RESUMO
BACKGROUND: Multiple Myeloma (MM) is a malignant hematologic disorder and the second most common blood cancer. Interleukin-6 (IL-6) has been identified as a crucial factor for the proliferation and survival of MM cells and the overexpression of IL-6 receptor is being studied as a molecular target for therapeutic and diagnostic use in myelomas and other comorbidities. Tocilizumab is a humanized monoclonal antibody that binds IL-6R. OBJECTIVE: We aim to label and evaluate Fab(Tocilizumab) with 99mTechnetium or Cy7 as potential MM imaging agents. METHODS: IL-6R distribution was analyzed by Laser Confocal Microscopy (LCM) in MM cell lines. Fab(Tocilizumab) was produced by the digestion of Tocilizumab with papain for 24h at 37°C, derivatized with NHS-HYNIC-Tfa and radiolabeled with 99mTc. Radiochemical stability and in vitro cell assays were evaluated. Biodistribution and SPECT/CT were performed. Also, Fab(Tocilizumab) was labeled with Cy7 for in vivo fluorescence imaging up to 72h. RESULTS: LCM analysis demonstrates IL-6R distribution on MM cell lines. Incubation with papain resulted in complete digestion of Tocilizumab and exhibited a good purity and homogeneity. Radiolabeling with 99mTc via NHS-HYNIC-Tfa was found to be fast, easy, reproducible and stable, revealing high radiochemical purity and without interfering with IL-6R recognition. Biodistribution and SPECT/CT studies showed a quick blood clearance and significant kidney and MM engrafted tumor uptake. Cy7-Fab(Tocilizumab) fluorescent imaging allowed MM1S tumor identification up to 72h p.i. CONCLUSION: These new molecular imaging agents could potentially be used in the clinical setting for staging and follow-up of MM through radioactive whole-body IL-6R expression visualization in vivo. The fluorescent version could be used for tissue sample evaluation and to guide surgical excision, if necessary.
Assuntos
Anticorpos Monoclonais Humanizados/química , Carbocianinas/química , Imagem Molecular , Mieloma Múltiplo/diagnóstico por imagem , Compostos de Organotecnécio/química , Compostos Radiofarmacêuticos/química , Humanos , Receptores de Interleucina-6/análiseRESUMO
This study investigated the expression and regulation of IL-6R in hepatitis B-associated moderate hepatic fibrosis and cirrhosis. Liver tissues, peripheral blood monocytes (PBMs) and serum were collected from 26 hepatitis B patients with liver fibrosis and 35 hepatitis B patients with liver cirrhosis. The levels of Il-6r mRNA expression in these samples were examined by quantitative real-time PCR and IL-6R protein levels were analyzed by western blot and ELISA. MiRNAs that regulate IL-6R expression were predicted by bioinformatics analysis, and validated by dual luciferase reporter assay. Compared with the hepatic fibrosis group, IL-6R was significantly upregulated at both mRNA and protein levels in liver tissues, PBMs and serum samples from the hepatic cirrhosis group (P<0.05). The 3'UTR of Il-6r mRNA was predicted to contain a miR-30b binding site and IL-6R was identified as a possible target of miR-30b. MiR-30b expression was significantly downregulated in samples from hepatic cirrhosis patients compared with hepatic fibrosis patients (P<0.05). In conclusion, IL-6R was upregulated while miR-30b was decreased in patients with liver cirrhosis. The miR-30 can directly regulate the expression of IL-6R.
Assuntos
Hepatite B/metabolismo , Cirrose Hepática/metabolismo , MicroRNAs/metabolismo , Receptores de Interleucina-6/metabolismo , Regiões 3' não Traduzidas , Adulto , Idoso , Regulação para Baixo , Feminino , Hepatite B/sangue , Humanos , Cirrose Hepática/sangue , Masculino , MicroRNAs/análise , MicroRNAs/química , Pessoa de Meia-Idade , Receptores de Interleucina-6/análise , Valores de Referência , Fatores de Tempo , Regulação para CimaRESUMO
This study investigated the expression and regulation of IL-6R in hepatitis B-associated moderate hepatic fibrosis and cirrhosis. Liver tissues, peripheral blood monocytes (PBMs) and serum were collected from 26 hepatitis B patients with liver fibrosis and 35 hepatitis B patients with liver cirrhosis. The levels of Il-6r mRNA expression in these samples were examined by quantitative real-time PCR and IL-6R protein levels were analyzed by western blot and ELISA. MiRNAs that regulate IL-6R expression were predicted by bioinformatics analysis, and validated by dual luciferase reporter assay. Compared with the hepatic fibrosis group, IL-6R was significantly upregulated at both mRNA and protein levels in liver tissues, PBMs and serum samples from the hepatic cirrhosis group (P<0.05). The 3′UTR of Il-6r mRNA was predicted to contain a miR-30b binding site and IL-6R was identified as a possible target of miR-30b. MiR-30b expression was significantly downregulated in samples from hepatic cirrhosis patients compared with hepatic fibrosis patients (P<0.05). In conclusion, IL-6R was upregulated while miR-30b was decreased in patients with liver cirrhosis. The miR-30 can directly regulate the expression of IL-6R.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Hepatite B/metabolismo , Cirrose Hepática/metabolismo , MicroRNAs/metabolismo , Receptores de Interleucina-6/metabolismo , Regulação para Baixo , Hepatite B/sangue , Cirrose Hepática/sangue , MicroRNAs/análise , MicroRNAs/química , Receptores de Interleucina-6/análise , Valores de Referência , Fatores de Tempo , Regulação para CimaRESUMO
BACKGROUND AND OBJECTIVES: The pathogenesis of essential thrombocythemia (ET), a disease characterized by megakaryocyte hyperplasia and persistent thrombocytosis, is not completely clarified. Interleukin-6 (IL-6), one of the cytokines related to megakaryocytic development, exerts its effect through binding to a cell surface receptor, IL-6Ra, and a signal transducing unit, gp130. Interestingly, the soluble form of the IL-6Ra, IL-6sR, is an agonist for IL-6 activity. In order to evaluate the possible participation of IL-6sR in ET we measured its levels in plasma, platelets and in the supernatant of a mononuclear cell culture. We also evaluated IL-6R on leukocyte membrane and IL-6R/IL-6sR mRNA expression in mononuclear cells. DESIGN AND METHODS: Fifty-five patients with ET were evaluated. IL-6sR and IL-6 were measured by an ELISA technique. Mononuclear cells were cultured for 48 hr and IL-6sR released into the supernatant was measured. IL-6R on leukocyte surfaces was evaluated by flow cytometry. IL-6R and IL-6sR mRNA levels were assessed by semi-quantitative reverse transcription polymerase chain reaction. RESULTS: Plasma IL-6sR levels were increased while intraplatelet levels were low in untreated ET patients. Plasma levels decreased during treatment. Non-stimulated mononuclear cells from ET patients released greater amounts of IL-6sR than did cells from normal controls in 48-hour culture. No abnormality was found in IL-6R or IL-6sR mRNA expression by mononuclear ET cells. IL-6R on leukocyte surfaces was normal. INTERPRETATION AND CONCLUSIONS: Increased plasma IL-6sR levels might have a role in the abnormal megakaryocytic proliferation seen in ET patients, while platelets and mononuclear cells could be the source of the above-mentioned high levels of plasma IL-6sR.
Assuntos
Receptores de Interleucina-6/análise , Trombocitemia Essencial/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Plaquetas/química , Células Cultivadas , Feminino , Humanos , Interleucina-6/sangue , Leucócitos Mononucleares , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Receptores de Interleucina-6/sangue , Receptores de Interleucina-6/genética , Solubilidade , Trombocitemia Essencial/etiologiaRESUMO
Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL-6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.