Katherine Li

Peripheral Blood Proteins Predict Mortality in Idiopathic Pulmonary Fibrosis

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology with a variable and unpredictable course. OBJECTIVES The aim of this study was to identify and validate plasma proteins that are predictive of outcome in IPF. METHODS Plasma samples were available for 241 patients with IPF (140 derivation and 101 validation). In the derivation cohort, concentrations of 92 proteins were analyzed using a multiplex bead-based immunoassay and concentrations of matrix metalloproteinase (MMP)-7, MMP-1, and surfactant protein D were assessed by ELISA. In the validation cohort concentrations of intercellular adhesion molecule (ICAM)-1, IL-8, and vascular cell adhesion molecule (VCAM)-1 were assessed by bead-based multiplex assay, and S100A12 and MMP-7 by ELISA. Associations of biomarkers with mortality, transplant-free survival, and disease progression were tested in the derivation and validation cohorts using nonparametric methods of survival analysis and the Cox proportional hazards model, and an integrated risk prediction score was derived and tested. MEASUREMENTS AND MAIN RESULTS High concentrations of MMP-7, ICAM-1, IL-8, VCAM-1, and S100A12 predicted poor overall survival, poor transplant-free survival, and poor progression-free survival in the derivation cohort. In the independent validation cohort high concentrations of all five were predictive of poor transplant-free survival; MMP-7, ICAM-1, and IL-8 of overall survival; and ICAM-1 of poor progression-free survival. The personal clinical and molecular mortality prediction index derived in the derivation cohort was highly predictive of mortality in the validation cohort. CONCLUSIONS Our results suggest that plasma proteins should be evaluated as a tool for prognosis determination in prioritization of patients for lung transplantation and stratification in drug studies.

Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis

Background and aims: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor α (anti-TNFα) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. Methods: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. Results: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. Conclusion: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.

Guselkumab (an IL-23–specific mAb) demonstrates clinical and molecular response in patients with moderate-to-severe psoriasis

BACKGROUND IL-23 expression is increased in psoriatic lesions and might regulate TH17 T-cell counts in patients with psoriasis. OBJECTIVES We sought to test a novel IL-23-specific therapeutic agent for the treatment of psoriasis. METHODS In this randomized, double-blind, placebo-controlled study the safety, tolerability, and clinical response of guselkumab, an anti-IL-23-specific mAb, were evaluated in patients with moderate-to-severe plaque psoriasis. A total of 24 patients were randomized to receive a single dose of placebo or 10, 30, 100, or 300 mg of guselkumab. Clinical response was assessed by using the Psoriasis Area and Severity Index (PASI). Additionally, histologic analysis and gene expression in skin biopsy specimens from guselkumab-treated patients were compared with those from placebo-treated patients. RESULTS At week 12, 50% (10 mg), 60% (30 and 100 mg), and 100% (300 mg) of guselkumab-treated patients, respectively, achieved a 75% improvement in PASI scores from baseline compared with 0% of placebo-treated patients. Improvements in PASI scores were generally maintained through week 24 in all guselkumab-treated patients. The proportion of patients experiencing an adverse event was comparable between the combined guselkumab (13/20 [65.0%]) and placebo (2/4 [50.0%]) groups through week 24. Analysis of lesional and nonlesional skin biopsy specimens demonstrated decreases in epidermal thickness and T-cell and dendritic cell expression in guselkumab-treated patients compared with values seen in placebo-treated patients. At week 12, significant reductions in psoriasis gene expression and serum IL-17A levels were observed in guselkumab-treated patients. CONCLUSION IL-23 inhibition with a single dose of guselkumab results in clinical responses in patients with moderate-to-severe psoriasis, suggesting that neutralization of IL-23 alone is a promising therapy for psoriasis.

Expanding the Psoriasis Disease Profile: Interrogation of the Skin and Serum of Patients with Moderate-to-Severe Psoriasis

Psoriasis is a complex disease with an expanding definition of its pathological features. We sought to expand/refine the psoriasis transcriptome using 85 paired lesional and non-lesional samples from a cohort of patients with moderate-to-severe psoriasis vulgaris who were not receiving active psoriasis therapy. This new analysis identified 4,175 probe sets (representing 2,725 unique known genes) as being differentially expressed in psoriasis lesions compared with matched biopsies of non-lesional skin when the following criteria were applied: >2-fold change and false discovery rate <0.05. These probe sets represent the largest and most comprehensive set of genes defining psoriasis at the molecular level and within the previously unidentified genes, a link to functional pathways associated with metabolic diseases/diabetes and to cardiovascular risk pathways is identified. In addition, we profiled the serum of moderate-to-severe psoriatics compared with healthy controls to assess the overlap of overexpressed lesional genes with overexpressed systemic proteins. We identified linkage of functional pathways in lesional skin associated with metabolic diseases/diabetes and cardiovascular risk with those pathways overexpressed in the serum, suggesting a potential linkage between altered gene transcription in the skin and comorbidities commonly seen in patients with moderate-to-severe psoriasis.

Meta-Analysis Derived (MAD) Transcriptome of Psoriasis Defines the “Core” Pathogenesis of Disease

The cause of psoriasis, a common chronic inflammatory skin disease, is not fully understood. Microarray experiments have been widely used in recent years to identify genes associated with psoriasis pathology, by comparing expression levels of lesional (LS) with adjacent non-lesional (NL) skin. It is commonly observed that the differentially expressed genes (DEGs) differ greatly across experiments, due to variations introduced in the microarray experiment pipeline. Therefore, a statistically based meta-analytic approach, which combines the results of individual studies, is warranted. In this study, a meta-analysis was conducted on 5 microarray data sets, including 193 LS and NL pairs. We termed this the Meta-Analysis Derived (MAD) transcriptome. In “MAD-5” transcriptome, 677 genes were up-regulated and 443 were down-regulated in LS skin compared to NL skin. This represents a much larger set than the intersection of DEGs of these 5 studies, which consisted of 100 DEGs. We also analyzed 3 of the studies conducted on the Affymetrix hgu133plus2 chips and found a greater number of DEGs (1084 up- and 748 down-regulated). Top canonical pathways over-represented in the MAD transcriptome include Atherosclerosis Signaling and Fatty Acid Metabolism, while several “new” genes identified are involved in Cardiovascular Development and Lipid Metabolism. These findings highlight the relationship between psoriasis and systemic manifestations such as the metabolic syndrome and cardiovascular disease. Then, the Meta Threshold Gradient Descent Regularization (MTGDR) algorithm was used to select potential markers distinguishing LS and NL skin. The resulting set (20 genes) contained many genes that were part of the residual disease genomic profile (RDGP) or “molecular scar” after successful treatment, and also genes subject to differential methylation in LS tissues. To conclude, this MAD transcriptome yielded a reference list of reliable psoriasis DEGs, and represents a robust pool of candidates for further discovery of pathogenesis and treatment evaluation.

Gene Expression Profiling and Response Signatures Associated With Differential Responses to Infliximab Treatment in Ulcerative Colitis

OBJECTIVES:Infliximab has been shown to induce clinical response and remission in ulcerative colitis (UC). To characterize the biological response of patients to infliximab, we analyzed the mRNA expression patterns of mucosal colonic biopsies taken from UC patients enrolled in the Active Ulcerative Colitis Trial 1 (ACT1) study.METHODS:Biopsies were obtained from 48 UC patients before treatment with 5 or 10 mg/kg infliximab, and at 8 and 30 weeks after treatment (n=113 biopsies). Global gene expression profiling was performed using Affimetrix GeneChip Human Genome U133 Plus 2.0 arrays. Expression profiling results for selected genes were confirmed using qPCR.RESULTS:Infliximab had a significant effect on mRNA expression in treatment responders, with both infliximab dose and duration of treatment having an effect. Genes affected are primarily involved with inflammatory response, cell-mediated immune responses, and cell-to-cell signaling. Unlike responders, non-responders do not effectively modulate TH1, TH2, and TH17 pathways. Gene expression can differentiate placebo and infliximab responders.CONCLUSIONS:Analysis of mRNA expression in mucosal biopsies following infliximab treatment provided insight into the response to therapy and molecular mechanisms of non-response.

Ustekinumab for patients with primary biliary cholangitis who have an inadequate response to ursodeoxycholic acid: A proof‐of‐concept study

The interleukin (IL)‐12 signaling cascade has been associated with primary biliary cholangitis (PBC). This multicenter, open‐label, proof‐of‐concept study evaluated the anti‐IL12/23 monoclonal antibody, ustekinumab (90 mg subcutaneous at weeks 0 and 4, then every 8 weeks through week 20), in adults with PBC and an inadequate response to ursodeoxycholic acid therapy (i.e., alkaline phosphatase [ALP] >1.67× upper limit of normal [ULN] after ≥6 months). ALP response was defined as a >40% decrease from baseline and ALP remission as ALP normalization (if baseline ALP 1.67×‐2.8× ULN) or <1.67× ULN (if baseline ALP >2.8× ULN). Changes in Enhanced Liver Fibrosis (ELF) scores and serum bile acids were also assessed. At baseline, patients had median disease duration of 3.2 years, median ELF score of 9.8, and highly elevated total bile acid concentration (median, 43.3 μmol/L); 13 of 20 (65%) patients had baseline ALP >3× ULN. Although steady‐state serum ustekinumab concentrations were reached by week 12, no patient achieved ALP response or remission. Median percent ALP reduction from baseline to week 28 was 12.1%. ELF score decreased slightly from baseline to week 28 (median reduction: 0.173), and total serum bile acid concentrations decreased from baseline to week 28 (median reduction: 8.8 μmol/L). No serious infections or discontinuations resulting from adverse events were reported through week 28. One patient had a serious upper gastrointestinal hemorrhage considered unrelated to test agent by the investigator. Conclusion: Open‐label ustekinumab therapy, though associated with a modest decrease in ALP after 28 weeks of therapy, did not otherwise appreciably change ALP and overt proof‐of‐concept was not established as per prespecified primary endpoint of proposed efficacy. No new ustekinumab safety signals were observed. (Hepatology 2016;64:189‐199)

Expression and activation of the oxytocin receptor in airway smooth muscle cells: Regulation by TNFα and IL-13

BackgroundDuring pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFα, cytokines known to be important in asthma.MethodExpression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma.ResultsPCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFα) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFα had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma.ConclusionTaken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.

Ustekinumab for patients with primary biliary cholangitis who have an inadequate response to ursodeoxycholic acid

The interleukin (IL)‐12 signaling cascade has been associated with primary biliary cholangitis (PBC). This multicenter, open‐label, proof‐of‐concept study evaluated the anti‐IL12/23 monoclonal antibody, ustekinumab (90 mg subcutaneous at weeks 0 and 4, then every 8 weeks through week 20), in adults with PBC and an inadequate response to ursodeoxycholic acid therapy (i.e., alkaline phosphatase [ALP] >1.67× upper limit of normal [ULN] after ≥6 months). ALP response was defined as a >40% decrease from baseline and ALP remission as ALP normalization (if baseline ALP 1.67×‐2.8× ULN) or <1.67× ULN (if baseline ALP >2.8× ULN). Changes in Enhanced Liver Fibrosis (ELF) scores and serum bile acids were also assessed. At baseline, patients had median disease duration of 3.2 years, median ELF score of 9.8, and highly elevated total bile acid concentration (median, 43.3 μmol/L); 13 of 20 (65%) patients had baseline ALP >3× ULN. Although steady‐state serum ustekinumab concentrations were reached by week 12, no patient achieved ALP response or remission. Median percent ALP reduction from baseline to week 28 was 12.1%. ELF score decreased slightly from baseline to week 28 (median reduction: 0.173), and total serum bile acid concentrations decreased from baseline to week 28 (median reduction: 8.8 μmol/L). No serious infections or discontinuations resulting from adverse events were reported through week 28. One patient had a serious upper gastrointestinal hemorrhage considered unrelated to test agent by the investigator. Conclusion: Open‐label ustekinumab therapy, though associated with a modest decrease in ALP after 28 weeks of therapy, did not otherwise appreciably change ALP and overt proof‐of‐concept was not established as per prespecified primary endpoint of proposed efficacy. No new ustekinumab safety signals were observed. (Hepatology 2016;64:189‐199)

Genes associated with intestinal permeability in ulcerative colitis: Changes in expression following infliximab therapy†

Background: Alterations in intestinal permeability have been implicated in ulcerative colitis (UC). Infliximab, a monoclonal anti‐tumor necrosis factor alpha (TNF&agr;) antibody, can induce clinical response in UC. Gene expression in colonic biopsies taken from responders and nonresponders to infliximab can provide insight into the mechanisms of the altered intestinal permeability at a molecular level. Methods: Colonic biopsies (n = 18 anti‐TNF&agr; naïve UC patients; n = 8 normal controls; n = 80 Active Ulcerative Colitis Trial [ACT] 1 patients) were analyzed for mRNA expression using gene expression microarrays. Computational reverse causal reasoning was applied to build causal network models of UC and response and nonresponse of UC to treatment. Quantitative reverse‐transcription polymerase chain reaction (qPCR) was used to confirm differentially expressed genes. Results: Reverse causal reasoning on mRNA expression data from anti‐TNF&agr;‐naïve UC and normal samples provided a mechanistic disease model of the biology of gene expression observed in UC. mRNA expression data from the ACT 1 study enabled construction of a mechanistic model describing the biology of nonresponders to infliximab, including evidence for increased intestinal permeability compared with normal and responder samples. Gene expression changes identified as central to intestinal permeability dysregulation were confirmed in normal, UC, and infliximab‐treated patients by qPCR analysis. Gene expression returned toward normal levels in infliximab responders, but not in nonresponders. Conclusion: Gene expression analysis and causal network modeling in combination showed that aberrant mRNA expression of genes involved in intestinal epithelial permeability for infliximab responders was restored toward levels observed in normal samples. Infliximab nonresponders showed no equivalent restoration in the expression of these genes. (Inflamm Bowel Dis 2012)