Low-density colloid centrifugation removes bacteria from boar semen doses after spiking with selected species.

Single-layer centrifugation (SLC) with a low-density colloid is an efficient method for removing contaminating microorganisms from boar semen while recovering most spermatozoa from the original sample. This study tested the performance of this technique, using 50-ml tubes, by spiking commercial semen doses prepared without antibiotics with selected bacterial species followed by storage at 17 °C. The doses were spiked up to 102/ml CFU (colony forming units) of the bacteria Burkholderia ambifaria, Pseudomonas aeruginosa, and Staphylococcus simulans. The semen was processed by SLC (15 ml of sample and 15 ml of colloid) with the colloid Porcicoll at 20% (P20) and 30% (P30), with a spiked control (CTL) and an unspiked control (CTL0), analyzing microbiology and sperm quality on days 0, 3 and 7. SLC completely removed B. ambifaria and S. simulans, considerably reducing P. aeruginosa and overall contamination (especially P30, ∼104 CFU/ml of total contamination on day 7, median). Sperm viability was lower in P20 and P30 samples at day 0, with higher cytoplasmic ROS. Still, results were similar in all groups on day 3 and reversed on day 7, indicating a protective effect of SLC (possibly directly by removal of damaged sperm and indirectly because of lower bacterial contamination). Sperm chromatin was affected by the treatment (lower DNA fragmentation and chromatin decondensation) and storage (higher overall condensation on day 7 as per chromomycin A3 and monobromobimane staining). In conclusion, SLC with low-density colloids can remove most bacteria in a controlled contamination design while potentially improving sperm quality and long-term storage at practical temperatures.

Bilateral neurosensory retinal detachment secondary to tadalafil treatment.

A 53-year-old male with no systemic disorders, other than controlled arterial hypertension, presented with asymptomatic, bilateral neurosensory retinal detachment (NRD) detected during a routine revision. The patient reported the use of tadalafil (a phosphodiesterase-5 inhibitor [PDE5I]) for erectile dysfunction. Following suspension of the drug, subretinal fluid reabsorption was confirmed, with the persistence of chronic alterations in the optical coherence tomography (OCT) and the visual field. PDE5Is have ocular side effects, including exudative retinal detachment. Although no direct causal relationship has been confirmed, PDE5 inhibition at chorioretinal level produces vasodilatation, increased choroid hydrostatic pressure, and exudation into the subretinal space. In cases of NRD, a thorough assessment of the drug treatment history is crucial. Patients who use PDE5I drugs should be alerted to their potential ocular side effects.

Low density Porcicoll separates spermatozoa from bacteria and retains sperm quality.

Antibiotics are added to semen extenders to control the growth of bacteria contaminating semen during collection but may contribute to the development of antibiotic resistance. An alternative would be physical separation of spermatozoa from bacteria. The objective of the present study was to evaluate two low densities of Porcicoll for removal of bacteria, and for their effect on sperm recovery and sperm quality. Semen was collected from boars at a commercial station. Aliquots of 8 extended ejaculates were subjected to colloid centrifugation through 20% Porcicoll (P20) and 30% Porcicoll (P30) in 500 mL tubes and then stored at 17 °C. Microbiological examination and sperm quality evaluation (computer assisted sperm analysis and flow cytometry) were carried out on controls and all colloid-selected samples immediately after preparation and again after storage for 3 and 7 days. The microorganisms found were mainly bacteria from the environment, gut or skin. There was a considerable reduction or complete removal of some bacteria by both colloids. Recovery rates were 86% for P20 and 81% for P30. Sperm quality was not adversely affected by colloid centrifugation on day 0, and thereafter showed a more gradual deterioration in colloid centrifuged samples than in controls, possibly due to lower bacterial contamination. There were no differences in sperm quality between the two colloid treatments. Thus, these results show that contaminating bacteria in semen can be controlled by centrifugation through low density colloids.

Detection of Natural Infection by Leishmania sp. in Bats (Chiroptera, Mammalia) that Inhabit the City of Corrientes, Argentina.

Leishmaniasis is a zoonotic disease of importance in Argentina. It is important to be acquainted with its possible natural hosts and reservoirs. Bats have several reservoir attributes, which means they could fulfill these roles. The aim of this work was to investigate the presence of Leishmania sp. in bats that inhabit the city of Corrientes. Bats were captured and identified. From the captured specimens, spleen samples were taken and Leishmania sp. detection techniques were applied, including both optical microscopy and PCR. It was possible to capture 95 bat specimens belonging to 10 species. Using cytological preparations, a prevalence of 1.29% was detected and using PCR a prevalence of 32% was found. Our results confirm the presence of natural infection by Leishmania sp. in bats of the city of Corrientes which reveals the possibility of these animal species fulfilling a reservoir role for this disease.

La leishmaniose est une zoonose importante en Argentine. La connaissance de ses réservoirs possibles est essentielle. Les chauves-souris ont plusieurs attributs pour remplir ce rôle. L'objectif de ce travail était d'étudier la présence de Leishmania sp. chez les chauves-souris qui habitent la ville de Corrientes. Des captures de chauves-souris ont été faites et celles-ci ont été identifiées. Des échantillons de rates ont été prélevés sur ces spécimens pour rechercher Leishmania sp. en utilisant la microscopie optique et la PCR. Il a été possible de capturer 95 spécimens de chauves-souris appartenant à dix espèces. En utilisant des préparations cytologiques, une prévalence de 1,29 % a été détectée et en utilisant la PCR, une prévalence de 32 % a été trouvée. Nos résultats confirment la présence d'une infection naturelle par Leishmania sp. chez les chauves-souris de la ville de Corrientes, ce qui révèle la possibilité que ces espèces animales jouent un rôle de réservoir pour cette maladie.

Removal of bacteria from boar semen using a low-density colloid.

Antibiotics are added to semen extenders when preparing commercial semen doses for artificial insemination according to national and international guidelines. However, this addition of antibiotics represents non-therapeutic usage and could be contributing to the development of antibiotic resistance. Colloid centrifugation was shown to reduce the load of bacteria present in boar semen and was capable of removing all bacteria if performed directly after semen collection, albeit with some loss of spermatozoa. The present experiment was conducted with a low density colloid to investigate whether it was possible to separate all of the spermatozoa from seminal plasma i.e. without selection for robust spermatozoa, or whether this would have a detrimental effect on sperm quality. Ejaculates from nine boars were extended in Beltsville Thawing Solution without antibiotics and were transported to the laboratory for Single Layer Centrifugation (SLC) on modified Porcicoll i.e. at a low density (S). A further modification was that a sterile inner tube was included inside some of the 50 mL centrifuge tubes to facilitate harvesting of the sperm pellet (M). Aliquots of all samples (control, S and M) were cultured for bacterial quantification and identification using standard microbiological methods. Sperm quality was evaluated daily. Three of the C and M samples and five of the S samples did not contain any bacteria. Mean bacterial counts for the remaining samples (colony forming units/mL) were as follows: C 259 ±â€¯216; S 30 ±â€¯22; M 33 ±â€¯15 (P < 0.01). Citrobacter spp., Staphylococcus simulans, Klebsiella variicola, Escherichia coli, Myroides odoratimimus, Proteus spp. and Enterococcus faecalis were identified in the control samples. There were marginal differences in sperm quality among treatments, with sperm velocity and linearity being higher in S and M samples than in C at all time points. However, sperm viability, capacitation and acrosome status were marginally better in controls than in S or M on day 0, but these differences disappeared during storage. Conclusions: centrifugation through a low density colloid can remove or reduce bacterial contamination in boar ejaculates without using antibiotics. Furthermore, it is possible to collect boar ejaculates without bacterial contamination by paying strict attention to hygiene.

Effect of Single Layer Centrifugation Porcicoll (70%, 80% and 90%) or supplementation with reduced glutathione, seminal plasma and bovine serum albumin on frozen-thawed boar sperm.

Selecting the optimal sperm population is essential for success with reproductive techniques. Porcicoll (formerly Androcoll-P) is a colloid formulation for selection of high-quality boar spermatozoa by single layer centrifugation (SLC). To date, most studies have been carried out with fresh semen and large volumes. We carried out 2 experiments to test the use of Porcicoll for thawed boar semen in small volumes. In Experiment 1, cryopreserved semen doses were thawed, split in 200-µL aliquots and layered on 1mL of Porcicoll 70%, 80% or 90%, or buffer without colloid. We assessed sperm recovery (the proportion of the loading dose that appeared in the pellet, %), and the physiology of the selected spermatozoa (flow cytometry: Viability, apoptotic changes, capacitation, mitochondrial activity, intracellular reactive oxygen species). The most suitable proportion was Porcicoll 80%, allowing acceptable sperm recovery (16.9±4.2%, compared to 70% (35.4%±3.0, p<0.001) and 90% (8.2%±3.0, P=0.001), and improved quality (mitochondrial activity: Porcicoll 80%: 77.7±1% vs Control: 60.3±0.7%, P<0.05). In Experiment 2, we compared 3 supplements to Porcicoll 80%: 500mM reduced glutathione (GSH), 20% seminal plasma (SP) and 0.5% bovine serum albumin (BSA). Supplementation with GSH or BSA did not cause relevant changes relative to Control. In contrast, SP induced membrane and acrosomal changes resembling capacitation, which might preclude its use in some applications, and decreased recovery (5.5%±1.9 vs. 24.3%±1.2 Control; P<0.001). However, it could be useful prior to other applications such as in vitro fertilisation. Overall, Porcicoll is an effective colloid for isolating a high-quality population from thawed boar sperm, 80% being a balanced option for good recovery and high quality. Supplements could be useful depending on the proposed use of the spermatozoa.

Total and mutated EGFR quantification in cell-free DNA from non-small cell lung cancer patients detects tumor heterogeneity and presents prognostic value.

Mutation analysis of epidermal growth factor receptor (EGFR) gene is essential for treatment selection in non-small cell lung cancer (NSCLC). Analysis is usually performed in tumor samples. We evaluated the clinical utility of EGFR analysis in plasma cell-free DNA (cfDNA) from patients under treatment with EGFR inhibitors. We selected 36 patients with NSCLC and EGFR-activating mutations. Blood samples were collected at baseline and during treatment with EGFR inhibitors. Wild-type EGFR, L858R, delE746-A750, and T790M mutations were quantified in cfDNA by droplet digital PCR. Stage IV patients had higher total circulating EGFR copy levels than stage I (3523 vs. 1003 copies/mL; p < 0.01). There was high agreement for activating mutations between baseline cfDNA and tumor samples, especially for L858R mutation (kappa index = 0.679; p = 0.001). In 34 % of advanced NSCLC patients, we detected mutations in cfDNA not previously detected in tumor samples and double mutations in 17 %. Patients with baseline total EGFR copy levels above the median presented decreased overall survival (OS) (341 vs. 870 days, p < 0.05) and progression-free survival (PFS) (238 vs. 783 days; p < 0.05) compared with those with total EGFR copy levels below the median. Patients with baseline concentrations of activating mutations above the median (94 copies/mL) had lower OS (317 vs. 805 days; p < 0.05) and PFS (195 vs. 724 days; p < 0.05). During follow-up, T790M resistance mutation was detected in 53 % of patients. Total and mutated EGFR analysis in cfDNA seems a relevant tool to characterize the molecular profile and prognosis of NSCLC patients harboring EGFR mutations.

Assessment of acrosome state in boar spermatozoa heads using n-contours descriptor and RLVQ.

This paper proposes a method for assessing the acrosome state of boar spermatozoa heads using digital image processing. We use gray level images in which spermatozoa have been labeled as acrosome-intact or acrosome damaged using the information of a coupled fluorescent image. The heads are segmented obtaining the outer head contour. A set of "n" inner contours separated by a logarithmic distance function is calculated later. For each point of the, in this case, seven contours a number of local texture features are computed. We have compared the classification performance of Relevance Learning Vector Quantization, class conditional means and KNN, employing cross-validation for the evaluation. Gradient magnitude data offer the best result with an overall test error of only 1%. This result outperforms previously applied methods and suggests this approach as an interesting automatized approach to this veterinarian problem.

Statistical approach to boar semen evaluation using intracellular intensity distribution of head images.

We propose a method for the classification of boar sperm heads based on their intracellular intensity distributions observed in microscopic images. The image pre-processing comprises segmentation of cell heads and normalization for brightness, contrast and size. Next, we define a model distribution of head intracellular intensity of an alive cell using a set of head images assumed to be alive by veterinary experts. We now consider two other sets of cell head images, one formed by heads assumed to be alive by experts and another formed by cells which present some abnormalities in their cytoplasm densities and are considered as dead by the experts. We define a measure of deviation from the model intensity distribution and for each head image of the two test sets we compute the deviation from the model. While the distributions of deviation values for alive and dead cells overlap, it is possible to choose an optimal value of a decision criterion for single cell classification in such a way that the error made in the estimation of the fraction of alive cells in a sample is minimal. In the range [0.7,1.0] of interesting values of the fraction of alive cells, the standard deviation of the fraction estimation error for samples of 100 head images is smaller than 0.04. Thus, in 95% of the cases the value of the fraction of alive cells in a sample estimated by a veterinary expert will be within 8% of the estimation made according to the proposed method. This result satisfies the requirements of veterinary practice.

Spontaneous resorption of a diabetic cataract in a geriatric dog.

Spontaneous cataract resorption is described in a geriatric cocker spaniel with a four-month history of diabetes mellitus. Resorption progressed to such a degree that vision was restored in that eye and almost all the cataract material disappeared. This is not common in geriatric dogs despite having been described with relative frequency in young animals with hereditary cataracts.

Does nitric oxide play a role in maternal tolerance towards the foetus?

In pregnancy there occurs maternal tolerance to the foetus. Several mechanisms have been proposed to explain this phenomenon. The main immune population in the decidua are macrophages and natural killer cells, but with some "special" suppressor characteristics. There is also a predominant TH2 response. The non classical MCH type I HLA-G is expressed by trophoblasts and can suppress lymphomononuclear cytotoxicity. Other system to avoid the immune system is the expression of indoleamine-2,3-dioxygenase, that suppresses T cell activation by degrading tryptophan. Even though in the placenta there is a high production of nitric oxide, a well-known immune modulator, low attention has been paid to its role in maternal tolerance. There are many data showing that NO affects the IDO, CD95/CD95-L and the balance between TH1/TH2. Maybe NO could interact with several mechanisms at the same time, which could modify the tolerogenic activity depending on the concentration and the presence of other factors in the medium.

Comparison of cyclosporin A and dexamethasone in the treatment of canine nictitans plasmacytic conjunctivitis.

Thirteen dogs with nictitans plasmocytic conjunctivitis were treated with 2.0 per cent cyclosporin drops in the right eye and with 0.1 per cent dexamethasone ointment in the left eye. The response to both therapies was monitored for six weeks, repeat biopsy specimens were taken, and the time for the clinical signs to recur recorded. Conjunctival cultures were taken before and after both therapies. There were no significant differences between the treatments in the remission of clinical signs, the reduction of inflammatory infiltrate in the biopsy specimens, or the time to recurrence of the condition or its subsequent severity. However, the eyes treated with 0.1 per cent dexamethasone tended to recover more rapidly than the eyes treated with 2.0 per cent cyclosporin, and the eyes treated with 2.0 per cent cyclosporin tended to be protected from a recurrence for longer than the eyes treated with 0.1 per cent dexamethasone.

Rapid screening of platelet donors for PlA1 (HPA-1a) alloantigen using a solid-phase microplate immunoassay.

PlA1 and PlA2 are alternative platelet-specific alloantigens in the PlA1 system. Sensitization to PlA1 underlies most cases of neonatal alloimmune thrombocytopenia (NAIT) and posttransfusion purpura (PTP) in white populations. A rapid and simple method for large-scale platelet phenotyping is desirable for identifying expectant mothers at risk of allosensitization and for identifying PlA1-negative donors when transfusions are indicated for treatment of NAIT or PTP. We investigated the effectiveness of a solid-phase microplate immunoassay for this purpose. Platelet-rich donor plasmas were tested using the Capture-P(R) kit (Immucor, Norcross, GA). Platelet monolayers in microtiter wells were incubated with anti-PlA1, washed, and exposed to red blood cells (RBCs) precoated with anti-human IgG. Adherence of RBCs in a diffuse pattern across the well surface indicated the attachment of anti-PlA1 to PlA1-positive platelets whereas sedimentation of unattached RBCs into a central pellet indicated the platelets were PlA1-negative. Of 520 donors, 15 (2.88%) tested PlA1-negative, which correlates well with the reported PlA2,2 frequency in whites of 2.25 percent. Results were confirmed by DNA genotyping and/or immunoblotting. This screening technique permits phenotyping donors for PlA1 alloantigen with minimal specialized equipment. Confirmatory testing for PlA2 alloantigen can be reserved for donors that test negative for PlA1.

Ischaemic renal injury.

Ischaemic renal injury is becoming one of the main causes of end-stage renal failure. The appearance of new diagnostic methods, as well as the evidence that surgery or percutaneous transluminal angioplasty techniques are capable of inducing a significant degree of recovery in a subset of patients, are encouraging events. The identification of the precise cellular pathogenetic mechanisms of ischaemic injury is an area for future research.

Clinical management of hyperglycaemic patients receiving total parenteral nutrition.

We evaluated the course of total parenteral nutrition (TPN) therapy in patients with hyperglycaemia. We studied 1) incidence of hyperglycaemia, 2) amount of glucose and insulin provided and 3) incidence of metabolic problems in patients receiving TPN who required insulin to attain metabolic control. The group included 91 patients, 38 women (64 +/- 15 years) and 53 men (64 +/- 12 years), who developed glycaemia higher than 200 mg/dl. Nine patients had a previous diagnosis of IDDM, 36 NIDDM and 46 secondary hyperglycaemia.. Total caloric requirements were initially supplied at 132 +/- 20% the basal energy expenditure (Harris-Benedict formula), and 1.4 +/- 0.3 g/kg of amino-acids. Initially, TPN provided 150-200 g/day of glucose (2.1 mg/kg/min). Regular insulin was added to the bag. The annual incidence of hyperglycaemia was estimated to be 121 per 1000 patients. Mean insulin requirements were 50 U/day (25 to 150 U/day), 0.7 +/- 0.3 U/kg. Comparing with prehospitalization insulin dose, 22% needed similar doses, and 11% lower doses. IDDM patients needed 1.7 times their pre-admission dose (1-4.5 times). The ratio of insulin:glucose in TPN was 0.3 +/- 0.1 U/g (0.1-1.2 U/g). Patients with renal failure had similar insulin requirements (56 +/- 26 U/day) than patients with normal renal function (49 +/- 19 U/day). None of the patients developed glycemic complications. In conclusion, diabetic patients receiving TPN have an acceptable metabolic control if individualized prescriptions and supplemental insulin are used.

[Influence of topical administration of diclofenac on the reduction of intraocular pressure induced by timolol in healthy subjects]. / Influence de l'administration topique de diclofénac sur la réduction de la pression intraoculaire produite par le timolol chez des sujets sains.

The presence or absence of an interaction between topical diclofenac (nonsteroidal antiinflammatory agent) and topical timolol (beta-blocker) was studied in 37 eyes. We instilled one drop of diclofenac several times into the patient's right eye and one drop of timolol into the same eye. We measured intraocular pressure in both eyes twice and compared the results in both eyes with the baseline intraocular pressure and with the pressure after administration of diclofenac alone and timolol alone. The joint use of both drugs did not modify the decrease in the ocular pressure induced by timolol. We can therefore use timolol and diclofenac together when a combination of antiinflammatory and beta-blocker agents is required.

Diclofenac sódico versus dexametasona en el pretratamiento de la trabeculoplastía con láser de argón / Diclofenac sodium vs dexametasone therapy before argon laser trabeculoplasty

Realizamos un estudio prospectivo, randomizado y doble ciego en 40 pacientes, para comparar la influencia que la administración tópica de un nuevo inhibidor de la síntesis de prostaglandinas -el diclofenac sódico al 0,1%- podría tener en el curso postoperatorio de ojos con glaucoma de ángulo abierto que fueron sometidos a una trabeculoplastia con láser de argón. No hallamos diferencias estadísticamente significativas entre los dos grupos en cuanto al incremento inmediato de la presión intraocular y la respuesta inflamatoria postoperatoria que siguieron a la laserterapia. Sin embargo, pensamos que el colirio de diclofenac sódico al 0,1% es útil como agente antiinflamatorio tópico después de la trabeculoplastia, por carecer de los efectos secundarios de los corticosteroides