RESUMO
BACKGROUND: Structural and biochemical changes in stored platelets are influenced by collection and processing methods. This international study investigates the effects of platelet (PLT) processing and storage conditions on HMGB1, sCD40L, and sCD62P protein levels in platelet concentrate supernatants (PCs). STUDY DESIGN/METHODS: PC supernatants (n = 3748) were collected by each international centre using identical centrifugation methods (n = 9) and tested centrally using the ELISA/Luminex platform. Apheresis versus the buffy coat (BC-PC) method, plasma storage versus PAS and RT storage versus cold (4°C) were investigated. We focused on PC preparation collecting samples during early (RT: day 1-3; cold: day 1-5) and late (RT: day 4-7; cold: day 7-10) storage time points. RESULTS: HMGB1, sCD40L, and sCD62P concentrations were similar during early storage periods, regardless of storage solution (BC-PC plasma and BC-PC PAS-E) or temperature. During storage and without PAS, sCD40L and CD62P in BC-PC supernatants increased significantly (+33% and +41%, respectively) depending on storage temperature (22 vs. 4°C). However, without PAS-E, levels decreased significantly (-31% and -20%, respectively), depending on storage temperature (22 vs. 4°C). Contrastingly, the processing method appeared to have greater impact on HMGB1 release versus storage duration. These data highlight increases in these parameters during storage and differences between preparation methods and storage temperatures. CONCLUSIONS: The HMGB1 release mechanism/intracellular pathways appear to differ from sCD62P and sCD40L. The extent to which these differences affect patient outcomes, particularly post-transfusion platelet increment and adverse events, warrants further investigation in clinical trials with various therapeutic indications.
Assuntos
Remoção de Componentes Sanguíneos , Proteína HMGB1 , Humanos , Remoção de Componentes Sanguíneos/métodos , Plaquetas/metabolismo , Preservação de Sangue/métodos , Ligante de CD40/metabolismo , Proteína HMGB1/metabolismo , Transfusão de PlaquetasRESUMO
Although SARS-CoV-2 mRNA vaccination has been shown to be safe and effective in the general population, immunocompromised solid organ transplant recipients (SOTRs) were reported to have impaired immune responses after one or two doses of vaccine. In this study, we examined humoral responses induced after the second and the third dose of mRNA vaccine in different SOTR (kidney, liver, lung, and heart). Compared to a cohort of SARS-CoV-2 naïve immunocompetent health care workers (HCWs), the second dose induced weak humoral responses in SOTRs, except for the liver recipients. The third dose boosted these responses but they did not reach the same level as in HCW. Interestingly, although the neutralizing activity against Delta and Omicron variants remained very low after the third dose, Fc-mediated effector functions in SOTR reached similar levels as in the HCW cohort. Whether these responses will suffice to protect SOTR from severe outcome remains to be determined.
RESUMO
Therapeutic uses of mesenchymal stromal cells (MSCs) have emerged over the past decade. Yet, their effect on tumor growth remains highly debated, particularly in an immune competent environment. In this study, we wanted to investigate the impact of human umbilical cord-derived MSCs (hUC-MSCs) on tumor growth in humanized mice generated by the human adoptive transfer of PBMCs or the cotransplantation of hematopoietic stem cells and human thymic tissue (human BLT [Hu-BLT]). Our results showed that the growth and immune rejection of engineered human fibroblastic tumors was not altered by the injection of hUC-MSCs in immune-deficient or humanized mice, respectively. This was observed whether tumor cells were injected s.c. or i.v. and independently of the injection route of the hUC-MSCs. Moreover, only in Hu-BLT mice did hUC-MSCs have some effects on the tumor-immune infiltrate, yet without altering tumor growth. These results demonstrate that hUC-MSCs do not promote fibroblastic tumor growth and neither do they prevent tumor infiltration and rejection by immune cells in humanized mice.
Assuntos
Linfócitos do Interstício Tumoral/imunologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Transferência Adotiva , Animais , Linhagem Celular Transformada/transplante , Fibroblastos/transplante , Vetores Genéticos , Rejeição de Enxerto/imunologia , Xenoenxertos , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Quimera por Radiação , Organismos Livres de Patógenos Específicos , Telomerase/genética , Telomerase/fisiologia , Timo/transplante , Geleia de Wharton/citologiaRESUMO
BACKGROUND: Immunologic abnormalities have been described in peripheral blood and central nervous system of patients suffering from Alzheimer's disease (AD), yet their role in the pathogenesis still remains poorly defined. AIM AND METHODS: We used the triple transgenic mouse model (3xTg-AD) to reproduce Aß (amyloid plaques) and tau (neurofibrillary tangles) neuropathologies. We analyzed important features of the adaptive immune system in serum, primary (bone marrow) as well as secondary (spleen) lymphoid organs of 12-month-old 3xTg-AD mice using flow cytometry and ELISPOT. We further investigated serum cytokines of 9- and 13-month-old 3xTg-AD mice using multiplex ELISA. Results were compared to age-matched non-transgenic controls (NTg). RESULTS: In the bone marrow of 12-month-old 3xTg-AD mice, we detected decreased proportions of short-term reconstituting hematopoietic stem cells (0.58-fold, P = 0.0116), while lymphocyte, granulocyte, and monocyte populations remained unchanged. Our results also point to increased activation of both B and T lymphocytes. Indeed, we report elevated levels of plasma cells in bone marrow (1.3-fold, P = 0.0405) along with a 5.4-fold rise in serum IgG concentration (P < 0.0001) in 3xTg-AD animals. Furthermore, higher levels of interleukin (IL)-2 were detected in serum of 9- and 13-month-old 3xTg-AD mice (P = 0.0018). Along with increased concentrations of IL-17 (P = 0.0115) and granulocyte-macrophage colony-stimulating factor (P = 0.0085), these data support helper T lymphocyte activation with Th17 polarization. CONCLUSION: Collectively, these results suggest that the 3xTg-AD model mimics modifications of the adaptive immunity changes previously observed in human AD patients and underscore the activation of both valuable and harmful pathways of immunity in AD.
Assuntos
Imunidade Adaptativa/fisiologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Citocinas/metabolismo , Linfócitos/patologia , Imunidade Adaptativa/genética , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Medula Óssea/patologia , Polaridade Celular/genética , Granulócitos/patologia , Humanos , Camundongos , Camundongos Transgênicos , Monócitos/patologia , Mutação/genética , Emaranhados Neurofibrilares , Presenilina-1/genética , Baço/patologia , Proteínas tau/genéticaRESUMO
BACKGROUND: We recently showed that transient warming effects decreased the functional and adhesion properties of mesenchymal stromal cells (MSC) while post-thaw viability remained high. In an attempt to better predict functional impairment of cryopreserved MSC, we further analysed the correlation between viability, immunosuppressive activity and adhesion of cells exposed or not to warming events. METHODS: MSC prepared from six umbilical cords were frozen to -130°C and immediately transferred in a dry ice container or exposed to room temperature for 2 to 10 min (warming events) prior to storage in liquid nitrogen. Viability, functionality (inhibition of T-cell proliferation), adhesion and expression of various integrins were evaluated. RESULTS: The monotonic loss of functional activity with time was proportional to the length of warming events to which MSC were subjected and correlated with the monotonic loss of adhesion capacity. In contrast, post-thaw viability assessment did not predict functional impairment. Interestingly, flow cytometry analyses revealed the emergence of a FSClow population present in the viable cell fraction of freshly thawed MSC, which displayed poor adhesion capacity and expressed low levels of integrin ß5. The prevalence of this FSClow population increased with the length of warming events and correlated with impaired functional and adhesion properties. DISCUSSION: Our results reveal that loss of functional activity (4-day test) induced by transient warming events could be predicted by evaluating adhesion (2-hr test) or FSC profile (10-min test) of MSC immediately post-thaw. These observations could lead to the development of surrogate tests for rapidly assessing the functional quality of cryopreserved MSC.
Assuntos
Temperatura Alta/efeitos adversos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Cordão Umbilical/citologia , Adesão Celular/fisiologia , Proliferação de Células , Tamanho Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Criopreservação/métodos , Citometria de Fluxo , Congelamento , HumanosRESUMO
BACKGROUND: Mesenchymal stromal cells (MSCs) have shown promising results for the treatment of refractory acute graft-versus-host disease. While safety of MSC infusion has been demonstrated, the use of cryopreserved MSCs in clinical trials has raised concerns regarding the retention of their functional activity. This has led to the recommendation by experts in the field to use freshly harvested MSCs, even though this approach is much less practical from a logistic point of view. In the present study, we revisited the impact of cryopreservation on MSC functionality and addressed the possibility that warming events on frozen cells rather than cryopreservation per se could impact MSC functionality. METHODS: Following controlled-rate freezing to -130°C, umbilical cord-derived MSCs were left at room temperature (RT) for 2-10 min or on dry ice for 10 min, before being transferred into liquid nitrogen (LqN2). MSCs of each group were subsequently tested (viability, functionality and cellular damage) and compared with their freshly harvested counterparts. RESULTS: We demonstrated that freshly harvested MSCs as well as cryopreserved MSCs that were left on dry ice following step-down freezing have comparable viability, functionality and integrity. In contrast, cryopreserved MSCs that were left at RT before being transferred into LqN2 were functionally impaired and showed cellular damage upon thawing even though they exhibited high viability. DISCUSSION: Warming events after freezing and not cryopreservation per se significantly impair MSC functionality, indicating that cryopreserved MSCs can be an advantageous alternative to freshly harvested cells for therapeutic purposes.
Assuntos
Preservação de Sangue/métodos , Criopreservação/métodos , Células-Tronco Mesenquimais/fisiologia , Cordão Umbilical/citologia , Proliferação de Células , Congelamento , Humanos , Células-Tronco Mesenquimais/imunologia , TemperaturaRESUMO
Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation.
Assuntos
Diferenciação Celular/genética , Ácido Láctico/metabolismo , Macrófagos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/farmacologia , Macrófagos/citologia , Camundongos Endogâmicos C57BL , Camundongos SCID , Monócitos/citologia , Monócitos/metabolismo , Cordão Umbilical/citologiaRESUMO
The immune tolerance induced by IVIg treatment is generally attributed to its capacity to modulate the functions of antigen presenting cells and to induce the expansion of regulatory T cells by mechanisms that are not well-defined. Herein, we investigated the contribution of the TNF-α/TGF-ß/IDO axis to IVIg-induced immune tolerance. We show that high dose IVIg is able to markedly increase the expression (>3 fold) of the well-known tolerogenic cytokine TGF-ß in monocytes. In addition, the expression of TNF-α, a pleiotropic cytokine that controls TGF-ß-induced tolerogenic effects, as well as of its cognate receptors (TNF-R1 and TNF-R2) is also significantly increased following IVIg treatment. Along with TNF-α, the expression of the enzyme and signaling protein IDO, known to mediate TGF-ß dependant tolerogenic effect, is similarly increased following IVIg treatment. We thus propose that the complex interplay between plasticity of immune cells and environmental modifications in which the TNF-α/TGF-ß/IDO axis may represent a new mechanism contributing to the development of tolerance in IVIg-treated patients.
Assuntos
Imunoglobulinas Intravenosas/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Monócitos/imunologia , Fator de Crescimento Transformador beta/imunologia , Fator de Necrose Tumoral alfa/imunologia , Células Cultivadas , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Humanos , Imunoglobulinas Intravenosas/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Allograft rejection and graft-versus-host disease (GvHD) are frequent complications following solid organ or stem cell transplantation in which T cell activation plays a central role. Despite the development of new immunosuppressive drugs that improve the success rate of transplantation, allograft survival continues to be a challenge. Recently, intravenous immunoglobulin (IVIg) has been proposed as prophylaxis and post-transplant treatment to reduce acute rejection episodes. IVIg is a therapeutic agent that is known to down-modulate T cell functions in patients with autoimmune disorders. To test the hypothesis that this immunomodulatory effect could be beneficial in the context of transplantation, we used mixed lymphocyte reactions (MLR) as an in vitro model of allograft rejection and GvHD. Our results show that IVIg strongly inhibits the MLR as evaluated by IL-2 secretion, a well-known marker of T cell activation. IVIg also modulates the secretion of other pro-(IL-6, IFN-γ) and anti-inflammatory (IL-1RA) cytokines. More importantly, we show that IVIg induces monocytes with a CD80(low) PD-L1(high) phenotype and that blockade of PD-L1 partially abrogates the inhibitory effect of IVIg. We have thus identified a new mechanism by which IVIg inhibits T cell functions in the context of transplantation, supporting the potential usefulness of IVIg in the prevention or treatment of graft rejection and GvHD.
Assuntos
Antígeno B7-H1/biossíntese , Imunoglobulinas Intravenosas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos , Monócitos/imunologia , Antígeno B7-H1/imunologia , Citometria de Fluxo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunoglobulinas Intravenosas/imunologia , Técnicas In Vitro , Ativação Linfocitária/imunologia , Monócitos/efeitos dos fármacos , Transplante HomólogoRESUMO
Strategies to enhance the expansion of umbilical cord blood hematopoietic stem and progenitor cells (HSPCs) are crucial to enable their widespread application to adults and to overcome important limitations, such as delayed engraftment. Osteoblasts regulate HSPCs under steady-state and also under stress conditions, when HSPCs undergo numerous cycles of expansion. We hypothesized that osteoblasts could provide better stimulation for the expansion of multipotent HSPCs and subsequent hematopoietic recovery than mesenchymal stromal cells. Hence, we assessed the growth and engraftment modulatory activities of mesenchymal stromal cell-derived osteoblasts (M-OSTs) on hematopoietic progenitors. Mesenchymal stromal cells and M-OSTs favored the maintenance of CD34(+) cells. The expansion of cord blood CD34(+) cells and myeloid progenitors was highest in cultures supplemented with unfiltered M-OST-conditioned medium (M-OST CM). In addition, increased expression of cell surface receptors important for the homing of progenitors to the bone marrow, C-X-C chemokine receptor type 4 and lymphocyte function-associated antigen 1, was observed in CM-based cultures. Additionally, M-OST CM positively modulated the engraftment properties of expanded progenitors. Most notably, although human platelet levels remained steady in the first 2 weeks in mice transplanted with HSPCs expanded in standard medium, levels in mice transplanted with M-OST CM HSPCs rose continuously. Consistent with this, short-term human progenitor reconstitution was consistently greater in M-OST recipients. Finally, cytokine array-based profiling revealed increases in insulin-like growth factor binding protein 2, chemokines, and myeloid stimulating cytokines in M-OST CM. In conclusion, this study suggests that M-OSTs represent a new underappreciated source of feeder cells for the expansion of HSPCs with enhanced thrombopoietic activity.
Assuntos
Proliferação de Células , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Multipotentes/metabolismo , Osteoblastos/metabolismo , Adulto , Animais , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Feminino , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Xenoenxertos , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Multipotentes/citologia , Osteoblastos/citologiaRESUMO
Intravenous immunoglobulin (IVIg) is successfully used in the treatment of autoimmune diseases involving self-reactive CD8(+) T cells. However, its direct influence on the cytotoxic response remains unknown. Using an antigen cross-presentation assay and a mouse model of ovalbumin (OVA) immunization, we showed that IVIg decreases the in vitro activation, proliferation and cytokine secretion of OVA-specific CD8(+) T cells (OT-I), as well as the in vivo generation of OVA-specific CD8(+) T cells. In addition, IVIg significantly decreases the proportion of perforin- and CD107a-expressing CD8(+) T cells, and inhibits the cytotoxic activity of OVA-activated OT-I cells. The interference of IVIg with the CD8(+) T-cell response is associated with T-cell receptor blockade, therefore reducing the interaction between effector and target cells. A similar blockade is observed on human CD8(+) T cells, suggesting that the observations reported here could apply to the IVIg-mediated improvement of CD8(+) T-cell-mediated autoimmune conditions in human patients.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Imunoglobulinas Intravenosas/farmacologia , Animais , Linfócitos T CD8-Positivos/imunologia , Feminino , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologiaAssuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Imunoglobulinas Intravenosas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Citometria de Fluxo , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Intravenous immunoglobulin (IVIg) is a therapeutic preparation of plasma-derived human IgG and is increasingly used for the treatment of several neurological inflammatory disorders. However, it is not clear whether the IgG molecules contained in IVIg can actually cross the BBB in treated patients. We recently showed that LRP1, an endocytic receptor involved in transcytosis of several proteins across the BBB was able to interact with IVIg. In the present study, we show that LRP1 is involved in IVIg internalization inside living cells. Our data also suggest that following internalization, IVIg is recycled to the cell surface, raising the possibility that LRP1 can mediate IVIg transcytosis across the BBB. Finally, we show that IVIg-LRP1 interaction leads to LRP1 tyrosine phosphorylation.
Assuntos
Barreira Hematoencefálica/metabolismo , Imunoglobulinas Intravenosas/metabolismo , Receptores de LDL/metabolismo , Transcitose , Proteínas Supressoras de Tumor/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Fosforilação/fisiologia , Tirosina/metabolismoRESUMO
Activation of self-reactive CD4(+) T cells plays a central role in the initiation and maintenance of autoimmune diseases. We recently reported that intravenous immunoglobulin (IVIg) inhibits the MHC II-restricted CD4(+) T cell activation induced by the presentation of immune complexes. Because native antigens can also play a role in the induction of several autoimmune diseases, we determined whether IVIg could also affect CD4(+) T cell activation following presentation of native antigens by APCs. Here we report that IVIg significantly reduces the activation of CD4(+) T cells by native ovalbumin. The inhibitory effect is FcγR-independent and occurs following internalization of IVIg inside APCs, where it interferes with the intracellular events leading to MHC II-dependent antigen presentation. The effect of IVIg on native antigen presentation could therefore contribute to dampen the autoimmune reaction by reducing CD4(+) T cell activation and the subsequent inflammatory response induced by these cells.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoglobulinas Intravenosas/imunologia , Ativação Linfocitária/imunologia , Animais , Linhagem Celular , Células Dendríticas/imunologia , Feminino , Imunoglobulinas Intravenosas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Ovalbumina/imunologiaAssuntos
Autoanticorpos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Células Mieloides/imunologia , Células Mieloides/fisiologia , Adulto , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/metabolismo , Autoanticorpos/metabolismo , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Saúde , Humanos , Imunidade Inata/fisiologia , Masculino , Modelos Biológicos , Proteinose Alveolar Pulmonar/sangue , Proteinose Alveolar Pulmonar/imunologia , Proteinose Alveolar Pulmonar/metabolismo , Transdução de Sinais/imunologia , Adulto JovemRESUMO
The mechanisms of therapeutic action of IVIg are still unclear in most autoimmune and inflammatory diseases. IVIg have been shown to bind to a variety of human proteins including BAFF, amyloid beta peptide and GM-CSF. It has been suggested that this autoreactivity could contribute to the therapeutic immunomodulatory effects of IVIg. In this work, we showed that native IgG purified from plasma under non-denaturing conditions were much less autoreactive than IVIg. However the native IgG autoreactivity with BAFF, amyloid beta peptide and GM-CSF was significantly increased by short incubation under the slightly denaturing conditions used during industrial plasma fractionation. We conclude that the relatively mild conditions used in industrial plasma fractionation are sufficiently denaturing to activate a significant amount of cryptic autoreactive plasma IgG which could be involved not only in the therapeutic immunomodulatory effects of IVIg but also in the adverse "allergic" reactions often observed in IVIg-infused patients.
Assuntos
Peptídeos beta-Amiloides/imunologia , Fator Ativador de Células B/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunoglobulina G/imunologia , Imunoglobulinas Intravenosas/imunologia , Etanol/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Imunoglobulinas Intravenosas/química , Desnaturação Proteica , Soro/química , Soro/imunologiaRESUMO
Previous work from our laboratory showed that IVIg could directly influence the fate of human B cells by inducing their differentiation. The initial goal of the present study was to identify the cell surface molecules recognized by IVIg on human B cells. Purified resting and CD40-activated human B cells were incubated with IVIg and lysed prior to immunoprecipitation. The immunoprecipitated proteins were identified by mass spectrometry (LC-MS). This analysis revealed that BCR, as well as other cell surface receptors or membrane associated proteins were the main targets of IVIg. Surprisingly, intracellular proteins were also found in the immunoprecipitates, suggesting that IVIg could penetrate inside living cells and interact with intracellular targets. We have further studied this unexpected phenomenon and obtained evidence indicating that a significant amount of IVIg was spontaneously internalized inside living cells. We showed that IVIg internalization could occur in a BCR- and FcgammaR-independent pathway. Furthermore, spontaneous IVIg internalization was also observed in whole blood incubated with therapeutic concentrations of IVIg, even in presence of the high endogenous IgG concentration. These observations first suggest that spontaneous internalization can occur in IVIg-treated patients and also that some of the observed alterations in the physiology of IVIg-treated cells may not be only dependent on extracellular interactions of IVIg with cell surface receptors or soluble plasma proteins but may also involve intracellular interactions.
Assuntos
Linfócitos B/metabolismo , Imunoglobulinas Intravenosas/farmacologia , Imunoglobulinas Intravenosas/farmacocinética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de IgG/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/imunologia , Antígenos CD40/metabolismo , Feminino , Humanos , Imunoprecipitação , Ativação Linfocitária , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologiaRESUMO
Human B cells can be cultured ex vivo for a few weeks, following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin-4 (IL-4). However, attempts to produce polyclonal antigen-specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen-specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells, recent data indicating that IL-4-activated human B cells are induced to express Toll-like receptor-4, the main LPS receptor, prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40-activated human B cells, accompanied by an increase in antigen-specific antibody secretion. This result suggested that some, but not all, B cells were able to differentiate into antibody-secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS-stimulated human B cells were induced to secrete higher amounts of IL-6, a pleiotropic cytokine well-known for its B-cell differentiation activity. In vivo, the effect of LPS on cytokine secretion by B cells may not only enhance B-cell differentiation but also help to sustain a local ongoing immune response to invading Gram-negative bacteria, until all pathogens have been cleared from the organism.
Assuntos
Linfócitos B/imunologia , Antígenos CD40/imunologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária/imunologia , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Epitopos de Linfócito B/imunologia , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Interleucina-10/biossíntese , Interleucina-4/imunologia , Interleucina-6/biossíntese , Camundongos , Sindecana-1/análiseRESUMO
BACKGROUND: The detection by EIA of antibodies (Abs) specific to HIV antigens in the serum of blood donors is important for transfusion safety. A small but significant number of donor sera (0.1-0.3%) yield false-positive results in EIA, and these donors must be permanently deferred from the blood donor list, causing operational and public relations problems. STUDY DESIGN AND METHODS: False-positive EIA reactions could be caused by the binding of low-affinity natural polyreactive Abs, which could be eluted by treatment with a chaotropic agent such as thiocyanate (SCN). The effect of the SCN elution on EIA specificity was studied using tetanus toxoid and HIV Abs. RESULTS: SCN elution preferentially reduced the binding of nonspecific Abs. Testing of 235 seronegative samples in HIV-1 and -2 EIA showed that SCN elution reduced the mean and dispersion of OD values obtained. In addition, SCN elution abolished the false-positive reactivity in HIV-1 and -2 EIA of 69 percent (299/435) of the repeatedly reactive specimens tested without impact on the reactivity of HIV-seropositive specimens. CONCLUSION: The SCN elution step increased the specificity of HIV-1 and -2 EIA and could facilitate the re-entry of previously deferred donors.