A functional role for Serum Amyloid A in the molecular regulation of autophagy in breast cancer.

It has been established that the acute phase protein, Serum amyloid A (SAA), which is usually synthesized by the liver, is also synthesized by cancer cells and cancer-associated cells in the tumor microenvironment. SAA also activates modulators of autophagy, such as the PI3K/Akt and MAPK signaling pathways. However, the role of SAA in autophagy in breast cancer still remains to be elucidated. The aim of this study was to investigate the role of SAA in the regulation of signaling pathways and autophagy in in vitro and in vivo models of breast cancer. The MDA-MB-231 and MCF7 cell lines were transiently transfected to overexpress SAA1. A tumor-bearing SAA1/2 knockout mouse model was also utilized in this study. SAA1 overexpression activated ERK signaling in the MDA-MB-231 cells, downregulated the PI3K pathway protein, PKB/Akt, in the MCF7 cell line, while SAA1/2 knockout also inhibited Akt. Furthermore, SAA1 overexpression in vitro downregulated autophagy, while the expression of SQSTM1/p62 was increased in the MCF7 cells, and SAA1/2 knockout induced autophagy in vivo. SAA overexpression in the MDA-MB-231 and MCF7 cells resulted in an increase in cell viability and increased the expression of the proliferation marker, MCM2, in the MCF7 cells. Furthermore, knockout of SAA1/2 resulted in an altered inflammatory profile, evident in the decrease of plasma IL-1ß, IL-6 and IL-10, while increasing the plasma levels of MCP-1 and TNF-α. Lastly, SAA1/2 knockout promoted resistance to apoptosis and necrosis through the regulation of autophagy. SAA thus regulates autophagy in breast cancer cells to promote tumorigenesis.

Five-Year Efficacy and Safety of Ustekinumab Treatment in Crohn's Disease: The IM-UNITI Trial.

BACKGROUND & AIMS: The IM-UNITI study and long-term extension (LTE) evaluated the long-term efficacy, safety, and immunogenicity of subcutaneous ustekinumab maintenance therapy in patients with Crohn's disease. Here, we report the final results of IM-UNITI LTE through 5 years. METHODS: Patients completing safety and efficacy evaluations at week 44 of the maintenance study were eligible to participate in the LTE and continue the treatment they were receiving. Unblinding occurred after completion of maintenance study analyses (August 2015), and patients receiving placebo were discontinued from the study after unblinding. No dose adjustment occurred in the LTE. Efficacy assessments were conducted every 12 weeks until unblinding and at dosing visits thereafter through week 252. Serum ustekinumab concentrations and antidrug antibodies were evaluated through weeks 252 and 272, respectively. RESULTS: Using an intent-to-treat analysis of all patients randomized to ustekinumab at maintenance baseline, 34.4% of patients in the every-8-weeks group and 28.7% in the every-12-weeks group were in clinical remission at week 252. Corresponding remission rates among patients who entered the LTE were 54.9% and 45.2%. Overall, adverse event rates (per 100 patient-years) from maintenance week 0 through the final visit generally were similar in the placebo and combined ustekinumab groups for all adverse events (440.3 vs 327.6), serious adverse events (19.3 vs 17.5), infections (99.8 vs 93.8), and serious infections (3.9 vs 3.4). Serum ustekinumab concentrations were maintained throughout the LTE. Antidrug antibodies occurred in 5.8% of patients who received ustekinumab during induction and maintenance and continued in the LTE. CONCLUSIONS: Patients receiving subcutaneous ustekinumab maintained clinical remission through 5 years. No new safety signals were observed. ClinicalTrials.gov number NCT01369355.

Serum Amyloid A Promotes Inflammation-Associated Damage and Tumorigenesis in a Mouse Model of Colitis-Associated Cancer.

BACKGROUND & AIMS: Identifying new approaches to lessen inflammation, as well as the associated malignant consequences, remains crucial to improving the lives and prognosis of patients diagnosed with inflammatory bowel diseases. Although it previously has been suggested as a suitable biomarker for monitoring disease activity in patients diagnosed with Crohn's disease, the role of the acute-phase protein serum amyloid A (SAA) in inflammatory bowel disease remains unclear. In this study, we aimed to assess the role of SAA in colitis-associated cancer. METHODS: We established a model of colitis-associated cancer in wild-type and SAA double-knockout (Saa1/2-/-) mice by following the azoxymethane/dextran sulfate sodium protocol. Disease activity was monitored throughout the study while colon and tumor tissues were harvested for subsequent use in cytokine analyses, Western blot, and immunohistochemistry +experiments. RESULTS: We observed attenuated disease activity in mice deficient for Saa1/2 as evidenced by decreased weight loss, increased stool consistency, decreased rectal bleeding, and decreased colitis-associated tissue damage. Macrophage infiltration, including CD206+ M2-like macrophages, also was attenuated in SAA knockout mice, while levels of interleukin 4, interleukin 10, and tumor necrosis factor-ɑ were decreased in the distal colon. Mice deficient for SAA also showed a decreased tumor burden, and tumors were found to have increased apoptotic activity coupled with decreased expression for markers of proliferation. CONCLUSION: Based on these findings, we conclude that SAA has an active role in inflammatory bowel disease and that it could serve as a therapeutic target aimed at decreasing chronic inflammation and the associated risk of developing colitis-associated cancer.

The Link Between Bacterial Inflammagens, Leaky Gut Syndrome and Colorectal Cancer.

There is a causal relationship between cancer (including colorectal cancer), chronic systemic inflammation and persistent infections, and the presence of dysregulated circulating inflammatory markers. It is known that aberrant clot formation and coagulopathies occur in systemic inflammation. In colorectal cancer, there is a close link between gut dysbiosis and an inflammatory profile. In this review, we present evidence of the connection between gut dysbiosis, the entry of bacteria into the internal environment, and the presence of their highly potent inflammagenic molecules, such as lipopolysaccharide and lipoteichoic acid, in circulation. These bacterial components may act as one of the main drivers of the inflammatory process (including hypercoagulation) in colorectal cancer. We review literature that points to the role of these bacterial inflammagens and how they contribute to colorectal carcinogenesis. Insight into the factors that promote carcinogenesis is crucial to effectively prevent and screen for colorectal cancer. Early diagnosis of an activated coagulation system and the detection of bacterial components in circulation and also in the tumour microenvironment, could therefore be important, and may also, together with modulation of the gut microbiota, serve as potential therapeutic targets.

Serum amyloid A and inflammasome activation: A link to breast cancer progression?

Breast cancer is the most frequently diagnosed cancer in women globally. Although there have been many significant advances made in the diagnosis and treatment of breast cancer, numerous unresolved challenges remain, which include prevention, early diagnosis, metastasis and recurrence. The role of inflammation in cancer development is well established and is believed to be one of the leading hallmarks of cancer progression. Recently, the role of the inflammasome, a cytosolic multiprotein complex, has received attention in different cancers. By contributing to the activation of inflammatory cytokines the inflammasome intensifies the inflammatory cascade. The inflammasome can be activated through several pathways, which include the binding of pattern associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) to toll-like receptors (TLRs). Serum amyloid A (SAA), a non-specific acute-phase protein, can function as an endogenous DAMP by binding to pattern recognition receptors like TLRs on both breast cancer cells and cancer associated fibroblasts (CAFs). SAA can thus stimulate the production of IL-1ß, thereby creating a favourable inflammatory environment to support tumour growth. The aim of this review is to highlight the possible role of SAA as an endogenous DAMP in the tumour microenvironment (TME) thereby promoting breast cancer growth through the activation of the NLRP3 inflammasome.

Colorectal cancer is associated with increased circulating lipopolysaccharide, inflammation and hypercoagulability.

Gut dysbiosis contributes to the development of a dysfunctional gut barrier, facilitating the translocation of bacteria and inflammagens, and is implicated in colorectal cancer (CRC) pathogenesis. Such 'leaky gut' conditions result in systemic inflammation, of which a hallmark is increased hypercoagulability. Fluorescence antibody confocal microscopy was used to determine circulating levels of lipopolysaccharide (LPS) in control and CRC populations. Here we showed that circulating levels of LPS are significantly elevated in the CRC population. We also showed that markers of inflammation and hypercoagulability are increased in this population. Furthermore, anomalous blood clotting and structural changes in blood components are presented. Importantly, the association between LPS levels, inflammation, and hematological dysfunction was analysed. Statistical regression models were applied to identify markers with strong association with CRC, and to investigate the correlation between markers. A core aim is enhanced biomarker discovery for CRC. We conclude that circulating LPS can promote systemic inflammation and contribute to the development of a pathological coagulation system, with resulting chronic inflammation and an activated coagulation system implicated in tumorigenesis. Blood-based screening tools are an emerging research area of interest for CRC screening. We propose the use of additional (novel) biomarkers to effectively screen for CRC.

IM-UNITI: Three-year Efficacy, Safety, and Immunogenicity of Ustekinumab Treatment of Crohn's Disease.

BACKGROUND AND AIMS: Following induction/maintenance treatment in the UNITI/IM-UNITI studies of ustekinumab for Crohn's disease, patients entered a long-term extension for up to 5 years from induction. Efficacy through 152 and safety through 156 weeks are reported. METHODS: At IM-UNITI Week 44, 567 ustekinumab-treated patients entered the long-term extension and continued to receive blinded subcutaneous ustekinumab on their assigned dose interval, without any subsequent dose adjustment. Placebo-treated patients discontinued after study unblinding [after IM-UNITI Week 44 analyses]. Efficacy data in the long-term extension [LTE] were collected every 12 weeks [q12w] before unblinding and then at q12w/q8w dosing visits. RESULTS: Through Week 156, 29.6% of ustekinumab-treated patients discontinued. In an intent-to-treat analysis of randomised patients from IM-UNITI Weeks 0-152, 38.0% of ustekinumab induction responders receiving the drug q12w and 43.0% q8w were in remission at Week 152. Among patients entering the long-term extension in their original randomised groups, 61.9% of q12w and 69.5% of q8w patients were in remission at Week 152. Across all ustekinumab-treated patients [randomised and non-randomised] entering the long-term extension, remission rates at Week 152 were 56.3% and 55.1% for q12w and q8w, respectively. Safety events [per 100 patient-years] were similar among all ustekinumab-treated patients entering the long-term extension and placebo [overall adverse events 389.70 vs 444.17; serious adverse events, 18.97 vs 19.54; serious infections, 4.21 vs 3.97]. Rates of antibodies to ustekinumab through Week 156 remained low, 4.6% in all randomised ustekinumab-treated patients; lowest among patients in the original randomised q8w group [2/82, 2.4%]. CONCLUSIONS: Continued treatment with subcutaneous ustekinumab maintained clinical response and remission through 3 years in a majority of patients who responded to induction therapy and was well-tolerated. ClinicalTrials.gov number NCT01369355.

Serum amyloid A binds to fibrin(ogen), promoting fibrin amyloid formation.

Complex associations exist between inflammation and thrombosis, with the inflammatory state tending to promote coagulation. Fibrinogen, an acute phase protein, has been shown to interact with the amyloidogenic ß-amyloid protein of Alzheimer's disease. However, little is known about the association between fibrinogen and serum amyloid A (SAA), a highly fibrillogenic protein that is one of the most dramatically changing acute phase reactants in the circulation. To study the role of SAA in coagulation and thrombosis, in vitro experiments were performed where purified human SAA, in concentrations resembling a modest acute phase response, was added to platelet-poor plasma (PPP) and whole blood (WB), as well as purified and fluorescently labelled fibrinogen. Results from thromboelastography (TEG) suggest that SAA causes atypical coagulation with a fibrin(ogen)-mediated increase in coagulation, but a decreased platelet/fibrin(ogen) interaction. In WB scanning electron microscopy analysis, SAA mediated red blood cell (RBC) agglutination, platelet activation and clumping, but not platelet spreading. Following clot formation in PPP, the presence of SAA increased amyloid formation of fibrin(ogen) as determined both with auto-fluorescence and with fluorogenic amyloid markers, under confocal microcopy. SAA also binds to fibrinogen, as determined with a fluorescent-labelled SAA antibody and correlative light electron microscopy (CLEM). The data presented here indicate that SAA can affect coagulation by inducing amyloid formation in fibrin(ogen), as well as by propelling platelets to a more prothrombotic state. The discovery of these multiple and complex effects of SAA on coagulation invite further mechanistic analyses.

Correlative Light-Electron Microscopy detects lipopolysaccharide and its association with fibrin fibres in Parkinson's Disease, Alzheimer's Disease and Type 2 Diabetes Mellitus.

Many chronic diseases, including those classified as cardiovascular, neurodegenerative, or autoimmune, are characterized by persistent inflammation. The origin of this inflammation is mostly unclear, but it is typically mediated by inflammatory biomarkers, such as cytokines, and affected by both environmental and genetic factors. Recently circulating bacterial inflammagens such as lipopolysaccharide (LPS) have been implicated. We used a highly selective mouse monoclonal antibody to detect bacterial LPS in whole blood and/or platelet poor plasma of individuals with Parkinson's Disease, Alzheimer's type dementia, or Type 2 Diabetes Mellitus. Our results showed that staining is significantly enhanced (P < 0.0001) compared to healthy controls. Aberrant blood clots in these patient groups are characterized by amyloid formation as shown by the amyloid-selective stains thioflavin T and Amytracker™ 480 or 680. Correlative Light-Electron Microscopy (CLEM) illustrated that the LPS antibody staining is located in the same places as where amyloid fibrils may be observed. These data are consistent with the Iron Dysregulation and Dormant Microbes (IDDM) hypothesis in which bacterial inflammagens such as LPS are responsible for anomalous blood clotting as part of the aetiology of these chronic inflammatory diseases.

The role of bile acids in nutritional support.

Nutritional support continues to receive much attention as a possible intervention to prevent loss of lean tissue mass, promote recovery and re-establish proper immune function in critical care patients. Yet there remains much controversy regarding the clinical efficacy of such interventions. In addition to the direct effect of nutrition in terms of micro- and macronutrient content, nutritional formulations may exert an effect via the physiological response to feeding. Here, we highlight the key role of postprandial reabsorbed bile acids in attenuating both the inflammatory response and autophagy. These observations suggest that not all patients would benefit from aggressive nutritional support.

Expediting red blood cell transfusions by syringing causes significant hemolysis.

BACKGROUND: Techniques commonly used to expedite blood transfusions include pneumatically pressurizing red blood cell (RBC) bags or manual syringing its contents. We compared these techniques on RBC hemolysis using a simulated transfusion model. STUDY DESIGN AND METHODS: Fifteen warmed RBC units that were 12.3 ± 4.3 (95% confidence interval [CI], 10.1-14.5) days old were each subjected to two experimental rapid transfusion techniques. RBCs from each technique were directed through 18- and 22-gauge cannulas attached to blood administration sets. One technique involved RBC bag pressurization to 300 mmHg. The other employed a 20-mL syringe to effect forceful, manual aspiration from the RBC bag followed by forceful, manual RBC injection. The control group was gravity driven without cannulas. Free hemoglobin (Hb) concentrations were measured and percent hemolysis was calculated. RESULTS: Free Hb concentrations and percent hemolysis (median [95% CI]) were similar in the control (0.05 [0.03-0.08] g/dL and 0.13% [0.09%-0.17%], respectively) and pressurized experiments (0.06 [0.05-0.09] g/dL; 0.14% [0.12%-0.22%]), respectively. Syringing resulted in 10-fold higher free Hb concentrations (0.55 [0.38-0.92] g/dL) and percent hemolysis (1.28% [1.03%-2.15%]) than when employing the control (p < 0.0001) or pressurization (p < 0.0001) techniques. Cannula sizes studied did not affect hemolysis. CONCLUSION: Forceful manual syringing caused significant hemolysis and high free Hb concentrations. Pressurizing RBC bags induced no more hemolysis than after gravity-facilitated transfusions. Syringing to expedite RBC transfusions should be avoided in favor of pneumatic RBC bag pressurization.

Ustekinumab as Induction and Maintenance Therapy for Crohn's Disease.

BACKGROUND: Ustekinumab, a monoclonal antibody to the p40 subunit of interleukin-12 and interleukin-23, was evaluated as an intravenous induction therapy in two populations with moderately to severely active Crohn's disease. Ustekinumab was also evaluated as subcutaneous maintenance therapy. METHODS: We randomly assigned patients to receive a single intravenous dose of ustekinumab (either 130 mg or approximately 6 mg per kilogram of body weight) or placebo in two induction trials. The UNITI-1 trial included 741 patients who met the criteria for primary or secondary nonresponse to tumor necrosis factor (TNF) antagonists or had unacceptable side effects. The UNITI-2 trial included 628 patients in whom conventional therapy failed or unacceptable side effects occurred. Patients who completed these induction trials then participated in IM-UNITI, in which the 397 patients who had a response to ustekinumab were randomly assigned to receive subcutaneous maintenance injections of 90 mg of ustekinumab (either every 8 weeks or every 12 weeks) or placebo. The primary end point for the induction trials was a clinical response at week 6 (defined as a decrease from baseline in the Crohn's Disease Activity Index [CDAI] score of ≥100 points or a CDAI score <150). The primary end point for the maintenance trial was remission at week 44 (CDAI score <150). RESULTS: The rates of response at week 6 among patients receiving intravenous ustekinumab at a dose of either 130 mg or approximately 6 mg per kilogram were significantly higher than the rates among patients receiving placebo (in UNITI-1, 34.3%, 33.7%, and 21.5%, respectively, with P≤0.003 for both comparisons with placebo; in UNITI-2, 51.7%, 55.5%, and 28.7%, respectively, with P<0.001 for both doses). In the groups receiving maintenance doses of ustekinumab every 8 weeks or every 12 weeks, 53.1% and 48.8%, respectively, were in remission at week 44, as compared with 35.9% of those receiving placebo (P=0.005 and P=0.04, respectively). Within each trial, adverse-event rates were similar among treatment groups. CONCLUSIONS: Among patients with moderately to severely active Crohn's disease, those receiving intravenous ustekinumab had a significantly higher rate of response than did those receiving placebo. Subcutaneous ustekinumab maintained remission in patients who had a clinical response to induction therapy. (Funded by Janssen Research and Development; ClinicalTrials.gov numbers, NCT01369329 , NCT01369342 , and NCT01369355 .).

EMerging BiomARKers in Inflammatory Bowel Disease (EMBARK) study identifies fecal calprotectin, serum MMP9, and serum IL-22 as a novel combination of biomarkers for Crohn's disease activity: role of cross-sectional imaging.

OBJECTIVES: In Crohn's disease (CD), clinical symptoms correspond poorly to inflammatory disease activity. Biomarkers reflective of mucosal and bowel wall inflammation would be useful to monitor disease activity. The EMBARK study evaluated disease activity in patients with ulcerative colitis (UC) and CD, and used endoscopy with or without cross-sectional imaging for biomarker discovery. METHODS: UC (n=107) and CD (n=157) patients were characterized and underwent ileocolonoscopy (ICO). A subset of CD patients (n=66) also underwent computed tomography enterography (CTE). ICO and CTE were scored by a gastroenterologist and radiologist who incorporated findings of inflammation into a single score (ICO-CTE) for patients that underwent both procedures. Serum and fecal biomarkers were evaluated for association with the Mayo Clinic endoscopy score in UC patients and with ICO alone or ICO-CTE in CD patients. Individual biomarkers with a moderate degree of correlation (P≤0.3) were evaluated using multivariate analysis with model selection using a stepwise procedure. RESULTS: In UC, ordinal logistic regression using Mayo Clinic endoscopy subscore selected the combination of fecal calprotectin and serum matrix metalloproteinase 9 (MMP9; pseudo R(2)=0.353). In CD, we found that use of the ICO-CTE increased specificity of known biomarkers. Using ICO-CTE as the dependent variable for biomarker discovery, the selected biomarkers were the combination of fecal calprotectin, serum MMP9, and serum IL-22 (r=0.699). CONCLUSIONS: Incorporation of both ICO and CTE into a single measure increased biomarker performance in CD. Combinations of fecal calprotectin and serum MMP9 for UC, and combinations of fecal calprotectin, serum MMP9, and serum interleukin-22 in CD, demonstrated the strongest association with imaging/endoscopy-defined inflammation.

Green Tea Polyphenols and Sulfasalazine have Parallel Anti-Inflammatory Properties in Colitis Models.

BACKGROUND: There is no cure for autoimmune chronic inflammatory bowel disease (IBD). IBD patients commonly use complementary and alternative medications of which the safety, efficacy, and interaction with standard-of-care therapies are not fully known. Thus the consequences can become life-threatening. Sulfasalazine commonly used in IBD, potentially has severe adverse effects, including infertility, pulmonary fibrosis, lack of response, and ultimately patients may require intestinal resection. We hypothesized that green tea polyphenols (GrTP, EGCG) and sulfasalazine have similar anti-inflammatory properties. METHODS: BALB/c mice received Dextran sodium sulfate (DSS) to induce colitis (ulcerative colitis model). Exposure of IL-10 deficient mice (BALB/c-background) to normal microbiota provoked enterocolitis (mimics Crohn's disease). Animals were treated with agents incorporated into daily diets. Control animals received sham treatment. RESULTS: DSS-treated animals developed severe bloody diarrhea and colitis (score 0-4, 3.2 ± 0.27). IL-10 deficient mice developed severe enterocolitis as manifested by diarrhea, rectal prolapse, and colonic lesions. Animals tolerated regimens (GrTP, EGCG, sulfasalazine) with no major side effects, and further developed less severe colitis. IL-10 deficient animals became moribund on high dose, while tolerated low and Mid doses with significant improved symptoms of enterocolitis. GrTP, EGCG, and sulfasalazine significantly ameliorated colonic damage and histological scores in treated animals in a similar manner (GrTP vs. DSS p < 0.05; EGCG, sulfasalazine vs. DSS p < 0.01). The inflammatory markers TNFα (3-fold), IL-6 (14-fold), and serum amyloid A (40-fold) increased in colitic animals and significantly decreased with treatment regiments. In contrast, circulatory leptin levels decreased in colitic animals (twofold). EGCG additionally reduced leptin levels (p < 0.01) while GrTP and sulfasalazine had no effect on leptin levels (p < 0.05). Hepatic and colonic antioxidants were significantly depleted in colitic animals and treatment regiments significantly restored antioxidants levels. CONCLUSION: GrTP and EGCG improved antioxidants levels and attenuated severity of colitis analogous to sulfasalazine. Future studies will reveal whether polyphenols can become an alternative/additive therapy for IBD therapy in humans.

Dietary medium-chain triglycerides promote oral allergic sensitization and orally induced anaphylaxis to peanut protein in mice.

BACKGROUND: The prevalence of peanut allergies is increasing. Peanuts and many other allergen sources contain significant amounts of triglycerides, which affect absorption of antigens but have unknown effects on sensitization and anaphylaxis. We recently reported that dietary medium-chain triglycerides (MCTs), which bypass mesenteric lymph and directly enter portal blood, reduce intestinal antigen absorption into blood compared with long-chain triglycerides (LCTs), which stimulate mesenteric lymph flow and are absorbed in chylomicrons through mesenteric lymph. OBJECTIVE: We sought to test how dietary MCTs affect food allergy. METHODS: C3H/HeJ mice were fed peanut butter protein in MCT, LCT (peanut oil), or LCT plus an inhibitor of chylomicron formation (Pluronic L81). Peanut-specific antibodies in plasma, responses of the mice to antigen challenges, and intestinal epithelial cytokine expression were subsequently measured. RESULTS: MCT suppressed antigen absorption into blood but stimulated absorption into Peyer patches. A single gavage of peanut protein with MCT, as well as prolonged feeding in MCT-based diets, caused spontaneous allergic sensitization. MCT-sensitized mice experienced IgG-dependent anaphylaxis on systemic challenge and IgE-dependent anaphylaxis on oral challenge. MCT feeding stimulated jejunal-epithelial thymic stromal lymphopoietin, Il25, and Il33 expression compared with that seen after LCT feeding and promoted T(H)2 cytokine responses in splenocytes. Moreover, oral challenges of sensitized mice with antigen in MCT significantly aggravated anaphylaxis compared with challenges with the LCT. Importantly, the effects of MCTs could be mimicked by adding Pluronic L81 to LCTs, and in vitro assays indicated that chylomicrons prevent basophil activation. CONCLUSION: Dietary MCTs promote allergic sensitization and anaphylaxis by affecting antigen absorption and availability and by stimulating T(H)2 responses.

Ustekinumab induction and maintenance therapy in refractory Crohn's disease.

BACKGROUND: In patients with Crohn's disease, the efficacy of ustekinumab, a human monoclonal antibody against interleukin-12 and interleukin-23, is unknown. METHODS: We evaluated ustekinumab in adults with moderate-to-severe Crohn's disease that was resistant to anti-tumor necrosis factor (TNF) treatment. During induction, 526 patients were randomly assigned to receive intravenous ustekinumab (at a dose of 1, 3, or 6 mg per kilogram of body weight) or placebo at week 0. During the maintenance phase, 145 patients who had a response to ustekinumab at 6 weeks underwent a second randomization to receive subcutaneous injections of ustekinumab (90 mg) or placebo at weeks 8 and 16. The primary end point was a clinical response at 6 weeks. RESULTS: The proportions of patients who reached the primary end point were 36.6%, 34.1%, and 39.7% for 1, 3, and 6 mg of ustekinumab per kilogram, respectively, as compared with 23.5% for placebo (P=0.005 for the comparison with the 6-mg group). The rate of clinical remission with the 6-mg dose did not differ significantly from the rate with placebo at 6 weeks. Maintenance therapy with ustekinumab, as compared with placebo, resulted in significantly increased rates of clinical remission (41.7% vs. 27.4%, P=0.03) and response (69.4% vs. 42.5%, P<0.001) at 22 weeks. Serious infections occurred in 7 patients (6 receiving ustekinumab) during induction and 11 patients (4 receiving ustekinumab) during maintenance. Basal-cell carcinoma developed in 1 patient receiving ustekinumab. CONCLUSIONS: Patients with moderate-to-severe Crohn's disease that was resistant to TNF antagonists had an increased rate of response to induction with ustekinumab, as compared with placebo. Patients with an initial response to ustekinumab had significantly increased rates of response and remission with ustekinumab as maintenance therapy. (Funded by Janssen Research and Development; CERTIFI ClinicalTrials.gov number, NCT00771667.).

Elevated IgG levels against specific bacterial antigens in obese patients with diabetes and in mice with diet-induced obesity and glucose intolerance.

High fat diets increase the risk for insulin resistance by promoting inflammation. The cause of inflammation is unclear, but germfree mouse studies have implicated commensal gut bacteria. We tested whether diet-induced obesity, diabetes, and inflammation are associated with anti-bacterial IgG. Blood from lean and obese healthy volunteers or obese patients with diabetes were analyzed by ELISA for IgG against extracts of potentially pathogenic and pro-biotic strains of Escherichia coli (LF-82 and Nissle), Bacteroides thetaiotaomicron, and Lactobacillus acidophilus, and for circulating tumor necrosis factor α (TNFα). C57Bl/6 mice were fed low- or high-fat diets (10% or 60% kcal from fat) for 10 weeks and tested for anti-bacterial IgG, bodyweight, fasting glucose, and inflammation. Obese diabetic patients had significantly more IgG against extracts of E. coli LF-82 compared with lean controls, whereas IgG against extracts of the other bacteria was unchanged. Circulating TNFα was elevated and correlated with IgG against the LF-82 extract. Mice fed high-fat diets had increased fasting glucose levels, elevated TNFα and neutrophils, and significantly more IgG against the LF-82 extracts. Diabetes in obesity is characterized by increased IgG against specific bacterial antigens. Specific commensal bacteria may mediate inflammatory effects of high-fat diets.

Pegylated arginine deiminase downregulates colitis in murine models.

UNLABELLED: Arginine deiminase (ADI), an arginine-metabolizing enzyme involved in cell signaling, is dysregulated in multiple inflammatory diseases and cancers. We hypothesized that pegylated ADI (ADI-PEG) provide protection against colitis. METHODS: Dextran sodium sulfate colitis was induced in IL-10-deficient and BALB/c (WT) mice. ADI-PEG was administered i.p., and inflammatory mediators and pathology were evaluated. RESULTS: Acute colitis in mice was manifested by increases in inflammatory biomarkers, such as serum amyloid A (SAA, P < 0.001), IL-12 p40, and disease index (3-Fold). In contrast, ADI-PEG significantly decreased clinical disease index, SAA levels, and inflammatory cytokines in blood as well as in colonic explants. Animals developed moderate (2.2 ± 0.3 WT) to severe (3.6 ± 0.5 IL-10 deficient) colonic pathology; and ADI-PEG treatment significantly improved the severity of colitis (P < 0.05). Marked infiltration of CD68+ macrophages and iNOS expression were detected in colonic submucosa in colitic animals but not detected in ADI-PEG-treated animals. CONCLUSION: ADI-PEG attenuated inflammatory responses by suppression of macrophage infiltration and iNOS expression in colitic animals. ADI-PEG can serve as a potential therapeutic value in IBD.

Long-term infliximab maintenance therapy for ulcerative colitis: the ACT-1 and -2 extension studies.

BACKGROUND: The aim was to evaluate long-term efficacy, quality of life, and safety in ulcerative colitis patients who received infliximab during the ACT-1 and -2 extension studies. METHODS: Adults with moderate-to-severely active ulcerative colitis in the 54-week ACT-1 and 30-week ACT-2 studies who achieved benefit from infliximab were eligible to participate in extension studies and receive up to 3 additional years of therapy. Patients received randomized study medication until all sites were unblinded; placebo-treated patients were discontinued. Patients receiving 5 or 10 mg/kg infliximab continued to receive open-label infliximab every 8 weeks. Patients receiving infliximab 10 mg/kg could decrease to 5 mg/kg; patients receiving infliximab 5 mg/kg could increase to 10 mg/kg if response was lost. RESULTS: A total of 229 of 484 infliximab-treated patients from the ACT-1 and ACT-2 main studies entered the long-term extensions. Overall, 70 (30.6%) patients discontinued infliximab infusions for adverse events (24 [10.5%]), lack of efficacy (11 [4.8%]), required a colectomy (1 [0.4%]), or for other reasons (34 [14.8%]). Proportions of patients whose Physician's Global Assessment scores were indicative of no or mild disease (score = 0 or 1) were maintained during the extension studies; 76.5% at Extension week 0 and ranged between 90.0% and 94.3% through Extension week 152. Improvement in Inflammatory Bowel Disease Questionnaire scores observed in the main studies was maintained. During the long-term extension, the infliximab safety profile was consistent with that of the main studies; no new or unexpected safety signals were observed. CONCLUSIONS: Long-term treatment with infliximab for up to 3 additional years was effective and well tolerated.

Osteopontin ablation attenuates progression of colitis in TNBS model.

INTRODUCTION: OPN has been implicated in the inflammatory response to Crohn's disease. We hypothesized that OPN deficiency protects against different stages of TNBS-induced colitis in a modified model that mimics Crohn's disease. MATERIAL AND METHODS: OPN-deficient and wildtype mice were treated intracolonically with TNBS and euthanized during acute, sub-acute and chronic colitis. RESULTS: TNBS-treated wildtype mice developed severe colitis, but OPN-deficient mice were significantly protected. Wildtype mice showed significant infiltration of inflammatory cells including macrophages, and colonic transmural thickening that progressed to strictures, increased matrix collagen deposits (X2 fold), and granuloma formation. These pathological findings were partially attenuated by OPN deficiency. The inflammatory marker, serum amyloid A (SAA), markedly increased in sub-acute stages regardless of OPN status. Conversely, OPN deficiency significantly reduced concentration of SAA in the acute and chronic stages. Secretory OPN was upregulated particularly in acute stage in wildtypes (P < 0.001) and as expected not present in OPN-deficient animals. Flow cytometry analysis of splenic macrophages revealed significant increases in scavenger receptors, macrosialin and F4/80 markers' expression in wildtypes. CONCLUSIONS: Our data support the role of OPN in induction of inflammation and establishment of chronic colitis. Therefore, OPN may represent a target for therapeutic intervention in Crohn's disease.