RESUMEN

Background Systematic lupus erythematosus (SLE) is characterized with various complications which can cause serious organ damage in the human body. Despite the significant improvements in disease management of SLE patients, the non-invasive diagnosis is entirely missing. In this study, we used urinary peptidomic biomarkers for early diagnosis of disease onset to improve patient risk stratification, vital for effective drug treatment. Methods Urine samples from patients with SLE, lupus nephritis (LN) and healthy controls (HCs) were analyzed using capillary electrophoresis coupled to mass spectrometry (CE-MS) for state-of-the-art biomarker discovery. Results A biomarker panel made up of 65 urinary peptides was developed that accurately discriminated SLE without renal involvement from HC patients. The performance of the SLE-specific panel was validated in a multicentric independent cohort consisting of patients without SLE but with different renal disease and LN. This resulted in an area under the receiver operating characteristic (ROC) curve (AUC) of 0.80 ( p < 0.0001, 95% confidence interval (CI) 0.65-0.90) corresponding to a sensitivity and a specificity of 83% and 73%, respectively. Based on the end terminal amino acid sequences of the biomarker peptides, an in silico methodology was used to identify the proteases that were up or down-regulated. This identified matrix metalloproteinases (MMPs) as being mainly responsible for the peptides fragmentation. Conclusions A laboratory-based urine test was successfully established for early diagnosis of SLE patients. Our approach determined the activity of several proteases and provided novel molecular information that could potentially influence treatment efficacy.

Asunto(s)

Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/orina , Péptidos/orina , Biomarcadores/orina , Estudios de Casos y Controles , Electroforesis Capilar , Humanos , Espectrometría de Masas , Proteoma

RESUMEN

The tumor necrosis factor like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 (Fn14), mediate inflammation and neuronal apoptosis in cerebral edema, ischemic stroke and multiple sclerosis. The downstream effectors and pathways linked to TWEAK-Fn14 signaling are strongly implicated in the pathology of Parkinson's disease (PD), thus indicating a putative role for TWEAK/Fn14 signaling in PD neurodegeneration. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, we aimed to determine whether genetic ablation or pharmacologic mitigation of the TWEAK protein and its Fn14 receptor affected substantia nigra and striatum Parkinsonian pathology. Changes in endogenous TWEAK protein expression were also quantified in tissue from both MPTP-treated mice and PD human samples. TWEAK protein expression was transiently increased in the striatal tissue but remained unaltered in substantia nigra tissue of MPTP-treated mice. There was also no change of TWEAK protein levels in the substantia nigra or the striatum of human PD patients as compared to matched control subjects. Mitigating the effects of endogenous TWEAK protein using neutralizing antibody did affect MPTP-mediated neurotoxicity in the substantia nigra using the sub-acute model of MPTP (30mg/kg i.p. over five consecutive days). Neither TWEAK nor Fn14 genetic ablation led to attenuation of MPTP-toxicity in the acute model. These findings suggest that TWEAK signaling might be an aspect of MPTP-mediated neuropathology and be involved in the overall neurodegenerative pathology of PD.

Asunto(s)

Trastornos Parkinsonianos/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factores de Necrosis Tumoral/metabolismo , Anciano , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Citocina TWEAK , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Intoxicación por MPTP , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/fisiología , Receptor de TWEAK

RESUMEN

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a TNF superfamily member, induces damage of the epithelial cells (ECs) and production of inflammatory mediaters through its receptor Fn14 in a model of acute colitis. In our current study of chronic colitis induced by repeated rectal injection of a hapten, we found that inflammation, fibrosis, and T helper 2 (Th2)-type immunity were significantly reduced in Fn14 gene knockout (KO) mice when compared with wild-type (WT) control mice. Expression of thymic stromal lymphopoietin (TSLP) was lower in Fn14 KO colon ECs than in WT ECs. TWEAK potentiates the induction of TSLP by interleukin-13 (IL-13) in colon explants from WT but not in Fn14 KO tissue. TSLP receptor KO mice exhibit milder chronic colitis, similar to that in Fn14 KO mice. TWEAK and IL-13 synergistically promote fibroblast proliferation. Thus we propose an IL-13-TWEAK/Fn14-TSLP axis as a key mechanism underlying chronic colitis with fibrosis.

Asunto(s)

Colitis/inmunología , Colon/patología , Fibroblastos/inmunología , Interleucina-13/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Células Th2/inmunología , Factores de Necrosis Tumoral/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Enfermedad Crónica , Colitis/inducido químicamente , Citocina TWEAK , Modelos Animales de Enfermedad , Femenino , Fibrosis , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Interleucina-13/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Técnicas de Cultivo de Órganos , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Receptor de TWEAK , Ácido Trinitrobencenosulfónico/administración & dosificación , Factores de Necrosis Tumoral/inmunología

RESUMEN

Embryonic pathways are often re-expressed in adult pathology. Here we investigated the role of the morphogen hedgehog (hh), which we found to be re-expressed in atherosclerotic plaques. Male ApoE - /- mice were treated for 12 weeks with an anti-hh antibody (5E1) or a control IgG (1E6) starting at the age of 6 or 18 weeks. Inhibition of hh signalling induced a significant increase in total plaque area in the aortic arch, a result of an increase (54% and 36%, respectively) in the area of advanced plaques (atheromata). In mice treated with anti-hh, plaques contained large (18-35% > ctrl), lipid-filled, sometimes multinucleated macrophage foam cells. Plasma cholesterol levels decreased after anti-hh treatment. In bone marrow-derived macrophages, foam cell formation was enhanced after inhibition of hh signalling. Anti-hh treatment caused a 54-75% increase in early oxLDL uptake (10-240 min), which was scavenger receptor-mediated. After 3-24 h of oxLDL incubation, intense Oil red O staining as well as increased amounts of cholesterol esters were present in these macrophages after anti-hh treatment. Activation of the HH-signalling cascade by recombinant Shh induced a decrease in oxLDL uptake. Here we show that the hh-signalling pathway is one of the morphogenic pathways that regulate plasma lipid levels and atherosclerosis development and progression.

Asunto(s)

Apolipoproteínas E/fisiología , Aterosclerosis/fisiopatología , Proteínas Hedgehog/fisiología , Lípidos/sangre , Macrófagos/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Apolipoproteínas E/deficiencia , Aterosclerosis/sangre , Aterosclerosis/patología , Peso Corporal , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Lipoproteínas LDL/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal

RESUMEN

BACKGROUND: Delayed xenograft rejection is associated with endothelial cell activation, platelet sequestration, and subsequent thrombosis. We evaluated whether human platelets could directly activate porcine endothelium (PEC), and if so, whether this was mediated by an interaction between platelet-bound CD154 and PEC CD40. METHODS: Platelet activation was achieved by thrombin exposure and confirmed by evaluation of up-regulated CD62P and CD154. Co-incubation of platelets or D1.1 cells with PEC was performed, and PEC activation was evaluated by up-regulation of CD62E. RESULTS: Co-incubation of resting platelets that lacked significant expression of CD62P and were void of CD154 did not activate PEC. In contrast, thrombin-activated human platelets expressing considerable amounts of both CD62P and CD154 induced PEC activation. This activation could be completely inhibited by coincubation with a humanized monoclonal antibody directed at human CD154 (hu5c8). Similarly, human D1.1 cells expressing CD154 were shown to activate PEC in a CD154-dependent manner. CONCLUSION: Human CD154 expressed on activated human platelets or on T cells interacts with CD40 expressed on PEC leading to PEC activation. This interaction can be inhibited by a monoclonal antibody directed against CD154, suggesting that an interaction between human CD154 and PEC CD40 is at least in part responsible for PEC activation seen in delayed xenograft rejection. These data strengthen the rationale for the use of CD154-directed therapy in discordant xenotransplantation.

Asunto(s)

Plaquetas/fisiología , Ligando de CD40/fisiología , Endotelio Vascular/fisiología , Transfusión de Plaquetas , Trasplante Heterólogo , Animales , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Células Cultivadas , Endotelio Vascular/citología , Humanos , Selectina-P/fisiología , Porcinos , Linfocitos T/inmunología , Linfocitos T/fisiología , Trombina/farmacología

RESUMEN

BACKGROUND: Allogeneic skin transplantation remains a rigorous test of any immune intervention designed to prevent allograft rejection. To date, no single, clinically available immunosuppressant has been reported to induce long-term primary skin allograft survival in primates. We have previously shown that treatment with the humanized CD154-specific monoclonal antibody, humanized 5C8 (hu5C8), induces long-term renal allograft survival in nonhuman primates. In this study, we evaluated the efficacy of hu5C8 in preventing primary skin allograft rejection in rhesus monkeys. METHODS: Ten rhesus monkeys were transplanted with full-thickness skin allografts mismatched at both class I and class II major histocompatibility loci. Of these, two were given no treatment, five were treated with hu5C8 alone, and three received hu5C8 combined with whole blood donor-specific transfusion (DST). All recipients also received skin autografts for comparison. Animals were followed by inspection, serial biopsy, mixed lymphocyte culture, and alloantibody determination. RESULTS: Treatment with hu5C8 alone or hu5C8 plus DST greatly prolonged allograft survival. Rejection occurred in the untreated group within 7 days. Mean allograft survival in the monotherapy hu5C8 group was >236 days and in the DST group was >202 days; these differences were not significant. Rejection eventually occurred in most animals. Allograft survival was not correlated with the development of T cell hyporesponsiveness in mixed lymphocyte culture. Rejection was not predicted by the development of donor-specific alloantibody. CONCLUSION: These results show that treatment with the CD154-specific monoclonal antibody, hu5C8, greatly delays the onset of acute skin allograft rejection.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Ligando de CD40/inmunología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/fisiología , Trasplante de Piel/inmunología , Enfermedad Aguda , Animales , Anticuerpos Monoclonales Humanizados , Formación de Anticuerpos , Especificidad de Anticuerpos , Supervivencia de Injerto/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Isoanticuerpos/sangre , Macaca mulatta , Trasplante de Piel/patología , Factores de Tiempo , Trasplante Homólogo

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Ligando de CD40/inmunología , Inmunoterapia/métodos , Cooperación Linfocítica/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Arteriosclerosis/inmunología , Arteriosclerosis/terapia , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Antígenos CD40/genética , Antígenos CD40/inmunología , Ligando de CD40/genética , Ligando de CD40/fisiología , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Comunicación Celular/inmunología , Quimiotaxis/fisiología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Regulación de la Expresión Génica , Rechazo de Injerto/prevención & control , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Activación de Linfocitos/inmunología , Macaca fascicularis , Ratones , Modelos Inmunológicos , Familia de Multigenes , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Células del Estroma/citología , Células del Estroma/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Virosis/inmunología

RESUMEN

BACKGROUND: Several conventional forms of immunosuppression have been shown to antagonize the efficacy of anti-CD154 monoclonal antibody- (mAb) based costimulatory molecule blockade immunotherapy. Our objective was to determine if allograft recipients treated with a conventional immunosuppressive regimen could be sequentially converted to anti-CD154 mAb monotherapy without compromising graft survival. METHODS: Outbred juvenile rhesus monkeys underwent renal allotransplantation from MHC-disparate donors. After a 60-day course of triple therapy immunosuppression with steroids, cyclosporine, and mycophenolate mofetil, monkeys were treated with: (1) cessation of all immunosuppression (control); (2) seven monthly doses of 20 mg/kg hu5C8 (maintenance), or; (3) 20 mg/kg hu5C8 on posttransplant days 60, 61, 64, 71, 79, and 88 followed by five monthly doses (induction+maintenance). Graft rejection was defined by elevation in serum creatinine>1.5 mg/dl combined with histologic evidence of rejection. RESULTS: Graft survival for the three groups were as follows: group 1 (control): 70, 75, >279 days; group 2 (maintenance): 83, 349, >293 days, and; group 3 (induction+maintenance): 355, >377, >314 days. Acute rejection developing in two of four monkeys after treatment with conventional immunosuppression was successfully reversed with intensive hu5C8 monotherapy. CONCLUSIONS: Renal allograft recipients can be successfully converted to CD154 blockade monotherapy after 60 days of conventional immunosuppression. An induction phase of anti-CD154 mAb appears to be necessary for optimal conversion. Therefore, although concurrent administration of conventional immunosuppressive agents including steroids and calcineurin inhibitors has been shown to inhibit the efficacy of CD154 blockade, sequential conversion from these agents to CD154 blockade appears to be effective.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Ligando de CD40/inmunología , Rechazo de Injerto/tratamiento farmacológico , Terapia de Inmunosupresión , Trasplante de Riñón , Animales , Supervivencia de Injerto/efectos de los fármacos , Humanos , Inmunohistoquímica , Riñón/metabolismo , Riñón/patología , Macaca mulatta , Retratamiento , Terapia Recuperativa , Trasplante de Piel , Trasplante Homólogo

RESUMEN

Autoimmunity results from a failure in central and/or peripheral tolerance; however, the events that initiate and maintain this dysfunction remain unclear. To better understand the mediators involved in autoimmunity, we investigated the cellular mechanisms maintaining disease in the (SWR x NZB)F(1) (SNF(1)) mouse model of systemic lupus erythematosus. Previously, we have shown that autoimmunity in this model is dependent on CD40-CD154 interactions. Herein, our studies reveal that the severity of disease in SNF(1) mice correlates with a marked increase in the frequency of apoptotic splenocytes, including a higher proportion of apoptotic dendritic cells (DC) in vivo. In addition, we demonstrate a significant disease-related increase in the absolute number of splenic CD11c(high) DC. The increased DC number appears to be attributable to DC proliferation and enhanced migration to the spleen, most likely induced by elevated splenic expression of secondary lymphoid chemokine. Importantly, these imbalances in apoptosis, secondary lymphoid chemokine expression, and DC homeostasis were reduced or normalized by anti-CD154 treatment. Thus, our data demonstrate CD154-dependent regulation of apoptosis and DC homeostasis in mice with lupus-like autoimmune disease. We suggest that these mechanisms comprise an autostimulatory loop, maintaining the cascade of autoimmunity by DC presentation of self-Ags derived from apoptotic cells and CD154-mediated costimulation.

Asunto(s)

Anticuerpos Monoclonales/administración & dosificación , Apoptosis/inmunología , Ligando de CD40/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Homeostasis/inmunología , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Animales , Apoptosis/genética , Recuento de Células , División Celular/genética , División Celular/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Quimiocina CCL21 , Quimiocinas CC/antagonistas & inhibidores , Quimiocinas CC/biosíntesis , Cruzamientos Genéticos , Regulación hacia Abajo/inmunología , Femenino , Homeostasis/genética , Inmunofenotipificación , Nefritis Lúpica/genética , Nefritis Lúpica/prevención & control , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos NZB , Índice de Severidad de la Enfermedad , Bazo/inmunología , Bazo/metabolismo , Bazo/patología

RESUMEN

We generated vascular cell adhesion molecule (VCAM)-1 "knock-in" mice and Cre recombinase transgenic mice to delete the VCAM-1 gene (vcam-1) in whole mice, thereby overcoming the embryonic lethality seen with conventional vcam-1-deficient mice. vcam-1 knock-in mice expressed normal levels of VCAM-1 but showed loss of VCAM-1 on endothelial and hematopoietic cells when interbred with a "TIE2Cre" transgene. Analysis of peripheral blood from conditional vcam-1-deficient mice revealed mild leukocytosis, including elevated immature B cell numbers. Conversely, the bone marrow (BM) had reduced immature B cell numbers, but normal numbers of pro-B cells. vcam-1-deficient mice also had reduced mature IgD+ B and T cells in BM and a greatly reduced capacity to support short-term migration of transferred B cells, CD4+ T cells, CD8+ T cells, and preactivated CD4+ T cells to the BM. Thus, we report an until now unappreciated dominant role for VCAM-1 in lymphocyte homing to BM.

Asunto(s)

Linfocitos B/fisiología , Médula Ósea/fisiología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Movimiento Celular/fisiología , Molécula 1 de Adhesión Celular Vascular/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Molécula 1 de Adhesión Celular Vascular/genética

RESUMEN

Allorejection and recurrence of autoimmunity are the major barriers to transplantation of islets of Langerhans for the cure of type 1 diabetes in humans. CD40-CD154 (CD40 ligand) interaction blockade by the use of anti-CD154 monoclonal antibody (mAb) has shown efficacy in preventing allorejection in several models of organ and cell transplantation. Here we report the beneficial effect of the chronic administration of a hamster anti-murine CD154 mAb, MR1, in prolonging islet graft survival in NOD mice. We explored the transplantation of C57BL/6 islets into spontaneously diabetic NOD mice, a combination in which both allogeneic and autoimmune components are implicated in graft loss. Recipients were treated either with an irrelevant control antibody or with MR1. MR1 administration was effective in prolonging allograft survival, but did not provide permanent protection from diabetes recurrence. The autoimmune component of graft loss was studied in spontaneously diabetic NOD mice that received syngeneic islets from young male NOD mice. In this combination, a less dramatic yet substantial delay in diabetes recurrence was observed in the MR1-treated recipients when compared with the control group. Finally, the allogeneic component was explored by transplanting C57BL/6 islets into chemically induced diabetic male NOD mice. In this setting, long-term graft survival (>100 days) was achieved in MR1-treated mice, whereas control recipients rejected their grafts within 25 days. In conclusion, chronic blockade of CD154 results in permanent protection from allorejection and significantly delays recurrence of diabetes in NOD mice.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Trasplante de Islotes Pancreáticos , Ratones Endogámicos NOD/fisiología , Linfocitos T/inmunología , Animales , Relación CD4-CD8 , Diabetes Mellitus/genética , Diabetes Mellitus/cirugía , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Experimental/cirugía , Supervivencia de Injerto/efectos de los fármacos , Inmunohistoquímica , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Trasplante de Islotes Pancreáticos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Subgrupos de Linfocitos T/efectos de los fármacos , Factores de Tiempo , Trasplante Homólogo

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Ligando de CD40/inmunología , Rechazo de Injerto/prevención & control , Trasplante de Riñón/inmunología , Animales , Relación Dosis-Respuesta Inmunológica , Inmunosupresores/uso terapéutico , Macaca mulatta , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/uso terapéutico

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Ligando de CD40/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Antígenos Comunes de Leucocito/inmunología , Animales , Trasplante de Islotes Pancreáticos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Factores de Tiempo , Trasplante Homólogo

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Ligando de CD40/inmunología , Rechazo de Injerto/inmunología , Trasplante de Piel/inmunología , Animales , Eritema/etiología , Rechazo de Injerto/prevención & control , Macaca mulatta , Factores de Tiempo , Trasplante Autólogo , Trasplante Homólogo

RESUMEN

The anti-inflammatory effects of IL-4 on activated monocytes differ from those on monocyte-derived macrophages (MDMac). While IL-4 suppresses LPS-induced IL-1beta , IL-12, IL-10 and TNFalpha production by monocytes, IL-4 suppresses only IL-1beta and IL-12 production by MDMac. The U937 and Mono Mac 6 cell lines have similar cytokine responses to IL-4 as monocytes and MDMac, respectively. The IL-4Ralpha and IL-2Rgamma (gammac) chains are well-characterized components of the IL-4 receptor. Cross-linking studies with 125I-IL-4 revealed that for monocytes and U937 cells, the binding of IL-4 to the receptor components was approximately 1:1 for IL-4Ralpha:gammac. In contrast, for MDMac and Mono Mac 6 cells that have a relative reduction in gammac surface expression, the binding of IL-4 to IL-4Ralpha:gammac was approximately 3:1. Furthermore, IL-4 induced IL-4Ralpha chain phosphorylation more rapidly in MDMac and Mono Mac 6 cells than in monocytes and U937 cells. This study identifies a correlation between altered 125I-IL-4 cross-linking to IL-4Ralpha:gammac, IL-4-induced signaling and regulation of pro-inflammatory cytokine production by IL-4.

Asunto(s)

Interleucina-4/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Receptores de Interleucina-4/química , Transducción de Señal , Western Blotting , Diferenciación Celular , Reactivos de Enlaces Cruzados/farmacología , Citocinas/metabolismo , Dimerización , Citometría de Flujo , Glicosilación , Humanos , Yodo/farmacología , Fosforilación , Pruebas de Precipitina , Factores de Tiempo , Células Tumorales Cultivadas , Células U937

RESUMEN

Using the murine model of hemophilia A, we have examined the role of CD154 in the secondary immune response to factor VIII (FVIII). We previously reported that repeated i.v. injection of FVIII in hemophilia A mice induces a T cell-dependent anti-FVIII antibody formation. Herein, blocking of CD154 by a monoclonal antibody in FVIII-primed hemophilia A mice resulted in the disappearance of pre-existing spleen germinal centers (GC) in the white pulp within 24 h of treatment. Moreover, further expansion of GC in response to FVIII challenge was completely inhibited. In parallel, anti-FVIII antibody titers were markedly reduced and T cell responses to FVIII were abolished. The rapid disappearance of the GC after anti-CD154 treatment was not accompanied by increased B cell apoptosis; instead B cells accumulated in the peripheral zone of the splenic white pulp. Interestingly, repeated exposure to FVIII with anti-CD154 antibody administration blocked anti-FVIII antibody formation but failed to induce long-lasting unresponsiveness. Our data demonstrate that the CD40-CD154 interaction is critical for B cell homeostasis and the secondary immune response to FVIII. For potential clinical application, the data also suggest that therapies targeting the CD154 molecule may be useful for the treatment of high titer FVIII inhibitors in hemophilia A.

Asunto(s)

Factor VIII/antagonistas & inhibidores , Centro Germinal/fisiología , Glicoproteínas de Membrana/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Apoptosis , Ligando de CD40 , Femenino , Hemofilia A/terapia , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunología

RESUMEN

The beta1 integrin very late antigen 4 (VLA-4) plays a central role in mobilization and homing of CD34+ cells. In this study, we examined the activation state of VLA-4 on CD34+ cells from bone marrow (BM) and peripheral blood (PB) by flow cytometry using a vascular cell adhesion molecule I-immunoglobulin (VCAM-I/IgG) fusion protein as soluble ligand. In an intraindividual analysis, we found a significantly reduced affinity and avidity of the VLA-4 receptor on CD34+ cells from PB during granulocyte colony-stimulating factor (G-CSF)-enhanced marrow recovery in comparison with steady-state BM. Moreover, the amount of circulating CD34+ cells during marrow recovery was inversely related to the activation state but not to the expression level of VLA-4, suggesting that a modulation of the functional state of VLA-4 is involved in the mobilization of CD34+ cells. Moreover, VLA-4 function on CD34+ cells from BM was associated with the maturation state of CD34+ cells as high-affinity VLA-4 receptors were observed on the vast majority of more primitive CD34+ cells. In addition, we found that Mg2+ ions as well as co-incubation of CD34+ cells with endothelial cells resulted in an activation of the VLA-4 receptor. In conclusion, modulation of the functional state of VLA-4 appears to be of relevance for the mobilization and homing of CD34+ haematopoietic stem cells.

Asunto(s)

Antígenos CD34 , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/inmunología , Integrinas/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Adhesión Celular/fisiología , Técnicas de Cocultivo , Endotelio/citología , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/citología , Humanos , Integrina alfa4beta1 , Magnesio/metabolismo , Unión Proteica , Proteínas Recombinantes , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

BACKGROUND: T-cell costimulatory blocking agents inhibit allospecific T-cell responses in vitro and prevent allograft rejection in vivo. Costimulatory requirements for discordant xenospecific cellular responses remain undefined. We have evaluated costimulatory molecule expression by porcine endothelial cells (PEC) after interaction with human cells and tested agents known to inhibit allospecific responses for their ability to inhibit xenospecific responses in vitro. METHODS: Human-specific agents were screened for their ability to bind porcine costimulatory molecules by FACS. Up-regulation of B7 molecules on PEC was evaluated by FACS after exposure to human cells or supernatants. The effect of human and/or porcine costimulatory blockade was tested in xeno-mixed lymphocyte reactions (XMLRs) and in natural killer (NK) cell cytotoxicity assays. RESULTS: B7 expression was induced on PEC after exposure to human T and NK cells or T cell-conditioned medium. The human XMLR was attenuated by human CTLA4-Ig and anti-human CD154 (hu5C8), and the combination was synergistic. Anti-human CD80 and CD86 antibodies alone had minor effects in the XMLR, but in combination with hu5C8 were as effective as human CTLA4-Ig plus hu5C8. Anti-hCD80 and hCD86 antibodies that did not cross-react with porcine CD80 or CD86 were as effective in blocking the MLR as those that did cross-react, indicating that the predominant costimulation in vitro was derived from the responding cells. None of the agents affected the xeno-NK response. CONCLUSIONS: We conclude that the costimulation-modulating agents block human anti-porcine T-cell responses in vitro predominantly through interruption of costimulation derived from responding cells. They have no effect on NK cell-mediated cytotoxicity.

Asunto(s)

Citotoxicidad Inmunológica , Inmunoconjugados , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Trasplante Heterólogo/inmunología , Abatacept , Animales , Antígenos CD/fisiología , Antígenos de Diferenciación/farmacología , Antígeno B7-1/fisiología , Antígeno B7-2 , Ligando de CD40 , Antígeno CTLA-4 , Células Cultivadas , Reacciones Cruzadas , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Humanos , Glicoproteínas de Membrana/fisiología , Porcinos

RESUMEN

In the present study, we investigated the role of the CD40L-CD40 pathway in a model of progressive atherosclerosis. ApoE-/- mice were treated with an anti-CD40L antibody or a control antibody for 12 wk. Antibody treatment started early (age 5 wk) or was delayed until after the establishment of atherosclerosis (age 17 wk). In both the early and delayed treatment groups, anti-CD40L antibody did not decrease plaque area or inhibit lesion initiation or age-related increase in lesion area. The morphology of initial lesions was not affected, except for a decrease in T-lymphocyte content. Effects of anti-CD40L antibody treatment on the morphology of advanced lesions were pronounced. In both the early and delayed treatment groups, T-lymphocyte content was significantly decreased. Furthermore, a pronounced increase in collagen content, vascular smooth muscle cell/myofibroblast content, and fibrous cap thickness was observed. In the delayed treatment group, a decrease in lipid core and macrophage content occurred. Interestingly, advanced lesions of anti-CD40L antibody-treated mice exhibited an increased transforming growth factor beta1 immunoreactivity, especially in macrophages. In conclusion, both early and delayed treatment with an anti-CD40L antibody do not affect atherosclerotic lesion initiation but do result in the development of a lipid-poor collagen-rich stable plaque phenotype. Furthermore, delayed treatment with anti-CD40L antibody can transform the lesion profile from a lipid-rich to a lipid-poor collagen-rich phenotype. Postulated mechanisms of this effect on plaque phenotype are the down-regulation of proinflammatory pathways and up-regulation of collagen-promoting factors like transforming growth factor beta.

Asunto(s)

Apolipoproteínas E/deficiencia , Arteriosclerosis , Antígenos CD40/inmunología , Glicoproteínas de Membrana/inmunología , Factores de Edad , Animales , Anticuerpos/administración & dosificación , Anticuerpos/inmunología , Apolipoproteínas E/genética , Arteriosclerosis/genética , Arteriosclerosis/inmunología , Ligando de CD40 , Modelos Animales de Enfermedad , Ratones , Linfocitos T/inmunología

RESUMEN

Members of the vertebrate hedgehog family (Sonic, Indian, and Desert) have been shown to be essential for the development of various organ systems, including neural, somite, limb, skeletal, and for male gonad morphogenesis. Sonic hedgehog and its cognate receptor Patched are expressed in the epithelial and/or mesenchymal cell components of the hair follicle. Recent studies have demonstrated an essential role for this pathway in hair development in the skin of Sonic hedgehog null embryos. We have further explored the role of the hedgehog pathway using anti-hedgehog blocking monoclonal antibodies to treat pregnant mice at different stages of gestation and have generated viable offspring that lack body coat hair. Histologic analysis revealed the presence of ectodermal placode and primodium of dermal papilla in these mice, yet the subsequent hair shaft formation was inhibited. In contrast, the vibrissae (whisker) development appears to be unaffected upon anti-hedgehog blocking monoclonal antibody treatment. Strikingly, inhibition of body coat hair morphogenesis also was observed in mice treated postnatally with anti-hedgehog monoclonal antibody during the growing (anagen) phase of the hair cycle. The hairless phenotype was reversible upon suspension of monoclonal antibody treatment. Taken together, our results underscore a direct role of the Sonic hedgehog signaling pathway in embryonic hair follicle development as well as in subsequent hair cycles in young and adult mice. Our system of generating an inducible and reversible hairless phenotype by anti-hedgehog monoclonal antibody treatment will be valuable for studying the regulation and mechanism of hair regeneration.

Asunto(s)

Proteínas de Drosophila , Cabello/embriología , Proteínas de Insectos/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Femenino , Cabello/fisiología , Proteínas Hedgehog , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Morfogénesis , Embarazo , Regeneración