Instrumentos de medida de resultados y de experiencias comunicadas por el paciente: directrices y herramientas para identificar y seleccionar los más adecuados / Outcome and patient-reported experience measurement instruments: Guidelines and tools to identify and select the most suitable

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Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa.

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z-VAD-FMK 100 µM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO2 and evaluated motility and kinetic parameters by computer-assisted semen analysis (CASA) and viability (propidium iodide), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), intracellular calcium (FLUO-3), membrane fluidity (M540) and ROS production (CM-H2 DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO-PRO-1+ and acrosome-reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca(2+) levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.

Estrous sheep serum enables in vitro capacitation of ram spermatozoa while preventing caspase activation.

Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3-7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1(+) and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase(-) spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation.

Effect of different media additives on capacitation of frozen-thawed ram spermatozoa as a potential replacement for estrous sheep serum.

Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin-hypotaurine alone, in combination with progesterone (P4), 17ß-estradiol (E2), or BSA, or just ß-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin-hypotaurine (alone), or in combination with P4, or E2, nor ß-cyclodextrin induced capacitation-related changes in frozen-thawed ram spermatozoa. Only the medium supplemented with heparin-hypotaurine-BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin-hypotaurine, P4, E2, ß-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa.

Fertility of cryopreserved ovine semen is determined by sperm velocity.

The present study aims to examine the predictive value of some sperm parameters on male fertility. Semen samples from six Manchega rams were collected and cryopreserved. Sperm quality was assessed after thawing and after 2h of incubation, either in the freezing extender (37°C) or after dilution in Synthetic Oviductal Fluid (SOF) (38°C, 5% CO2), attempting to mimic the physiological conditions of the female reproductive tract. The following sperm parameters were evaluated: motility and kinetic parameters by computer-assisted semen analyzer (CASA), and sperm viability (propidium iodide), mitochondrial membrane potential (JC-1), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), and intracellular calcium (fluo-3) by flow cytometry. Results showed no significant differences between incubation media neither after thawing nor after incubation. There were no significant correlations between fertility and sperm parameters assessed by flow cytometry. However, after incubation in the freezing extender, sperm samples from males with poor fertility yielded less linearity and velocity (P<0.05) as indicated by motility parameters analyzed by CASA. These results indicate that kinematic sperm motility parameters evaluation by CASA might be useful to identify samples with poor fertility.

Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants.

Antioxidants may be useful for supplementing sperm extenders. We have tested dehydroascorbic acid (DHA), TEMPOL, N-acetyl-cysteine (NAC) and rutin on epididymal spermatozoa from red deer, during incubation at 37 °C. Cryopreserved spermatozoa were thawed, washed and incubated with 1 mM or 0.1 mM of each antioxidant, including oxidative stress (Fe(2+)/ascorbate). Motility (CASA and clustering of subpopulations), viability, mitochondrial membrane potential, and acrosomal status were assessed at 2 and 4 h. Lipoperoxidation, intracellular reactive oxygen species (ROS) and DNA damage (DNA) status (TUNEL) were checked at 4 h. Oxidative stress increased ROS, lipoperoxidation and DNA damage. Overall, antioxidants negatively affected motility and physiological parameters. Only DHA 1 mm protected motility, increasing the fast and progressive subpopulation. However, it had a detrimental effect on acrosomal and DNA status, in absence of oxidative stress. Tempol and rutin efficiently reduced lipoperoxidation, ROS, and DNA damage in presence of oxidative stress. NAC was not as efficient as TEMPOL or rutin reducing lipoperoxidation or protecting DNA, and did not reduce ROS, but its negative effects were lower than the other antioxidants when used at 1 mm, increasing the subpopulation of hyperactivated-like spermatozoa at 2 h. Our results show that these antioxidants have mixed effects when spermatozoa are incubated at physiological temperatures. DHA may not be suitable because of prooxidant effects, but TEMPOL, NAC and rutin may be considered for cryopreservation trials. In general, exposure of red deer spermatozoa to these antioxidants should be limited to low temperatures, when only protective effects may develop.

Effect of semen collection method (artificial vagina vs. electroejaculation), extender and centrifugation on post-thaw sperm quality of Blanca-Celtibérica buck ejaculates.

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl(®), Andromed(®) and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl(®) and Andromed(®) resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl(®) or Andromed(®) are used as freezing extenders.

Angiotensin-converting enzyme gene single polymorphism as a genetic biomarker of diabetic peripheral neuropathy: longitudinal prospective study.

BACKGROUND: Identifying patients at risk of developing diabetic peripheral neuropathy (DPN) is of paramount importance in those with type 2 diabetes mellitus (T2DM) to provide and anticipate secondary prevention measures as well as intensify action on risk factors, particularly so in primary care. Noteworthy, the incidence of DPN remains unknown in our environment. AIMS: (i) To analyze a single angiotensin-converting enzyme (ACE) gene polymorphism (D/I) as a genetic marker of risk of developing DPN, and (ii) to determine the incidence of DPN in our environment. RESEARCH DESIGN AND METHODS: Longitudinal study with annual follow-up for 3years involving a group of T2DM (N=283) randomly selected. ACE gene polymorphism distribution (I=insertion; D=deletion) was determined. DPN was diagnosed using clinical and neurophysiology evaluation. RESULTS: Baseline DPN prevalence was 28.97% (95% CI, 23.65-34.20). ACE polymorphism heterozygous genotype D/I presence was 60.77% (95% CI, 55.05-66.5) and was independently associated with a decreased risk of DPN (RR, 0.51; 95% CI, 0.30-0.86). DPN correlated with age (P<0.001) but not with gender (P=0.466) or time of evolution of T2DM (P=0.555). Regarding end point, DPN prevalence was 36.4% (95% CI, 30.76-42.04), and accumulated incidence was 10.4% 3years thereafter. In the final Poisson regression analysis, the presence of heterozygous genotype remained independently associated with a decreased risk of DPN (RR, 0.71; (95% CI, 0.53-0.96). DPN presence remained correlated with age (P=0.002), but not with gender (P=0.490) or time of evolution (P=0.630). CONCLUSIONS: In our series, heterozygous ACE polymorphism (D/I) stands as a protective factor for DPN development. Accumulated incidence of DPN was relevant. Further prospective studies are warranted.

[Prevalence of food habits and nutritional status in adult population served in primary care]. / Estudio de prevalencia sobre los hábitos alimentarios y el estado nutricional en población adulta atendida en atención primaria.

OBJECTIVE: To identify dietary patterns and nutritional status of the population between 18-44 years visited at a health center (HC) and explore the associated factors. METHODS: Cross-sectional study conducted in an urban HC in a random sample of 201 people. Were analyzed socio-demographic data, anthropometric measurements, 24 hours latest reminder intake, weekly food frequency questionnaire. RESULTS: The mean age of participants was 32.81 years (standard deviation 6.72). 63.68% (128) were women and 62.69% (126) immigrants. 45.27% (91) expressed no exercise. About 60% of participants related a deficit in consumption milk, fruits, meat and farinaceous and 80% vegetables, 58% related excessive fat. 44.4% (56) of immigrants was 20.3% with respect to obese (15) of native (P = 0.001). The proportion of people with "waist-risk" was higher among immigrants as compared to the native (19.0% vs 6.8%, P = 0.017) and among those who did not exercise with respect to each other (17.6% vs 8.1%, P = 0.049). Immigrants perform less physical activity related to the native (42.1% vs 62.7%, P = 0.013). Being an immigrant was the variable explaining of distribution acceptable daily macronutrient intake (Odds Ratio 4.7, Confidence Interval 95% 2.02-11.03). DISCUSSION: The participants related excessive consumption of fats and deficient in the rest of food groups. Obesity and the "risk waist" were more common among immigrants and sedentary population. Immigrants talked of a better distribution of nutrients in your daily diet.

Estudio de prevalencia sobre los hábitos alimentarios y el estado nutricional en población adulta atendida en atención primaria / Prevalence of food habits and nutritional status in adult population served in primary care

Introducción: Identificar los hábitos alimentarios y el estado nutricional de la población entre 18-44 años atendida en un centro de salud (CS) y estudiar los factores asociados. Métodos: Estudio transversal realizado en un CS urbano en el que se seleccionó una muestra aleatoria de 201 personas. Se analizaron datos socio-demográficos, medidas antropométricas, recordatorio ingesta últimas 24 horas, cuestionario de frecuencia alimentaria semanal. Resultados: La edad media de los participantes fue de 32,81 años (Desviación Estándar 6,72). El 63,68%(128) eran mujeres y 62,69%(126) inmigrantes. El 45,27%(91) manifestaba no realizar ejercicio. Alrededor del 60% de los participantes refería un consumo deficitario de lácteos, frutas, cárnicos y farináceos y el 80% de verduras; el 58% lo refería excesivo de lípidos. El 44,4%(56) de inmigrantes era obeso respecto al 20,3%(15) de autóctonos (P= 0,001). La proporción de personas con «cintura de riesgo» era superior entre los inmigrantes respecto a los autóctonos (19,0% vs 6,8%; P= 0,017) y entre los que no hacían ejercicio respecto a los que sí (17,6% vs 8,1%; P= 0,049). Los inmigrantes referían realizar menos ejercicio físico que los autóctonos (42,1% vs 62,7%; P= 0,013). Ser inmigrante era la variable más explicativa de la distribución aceptable de macronutrientes en la ingesta diaria (Odds Ratio 4,7; Intervalo de Confianza del 95% 2,02-11,03). Discusión: Los participantes referían un consumo excesivo de lípidos y deficitario del resto de grupos alimentarios. La obesidad y la «cintura de riesgo» eran más frecuentes entre los inmigrantes y los participantes más sedentarios. Los inmigrantes referían una mejor distribución de macronutrientes en su alimentación diaria (AU)

Objective: To identify dietary patterns and nutritional status of the population between 18-44 years visited at a health center (HC) and explore the associated factors. Methods: Cross-sectional study conducted in an urban HC in a random sample of 201 people. Were analyzed socio-demographic data, anthropometric measurements, 24 hours latest reminder intake, weekly food frequency questionnaire. Results: The mean age of participants was 32.81 years (standard deviation 6.72). 63.68% (128) were women and 62.69% (126) immigrants. 45.27% (91) expressed no exercise. About 60% of participants related a deficit in consumption milk, fruits, meat and farinaceous and 80% vegetables, 58% related excessive fat. 44.4% (56) of immigrants was 20.3% with respect to obese (15) of native (P = 0.001). The proportion of people with «waist-risk» was higher among immigrants as compared to the native (19.0% vs 6.8%, P = 0.017) and among those who did not exercise with respect to each other (17.6% vs 8.1%, P = 0.049). Immigrants perform less physical activity related to the native (42.1% vs 62.7%, P = 0.013). Being an immigrant was the variable explaining of distribution acceptable daily macronutrient intake (Odds Ratio 4.7, Confidence Interval 95% 2.02-11.03). Discussion: The participants related excessive consumption of fats and deficient in the rest of food groups. Obesity and the «risk waist» were more common among immigrants and sedentary population. Immigrants talked of a better distribution of nutrients in your daily diet (AU)

El filtrado glomerular reducido según las fórmulas de Cockcroft-Gault y MDRD no siempre predice la morbimortalidad cardiovascular en los pacientes hipertensos atendidos en atención primaria / Decreased glomerular filtration rate using the Cockgroft-Gaultand MDRD formulas does not always predict cardiovascular morbidity and mortality in hypertensive primary care patients

Antecedentes: El deterioro de la función renal se ha asociado con un incremento de la morbimortalidad cardiovascular. El objetivo del estudio fue analizar la asociación del filtrado glomerular (FG) basal, según las fórmulas de Cockcroft-Gault y MDRD, con la incidencia de eventos cardiovasculares (ECV) en una cohorte de personas hipertensas seguida durante 12 años. Métodos: Estudio prospectivo de una muestra aleatoria de 223 hipertensos libres de ECV atendidos en un centro de atención primaria urbano. Se estimó el FG mediante ambas fórmulas. Se consideró ECV la aparición de cardiopatía isquémica, insuficiencia cardíaca, accidente cerebrovascular, vasculopatía periférica o muerte por ECV. Se analizaron los datos mediante el método actuarial y modelos de regresión de Cox. Resultados: La mediana de tiempo de seguimiento fue de 10,7 años (rango intercuartílico,6,5-12,1). El seguimiento fue completo en 191 participantes (85,7%). La supervivencia acumulada fue del 64,7% (intervalo de confianza [IC] del 95%: 57,9-71,6%). La tasa media de incidencia de ECV durante todo el período de seguimiento fue de 3,6 (IC del 95%, 2,7-4,4%) por 100 personas hipertensas/año. El modelo multivariable final mostró que las variables con mayor poder predictivo de ECV en la población de estudio fueron la diabetes y la estimación del FG >60ml/min/1,73 m2mediante fórmula MDRD. Conclusiones: Se observó una relación entre la aparición de ECV y los valores de FG estimados por la fórmula MDRD al inicio del seguimiento superiores a 60 ml/min/1,73 m2, inversa a la eserada. La estimación del FG mediante fórmula de Cockcroft-Gault no se asoció con el riesgo cardiovascular (AU)

Background: Renal function decrease is associated with cardiovascular morbidity and mortality. The aim was to analyze the association of cardiovascular morbidity and mortality with baseline glomerular filtration rate (GFR), according Cockcroft-Gault and MDRD formulas, with incidence of major adverse cardiovascular events (MACEs) in a cohort of hypertensive individuals followed for 12 years. Methods: We performed a prospective study of a random sample of 223 hypertensive patients free of MACEs followed in an urban Primary Care Center. GFR was estimated using both formulas. MACEs were considered the onset of ischemic heart disease, heart failure, stroke, peripheral vascular disease or cardiovascular death. Data were analyzed using the life-table method and Cox regression modeling. Results: Follow-up median was 10.7 (interquartile range, 6.5-12.1) years. Follow-up was complete in 191 participants (85.7%). The cumulative survival was 64.7% (95%Confidence Interval (CI) 57.9%-71.6%). The incidence of MACEs during the follow-up period was 3.6 (95% CI, 2.7-4.4) per 100 subject-years. The final multivariable model showed that the most predictive variables of MACEs in the study population were the presence of diabetes and the estimation of GFR >60 ml/min/1.73 m2by MDRD equation. Conclusions: There was a relationship between the occurrence of MACEs and MDRD formula estimated GFR above60 ml/min/1.73 m2at study entry, inversely to what expected. C-G formula estimated GFR by was not associated with cardiovascular risk (AU)

[Decreased glomerular filtration rate using the Cockckroft-Gault and MDRD formulas does not always predict cardiovascular morbidity and mortality in hypertensive primary care patients]. / El filtrado glomerular reducido según las fórmulas de Cockcroft-Gault y MDRD no siempre predice la morbimortalidad cardiovascular en los pacientes hipertensos atendidos en atención primaria.

BACKGROUND: A decrease in renal function is associated with cardiovascular morbidity and mortality. The aim of this study was to analyse the association of cardiovascular morbidity and mortality with baseline glomerular filtration rate (GFR), calculated according to the Cockcroft-Gault and MDRD formulas, with the incidence of major adverse cardiovascular events (MACEs) in a cohort of hypertensive individuals followed for 12 years. METHOD: We performed a prospective study of a random sample of 223 hypertensive patients free of MACEs, who were followed in an urban Primary Care Centre. GFR was estimated using both formulas. MACEs were considered as the onset of ischaemic heart disease, heart failure, heart attacks, peripheral vascular disease or cardiovascular death. Data were analysed using the life-table method and Cox regression modeling. RESULTS: The median follow-up was 10.7 (interquartile range, 6.5-12.1) years. Follow-up was completed in 191 participants (85.7%). The cumulative survival was 64.7% (95% Confidence Interval (CI), 57.9-71.6). The incidence of MACEs during the follow-up period was 3.6 (95% CI, 2.7-4.4) per 100 subject-years. The final multivariable model showed that the most predictive variables of MACEs in the study population were the presence of diabetes mellitus and the estimation of GFR ≥60 ml/min/1.73 m2 by MDRD equation. CONCLUSIONS: There was a relationship between the occurrence of MACEs and an estimated GFR by MDRD above 60 ml/min/1.73 m2 at study entry, inversely to what was expected. GFR estimated by the C-G formula was not associated with cardiovascular risk.

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa.

The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility.

Comparison of the TBARS assay and BODIPY C11 probes for assessing lipid peroxidation in red deer spermatozoa.

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C(11) (B581) and BODIPY 665/676 C(11) (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H(2)O(2) ) 0.1 mM or 1 mM, or tert-butyl hydroperoxide (TBH) 0.1 mM or 1 mM. LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mM of TBH or H(2)O(2). Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.

Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulations.

In the present study, we have related mitochondrial membrane potential (DeltaPsim) and forward scatter (FSC) to apoptotic-related changes in spermatozoa. Thawed red deer spermatozoa were incubated in synthetic oviductal fluid medium (37 degrees C, 5% CO2), with or without antioxidant (100 microm Trolox). At 0, 3, 6 and 9 h, aliquots were assessed for motility and were stained with a combination of Hoechst 33342, propidium ioide (PI), YO-PRO-1 and Mitotracker Deep Red for flow cytometry. The proportion of spermatozoa YO-PRO-1+ and PI+ (indicating a damaged plasmalemma; DEAD) increased, whereas that of YO-PRO-1- and PI- (INTACT) spermatozoa decreased. The proportion of YO-PRO-1+ and PI- spermatozoa (altered plasmalemma; APOPTOTIC) did not change. Both DEAD and APOPTOTIC spermatozoa had low DeltaPsim. Most high-DeltaPsim spermatozoa were INTACT, and their proportion decreased with time. The FSC signal also differed between different groups of spermatozoa, in the order APOPTOTIC > DEAD > INTACT/low DeltaPsim > INTACT/high DeltaPsim; however, the actual meaning of this difference is not clear. APOPTOTIC spermatozoa seemed motile at 0 h, but lost motility with time. Trolox only slightly improved the percentage of INTACT spermatozoa (P < 0.05). The population of APOPTOTIC spermatozoa in the present study may be dying cells, possibly with activated cell death pathways (loss of DeltaPsim). We propose that the sequence of spermatozoon death here would be: (1) loss of DeltaPsim; (2) membrane changes (YO-PRO-1+ and PI-); and (3) membrane damage (PI+). INTACT spermatozoa with low DeltaPsim or altered FSC may be compromised cells. The present study is the first that directly relates membrane integrity, apoptotic markers and mitochondrial status in spermatozoa. The results of the present study may help us understand the mechanisms leading to loss of spermatozoon viability after thawing.

Genetic and immunological characterization of the 14-3-3xi molecule from Schistosoma bovis.

Currently available candidate vaccines against schistosomiasis elicit only partial protection. In addition, the type of immune response that could lead to the highest level of protection against schistosomes has not yet been described. Thus, efforts should be made in both the identification of novel proteins essential for the parasite cycle and in the modulation of immune responses against these novel candidates through the combined use of immunomodulatory molecules. Several parasites have 14-3-3 proteins, and these proteins are known to play a key role in parasite biology. In the present work, we report the isolation and characterization of a new 14-3-3 gene from Schistosoma bovis and offer new information regarding the genetic structure of the gene. In addition, we have produced the corresponding recombinant protein. Finally, we describe the immune responses elicited by this protein when combined with 4 different immunomodulators in immunized mice.

The Sb14-3-3zeta recombinant protein protects against Schistosoma bovis in BALB/c mice.

Schistosoma bovis is a trematode parasite mainly affecting cattle and sheep. Evidences about the arise of drug resistance and the high rates of re-infection of animals in endemic areas have pointed out the need of developing new control tools, e.g., effective vaccines. Schistosomes 14-3-3 proteins have been defined as vaccine candidates against respective infections. We have therefore investigated the protective capacity of the 14-3-3 protein from S. bovis - Sb14zeta - against S. bovis in mice. In addition, we have addressed the influence of the co-administration of four different immunomodulators with the 14-3-3 polypeptide. The values of protection against S. bovis were statistically significant when the Sb14zeta was combined in two independent experiments with the AA0029 (61.0% and 40.31%), AA2829 (49% and 36.3%) and PAL (49% and 40.075%) immunomodulatory molecules. Immune responses from vaccinated animals showed that the highest protection rates do not necessarily match with a dominant Th1-type response.

Réplica. Tuberculosis en zona de gran incidencia y con elevado porcentaje de inmigrantes / Reply. Tuberculosis in an area high incidence and a large percentage of immigrants

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Synthesis and cytotoxicity of hydrophobic esters of podophyllotoxins.

Diverse norbornenecarboxylate esters of podophyllotoxin and its epimers and diastereoisomers have been prepared through Diels-Alder cycloaddition by treating the dienophilic acrylates of cyclolignans with cyclopentadiene. Their cytotoxicities against several cancer cell lines have been evaluated and the results compared with those found for other lignan esters. Podophyllotoxin adducts showed a one-fold increase in activity when compared to the natural product. The preparation of more hydrophobic esters, which showed less cytotoxicity, demonstrated that this activity is not primarily due to the lipophilic factor, but mainly to the spatial arrangement of the bulky moiety, which could contribute to increase the binding to the target site.