Targeted Knockdown of Genes in the Choroid Plexus.

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and pathological conditions. Recent research has shown that regulating ChP activity may offer protection against CNS disorders. However, studying the biological function of the ChP without affecting other brain regions is challenging due to its delicate structure. This study presents a novel method for gene knockdown in ChP tissue using adeno-associated viruses (AAVs) or cyclization recombination enzyme (Cre) recombinase protein consisting of TAT sequence (CRE-TAT). The results demonstrate that after injecting AAV or CRE-TAT into the lateral ventricle, the fluorescence was exclusively concentrated in the ChP. Using this approach, the study successfully knocked down the adenosine A2A receptor (A2AR) in the ChP using RNA interference (RNAi) or Cre/locus of X-overP1 (Cre/LoxP) systems, and showed that this knockdown could alleviate the pathology of experimental autoimmune encephalomyelitis (EAE). This technique may have important implications for future research on the ChP's role in CNS disorders.

HERV-W ENV Induces Innate Immune Activation and Neuronal Apoptosis via linc01930/cGAS Axis in Recent-Onset Schizophrenia.

Schizophrenia is a severe neuropsychiatric disorder affecting about 1% of individuals worldwide. Increased innate immune activation and neuronal apoptosis are common findings in schizophrenia. Interferon beta (IFN-ß), an essential cytokine in promoting and regulating innate immune responses, causes neuronal apoptosis in vitro. However, the precise pathogenesis of schizophrenia is unknown. Recent studies indicate that a domesticated endogenous retroviral envelope glycoprotein of the W family (HERV-W ENV, also called ERVWE1 or syncytin 1), derived from the endogenous retrovirus group W member 1 (ERVWE1) locus on chromosome 7q21.2, has a high level in schizophrenia. Here, we found an increased serum IFN-ß level in schizophrenia and showed a positive correlation with HERV-W ENV. In addition, serum long intergenic non-protein coding RNA 1930 (linc01930), decreased in schizophrenia, was negatively correlated with HERV-W ENV and IFN-ß. In vitro experiments showed that linc01930, mainly in the nucleus and with noncoding functions, was repressed by HERV-W ENV through promoter activity suppression. Further studies indicated that HERV-W ENV increased IFN-ß expression and neuronal apoptosis by restraining the expression of linc01930. Furthermore, HERV-W ENV enhanced cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes protein (STING) expression and interferon regulatory factor 3 (IRF3) phosphorylation in neuronal cells. Notably, cGAS interacted with HERV-W ENV and triggered IFN-ß expression and neuronal apoptosis caused by HERV-W ENV. Moreover, Linc01930 participated in the increased neuronal apoptosis and expression level of cGAS and IFN-ß induced by HERV-W ENV. To summarize, our results suggested that linc01930 and IFN-ß might be novel potential blood-based biomarkers in schizophrenia. The totality of these results also showed that HERV-W ENV facilitated antiviral innate immune response, resulting in neuronal apoptosis through the linc01930/cGAS/STING pathway in schizophrenia. Due to its monoclonal antibody GNbAC1 application in clinical trials, we considered HERV-W ENV might be a reliable therapeutic choice for schizophrenia.

Domesticated HERV-W env contributes to the activation of the small conductance Ca2+-activated K+ type 2 channels via decreased 5-HT4 receptor in recent-onset schizophrenia.

The human endogenous retroviruses type W family envelope (HERV-W env) gene is located on chromosome 7q21-22. Our previous studies show that HERV-W env is elevated in schizophrenia and HERV-W env can increase calcium influx. Additionally, the 5-HTergic system and particularly 5-hydroxytryptamine (5-HT) receptors play a prominent role in the pathogenesis and treatment of schizophrenia. 5-hydroxytryptamine receptor 4 (5-HT4R) agonist can block calcium channels. However, the underlying relationship between HERV-W env and 5-HT4R in the etiology of schizophrenia has not been revealed. Here, we used enzyme-linked immunosorbent assay to detect the concentration of HERV-W env and 5-HT4R in the plasma of patients with schizophrenia and we found that there were decreased levels of 5-HT4R and a negative correlation between 5-HT4R and HERV-W env in schizophrenia. Overexpression of HERV-W env decreased the transcription and protein levels of 5-HT4R but increased small conductance Ca2+-activated K+ type 2 channels (SK2) expression levels. Further studies revealed that HERV-W env could interact with 5-HT4R. Additionally, luciferase assay showed that an essential region (-364 to -176 from the transcription start site) in the SK2 promoter was required for HERV-W env-induced SK2 expression. Importantly, 5-HT4R participated in the regulation of SK2 expression and promoter activity. Electrophysiological recordings suggested that HERV-W env could increase SK2 channel currents and the increase of SK2 currents was inhibited by 5-HT4R. In conclusion, HERV-W env could activate SK2 channels via decreased 5-HT4R, which might exhibit a novel mechanism for HERV-W env to influence neuronal activity in schizophrenia.

A high salt diet protects interleukin 10-deficient mice against chronic colitis by improving the mucosal barrier function.

A high salt diet (HSD) is often associated with a high risk for a variety of diseases, such as obesity and cardiovascular disease. Previous studies have demonstrated that an HSD enhances Th17 responses and increases the severity of autoimmune diseases. In this study, we investigated the effects of HSD (4% NaCl w/w) on colitis in IL-10-/- mice by comparing it with IL-10-/- mice on a normal salt diet (NSD, 1% NaCl w/w). The colonic epithelial barrier integrity in IL-10-/- mice, as well as differentiated Caco-2 cells exposed to high NaCl and proinflammatory cytokines, was also evaluated. Surprisingly, an HSD significantly ameliorated macroscopic colitis, improved the intestinal permeability of FITC-dextran, and decreased multiple proinflammatory cytokines in the colonic mucosa of IL-10-/- mice. While occludin and claudin-1, two major tight-junction proteins, were markedly down-regulated in IL-10-/- mice, HSD effectively restored their expressions. In Caco-2 cells, proinflammatory cytokines (TNF-α and IL-1ß) potently decreased the expression of occludin and claudin-1 regardless of salt conditions [0.9% (standard), 1.2%, or 1.5% NaCl]. Under high salt conditions (1.5% NaCl), transepithelial electrical resistance (TEER) was elevated, while the addition of IL-10 further downregulated occludin and claudin-1 expressions by ~50% and lowered TEER. These findings suggest that, in the absence of IL-10, HSD promotes intestinal epithelial integrity and exerts an anti-inflammatory role as demonstrated by alleviated colitis in IL-10-/- mice. Moreover, Caco-2 data indicate that, in an inflammatory environment and under high NaCl conditions, IL-10 may play a proinflammatory role by disrupting colonic epithelial integrity and thus further promoting inflammation.

HERV-W Envelope Triggers Abnormal Dopaminergic Neuron Process through DRD2/PP2A/AKT1/GSK3 for Schizophrenia Risk.

An increasing number of studies have begun considering human endogenous retroviruses (HERVs) as potential pathogenic phenomena. Our previous research suggests that HERV-W Envelope (HERV-W ENV), a HERV-W family envelope protein, is elevated in schizophrenia patients and contributes to the pathophysiology of schizophrenia. The dopamine (DA) hypothesis is the cornerstone in research and clinical practice related to schizophrenia. Here, we found that the concentration of DA and the expression of DA receptor D2 (DRD2) were significantly higher in schizophrenia patients than in healthy individuals. Intriguingly, there was a positive correlation between HERV-W ENV and DA concentration. Depth analyses showed that there was a marked consistency between HERV-W ENV and DRD2 in schizophrenia. Studies in vitro indicated that HERV-W ENV could increase the DA concentration by regulating DA metabolism and induce the expression of DRD2. Co-IP assays and laser confocal scanning microscopy indicated cellular colocalization and a direct interaction between DRD2 and HERV-W ENV. Additionally, HERV-W ENV caused structural and functional abnormalities of DA neurons. Further studies showed that HERV-W ENV could trigger the PP2A/AKT1/GSK3 pathway via DRD2. A whole-cell patch-clamp analysis suggested that HERV-W ENV enhanced sodium influx through DRD2. In conclusion, we uncovered a relationship between HERV-W ENV and the dopaminergic system in the DA neurons. Considering that GNbAC1, a selective monoclonal antibody to the MSRV-specific epitope, has been promised as a therapy for treating type 1 diabetes and multiple sclerosis (MS) in clinical trials, understanding the precise function of HERV-W ENV in the dopaminergic system may provide new insights into the treatment of schizophrenia.

Caenorhabditis elegans methionine/S-adenosylmethionine cycle activity is sensed and adjusted by a nuclear hormone receptor.

Vitamin B12 is an essential micronutrient that functions in two metabolic pathways: the canonical propionate breakdown pathway and the methionine/S-adenosylmethionine (Met/SAM) cycle. In Caenorhabditis elegans, low vitamin B12, or genetic perturbation of the canonical propionate breakdown pathway results in propionate accumulation and the transcriptional activation of a propionate shunt pathway. This propionate-dependent mechanism requires nhr-10 and is referred to as 'B12-mechanism-I'. Here, we report that vitamin B12 represses the expression of Met/SAM cycle genes by a propionate-independent mechanism we refer to as 'B12-mechanism-II'. This mechanism is activated by perturbations in the Met/SAM cycle, genetically or due to low dietary vitamin B12. B12-mechanism-II requires nhr-114 to activate Met/SAM cycle gene expression, the vitamin B12 transporter, pmp-5, and adjust influx and efflux of the cycle by activating msra-1 and repressing cbs-1, respectively. Taken together, Met/SAM cycle activity is sensed and transcriptionally adjusted to be in a tight metabolic regime.

Avertin affects murine colitis by regulating neutrophils and macrophages.

Anesthetics are thought to be involved in immunomodulation. Avertin is one of the safest and most commonly used intravenous anesthetics in rodent experiments; it is also widely used in euthanasia of inflammatory bowel disease (IBD) models. This study aimed to define the role and mechanism of action of Avertin on murine colitis. We assessed the effects of a single Avertin injection on colitis using the disease activity index (DAI), pathology, enzyme-linked immunosorbent assay (ELISA), multiplex-ELISA, flow cytometry, and routine blood examination in wild-type (WT) and dextran sodium sulphate (DSS)-treated mice. Although Avertin caused acute cecitis in WT mice after 24 h and aggravated inflammation in the medium term, it alleviated inflammation in the late stage of DSS-induced colitis according to the DAI. Avertin upregulated MPO production and induced the accumulation of neutrophils and macrophages in intestinal mucosa of both WT and DSS-treated mice; the altered MPO might indicate a change in respiratory burst. However, it exhibited a more effective suppression of inflammatory factors secreted by macrophages as the colitis progressed. Avertin led to an increase in neutrophils and decrease in monocytes in both WT and DSS-treated mice blood. Our findings suggest that Avertin aggravates inflammation in the early and medium terms, but alleviates inflammation in the late stage of colitis by regulating neutrophils and macrophages.

Unexpected mechanism of colitis amelioration by artesunate, a natural product from Artemisia annua L.

BACKGROUND: Artemisinin and its derivatives are known to exert immunosuppressive effects through modulating adaptive immunity. We investigated a novel role of artesunate in regulating innate immunity, including both macrophages (MΦ) and dendritic cells (DCs), which are known to involve in DSS-induced colitis. METHODS: Effects of artesunate on innate immunity were extensively evaluated, both in vivo using DSS-colitis model with WT and T cell-deficient RAG mice (RAG-/-) and in vitro using cell culture models, including in-depth analyses of MΦ/DC apoptosis and cytokine expression by flow cytometry, Western blot, or immunohistology. RESULTS: Unexpectedly, artesunate significantly ameliorated the DSS colitis of both WT and RAG1-/- mice with similar potency, suggesting a mechanism that involves primarily innate rather than adaptive immunity. In vivo mechanistic studies revealed that artesunate markedly induced apoptosis of lamina propria MΦs and DCs and suppressed mucosal TNF-α and IL-12p70 in DSS-colitis. In vitro, artesunate potently induced a dose- and time-dependent apoptosis of murine bone marrow-derived DCs and human THP-1 MΦs, through the caspases-9-mediated intrinsic pathway. Artesunate significantly decreased the secretion of IL-12p40/70 by DCs and TNF-α by MΦs. Furthermore, a combination of artesunate with an immunomodulator (methotrexate/triptolide/azathioprine) exhibited superior potency in promoting apoptosis of MΦs than any individual drug alone. CONCLUSIONS: The immunomodulatory mechanism of artesunate in colitis involves a novel and potent induction of the intrinsic apoptosis pathway of proliferating MΦs and DCs and suppression of IL-12 and TNF-α. Artemisinin and its derivatives are promising new therapeutic alternatives for IBD, either alone or in combination with other immunomodulators.

Deletion of IL-6 Exacerbates Colitis and Induces Systemic Inflammation in IL-10-Deficient Mice.

BACKGROUND AND AIMS: Interleukin 6 [IL-6] or its receptor is currently a candidate for targeted biological therapy of inflammatory bowel disease [IBD]. Thus, a comprehensive understanding of the consequences of blocking IL-6 is imperative. We investigated this by evaluating the effects of IL-6 deletion on the spontaneous colitis of IL-10-deficient mice [IL-10-/-]. METHODS: IL-6/IL-10 double-deficient mice [IL-6-/-/IL-10-/-] were generated and analysed for intestinal inflammation, general phenotypes and molecular/biochemical changes in the colonic mucosa compared with wild-type and IL-10-/- mice. RESULTS: Unexpectedly, the IL-6-/-/IL-10-/- mice showed more pronounced gut inflammation and earlier disease onset than IL-10-/- mice, both locally [colon and small bowel] and systemically [splenomegaly, ulcerative dermatitis, leukocytosis, neutrophilia and monocytosis]. IL-6-/-/IL-10-/- mice exhibited elevations of multiple cytokines [IL-1ß, IL-4, IL-12, TNFα] and chemokines [MCP-1 and MIG], but not IFN-γ [Th1], IL-17A and IL-17G [Th17], or IL-22 [Th22]. FOXP3 and TGF-ß, two key factors for regulatory T [Treg] cell differentiation, were significantly down-regulated in the colonic mucosa, but not in the thymus or mesenteric lymph nodes, of IL-6-/-/IL-10-/- mice. CTLA-4 was diminished while iNOS was up-regulated in the colonic mucosa of IL-6-/-/IL-10-/- mice. CONCLUSION: In IL-10-/- mice, complete IL-6 blockade significantly aggravates gut inflammation, at least in part by suppressing Treg/CTLA-4 and promoting the IL-1ß/Th2 pathway. In addition, the double mutant exhibits signs of severe systemic inflammation. Our data define a new function of IL-6 and suggest that caution should be exercised when targeting IL-6 in IBD patients, particularly those with defects in IL-10 signalling.

A Delicate Balance between Bacterial Iron and Reactive Oxygen Species Supports Optimal C. elegans Development.

Iron is an essential micronutrient for all forms of life; low levels of iron cause human disease, while too much iron is toxic. Low iron levels induce reactive oxygen species (ROS) by disruption of the heme and iron-sulfur cluster-dependent electron transport chain (ETC). To identify bacterial metabolites that affect development, we screened the Keio Escherichia coli collection and uncovered 244 gene deletion mutants that slow Caenorhabditis elegans development. Several of these genes encode members of the ETC cytochrome bo oxidase complex, as well as iron importers. Surprisingly, either iron or anti-oxidant supplementation reversed the developmental delay. This suggests that low bacterial iron results in high bacterial ROS and vice versa, which causes oxidative stress in C. elegans that subsequently impairs mitochondrial function and delays development. Our data indicate that the bacterial diets of C. elegans provide precisely tailored amounts of iron to support proper development.

Antibiotics induce sustained dysregulation of intestinal T cell immunity by perturbing macrophage homeostasis.

Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (TH1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring TH17 and TH2 responses for clearance (bacterial Citrobacter rodentium and helminth Trichuris muris infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell-mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use.

Mismatch Repair Protein hMLH1, but not hMSH2, Enhances Estrogen-Induced Apoptosis of Colon Cancer Cells.

BACKGROUND: Epidemiological studies suggest a protective role of estrogen against colon carcinogenesis; this effect appears to be dependent on mismatch repair (MMR) status. However, the underlying mechanism remains unclear. This study investigated the role of MMR proteins in apoptosis of colon cancer cells in the presence or absence of estrogen. METHODS: Two major MMR proteins, human mutL homolog 1 (hMLH1) and mutS homolog 2 (hMSH2), as well as estrogen receptor-ß (ERß), were transiently expressed in either hMLH1-deficient HCT116 cells or hMSH2-deficient LoVo cells. Effects of estradiol on cell viability and apoptosis were assessed. Furthermore, we examined the apoptotic status of epithelial cells in colonic mucosa taken from previous healthy female subjects with menopausal syndrome before and after 6-month hormone replacement therapy (HRT). RESULTS: In hMLH1-deficient HCT116 cells, re-expression of hMLH1 led to a significantly decreased cell viability and increased apoptosis, which were further enhanced by estradiol, including marked increase of activated caspase-3 and caspase-9, as well as Bax and P53. The effect of hMLH1 overexpression in LoVo cells resulted in a similar increase in apoptosis that was greatly stimulated by estradiol. The enhanced apoptosis by hMLH1 and estradiol was further validated by FACS analyses of Annexin V expression. Re-expression of hMSH2 or overexpression of ERß in HCT116 cells also enhanced apoptosis; however, the effects were independent of estradiol. Furthermore, studies on healthy menopausal women before and after 6-month HRT demonstrated a significant HRT-mediated upregulation of the hMLH1 expression, with concomitant elevation of caspase-3 and caspase-9 activation in the colonic mucosa. CONCLUSION: We present the first evidence that hMLH1 and hMSH2 have similar but distinct roles in the apoptosis of colon cancer cells: an increased expression of either one can promote apoptosis, while only the effect of hMLH1 but not hMSH2 is estradiol-dependent. Our data suggest that MMR status should be assessed before hormone replacement therapy or future application of estrogen-based chemoprevention.

The Pathogenic Role of NLRP3 Inflammasome Activation in Inflammatory Bowel Diseases of Both Mice and Humans.

BACKGROUND AND AIMS: NLRP3 inflammasome is known to be involved in inflammatory bowel diseases. However, it is controversial whether it is pathogenic or beneficial. This study evaluated the roles of NLRP3 inflammasome in the pathogenesis of inflammatory bowel disease in IL-10-/- mice and humans. METHODS: NLRP3 inflammasome in colonic mucosa, macrophages, and colonic epithelial cells were analysed by western blotting. The NLRP3 inflammasome components were studied by sucrose density gradient fractionation, chemical cross-linking, and co-immunoprecipitation. The role of NLPR3 inflammasome in the pathogenesis of colitis was extensively evaluated in IL-10-/- mice, using a specific NLPR3 inflammasome inhibitor glyburide. RESULTS: NLRP3 inflammasome was upregulated in colonic mucosa of both IL-10-/- mice and Crohn's patients. NLRP3 inflammasome activity in IL-10-/- mice was elevated prior to colitis onset; it progressively increased as disease worsened and peaked as macroscopic disease emerged. NLRP3 inflammasome was found in both intestinal epithelial cells and colonic macrophages, as a large complex with a molecular weight of ≥ 360 kDa in size. In the absence of IL-10, NLRP3 inflammasome was spontaneously active and more robustly responsive when activated by LPS and nigericin. Glyburide markedly suppressed NLRP3 inflammasome expression/activation in IL-10-/- mice, leading to not only alleviation of ongoing colitis but also prevention/delay of disease onset. Glyburide also effectively inhibited the release of proinflammatory cytokines/chemokines by mucosal explants from Crohn's patients. CONCLUSIONS: Abnormal activation of NLRP3 inflammasome plays a major pathogenic role in the development of chronic colitis in IL-10-/- mice and humans. Glyburide, an FDA-approved drug, may have great potential in the management of inflammatory bowel diseases.

Compartment-specific immunity in the human gut: properties and functions of dendritic cells in the colon versus the ileum.

OBJECTIVE: Dendritic cells (DC) mediate intestinal immune tolerance. Despite striking differences between the colon and the ileum both in function and bacterial load, few studies distinguish between properties of immune cells in these compartments. Furthermore, information of gut DC in humans is scarce. We aimed to characterise human colonic versus ileal DC. DESIGN: Human DC from paired colonic and ileal samples were characterised by flow cytometry, electron microscopy or used to stimulate T cell responses in a mixed leucocyte reaction. RESULTS: A lower proportion of colonic DC produced pro-inflammatory cytokines (tumour necrosis factor-α and interleukin (IL)-1ß) compared with their ileal counterparts and exhibited an enhanced ability to generate CD4(+)FoxP3(+)IL-10(+) (regulatory) T cells. There were enhanced proportions of CD103(+)Sirpα(-) DC in the colon, with increased proportions of CD103(+)Sirpα(+) DC in the ileum. A greater proportion of colonic DC subsets analysed expressed the lymph-node-homing marker CCR7, alongside enhanced endocytic capacity, which was most striking in CD103(+)Sirpα(+) DC. Expression of the inhibitory receptor ILT3 was enhanced on colonic DC. Interestingly, endocytic capacity was associated with CD103(+) DC, in particular CD103(+)Sirpα(+) DC. However, expression of ILT3 was associated with CD103(-) DC. Colonic and ileal DC differentially expressed skin-homing marker CCR4 and small-bowel-homing marker CCR9, respectively, and this corresponded to their ability to imprint these homing markers on T cells. CONCLUSIONS: The regulatory properties of colonic DC may represent an evolutionary adaptation to the greater bacterial load in the colon. The colon and the ileum should be regarded as separate entities, each comprising DC with distinct roles in mucosal immunity and imprinting.

In Vitro Conditioned Bone Marrow-Derived Mesenchymal Stem Cells Promote De Novo Functional Enteric Nerve Regeneration, but Not Through Direct-Transdifferentiation.

Injury or neurodegenerative disorders of the enteric nervous system (ENS) cause gastrointestinal dysfunctions for which there is no effective therapy. This study, using the benzalkonium chloride-induced rat gastric denervation model, aimed to determine whether transplantation of bone marrow-derived mesenchymal stem cells (BMSC) could promote ENS neuron regeneration and if so, to elucidate the mechanism. Fluorescently labeled BMSC, isolated from either WT (BMSC labeled with bis-benzimide [BBM]) or green fluorescent protein (GFP)-transgenic rats, were preconditioned in vitro using fetal gut culture media containing glial cell-derived neurotrophic factor (GDNF), and transplanted subserosally into the denervated area of rat pylorus. In the nerve-ablated pylorus, grafted BMSC survived and migrated from the subserosa to the submucosa 28 days after transplantation, without apparent dedifferentiation. A massive number of PGP9.5/NSE/HuC/D/Tuj1-positive (but GFP- and BBM-negative) neurons were effectively regenerated in denervated pylorus grafted with preconditioned BMSC, suggesting that they were regenerated de novo, not originating from trans-differentiation of the transplanted BMSC. BMSC transplantation restored both basal pyloric contractility and electric field stimulation-induced relaxation. High levels of GDNF were induced in both in vitro-preconditioned BMSC as well as the previously denervated pylorus after transplantation of preconditioned BMSC. Thus, a BMSC-initiated GDNF-positive feedback mechanism is suggested to promote neuron regeneration and growth. In summary, we have demonstrated that allogeneically transplanted preconditioned BMSC initiate de novo regeneration of gastric neuronal cells/structures that in turn restore gastric contractility in pylorus-denervated rats. These neuronal structures did not originate from the grafted BMSC. Our data suggest that preconditioned allogeneic BMSC may have therapeutic value in treating enteric nerve disorders.

Molecular Analysis of Inflammatory Bowel Disease: Clinically Useful Tools for Diagnosis, Response Prediction, and Monitoring of Targeted Therapy.

Biomarkers of inflammatory bowel disease (IBD) are non-invasive or minimally invasive tests for IBD diagnosis and/or prognosis as well as assessment of disease activity and response to therapy. Here, we update the current status of IBD biomarkers, including serological, fecal, and genetic (DNA and microRNA [miRNA]) biomarkers. As an update, the classical serological biomarkers, including ASCA, pANCA, anti-OmpC, anti-Cbir, anti-I2, and other anti-glycan antibodies, are discussed only briefly. Emphasis in this article is given to those that have been recently identified or extensively characterized, as well as to the clinical utilities of biomarkers, with special attention to prediction of disease complication and activity assessment, mucosal healing, and response to therapies. In particular, we discuss the utilities of blood-based biomarkers predicting therapeutic response to anti-tumor necrosis factor (TNF)-α, such as anti-anti-TNFα antibodies or anti-drug antibodies (ADA) and trough level of anti-TNFα. Fecal biomarkers, which have recently attracted substantial attention, are also discussed extensively, including the well-characterized calprotectin and lactoferrin, as well as the recently characterized M2-PK, CHI3L1, neopterin, MMP-9, and HMGB1. Genome and exome sequencing enables the discovery of a number of rare yet disease-defining IBD-susceptible genes, particularly those involved in very early onset IBD, providing rare yet extremely useful biomarkers at genetic levels for IBD diagnosis/prognosis. Finally, we briefly summarize the discovery, characterization, and potential implications of miRNA as potential IBD biomarkers.

Activation of the Liver X Receptor by Agonist TO901317 Improves Hepatic Insulin Resistance via Suppressing Reactive Oxygen Species and JNK Pathway.

Activation of Liver X receptors (LXRs), key transcriptional regulators of glucose metabolism, normalizes glycemia and improves insulin sensitivity in rodent models with insulin resistance. However, the molecular mechanism is unclear. This study is aimed to elucidate the mechanism of LXRs-mediated liver glucose metabolic regulation in vitro and in vivo. Db/db mice were used as an in vivo model of diabetes; palmitate (PA)-stimulated HepG2 cells were used as an in vitro cell model with impairment of insulin signaling. TO901317 (TO) was chosen as the LXRs agonist. We demonstrated that TO treatment for 14 days potently improved the hepatic glucose metabolism in db/db mice, including fasting blood glucose, fasting insulin level, and HOMA-IR. TO had no effect on the glucose metabolism in normal WT mice. TO-mediated activation of hepatic LXRs led to strong inhibition of ROS production accompanied by inactivation of JNK pathway and re-activation of Akt pathway. TO also suppressed the expression of gluconeogenic genes such as PEPCK and G-6-pase in db/db mice, but not in WT mice. In HepG2 cells, TO almost completely restored PA-induced Akt inactivation, and suppressed PA-stimulated ROS production and JNK activation. Interestingly, basal level of ROS was also inhibited by TO in HepG2 cells. TO significantly inhibited PA-stimulated expressions of gluconeogenic genes. Finally, we found that anti-oxidative genes, such as Nrf2, were up-regulated after LXRs activation by TO. These results strongly support the notion that activation of LXRs is critical in suppression of liver gluconeogenesis and improvement of insulin sensitivity in diabetic individuals. At molecular levels, the mode of action appears to be as fellows: under diabetic condition, ROS production is increased, JNK is activated, and Akt activity is inhibited; TO-mediated LXR activation potently inhibits ROS production, increases anti-oxidative gene expressions, suppresses JNK activation, and restores Akt activity. Our data provide new evidence to support LXRs as promising therapeutic targets for anti-diabetic drug development.

In vivo sodium tungstate treatment prevents E-cadherin loss induced by diabetic serum in HK-2 cell line.

Diabetic nephropathy (DN) is characterized by interstitial inflammation and fibrosis, which is the result of chronic accumulation of extracellular matrix produced by activated fibroblasts in the renal tubulointerstitium. Renal proximal tubular epithelial cells (PTECs), through the process of epithelial-to-mesenchymal transition (EMT), are the source of fibroblasts within the interstitial space, and loss of E-cadherin has shown to be one of the earliest steps in this event. Here, we studied the effect of the anti-diabetic agent sodium tungstate (NaW) in the loss of E-cadherin induced by transforming growth factor (TGF) ß-1, the best-characterized in vitro EMT promoter, and serum from untreated or NaW-treated diabetic rats in HK-2 cell line, a model of human kidney PTEC. Our results showed that both TGFß-1 and serum from diabetic rat induced a similar reduction in E-cadherin expression. However, E-cadherin loss induced by TGFß-1 was not reversed by NaW, whereas sera from NaW-treated rats were able to protect HK-2 cells. Searching for soluble mediators of NaW effect, we compared secretion of TGFß isoforms and vascular endothelial growth factor (VEGF)-A, which have opposite actions on EMT. One millimolar NaW alone reduced secretion of both TGFß-1 and -2, and stimulated secretion of VEGF-A after 48 h. However, these patterns of secretion were not observed after diabetic rat serum treatment, suggesting that protection from E-cadherin loss by serum from NaW-treated diabetic rats originates from an indirect rather than a direct effect of this salt on HK-2 cells, via a mechanism independent of TGFß and VEGF-A functions.

Intestinal antigen-presenting cells in mucosal immune homeostasis: crosstalk between dendritic cells, macrophages and B-cells.

The intestinal immune system maintains a delicate balance between immunogenicity against invading pathogens and tolerance of the commensal microbiota. Inflammatory bowel disease (IBD) involves a breakdown in tolerance towards the microbiota. Dendritic cells (DC), macrophages (MΦ) and B-cells are known as professional antigen-presenting cells (APC) due to their specialization in presenting processed antigen to T-cells, and in turn shaping types of T-cell responses generated. Intestinal DC are migratory cells, unique in their ability to generate primary T-cell responses in mesenteric lymph nodes or Peyer's patches, whilst MΦ and B-cells contribute to polarization and differentiation of secondary T-cell responses in the gut lamina propria. The antigen-sampling function of gut DC and MΦ enables them to sample bacterial antigens from the gut lumen to determine types of T-cell responses generated. The primary function of intestinal B-cells involves their secretion of large amounts of immunoglobulin A, which in turn contributes to epithelial barrier function and limits immune responses towards to microbiota. Here, we review the role of all three types of APC in intestinal immunity, both in the steady state and in inflammation, and how these cells interact with one another, as well as with the intestinal microenvironment, to shape mucosal immune responses. We describe mechanisms of maintaining intestinal immune tolerance in the steady state but also inappropriate responses of APC to components of the gut microbiota that contribute to pathology in IBD.

Fractionation of subcellular membrane vesicles of epithelial and non-epithelial cells by OptiPrep™ density gradient ultracentrifugation.

Density gradient ultracentrifugation (DGUC) is widely used for physical isolation (enrichment rather than purification) of subcellular membrane vesicles. It has been a valuable tool to study specific subcellular localization and dynamic trafficking of proteins. While sucrose has been the main component of density gradients, several years ago, synthetic OptiPrep™ (iodixanol) began being used for separation of organelles due to its iso-osmotic property. Here, we describe a detailed protocol for density gradient fractionation of various mammalian subcellular vesicles, including endoplasmic reticulum (ER), Golgi apparatus, endosomes, and lipid rafts, as well as apical and basolateral membranes of polarized epithelial cells.