RESUMO
Stem cells reside in a specialized regulatory microenvironment or niche, where they receive appropriate support for maintaining self-renewal and multi-lineage differentiation capacity. The niche may also protect stem cells from environmental insults including cytotoxic chemotherapy and perhaps pathogenic immunity. The testis, hair follicle and placenta are all sites of residence for stem cells and are immune-suppressive environments, called immune-privileged sites, where multiple mechanisms cooperate to prevent immune attack, even enabling prolonged survival of foreign allografts without immunosuppression. We sought to determine if somatic stem-cell niches more broadly are immune-privileged sites by examining the haematopoietic stem/progenitor cell (HSPC) niche in the bone marrow, a site where immune reactivity exists. We observed persistence of HSPCs from allogeneic donor mice (allo-HSPCs) in non-irradiated recipient mice for 30 days without immunosuppression with the same survival frequency compared to syngeneic HSPCs. These HSPCs were lost after the depletion of FoxP3 regulatory T (T(reg)) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with T(reg) cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. T(reg) cells seem to participate in creating a localized zone where HSPCs reside and where T(reg) cells are necessary for allo-HSPC persistence. In addition to processes supporting stem-cell function, the niche will provide a relative sanctuary from immune attack.
Assuntos
Sobrevivência de Enxerto/imunologia , Células-Tronco Hematopoéticas/imunologia , Imageamento Tridimensional , Nicho de Células-Tronco/imunologia , Linfócitos T Reguladores/imunologia , Animais , Sobrevivência Celular/imunologia , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-10/deficiência , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nicho de Células-Tronco/citologia , Linfócitos T Reguladores/metabolismo , Fatores de Tempo , Transplante Homólogo/imunologiaRESUMO
Cell differentiation is typically directed by external signals that drive opposing regulatory pathways. Studying differentiation under polarizing conditions, with only one input signal provided, is limited in its ability to resolve the logic of interactions between opposing pathways. Dissection of this logic can be facilitated by mapping the system's response to mixtures of input signals, which are expected to occur in vivo, where cells are simultaneously exposed to various signals with potentially opposing effects. Here, we systematically map the response of naïve T cells to mixtures of signals driving differentiation into the Th1 and Th2 lineages. We characterize cell state at the single cell level by measuring levels of the two lineage-specific transcription factors (T-bet and GATA3) and two lineage characteristic cytokines (IFN-γ and IL-4) that are driven by these transcription regulators. We find a continuum of mixed phenotypes in which individual cells co-express the two lineage-specific master regulators at levels that gradually depend on levels of the two input signals. Using mathematical modeling we show that such tunable mixed phenotype arises if autoregulatory positive feedback loops in the gene network regulating this process are gradual and dominant over cross-pathway inhibition. We also find that expression of the lineage-specific cytokines follows two independent stochastic processes that are biased by expression levels of the master regulators. Thus, cytokine expression is highly heterogeneous under mixed conditions, with subpopulations of cells expressing only IFN-γ, only IL-4, both cytokines, or neither. The fraction of cells in each of these subpopulations changes gradually with input conditions, reproducing the continuous internal state at the cell population level. These results suggest a differentiation scheme in which cells reflect uncertainty through a continuously tuneable mixed phenotype combined with a biased stochastic decision rather than a binary phenotype with a deterministic decision.
Assuntos
Linfócitos T/citologia , Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Separação Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Fator de Transcrição GATA3/metabolismo , Interferon gama/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Proteínas com Domínio T/metabolismoRESUMO
The promise of islet cell transplantation cannot be fully realized in the absence of improvements in engraftment of resilient islets. The marginal mass of islets surviving the serial peritransplant insults may lead to exhaustion and thereby contribute to an unacceptably high rate of intermediate and long-term graft loss. Hence, we have studied the effects of treatment with alpha 1-antitrypsin (AAT) in a syngeneic nonautoimmune islet graft model. A marginal number of syngeneic mouse islets were transplanted into nonautoimmune diabetic hosts and islet function was analyzed in control and AAT treated hosts. In untreated controls, marginal mass islet transplants did not restore euglycemia. Outcomes were dramatically improved by short-term AAT treatment. Transcriptional profiling identified 1,184 differentially expressed transcripts in AAT-treated hosts at 3 d posttransplantation. Systems-biology-based analysis revealed AAT down-regulated regulatory hubs formed by inflammation-related molecules (e.g., TNF-α, NF-κB). The conclusions yielded by the systems-biology analysis were rigorously confirmed by QRT-PCR and immunohistology. These data suggest that short-term AAT treatment of human islet transplant recipients may be worthy of a clinical trial.
Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , alfa 1-Antitripsina/metabolismo , Animais , Sobrevivência Celular , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Sistema Imunitário , Inflamação , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Resultado do TratamentoRESUMO
Effective means to identify anti-donor immune activity before the transplant organ is damaged and rejected has been an important goal in transplantation research. Development of sensitive and non-invasive diagnostic methods that probe the immune status of the recipient as well as the resilience of the donor organ should enable personalized application of immunosuppressive drugs. With a non-invasive biomarker for rejection, it should be possible to selectively treat the patients that are rejecting the graft and wean the tolerant patients from immunosuppression. Although A20 is also expressed by activated CD4+ T cells and CD8+ T cells, its expression by mouse tubular cells has been shown to play an important role in protecting allografts from ischemia/reperfusion (I/R) injury and rejection. Using quantitative (real-time) reverse transcriptase polymerase chain reaction (qt-RT-PCR), we showed that expression levels of A20, heme oxygenase (HO)-1, other anti-apoptotic molecules, granzyme-B (GZMB), perforin (PRF1), CD3 and other immune molecules in renal transplant biopsies, urinary cells and peripheral blood cells are predictive of transplantation outcomes. Measuring A20 at mRNA and protein levels has the potentiality to be diagnostic and prognostic of transplantation outcomes and thereby help in timely therapeutic interventions to prolong graft life.
Assuntos
Biomarcadores/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transplante de Rim , Proteínas Nucleares/metabolismo , Apoptose , Humanos , Monitorização Fisiológica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Transplante Homólogo , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
Purinergic signaling and associated ectonucleotidases, such as CD39 and CD73, have been implicated in the pathogenesis of inflammatory bowel disease (IBD). CD39 is known to be a Treg memory cell marker, and here we determine the phenotype and function of CD73(+) CD4(+) T lymphocytes in patients with IBD. We describe elevated levels of CD73(+) CD4(+) T cells in the peripheral blood and intestinal lamina propria of patients with active IBD. The functional phenotype of these CD73(+) CD4(+) T cells was further determined by gene expression, ecto-enzymatic activity, and suppressive assays. Increased numbers of CD73(+) CD4(+) T cells in the periphery and lamina propria were noted during active inflammation, which returned to baseline levels following anti-TNF treatment. Peripheral CD73(+) CD4(+) T cells predominantly expressed CD45RO, and were enriched with IL-17A(+) cells. The CD73(+) CD4(+) cell population expressed higher levels of RORC, IL-17A, and TNF, and lower levels of FOXP3 and/or CD25, than CD73(-) CD4(+) T cells. Expression of CD73 by peripheral CD4(+) T cells was increased by TNF, and decreased by an anti-TNF monoclonal antibody (infliximab). In vitro, these peripheral CD73(+) CD4(+) T cells did not suppress proliferation of CD25(-) effector cells, and expressed higher levels of pro-inflammatory markers. We conclude that the CD73(+) CD4(+) T-cell population in patients with active IBD are enriched with cells with a T-helper type 17 phenotype, and could be used to monitor disease activity during treatment.
Assuntos
5'-Nucleotidase/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa/imunologia , Células Th17/imunologia , 5'-Nucleotidase/sangue , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Citometria de Fluxo , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/imunologia , Fármacos Gastrointestinais/farmacologia , Fármacos Gastrointestinais/uso terapêutico , Humanos , Imunofenotipagem , Doenças Inflamatórias Intestinais/sangue , Infliximab , Leucócitos Mononucleares/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Inflammation induces the NF-κB dependent protein A20 in human renal proximal tubular epithelial cells (RPTEC), which secondarily contains inflammation by shutting down NF-κB activation. We surmised that inducing A20 without engaging the pro-inflammatory arm of NF-κB could improve outcomes in kidney disease. We showed that hepatocyte growth factor (HGF) increases A20 mRNA and protein levels in RPTEC without causing inflammation. Upregulation of A20 by HGF was NF-κB/RelA dependent as it was abolished by overexpressing IκBα or silencing p65/RelA. Unlike TNFα, HGF caused minimal IκBα and p65/RelA phosphorylation, with moderate IκBα degradation. Upstream, HGF led to robust and sustained AKT activation, which was required for p65 phosphorylation and A20 upregulation. While HGF treatment of RPTEC significantly increased A20 mRNA, it failed to induce NF-κB dependent, pro-inflammatory MCP-1, VCAM-1, and ICAM-1 mRNA. This indicates that HGF preferentially upregulates protective (A20) over pro-inflammatory NF-κB dependent genes. Upregulation of A20 supported the anti-inflammatory effects of HGF in RPTEC. HGF pretreatment significantly attenuated TNFα-mediated increase of ICAM-1, a finding partially reversed by silencing A20. In conclusion, this is the first demonstration that HGF activates an AKT-p65/RelA pathway to preferentially induce A20 but not inflammatory molecules. This could be highly desirable in acute and chronic renal injury where A20-based anti-inflammatory therapies are beneficial.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Inflamação/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Células Cultivadas , Proteínas de Ligação a DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Túbulos Renais Proximais/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Regulação para Cima/efeitos dos fármacosRESUMO
Complement dependent cytotoxicity crossmatch (CDC-XM) has been the original standard crossmatch test, whereas, flow cytometry crossmatch (FCXM) is an enhanced and highly sensitive crossmatch assay performed to detect donor specific anti-HLA antibodies (DSA). We analyzed American Society for Histocompatibility and Immunogenetics (ASHI) proficiency testing data (2011-2020) and examined the number of laboratories performing CDC-XM vs. FCXM, the overall efficiency of laboratories in reporting ≥80% consensus CDC-XM vs. FCXM result, and reasons for non-consensus results in the two assays. Of 600 crossmatches in each crossmatch category, the percentage of laboratories reporting T cell CDC-XMs reduced from 40% in 2011 to 13% in 2020, T cell anti-human globulin (AHG) CDC-XM reduced from 56% in 2011 to 21% in 2020, and B cell CDC-XM reduced from 51% in 2011 to 20% in 2020. The percentage of laboratories performing T cell and B cell FCXM remained at approximately 80% throughout. CDC-XM performed on par with FCXM in providing a consensus negative result using negative DSA serum, but under-performed in comparison to FCXM in providing a consensus positive result using positive DSA serum. In addition, only minority of CDC-XMs was reported positive in presence of complement fixing DSA. This study shows that non-consensus CDC-XM was always in presence of HLA IgG DSA and that laboratories may be struggling to interpret the low sensitive CDC-XM results, where highly sensitive solid phase multi-antigen or single antigen assay shows the presence of HLA IgG DSA in serum.
Assuntos
Transplante de Rim , Laboratórios , Citometria de Fluxo , Rejeição de Enxerto , Antígenos HLA , Teste de Histocompatibilidade/métodos , Humanos , Imunoglobulina G , IsoanticorposRESUMO
OBJECTIVES: A T-cell-positive and B-cell-negative flow cytometry crossmatch result remains a conundrum since HLA class I antigens are expressed on both T and B cells. We investigated the frequency, donor HLA specificity of the antibodies, and mechanisms for these crossmatch results. MATERIALS AND METHODS: We analyzed 3073 clinical flow cytometry crossmatch tests performed in an American Society of Histocompatibility and Immunogeneticsaccredited histocompatibility laboratory. The sera associated with the T-cell positive and B-cell negative flow cytometry crossmatches were also tested for donor HLA immunoglobulin G antibodies using LABScreen single antigen assays. RESULTS: Among the 3073 test results, 1963 were T-cell negative and B-cell negative, 811 were T-cell negative and B-cell positive, 274 were T-cell positive and B-cell positive, and 25 were T-cell positive and B-cell negative. The LABScreen single antigen assay detected HLA class I immunoglobulin G donor-specific antibodies in 23 of 25 sera associated with a T-cell positive and B-cell negative flow cytometry crossmatch result, and donorspecific antibodies directed at not only HLA-Cw but also at HLA-A or HLA-B were observed. In addition, we identified that the B-cell channel shift threshold used to classify a B-cell flow cytometry crossmatch was a potential contributor to a T-cell-positive and B-cellnegative flow cytometry crossmatch result. CONCLUSIONS: Our analysis of 3073 flow cytometry crossmatches, in addition to demonstrating that HLA antibodies directed at the HLA-A, -B, or -Cw locus were associated with a T-cell-positive and B-cell-negative result, identified mechanisms for the surprising T-cell-positive and B-cell-negative flow cytometry crossmatch result.
Assuntos
Antígenos HLA , Linfócitos T , Citometria de Fluxo/métodos , Antígenos HLA-A , Teste de Histocompatibilidade/métodos , Humanos , Imunoglobulina G , Resultado do TratamentoRESUMO
Invasive insulitis is a destructive T cell-dependent autoimmune process directed against insulin-producing beta cells that is central to the pathogenesis of type 1 diabetes mellitus (T1DM) in humans and the clinically relevant nonobese diabetic (NOD) mouse model. Few therapies have succeeded in restoring long-term, drug-free euglycemia and immune tolerance to beta cells in overtly diabetic NOD mice, and none have demonstrably enabled enlargement of the functional beta cell mass. Recent studies have emphasized the impact of inflammatory cytokines on the commitment of antigen-activated T cells to various effector or regulatory T cell phenotypes and insulin resistance and defective insulin signaling. Hence, we tested the hypothesis that inflammatory mechanisms trigger insulitis, insulin resistance, faulty insulin signaling, and the loss of immune tolerance to islets. We demonstrate that treatment with alpha1-antitrypsin (AAT), an agent that dampens inflammation, does not directly inhibit T cell activation, ablates invasive insulitis, and restores euglycemia, immune tolerance to beta cells, normal insulin signaling, and insulin responsiveness in NOD mice with recent-onset T1DM through favorable changes in the inflammation milieu. Indeed, the functional mass of beta cells expands in AAT-treated diabetic NOD mice.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , alfa 1-Antitripsina/farmacologia , Idade de Início , Animais , Diabetes Mellitus Tipo 1/imunologia , Feminino , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Inflamação/imunologia , Insulina/metabolismo , Resistência à Insulina , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de TempoRESUMO
CD4+ T helper (Th) cell subset generation in vivo requires T cell receptor activation and surface CD28 co-stimulation in the presence of one or more cytokines. Similarly, Th cells can be generated in vitro by activating naïve CD4+CD25- T cells with plate bound-anti-CD3 monoclonal antibody (mAb) (pbCD3) and soluble-anti-CD28 mAb (sCD28) in the presence of polarizing recombinant (r) cytokines and anti-cytokine mAbs. In comparison to in vitro CD4+CD25- T cells, memory CD4+CD25-CD45RO+ T cells have been shown to convert to Th9 cells more efficiently. Here, protocol for in vitro generation of human Th9 cells by activating CD4+CD25-CD45RO+ memory T cells with pbCD3 and sCD28 in the presence of polarizing recombinant interleukin-4 (rIL-4) and transforming growth factor (rTGF-ß) is described.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Anticorpos Monoclonais/metabolismo , Antígenos CD4/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-4/metabolismo , Interleucina-9/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
The relationship between circulating pre-transplant immunoglobulin G (IgG) antibodies to donor human leukocyte antigen (HLA) -C locus determined antigens alone and acute rejection, kidney allograft function, and graft survival is not fully defined. Also, the impact of circulating pre-transplant IgG antibodies to donor HLA-C locus antigens alone on these outcomes has not been compared with the impact of circulating pre-transplant IgG antibodies to donor HLA-A or -B locus antigens. We conducted a retrospective review of records of 1252 kidney allograft recipients transplanted at our center between January 2010 and January 2016 to identify patients with circulating pre-transplant IgG antibodies directed at kidney donor HLA-A, -B, or -C locus determined antigens. Antibodies were detected and reported using the LABScreen Single Antigen Bead assay with microbeads coated with single HLA class I antigens. Pre-transplant and post-transplant data were collected and the graft outcomes of 16 kidney graft recipients with antibodies to HLA-C locus antigens were compared to the outcomes in 56 recipients with antibodies to HLA-A or -B locus determined antigens. The one-year acute rejection rate was 6% in those with donor-specific antibodies (DSA) to HLA-C locus antigens and 20% in those with DSA to HLA-A or -B locus antigens. The graft survival rate was 100% in those with DSA to HLA-C locus antigens and 95% in those with DSA to HLA-A or -B locus antigens. None of the numerical differences were statistically significant (p>0.05). The presence of circulating pre-transplant IgG antibodies directed at kidney donor HLA-C locus antigens alone may not be associated with an increased risk of acute rejection or a decreased graft survival rate. Our observations support the concept that circulating pre-transplant IgG antibodies directed at kidney donor HLA-C locus antigens alone do not negatively impact kidney allograft outcomes and that the mean fluorescence intensities of the antibodies directed at HLA-C locus alone should not be used to list unacceptable HLA-C locus antigens for kidney allocation. A study with a larger cohort is needed to investigate our hypothesis.
Assuntos
Sobrevivência de Enxerto , Antígenos HLA , Antígenos HLA-A , Isoanticorpos , Transplante de Rim , Aloenxertos , Rejeição de Enxerto , Humanos , Imunoglobulina G , Estudos Retrospectivos , Doadores de TecidosRESUMO
CD4(+)CD25(high) T regulatory (Tr) cells, representing high IL-2 receptor alpha chain expressing cells, have been shown to inhibit proliferation and cytokine secretion by CD4(+) T cells that are assumed to represent important effector cells in auto-aggressive immunity. Tr cells may therefore be considered of importance in the pathogenesis of multiple sclerosis (MS). Glatiramer acetate (GA; Copaxone) is approved as a disease-modulating agent that ameliorates the course of MS. The goal of this study was to examine in vitro effects of GA on Tr cells from MS patients subgrouped according to treatment without or with disease-modulating drugs, and healthy controls (HC). Three-colour flow cytometry was used to investigate in vitro influence of GA, and of the encephalitogenic myelin basic protein (MBP) peptide 83-89 as control, on the blood Tr cell proportion and on their functionally important cell surface molecules CD45RO, CD69, CD95 and HLA-DR, and on intracellular CTLA-4 and IL-10. Irrespective of exposure to GA or MBP((83-99)), levels of blood Tr cells expressing HLA-DR remained low in untreated MS patients and HC compared to the three treated MS patient groups. In vitro exposure to GA resulted in elevated levels of IL-10 producing Tr cells in all MS patient groups irrespective of receiving treatment as well as in HC. Exposure to GA or MBP((83-99)) had no effects on levels of Tr cells expressing other above-mentioned molecules. We conclude that GA induces elevated IL-10 production by Tr cells that is uniform and independent of ongoing MS treatment with IFN-beta or GA or IFN-beta+GA.
Assuntos
Adjuvantes Imunológicos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Interleucina-10/biossíntese , Esclerose Múltipla/imunologia , Peptídeos/farmacologia , Receptores de Interleucina-2/biossíntese , Subpopulações de Linfócitos T/efeitos dos fármacos , Adjuvantes Imunológicos/uso terapêutico , Adulto , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Feminino , Acetato de Glatiramer , Antígenos HLA-DR/biossíntese , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Proteína Básica da Mielina/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos/uso terapêutico , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Magnetic resonance imaging (MRI) allows examining inflammation and central nervous system (CNS) tissue damage in patients suffering from multiple sclerosis (MS), a demyelinating disease of the CNS. Using real-time PCR, we quantified mRNA levels of apoptosis regulators CD95, CD95 ligand, caspase-8, -10 and cellular FLICE-inhibitory protein (cFLIP), and cytokines IL-10 and TNF-alpha in blood mononuclear cells of MS patients at the time of MRI examination. Patients with detectable gadolinium-enhancing lesions had lower expression of CD95 and caspase-8 (P<0.05). Lesion load and brain atrophy did not correlate with expression levels of any of the target molecules studied. Disease duration correlated positively with both FLIP/caspase-8 and CD95/CD3 ratios (P<0.05). These results support the notion that the CD95-dependent pathway plays a complex role in the regulation of survival of activated immune cells in MS.
Assuntos
Caspases/biossíntese , Caspases/genética , Gadolínio , Imageamento por Ressonância Magnética/métodos , Esclerose Múltipla/metabolismo , Receptor fas/biossíntese , Receptor fas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Caspase 8 , Caspase 9 , Caspases/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/enzimologia , Esclerose Múltipla/patologia , RNA Mensageiro/biossíntese , Estatísticas não Paramétricas , Receptor fas/sangueRESUMO
The predominant anti-HLA IgG subclass during antibody mediated rejection episodes is IgG1. Class I DSA IgG subclasses respond to single cycle of bortezomib-based therapy more frequently as compared to Class II DSA IgG subclasses. High titer Class II DSA IgG subclasses are less likely to respond to single cycle bortezomib-based therapy. Future studies are needed to determine if the response to bortezomib-based therapy is dependent on the type of DSA and/or the strength of the DSA at the time of antibody mediated rejection episode.
Assuntos
Ácidos Borônicos/uso terapêutico , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/imunologia , Antígenos HLA/imunologia , Imunoglobulina G/imunologia , Transplante de Órgãos , Pirazinas/uso terapêutico , Adulto , Antineoplásicos/uso terapêutico , Bortezomib , Feminino , Humanos , Imunoglobulina G/sangue , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Plasmaferese , Esteroides/uso terapêutico , Doadores de TecidosRESUMO
BACKGROUND: Previously, we have demonstrated that short-term treatment of new onset diabetic Non-obese diabetic (NOD) mice, mice that are afflicted with both type 1 (T1D) and type 2 (T2D) diabetes with either Power Mix (PM) regimen or alpha1 antitrypsin (AAT) permanently restores euglycemia, immune tolerance to self-islets and normal insulin signaling. METHODOLOGY AND PRINCIPAL FINDINGS: To search for relevant therapeutic targets, we have applied genome wide transcriptional profiling and systems biology oriented bioinformatics analysis to examine the impact of the PM and AAT regimens upon pancreatic lymph node (PLN) and fat, a crucial tissue for insulin dependent glucose disposal, in new onset diabetic non-obese diabetic (NOD) mice. Systems biology analysis identified tumor necrosis factor alpha (TNF-α) as the top focus gene hub, as determined by the highest degree of connectivity, in both tissues. In PLNs and fat, TNF-α interacted with 53% and 32% of genes, respectively, associated with reversal of diabetes by previous treatments and was thereby selected as a therapeutic target. Short-term anti-TNF-α treatment ablated a T cell-rich islet-invasive and beta cell-destructive process, thereby enhancing beta cell viability. Indeed anti-TNF-α treatment induces immune tolerance selective to syngeneic beta cells. In addition to these curative effects on T1D anti-TNF-α treatment restored in vivo insulin signaling resulting in restoration of insulin sensitivity. CONCLUSIONS: In short, our molecular analysis suggested that PM and AAT both may act in part by quenching a detrimental TNF-α dependent effect in both fat and PLNs. Indeed, short-term anti-TNF-α mAb treatment restored enduring euglycemia, self-tolerance, and normal insulin signaling.
Assuntos
Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 2/etiologia , Fator de Necrose Tumoral alfa/fisiologia , Tecido Adiposo/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Diabetes Mellitus Tipo 2/terapia , Feminino , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Tolerância Imunológica , Insulina/sangue , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/fisiologia , Transplante das Ilhotas Pancreáticas , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Biologia de Sistemas , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , alfa 1-Antitripsina/farmacologiaRESUMO
It has been reported that rapamycin (RPM) can induce de novo conversion of the conventional CD4(+)Foxp3(-) T cells into CD4(+)Foxp3(+) regulatory T cells (iTregs) in transplantation setting. It is not clear whether RPM can similarly generate suppressor CD8(+) T cells to facilitate graft acceptance. In this study, we investigated the ability of short-term RPM treatment in promoting long-term acceptance (LTA) of MHC-mismatched skin allografts by generating a CD8(+) suppressor T-cell population. We found that CD4 knockout (KO) mice (in C57BL/6 background, H-2(b)) can promptly reject DBA/2 (H-2(d)) skin allografts with mean survival time (MST) being 13 days (p < 0.01). However, a short course RPM treatment in these animals induced LTA with graft MST longer than 100 days. Adoptive transfer of CD8(+) T cells from LTA group into recombination-activating gene 1 (Rag-1)-deficient mice provided donor-specific protection of DBA/2 skin grafts against cotransferred conventional CD8(+) T cells. Functionally active immunoregulatory CD8(+) T cells also resided in donor skin allografts. Eighteen percent of CD8(+) suppressor T cells expressed CD28 as measured by flow cytometry, and produced reduced levels of IFN-γ, IL-2, and IL-10 in comparison to CD8(+) effector T cells as measured by ELISA. It is unlikely that CD8(+) suppressor T cells mediated graft protection via IL-10, as IL-10/Fc fusion protein impaired RPM-induced LTA in CD4 KO mice. Our data supported the notion that RPM-induced suppressor CD8(+) T cells home to the allograft and exert donor-specific graft protection.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Rejeição de Enxerto/prevenção & controle , Imunossupressores/farmacologia , Sirolimo/farmacologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Rejeição de Enxerto/mortalidade , Sobrevivência de Enxerto/efeitos dos fármacos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pele/patologia , Transplante de Pele , Transplante HomólogoRESUMO
PURPOSE: Resistance to antiangiogenic therapy is an important clinical problem. We examined whether resistance occurs at least in part via reversible, physiologic changes in the tumor, or results solely from stable genetic changes in resistant tumor cells. EXPERIMENTAL DESIGN: Mice bearing two human RCC xenografts were treated with sorafenib until they acquired resistance. Resistant 786-O cells were harvested and reimplanted into naïve mice. Mice bearing resistant A498 cells were subjected to a 1 week treatment break. Sorafenib was then again administered to both sets of mice. Tumor growth patterns, gene expression, viability, blood vessel density, and perfusion were serially assessed in treated vs control mice. RESULTS: Despite prior resistance, reimplanted 786-O tumors maintained their ability to stabilize on sorafenib in sequential reimplantation steps. A transcriptome profile of the tumors revealed that the gene expression profile of tumors upon reimplantation reapproximated that of the untreated tumors and was distinct from tumors exhibiting resistance to sorafenib. In A498 tumors, revascularization was noted with resistance and cessation of sorafenib therapy and tumor perfusion was reduced and tumor cell necrosis enhanced with re-exposure to sorafenib. CONCLUSIONS: In two RCC cell lines, resistance to sorafenib appears to be reversible. These results support the hypothesis that resistance to VEGF pathway therapy is not solely the result of a permanent genetic change in the tumor or selection of resistant clones, but rather is due to a great extent to reversible changes that likely occur in the tumor and/or its microenvironment.
Assuntos
Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/tratamento farmacológico , Piridinas/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Niacinamida/análogos & derivados , Compostos de Fenilureia , SorafenibeRESUMO
BACKGROUND: IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL-4+TGF-beta directs mouse CD4(+)CD25(-)CD62L(+) T cells to commit to inflammatory IL-9 producing CD4(+) T cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-beta induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4(+)CD25(-)CD45RO(+) T cells as compared to naïve CD4(+)CD25(-)CD45RA(+) T cells. In addition, as compared to pbCD3/sCD28 plus TGF-beta stimulation, IL-4+TGF-beta stimulated memory CD4(+)CD25(-)CD45RO(+) T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4(+)IL-9(+) T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNgamma or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-beta stimulated resting memory CD4(+) T cells demonstrated that the addition of IL-1beta, IL-12, and IL-21 further enhance IL-9 production. CONCLUSIONS/SIGNIFICANCE: Taken together these data show both the differences and similarities between mouse and human CD4(+)IL9(+) T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4(+) T cells to antigen.
Assuntos
Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica , Interleucina-9/imunologia , Citocinas/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Humanos , Reação em Cadeia da PolimeraseRESUMO
We have previously demonstrated that biomarkers of inflammation and immune activity detected within intraoperative renal transplant allograft biopsies are linked to adverse short-term post-transplantation clinical outcomes. Now we provide a post hoc analysis of our earlier data in the light of longer clinical follow-up. A total of 75 consecutively performed renal allografts were analyzed for gene expression of proinflammatory molecules, inflammation-induced adhesion molecules, and antiapoptotic genes expressed 15 minutes after vascular reperfusion to determine whether this analysis can aid in predicting long-term quality of renal function, proteinuria, graft loss, and death-censored graft. We have built predictive models for proteinuria (area under the curve = 0.859, p = 0.0001) and graft loss (area under the curve = 0.724, p = 0.027) 2 years post-transplantation using clinical variables in combination with intragraft gene expression data of tumor necrosis factor-alpha, interleukin-6, CD40, CD3, and tumor necrosis factor-alpha, Bcl-2, and interferon-gamma, respectively. This post hoc analysis demonstrates that hypothesis-driven, targeted polymerase chain reaction profiling of gene expression in the donor kidney at the time of engraftment can predict 2-year post-transplantation clinical outcomes.