A protein isolated from human oviductal tissue in vitro secretion, identified as human lactoferrin, interacts with spermatozoa and oocytes and modulates gamete interaction.

STUDY QUESTION: Is lactoferrin (LF) (detected in oviductal secretion) able to bind to oocytes and sperm and modulate gamete interaction? SUMMARY ANSWER: LF binds to zona pellucida (ZP) and spermatozoa (depending upon the capacitation stage and acrosome status) and inhibits gamete interaction in vitro. WHAT IS KNOWN ALREADY: Proteins from human oviductal tissue secretion modulate gamete interaction and parameters of sperm function in vitro and some of them bind to sperm, but they remain to be isolated and identified. STUDY DESIGN, SIZE, DURATION: Proteins were isolated from human oviductal tissue secretion using their sperm membrane binding ability. One of the isolated proteins was identified as human LF and immunolocalized in tubal tissues. LF expression was analyzed in native oviductal fluid and oviduct epithelial cells (at different phases of the menstrual cycle: proliferative, periovulatory and secretory). In addition, the LF binding sites on spermatozoa (at different capacitation and acrosome reaction stages) and on ZP and the dose-dependent effect of LF on gamete interaction were investigated. All experiments were performed at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Tubal tissues obtained from premenopausal patients (scheduled for hysterectomy, n = 23) were cultured in DMEM/Ham's F12 medium and conditioned media (CM) were collected. Motile spermatozoa were obtained by swim-up from normozoospermic semen samples from healthy donors (n = 4). An affinity chromatography with sperm membrane extracts was used to isolate proteins from CM. Isolated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophresis and further identified by nano liquid chromatography tandem mass spectrometry peptide sequencing. The presence of LF in oviductal tissue was investigated by immunohistochemistry and immunofluorescence and was detected in native oviductal fluid and oviduct epithelial cells homogenates by western blot. LF binding sites on gametes were investigated by incubating gametes with the protein coupled to fluorescein isothiocyanate (FITC). The acrosome reaction was assessed with Pisum sativum agglutinin conjugated with rhodamine. The effect of increasing concentrations of LF (0.1-100 µg/ml) on gamete interaction was evaluated by a sperm-ZP binding assay, using human oocytes donated by women undergoing IVF procedures. MAIN RESULTS AND THE ROLE OF CHANCE: A protein isolated by the affinity column was identified as human LF. LF was immunolocalized in human oviductal tissue and detected in oviductal fluid and oviduct epithelial cell homogenates. In the latter case, LF expression was highest at the periovulatory phase of the menstrual cycle (P < 0.01). Different LF binding patterns were observed on spermatozoa depending upon capacitation stage and if the acrosome reaction had occurred. Unstained sperm were most prevalent before capacitation, but after incubation for 6 h under capacitating conditions and in acrosome-reacted sperm LF binding was observed, mainly localized in the equatorial segment and post-acrosomal region of the sperm head. LF binding studies on ZP showed homogenous staining. LF caused a dose-dependent significant inhibition of sperm-ZP interaction, and the effect was already significant (P < 0.01) with the lowest LF concentration used. LIMITATIONS, REASONS FOR CAUTION: This study has investigated the effect of LF only on human gamete interaction in vitro and thus has some limitations. Further investigations of the potential mechanisms involved in LF action both on gamete function in vitro and in vivo in animal models are needed to confirm the role of this protein in the reproductive process. WIDER IMPLICATIONS OF THE FINDINGS: The present data indicate that human oviductal LF expression is cycle dependent and inhibited gamete interaction in vitro. No previous data were available about potential direct effects of LF on gamete interaction. It could be thought that the protein is involved in the regulation of the reproductive process, perhaps contributing to prevent polyspermy. Thus, further research is needed to clarify the potential role of LF in the regulation of the fertilization process. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from FONCYT (PICT 01095, S.A.G., M.J.M) and SECyT UNR (PIDBIO238, S.A.G). The authors have no conflict of interest to declare.

Integrated approach for detection of nonculturable cells of Ralstonia solanacearum in asymptomatic Pelargonium spp. cuttings.

Ralstonia solanacearum (biovar 2, race 3) is a soil and water-borne pathogen that causes serious diseases in several solanaceous hosts. It can also infect geranium plants, posing an important threat to their culture when latently infected cuttings are imported from countries where the pathogen is endemic. R. solanacearum can be present in very low numbers in asymptomatic geranium cuttings, and/or in a particular stressed physiological state that escapes direct isolation on the solid media usually employed. Consequently, an integrated protocol has been developed to analyze asymptomatic geranium cuttings routinely. The first screening tests include isolation and co-operational-polymerase chain reaction (Co-PCR), based on the simultaneous and co-operational action of three primers from 16S rRNA of R. solanacearum. This method was selected as the most sensitive one, able to detect only 1 cell/ml including nonculturable cells. When isolation is negative but Co-PCR is positive, the bioassay in tomato plants is proposed, since stressed bacterial cells or those present in low numbers that do not grow on solid media can be recovered from inoculated tomato plants and retain pathogenicity. This methodology has been demonstrated to be useful and has allowed us to assess the relevance of the physiological status of bacterial cells and its implications in detection. It also reveals the risk of introducing R. solanacearum through asymptomatic geranium material when relying only on bacterial isolation.

Immunosuppression with generic tacrolimus and mycophenolate mofetil in renal transplant recipients: preliminary report in Chile.

The association of tacrolimus (TAC) and mycophenolate mofetil (MMF) in renal transplant patients has diminished the incidence of acute rejection. We evaluated the use of generic TAC and MMF as primary immunosuppression in 6 living related (LR) and 11 cadaveric (C) donor renal transplant recipients (9 men, 8 women) of mean age 37 +/- 12 years (range, 17-56 years) between May 2006 and June 2007. From day 0 all patients received TAC, MMF, and prednisone without antibody induction. They were followed for the development of acute rejection, graft loss, side effects, and mortality. Mean follow-up was 7.6 months (range, 2-15 months). No biopsy-proven acute rejection episodes, graft loss, or recipient deaths were observed. Creatinine levels at the end of the study were 1.90 +/- 1.0 mg/dL (range, 0.62-4.25 mg/dL for C recipients and 1.19 +/- 0.15 mg/dL (range, 0.91-1.35 mg/dL) for LR recipients. Mean systolic and diastolic blood pressures were 130/73 mm Hg with 12 patients (70.5%) on antihypertensive therapy with calcium antagonists and beta-blockers. Mean (range) of total cholesterol, triglycerides, and glucose were 172 (110-244) mg/dL, 139 (69-277) mg/dL, and 89 (63-129) mg/dL, respectively. MMF was suspended in 1 patient due to diarrhea and 1 other because of leukopenia. We observed that generic TAC and MMF yielded effective and safe immunosuppression in terms of mortality, biopsy-proven acute rejection, and graft loss with a low incidence of adverse effects during the study period.

[Factors that affect the prescription of benzodiazepines and actions to improve their use: a Delphi study of primary care doctors]. / Factores que influyen en la prescripción de benzodiacepinas y acciones para mejorar su uso: un estudio Delphi en médicos de atención primaria.

OBJECTIVES: To identify the factors that affect the prescription of benzodiazepines and similar drugs and the actions that can be taken to reduce their prescription.Design. Consensus method. Delphi technique. SETTING: Four primary care areas in Asturias. Participants. 39 doctors from primary care teams agreed voluntarily to take part in the study, and 32 completed the study. They belonged to 20 health centres. METHOD: They were sent by mail three questionnaires one after the other. The second and third questionnaires were worked out on the basis of the analysis of the information from the replies to the preceding questionnaire. Those who did not send in a reply were reminded by phone. RESULTS: The 5 most influential factors in benzodiazepine prescription were agreed: reduction in the threshold of tolerance of emotional discomfort; increase of the prevalence of pathologies; lack of time in the consulting-room; social and economic conditioning factors; properties of the benzodiazepine family. The 5 most important actions that could reduce prescription of these drugs were agreed: general health education; reduction in case loads; making doctors more conscious of prescribing correctly; strengthening the social support network; doctors fomenting use of effective alternative treatments. CONCLUSIONS: The prescription of benzodiazepines and their analogues is a multi-factorial action with social and psychological roots. The action most voted on to reduce their prescription was general health education.

A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction.

A simple and rapid method for extracting DNA from plants based on the use of an extraction buffer and precipitation with isopropanol was assayed to see its usefulness in detecting pathogenic bacteria in plant material. The method was compared with a phenol-chloroform standard procedure obtaining higher sensitivity levels of detection. The protocol developed was efficient for detecting a Gram-positive bacterium, Clavibacter michiganensis subsp. sepedonicus and several Gram-negative pathogenic bacteria (Ralstonia solanacearum, Erwinia amylovora, Xanthomonas axonopodis pv. citri) with a sensitivity of 10(2)-10(3) cfu/ml in spiked samples. It was also efficient to specifically identify such bacteria in naturally infected plant material. This procedure is proposed as a routine tool for detection of plant pathogenic bacteria, as well as in environmental microbiology and biotechnology studies.

Relación LH/progesterona como índice pronóstico de la respuesta ovárica a la HCG / LH/progesterone ratio as prognostic index of ovaric response to HCG

Objetivo: Seleccionar un parámetro hormonal como mejor predictor de la respuesta a la HCG, en pacientes bajo Hiperestimulación Ovárica Controlada (H.O.C.). Material y Método: Se estudiaron 43 pacientes que iniciaron un ciclo para FIV o ICSI. La descarga ovulatoria se efectuó con 10.000 IU de HCG (Profasi, Serono; Endocorion, Elea, Pregnyl, Organón), en forma prospectiva y randomizada. En todas las pacientes se extrajo sangre a las 8 a.m. de los días 0 (día de la HCG), + 1 y + 2. En cada muestra se determinaron las concentraciones de LH, P y HCG. Todas fueron medidas en el mismo ensayo (RIA e IRMA). Otras variantes analizadas fueron edad, FSH y E2 al 3er. día del ciclo previo, E2 en el día 0, número de folículos punzados y ovocitos maduros (M II y M1B) recuperados. Las pacientes se dividieron en tres grupos según su respuesta ovárica: 1) Buena, relación ovocito recuperado/folículo punzado = 60 por ciento y = 5 ovocitos maduros obtenidos; 2) Moderada, < 60 por ciento de recuperación y 3) Pobre, < 5 ovocitos maduros recuperados. Análisis estadístico: Test no paramétrico U de Mann Whitney. Resultados: La edad promedio fue 35 años (r = 25-35). Las 43 pacientes se distribuyeron en Grupo I = 24, Grupo II = 7 y Grupo III = 12. Los tres preparados de HCG utilizados no mostraron diferencias entre sí. En los 3 días, la LH del G.I fue menor al G.III (p < 0,05). En los días + 1 y + 2, la P del G.I fue mayor a los G.II y G.III (p < 0,05). El incremento de P desde el día 0 al + 1 fue mayor en el G.I vs. G.III (p 0,05). El cociente LH/P arrojó diferencia entre G.I y III (p < 0,001 en el día + 1 y p < 0,007 en el día + 2). En la distribución de frecuencias, un cociente LH/P = 0,03 en el día + 1, separó los grupos I y III en un 90 por ciento. Conclusiones: Las diferencias de LH y P estrían reflejando una mejor sincronización de las células granulosas a la estimulación exógena, en el G.I. El índice LH/P en el día + 1, fue eficaz en separar los grupos de buena y pobre respuesta; un cociente = 0,03, predice en un 90 por ciento una respuesta ovárica adecuada

The effect of different methods of ovarian stimulation for human in vitro fertilization and embryo replacement.

Many types of medication regimens have been used to produce ovarian stimulation for in vitro fertilization and embryo replacement. However, few comparative studies have been reported. In this investigation, 105 patients were given either clomiphene citrate alone, human menopausal gonadotropins alone, or a combination of these drugs; and the resulting number of oocytes collected, embryos produced, and pregnancies initiated are reported. The data demonstrate optimal follicle development and fertilization rates with combinations of drugs, rather than the use of a single agent. Further studies are needed to determine whether there is a statistically significant difference in pregnancy rates between groups.

Pituitary response to insulin-induced hypoglycemia in patients with amenorrhea of different etiologies.

The insulin-induced hypoglycemia test was used to study the hypothalamic-pituitary function of nine normal control subjects and 49 patients with amenorrhea. There were 10 patients with secondary amenorrhea due to hypothalamic dysfunction, eight with hypothalamic failure, eight with primary amenorrhea due to hypogonadotropic hypogonadism, 19 with a prolactin-secreting pituitary adenoma, and four with Sheehan's syndrome. After the administration of insulin (0.15 unit/kg), a significant increase in plasma levels of prolactin, growth hormone, and cortisol occurred in all normal subjects. Of the 10 patients with hypothalamic dysfunction, two had a blunted prolactin response, six had an abnormal growth hormone response, and all had a normal cortisol response. Of those with hypothalamic failure, abnormal responses for prolactin were seen in two patients, for growth hormone in four patients, and all had a normal increase in cortisol. Five of the eight patients with primary amenorrhea had a blunted response for prolactin, six for growth hormone, and there were no abnormalities for plasma cortisol. All 19 patients with pituitary adenomas had a blunted increase in prolactin, 16 had an abnormal growth hormone response, and two had an abnormal cortisol response. Prolactin, growth hormone, and cortisol responses were blunted in all patients with Sheehan's syndrome. These results demonstrate that the release of growth hormone subsequent to insulin-induced hypoglycemia is the most common abnormality seen in women with amenorrhea. The high frequency of abnormal release of prolactin indicates that serum prolactin should be measured when this test is performed. In addition, patients with prolactin-secreting adenomas and those with Sheehan's syndrome should be given an insulin tolerance test before treatment is instituted, so that patients with secondary adrenal insufficiency can be identified.