Mouse breeding facilities in Argentina: Current state, challenges, and strengths in relation to animal welfare.

The science and technology of laboratory animals has come a long way worldwide, but for reasons related to the development of the countries, this journey started later in some Latin American countries, as is the case of Argentina. Without a specific legal framework to conduct animal experimentation, local strengths to promote animal welfare are based on professionals specifically trained in the care of laboratory animals as well as an extended network of ethics committees that ensures compliance with the ethical principles applied to animal experimentation. Nevertheless, there are no updated reports showing welfare indicators in rodent facilities. Therefore, we conducted a survey on mice breeding facilities enrolled in a national record elaborated by the National Ministry of Science. Questions related to four of the Five Domains Model of Mellor, concerning (1) nutrition, (2) physical environment, (3) health, and (4) behavioral interactions with the environment, other animals, and humans, were included as well as information concerning general aspects of the establishments. Data obtained from 25 mice breeder facilities localized all over the country were summarized, providing for the first time a clear picture of the national situation about the welfare of laboratory mice in these establishments. This data will be essential to design future policy as well as for deciding priorities aiming to improve the welfare of mice bred in Argentinian facilities.

Hemophagocytic lymphohistiocytosis as a presentation of T-cell lymphoma. / [Linfohistiocitosis hemofagocítica como presentación de Linfoma de células-T.]

Introduction: Hemophagocytic lymphohistiocytosis (HHL), a severe hyperinflammatory syndrome caused by aberrant activation of macrophages and cytotoxic T cells, is clinically manifested as a febrile onset along with cytopenias, high ferritin serum level and splenomegaly. In adult patients, secondary causes of it should be looked for, such as autoimmune and infectious diseases and neoplasms. Prompt initiation of treatment is important due to the high mortality of this syndrome. Methods: A case of a 53-year-old patient diagnosed with HHL associated with T-cell lymphoma is presented as a sudden onset of several symptoms and signs of HHL, along with infectious complications. Results: Results: Once the diagnosis of HHL was made, treatment with Etoposide and Dexamethasone was started, and with the diagnosis of T-cell lymphoma established, chemotherapy treatment with a CHOEP scheme was started, with a favorable initial evolution. Main conclusion: As HHL is a rare entity associated with high mortality, initial suspicion must be high when facing an acute onset of fever, cytopenia and splenomegaly. Nonetheless, diagnosing HHL is challenging and often appear superimposed on multiple infectious diseases. The early initiation of treatmbent is important given the high mortality of this pathology

Introducción: La linfohistiocitosis hemofagocítica (LHH) es un síndrome hiperinflamatorio severo causado por activación aberrante de macrófagos y células T citotóxicas que se manifiesta clínicamente como un cuadro febril asociado a citopenias, hiperferritininemia y esplenomegalia. Ante su diagnóstico en adultos se deben buscar causas secundarias como neoplasias hematológicas. El inicio precoz del tratamiento es importante debido a su elevada mortalidad. Métodos: Se presenta el caso de una paciente de 53 años de edad que se le realizó diagnóstico de LHH asociada a Linfoma de células T. La forma de presentación de dicho cuadro fue de manera abrupta con varios síntomas y signos de dicho síndrome, además de múltiples complicaciones infecciosas asociadas. Resultados: Ante el diagnóstico inicial de LHH se inició tratamiento con etopósido y dexametasona, y al obtener el diagnóstico de Linfoma de células T, se instauró tratamiento quimioterápico con esquema de CHOEP, con evolución inicial favorable. Conclusiones: La LHH es una entidad poco frecuente asociada a una elevada mortalidad, por lo que su índice de sospecha debe de ser elevado ante un cuadro clínico y analítico compatible. Sin embargo, su diagnóstico constituye un gran desafío clínico, pudiendo presentarse en algunas ocasiones superpuesto a cuadros infecciosos múltiples. El inicio precoz del tratamiento es importante debido a su alta mortalidad.

Comparison of serological techniques for the diagnosis of equine infectious Anemia in an endemic area of Argentina.

Equine Infectious Anemia is a transmissible viral disease present worldwide, caused by an RNA virus. Viral transmission is mainly mechanical through blood or its products most frequently by blood-sucking arthropods and iatrogenesis as well. OIE recommends Coggins Test as the diagnostic method of choice. Some ELISA tests detect antibodies earlier than the Coggins test, but may produce false-positive results. Currently, new techniques for EIA diagnosis are being developed, such as fluorescence polarization assay which is a simple method for measuring antigen-antibody interaction. The aim of this study was to assess cELISA and Fluorescence Polarization Assay performance for the serological diagnosis of EIA by comparing their results with those of the Coggins test. Tests were performed on 91 workhorses from an endemic zone in the northeast region of Argentina. From the total samples analyzed, 42 tested negative and 49 tested positive in the Coggins test. Same results were obtained using FPA. Using the cELISA, 41 negative results and 50 positive results were obtained. The Receiver Operating Characteristic analysis showed that FPA performance was excellent. Therefore FPA is proposed as an outstanding EIA diagnostic test to be validated in the near future by its simplicity, speed, and objective interpretation of results.

Lithium in drinking water, altitude, and suicide rates in rural areas of Argentinean Andes.

The Lithium Triangle in the Andean plateau involves high altitude (>3,000 m asl) hydrological systems having high lithium graded waters. This research was carried-out in rural areas of north westernmost Argentinean Andes and was aimed: 1) to determine concentrations of lithium in drinking waters; 2) to calculate suicide mortality rates based on available official data (2003-2013); 3) to analyze bivariate differences between lithium concentrations in drinking water, mean rates of suicide mortality, altitude of sampling sites, and water sources; 4) to analyze bivariate correlations between lithium concentrations in drinking water, mean rates of suicide mortality, and altitude; 5) to test predictive models for mean rates of suicide mortality, when considering the predictors lithium concentrations in drinking water, altitude, and water sources. Lithium determinations in drinking waters were performed by Microwave Plasma-Atomic Emission Spectrometer. Nonparametric tests were applied to analyze differences and correlations. Generalized linear models (GLM) were used to fitting models for mean rates of suicide. Drinking waters contained up to 2.98 mg L-1 of lithium. Mean rates of suicide mortality (per 100,000 inhabitants) were high, ranging from 19.12 (± 19.83) to 30.22 (± 16.70). Lithium but not altitude was positively correlated with suicide mortality when analyzing bivariate correlations (Li: rho = 0.76, p-value < 0.001). However, when GLM were calculated, a significant interaction effect was found between lithium and altitude (p-value < 0.001). This interaction effect would act in some way restraining the suicide mortality rates.

Genetic Characterization of Mangalarga Marchador Breed Horses Based on Microsatellite Molecular Markers.

The Mangalarga Marchador (MM) horse breed has expressive importance in the Brazilian economy. Thus, the aim of this study was to investigate diversity in the MM breed. A database with a total of 3,193 genotyped horses was used (MM, n = 2,829; Andalusian - AND, n = 67; Pure Blood Lusitano - LUS, n = 43; English Thoroughbred - THO, n = 54; Arabian - ARA, n = 99; Campolina - CAM, n = 61; and Mangalarga - MAN, n = 40) for 13 microsatellite. Diversity parameters were estimates, such as mean number of alleles (Nma) and the number of rare alleles (AR), expected heterozygosity (He), F statistics, genetic distances, Hardy-Weinberg equilibrium test (HWE), population structure, and others. The Nma was 10.85, the AR was prevalent in the MM, and the He was 0.7402. In MM, the values of Fis (-0.0195), Fit (0.0566), Fst (0.0748), and deviations of HWE were observed. The genetic distances of the ARA and THO breeds with the other breeds were greater than the distances between the Brazilian breeds and between these and the breeds in the Iberian Peninsula. The population structure indicated that MM was substructured, yet there were some more genetically defined breeding farms. The genetic diversity is satisfactory for MM conservation, but the population is substructure, and parameters indicate moderate gene flow and the existence, though few, of crosses with other horse breeds. Immediate implementation of a genetic breeding program is required, especially seeking to conserve the structure of the MM breed as a well-defined genetic entity.

Neurochemical, Behavioral, and Neurogenic Validation of a Hyposerotonergic Animal Model by Voluntary Oral Consumption of para-Chlorophenylalanine.

The inhibitor of tryptophan hydroxylase, para-chlorophenylalanine (PCPA), has been classically employed as a pharmacological tool to deplete serotonin (5-HT) in animal models and to evaluate whether this neurotransmitter is involved in the action of pharmacological compounds. PCPA is usually administrated by intraperitoneal (ip) injections, which are stressful and painful. To avoid ip injections, we designed and validated a protocol for PCPA oral administration. C57BL/6 elite male mice received PCPA during 7 days either ip or by giving the drug inside jelly cubes at an estimated dose of 500 mg/kg on days 1 and 2 and 250 mg/kg for the rest of the treatment. 5-HT levels decreased by 85% and 55% in the hippocampus of mice treated with oral or ip PCPA, respectively, whereas in the prefrontal cortex, 5-HT levels decreased by 65% (oral) and 50% (ip). Behavioral tests, like the forced swimming test (FST), the nestlet shredding test (NST), and the marble burying test (MBT), were performed. In the FST, mice received fluoxetine ip 30 min before the test. In mice with oral PCPA treatment, fluoxetine did not induce significant reductions of immobility, indicating that reduction of 5-HT levels was effective. No effect of ip or oral 5-HT depletion was observed in the NST nor in the MBT. In a second experiment, mice received oral PCPA for 8 weeks: again, serotonin levels were significantly decreased in both hippocampus and cortex, and effects on hippocampal neurogenesis replicated previous observations in hyposerotonergic mice. Therefore, neurochemical, behavioral, and neurogenic results allow us to validate this refined protocol for voluntary oral consumption of PCPA.

Reproductive hormones influence zinc homeostasis in the bovine cumulus-oocyte complex: Impact on intracellular zinc concentration and transporters gene expression.

Zinc (Zn) is a vital trace element for the body and its bioavailability influences numerous reproductive events. However, the mechanisms that regulate Zn homeostasis in the cumulus-oocyte complex (COC) are yet to be elucidated. The aim of this study was to investigate the role of estradiol 17-beta (E2), FSH and LH in Zn homeostasis regulation in bovine COC matured in vitro and Zn transporters gene expression. For this purpose, intracellular Zn levels in oocytes and cumulus cells (CC) were assessed using a Zn-specific fluorescent indicator. In addition, gene expression and sequencing of six Zn transporters (Slc39a6, Slc39a8, Slc39a14, Slc30a3, Slc30a7 and Slc30a9) were assessed. Our results demonstrated that the simultaneous presence of E2, FSH, and LH during oocyte maturation altered intracellular zinc levels and transporters expression in both oocytes and CC. Transporter's gene expression was different in oocytes and CC, possibly due to cell-specific changes in Zn levels during maturation. The interaction effects of Zn with hormonal treatments influenced the results. This study emphasizes that Slc39a6 is highly sensitive to hormone induction. Overall, the hormonal modulation of Zn homeostasis in the COC was evidenced. Also, a preponderant role of FSH as a modulator of Zn intracellular levels and transporter gene expression is suggested.

Conducting and reporting animal experimentation: Quo vadis?

Most scientific journals ask authors to include a statement in their articles that animal studies have been carried out in agreement with international regulations on the use and care of laboratory animals. This statement implies that all the experiments conducted on animals have been evaluated and accepted by an Ethical Committee and, that animal welfare has been put as a priority throughout the experimental protocol. Nevertheless, discrepancies are commonly found between the described procedures and the guidelines that are claimed to have been followed; this reveals a double dilemma. First, animal welfare is not always considered, implicating discomfort or even worse, suffering to animals involved. Secondly, revisions of manuscripts are sometimes done without taking into account ethical and regulatory aspects concerning the use of animals. Underestimation of pain or suffering, disregard for physiological parameters, and other examples recently reported in scientific journals by neuroscientists from all over the world are discussed in this article. In a period of great debate about the ethical use of animals, with society being involved and engaged in the discussion, this Neuro-Opinion intends to call the attention of researchers, ethical committee members, and journal editors about the need of strictly endorsing international regulations and placing animal welfare as the top priority.

Differential Hippocampal Expression of BDNF Isoforms and Their Receptors Under Diverse Configurations of the Serotonergic System in a Mice Model of Increased Neuronal Survival.

Neurotrophic factors are relevant regulators of the neurogenic process at different levels. In particular, the brain-derived neurotrophic factor, BDNF, is highly expressed in the hippocampus (HC) of rodents and participates in the control of neuronal proliferation, and survival in the dentate gyrus (DG). Likewise, serotonin is also involved in the regulation of neurogenesis, though its role is apparently more complex. Indeed, both enhancement of serotonin neurotransmission as well as serotonin depletion, paradoxically increase neuronal survival in the HC of mice. In this study, we analyzed the protein expression of the BDNF isoforms, i.e., pro- and mature-BDNF, and their respective receptors p75 and TrkB, in the HC of mice chronically treated with para-chloro-phenyl-alanine (PCPA), an inhibitor of serotonin synthesis. The same analysis was conducted in hyposerotonergic mice with concomitant administration of the 5-HT1 A receptor agonist, 8-Hydroxy-2-(di-n- propylamino) tetralin (8-OH-DPAT). Increased expression of p75 receptor with decreased expression of pro-BDNF was observed after chronic PCPA. Seven-day treatment with 8-OH-DPAT reestablished the expression of pro-BDNF modified by PCPA, and induced an increase in the expression of p75 receptor. It has been demonstrated that PCPA-treated mice have higher number of immature neurons in the HC. Given that immature neurons participate in the pattern separation process, the object pattern separation test was conducted. A better performance of hyposerotonergic mice was not confirmed in this assay. Altogether, our results show that molecules in the BDNF signaling pathway are differentially expressed under diverse configurations of the serotonergic system, allowing for fine-tuning of the neurogenic process.

Genomic structural diversity in Criollo Argentino horses: Analysis of copy number variations.

Copy number variation (CNV) has been proved to be widespread in human, animal and plant genomes. Together with single nucleotide polymorphisms (SNPs), CVNs play a key role in genetic diversity. In this study, genome-wide detection of CNVs was performed based on SNP data from 24 Criollo Argentino horses genotyped with the GGP Equine70k array. Overall, 165 CNVs meeting stringent quality control criteria were identified and then aggregated into 87 CNV regions (CNVRs), representing a horse genome coverage of 13.69 Mb. Functional analysis of CNVRs allowed the identification of 337 genes implicated in a wide range of biological functions such as signal transducer activity (olfactory receptors), receptor activities and binding. Furthermore, enrichment analysis showed that the most represented protein classes (over 25%) were immunoglobulin receptor subfamily, immunoglobulins and major histocompatibility complex antigen (beta-2-microglobulin). To the best of our knowledge, this is the first report of CNV in Criollo Argentino horses.

Non-aversive photographic measurement method for subcutaneous tumours in nude mice.

We have developed a new method for the measurement of subcutaneous tumour volume which consists in taking photographs of mice in their home cages, to refine the standard method of measurement with calipers. We consider this new method to be non-aversive, as it may be more compatible with mice behavioural preferences and, therefore, improve their welfare. Photographs are captured when mice voluntarily go into an acrylic tube containing graph paper that is later used as a scale. Tumour volumes measured with the caliper and the non-aversive photographic method were compared to those obtained by water displacement volume and weight. Behavioural and physiological changes were evaluated to assess animal welfare. Significant differences were found between measurements obtained with the caliper and the non-aversive photographic method, v. the reference volume acquired by water displacement (P < 0.001). Nevertheless, there was good consistency for these measurements when tumours were measured repeatedly, with all Intra-Class Correlation Coefficients above 0.95. Mice on which the non-aversive photographic method was employed were significantly less reluctant to establish contact with the experimenter (P < 0.001) and behaved less anxiously in a modified-Novelty Suppressed Feeding test. Particularly, statistically significant differences were found in connection with the latency to eat an almond piece (P < 0.05), the frequency of grooming (P < 0.001) and the frequency of defecation (P < 0.001). Corticosterone concentration in faeces and blood glucose were determined and no significant changes were found. Therefore, we propose the non-aversive photographic method to measure subcutaneous tumours as a way to refine methodologies in the field of experimental oncology.

Estimación de riesgo carcinógeno por exposición crónica al arsénico a través del agua de consumo en la Puna, Jujuy / Estimation of Carcinogenic Risk due to Chronic Exposure to Arsenic via Water for Human Consumption in Puna, Jujuy

INTRODUCCIÓN: La ingesta crónica de arsénico (As) se asocia con cáncer. Se sabe que las cuencas hídricas de la Puna de Jujuy contienen As y que muchos hogares no tienen agua potable de red, pero aún no se había estimado el riesgo carcinógeno para los pobladores por ingerir el agua local. OBJETIVOS: Estimar el nivel de riesgo carcinógeno para los pobladores locales debido a exposición crónica al As a través del agua de bebida. MÉTODOS: Se determinaron las concentraciones de As en muestras de agua de consumo de poblados de Cochinoca, Susques y Tumbaya con espectroscopía de emisión atómica de plasma acoplado inductivamente. Se calculó el riesgo carcinógeno con modelos matemáticos de la United States Environmental Protection Agency. RESULTADOS: Se halló As en todas las muestras (rango 0,041-0,34 mg/l/As), y el 83% superó el máximo permitido para agua potable (0,05 mg/l/As). Según las concentraciones medias de As por departamento, el riesgo carcinógeno para los pobladores estuvo entre 2,44 x 10-3 y 5,89 x 10-3. El riesgo carcinógeno para quienes consumen agua potable de red fue de 2,36 x 10-3 y para quienes consumen agua de otras procedencias, de 4,76 x 10-3. Todos los valores hallados superaron el máximo de aceptabilidad del riesgo asociado a la exposición a un carcinógeno (10-5). CONCLUSIONES: Es necesario implementar programas de comunicación de riesgos y políticas en salud para disminuir riesgos debidos a ingesta de agua con contenidos arsenicales en esta región.

BDNF isoforms: a round trip ticket between neurogenesis and serotonin?

The brain-derived neurotrophic factor, BDNF, was discovered more than 30 years ago and, like other members of the neurotrophin family, this neuropeptide is synthetized as a proneurotrophin, the pro-BDNF, which is further cleaved to yield mature BDNF. The myriad of actions of these two BDNF isoforms in the central nervous system is constantly increasing and requires the development of sophisticated tools and animal models to refine our understanding. This review is focused on BDNF isoforms, their participation in the process of neurogenesis taking place in the hippocampus of adult mammals, and the modulation of their expression by serotonergic agents. Interestingly, around this triumvirate of BDNF, serotonin, and neurogenesis, a series of recent research has emerged with apparently counterintuitive results. This calls for an exhaustive analysis of the data published so far and encourages thorough work in the quest for new hypotheses in the field. BDNF is synthetized as a pre-proneurotrophin. After removal of the pre-region, proBDNF can be cleaved by intracellular or extracellular proteases. Mature BDNF can bind TrkB receptors, promoting their homodimerization and intracellular phosphorylation. Phosphorylated-TrkB can activate three different signaling pathways. Whereas G-protein-coupled receptors can transactivate TrkB receptors, truncated forms can inhibit mBDNF signaling. Pro-BDNF binds p75(NTR) by its mature domain, whereas the pro-region binds co-receptors.

Autoinmunidad contra la célula beta en el adulto mayor con diabetes tipo 2: impacto clínico, metabólico y terapéutico / Beta cell autoimmunity in elederly with type 2 diabetes: clinical, metabolic and therapeutic impact

Introducción: se estudiaron 111 pacientes con diabetes diagnosticada después de los 65 ó más años de edad no obesos (IMC<30 kg/m2). Materiales y métodos: se determinaron cuatro anticuerpos marcadores de autoinmunidad para células ß pancreáticas: GADA, ZnT8, IA2A y PAA. Se midieron variables metabólicas, bioquímicas, antropométricas, clínicas y terapéuticas. Resultados: el 32% de los pacientes presentó autoanticuerpos. El autoanticuerpo más hallado fue el PAA 58,3%, seguido del GADA 30,5%, el ZnT8 27,7% y el IA2A 5,5%. Los pacientes con anticuerpos presentaron mayor circunferencia de cintura, insulinorresistencia (HOMA-IR), secreción de insulina (HOMAß) y complicaciones microangiopáticas. Entre ellos también fue más común que requirieran de insulina pero la diferencia no fue significativa. Conclusiones: estos resultados son diferentes de los que se esperaría tanto en diabéticos 2 como en diabéticos LADA más jóvenes, lo que sugiere que esta población tendría rasgos fenotípicos particulares y merece ser analizada con mayor extensión y profundidad. Proponemos la denominación LADE (latent autoimmune diabetes in elderly) para estos pacientes mayores de 65 años no obesos DM2 Au+

Development of a peptide ELISA for the diagnosis of Equine arteritis virus.

A peptide-based indirect ELISA was developed to detect antibodies against Equine arteritis virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein M were designed, synthesized, purified and used as antigens either alone or combined. Ninety-two serum samples obtained from the 2010 Equine viral arteritis outbreak, analyzed previously by virus neutralization, were evaluated by the ELISA here developed. The best resolution was obtained using peptide GP5. The analysis of the inter- and intraplate variability showed that the assay was robust. The results allow concluding that this peptide-based ELISA is a good alternative to the OIE-prescribed virus neutralization test because it can be standardized between laboratories, can serve as rapid screening, can improve the speed of diagnosis of EAV-negative horses and can be particularly useful for routine surveillance in large populations.

Equine arteritis virus gP5 protein induces apoptosis in cultured insect cells.

Equine Arteritis Virus (EAV) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. In this work, we found a cytotoxicity effect of the EAV gP5 protein on baculovirus-insect cells and a low yield of protein recovery. Besides, different morphological features by electron transmission microscopy, DNA fragmentation in agarose gel, TUNEL analysis and caspase 3 activity were found. All these findings indicate that the EAV gP5 protein induces apoptosis in insect cells.

A study of the association between chronic superficial keratitis and polymorphisms in the upstream regulatory regions of DLA-DRB1, DLA-DQB1 and DLA-DQA1.

Canine chronic superficial keratitis (CSK) is an inflammatory corneal disease that primarily occurs in German shepherd dogs (GSDs). Several studies support the hypothesis that CSK is an immune-mediated disease. To investigate the genetic factors associated with CSK development, the upstream regulatory regions (URRs) of the DLA-DRB, -DQA and -DQB genes were genotyped in 60 dogs, including 32 CSK animals. LD analysis identified two blocks (r(2)≤45), with two DLA-DRB1 and five DLA-DQB1 haplotypes. Analysis of DLA-URR alleles/haplotypes showed a significant association between DQB1*-154 [C/T] (p=0.016) and CSK, suggesting that the T variant may increase the risk for developing CSK disease (OR=3, 95% CI=1.25-7.68). When haplotype associations were performed, the URR-DQB*CATT haplotype was significantly associated with CSK (p=0.016), increasing the risk of develop this disease over two-fold (OR=3, 95%, CI=1.25-7.68). These results showed that dogs homozygous at DRB1*69 [C/T] had a risk for developing CSK disease that was over four times the risk for heterozygotes. This genetic association supports the previous clinical, histological and pharmacological studies that suggest that CSK is an immune-mediated disease, and this association could potentially be used to identify susceptible animals.

Substitution of human for horse urine disproves an accusation of doping*.

In order to detect switching and/or manipulation of samples, the owner of a stallion asked our lab to perform a DNA test on a positive doping urine sample. The objective was to compare the urine DNA profile versus blood and hair DNA profiles from the same stallion. At first, 10 microsatellite markers were investigated to determine the horse identity. No results were obtained when horse specific markers were typed in the urine sample. In order to confirm the species origin of this sample we analyzed the mitochondrial cytochrome b gene. This analysis from blood and hair samples produced reproducible and clear PCR-RFLP patterns and DNA sequence match with those expected for horse, while the urine sample results were coincident with human. These results allowed us to exclude the urine sample from the questioned stallion and determine its human species origin, confirming the manipulation of urine sample.