Lori A. Coburn

The Successful Use of Adalimumab to Treat Active Crohn's Disease of an Ileoanal Pouch During Pregnancy

The use of immunosuppressive medications during pregnancy in patients with Crohn’s disease is controversial. However, most clinicians would agree that both the unborn baby and the mother will do better if the mother’s inflammatory bowel disease (IBD) can be controlled. Studies have shown that patients with active Crohn’s disease are more likely to experience preterm labor and have babies that are small for gestational age [1]. Currently, the most potent agent approved for Crohn’s disease is infliximab. This is a chimeric anti– tumor necrosis factor-alpha (TNF-α) antibody. Recently, adalimumab (Humira), a fully humanized anti–TNF-α antibody, was approved for the treatment of rheumatoid arthritis (RA) [2–5]. Preliminary studies have demonstrated efficacy in patients with Crohn’s disease, including those who have had a reaction to or lost their response to infliximab because of the development of antibodies to the murine component of this agent [6, 7]. The safety of adalimumab in pregnancy is unknown. There has only been 1 previous report of the use of adalimumab during pregnancy [8]. Herein, we report the first use of adalimumab during pregnancy to treat severe Crohn’s disease of the ileoanal pouch.

L-arginine Supplementation Improves Responses to Injury and Inflammation in Dextran Sulfate Sodium Colitis

Inflammatory bowel disease (IBD), consisting of Crohns disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y + cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS−/− mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.

Tumor Suppressor Function of the Plasma Glutathione Peroxidase Gpx3 in Colitis-Associated Carcinoma

The glutathione peroxidases, a family of selenocysteine-containing redox enzymes, play pivotal roles in balancing the signaling, immunomodulatory, and deleterious effects of reactive oxygen species (ROS). The glutathione peroxidase GPX3 is the only extracellular member of this family, suggesting it may defend cells against ROS in the extracellular environment. Notably, GPX3 hypermethylation and underexpression occur commonly in prostate, gastric, cervical, thyroid, and colon cancers. We took a reverse genetics approach to investigate whether GPX3 would augment inflammatory colonic tumorigenesis, a process characterized by oxidative stress and inflammation, comparing Gpx3(-/-) mice in an established two-stage model of inflammatory colon carcinogenesis. Gpx3-deficient mice exhibited an increased tumor number, though not size, along with a higher degree of dysplasia. In addition, they exhibited increased inflammation with redistribution toward protumorigenic M2 macrophage subsets, increased proliferation, hyperactive WNT signaling, and increased DNA damage. To determine the impact of acute gene loss in an established colon cancer line, we silenced GPX3 in human Caco2 cells, resulting in increased ROS production, DNA damage and apoptosis in response to oxidative stress, combined with decreased contact-independent growth. Taken together, our results suggested an immunomodulatory role for GPX3 that limits the development of colitis-associated carcinoma.

The Apolipoprotein E-Mimetic Peptide COG112 Inhibits NF-κB Signaling, Proinflammatory Cytokine Expression, and Disease Activity in Murine Models of Colitis

Inflammatory bowel disease (IBD), consisting of Crohns disease and ulcerative colitis, is a source of substantial morbidity and remains difficult to treat. New strategies for beneficial anti-inflammatory therapies would be highly desirable. Apolipoprotein (apo) E has immunomodulatory effects and synthetically derived apoE-mimetic peptides are beneficial in models of sepsis and neuroinflammation. We have reported that the antennapedia-linked apoE-mimetic peptide COG112 inhibits the inflammatory response to the colitis-inducing pathogen Citrobacter rodentium in vitro by inhibiting NF-κB activation. We now determined the effect of COG112 in mouse models of colitis. Using C. rodentium as an infection model, and dextran sulfate sodium (DSS) as an injury model, mice were treated with COG112 by intraperitoneal injection. With C. rodentium, COG112 improved the clinical parameters of survival, body weight, colon weight, and histologic injury. With DSS, COG112 ameliorated the loss of body weight, reduction in colon length, and histologic injury, whether administered concurrently with induction of colitis, during induction plus recovery, or only during the recovery phase of disease. In both colitis models, COG112 inhibited colon tissue inducible nitric-oxide synthase (iNOS), KC, TNF-α, IFN-γ, and IL-17 mRNA expression, and reduced nuclear translocation of NF-κB, as determined by immunoblot and immunofluorescence confocal microscopy. IκB kinase (IKK) activity was also reduced, which is necessary for activation of the canonical NF-κB pathway. Isolated colonic epithelial cells exhibited marked attenuation of expression of iNOS and the CXC chemokines KC and MIP-2. These studies indicate that apoE-mimetic peptides such as COG112 are novel potential therapies for IBD.

STAT6 Deficiency Ameliorates Severity of Oxazolone Colitis by Decreasing Expression of Claudin-2 and Th2-Inducing Cytokines

Patients suffering from ulcerative colitis (UC) exhibit chronic colonic inflammation caused by a dysregulated mucosal immune response and epithelial barrier disruption. Th2 cytokines, including IL-13, have been implicated in the pathogenesis of UC. IL-13 induces phosphorylation of STAT6, and we previously demonstrated increased epithelial p-STAT6 in children with UC. In this study, we investigated the role of STAT6 in oxazolone colitis, a murine model of UC, by inducing colitis in STAT6-deficient (STAT6−/−) and wild type (WT) mice. We observed increased epithelial cell, T cell, macrophage, and NKT cell STAT6 phosphorylation, as well as increased p-STAT6+ IL-13–producing NKT cells, in colitic WT mice. Colitis was attenuated in STAT6−/− mice, with improvements in weight, colon length, and histopathology. There was decreased induction of the pore-forming tight junction protein claudin-2 in STAT6−/− mice. Similarly, short hairpin RNA STAT6 knockdown reduced claudin-2 induction and transepithelial resistance decrease in IL-13–treated human T84 cells. Tissue expression of IL-13, IFN-γ, IL-17, and IL-10 mRNA was similarly induced in WT and STAT6−/− colitic mice; however, we observed increased mRNA expression for the Th2-inducing cytokines IL-33 and thymic stromal lymphopoietin in WT mice with colitis, which was abrogated in STAT6−/− mice. Mesenteric lymph node cells from STAT6−/− mice with colitis exhibited reduced secretion of IL-4, IL-5, IL-13, and IFN-γ. IL-33 augmented mesenteric lymph node cell secretion of IL-5, IL-13, IL-6, and IFN-γ. These data implicate STAT6 in the pathogenesis of colitis in vivo with important roles in altering epithelial barrier function and regulating Th2-inducing cytokine production.

Increased Expression and Cellular Localization of Spermine Oxidase in Ulcerative Colitis and Relationship to Disease Activity

Background: Polyamines are important in cell growth and wound repair, but have also been implicated in inflammation‐induced carcinogenesis. Polyamine metabolism includes back‐conversion of spermine to spermidine by the enzyme spermine oxidase (SMO), which produces hydrogen peroxide that causes oxidative stress. In ulcerative colitis (UC), levels of spermine are decreased compared to spermidine. Therefore, we sought to determine if SMO is involved in UC. Methods: Colon biopsies and clinical information from subjects undergoing colonoscopy for evaluation of UC or colorectal cancer screening were utilized from 16 normal controls and 53 UC cases. Histopathologic disease severity was graded and the Mayo Disease Activity Index (DAI) and endoscopy subscore assessed. SMO mRNA expression was measured in frozen biopsies by TaqMan‐based real‐time polymerase chain reaction (PCR). Formalin‐fixed tissues were used for SMO immunohistochemistry. Results: There was a 3.1‐fold upregulation of SMO mRNA levels in UC patients compared to controls (P = 0.044), and a 3.7‐fold increase in involved left colon versus paired uninvolved right colon (P < 0.001). With worsening histologic injury in UC there was a progressive increase in SMO staining of mononuclear inflammatory cells. There was a similar increase in SMO staining with worsening endoscopic disease severity and strong correlation with the DAI (r = 0.653, P < 0.001). Inflammatory cell SMO staining was increased in involved left colon versus uninvolved right colon. Conclusions: SMO expression is upregulated in UC tissues, deriving from increased levels in mononuclear inflammatory cells. Dysregulated polyamine homeostasis may contribute to chronic UC by altering immune responses and increasing oxidative stress. (Inflamm Bowel Dis 2010)

High-throughput multi-analyte Luminex profiling implicates eotaxin-1 in ulcerative colitis.

Accurate and high-throughput technologies are needed for identification of new therapeutic targets and for optimizing therapy in inflammatory bowel disease. Our aim was to assess multi-analyte protein-based assays of cytokines/chemokines using Luminex technology. We have reported that Luminex-based profiling was useful in assessing response to L-arginine therapy in the mouse model of dextran sulfate sodium colitis. Therefore, we studied prospectively collected samples from ulcerative colitis (UC) patients and control subjects. Serum, colon biopsies, and clinical information were obtained from subjects undergoing colonoscopy for evaluation of UC or for non-UC indications. In total, 38 normal controls and 137 UC cases completed the study. Histologic disease severity and the Mayo Disease Activity Index (DAI) were assessed. Serum and colonic tissue cytokine/chemokine profiles were measured by Luminex-based multiplex testing of 42 analytes. Only eotaxin-1 and G-CSF were increased in serum of patients with histologically active UC vs. controls. While 13 cytokines/chemokines were increased in active UC vs. controls in tissues, only eotaxin-1 was increased in all levels of active disease in both serum and tissue. In tissues, eotaxin-1 correlated with the DAI and with eosinophil counts. Increased eotaxin-1 levels were confirmed by real-time PCR. Tissue eotaxin-1 levels were also increased in experimental murine colitis induced by dextran sulfate sodium, oxazolone, or Citrobacter rodentium, but not in murine Helicobacter pylori infection. Our data implicate eotaxin-1 as an etiologic factor and therapeutic target in UC, and indicate that Luminex-based assays may be useful to assess IBD pathogenesis and to select patients for anti-cytokine/chemokine therapies.

Increased serum levels of L‐arginine in ulcerative colitis and correlation with disease severity

Background: L‐arginine (L‐Arg) is a semi‐essential amino acid that is the substrate for both nitric oxide and polyamine synthesis. Cellular uptake of L‐Arg is an active transport process that is subject to competitive inhibition by L‐ornithine (L‐Orn) and L‐lysine (L‐Lys). We investigated L‐Arg utilization in patients with ulcerative colitis (UC). Methods: Serum was collected from 14 normal controls and 22 UC patients with pancolitis of moderate or severe activity by histopathology score. The Mayo Disease Activity Index (DAI) and endoscopy subscore were assessed. Serum amino acid levels were measured by high‐performance liquid chromatography. Arginine availability index (AAI) was defined as [L‐Arg]/([L‐Orn] + [L‐Lys]). Results: Serum L‐Arg levels were significantly associated with histopathologic grade (P = 0.001). L‐Arg levels were increased in subjects with severe colitis when compared to those with moderate colitis or normal mucosa. L‐Orn + L‐Lys levels were also increased in severe colitis, so that AAI was not significantly increased. L‐Arg levels were also strongly associated with the endoscopy subscore (P < 0.001). There was a strong correlation between DAI and L‐Arg levels (r = 0.656, P < 0.001). Conclusions: Serum L‐Arg levels correlate with UC disease severity but availability is not increased due to competitive inhibition by L‐Orn and L‐Lys. Our findings suggest that L‐Arg uptake by cells in the inflamed colon is defective, which may contribute to the pathogenesis of UC. Studies delineating the mechanism of uptake inhibition could enhance our understanding of UC or lead to novel treatment options. Inflamm Bowel Dis 2010

The Role of the Microbiome in Gastrointestinal Cancer.

Humans are host to complex microbial communities previously termed normal flora and largely overlooked. However, resident microbes contribute to both health and disease. Investigators are beginning to define microbes that contribute to the development of gastrointestinal malignancies and the mechanisms by which this occurs. Resident microbes can induce inflammation, leading to cell proliferation and altered stem cell dynamics, which can lead to alterations in DNA integrity and immune regulation and promote carcinogenesis. Studies in human patients and rodent models of cancer have identified alterations in the microbiota of the stomach, esophagus, and colon that increase the risk for malignancy.

Cationic amino acid transporter 2 enhances innate immunity during Helicobacter pylori infection.

Once acquired, Helicobacter pylori infection is lifelong due to an inadequate innate and adaptive immune response. Our previous studies indicate that interactions among the various pathways of arginine metabolism in the host are critical determinants of outcomes following infection. Cationic amino acid transporter 2 (CAT2) is essential for transport of l-arginine (L-Arg) into monocytic immune cells during H. pylori infection. Once within the cell, this amino acid is utilized by opposing pathways that lead to elaboration of either bactericidal nitric oxide (NO) produced from inducible NO synthase (iNOS), or hydrogen peroxide, which causes macrophage apoptosis, via arginase and the polyamine pathway. Because of its central role in controlling L-Arg availability in macrophages, we investigated the importance of CAT2 in vivo during H. pylori infection. CAT2−/− mice infected for 4 months exhibited decreased gastritis and increased levels of colonization compared to wild type mice. We observed suppression of gastric macrophage levels, macrophage expression of iNOS, dendritic cell activation, and expression of granulocyte-colony stimulating factor in CAT2−/− mice suggesting that CAT2 is involved in enhancing the innate immune response. In addition, cytokine expression in CAT2−/− mice was altered from an antimicrobial Th1 response to a Th2 response, indicating that the transporter has downstream effects on adaptive immunity as well. These findings demonstrate that CAT2 is an important regulator of the immune response during H. pylori infection.