Validation of predictive models for disease outcomes in paediatric ulcerative colitis: A multicentre prospective inception cohort.

BACKGROUND: Several studies have proposed models to predict disease outcomes in paediatric ulcerative colitis (UC), notably PROTECT, Schechter and PIBD-ahead, but none has been validated by external cohorts AIM: To explore these models in a prospective multicentre inception cohort METHODS: Children newly diagnosed with UC in 17 centres were followed at disease onset and 3 and 12 months thereafter, as well as at last visit. Outcomes included steroid-free remission (SFR) and acute severe colitis (ASC). RESULTS: Of the 223 included children, 74 (34%), 97 (43%) and 52 (23%) presented with mild, moderate and severe disease, respectively. SFR rate was 35% at 3 months and 47% at 12 months (62% of those with mild disease at diagnosis vs. 41% in moderate-severe disease; p = 0.01). Thirty-six (16%) children developed ASC during the first month after diagnosis, and 53 (24%) during the first year. The AUC of the PROTECT model for predicting SFR at 3 and 12 months was 0.78 [95% CI 0.65-0.92] and 0.57 [95% CI 0.47-0.66], respectively. The sensitivity/specificity/PPV/NPV of Schechter's criteria to predict sustained SFR at 12 months was 50%/60%/35%/74%. ASC was predicted only by the PUCAI score at diagnosis and at 3 months. CONCLUSIONS: The PROTECT model had a good predictive utility for SFR at 3 months, but not at 12 months. The other predictive models did not achieve sufficient accuracy, which was far from that reported in the original studies. This highlights the necessity for external validation of any prediction model prior to its implementation into clinical practice.

The Role and Function of Mucins and Its Relationship to Inflammatory Bowel Disease.

Mucus is present throughout the gastrointestinal tract and is essential for regulating gut microbiota homeostasis and preventing disease by protecting the gastrointestinal barrier from microorganisms, pathogens and toxins or other irritants. Mucin (MUC)-2 is a secreted protein produced by epithelial goblet cells as the main component of mucus. Defects in the gastrointestinal tract, such as inflammation and ulcers, cause damage to the mucus barrier, which can worsen mucus quality and reduce mucus production. Therefore, we would like to review the characteristics of MUC2 and its role in intestinal disorders and highlight the importance of further studies. We also investigated whether the role of MUC2 differs between children and adults, ulcerative colitis (UC) and Crohn's disease (CD).

Existing Prediction Models of Disease Course in Paediatric Crohn's Disease Are Poorly Replicated in a Prospective Inception Cohort.

BACKGROUND: Several groups have proposed models to predict disease outcomes in paediatric Crohn's disease [CD], notably the RISK, GROWTH, and the Porto group, but none were externally validated. We aimed to explore these predictive models and individual predictors summarised by the PIBD-ahead project in a prospective inception cohort of paediatric CD. METHODS: We included children who were diagnosed with CD at two medical centres and followed them at 3 and 12 months thereafter as well as at the last follow-up. Outcomes included steroid-free remission [SFR], surgery, and stricturing/fistulising disease. RESULTS: In all 155 children were included (median follow-up of 31 [16-48] months, 107 [71%] had moderate-to-severe disease). Stricturing and penetrating disease at diagnosis were noted in 34 [22%] and two [1.3%] children, respectively, and these were excluded from the relevant analyses. At 1 year, 10 [8.3%] developed new stricturing disease, two [1.7%] developed penetrating disease, seven [5%] required intestinal surgery, and 15 [10%] required perianal surgery. The sensitivity/specificity/positive predictive value [PPV]/negative predictive value [NPV] of the GROWTH criteria for predicting SFR at 12 months [occurring in 70% of children] were 20%/85%/76%/31% and for surgery at 2 years were 96%/20%/16%/96%, respectively. Strictures were predicted by the RISK model with sensitivity/specificity/PPV/NPV of 33%/73%/18%/86%, respectively. The sensitivity/specificity/PPV/NPV of the Porto criteria to predict surgery were 86%/10%/4%/94%, respectively. None of the Pediatric Inflammatory Bowel Disease-ahead [PIBD-ahead] predictors were associated with surgery or stricturing disease. CONCLUSIONS: None of the three main predictive models in paediatric CD achieved sufficient accuracy, far from that reported in the original cohorts. This highlights the necessity of external validation in any prediction model prior to its implementation in clinical practice.

Development of Spinal Tuberculosis in an Adolescent With Crohn's Disease After Infliximab Therapy: A Case Report With Literature Review.

INTRODUCTION: Tuberculosis (TB) spondylitis, also known as Pott's disease, is a severe form of extrapulmonary TB. Infliximab treatment for Crohn's disease (CD) patients increases the risk of TB, and is likely to increase the risk of TB spondylitis as well. CASE PRESENTATION: We report a rare case of TB spondylitis development in a 16-year-old female with CD. She had a close household contact of active pulmonary TB and received contact investigation. She was diagnosed with latent TB 1 month before the diagnosis of CD, and had started a latent TB treatment regimen with isoniazid for 9 months. At 5 months from the start of latent TB treatment, infliximab was started. Approximately 1 year after infliximab treatment, her infusion interval was shortened from every 8 weeks to every 4 weeks owing to secondary loss of response due to nonimmunogenic pharmacokinetic failure. One month later, miliary TB developed and infliximab was stopped. She received a miliary TB treatment regimen for 6 months, curing the disease. Three months later, spinal TB was incidentally detected on abdominal computed tomography. She received a TB treatment regimen for 12 months, curing spinal TB. Currently, she is receiving vedolizumab to treat CD and is in clinical remission. Although this patient has sufficiently been treated at each stage of TB development, particularly for latent TB and miliary TB, TB spondylitis still developed. CONCLUSION: Considering that TB spondylitis developed despite sufficient treatment at each stage, pediatric gastroenterologists should stay cautious when using anti-tumor necrosis factor agents in patients with inflammatory bowel disease with a history of latent TB.

Phospholipase D1 is upregulated by vorinostat and confers resistance to vorinostat in glioblastoma.

Glioblastoma (GBM) is an aggressive brain tumor and drug resistance remains a major barrier for therapeutics. Epigenetic alterations are implicated in GBM pathogenesis, and epigenetic modulators including histone deacetylase (HDAC) inhibitors are exploited as promising anticancer therapies. Here, we demonstrate that phospholipase D1 (PLD1) is a transcriptional target of HDAC inhibitors and confers resistance to HDAC inhibitor in GBM. Treatment of vorinostat upregulates PLD1 through PKCζ-Sp1 axis. Vorinostat induces dynamic changes in the chromatin structure and transcriptional machinery associated with PLD1 promoter region. Cotreatment of vorinostat with PLD1 inhibitor further attenuates invasion, angiogenesis, colony-forming capacity, and self-renewal capacity, compared with those of either treatment. PLD1 inhibitor overcomes resistance to vorinostat in GBM cells intracranial GBM tumors. Our finding provides new insight into the role of PLD1 as a target of resistance to vorinostat, and PLD1 inhibitor might provide the basis for therapeutic combinations with improved efficacy of HDAC inhibitor.

Inhibition of phospholipase D2 augments histone deacetylase inhibitor-induced cell death in breast cancer cells.

BACKGROUND: Histone deacetylase (HDAC) inhibitors are promising anticancer drugs but their effect on tumor treatment has been disappointing mainly due to the acquisition of HDAC inhibitor resistance. However, the mechanisms underlying such resistance remain unclear. METHODS: In this study, we performed Western blot, q-PCR, and promoter assay to examine the expression of HDAC inhibitor-induced phospholipase D2 (PLD2) in MDA-MB231and MDA-MB435 breast cancer cells. Apoptosis and proliferation were analyzed by flow cytometry. In addition to invasion and migration assay, angiogenesis was further measured using in vitro tube formation and chick embryo chorioallantoic membrane model. RESULTS: HDAC inhibitors including suberoylanilide hydroxamic acid (SAHA), trichostatin, and apicidin, induce expression of PLD2 in a transcriptional level. SAHA upregulates expression of PLD2 via protein kinase C-ζ in breast cancer cells and increases the enzymatic activity of PLD. The combination treatment of SAHA with PLD2 inhibitor significantly enhances cell death in breast cancer cells. Phosphatidic acid, a product of PLD activity, prevented apoptosis promoted by cotreatment with SAHA and PLD2 inhibitor, suggesting that SAHA-induced PLD2 expression and subsequent activation of PLD2 might confers resistance of breast cancer cells to HDAC inhibitor. The combinational treatment of the drugs significantly suppressed invasion, migration, and angiogenesis, compared with that of either treatment. CONCLUSION: These findings provide further insight into elucidating the advantages of combination therapy with HDAC and PLD2 inhibitors over single-agent strategies for the treatment of cancer.

Author Correction: Sirt1 negatively regulates FcεRI-mediated mast cell activation through AMPK- and PTP1B-dependent processes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Activation of Mevalonate Pathway via LKB1 Is Essential for Stability of Treg Cells.

The function of regulatory T (Treg) cells depends on lipid oxidation. However, the molecular mechanism by which Treg cells maintain lipid metabolism after activation remains elusive. Liver kinase B1 (LKB1) acts as a coordinator by linking cellular metabolism to substrate AMP-activated protein kinase (AMPK). We show that deletion of LKB1 in Treg cells exhibited reduced suppressive activity and developed fatal autoimmune inflammation. Mechanistically, LKB1 induced activation of the mevalonate pathway by upregulating mevalonate genes, which was essential for Treg cell functional competency and stability by inducing Treg cell proliferation and suppressing interferon-gamma and interleukin-17A expression independently of AMPK. Furthermore, LKB1 was found to regulate intracellular cholesterol homeostasis and to promote the mevalonate pathway. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate inhibited conversion of Treg cells and enhanced survival of LKB1-deficient Treg mice. Thus, LKB1 is a key regulator of lipid metabolism in Treg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.

Epicatechin Prevents Methamphetamine-Induced Neuronal Cell Death via Inhibition of ER Stress.

Methamphetamine (METH) acts strongly on the nervous system and damages neurons and is known to cause neurodegenerative diseases such as Alzheimer's and Parkinson's. Flavonoids, polyphenolic compounds present in green tea, red wine and several fruits exhibit antioxidant properties that protect neurons from oxidative damage and promote neuronal survival. Especially, epicatechin (EC) is a powerful flavonoid with antibacterial, antiviral, antitumor and antimutagenic effects as well as antioxidant effects. We therefore investigated whether EC could prevent METH-induced neurotoxicity using HT22 hippocampal neuronal cells. EC reduced METH-induced cell death of HT22 cells. In addition, we observed that EC abrogated the activation of ERK, p38 and inhibited the expression of CHOP and DR4. EC also reduced METH-induced ROS accumulation and MMP. These results suggest that EC may protect HT22 hippocampal neurons against METH-induced cell death by reducing ER stress and mitochondrial damage.

Allergen­removed Rhus verniciflua Stokes suppresses invasion and migration of pancreatic cancer cells through downregulation of the JAK/STAT and Src/FAK signaling pathways.

Pancreatic cancer is a leading cause of mortality and morbidity worldwide. Due to drug resistance, and the high toxicity and adverse side effects of existing chemotherapeutic drugs, the current treatment of highly aggressive pancreatic cancer is considered inadequate. Allergen­removed Rhus verniciflua Stokes (aRVS) has a strong antiproliferative effect in various cancer cells, and due to its low toxicity, it has emerged as an attractive candidate for cancer treatment. However, the potential use of aRVS as a treatment for pancreatic cancer is relatively unexplored. The present study examined the effects of aRVS on the invasion and migration of pancreatic cancer cells, and identified the molecular mechanisms underlying its anticancer effects. aRVS inhibited the Janus kinase/signal transducer and activator of transcription pathway in pancreatic cancer cells, and decreased the protein expression of mucin 4. In addition, it inhibited the activation of focal adhesion kinase and Src signaling, and decreased the expression of matrix metalloproteinase 9, which may reduce the migration and invasion of pancreatic cancer cells. In conclusion, the present study suggested that aRVS may be a potential treatment for aggressive pancreatic cancer.

A ß-galactosidase-expressing E. coli culture as an alternative test to identify skin sensitizers and non-sensitizers.

Although the Organization for Economic Cooperation and Development (OECD) has adopted several in vitro methods with reasonable predictive capacity, alternative methods for identifying skin sensitizers and non-sensitizers with reliability and simplicity are still required for more efficient and economic prediction. The present study was to design an in vitro system with the use of a ß-galactosidase-expressing E. coli culture for simpler but sufficiently accurate classification of skin sensitizers and non-sensitizers. A LacZ gene-containing E. coli strain that is capable of producing ß-galactosidase enzyme was induced by isopropyl ß-D-1-thiogalactopyranoside with concomitant treatment with test chemicals. After 6-hr incubation, cells were lysed and ß-galactosidase enzyme activity was monitored colorimetrically by using O-nitrophenyl-D-galactopyranoside as a substrate. Following optimization of several experimental conditions, 22 skin sensitizers and 11 non-sensitizers were examined to assess predictive capacity of this method. The results indicated that predictivity was as follows: 90.9% sensitivity, 81.8% specificity, and 87.9% accuracy, when 17.3% of control activity was used as the cut-off value to separate sensitizers from non-sensitizers. Data suggested that the current bacterial system expressing ß-galactosidase may serve as a useful alternative test for classifying skin sensitizers and non-sensitizers, without the utilization of animals or mammalian cell cultures.

Phase I and phase II metabolite identification of rutaecarpine in freshly isolated hepatocytes from male Sprague-Dawley rats.

Rutaecarpine, an alkaloid originally isolated from Evodia rutaecarpa, has been used for the treatment of gastrointestinal disorders in Asia. In the present study, the phase I and phase II metabolites of rutaecarpine were investigated in freshly isolated hepatocytes from male Sprague-Dawley rats. The individual metabolites were characterized via liquid chromatography-tandem mass spectrometry. The incubation of rutaecarpine with freshly isolated hepatocytes for 2 h yielded five major phase I metabolites. In addition, three glucuronide conjugates and four sulfate conjugates were observed. Because the majority of metabolites observed in vivo were identified, freshly isolated hepatocytes might be useful for the identification of certain metabolites formed from drug candidates from a reduced number of experimental animals.

Sirt1 negatively regulates FcεRI-mediated mast cell activation through AMPK- and PTP1B-dependent processes.

Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both in vitro and in vivo. Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.

BJ-1108, a 6-Amino-2,4,5-trimethylpyridin-3-ol analogue, regulates differentiation of Th1 and Th17 cells to ameliorate experimental autoimmune encephalomyelitis.

BACKGROUND: CD4+ T cells play an important role in the initiation of an immune response by providing help to other cells. Among the helper T subsets, interferon-γ (IFN-γ)-secreting T helper 1 (Th1) and IL-17-secreting T helper 17 (Th17) cells are indispensable for clearance of intracellular as well as extracellular pathogens. However, Th1 and Th17 cells are also associated with pathogenesis and contribute to the progression of multiple inflammatory conditions and autoimmune diseases. RESULTS: In the current study, we found that BJ-1108, a 6-aminopyridin-3-ol analogue, significantly inhibited Th1 and Th17 differentiation in vitro in a concentration-dependent manner, with no effect on proliferation or apoptosis of activated T cells. Moreover, BJ-1108 inhibited differentiation of Th1 and Th17 cells in ovalbumin (OVA)-specific OT II mice. A complete Freund's adjuvant (CFA)/OVA-induced inflammatory model revealed that BJ-1108 can reduce generation of proinflammatory Th1 and Th17 cells. Furthermore, in vivo studies showed that BJ-1108 delayed onset of disease and suppressed experimental autoimmune encephalomyelitis (EAE) disease progression by inhibiting differentiation of Th1 and Th17 cells. CONCLUSIONS: BJ-1108 treatment ameliorates inflammation and EAE by inhibiting Th1 and Th17 cells differentiation. Our findings suggest that BJ-1108 is a promising novel therapeutic agent for the treatment of inflammation and autoimmune disease.

BJ-3105, a 6-Alkoxypyridin-3-ol Analog, Impairs T Cell Differentiation and Prevents Experimental Autoimmune Encephalomyelitis Disease Progression.

CD4+ T cells are essential in inflammation and autoimmune diseases. Interferon-γ (IFN-γ) secreting T helper (Th1) and IL-17 secreting T helper (Th17) cells are critical for several autoimmune diseases. To assess the inhibitory effect of a given compound on autoimmune disease, we screened many compounds with an in vitro Th differentiation assay. BJ-3105, a 6-alkoxypyridin-3-ol analog, inhibited IFN-γ and IL-17 production from polyclonal CD4+ T cells and ovalbumin (OVA)-specific CD4+ T cells which were activated by T cell receptor (TCR) engagement. BJ-3105 ameliorated the experimental autoimmune encephalomyelitis (EAE) model by reducing Th1 and Th17 generation. Notably, Th cell differentiation was significantly suppressed by BJ-3105 treatment without inhibiting in vitro proliferation of T cells or inducing programmed cell death. Mechanistically, BJ-3105 inhibited the phosphorylation of JAK and its downstream signal transducer and activator of transcription (STAT) that is critical for Th differentiation. These results demonstrated that BJ-3105 inhibits the phosphorylation of STAT in response to cytokine signals and subsequently suppressed the differentiation of Th cell responses.

BJ-1108, a 6-Amino-2,4,5-trimethylpyridin-3-ol analogue, regulates differentiation of Th1 and Th17 cells to ameliorate experimental autoimmune encephalomyelitis

BACKGROUND: CD4+ T cells play an important role in the initiation of an immune response by providing help to other cells. Among the helper T subsets, interferon-γ (IFN-γ)-secreting T helper 1 (Th1) and IL-17-secreting T helper 17 (Th17) cells are indispensable for clearance of intracellular as well as extracellular pathogens. However, Th1 and Th17 cells are also associated with pathogenesis and contribute to the progression of multiple inflammatory conditions and autoimmune diseases. RESULTS: In the current study, we found that BJ-1108, a 6-aminopyridin-3-ol analogue, significantly inhibited Th1 and Th17 differentiation in vitro in a concentration-dependent manner, with no effect on proliferation or apoptosis of activated T cells. Moreover, BJ-1108 inhibited differentiation of Th1 and Th17 cells in ovalbumin (OVA)-specific OT II mice. A complete Freund's adjuvant (CFA)/OVA-induced inflammatory model revealed that BJ-1108 can reduce generation of proinflammatory Th1 and Th17 cells. Furthermore, in vivo studies showed that BJ-1108 delayed onset of disease and suppressed experimental autoimmune encephalomyelitis (EAE) disease progression by inhibiting differentiation of Th1 and Th17 cells. CONCLUSIONS: BJ-1108 treatment ameliorates inflammation and EAE by inhibiting Th1 and Th17 cells differentiation. Our findings suggest that BJ-1108 is a promising novel therapeutic agent for the treatment of inflammation and autoimmune disease.

Role of Intestinal Microbiota in Baicalin-Induced Drug Interaction and Its Pharmacokinetics.

Since many glycoside compounds in natural products are hydrolyzed by intestinal microbiota when administered orally, it is of interest to know whether their pharmacological effects are derived from the glycoside itself or from the aglycone form in vivo. An interesting example is baicalin versus baicalein, the aglycone of baicalin, which is contained in some herbs from Labiatae including Scutellaria baicalensis Georgi and Scutellaria lateriflora Linne. The herbs have been extensively used for treatment of inflammatory diseases in Asia. Although there have been numerous reports regarding the pharmacological effects of baicalin and baicalein in vivo and in vitro, some reports indicated that the glycoside form would hardly be absorbed in the intestine and that it should be hydrolyzed to baicalein in advance for absorption. Therefore, the role of metabolism by intestinal microbiota should also be considered in the metabolism of baicalin. In addition, baicalin contains a glucuronide moiety in its structure, by which baicalin and baicalein show complex pharmacokinetic behaviors, due to the interconversion between them by phase II enzymes in the body. Recently, concerns about drug interaction with baicalin and/or baicalein have been raised, because of the co-administration of Scutellaria species with certain drugs. Herein, we reviewed the role of intestinal microbiota in pharmacokinetic characteristics of baicalin and baicalein, with regards to their pharmacological and toxicological effects.

Anti-angiogenic activity of macrolactin A and its succinyl derivative is mediated through inhibition of class I PI3K activity and its signaling.

In the current study, macrolactin compounds, macrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), were investigated for their anti-angiogenic activities and action mechanism. MA and SMA inhibited in vitro and in vivo angiogenesis induced by three different classes of pro-angiogenic factors, VEGF, IL-8, and TNF-α. SMA exhibited stronger anti-angiogenic activity than MA, and such anti-angiogenic activity of SMA was consistently observed in MDA-MB-231 human breast cancer cell-inoculated CAM assay showing dose-dependent suppression of tumor growth and tumor-induced angiogenesis. In an in vitro PI3K competitive activity assay, SMA induced concentration-dependent inhibition of class I PI3K isoforms, p110α, p110ß, p110δ, and p110γ. In addition, non-receptor tyrosine kinase c-Src, which is involved in the activation of PI3K heterodimer, was suppressed by MA and SMA. Correspondingly, MA and SMA significantly inhibited the stimulus-induced phosphorylation of Akt, mTOR, p70S6K, and ribosomal S6 in human umbilical vein endothelial cells (HUVECs). At the same time, the stimulus-induced production of reactive oxygen species (ROS) and activation of NF-κB were significantly suppressed by MA and SMA. Moreover, the macrolactins suppressed NF-κB-regulated HSP90 protein expression, which stabilizes phosphorylated Akt and NADPH oxidase. Suppression of NF-κB in macrolactin-treated HUVECs with concurrent inhibition of rS6 indicates that MAs effectively block angiogenesis through down-regulation of genes related to angiogenesis at both transcriptional and translational levels. Taken together, the results demonstrate that anti-angiogenic effect of MA and SMA is mediated through inhibition of PI3K/Akt and NADPH oxidase-derived ROS/NF-κB signaling pathways. These results further indicate that MA and SMA may be applicable for treatment of various diseases associated with angiogenesis.

6-Amino-2,4,5-trimethylpyridin-3-ols: a new general synthetic route and antiangiogenic activity.

A new synthetic strategy for preparation of a wide range of 6-amino-2,4,5-trimethylpyridin-3-ols from pyridoxine·HCl via a six-step sequence has been developed. This approach features an introduction of various amino groups to C(6)-position of 3-benzyloxy-6-bromo-2,4,5-trimethylpyridine (13), a key intermediate, by a Buchwald-Hartwig amination reaction using palladium(0) transition metal, which certainly renders an expanded scope of amino substituents. Some analogs prepared using the methods described here showed high level of antiangiogenic and antitumor activities in chick chorioallantoic membrane (CAM) assay, demonstrating the potential of these new aminopyridinols as antiangiogenic agents.