in all evaluations. The usefulness of this method was demonstrated in a pharmacokinetic study of levofloxacin in healthy adult volunteers. The present method offers two main advantages: (a) the use of microsamples reduces the total volume of blood to be collected from patients; and (b) it provides a good cost-effectiveness ratio. It is concluded that the method is rapid, simple, sensitive, economical and suitable for the determination of levofloxacin in human plasma using a small volume of sample.

[Intracellular calcium-dependent contractile effects of serotonin in the human umbilical artery]. / Efectos contráctiles dependientes del calcio intracelular inducidos por la serotonina en la arteria umbilical humana.

BACKGROUND: Absence of innervation is a hallmark of human umbilical vessels. Intervillous space blood flow is regulated by vasoactive substances and calcium dependent contractility, both in normal and pathological conditions such as preeclampsia-eclampsia. OBJECTIVE: To obtain additional information on the intracellular calcium contractile effects of serotonin in human umbilical arteries. MATERIALS AND METHODS: Umbilical arteries from normal pregnancies were dissected, cut in 5 mm rings and mounted in a temperature-controlled isolated organ chamber, using calcium-free Krebs solution. The contractile effects of serotonin, lantane, verapamil and cyclopiazonic acid were evaluated at different concentrations using a computer coupled biopac polygraph. RESULTS: No differences in response were observed in the presence and absence of intracellular calcium. The positive contractile effects observed with serotonin were significantly decreased with repeated stimulation. An increase in the basal tone of the vessel was observed after calcium supplementation was added to the solution. This effect was minimized in the presence of verapamil and lantane. The contractile effects of serotonin in the calcium-free solution were not affected by the presence of cyclopiazonic acid. CONCLUSIONS: Serotonin contractile effects in the human umbilical artery depend mainly on intracellular calcium levels which favor the gradual entrance of this ion over time. Calcium influx induced by serotonin is possible through L and Non-L channels apparently insensitive to ciclopiozonic acid.

Efectos contráctiles dependientes del calcio intracelular inducidos por la serotonina en la arteria umbilical humana / Intracellular calcium-dependent contractile effects of serotonin in the human umbilical artery

Antecedentes: Los vasos umbilicales humanos se caracterizan por la ausencia de inervación y por consecuencia, las diferentes sustancias vasoactivas y la respuesta contráctil a través de los iones de calcio son los factores que determinan el flujo sanguíneo al espacio intervelloso en condiciones normales y patológicas, como es el caso de la preeclampsia eclampsia. Objetivo: Aportar información adicional de la respuesta contráctil de la serotonina en relación al calcio intracelular en la arteria umbilical humana. Material y métodos: Se utilizaron vasos umbilicales procedentes de mujeres con embarazo normoevolutivo, los cuales una vez disecados se cortaron en anillos de 5 mm y se montaron en cámara de órgano aislado, utilizando solución de Krebs con y sin calcio, burbujeada con carbógeno y la temperatura controlada. Se evaluó el efecto contráctil inducido por serotonina a diferentes concentraciones molares y se contrastó con verapamil, lantano y ácido ciclopiazónico, cuantificando la respuesta contráctil mediante un polígrafo biopac acoplado a un sistema computacional. Resultados: No se observaron diferencias significativas en la magnitud de la respuesta obtenida en presencia y ausencia de calcio extracelular. Se apreció el efecto contráctil a la serotonina que disminuyó significati vamente como respuesta a la estimulación repetida a la misma; así mismo, se incrementó el tono basal posterior a la adición del calcio al medio de incubación, lo que dependió del tiempo de exposición. También se observó la inhibición parcial del incremento en el tono basal con vera pamil y lantano. Finalmente, el pretratamiento con ácido ciclopiazónico no modificó la respuesta contráctil a la serotonina en un medio sin calcio. Conclusiones: La contracción inducida por serotonina en la arteria umbilical humana, depende principalmente de calcio intracelular y favorece el ingreso capacitativo de este ion, el cual se incrementa gradualmente a través del tiempo. El ingreso capacitativo del calcio secundario al vaciamiento de los depósitos intracelulares de este ion con serotonina se efectúa a través de canales tipo L y no-L, y no parecen ser sensibles al ácido ciclopiazónico.

Background: Absence of innervation is a hallmark of human umbilical vessels. Intervillous space blood flow is regulated by vasoactive substances and calcium dependent contractility, both in normal and pathological conditions such as preeclampsia eclampsia. Objective: To obtain additional information on the intracellular calcium contractile effects of serotonin in human umbilical arteries. Materials and Methods: Umbilical arteries from normal pregnancies were dissected, cut in 5 mm rings and mounted in a temperature controlled isolated organ chamber, using calciumfree Krebs solution. The contractile effects of serotonin, lantane, verapamil and cyclopiazonic acid were evaluated at different concentrations using a computer coupled biopac polygraph. Results: No differences in response were observed in the presence and absence of intracellular calcium. The positive contractile effects observed with serotonin were significantly decreased with repeated stimulation. An increase in the basal tone of the vessel was observed after calcium supplementation was added to the solution. This effect was minimized in the presence of verapamil and lantane. The contractile effects of serotonin in the calcium free solution were not affected by the presence of cyclopiazonic acid. Conclusions: Serotonin contractile effects in the human umbilical artery depend mainly on intracellular calcium levels which favor the gradual entrance of this ion over time. Calcium influx induced by serotonin is possible through L and Non-L channels apparently insensitive to cyclopiozonic acid.