RESUMEN
Stimulation of naïve CD4(+) T cells through engagement of the T-cell receptor (TCR) and the CD28 co-receptor initiates cell proliferation which critically depends on interleukin (IL)-2 secretion and subsequent autocrine signalling via the IL-2 receptor. However, several studies indicate that in CD28-costimulated T cells additional IL-2-independent signals are also required for cell proliferation. In this study, using a neutralizing anti-human IL-2 antibody and two selective, structurally unrelated, cell-permeable I-κB kinase (IKK) inhibitors, BMS-345541 and PS-1145, we show that in human naïve CD4(+) T cells stimulated through a short engagement of the TCR and the CD28 co-receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin-dependent kinase 2 (CDK2) and the stability of the F-box protein S-phase kinase-associated protein 2 (SKP2) and its co-factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL-2-independent mechanisms.
Asunto(s)
Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulación de la Expresión Génica/inmunología , Quinasa I-kappa B/antagonistas & inhibidores , Interleucina-2/metabolismo , Activación de Linfocitos/inmunología , Transporte Activo de Núcleo Celular , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Quinasas CDC2-CDC28 , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Ciclina D3/genética , Ciclina D3/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Proteínas I-kappa B/metabolismo , Imidazoles/farmacología , Interleucina-2/antagonistas & inhibidores , Interleucina-2/genética , Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación de Linfocitos/efectos de los fármacos , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Quinoxalinas/farmacología , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The reaction of mesangial cells with aberrantly glycosylated IgA1 has been implicated in the etiology of IgA nephropathy (IgAN). Tumor necrosis factor, which is assumed to mediate the interaction between mesangial cells and podocytes, also induces the expression of platelet-activating factor (PAF). In this study, we determined whether PAF affects the expression of nephrin (an adhesion molecule critical to glomerular permselectivity) and cytoskeletal F-actin organization in podocytes. We treated human mesangial cells with atypically glycosylated IgA1 either prepared in vitro or derived from the sera of patients with IgAN. We then prepared conditioned media from these cells and added them to cultured human podocytes in the presence of PAF receptor antagonists. Podocytes transfected to overexpress acetylhydrolase, the main catabolic enzyme of PAF, served as controls. Downregulation of nephrin expression and F-actin reorganization occurred when podocytes were cultured with mesangial cell-conditioned medium. Preincubation of podocytes with a PAF receptor antagonist prevented the loss and redistribution of nephrin. In control podocytes overexpressing acetylhydrolase, nephrin loss was abrogated. Our results suggest that atypically glycosylated IgA-induced PAF from mesangial cells is a mediator of podocyte changes, which, when more directly tested elsewhere, were found to be associated with proteinuria. Hence, it is possible that these in vitro findings may be relevant to the proteinuria of IgAN.
Asunto(s)
Inmunoglobulina A/metabolismo , Proteínas de la Membrana/fisiología , Células Mesangiales/metabolismo , Factor de Activación Plaquetaria/metabolismo , Podocitos/metabolismo , Adolescente , Adulto , Estudios de Casos y Controles , Células Cultivadas , Niño , Medios de Cultivo Condicionados , Técnica del Anticuerpo Fluorescente Indirecta , Glicosilación , Humanos , Factor de Activación Plaquetaria/genética , Adulto JovenRESUMEN
BACKGROUND: Patients with 22q11.2 deletion syndrome (22q11.2DS) may develop severe thrombocytopenic purpura and hemolytic anemia. There are no reliable predictors for the development of hematologic autoimmunity (HA) in these patients. OBJECTIVE: To describe the peculiar B and T subpopulation defects in patients with 22q11DS who have developed HA and test if these defects precede the development of HA. METHODS: We performed a case-control multicenter study. Patients with HA were compared with a control population of 22q11.2DS without HA (non-HA). A complete immunological evaluation was performed at diagnosis and at the last follow-up including extensive T and B phenotypes. RESULTS: Immunophenotype at the last follow-up was available in 23 HA and 45 non-HA patients. HA patients had significantly decreased percentage of naïve CD4+ cells (26.8% vs 43.2%, P = .003) and recent thymic emigrants (48.6% vs 80.5%, P = .046); decreased class-switched B cells (2.0% vs 5.9%, P = .04) and increased naive B cells (83.5% vs 71.4%, P = .02); increased CD16+/56+ both in absolute number (312 vs 199, P = .009) and percentage (20.0% vs 13.0%, P = .03). Immunophenotype was performed in 36 patients (11 HA and 25 non-HA) at diagnosis. Odds ratio (OR) of immune cytopenia were estimated for both CD4 naïve ≤30% (OR 14.0, P = .002) and switched memory B cells ≤2% (OR 44.0, P = .01). The estimated survival curves reached statistical significance, respectively, P = .0001 and P = .002. CONCLUSIONS: Among patients with 22q11.2DS, those with HA have characteristic lymphocyte anomalies that appear considerably before HA onset. Systematic immunophenotyping of patients with 22q11.2DS at diagnosis is advisable for early identification of patients at risk for this severe complication.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos B/inmunología , Síndrome de DiGeorge/inmunología , Linfopenia/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Enfermedades Autoinmunes/epidemiología , Estudios de Casos y Controles , Niño , Preescolar , Síndrome de DiGeorge/epidemiología , Femenino , Humanos , Inmunofenotipificación , Linfopenia/epidemiología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Oligoarticular juvenile idiopathic arthritis (oJIA) is the most frequent form of chronic arthritis in children; the clinical course is extremely variable. In this study we have characterized by flow cytometry synovial B and T cells subsets in patients with oJIA in order to identify any parameters that could predict a more aggressive course of disease. METHODS: B and T cells from synovial fluid (SF) of 39 patients with oJIA were characterized by flow cytometry. In 22 patients SF was analysed at the onset of the disease (GroupA), in 17 SF was analysed at articular relapse (Group B). All patients in Group A were followed up for at least for 2 years after SF analysis: 13 patients relapsed during the follow-up period. RESULTS: Comparison of SF from Group A and Group B demonstrated an activated phenotype in relapsed patients, with higher Switched Memory B cells (58.53 vs 36.07% of CD19+, P-value 0.004) and lower Naïve B cells (8.53 vs 25.9 of CD19+, P-value 0.002) in Group B. Furthermore, patients from Group A who did not relapse showed lower percentages of synovial DNT (2.38 vs 1.50% of CD3 + TCRalpha/beta+, P-value 0.025) and γδ T cells (19.1 vs 15.0% of CD3+ cells, P-value 0.004) at the onset, if compared with other Group A patients. CONCLUSIONS: In oJIA relapse SF present an activated B phenotype. Patients at disease onset with DNTs <1.8% and/or γδ T cells <16% of CD3+ in synovial fluid have longer free-disease survival. © 2017 International Clinical Cytometry Society.
Asunto(s)
Artritis Juvenil/inmunología , Artritis Juvenil/mortalidad , Líquido Sinovial/inmunología , Subgrupos de Linfocitos T/inmunología , Antígenos CD19/inmunología , Linfocitos B/inmunología , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Citometría de Flujo/métodos , Humanos , MasculinoRESUMEN
UNLABELLED: Immunological and clinical benefits of highly active antiretroviral therapy (HAART) do not always correlate with its efficacy in controlling HIV replication. This could be due to biological effects on the cell of retroviral agents. DESIGN: To this view we evaluated possible direct immunomodulatory effects of two HIV protease inhibitors, such as Saquinavir (SQ) and Ritonavir (RIT). METHODS: In particular we assessed the PHA- and anti-CD3-driven T cell proliferation, mixed lymphocyte reaction (MLR) and cytokine production on PBMCs from HIV-uninfected subjects incubated with increasing concentrations (2, 5, 10 and 20 microM) of SQ or RIT. RESULTS: Treatment of PBMCs with RIT resulted in a dose-dependent reduction of lymphoproliferative responses. Such an effect was also marked with SQ. MLR was significantly reduced in a concentration-dependent fashion after incubation with either drug. The percentages of stimulated PBMCs and mostly of CD4+ cells expressing TNF-alpha, IL-2 and IFN-gamma were also reduced by SQ or RIT. CONCLUSION: At therapeutic doses both SQ and RIT exhibit potent immunomodulatory activity, which may contribute to correct the HIV-driven cytokine dysregulation and account for some clinical and immunological benefits of therapy in patients with virologic failure. In view of autoreactive immunopathology occurring in AIDS, these direct biologic effects raise intriguing speculations on anti-HIV strategy.
Asunto(s)
Citocinas/biosíntesis , Inhibidores de la Proteasa del VIH/farmacología , Linfocitos/inmunología , Ritonavir/farmacología , Saquinavir/farmacología , Proliferación Celular/efectos de los fármacos , Citocinas/inmunología , Relación Dosis-Respuesta a Droga , Humanos , Activación de Linfocitos/efectos de los fármacos , Prueba de Cultivo Mixto de Linfocitos , Linfocitos/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
CREB has been described as critical for leukemia progress. We investigated CREB expression in ALL and AML pediatric patients. CREB protein was significantly high (p<0.001) at diagnosis but not during remission. This study underlines the role of CREB in leukemia and suggests new insights into the transformation process.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Leucemia/metabolismo , Leucemia/patología , Adolescente , Adulto , Anciano , Niño , Preescolar , Cromosomas Humanos/genética , Progresión de la Enfermedad , Humanos , Lactante , Leucemia/genética , Persona de Mediana EdadRESUMEN
We evaluated the effect of the human immunodeficiency virus (HIV) protease inhibitor saquinavir on the imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia cell lines. Saquinavir, which is also a proteasome blocker, showed dose- and time-related anti-proliferative activity, particularly on the imatinib-resistant lines and a pro-apoptotic effect. Association with imatinib caused a significant increase of activity.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Piperazinas/farmacología , Inhibidores de Proteasas/farmacología , Pirimidinas/farmacología , Saquinavir/farmacología , Adolescente , Apoptosis/efectos de los fármacos , Benzamidas , Crisis Blástica/tratamiento farmacológico , Crisis Blástica/patología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Mesilato de Imatinib , Interferones/uso terapéutico , Células K562/efectos de los fármacos , Células K562/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mieloide de Fase Crónica/tratamiento farmacológico , Leucemia Mieloide de Fase Crónica/patología , Masculino , Persona de Mediana Edad , Inhibidores de ProteasomaRESUMEN
BACKGROUND: In HIV-infected patients some clinical and immunological benefits of antiretroviral therapy, which frequently include a combination of HIV protease inhibitors (PIs) and reverse transcriptase inhibitors (RTIs), cannot be solely explained by the drugs' action on viral enzymes. Proteasomes constitute the central protease of the ubiquitin ATP-dependent pathway involved in many cellular processes, as well as in HIV maturation and aggressiveness. OBJECTIVE: To explore whether the PIs nelfinavir and saquinavir and the RTIs abacavir, nevirapine, delavirdine, stavudine and didanosine affect proteasome function in vitro and in vivo. METHODS: Peptidase activity of purified human 26S and 20S proteasomes was assayed with and without the drugs at different concentrations. Intracellular proteasome proteolytic activity was evaluated by searching for ubiquitin-tagged proteins in HL60 cells incubated with and without the drugs. RESULTS: At therapeutic dosages, nelfinavir and saquinavir inhibited proteasome peptidase activity and caused intracellular accumulation of polyubiquitinated proteins, a hallmark of proteasome proteolytic inhibition in vivo; the RTIs failed to evoke either effect. CONCLUSION: Proteasomes are targeted by the two PIs but not the RTIs. Therefore, in HIV-infected patients the beneficial effect of a therapy including one of the two PIs should partly rely on inhibition of host proteasome function.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Nelfinavir/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Saquinavir/farmacología , Células HL-60 , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Ubiquitina/metabolismoRESUMEN
BACKGROUND: Proteasomes constitute the degradative machinery of the ubiquitin/adenosine triphosphate-dependent proteolytic pathway, which is involved in many cell functions, including immune response and apoptosis, and in HIV maturation and infectivity. OBJECTIVE: To examine whether proteasomes are targeted by antiretroviral agents. METHODS: Chymotrypsin-like, trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of purified human 26S and 20S proteasomes, the latter depleted or enriched in 11S regulator, were assayed after incubation with indinavir, lamivudine and zidovudine at 1-80 microM alone and in combination. To assess the drug effects on cellular functions regulated by proteasomes, the accumulation of ubiquitin-tagged proteins, the processing of the nuclear factor kappa B precursor p105, and the degradation of the inhibitor of nuclear factor kappa B, isoform alpha (IkappaBalpha) were evaluated by Western immunoblotting in Jurkat cells after incubation for 6 h with the drugs above. RESULTS: Trypsin-like and mostly chymotrypsin-like activities of purified 26S proteasome were inhibited by each drug from 10 to 80 microM, more by double combinations and mostly by the triple combination. The peptidyl-glutamyl-peptide hydrolysing activity of the 26S proteasome and the three peptidase activities of the 20S proteasome, depleted or enriched in 11S regulator, were unaffected. The accumulation of ubiquitin-tagged proteins, reduced IkappaBalpha degradation and p105 processing were appreciable in intact cells with the triple drug combination. CONCLUSION: The human 26S proteasome is a target of antiretroviral agents. This suggests that the antiviral action and some clinical and immunological benefits of combined antiretroviral therapy rely not only on its known effects on viral enzymes, but also on host cell components.
Asunto(s)
Fármacos Anti-VIH/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Proteínas I-kappa B , Indinavir/farmacología , Lamivudine/farmacología , Péptido Hidrolasas/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal , Inhibidores de la Transcriptasa Inversa/farmacología , Zidovudina/farmacología , Proteínas de Unión al ADN/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Células Jurkat , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Ubiquitina/metabolismoRESUMEN
A therapeutic role of STI571 (imatinib mesylate) has been anticipated in patients with c-Kit positive neuroectodermal tumors. We examined the efficacy of STI571 to inhibit expansion of c-Kit positive neuroectodermal tumor cell lines in vitro and in a mouse model inoculated with ES (Ewing sarcoma) derived tumor cells, and investigated the molecular mechanism of STI571 action. Eleven tumor lines of ES, PNET (primitive neuroectodermal tumors) and NB (neuroblastoma) were assayed in the presence of 1, 5, 10, 15, 20 or 30 micro M STI571 for 24, 48, 72 h and 7 days. The mechanism of STI571 action was investigated using a microphysiometer cytosensor that determines cellular metabolic rates in the presence of STI571. c-Kit and global protein phosphorylation was assayed by immunoprecipitation and a direct enzyme-linked immunoadsorbent assay after 72 h of 10 micro M STI571. Apoptosis was investigated by propidium iodide (PI), Annexin V staining and by enzymatic activity of caspase-3. Moreover, apoptotic gene expression was investigated using microarray technology. In nude mice, tumor volume and histology were analyzed in STI571 treated and untreated mice, and apoptotic gene expression analysis was performed on tumor masses. A decrease in cell proliferation and increase of cell apoptosis was caused by STI571 in a concentration- and time-dependent manner. Cytosensor microphysiometer and immunoprecipitation experiments demonstrated a time- and concentration-dependent decrease of cellular metabolic activity and global protein dephosphorylation after STI571 exposure. The inhibition by STI571 appeared at least to some extent independent of c-Kit inhibition since cells remained sensitive to SCF stimulation. Tumor volume was significantly reduced in STI571-treated mice compared to tumors from control inoculated non-treated mice. The apoptosis pathway in response to STI571 appeared not to be dependent on caspase activation, while gene expression profiles suggested accumulation of reactive oxygen species (ROS) resulting in cell death after exposure to STI571. The results point to the potential relevance of STI157 for neuroectodermal tumor therapy in view of its inhibitory effect on tumor cell growth, in spite of the observation that the inhibition of the c-Kit signaling pathway is not critically involved.
Asunto(s)
Antineoplásicos/farmacología , Tumores Neuroectodérmicos/tratamiento farmacológico , Tumores Neuroectodérmicos/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/farmacología , Animales , Anexina A5/metabolismo , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Benzamidas , Caspasa 3 , Caspasas/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Trasplante de Células , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mesilato de Imatinib , Ratones , Ratones Desnudos , Ratones SCID , Trasplante de Neoplasias , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/administración & dosificación , Pirimidinas/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/metabolismo , Factores de Tiempo , Trasplante Heterólogo , Carga Tumoral/efectos de los fármacosRESUMEN
Neuroblastoma (NB) has a poor prognosis when in advanced stages, highlighting the need for new therapeutic options. The human immunodeficiency virus (HIV) protease inhibitor saquinavir is active in vitro against chronic myeloid leukaemia cells, in synergy with the tyrosine kinase inhibitor imatinib. Here, we evaluated the effects of saquinavir, alone or in association with imatinib, on cell proliferation (count of viable cells after trypan blue exclusion), apoptosis (Annexin V binding) and invasion (through a transwell membrane coated with Matrigel) in SJ-N-KP, IMR5, AF-8, SK-N-SH and SK-N-BE NB lines, all expressing c-kit and PDGF-R (determined by flow cytometry). Saquinavir showed a dose-dependent anti-proliferative and anti-invasive activity on NB lines, increased by the association with imatinib when the two drugs were utilized at clinically attainable concentrations. The same low saquinavir concentrations inhibited in NB cells the nuclear activation of NF-κB (Western immuno-blotting for nuclear NF-κB p50 and p65). Saquinavir at high concentrations also exerted a pro-apoptotic activity on NB lines, significantly increased by the association with imatinib. In conclusion, saquinavir and imatinib are both drugs utilized for long-term therapies, with good oral bioavailability and a well-known toxicity profile. The anti-NB activity of saquinavir and of its association with imatinib suggests a potential usefulness in the treatment of NB, particularly for remission maintenance.