Development and Validation of a Test to Monitor Endoscopic Activity in Patients With Crohn's Disease Based on Serum Levels of Proteins.

BACKGROUND & AIMS: Noninvasive tests to measure endoscopic activity in patients with Crohn's disease (CD) have limitations. We aimed to develop a test to identify patients in remission, based on endoscopic analysis, and monitor CD activity based on serum levels of proteins. METHODS: We developed a test to measure 13 proteins in blood (ANG1, ANG2, CRP, SAA1, IL7, EMMPRIN, MMP1, MMP2, MMP3, MMP9, TGFA, CEACAM1, and VCAM1), called the endoscopic healing index [EHI], using samples from 278 patients with CD from a multinational training cohort. We validated the test using 2 independent cohorts of patients with CD: 116 biologic-naive patients with early-stage CD (validation cohort 1) and 195 biologic-exposed patients with chronic CD (validation cohort 2). The ability of the test to identify patients with active disease vs patients in remission (defined as a simple endoscopic score for CD of ≤2 and ≤1 in each segment, or a total CD endoscopic index of severity score <3) was assessed by using area under receiver operating characteristic curve (AUROC) analysis. The diagnostic accuracy of the test was compared with that of measurement of serum C-reactive protein (CRP) and fecal calprotectin. RESULTS: The EHI scores range from 0 to 100 units; higher scores indicate more severe CD activity, based on endoscopy findings. The EHI identified patients in remission with an AUROC of 0.962 in validation cohort 1 (95% confidence interval, 0.942-0.982) and an AUROC of 0.693 in validation cohort 2 (95% confidence interval, 0.619-0.767), regardless of CD location or phenotype. A cutoff value of 20 points identified patients in remission with the highest level of sensitivity (97.1% in validation cohort 1 and 83.2% in validation cohort 2), with specificity values of 69.0% and 36.6%, respectively. A cutoff value of 50 points identified patients in remission with the highest level of specificity (100% in validation cohort 1 and 87.8% in validation cohort 2), with sensitivity values of 37.3% and 30.0%, respectively. The EHI identified patients in remission with a significantly higher AUROC value than the test for CRP (0.876, P < .001 in validation cohort 1 and 0.624, P = .109 in validation cohort 2). In analysis of patients with available FC measurements, the AUROC value for the EHI did not differ significantly from that of measurement of FC (AUROC, 0.950 for EHI vs AUROC, 0.923 for FC; P = .147 in validation cohort 1 and AUROC, 0.803 for EHI vs AUROC, 0.854 for FC; P = .298 in validation cohort 2). CONCLUSIONS: We developed an index called the EHI to identify patients with CD in endoscopic remission based on blood levels of 13 proteins. The EHI identified patients with resolution of endoscopic disease activity, with good overall accuracy, although with variation between the 2 cohorts assessed. The EHI AUROC values were comparable to measurement of FC and higher than measurement of serum CRP. The test might be used in practice to assess endoscopic activity in patients with CD.

Biomarkers Are Associated With Clinical and Endoscopic Outcomes With Vedolizumab Treatment in Ulcerative Colitis.

Background: Vedolizumab inhibits α4ß7-mediated lymphocyte trafficking and is effective in ulcerative colitis (UC). This study evaluated drug and biomarker concentrations and patient outcomes during vedolizumab treatment in UC. Methods: Prospectively scored maintenance clinical (26.5 weeks; interquartile range [IQR], 16.3-37.0 weeks) and endoscopic (23.5 weeks; IQR, 16.8-35.6 weeks) outcomes were compared with serum vedolizumab concentrations, antivedolizumab antibodies, and serum biomarkers at baseline and weeks 2, 6, 14, and 26. A linear mixed-effects model compared biomarker trajectories over time between clinical and endoscopic remitters and nonremitters. Results: Thirty-two patients were included. Soluble (s)-tumor necrosis factor (TNF)-α, s-α4ß7, s-mucosal addressin cell adhesion molecule (s-MAdCAM-1), and s-amyloid A (s-AA) significantly changed with treatment. A linear mixed-effects model demonstrated that s-α4ß7 (P = 0.044) increased and s-MAdCAM-1 (P = 0.006) and s-vascular cell adhesion molecule-1 (s-VCAM-1, P = 0.001) decreased more rapidly in patients achieving clinical remission in maintenance. S-MAdCAM-1 (P = 0.005), s-intracellular adhesion molecule-1 (ICAM-1; P = 0.014), s-VCAM-1 (P < 0.001), and s-TNF (P = 0.052) decreased more rapidly in endoscopic remitters. In clinical remitters, higher week 14 (20.3 ng/mL vs 6.0 ng/mL; P = 0.013) and week 26 (14.1 ng/mL vs 8.6 ng/mL; P = 0.05) s-α4ß7 were observed. In endoscopic remitters, week 2 (6.7 pg/mL vs 17.8 pg/mL; P = 0.038) and week 6 (3.9 pg/mL vs 15.6 pg/mL; P = 0.005) s-TNF and week 14 s-VCAM (589.1 ng/mL vs 746.0 ng/mL; P = 0.05) were lower. Conclusion: Serum biomarkers were associated with outcomes in vedolizumab-treated UC patients. s-α4ß7 increased, whereas s-MAdCAM-1, s-VCAM-1, s-ICAM-1, and s-TNF decreased more rapidly in remitters. At individual time points, induction s-TNF and maintenance s-VCAM-1 concentrations were lower, whereas maintenance s-α4ß7 concentrations were higher in remitters.

Validation of Serum Test for Advanced Liver Fibrosis in Patients With Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: We analyzed markers of fibrosis in serum samples from patients with nonalcoholic fatty liver disease (NAFLD), assessed by liver biopsy. We used serum levels of markers to develop an algorithm to discriminate patients with advanced fibrosis from those with mild or moderate fibrosis and validated its performance in 2 independent cohorts of patients with NAFLD. METHODS: We performed a retrospective analysis of serum samples from 396 patients with NAFLD and different stages of fibrosis (F0-F4), collected from 2007 through 2017 on the day of liver biopsy (training cohort 1). We measured serum concentrations of alpha-2 macroglobulin (A2M), hyaluronic acid (HA), and TIMP metallopeptidase inhibitor 1 (TIMP1), and used measurements to develop an algorithm that could discriminate patients with NAFLD with advanced fibrosis (F3-F4; 24.1% of cohort) from those with mild or moderate fibrosis (F0-F2; 79.5% of cohort). We validated the algorithm using serum samples collected from a separate 396 patients from the same time period and location (validation cohort 1), as well as 244 patients with NAFLD evaluated at a separate location, from 2011 through 2017, within a median of 11 days of liver biopsy (cohort 2). RESULTS: The algorithm identified patients with advanced fibrosis vs mild or moderate fibrosis in training cohort 1 with an area under the receiver operating characteristic (AUROC) curve of 0.867 (95% CI, 0.827-0.907), 84.8% sensitivity (95% CI, 75.5%-91.0%), and 72.3% specificity (95% CI, 66.9%-77.3%), at a cutoff score of 17. The AUROC for the combined validation cohorts 1 and 2 (n=640) was 0.856 (95% CI, 0.820-0.892), identifying patients with 79.7% sensitivity (95% CI, 71.9%-86.2%) and 75.7% specificity (95% CI, 71.8%-79.4%) at the predetermined cutoff score of 17. The algorithm had negative predictive values that ranged from 92.5% to 94.7% in the validation cohorts; it correctly classified 90.0% of F0 samples, 75.0% of F1 samples, 77.4% of F3 samples, and 94.4% of F4 samples. CONCLUSION: We developed an algorithm that identifies patients with advanced fibrosis from those with mild to moderate fibrosis in patients with NAFLD with an AUROC value of approximately 0.86, based on levels of serum biomarkers. We validated the findings in 2 separate sets of patients with biopsy-proven NAFLD. The algorithm can be used non-invasively to determine risk of advanced fibrosis in patients with NAFLD.

Serum Concentrations of 7α-hydroxy-4-cholesten-3-one Are Associated With Bile Acid Diarrhea in Patients With Crohn's Disease.

BACKGROUND & AIMS: Patients with Crohn's disease (CD) often have bile acid diarrhea (BAD), due to bile acid malabsorption following ileal resection (IR). Bile acid malabsorption increases production of 7α-hydroxy-4-cholesten-3-one (C4), a bile acid precursor. We investigated relationships between serum concentrations of C4 and BAD in patients with CD. METHODS: We collected demographic data, serum samples, and information on the presence of diarrhea (>3 liquid bowel movements/day), as well as clinical, endoscopic, and histologic scores from 26 patients with CD and IR, 21 patients with CD without IR, and 37 patients with ulcerative colitis (UC). We compared serum concentrations of C4 and fibroblast growth factor 19 (FGF19) between groups. We performed area under the receiver operating characteristic curve (AUROC) analysis to identify the optimal cutoff C4 concentrations for the diagnosis of diarrhea attributable to bile acid malabsorption (BAD), defined as diarrhea and a serum concentration of FGF19 <60 pg/mL. RESULTS: Patients with UC had a median serum C4 concentration of 11.8 ng/mL, whereas patients with CD and IR with ileitis (documented endoscopically) had a median concentration of 100.0 ng/mL (P compared to UC < .0001) and patients with CD and IR without ileitis had a median concentration of 51.6 ng/mL (P compared to UC < .001). Patients with CD without IR did not have a significantly higher median concentration of C4 than patients with UC (P = .71), regardless of ileitis (P = .34). When endoscopic findings were confirmed histologically, similar results were found to analyses using endoscopic findings alone. A higher proportion of patients with active UC had diarrhea (72.0% vs 0 patients with inactive UC; P < .001), but their median concentrations of C4 did not differ significantly from that of patients with inactive UC (12.1 ng/mL vs 9.7 ng/mL; P = .3). A cutoff concentration of C4 of 48.3 ng/mL or greater identified patients with diarrhea attributable to bile acid malabsorption with 90.9% sensitivity, 84.4% specificity, and an AUROC 0.94. A significantly higher proportion of patients with concentrations of C4 above this cutoff had BAD (50.0%) than below this cutoff (1.8%) (P < .001). When we analyzed only patients with diarrhea, a C4 cutoff of 48.3 ng/mL identified those with low FGF19 concentrations (<60 pg/mL) with 91% sensitivity and 95.5% specificity (AUROC, 0.99). Above this cutoff, 83.3% of patients had a serum concentration of FGF19 <60 pg/mL compared to 4.5% below this threshold (P < .0001). C4 concentrations correlated with the number of daily bowel movements (r = 0.41; P = .004) and correlated inversely with FGF19 concentrations (r = -0.72; P<.0001). CONCLUSION: We observed significantly increased serum concentrations of C4 in patients with CD with IR, compared to patients with UC. A cutoff concentration of C4 above 48.3 ng/mL identifies patients with diarrhea likely attributable to bile acid malabsorption (BAD) with an AUROC value of 0.94. Increased serum levels of bile acid precursors identify patients with diarrhea and a low serum concentration of FGF19, and concentrations of C4 correlate with daily liquid bowel movements and correlate inversely with FGF19 concentrations. C4 may be a biomarker to identify patients with diarrhea attributable to bile acid malabsorption.

Keratinocyte-derived chemokine in obesity: expression, regulation, and role in adipose macrophage infiltration and glucose homeostasis.

Obese adipose tissue (AT) is associated with chronic inflammation, and we hypothesized that the keratinocyte-derived chemokine (KC), the mouse ortholog of human interleukin-8, plays a role in obesity-mediated AT inflammation and the subsequent manifestation of insulin resistance. KC expression is increased in the AT and plasma of genetically (ob/ob) and high fat diet-induced obese mouse models, and this increase may be mediated by the elevated leptin and tumor necrosis factor-alpha levels associated with obesity. Obesity-induced KC expression occurs primarily in stromal vascular cells and not in adipocytes, and it is high in preadipocytes and decreases during adipogenesis. Although KC has no effect on adipogenesis, it induces adipocyte expression of inflammatory factors and the insulin resistance mediator, suppressor of cytokine signaling 3. Using chimeric mice deficient in the KC receptor CXCR2 in their bone marrow, we show that the lack of CXCR2 in hematopoietic cells is sufficient to protect from adipose and skeletal muscle macrophage recruitment and development of insulin resistance in diet-induced obese mice. These studies suggest that KC and its receptor CXCR2 are potential targets for the development of new therapeutic approaches for treatment of obesity-related insulin resistance, type II diabetes, and related cardiovascular diseases.

Differential repression of c-myc and cdc2 gene expression by ERF and PE-1/METS.

The molecular mechanisms that control the proliferation and differentiation of specific cell types remain poorly understood. Positive ETS factors play important roles in mediating proliferative responses to Ras/MAPK signaling in many cell types following mitogenic stimulation. PE-1/METS, a member of the ETS-domain family transcription factors that functions as a transcriptional repressor, can block mitogenic responses mediated by positively acting Ets factors. The anti-proliferative functions of PE-1/METS require its interaction with DP103, a multifunctional DEAD-box protein that mediates interactions with corepressor proteins and acts in a cooperative manner with Rb family members and to repress cell cycle control genes. ETS-2 repressor factor (ERF) is structurally related to and also functions as a transcriptional repressor, but endogenous target genes and mechanisms of repression remain unknown. Here, we demonstrate that like PE-1/METS, ERF-mediated repression also requires DP103, and that ERF negatively regulates the c-myc and cdc2 genes. In contrast to PE-1/METS, however, ERF-mediated repression of these genes is inactivated by MAPK signaling through phosphorylation sites that are ERF-specific. Furthermore, constitutive activation of the Ras/MAPK pathway in RAW 264.7 cells transformed by the v-Abelson leukemia virus is associated with constitutive inactivation of ERF in this cell type. We propose that ERF and PE-1/METS function to impose 'repression checkpoints' on a subset of cell cycle control genes that are differentially regulated by growth factor signaling pathways that control proliferation and differentiation and that ERF is targeted for inactivation by transforming oncogenes such as vAbl.

Altered adipose and plasma sphingolipid metabolism in obesity: a potential mechanism for cardiovascular and metabolic risk.

The adipose tissue has become a central focus in the pathogenesis of obesity-mediated cardiovascular and metabolic disease. Here we demonstrate that adipose sphingolipid metabolism is altered in genetically obese (ob/ob) mice. Expression of enzymes involved in ceramide generation (neutral sphingomyelinase [NSMase], acid sphingomyelinase [ASMase], and serine-palmitoyl-transferase [SPT]) and ceramide hydrolysis (ceramidase) are elevated in obese adipose tissues. Our data also suggest that hyperinsulinemia and elevated tumor necrosis factor (TNF)-alpha associated with obesity may contribute to the observed increase in adipose NSMase, ASMase, and SPT mRNA in this murine model of obesity. Liquid chromatography/mass spectroscopy revealed a decrease in total adipose sphingomyelin and ceramide levels but an increase in sphingosine in ob/ob mice compared with lean mice. In contrast to the adipose tissue, plasma levels of total sphingomyelin, ceramide, sphingosine, and sphingosine 1-phosphate (S1P) were elevated in ob/ob mice. In cultured adipocytes, ceramide, sphingosine, and S1P induced gene expression of plasminogen activator inhibitor-1, TNF-alpha, monocyte chemoattractant protein-1, interleukin-6, and keratinocyte-derived chemokine. Collectively, our results identify a novel role for sphingolipids in contributing to the prothrombotic and proinflammatory phenotype of the obese adipose tissue currently believed to play a major role in the pathogenesis of obesity-mediated cardiovascular and metabolic disease.

PE-1/METS, an antiproliferative Ets repressor factor, is induced by CREB-1/CREM-1 during macrophage differentiation.

The molecular mechanisms involved in regulating the balance between cellular proliferation and differentiation remain poorly understood. Members of the Ets-domain family of transcription factors are candidates for proteins that might differentially regulate cell cycle control and cell type-specific genes during the differentiation of myeloid progenitor cells. The Ets repressor PE-1/METS has been suggested to contribute to growth arrest during terminal macrophage differentiation by repressing Ets target genes involved in Ras-dependent proliferation. An important feature of this regulatory model is that PE-1/METS is itself induced by the program of macrophage differentiation elicited by M-CSF. Here, we present evidence that the PE-1/METS gene is a transcriptional target of the cyclic AMP response element-binding protein-1 (CREB-1). CREB-1 expression is dramatically up-regulated during macrophage differentiation and phosphorylation of CREB-1 and the related factor CREM-1 are stimulated by M-CSF in a SAPK2/p38-dependent manner. Chromatin immunoprecipitation experiments demonstrate that CREB-1/CREM-1 are recruited to the PE-1/METS promoter as well as to the promoters of other genes that are up-regulated during terminal macrophage differentiation. Overexpression of CREB-1 stimulates the activities of the PE-1/METS, and macrosialin promoters, while expression of a dominant negative form of CREB-1 during macrophage differentiation inhibits expression of the PE-1/METS and macrosialin genes. Inhibition of CREB function also results in reduced expression of CD54 and impaired cell adhesion. Taken together, these findings reveal new roles of CREB-1/CREM-1 as regulators of macrophage differentiation.