The pseudo-caspase FLIP(L) regulates cell fate following p53 activation.

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

Review of recent evidence on the management of heartburn in pregnant and breastfeeding women.

Gastroesophageal reflux disease (GERD) is one the most common medical complaints in pregnant women. Some women continue to experience GERD symptoms after delivery. Effective management of GERD symptoms is important to improve productivity and quality of life. Management of heartburn in pregnant and breastfeeding women involves lifestyle modifications, dietary modifications, non-pharmaceutical remedies and pharmaceutical drugs. For most patients, lifestyle/dietary modifications are helpful in reducing GERD symptoms. For patients who require a more intense intervention, various types of pharmaceutical drugs are available. However, the suitability of each treatment for use during pregnancy and lactation must be taken into consideration. This article explores the reported efficacy and safety of these treatment options in pregnant and breastfeeding women. Recommended treatment algorithm in pregnant and breastfeeding women have been developed.

Inhibition of anti-viral responses in intestinal epithelial cells by epigenetic modifying drugs is mediated by a reduction in viral pattern recognition receptor expression and activity.

Background: Pattern recognition receptors form an essential part of the host defenses against pathogens, in particular in the intestinal epithelium. However, despite their importance relatively little is understood about the regulation of their expression. Increasing evidence suggesting that epigenetic mechanisms such as DNA methylation and histone acetylation have substantial effects on gene expression and regulation. Epigenetic modifying drugs are now used to treat certain cancers but not a lot is known about their effects on the innate immune system. Thus, we set out to examine the role of such drugs in the expression and function of Toll-like receptors. Methods: Using the HCT116 epithelial cell line, we determined the effects of genetic knockout of the DNA methyltransferases enzymes (DNMTs), as well as pharmacological inhibition of the DNMTs and histone deacetylase complexes (HDACs) on TLR responses to their ligands. Results: Our initial results showed that anti-viral responses were affected by changes in the epigenome, with TLR3 responses showing the most dramatic differences. We determined that inhibition of methylation and acetylation inhibited poly I:C induced increases in signaling protein phosphorylation, as well as increases in cytokine mRNA expression and release. We also observed that treatment with epigenetic modifying drugs were leading to large increases in IRF8 expression, a protein that is a known negative regulator of TLR3. When we overexpressed IRF8 in our WT cells we noticed inhibition of poly I:C responses. Conclusion: This research highlighted the potential immunoregulatory role of epigenetic modifying drugs specifically in response to viral stimulation.

Differential DNA methylation patterns of homeobox genes in proximal and distal colon epithelial cells.

Region and cell-type specific differences in the molecular make up of colon epithelial cells have been reported. Those differences may underlie the region-specific characteristics of common colon epithelial diseases such as colorectal cancer and inflammatory bowel disease. DNA methylation is a cell-type specific epigenetic mark, essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. Little is known about any region-specific variations in methylation patterns in human colon epithelial cells. Using purified epithelial cells and whole biopsies (n= 19) from human subjects, we generated epigenome-wide DNA methylation data (using the HELP-tagging assay), comparing the methylation signatures of the proximal and distal colon. We identified a total of 125 differentially methylated sites (DMS) mapping to transcription start sites of protein-coding genes, most notably several members of the homeobox (HOX) family of genes. Patterns of differential methylation were validated with MassArray EpiTYPER. We also examined DNA methylation in whole biopsies, applying a computational technique to deconvolve variation in methylation within cell types and variation in cell-type composition across biopsies. Including inferred epithelial proportions as a covariate in differential methylation analysis applied to the whole biopsies resulted in greater overlap with the results obtained from purified epithelial cells compared with when the covariate was not included. Results obtained from both approaches highlight region-specific methylation patterns of HOX genes in colonic epithelium. Regional variation in methylation patterns has implications for the study of diseases that exhibit regional expression patterns in the human colon, such as inflammatory bowel disease and colorectal cancer.

The intestinal epithelial cell cycle: uncovering its 'cryptic' nature.

PURPOSE OF REVIEW: To discuss the recent landmark findings that have increased our understanding not only of the role of the epithelial cell cycle in the homeostasis of the small intestine, but also its relevance to inflammation and cancer. RECENT FINDINGS: Recent data have unveiled novel information on protein interactions directly involved in the cell cycle as well as in the pathways that transduce external environmental signals to the cell cycle. A growing body of the recent evidence confirms the importance of food as well as hormonal regulation in the gut on cell cycle. Information on the contribution of the epithelial microenvironment, including the microbiota, has grown substantially in the recent years as well as on the gene-environment interactions and the multiple epigenetic mechanisms involved in regulating cell-cycle proteins and signalling. Finally, further studies investigating the dysregulation of the cell cycle during inflammation and proliferation have increased our understanding of the pathophysiology of chronic inflammatory diseases and cancer. SUMMARY: This review highlights some of the most recent advances that further emphasize the importance of the cell cycle in the small intestine during homeostasis as well as in inflammation and cancer.

Targeting stromal cell sialylation reverses T cell-mediated immunosuppression in the tumor microenvironment.

Immunosuppressive tumor microenvironments (TMEs) reduce the effectiveness of immune responses in cancer. Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote tumor progression by enhancing immune cell suppression in colorectal cancer (CRC). Hyper-sialylation of glycans promotes immune evasion in cancer through binding of sialic acids to their receptors, Siglecs, expressed on immune cells, which results in inhibition of effector functions. The role of sialylation in shaping MSC/CAF immunosuppression in the TME is not well characterized. In this study, we show that tumor-conditioned stromal cells have increased sialyltransferase expression, α2,3/6-linked sialic acid, and Siglec ligands. Tumor-conditioned stromal cells and CAFs induce exhausted immunomodulatory CD8+ PD1+ and CD8+ Siglec-7+/Siglec-9+ T cell phenotypes. In vivo, targeting stromal cell sialylation reverses stromal cell-mediated immunosuppression, as shown by infiltration of CD25 and granzyme B-expressing CD8+ T cells in the tumor and draining lymph node. Targeting stromal cell sialylation may overcome immunosuppression in the CRC TME.

Inflammation, DNA methylation and colitis-associated cancer.

Inflammation can result from a range of sources including microbial infections, exposure to allergens and toxic chemicals, autoimmune disease and obesity. A well-balanced immune response can be anti-tumorigenic; however, a sustained or chronic inflammatory response is generally harmful as the immune response becomes distorted. A causal link between chronic inflammation and cancer is now well accepted and many chronically inflamed organs of the gastrointestinal tract show this association. For example, patients with inflammatory bowel disease (IBD), including both ulcerative colitis and Crohn's disease, have a 2- to 3-fold greater lifetime risk of developing colorectal cancer compared with the general population. The development of colitis-associated cancer (CAC) is thought to be multifaceted and is probably due to a combination of genetic factors, epigenetic factors and the duration, extent and severity of disease. Recently, epigenetic alterations, in particular alterations in DNA methylation, have been observed during inflammation and inflammation-associated carcinogenesis. The mediators of this, the significance of these changes in DNA methylation and the effect this has on gene expression and the malignant transformation of the epithelial cells during IBD and CAC are discussed in this review. The recent advances in technologies to study genome-wide DNA methylation and the therapeutic potential of understanding these molecular mechanisms are also highlighted.

Autophagosomal IkappaB alpha degradation plays a role in the long term control of tumor necrosis factor-alpha-induced nuclear factor-kappaB (NF-kappaB) activity.

Transcription factor NF-κB is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-κB is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-α on NF-κB activity for up to 48 h in intestinal epithelial cells. Results show that NF-κB remained persistently activated up to 48 h after TNF-α and that the long term activation of NF-κB was accompanied by a biphasic degradation of IκBα. The first phase of IκBα degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-α stimulated formation of autophagosomes in intestinal epithelial cells and that IκBα co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-α-induced degradation of IκBα and lowered NF-κB target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-κB activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-κB may mediate this effect.

Inflammation-specific targeted carriers for local drug delivery to inflammatory bowel disease.

Delivering drugs directly to the inflamed intestinal sites to treat inflammatory bowel disease (IBD), particularly Crohn's and ulcerative colitis, is highly challenging. Recent advances in colitis therapy medications are expanding opportunities for improving local on-site drug availability by minimising the associated systemic side-effects. Drug delivery with targeted carrier systems has shown the potential to increase site-specificity, stability, and therapeutic efficacy. Herein, we report the development of a strong anionic charged inflammation targeted nanocarriers (IT-NCs) loaded with an immunosuppressant model drug. This system showed preferential adhesion on a charge-modified surface in vitro, and in both dextran sulfate sodium (DSS) and TNBS colitis mice in vivo models. IT-NCs showed improved colitis phenotype therapeutic efficacy in both animal models compared to free drug. Furthermore, ex vivo study of colon tissue biopsies from patients with colitis revealed that IT-NCs adhered preferentially to inflamed biopsies compared to normal. Together, our results suggest that IT-NCs have promising therapeutic potential as delivery carriers' in colitis management.

Mechanisms of drug toxicity or intolerance.

Classically, adverse drug reactions had been considered as type A reactions which are related to the main pharmacological action of the drug and therefore are predictable. Such reactions are predictable, reversible, and usually can be managed by lowering the dose of the offending drug. However, other adverse effects of drugs can occur which are unrelated to the main pharmacological action of the drug - type B reactions. Such adverse effects are termed idiosyncratic and are often initiated by metabolites of the parent drug or by other indirect mechanisms. The detailed understanding of adverse drug events has become a major focus of the regulatory agencies throughout the world. The pharmacotherapy of gastrointestinal and liver disorders is becoming increasingly complex. In recent years, with the advent of novel therapeutic agents to treat a host of disorders, including viral hepatitis, gastrointestinal motility disorders, inflammatory bowel disease and others, the potential for serious clinically relevant drug reactions has increased. In the pharmacotherapy of gastrointestinal and liver diseases, a significant number of adverse events that occur can be explained by drug interactions. Some pharmacokinetic drug interactions are based on the competitive inhibition of the rate of drug metabolism of one of the drugs, leading to an increased concentration of the drug which was not intended. In other examples, the interaction can be mechanistic in which one or more drugs when co-administered potentiate each other's actions without any change in drug levels, termed pharmacodynamic interactions.

Comparison of medium to long-term outcomes of acute severe ulcerative colitis patients receiving accelerated and standard infliximab induction.

INTRODUCTION: Accelerated dose infliximab (IFX) induction is associated with reduced short-term colectomy rate in acute severe ulcerative colitis (ASUC). Data on medium/long-term outcomes of this strategy are limited. AIMS: Evaluate medium/long-term outcomes in patients receiving IFX induction for ASUC, comparing accelerated dose (AD) and standard dose (SD) induction. METHODS: Retrospective study of consecutive patients admitted with corticosteroid-refractory ASUC in four tertiary referral centres within INITIative IBD research network (www.initiativeibd.ie). IFX rescue was given either as SD (weeks 0, 2, 6) or AD (<28 days) from January 2010 to September 2017. AD induction has been utilised in participating centres since 2014. Consequently SD patients were subdivided based on time period of IFX rescue: historical SD group (SD1) (2010-2013) and current SD group (SD2) (2014-2017). Primary endpoint was time to colectomy; secondary endpoint was time to IFX discontinuation if induction was complete. RESULTS: 145 patients received rescue IFX (AD=58, SD1=32, SD2=55). Disease severity at induction was comparable between AD and SD1 groups; however, SD2 group had less severe disease: median C-reactive protein (CRP) 39, 44 and 20 mg/L for AD, SD1 and SD2 groups, respectively (p=0.026, Kruskal-Wallis); median CRP: albumin ratio was 1.4, 1.8 and 0.6 (p=0.016). Median follow-up for AD, SD1 and SD2 groups was 1.6 (IQR 1.1-3.1), 4.9 (IQR 2.6-5.5) and 1.5 (IQR 0.9-2.3) years. Time to colectomy was shorter in SD1 (log rank p=0.0013); no significant difference in time to colectomy was observed comparing AD and SD2 groups (log rank p=0.32). 123 patients (84%) completed IFX induction and received maintenance therapy. Time to IFX discontinuation was shorter in SD1 (log rank p=0.009). CONCLUSION: Time to colectomy is significantly prolonged with use of AD IFX in selected ASUC patients with more severe disease. Historical use of standard IFX induction for all ASUC patients is associated with inferior long-term outcomes.

Risk factors for opportunistic infections in patients with inflammatory bowel disease.

BACKGROUND & AIMS: We sought to identify and quantify the clinical factors that were associated with opportunistic infections in inflammatory bowel disease patients. METHODS: We identified 100 consecutive IBD patients with opportunistic infections. For each case, 2 matched IBD patients who did not have a history of opportunistic infection were selected as controls. Conditional logistic regression was used to assess associations between putative risk factors and opportunistic infections, presented as odds ratios (OR) and 95% confidence intervals (CIs). RESULTS: In univariate analysis, use of corticosteroids (OR, 3.4; 95% CI, 1.8-6.2), azathioprine/6-mercaptopurine (OR, 3.1; 95% CI, 1.7-5.5), and infliximab (OR, 4.4; 95% CI, 1.2-17.1) were associated individually with significantly increased odds for opportunistic infection. Multivariate analysis indicated that use of any one of these drugs yielded an OR of 2.9 (95% CI, 1.5-5.3), whereas use of 2 or 3 of these drugs yielded an OR of 14.5 (95% CI, 4.9-43) for opportunistic infection. The relative risk of opportunistic infection was greatest in IBD patients seen at older than 50 years of age (OR, 3.0; 95% CI, 1.2-7.2, relative to those 24 years or younger). No patient died from opportunistic infection. CONCLUSIONS: Immunosuppressive medications, especially when used in combination, and older age are associated with increased risk of opportunistic infections. The absolute risk of opportunistic infection in IBD patients remains to be determined, as does any potential benefit of any preventive strategy.

Small-bowel imaging in Crohn's disease: a prospective, blinded, 4-way comparison trial.

BACKGROUND: With the introduction of new techniques to image the small bowel, there remains uncertainty about their role for diagnosing Crohn's disease. OBJECTIVE: To assess the sensitivity and specificity of capsule endoscopy (CE), CT enterography (CTE), ileocolonoscopy, and small-bowel follow-through (SBFT) in the diagnosis of small bowel Crohn's disease. METHODS: Prospective, blinded trial. SETTING: Inflammatory bowel disease clinic at an academic medical center. PATIENTS: Known or suspected Crohn's disease. Exclusion criteria included known abdominal abscess and non-steroidal anti-inflammatory drug (NSAID) use. Partial small-bowel obstruction (PSBO) at CTE excluded patients from subsequent CE. INTERVENTIONS: Patients underwent all 4 tests over a 4-day period. MAIN OUTCOME MEASUREMENTS: Sensitivity, specificity, and accuracy of each test to detect active small-bowel Crohn's disease. The criterion standard was a consensus diagnosis based upon clinical presentation and all 4 studies. RESULTS: Forty-one CTE examinations were performed. Seven patients (17%) had an asymptomatic PSBO. Forty patients underwent colonoscopy, 38 had SBFT studies, and 28 had CE examinations. Small-bowel Crohn's disease was active in 51%, absent in 42%, inactive in 5%, and suspicious in 2% of patients. The sensitivity of CE for detecting active small-bowel Crohn's disease was 83%, not significantly higher than CTE (83%), ileocolonoscopy (74%), or SBFT (65%). However, the specificity of CE (53%) was significantly lower than the other tests (P < .05). One patient developed a transient PSBO due to CE, but no patients had retained capsules. LIMITATION: Use of a consensus clinical diagnosis as the criterion standard-but this is how Crohn's disease is diagnosed in practice. CONCLUSIONS: The sensitivity of CE for active small-bowel Crohn's disease was not significantly different from CTE, ileocolonoscopy, or SBFT. However, lower specificity and the need for preceding small-bowel radiography (due to the high frequency of asymptomatic PSBO) may limit the utility of CE as a first-line test for Crohn's disease.

Stromal Cell PD-L1 Inhibits CD8+ T-cell Antitumor Immune Responses and Promotes Colon Cancer.

Stromal cells of mesenchymal origin reside below the epithelial compartment and provide structural support in the intestine. These intestinal stromal cells interact with both the epithelial cell compartments, as well as infiltrating hematopoietic immune cells. The importance of these cells in regulating immune homeostasis during inflammation is well recognized. However, little is known about their function and phenotype in the inflammatory tumor microenvironment. Using a syngeneic, immunogenic model of colorectal cancer, we showed that TNFα-initiated inflammatory signaling in CT26 colorectal cancer cells selectively induced PD-L1 expression in stromal cells. Using CD274 shRNA and antibody-mediated approaches, we showed that stromal cell PD-L1 potentiated enhanced immunosuppression, characterized by inhibition of activated CD8+ granzyme B-secreting T cells in vitro, and the inhibition of CD8+ effector cells was associated with enhanced tumor progression. Stromal cell immunosuppressive and tumor-promoting effects could be reversed with administration of anti-PD-1 in vivo We validated our findings of stromal cell CD274 expression in two cohorts of clinical samples and also observed PD-L1 induction on human stromal cells in response to exposure to the inflammatory secretome from human colon cancer cells, irrespective of microsatellite instability. Collectively, our data showed that tumor-associated stromal cells support T-cell suppression by PD-L1 induction, which is dependent on colon cancer inflammatory signaling. Our findings reveal a key role of mesenchymal stromal cells PD-L1 in suppression of CD8+ antitumor immune responses and potentiation of colorectal cancer progression. Cancer Immunol Res; 6(11); 1426-41. ©2018 AACR.

Gastroesophageal reflux disease in pregnancy.

Gastroesophageal reflux disease during pregnancy is common. Altered structure and function of the normal physiological barriers to reflux of gastric contents into the oesophagus explain the high incidence of this problem in pregnant women. For the majority of patients, life-style modifications are helpful, but are not sufficient to control symptoms and medication is required. The optimum management of reflux in pregnant patients requires special attention and expertise, since the safety of the mother, foetus and neonate remain the primary focus. Gastroenterologists and obstetricians should work together to optimise treatment. Typically, one utilises a step-up program that starts with life-style modifications and antacids. If those methods fail, histamine-2 receptor antagonists and proton pump inhibitors are tried. Rarely, promotility agents are used. Initiation of these medications must be undertaken after a careful discussion of risks and benefits with patients. In patients without a prior history of reflux, symptoms usually abate after delivery.

Inflammation-associated DNA methylation patterns in epithelium of ulcerative colitis.

Aberrant DNA methylation patterns have been reported in inflamed tissues and may play a role in disease. We studied DNA methylation and gene expression profiles of purified intestinal epithelial cells from ulcerative colitis patients, comparing inflamed and non-inflamed areas of the colon. We identified 577 differentially methylated sites (false discovery rate <0.2) mapping to 210 genes. From gene expression data from the same epithelial cells, we identified 62 differentially expressed genes with increased expression in the presence of inflammation at prostate cancer susceptibility genes PRAC1 and PRAC2. Four genes showed inverse correlation between methylation and gene expression; ROR1, GXYLT2, FOXA2, and, notably, RARB, a gene previously identified as a tumor suppressor in colorectal adenocarcinoma as well as breast, lung and prostate cancer. We highlight targeted and specific patterns of DNA methylation and gene expression in epithelial cells from inflamed colon, while challenging the importance of epithelial cells in the pathogenesis of chronic inflammation.

Budesonide in the treatment of inflammatory bowel disease: the first year of experience in clinical practice.

BACKGROUND: Delayed release budesonide was approved by the FDA for the treatment of mildly to moderately active Crohn's disease involving the ileum and ascending colon. Controlled trials have demonstrated that budesonide is effective in inducing remission and for maintenance of remission, with less frequent steroid side effects than conventional steroids. We sought to determine the benefit of this medication in clinical practice and to identify any non-FDA-approved uses that may warrant further study. METHODS: Patients in whom oral budesonide was prescribed between November 1, 2001 and October 31, 2002, were identified and medical records were reviewed. Patients were categorized by indication for therapy: ileocolonic Crohn's disease (group 1), Crohn's disease elsewhere (group 2), and other conditions (group 3). RESULTS: A total of 225 patients were identified (108 in group 1, 62 in group 2, and 55 in group 3). Group 3 included patients with microscopic colitis (n = 28), pouchitis (n = 13), ulcerative colitis (n = 12), and celiac disease (n = 2). A favorable outcome occurred in 61% of group 1 patients but only 24% of patients in group 2. In group 3, only microscopic colitis patients and pouchitis patients experienced response rates >50% (77% and 60%, respectively). CONCLUSION: Budesonide is effective in a majority of patients with ileocolonic Crohn's disease and microscopic colitis, which is consistent with results reported from clinical trials. A majority of patients with pouchitis also benefit from budesonide therapy, but prospective controlled trials are necessary to clarify the benefit in this group.

NF-kappaB signaling: pros and cons of altering NF-kappaB as a therapeutic approach.

The transcription factor, nuclear factor-kappaB (NF-kappaB), is a dominant regulator of the expression of hundreds of genes, many of which play important roles in the regulation of inflammation and programmed cell death (apoptosis). Since the discovery of NF-kappaB in the mid 1980s, this transcription factor has been the subject of intense investigation. Excess or inappropriate activation of NF-kappaB has been observed in human inflammatory bowel disease and in a host of other inflammatory diseases and type of cancer. Functional studies in animals have shed light on the role of NF-kappaB in broader pathophysiological contexts. From such studies, it has become quite clear that NF-kappaB plays unique and distinct functions in different cell types. Because of the importance of NF-kappaB in signaling inflammation, and in inhibiting programmed cell death, many pharmaceutical companies are developing small-molecule inhibitors of this pathway. In this article, we evaluate the relative pros and cons of blocking NF-kappaB as a therapeutic approach for inflammatory bowel disease. On the basis of the results of studies in animals that have primarily used genetic approaches to inhibit NF-kappaB activity, we suggest that there are certain niche indications for blocking NF-kappaB in inflammatory bowel disease that offer particular promise.

Infliximab Selectively Modulates the Circulating Blood Monocyte Repertoire in Crohn's Disease.

BACKGROUND: Infliximab (IFX), an anti-tumour necrosis factor alpha (TNFα) monoclonal antibody, provides clinical benefits in treating Crohn's disease (CD) but its mechanisms of action are not fully elucidated. This study investigated blood monocyte repertoires and the acute effects of IFX infusion on monocyte subset phenotype and function in IFX-treated patients with CD. METHODS: Monocytes and monocyte subsets were enumerated and phenotypically characterized by multicolor flow cytometry in freshly isolated blood from healthy controls (n = 21) and patients with CD treated with (IFX, n = 24) and without (non-IFX, n = 20) IFX. For the IFX-CD group, blood was sampled immediately before (tough-IFX) and after (peak-IFX) infusion. Monocyte responses to lipopolysaccharide were analyzed by whole-blood intracellular cytokine staining. RESULTS: Non-IFX and IFX-CD patients had increased numbers of intermediate (CD14CD16) monocytes compared with healthy controls, whereas classical (CD14CD16) and nonclassical (CD14CD16) monocytes were numerically reduced in the IFX-CD group alone. In all groups, monocyte subsets expressed high surface levels of transmembrane (tm)TNFα. After IFX infusion, a significant reduction in monocyte numbers occurred. Post-IFX monocytopenia was proportionately greatest for classical and intermediate subsets, correlated with postinfusion IFX levels and was not associated with monocyte apoptosis. In contrast, lipopolysaccharide-induced production of TNFα and IL-12 by monocytes was significantly reduced in peak-IFX compared with trough-IFX blood samples. CONCLUSIONS: Actively managed CD is associated with monocyte repertoire skewing suggestive of chronic inflammatory stimulation. Infused IFX acutely targets monocytes, likely by binding to tmTNFα, resulting in a non-apoptosis-related decline in circulating monocyte numbers and blunting of the inflammatory response of monocytes remaining in the blood.