RESUMO
Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various intestinal and systemic inflammatory and infectious diseases, including inflammatory bowel disease (IBD) and tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility and convenience. In this study, we introduce the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 and CP17, that bind to different epitopes of the calprotectin complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. FESCA's output luminescence can be measured with standard laboratory equipment as well as consumer-grade cell phone cameras. FESCA can detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, and whole blood. Notably, FESCA can detect abnormally elevated native calprotectin from TB patients. In summary, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, with the potential to improve the diagnosis and monitoring of inflammatory diseases, especially in at-home or point-of-care settings.
Assuntos
Técnicas Biossensoriais , Complexo Antígeno L1 Leucocitário , Medições Luminescentes , Complexo Antígeno L1 Leucocitário/análise , Humanos , Técnicas Biossensoriais/métodos , Medições Luminescentes/métodos , Luciferases/metabolismo , Luciferases/química , Biomarcadores/análise , Biomarcadores/sangue , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/metabolismo , Tuberculose/diagnóstico , Tuberculose/sangue , LuminescênciaRESUMO
Inflammatory bowel disease-associated colorectal carcinomas (IBD-CRCs) develop in a background of chronic inflammation, and thus, the molecular landscape of these tumors likely differs from that of sporadic colorectal cancer. To add to emerging data on molecular alterations present in these tumors, we analyzed our institution's cohort of IBD-CRCs. CRCs resected from patients with IBD underwent molecular analysis via a 50-gene hot-spot solid tumor panel (OncoScreen ST2.0). In-house sporadic CRCs and The Cancer Genome Atlas project data were used for comparison. Fifty-five IBD-CRCs from 48 patients were successfully analyzed. Mutations in TP53 were most common and were present in 69% of IBD-CRCs; a similar percentage of TP53 mutations was detected in sporadic colorectal carcinomas (70%). APC and KRAS mutations were significantly less common in IBD-CRCs than in sporadic CRCs (15% versus 53%, Pâ¯<â¯.001 and 20% versus 38%, Pâ¯=â¯.02, respectively). Additionally, the potentially targetable IDH1 R132 mutation was present in 7% of IBD-CRCs but only 1% of sporadic CRCs and The Cancer Genome Atlas CRCs; alterations in other genes with potential targeted therapies were very rare. In conclusion, IBD-CRCs exhibit molecular differences when compared to sporadic CRCs, suggesting different pathways of carcinogenesis, although certain alterations are common to both types of tumors. IDH1 mutations are present in a subset of IBD-CRCs, which may expand therapeutic options in the future.
Assuntos
Adenocarcinoma/genética , Carcinogênese/genética , Neoplasias Colorretais/genética , Doenças Inflamatórias Intestinais/complicações , Proteína da Polipose Adenomatosa do Colo/genética , Adulto , Idoso , Análise Mutacional de DNA , Feminino , Humanos , Isocitrato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Background and Aims: Mucosal appearance on endoscopy is an important indicator of inflammatory burden and determines prognosis in ulcerative colitis (UC). Inflammation induces tryptophan metabolism along the kynurenine pathway (KP) and yields immunologically relevant metabolites. We sought to examine whether changes in serum tryptophan metabolites and tissue expression of KP enzymes are associated with UC endoscopic and histologic disease severity. Methods: Serum and mucosal samples were prospectively obtained at colonoscopy in patients with UC. Mayo disease activity scores, demographics, smoking status, medications, and outcomes were collected. Serum tryptophan metabolites were analyzed using ultra-high performance liquid chromatography (uHPLC), and gas chromatography-mass spectrometry (GC-MS), and enzyme expression was determined by quantitative real-time polymerase chain reaction. Metabolite and enzyme levels were compared by endoscopic subscore, clinical disease activity, time to surgery, and hospitalization. Results: This study included 99 patients with Mayo endoscopic subscores 0-3. Kynurenic acid/tryptophan ratio (KYNA/T) and expression of indolamine 2,3-dioxygenase 1 (IDO1), tryptophan 2,3-dioxygenase, kynurinase, and kynurenine monooxygenase correlated positively with endoscopic subscore. Adjusting for age of diagnosis, smoking status, disease extent, and medications yielded significant odds of endoscopic inflammation with increasing KYNA/T (OR 1.0015, P = 0.0186) and IDO1 expression (OR 1.0635, P = 0.0215). The highest tertile ratio of KYNA/T had shorter time to surgery (P = 0.009) and hospitalization (P = 0.01) than the lowest. Conclusions: Increasing KYNA/T is closely associated with endoscopic inflammation and predictive of disease outcomes in patients with UC. These findings identify this novel metabolic association and further support the role of the KP in regulating mucosal inflammation in UC. 10.1093/ibd/izy103_video1izy103.video15788135676001.
Assuntos
Colite Ulcerativa/metabolismo , Cinurenina/metabolismo , Triptofano/metabolismo , Adolescente , Adulto , Idoso , Biomarcadores/análise , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Colite Ulcerativa/patologia , Colonoscopia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Inflamação/metabolismo , Cinurenina/sangue , Modelos Logísticos , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Análise Multivariada , Sensibilidade e Especificidade , Triptofano/sangue , Adulto JovemRESUMO
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene and protein expression. miRNAs are critical to a normal immune response and have altered expression in multiple immune-mediated disorders. This emerging role of miRNAs in the pathogenesis of multiple disease states has led to investigations into miRNA expression profiles in inflammatory bowel disease (IBD). The discovery of miRNAs in IBD is likely to contribute to our understanding of IBD pathogenesis and lead to clinical advances in IBD. This review focuses on miRNA expression in inflammation, autoimmune disorders, and inflammation-associated cancer, as well as their function in the biology and management of IBD.
Assuntos
Regulação da Expressão Gênica , Doenças Inflamatórias Intestinais/genética , MicroRNAs/fisiologia , HumanosRESUMO
BACKGROUND: miR-143 and miR-145 are believed to function as colon cancer tumor suppressors, as they inhibit colon cancer cell growth and are downregulated in sporadic colonic tumors. We speculated that miR-143 and miR-145 might also be downregulated and contribute to malignant transformation of colonic epithelium in longstanding ulcerative colitis (UC). METHODS: Biopsies were obtained 20 cm proximal to the anus from individuals with quiescent UC and from normal controls. RNA and proteins were extracted and measured. miR-143 and miR-145 were quantified by real-time polymerase chain reaction (PCR) and miR-145 was also assessed by in situ hybridization. Putative targets of these miRNAs, K-RAS, API5, MEK-2 (miR-143), and IRS-1 (miR-145) were determined by western blotting. To assess the effects of miR-143 and miR-145 on these predicted targets, HCT116 and HCA-7 colorectal cancer cells were transfected with miR-143 and miR-145 and expression levels of these proteins were measured. RESULTS: In UC, miR-143 and miR-145 were significantly downregulated 8.3-fold (3.4-20.1) (P < 0.0001) and 4.3-fold (2.3-7.8) (P < 0.0001), respectively, compared to normal colon. In contrast, IRS-1, K-RAS, API5, and MEK-2 were upregulated in UC, consistent with their assignments as targets of these miRNAs. Furthermore, transfected miR-143 and miR-145 significantly downregulated these proteins in HCT116 or HCA-7 cells. CONCLUSIONS: Compared to normal colonic mucosa, in chronic UC miR-143 and miR-145 were significantly downregulated and their predicted targets, IRS-1, K-RAS, API5, and MEK-2 were upregulated. We postulate that loss of these tumor suppressor miRNAs predispose to chronic inflammation and neoplastic progression in IBD.
Assuntos
Colite Ulcerativa/genética , Inflamação/genética , MicroRNAs/genética , Proto-Oncogenes , Adulto , Western Blotting , Estudos de Casos e Controles , Doença Crônica , Colite Ulcerativa/patologia , Colo/metabolismo , Colo/patologia , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
BACKGROUND: The management of low-grade (LGD) and indefinite dysplasia (IND) in patients with ulcerative colitis (UC) remains controversial, as outcomes after a diagnosis of LGD or IND in previous studies vary widely. METHODS: All patients evaluated were from a single institution referral center who had a history of UC and a diagnosis of either LGD or IND between 1994 and 2008 as confirmed by 2 expert gastrointestinal (GI) pathologists. Data were collected by chart review of electronic and paper medical records. All patients who did not undergo a colectomy within 90 days of their dysplasia diagnosis were included in the final analysis. Hazard ratios for risk factors as well as incidence rates and Kaplan-Meier estimates were used to calculate the progression to high-grade dysplasia (HGD) or colorectal cancer (CRC). RESULTS: Thirty-five patients were included in the analysis, of whom 2 patients with IND and 2 patients with LGD developed HGD or CRC over a mean duration of 49.8 months. In total, the incident rate for advanced neoplasia for all patients was 2.7 cases of HGD or CRC per 100 person-years at risk. For flat and polypoid LGD the incident rate of advanced neoplasia was 4.3 and 1.5 cases per 100 person-years at risk, respectively. Patients with primary sclerosing cholangitis (PSC) had an incident rate of 10.5 cases per 100 years of patient follow-up. CONCLUSIONS: We report a low rate of progression to HGD or CRC in patients who underwent surveillance for LGD or IND; polypoid dysplasia showed less risk of progression than flat dysplasia.
Assuntos
Adenocarcinoma/epidemiologia , Adenocarcinoma/etiologia , Colite Ulcerativa/complicações , Colite Ulcerativa/patologia , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/etiologia , Adenocarcinoma/patologia , Adolescente , Adulto , Transformação Celular Neoplásica/patologia , Colangite Esclerosante/complicações , Colangite Esclerosante/epidemiologia , Colangite Esclerosante/cirurgia , Colectomia , Colite Ulcerativa/cirurgia , Colonoscopia , Neoplasias Colorretais/patologia , Progressão da Doença , Humanos , Incidência , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Vigilância de Evento Sentinela , Adulto JovemRESUMO
BACKGROUND: Chronic hepatitis C can result in fatty changes in the liver. Previous studies have suggested that hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with hepatitis C virus (HCV) infection. The authors sought to determine whether hepatic steatosis is associated with hepatocellular carcinoma (HCC) in a cohort of patients with hepatitis C-related cirrhosis. METHODS: The authors retrospectively identified 94 consecutive patients with hepatitis C cirrhosis who underwent liver transplantation from 1992 to 2005 and had pathology available for review. Of these, 32 had evidence of HCC, and 62 had no HCC on explant histology. All explant specimens were graded again for steatosis by a single, blinded pathologist. Steatosis, age, sex, body mass index, HCV RNA, HCV genotype, Model for End-Stage Liver Disease (MELD) score, chronic alcohol use, and diabetes were examined in univariate and multivariate analyses for association with HCC. RESULTS: In total, 69% of patients in the HCC group and 50% of patients in the control group had evidence of steatosis (1+) on histology. Odds ratios for the development of HCC for each grade of steatosis compared with grade 0 were as follows: grade 1 (1.61 [0.6-4.3]), grade 2 (3.68 [1.1-12.8]), and grade 3 or 4 (8.02 [0.6-108.3]) (P = .03 for the trend). In univariate analysis, there was a significant association between increasing steatosis grade (P = .03), older age (56 years vs 49 years; P < .02), higher aspartate aminotransferase (122.5 U/L vs 91.5 U/L; P = .005), higher alanine aminotransferase (95.8 U/L vs 57.2 U/L; P = .002), higher alpha-fetoprotein (113.5 ng/mL vs 17.8 ng/mL; P < .001), lower median HCV RNA (239,000 IU/mL vs 496,500 IU/mL; P = .02), higher biologic MELD score (21.8 vs 20.3; P = .03), and risk of HCC. In multivariate analysis, age (P = .02), AFP (P = .007), and steatosis (P = .045) were significantly associated with HCC. CONCLUSIONS: In patients with HCV-related cirrhosis, the presence of hepatic steatosis is independently associated with the development of hepatocellular carcinoma. These findings suggest that steatosis poses an additional risk for HCC and that increased vigilance should be practiced in surveillance of persons with both HCV and steatosis.