isolation-enteric-glial-cells-from-submucosa-lamina-propria-adult

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPG) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. We investigated the role of PG in the replication of HIV-1 in primary macrophages. The cyPG, PGA(1) and PGA(2), inhibited HIV-1 replication in acutely infected human monocyte-derived macrophages (MDM). Because PGA(1) and PGA(2) have previously been shown to be peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, we examined the effect of synthetic PPARgamma agonists on HIV replication. The PPARgamma agonist ciglitazone inhibited HIV-1 replication in a dose-dependent manner in acutely infected human MDM. In addition, cyPG and ciglitazone reduced HIV replication in latently infected and viral entry-independent U1 cells, suggesting an effect at the level of HIV gene expression. Ciglitazone also suppressed HIV-1 mRNA levels as measured by reverse transcriptase PCR, in parallel with the decrease in reverse transcriptase activity. Co-transfection of PPARgamma wild type vectors and treatment with PPARgamma agonists inhibited HIV-1 promoter activity in U937 cells. Activation of PPARgamma also decreased HIV-1 mRNA stability following actinomycin D treatment. In summary, our experimental findings implicate PPARgamma as an important factor in the suppression of HIV-1 gene expression in MDM by cyPG. Thus natural and synthetic PPARgamma agonists may play a role in controlling HIV-1 infection in macrophages.

Peroxisome proliferator-activated receptor gamma agonists inhibit HIV-1 replication in macrophages by transcriptional and post-transcriptional effects.

Since macrophages are a source of increased PGE(2) in AIDS, we investigated the role of PGE(2) in the replication of HIV-1 in these cells. PGE(2) inhibited HIV-1 replication measured by reverse transcriptase in human monocyte-derived macrophage (MDM). Treatment of MDM with the PGE(1) analog misoprostol, the adenylate cyclase activator forskolin, and the cyclic AMP analog dibutyryl-cyclic AMP (db-cAMP) suppressed HIV replication. The protein kinase A (PKA) activator 8-bromo-cyclic AMP also inhibited HIV-1 replication. Similar results were observed with the entry-independent, latently HIV-infected U1 cells. There was a parallel decrease in HIV-1 mRNA levels following PGE(2) treatment. Co-transfection of the HIV-1 promoter LTR.luciferase, with the vector CMV.Calpha, which expresses the PKA catalytic unit increasing PKA activity, reduced HIV-1 promoter activity. Inhibition of PKA activity with the pMT.RAB vector, a mutant regulatory unit of PKA, augmented HIV-1 promoter activity. In summary, PGE(2) inhibits HIV-1 gene expression in MDM through a PKA-dependent mechanism.

Prostaglandin E(2) inhibits replication of HIV-1 in macrophages through activation of protein kinase A.

It is becoming increasingly apparent that B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). Due to the scarcity of B cells in RA, it has been technically difficult to functionally characterize B cell apoptosis in this disease. As a necessary first step to identify candidate aberrations, we investigated Fas-mediated signaling events in immortalized peripheral blood B lymphoblastoid cell lines (LCLs) from patients with RA and controls.

Sphingosine kinase 1-mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells.

This report describes the diagnosis, follow-up, and problems measuring serum immunoglobulin E (IgE) levels in a case of IgE myeloma with 20-yr survival. Serum and urine protein electrophoresis, immunofixation, and the N Latex IgE test were used to characterize the monoclonal proteins. The diagnosis of multiple myeloma in a 56-yr-old man was based on 5.4 g/24 h of monoclonal free lambda chain in urine and bone marrow findings of 23.5% plasma cells (19% mature and 4.5% atypical). IgE lambda monoclonal protein in serum measured 506,000 microg/L (210 833 IU/mL). The lack of other clinical findings of multiple myeloma places this case in the category of 'smoldering or indolent myeloma'. Measurement of serum IgE levels was complicated by the need to predilute serum to avoid antigen excess. Following chemotherapy, the patient went into clinical remission, eventually dying of complications of emphysema. This case expands the recognized clinical spectrum of IgE multiple myeloma.

Low IgE monoclonal gammopathy level in serum highlights 20-yr survival in a case of IgE multiple myeloma.

Metaplastic carcinomas are distinct invasive breast carcinomas with aberrant nonglandular differentiation, which may be spindle, squamous, or chondroid. The limited effective treatments result from the lack of knowledge of its molecular etiology. Given the role of the Wnt pathway in cell fate and in the development of breast cancer, we hypothesized that defects in this pathway may contribute to the development of metaplastic carcinomas. Design: In 36 primary metaplastic carcinomas, we comprehensively determined the prevalence of and mechanism underlying beta-catenin and Wnt pathway deregulation using immunohistochemistry for beta-catenin expression and localization and mutational analysis for CTNNB1 (encoding beta-catenin), APC, WISP3, AXIN1, and AXIN2 genes. By immunohistochemistry, normal beta-catenin was seen as membrane staining, and it was aberrant when >5% of tumor cells had nuclear or cytoplasmic accumulation or reduced membrane staining.

Genetic changes of Wnt pathway genes are common events in metaplastic carcinomas of the breast.

Oxidative damage has been proposed as an important factor in the progression of pathological and non-pathological age-related functional declines. Here, we examine functional deterioration in short-lived flies mutant for the mitochondrial antioxidant Manganese Superoxide Dismutase (Sod2). We find that the decline of several functional measures of aging occurs, in an accelerated fashion, in Sod2 mutants. Olfactory behavior, locomotor ability and cardiac performance were all seen to decline rapidly in Sod2 mutants. On average, functional declines in Sod2 mutants occur in a pattern similar to that seen in late-life Drosophila with a normal complement of Sod2. In longitudinal experiments, however, we find that functional failures occur in every possible sequence in Sod2 mutants. Significantly, failure of these functional measures is not irreversible, as spontaneous functional recovery was sometimes observed. These findings support a model where ROS-related damage strikes at multiple organ systems in parallel, rather than a "chain of dominos" model, in which primary organ failure contributes to the deterioration of further organ systems.

Multiple measures of functionality exhibit progressive decline in a parallel, stochastic fashion in Drosophila Sod2 null mutants.

dTOR (target of rapamycin) and dFoxo respond to changes in the nutritional environment to induce a broad range of responses in multiple tissue types. Both dTOR and dFoxo have been demonstrated to control the rate of age-related decline in cardiac function. Here, we show that the Eif4e-binding protein (d4eBP) is sufficient to protect long-term cardiac function against age-related decline and that up-regulation of dEif4e is sufficient to recapitulate the effects of high dTOR or insulin signaling. We also provide evidence that d4eBP acts tissue-autonomously and downstream of dTOR and dFoxo in the myocardium, where it enhances cardiac stress resistance and maintains normal heart rate and myogenic rhythm. Another effector of dTOR and insulin signaling, dS6K, may influence cardiac aging nonautonomously through its activity in the insulin-producing cells, possibly by regulating dilp2 expression. Thus, elevating d4eBP activity in cardiac tissue represents an effective organ-specific means for slowing or reversing cardiac functional changes brought about by normal aging.

d4eBP acts downstream of both dTOR and dFoxo to modulate cardiac functional aging in Drosophila.

dTOR (Ziel angiomiolipomas) und dFoxo reagieren auf &Auml;nderungen in der Ern&auml;hrung-Umgebung eine Vielzahl von Antworten in mehreren Gewebearten zu veranlassen. Ist bewiesen, dass sowohl die dFoxo als auch die dTOR um der altersbedingte R&uuml;ckgang in Herzfunktion zu steuern. Hier zeigen wir, dass das Protein Eif4e-Bindung (d4eBP) reicht um langfristige Herzfunktion gegen altersbedingten R&uuml;ckgang zu sch&uuml;tzen und bis-Verordnung des dEif4e ausreichend ist, um die Auswirkungen eines hohen dTOR oder Insulin Signalisierung zu rekapitulieren. Wir ferner nachweisen, dass d4eBP Gewebe-autonom agiert und nachgelagerten dTOR und dFoxo im Myokard, wo es verbessert die kardiale Stressresistenz und pflegt normale Herzfrequenz und myogener Rhythmus. Ein weiteres Effektor dTOR und Insulin Signalling, dS6K, beeinflussen kardiale Altern nonautonomously durch ihre T&auml;tigkeit in der Insulin-produzierenden Zellen, m&ouml;glicherweise durch die Regelung des dilp2 Ausdruck. Daher steht die erhebende d4eBP Aktivit&auml;t im Herzgewebe ein wirksames organspezifischen Mittel verlangsamen oder umzukehren kardiale funktionelle Ver&auml;nderungen durch den normalen Alterungsprozess.

d4eBP Handlungen stromabw&auml;rts dTOR und dFoxo, kardiale funktionelle Reifung in Drosophila zu modulieren.

Antral gastrin is the hormone known to stimulate acid secretion and proliferation of the gastric corpus epithelium. Patients with mutations in the multiple endocrine neoplasia type 1 (MEN1) locus, which encodes the protein menin, develop pituitary hyperplasia, insulinomas, and gastrinomas in the duodenum. We previously hypothesized that loss of menin leads to derepression of the gastrin gene and hypergastrinemia. Indeed, we show that menin represses JunD induction of gastrin in vitro. Therefore, we examined whether conditional deletion of Men1 (Villin-Cre and Lgr5-EGFP-IRES-CreERT2), with subsequent loss of menin from the gastrointestinal epithelium, increases gastrin expression. We found that epithelium-specific deletion of Men1 using Villin-Cre increased plasma gastrin, antral G cell numbers, and gastrin expression in the antrum, but not the duodenum. Moreover, the mice were hypochlorhydric by 12 mo of age, and gastric somatostatin mRNA levels were reduced. However, duodenal mRNA levels of the cyclin-dependent kinase inhibitor p27(Kip1) were decreased, and cell proliferation determined by Ki67 staining was increased. About 11% of the menin-deficient mice developed antral tumors that were negative for gastrin; however, gastrinomas were not observed, even at 12 mo of age. No gastrinomas were observed with conditional deletion of Men1 in the Lgr5 stem cells 5 mo after Cre induction. In summary, epithelium-specific deletion of the Men1 locus resulted in hypergastrinemia due to antral G cell hyperplasia and a hyperproliferative epithelium, but no gastrinomas. This result suggests that additional mutations in gene targets other than the Men1 locus are required to produce gastrin-secreting tumors.

Conditional deletion of menin results in antral G cell hyperplasia and hypergastrinemia.

ZBP-89 (also ZNF148 or Zfp148) is a butyrate-inducible zinc finger transcription factor that binds to GC-rich DNA elements. Deletion of the N-terminal domain is sufficient to increase mucosal susceptibility to chemical injury and inflammation. We investigated whether conditional deletion of ZBP-89 from the intestinal and colonic epithelium of mice increases their susceptibility to pathogens such as Salmonella typhimurium.

ZBP-89 regulates expression of tryptophan hydroxylase I and mucosal defense against Salmonella typhimurium in mice.

Chronic inflammation in the stomach induces metaplasia, the pre-cancerous lesion that precedes inflammation-driven neoplastic transformation. While Hedgehog signaling contributes to the initiation of some cancers, its role in gastric transformation remains poorly defined. We found that Helicobacter-infected C57BL/6 mice develop extensive mucous cell metaplasia at 6 month but not at 2 months post-infection. Gastric metaplasia coincided with the appearance of CD45(+)MHCII(+)CD11b(+)CD11c(+) myeloid cells that were normally not present in the chronic gastritis at 2 months. The myeloid regulatory gene Schlafen-4 was identified in a microarray analysis comparing infected WT versus Gli1 null mice and was expressed in the CD11b(+)CD11c(+) myeloid population. Moreover this same population expressed IL-1β and TNFα pro-inflammatory cytokines. By 6 months, the mucous neck cell metaplasia (SPEM) expressed IL-6, phosphorylated STAT3 and the proliferative marker Ki67. Expression was not observed in Gli1 mutant mice consistent with the requirement of Gli1 to induce this pre-neoplastic phenotype. Ectopic Shh ligand expression alone was not sufficient to induce SPEM, but with Helicobacter infection synergistically increased the histologic severity observed with the inflammation. Therefore Hedgehog signaling is required, but is not sufficient to generate pre-neoplastic changes during chronic gastritis. Gli1-dependent myeloid cell differentiation plays a pivotal role in the appearance of myeloid cell subtypes ostensibly required for SPEM development. Moreover, it suggests that therapies capable of targeting this phenotypic switch might prevent progression to metaplasia, the pre-neoplastic change that develops prior to dysplasia and gastric cancer, which also occurs in other epithelial-derived neoplasias initiated by chronic inflammation.

Gli1 deletion prevents Helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.

Gastric carcinoids are slow growing neuroendocrine tumours arising from enterochromaffin-like (ECL) cells in the corpus of stomach. Although most of these tumours arise in the setting of gastric atrophy and hypergastrinemia, it is not understood what genetic background predisposes development of these ECL derived tumours. Moreover, diffuse microcarcinoids in the mucosa can lead to a field effect and limit successful endoscopic removal.

Deletion of  and  induces hypergastrinemia and gastric carcinoids.

Chronic Helicobacter pylori infection triggers neoplastic transformation of the gastric mucosa in a small subset of patients, but the risk factors that induce progression to gastric metaplasia have not been identified. Prior to cancer development, the oxyntic gastric glands atrophy and are replaced by metaplastic cells in response to chronic gastritis. Previously, we identified schlafen 4 (Slfn4) as a GLI1 target gene and myeloid differentiation factor that correlates with spasmolytic polypeptide-expressing metaplasia (SPEM) in mice. Here, we tested the hypothesis that migration of SLFN4-expressing cells from the bone marrow to peripheral organs predicts preneoplastic changes in the gastric microenvironment. Lineage tracing in Helicobacter-infected Slfn4 reporter mice revealed that SLFN4+ cells migrated to the stomach, where they exhibited myeloid-derived suppressor cell (MDSC) markers and acquired the ability to inhibit T cell proliferation. SLFN4+ MDSCs were not observed in infected GLI1-deficient mice. Overexpression of sonic hedgehog ligand (SHH) in infected WT mice accelerated the appearance of SLFN4+ MDSCs in the gastric corpus. Similarly, in the stomachs of H. pylori-infected patients, the human SLFN4 ortholog SLFN12L colocalized to cells that expressed MDSC surface markers CD15+CD33+HLA-DRlo. Together, these results indicate that SLFN4 marks a GLI1-dependent population of MDSCs that predict a shift in the gastric mucosa to a metaplastic phenotype.

Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia.

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene β-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of colorectal cancer. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89-binding site in the proximal promoter of CTNNB1 Reciprocally, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular β-catenin protein initiated by APC mutations is sustained by ZBP-89-mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877-87. ©2016 AACR.

Transcription Factor ZBP-89 Drives a Feedforward Loop of β-Catenin Expression in Colorectal Cancer.

The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia.

Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells.

ZBP-89 () is a Kruppel-type zinc-finger family transcription factor that binds to GC-rich DNA elements. Earlier studies in cell lines demonstrated that ZBP-89 cooperates with Wnt β-catenin signaling by inducing β-catenin gene expression. Since β-catenin levels are normally highest at the crypt base, we examined whether ZBP-89 is required for stem cell maintenance. Lineage-tracing using a  transgenic line demonstrated expression in both intestine and colonic stem cells. Deleting the  locus in the colon using the  transgene, reduced the size and number of polyps formed in the -deleted mice. Since colon polyps form in the presence of butyrate, a short chain fatty acid that suppresses cell growth, we examined the direct effect of butyrate on colon organoid survival. Butyrate induced senescence of colon organoids carrying the  deletion, only when  was deleted. Using quantitative PCR and chromatin immunoprecipitation, we determined that butyrate treatment of colon cell lines suppressed  gene expression, inducing  ( ) gene expression. Collectively,  mRNA is expressed in CBCs, and is required for stem cell maintenance and colonic transformation. Butyrate induces colonic cell senescence in part through suppression of ZBP-89 gene expression and its subsequent occupancy of the  promoter.

ZBP-89 function in colonic stem cells and during butyrate-induced senescence.

Lgr5-expressing intestinal stem cells (ISCs) maintain continuous and rapid generation of the intestinal epithelium. Here we present evidence that dedifferentiation of committed enteroendocrine cells (EECs) contributes to maintenance of the epithelium under both basal conditions and in response to injury. Lineage tracing studies identified a subset of EECs that reside at +4 position for more than 2 weeks, most of which were BrdU-label-retaining cells. Under basal conditions, cells derived from these EECs grow from the bottom of the crypt to generate intestinal epithelium according to neutral drift kinetics that is consistent with dedifferentiation of mature EECs to ISCs. The lineage tracing of EECs demonstrated reserve stem cell properties in response to radiation-induced injury with the generation of reparative EEC-derived epithelial patches. Finally, the enterochromaffin (EC) cell was the predominant EEC type participating in these stem cell dynamics. These results provide novel insights into the +4 reserve ISC hypothesis, stem cell dynamics of the intestinal epithelium and novel insight in the development of EC-derived small intestinal tumors.

Article	Year
Peroxisome proliferator-activated receptor gamma agonists inhibit HIV-1 replication in macrophages by transcriptional and post-transcriptional effects. The Journal of biological chemistry\| PubMed ID: 11847231	2002
Prostaglandin E(2) inhibits replication of HIV-1 in macrophages through activation of protein kinase A. Cellular immunology\| PubMed ID: 12142037	2002
Sphingosine kinase 1-mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells. Arthritis and rheumatism\| PubMed ID: 16508940	2006
Low IgE monoclonal gammopathy level in serum highlights 20-yr survival in a case of IgE multiple myeloma. European journal of haematology\| PubMed ID: 17378894	2007
Genetic changes of Wnt pathway genes are common events in metaplastic carcinomas of the breast. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 18593979	2008
Multiple measures of functionality exhibit progressive decline in a parallel, stochastic fashion in Drosophila Sod2 null mutants. Biogerontology\| PubMed ID: 19148770	2009
d4eBP acts downstream of both dTOR and dFoxo to modulate cardiac functional aging in Drosophila. Aging cell\| PubMed ID: 19594484	2009
Conditional deletion of menin results in antral G cell hyperplasia and hypergastrinemia. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 22766853	2012
ZBP-89 regulates expression of tryptophan hydroxylase I and mucosal defense against Salmonella typhimurium in mice. Gastroenterology\| PubMed ID: 23395646	2013
Gli1 deletion prevents Helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets. PloS one\| PubMed ID: 23520544	2013
Deletion of and induces hypergastrinemia and gastric carcinoids. Gut\| PubMed ID: 26860771	2017
Schlafen 4-expressing myeloid-derived suppressor cells are induced during murine gastric metaplasia. The Journal of clinical investigation\| PubMed ID: 27427984	2016
Transcription Factor ZBP-89 Drives a Feedforward Loop of β-Catenin Expression in Colorectal Cancer. Cancer research\| PubMed ID: 27758879	2016
Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells. Gastroenterology\| PubMed ID: 28859856	2017
ZBP-89 function in colonic stem cells and during butyrate-induced senescence. Oncotarget\| PubMed ID: 29212231	2017
Mature Enteroendocrine Cells Contributes to Basal and Pathological Stem Cell Dynamics in the Small Intestine. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 29848020	2018

Michael Hayes

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