RESUMEN
Universal prophylaxis for cytomegalovirus (CMV) prevention is viable but, compared with a preemptive strategy, leads to higher incidence of late-onset disease (LOD) associated with poor patient and graft survival. The purpose of this study was to compare LOD with early onset disease (EOD), with a focus on the highest risk kidney transplant recipients (KTRs): CMV seronegative recipients transplanted from seropositive donors (D+R-). Since CMV control depends on both antiviral treatment and specific immune response, we also compared Vδ2-negative (Vδ2(neg) ) γδ T cell expansion involved in CMV infection resolution. EOD was defined as occurring <3 mo and LOD as occurring >3 mo after transplantation. Depending on the period, universal prophylaxis or preemptive treatment was used. Overall, 168 D+R- KTRs were included between 2003 and 2011. LOD was associated with a lower peak DNAemia (p = 0.04), fewer recurrences (odds ratio 0.16; 95% confidence interval 0.05-0.55; p = 0.01) and shorter anti-CMV curative treatment (40 vs. 60 days, p < 0.0001). As a corollary, we found that Vδ2(neg) γδ T cell expansion was faster in LOD than in EOD (31 vs. 168 days after the beginning of CMV disease, p < 0.0001). In D+R- KTRs, universal prophylaxis is associated with more LOD, which had better infection management and a faster immune response. These results support the use of universal prophylaxis over a preemptive strategy and reappraise outcomes of LOD.
Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Supervivencia de Injerto/inmunología , Fallo Renal Crónico/cirugía , Trasplante de Riñón , Linfocitos T/inmunología , Edad de Inicio , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/virología , Femenino , Estudios de Seguimiento , Tasa de Filtración Glomerular , Supervivencia de Injerto/efectos de los fármacos , Humanos , Pruebas de Función Renal , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Linfocitos T/efectos de los fármacos , Donantes de TejidosRESUMEN
BACKGROUND: The incidence and the impact of asymptomatic cytomegalovirus (CMV) DNAemia occurring after the first year post transplantation is unknown. METHODS: In this retrospective cross-sectional study, we analyzed the incidence, risk factors, and impact of 2-year post-transplantation asymptomatic CMV DNAemia (2YCD) on graft function. We included 892 consecutive asymptomatic kidney transplant recipients transplanted for at least 2 years and all were monitored using whole blood CMV quantitative nucleic acid amplification testing (CMV-QNAT). RESULTS: Twenty-eight patients displayed 2YCD (3.1%). Using multivariate analysis in 578 patients, we found that female gender (odds ratio [OR] = 2.57, P = 0.02), a past history of CMV drug-resistance mutation (OR = 8.73, P = 0.005), and corticosteroid use (OR = 2.37, P = 0.03) were independently associated with an increased risk of 2YCD. 2YCD was associated with an increased incidence of subsequent CMV disease over the year following its diagnosis (7% vs. 0.6%, P = 0.02). Patients with 2YCD also exhibited a declining estimated glomerular filtration rate more frequently (77%) than patients with a negative CMV-QNAT (56%, P = 0.02). CONCLUSION: 2YCD appears to be a rare entity, which appears to be associated with chronic graft dysfunction.
Asunto(s)
Infecciones Asintomáticas , Infecciones por Citomegalovirus/etiología , Citomegalovirus/aislamiento & purificación , Trasplante de Riñón , Complicaciones Posoperatorias , Anciano , Infecciones Asintomáticas/epidemiología , Estudios Transversales , Citomegalovirus/genética , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/epidemiología , Femenino , Estudios de Seguimiento , Supervivencia de Injerto , Humanos , Incidencia , Modelos Logísticos , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/virología , Estudios Retrospectivos , Factores de Riesgo , Factores SexualesRESUMEN
Celiac disease is an auto-immune enteropathy involving genetic factors. It is associated in almost all the patients, to specific susceptibility alleles encoding histocompatibility antigens (HLA for human leucocyte antigen), specifically certain variants of the HLA-DQ2, and the HLA-DQ8 HLA class II molecules. Its estimated prevalence is 1% in the european and north-american populations. However, although these alleles represent the main genetic factor for this disease, they do not explain it on their own, as they are expressed by up to 30% of the population. Recent immunological advances allowed identifying the immunodominant epitopes of gluten, to establish the role of tissue transglutaminase in the disease and to define at the atomic level the presentation of these antigens by the HLA-DQ molecule. It is noteworthy that the HLA susceptibility alleles only account for 40% of the whole genetic risk, and the challenge is now to explain the remaining 60%. Genome-wide association studies using the DNA arrays technology to screen single nucleotide polymorphisms to pinpoint candidate regions and genes, have started to provide answers, but contradictory results sometimes still persist. Most of the genes emerging as statistically significantly associated with celiac disease are involved in the immune response, and suggest that the situation is complex.
Asunto(s)
Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Fenómenos Inmunogenéticos , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Glútenes/efectos adversos , Glútenes/inmunología , Antígenos HLA/genética , Antígenos HLA/inmunología , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Modelos BiológicosRESUMEN
Screening studies using high-sensitivity and specificity markers indicate a prevalence of celiac disease of up to 1% in European and North-American populations. Celiac disease is a frequent condition that has become an important public health issue. Yet the majority of cases remain undiagnosed due to the polymorphism of its clinical manifestations. The new insight in the pathogenesis of celiac disease has lead to the development of new diagnostic tools. Early screening of symptomatic patients and pre-identified at-risk groups significantly improves the quality of life while reducing morbidity and mortality. However, prophylactic benefits of early diagnosis by assessing the general population have not been shown in any study. French and Northern American scientific societies have introduced serological testing in their newly revised strategies to diagnose celiac disease. Older markers judged insufficiently accurate like anti-gliadin and anti-reticulin antibodies have recently been withdrawn from the list of reimbursed medical expenses in France. Anti-endomysium and tissue transglutaminase IgA antibodies have proven to be at this day the most sensitive and specific markers for the diagnosis and follow-up of patients on gluten-free diet, at the exception of IgA-deficient patients. Assays testing for IgG antibodies are recommended upon IgA-deficiency. Although very accurate, a better standardisation of current assays may enable serological testing to replace in a near future histological confirmation brought by small bowel biopsies which remains today the gold standard test to diagnose celiac disease. Indeed, serological testing represents and attractive alternative as it is less invasive, less expansive, laboursaving and more objective in interpretation.
Asunto(s)
Enfermedad Celíaca/diagnóstico , Enfermedad Celíaca/inmunología , Autoanticuerpos/inmunología , Enfermedad Celíaca/epidemiología , Proteínas de Unión al GTP/fisiología , Gliadina/inmunología , Humanos , Tamizaje Masivo/métodos , Prevalencia , Proteína Glutamina Gamma Glutamiltransferasa 2 , Reticulina/inmunología , Pruebas Serológicas , Transglutaminasas/fisiologíaRESUMEN
Mechanically harvested lymphocytes invading an irreversibly rejected human kidney allograft were seeded at limiting dilution to calculate the frequency of growing precursors. Optimal growth frequency (1/13) was obtained when Epstein-Barr virus (EBV)-transformed donor B lymphocytes were used as stimulators (D-BLCL) in the presence of interleukin 2 (IL-2). The 55 clones analyzed were all T11+ and T3+, and all expressed DR antigens (45% were T8+ and 55% T4+). Only one clone had a double-labeled (T4+ T8+) surface. All cells proliferated significantly against D-BLCL, although T4+ clones had a significantly shorter average doubling time than T8+ clones. Nearly all T8+ clones were specifically cytotoxic for D-BLCL, while both T4 and T8 did not react against K562, autologous EBV-BLCL, and third-party EBV-BLCL. Detectable IL-2 was found in the culture supernatants of only a minority of clones (all T4+).
Asunto(s)
Rechazo de Injerto , Trasplante de Riñón , Linfocitos T/citología , Anticuerpos Monoclonales/análisis , División Celular , Supervivencia Celular , Transformación Celular Viral , Células Clonales/citología , Femenino , Herpesvirus Humano 4 , Humanos , Interleucina-2/biosíntesis , Riñón/inmunología , Persona de Mediana Edad , FenotipoRESUMEN
In normal individuals, gammadelta T cells account for less than 6% of total peripheral T lymphocytes and mainly express T-cell receptor (TCR) Vdelta2-Vgamma9 chains. We have previously observed a dramatic expansion of gammadelta T cells in the peripheral blood of renal allograft recipients only when they developed cytomegalovirus (CMV) infection. This increase was long lasting (more than 1 year), was associated with an activation of gammadelta T cells, and concerned only Vdelta1 or Vdelta3 T-cell subpopulations. Analysis of gammadelta TCR junctional diversity revealed that CMV infection in these patients was accompanied by (a) a marked restriction of CDR3 size distribution in Vdelta3 and, to a lesser extent, in Vdelta1 chains; and (b) a selective expansion of Vdelta1 cells bearing recurrent junctional amino acid motifs. These features are highly suggestive of an in vivo antigen-driven selection of gammadelta T-cell subsets during the course of CMV infection. Furthermore, Vdelta1 and Vdelta3 T cells from CMV-infected kidney recipients were able to proliferate in vitro in the presence of free CMV or CMV-infected fibroblast lysates but not uninfected or other herpes virus-infected fibroblast lysates. This in vitro expansion was inhibited by anti-gammadelta TCR mAb's. These findings suggest that a population of gammadelta T cells might play an important role in the immune response of immunosuppressed patients to CMV infection.
Asunto(s)
Citomegalovirus/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Infecciones por Citomegalovirus/etiología , Infecciones por Citomegalovirus/inmunología , Cartilla de ADN/genética , Femenino , Humanos , Técnicas In Vitro , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/inmunología , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Factores de TiempoRESUMEN
The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated by binding either erythropoietin (EPO) or gp55, the Friend spleen focus-forming virus glycoprotein. The highly specific interaction between gp55 and EPO-R triggers cell proliferation and thereby causes the first stage of Friend virus-induced erythroleukemia. We have generated functional chimeric receptors containing regions of the EPO-R and the interleukin-3 receptor (AIC2A polypeptide), a related cytokine receptor which does not interact with gp55. All chimeric receptors were expressed at similar levels, had similar binding affinities for EPO, and conferred EPO-dependent cell growth. Only those chimeric receptors which contained the EPO-R transmembrane region were activated by gp55. These results demonstrate that the transmembrane region of the EPO-R is critical for activation by gp55. In addition, analysis of a soluble, secreted EPO-R and cysteine point mutants of the EPO-R show that the extracytoplasmic region of the EPO-R specifically interacts with gp55.
Asunto(s)
Leucemia Experimental/metabolismo , Receptores de Superficie Celular/metabolismo , Virus Formadores de Foco en el Bazo/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Sitios de Unión/genética , Células Cultivadas , Análisis Mutacional de ADN , Conformación Proteica , Receptores de Superficie Celular/genética , Receptores de Eritropoyetina , Receptores de Interleucina-3/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , TransfecciónRESUMEN
FasL and TRAIL are apoptotic ligands of the TNF-like cytokines family, acting via activation of the transmembrane death domain containing receptors Fas for FasL, and DR4 or DR5 for TRAIL. A glycosylphosphatidylinositol-linked TRAIL receptor called DcR1 behaves as a decoy receptor inhibiting TRAIL-mediated cell death in several cellular systems. We engineered and stably expressed a chimeric GPI-linked Fas receptor (Fas-GPI) in T-lymphocyte cell lines constitutively expressing functional transmembrane Fas. Surprisingly, despite lacking the death domain region of functional Fas, Fas-GPI was able to significantly increase Fas-mediated cell death triggered by membrane bound or soluble FasL, whereas engagement of Fas-GPI alone did not trigger apoptosis. This potentiating effect, but not transmembrane Fas activation, was selectively inhibited by protein kinase C activation with phorbol esters, demonstrating that Fas-GPI activated a specific synergistic signal transduction pathway. Fas-GPI and transmembrane Fas were localized in distinct membrane compartments, since Fas-GPI, but not transmembrane Fas, was found in the glycolipid-rich membrane microdomains. These results suggest that apoptosis induced by members of this ligand/receptors family may be differentially modulated through other and parallel signalling pathways.
Asunto(s)
Apoptosis/fisiología , Glicosilfosfatidilinositoles/metabolismo , Glicoproteínas de Membrana/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Receptor fas/fisiología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Células Cultivadas , Fragmentación del ADN/fisiología , Activación Enzimática/efectos de los fármacos , Proteína Ligando Fas , Humanos , Células Jurkat , Microdominios de Membrana/metabolismo , Ratones , Ingeniería de Proteínas , Proteína Quinasa C/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL hybrid gene. Two types of hybrid BCR-ABL mRNA have been found, B2A2 and B3A2. As the BCR-ABL rearrangement is specific to leukemic cells, selective inhibition of leukemic cell growth by BCR-ABL antisense oligonucleotides (ASO) has been reported in vitro for CML patients and cell lines. However, controversial results have been obtained from preclinical studies using anti-BCR-ABL ASO, as nonspecific inhibition of leukemic cell growth was evidenced in some cases. B3 exon secondary structure was deduced from its sequence and found to be a loop. According to this predictive structure of exon B3, a 56-mer antisense oligonucleotide targeting the polypurine bases from the B2A2 junction was devised which would inhibit proliferation (MTT assay) of B3A2 junction cell lines (K562 and a murine cell line Ba/F3 transfected with the B3A2 junctional sequence). This ASO had a hairpin-like secondary structure and was found to be much more resistant to the action of nucleases than control 18-mer standard oligonucleotides. Hybridization to its target mRNA occurs via formation of a triplex structure. A concentration of 5 microM of specific 56-mer B2A2 ASO was necessary to demonstrate 50% optical density (OD) reduction for K562 cell line and Ba/F3 transformed by B3A2 cDNA. Sense and non-sense 56-mer sequence or 18-mer linear ASO showed no effect for these concentrations. Western blot showed a partial inhibition of P210 protein; expression of P145abl remains unchanged. The 56-mer ASO also inhibited the proliferation of B2A2 junction cell line BV173 at the same concentration and showed no effect on the HL60 cell line used as control.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Oligonucleótidos Antisentido/farmacología , Secuencia de Bases , División Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Estabilidad de Medicamentos , Exones , Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mensajero/química , Transfección , Células Tumorales CultivadasRESUMEN
Human Interleukin for DA cells (HILDA), a cytokine also known as leukemia inhibitory factor (LIF), induces proliferation without concurrent differentiation of murine embryonic stem cells. Therefore, we investigated the effects of recombinant HILDA/LIF on the proliferation and differentiation of human hematopoietic progenitor cells (HPC) grown in long-term bone marrow cultures (LTBMC). Pre-established stromal cell layers were reinoculated with autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells in the presence or absence of HILDA/LIF (200 U/ml). At weekly intervals cultures were sacrificed, and the cells in the adherent and the nonadherent cell fractions were counted. The numbers of HPC were determined by culturing these cells in semisolid medium stimulated with phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), and LTBMC supernatants were assayed in semisolid cultures for the presence of colony-stimulating activity (CSA). The total number of cells, their differential counts, the number of HPC, and the concentrations of CSA in culture supernatants were similar for long-term cultures containing HILDA/LIF and for controls. These data suggest that HILDA/LIF may not play a role in the proliferation and differentiation of normal human (early) HPC in LTBMC. Moreover, HILDA/LIF did not stimulate the proliferation of relatively mature progenitor cells in semisolid cultures, not did it influence the colony formation induced by other colony-stimulating factors (CSF). Finally, using a [3H]thymidine suicide test we could not find an effect of HILDA/LIF on the cell-cycle status of HPC.
Asunto(s)
Células de la Médula Ósea , Inhibidores de Crecimiento/farmacología , Células Madre Hematopoyéticas/citología , Interleucina-6 , Linfocinas/farmacología , Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Granulocitos/citología , Granulocitos/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Factor Inhibidor de Leucemia , Macrófagos/citología , Macrófagos/efectos de los fármacos , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Timidina/metabolismo , Factores de Tiempo , TritioRESUMEN
OBJECTIVE: To study the relationship between the CD4+ cell response after initiation of protease inhibitors and the occurrence of opportunistic infections and survival. DESIGN: Prospective observational cohort study. METHODS: HIV-1-seropositive subjects followed-up in HIV centres of Bordeaux University Hospital, Southwest France who were prescribed at least one available protease inhibitor between January and December 1996 were included in this analysis. A Cox model estimated the independent effect of baseline covariates and CD4+ cell response, considered as a time-dependent covariate, on the occurrence of new AIDS-defining opportunistic infection, new AIDS-defining events, new AIDS-defining opportunistic infection or death. RESULTS: A total of 556 HIV-positive patients were prescribed at least one protease inhibitor: 34% saquinavir, 52% indinavir, and 14% ritonavir. Median CD4+ cell count at baseline was 95 x 10(6)/l and mean plasma HIV RNA was 5.0 log10 copies/ml. After a median follow-up of 230 days, 65 patients experienced a new episode of opportunistic infection, 79 patients experienced at least one AIDS-defining event, and 24 had died. On average, the increase in CD4+ cell count was 42 x 10(6)/l (SD, 74) after a median of 49 days. In the multivariate analysis of opportunistic infection or death, each 50% higher CD4+ cell count at baseline was associated with a 23% reduction [95% confidence interval (CI), 14-30] of risk. Each 50% increase in CD4+ cell count during follow-up was associated with a 9% reduction (95% CI, 2-15) of risk, adjusted for the presence of AIDS prior to protease inhibitor therapy (hazard ratio, 3.76 versus absence of AIDS; P < 0.01) and haemoglobin level (hazard ratio, 0.48 if > 11 g/dl versus <11 g/dl; P < 0.01). CONCLUSION: Our results show, at least indirectly, how protease inhibitors might produce clinical stabilization. This result may be due to improved functionality of CD4+ cells in patients started on protease inhibitors.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/prevención & control , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Inhibidores de la Proteasa del VIH/uso terapéutico , Adulto , Recuento de Linfocito CD4 , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Infecciones por VIH/mortalidad , Humanos , Indinavir/efectos adversos , Indinavir/uso terapéutico , Masculino , Valor Predictivo de las Pruebas , Estudios Prospectivos , Factores de Riesgo , Ritonavir/efectos adversos , Ritonavir/uso terapéutico , Saquinavir/efectos adversos , Saquinavir/uso terapéuticoRESUMEN
In a retrospective study, an increase in double-negative (CD3+ CD4- CD8-) (DN) T lymphocytes has been shown to be an independent predictor of disseminated Mycobacterium avium complex (D.MAC) infection in patients with less than 100 CD4+ T cells per mm3. To better characterize this cell expansion, a prospective study was designed. From July 1995 to April 1997, 206 HIV-infected patients with less than 100 CD4+ T cells per mm3 were prospectively followed up and immunophenotyped. The median followup was 1.1 year (+/-0.5 year), and 14 new D.MAC infections were diagnosed among 84 first AIDS-defining events. In univariate and multivariate analyses, D.MAC infections were the only opportunistic infection with a significant increase in DN T-cell percentage (median = 6.6; range = 1.7 to 24.5, P = 0.004) compared with patients without any opportunistic infection. This alteration in T-lymphocyte count could constitute a predictor for D.MAC infection in clinical practice.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Infección por Mycobacterium avium-intracellulare/inmunología , Subgrupos de Linfocitos T/inmunología , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Infecciones Oportunistas Relacionadas con el SIDA/patología , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Adulto , Anciano , Estudios de Cohortes , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Infección por Mycobacterium avium-intracellulare/complicaciones , Estudios ProspectivosRESUMEN
The concentration of the immunosuppressive drugs cyclosporine A (CSA) and FK506 in biological fluids is routinely determined by antibody-based assays, which for several reasons do not give accurate information on the actual level of immunosuppression in the patient. To alleviate this problem, we developed a functional reporter gene assay which uses the enhancer fragment of the interleukin-2 promoter region driving the expression of the green fluorescent protein (GFP). This construct was stably transfected in the Jurkat human T lymphoblastoid cell line. Upon stimulation of the cell recipient, the GFP was produced and evaluated by flow cytometry. Immunosuppressants acting via inhibition of interleukin-2 synthesis, such as CSA or FK506, inhibited the production of GFP in a dose-dependent manner. This assay can be performed within a working day with a good reproducibility and was more sensitive than the antibody-based assays, since its detection limit was as low as 10 ng/ml for CSA and 0.5 ng/ml for FK506. We used it for the follow up of drug level present in the blood of transplanted patients, and compared the results with those obtained with the antibody-based assay routinely carried out in our hospital. The conclusions suggest that this assay is a valuable alternative to the presently available assays for the measurement of the immunosuppressive activity found in body fluids.
Asunto(s)
Ciclosporina/sangre , Citometría de Flujo/métodos , Inmunosupresores/sangre , Interleucina-2/genética , Proteínas Luminiscentes/genética , Tacrolimus/sangre , Anticuerpos Monoclonales/inmunología , Relación Dosis-Respuesta a Droga , Genes Reporteros , Glucocorticoides/farmacología , Proteínas Fluorescentes Verdes , Humanos , Células Jurkat , Trasplante de Riñón , Proteínas Luminiscentes/metabolismo , Metilprednisolona/farmacología , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Leukemia inhibitory factor (LIF), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT1) act through transmembrane receptors which share the gp190 glycoprotein chain. The understanding of its involvement in the biology of these cytokines is of importance since these systems have recently been shown to participate in major inflammatory and neoplastic processes such as myelomatosis (Rose-John, S., Heinrich, P.C., 1994. Soluble receptors for cytokines and growth factors: generation and biological function. Biochem. J. 300, 281). In addition, this family of receptors also shares the gp130 transducing chain, with the IL6 and IL11 receptors. Because IL6 and gp130 were the first members to be discovered, most of the available reagents are directed at them. In this respect, monoclonal antibodies have played a major role in elucidating these receptor/ligand interactions and exploring the pathophysiological aspects of their biology. So far, no such reagents have been described for the gp190. We now report the production and characterization of 16 monoclonal antibodies directed against human gp190. They were obtained using recombinant chimeric or truncated proteins produced in a eukaryotic CHO cell line. One was able to block the biological activity of LIF. Because gp190 comprises two hematopoietin binding domains, crude epitope mapping was possible using the same reagents. While more of these antibodies are required, the present set validate the technological approach used for their preparation and should improve our understanding of this class of cytokines.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Interleucina-6 , Receptores de Citocinas/inmunología , Animales , Anticuerpos Bloqueadores/biosíntesis , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Secuencia de Bases , Células CHO , Cricetinae , Cartilla de ADN/genética , Inhibidores de Crecimiento/metabolismo , Humanos , Inmunización , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Linfocinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Receptores de Citocinas/antagonistas & inhibidores , Receptores de Citocinas/genética , Receptores OSM-LIF , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Interleukin 2 (IL-2) has been purified by a protocol using gel filtration high performance liquid chromatography (HPLC) and hydrophobic affinity chromatography with blue-trisacryl M. Peripheral blood lymphocytes or tonsillar lymphocytes were stimulated with phytohemagglutinin (PHA). Serum free conditioned medium (CM) containing IL-2, other lymphokines and residual PHA molecules was analyzed after 3 variations of ammonium sulfate (AS) precipitation: (1) precipitation of CM with 50% AS yielded a precipitate containing most of the residual PHA but also a fraction of IL-2. (2) Precipitation with direct 80% AS of crude CM yielded both IL-2 and residual PHA. (3) A double step procedure (50% AS followed by 80% AS) yielded a precipitate containing IL-2 but free of residual lectin. HPLC purification of these various AS-precipitated materials or of lyophilized crude CM yielded 2 peaks with mitogenic activity as assayed with the CTLL2 murine clone or IL-2-dependent human Con A-stimulated lymphoblasts. IFN was easily separated from IL-2 and PHA, but BCGF still copurified with IL-2. Peak I (25 kDa) was enriched 400-fold for IL-2 while peak II (68 kDa) contained the residual PHA. The IL-2-containing fractions eluted from HPLC were further purified by blue-trisacryl M chromatography. The IL-2 eluted with 0.4 M NaCl. The entire protocol (HPLC followed by blue-trisacryl) led routinely to 8000-fold IL-2 enrichment. Preparative HPLC directly applied to lyophilized crude (CM) enriched IL-2 activity 400-fold with yield averaging 60% of the IL-2 input. The final material was free from interferon and IL-1, but BCGF still copurified with IL-2. The 2-step purified material (HPLC and blue-trisacryl) gave 2 bands in SDS-PAGE both of which contained IL-2.
Asunto(s)
Interleucina-2/aislamiento & purificación , Linfocitos/análisis , Sulfato de Amonio/farmacología , Transformación Celular Neoplásica/inmunología , Precipitación Química , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Humanos , Interleucina-2/fisiología , Leucemia Linfoide/inmunología , Activación de Linfocitos , Linfocitos/inmunología , Fitohemaglutininas/farmacologíaRESUMEN
The effect of cyclosporine and metabolite 17 (M17) as well as other CsA-related compounds (CsG, dihydro-CsC, dihydro-CsD, CsH, B5.49, and H7.94) was tested on T lymphocyte clone proliferation. In these experiments, antigen and interleukin 2 (IL-2) dependent long-term T lymphocyte clones derived from a rejected human kidney graft infiltrate were used. They were specifically committed (proliferation and cytotoxicity) for the donor Epstein-Barr virus (EBV)-transformed cells. CsA strongly inhibited clone T cell proliferation induced by the antigen. Inhibition of antigen-driven proliferation was reversed by pure recombinant IL-2 (rec-IL-2) only when low amounts of CsA (less than 25 ng/ml) were used, whereas this lymphokine was ineffective at higher but still pharmacological CsA concentrations (50-500 ng/ml). Increasing rec-IL-2 concentrations did not modify this finding. In addition, CsA, did not inhibit the growth signal(s) induced by rec-IL-2/IL-2 receptor interactions when R-IL-2 is pre-expressed on clone cells. M17 was far less effective in inhibiting antigen-induced clone cell proliferation (50% inhibition at 16 ng/ml versus 500 ng/ml with, respectively, CsA and M17) but was nevertheless inhibitory. This observation, if extended to other metabolites, could be important for interpretation of the relevance of "CsA" concentration through radio-immunoassay monitoring of recipients' blood. Although CsA appeared to display the major inhibitory effect, dihydro-CsC and CsG, as well as B5.49 and H7.94 CsA-related compounds, also exhibited strong activity. Dihydro-CsD was less inhibitory, and CsH had no effect.
Asunto(s)
Ciclosporinas/farmacología , Antígenos HLA-B , Trasplante de Riñón , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Células Clonales/efectos de los fármacos , Depresión Química , Rechazo de Injerto , Antígenos HLA/inmunología , Antígeno HLA-B18 , Antígenos HLA-DR/inmunología , Subtipos Serológicos HLA-DR , Humanos , Interleucina-2/farmacología , Riñón/patología , Proteínas Recombinantes/farmacologíaRESUMEN
HILDA/LIF, a recently described glycoprotein, has been characterized from supernatants of alloreactive T cell clones (CD4 and CD8) extracted from a human rejected kidney graft. This suggests a possible role for HILDA/LIF in the rejection process. In order to further investigate this possible role and the role of other cytokines in allograft rejection, we tested HILDA/LIF, G.CSF, IL-6, TNF alpha, and IL-1 beta in supernatants of cultured mononucleated cells from patients during rejection and from stable grafted patients. In addition, we also tested HILDA/LIF in urine of the same patients. No significant differences were directly observed in the production of HILDA/LIF, TNF alpha, and IL-1 beta in supernatants from mononucleated cells between rejecting and stable patients. However, when antibodies were used to block the TNF alpha and the IL-1 beta receptors, an increase of both cytokines was detected in cells from rejecting patients suggesting that an over-expression of both receptors and cytokines occurred during rejection. A significant increase was also observed for both G.CSF and IL-6 during the rejection compared to stable grafts. In addition, HILDA/LIF was detected in urine of patients during rejection and not in urine of stable patients, suggesting that this cytokine may indeed play a role in rejection.
Asunto(s)
Rechazo de Injerto/metabolismo , Factor Estimulante de Colonias de Granulocitos/metabolismo , Inhibidores de Crecimiento/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Trasplante de Riñón/inmunología , Linfocinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos , División Celular , Citocinas/metabolismo , Rechazo de Injerto/orina , Inhibidores de Crecimiento/orina , Humanos , Interleucina-1/inmunología , Factor Inhibidor de Leucemia , Linfocinas/orina , Receptores del Factor de Necrosis Tumoral/inmunologíaRESUMEN
The cytokine human interleukin for Da cells/leukemia inhibitory factor (HILDA/LIF) exerts multiple biological effects in vitro. In mice, high circulating levels of HILDA/LIF induce a wide range of pathophysiological events, some of them closely involved with immunological and inflammatory responses. Using a sandwich ELISA recognizing the natural human HILDA/LIF molecule with a threshold of 50 pg/ml in urine and 150 pg/ml in plasma, we monitored the urine and plasma HILDA/LIF levels of 22 patients in their first year after a kidney transplant. HILDA/LIF urine excretion is increased during acute rejection, and infections also trigger heavy HILDA/LIF plasma concentrations or urine excretion. In addition, this study raises the question of HILDA/LIF involvement in post-kidney-transplant phenomena such as hypercalcemia, osteoporosis, or the reversal of anemia.
Asunto(s)
Inhibidores de Crecimiento/sangre , Inhibidores de Crecimiento/orina , Interleucina-6 , Trasplante de Riñón/fisiología , Linfocinas/sangre , Linfocinas/orina , Adolescente , Adulto , Anuria/etiología , Femenino , Rechazo de Injerto , Humanos , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/inmunología , Factor Inhibidor de Leucemia , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Procedimientos Quirúrgicos Operativos , Factores de TiempoRESUMEN
Recently, a new lymphokine called HILDA (human interleukin for DA cells) has been described and cloned. This cytokine, initially described to be produced by alloreactive T lymphocyte clones grown from a rejected human kidney allograft, is identical to other factors termed D-factor, differentiation-inducing factor, differentiation inhibitory activity, hepatocyte-stimulating factor III, and leukemia inhibitory factor. HILDA/LIF induces various effects on neural, hemopoietic, embryonic cells as well as on bone remodeling and acute phase protein synthesis in hepatocyte. In this study we demonstrate the presence of HILDA/LIF in the urine but not in the serum of kidney graft recipients during acute rejection episodes, whereas this lymphokine was detectable neither in the serum nor in the urine of kidney transplanted patients with stable renal function. These data reinforce the notion of a possible role for this lymphokine in the inflammatory and/or the immune response.
Asunto(s)
Inhibidores de Crecimiento/orina , Interleucina-6 , Trasplante de Riñón/inmunología , Linfocinas/orina , Anticuerpos Monoclonales/uso terapéutico , Creatinina/sangre , Femenino , Rechazo de Injerto , Inhibidores de Crecimiento/sangre , Humanos , Inmunosupresores/uso terapéutico , Riñón/fisiología , Factor Inhibidor de Leucemia , Linfocinas/sangre , Masculino , Metilprednisolona/uso terapéuticoRESUMEN
One line of investigation of cellular events leading to rejection of an allograft has been to collect the cells infiltrating the rejected allograft and to subject them to in vitro functional and cell marker analysis. Outlined in this article is a description of the limiting dilution analysis technique applied to mechanically disrupted cells obtained from three different rejected human kidney allografts and a phenotypic cell surface marker, as well as a functional study of the progeny of such cells at a clonal level. Of the harvested cells, 65% were T11+, 58% were OKT8+, and 14% were OKT4+. The frequencies of colony forming cells (CFC) were assessed in liquid medium supplemented with lectin-free purified IL-2 (selecting in vivo activated cells) or with IL-2 plus PHA. The CFC frequencies ranged from 1/777 to 1/120 cells plated and from 1/250 to 1/90 cells plated in, respectively, IL-2 and IL-2 plus PHA. Only colonies with a probability of monoclonality more than 80% were further expanded in the presence of lectin-free IL-2. Among 31 colonies grown in the presence of IL-2 alone, all colony-cells were OKT11+, 4/31 were T4+T8-, 24/31 T4-T8+--and, finally, 3/31 were T4+T8+. On the other hand, 122 colonies grown in the presence of IL-2 plus PHA were also all OKT11+, but 47/122 were T4+T8-, and 62/122 were T4-T8+. In addition, expression of DR molecules was highly variable from colony to colony. Significant antidonor cytotoxicity was recorded in 24 colonies, most of them expressing T8 molecules at their surface. Moreover cytotoxic antidonor colonies seemed to recognize an antigen determinant different from the known HLA incompatibilities between donor and recipient. In three long-term-cultured colonies, we noticed a shift in surface marker: from the expression of T8 to either the coexpression of T4 or the loss of the T8 to the sole expression of the T4 molecules. This methodology is a step forward in the elaboration of techniques for determining the relationships between the surface marker identity and the immune function of cells activated in vivo, which are found in a rejected human kidney.