RESUMEN
BACKGROUND: More accurate predictive factors for colorectal cancer (CRC) are urgently needed. This study aimed to assess the potential prognostic roles of circulating tumor cells (CTCs), neutrophil-lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR) in CRC patients. METHODS: Between 2014 and 2017, 118 CRC patients newly diagnosed at the Affiliated Zhongshan Hospital of Dalian University were retrospectively analyzed, including 72 (61%) patients that underwent radical resection (resectable CRC) and 46 (39%) advanced patients with metastatic CRC (mCRC). The CellSearch System was used to detect CTCs, and Spearman's correlation analyses tested the correlations between CTC counts and both NLR and PLR. Statistical analyses were performed using the Kaplan-Meier method, log-rank tests, and Cox proportional hazards models. RESULTS: Of the resectable cohort, 24% were positive for CTCs. Of the advanced cohort, 49% were positive for CTCs. The presence of CTCs was associated with advanced age (≥63 years old; P=0.037), a high PLR value (P=0.008), and a high NLR value (P=0.034). Additionally, baseline NLR [hazard ratio (HR) =0.423; 95% confidence intervals (CI), 0.223-0.803; P=0.008], PLR (HR =0.513; 95% CI, 0.276-0.954; P=0.035), and CTC counts (HR =2.155; 95% CI, 1.152-4.032; P=0.016) were significantly associated with progression-free survival (PFS) in a univariate analysis of mCRC patients that received chemotherapy. Multivariate analysis further showed that NLR (P=0.044) and CTCs (P=0.047) were independent prognostic factors for mCRC patients. CONCLUSIONS: This study provided evidence that NLR and CTC counts could serve as robust prognostic factors for patients with mCRC.
RESUMEN
BACKGROUND: The activation of inflammation and coagulation cascades plays an essential role in the development of various malignancies, including metastatic breast cancer (MBC). This retrospective study aimed to investigate the prognostic role of the combination of fibrinogen and the inflammation-based index in patients with MBC. METHODS: A total of 176 patients with MBC were retrospectively reviewed. The clinical and pathological data of included patients were followed-up and analyzed. The plasma fibrinogen concentration (FIB), neutrophil-lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR) were measured. Dynamic variations in the FIB, NLR, and PLR values were collected from 56 MBC patients before and after first-line therapy. Receiver operating characteristic (ROC) curves were constructed to assess the optimal cut-off values. Correlations between FIB and NLR or PLR were evaluated using Spearman correlation analysis. The Kaplan-Meier method, two-tailed log-rank test, and Cox proportional hazard model were used for statistical analysis. RESULTS: Baseline FIB was positively correlated with NLR and PLR values in MBC patients (P<0.05). Additionally, multivariable analysis proved that the ERBB2 + subtype (P=0.023), basal-like subtype (P=0.032), targeted therapy (P=0.033), other regimens (P=0.005), and baseline FIB level (P=0.004) were independent prognostic variables for progression-free survival (PFS) in MBC patients. Furthermore, ERBB2+, basal-like subtypes, and baseline hyperfibrinogenemia were independent factors for poor prognosis in MBC patients [hazard ratio (HR): 3.717, 95% confidence interval (CI): 1.561-8.851, P=0.003; HR: 3.245, 95% CI: 1.368-7.698, P=0.008; HR: 2.069, 95% CI: 1.352-3.167, P=0.001, respectively]. Most importantly, the FIB level increased significantly after first-line therapy in patients with disease progression (3.73±0.63 vs. 5.32±0.52 g/L, P=0.042) and also decreased markedly in stable disease (3.42±1.05 vs. 3.03±0.73 g/L, P=0.036). However, PFS and overall survival (OS) were not significantly correlated with the dynamic changes of FIB and the inflammation-based index. CONCLUSIONS: The present study provided evidence that baseline FIB combined with NLR and PLR could serve as prognostic predictors for MBC patients. Dynamic change of FIB before and after first-line therapy could also be used as a potential predictor of therapeutic response in MBC patients.
RESUMEN
A constant provision of ATP is of necessity for cardiac contraction. As the heart progresses toward failure following a myocardial infarction (MI), it undergoes metabolic alterations that have the potential to compromise the ability to meet energetic demands. This study evaluated the efficacy of mesenchymal stem cell (MSC) transplantation into the infarcted heart to minimize impairments in the metabolic processes that contribute to energy provision. Seven and twenty-eight days following the MI and MSC transplantation, MSC administration minimized cardiac systolic dysfunction. Hyperinsulinemic-euglycemic clamps, coupled with 2-[(14)C]deoxyglucose administration, were employed to assess systemic insulin sensitivity and tissue-specific, insulin-mediated glucose uptake 36 days following the MI in the conscious, unrestrained, C57BL/6 mouse. The improved systolic performance in MSC-treated mice was associated with a preservation of in vivo insulin-stimulated cardiac glucose uptake. Conserved glucose uptake in the heart was linked to the ability of the MSC treatment to diminish the decline in insulin signaling as assessed by Akt phosphorylation. The MSC treatment also sustained mitochondrial content, ADP-stimulated oxygen flux, and mitochondrial oxidative phosphorylation efficiency in the heart. Maintenance of mitochondrial function and density was accompanied by preserved peroxisome proliferator-activated receptor-γ coactivator-1α, a master regulator of mitochondrial biogenesis. These studies provide insight into mechanisms of action that lead to an enhanced energetic state in the infarcted heart following MSC transplantation that may assist in energy provision and dampen cardiac dysfunction.
Asunto(s)
Adenosina Difosfato/farmacología , Glucosa/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Mitocondrias Cardíacas/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/cirugía , Animales , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiologíaRESUMEN
BACKGROUND: This study aimed to evaluate the efficacy of mesenchymal stem cell (MSC) transplantation to mitigate abnormalities in cardiac-specific and systemic metabolism mediated by a combination of a myocardial infarction and diet-induced insulin resistance. METHODS: C57BL/6 mice were high-fat fed for eight weeks prior to induction of a myocardial infarction via chronic ligation of the left anterior descending coronary artery. MSCs were administered directly after myocardial infarction induction through a single intramyocardial injection. Echocardiography was performed prior to the myocardial infarction as well as seven and 28 days post-myocardial infarction. Hyperinsulinemic-euglycemic clamps coupled with 2-[14C]deoxyglucose were employed 36 days post-myocardial infarction (13 weeks of high-fat feeding) to assess systemic insulin sensitivity and insulin-mediated, tissue-specific glucose uptake in the conscious, unrestrained mouse. High-resolution respirometry was utilized to evaluate cardiac mitochondrial function in saponin-permeabilized cardiac fibers. RESULTS: MSC administration minimized the decline in ejection fraction following the myocardial infarction. The greater systolic function in MSC-treated mice was associated with increased in vivo cardiac glucose uptake and enhanced mitochondrial oxidative phosphorylation efficiency. MSC therapy promoted reductions in fasting arterial glucose and fatty acid concentrations. Additionally, glucose uptake in peripheral tissues including skeletal muscle and adipose tissue was elevated in MSC-treated mice. Enhanced glucose uptake in these tissues was associated with improved insulin signalling as assessed by Akt phosphorylation and prevention of a decline in GLUT4 often associated with high-fat feeding. CONCLUSIONS: These studies provide insight into the utility of MSC transplantation as a metabolic therapy that extends beyond the heart exerting beneficial systemic effects on insulin action.
Asunto(s)
Dieta Alta en Grasa , Metabolismo Energético , Resistencia a la Insulina , Trasplante de Células Madre Mesenquimatosas , Infarto del Miocardio/cirugía , Miocardio/metabolismo , Tejido Adiposo/metabolismo , Animales , Glucemia/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Ácidos Grasos/sangre , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Insulina/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/metabolismo , Músculo Esquelético/metabolismo , Infarto del Miocardio/sangre , Infarto del Miocardio/fisiopatología , Fosforilación Oxidativa , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Recuperación de la Función , Volumen Sistólico , Sístole , Factores de TiempoRESUMEN
Intense interest has been focused on cell-based therapy for the infarcted heart given that stem cells have exhibited the ability to reduce infarct size and mitigate cardiac dysfunction. Despite this, it is unknown whether mesenchymal stem cell (MSC) therapy can prevent metabolic remodeling following a myocardial infarction (MI). This study examines the ability of MSCs to rescue the infarcted heart from perturbed substrate uptake in vivo. C57BL/6 mice underwent chronic ligation of the left anterior descending coronary artery to induce a MI. Echocardiography was performed on conscious mice at baseline as well as 7 and 23 days post-MI. Twenty-eight days following the ligation procedure, hyperinsulinemic euglycemic clamps assessed in vivo insulin sensitivity. Isotopic tracer administration evaluated whole body, peripheral tissue, and cardiac-specific glucose and fatty acid utilization. To gain insight into the mechanisms by which MSCs modulate metabolism, mitochondrial function was assessed by high-resolution respirometry using permeabilized cardiac fibers. Data show that MSC transplantation preserves insulin-stimulated fatty acid uptake in the peri-infarct region (4.25 ± 0.64 vs. 2.57 ± 0.34 vs. 3.89 ± 0.54 µmol·100 g(-1)·min(-1), SHAM vs. MI + PBS vs. MI + MSC; P < 0.05) and prevents increases in glucose uptake in the remote left ventricle (3.11 ± 0.43 vs. 3.81 ± 0.79 vs. 6.36 ± 1.08 µmol·100 g(-1)·min(-1), SHAM vs. MI + PBS vs. MI + MSC; P < 0.05). This was associated with an enhanced efficiency of mitochondrial oxidative phosphorylation with a respiratory control ratio of 3.36 ± 0.18 in MSC-treated cardiac fibers vs. 2.57 ± 0.14 in the infarct-only fibers (P < 0.05). In conclusion, MSC therapy exhibits the potential to rescue the heart from metabolic aberrations following a MI. Restoration of metabolic flexibility is important given the metabolic demands of the heart and the role of energetics in the progression to heart failure.
Asunto(s)
Metabolismo Energético/fisiología , Trasplante de Células Madre Mesenquimatosas , Mitocondrias/metabolismo , Infarto del Miocardio/terapia , Miocardio/metabolismo , Remodelación Ventricular/fisiología , Animales , Corazón/fisiopatología , Ratones , Mitocondrias/patología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Fosforilación/fisiologíaRESUMEN
Liver allografts are spontaneously accepted in the liver transplantation mouse model; however, the basis for this tolerance and the conditions that abrogate spontaneous tolerance to liver allografts are incompletely understood. We examined the role of CpG oligodeoxynucleotide (ODN) in triggering the liver inflammatory reaction and allograft rejection. Bioluminescence imaging quantified the activation of nuclear transcriptional factor kappaB (NF-kappaB) at different time points post-transplantation. Intrahepatic lymphocyte subsets were analyzed by immunofluorescence assay and flow cytometry. The results showed that liver allografts survived for more than 100 days without a requirement for any immunosuppressive therapy. Donor-matched cardiac allografts were permanently accepted, whereas third-party cardiac grafts were rejected with delayed kinetics; this confirmed donor-specific tolerance. NF-kappaB activation in the liver allografts was transiently increased on day 1 and diminished by day 4; in comparison, it was elevated up to 10 days post-transplantation in the cardiac allografts. When CpG ODN was administered at a high dose (50 microg per mouse x 1) to the recipients on day 7 post-transplantation, it induced an acute liver inflammatory reaction with elevated NF-kappaB activation in both allogeneic and syngeneic liver grafts. Multiple doses of CpG ODN (10 microg per mouse x 3) elicited acute rejection of the liver allografts with significant T cell infiltration in the liver allografts, reduced T regulatory cells, and enhanced interferon gamma-producing cells in the intrahepatic infiltrating lymphocytes. These data demonstrate that CpG ODN initiates an inflammatory reaction and abrogates spontaneous tolerance in the liver transplantation mouse model. Liver Transpl 15:915-923, 2009. (c) 2009 AASLD.
Asunto(s)
Islas de CpG , Trasplante de Hígado/inmunología , Hígado/patología , Oligonucleótidos/genética , Animales , Citometría de Flujo/métodos , Tolerancia Inmunológica , Inflamación , Cinética , Trasplante de Hígado/métodos , Linfocitos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Microscopía Fluorescente/métodos , FN-kappa B/metabolismo , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Protein kinase C-theta (PKCtheta) mediates critical T-cell receptor signals required for T-cell activation. We have recently shown that PKCtheta knockout (PKCtheta, H-2b) T cells, when transferred into T/B cell-deficient mice, failed to reject fully allogeneic (H-2d) cardiac grafts and that transgenic expression of antiapoptotic Bcl-xL gene in PKCtheta T cells restored allograft rejection. METHODS: We used PKCtheta mice as recipients of cardiac allografts, compared with wild-type (WT) cardiac allograft transplantation. Anti-CD154 monoclonal antibody (MR1) and human CTLA4Ig were sued to induce donor-specific tolerance. T-cell proliferation, T-cell subsests, nuclear factor kappa B (NF-kappaB) activation, and Bax and Bcl-xL were analyzed. RESULTS: Although suboptimal anti-CD154 monoclonal antibody or human CTLA4Ig failed to delay cardiac allograft rejection in WT mice, the same therapy induced long-term survival of cardiac allografts in PKCtheta mice. Donor-type second cardiac allografts (H-2d) were accepted, and third-party heart allografts (H-2k) were rejected by tolerant PKCtheta mice. However, tolerance state could not be effectively transferred with T cells from tolerance PKCtheta mice. Compared with WT mice, reduced NF-kappaB activation, T-cell proliferation, and T-cell infiltration in PKCtheta spleens were observed. PKCtheta mice reveal reduced CD4/CD25/FoxP3, Th1/Th17 subsets, and mouse MHC class II (IE)-reactive CD4Vbeta11 T cells. Apoptotic molecule, Bax, was increased and antiapoptotic molecule, Bcl-xL, was reduced in PKCtheta spleen cells. CONCLUSION: We concluded that PKCtheta mice have a defected alloimmune response and are susceptible to tolerance induction, which is associated with a clonal deletion of T-cell subsets.
Asunto(s)
Trasplante de Corazón/inmunología , Isoenzimas/deficiencia , Proteína Quinasa C/deficiencia , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/inmunología , Ligando de CD40/inmunología , Antígeno CTLA-4 , Humanos , Tolerancia Inmunológica , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Linfocitos/citología , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa C-theta , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Trasplante HomólogoRESUMEN
The impact of memory B cells and alloantibodies on the ability to induce transplantation tolerance has not been elucidated. We have developed a murine heart transplant model that isolates the contributions of functional memory B cells from memory T cells in allograft rejection. Memory 3-83 B cells with dual specificity for H-2K(k) and H-2K(b) were generated in 3-83 Igi BCR knockin (BALB/c background) mice by the transplantation of C3H (H-2K(k)) hearts in the absence of immunosuppression. To test the effect of functional memory 3-83 B cells, C3H-primed 3-83 Igi recipients were challenged with C57BL/6 hearts (H-2K(b)) at 60-90 days post-C3H heart transplant and treated with anti-CD154 mAbs. Despite immunosuppression, the C57BL/6 hearts were acutely rejected within 10-13 days and graft rejection was associated with increased frequencies of C57BL/6-specific IFN-gamma-producing T cells. Histology revealed significant numbers of infiltrating T cells, consistent with acute T cell-mediated rejection. The resistance to tolerance induction was dependent on the synergistic effects of memory 3-83 B cells and alloantibodies, whereas memory T cells are not necessary. We conclude that the combined effects of functional memory B cells and alloantibodies prevent anti-CD154-mediated graft acceptance by facilitating the CD40-CD154-independent activation of alloreactive T cells. This study provides insight into the potential ability of memory B cells and alloantibodies to prevent anti-CD154-mediated graft acceptance.
Asunto(s)
Anticuerpos Monoclonales/fisiología , Subgrupos de Linfocitos B/inmunología , Ligando de CD40/inmunología , Rechazo de Injerto/inmunología , Trasplante de Corazón/inmunología , Memoria Inmunológica , Isoanticuerpos/metabolismo , Isoantígenos/metabolismo , Enfermedad Aguda , Animales , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/trasplante , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Ligando de CD40/antagonistas & inhibidores , Femenino , Técnicas de Sustitución del Gen , Rechazo de Injerto/patología , Supervivencia de Injerto/inmunología , Trasplante de Corazón/patología , Tolerancia Inmunológica , Terapia de Inmunosupresión , Isoanticuerpos/biosíntesis , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante HeterotópicoRESUMEN
BACKGROUND: We characterized the role of T cell subsets and major histocompatibility complex molecules in allograft rejection and recurrence of autoimmune diabetes. METHODS: Adoptive cell transfer and vascularized segmental pancreas transplantation were performed in mice. RESULTS: In an alloimmune response model, transfer of nondiabetic CD4, but not CD8 T cells, elicited pancreas allograft rejection in streptozotocin (STZ)-induced diabetic NOD/scid mice. Pancreas allografts were acutely rejected in STZ-induced diabetic NOD/beta2m mice (confirmed the absence of major histocompatibility complex [MHC] class I and CD8 T cells) and permanently accepted in NOD/CIIT mice (confirmed the absence of MHC class II and CD4 T cells). The results suggest that rejection of pancreas allograft is CD4-dependent and MHC class I-independent. In the autoimmune diabetes model, whole spleen cells obtained from diabetic NOD mice induced autoimmune diabetes in NOD/scid and NOD/CIIT mice, but the onset of diabetes was delayed in NOD/beta2m mice. However, the purified diabetic T cells failed to elicit autoimmune diabetes in NOD/beta2m mice. NOD/scid and NOD/CIIT pancreas grafts were acutely destroyed whereas four of six NOD/beta2m pancreas grafts were permanently accepted in autoimmune diabetic NOD mice. CONCLUSION: CD4 T cells are sufficient for the induction of allograft rejection, and MHC class I molecule is required to induce recurrent autoimmune diabetes after pancreas transplantation in mice.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/etiología , Rechazo de Injerto/etiología , Antígenos de Histocompatibilidad Clase I/fisiología , Trasplante de Páncreas/efectos adversos , Animales , Linfocitos T CD8-positivos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Recurrencia , Trasplante HomólogoRESUMEN
Exposure to certain viruses and parasites has been shown to prevent the induction of transplantation tolerance in mice via the generation of cross-reactive memory T cell responses or the induction of bystander activation. Bacterial infections are common in the perioperative period of solid organ allograft recipients in the clinic, and correlations between bacterial infections and acute allograft rejection have been reported. However, whether bacterial infections at the time of transplantation have any effect on the generation of transplantation tolerance remains to be established. We used the Gram-positive intracellular bacterium Listeria monocytogenes (LM) as a model pathogen because its effects on immune responses are well described. Perioperative LM infection prevented cardiac and skin allograft acceptance induced by anti-CD154 and donor-specific transfusion in mice. LM-mediated rejection was not due to the generation of cross-reactive T cells and was largely independent of signaling via MyD88, an adaptor for most TLRs, IL-1, and IL-18. Instead, transplant rejection following LM infection was dependent on the expression of the phagosome-lysing pore former listeriolysin O and on type I IFN receptor signaling. Our results indicate that bacterial exposure at the time of transplantation can antagonize tolerogenic regimens by enhancing alloantigen-specific immune responses independently of the generation of cross-reactive memory T cells.
Asunto(s)
Rechazo de Injerto/inmunología , Trasplante de Corazón/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Transducción de Señal/inmunología , Trasplante de Piel/inmunología , Tolerancia al Trasplante , Animales , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/metabolismo , Efecto Espectador/inmunología , Rechazo de Injerto/metabolismo , Rechazo de Injerto/microbiología , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/metabolismo , Humanos , Memoria Inmunológica , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Interleucina-1/inmunología , Interleucina-1/metabolismo , Interleucina-18/inmunología , Interleucina-18/metabolismo , Isoantígenos/inmunología , Isoantígenos/metabolismo , Listeria monocytogenes/metabolismo , Listeriosis/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/inmunología , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Trasplante HomólogoRESUMEN
Alloantibodies can play a key role in acute and chronic allograft rejection. However, relatively little is known of factors that control B cell responses following allograft tolerance induction. Using 3-83 Igi mice expressing an alloreactive BCR, we recently reported that allograft tolerance was associated with the sustained deletion of the alloreactive B cells at the mature, but not the immature, stage. We have now investigated the basis for the long-term control of alloreactive B cell responses in a non-BCR-transgenic model of C57BL/6 cardiac transplantation into BALB/c recipients treated with anti-CD154 and transfusion of donor-specific spleen cells. We demonstrate that the long-term production of alloreactive Abs by alloreactive B cells is actively regulated in tolerant BALB/c mice through the dominant suppression of T cell help. Deletion of CD25(+) cells resulted in a loss of tolerance and an acquisition of the ability to acutely reject allografts. In contrast, the restoration of alloantibody responses required both the deletion of CD25(+) cells and the reconstitution of alloreactive B cells. Collectively, these data suggest that alloreactive B cell responses in this model of tolerance are controlled by dominant suppression of T cell help as well as the deletion of alloreactive B cells in the periphery.
Asunto(s)
Linfocitos B/inmunología , Rechazo de Injerto/inmunología , Trasplante de Corazón/inmunología , Isoanticuerpos/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Linfocitos T/inmunología , Tolerancia al Trasplante/inmunología , Animales , Linfocitos B/patología , Ligando de CD40/inmunología , Rechazo de Injerto/genética , Rechazo de Injerto/patología , Depleción Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de Antígenos de Linfocitos B/genética , Linfocitos T/patología , Factores de Tiempo , Tolerancia al Trasplante/genética , Trasplante HomólogoRESUMEN
BACKGROUND: The purpose of the current study was to evaluate the role of bioluminescence imaging (BLI) in the determination of nuclear factor (NF)-kappaB activation in cardiac allograft rejection and ischemia-reperfusion injury. METHODS: To visualize NF-kappaB activation, luciferase transgenic mice under the control of a mouse NF-kappaB promoter (NF-kappaB-Luc) were used as donors or recipients of cardiac grafts. Alternatively, NF-kappaB-Luc spleen cells were adoptively transferred into Rag2 -/- mice with or without cardiac allografts. BLI was performed posttransplantation to detect luciferase activity that represents NF-kappaB activation. RESULTS: The results show that luciferase activity was significantly increased in the cardiac allografts when NF-kappaB-Luc mice were used as recipients as well as donors. Luciferase activity was also elevated in the wild-type cardiac allografts in Rag2 -/- mice that were transferred with NF-kappaB-Luc spleen cells. CD154 monoclonal antibody (mAb) therapy inhibited luciferase activity and induced long-term survival of cardiac allografts. toll-like receptor-9 ligand, CpG DNA, enhanced luciferase activity and abrogated tolerance induction by CD154 mAb. Luciferase activity was also increased in ischemia-reperfusion injury of the cardiac grafts. CONCLUSION: BLI using Luc-NF-kappaB mice is a noninvasive approach to visualize the activation of NF-kappaB signaling in mouse cardiac allograft rejection and ischemia-reperfusion injury. CD154 mAb can inhibit NF-kappaB activation, which is reversed by toll-like receptor engagement.
Asunto(s)
Trasplante de Corazón/inmunología , Luminiscencia , FN-kappa B/fisiología , Traslado Adoptivo , Animales , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Bazo/inmunología , Trasplante Homólogo/inmunologíaRESUMEN
We have previously reported that anti-Gal-alpha1,3Gal (Gal) IgG3 mAbs mediate a classical complement-dependent hyperacute rejection (HAR), while anti-Gal IgG1 mAbs mediate HAR that is dependent on complement, the Fc-gamma receptors FcgammaRII/III (CD32/CD16), and NK cells. IgG2a and IgG2b subclasses can activate complement and have FcgammaR binding properties in vitro. Whether these IgG subclasses can mediate HAR in vivo and the mechanisms by which they would do so are not known. In this study, we isolated spontaneous IgG switch mutants from an anti-Gal IgG1 hybridoma. In vitro complement-mediated hemolytic assays with mouse complement indicate that both anti-Gal IgG2a and IgG2b mAbs were more potent compared with the parent anti-Gal IgG1. In vivo administration of anti-Gal IgG2a and IgG2b mAbs into Gal-/- mice induced HAR of rat cardiac xenografts. HAR induced by anti-Gal IgG2a and IgG2b was dependent on complement activation and the presence of NK cells. Using FcgammaRIII-deficient (Gal-/-CD16-/-) recipients, we observed that HAR mediated by different anti-Gal IgG subclasses was variably dependent on FcgammaRIII, with IgG1>IgG2b>>IgG2a=IgG3. Using FcgammaRI-deficient (Gal-/-CD64-/-) recipients, we observed that HAR mediated by anti-Gal IgG1, IgG2a, and IgG2b, but not by anti-Gal IgG3, was dependent on FcgammaRI. Collectively, these studies demonstrate the necessity and sufficiency of complement in IgG3-mediated HAR and the necessity of both complement and FcgammaR, especially FcgammaRI, in IgG1-, IgG2a-, and IgG2b-mediated HAR.
Asunto(s)
Activación de Complemento , Disacáridos/inmunología , Rechazo de Injerto/inmunología , Inmunoglobulina G/inmunología , Receptores de IgG/fisiología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Proteínas del Sistema Complemento/metabolismo , Disacáridos/genética , Rechazo de Injerto/genética , Trasplante de Corazón , Hibridomas/metabolismo , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Células Asesinas Naturales/inmunología , Ratones , Ratones Noqueados , Ratas , Ratas Endogámicas Lew , Receptores de IgG/genéticaRESUMEN
Alloreactive B cells can contribute to graft rejection. Anti-CD154 treatment together with donor-specific transfusion (DST) results in the long-term survival of MHC-mismatched mouse heart grafts and inhibition of alloantibody production. To characterize the mechanism of B cell tolerance induced by the anti-CD154 and DST, we used 3-83Igi mice, on BALB/c (H-2K(d)) background, that express a B cell receptor that reacts with MHC class I antigens H-2K(b). Transplanting C57BL/6 (H-2K(b)) hearts into 3-83Igi mice, followed by tolerance induction, resulted in the peripheral deletion of mature but not immature 3-83 B cells. The sustained deletion of mature alloreactive B cells required the presence of the allograft and can be explained by the absence of T cell help.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Supresión Clonal/inmunología , Animales , Antígenos CD4/inmunología , Ligando de CD40/inmunología , Muerte Celular , Epítopos/inmunología , Trasplante de Corazón/inmunología , Ratones , Bazo/citología , Trasplante HomólogoRESUMEN
BACKGROUND: FK778 is a malononitrilamide, a class of immune suppressive compounds with antiviral features and experimental activity in chronic rejection, a potentially interesting combination for organ transplantation. The goal of this project was to study the tolerability, immune suppressive efficacy, and anti-cytomegalovirus (CMV) activity of FK778 and to assess the in vivo relevance of its previously described inhibition of de novo pyrimidine synthesis. METHODS: Heart transplants were performed in rats (Brown Norway [BN] to Lewis) and treated with varying doses of FK778 or leflunomide for 28 days. At 28 days, at the time of rejection or at the death of the animal, the allograft and other vital organs were obtained for study by light microscopy and immunohistochemistry. In separate experiments, Lewis rats were given sublethal irradiation, inoculated with rat CMV (Maastricht strain), and treated with varying doses of FK778 and leflunomide. In both the transplant and CMV studies, IP uridine was given at 250 mg/kg to cohorts or animals receiving FK778 and leflunomide. RESULTS: FK778 controls acute rejection and inhibits CMV replication at 20 mg/kg but is toxic at 25 mg/kg. Toxicity is manifested as anemia, changes in hepatic and intestinal histology, and mortality. The toxicity but not the immune suppressive or antiviral efficacy, is reduced significantly by exogenous uridine administration. CONCLUSION: FK778 has both immune suppressive and antiviral activities, neither of which is entirely dependent on inhibition of pyrimidine synthesis. These, and other published observations, suggest that the antiviral activity and a considerable part of the efficacy of the malononitrilamide family of drugs is attributable to activities other than drug induced pyrimidine deficiency.
Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/tratamiento farmacológico , Trasplante de Corazón , Inmunosupresores/farmacología , Isoxazoles/farmacología , Alquinos , Animales , Peso Corporal/efectos de los fármacos , Rechazo de Injerto/prevención & control , Nitrilos , Ratas , Ratas Endogámicas BN , Trasplante Homólogo , Uridina/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Classic hyperacute rejection is dependent on the activation of the terminal components of complement. Recently, xenoantibodies with limited abilities to activate the classical pathway of complement in vitro have been implicated in the acute vascular rejection of xenografts. It is unclear how these Abs affect their pathogenic activities in vivo. In this study, we demonstrate the ability of an anti-Gal-alpha1,3Gal (Gal) IgG1, with modest complement-activating abilities in vitro, to induce xenograft rejection. This rejection was dependent on the activation of complement, on FcgammaR-mediated interactions, and on the presence of NK cells. Inhibition of any one of these factors resulted in the abrogation of IgG1-mediated rejection. In contrast, an anti-Gal IgG3 mAb induced classic, hyperacute rejection that was solely dependent on complement activation. Our observations implicate two types of IgG-mediated rejection; one that is dependent on complement activation, and a second that is uniquely dependent on complement, FcgammaR, and NK cells.
Asunto(s)
Rechazo de Injerto/inmunología , Inmunoglobulina G/inmunología , Células Asesinas Naturales/inmunología , Trasplante Heterólogo/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Activación de Complemento/inmunología , Disacáridos/inmunología , Trasplante de Corazón/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Endogámicas Lew , Receptores de IgG/inmunologíaRESUMEN
We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Trasplante Óseo/inmunología , Ligando de CD40/inmunología , Interferón gamma/biosíntesis , Interleucina-10/fisiología , Tolerancia al Trasplante/inmunología , Animales , Supresión Clonal/genética , Trasplante de Corazón/inmunología , Sueros Inmunes/administración & dosificación , Sueros Inmunes/farmacología , Terapia de Inmunosupresión , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Interferón gamma/antagonistas & inhibidores , Interleucina-10/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de Piel/inmunología , Donantes de Tejidos , Trasplante Homólogo/inmunologíaRESUMEN
BACKGROUND: We studied the ability of CD4 and CD8 T cells to induce rejection of pancreas xenografts in a concordant combination using rat pancreas xenografts as donors and chemically induced diabetic mice as recipients. METHODS: Lewis rat (2 to 3 weeks old) pancreas xenografts were transplanted into streptozotocin (STZ)-induced diabetic mice. Lymphocyte proliferation and cytokine production were analyzed in vitro. All pancreas xenografts were assessed by functional (blood glucose) and histopathologic examinations. RESULTS: Lewis rat pancreas grafts were rejected within 10 to 13 days, with mononuclear cell infiltrate and tissue necrosis in STZ-induced diabetic mice. A predominant T cell receptor alphabeta -CD4 cell (on day 4) and T cell receptor alphabeta -CD8 cell (on day 8) infiltrate and IgM deposition were found in the pancreas xenografts after transplantation. Anti-CD4 (GK1.5), but not anti-CD8 (YTS169.4), monoclonal antibodies resulted in a prolonged survival of Lewis rat pancreas xenografts. Lewis pancreas xenografts were permanently accepted by CD4 knockout mice but not by CD8 knockout mice. The pancreas xenografts were acutely rejected with a mean survival time of 15.3 days in B cell-deficient mice (microMT/microMT). Transfer of CD4 but not CD8 spleen cells from naïve C57BL/6 mice into Rag2 mice led to acute rejection of transplanted pancreas xenografts. However, activated CD8 spleen cells elicited rejection of Lewis rat pancreas xenografts in SZT-induced diabetic mice. CONCLUSION: The current results show that CD4 T cells are necessary and sufficient for mediating the rejection of Lewis rat pancreas xenografts in STZ-induced diabetic mice. However, CD8 cells, when activated, can also induce acute rejection of concordant pancreas xenografts.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Rechazo de Injerto/fisiopatología , Trasplante de Páncreas , Trasplante Heterólogo , Animales , Diabetes Mellitus Experimental/cirugía , Ratones , Ratones Endogámicos , Ratas , Ratas Endogámicas LewRESUMEN
BACKGROUND: One of the most promising approaches to achieving allograft tolerance involves the transient inhibition of co-stimulatory signals in T cells. There is, however, increasing evidence that this approach alone cannot universally elicit allograft tolerance and that adjunct therapies capable of synergizing with co-stimulation blockade may be necessary. METHODS: We developed a novel tolerance strategy involving co-transplantation of intact allogeneic bone fragments containing active bone marrow (intact active bone [IAB]) with heart allograft and transient anti-CD40L monoclonal antibody therapy. RESULTS: Mice treated with IAB and anti-CD40L were tolerant to major histocompatibility complex and minor antigen-mismatched cardiac and skin allografts. Heart allografts had normal histology up to 270 days posttransplantation, and the production of graft-reactive antibodies was inhibited. Microchimerism, but no macrochimerism, of donor cells was detected in the peripheral blood or lymphoid organs of tolerant mice receiving IAB and anti-CD40L. Lymphocytes from tolerant mice retained normal proliferative responsiveness to donor cells in vitro but demonstrated a donor-specific loss in the priming of interferon-gamma responses. The ability to produce interleukin-2 or -4 when stimulated with donor cells was normal. CONCLUSIONS: Contrary to previous reports of the ability of bone marrow cells to induce central deletional tolerance, our data suggest that the regimen involving co-transplantation of IAB on the day of heart allograft transplantation and transient anti-CD40L therapy induces a robust donor-specific peripheral tolerance.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Trasplante de Médula Ósea/inmunología , Ligando de CD40/inmunología , Galactosiltransferasas/fisiología , Trasplante de Corazón/inmunología , Terapia de Inmunosupresión/métodos , Trasplante Homólogo/inmunología , Animales , Galactosiltransferasas/deficiencia , Galactosiltransferasas/genética , Supervivencia de Injerto/inmunología , Isoanticuerpos/inmunología , Ratones , Ratones Noqueados , Linfocitos T/inmunología , Quimera por TrasplanteRESUMEN
The generation of GT-Ko mice has provided unique opportunities to study allograft and xenograft rejection in the context of anti-alpha1,3-Gal antibody (anti-Gal Ab) responses. In this study we used the allotransplantation model of C3H hearts into galactosyltransferase-deficient (GT-Ko) mice and the xenotransplantation model of baby Lewis rat hearts into GT-Ko mice to investigate the ability of CTLA-41g in combination with anti-CD40L mAb to control graft rejection and anti-Gal Ab production. Murine CTLA-41g or anti-CD40L monotherapy prolonged allograft survival, and the combination of these reagents was most immunosuppressive. However short-term treatment with murine cytotoxic T lymphocyte associated antigen-4 (muCTLA-41g) and/or CD40 ligand (CD154) monoclonal antibodies (anti-CD40L mAbs) was unable to induce indefinite allograft survival. CTLA-4-immunoglobulin fusion protein (CTLA-41g) or anti-CD40L monotherapy only marginally prolonged xenograft survival; the combination of human CTLA-41g and anti-CD40L significantly prolonged xenograft survival (74days), while the combination of murine CTLA-41g and anti-CD40L resulted in graft survival of >120days. CTLA-41g or anti-CD40L monotherapy or the combination of these agents inhibited the production of alloAbs, including anti-Gal Abs. CTLA-41g or anti-CD40L monotherapy partially controlled xenoAb and anti-Gal Ab production, while the combination was more effective. These observations corroborate our previous observations that humoral, including anti-Gal Ab, responses and rejection following allograft or concordant xenograft transplantation in GT-Ko mice are T-cell dependent and can be controlled by costimulation blockade.