Swine spermatozoa trigger aggregated neutrophil extracellular traps leading to adverse effects on sperm function.

In pigs, the number of PMN in uterus lumen increases within few hours after natural or artificial AI resulting in early PMN-derived innate immune reactions. Sperm-NETs formation was recently reported to occur in various mammalian species. Aim of this study was to investigate direct interactions of boar spermatozoa with swine PMN, the release of sperm-mediated NETs, and to assess NET-derived effects on sperm functionality. Sperm-triggered NETs were visualized by SEM- and immunofluorescence analyses. Sperm-mediated NETosis was confirmed by presence of extruded DNA with global histones and NE. Largest sizes of sperm-mediated aggNETs were detected after 5 h thereby resulting in effective massive sperm entrapment. The number of aggNETs increased from 3 h onwards. Kinetic studies of swine sperm-mediated NETosis showed to be a time-dependent cellular process. In addition, number of NETs-entrapped spermatozoa increased at 3 h of exposure whilst few free spermatozoa were detected after 3 h. Anchored NETs also increased from 3 h onwards. The cytotoxicity of NETs was confirmed by diminution of the total motility and the progressive motility. Spermatozoa membrane integrity and function loss exposed to NETs was confirmed from 3 h. Experiments revealed NETs-derived damaging effects on swine spermatozoa in membrane integrity, motility and functionality. We hypothesize that swine sperm-triggered aggNETs might play a critical role in reduced fertility potential in swine reproductive technique. Thus, aggNETs formation needs to be considered in future studies about uterine environment as well as advance of sperm in the porcine female reproductive tract.

Evaluation of nutritional values of wild mushrooms and spent substrate of Lentinus crinitus (L.) Fr.

In Colombia, despite the great diversity of mushrooms, most are yet unknown from the taxonomic point of view, and even less known from their nutritional composition or their possible application to obtain value-added products from agro-waste. The mycelial growth of Lentinus crinitus (L.) Fr strain was investigated on agro-waste in culture media agar and correlation analyses were performed. The proximate and mineral element composition was determinate in wild mushrooms and spent substrate of L. crinitus, obtained in the solid-state fermentation. The evaluation of the mycelial growth of the L. crinitus strain confirmed that it can grow on agro-waste. The treatment T6 (Orange peel and brand) was determined to be the best for the mycelial growth of L. crinitus (0.0790 cm/h), T7 (Bran, Orange peel and rice husk) and T5 (Rice hush and orange peel) followed, with mycelial growth rates of 0.0753 cm/h and .0720 cm/h, respectively. The growth rate was positively correlated with C/N ratios but negatively correlated with Zn, N and protein. The combination of the agro-waste (T6, T7 and T5) were used to obtain the spent substrate and assess its nutritional potential. The results showed that wild mushrooms of L. crinitus had protein contents of 14.42%, and fiber of 57.18%. The spent substrate of L. crinitus increased their protein content (10.5-11.22%), fiber (44.1-56%) and nitrogen (1.64-1.28%). These advances are promising for the use of L. crinitus as degrader of agro-waste to obtain different products of food and agro-industrial interest.

Gradient sperm selection for reproductive techniques in cattle: Is Isolate a suitable replacement for Percoll?

In assisted reproductive techniques, it is essential to perform a sperm selection to obtain spermatozoa with high motility and membrane integrity for in vitro fertilisation (IVF) and high-DNA integrity for intracytoplasmic sperm injection (ICSI). In this study, we evaluated whether Isolate® was a suitable substitute for Percoll® for assisted reproductive techniques. Commercial cryopreserved bovine semen was used after selection in both gradients, and plasma and acrosome membrane integrity, reactive oxygen species (ROS) levels, DNA integrity and mitochondrial membrane potential (ΔΨm) were assessed by flow cytometry. Motility parameters were also evaluated by CASA system. A similar percentage of spermatozoa with intact plasma membrane, acrosome integrity and high ΔΨm was observed in both sperm selection methods, but only Percoll® showed higher percentage of spermatozoa with intact plasma and acrosome membrane compared to the post-thawing group. No differences were observed in the motility, ROS, DNA fragmentation and on the in vitro embryo production in all experimental groups. In conclusion, the selection of bovine spermatozoa with Isolate® generates spermatozoa with similar quality parameters and embryonic development compared to Percoll® providing a suitable alternative sperm selection method for assisted reproductive techniques in this species.

Effect of sperm pretreatment with glutathione and membrane destabilizing agents lysolecithin and Triton X-100, on the efficiency of bovine intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an assisted reproduction tool with several applications. Its effectiveness in bovines is lower than that in other species, mainly because of difficulties in the decondensation of the sperm nucleus after injection, and the presence of the acrosome and the plasma membrane which remain intact in this procedure. In this study, we assessed the effect of lysolecithin (LL) and Triton X-100 (TX), in combination with glutathione (GSH) as sperm pretreatments prior to ICSI. The GSH-LL and GSH-TX groups showed 0% of spermatozoa with intact membrane (SYBR 14+/PI), in comparison with the control (63.3%) and GSH (65.7%) groups. The proportions of spermatozoa with damaged acrosome membrane in the GSH-LL, GSH-TX, GSH and control groups were 46%, 35.9%, 10.5% and 7.5%, respectively. Sperm chromatin decondensation analysis showed that the groups incubated for 3 hr with GSH presented greater decondensation (p < .05). Although fertilization was improved in all treatment groups evaluated, no differences were observed in the cleavage rate 72 hr after activation in the GSH (73.7%), GSH-LL (80.2%) and GSH-TX (77.8%) groups compared to the control (66.3%), neither in the blastocyst rate on day 8 (24.0%, 26.2%, 27.1% and 28.4% for the control, GSH, GSH-LL and GSH-TX groups, respectively). No differences were also observed in the total number of cells in all groups. In conclusion, although these sperm treatments promoted nuclear decondensation and induced plasma membrane disruption, these effects were not sufficient to improve bovine embryonic development after ICSI.

Methyl-ß-Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida-Binding Ability of Frozen/Thawed Bovine Spermatozoa.

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein-free media. However, the additive capacitating effect of methyl-ß-cyclodextrin (MßCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen-thawed bovine spermatozoa in a BSA-containing medium supplemented with MßCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MßCD in a dose-dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MßCD. However, pre-incubation of spermatozoa in MßCD-supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)-binding ability (p < 0.05). Thus, we conclude that MßCD supplementation is able to enhance the capacitation status of frozen-thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.

Aspergilosis necrotizante crónica en un paciente con secuelas de tuberculosis pulmonar / Chronic necrotizing aspergillosis in a patient with pulmonary tuberculosis sequelae

Aspergillus fumigatus is the causative agent of differents pathologies in the human being: aspergilloma, allergic bronchopulmonary aspergillosis, chronic necrotizing aspergillosis and invasive aspergillosis. In chronic necrotizing aspergillosis there is local invasion of the lung tissue and parenchyma destruction. Chronic necrotizing aspergillosis is different from invasive aspergillosis, because the abscence of vascular invasion or dissemination. Chronic necrotizing aspergillosis is seen in middle-aged and elderly with underlying lung diseases: COPD, tuberculosis sequelae, lung resection, pneumoconiosis, radiotherapy, lung infarction or sarcoidosis. Clinical manifestations are non specific, being the most usual fever, cough, sputum production and weight loss. Incidence of chronic necrotizing aspergillosis is unknown in Chile. Chronic necrotizing aspergillosis can produce death. It requires early diagnosis and treatment. In a patient with a predisposing disease and with prolonged fever and consuntive status, diagnosis of chronic necrotizing aspergillosis should be considered. We present a patient with chronic necrotizing aspergillosis attended at Instituto Nacional del Tórax (Thorax National Institute) in Santiago.

Aspergillus fumigatus puede causar diferentes patologías en el ser humano: aspergiloma, aspergilosis broncopulmonar alérgica, aspergilosis necrotizante crónica, aspergilosis invasora. En la aspergilosis necrotizante crónica hay invasión local del parénquima y destrucción. A diferencia de la aspergilosis invasora no invade vasos sanguíneos ni se disemina a otros órganos. La aspergilosis necrotizante crónica se presenta en pacientes de edad media o ancianos con patología pulmonar previa: EPOC, secuelas de tuberculosis, resección pulmonar, neumoconiosis, radioterapia, infarto pulmonar o sarcoidosis. La clínica es indolente e inespecífica, con fiebre, tos, expectoración y baja de peso. Se desconoce la incidencia de aspergilosis necrotizante crónica en nuestro medio. La aspergilosis necrotizante crónica es potencialmente fatal, por lo que requiere de un diagnóstico y tratamiento oportuno. Creemos que, debe considerarse esta entidad ante un cuadro consuntivo y febril prolongado, en pacientes con enfermedades predisponentes que producen una leve baja de la inmunidad. Describimos el caso de un paciente atendido en el Instituto Nacional del Tórax.

Desenlace fatal por loxoscelismo cutáneo visceral / Severe loxoscelism with lethal outcome. Report of one case

Loxoscelism, is caused by the bite of Loxosceles laeta spiders. It has two clinical forms: cutaneous loxoscelism (CL) and viscerocutaneous loxoscelism (VCL). VCL is characterized by hematuria, hemoglobinuria, jaundice, fever and sensorial involvement. In severes cases there is massive hemolysis and renal failure, with high letality. We report a 71 year-old man, brought to the hospital five days after suffering a spider bite. The patient was admitted with a severe kidney failure, hemolysis, metabolic acidosis and clotting disorder. The patient was managed with adrenal steroids and dialisys but died five weeks after hospital admission. This particualr patient consulted late and had multiple factors of poor prognosis.

Tuberculosis miliar en paciente inmunocompetente como causa rara de fiebre nosocomial / Miliary tuerculoosis an uncommon cause of nosocomial fever in an immunocompetent patient

A progresive decrease of tuberculosis incidence has been shown in the last years in Chile. Presently Chile is considered as a country with a low incidence of tuberculosis (< 20 cases per 10(5) inhabitants). Miliary tuberculosis is an uncommon form of tuberculosis in immunocompetent patients. Miliary tuberculosis is tipically observed in immunocompromised patients. It is an infrequent cause of nosocomial fever. We report a case of miliary tuberculosis in an immunocompetent patient, that caused intrahospitalary fever, in the course of a long term hospitalization.

La tuberculosis ha disminuido su incidencia en los últimos años en Chile, siendo considerado actualmente como un país con baja incidencia de tuberculosis (< 20 casos por 10(5) habitantes). Dentro de las diferentes formas de presentación clínica de la tuberculosis, la diseminación miliar es rara en pacientes inmunocompetentes, estando restringida prácticamente a determinados grupos de riesgo. Más excepcional aún es considerar a la tuberculosis miliar como causa de fiebre intrahospitalaria. Comunicamos un caso de tuberculosis miliar en un paciente inmunocompetente, como causa de fiebre nosocomial, durante una hospitalización prolongada.

Susceptibilidad antimicrobiana de cepas de Pseudomonas aeruginosa aisladas en el laboratorio del Hospital Regional Dr. Leonardo Guzmán de Antofagasta, Chile / In vitro antimicrobial susceptibility of Pseudomonas aeruginosa strains isolated at Hospital Dr. Leonardo Guzmán, Antofagasta, Chile

Pseudomonas aeruginosa es un patógeno nosocomial frecuente que presenta elevada resistencia a los antimicrobianos y causa infecciones graves cuando hay alteración de los mecanismos defensivos del paciente. Así, conocer los patrones locales de sensibilidad es importante para la elección del tratamiento antimicrobiano adecuado en cada institución. En este trabajo determinamos la susceptibilidad antimicrobiana de cepas de P. aeruginosa aisladas desde pacientes atendidos en el Hospital Regional de Antofagasta. La mayoría de los pacientes tenía alguna condición predisponente a la infección y 48% tenía una infección grave. Las cepas mostraron mayor resistencia a los antimicrobianos que lo reportado en trabajos nacionales previos. Las cepas fueron altamente resistentes a amikacina (36,8%), ceftazidima (36,8%) y ciprofloxacina (68,4%), moderadamente resistentes a imipenem (26,3%), mientras que eran escasamente resistentes a piperacilina/tazobactam (5,3%) y cefoperazona/sulbactam (15,8%), Este es el primer trabajo, realizado en nuestra región, que estudia la susceptibilidad de P. aeruginosa frente a distintos grupos de antimicrobianos utilizados en clínica.

Pseudomonas aeruginosa is a nosocomial pathogen that often displays a high degree of antibiotic resistance. This pathogen causes also serious infections specially in patients with severe diseases or immunodeficiency. To offer the best treatment in every institution it is necessary to know the local pattern of antimicrobial susceptibility, then we studied the antibiotic susceptibility of P. aeruginosa strains isolated from patients attended in the Regional Hospital of Antofagasta. Most of them had an underlying disease that predisposed them to the infection and 48% had a severe infection. The strains showed higher drug resistance than that reported by other chilean researchers. P. aeruginosa displayed high resistance to amikacin (36,8%), ceftazidime (36,8%) and ciprofloxacin (68,4%) intermediate resistance to imipenem (26,3%), but low resistance to piperacillin/tazobactam (5,3%) and cefoperazone/sulbactam (15,8%). This is the first drug susceptibility study conducted in the Second Region of Chile, where P. aeruginosa was assayed against those antibiotics used in the clinical practice.

Genetic effects induced by neutrons in Drosophila melanogaster I. Determination of absorbed dose.

A method to obtain the absorbed dose in Drosophila melanogaster irradiated in the thermal column facility of the Triga Mark III Reactor has been developed. The method is based on the measurements of neutron activation of gold foils produced by neutron capture to obtain the neutron fluxes. These fluxes, combined with the calculations of kinetic energy released per unit mass, enables one to obtain the absorbed doses in Drosophila melanogaster.

Effects of microcystin-LR on the partial reactions of the Na(+)-K+ pump of the gill of carp (Cyprinus carpio Linneo).

Several partial reactions of the Na(+)-K+ pump enzyme were studied in a microsomal fraction derived from the gill of carp (Cyprinus carpio Linneo). We tested the effect of three toxins [(i) microcystin-LR, (ii) microcystin-LR-like toxin component isolated from Microcystis aeruginosa culture and (iii) okadaic acid] on the phosphorylation, ouabain binding and ATPase activity of the Na(+)-K+ pump. The K(+)-dependent hydrolysis of the Na(+)-dependent phosphorylation of Na(+)-K+ pump, as well the release of bound ouabain were inactivated in direct proportion to the amount of each toxin treatment. These results indicate that these toxins not only block the hydrolysis of phosphorylated protein at serine and threonine residues, but also inhibit the aspartic dephosphorylation step of the sodium pump enzymes. This inactivation could disrupt the ion homeostasis of the internal medium by blocking the gill function. The blockage of gill activity could be the cause of the massive fish death during blooms of M. aeruginosa.

Inhibitory effects of Microcystis aeruginosa toxin on ion pumps of the gill of freshwater fish.

A microsomal fraction enriched in ion pump enzymes was isolated from the gill of the carp (Cyprinus carpio Linneo). Mg(2+)-dependent (Na+ + K+), Na+, HCO3- and Ca(2+)-stimulated ATPase activities were studied following treatment with microcystin-LR-like toxin, the major toxic component isolated from Microcystis aeruginosa culture. These enzyme activities were inhibited in a dose-dependent manner. The maximum inhibition of each enzyme, induced with nM concentration of the toxin, was similar to that produced by inhibitors specific for each ATPase activity. The Mg(2+)-ATPase activity and non-specific hydrolysis of ATP were unaffected. These results strongly suggest that the massive fish death during M. aeruginosa blooms may result from the loss of ion homeostatic processes produced by the inhibitory action of microcystin on the ion pumps of gill chloride cells.

Inhibitory effect of a Microcystis sp (cyanobacteria) toxin on development of preimplantation mouse embryos.

1. A soluble toxin, purified from the algae bloom of an eutrophic lake dominated by Microcystis, is a very effective inhibitor of early embryo development in a dose-response relationship. 2. Two- and 8-cell mouse embryos under the influence of Microcystis toxin do not reach the developmental stages of morula and blastocyst, respectively. 3. Actin cortex is disorganized without change in the microtubules structure. 4. Results are discussed in terms of the possible mechanisms by which the toxin arrests development considering, specifically, effects on the cytoskeleton and/or on voltage-insensitive transmembrane Ca2+ channels.

[Morphological and functional parameters of the left ventricle (mass, wall thickness and end-systolic stress) in school children with different levels of blood pressure, at rest and during maximal exercise]. / Parametros morfológicos y funcionales del ventrículo izquierdo (masa, grosor parietal y stress parietal telesistólico) en escolares con diferentes niveles de presión arterial, en reposo y durante el ejercicio máximo.

Echocardiographically determined left ventricular mass, diastolic septal and posterior wall thickness and end-systolic wall stress, as well as electrocardiographic indexes of left ventricular enlargement (Sokolow-Lyon index and Romhilt-Estes score) and of left atrial enlargement (P terminal index) were correlated with resting and exercise systolic and diastolic blood pressures, and with several parameters of body size (weight, height, body surface area, Quetelet index), in 130 school children (61 boys, 69 girls) 6 to 15 years of age. Parameters of body size had a positive correlation both with systolic and diastolic blood pressures and with parameters of left ventricular size. Thus, the latter were adjusted for body surface area, for correlation with blood pressure. Left ventricular mass and diastolic septal and posterior wall thickness had a very poor correlation with resting and exercise diastolic blood pressures. Left ventricular mass and diastolic posterior wall thickness had a significantly higher correlation with peak exercise systolic blood pressure than with resting systolic blood pressure. End-systolic wall stress had a positive correlation with resting diastolic and systolic blood pressures. Electrocardiographic parameters of left ventricular and left atrial enlargement had a very poor correlation with resting and exercise blood pressure. Our findings suggest that early in life left ventricular mass and wall thickness are more closely related to maximal systolic blood pressure during physical exercise than to blood pressure in basal conditions. The electrocardiogram is an insensitive method to detect early modifications of left ventricular size in relation to different levels of blood pressure. The echocardiogram is the method of choice for this purpose.

Inhibitory effect of a toxic peptide isolated from a waterbloom of Microcystis sp. (Cyanobacteria) on iron uptake by rabbit reticulocytes.

The effect of a soluble toxin purified from the algae bloom of a eutrophic lake dominated by Microcystis on the receptor-mediated endocytosis of ferro-transferrin in rabbit reticulocytes was studied. The toxin was a very effective inhibitor of cell iron uptake. Kinetic studies using 125I, 59Fe-labeled transferrin indicated that the step of ferrotransferrin internalization was selectively inhibited by the toxin while the surface receptor-binding capacity, the externalization of previously internalized transferrin, and the cellular ATP levels were not affected. These findings indicate that the reduction of iron uptake caused by the toxin is due to inhibition of the internalization of surface-located transferrin-transferrin receptor complexes, perhaps due to a disruption of cytoskeleton integrity.

Sulphatide content in a membrane fraction isolated from rabbit gastric mucosal: its possible role in the enzyme involved in H+ pumping.

The sulphatide content of vesicular membrane fraction from rabbit mucosal gastric microsomes was analyzed. This vesicular membrane fraction, in addition to a high sulphatide content, was enriched in an ouabain-insensitive (H+ + K+)-ATPase, a (Mg+2 + K+)-activated phosphatase, and a H+ pumping activity. The enzyme system involved in the process of acid secretion and the translocation of K+ was studied in these membrane preparations treated with arylsulphatase A, an enzyme that specifically hydrolyzes sulphatide. The results indicate that the breakdown of sulphatides of the vesicular membrane fraction inactivated both the (H+ + K+)-ATPase activity and the H+ pumping. Both activities were partially restored by the sole addition of sulphatide. The K+-stimulated ouabain-insensitive phosphatase activity, suggested as a partial reaction of the (H+ + K+)-ATPase sequence, was unaffected by arylsulphatase. These results suggest that sulphatides may play a function in the high activity binding site for K+ of the enzyme involved in H+ pumping.

Adsorption of uni and divalent cations to planar bilayer membranes containing sulphatides or phosphatidylserine.

It has been postulated that sulphatides may be the K+ binding site of the sodium pump. In order to test this hypothesis we studied the binding of K+ to bilayer membranes containing sulphatides or phosphatidylserine. The adsorption constants of Na+, K+ and Ca2+ to planar bilayers containing these acidic lipids were determined from changes in the electrostatic potential at the membrane surface. Our results indicate that univalent cations adsorb weakly to both lipids and Ca2+ binds more strongly. The sequence of ion binding was Ca2+ greater than Na+ greater than K+. These results indicate that K+ does not bind specifically to sulphatides or phosphatidylserine and rule out the proposal that sulphatides by themselves provide the K+ binding site of the sodium pump.

Role of sulfatide on phosphoenzyme formation and ouabain binding of the (Na+ + K+)ATPase.

A microsomal fraction rich in (Na+ + K+)ATPase activity has been isolated from the outer medulla of pig kidney. The ability of this preparation to form phosphoenzyme on incubation with [gamma-32P]ATP and to bind [3H]ouabain was studied when its sulfatide was hydrolyzed by arylsulfatase treatment. The K+-dependent hydrolysis of the Na+-dependent phosphorylated intermediate as well as the ouabain binding were inactivated in direct relation to the breakdown of sulfatide. Both characteristics of the (Na+ + K+)ATPase preparation, lost by arylsulfatase treatment, were partially restored by the sole addition of sulfatide. These experiments indicate that sulfatide may play a role in sodium ion transport either in the conformational transition of the K+-insensitive phosphointermediate, E1P, to the K+-sensitive intermediate, E2P, or in the configuration of the high-affinity binding site for K+ of the E2P form. In addition, this glycolipid may have a specific role in the proteolipidic subunit that binds ouabain.

Possible role of sulphatide in the K+-activated phosphatase activity.

A microsomal fraction rich in (Na+ + K+)-ATPase has been isolated from the outer medulla of pig kidney. (Mg2+ + K+)-activated ouabain-sensitive phosphatase activity was studied in this preparation treated with arylsulphatase, an enzyme that specifically hydrolyzes ceramide galactose-3-sulphate. The activity of phosphatase was inactivated in proportion to the amount of sulphatide hydrolyzed. A maximum inactivation of ouabain-sensitive activity was obtained with 60% of the sulphatide content hydrolyzed. The inactivation caused by arylsulphatase was partially reversed by the sole addition of sulphatide. The evidence offered in this paper about sulphatide function in the sodium pump mechanism supports the idea that sulphatides are involved in the K+-activated phosphatase, a partial reaction of the (Na+ + K+)-ATPase.