Immunohistochemical Characterization of Tumor-Associated Macrophages in Canine Lymphomas.

Macrophages have been confirmed to play a significant role in the behavior of human lymphomas, albeit no consistent data are so far available in canine lymphomas. The present study characterizes the macrophages present in cases of canine nodal lymphoma and their relationship with the histological grade and the immunophenotype. Samples from the lymph nodes of 25 dogs diagnosed with lymphoma were selected. Immunohistochemistry was used to determine the tumor immunophenotype (CD3 and CD20 antibodies) and macrophage characterization (Iba1, MAC387, CD204, CD163 and iNOS antibodies). Macrophage counting was performed in 10 randomly selected, high-power fields per sample. Generalized linear models with Poisson distribution were used for statistical analysis. A significantly greater number of macrophages (Iba1+) were detected in high-grade and B-cell lymphomas. The highest amount of both M1 (iNOS+) and M2 (CD204+ and CD163+) subtypes were observed in B-cell lymphomas. High-grade lymphomas showed a greater number of CD204+ and CD163+ cells and recently recruited MAC387+ macrophages. The latter were most abundant in T than in B-cell lymphomas. In conclusion, a significant population of macrophages is present in canine lymphomas, which constitute a heterogeneous population that shows variations in the amount and immunohistochemical profile according to the histological grade and immunophenotype.

Case Report: Multiple Cavernous Pericardial Lymphangioma (Pericardial Lymphangiomatosis) in a Captive Peregrine Falcon (Falco peregrinus brookei).

A 12-year-old female peregrine falcon (Falco peregrinus brookei) from a private raptor breeding facility that presented a good body condition, died suddenly without showing previous symptoms. At necropsy, in the coelomic cavity, multiple cystic structures demarcated by a thin transparent wall and filled with a serous content were observed. They were firmly adhered to the cranial part of the epicardium and adjacent tissues and occupied the entire thoracic area of the coelomic cavity. Microscopically, emerging simultaneously from several areas the epicardium, multiple irregular channels and cystic spaces, lined by a single endothelial cell layer and separated by fibrovascular septa containing smooth muscle tissue, were observed. Immunohistochemical examination revealed that the neoplastic endothelial cells positively immunolabelled for the pan-endothelial marker factor VIII-related antigen but immunostained negative for cytokeratins (PCK26) while strong positivity for sarcomeric α-smooth muscle actin (α-SMA) was detected in the cystic walls. Based on the morphological and immunohistochemical findings, lesions were determined as consistent with a multiple cavernous pericardial lymphangioma, or pericardial lymphangiomatosis, a rare vascular neoplasm. The animal also showed a diffuse chronic perihepatitis, a necrotic area in the liver and foci of cartilaginous metaplasia and calcification in the aorta and vena cava. Literature review, particularly on the epidemiology of lymphangioma, demonstrated the rarity of this tumor in the different animal species and in this location, particularly in birds, being the first report of this type of tumor in a peregrine falcon.

Early response of monocyte-derived macrophages from vaccinated and non-vaccinated goats against in vitro infection with Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis is a disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Vaccination is the most cost-effective control method. However, despite the fact that macrophages are the main target cells for this pathogen, the precise mechanisms behind the response of the macrophage to Map infection and how it is modified by vaccination are yet poorly understood. The aim of this study was to investigate the effect of Silirum® vaccination in the early immune response of caprine monocyte-derived macrophages (CaMØs). Peripheral blood mononuclear cells (PBMCs) were obtained from vaccinated and non-vaccinated goats, cultured in vitro until differentiation to macrophages and infected with Map. After a 24 h incubation, Map viability and DNA were assessed in culture by viable colony count and real time quantitative polymerase chain reaction (qPCR). In addition, Map phagocytosis and expression of IL-10, IL-12, IFN-γ, TNF-α, IL-17A, IL-1ß, iNOS, IL-6 and MIP-1ß were also evaluated through immunofluorescence labelling and reverse transcriptase qPCR (RT-qPCR), respectively. A significant reduction of Map viability was observed in both supernatants (P < 0.05) and CaMØs (P < 0.001) from the vaccinated group. Similarly, the percentage of infected CaMØs and the number of internalized Map by CaMØs (P < 0.0001) was higher in the vaccinated group. Finally, iNOS (P < 0.01) and IL-10 were significantly up-regulated in CaMØs from vaccinated goats, whereas only MIP-1ß was up-regulated in non-vaccinated animals (P < 0.05). These results show that vaccination modifies the immune response of CaMØs, suggesting that the phagocytosis and microbiocidal activity of macrophages against Map is enhanced after vaccination.

Influence of Heterologous and Homologous Vaccines, and Their Components, on the Host Immune Response and Protection Against Experimental Caprine Paratuberculosis.

Vaccination against paratuberculosis, a chronic disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), has been considered as the most effective control method. However, protection is incomplete, and the mechanisms operating in the response of the animals to vaccination are not fully understood. Therefore, this study analyzed the immune response and the effects on protection against Map infection, elicited by paratuberculosis (Silirum®) and tuberculosis (heat-inactivated M. bovis [HIMB]) vaccines and their components in a caprine experimental model. Fifty goat kids were divided into 10 groups (n = 5) according to their vaccination (Silirum®, HIMB and nonvaccinated), immunization (inactivated bacteria or adjuvant), and/or infection. Oral challenge with Map was performed 45 days postvaccination/immunization (dpv), and animals were euthanized at 190 dpv. Peripheral immune response and proportion of lymphocyte subpopulations were assessed monthly by enzyme-linked immunosorbent assay and flow cytometry analysis, respectively. Local immune response, proportion of tissue lymphocyte subpopulations, Map detection (polymerase chain reaction), and histological examination were conducted in gut-associated lymphoid tissues. All infected groups developed paratuberculosis granulomatous lesions despite vaccination or immunization. The Silirum® and HIMB-vaccinated groups showed a considerable lesion reduction consistent with a significant peripheral cellular and humoral immune response. Besides, a lower number of granulomas were observed in groups immunized with inactivated bacteria and adjuvants in comparison to nonvaccinated and infected group. However, despite not being significant, this reduction was even higher in adjuvant immunized groups, which developed milder granulomatous lesion with no detectable peripheral immune responses associated with immunization. No changes in the peripheral and local proportion of lymphocyte subsets or local immune response were detected in relation to either vaccination/immunization or infection. Despite that paratuberculosis and tuberculosis vaccination showed a partial and cross-protection against Map infection, respectively, only histological examination could assess the progression of infection in these animals. In addition, the pattern observed in the reduction of the lesions in adjuvant immunized groups suggests the possible involvement of a nonspecific immune response that reduces the development of granulomatous lesions.

Optimized in vitro isolation of different subpopulation of immune cells from peripheral blood and comparative techniques for generation of monocyte-derived macrophages in small ruminants.

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.

Factores asociados a la donación voluntaria de sangre en internos de medicina de Perú / Factors associated with voluntary blood donation among medical interns in Peru

Introducción: Perú ocupa el penúltimo lugar en el número de transfusiones de unidades de sangre y glóbulos rojos por cada 1000 habitantes en 17 países de América. Objetivo: determinar la frecuencia en donación voluntaria de sangre y sus factores asociados en internos de medicina en 12 ciudades del Perú, 2016. Material y Métodos: estudio transversal analítico. La población de estudio fue 1229 internos de medicina de 35 hospitales en 12 ciudades. La muestra final estuvo compuesta por 637 (51,8 por ciento del total). Se utilizaron modelos de regresión logística de efectos mixtos para los conglomerados formados por el hospital en el que el estudiante realizaba el internado, se calcularon odds ratios ajustados (ORa) e intervalos de confianza al 95 por ciento. Resultados: Se encontró que 40,2 por ciento donó sangre voluntariamente al menos una vez y 7,1 por ciento al menos tres veces en su vida. En los análisis ajustados, tener más de 25 años en comparación con tener menos de 24 años (ORa: 2,00, IC 95 por ciento: 1,05 - 3,83) estuvo asociado a haber donado sangre alguna vez; en tanto que ser de sexo femenino (ORa: 0,15, IC 95 por ciento: 0,05 - 0,43) estuvo inversamente asociado a haber donado sangre al menos tres veces. Conclusión: Si bien cuatro de cada diez internos han donado sangre voluntariamente alguna vez, menos de uno de cada diez lo han hecho al menos tres veces. La mayor edad estuvo asociada a haber donado sangre voluntariamente alguna vez. Las mujeres tuvieron menor frecuencia de haberlo hecho al menos tres veces(AU)

Introduction: Peru ranks next to last in the number of transfusions of units of red blood cells per 1000 inhabitants in 17 countries of America. Objective: To determine the frequency of voluntary blood donation and its associated factors in medical interns in 12 cities of Peru, 2016. Material and Methods: An analytical cross-sectional study was conducted. The study population consisted of 1229 medical interns from 35 hospital sites distributed in 12 cities. The final sample was made up of 637 interns (51.8 percent of the total). To do this, mixed effect logistic regression models were used for the conglomerates formed by the hospital in which the student performed the internship. Adjusted odds ratios (ORa) and 95 percent confidence intervals were calculated. Results: The results show that 40.2 percent of medical interns donated blood voluntarily at least once in their lifetime and 7.1 percent did it at least three times in their lifetime. In the adjusted analyzes, being over 25 years old compared to be under 24 years old (ORa: 2.00, 95 percent CI: 1.05 - 3.83) was associated with history of one blood donation; while being female (ORa: 0.15, 95 percent CI: 0.05 - 0.43) was inversely associated with history of at least three blood donations. Conclusion: Although four in ten interns have donated blood at least once, less than one in ten have donated blood at least three times. The older age was associated with having donated blood voluntarily at least once. Women were less likely to have donated blood at least three times(AU)

Local assessment of WC1+ γδ T lymphocyte subset in the different types of lesions associated with bovine paratuberculosis.

The local expression of WC1+ γδ T lymphocytes subset has been evaluated by immunohistochemical methods at the different types of lesions present in cows naturally infected with Mycobacterium avium subsp. paratuberculosis (Map) and in non-infected control animals. Infected cattle were either in the latent/subclinical (focal lesions) or clinical (diffuse paucibacillary and multibacillary forms) stage of paratuberculosis. To assess the cell distribution, a differential cell count was carried out at the lamina propria, gut-associated lymphoid tissue and submucosa. A significant increase in the number of WC1+ γδ T cells was observed in all the infected animals, regardless of the type of lesion. Cows with focal lesions showed higher number of labeled cells than those with diffuse forms, where no differences were found between the two types. This increase in the number of positively immunolabelled lymphocytes in infected animals was seen in the lamina propria, with higher values in those with focal lesions. While in the lymphoid tissue no differences in the numbers were observed, in animals with focal lesions, WC1+ γδ T cells tended to be located at the periphery of the granulomas. These findings suggest a proinflammatory action of WC1+ γδ T lymphocytes in bovine paratuberculosis, which might play an important role in the containment of the Map-infection in the focal granulomas located in the lymphoid tissue, helping to prevent the progression toward diffuse forms responsible for the clinical signs.

Immunohistochemical characterization of tuberculous lesions in sheep naturally infected with Mycobacterium bovis.

BACKGROUND: Sheep have been traditionally considered as less susceptible to Mycobacterium bovis (Mbovis) infection than other domestic ruminants such as cattle and goats. However, there is increasing evidence for the role of this species as a domestic Mbovis reservoir, mostly when sheep share grazing fields with infected cattle and goats. Nevertheless, there is a lack of information about the pathogenesis and the immune response of Mbovis infection in sheep. The goals of this study were to characterize the granuloma stages produced by the natural infection of Mbovis in sheep, to compare them with other species and to identify possible differences in the sheep immune response. Samples from bronchial lymph nodes from twelve Mbovis-naturally infected sheep were used. Four immunohistochemical protocols for the specific detection of T-lymphocytes, B-lymphocytes, plasma cells and macrophages were performed to study the local immune reaction within the granulomas. RESULTS: Differences were observed in the predominant cell type present in each type of granuloma, as well as differences and similarities with the development of tuberculous granulomas in other species. Very low numbers of T-lymphocytes were observed in all granuloma types indicating that specific cellular immune response mediated by T-cells might not be of much importance in sheep in the early stages of infection, when macrophages are the predominant cell type within lesions. Plasma cells and mainly B lymphocytes increased considerably as the granuloma developed being attracted to the lesions in a shift towards a Th2 response against the increasing amounts of mycobacteria. Therefore, we have proposed that the granulomas could be defined as initial, developed and terminal. CONCLUSIONS: Results showed that the study of the lymphoid tissue granulomata reinforces the view that the three different types of granuloma represent stages of lesion progression and suggest an explanation to the higher resistance of sheep based on a higher effective innate immune response to control tuberculosis infection.