BAFF promotes heightened BCR responsiveness and manifestations of chronic GVHD after allogeneic stem cell transplantation.

Patients with chronic graft-versus-host disease (cGVHD) have increased B cell-activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major histocompatibility complex-mismatched model with cGVHD-like manifestations, we first examined B-lymphopenic µMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B-cell number. Mice that later developed cGVHD had significantly increased numbers of recipient fibroblastic reticular cells with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD, because BAFF transcript in CD4+ T cells from diseased mice and patients was increased. cGVHD manifestations in mice were associated with high BAFF/B-cell ratios and persistence of B-cell receptor (BCR)-activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR responsiveness to surrogate antigen and NOTCH ligand. BAFF Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR activation or when alloantigen was present in vivo. Using T cell-depleted (BM only) BAFF Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells, and alloantibody production. We demonstrate that pathologic production of BAFF promotes an altered B-cell compartment and augments BCR responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in patients with cGVHD.

Insect Immune Evasion by Dauer and Nondauer Entomopathogenic Nematodes.

The immune response of animals, including insects, is overcome by some parasites. For example, dauer larvae (DL) of the obligate entomopathogenic nematodes (EPNs) Heterorhabditis and Steinernema can invade insects, evade their defenses, and cause death. Although DL were long assumed to be the only infective stage of nematodes, recent reports suggest that L2-L3 larvae of facultative EPNs are also capable of killing insects. There are no studies, to our knowledge, about the role of nonimmunological barriers (the exoskeleton and its openings) in avoiding infection by DL and L2-L3 larvae, or whether these larval stages evade the host immune system in the same way. The objective of this study was to examine these questions by infecting Galleria mellonella with the facultative parasitic nematode Rhabditis regina. DL or L2-L3 larvae were either deposited on or near the moths or injected into their hemocoel. Once nematodes reached the hemocoel, the following host immune response parameters were quantified: prophenoloxidase, phenoloxidase, lytic activity, and the number of granular hemocytes. DL showed a greater ability to penetrate the exoskeleton than L2-L3 larvae. Once inside, however, both went unnoticed by the immune system and killed the insect. A higher number of granular hemocytes was activated by L2-L3 larvae than DL. We show for the first time that L2-L3 larvae can penetrate and evade the insect immune system. Further research is needed to compare facultative and specialized EPNs to determine which is more likely, with both DL and L2-L3 larvae, to evade insect defense barriers and produce death. The results will contribute to understanding the evolution of virulence in entomopathogenic nematodes.

Lactobacillus pentosus ABHEAU-05: An in vitro digestión resistant lactic acid bacterium isolated from a traditional fermented Mexican beverage / Lactobacillus pentosus ABHEAU-05: una bacteria ácido láctica resistente a la digestión in vitro aislada de una bebida fermentada tradicional mexicana

Abstract The health benefits attributed to probiotics generate interest in the search of competent strains adapted to several ecological niches, especially those related to traditional beverages and foods of each country. Pineapple tepache, a traditional Mexican fermented beverage, was used for the isolation of lactic acid bacteria with probiotic potential, one of which withstood the in vitro tests. The isolated strain AB-05, which exhibited the tested probiotic functional properties, was designated as Lactobacillus pentosus ABHEAU-05. The sequence was registered in GenBank under access code MK587617. This study is the first report of a lactic acid bacterium with in vitro digestion resistance isolated from pineapple tepache. The survival of L. pentosus ABHEAU-05 in a symbiotic medium was proven using fermented milk enriched with inulin. The in vitro digestion-resistant probiotic activity of lactobacilli was measured through analysis of pH and proteolysis. Results showed that L. pentosus grew properly in fermented milk; therefore, this microorganism could be used in the manufacture of this kind of products. The concentration of L. pentosus reached up to 8.5 logCFU/ml after 40 h of fermentation. In addition, the production of peptides and the decrease in pH indicated the vigorous and active metabolic state of the lactic acid bacterium tested. The activity and the concentration of this microorganism were maintained during refrigeration. The results of this research conclude that L. plantarum ABHEAU-05 is an in vitro digestion-resistant microorganism that can be used as a starter culture for the production of functional foods of dairy origin.

Resumen Los beneficios a la salud atribuidos a los probióticos generan interés en la búsqueda de cepas competentes adaptadas a varios nichos ecológicos, especialmente los relacionados con bebidas y alimentos tradicionales de cada país. En este estudio, se aisló del tepache de pina, una bebida fermentada tradicional mexicana, una bacteria láctica resistente a la digestión in vitro. Entre 5 bacterias aisladas, una de ellas soportó las pruebas simuladas de digestión gastrointestinal. Se analizó la resistencia a sales biliares, a condiciones ácidas y al ataque enz-imático con pepsina. La bacteria aislada, que exhibió las propiedades funcionales probióticas referidas, fue identificada como Lactobacillus pentosus y designada como L. pentosus ABHEAU-05. La secuencia fue depositada en GenBank (acceso MK587617). Se comprobó la supervivencia de L. pentosus ABHEAU-05 en una leche fermentada adicionada con inulina y su resistencia a la digestión in vitro mediante el análisis del pH y la proteólisis. Los resultados muestran que la leche fermentada es una matriz adecuada, donde L. pentosus ABHEAU-05 se desarrolla sin inconvenientes, alcanzando un título de 8,5 logufc/ml después de 40 h de fermentación. Además, la producción de péptidos y el descenso del pH indicaron el estado metabólico vigoroso y activo del microorganismo probiótico. Se concluye que L. pentosus ABHEAU-05 es un microorganismo resistente a la digestión in vitro, que puede servir como cultivo iniciador para la producción de alimentos de origen lácteo. Este es el primer informe acerca del aislamiento de una bacteria ácido láctica resistente a la digestión in vitro a partir del tepache de piña.

Fate Mapping In Vivo to Distinguish Bona Fide Microglia Versus Recruited Monocyte-Derived Macrophages in Retinal Disease.

With the new understanding that adult microglia in mice have embryonic origins and are maintained in situ throughout life, it has become pertinent to now understand how these unique cells differ from monocyte-derived macrophages. The latter are recruited into the neural retina (and elsewhere in CNS) in certain diseased states, such as in various forms of retinal degeneration. However, phenotypic markers expressed by microglia and monocyte-derived macrophages largely overlap, thereby making it technically challenging to distinguish the two cell types in disease. To address this problem in mice, we have established an in vivo fate mapping system that enables distinguishing these two cell types in retinal disease models. Our approach leverages the seminal work that originally developed Cx3cr1-CreER mice and is based on commercially available mouse strains. Here, we detail our protocol and how to apply this fate mapping method paired with flow cytometry (or immunohistochemistry) to faithfully distinguish and examine microglia vs. monocyte-derived macrophages in a mutually exclusive manner. This approach will henceforth empower new efforts to identify functional specializations of these two populations in the pathobiology of retinal degenerative diseases and possibly other conditions of the retina where monocyte recruitment is observed, such as in glaucoma, diabetic retinopathy, ischemia reperfusion, retinal detachment, and so on.

SYK inhibitor entospletinib prevents ocular and skin GVHD in mice.

Graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HCT). The tyrosine kinase SYK contributes to both acute and chronic GVHD development, making it an attractive target for GVHD prevention. Entospletinib (ENTO) is a second-generation highly selective SYK inhibitor with a high safety profile. Potential utility of ENTO as GVHD prophylaxis in patients was examined using a preclinical mouse model of eye and skin GVHD and ENTO-compounded chow. We found that early SYK inhibition improved blood immune cell reconstitution in GVHD mice and prolonged survival, with 60% of mice surviving to day +120 compared with 10% of mice treated with placebo. Compared with mice receiving placebo, mice receiving ENTO had dramatic improvements in clinical eye scores, alopecia scores, and skin scores. Infiltrating SYK+ cells expressing B220 or F4/80, resembling SYK+ cells found in lichenoid skin lesions of chronic GVHD patients, were abundant in the skin of placebo mice but were rare in ENTO-treated mice. Thus, ENTO given early after HCT safely prevented GVHD.

Intoxicación letal con aldicarb: análisis de sangre post mortem mediante LC-ESI-MS/MS / Fatal intoxication with aldicarb: Analysis in post mortem blood by LC-ESI-MS/MS

Antecedentes. El aldicarb es un plaguicida carbamato de alta toxicidad asociado a intoxicaciones agudas fatales en el ser humano. Su mecanismo de acción consiste en la inhibición de la enzima acetilcolinesterasa (AChE) que ocasiona la acumulación del neurotransmisor acetilcolina en la hendidura sináptica. Esta acumulación provoca síntomas colinérgicos y, dependiendo de la dosis de exposición, puede paralizar los sistemas respiratorio y nervioso hasta llegar a la muerte. Objetivo. Determinar el nivel de aldicarb en sangre post mortem en casos de intoxicación aguda. Materiales y métodos. Investigación de tipo experimental empleando un cromatógrafo líquido con espectrometría de masas, con ionización electrospray y análisis en modo tándem (LC-ESI-MS/MS). Los estándares de aldicarb y el aldicarb-d3 fueron comprados de Dr. Ehrenstorfer GmbH. El método consiste en una precipitación de proteínas de la sangre y su posterior análisis por LC-ESI-MS/MS, utilizando el aldicarb-d3 como estándar interno. El método fue aplicado a siete casos de intoxicación letal por presunta acción del aldicarb. Resultados. El aldicarb se encontró en la sangre de seis de los casos estudiados, en niveles desde 0.12 a 1.90 µg/mL. Solo en uno de los casos no se detectó aldicarb. En cuanto la presunta manera de muerte, en seis de los casos analizados fue el suicidio y en un caso se clasificó como muerte en estudio. Conclusiones. Los resultados obtenidos con la metodología analítica y la técnica LC-ESI-MS/MS son satisfactorios en términos de la determinación cuantitativa de aldicarb en sangre total post mortem. La aplicación de la metodología descrita en toxicología forense evidencia el empleo de este plaguicida en actos suicidas.

Background. Aldicarb is a high toxicity carbamate pesticide associated to human fatal acute intoxications. Its mechanism of action consists of the inhibition of the acetylcholinesterase enzyme (AChE), which induces the accumulation of the neurotransmitter acetylcholine in the synaptic cleft. This accumulation induces cholinergic symptoms and, depending on the exposition dose, it can paralyze the respiratory and nervous systems, leading to death. Objective. To determine aldicarb levels in post mortem blood in cases of acute intoxication. Materials and methods. An experimental research was conducted using liquid chromatography tandem-mass spectrometry (LC-ESI-MS/MS) with electrospray ionization. The aldicarb and aldicarb-d3 standards were purchased from Dr. Ehrenstorfer GmbH corporation. This method carries out a protein precipitation of blood and its analysis using LC-ESI-MS/MS, using aldicarb-d3 as internal standard. This method was applied to seven cases of fatal intoxication by presumable action of aldicarb. Results. Aldicarb was found in six of the studied cases on levels between 0.12 and 1.90 µg/mL. Aldicarb was not detected in blood only in one case. Six of the cases were associated to suicide as a manner of death and in one of them it remained under study. Conclusions. The results obtained with the analytical methodology and the use of the LC-ESI-MS/MS technique are satisfactory in terms of the quantitative determination of aldicarb in post mortem total blood. The application of the described methodology in forensic toxicology evidences the use of this pesticide in suicidal practices.

Matched sibling donors versus alternative donors in allogeneic hematopoietic stem cell transplantation for pediatric severe aplastic anemia in México.

Objectives Hematopoietic stem cell transplantation (HSCT) from a matched sibling donor (MSD) is the preferred initial treatment for children with severe aplastic anemia (SAA). Unfortunately, only about 30% of patients have a suitable human leukocyte antigen-matched sibling. Methods We have analyzed the outcome of 42 patients who received HSCT (22 MSD and 20 alternative donors (AD)) for SAA at the seven major pediatric HSCT centers in Mexico between 2001 and 2013. Results With a median follow-up of 30 months (range, 0.4-144), the 5-year overall survival in children transplanted from MSD was 86.4 + 7.3 vs. 49.5 + 11% for children after AD-HSCT (P = 0.013). The cumulative incidence of treatment-related mortality (TRM) was in the MSD-HSCT 9.1 + 3.9% vs. 47.6 + 9.1% in the AD-HSCT context (P = 0.007). Infectious complications contributed to death (91%) of most patients who received AD-HSCT. Discussion Even when the results of patients given MSD-HSCT are adequate, there is still much room for improvement particularly in children allografted with AD and in the supportive care. The development of an economicwise designed prospective project with MSD or matched unrelated donor HSCTs as a first line of treatment of children with SAA as a unified national trial could address these issues.

Prevalencia de mercurio y plomo en población general de Bogotá 2012/2013 / Mercury and lead prevalence in a sample of people living in Bogotá, 2012-2013

Objetivos Establecer la prevalencia de mercurio y plomo en población general de Bogotá, posibles efectos en salud y relación con zonas de exposición ambiental. Métodos Estudio transversal, muestra de 401 individuos de población general de Bogotá, distribuida aleatoria y proporcionalmente según localidad y zonas de exposición ambiental de alto, medio y bajo riesgo. Se realizó valoración médica y cuantificación de plomo (sangre) y mercurio (sangre, cabello, orina). Se realizó análisis descriptivo preliminar. Resultados Edad de los participantes: 3-91 años (media 46), ocupación más frecuente ama de casa (45,1 %, n=181); promedios de concentraciones: mercurio en cabello: 1,00 µg/g, sangre: 3,13 µg/L, y orina: 0,29 µg/L; plomo en sangre: 8,62 µg/dL. Individuos con concentraciones superiores a los valores de referencia internacionales: 54 (13,5 %) para mercurio (OMS); 10 (2,5 %) para plomo (CDC). Los hallazgos clínicos son inespecíficos, las zonas de exposición no parecen relacionarse con las concentraciones encontradas. Discusión Un porcentaje importante de individuos presentan concentraciones elevadas de los metales estudiados, lo que resalta la necesidad de identificar y controlar las fuentes ambientales de mercurio y plomo que están afectando a la población general de Bogotá, con posibles consecuencias en su salud.(AU)

Objectives Establishing mercury and lead prevalence in a sample of people living in Bogotá, potential effects on their health and the relationship with areas of environmental exposure. Methods This was a cross-sectional study involving a randomly and proportionally distributed sample of 401 people living in Bogotá, according to locality and their high, medium and low risk environmental exposure. A medical assessment was made and lead (in blood) and mercury levels (blood, hair, urine) quantified. A preliminary descriptive analysis was made. Results The participants were aged 3-91 years-old (mean 46), the most frequently occurring occupation was that of housewife (45.1%, n=181). Mean mercury concentration in hair was 1µg/g, 3.13 µg/L in blood and 0.29 µg/L in urine; lead in blood was found to be 8.62 µg/dL. Fifty-four people (13.5%) had higher concentrations than international reference values for mercury (WHO) and 10 (2.5%) for lead (CDC). Clinical findings were non-specific; exposure areas did not seem to relate to the concentrations found. Discussion A significant percentage of the study population had high mercury and/or lead levels and such high prevalence highlights the need for identifying and controlling sources of exposure to mercury and lead which could have adverse consequences regarding the health of Bogota’s general population.(AU)

Determinación de mercurio en pescado fresco de mayor consumo en Bogotá marzo-abril 2003 / Determination of mercury in fresh fish of highest consumption in Bogotá March-April 2003

En el mundo son varios los episodios de intoxicaciones crónicas masivas debidas al consumo de pescado contaminado con mercurio, por los desechos químicos industriales en aguas de ríos y océanos en donde se capturan los peces para consumo humano. En consecuencia, se consideró importante determinar la concentración de mercurio en pescados de mayor consumo en Bogotá, como insumo en el proceso de vigilancia epidemiológica de metales pesados en alimentos, realizándose un estudio descriptivo que cuantificó la concentración de mercurio en pescado fresco de mayor consumo en la Capital, como lo son el bagre, bocachico y nicuro, procedentes del río Magdalena, mediante la técnica de absorción atómica por vapor frío, por parte de Toxicología del Laboratorio de Salud Pública de la Secretaría Distrital de Salud, entre marzo-abril de 2003. Se tuvieron en cuenta variables como la especie, la época del año y la procedencia. El universo: quince toneladas de pescado semanal, cantidad que llega aproximadamente al puerto pesquero nacional. La unidad muestral correspondió a 88 muestras de pescado (nicuro, bocachico y bagre). El cálculo del tamaño de la muestra se hizo con un nivel de confianza de 99% y un margen de error de 2%, mediante el programa EPIINFO 6.0, estableciendo un número de muestras mínimo representativo de 75 unidades de pescado fresco; se midió la concentración de mercurio. Para el respectivo análisis de estas muestras se comparó el resultado con la Norma Técnica Colombiana, NTC 1443, que hace referencia a la concentración máxima admisible de mercurio en pescado (0,5 p.p.m) y la Norma Internacional de Suecia (0,2 p.p.m). Con base en esto se halló un 7,95% con concentraciones por encima a lo sugerido por la Norma colombiana, y un 20,45% con concentraciones por encima de lo sugerido por la Norma de Suecia. De acuerdo a las especies analizadas la mayor concentración de mercurio la presentó el bagre, seguido de bocachico, y después por el nicuro; esto se relacionó con el contenido de masa muscular que tenían estos pescados por cuanto entre más grande, o mayor masa tenía, mayor fue la concentración hallada.

In the world there are several episodes of massive chronic poisoning due to the consumption of fish contaminated with mercury, due to industrial chemical waste in river and ocean waters where fish are caught for human consumption. Consequently, it was considered important to determine the concentration of mercury in the most consumed fish in Bogota, as an input in the epidemiological surveillance process of heavy metals in food. A descriptive study was carried out to quantify the concentration of mercury in the most consumed fresh fish in the Capital, such as catfish, bocachico and nicuro, from the Magdalena River, using the cold vapor atomic absorption technique, by the Toxicology Department of the Public Health Laboratory of the District Health Secretariat, between March and April 2003. Variables such as species, time of year and origin were taken into account. The universe: fifteen tons of fish per week, an amount that arrives approximately at the national fishing port. The sample unit corresponded to 88 fish samples (nicuro, bocachico and catfish). The sample size was calculated with a confidence level of 99% and a margin of error of 2%, using the EPIINFO 6.0 program, establishing a minimum representative number of samples of 75 units of fresh fish; the mercury concentration was measured. For the respective analysis of these samples, the result was compared with the Colombian Technical Standard, NTC 1443, which refers to the maximum allowable concentration of mercury in fish (0.5 p.p.m) and the Swedish International Standard (0.2 p.p.m). Based on this, 7.95% were found with concentrations above those suggested by the Colombian Standard, and 20.45% with concentrations above those suggested by the Swedish Standard. According to the species analyzed, the highest concentration of mercury was found in catfish, followed by bocachico, and then by nicuro; this was related to the muscle mass content of these fish, since the larger or greater the mass, the higher the concentration found.

Colon epithelial cell differentiation is inhibited by constitutive c-myb expression or mutant APC plus activated RAS.

Blocked differentiation is a hallmark of cancer cells and the restoration of differentiation programs in vivo is an actively pursued clinical aim. Understanding the key regulators of cyto-differentiation may focus therapies on molecules that reactivate this process. c-myb expression declines rapidly when human colon cancer epithelial cells are induced to differentiate with the physiologically relevant short-chain fatty acid, sodium butyrate. These cells show increased expression of alkaline phosphatase and cytokeratin 8. Similarly, murine Immorto-epithelial cells derived from wild-type colon cells also show c-myb mRNA declines when induced to differentiate with sodium butyrate. Immorto-cells harboring a single APC mutation are indistinguishable from wild-type cells with regard to differentiation, while addition of activated RAS alone markedly enhances differentiation. In marked contrast, complete differentiation arrest occurs when both APC and RAS are mutated. Expression of MybER, a 4-hydroxytamoxifen-activatable form of c-Myb, blocks differentiation in wildtype and APC mutant Immorto-cell lines as well as LIM1215 human colon carcinoma cells. These data identify two pathways of oncogenic change that lead to retarded epithelial cell differentiation, one involving the presence of a single APC mutation in conjunction with activated RAS or alternatively constitutive c-myb expression.