Determinação de parâmetros de qualidade para formas farmacêuticas homeopáticas

All medicine, whether allopathic or homeopathic, must go through strict quality control, which must ratify their characteristics throughout the period of validity. During the time of preparation and storage, solutions of the drugs are in permanent contact with packaging materials that can release undesirable substances to the solution. Several factors may influence the release of packing materials, and factorial design (FD) is a useful tool for analyzing the phenomenon. The aim of this study was the determination of quality parameters for Homeopathic solid (globules) and liquid (drops) dosage forms. It was carried out analysis in homeopathic globules for weight variation, mechanical strength, and moisture content uniformity. For liquid preparations, standard solutions were prepared from natural rubber bulbs, which were subjected to exhaustive extraction with two ethanol solutions (30 and 70%) in the ultrasonic bath for 20 minutes at 25°C and 50°C in three successive cycles. Studies of transfer have been made within five days, by spectrophotometric analysis in the UV region at 312 nm with λmáx and 323 nm for samples in 70% ethanol and 30% respectively. PH values were analyzed. We also conducted two FD studies, where the first, the three-level variables were solvent (chloroform, ethanol and nhexane), sample mass (30, 60 and 90mg), particle size (large disk, small disk and powder sample). In the second study, the solvent level variables were different ethanolic degrees (EtOH 30%, 70% and pure). The percentage of lending in the solutions was 5.5%, 12.4%, 24.2% and 41% of the total estimated in the reference solution. The values of rate constants of transfer were determined in the order of 0.0134 days-1 and 0.0232 days-1 in absorbance values, the solutions in ethanol at 30% and 70% respectively. These results suggest that the speed of transfer of materials from rubber is affected both by the nature of the vehicle as by the temperatureTodo medicamento, quer seja alopático ou homeopático, deve passar por rigoroso controle de qualidade, o qual deve ratificar as suas características ao longo de todo o período de validade. Durante o tempo de preparo e armazenamento, as soluções dos medicamentos estão em contato permanente com os materiais de embalagem que podem liberar substâncias indesejáveis para a solução. Vários fatores podem influenciar a liberação de materiais da embalagem, e planejamento fatorial (PF) é uma ferramenta útil para analisar o fenômeno. O objetivo deste trabalho foi a determinação de parâmetros de qualidade para Formas Farmacêuticas Homeopáticas sólidas (glóbulos) e liquidas (gotas). Foi efetuada a avaliação dos glóobulos homeopáticos no que diz respeito à sua variação de peso, testes de resistência mecânica, uniformidade de conteúdo e umidade. Para as preparações liquidas, preparamos soluções de referência a partir de bulbos de borracha natural, os quais foram submetidos à extração exaustiva com duas soluções de etanol (30 e 70%), em banho de ultrassom durante 20 minutos a 25°C e 50°C, em três ciclos sucessivos de 24 horas. Os estudos de cedência foram efetuados num tempo de cinco dias, através de análise espectrofotométrica na região UV com λmáx em 312 nm e 323 nm para as amostras em etanol a 70% e 30%, respectivamente. Foram verificados valores de pH. Foram também realizados dois estudos de PF, onde no primeiro, as três variáveis de nível foram solvente (clorofórmio, etanol e n-hexano), massa de amostra (30, 60 e 90mg), a forma de amostra (grande disco, disco pequeno, a amostra em pó). No segundo estudo, as variáveis de nível solvente foram graduações etanólicas diferentes (EtOH 30%, 70% e puro). O percentual de cedência nas soluções foi de 5,5%, 12,4%, 24,2% e 41% do total estimado na solução de referência. Os valores das constantes de velocidade de cedência foram determinados na ordem de 0,0134 dia-1 e 0,0232 dia-1, em valores de absorbância, nas soluções em etanol a 30% e 70%, respectivamente. Esses resultados sugerem que a velocidade de cedência de materiais a partir da borracha é afetada tanto pela natureza do veículo como pela temperatur

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

International audienceSingle-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution.Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell.The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions.This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data

IL-7-dependent chemokine production by intestinal endothelial cells during acute HIV/SIV infection

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Mucosal IL-7 response in the gut during HIV/SIV acute infection

International audienceBackground: The very early events of the mucosal immune response to HIV/SIV infection still remain poorly understood. Our previous results demonstrate that IL-7 is expressed in the gut as part of the cytokine storm that occurs during initial virus dissemination, coincidently with viral spread and associated with an increased production of chemokines, leading to immune cell homing.The aim of this work was to identify mucosal cells responsible for this early IL-7 production as well as those responding to IL-7 by chemokine production in the gut.Methods: We thus analysed separately different cell types, known to be present in the gut mucosa to find out how much they can up-regulate their IL-7 production upon inflammatory conditions or how much they are able to secrete chemokines upon IL-7 stimulation. We used epithelial and endothelial cells isolated from gut mucosa of healthy macaques, as well as human endothelial cells differentiated from circulating endothelial cell precursors. These cells were cultured with the supernatant of SIV-infected or non-infected activated PBL or stimulated by pro-inflammatory cytokines with or without IL-7. IL-7 production was measured by RT-qPCR and ELISA in the supernatant of cultured cells. CD127 expression was analysed by RT-qPCR and FACS analysis. Chemokine production was measured by RT-qPCR and MSD.Results: We demonstrated that supernatants of infected activated PBLs boosted IL-7 production by epithelial cells, contrarily to the supernatants of non-infected PBLs. These cells also up-regulated their IL-7 production when stimulated by Interferons (IFNs). Endothelial cells showed an increased expression of CD127 when stimulated by TNF or IFNs, with higher expression when co-stimulated by IL-7, suggesting an increased responsiveness of cells to the IL-7 present in the gut environment during the SIV infection driven inflammatory status. In addition, IL-7 combined to inflammatory cytokines induced higher expression of chemokines by endothelial cells, such as IP-10, IL-8 and RANTES, which are important chemo-attractive molecules for immune cells.Conclusions: This work demonstrates that IL-7 production by intestinal epithelial cells is up regulated by inflammatory signals and identifies endothelial cells as one of the chemokine-expressing cell types under IL-7 stimulation

IL-7 as an adjuvant for mucosal vaccine development

International audienceBackground: Despite considerable research efforts, mucosal immunity remains particularly difficult to stimulate through vaccines. Systemic injection of IL-7 stimulates chemokines-induced recruitment of circulating T-cells into mucosae.Methods: The optimal dose of IL-7 to be sprayed on mucosal surface was defined on 14 rhesus macaques. On mucosal biopsies collected after IL-7 administration, we quantified local transcription of 19 chemokines by qRT-PCR and cell infiltration by immunochemistry plus image analysis. Six macaques were immunized with antigens (DT and the HIV-1 gp41-P1 peptide) applied directly on the vaginal mucosa, two days after either IL-7 or PBS administration. The immunizations were repeated thrice, four months apart, and the macaques were euthanized 2 weeks after the last immunization. Antigen-specific IgA and IgG productions were quantified in vaginal secretions by ELISA. Antigen-specific plasma cells were detected by reverse immunohistochemistry in tissue, and by B-cell ELISPOT in PBMCs.Results: A significant overexpression of twelve chemokines was observed 48 hours after mucosal administration of 10µg of IL-7. Subsequently, mDC, macrophage, NK, B- and T-cell numbers significantly raised in the IL-7-treated mucosae, suggesting massive chemokine-driven infiltration. Administration of antigens led to a stronger mucosal immune response in the IL-7-treated macaques as compared to animals immunized with antigens alone. Robust production of antigen-specific IgAs and IgGs was detected in vaginal secretions. The immunizations repeated thrice sustained mucosal specific immune responses. Antigen-specific-antibody secreting cells were recovered from PBMC and more DT-specific plasma cells were found in the vaginal mucosae (IgA isotype) and the draining lymph nodes (IgG isotype) of IL-7-treated macaques. Tertiary lymphoid organs were observed in vaginal mucosae from IL-7-treated macaques only.Conclusions: Pre-treatment by non-traumatic vaginal administration of IL-7 (10µg by spray), allows for the development of a strong mucosal immune response in macaques following subsequent mucosal vaccination, through local chemokine expression and the recruitment of immune cells in the vaginal mucosa. The mucosal localization of IgA-specific plasma cells argues for their main contribution in the high levels of specific-IgAs evidenced in the vaginal secretions. These data suggest that IL-7 could be used as a mucosal adjuvant to elicit vaginal antibody response, the most promising way to confer protection to numerous STD

Altered Immune Phenotypes and HLA-DQB1 Gene Variation in Multiple Sclerosis Patients Failing Interferon β Treatment

International audienceBackground Interferon beta (IFN β ) has been prescribed as a first-line disease-modifying therapy for relapsing-remitting multiple sclerosis (RRMS) for nearly three decades. However, there is still a lack of treatment response markers that correlate with the clinical outcome of patients. Aim To determine a combination of cellular and molecular blood signatures associated with the efficacy of IFN β treatment using an integrated approach. Methods The immune status of 40 RRMS patients, 15 of whom were untreated and 25 that received IFN β 1a treatment (15 responders, 10 non-responders), was investigated by phenotyping regulatory CD4 + T cells and naïve/memory T cell subsets, by measurement of circulating IFN α / β proteins with digital ELISA (Simoa) and analysis of ~600 immune related genes including 159 interferon-stimulated genes (ISGs) with the Nanostring technology. The potential impact of HLA class II gene variation in treatment responsiveness was investigated by genotyping HLA - DRB1, -DRB3,4,5, -DQA1 , and - DQB1 , using as a control population the Milieu Interieur cohort of 1,000 French healthy donors. Results Clinical responders and non-responders displayed similar plasma levels of IFN β and similar ISG profiles. However, non-responders mainly differed from other subject groups with reduced circulating naïve regulatory T cells, enhanced terminally differentiated effector memory CD4 + T EMRA cells, and altered expression of at least six genes with immunoregulatory function. Moreover, non-responders were enriched for HLA-DQB1 genotypes encoding DQ8 and DQ2 serotypes. Interestingly, these two serotypes are associated with type 1 diabetes and celiac disease. Overall, the immune signatures of non-responders suggest an active disease that is resistant to therapeutic IFN β , and in which CD4 + T cells, likely restricted by DQ8 and/or DQ2, exert enhanced autoreactive and bystander inflammatory activities