RESUMO
OBJECTIVES: To test the potential efficacy of recombinant macrophage inhibitory cytokine-1 (MIC-1/GDF15) as an obesity therapeutic. METHODS: Male C57BL/6 J mice, either fed on normal chow or high-fat diet for 16 weeks to induce diet-induced obesity, were infused with either recombinant MIC-1/GDF15 or vehicle for 34 days by osmotic minipump. During the experimental period metabolic parameters were measured. Blood and tissue were collected for analysis of inflammatory markers. RESULTS: MIC-1/GDF15 decreased food intake and body weight of high-fat-fed and chow-fed mice compared with their vehicle-treated control mice. MIC-1/GDF15 reduced body weight, accompanied by greater reduction in fat mass in high-fat-fed mice compared to its effect on chow-fed mice. Further, whilst MIC-1/GDF15-treated chow-fed mice lost lean as well as fat mass, MIC-1/GDF15-treated high-fat-fed mice lost fat mass alone. This reduction in body weight and adiposity was due largely to reduced food intake, but MIC-1/GDF15-treated high-fat-fed mice also displayed increased energy expenditure that may be due to increased thermogenesis. MIC-1/GDF15-treated high-fat-fed mice also had higher circulating level of adiponectin and lower tissue expression, and circulating levels of leptin and inflammatory mediators associated with insulin resistance. Peripheral insulin and glucose intolerance were improved in both MIC-1/GDF15-treated high-fat-fed and chow-fed mice compared to that of their vehicle-treated control mice. CONCLUSIONS: MIC-1/GDF15 is highly effective in reducing adiposity and correcting the metabolic dysfunction of mice with high-fat fed. These studies suggest that MIC-1/GDF15 may be a candidate anti-obesity therapeutic.
Assuntos
Adiposidade/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Fator 15 de Diferenciação de Crescimento/farmacologia , Obesidade/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/fisiopatologia , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Serum macrophage inhibitory cytokine-1 (MIC-1/GDF15) concentration has been associated with colonic adenomas and carcinoma. AIMS: To determine whether circulating MIC-1/GDF15 serum concentrations are higher in the presence of adenomas and whether the level decreases after excision. METHODS: Patients were recruited prospectively from a single centre and stratified into five groups: no polyps (NP); hyperplastic polyps (HP); sessile serrated ademona (SSA); adenomas (AP); and colorectal carcinoma (CRC). Blood samples were collected immediately before and 4 weeks after colonoscopy. MIC-1/GDF15 serum levels were quantified using ELISA. RESULTS: Participants (n=301) were stratified as: NP; n=116 (52%), HP; n=37 (12%), SSA; n=19 (7%), AP; n=68 (23%); and CRC; n=3 (1%). Patients were excluded from the study due to nondiagnostic pathology (n=9, 3%) and exclusion criteria (n=20, 6%). In the 272 remaining subjects (M=149; F=123), age (P=.005), history of colonic polyps (P=.003) and family history of colonic polyps (P=.002) were associated with presence of adenomas. Baseline median MIC-1/GDF15 serum levels increased significantly from NP 609 (460-797) pg/mL, HP 582 (466-852) pg/mL, SSA 561 (446-837) pg/mL to AP 723 (602-1122) pg/mL and CRC 1107 (897-1107) pg/mL; (P<.001). In the pre- and postpolypectomy paired adenoma samples median MIC-1/GDF15 reduced significantly from 722 (603-1164) pg/mL to 685 (561-944) pg/mL (P=.002). A ROC analysis for serum MIC-1/GDF15 to identify adenomatous polyps indicated an area under the curve of 0.71. CONCLUSIONS: Our data suggest that serum MIC-1/GDF15 has the diagnostic characteristics to increase the detection of colonic neoplasia and improve screening.
Assuntos
Adenoma/diagnóstico , Neoplasias do Colo/patologia , Pólipos do Colo/diagnóstico , Fator 15 de Diferenciação de Crescimento/sangue , Pólipos Adenomatosos/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Colonoscopia , Neoplasias Colorretais/diagnóstico , Feminino , Humanos , Hiperplasia/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
Anorexia-cachexia associated with cancer and other diseases is a common and often fatal condition representing a large area of unmet medical need. It occurs most commonly in advanced cancer and is probably a consequence of molecules released by tumour cells, or tumour-associated interstitial or immune cells. These may then act directly on muscle to cause atrophy and/or may cause anorexia, which then leads to loss of both fat and lean mass. Although the aetiological triggers for this syndrome are not well characterized, recent data suggest that MIC-1/GDF15, a transforming growth factor-beta superfamily cytokine produced in large amounts by cancer cells and as a part of other disease processes, may be an important trigger. This cytokine acts on feeding centres in the hypothalamus and brainstem to cause anorexia leading to loss of lean and fat mass and eventually cachexia. In animal studies, the circulating concentrations of MIC-1/GDF15 required to cause this syndrome are similar to those seen in patients with advanced cancer, and at least some epidemiological studies support an association between MIC-1/GDF15 serum levels and measures of nutrition. This article will discuss its mechanisms of central appetite regulation, and the available data linking this action to anorexia-cachexia syndromes that suggest it is a potential target for therapy of cancer anorexia-cachexia and conversely may also be useful for the treatment of severe obesity.
Assuntos
Anorexia/etiologia , Caquexia/etiologia , Fator 15 de Diferenciação de Crescimento/metabolismo , Terapia de Alvo Molecular , Neoplasias/complicações , Obesidade/complicações , Fator de Crescimento Transformador beta1/metabolismo , Animais , Anorexia/psicologia , Anorexia/terapia , Apetite/efeitos dos fármacos , Apetite/genética , Biomarcadores/metabolismo , Caquexia/psicologia , Caquexia/terapia , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular/tendências , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/psicologia , Obesidade/genética , Obesidade/metabolismo , Obesidade/psicologia , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Aumento de Peso/efeitos dos fármacosRESUMO
Few reports have compared available serum free light chain (SFLC) assays. Here, a retrospective audit of the Freelite SFLC assay compared results to electrophoresis (EP)/immunofixation (IFX) and the N Latex FLC assay.A total of 244 samples collected over 3.5 months were studied using the Freelite and N Latex FLC nephelometry assays. Results were compared with serum and/or urine EP/IFX. The precision and linearity of the N Latex FLC assay was examined.Detectable paraprotein by serum or urine EP/IFX was present in 94% of samples with kappa and 100% with lambda FLC restriction. The correlation between the assays was higher for kappa (rhoâ=â0.97) than lambda (rhoâ=â0.89) especially when lambda results were above the upper limit of normal (rhoâ=â0.62). Agreement in the categorical diagnosis as measured by the Cohen's kappa statistic was good (0.70). The N Latex FLC assay displayed good precision and linearity. In discordant samples the Freelite and N Latex FLC assays had equivalent agreement with IFX.Traditional methods of EP/IFX detected paraproteins in the majority of cases. Correlation between the Freelite and N Latex FLC assay is better for kappa than lambda FLC. The two assays are not entirely equivalent. Care should be taken by interpreting physicians and laboratories considering switching assays.
Assuntos
Eletroforese/métodos , Imunoensaio/métodos , Cadeias Leves de Imunoglobulina/sangue , Cadeias Leves de Imunoglobulina/urina , Paraproteinemias/diagnóstico , Idoso , Feminino , Humanos , Látex , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
BACKGROUND: Docetaxel improves symptoms and survival in metastatic castration-resistant prostate cancer (CRPC). However, â¼50% of patients are chemoresistant. This study examined whether changes in cytokine levels predict for docetaxel resistance in vitro and in a clinical cohort. METHODS: PC3 cells or their docetaxel-resistant subline (PC3Rx) were co-cultured with U937 monocytes, with and without docetaxel treatment, and cytokine levels were measured. The circulating levels of 28 cytokines were measured pre-/post cycle 1 of docetaxel from 55 men with CRPC, and compared with prostate-specific antigen (PSA) response. RESULTS: PC3Rx-U937 co-culture expressed more cytokines, chiefly markers of alternative macrophage differentiation, compared with PC3-U937 co-culture. Docetaxel treatment enhanced cytokine production by PC3Rx-U937 co-culture, while reducing cytokine levels in PC3-U937. In patients, changes in the levels of seven circulating cytokines (macrophage inhibitory cytokine 1 (MIC1), interleukin (IL)-1ra, IL-1ß, IL-4, IL-6, IL-12 and IFNγ) after cycle 1 of docetaxel were associated with progressive disease (all P<0.05). The combination of changes in MIC1, IL-4 and IL-6 most strongly predicted PSA response (P=0.002). CONCLUSIONS: In vitro studies suggest docetaxel resistance is mediated, at least in part, by cytokines induced by the interaction between the docetaxel-resistant tumour cells and macrophages. Early changes in circulating cytokine levels were associated with docetaxel resistance in CRPC patients. When considered together, these data suggest a significant role for the inflammatory response and macrophages in the development of docetaxel resistance in CRPC.
Assuntos
Citocinas/sangue , Resistencia a Medicamentos Antineoplásicos , Calicreínas/sangue , Macrófagos/metabolismo , Antígeno Prostático Específico/sangue , Neoplasias de Próstata Resistentes à Castração/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Docetaxel , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Taxoides/farmacologiaRESUMO
BACKGROUND: Biomarkers are needed to improve current diagnosis and surveillance strategies for patients with Barrett's oesophagus (BO) and oesophageal adenocarcinoma (OAC). Macrophage inhibitory cytokine 1/growth differentiation factor 15 (MIC-1/GDF15) tissue and plasma levels have been shown to predict disease progression in other cancer types and was therefore evaluated in BO/OAC. METHODS: One hundred thirty-eight patients were studied: 45 normal oesophagus (NE), 37 BO, 16 BO with low-grade dysplasia (LGD) and 40 OAC. RESULTS: Median tissue expression of MIC-1/GDF15 mRNA was ⩾25-fold higher in BO and LGD compared to NE (P<0.001); two-fold higher in OAC vs BO (P=0.039); and 47-fold higher in OAC vs NE (P<0.001). Relative MIC-1/GDF15 tissue expression >720 discriminated between the presence of either OAC or LGD vs NE with 94% sensitivity and 71% specificity (ROC AUC 0.86, 95% CI 0.73-0.96; P<0.001). Macrophage inhibitory cytokine 1/growth differentiation factor 15 plasma values were also elevated in patients with OAC vs NE (P<0.001) or BO (P=0.015).High MIC-1/GDF15 plasma levels (⩾1140 pg ml(-1)) were an independent predictor of poor survival for patients with OAC (HR 3.87, 95% CI 1.01-14.75; P=0.047). CONCLUSIONS: Plasma and tissue levels of MIC-1/GDF15 are significantly elevated in patients with BO, LGD and OAC. Plasma MIC-1/GDF15 may have value in diagnosis and monitoring of Barrett's disease.
Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Neoplasias Esofágicas/genética , Fator 15 de Diferenciação de Crescimento/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Estudos de Casos e Controles , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
BACKGROUND: Macrophage inhibitory cytokine-1(MIC-1) is a potential modulator of systemic inflammation and nutritional depletion, both of which are adverse prognostic factors in oesophago-gastric cancer (OGC). METHODS: Plasma MIC-1, systemic inflammation (defined as plasma C-reactive protein (CRP) of > or =10 mg l(-1) or modified Glasgow prognostic score (mGPS) of > or =1), and nutritional status were assessed in newly diagnosed OGC patients (n=293). Healthy volunteers (n=35) served as controls. RESULTS: MIC-1 was elevated in patients (median=1371 pg ml(-1); range 141-39 053) when compared with controls (median=377 pg ml(-1); range 141-3786; P<0.001). Patients with gastric tumours (median=1592 pg ml(-1); range 141-12 643) showed higher MIC-1 concentrations than patients with junctional (median=1337 pg ml(-1); range 383-39 053) and oesophageal tumours (median=1180 pg ml(-1); range 258-31 184; P=0.015). Patients showed a median weight loss of 6.4% (range 0.0-33.4%), and 42% of patients had an mGPS of > or =1 or plasma CRP of > or =10 mg l(-1) (median=9 mg l(-1); range 1-200). MIC-1 correlated positively with disease stage (r(2)=0.217; P<0.001), age (r(2)=0.332; P<0.001), CRP (r(2)=0.314; P<0.001), and mGPS (r(2)=0.336; P<0.001), and negatively with Karnofsky Performance Score (r(2)=-0.269; P<0.001). However, although MIC-1 correlated weakly with dietary intake (r(2)=0.157; P=0.031), it did not correlate with weight loss, BMI, or anthropometry. Patients with MIC-1 levels in the upper quartile showed reduced survival (median=204 days; 95% CI 157-251) when compared with patients with MIC-1 levels in the lower three quartiles (median=316 days; 95% CI 259-373; P=0.036), but MIC-1 was not an independent prognostic indicator. CONCLUSIONS: There is no independent link between plasma MIC-1 levels and depleted nutritional status or survival in OGC.
Assuntos
Adenocarcinoma/mortalidade , Neoplasias Esofágicas/mortalidade , Fator 15 de Diferenciação de Crescimento/sangue , Inflamação/sangue , Estado Nutricional/fisiologia , Neoplasias Gástricas/mortalidade , Adenocarcinoma/sangue , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Neoplasias Esofágicas/sangue , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/patologia , Feminino , Humanos , Mediadores da Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Neoplasias Gástricas/sangue , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologia , Análise de SobrevidaRESUMO
Macrophage inhibitory cytokine-1 (MIC-1), a divergent member of transforming growth factor-beta superfamily, has been recently shown to be produced by the human placenta with detectable levels in maternal serum. In this study, using immunohistochemistry, we have localized MIC-1 in placenta, decidua and foetal membranes across pregnancy and, using an enzyme-linked immunoassay, measured MIC-1 in maternal serum in normal pregnancy, in association with labour and pre-eclampsia. In the placenta MIC-1 was principally localized to the syncytiotrophoblast while in the foetal membranes MIC-1 was present in the amniotic epithelium, chorionic trophoblasts and adherent decidual cells. There were no differences in MIC-1 staining distribution or intensity in the placentae between women in labour and not in labour, or between healthy and pre-eclamptic pregnancies. MIC-1 staining in the foetal membranes was slightly stronger after a labour and delivery compared to those delivered by elective Caesarean section. MIC-1 levels in the maternal serum increased with advancing gestation but there were no significant differences in maternal serum levels associated with either labour or pre-eclampsia.These observations would be consistent with MIC-1 having roles at the maternal-foetal interface, perhaps in the establishment and/or maintenance of pregnancy. Our data argue against MIC-1 having a significant role in the regulation of labour or in the pathophysiology of pre-eclampsia.
Assuntos
Citocinas/metabolismo , Decídua/metabolismo , Membranas Extraembrionárias/metabolismo , Trabalho de Parto/sangue , Pré-Eclâmpsia/sangue , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Fator 15 de Diferenciação de Crescimento , Humanos , Técnicas Imunoenzimáticas , Gravidez , Primeiro Trimestre da Gravidez , Segundo Trimestre da GravidezRESUMO
Macrophages play an important role in immune and inflammatory responses, largely through secretion of bioactive molecule such as cytokines. While calcium is known to be an important regulator of this process, less is known about the role of other ions and the ion channels that regulate them. We have previously implicated an outwardly rectifying potassium channel (Kor) in this process and for this reason we have investigated the role of potassium (K+) and K+ channels in the regulation of tumour necrosis factor-alpha (TNF-alpha)and interleukin (IL)-8 production by activated human culture-derived macrophages. The effect of blockade of Kor is to inhibit phorbol myristate acetate (PMA)-induced cytokine production by translational or post-translational mechanisms, an effect that is duplicated by increasing extracellular K+. By contrast, the effects of K+ on LPS-stimulated cells are far more complex and are probably mediated through the change of osmolality and occur largely at the mRNA level. This data directly implicates K+, and its regulation through Kor, in early events following PMA stimulation of these cells.
Assuntos
Interleucina-8/biossíntese , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Canais de Potássio/fisiologia , Potássio/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , 4-Aminopiridina/farmacologia , Cálcio/farmacologia , Células Cultivadas/efeitos dos fármacos , Charibdotoxina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/genética , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Concentração Osmolar , Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/biossíntese , Selenito de Sódio/farmacologia , Sotalol/farmacologia , Estresse Mecânico , Sacarose/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tetraetilamônio/farmacologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the TGF-beta superfamily. There are at least two known alleles of MIC-1 that are due to a G-->C point substitution at position 6 of the mature protein, which alters a histidine to an aspartic acid (MIC-1 H and MIC-1 D). We have determined the phenotype of MIC-1 circulating in serum by exploiting the differences in the affinity of the two monoclonal antibodies to the H and D alleles of MIC-1. A PCR-RFLP-based method for genotyping MIC-1 is also described. We validate these two assays using DNA sequencing of 19 subjects as the standard. We then used the validated assay to determine the frequency of the two MIC-1 alleles in a population of 261 adult blood donors. Inter-assay and sequencing concordance was 100%. The frequency of the three common MIC-1 genotypes was homozygous (HH), 54%; heterozygous (HD), 39%; and homozygous (DD), 7%. This novel antibody-based assay confidently determines the genotype of MIC-1. It offers the advantages of an ELISA-ease of automation, high-volume throughput of samples, and ease of use in a routine, clinical laboratory.
Assuntos
Citocinas/sangue , Citocinas/genética , Análise Mutacional de DNA/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Adolescente , Adulto , Idoso , Alelos , Animais , Sequência de Bases , Feminino , Genótipo , Fator 15 de Diferenciação de Crescimento , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodosRESUMO
We and others have recently cloned a new member of the transforming growth factor-beta superfamily, growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF-15/MIC-1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF-15/MIC-1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF-15/MIC-1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF-15/MIC-1-producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Fluorescent microscopy revealed both intra- and extracellular GDF-15/MIC-1 ir. Up-regulation of GDF-15/MIC-1 in activated macrophages (Mstraight phi) is also supported by RT-PCR, ICC, and Western blot experiments showing pronounced induction of GDF-15/MIC-1 expression (mRNA and protein) in retinoic acid/phorbol ester-stimulated human M phi. Our data suggest that 1) GDF-15/MIC-1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme-epithelial interactions. Finally, GDF-15/MIC-1 may also act within the antiinflammatory cytokine network activated in CNS lesions.
Assuntos
Envelhecimento/metabolismo , Animais Recém-Nascidos/metabolismo , Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Citocinas , Ratos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Ventrículos Cerebrais , Plexo Corióideo/metabolismo , Epêndima/citologia , Epêndima/metabolismo , Fator 15 de Diferenciação de Crescimento , Humanos , Macrófagos/fisiologia , RNA Mensageiro/metabolismo , Ratos Wistar , Fator de Crescimento Transformador beta/genéticaRESUMO
CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.
Assuntos
Canais de Cloreto/química , Cloro/química , Sequência de Aminoácidos , Sítios de Ligação , Membrana Celular/metabolismo , Cloro/metabolismo , Cisteína/química , Eletrofisiologia , Escherichia coli/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Técnicas de Patch-Clamp , Mutação Puntual , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily. While it is synthesized in a pre-pro form, it is unique among superfamily members because it does not require its propeptide for correct folding or secretion of the mature peptide. To investigate factors that enable these propeptide independent events to occur, we constructed MIC-1/TGF-beta1 chimeras, both with and without a propeptide. All chimeras without a propeptide secreted less efficiently compared with the corresponding constructs with propeptide. Folding and secretion were most affected after replacement of the predicted major alpha-helix in the mature protein, residues 56-68. Exchanging the human propeptide in this chimera with either the murine MIC-1 or TGF-beta1 propeptide resulted in secretion of the unprocessed, monomeric chimera, suggesting a specific interaction between the human MIC-1 propeptide and mature peptide. Propeptide deletion mutants enabled identification of a region between residues 56 and 78, which is important for the interaction between the propeptide and the mature peptide. Cotransfection experiments demonstrated that the propeptide must be in cis with the mature peptide for this phenomenon to occur. These results suggest a model for TGF-beta superfamily protein folding.
Assuntos
Citocinas/fisiologia , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Ativinas , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/química , Células CHO , Cricetinae , Citocinas/química , Citocinas/genética , Primers do DNA , Glicosilação , Fator 15 de Diferenciação de Crescimento , Humanos , Inibinas/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/genéticaRESUMO
Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily whose increased expression is associated with macrophage activation and which is expressed highly in placenta as compared to other tissues. There are two known allelic forms of human MIC-1 due an amino acid substitution at position 6 of the mature protein. We have raised four monoclonal antibodies (MAbs) and one polyclonal antiserum to the mature protein region of human MIC-1 and have used an extensive panel of MIC-1 relatives, mutants, and chimeras to map their epitopes. None of the MAbs were able to cross-react with either the murine homologue of MIC-1 or with hTGF-beta1, and all of the MAb epitopes were conformation-dependent. A distinct cross-reactivity pattern with the various antigens was observed for each of the monoclonal and polyclonal antibodies suggesting the presence of at least five immunogenic regions on the MIC-1 surface. One of the MAbs is directed against the amino terminus of the protein and can distinguish between the two allelic forms of MIC-1. The epitopes for the other three MAbs were located near the tips of the so-called "fingers" of the protein and appeared to be partially overlapping as each involved amino acids in the region 24-37. In one case, it was possible to mutate murine MIC-1 so that it could be recognized by one of the MAbs. Finally, the use of another mutant in which Cys 77 was replaced by serine enabled confirmation of the location of the MIC-1 interchain disulfide bond.
Assuntos
Citocinas/química , Citocinas/imunologia , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/genética , Células CHO , Cricetinae , Reações Cruzadas/genética , Citocinas/genética , Citocinas/metabolismo , Epitopos/genética , Epitopos/metabolismo , Vetores Genéticos/síntese química , Vetores Genéticos/imunologia , Fator 15 de Diferenciação de Crescimento , Humanos , Soros Imunes/biossíntese , Soros Imunes/química , Soros Imunes/genética , Soros Imunes/metabolismo , Camundongos , Dados de Sequência Molecular , Família Multigênica/imunologia , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Recent studies have suggested that dermal dendritic cells (DDCs) may play a part in maintaining the structure of the dermis and in dermal immune modulation. Alteration in the population of DDCs has been noted in localized and systemic scleroderma, particularly a decline in the number of CD34+ DDCs. Objectives To define the alteration of the DDC populations with respect to the histological stage of morphoea. METHODS: We examined 33 biopsies of morphoea, categorized into four histological stages, and examined the DDC population (CD34+ DDCs and factor XIIIa+ DDCs), the lymphocytic infiltrate, and tenascin (extracellular matrix glycoprotein) and transforming growth factor (TGF)-beta1 expression in each biopsy. RESULTS: As the dermis became less inflammatory and more sclerotic, there was a significant decline in the number of CD34+ DDCs and an increase in the number of factor XIIIa+ DDCs. The pan-T-cell infiltrate (UCHL-1/CD45RO) and tenascin deposition exhibited a similar pattern, with elevated expression in inflammatory stages and a decrease in expression as the dermis became sclerotic. TGF-beta1 was significantly elevated in three of the four histological stages of morphoea, in both the inflammatory and sclerotic stages. The proposed four-stage histological analysis of morphoea biopsies was a useful basis for studying dendritic cells and mediators in cutaneous sclerosis. CONCLUSIONS: Our study indicates that there is a reciprocal relationship between CD34+ DDCs and factor XIIIa+ DDCs in morphoea that correlates with the relative degrees of inflammation and sclerosis.
Assuntos
Células Dendríticas/patologia , Esclerodermia Localizada/patologia , Adolescente , Adulto , Idoso , Antígenos CD34/metabolismo , Biópsia , Criança , Células Dendríticas/metabolismo , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Esclerodermia Localizada/metabolismo , Tenascina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1RESUMO
OBJECTIVE: To evaluate the effects of azimilide and ambasilide on the biophysical properties of the human-ether-a-go-go-related (HERG) channel. METHODS: HERG was stably transfected into Chinese hamster ovary (CHO-K1) cells and currents were measured using a whole cell, voltage-clamp technique. RESULTS: Azimilide had a 'dual effect', inhibiting current at voltage steps above -40 mV and augmenting current at -40 and -50 mV. Tail current inhibition following a step to +30 mV did not vary with temperature (IC(50) 610 nM at 22 degrees C and 560 nM at 37 degrees C). The agonist effect at -50 mV was concentration-dependent and correlated with a hyperpolarizing shift in the V(1/2) of activation (r=0.98, P<0.05). Time constants of inactivation were faster and there was a -10 mV shift in the V(1/2) of steady state inactivation suggestive of open and inactivated state binding. By comparison, ambasilide inhibited HERG channels with lower potency (IC(50) 3.6 microM), in a voltage- and time-dependent but frequency-independent manner (0.03-1 Hz). Ambasilide had no effect on activation or inactivation gating but prolonged both fast and slow components of deactivation consistent with unbinding from the open state. The net effect of both drugs was similar during a voltage ramp which simulated a cardiac action potential. CONCLUSIONS: Inhibition of HERG channels by azimilide and ambasilide exhibits a similar time and voltage-dependence. While both exhibit affinity for the open state, azimilide also binds to inactivated channels.
Assuntos
Aminobenzoatos/farmacologia , Antiarrítmicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas de Transporte de Cátions , Proteínas de Ligação a DNA , Imidazóis/farmacologia , Imidazolidinas , Piperazinas/farmacologia , Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio , Transativadores , Animais , Células CHO , Cricetinae , Depressão Química , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Humanos , Hidantoínas , Técnicas de Patch-Clamp , Regulador Transcricional ERGRESUMO
The methylotrophic yeast, Pichia pastoris, has been used to express both human and murine macrophage inhibitory cytokine-1 (MIC-1), a transforming growth factor beta (TGF-beta) superfamily cytokine. This is the first report of the expression of a correctly folded TGF-beta superfamily protein in a microbial organism. The protein is secreted in its correctly folded dimeric form at milligram per litre quantities, which are significantly higher than we have been able to achieve using mammalian expression systems. Purification schemes are described, and the purified protein is immunologically identical to protein produced in a mammalian expression system. Protein expression was influenced by a number of factors, most significantly by the concentration of methanol used during the induction phase. However, with very high levels of MIC-1 induction, substantial amounts of MIC-1 monomer were also secreted.
Assuntos
Citocinas/genética , Fator de Crescimento Transformador beta/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Células CHO , Cricetinae , Citocinas/química , Citocinas/imunologia , DNA Recombinante/genética , DNA Recombinante/isolamento & purificação , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Fator 15 de Diferenciação de Crescimento , Humanos , Metanol/farmacologia , Camundongos , Dados de Sequência Molecular , Radioimunoensaio , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Halofantrine is a widely used antimalarial agent which has been associated with prolongation of the 'QT interval' of the electrocardiogram (ECG), torsades de pointes and sudden death. Whilst QT prolongation is consistent with halofantrine-induced increases in cardiac ventricular action potential duration, the cellular mechanism for these observations has not been previously reported. The delayed rectifier potassium channel, I(Kr), is a primary site of action of drugs causing QT prolongation and is encoded by the human-ether-a-go-go-related gene (HERG). We examined the effects of halofantrine on HERG potassium channels stably expressed in Chinese hamster ovary (CHO-K1) cells. Halofantrine blocked HERG tail currents elicited on repolarization to -60 mV from +30 mV with an IC(50) of 196.9 nM. The therapeutic plasma concentration range for halofantrine is 1.67-2.98 microM. Channel inhibition by halofantrine exhibited time-, voltage- and use-dependence. Halofantrine did not alter the time course of channel activation or deactivation, but inactivation was accelerated and there was a 20 mV hyperpolarizing shift in the mid-activation potential of steady-state inactivation. Block was enhanced by pulses that render channels inactivated, and channel blockade increased with increasing duration of depolarizing pulses. We conclude that HERG channel inhibition by halofantrine is the likely underlying cellular mechanism for QT prolongation. Our data suggest preferential binding of halofantrine to the open and inactivated channel states.