Colon Lymph Nodes Research Articles

P1-02-03: The Reciprocal Roles of E-Cadherin and ZEB1 Demonstrate the Mesenchymal-Epithelial Transition as a Primary Characteristic of Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is a rare but very aggressive form of breast cancer. IBC is characterized by nests of tightly aggregated cells, defined as tumor emboli, that exhibit characteristics of cancer stem cells (CSCs). IBC tumor emboli express E-cadherin which is required to maintain their integrity and our recent evidence demonstrates that expression of E-cadherin by tumor emboli is associated with lack of ZEB1 expression, a transcriptional repressor of E-cadherin. This is at odds with the current hypothesis that metastatic progression is associated with the process of epithelial mesenchymal transition (EMT), with loss of E-cadherin and gain of transcription factors including ZEB1, acquisition of CSC characteristics and enhanced invasive capabilities. Materials and Methods: shRNA knockdown and over-expression methods, real time PCR arrays, western blotting, and in vitro assays to evaluate proliferation, invasion, growth in soft agar and clonogenicity and in vivo animal studies were used. Results: Expression of E-cadherin was reduced by shRNA and ZEB1 was expressed in SUM149 IBC tumor cells. Numerous EMT-related genes were upregulated with loss of E-cadherin and gain of ZEB1, including N-cadherin and vimentin. However, there were marginal differences in the in vitro parameters of proliferation, Matrigel invasion and anchorage independent growth in soft agar between SUM149-shECad or SUM149-ZEB1 clones and their respective vector control cells. The loss of E-cadherin and gain of ZEB1 altered the morphology of SUM149 cells when cultured under low adherence conditions permissive for the enrichment of CSC, exhibiting a reversion in grape-like morphology to more well defined spheres, which was accompanied by increased clonogenicity in both SUM149-shECad and SUM149-ZEB1 cells. The loss of E-cadherin and the gain of ZEB1 significantly inhibited tumor growth of cells injected in the mammary fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumor volume at 56 days for E-cadherin vector control cells was 771.9 mm3 +/− 185.6 compared to shECadherin tumors, which was 13.6 mm3 +/− 7.2. Tumor volume of ZEB1 vector control tumors was 346.1 mm3 +/− 96 compared to volume of ZEB1 expressing tumors, which was 21.5 mm3 +/− 7.2.Conclusions: E-cadherin with lack of ZEB1 expression in IBC is consistent with a mesenchymal-epithelial transition (MET), consistent with the retention of the epithelial phenotype while maintaining a program of rapid metastasis and colonization of lymph nodes and distant organ sites. Furthermore, we demonstrate that the E-cadherin-ZEB1 axis is critical for the in vivo growth of IBC tumor cells. Although SUM149 cells are fully capable of undergoing an EMT process, which is under negative regulation by E-cadherin, the process of EMT does not drive in vivo tumor growth in IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-03.

Colonic and gastric cancer metastatic lymph nodes: applications of autofluorescence-based techniques

Colonic and gastric cancer metastatic lymph nodes: applications of autofluorescence-based techniques

Colonic and gastric cancer metastatic lymph nodes: applications of autofluorescence-based techniques

Lymphadenectomy and extended lymphadenectomy are still controversial issues in surgical oncology. The detection methods for metastatic lymph nodes include lymphoscintigraphy and radiolabelled antibody, but immunohistochemical sentinel lymph node (SLN) identification is commonly used. The potential diagnostic use of cell and tissue autofluorescence (AF) is well known. Here we review our studies on the application of AF-based techniques for diagnosing metastatic lymph nodes. We had previously demonstrated that AF imaging allows discrimination between hyperplastic and primary neoplastic lymph nodes. Our more recent studies show that the combination of autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy can be applied to the diagnosis of secondary neoplastic lymph nodes in gastric and colorectal cancer. These techniques have been validated by histochemical and immunohistochemical methods. Studies are in progress to select the best dye tracer for SLN identification, to improve image quality and to develop a software for automatic analysis of tissue AF to help the decision-making process during surgery.

Colonic and gastric cancer metastatic lymph nodes: applications of autofluorescence-based techniques

Lymphadenectomy and extended lymphadenectomy are still controversial issues in surgical oncology. The detection methods for metastatic lymph nodes include lymphoscintigraphy and radiolabelled antibody, but immunohistochemical sentinel lymph node (SLN) identification is commonly used. The potential diagnostic use of cell and tissue autofluorescence (AF) is well known. Here we review our studies on the application of AF-based techniques for diagnosing metastatic lymph nodes. We had previously demonstrated that AF imaging allows discrimination between hyperplastic and primary neoplastic lymph nodes. Our more recent studies show that the combination of autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy can be applied to the diagnosis of secondary neoplastic lymph nodes in gastric and colorectal cancer. These techniques have been validated by histochemical and immunohistochemical methods. Studies are in progress to select the best dye tracer for SLN identification, to improve image quality and to develop a software for automatic analysis of tissue AF to help the decision-making process during surgery.

Antibiotics induced commensal flora disruption favours escherichia coli AIEC colonization and mesenteric translocation in NOD2 knock-out mice

Methods Disruption of commensal microbiota was induced by a 3 days antibiotic treatment orally administered in WT and NOD2KO mice. At day 3, mice were infected with 10 CFU AIEC once a day for 2 days. Animals were sacrificed at day 1, 5, 30 and 60 after AIEC administration. Ileum, colon and mesenteric lymph nodes (MLN) were sampled for AIEC quantification in ileal and colonic tissues, bacterial translocation in MLN, and evaluation of histological abnormalities and intestinal inflammation.

Abstract C44: Metastatic potential of prostate tumor cells is affected by hyaluronan metabolism.

Abstract Introduction: Accumulation of hyaluronan (HA) and elevation of the HA hydrolytic enzyme Hyal1 have been shown to promote prostate cancer progression. Immunohistochemical detection of HA and Hyal1 is diagnostic in biopsies and resected tissue, and predicts biochemical recurrence. We previously found that stable co-overexpression of Hyal1 and HAS, the HA biosynthetic enzyme, in 22Rv1 prostate tumor cells accelerates orthotopic tumor growth and spontaneous lymph node (LN) metastasis. Furthermore, the presence of cell-associated HA is required for adhesion of prostate tumor cells to bone marrow derived endothelial cells in vitro, suggesting surface HA could impact metastatic preference and/or efficiency of tumor cells by promoting arrest in specific vasculatures such as that of the bone. Here, we tested this hypothesis using intracardiac (IC) injections to model the hematogenous metastatic spread of tumor cells. Experimental Procedures: Male nu/nu, nude/SCID and NOD/SCID mice were injected via the left ventricle with 1×105 tumor cells in 0.1 ml HBSS containing IRDye 800CW carboxylate, a non-reactive near infrared (NIR) imaging agent, as an indicator of successful injection. Mice (n=75) were monitored over a 10 week period by non-invasive fluorescent optical imaging of dual probes in the 700 and 800 channels of the Pearl Impulse Small Animal Imager. Probes were designed to identify tumor tissue (IRDye 800CW conjugated to EGF, 2DG, or RGD), lymphatic tissue (IRDye 680RD hyaluronan (HA)) or bone tissue (IRDye 680RD BoneTag). Animals were imaged at the study endpoint and metastases noted. Tissues were excised, imaged ex vivo, and metastasis confirmed by histology. Results: PC3M-LN4 highly metastatic HA-encapsulated prostate tumor cells produced macrometastases at a higher rate (33%) after 10 wks than 22Rv1 HA-deficient cells (14%) in nu/nu and NOD/SCID mice. Suspect lesions were present in lung, bone, liver, adrenals, kidney, brain, and LN. Hyal1 overexpressing 22Rv1 cells were more frequently metastatic to brain and lungs and more rapidly growing, since that group exhibited a higher mortality rate. However, the frequency of metastasis observed in LN and bone was significantly higher for HAS transfectants, which also showed greater overall tumor burden in LN. Conclusions: Although Hyal1 expression promotes LN growth via lymphatic invasion from prostate, HAS expression and surface HA retention provide a greater LN colonization advantage for tumor cells already in circulation. Use of IRDye 800CW and IRDye 680RD optical imaging agents in a multiplex fashion provided strong evidence of macro- and micro-metastases along with additional detail relevant to the microenvironment upon intraoperative evaluation. In several instances the dual optical agent evaluation alerted us to the presence of suspect lesions subsequently confirmed by H&E staining. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C44.

Colonization Kinetics of Different Methicillin-Resistant Staphylococcus aureus Sequence Types in Pigs and Host Susceptibilities

In this study we investigated the kinetics of colonization, the host susceptibility and transmissibility of methicillin-resistant Staphylococcus aureus (MRSA) after nasal treatment of pigs with three different MRSA strains of distinctive clonal lineages (sequence type 398 [ST398], ST8, and ST9), and origin in weaning piglets. The colonization dose of 5.0 × 10(8) CFU/animal was determined in preliminary animal studies. A total of 57 piglets were randomly divided into four test groups and one control group. Each of three test groups was inoculated intranasally with either MRSA ST8, MRSA ST9, or MRSA ST398. The fourth group was a mixture of animals inoculated with MRSA ST398 and noninoculated "sentinel" animals. Clinical signs, the nasal, conjunctival, and skin colonization of MRSA, fecal excretion, and organ distribution of MRSA, as well as different environmental samples were examined. After nasal inoculation with MRSA piglets of all four test groups showed no clinical signs of an MRSA infection. MRSA was present on the nasal mucosa, skin, and conjunctiva in all four test groups, including sentinel animals. Likewise, fecal excretion and internal colonization of MRSA ST8, ST9, and ST398 could be shown in each group. However, fecal excretion and the colonization rate of the nasal mucosa with MRSA ST9 were significantly lower in the first days after infection than in test groups infected with ST8 and ST398. The results of this study suggest differences in colonization potential of the different MRSA types in pigs. Furthermore, colonization of lymph nodes (e.g., the ileocecal lymph node) with MRSA of the clonal lineage ST398 was demonstrated.

Expression of mammalian target of rapamycin in colon cancer and its effect on proliferation and apoptosis of human colon cancer HT-29 cells

To explore the expression of mammalian target of rapamycin (mTOR) in colon cancer and the inhibitory effect of mTOR siRNA on the proliferation and apoptosis of human colon cancer HT-29 cells. Immunohistochemistry was employed to detect the expression of phosphorylated mTOR (p-mTOR) in colon cancer tissues (n = 50) and normal colon tissues (n = 50) from First Affiliated Hospital of Zhengzhou University (October 2009 to June 2010). The correlations were examined between the expression of p-mTOR and such clinical pathological data as colon cancer staging and lymph node metastasis. The oligonucleotide templates of mTOR siRNA were designed and employed to transfect HT-29. The nonsense control groups were established accordingly by siRNA with an insignificant order. And the blank group had no transfection. The protein level of mTOR was measured by Western blotting. The method of MTT was used to detect the cell proliferation. And the method of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was employed to detect the apoptosis. The expression of p-mTOR was higher in the colon cancer group than the control group [60% (30 cases) vs 14% (7 cases), P < 0.05]. And the expression level was correlated with the degree of lymph node metastasis and tumor differentiation (r = 0.311, 0.427, both P < 0.05). The expression of mTOR protein in the transfection group was lower than the blank and the nonsense control groups (0.39 ± 0.25 vs 1.18 ± 0.05, 1.46 ± 0.09, both P < 0.05). The proliferation was lower than those of the blank and the nonsense control groups (0.275 ± 0.033 vs 0.460 ± 0.028, 0.450 ± 0.037, both P < 0.05). The apoptotic indices were higher than those of the blank and control groups (12.33 ± 1.53 vs 0.33 ± 0.31, 1.67 ± 0.58, both P < 0.05). The expression of mTOR was higher in colon cancer tissues than that in normal colon tissues. The RNA interference of mTOR in HT-29 cell line can effectively knock down the expression of mTOR so as to significantly inhibit cell proliferation and promote cell apoptosis.

Predictive value of immunohistochemical expression of claudin-1 in colonic carcinoma

ObjectiveColonic carcinoma is one of the most common cancers worldwide. Recently, the possible involvement of claudin-1, one of the major tight junction proteins, in the process of tumorigenesis has been suggested. Also, claudin-1 has emerged as a potential prognostic factor in different types of tumors. The aim of this study was to detect caludin-1 expression in colonic carcinoma and to correlate its expression with clinicopathological variables in an attempt to delineate its role as a potential new prognostic marker. Material and methodsImmunohistochemical expression of claudin-1 was assessed in 50 Egyptian patients with colonic adenocarcinoma. The predictive performance of claudin-1 expression was statistically evaluated. ResultsDecreased claudin-1 expression was found in 62% of colonic adenocarcinoma cases while similar expression was found in 38% of the cases. Statistical analysis showed a statistically significant inverse correlation between claudin-1 expression and tumor grade, depth of invasion, lymph node involvement, and tumor stage. Regression analysis showed that claudin-1 decreased expression significantly predicts that the tumor is of a high grade, high stage, and is associated with lymph node involvement. ROC curve analysis showed that claudin-1 had a sensitivity of 88.24% and a specificity of 81.25% for the prediction of tumor stage and a sensitivity of 73.33% and a specificity of 82.86% for the prediction of lymph node involvement. ConclusionsClaudin-1 decreased expression in colonic carcinoma contributes to tumor dedifferentiation, invasion and metastasis. Claudin-1 expression could be used as a predictor of colonic carcinoma stage and lymph node status with a high sensitivity and specificity.

Is the Easier Way Ever the Better Way?

For the past four decades, the standard management of patients with advanced ovarian cancer has been primary cytoreductive surgery followed by chemotherapy. Numerous studies have demonstrated that patientswhoareoptimallycytoreducedenjoyprolongedprogression-free and overall survival compared with those who are suboptimally debulked. Although the data in support of optimal cytoreduction are almost all retrospective, improved patient outcome with more extensive surgical debulking has been consistently observed, leading to the ideal goal of tumor cytoreduction to no macroscopic visible disease after initial diagnostic/therapeutic surgery. During the same four decades, the gynecologic oncology community has significantly improved the outcomes of patients with advanced ovarian cancer through the development of newer cytotoxic agents, improved schedules and routes of administration, and, more recently, advances in targeted therapies. Surgical improvements have paralleled the development of better medical therapies, with the evolution of the concept, principle, and proof of principle that patients left with no or minimal disease after cytoreduction have prolonged survival compared with those left with large-volume disease. Improved surgical instrumentation and supportive management (antibiotics, blood banking, and intensive care) have significantly reduced postoperative morbidity and mortality. The impact of debulking on patient survival has been shown to be equivalent whether complete debulking is attained with easily resectable disease involving the omentum and ovaries or whether it requires more extensive surgery involving the peritoneal diaphragm, rectosigmoid colon, or paraaortic lymph nodes. Furthermore, contemporary studies have demonstrated that the impact of potentially negative biologic factors such as grade and histology can be overcome by surgical debulking. Although initial tumor load and tumor biology will always have some influence on survival, the expansion of the surgical armamentarium available for cytoreductive procedures has led to an increase in the percentage of optimally debulked patients at many centers, with a commensurate improvement in chemotherapy response and progression-free and overall survival. Advocates of a less-is-more approach in this setting point to the potential of excessive morbidity associated with extensive surgery and conclude that anything more than hysterectomy, bilateral salpingooophorectomy, and omentectomy cannot be justified. It is true that surgeons not trained, capable, or willing to perform extensive surgical cytoreduction for advanced ovarian cancer should not attempt it. However, just as the gynecologic oncologist chides the general obstetrician/gynecologist for operating on a patient with a suspicious complex adnexal mass and elevated CA125 without gynecologic oncologic backup for surgical staging, gynecologic oncologists should be similarly chided for attempting advanced ovarian cancer debulking without the requisite tools for success. These procedures require the expertise of the gynecologic oncologist but may also necessitate the assistance of general surgeons, surgical oncologists, or hepatobiliary surgeons, particularly when there is extensive upper abdominal disease. It is sobering to consider that in these examples, the risk that the general obstetrician/gynecologist would encounter an ovarian cancer is less than 5%, whereas the likelihood that a gynecologic oncologist operating on a patient with advanced ovarian cancer would identify extensive upper abdominal disease is greater than 40%. Given the extended survival associated with achieving successful surgical resection to no gross residual disease in patients with advanced ovarian cancer, it is the duty of the treating clinician to give the patient the best opportunity to attain this result, irrespective of whether it falls within that surgeon’s current skill set. One must either obtain the necessary skills, through additional training or repeated multispecialty intraoperative consultation, or refer the patient to a surgeon capable of performing the necessary procedures. The question of the best timing for surgery, or what some have termed the less-is-more approach, regarding patients with advanced ovarian cancer was evaluated by the European Organization for Research and Treatment of Cancer–Gynecologic Cancer Group and the National Cancer Institute of Canada Clinical Trials Group and was recently reported by Vergote et al. In that trial, patients with stage IIIC or IV ovarian cancer were randomly assigned to primary debulking surgery (PDS) followed by platinum-based chemotherapy or to neoadjuvant platinum-based chemotherapy (NACT) followed by interval debulking surgery. After debulking surgery, the largest residual tumor was 1 cm or smaller in diameter in 41.6% of patients in the PDS arm and in 80.6% of those in the NACT arm, with a trend toward more postoperative adverse events and mortality in the PDS arm. Progressionfree and overall survival were the same between the two arms. Complete resection of all macroscopic disease, at PDS or after NACT, was the strongest independent variable in predicting overall survival. These results were initially reported at the International Gynecologic Cancer Society meeting in Bangkok, Thailand, 2 years ago. Since JOURNAL OF CLINICAL ONCOLOGY COMMENTS AND CONTROVERSIES VOLUME 29 NUMBER 31 NOVEMBER 1 2011

CT Findings in Adult Celiac Disease

Celiac disease is now recognized as a common disease, occurring in about one in every 200 Americans. However, less than 10% of cases are currently diagnosed, with a diagnostic delay of more than 10 years from onset of symptoms. In the past, barium examination of the small bowel demonstrated a pattern of abnormal findings caused by the pathophysiologic changes induced by malabsorption, thus leading to diagnosis of celiac disease and other diseases of malabsorption. Although not specific, that pattern prompted further patient evaluation. The number of barium examinations performed and the skill necessary to interpret their results are both in decline. Abdominal pain in celiac disease is a common early complaint that often leads to computed tomography (CT). Improved CT resolution now permits better depiction of the small bowel, colon, and mesenteric lymph nodes, all of which are affected by celiac disease. Detection of celiac disease with CT will allow treatment to be initiated to prevent the significant morbidity and increased mortality associated with a delay in diagnosis. The abnormal CT findings seen over the past decade during review of more than 200 cases of celiac disease demonstrate that CT depicts more features of celiac disease than did barium examination. Pattern recognition for the diagnosis of small bowel diseases that create structural changes in the bowel wall is well accepted. Because it demonstrates features of celiac disease not detected with barium examination, CT may be more sensitive than barium examination for diagnosis of this disease.

KLF4-Mediated Negative Regulation of IFITM3 Expression Plays a Critical Role in Colon Cancer Pathogenesis

IFITM3, an IFN-inducible gene, is overexpressed in human colorectal cancer. In this study, we sought to determine the clinical significance and underlying mechanisms of its dysregulated expression in human colon tumor specimens and murine models of this disease. IFITM3 expression in a tissue microarray of tumor and matched normal colon tissue specimens and lymph node metastasis specimens obtained from 203 patients with colon cancer was measured immunohistochemically. IFITM3 was expressed at higher levels in colon tumors and, particularly, nodal metastases than in normal colon tissue. A Cox proportional hazards model showed that IFITM3 expression was an independent prognostic factor for disease-free survival in patients with colon cancer. Knockdown of IFITM3 expression by a specific siRNA significantly suppressed the proliferation, colony formation, migration, and invasion of colon cancer cells in vitro and tumor growth and metastasis in a xenograft model. Restored expression of KLF4, a putative tumor suppressor, downregulated IFITM3 expression in colon cancer cells in vitro. Two KLF4-binding sites in the IFITM3 promoter bound specifically to KLF4 protein in a chromatin immunoprecipitation assay and promoter mutagenesis analyses. Specific deletion of KLF4 led to IFITM3 overexpression in colon mucosa in Villin-Cre(+);Klf4(fl/fl) mice. An inverse correlation between loss of KLF4 expression and IFITM3 overexpression was evident in human colon tumors. These clinical and mechanistic findings indicate that IFITM3 is a direct transcriptional target of KLF4 and that dysregulated KLF4 expression leads to aberrant IFITM3 expression, thus contributing to colon cancer progression and metastasis.

Histochemical localization of caldesmon isoforms in colon adenocarcinoma and lymph node metastases

Alternative splicing of the caldesmon gene results in high (h-caldesmon) and low (l-caldesmon) molecular weight isoforms of the cytoskeleton-associated protein caldesmon. h-Caldesmon is predominantly expressed not only in smooth-muscle cells but also in pericryptal fibroblasts in colon. l-Caldesmon is widely expressed and localized in podosomes/invadopodia. Studies with transformed and cancer cell lines suggest that a reduction in l-caldesmon facilitates podosome/invadopodia formation and metastasis. We investigated caldesmon isoforms in colon adenocarcinoma and their lymph node metastases using immunohistochemistry. Caldesmon immunoreactivity of colon adenocarcinoma primary tumors and lymph node metastases was very similar. l-Caldesmon immunoreactivity of cancer epithelial cells in primary tumors and lymph node metastases varied ranging from reduced to stronger as compared to immunoreactivity of normal areas. The variation did not show a consistent relation to the tumor center or invasive margin. While h-caldesmon immunoreactivity of pericryptal fibroblasts and blood vessels in the stroma of primary tumors and lymph node metastases was markedly reduced, cancer-associated fibroblasts and blood vessels in the stroma were strongly immunoreactive for l-caldesmon. Our results show a differential behavior of h- and l-caldesmon isoforms in epithelium and stroma of colon adenocarcinoma and lymph node metastases.

Positive RAR/RXR and Negative STAT3 Regulation of FOXP3 Transcription is Mediated by Control of SMAD3 Access and Binding to an Enhancer

Some clinical trials and epidemiologic studies revealed improvements in symptoms of ulcerative colitis after smoking and nicotine patch treatment. However, mechanisms underlying the ameliorating effect of nicotine remain poorly understood. We have previously reported that nicotine administration ameliorates murine oxazolone (OXZ)-induced colitis through alpha7 nicotine acetylcholine receptors (α7nAChRs). In addition, we have shown that mRNA of α7nAChRs was expressed in plasmacytoid dendritic cells (pDCs) of OXZ colitis mouse colon. The aim of this study was to investigate a pathophysiological role of pDCs and immunological effects of nicotine on pDCs in ulcerative colitis. Method: OXZ-induced colitis was developed in BALB/c mice. Lamina propria mononuclear cells (LPMCs) of the colon and mesenteric lymph node (MLN) were collected in OXZ colitis mice. pDCs were isolated from LPMCs by using magnetic cell separation system (BD biosciences). In addition, mouse bone marrow (BM) cells were differentiated to pDC-like cells (BMpDC) by Flt3 ligand. Furthermore, immunological effects of nicotine on BMpDCwere examined by flow cytometry and BMpDC migration was examined by chemotaxis assay using EZ-TAXIScan. Moreover, the effect of nicotine administration on pDC subset proportion was investigated in the MLN of OXZ colitis mice. Result: α7nAChRs mRNA was expressed in pDC of OXZ colitis mouse colon and BMpDC, but not BM conventional dendritic cells (cDC). Flow cytometry analysis revealed that neither CD80, CD86 nor MHC class II expression in BMpDC was affected with nicotine in the induction of pDC maturation by CpG-DNA. On the other hand, CCL21induced migration of mature BMpDC was inhibited by nicotine and α7nAChRs selective agonist GTS-21, and α7nAChRs selective antagonist methyllycaconitine (MLA) blocked the inhibitory effect of nicotine and GTS-21. Furthermore, nicotine administration downregulated pDC subset proportion in the MLN of OXZ colitis mice. Conclusion: These data suggest that the activation of α7nAChRs reduces pDC-mediated antigen presentation to naive CD4 T cells by inhibiting pDC migration from colon to MLN, thereby alleviating the symptoms in OXZ colitis. Our results may contribute to the development of therapeutic medicines for ulcerative colitis by activating the cholinergic anti-inflammatory pathway.

Nicotine Improves Symptoms in Murine Oxazolone-Induced Ulcerative Colitis by Inhibiting Migration of Plasmacytoid Dendritic Cells Through Alpha7 Nicotinic Acetylcholine Receptors

Some clinical trials and epidemiologic studies revealed improvements in symptoms of ulcerative colitis after smoking and nicotine patch treatment. However, mechanisms underlying the ameliorating effect of nicotine remain poorly understood. We have previously reported that nicotine administration ameliorates murine oxazolone (OXZ)-induced colitis through alpha7 nicotine acetylcholine receptors (α7nAChRs). In addition, we have shown that mRNA of α7nAChRs was expressed in plasmacytoid dendritic cells (pDCs) of OXZ colitis mouse colon. The aim of this study was to investigate a pathophysiological role of pDCs and immunological effects of nicotine on pDCs in ulcerative colitis. Method: OXZ-induced colitis was developed in BALB/c mice. Lamina propria mononuclear cells (LPMCs) of the colon and mesenteric lymph node (MLN) were collected in OXZ colitis mice. pDCs were isolated from LPMCs by using magnetic cell separation system (BD biosciences). In addition, mouse bone marrow (BM) cells were differentiated to pDC-like cells (BMpDC) by Flt3 ligand. Furthermore, immunological effects of nicotine on BMpDCwere examined by flow cytometry and BMpDC migration was examined by chemotaxis assay using EZ-TAXIScan. Moreover, the effect of nicotine administration on pDC subset proportion was investigated in the MLN of OXZ colitis mice. Result: α7nAChRs mRNA was expressed in pDC of OXZ colitis mouse colon and BMpDC, but not BM conventional dendritic cells (cDC). Flow cytometry analysis revealed that neither CD80, CD86 nor MHC class II expression in BMpDC was affected with nicotine in the induction of pDC maturation by CpG-DNA. On the other hand, CCL21induced migration of mature BMpDC was inhibited by nicotine and α7nAChRs selective agonist GTS-21, and α7nAChRs selective antagonist methyllycaconitine (MLA) blocked the inhibitory effect of nicotine and GTS-21. Furthermore, nicotine administration downregulated pDC subset proportion in the MLN of OXZ colitis mice. Conclusion: These data suggest that the activation of α7nAChRs reduces pDC-mediated antigen presentation to naive CD4 T cells by inhibiting pDC migration from colon to MLN, thereby alleviating the symptoms in OXZ colitis. Our results may contribute to the development of therapeutic medicines for ulcerative colitis by activating the cholinergic anti-inflammatory pathway.

Production of IFN-β during Listeria monocytogenes Infection Is Restricted to Monocyte/Macrophage Lineage

The family of type I interferons (IFN), which consists of several IFN-α and one IFN-β, are produced not only after stimulation by viruses, but also after infection with non-viral pathogens. In the course of bacterial infections, these cytokines could be beneficial or detrimental. IFN-β is the primary member of type I IFN that initiates a cascade of IFN-α production. Here we addressed the question which cells are responsible for IFN-β expression after infection with the intracellular pathogen Listeria monocytogenes by using a genetic approach. By means of newly established reporter mice, maximum of IFN-β expression was observed at 24 hours post infection in spleen and, surprisingly, 48 hours post infection in colonized cervical and inguinal lymph nodes. Colonization of lymph nodes was independent of the type I IFN signaling, as well as bacterial dose and strain. Using cell specific reporter function and conditional deletions we could define cells expressing LysM as the major IFN-β producers, with cells formerly defined as Tip-DCs being the highest. Neutrophilic granulocytes, dendritic cells and plasmacytoid dendritic cells did not significantly contribute to type I IFN production.

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Pathogenesis of a Bovine Enterovirus-1 Isolate in Experimentally Infected Calves

The pathogenesis and virulence of Bovine enterovirus-1 (BEV-1) in cattle is largely unknown. Reports concerning its virulence suggest that there might be an association between BEV-1 infections and a range of diseases in cattle that vary from respiratory to enteric to reproductive disease and infertility. In the current study, the pathogenesis associated with acute infection of BEV-1 in calves experimentally inoculated with the Oklahoma isolate of BEV-1 was described. Although interpretation of the study was limited by lack of an effective control group, results suggest that an association between inoculation of BEV-1, virus localization, and the potential development of lesions in the brain and heart probably exists. In the experiment, BEV-1 virus localized to the terminal ileum, ileocecal and cecocolonic junctions, spiral colon, and ileocecal lymph nodes; BEV-1 virus was detected in the cytoplasm of enterocytes, lamina propria macrophages, endothelium, neurons of the submucosal and myenteric plexi, and lymphocytes of the submucosal lymphoid tissue. Although no clinical signs were noted following acute infection, BEV-1 was localized in the cerebellar white matter of a calf with encephalitis and in the heart of another calf with coronary arteritis. The current study suggests that the BEV-1 isolate is infectious to young calves and that BEV-1 potentially can have a similar pathogenesis to that observed in natural or experimental enterovirus infections in other species.

In vivo analysis of Yersinia enterocolitica infection using luxCDABE

Yersiniae expressing an L-arabinose-inducible luxCDABE reporter were used to analyze the colonization of mice. Infection of live mice was followed over a period of 6 days. These experiments revealed frequent colonization of cervical lymph nodes after oral, but not intravenous infection. Furthermore, the well-known colonization of the small intestine, Peyer's patches (PPs) of the ileum, the cecal lymph follicle, mesenteric lymph nodes, liver, and spleen was easily detectable. Removal of the intestinal tract of mice revealed that the number of abscessed PPs and other tissues can be easily quantified. Experiments with an invasin mutant expressing luxCDABE revealed a significantly reduced number of abscessed PPs, cecal lymph follicles, and lymph nodes in yersiniae lacking invasin.

American Ginseng Suppresses Colitis through p53-Mediated Apoptosis of Inflammatory Cells

Ulcerative colitis is a dynamic, chronic inflammatory condition associated with an increased colon cancer risk. Inflammatory cell apoptosis is a key mechanism regulating ulcerative colitis. American ginseng (AG) is a putative antioxidant that can suppress hyperactive immune cells. We have recently shown that AG can prevent and treat mouse colitis. Because p53 levels are elevated in inflammatory cells in both mouse and human colitis, we tested the hypothesis that AG protects from colitis by driving inflammatory cell apoptosis through a p53 mechanism. We used isogenic p53(+/+) and p53(-/-) inflammatory cell lines as well as primary CD4(+)/CD25(-) effector T cells from p53(+/+) and p53(-/-) mice to show that AG drives apoptosis in a p53-dependent manner. Moreover, we used a dextran sulfate sodium (DSS) model of colitis in C57BL/6 p53(+/+) and p53(-/-) mice to test whether the protective effect of AG against colitis is p53 dependent. Data indicate that AG induces apoptosis in p53(+/+) but not in isogenic p53(-/-) cells in vitro. In vivo, C57BL/6 p53(+/+) mice are responsive to the protective effects of AG against DSS-induced colitis, whereas AG fails to protect from colitis in p53(-/-) mice. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of inflammatory cells within the colonic mesenteric lymph nodes is elevated in p53(+/+) mice consuming DSS + AG but not in p53(-/-) mice consuming DSS + AG. Results are consistent with our in vitro data and with the hypothesis that AG drives inflammatory cell apoptosis in vivo, providing a mechanism by which AG protects from colitis in this DSS mouse model.