Elevated miR-124-3p in the aging colon disrupts mucus barrier and increases susceptibility to colitis by targeting T-synthase.

The risk of colitis and colorectal cancer increases markedly throughout adult life, endangering the health and lives of elderly individuals. Previous studies have proposed that bacterial translocation and infection are the main risk factors for these diseases. Therefore, in the present study, we aimed to identify the underlying mechanism by focusing on the mucus barrier function and mucin-type O-glycosylation. We evaluated alterations in the colon mucus layer in 2-, 16-, and 24-month-old mice and aged humans. Aged colons showed defective intestinal mucosal barrier and changed mucus properties. The miR-124-3p expression level was significantly increased in the aged distal colonic mucosa, which was accompanied by an increase in pathogens and bacterial translocation. Meanwhile, T-synthase, the rate-limiting enzyme in O-glycosylation, displayed an age-related decline in protein expression. Further experiments indicated that miR-124-3p modulated O-glycosylation by directly targeting T-synthase. Moreover, young mice overexpressing miR-124-3p exhibited abnormal glycosylation, early-onset, and more severe colitis. These data suggest that miR-124-3p predisposes to senile colitis by reducing T-synthase, and the miR-124-3p/T-synthase/O-glycans axis plays an essential role in maintaining the physiochemical properties of colonic mucus and intestinal homeostasis.

KITLG is a novel target of miR-34c that is associated with the inhibition of growth and invasion in colorectal cancer cells.

MiR-34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR-34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR-34c and KITLG mRNA expression levels in CRC cell lines, including HT-29, HCT-116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR-34c in the 3' untranslated region (3'UTR) of KITLG mRNA. A dual-luciferase reporter assay further confirmed that KITLG is a direct target of miR-34c. Then, the cell lines were infected with lentiviruses expressing miR-34c or a miR-34c specific inhibitor. Restoration of miR-34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR-34c increased the expression of KITLG protein. The miR-34c-mediated down-regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down-regulation for the tumour-suppressive effects of miR-34c in CRC cells. In conclusion, our results demonstrated that miR-34c might interfere with KITLG-related CRC and could be a novel molecular target for CRC patients.

Conditioned medium from human amniotic epithelial cells may induce the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells.

Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron-like cells from hUCB-MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB-MSCs with k252a, the Trk receptor inhibitor of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron-like cells was significantly decreased compared with ACM-treated hUCB-MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron-like cell differentiation compared with control. The transplantation of ACM-induced hUCB-MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron-like cells. In conclusion, we propose that hUCB-MSCs are a good source of DA neuron-like cells and that ACM is a potential inducer to obtain DA neuron-like cells from hUCB-MSCs in vitro for an ethical and legal cell therapy for PD.

Pleiotrophin is involved in the amniotic epithelial cell-induced differentiation of human umbilical cord blood-derived mesenchymal stem cells into dopaminergic neuron-like cells.

We have reported that human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are capable of differentiating into dopaminergic (DA) neuron-like cells upon being induced by amniotic epithelial cells (AECs). However, what factor(s) is involved in the differentiation process has not been explored out thoroughly. Because pleiotrophin (PTN) is known to exert important trophic effects on DA neurons, in the present study, we investigated whether PTN is released by AECs and whether it is involved in the differentiation of hUCB-MSCs into DA neuron-like cells. The expression and secretion of PTN by AECs were detected by immunofluorescence, RT-PCR and ELISA. The hUCB-MSCs were isolated and treated with AEC-conditioned medium (ACM) or recombinant human PTN. Compared to the controls, a higher proportion of treated cells differentiated into DA neuron-like cells, indicated by the increased expression of TH and DAT and the increased dopamine content. These results indicate that PTN released by AECs acts as a synergetic factor with other neurotrophic factors and is involved in the differentiation of hUCB-MSCs into DA neuron-like cells. We suggest that ACM, which contains PTN and other neurotrophic factors, could potentially be used as an agent to promote the differentiation of DA neuron-like cells from hUCB-MSCs for cell therapy of Parkinson's disease without creating legal or ethical issues.

[Surgical treatment for cancer of the pancreatic head].

OBJECTIVE: To explore and evaluate the therapeutic efficacy of surgical treatment for cancer of the pancreatic head. METHODS: The clinical data of 96 patients with cancer of the pancreatic head admitted in our hospital from January 2002 to December 2009 were retrospectively analyzed. pancreatoduodenectomy was performed in 48 cases, extended pancreatoduodenectomy in 30 cases, and Roux-Y cholangiojejunostomy in 18 cases. RESULTS: The 1, 2 and 3-year survival rates were 59.2%, 41.8% and 13.2%, respectively, in the patients treated with pancreatoduodenectomy, and 73.2%, 58.2% and 24.1%, respectively, in the patients treated with extended pancreatoduodenectomy. The 1, 2 and 3-year survival rates were 36.8%, 15.8% and 5.3%, respectively, in the patients with unresectable tumor who received radiotherapy and (or) chemotherapy in Roux-Y cholangiojejunostomy. The postoperative morbidity was 29.2%, 30.0% and 27.8% in the patients treated with pancreatoduodenectomy, extended pancreatoduodenectomy and Roux-Y cholangiojejunostomy, respectively. CONCLUSIONS: Pancreatoduodenectomy is the most effective treatment. Extended pancreatoduodenectomy can improve the surgical resection rate, reduce the recurrence rate and improve the survival rate. Internal drainage is an important palliative measure.

Co-expression of Japanese encephalitis virus prM-E-NS1 antigen with granulocyte-macrophage colony-stimulating factor enhances humoral and anti-virus immunity after DNA vaccination.

Japanese encephalitis virus (JEV) is an agent of Japanese encephalitis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) is an attractive DNA vaccine adjuvant for its antigen presentation. In the present study, we have constructed DNA vaccines that carried JEV prM-E-NS1 genes with or without the GM-CSF gene. Immunization with the bicistronic plasmid pCAG-JEGM that co-expresses GM-CSF and viral prM-E-NS1, resulted in the highest IgG response and sufficient protection against virus-challenged BALB/c mice. However, much to our surprise, co-inoculation of the GM-CSF plasmid with the pCAG-JE plasmid expressing viral prM-E-NS1 lead to a low antibody titer and a relatively low survival rate. Moreover, anamnestic antibody-mediated protection played a dominant role in the mice JEV challenge model, according to the enhancement of post-challenge neutralizing antibody titers and further adoptive transfer experiments. Taken together, this study should encourage further development of JEV DNA vaccine strategies and caution against the use of cytokines as an adjuvant.

Plasticity of interstitial cells of cajal: a study in the small intestine of adult Guinea pigs.

Although it is well known that the reduction of interstitial cells of Cajal (ICCs) is associated with several gastrointestinal motility disorders in clinic, it is unknown whether the mature ICCs still have an active plasticity in adult mammals. This study focused on the issues of the reduction of ICCs during Imatinib administration and the recovery of ICCs following drug withdrawal in the small intestine of adult guinea pigs. ICCs were revealed by immunofluorescence on whole mount preparations with anti-Kit, alpha-smooth muscle actin, (alpha-SMA), and 5-bromo-2'-deoxyuridine (BrdU) antibodies. Moreover, the occurrence of apoptosis was also assayed. Imatinib treatment led to a gradual reduction of ICCs in number around the myenteric plexus and deep muscular plexus, which was dependent on the time but no apoptosis of ICCs was detected with the TUNEL method. During Imatinib treatment, some ICC-like cells were double labeled for Kit and alpha-SMA and a few ICC-like cells were only stained with alpha-SMA. When Imatinib was discontinued, the number of ICCs recovered to normal within 32 days. During this time, some proliferating ICCs were demonstrated by double labeling with Kit and BrdU antibodies. Our results indicated that Kit signaling was essential for the maintenance of survival and proliferation of the mature ICCs in the small intestine of adult guinea pigs. Moreover, ICCs might transdifferentiate to a type of alpha-SMA(+) cells, perhaps a phenotype of smooth muscle cells, when there is a loss-of-function of Kit.

Distribution of the interstitial Cajal-like cells in the gallbladder and extrahepatic biliary duct of the guinea-pig.

It has been suggested that interstitial Cajal-like cells (ICLC) may be involved in the spontaneous rhythmic electrical activities of the extrahepatic bile duct system. The present study investigated the distribution and characteristics of ICLC, which are immunopositive for CD117/ Kit receptor tyrosine kinase, using immunohistochemistry employing a monoclonal antibody raised against CD117/Kit on whole-mount preparations. The Kit-positive ICLC were examined using confocal laser scanning microscopy or fluorescence microscopy. ICLC, immunoreactive for Kit, were pleiomorphic and/or spindle-shaped cells with a few bipolar processes and distributed in the smooth muscle layers of the gallbladder and bile duct system. They were scattered in the hepatic duct, cystic duct and gallbladder as well as in the upper part of the common bile duct. The ICLC gradually increased in number and formed a completed cellular network in the lower part of the common bile duct and ampulla. The numbers of ICLC in the ampulla were similar to that of the duodenum and significantly much greater in number than in the gallbladder and bile ducts. The density of the ICLC in the common bile duct was significantly higher than that of other bile ducts. Our results suggested that the ICLC might contribute to the regulation of the spontaneous rhythmic contraction and development of motility disorders of the bile duct system.

Interstitial cells of Cajal could regenerate and restore their normal distribution after disrupted by intestinal transection and anastomosis in the adult guinea pigs.

Surgical manipulations of the gastrointestinal (GI) tract usually lead to loss of interstitial cells of Cajal (ICCs). The present study prepared to investigate whether ICCs can regenerate and restore their normal distribution up to 5 months after semitransection and end-to-end anastomosis of small intestines of adult guinea pigs. The segments of anastomosis were studied by immunohistochemistry with anti-KIT, 5-bromo-2'-deoxyuridine (BrdU), stem cell factor (SCF), and neurofilament 200 antibodies and also by transmission electron microscopy (TEM). At early stage, intestinal surgery led to intestinal wall impairment and ICCs loss, and ICCs near the site of anastomosis gradually increased in numbers. About 150 days postoperation, the distribution of ICCs and the microstructure of intestinal wall appeared to be similar with those of the control. By double immunostaining with BrdU and KIT antibodies, a number of proliferated ICCs were seen near the site of transection/anastomosis. Furthermore, KIT ligand, SCF, was mainly observed in the smooth muscle cells (SMCs), which are located close to ICCs. TEM observation revealed a number of immature and mature ICCs in this region. Our results indicated that ICCs could regenerate and restore their normal distribution after intestinal surgery and SMCs might be involved in the regenerated events of ICCs in the adult guinea pig GI tract.

JAK-STAT signaling pathway in pulmonary arterial smooth muscle cells is activated by hypoxia.

Pulmonary arterial smooth muscle cells (PASMC) were divided into a normoxic group (N), 2, 8 and 12 h hypoxic groups (H2, H8 and H12) and an AG490 plus 8 h hypoxic group (AG490). The expression of JAK1, JAK2, JAK3 and TYK2 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). STAT1 and STAT3 protein expressions were determined by Western blotting. The results showed that the levels of JAK1, JAK2 and JAK3 mRNA did not change significantly in the N group but were increased after 2 h exposure to hypoxia. JAK1, JAK2 and JAK3 mRNA expressions peaked at 8 h. It decreased at 12 h but remained above those in the N group. TYK2 mRNA was not found in either hypoxic or normal PASMC. The phospho-STAT1 and -STAT3 protein levels increased after 2 h exposure to hypoxia. They were about 2.8 and 4.1 times the N group, respectively, after 8 h. They decreased at 12 h but remained above those in the N group. AG490 inhibited phospho-STAT3 protein expression by about 25.5% at 8 h but did not block the expression of phospho-STAT1 protein. These findings suggest that hypoxia induces the expression of JAK1, JAK2, JAK3 and phospho-STAT1 and -STAT3 in PASMC. Hypoxia activates the JAKs-STATs signaling pathway, which may participate in the pathogenesis of hypoxic PASMC injury.

Distribution and differences of estrogen receptor beta immunoreactivity in the brain of adult male and female rats.

Studies have shown that estrogen plays important roles in regulating neural structure and function in the brain, but the mechanism remains unclear. The actions of estrogen were thought to be mediated by a single estrogen receptor until the identification of another estrogen receptor, namely estrogen receptor-beta (ER-beta). Here we report a comprehensive study of the localization of ER-beta immunoreactivity and differences in the brains of adult male and female rats on the basis of a nickel ammonium sulfate-enhanced immunocytochemical method using a polyclonal antiserum sc-8974. The results of these studies revealed: (1) ER-beta immunoactive material was mainly localized in the neuronal nucleus, but it was also detectable in the cytoplasm and neuronal processes; (2) in both male and female rats, high levels of ER-beta immunopositive signals were detected in the anterior olfactory nucleus, cerebral cortex, Purkinje cells, vertical limb of the diagonal band, red nucleus, locus ceruleus, and motor trigeminal nucleus. Moderate levels were found in the medial septum, lateral amygdaloid nucleus, substantia nigra, and central gray. Weak signals were localized in other subregions of the hypothalamus and amygdaloid complex; (3) there was an obvious difference of ER-beta immunoreactivity between male and female rats, and its intracellular distribution also showed a sex difference. The above results provide the first detailed evidence that ER-beta protein is widely distributed in both male and female rat brains, but that distinctive sex differences also exist. Estrogen may exert its function in different brain regions in a gender-specific manner.

Immunocytochemical localization of estrogen receptor beta in the rat brain.

The localization of the new estrogen receptor, ER-beta, in the rat brain was studied by immunocytochemical technique, and the results revealed that ER-beta immunoactive material was predominantly localized in the neuronal nucleus, but it was also detectable in the cytoplasm and neuronal processes. High levels of ER-beta immunopositive signals were detected in the cerebral cortex, vertical limb of the diagonal band, Purkinje cells, locus ceruleus, and motor trigeminal nucleus. Moderate levels were found in the medial septum, lateral amygdaloid nucleus, substantia nigra, and central gray. Weak signals were localized in some subregions of the hypothalamus and amygdaloid complex. Some differences of the expression of ER-beta immunoreactivity between male and female rats were also noticed. The above results provide the first detailed evidence that ER-beta protein is widely distributed in the rat brain, and ER-beta may be involved in some important brain function such as learning and memory.