Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

The inflammasome components NLRP3 and ASC are cytosolic proteins, which upon sensing endotoxins or danger cues, form multimeric complexes to process interleukin (IL)-1ß for secretion. Here we found that antigen (Ag)-triggered degranulation of IgE-sensitized mast cells (MCs) was mediated by NLRP3 and ASC. IgE-Ag stimulated NEK7 and Pyk2 kinases in MCs to induce the deposition of NLRP3 and ASC on granules and form a distinct protein complex (granulosome) that chaperoned the granules to the cell surface. MCs deficient in NLRP3 or ASC did not form granulosomes, degranulated poorly in vitro and did not evoke systemic anaphylaxis in mice. IgE-Ag-triggered anaphylaxis was prevented by an NLRP3 inhibitor. In endotoxin-primed MCs, pro-IL-1ß was rapidly packaged into granules after IgE-Ag stimulation and processed within granule remnants by proteases after degranulation, causing lethal anaphylaxis in mice. During IgE-Ag-mediated degranulation of endotoxin-primed MCs, granulosomes promoted degranulation, combined with exteriorization and processing of IL-1ß, resulting in severe inflammation.

Recurrent infections drive persistent bladder dysfunction and pain via sensory nerve sprouting and mast cell activity.

Urinary tract infections (UTIs) account for almost 25% of infections in women. Many are recurrent (rUTI), with patients frequently experiencing chronic pelvic pain and urinary frequency despite clearance of bacteriuria after antibiotics. To elucidate the basis for these bacteria-independent bladder symptoms, we examined the bladders of patients with rUTI. We noticed a notable increase in neuropeptide content in the lamina propria and indications of enhanced nociceptive activity. In mice subjected to rUTI, we observed sensory nerve sprouting that was associated with nerve growth factor (NGF) produced by recruited monocytes and tissue-resident mast cells. Treatment of rUTI mice with an NGF-neutralizing antibody prevented sprouting and alleviated pelvic sensitivity, whereas instillation of native NGF into naïve mice bladders mimicked nerve sprouting and pain behavior. Nerve activation, pain, and urinary frequency were each linked to the presence of proximal mast cells, because mast cell deficiency or treatment with antagonists against receptors of several direct or indirect mast cell products was each effective therapeutically. Thus, our findings suggest that NGF-driven sensory sprouting in the bladder coupled with chronic mast cell activation represents an underlying mechanism driving bacteria-independent pain and voiding defects experienced by patients with rUTI.

A humanized mouse model to study mast cells mediated cutaneous adverse drug reactions.

Recently a G-protein-coupled receptor, MAS Related GPR Family Member X2 (MRGPRX2), was identified as a specific receptor on human mast cells responsible for IgE independent adverse drug reactions (ADR). Although a murine homologue, Mrgprb2, has been identified for this receptor, its affinity for many ADR-causing drugs is poor making it difficult to undertake in vivo studies to examine mechanisms of ADR and to develop therapeutic strategies. Here, we have created humanized mice capable of generating MRGPRX2-expressing human MCs allowing for the study of MRGPRX2 MCs-mediated ADR in vitro as well as in vivo. Humanized mice were generated by hydrodynamic-injection of plasmids expressing human GM-CSF and IL-3 into NOD-scid IL2R-γ-/- strain of mice that had been transplanted with human hematopoietic stem cells. These GM/IL-3 humice expressed high numbers of tissue human MCs but the MRGPRX2 receptor expressed in MCs were limited to few body sites including the skin. Importantly, large numbers of MRGPRX2-expressing human MCs could be cultured from the bone marrow of GM/IL-3 humice revealing these mice to be an important source of human MCs for in vitro studies of MRGPRX2-related MCs activities. When GM/IL-3 humice were exposed to known ADR causing contrast agents (meglumine and gadobutrol), the humice were found to experience anaphylaxis analogous to the clinical situation. Thus, GM/IL-3 humice represent a valuable model for investigating in vivo interactions of ADR-causing drugs and human MCs and their sequelae, and these mice are also a source of human MRGPRX2-expressing MCs for in vitro studies.

The Inflammasome Adaptor ASC Intrinsically Limits CD4+ T-Cell Proliferation to Help Maintain Intestinal Homeostasis.

The inflammasome is a multi-protein complex that mediates proteolytic cleavage and release of the pro-inflammatory cytokines IL-1ß and IL-18, and pyroptosis-a form of cell death induced by various pathogenic bacteria. Apoptosis-associated speck-like protein containing a CARD (ASC) has a pivotal role in inflammasome assembly and activation. While ASC function has been primarily implicated in innate immune cells, its contribution to lymphocyte biology is unclear. Here we report that ASC is constitutively expressed in naïve CD4+ T cells together with the inflammasome sensor NLRP3 and caspase-1. When adoptively transferred in immunocompromised Rag1-/- mice, Asc-/- CD4+ T cells exacerbate T-cell-mediated autoimmune colitis. Asc-/- CD4+ T cells exhibit a higher proliferative capacity in vitro than wild-type CD4+ T cells. The increased expansion of Asc-/- CD4+ T cells in vivo correlated with robust TCR-mediated activation, inflammatory activity, and higher metabolic profile toward a highly glycolytic phenotype. These findings identify ASC as a crucial intrinsic regulator of CD4+ T-cell expansion that serves to maintain intestinal homeostasis.

Calcineurin-mediated IL-2 production by CD11chighMHCII+ myeloid cells is crucial for intestinal immune homeostasis.

The intestinal immune system can respond to invading pathogens yet maintain immune tolerance to self-antigens and microbiota. Myeloid cells are central to these processes, but the signaling pathways that underlie tolerance versus inflammation are unclear. Here we show that mice lacking Calcineurin B in CD11chighMHCII+ cells (Cnb1 CD11c mice) spontaneously develop intestinal inflammation and are susceptible to induced colitis. In these mice, colitis is associated with expansion of T helper type 1 (Th1) and Th17 cell populations and a decrease in the number of FoxP3+ regulatory T (Treg) cells, and the pathology is linked to the inability of intestinal Cnb1-deficient CD11chighMHCII+ cells to express IL-2. Deleting IL-2 in CD11chighMHCII+ cells induces spontaneous colitis resembling human inflammatory bowel disease. Our findings identify that the calcineurin-NFAT-IL-2 pathway in myeloid cells is a critical regulator of intestinal homeostasis by influencing the balance of inflammatory and regulatory responses in the mouse intestine.

Calcineurin B in CD4+ T Cells Prevents Autoimmune Colitis by Negatively Regulating the JAK/STAT Pathway.

Calcineurin (Cn) is a protein phosphatase that regulates the activation of the nuclear factor of activated T-cells (NFAT) family of transcription factors, which are key regulators of T-cell development and function. Here, we generated a conditional Cnb1 mouse model in which Cnb1 was specifically deleted in CD4+ T cells (Cnb1CD4 mice) to delineate the role of the Cn-NFAT pathway in immune homeostasis of the intestine. The Cnb1CD4 mice developed severe, spontaneous colitis characterized at the molecular level by an increased T helper-1-cell response but an unaltered regulatory T-cell compartment. Antibiotic treatment ameliorated the intestinal inflammation observed in Cnb1CD4 mice, suggesting that the microbiota contributes to the onset of colitis. CD4+ T cells isolated from Cnb1CD4 mice produced high levels of IFNγ due to increased activation of the JAK2/STAT4 pathway induced by IL-12. Our data highlight that Cn signaling in CD4+ T cells is critical for intestinal immune homeostasis in part by inhibiting IL-12 responsiveness of CD4+ T cells.

Genetic and Pharmacological Dissection of the Role of Spleen Tyrosine Kinase (Syk) in Intestinal Inflammation and Immune Dysfunction in Inflammatory Bowel Diseases.

Background: The DNAX adaptor protein 12 (DAP12) is a transmembrane adaptor molecule that signals through the activation of Syk (Spleen Tyrosine Kinase) in myeloid cells. The purpose of this study is to investigate the role of DAP12 and Syk pathways in inflammatory bowel diseases (IBDs). Methods: DAP12 deficient and DAP12 transgenic, overexpressing an increased amount of DAP12, mice and Syk deficient mice in the C57/BL6 background were used for these studies. Colitis was induced by administering mice with dextran sulfate sodium (DSS), in drinking water, or 2,4,6-trinitrobenzene sulfonic acid (TNBS), by intrarectal enema. Results: Abundant expression of DAP12 and Syk was detected in colon samples obtained from Crohn's disease patients with expression restricted to immune cells infiltrating the colonic wall. In rodents development of DSS colitis as measured by assessing severity of wasting diseases, global colitis score,and macroscopic and histology scores was robustly attenuated in DAP12-/- and Syk-/- mice. In contrast, DAP12 overexpression resulted in a striking exacerbation of colon damage caused by DSS. Induction of colon expression of proinflammatory cytokines and chemokines in response to DSS administration was attenuated in DAP12-/- and Syk-/- mice, whereas opposite results were observed in DAP12 transgenic mice. Treating wild-type mice with a DAP-12 inhibitor or a Syk inhibitor caused a robust attenuation of colitis induced by DSS and TNBS. Conclusions: DAP12 and Syk are essential mediators in inflammation-driven immune dysfunction in murine colitides. Because DAP12 and Syk expression is upregulated in patients with active disease, present findings suggest a beneficial role for DAP12 and Syk inhibitors in IBD.

Highly specific blockade of CCR5 inhibits leukocyte trafficking and reduces mucosal inflammation in murine colitis.

Targeted disruption of leukocyte trafficking to the gut represents a promising approach for the treatment of inflammatory bowel diseases (IBDs). CCR5, the shared receptor for MIP1α and ß and RANTES, is expressed by multiple leukocytes. Here, we aimed to determine the role of CCR5 in mediating leukocyte trafficking in models of colitis, and evaluate the therapeutic potential of maraviroc, an orally active CCR5 antagonist used in the treatment of CCR5-tropic HIV. Acute and chronic colitis were induced by administration of DSS or TNBS to wild-type and CCR5(-/-) mice or adoptive transfer of splenic naïve CD4(+) T-cells from wild type or CCR5(-/-) mice into RAG-1(-/-). CCR5 gene ablation reduced the mucosal recruitment and activation of CCR5-bearing CD4(+) and CD11b(+) leukocytes, resulting in profound attenuation of signs and symptoms of inflammation in the TNBS and transfer models of colitis. In the DSS/TNBS colitis and in the transfer model, maraviroc attenuated development of intestinal inflammation by selectively reducing the recruitment of CCR5 bearing leukocytes. In summary, CCR5 regulates recruitment of blood leukocytes into the colon indicating that targeting CCR5 may offer therapeutic options in IBDs.

In vivo administration of ritonavir worsens intestinal damage caused by cyclooxygease inhibitors.

The protease inhibitor ritonavir is part of the highly active anti-retroviral therapy (HAART) successfully used in the treatment of human immunodeficiency virus (HIV)-1 infection. There is evidence that ritonavir alters intestinal permeability and induces damage to the small intestine. Because HIV infected patients taking HAART are at high risk for developing cardiovascular complications, there might be a need for the use of low dose of aspirin (ASA) to prevent ischemic events. Similarly, long term survival exposes HIV infected persons to detrimental interactions of ritonavir with non-steroidal anti-inflammatory drugs (NSAIDs). In the present work we tested whether ritonavir worsens intestinal injury caused by NSAIDs and ASA. C57BL6 mice were treated for 25 days with ritonavir and for a further 5 days with the combination of ritonavir plus ASA or ritonavir plus naproxen. In a second set of experiments C57BL6 mice were cotreated with ritonavir plus misoprostol, a PGE1 analog. We found that ritonavir administration caused intestinal damage and its co-administration with naproxen or ASA exacerbated the severity of injury and intestinal inflammation, as assessed by measuring haematocrit, MPO, mucosal levels of PGE2 and mRNA levels of iNOS, MCP-1 and VLA-1. Co-administration of misoprostol protected against intestinal damage induced by naproxen and ritonavir. In conclusion we demonstrated that ritonavir causes intestinal damage and that its association with NSAIDs or ASA worsens the damage caused by COX-inhibitors. Misoprostol rescues from intestinal damage caused by ritonavir. Further studies are need to clarify whether this observation has a clinical readout.

Solomonsterol A, a marine pregnane-X-receptor agonist, attenuates inflammation and immune dysfunction in a mouse model of arthritis.

In the present study we provide evidence that solomonsterol A, a selective pregnane X receptor (PXR) agonist isolated from the marine sponge Theonella swinhoei, exerts anti-inflammatory activity and attenuates systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. Solomonsterol A was effective in protecting against the development of arthritis induced by injecting transgenic mice harboring a humanized PXR, with anti-collagen antibodies (CAIA) with beneficial effects on joint histopathology and local inflammatory response reducing the expression of inflammatory markers (TNFα, IFNγ and IL-17 and chemokines MIP1α and RANTES) in draining lymph nodes. Solomonsterol A rescued mice from systemic inflammation were assessed by measuring arthritis score, CRP and cytokines in the blood. In summary, the present study provides a molecular basis for the regulation of systemic local and systemic immunity by PXR agonists.

FXR mediates a chromatin looping in the GR promoter thus promoting the resolution of colitis in rodents.

Glucocorticoids (GCs) are important endocrine regulators of a wide range of physiological processes ranging from immune function to glucose and lipid metabolism. For decades, synthetic glucocorticoids such as dexamethasone have been the cornerstone for the clinical treatment of inflammatory bowel diseases (IBD). A previous study has shown that farnesoid X receptor (FXR) enhances the transcription of NR3C1 gene, which encodes for human GR, by binding to a conserved FXR response element (FXRE) in the distal promoter of this gene. In the present study we demonstrate that FXR promotes the resolution of colitis in rodents by enhancing Gr gene transcription. We used the chromatin conformation capture (3C) assay to demonstrate that this FXRE is functional in mediating a head-to-tail chromatin looping, thus increasing Gr transcription efficiency. These findings underscore the importance of FXR/GR axis in the control of intestinal inflammation.

Dissociation of intestinal and hepatic activities of FXR and LXRα supports metabolic effects of terminal ileum interposition in rodents.

The farnesoid X receptor (FXR) and the liver x receptors (LXRs) are bile acid-activated receptors that are highly expressed in the enterohepatic tissues. The mechanisms that support the beneficial effects of bariatric surgery are only partially defined. We have investigated the effects of ileal interposition (IT), a surgical relocation of the distal ileum into the proximal jejunum, on FXR and LXRs in rats. Seven months after surgery, blood concentrations of total bile acids, taurocholic acid, an FXR ligand, and taurohyocholic acid, an LXRα ligand, were significantly increased by IT (P < 0.05). In contrast, liver and intestinal concentrations of conjugated and nonconjugated bile acids were decreased (P < 0.05). These changes were associated with a robust induction of FXR and FXR-regulated genes in the intestine, including Fgf15, a negative regulator of bile acid synthesis. IT repressed the liver expression of glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase (Pepck), two gluconeogenetic genes, along with the expression of LXRα and its target genes sterol regulatory element-binding protein (Srebp) 1c and fatty acid synthase (Fas) in the liver. Treating IT rats with chenodeoxycholic acid ameliorated insulin signaling in the liver. Whether confirmed in human settings, these results support the association of pharmacological therapies with bariatric surgeries to exploit the selective activation of intestinal nuclear receptors.

Probiotics VSL#3 protect against development of visceral pain in murine model of irritable bowel syndrome.

BACKGROUND AND AIMS: Irritable bowel syndrome (IBS) is linked to post-inflammatory and stress-correlated factors that cause changes in the perception of visceral events. Probiotic bacteria may be effective in treating IBS symptoms. Here, we have investigated whether early life administration of VSL#3, a mixture of 8 probiotic bacteria strains, protects against development of visceral hypersensitivity driven by neonatal maternal separation (NMS), a rat model of IBS. METHODS: Male NMS pups were treated orally with placebo or VSL#3 from days 3 to 60, while normal, not separated rats were used as controls. After 60 days from birth, perception of painful sensation induced by colorectal distension (CRD) was measured by assessing the abdominal withdrawal reflex (score 0-4). The colonic gene expression was assessed by using the Agilent Whole Rat Genome Oligo Microarrays platform and confirmed by real time PCR. RESULTS: NMS rats exhibited both hyperalgesia and allodynia when compared to control rats. VSL#3 had a potent analgesic effect on CRD-induced pain without changing the colorectal compliance. The microarray analysis demonstrated that NMS induces a robust change in the expression of subsets of genes (CCL2, NOS3, THP1, NTRK1, CCR2, BDRKRB1, IL-10, TNFRSF1B, TRPV4, CNR1 and OPRL1) involved in pain transmission and inflammation. TPH1, tryptophan hydroxylase 1, a validated target gene in IBS treatment, was markedly upregulated by NMS and this effect was reversed by VSL#3 intervention. CONCLUSIONS: Early life administration of VSL#3 reduces visceral pain perception in a model of IBS and resets colonic expression of subsets of genes mediating pain and inflammation. TRANSCRIPT PROFILING: Accession number of repository for expression microarray data is GSE38942 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38942).

Efficacy of the CCR5 antagonist maraviroc in reducing early, ritonavir-induced atherogenesis and advanced plaque progression in mice.

BACKGROUND: CCR5 plays an important role in atherosclerosis and ischemic cardiovascular diseases, as well as in HIV replication and diffusion. HIV infection is characterized by a high burden of cardiovascular diseases, particularly in subjects exposed to ritonavir-boosted protease inhibitors. Maraviroc, a CCR5 antagonist antiretroviral drug, might provide benefit for patients with M-tropic HIV infections at high risk for cardiovascular diseases. METHODS AND RESULTS: Exposure to maraviroc limits the evolution and associated systemic inflammation of ritonavir-induced atherosclerotic in ApoE(-/-) mice and inhibits plaques development in a late model of atherosclerosis in which dyslipidemia plays the main pathogenic role. In ritonavir-treated mice, maraviroc reduced plaque areas and macrophage infiltration; downregulated the local expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and interleukin-17A; and reduced tumor necrosis factor-α and RANTES (regulated on activation, normal T cell expressed, and secreted). Moreover, maraviroc counterregulated ritonavir-induced lipoatrophy and interlelukin-6 gene expression in epididymal fat, along with the splenic proinflammatory profile and expression of CD36 on blood monocytes. In the late model, maraviroc inhibited atherosclerotic progression by reducing macrophage infiltration and lowering the expression of adhesion molecules and RANTES inside the plaques. However, limited systemic inflammation was observed. CONCLUSIONS: In a mouse model of genetic dyslipidemia, maraviroc reduced the atherosclerotic progression by interfering with inflammatory cell recruitment into plaques. Moreover, in mice characterized by a general ritonavir-induced inflammation, maraviroc reversed the proinflammatory profile. Therefore, maraviroc could benefit HIV-positive patients with residual chronic inflammation who are at a high risk of acute coronary disease despite a suppressive antiretroviral therapy. To determine these benefits, large clinical studies are needed.

The bile acid sensor FXR is required for immune-regulatory activities of TLR-9 in intestinal inflammation.

BACKGROUND: Toll like receptors (TLRs) sense the intestinal microbiota and regulate the innate immune response. A dysregulation of TLRs function participates into intestinal inflammation. Farnesoid X Receptor (FXR) is a nuclear receptor and bile acid sensor highly expressed in entero-hepatic tissues. FXR regulates lipid metabolism and innate immunity. METHODOLOGY/PRINCIPAL FINDINGS: In this study we have investigated whether FXR gene expression/function in the intestine is modulated by TLRs. We found that in human monocytes activation of membrane TLRs (i.e. TLR2, 4, 5 and 6) downregulates, while activation of intracellular TLRs (i.e. TLR3, 7, 8 and 9) upregulates the expression of FXR and its target gene SHP, small heterodimer partner. This effect was TLR9-dependent and TNFα independent. Intestinal inflammation induced in mice by TNBS downregulates the intestinal expression of FXR in a TLR9-dependent manner. Protection against TNBS colitis by CpG, a TLR-9 ligand, was lost in FXR(-/-) mice. In contrast, activation of FXR rescued TLR9(-/-) and MyD88(-/-) mice from colitis. A putative IRF7 response element was detected in the FXR promoter and its functional characterization revealed that IRF7 is recruited on the FXR promoter under TLR9 stimulation. CONCLUSIONS/SIGNIFICANCE: Intestinal expression of FXR is selectively modulated by TLR9. In addition to its role in regulating type-I interferons and innate antiviral immunity, IRF-7 a TLR9-dependent factor, regulates the expression of FXR, linking microbiota-sensing receptors to host's immune and metabolic signaling.

Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non-steroidal anti-inflammatory drugs and aspirin in mice.

BACKGROUND AND PURPOSE: Low doses of aspirin (acetylsalicylic acid; ASA) and non-steroidal anti-inflammatory drugs (NSAIDs) increase the risk of gastrointestinal bleeding. GPBAR1 is a bile acid receptor expressed in the gastrointestinal tract. Here, we have investigated whether GPBAR1 was required for mucosal protection in models of gastrointestinal injury caused by ASA and NSAIDs. EXPERIMENTAL APPROCH: GPBAR1(+/+) and GPBAR1(-/-) mice were given ASA (10-50 mg.kg(-1)) or naproxen. Gastric and intestinal mucosal damage was assessed by measuring lesion scores. KEY RESULTS: Expression of GPBAR1, mRNA and protein, was detected in mouse stomach. Mice lacking GPBAR1 were more sensitive to gastric and intestinal injury caused by ASA and NSAIDs and exhibited a markedly reduced expression of cystathionine-γ-liase (CSE), cystathionine-ß-synthase (CBS) and endothelial NOS enzymes required for generation of H(2)S and NO, in the stomach. Treating GPBAR1(+/+) mice with two GPBAR1 agonists, ciprofloxacin and betulinic acid, rescued mice from gastric injury caused by ASA and NSAIDs. The protective effect of these agents was lost in GPBAR1(-/-) mice. Inhibition of CSE by DL-propargylglycine completely reversed protection afforded by ciprofloxacin in wild type mice, whereas treating mice with an H(2)S donor restored the protective effects of ciprofloxacin in GPBAR1(-/-) mice. Deletion of GPBAR1 altered the morphology of the small intestine and increased sensitivity to injury caused by naproxen. CONCLUSION AND IMPLICATIONS: GPBAR1 is essential to maintain gastric and intestinal mucosal integrity. GPBAR1 agonists protect against gastrointestinal injury caused by ASA and NSAIDs by a COX-independent mechanism.

Epigenetic modulation by methionine deficiency attenuates the potential for gastric cancer cell dissemination.

INTRODUCTION: Methionine dependency occurs frequently in tumor cells. Here we have investigated the effect of methionine deficiency on metastatic potential of gastric cancer cells in vitro and in vivo. MATERIALS AND METHODS: Model of peritoneal carcinomatosis and xenograft was generated by intraperitoneal or subcutaneous implantation of gastric cancer cells in NOD-SCID mice. In comparison to control medium, 3-day culture of MKN45, MKN74, and KATOIII cells in a methionine-deficient medium inhibited cell proliferation, increased the rate of cell apoptosis, and reduced cell adhesion and migration. In the xenograft model induced by implantation of MNK45 and MNK74 cells, two cycles of methionine-deficient diet reduced the tumor growth. Further on, a 10-day cycle of methionine-deficient diet reduced the number of peritoneal nodules in the model of peritoneal carcinomatosis induced by MKN45 cells injection. Finally, a microarray analysis of the methylation of promoter CpG islets demonstrated that methionine deficiency reduced the promoter methylation of E-cadherin whose expression was markedly increased in vivo and in vitro. RESULTS: In summary, we have provided evidence that a methionine-deficient diet modulates the growth of gastric tumor cells and in vitro deficiency of methionine increased apoptosis and decreased cellular adhesion and migration associated to epigenetic change of E-cadherin gene, in vivo and in vitro.

CCR5 Antagonism by Maraviroc Reduces the Potential for Gastric Cancer Cell Dissemination.

The chemokine (C-C motif) receptor 5 (CCR5) that belongs to the family of G protein-coupled receptors is exploited by macrophage tropic (R5) human immunodeficiency virus type 1 (HIV-1) to enter cells. Maraviroc, a small molecule CCR antagonist, is used as a part of combination antiretroviral therapy to treat persons infected by R5 HIV-1. CCR5 is expressed in various cancers, and its level of expression is a negative predictor of patients' survival in gastric cancers. Here, we report MKN45, MKN74, and KATOIII cells, three human gastric cancer cell lines with different stages of differentiation, which express CCR5 as detected by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR), and its ligand RANTES. In vitro experiments demonstrate that CCR5 antagonism reduces gastric cancer cell migration induced by macrophage inflammatory protein 1α (MIP-1α), MIP-1ß, and RANTES and adhesion to the ex-planted murine peritoneum. Administration of maraviroc from days 3 to 10 after MKN45 cell inoculation to severe combined immunodeficient (SCID) mice effectively reduced the extent of peritoneal disease and increased survival. Maraviroc treatment also reduced the tumor burden in a xenograft model. Gene expression and RT-PCR analyses revealed that CCR5 antagonism in vivo modulates the expression of genes known for their role in cancer growth including interleukin-10 receptor B; hepatocyte growth factor receptor (MET); the homolog of the atypical cadherin gene, FAT1; Nm23-H1; and lymphotoxin ß receptor. In summary, we have shown that CCR5 is mechanistically involved in dissemination of gastric cancer cells, suggesting that small molecule inhibitors of CCR5 might be exploited for their anticancer potential.

VSL#3 resets insulin signaling and protects against NASH and atherosclerosis in a model of genetic dyslipidemia and intestinal inflammation.

BACKGROUND: Signals generated by the inflammed intestine are thought to contribute to metabolic derangement. The intestinal microbiota contributes to instructing the immune system beyond the intestinal wall and its modulation is a potential target for treating systemic disorders. AIMS: To investigate the pathogenetic role of low grade intestinal inflammation in the development of steatohepatitis and atherosclerosis in a model of genetic dyslipidemia and to test the therapeutic potential of a probiotics intervention in protecting against development of these disorders. RESULTS: ApoE(-/-) mice were randomized to receive vehicle or VSL#3, a mixture of eight probiotics, at the dose of 20×10(9) colony-forming units/kg/day for three months alone or in combination with 0.2% of dextran sulfate sodium (DSS) in drinking water. Administering DSS to ApoE(-/-) mice failed to induce signs and symptoms of colitis but increased intestinal permeability to dextran FITC and, while had no effect on serum lipids, increased the blood levels of markers of liver injury and insulin resistance. DSS administration associated with low level inflammation of intestinal and mesenteric adipose tissues, caused liver histopathology features of steatohepatitis and severe atherosclerotic lesions in the aorta. These changes were prevented by VSL#3 intervention. Specifically, VSL#3 reversed insulin resistance, prevented development of histologic features of mesenteric adipose tissue inflammation, steatohepatitis and reduced the extent of aortic plaques. Conditioned media obtained from cultured probiotics caused the direct transactivation of peroxisome proliferator-activated receptor-γ, Farnesoid-X-receptors and vitamin D receptor. CONCLUSIONS: Low grade intestinal inflammation drives a transition from steatosis to steatohepatitis and worsens the severity of atherosclerosis in a genetic model of dyslipidemia. VSL#3 intervention modulates the expression of nuclear receptors, corrects for insulin resistance in liver and adipose tissues and protects against development of steatohepatitis and atherosclerosis.

Plakilactones from the marine sponge Plakinastrella mamillaris. Discovery of a new class of marine ligands of peroxisome proliferator-activated receptor γ.

In this paper we report the isolation and the molecular characterization of a new class of PPARγ ligands from the marine environment. Biochemical characterization of a library of 13 oxygenated polyketides isolated from the marine sponge Plakinastrella mamillaris allowed the discovery of gracilioether B and plakilactone C as selective PPARγ ligands in transactivation assays. Both agents covalently bind to the PPARγ ligand binding domain through a Michael addition reaction involving a protein cysteine residue and the α,ß-unsaturated ketone in their side chains. Additionally, gracilioether C is a noncovalent agonist for PPARγ, and methyl esters 1 and 2 are noncovalent antagonists. Structural requirements for the interaction of these agents within the PPARγ ligand binding domain were obtained by docking analysis. Gracilioether B and plakilactone C regulate the expression of PPARγ-dependent genes in the liver and inhibit the generation of inflammatory mediators by macrophages.