Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis.

Commensal gut microflora and dietary fiber protect against colonic inflammation and colon cancer through unknown targets. Butyrate, a bacterial product from fermentation of dietary fiber in the colon, has been implicated in this process. GPR109A (encoded by Niacr1) is a receptor for butyrate in the colon. GPR109A is also a receptor for niacin, which is also produced by gut microbiota and suppresses intestinal inflammation. Here we showed that Gpr109a signaling promoted anti-inflammatory properties in colonic macrophages and dendritic cells and enabled them to induce differentiation of Treg cells and IL-10-producing T cells. Moreover, Gpr109a was essential for butyrate-mediated induction of IL-18 in colonic epithelium. Consequently, Niacr1(-/-) mice were susceptible to development of colonic inflammation and colon cancer. Niacin, a pharmacological Gpr109a agonist, suppressed colitis and colon cancer in a Gpr109a-dependent manner. Thus, Gpr10a has an essential role in mediating the beneficial effects of gut microbiota and dietary fiber in colon.

Transgelin increases metastatic potential of colorectal cancer cells in vivo and alters expression of genes involved in cell motility.

BACKGROUND: Transgelin is an actin-binding protein that promotes motility in normal cells. Although the role of transgelin in cancer is controversial, a number of studies have shown that elevated levels correlate with aggressive tumor behavior, advanced stage, and poor prognosis. Here we sought to determine the role of transgelin more directly by determining whether experimental manipulation of transgelin levels in colorectal cancer (CRC) cells led to changes in metastatic potential in vivo. METHODS: Isogenic CRC cell lines that differ in transgelin expression were characterized using in vitro assays of growth and invasiveness and a mouse tail vein assay of experimental metastasis. Downstream effects of transgelin overexpression were investigated by gene expression profiling and quantitative PCR. RESULTS: Stable overexpression of transgelin in RKO cells, which have low endogenous levels, led to increased invasiveness, growth at low density, and growth in soft agar. Overexpression also led to an increase in the number and size of lung metastases in the mouse tail vein injection model. Similarly, attenuation of transgelin expression in HCT116 cells, which have high endogenous levels, decreased metastases in the same model. Investigation of mRNA expression patterns showed that transgelin overexpression altered the levels of approximately 250 other transcripts, with over-representation of genes that affect function of actin or other cytoskeletal proteins. Changes included increases in HOOK1, SDCCAG8, ENAH/Mena, and TNS1 and decreases in EMB, BCL11B, and PTPRD. CONCLUSIONS: Increases or decreases in transgelin levels have reciprocal effects on tumor cell behavior, with higher expression promoting metastasis. Chronic overexpression influences steady-state levels of mRNAs for metastasis-related genes.

Constitutively CD40-activated B cells regulate CD8 T cell inflammatory response by IL-10 induction.

B cells are exposed to high levels of CD40 ligand (CD40L, CD154) in chronic inflammatory diseases. In addition, B cells expressing both CD40 and CD40L have been identified in human diseases such as autoimmune diseases and lymphoma. However, how such constitutively CD40-activated B cells under inflammation may impact on T cell response remains unknown. Using a mouse model in which B cells express a CD40L transgene (CD40LTg) and receive autocrine CD40/CD40L signaling, we show that CD40LTg B cells stimulated memory-like CD4 and CD8 T cells to express IL-10. This IL-10 expression by CD8 T cells was dependent on IFN-I and programmed cell death protein 1, and was critical for CD8 T cells to counterregulate their overactivation. Furthermore, adoptive transfer of naive CD8 T cells in RAG-1(-/-) mice normally induces colitis in association with IL-17 and IFN-γ cytokine production. Using this model, we show that adoptive cotransfer of CD40LTg B cells, but not wild-type B cells, significantly reduced IL-17 response and regulated colitis in association with IL-10 induction in CD8 T cells. Thus, B cells expressing CD40L can be a therapeutic goal to regulate inflammatory CD8 T cell response by IL-10 induction.

Engineering DNA nanoparticles as immunomodulatory reagents that activate regulatory T cells.

Nanoparticles containing DNA complexed with the cationic polymer polyethylenimine are efficient vehicles to transduce DNA into cells and organisms. DNA/polyethylenimine nanoparticles (DNPs) also elicit rapid and systemic release of proinflammatory cytokines that promote antitumor immunity. In this study, we report that DNPs possess previously unrecognized immunomodulatory attributes due to rapid upregulation of IDO enzyme activity in lymphoid tissues of mice. IDO induction in response to DNP treatment caused dendritic cells and regulatory T cells (Tregs) to acquire potent regulatory phenotypes. As expected, DNP treatment stimulated rapid increase in serum levels of IFN type I (IFN-αß) and II (IFN-γ), which are both potent IDO inducers. IDO-mediated Treg activation was dependent on IFN type I receptor signaling, whereas IFN-γ receptor signaling was not essential for this response. Moreover, systemic IFN-γ release was caused by TLR9-dependent activation of NK cells, whereas TLR9 signaling was not required for IFN-αß release. Accordingly, DNPs lacking immunostimulatory TLR9 ligands in DNA stimulated IFN-αß production, induced IDO, and promoted regulatory outcomes, but did not stimulate potentially toxic, systemic release of IFN-γ. DNP treatment to induce IDO and activate Tregs blocked Ag-specific T cell responses elicited in vivo following immunization and suppressed joint pathology in a model of immune-mediated arthritis. Thus, DNPs lacking TLR9 ligands may be safe and effective reagents to protect healthy tissues from immune-mediated destruction in clinical hyperimmune syndromes.

NF-κB directly regulates Fas transcription to modulate Fas-mediated apoptosis and tumor suppression.

Fas is a member of the death receptor family. Stimulation of Fas leads to induction of apoptotic signals, such as caspase 8 activation, as well as "non-apoptotic" cellular responses, notably NF-κB activation. Convincing experimental data have identified NF-κB as a critical promoter of cancer development, creating a solid rationale for the development of antitumor therapy that suppresses NF-κB activity. On the other hand, compelling data have also shown that NF-κB activity enhances tumor cell sensitivity to apoptosis and senescence. Furthermore, although stimulation of Fas activates NF-κB, the function of NF-κB in the Fas-mediated apoptosis pathway remains largely undefined. In this study, we observed that deficiency of either Fas or FasL resulted in significantly increased incidence of 3-methylcholanthrene-induced spontaneous sarcoma development in mice. Furthermore, Fas-deficient mice also exhibited significantly greater incidence of azoxymethane and dextran sodium sulfate-induced colon carcinoma. In addition, human colorectal cancer patients with high Fas protein in their tumor cells had a longer time before recurrence occurred. Engagement of Fas with FasL triggered NF-κB activation. Interestingly, canonical NF-κB was found to directly bind to the FAS promoter. Blocking canonical NF-κB activation diminished Fas expression, whereas blocking alternate NF-κB increased Fas expression in human carcinoma cells. Moreover, although canonical NF-κB protected mouse embryo fibroblast (MEF) cells from TNFα-induced apoptosis, knocking out p65 diminished Fas expression in MEF cells, resulting in inhibition of FasL-induced caspase 8 activation and apoptosis. In contrast, knocking out p52 increased Fas expression in MEF cells. Our observations suggest that canonical NF-κB is a Fas transcription activator and alternate NF-κB is a Fas transcription repressor, and Fas functions as a suppressor of spontaneous sarcoma and colon carcinoma.

Functional alteration of PARL contributes to mitochondrial dysregulation in Parkinson's disease.

Molecular genetics has linked mitochondrial dysfunction to the pathogenesis of Parkinson's disease by the discovery of rare, inherited mutations in gene products that associate with the mitochondria. Mutations in PTEN-induced kinase-1 (PINK1), which encodes a mitochondrial kinase, and PARKIN, encoding an E3 ubiquitin ligase, are the most frequent causes of recessive Parkinson's disease. Recent functional studies have revealed that PINK1 recruits PARKIN to mitochondria to initiate mitophagy, an important autophagic quality control mechanism that rids the cell of damaged mitochondria. PINK1 is post-translationally processed into a cleaved form whose levels are tightly regulated, although the significance of this processing is unknown. Here we demonstrate that the mitochondrial protease presenilin-associated rhomboid-like (PARL) can affect the proteolytic processing of PINK1 and that normal PINK1 localization and stability requires PARL's catalytic activity. PARL deficiency impairs PARKIN recruitment to mitochondria, suggesting PINK1's processing and localization are important in determining its interaction with PARKIN. We sequenced the PARL gene in Parkinson's disease patients and discovered a novel missense mutation in a functional domain of PARL's N-terminus. This PARL mutant is not sufficient to rescue PARKIN recruitment, suggesting that impaired mitophagy may be an underlying mechanism of disease pathogenesis in patients with PARL mutations.

Connexin 43 signaling enhances the generation of Foxp3+ regulatory T cells.

Despite their importance for the functioning of the immune system, thymic development and peripheral maintenance of Foxp3(+) regulatory T (T(R)) cells are poorly understood. We have found that connexin 43 (Cx43), expressed by thymic T(R) cells progenitors, supports T(R) development. Mice with deletion of the Cx43 gene induced in T cells produce only few T(R) cells and had increased proportion of activated T cells in the lymph nodes, suggesting impaired peripheral tolerance. Reduction of the T(R) cell numbers was accompanied by increased presence of CD4(+)CD25(+)GITR(+)Foxp3(-) T cells, which did not produce inflammatory cytokines and lost suppressor function. These results strongly argue that we have discovered a novel signaling pathway, controlled by Cx43, that enhances the generation of T(R) cells. We propose that a possible mechanism of Cx43 activity is by regulating Foxp3 expression in T(R) lineage cells.

Primary leiomyosarcomas of the gastrointestinal tract in the post-gastrointestinal stromal tumor era.

Most mesenchymal neoplasms of the gastrointestinal tract are currently classified as gastrointestinal stromal tumors (GIST). Gastrointestinal stromal tumors are diagnosed by immunopositivity for CD117, CD34, and DOG1.1, with or without molecular analyses. According to the World Health Organization classification, the diagnosis of primary leiomyosarcomas of the gastrointestinal tract is so rare that there are no significant data on demographic, clinical, or gross features of this tumor. A comprehensive literature search was performed to identify gastrointestinal leiomyosarcomas. Searches were limited to the past 12 years because definitive tools to differentiate leiomyosarcomas from GIST were introduced in the late 1990s. Cases were included only if convincing data were presented. Six cases of esophageal leiomyosarcoma and 5 cases of gastric leiomyosarcoma were confirmed. Furthermore, 26 cases of leiomyosarcoma of the small bowel, 11 cases of the colon, and 8 cases arising in the rectum were identified. Finally, 28 cases of infantile and adolescent leiomyosarcoma were reviewed. Although survival analysis is precluded by small case numbers and limited survival data availability, the trend identifies that increased size and mitotic activity portends to a worse prognosis in small bowel leiomyosarcomas. Colonic leiomyosarcomas appear to be aggressive tumors, regardless of tumor size and mitotic activity. Rectal leiomyosarcomas present as smaller tumors with favorable prognosis. Leiomyosarcomas in post-GIST era are rare tumors of the gastrointestinal tract with distinctive clinicopathologic characteristics. Owing to different treatment options, it is necessary to accurately differentiate these from GIST, using a combination of histologic appearance, presence of smooth muscle antigens, and absence of specific GIST immunomarkers.

Calretinin immunostaining as an adjunct in the diagnosis of Hirschsprung disease.

Historically, the diagnosis of Hirschsprung disease was made by evaluating multiple hematoxylin and eosin-stained slides and performing acetylcholinesterase histochemical staining. Recently, calretinin immunohistochemical staining has been reported and found to be superior to acetylcholinesterase staining in the confirmation of aganglionosis. We retrieved tissue blocks from 23 patients with proven Hirschsprung disease from the archives of the Medical College of Georgia. In addition, we selected 23 control patients with ganglion cells. All cases were stained with calretinin, and the presence or absence of both intrinsic nerve fibers (INFs) and ganglion cells was scored by 4 pathologists with fairly strong agreement (κ = 0.858). All cases of proven Hirschsprung disease were negative for INFs. Eighty-three percent of non-Hirschsprung patients were positive for INFs. Based on statistical analysis, the association between disease status and pathologist rating was statistically significant (P < .0001). We also found calretinin immunostaining to be a useful adjunctive modality in the diagnosis of Hirschsprung disease.

A cluster of Achromobacter xylosoxidans led to identification of Pseudomonas aeruginosa and Serratia marcescens contamination at a long-term-care facility.

A cluster of Achromobacter xylosoxidans, an emerging multidrug-resistant aquaphilic bacterium, was identified in 3 long-term-care facility residents. As Pseudomonas aeruginosa and Serratia marcescens were also present in clinical specimens, we conducted an investigation of all 3 water-associated species and identified P. aerguniosa and S. marcescens contamination at the facility. Sequencing analysis linked P. aeruginosa to a clinical isolate. Findings highlight the need for precautionary measures to prevent transmission of water-associated multidrug-resistant bacteria in long-term-care facilities.

Utilization of peripherin and S-100 immunohistochemistry in the diagnosis of Hirschsprung disease.

Evaluation of rectal biopsies for ganglion cells is performed for patients suspected of having Hirschsprung disease. At times, identification of ganglion cells can be difficult, especially in newborns. To assist in diagnosis, frozen tissue can be collected for acetylcholinesterase histochemical staining. At our institution, we developed a protocol using peripherin and S-100 immunostaining as an adjunct to hematoxylin and eosin (H&E) for the identification of ganglion cells. Further, at the time of frozen section, we performed Diff Quik staining to highlight ganglion cells. One hundred and thirty eight rectal biopsies submitted for evaluation of Hirschsprung disease were compiled from the archives of the Medical College of Georgia from 2002 to 2009. Initial evaluation consisted of eight levels of H&E-stained slides and two unstained slides each for immunostaining with peripherin and S-100. If on initial evaluation, ganglion cells were not identified, additional H&E and peripherin immunostains were performed. Peripherin immunostaining was unequivocally identified in the cytoplasm of ganglion cells of patients at all ages. Of the 136 patients with diagnostic biopsies, 80% had ganglion cells. Of these, 93% of cases were diagnosed on the original eight levels. Twenty-seven cases were devoid of ganglion cells, and of these, 81% showed submucosal neural hypertrophy on S-100 staining. Twenty-six patients had confirmed aganglionic segments at the time of colonic resection. One patient had colostomy only. A total of 54 frozen sections were performed on 25 patients over this same period of time. Diff Quick staining was found to be very useful. In this study, our protocol proved to be very sensitive, specific, and efficient for the diagnosis of Hirschsprung disease.

Large pedunculated eccrine hidrocystoma: a case report.

Eccrine hidrocystomas are benign, small, cystic growths that characteristically occur on the eyelids, but away from the lid margin. The size of these growths are generally reported as being between 1 and 5 mm. The case of a patient with a very large hidrocystoma of the eyelid with a previously undescribed pedunculated morphology is presented. The pathologic features of this entity and the different diagnosis of cystic lesions are also discussed.

Normal mitochondrial dynamics requires rhomboid-7 and affects Drosophila lifespan and neuronal function.

In addition to being energy generators, mitochondria control many cellular processes including apoptosis. They are dynamic organelles, and the machinery of membrane fusion and fission is emerging as a key regulator of mitochondrial biology. We have recently identified a novel and conserved mitochondrial rhomboid intramembrane protease that controls membrane fusion in Saccharomyces cerevisiae by processing the dynamin-like GTPase, Mgm1, thereby releasing it from the membrane. The genetics of mitochondrial membrane dynamics has until now focused primarily on yeast. Here we show that in Drosophila, the mitochondrial rhomboid (Rhomboid-7) is required for mitochondrial fusion during fly spermatogenesis and muscle maturation, both tissues with unusual mitochondrial dynamics. We also find that mutations in Drosophila optic atrophy 1-like (Opa1-like), the ortholog of yeast mgm1, display similar phenotypes, suggesting a shared role for Rhomboid-7 and Opa1-like, as with their yeast orthologs. Loss of human OPA1 leads to dominant optic atrophy, a mitochondrial disease leading to childhood onset blindness. rhomboid-7 mutant flies have severe neurological defects, evidenced by compromised signaling across the first visual synapse, as well as light-induced neurodegeneration of photoreceptors that resembles the human disease. rhomboid-7 mutant flies also have a greatly reduced lifespan.

The current state of proteomics in GI oncology.

Proteomics refers to the study of the entire set of proteins in a given cell or tissue. With the extensive development of protein separation, mass spectrometry, and bioinformatics technologies, clinical proteomics has shown its potential as a powerful approach for biomarker discovery, particularly in the area of oncology. More than 130 exploratory studies have defined candidate markers in serum, gastrointestinal (GI) fluids, or cancer tissue. In this article, we introduce the commonly adopted proteomic technologies and describe results of a comprehensive review of studies that have applied these technologies to GI oncology, with a particular emphasis on developments in the last 3 years. We discuss reasons why the more than 130 studies to date have had little discernible clinical impact, and we outline steps that may allow proteomics to realize its promise for early detection of disease, monitoring of disease recurrence, and identification of targets for individualized therapy.

Protective effects of the carotenoid zeaxanthin in experimental nonalcoholic steatohepatitis.

Fat infiltration and inflammation cause liver injury and fibrosis and may progress to nonalcoholic steatohepatitis (NASH) and end-stage liver disease. Currently, there are no effective treatments for NASH. Zeaxanthin is a carotenoid which has been shown to be preferentially accumulated in the adipose tissue and liver. We hypothesized that treatment with zeaxanthin may decrease oxidative stress in the liver and, possibly, halt the inflammation and fibrosis associated with NASH. Here we tested zeaxanthin effects in preventing progression of liver injury in a model of NASH. Mongolian gerbils, fed a methionine-choline-deficient diet, were treated with different doses of zeaxanthin. We assessed histopathological changes by hematoxylin-eosin and Masson trichrome staining and determined oxidative stress by measuring lipid peroxidation. The obtained results show that zeaxanthin significantly prevented NASH progression by decreasing oxidative stress and liver fibrosis, thus suggesting a potential therapeutic application for this carotenoid in the management of NASH.

Synthesis, nicotinic acetylcholine receptor binding, and pharmacological properties of 3'-(substituted phenyl)deschloroepibatidine analogs.

A series of 3'-(substituted phenyl)deschloroepibatidine analogs (5a-j) were synthesized. The alpha4beta2( *) and alpha7 nicotinic acetylcholine receptor (nAChR) binding properties and functional activity in the tail-flick, hot-plate, locomotor, and body temperature tests in mice of 5a-j were compared to those of the nAChR agonist, nicotine (1), epibatidine (4), and deschloroepibatidine (13), the partial agonist, varenicline (3), and the antagonist 2'-fluoro-3'-(substituted phenyl)deschloroepibatidine analogs (7a-j). Unlike epibatidine and deschloroepibatidine, which are potent agonists in the tail-flick test, 5a-k show no or very low antinociceptive activity in the tail-flick or hot-plate test. However, they are potent antagonists in nicotine-induced antinociception in the tail-flick test, but weaker than the corresponding 2'-fluoro-3'-(substituted phenyl)deschloroepibatidines.

Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes.

One mechanism contributing to immunologic unresponsiveness toward tumors may be presentation of tumor antigens by tolerogenic host APCs. We show that mouse tumor-draining LNs (TDLNs) contained a subset of plasmacytoid DCs (pDCs) that constitutively expressed immunosuppressive levels of the enzyme indoleamine 2,3-dioxygenase (IDO). Despite comprising only 0.5% of LN cells, these pDCs in vitro potently suppressed T cell responses to antigens presented by the pDCs themselves and also, in a dominant fashion, suppressed T cell responses to third-party antigens presented by nonsuppressive APCs. Adoptive transfer of DCs from TDLNs into naive hosts created profound local T cell anergy, specifically toward antigens expressed by the transferred DCs. Anergy was prevented by targeted disruption of the IDO gene in the DCs or by administration of the IDO inhibitor drug 1-methyl-D-tryptophan to recipient mice. Within the population of pDCs, the majority of the functional IDO-mediated suppressor activity segregated with a novel subset of pDCs coexpressing the B-lineage marker CD19. We hypothesize that IDO-mediated suppression by pDCs in TDLNs creates a local microenvironment that is potently suppressive of host antitumor T cell responses.

Association of nuclear, cytoplasmic expression of galectin-3 with beta-catenin/Wnt-pathway activation in thyroid carcinoma.

OBJECTIVES: To characterize the localization of galectin-3 in benign and malignant thyroid neoplasms and to correlate this with alterations in beta-catenin and cyclin D1 expression. DESIGN: Immunohistochemical study of 116 paraffin-embedded archival specimens from 113 patients who had undergone thyroidectomy and tissue placed into a commercially available tissue microarray. SETTING: Tertiary care hospital. INTERVENTIONS: Thyroid tissue microarrays were stained by standard immunohistochemical protocols with monoclonal antibodies against galectin-3, beta-catenin, and cyclin D1. MAIN OUTCOME MEASURES: Nuclear and cytoplasmic expression of galectin-3 was correlated with clinical parameters, beta-catenin, and cyclin D1 expression. RESULTS: Both cytoplasmic (56%) and nuclear (42%) galectin-3 expression was observed in most malignant neoplasms but was absent in benign thyroid specimens (P<.001). Among carcinomas, cytoplasmic galectin-3 expression was observed in papillary thyroid carcinomas (82%) and follicular (33%) and medullary (9%) carcinomas but was absent in anaplastic carcinomas (P<.001). Galectin-3 nuclear expression was observed in papillary thyroid carcinomas (62%) and follicular carcinomas (33%) but was undetectable in medullary, anaplastic carcinomas (P<.001). Cytoplasmic but not nuclear galectin-3 was inversely correlated with American Joint Committee on Cancer TNM stage (P = .02). There was a strong correlation between cytoplasmic and nuclear beta-catenin expression and both nuclear (P = .04) and cytoplasmic (P = .003) galectin-3 expression. Similarly, there was a strong association between galectin-3 nuclear (P<.001) and cytoplasmic (P<.001) expression and cyclin D1 expression. CONCLUSION: Cytoplasmic and nuclear galectin-3 expression seem to be associated with activation of the Wnt-signaling pathway in well-differentiated thyroid neoplasms, suggesting that galectin-3 plays a role in thyroid carcinogenesis.

The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells.

Programmed death-ligand 1 (PD-L1) is an inhibitory ligand that binds to PD-1 to suppress T cell activation. PD-L1 is constitutively expressed and inducible in tumor cells, but the expression profiles and regulatory mechanism of PD-L1 in myeloid-derived suppressor cells (MDSCs) are largely unknown. We report that PD-L1 is abundantly expressed in tumor-infiltrating leukocytes in human patients with both microsatellite instable and microsatellite stable colon cancer. About 60% CD11b+CD33+HLA-DR- MDSCs from peripheral blood of human colon cancer patients are PD-L1+. PD-L1+ MDSCs are also significantly higher in tumor-bearing mice than in tumor-free mice. Interestingly, the highest PD-L1+ MDSCs were observed in the tumor microenvironment in which 56-71% tumor-infiltrating MDSCs are PD-L1+in vivo. In contrast, PD-L1+ MDSCs are significantly less in secondary lymphoid organs and peripheral blood as compared to the tumor tissues, whereas bone marrow MDSCs are essentially PD-L1- in tumor-bearing mice. IFNγ is highly expressed in cells of the tumor tissues and IFNγ neutralization significantly decreased PD-L1+ MDSCs in the tumor microenvironment in vivo. However, IFNγ-activated pSTAT1 does not bind to the cd274 promoter in MDSCs. Instead, pSTAT1 activates expression of IRF1, IRF5, IRF7 and IRF8 in MDSCs, and only pSTAT1-activated IRF1 binds to a unique IRF-binding sequence element in vitro and chromatin in vivo in the cd274 promoter to activate PD-L1 transcription. Our data determine that PD-L1 is highly expressed in tumor-infiltrating MDSCs and in a lesser degree in lymphoid organs, and the pSTAT1-IRF1 axis regulates PD-L1 expression in MDSCs.