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1.
Cytotechnology ; 70(1): 31-44, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29322348

RESUMO

Clinical use of multipotent Mesenchymal Stromal Cell (MSC)-based medicinal products requires their production in compliance with Good Manufacturing Practices, thus ensuring that the final drug product meets specifications consistently from batch to batch in terms of cell viability, identity, purity and potency. Potency relates to the efficacy of the medicine in its target clinical indication, so adequate release tests need to be defined and validated as quality controls. Herein we report the design and optimisation of parameters affecting the performance of an in vitro cell-based assay for assessing immunomodulatory potential of clinical grade MSC for human use, based on their capacity to inhibit proliferation of T lymphocytes under strong polyclonal stimuli. The resulting method was demonstrated to be reproducible and relatively simple to execute. Two case studies using clinical grade MSC are presented as examples to illustrate the applicability of the methodology described in this work.

2.
N Biotechnol ; 35: 19-29, 2017 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-27810336

RESUMO

Umbilical cord blood (UCB) transplantation is associated with long periods of aplastic anaemia. This undesirable situation is due to the low cell dose available per unit of UCB and the immaturity of its progenitors. To overcome this, we present a cell culture strategy aimed at the expansion of the CD34+ population and the generation of granulocyte lineage-committed progenitors. Two culture products were produced after either 6 or 14days of in vitro expansion, and their characteristics compared to non-expanded UCB CD34+ controls in terms of phenotype, colony-forming activity and multilineage repopulation potential in NOD-scid IL2Rγnull mice. Both expanded cell products maintained rapid SCID repopulation activity similar to the non-expanded control, but 14-day cultured cells showed impaired long term SCID repopulation activity. The process was successfully scaled up to clinically relevant doses of 89×106 CD34+ cells committed to the granulocytic lineage and 3.9×109 neutrophil precursors in different maturation stages. Cell yields and biological properties presented by the cell product obtained after 14days in culture were superior and therefore this is proposed as the preferred production setup in a new type of dual transplant strategy to reduce aplastic periods, producing a transient repopulation before the definitive engraftment of the non-cultured UCB unit. Importantly, human telomerase reverse transcriptase activity was undetectable, c-myc expression levels were low and no genetic abnormalities were found, as determined by G-banding karyotype, further confirming the safety of the expanded product.


Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Sangue Fetal/citologia , Anemia Aplástica/sangue , Anemia Aplástica/etiologia , Anemia Aplástica/prevenção & controle , Animais , Antígenos CD34/sangue , Biotecnologia , Diferenciação Celular , Linhagem da Célula , Ensaio de Unidades Formadoras de Colônias , Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Feminino , Sangue Fetal/imunologia , Facilitação Imunológica de Enxerto/métodos , Granulócitos/citologia , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neutrófilos/citologia
3.
PLoS One ; 8(5): e63296, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23691013

RESUMO

INTRODUCTION: Efferocytosis is a crucial process by which apoptotic cells are cleared by phagocytes, maintaining immune tolerance to self in the absence of inflammation. Peripheral tolerance, lost in autoimmune processes, may be restored by the administration of autologous dendritic cells loaded with islet apoptotic cells in experimental type 1 diabetes. OBJECTIVE: To evaluate tolerogenic properties in dendritic cells induced by the clearance of apoptotic islet cells, thus explaining the re-establishment of tolerance in a context of autoimmunity. METHODS: Bone marrow derived dendritic cells from non-obese diabetic mice, a model of autoimmune diabetes, were generated and pulsed with islet apoptotic cells. The ability of these cells to induce autologous T cell proliferation and to suppress mature dendritic cell function was assessed, together with cytokine production. Microarray experiments were performed using dendritic cells to identify differentially expressed genes after efferocytosis. RESULTS: Molecular and functional changes in dendritic cells after the capture of apoptotic cells were observed. 1) Impaired ability of dendritic cells to stimulate autologous T cell proliferation after the capture of apoptotic cells even after proinflammatory stimuli, with a cytokine profile typical for immature dendritic cells. 2) Suppressive ability of mature dendritic cell function. 3) Microarray-based gene expression profiling of dendritic cells showed differential expression of genes involved in antigen processing and presentation after efferocytosis. 4) Prostaglandin E2 increased production was responsible for immunosuppressive mechanism of dendritic cells after the capture of apoptotic cells. CONCLUSIONS: The tolerogenic behaviour of dendritic cells after islet cells efferocytosis points to a mechanism of silencing potential autoreactive T cells in the microenvironment of autoimmunity. Our results suggest that dendritic cells may be programmed to induce specific immune tolerance using apoptotic cells; this is a viable strategy for a variety of autoimmune diseases.


Assuntos
Autoimunidade , Células Dendríticas/metabolismo , Dinoprostona/biossíntese , Fagocitose , Animais , Proliferação de Células , Células Dendríticas/citologia , Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T Reguladores/citologia
4.
J Nutr ; 138(12): 2392-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19022963

RESUMO

Group A rotaviruses (RV) are the most common causative agents of acute gastroenteritis in children <2 y. The present study was designed to establish the effect of a bovine whey protein concentrate (WPC) in a RV infection model in suckling rats. From d 3 of life, suckling Lewis rats received daily supplements of WPC, WPC plus lactoferrin (LF), standard infant formula (SIF), or water (RV-infected group and an untreated, uninfected reference group). On d 8 of life, heterologous simian RV SA-11 was inoculated orally in the WPC-RV, WPC+LF-RV, SIF-RV, and RV groups. WPC and WPC+LF reduced diarrhea incidence from approximately 90% in RV group to approximately 60% in WPC-RV and WPC+LF-RV groups (P < 0.05), whereas the area under the curve (AUC) of severity along time diminished from approximately 10 AUC in the RV group to approximately 6 AUC in both supplemented groups (P < 0.05). Serum levels of anti-RV antibodies, splenocyte proliferation, and interferon-gamma secretion after specific stimulation were significantly lower in the WPC-RV and WPC+LF-RV groups than in the SIF-RV and RV groups. In the intraepithelial intestinal compartment, RV infection increased the proportion of typical mucosal T cells (IE-T CD8alphaalpha+); however, this modification was controlled by WPC and WPC+LF supplementation. In general, for most of the parameters studied, the SIF-RV and RV groups did not differ. In summary, daily supplementation with WPC or WPC+LF in early life considerably reduces the severity of RV-induced acute gastroenteritis and modulates the immune response against the pathogen.


Assuntos
Diarreia/dietoterapia , Fatores Imunológicos/administração & dosagem , Proteínas do Leite/administração & dosagem , Infecções por Rotavirus/dietoterapia , Animais , Animais Lactentes , Anticorpos Antivirais/sangue , Diarreia/imunologia , Suplementos Nutricionais , Feminino , Imunidade Inata , Imunidade nas Mucosas , Técnicas In Vitro , Lactoferrina/administração & dosagem , Lactoferrina/imunologia , Masculino , Proteínas do Leite/imunologia , Ratos , Ratos Endogâmicos Lew , Rotavirus/imunologia , Infecções por Rotavirus/imunologia , Proteínas do Soro do Leite
5.
Dev Comp Immunol ; 32(12): 1405-8, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18638501

RESUMO

Natural killer T (NKT) cells have been described in the liver and spleen of adult rats, but their presence and function in other tissues and in early life remains uncertain. This study was designed to determine the proportion of NK cells and NKT cells among small intestine intraepithelial (IE) lymphocytes in suckling rats and adult animals by flow cytometry. Very few intestinal IE-NKT cells (NKR-P1A+ TCRalphabeta+) were present in adult rats ( approximately 1%), but a high proportion of this population was found during early life ( approximately 40% of IE lymphocytes in 9-day-old rats), with a marked age-decreasing pattern. Most of these cells presented the CD8alphabeta+ phenotype. Intestinal IE-NK cells (NKR-P1A+ TCRalphabeta-) were also present in a relatively high proportion during the suckling period ( approximately 30% of IE lymphocytes). Thus, a predominance of both NK and NKT cell subpopulations in small intestine epithelium is characteristic in the early life of rats and may have a protective role during the suckling period.


Assuntos
Diferenciação Celular/imunologia , Mucosa Intestinal/citologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Animais Recém-Nascidos , Feminino , Mucosa Intestinal/imunologia , Células Matadoras Naturais/citologia , Ratos , Ratos Endogâmicos Lew , Subpopulações de Linfócitos T/citologia
6.
Br J Nutr ; 98 Suppl 1: S80-4, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17922966

RESUMO

During neonatal life, challenges from breast milk and microbial flora promote immune system maturation. Immunonutrition in these stages may become an important way to increase natural defence systems. The aim of this study was to determine the effect of a daily bovine milk whey protein concentrate (WPC) supplement on the intestinal and systemic immune systems in suckling rats. The composition of intraepithelial and lamina propria lymphocytes (IEL and LPL) was analysed by flow cytometry. Systemic and intestinal humoral immune responses were determined by sera Ig levels and Ig-secreting cell quantification by ELISA and ELISPOT, respectively. From birth, suckling Wistar rats were supplemented with WPC or standard infant formula (SIF). The WPC group showed the same proportion of most of the main mucosal cell subsets as the reference animals. However, in the first days of life WPC enhanced the innate immunity by increasing the NK cell proportion in both epithelial and lamina propria (LP) compartments. A rise in intestinal CD8alphaalpha+ IEL was also induced by WPC supplementation. A time-course of sera Ig levels and spontaneous IgA, IgM and IgG production by LPL and mononuclear cells from blood and spleen, in the WPC group, exhibited a similar pattern to those pups fed only by dam's milk. In summary, the present results show the effects of WPC on enhancing mucosal innate immunity during early life.


Assuntos
Suplementos Nutricionais , Intestino Delgado/imunologia , Proteínas do Leite/imunologia , Envelhecimento/imunologia , Fenômenos Fisiológicos da Nutrição Animal/imunologia , Animais , Animais Recém-Nascidos , Animais Lactentes , Bovinos , Crescimento , Imunidade nas Mucosas , Imunoglobulinas/biossíntese , Mucosa Intestinal/imunologia , Subpopulações de Linfócitos/imunologia , Ratos , Ratos Wistar , Proteínas do Soro do Leite
7.
Dev Comp Immunol ; 31(12): 1264-77, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17459475

RESUMO

The aim of this study is to characterize the rat spleen lymphoid tissue during the suckling period by means of lymphocyte composition and their functionality. Lymphocyte phenotype was determined by immunofluorescence and flow cytometry. The proliferative ability and the antibody secretion activity were considered as functional markers. During the first 2 weeks of life, rat spleen mainly contained B cells (CD45RA+ or Igkappa+). In this period, T (TCRalphabeta+CD4+, TCRalphabeta+CD8+ and TCRgammadelta+CD8+) and NK/NKT (NKR-P1A+) cell proportions were far less than those of adult rats. Moreover, the spleen immune functionality proved to be very low. In the second half of the suckling period, CD4+ and CD8+ cells in the spleen increased in number and proportion, with immature cells progressively displaced by phenotypic mature lymphocytes containing CD3, TCRalphabeta, CD5 and CD2 molecules on their surface. Additionally, although B and T lymphocyte developed their proliferative ability during this period, it was not fully developed at weaning.


Assuntos
Linfócitos B/imunologia , Células Matadoras Naturais/imunologia , Baço/imunologia , Linfócitos T/imunologia , Animais , Animais Recém-Nascidos , Animais Lactentes , Linfócitos B/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Citometria de Fluxo , Imunofenotipagem , Células Matadoras Naturais/citologia , Lactação , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/imunologia , Ratos , Baço/citologia , Linfócitos T/citologia
8.
Pediatr Res ; 58(1): 164-9, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15774849

RESUMO

We characterized the lymphocyte phenotype and the ability to produce Ig by lamina propria (LP) cells from rat ileum throughout the suckling period. In the first week after birth, <10% of LP lymphocytes were B cells, but at weaning, this figure rose to >30% as found in the adult. These B cells did not bear surface IgA (sIgA-). However, the number of sIgA+, which may correspond to B blast cells because they were outside lymphocyte cytometer gate, increased. In LP, IgM-secreting cells (SC) appeared during the second week of life, and IgA-SC were detected later but at a lower number. Regarding LP T cells, CD8+ cells were more abundant than CD4+ cells along the first 2 postnatal weeks, and CD3+CD8alphaalpha+TCRalphabeta+CD5-CD25- was their predominating phenotype. In this 2-wk period, between 8 and 20% of LP were natural killer cells. LP CD4+ lymphocytes in neonatal rats showed increasing co-expression of TCRalphabeta, whereas the co-expression of CD90 decreased and the CD4+CD25+ cell percentage did not achieve adult values. In conclusion, in the first 2 wk of the rat life, the gut LP immune system shows abundant CD8alphaalpha+ cells, including NK cells. Thereafter, LP B cells increase dramatically and Ig-SC appear, with IgM-SC being more abundant than IgA-SC. CD4+ LP lymphocytes acquire a mature phenotype and adult proportions later after weaning.


Assuntos
Imunoglobulinas/metabolismo , Intestino Delgado/imunologia , Intestino Delgado/metabolismo , Linfócitos/citologia , Animais , Animais Recém-Nascidos , Complexo CD3/biossíntese , Linfócitos T CD4-Positivos/citologia , Antígenos CD5/biossíntese , Antígenos CD8/biossíntese , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunoglobulina A/química , Imunoglobulina M/química , Células Matadoras Naturais/citologia , Antígenos Comuns de Leucócito/biossíntese , Lipase Lipoproteica/metabolismo , Subpopulações de Linfócitos/química , Linfócitos/metabolismo , Masculino , Microscopia de Fluorescência , Fenótipo , Ratos , Ratos Endogâmicos Lew , Receptores de Interleucina-2/biossíntese , Antígenos Thy-1/biossíntese , Fatores de Tempo
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