Signaling pathways regulating FSH- and amphiregulin-induced meiotic resumption and cumulus cell expansion in the pig.

To define signaling pathways that drive FSH- and epidermal growth factor (EGF)-like peptide-induced cumulus expansion and oocyte meiotic resumption, in vitro cultured pig cumulus-oocyte complexes were treated with specific protein kinase inhibitors. We found that FSH-induced maturation of oocytes was blocked in germinal vesicle (GV) stage by protein kinase A (PKA), MAPK14, MAPK3/1, and EGF receptor (EGFR) tyrosine kinase inhibitors (H89, SB203580, U0126, and AG1478 respectively) whereas phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog (PI3K/AKT) inhibitor (LY294002) blocked maturation of oocytes in metaphase I (MI). Amphiregulin (AREG)-induced maturation of oocytes was efficiently blocked in GV by U0126, AG1478, and low concentrations of LY294002; H89, SB203580, and high concentrations of LY294002 allowed the oocytes to undergo breakdown of GV and blocked maturation in MI. Both FSH- and AREG-induced cumulus expansion was incompletely inhibited by H89 and completely inhibited by SB203580, U0126, AG1478, and LY294002. The inhibitors partially or completely inhibited expression of expansion-related genes (HAS2, PTGS2, and TNFAIP6) with two exceptions: H89 inhibited only TNFAIP6 expression and LY294002 increased expression of PTGS2. The results of this study are consistent with the idea that PKA and MAPK14 pathways are essential for FSH-induced transactivation of the EGFR, and synthesis of EGF-like peptides in cumulus cells and MAPK3/1 is involved in regulation of transcriptional and posttranscriptional events in cumulus cells required for meiotic resumption and cumulus expansion. PI3K/AKT signaling is important for regulation of cumulus expansion, AREG-induced meiotic resumption, and oocyte MI/MII transition. The present data also indicate the existence of an FSH-activated and PKA-independent pathway involved in regulation of HAS2 and PTGS2 expression in cumulus cells.

[Early prediction of severity in acute pancreatitis]. / Predicción precoz de severidad en pancreatitis aguda.

OBJECTIVE: To identify the score that best predicts early severity in patients with acute pancreatitis. MATERIALS AND METHODS: A prospective cohort study was performed from December 2009 to December 2011. Cases of pancreatitis were classified as mild or severe based on the organ failure criteria and/or local complications according to the Atlanta Symposium. APACHE-II, BISAP and urea were calculated using data from the first 24 hours. Ranson was calculated using data from the first 48 hours. Hemoconcentratión was assessed 24 hours from admission, after adequate hydration. To assess the predictive value of the different scores the area under the curve ROC was used. RESULTS: 329 patients with acute pancreatitis were included: 214 women (65%) and 115 males (35%). The most common etiology was biliary 245 (74.3%). Two hundred and forty were mild cases of pancreatitis (73%) and 89 were severe (27%). The areas under the curve ROC were: APACHE-II: 0.74; BISAP: 0.78; Ranson: 0.48; urea: 0.70; hemoconcentratión: 0.73. There were 10 deaths (mortality: 3%). There were no significant differences between BISAP and APACHE-II, urea or hemoconcentration, but a significant difference existed with Ranson score. CONCLUSION: BISAP is a simple score that applied early in the emergency room identifies patients with severe pancreatitis with an efficacy similar to APACHE-II, urea and hemoconcentration, and KEY WORDS: acute pancreatitis, BISAP, severity, prognostic scoring.

Composite hyaluronate-type I collagen-fibrin scaffold in the therapy of osteochondral defects in miniature pigs.

The potential of novel scaffold containing sodium hyaluronate, type I collagen, and fibrin was investigated in the regeneration of osteochondral defects in miniature pigs. Both autologous chondrocyte-seeded scaffolds and non-seeded scaffolds were implanted into two defects located in the non-weight-bearing zone of the femoral trochlea (defect A was located more distally and medially, defect B was located more proximally and laterally). Control defects were left untreated. Twelve weeks after the operation, the knees were evaluated in vivo using MRI. Six months after the implantation, the defects were analyzed using MRI, histological, and immunohistochemical analysis. In the A defects of chondrocyte-seeded scaffold group, hyaline cartilage and fibrocartilage was formed, containing type II collagen, acidic and neutral glycosaminoglycans while the non-seeded scaffold group was predominantly filled with fibrocartilage. Defects in the control group were predominantly filled with fibrous tissue. Histomorphometric analysis of photomicrographs revealed a significantly higher amount of hyaline cartilage in the cell-seeded scaffold group in A defects than in other groups. Both scaffold groups in A defects showed significantly less fibrous tissue than cell-seeded defects B and the control group. Both histological and MRI analysis proved that the novel composite scaffold has a potential to regenerate osteochondral defects within six months.

Effect of bone morphogenetic protein-15 on gonadotropin-stimulated synthesis of hyaluronan and progesterone in porcine ovarian follicle.

Bone morphogenetic protein-15 (BMP-15), an oocyte-derived growth factor, has been shown to play integral roles in regulation of ovarian follicular function in mammals. Despite the recognition of the physiological importance of the BMP system in regulation of gonadotropin action in the ovary, molecular mechanisms of BMP-15 effect on oocyte and somatic follicular cell functions remain poorly understood. The objective of this study was to determine the effect of BMP-15 on the FSH/LH-stimulated synthesis of hyaluronan (HA) by oocyte cumulus complexes (OCC) and progesterone by OCC and granulosa cells (GC) in the presence or absence of serum using primary porcine cultures. In addition, the effect of BMP-15 on oocyte maturation- and steroidogenesis-related transcripts after 4, 8, 16, and 24 hours of cultivation was evaluated using real-time RT-PCR. We demonstrated that the FSH/LH-induced cumulus expansion was accompanied by a significant increase in CD44, PTGS2, CYP11A1 (at 4 h) and AREG, HAS2, TNFAIP6, STAR (at 8 h) mRNAs. While FSH/LH-stimulated total HA synthesis by OCC was not affected by BMP-15 in serum-supplemented medium, its retention within the complex was significantly increased after the action of BMP-15 in comparison to FSH/LH alone (P < 0.001; 65% versus 35%, respectively). Moreover, we detected a significant increase in the expression of AREG and TNFAIP6 (both at 16 h), and CYP11A1 (at 24 h) in FSH/LH-stimulated OCC due to the action of BMP-15 compared to complexes cultured only with FSH/LH. In the presence of serum, BMP-15 markedly increased FSH/LH-stimulated progesterone secretion by OCC (about 69%) and induced a significant decrease in FSH/LH-induced progesterone release by GC (about 35%) compared to FSH/LH alone. The present results indicate that the addition of BMP-15 to the gonadotropin-stimulated OCC cultured in serum-supplemented medium might improve oocyte-cumulus maturation.

[Questionnaire study of general practitioners regarding experience and attitude to opioid substitution therapy]. / Dotazníková studie mezi praktickými lékari zamerená na zkusenosti a postoje k substituci opioidy.

UNLABELLED: General practitioners (GPs) in their surgeries and substitution treatment centres are the major providers of opioid maintenance treatment in a number of European countries. Although in the Czech Republic any GP has been allowed to prescribe buprenorphine (Subutex) since 2001, the opioid substitution treatment provided by primary care professionals has not been the subject of research to date. OBJECTIVE: To collect and analyze data on GPs' experience gained with opioid maintenance treatment in the Czech Reupblic, their attitudes and needs. METHOD: A structured questionnaire was distributed via the Bulletin of the Association of General Practitioners and district Association representatives. The validity of study results may be affected by a low respondence rate (10%) with 398 questionnaires only returned by mail. RESULTS: Twenty-eight (7%) GPs reported to have gained experience with buprenorphine which was most frequently prescribed in the regions with the highest prevalence of heroin users, i.e. in Prague and the Ustí nad Labem region (27% and 12%, respectively). Other regions, including wes- tern and southern Bohemia with relatively high prevalence of heroin users, showed lower buprenorphine prescription rates (0-6%). Most buprenorphine prescribers (78%) rated their experience as positive or highly positive. Availability and effectiveness were seen as the main pros of the substitution treatment. One third of the GPs who have not prescribed opioid maintenance treatment yet are considering doing so in the future. Greater awareness of drug abuse issues and availability of methodical guidance and consulting in opioid substitution treatment are going to become the most relevant factors in the future. Possible reportability of data on opioid maintenance treatment to a central registry does not seem to be a major obstacle to implementing the substitution treatment in the GPs' surgeries. Decision makers should take advantage of the GPs' potential to promote the opioid maintenance treatment in the Czech Republic.

Lack of effect of boar seminal vesicle fluid immunosuppressor factor on embryo development.

The effects of an immunosuppressive factor (ISF) isolated from boar seminal vesicle fluid on in vitro and in vivo mouse development were investigated. It was found that the zona pellucida of porcine and mouse oocytes pre-incubated with ISF reacted in indirect immunofluorescence with antibodies against ISF. Further results indicated that the boar ISF had no effect on embryo development.

Effect of intraovarian factors on porcine follicular cells: cumulus expansion, granulosa and cumulus cell progesterone production.

The role of granulosa cell conditioned media (CM) containing luteinization stimulator (LS), and the role of EGF in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral follicles was investigated. The CM were prepared by incubation of granulosa cells isolated from large antral follicles. After 24h incubation, more than 61 or 64% of OCC expanded to the +3 and +4 stage in the presence of CM (50%) or EGF (10ng/ml), respectively. The stimulatory effect of LS and EGF on the cumulus expansion was accompanied by the enhanced hyaluronic acid synthesis. Complete suppression of cumulus expansion stimulated by LS and EGF was observed in the presence of 10 micromol/l genistein (tyrosine kinase inhibitor), in the presence of 10mmol/l LiCl (the inhibitor of inositol 1,4,5-trisphosphate metabolism), and 100 micromol/l gallopamil, verapamil and norverapamil (calcium channel blockers). Stimulatory effect of EGF on the cumulus expansion of OCC isolated from large follicles was accompanied by the increased cumulus cell progesterone production. However, EGF did not affect the progesterone production by OCC isolated from small follicles. To determine whether EGF could modulate the granulosa cell steroidogenesis also, the effect of EGF on granulosa cells isolated from large (LGC) and small (SGC) follicles was compared. EGF (10ng/ml) failed to affect the progesterone synthesis during 72h culture of SGC but significantly enhanced the LGC progesterone production. Our results indicate that luteinization factor stimulates the cumulus expansion and hyaluronic acid synthesis by the OCC isolated from large antral follicles. The mechanism of LS- and EGF-induced cumulus expansion may involve tyrosine kinase activation and calcium mobilization. In addition, these results indicate the different response of porcine cumulus and granulosa cells originating from small and large follicles on the stimulatory effect of EGF.

Development of in vivo derived diploid and tetraploid pig embryos in a modified medium NCSU 37.

The aim of this study was to assess development of diploid and tetraploid in vivo derived pig embryos cultured in a modified medium NCSU 37 in an atmosphere with reduced concentration of oxygen. The tetraploid embryos were produced by electrofusion of two-cell embryos that had been cultured in vitro from the one-cell stage before fusion (cultured two-cell embryos) or by fusion of freshly recovered two-cell embryos. Development to blastocyst stage of tetraploid embryos, generated from the cultured two-cell embryos was significantly inferior to the development of control one-cell embryos (29.1 +/- 9.7% versus 66.8 +/- 9.7%; P < 0.05). However, development of tetraploid embryos produced from the freshly recovered two-cell embryos and control two-cell embryos was very similar (89.9 +/- 6.1% versus 81.3 +/- 3.4%). Detection of chromosomes 1 and 10 by in situ hybridization showed that more than 85% of the cultured control embryos were diploid while 15% of the embryos were mosaic. Among the fused embryos 50% were tetraploid, 29% mosaic and 21% diploid. These data indicate that the modified medium NCSU 37 provides optimum environment for pre-implantation development of pig diploid and tetraploid embryos.

Parthenogenetic development of activated in vitro matured bovine oocytes.

Bovine oocytes matured in vitro for 26 hours were electrically stimulated 1) by a single pulse (Treatment A); 2) by 3 pulses 30 minutes apart (Treatment B); 3) by a single pulse followed by 5 minutes of incubation in the stimulation medium (Treatment C); or 4) by a single pulse at 27 hours of maturation (Treatment D). The oocytes were then cultured for up to 8 days to assess parthenogenetic activation and development. Each electrical stimulation consisted of a 60-mus square wave pulse of 2.5 or 3.6 kV/cm. Treatment A was less effective than the other treatments (P<0.05), activating 47 or 59% of oocytes at 2.5 or 3.6 kV/cm, respectively. However, there were no differences due to voltage nor among the other treatments, which activated 64 to 78% of the oocytes. The cleavage rate, 28 to 38%, was not affected by the activation treatment, but development to the 8-cell stage or beyond was greater after activation with the higher voltage. While the numbers of morulae or blastocysts resulting from any given treatment were too small to support meaningful statistical comparison, the results indicate that bovine parthenogenotes produced in vitro are capable of development to the blastocyst stage.

RNA synthesis in porcine blastomere nuclei introduced into in vitro matured ooplasm.

The objective was to investigate the RNA synthesis in porcine blastomere nuclei upon transplantation into in vitro matured enucleated oocytes. Nuclei from 2- to 8-cell porcine embryos were introduced into the ooplasm of in vitro matured and enucleated porcine oocytes by electrofusion, and the resultant reconstructed embryos were cultured in vitro. Before fusion or at different intervals after this event embryos were incubated with [3H]-uridine, fixed, and histologically processed for autoradiography in order to detect RNA synthesis. About two thirds of the embryos were considered to depict normal development. All blastomeres displayed pronounced RNA synthesis before fusion, at 3 and 9 h after fusion the synthesis decreased or ceased, and at 24-49 h some embryos resumed synthesis at the 1- to 2-cell stage.

[Our experience with bone scintigraphy in the diagnosis of wrist injuries.]. / Nase zkusenosti s kostní scintigrafií pri diagnostice poranení zápestí

The authors evaluate, based on a group of 40 patients, the contribution of bone scintigraphy to the diagnosis of injuries of the wrist; scintigraphy has a high sensitivity and specificity in fractures of the navicular bone. In addition to injury of that bone the scintigraphic picture of both wrists makes it possible to focus attention also on affections of others structures and thus to contribute to a better diagnosis and treatment of the wrist. The authors found in fractures of the navicular bone in 70 % agreement with the X-ray examination made several weeks after the injury. As regards injuries of other bones of the wrist positive on scintigraphy, the X-ray examination was negative. In the authors' opinion the discrepancy between scintigraphy and X-ray examination is due to the limited possibilities of clasical X-ray pictures. By introduction of more perfect examination methods such as computer tomography and nuclear magnetic resonance in the diagnosis of wrist injuries the number of falsely positive scintigraphic findings should decline. The authors recommend to make scintigraphic examinations during the period between the third day to the end of the third week after the first examination at the ambulatory department. A negative scintigraphic finding rules out injuries of the wrist. Where it is not possible to make a scintigraphic examination, the authors recommend follow-up of the patients for at least six weeks following the first visit of the ambulatory department. Key words: bone scintigraphy, wrist injury, navicular bone of the wrist.

[Results of using high porosity aortofemoral bifurcation prostheses]. / Výsledky pouzitia ultrapórovitých aortofemorálnych bifurkacných protéz.

The authors report on their four-year experience with the use of extremely highly porous vascular polyester prostheses of Czechoslovak proveniencè, whose wall was sealed with fibrin. Aortofemoral bifurcated prostheses with a permeability of 6 100 ml/cm2/min were implanted in 102 patients. The method of fibrination appears to be simple and safe. No hemorrhage through the prosthetic wall was observed. The authors expect the high porosity of the prostheses to result in longer lasting patency of the grafts and thus in improvement of long-term results.

[Sealing of ultraporous vascular prostheses with autologous fibrinogen]. / Utesnovanie ultrapórovitých cievnych protéz autológnym fibrinogénom.

After their long lasting experience with commercially prepared fibrinogen for sealing off ultraporous vascular prostheses, the authors have introduced the use of freshly prepared fibrinogen from the patient's own blood. This approach provides an additional advantage by allowing to substitute the blood losses at the operation with shelfed autologous blood. The preparation of autologous fibrinogen is described and the advantages of its use are discussed.

[Choledochal cyst in pregnancy and puerperium: report on two cases and review of literature]. / Quiste de colédoco en la gestación y puerperio: reporte de dos casos y revisión de la literatura.

A choledochal cyst is a cystic dilation of the intrahepatic or extrahepatic biliary tract. According to the most accepted theory, it is caused by an anomalous pancreatobiliary junction. The most important complications are cholangiocarcinoma, lithiasis, and pancreatitis. Current therapy is surgical resection. Only 20% to 30% of cases are diagnosed in adult life. Two cases of choledochal cysts are reported in female adult patients, one of them in late pregnancy and the other in puerperium. Diagnosis of choledochal cyst in pregnancy and puerperium is an uncommon event, entailing particular considerations regarding symptoms and treatment.

Predicción precoz de severidad en pancreatitis aguda / Early prediction of severity in acute pancreatitis

OBJETIVO: Identificar el indicador que mejor predice la severidad en forma temprana en pacientes con pancreatitis aguda. MATERIALES Y MÉTODOS: Se realizó un estudio de cohorte prospectiva entre diciembre del 2009 y diciembre del 2011. Se clasificó los cuadros de pancreatitis aguda en leve y severa en base a los criterios de falla orgánica o complicaciones locales según el Simposio de Atlanta. Los indicadores de APACHE-II, BISAP y urea fueron calculados usando los datos de las primeras 24 horas de admisión. El indicador de Ranson se calculó con datos de las primeras 48 horas. La hemoconcentración se calculó a las 24 horas de la admisión después de una adecuada hidratación. Para evaluar la capacidad predictiva de los diferentes indicadores se utilizó el área bajo la curva ROC. RESULTADOS: Ingresaron al estudio 329 pacientes con pancreatitis aguda: doscientos catorce mujeres (65%) y 115 varones (35%). La etiología más frecuente fue la biliar 245 (74.3%). Doscientos cuarenta fueron pancreatitis leves (73%) y 89 pancreatitis severas (27%). Las áreas bajo la curva ROC de los indicadores fueron: APACHE-II: 0.74; BISAP: 0.78; Ranson: 0.48; urea: 0.70; hematocrito a las 24 horas: 0.73. Se registraron 10 muertes (mortalidad: 3%). No se observó diferencias significativas del indicador BISAP con el indicador de APACHE-II, con la urea ni la hemoconcentración, pero sí con el puntaje de Ranson. CONCLUSIÓN: BISAP es un score sencillo que aplicado tempranamente en la emergencia identifica pacientes con cuadro clínico severo con eficacia similar al APACHE-II, urea y hemoconcentración; y mejor que Ranson.

OBJECTIVE: To identify the score that best predicts early severity in patients with acute pancreatitis. MATERIALS AND METHODS: A prospective cohort study was performed from December 2009 to December 2011. Cases of pancreatitis were classified as mild or severe based on the organ failure criteria and/or local complications according to the Atlanta Symposium. APACHE-II, BISAP and urea were calculated using data from the first 24 hours. Ranson was calculated using data from the first 48 hours. Hemoconcentratión was assessed 24 hours from admission, after adequate hydration. To assess the predictive value of the different scores the area under the curve ROC was used. RESULTS: 329 patients with acute pancreatitis were included: 214 women (65%) and 115 males (35%). The most common etiology was biliary 245 (74.3%). Two hundred and forty were mild cases of pancreatitis (73%) and 89 were severe (27%). The areas under the curve ROC were: APACHE-II: 0.74; BISAP: 0.78; Ranson: 0.48; urea: 0.70; hemoconcentratión: 0.73. There were 10 deaths (mortality: 3%). There were no significant differences between BISAP and APACHE-II, urea or hemoconcentration, but a significant difference existed with Ranson score. CONCLUSION: BISAP is a simple score that applied early in the emergency room identifies patients with severe pancreatitis with an efficacy similar to APACHE-II, urea and hemoconcentration, and better than Ranson.

Activity of maturation promoting factor in pig oocytes after microinjection and serial transfer of maturing cytoplasm.

Microinjection of 120 pl homologous or heterologous (mouse) maturing cytoplasm into immature pig oocytes resulted in germinal vesicle breakdown (GVBD) and chromosome condensation (CC) after 8 h of culture. In contrast, nearly all control oocytes remained at the germinal vesicle stage. Immature cytoplasm did not induce nuclear maturation of recipient oocytes when injected. The cytoplasm of the oocytes injected with maturing cytoplasm preserved the capacity to cause GVBD in the second recipients. These results suggest that the level of injected maturation promoting factor (MPF) must rise before GVBD can occur. When the pig oocytes injected with 120 pl of mouse maturing cytoplasm were cultured in cycloheximide for 8 h, the presence of GVBD was dramatically reduced. Thus, in pig oocytes the increase in active MPF up to the effective concentration requires protein synthesis.