RESUMO
Viral hepatitis infection is a major global issue and a leading cause of liver disease and associated deaths. Over time, patients infected with hepatitis B (HBV) or C virus (HCV) develop cirrhosis and, eventually, hepatocellular carcinoma (HCC). For this reason, they need to be constantly monitored. Current Asian guidelines recommend the determination of serum alpha-fetoprotein (AFP) together with liver ultrasounds every six months to detect HCC nodules. However, both methods have several limitations, and other biomarkers have been studied for monitoring cirrhosis, including SCCA-IgM, an immune-complex formed by Squamous Cell Carcinoma Antigen and IgM. To date, SCCA-IgM has been validated as a novel biomarker for liver diseases only in European populations. The aim of our study was to analyze SCCA-IgM as a biomarker to monitor cirrhosis evolution in an Asian cohort of patients and to compare its performance to that of AFP. We analyzed the concentration of AFP and SCCA-IgM in serum samples obtained from a group of Asian adult patients with cirrhosis or HCC and a control group of patients admitted for gastrointestinal disorders. In untreated patients and similarly to AFP, SCCA-IgM levels were significantly higher in patients with cirrhosis compared to those with HCC. In addition, SCCA-IgM, but not AFP serological levels, were significantly lower in HCC patients who were treated with surgical resection compared to those who received a different therapy.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Imunoglobulina M/sangue , Cirrose Hepática/diagnóstico , Neoplasias Hepáticas/diagnóstico , Serpinas/sangue , Adulto , Idoso , Área Sob a Curva , Povo Asiático , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/etnologia , Carcinoma Hepatocelular/cirurgia , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Humanos , Cirrose Hepática/sangue , Cirrose Hepática/etnologia , Cirrose Hepática/cirurgia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/etnologia , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Curva ROC , alfa-Fetoproteínas/metabolismoRESUMO
INTRODUCTION: Hepatocellular carcinoma (HCC) develops in about 3-4% of cirrhotic patients every year. The squamous cell carcinoma antigen (SCCA) has been found elevated in liver cancer specimens by immunohistochemistry, and detected in complex with IgM (SCCA-IgM) in the serum of patients with HCC. The aim of this study was to evaluate the ability of serological SCCA-IgM levels to predict the efficacy of HCC therapy. MATERIALS AND METHODS: From April 2012 to April 2014, 131 patients with a new diagnosis of HCC were enrolled. The HCC diagnosis was made according to the EASL guidelines. The patients were staged and treated according to the BCLC Staging System: BCLC stages A and B were treated with locoregional therapy, and BCLC stage C was treated with Sorafenib. Response to therapy was evaluated according to the mRECIST criteria. Serum SCCA-IgM levels were determined by a commercially available ELISA kit at basal time (T0) and after one month of treatment (T1). RESULTS: At baseline and one month into therapy, SCCA-IgM levels were significantly lower (p value <.05) in patients who responded to therapy compared to those who did not respond (median SCCA-IgM level [25th + 75th percentile] at T0:115.1 AU/mL [50.0 + 174.4] vs. 149.1 AU/mL [111.3 + 198.8]; median SCCA-IgM level [25th + 75th percentile] at T1: 113.4 AU/mL [50.0 + 194.2] vs. 170.6 AU/mL [111.7 + 344.2]). CONCLUSION: Our study suggests that the SCCA-IgM determination could be helpful in predicting the response to therapy in patients with HCC.
Assuntos
Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/tratamento farmacológico , Imunoglobulina M/sangue , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/tratamento farmacológico , Serpinas/sangue , Adulto , Área Sob a Curva , Índice de Massa Corporal , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/patologia , Estadiamento de Neoplasias , Curva ROC , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
IgM antibodies bound to different cancer antigens have shown recently a higher diagnostic value, compared with the corresponding free molecule, giving rise to a new family of biomarkers. High or increasing levels of Squamous Cell Carcinoma Antigen (SCCA)-IgM immune complexes were associated with more advanced liver disease and increased risk of development of HCC. Rheumatoid factor (RF) represents a long-standing problem of interference for immunometric assays. The aim of the present study was to examine the specificity of SCCA-IgM in relation to the presence of RF reactivity in patients infected with hepatitis C virus (HCV). Sera of 73 patients with cirrhosis, infected with HCV, (mean age ± SD: 66 ± 13 years; M/F: 45/28), including 21 patients with HCC, were studied. SCCA-IgM immune complexes levels were measured by a commercial ELISA. To evaluate the possible interfering effect of RF, the standard calibrator, positive for SCCA-IgM, was spiked with serial dilutions of a RF positive or negative serum. SCCA-IgM immune complexes were positive in 35 out of 73 (48%) patients, while RF activity was found in 10 out of 73 (14%) patients. Patients with cirrhosis with RF activity had significantly higher levels of SCCA-IgM, compared to RF negative cases; however, no significant correlation between SCCA-IgM and RF values was observed. In samples created artificially the same results in terms of reactivity for SCCA-IgM were obtained, regardless of the presence of RF activity. These findings support the lack of correlation between the two parameters found in sera of patients infected with HCV.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Antígenos de Neoplasias/sangue , Carcinoma Hepatocelular/diagnóstico , Hepatite C/diagnóstico , Cirrose Hepática/diagnóstico , Neoplasias Hepáticas/diagnóstico , Fator Reumatoide/sangue , Serpinas/sangue , Idoso , Antígenos de Neoplasias/imunologia , Calibragem , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hepacivirus/imunologia , Hepatite C/sangue , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Imunoglobulina M/sangue , Cirrose Hepática/sangue , Cirrose Hepática/imunologia , Cirrose Hepática/virologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Fator Reumatoide/imunologia , Sensibilidade e Especificidade , Serpinas/imunologiaRESUMO
Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.
Assuntos
Antivirais/química , Antivirais/farmacologia , Vírus da Influenza A/enzimologia , Influenza Aviária/tratamento farmacológico , Neuraminidase/antagonistas & inibidores , Oseltamivir/análogos & derivados , Oseltamivir/farmacologia , Animais , Antivirais/síntese química , Sítios de Ligação , Aves/virologia , Vírus da Influenza A Subtipo H7N1/química , Vírus da Influenza A Subtipo H7N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H7N1/enzimologia , Vírus da Influenza A Subtipo H7N3/química , Vírus da Influenza A Subtipo H7N3/efeitos dos fármacos , Vírus da Influenza A Subtipo H7N3/enzimologia , Vírus da Influenza A/química , Vírus da Influenza A/efeitos dos fármacos , Influenza Aviária/enzimologia , Modelos Moleculares , Neuraminidase/química , Neuraminidase/metabolismo , Oseltamivir/síntese químicaRESUMO
CONTEXT: Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors. OBJECTIVE: Validate the use of recombinant neuraminidase expressed in baculovirus located on the viral surface capsule to develop a neuraminidase inhibitor screening assay. MATERIALS AND METHODS: Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2'-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor. RESULTS: The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments. DISCUSSION AND CONCLUSIONS: The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.
Assuntos
Baculoviridae/genética , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A Subtipo H7N1/enzimologia , Vírus da Influenza A Subtipo H7N3/enzimologia , Neuraminidase/antagonistas & inibidores , Oseltamivir/análogos & derivados , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Vírus da Influenza A Subtipo H7N1/genética , Vírus da Influenza A Subtipo H7N3/genética , Cinética , Neuraminidase/metabolismo , Oseltamivir/química , Oseltamivir/farmacologia , Relação Estrutura-AtividadeRESUMO
A novel dendrimeric peptide ligand dubbed D-PAM-Φ was designed to achieve a high capacity for human IgG through the decoration of the D-PAM scaffold. The design criteria based on the introduction of small hydrophobic groups on the D-PAM structure were supported by the recently published solid-state structure of D-PAM complexed to the Fc fragment of a recombinant human IgG1 and by molecular dynamic simulations that provided information on the mode of binding of phenylacetyl-D-PAM (D-PAM-Φ). D-PAM-Φ was immobilised on an activated solid support and compared with the parent D-PAM affinity matrix. The newly obtained affinity sorbent was evaluated for its capacity to selectively capture polyclonal human IgG; the binding capacity was approximately 10 mg/ml, an almost 10-fold enhancement with respect to the D-PAM-functionalised matrices without the specificity of binding being reduced. The new ligand was also effective in the capturing of recombinant humanised IgG1 from a clarified cell culture supernatant. Under a typical laboratory-scale affinity chromatography assembly and preliminarily optimised binding conditions, the affinity purification of humanised IgG1 from culture supernatants rendered the desired product, with purity higher than 90%. The results suggest that the application of the computational approach on the structure of the D-PAM-Fc complex may be very valuable in the development of novel lead molecules for the downstream processing of human or humanised antibodies used in therapy.
Assuntos
Materiais Biomiméticos/química , Dendrímeros/química , Imunoglobulina G/isolamento & purificação , Peptídeos/química , Proteína Estafilocócica A/química , Humanos , Imunoglobulina G/sangue , Ligantes , Peptídeos/imunologia , Conformação Proteica , Proteína Estafilocócica A/imunologiaRESUMO
Epithelial-mesenchymal transition is believed to facilitate invasion and metastasis formation of epithelial tumour cells. SERPINB3 is a serine protease inhibitor, physiologically found in normal squamous epithelium but over-expressed in epithelial tumours and known to inhibit apoptosis. We tested the hypothesis that SERPINB3 has a role in invasion by modulating the epithelial-mesenchymal transition programme, using morphological, molecular and cell biology techniques on HepG2 cell clones transfected with the human SERPINB3 gene. The paracrine effect of this serpin was determined by the addition of exogenous recombinant SERPINB3 protein to HepG2 and MDCK cell line. SERPINB3 expression leads to changes in transfected cells morphology, characterized by clusters of loosely connected cells with elongated shape. Ultrastructural analysis confirmed the decrease of desmosomal junctions and widening of intercellular spaces. These alterations were associated with a reduction of E-cadherin and an increase of beta-catenin, with a parallel increase of cell proliferation. SERPINB3 clones, untransfected HepG2 and MDCK cells treated with exogenous SERPINB3 expressed vimentin, undetectable in controls. SERPINB3 induced significant cell scattering, migration and invasiveness in untransfected cells. These effects were not dependent on the anti-protease activity of the protein, as documented by the results obtained with an active loop-deleted recombinant SERPINB3 protein. Scatter activity was inhibited by an anti-SERPINB3 antibody in a dose-dependent manner and SERPINB3-transfected cells formed a significantly higher number of colonies on soft agar than controls. In conclusion, the observed results indicate that SERPINB3 induces deregulation of adhesion processes and increases the invasiveness potential supported by features of epithelial-mesenchymal transition, acting at both the autocrine and the paracrine level.
Assuntos
Antígenos de Neoplasias/fisiologia , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/fisiologia , Serpinas/fisiologia , Antígenos de Neoplasias/metabolismo , Adesão Celular/fisiologia , Células Epiteliais/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/ultraestrutura , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Mesoderma/patologia , Microscopia Eletrônica , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Comunicação Parácrina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Serpinas/metabolismoRESUMO
BACKGROUND: The combination of alpha-fetoprotein (AFP) and squamous cell carcinoma antigen immunocomplex (SCCA-IgM) have been proposed for its use in the screening of hepatocellular carcinoma (HCC). Current screening programs for all cirrhotic patients are controversial and a personalized screening is an unmet need in the precision medicine era. AIM: To determine the role of the combination of SCCA-IgM and AFP in predicting mid- and long-term appearance of HCC. METHODS: Two-hundred and three cirrhotic patients (Child A 74.9%, B 21.2%, C 3.9%) were followed-up prospectively every six months to screen HCC by ultrasound and AFP according to European Association for the Study of the Liver guidelines. The estimation cohort was recruited in Italy (30.5%; 62/203) and validation cohort from Spain (69.5%; 141/203). Patients underwent to evaluate SCCA-IgM by enzyme-linked immunosorbent assay (Hepa-IC, Xeptagen, Italy) and AFP levels at baseline. Patients were followed-up for 60 mo, being censored at the time of the appearance of HCC. RESULTS: There were 10.8% and 23.1% of HCC development at two- and five-years follow-up. Patients with HCC showed higher levels of SCCA-IgM than those without it (425.72 ± 568.33 AU/mL vs 195.93 ± 188.40 AU/mL, P = 0.009) during the five-year follow-up. In multivariate analysis, after adjusting by age, sex, aspartate transaminase and Child-Pugh, the following factors were independently associated with HCC: SCCA-IgM [Hazard ratio (HR) = 1.001, 95%CI: 1.000-1.002; P = 0.003], AFP (HR = 1.028, 95%CI: 1.009-1.046; P = 0.003) and creatinine (HR = 1.564 95%CI: 1.151-2.124; P = 0.004). The log-rank test of the combination resulted in 7.488 (P = 0.024) in estimation cohort and 11.061 (P = 0.004) in the validation cohort, and a 100% of correctly classified rate identifying a low-risk group in both cohorts in the two-year follow-up. CONCLUSION: We have constructed a predictive model based on the combination of SCCA-IgM and AFP that provides a new HCC screening method, which could be followed by tailored HCC surveillance for individual patients, especially for those cirrhotic patients belonging to the subgroup identified as low-risk of HCC development.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Antígenos de Neoplasias , Biomarcadores Tumorais , Humanos , Imunoglobulina M , Cirrose Hepática/complicações , Cirrose Hepática/diagnóstico , Serpinas , alfa-FetoproteínasRESUMO
Transforming growth factor-beta1 (TGF-beta1) is the master cytokine in the pathogenesis of liver fibrosis. TGF-beta1 and extent of fibrosis were correlated recently to the serpin SERPINB3 in idiopathic pulmonary fibrosis, a chronic disease recalling liver cirrhosis. The aim of this study was to assess the relation between SERPINB3, TGF-beta1 and fibrosis in chronic liver diseases and to determine the effect of this serpin on TGF-beta1 expression using in vitro models. SERPINB3 and TGF-beta1 were evaluated in liver biopsies of 94 patients with chronic liver disease. The effect of SERPINB3 on TGF-beta1 expression was determined in primary human hepatocytes, HepG2 and Huh7 cells transfected with intact SERPINB3 human gene or with reactive site loop deleted mutants. A significant correlation between TGF-beta1 and SERPINB3 at the protein level was observed in liver biopsies, confirmed by a positive correlation at mRNA level. Both proteins were correlated to the extent of liver fibrosis. All transfected cells showed increased TGF-beta1 mRNA and protein production and the integrity of the reactive site loop of the serpin was crucial to achieve this effect. In conclusion, chronically damaged hepatocytes produce SERPINB3 and TGF-beta, and the anti-protease activity of this serpin might be implicated in TGF-beta1 induction.
Assuntos
Antígenos de Neoplasias/metabolismo , Cirrose Hepática/metabolismo , Serpinas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Idoso , Antígenos de Neoplasias/genética , Domínio Catalítico , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Expressão Gênica , Humanos , Fígado/patologia , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Serpinas/genética , TransfecçãoRESUMO
Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies.
Assuntos
Complexos Multiproteicos/isolamento & purificação , Extração em Fase Sólida/métodos , Anticorpos Imobilizados/análise , Anticorpos Imobilizados/química , Anticorpos Imobilizados/metabolismo , Complexo Antígeno-Anticorpo/análise , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/isolamento & purificação , Complexo Antígeno-Anticorpo/metabolismo , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/isolamento & purificação , Antígenos de Neoplasias/metabolismo , Avidina/química , Avidina/metabolismo , Biotina/química , Biotina/metabolismo , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoglobulina G/análise , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Complexos Multiproteicos/análise , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Serpinas/imunologia , Serpinas/isolamento & purificação , Serpinas/metabolismo , alfa-Fetoproteínas/análise , alfa-Fetoproteínas/imunologia , alfa-Fetoproteínas/isolamento & purificação , alfa-Fetoproteínas/metabolismoRESUMO
BACKGROUND: Squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) is a useful biomarker for the risk of development of hepatocellular carcinoma (HCC) in patients with cirrhosis due to its progressive increase associated to HCC evolution. In patients with cirrhosis, other assays have been affected by interfering reactivities of IgM. In this study, the analytical specificity of the SCCA-IgM assay was assessed by evaluating SCCA-IgM measurement dependence on different capture phases, and by measuring the recovery of SCCA-IgM reactivity following serum fractionation. METHODS: Serum samples from 82 patients with cirrhosis were analyzed. SCCA-IgM was measured using the reference test (Hepa-IC, Xeptagen, Italy) that is based on rabbit oligoclonal anti-squamous cell carcinoma antigen (SCCA) and a dedicated ELISA with a mouse monoclonal anti-SCCA as the capture antibody. RESULTS: SCCA-IgM concentrations measured with the reference assay (median value=87 AU/mL) were higher than those measured with the mouse monoclonal test (median value=78 AU/mL). However, the differences in the SCCA-IgM distribution were not statistically significant (p>0.05). When SCCA-IgM concentrations measured with both tests were compared, a linear correlation was found (r=0.77, p<0.05). Fractionation of the most reactive sera by gel-filtration chromatography showed that total recovery of SCCA-IgM reactivity was seen only in the fractions corresponding to components with a molecular weight higher than IgM and SCCA (>2000 kDa) with both tests. CONCLUSIONS: The equivalence of both SCCA-IgM assays and the absence of reactivity not related to immune complexes support the analytical specificity of SCCA-IgM measurements. The results validate the assessment of SCCA-IgM for prognostic purposes in patients with cirrhosis.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Antígenos de Neoplasias/sangue , Bioensaio , Carcinoma Hepatocelular/sangue , Fibrose/sangue , Fibrose/diagnóstico , Imunoglobulina M/sangue , Serpinas/sangue , Idoso , Complexo Antígeno-Anticorpo/imunologia , Antígenos de Neoplasias/imunologia , Biomarcadores/sangue , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina M/imunologia , Pessoa de Meia-Idade , Prognóstico , Sensibilidade e Especificidade , Serpinas/imunologiaRESUMO
Complications of chronic liver diseases - particularly hepatocellular carcinoma (HCC) - are a major cause of mortality worldwide. Several studies have shown that high or increasing levels of serum Squamous Cell Carcinoma Antigen-Immunoglobulin M complex (SCCA-IgM) are associated with development of HCC in patients with advanced liver disease and worse survival in patients with liver cancer. The aim of the present study was to assess, in patients with advanced liver disease, differences in long-term clinical outcomes in relation to baseline levels of serum SCCA-IgM. Ninety one consecutive outpatients with liver cirrhosis of different etiologies, without hepatocellular carcinoma at presentation, were enrolled from April 2007 to October 2012 in a prospective study. For a median time of 127 months, patients were bi-annually re-evaluated. SCCA-IgM complex levels were determined with a validated enzyme-linked immunosorbent assay. The results provided evidence that serum SCCA-IgM is a predictor of overall survival. The best cut-off to discriminate both HCC-free and overall survival rates was 120 AU/mL. Patients with baseline values higher than this threshold showed a substantial increase in both HCC incidence rate and all-cause mortality rate. In conclusion, a single measurement of serum SCCA-IgM helps to identify those patients with liver cirrhosis with increased risks of HCC development and mortality.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/imunologia , Antígenos de Neoplasias/imunologia , Imunoglobulina M/imunologia , Cirrose Hepática/sangue , Cirrose Hepática/mortalidade , Serpinas/imunologia , Adulto , Idoso , Antígenos de Neoplasias/sangue , Biomarcadores , Carcinoma Hepatocelular , Feminino , Humanos , Imunoglobulina M/sangue , Cirrose Hepática/etiologia , Hepatopatias , Neoplasias Hepáticas , Masculino , Pessoa de Meia-Idade , Prognóstico , Serpinas/sangue , Análise de SobrevidaRESUMO
BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has a high prevalence in obesity and its presence should be screened. Laparoscopic sleeve gastrectomy (LSG) is an effective treatment for obesity, but its effects on NAFLD are still to be firmly established. The diagnosis of non-alcoholic steatohepatitis (NASH) is currently performed by liver biopsy, a costly and invasive procedure. Squamous cell carcinoma antigen-IgM (SCCA-IgM) is a biomarker of viral hepatitis to hepatocellular carcinoma development and its role in NAFLD to NASH progression has not yet been investigated. OBJECTIVE: The aim of this study was to evaluate SCCA-IgM as a non-invasive biomarker of NAFLD/NASH in patients with different degrees of metabolic-complicated obesity before and after LSG. METHOD: Fifty-six patients with obesity were studied before and 12 months after LSG; anthropometric, biochemical, clinical, and imaging data were collected. RESULTS: At baseline steatosis was strongly associated with the glycaemic profile (p = 0.016) and was already present in prediabetic patients with obesity (82%). Only 3 patients had an SCCA-IgM level above the normal cut-off. SCCA-IgM titre did not change according to glycaemic profile or steatosis. Metabolic and inflammatory factors and transaminases significantly reduced after LSG-induced weight loss, except for SCCA-IgM. The ALT/AST ratio decreased post-LSG correlated with BMI (r = 0.297, p = 0.031), insulin (r = 0.354, p = 0.014), and triglycerides (r = 0.355, p = 0.009) reduction. CONCLUSIONS: Our results confirm the tight link between NAFLD and metabolic complications, suggesting prediabetes as a new risk factor of steatosis. SCCA-IgM does not seem to have a role in the identification and prognosis of NAFLD.
Assuntos
Antígenos de Neoplasias/imunologia , Biomarcadores/sangue , Diabetes Mellitus Tipo 2/sangue , Imunoglobulina M/sangue , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Obesidade/sangue , Estado Pré-Diabético/sangue , Serpinas/imunologia , Adulto , Antígenos de Neoplasias/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/cirurgia , Feminino , Seguimentos , Gastrectomia/métodos , Gastrectomia/reabilitação , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/cirurgia , Obesidade/complicações , Obesidade/diagnóstico , Obesidade/cirurgia , Obesidade Mórbida/sangue , Obesidade Mórbida/complicações , Obesidade Mórbida/diagnóstico , Obesidade Mórbida/cirurgia , Estado Pré-Diabético/complicações , Estado Pré-Diabético/diagnóstico , Estado Pré-Diabético/cirurgia , Prognóstico , Fatores de Risco , Serpinas/sangue , Resultado do Tratamento , Redução de PesoRESUMO
BACKGROUND: Prostate biopsy is the gold standard for prostate cancer (PCa) diagnosis, but it's invasive and associated with adverse events. Novel reliable tumor biomarkers and accurate non-invasive tests are required to avoid biopsies. The immune complex PSA-IgM is a new marker for PCa, and it has been included in an algorithm to generate the diagnostic index iXip, which determines the probability of having PCa. In this study we evaluated the ability of iXip to reduce the number of repeat biopsies in patients with a previous negative biopsy and suspicious for PCa. PATIENTS AND METHODS: 219 patients referred for prostate rebiopsy were included in the study. Each patient underwent a trans-rectal ultrasound-guided prostate biopsy and prostate volume examination. Blood samples were collected before any prostatic manipulation to determine the serological levels of PSA-IgM and PSA. The iXip index was calculated as previously reported using an online calculator. RESULTS: iXip values in patients with a positive biopsy were significantly higher than the ones observed in negative patients (p-valueâ¯=â¯0.001). Based on iXip values, patients were divided in five risk groups: those with iXipâ¯<â¯0.2 had 0% probability of having PCa. High values of the Gleason score (≥7) were observed mostly in patients with iXip 0.3-0.8. CONCLUSION: Our preliminary results show that iXip identifies a sub-group of patients who can safely avoid rebiopsy because they do not have PCa. The index is a promising tool that could reduce the number of unnecessary prostate biopsies and the relative clinical complications and expenses.
RESUMO
Non-alcoholic steato-hepatitis (NASH) is a severe disease characterised by liver inflammation and progressive hepatic fibrosis, which may progress to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests that in hepatitis C virus patients steatosis and NASH are associated with faster fibrosis progression and hepatocellular carcinoma. A safe and reliable non-invasive diagnostic method to detect NASH at its early stages is still needed to prevent progression of the disease. We prospectively enrolled 91 hepatitis C virus-positive patients with histologically proven chronic liver disease: 77 patients were included in our study; of these, 10 had NASH. For each patient, various clinical and serological variables were collected. Different algorithms combining squamous cell carcinoma antigen-immunoglobulin-M (SCCA-IgM) levels with other common clinical data were created to provide the probability of having NASH. Our analysis revealed a statistically significant correlation between the histological presence of NASH and SCCA-IgM, insulin, homeostasis model assessment, haemoglobin, high-density lipoprotein and ferritin levels, and smoke. Compared to the use of a single marker, algorithms that combined four, six or seven variables identified NASH with higher accuracy. The best diagnostic performance was obtained with the logistic regression combination, which included all seven variables correlated with NASH. The combination of SCCA-IgM with common clinical data shows promising diagnostic performance for the detection of NASH in hepatitis C virus patients.
Assuntos
Antígenos de Neoplasias/sangue , Hepatite C/sangue , Imunoglobulina M/sangue , Hepatopatia Gordurosa não Alcoólica/sangue , Serpinas/sangue , Adulto , Algoritmos , Biomarcadores Tumorais/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Hepatite C/complicações , Hepatite C/patologia , Hepatite C/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/virologiaRESUMO
BACKGROUND: Squamous cell carcinoma antigen (SCCA)-IgM complex has been described as a promising tool to identify patients with progressive liver disease at higher risk of hepatocellular carcinoma (HCC) development in retrospective studies. AIM: To assess the clinical value of this biomarker in patients with cirrhosis in a prospective study. METHODS: Patients with overt cirrhosis were prospectively evaluated at 6-month intervals for HCC development and decompensation with clinical examination, liver ultrasound, α-fetoprotein measurement. SCCA-IgM was measured in serum by immunoenzymatic assay. Median follow-up duration was 52 months (range 12-68 months). RESULTS: 70 patients (26% male; mean age 56±10 years) were enrolled. The main aetiological factors were alcohol (44%) and hepatitis C (34%). Baseline values of SCCA-IgM were significantly higher in patients who developed HCC. Positivity of the biomarker at baseline was associated with a significantly shorter HCC-free survival, while α-fetoprotein (cut off >20 ng/ml) was not significant. SCCA-IgM positivity and hepatitis C were significant prognostic factors for HCC development. The biomarker was not associated with the development of clinical complications of cirrhosis. CONCLUSION: This prospective study demonstrates that in patients with cirrhosis SCCA-IgM is associated with HCC development and may be useful for clinical management of cirrhotic patients at higher risk of HCC development.
Assuntos
Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/imunologia , Carcinoma Hepatocelular/imunologia , Imunoglobulina M/imunologia , Neoplasias Hepáticas/imunologia , Serpinas/imunologia , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/metabolismo , Estudos de Coortes , Feminino , Seguimentos , Hepatite C Crônica/complicações , Humanos , Cirrose Hepática/complicações , Cirrose Hepática Alcoólica/complicações , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , alfa-Fetoproteínas/metabolismoRESUMO
Antineutrophil cytoplasm autoantibodies with specificity for proteinase 3 (PR3) are thought to play a major role in the pathogenesis of Wegener's granulomatosis (WG), presumably by their potential to activate neutrophils. In patients with WG, high expression of PR3 on the surface of nonprimed neutrophils is associated with an increased incidence and rate of relapse. In this study, we analyzed the functional significance of constitutive PR3 expression for neutrophil activation as induced by anti-PR3 antibody. Therefore, primed and nonprimed neutrophils were stimulated with the monoclonal anti-PR3 antibody PR3G-3. Activation was measured as actin polymerization by the phalloidin assay as an early, detectable activation event and oxidative burst by the dihydrorhodamine assay, as a late, detectable activation event. In contrast to the oxidative burst, we found that anti-PR3 antibody-induced actin polymerization could be triggered in neutrophils without priming with tumor necrosis factor alpha (TNF-alpha). In addition, a correlation was found between the level of PR3 expression on the surface of these nonprimed neutrophils and the degree of actin polymerization. However, after priming with TNF-alpha, no correlation was found between membrane expression of PR3 and the level of actin polymerization or respiratory burst as induced by anti-PR3 antibody. These data suggest that the presence of PR3 on the surface of nonprimed neutrophils has consequences for their susceptibility to the initial activation step by anti-PR3 antibodies. These data may be relevant in view of the observed relation between membrane expression of PR3 on nonprimed neutrophils of patients with WG and their susceptibility for relapses.
Assuntos
Membrana Celular/imunologia , Quimiotaxia de Leucócito/imunologia , Granulomatose com Poliangiite/imunologia , Neutrófilos/imunologia , Serina Endopeptidases/imunologia , Actinas/biossíntese , Anticorpos Anticitoplasma de Neutrófilos/sangue , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Autoanticorpos/imunologia , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Predisposição Genética para Doença/genética , Humanos , Mieloblastina , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/imunologia , Recidiva , Explosão Respiratória/efeitos dos fármacos , Explosão Respiratória/imunologia , Serina Endopeptidases/biossíntese , Serina Endopeptidases/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Sequence-simplified variants of a 15-mer peptide antigen, identified by amino acid side chains in alternating positions were synthesized introducing glycine residues alternatively in the parent peptide sequence and used to induce antibodies in rabbit. They reacted to a significant extent with anti-parent peptide antibodies, and in addition, affinity purified antibodies against these halved forms recognized with similar affinity and specificity, the starting peptide in affinity chromatography, optical biosensor and enzyme linked immunosorbent assay (ELISA) experiments, while no cross-reactivity was detected between reduced antigens. These findings suggest that a peptide antigen can display two molecular surfaces of recognition, identified by side chains of residues in alternating positions. Each surface can even take part in antigen/antibody interaction independently, thus indicating the possibility to select and assembly sequence-simplified forms belonging to different epitopes, also deriving from different molecules, to generate new structures incorporating a two-fold antigen/antibody specificity. Two "chimeric" forms were then synthesized starting from the P15 and P13 complementary peptides, both able to bind interleukin 2. These structures, showing simultaneously trans-surfaces of recognition belonging to both parent forms, have been found to retain antigenic properties against antibodies of simplified P15 derivatives showing the same molecular surface of recognition. In addition, anti-chimeric antibodies recognized both P15 and P13 starting peptides, while no cross-antibody recognition was observed between chimeric antigens.
Assuntos
Reações Antígeno-Anticorpo , Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Cinética , Dados de Sequência Molecular , Peptídeos/química , CoelhosRESUMO
Hepatitis C virus (HCV) is the main cause of chronic liver disease and cirrhosis in Western countries. Over time, the majority of cirrhotic patients develop hepatocellular carcinoma (HCC), one of the most common fatal cancers worldwide - fourth for incidence rate. A high public health priority need is the development of biomarkers to screen for liver disease progression and for early diagnosis of HCC development, particularly in the high risk population represented by HCV-positive patients with cirrhosis. Several studies have shown that serological determination of a novel biomarker, squamous cell carcinoma antigen-immunoglobulins M (SCCA-IgM), might be useful to identify patients with progressive liver disease. In the initial part of this review we summarize the main clinical studies that have investigated this new circulating biomarker on HCV-infected patients, providing evidence that in chronic hepatitis C SCCA-IgM may be used to monitor progression of liver disease, and also to assess the virological response to antiviral treatment. In the last part of this review we address other, not less important, clinical applications of this biomarker in hepatology.
RESUMO
For a long time the complement cascade has been believed to be the predominant pathway to inflammation and tissue destruction in autoimmune diseases such as systemic lupus erythematosus. Recently, new evidences show that FcRs may share the primacy with complement cascade, playing an equal or greater role in the disease process. The generation of specific mouse strains deficient in individual components has clarified the different role played by complement and Fc receptors in their interaction with ICs, illustrating that complement is essential for innate immunity against microbial pathogens, requiring natural antibodies to mediate its protective effects, whereas FcyRs have evolved as the principal system for coupling antigen-antibody complexes to effector cells and initiate the inflammatory cascade. Validation of FcRs as new therapeutic targets for autoimmune diseases, in particular for Systemic Lupus Erythematosus (SLE), has been provided by a large number of studies where the biological action of soluble forms of FcyRs or of monoclonal antibodies targeting Fc receptors has been assessed. Additional support to the role of FcRs in SLE has been provided by data obtained with compounds derived from combinatorial chemistry, such as TG19320, a tetrameric tripeptide which interferes with IgG/FcgammaR interaction in vitro and prevents glomerulonephritis in vivo in a SLE susceptible mouse strain. These findings might open the way to new therapeutic approaches for disorders where the role of FcRs has been established, including not only autoimmune diseases like systemic lupus erythematosus, rheumatoid arthritis, multiple myeloma, but also acquired immunodeficiency syndrome (AIDS).