MicroRNA-496 and Mechanistic Target of Rapamycin Expression are Associated with Type 2 Diabetes Mellitus and Obesity in Elderly People.

BACKGROUND: Mechanistic target of rapamycin (mTOR) regulates lipid and glucose metabolism thus playing a key role in metabolic diseases like type 2 diabetes mellitus (T2DM). Recently, we demonstrated a functional interaction of microRNA-496 (miR-496) with mTOR and its impact on the regulation of human ageing. OBJECTIVES: As T2DM is most prevalent in older adults, we hypothesized that miR-496 may also have an impact on mTOR regulation in T2DM. METHODS: Based on real-time PCR and enzyme-linked immunosorbent assay, mTOR gene and protein expression as well as miR-496 expression were monitored in peripheral blood mononuclear cells (PBMC) from T2DM patients (median age: 71) and healthy age- and BMI matched controls (median age: 69). -Results: We demonstrated significant upregulation of phospho-mTOR and P70S6 Kinase (P70S6K) levels and significant downregulation of miR-496 in PBMC from elderly T2DM patients in comparison to a BMI and age-matched control cohort. Moreover, significant upregulation of phospho-mTOR protein and significant downregulation of miR-496 were observed in advanced stages of obesity. CONCLUSIONS: BMI-dependent upregulation of mTOR and the inverse expression profile of miR-496 observed in elderly T2DM patients suggest a correlation with T2DM. Hence, our results indicate a potential association of miR-496 with mTOR expression in elderly T2DM patients and obesity. Since phosphorylation of P70S6K was also elevated in T2DM patients, we conclude that mTOR signaling through TORC1 may be affected in the regulation of T2DM.

Chemokine receptor CCR6 expression is regulated by miR-518a-5p in colorectal cancer cells.

BACKGROUND: Recently, involvement of the chemokine/receptor system CCL20/CCR6 in colorectal cancer (CRC) progression was shown. Here, we analyzed the functional interaction of miRNA-518-5p (miR-518a-5p) with CCR6 and its impact on CCR6 expression in CRC cells. METHODS: MiR-518a-5p was identified by computer software to potentially interact with CCR6. Hence, functional implications of miR-518a-5p with the 3'UTR of CCR6 were analyzed using the Dual Luciferase Reporter assay system. Confirmation of the predicted target site for miR-518a-5p was achieved by site-directed mutagenesis of the seed sequence in the 3'UTR of CCR6 and subsequent application of the mutated seed sequence in a luciferase assay with miR-518a-5p mimics. Accordingly, two CRC cell lines (Caco-2 and HT-29) were transfected with miR-518a-5p miRNA mimics and gene and protein expression of CCR6 was monitored using qRT PCR and immunocytochemistry, respectively. RESULTS: Addition of miR-518a-5p led to significant down-regulation of luciferase activity (P < 0.05), which was significantly reversed in a reporter test system containing the mutated seed sequences in the 3'UTR of CCR6. Following transfection of CRC cell lines with miR-518a-5p mimics and subsequent monitoring of CCR6 expression showed significant down-regulation of CCR6 mRNA and CCR6 protein expression in both CRC cell lines under investigation (P < 0.05). CONCLUSIONS: We have shown that miR-518a-5p functionally interacts with CCR6 and that transfection of CRC cells with miR-518a-5p leads to significant CCR6 down-regulation. Consequently, CCR6 expression is regulated by miR-518a-5p in CRC cells indicating that regulation of CCR6 expression by miR-518a-5p might be a regulatory mechanism involved in CRC pathogenesis.

Lung function early after lung transplantation is correlated with the frequency of regulatory T cells.

PURPOSES: Outcomes following lung transplantation are limited by bronchiolitis obliterans syndrome (BOS). As the number of circulating regulatory T cells (Treg) is lower in lung recipients with BOS than in stable lung recipients, we hypothesized that Treg is also correlated with lung function in the early post-transplantation period. METHODS: This prospective study included 18 consecutive patients whose lung function parameters were recorded 3 weeks and 3 months after transplantation, between February and July 2007. Peripheral blood mononuclear cells were stained with anti-CD3, -CD4, -CD8, -CD19, -CD25, -CD28, -CD45RA, -CD45RO, -CD69, -CD127, -CTLA4, and -Foxp3 antibodies and FACS assays were performed. In addition, intracellular cytokines were stained for FACS. RESULTS: Treg-specific markers (Foxp3, CD127(lo), and CTLA4) in the CD25+ CD4+ population were correlated with both forced expiratory volume in 1 s and forced vital capacity. Th1-cytokine secretion was more dominant in CD4+ CD25+ T cells than in CD4+ CD25- T cells. In contrast, Th2 and Treg cytokine secretion was the dominant response in stable recipients. CONCLUSIONS: The frequency of Treg cells was positively correlated with good lung function in the early period after lung transplantation.

Correlation of donor leukocyte chimerism with pulmonary allograft survival after immunosuppressive drug withdrawal in a porcine model.

BACKGROUND: This study was designed to analyze the role of postoperative donor cell chimerism for the induction and maintenance of transplantation tolerance in a porcine lung transplantation model. METHODS: Left-sided single lung transplantation from major histocompatibility mismatched male donors was performed in 27 female minipigs. All received a 28-day course of pharmacologic immunosuppression using various agents, some in combination with preoperative irradiation. Groups for eventual analysis were strictly defined by outcome, that is, pigs with acute rejection before postoperative day 178 (n=16) were allocated into one group, long-term surviving animals (n=11) into the other. Peripheral blood chimerism was monitored by flow cytometry and real-time polymerase chain reaction. Intragraft chimerism was detected from bronchoalveolar lavage fluid (BALF) by fluorescent in situ hybridization. RESULTS: Blood chimerism peaked 1 hour after transplantation and was significantly higher in the group of long-term survivors at that time. Thereafter chimerism rapidly decreased, but tended to remain higher in long-term survivors. In case of acute rejection donor cells were lost, but remained detectable for up to 36 postoperative months in tolerant animals. In BALF, the percentage of male nuclei was equally high under immunosuppression in both groups. Rejecting animals showed a rapid decrease of Y-bearing cells in BALF after drug withdrawal and an almost complete loss when acute rejection occurred. In tolerant pigs, intragraft chimerism remained detectable throughout the follow-up. CONCLUSIONS: This study demonstrates a clear correlation of donor leukocyte chimerism with long-term allograft survival in a porcine allogeneic lung transplantation model.

Postoperative intravenous infusion of donor-derived transplant acceptance-inducing cells as an adjunct immunosuppressive therapy in a porcine pulmonary allograft model.

There is very limited published information testifying to the safety and possible complications of cell-based therapies. Accurately assessing the potential risks of translating novel, cell-based immunosuppressive protocols into clinical trials is therefore extremely difficult. This report describes the use of a pulmonary allograft model in outbred miniature pigs as a preliminary step in the development of a safe, clinically feasible, cell-based immunosuppressive protocol. Single lung transplants were performed in 22 MHC Class I-mismatched donor-recipient pairs, which were randomized between four treatment groups. For the first 28 days postoperatively, all animals were immunosuppressed with methylprednisilone and tacrolimus, with or without preoperative irradiation; subsequently, pharmacological immunosuppression was stopped in all treatment groups. Animals in two groups also received a central venous infusion of donor-derived 'transplant acceptance-inducing cells' (TAICs) on the seventh and 14th days postoperatively. Allograft survival was monitored by sequential chest X-rays, bronchoscopies and transbronchial biopsy histologies. No acute adverse events were associated with the administration of TAICs and there was no evidence of accelerated graft rejection. The observations presented in this report represent an important first step towards the development of a clinically applicable protocol for the use of TAIC therapy in lung transplantation.

Latent transforming growth factor binding protein 4 (LTBP-4) is downregulated in human mammary adenocarcinomas in vitro and in vivo.

Transforming growth factor beta (TGF-ss) is able to inhibit proliferation of epithelial cells and is involved in the carcinogenesis of human mammary tumours. Three latent transforming growth factor-beta binding proteins (LTBP-1, -3 and -4) are involved in TGF-beta function. The aim of the study was to analyze the expression profiles of TGF-beta 1 and 2 and LTBP-4 in human mammary carcinoma cell lines as well as in human mammary tumours. Expression analysis was performed at the transcription and protein level under in vivo and in vitro conditions. LTBP-4 expression was quantitatively analysed in human carcinomas of the mammary gland and in healthy mammary tissues of the same patients. Downregulation of LTBP-4 in all investigated human mammary tumours compared to normal tissues could be demonstrated. Results also revealed that protein levels of TGF-beta 1 are downregulated and of TGF-beta 2 are upregulated in human mammary carcinoma cell lines compared to primary (normal) human mammary epithelial cells. LTBP-4 reduction in neoplasms leads to a possible decrease of TGF-beta 1 extracellular deposition with reduced TGF-beta 1 bioavailability. TGF-beta 2 was upregulated, which indicates a possible compensatory mechanism. This study demonstrated a possible functional role of LTBP-4 for TGF-beta bioavailability with respect to carcinogenesis of human mammary tumours in vivo and in vitro.

Chemokine expression in hepatocellular carcinoma versus colorectal liver metastases.

AIM: To evaluate and compare the expression profiles of CXCL12 (SDF-1), CCL19 (MIP-3beta), CCL20 (MIP-3alpha) and CCL21 (6Ckine, Exodus2) and their receptors on RNA and protein levels in hepatocellular carcinoma (HCC) versus colorectal liver metastases (CRLM) and to elucidate their impact on the carcinogenesis and progression of malignant liver diseases. METHODS: Chemokine expression was analyzed by RT-PCR and ELISA in 11 cases of HCC specimens and in 23 cases of CRLM and corresponding adjacent non-tumorous liver tissues, respectively. Expressions of their receptors CXCR4, CCR6 and CCR7 were analyzed by RT-PCR and Western blot analysis in the same cases of HCC and CRLM. RESULTS: Significant up-regulation for CCL20/CCR6 was detected in both cancer types. Moreover, CCL20 demonstrated significant overexpression in CRLM in relation to the HCC tissues. Being significantly up-regulated only in CRLM, CXCR4 displayed an aberrant expression pattern with respect to the HCC tissues. CONCLUSION: Correlation of CXCR4 expression with CRLM suggests CXCR4 as a potential predictive factor for CRLM. High level expression of CCL20 and its receptor CCR6 in HCC and CRLM with marked up-regulation of CCL20 in CRLM in relation to HCC tissues indicates involvement of the CCL20/CCR6 ligand-receptor pair in the carcinogenesis and progression of hepatic malignancies.

Preoperative low-dose irradiation promotes long-term allograft acceptance and induces regulatory T cells in a porcine model of pulmonary transplantation.

BACKGROUND: A simplified conditioning protocol including single-dose preoperative thymic and low-dose whole body irradiation with or without subsequent donor bone marrow transplantation (BMTx) can be applied in porcine lung transplantation. We hypothesized that this protocol would prolong allograft survival. METHODS: Left-sided single lung transplantation from major histocompatibility complex (MHC)-mismatched donors was performed in 27 minipigs. Recipients received whole body (1.5 Gy) and thymic irradiation (7 Gy) before transplantation (IRR group, n=6), intravenous immunosuppression with methylprednisolone, cyclosporine, and azathioprine for 27 postoperative days (IS group, n=5) or both (IRR+IS group, n=10). BMTx group recipients were treated with irradiation, immunosuppression and a donor bone marrow infusion on postoperative day 1. Peripheral blood leukocyte phenotype and donor cell chimerism were monitored by flow cytometry. Purified CD25+ T cells from long-term survivors or rejecting animals were used for in vitro MLR suppression assays. RESULTS: Median graft survival was: IRR 12 days, IS 55 days, IRR+IS 239 days, and BMTx 80 days (P<0.0001). Early peripheral blood macrochimerism was substantial in both the IRR+IS and the BMTX group but was lost in all groups after day 80. The frequency and suppressive function of CD4+CD25+ T cells were enhanced in IRR+IS group long-term survivors. CONCLUSION: Although donor bone marrow infusion was not beneficial in our model, a substantial proportion of the animals treated with irradiation and a 28-day course of immunosuppression accepted their lung allografts long term. The mechanism involved in maintaining allograft tolerance may be based on peripheral T-cell regulation.

Involvement of chemokine receptor CCR6 in colorectal cancer metastasis.

Various chemokine receptors, namely CXCR4, CCR6 and CCR7, have recently been shown to be involved in the regulation of metastasis in malignant tumors. However, little is known about the role of these receptors in promoting tumor metastasis of colorectal cancer (CRC) to the primary site of CRC metastasis in the liver. To investigate this issue, we analyzed the expression of the chemokine receptors CXCR4, CCR6 and CCR7 in colorectal tumors and colorectal liver metastases. In the present study, 30 human cancer samples from colorectal tissue, 30 human samples from colorectal liver metastases and the adjacent nontumorous liver tissues were screened using quantitative real-time PCR, Western blot analysis, histochemistry, microdissection and the enzyme-linked immunosorbent assay (ELISA). While an overexpression of all the chemokine receptors was found in CRC, in colorectal liver metastases only the chemokine receptors CXCR4 and CCR6 were significantly upregulated. Consequently, we investigated the expression of the corresponding ligands CXCL12/SDF1alpha, CCL20/MIP3alpha, CCL19/MIP3beta and CCL21/6Ckine in various organs, such as the stomach, esophagus, pancreas, colon and rectum, in comparison with their expression in the liver as the primary site of metastatic spread in CRC. We found that only CCL20 exhibits peak levels of expression in the liver, thus indicating that an increased production of CCL20 may contribute to the selective recruitment of CCR6-expressing cancer cells in CRC. Furthermore, we could demonstrate that CRC patients who developed liver metastases express significantly more CCL20 and CCL21 in the liver in comparison with an unaffected control group. Therefore, our findings strongly suggest an association between CCL20/CCR6 expression in human CRC and the promotion of colorectal liver metastasis.