Increased Foxp3+ regulatory T cells in poly(ADP-Ribose) polymerase-1 deficiency.

Growing evidence is unveiling a role for poly(ADP-ribose) polymerase (PARP)-1 in the regulation of inflammatory/immune responses. In the current study, we investigated the effects of PARP-1 deficiency on regulatory T cell differentiation. Increased numbers of regulatory CD4(+)CD25(+)/Foxp3(+) T cells were found in thymus, spleen, and lymph nodes of PARP-1 knockout (KO) mice compared with wild-type (WT) controls. The increased frequency of regulatory T cells in the periphery resulted in impaired CD4 cell proliferation and IL-2 production, which could be restored by CD25(+) cell depletion. Phenotype and inhibitory functions of PARP-1 KO regulatory T cells were similar to WT cells, indicating that PARP-1 affects regulatory T cell differentiation rather than function. Purified naive CD4 cells from PARP-1 KO mice stimulated in vitro expressed forkhead box p3 mRNA at higher levels and generated a greater number of Foxp3(+) cells (inducible regulatory T [iTreg] cells) than the WT counterpart. This finding was due to a higher rate of naive CD4 cell to Foxp3(+) iTreg cell conversion rather than to higher resistance to apoptosis induction. Interestingly, PARP-1 deficiency did not affect retinoid-related orphan receptor-gammat mRNA expression and differentiation of purified naive CD4 cells to Th17 cells. PARP-1 KO iTreg cells showed features similar to WT regulatory T cells, suggesting that modulation of PARP-1 during the immune response might be used to induce greater numbers of functional regulatory T cells. In conclusion, our findings represent the first evidence that PARP-1 can affect regulatory T cell differentiation and open new perspectives on potential targets for modulating immune responses.

Effect of CaS Nanostructures in the Proliferation of Human Breast Cancer and Benign Cells In Vitro.

We report on the effect of naked CaS nanostructures on the proliferation of carcinoma cancer cells and normal fibroblasts in vitro. The CaS nanostructures were prepared via the microwave-mediated decomposition of dimethyl sulfoxide (DMSO) in the presence of calcium acetate Ca ( CH 3 CO 2 ) 2 . Light scattering measurements revealed that dispersions contain CaS nanostructures in the size range of a few Å to about 1 nanometer, and are formed when DMSO is decomposed in the presence of Ca ( CH 3 CO 2 ) 2 . Theoretical calculations at the DFT/B3LYP/DGDZVP level of theory on ( C a S ) n clusters ( n = 1 , 2 , 3 , and 4) are consistent with clusters in this size range. The absorption spectra of the CaS nanostructures are dominated by strong bands in the UV, as well as weaker absorption bands in the visible. We found that a single dose of CaS nanoclusters smaller than 0.8 nm in diameter does not affect the survival and growth rate of normal fibroblasts and inhibits the proliferation rate of carcinoma cells in vitro. Larger CaS nanostructures, approximately (1.1 ± 0.2) nm in diameter, have a similar effect on carcinoma cell proliferation and survival rate. The CaS nanoclusters have little effect on the normal fibroblast cell cycle. Human carcinoma cells treated with CaS nanocluster dispersion exhibited a decreased ability to properly enter the cell cycle, marked by a decrease in cell concentration in the G0/G1 phase in the first 24 h and an increase in cells held in the SubG1 and G0/G1 phases up to 72 h post-treatment. Apoptosis and necrotic channels were found to play significant roles in the death of human carcinoma exposed to the CaS nanoclusters. In contrast, any effect on normal fibroblasts appeared to be short-lived and non-detrimental. The interaction of CaS with several functional groups was further investigated using theoretical calculations. CaS is predicted to interact with thiol ( R-SH ), hydroxide ( R - OH ), amino ( R - NH 2 ), carboxylic acid ( R - COOH ), ammonium ( R-NH 3 + ), and carboxylate ( R-COO - ) functional groups. None of these interactions are predicted to result in the dissociation of CaS. Thermodynamic considerations, on the other hand, are consistent with the dissociation of CaS into Ca 2 + ions and H 2 S in acidic media, both of which are known to cause apoptosis or cell death. Passive uptake and extracellular pH values of carcinoma cells are proposed to result in the observed selectivity of CaS to inhibit cancer cell proliferation with no significant effect on normal fibroblast cells. The results encourage further research with other cell lines in vitro as well as in vivo to translate this nanotechnology into clinical use.

Pharmacological inhibition of TLR9 activation blocks autoantibody production in human B cells from SLE patients.

OBJECTIVES: Toll-like receptor 9 (TLR9), which recognizes hypomethylated DNA [cytosine-phosphate-guanine (CpG)], plays a role in the maintenance of serological memory and has been recently implicated in the pathogenesis of SLE. We previously reported that in vitro TLR9 triggers memory B-cell differentiation into antibody-producing cells, and that the MyD88-inhibitor ST2825 blocks TLR9-induced plasma cell (PC) generation. Here, we investigated whether memory B cells produce autoantibodies in SLE patients with active disease or in clinical remission, and whether ST2825 could inhibit PC generation in SLE patients. METHODS: Peripheral blood mononuclear cells from 10 SLE patients in clinical remission and 2 with active SLE were cultured in the presence of CpG with or without ST2825. Phenotypical analysis of CpG-stimulated cells was performed by flow cytometry. Supernatants were collected to measure antibody production by ELISA and to detect autoantibodies by IF. RESULTS: CpG-induced TLR9 stimulation caused autoantibody secretion in patients with active disease and in the majority of patients in clinical remission. Inhibition of MyD88 completely blocked the de novo generation of PCs and the secretion of autoantibodies. CONCLUSIONS: Autoreactive B cells persist in SLE patients during disease remission in the circulating B-cell memory pool. TLR9-dependent activation of memory B cells by pathogens could be one of the mechanisms triggering relapses in SLE. Compounds targeting the TLR/MyD88 pathway may be used as novel therapeutic tools to treat acute disease and to prevent relapses in SLE patients.

Infectious Agents and Inflammation: The Role of Microbiota in Autoimmune Arthritis.

In higher vertebrates, mucosal sites at the border between the internal and external environments, directly interact with bacteria, viruses, and fungi. Through co-evolution, hosts developed mechanisms of tolerance or ignorance toward some infectious agents, because hosts established "gain of function" interactions with symbiotic bacteria. Indeed, some bacteria assist hosts in different functions, among which are digestion of complex carbohydrates, and absorption and supply of vitamins. There is no doubt that microbiota modulate innate and acquired immune responses starting at birth. However, variations in quality and quantity of bacterial species interfere with the equilibrium between inflammation and tolerance. In fact, correlations between gut bacteria composition and the severity of inflammation were first described for inflammatory bowel diseases and later extended to other pathologies. The genetic background, environmental factors (e.g., stress or smoking), and diet can induce strong changes in the resident bacteria which can expose the intestinal epithelium to a variety of different metabolites, many of which have unknown functions and consequences. In addition, alterations in gut permeability may allow pathogens entry, thereby triggering infection and/or chronic inflammation. In this context, a local event occurring at a mucosal site may be the triggering cause of an autoimmune reaction that eventually involves distant sites or organs. Recently, several studies attributed a pathogenic role to altered oral microbiota in rheumatoid arthritis (RA) and to gut dysbiosis in spondyloarthritis (SpA). There is also growing evidence that different drugs, such as antibiotics and immunosuppressants, can influence and be influenced by the diversity and composition of microbiota in RA and SpA patients. Hence, in complex disorders such RA and SpA, not only the genetic background, gender, and immunologic context of the individual are relevant, but also the history of infections and the structure of the microbial community at mucosal sites should be considered. Here the role of the microbiota and infections in the initiation and progression of chronic arthritis is discussed, as well as how these factors can influence a patient's response to synthetic and biologic immunosuppressive therapy.

Monitoring Perinatal Gut Microbiota in Mouse Models by Mass Spectrometry Approaches: Parental Genetic Background and Breastfeeding Effects.

At birth, contact with external stimuli, such as nutrients derived from food, is necessary to modulate the symbiotic balance between commensal and pathogenic bacteria, protect against bacterial dysbiosis, and initiate the development of the mucosal immune response. Among a variety of different feeding patterns, breastfeeding represents the best modality. In fact, the capacity of breast milk to modulate the composition of infants' gut microbiota leads to beneficial effects on their health. In this study, we used newborn mice as a model to evaluate the effect of parental genetic background (i.e., IgA-producing mice and IgA-deficient mice) and feeding modulation (i.e., maternal feeding and cross-feeding) on the onset and shaping of gut microbiota after birth. To investigate these topics, we used either a culturomic approach that employed Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MS), or bottom-up Liquid Chromatography, with subsequent MSMS shotgun metaproteomic analysis that compared and assembled results of the two techniques. We found that the microbial community was enriched by lactic acid bacteria when pups were breastfed by wild-type (WT) mothers, while IgA-deficient milk led to an increase in the opportunistic bacterial pathogen (OBP) population. Cross-feeding results suggested that IgA supplementation promoted the exclusion of some OBPs and the temporary appearance of beneficial species in pups fed by WT foster mothers. Our results show that both techniques yield a picture of microbiota from different angles and with varying depths. In particular, our metaproteomic pipeline was found to be a reliable tool in the description of microbiota. Data from these studies are available via ProteomeXchange, with identifier PXD004033.

A metaproteomic pipeline to identify newborn mouse gut phylotypes.

In order to characterize newborn mouse gut microbiota phylotypes in very early-life stages, an original metaproteomic pipeline, based on LC-MS(2)-spectra and Mascot driven NCBI non-redundant repository database interrogation was developed. An original computational analysis assisted in the generation of a taxonomic gut architecture from protein hits to operational taxonomic units (OTUs) and related functional categories. Regardless of the mouse's genetic background, a prevalence of Firmicutes (Lactobacillaceae) and Proteobacteria (Enterobacteriaceae) was observed among the entire Eubacteria taxonomic node. However, a higher abundance of Firmicutes was retrieved for Balb/c gut microbiota compared to Rag2(ko) mice, the latter was mainly characterized by a Proteobacteria enriched microbiota. The metaproteomic-obtained OTUs were supported, for the identification (ID) of the cultivable bacteria fraction, corroborated by axenic culture-based MALDI-TOF MS IDs. Particularly, functional analysis of Rag2(ko) mice gut microbiota proteins revealed the presence of abundant glutathione, riboflavin metabolism and pentose phosphate pathway components, possibly related to genetic background. The metaproteomic pipeline herein presented may represent a useful tool to investigate the highly debated onset of the human gut microbiota in the first days of life, when the bacterial composition, despite its very low diversity (complexity), is still very far from an exhaustive description and other complex microbial consortia. BIOLOGICAL SIGNIFICANCE: The manuscript deals with a "frontier" topic regarding the study of the gut microbiota and the application of a metaproteomic pipeline to unveil the complexity of this fascinating ecosystem at the very early stages of life. Indeed during these phases, its diversity is very low but the bacterial content is highly "instable", and the relative balance between mucosal and fecal bacteria starts its dynamics of "fight" to get homeostasis. However, in the neonatal period, especially immediately after birth, a comprehensive description of this microbial eco-organ is still lacking, while it should be mandatory to highlight its first mechanisms of homeostasis and perturbation, while it co-develops with and within the host species. In order to unravel its low but almost unknown microbial community multiplicity, the newborn mouse gut, characterized by a "very" low complexity, was herein selected as model to design a LC-MS(2)-based shotgun metaproteomic approach, potentially suitable to study onset and shaping in human newborns. A microbiological semi-automatic computational analysis was performed to infer gut phylotypes; such as proof of evidence, related OTUs were compared to axenic-culture-based MALDI-TOF MS IDs showing consistency at family and phyla levels for the bacterial cultivable fraction. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

¿Se puede controlar el Formaldehído? / Is it possible to control formaldehyde?

El Formaldehido es utilizado ampliamente en Anatomía Patológica para fijar y conservar las muestras, desde el 1 de Enero de 2016 ha entrado en vigor la nueva clasificación de este agente como cancerígeno, por ello es importante la evaluación de riesgo ante tal exposición. Objetivo: demostrar la efectividad de la aplicación de medidas preventivas para disminuir la exposición al formaldehido en salas de tallado de laboratorios de Anatomía Patológica. Metodología: se realizó una valoración ambiental de la exposición al formaldehido, en la sala de tallado del Hospital Universitario de Fuenlabrada, en los puestos de trabajo de técnico y facultativo de Anatomía Patológica, en el año 2008 y posteriormente en el 2012 tras aplicar medidas preventivas. El método de muestreo seguido fue el Método NIOSH 2016 Determinación de Formaldehido en Aire. Método de absorción en Gel de sílice tratado con 2,4- dinitrophenylhydrazine y se compararon los resultados con el Valor Limite Ambiental de Corta Exposición (VLA-EC: 0.37 mg/ m3) establecido en la guía de «Limites de Exposición Profesional para Agentes Químicos» del Instituto Nacional de Seguridad e Higiene en el Trabajo. Resultados: el índice de exposición en 2012 después de incrementar la ventilación (22 renovaciones por hora en sala de tallado y a 15 renovaciones por hora en almacén de muestras) y de efectuar almacenamiento independiente con extracción localizada, es de una Media Geométrica (MG) ￡ 0.5 siendo el valor aceptable. Conclusiones: La ventilación general y el almacenamiento independiente son medidas preventivas con gran impacto, sin embargo lo recomendado para controlar tal exposición es la sustitución del formaldehido por otro producto de menor toxicidad y posteriormente las modificaciones de los procedimientos de trabajo que esto conlleve (AU)

Formaldehyde is widely used in Pathology to set and store samples. From January 1, 2016 this agent was classified as carcinogen, so it is important to risk assessment to such exposure. Objective: To demonstrate the effectiveness of preventive measures to reduce exposure to formaldehyde in rooms carved Laboratory of Pathology. Methodology: We made an environmental assessment of formaldehyde exposure in the room carved from Hospital Universitario de Fuenlabrada, in the jobs of technician and pathologist, in 2008 and later in 2012 after preventive measures. The sampling method was followed NIOSH Method 2016 Determination of Formaldehyde in Air. Absorption method silica gel treated with 2,4- dinitrophenylhydrazine and the results were compared with the Environmental Exposure Limit Value Short (STEL: 0.37 mg / m3) established in the guide «Occupational Exposure Limits for Chemical Agents» National Institute for Safety and Health at Work. Results: the exposure index in 2012 after increasing ventilation (22 air changes per hour in room carved and 15 air changes per hour in store samples) and perform independent storage localized extraction is a geometric mean (GM) 0.5 being the acceptable value. Conclusions: General ventilation and separate storage are preventive measures with great impact, however recommended to control such exposure is the substitution of formaldehyde by another product of lower toxicity and subsequent changes in work procedures that this entails (AU)

Implantación y coordinación del protocolo de accidente biológico para tres áreas sanitarias desde un servicio de prevención / Establishment and co-ordination of the accidental biologic exposure protocol for three health care areas from a single prevention service

Dada la frecuencia del accidente biológico entre los sanitarios, la jornada continuada en este sector y la amplitud geográfica que nuestro SPRL debe atender, hemos elaborado un protocolo de actuación asistencial coordinando los distintos centros y servicios implicados. El propósito es suplir la falta de atención continuada por parte de nuestro servicio, por ello hemos diseñado un sobre de accidente biológico, en el que se incluyen una serie de modelos específicos para la atención de estos accidentes. Los sobres se han puesto a disposición de los servicios de urgencias y centros de salud, informando a todos los trabajadores (AU)