The ideal treatment and recovery of osteoarticular injury remain to be resolved. Small intestinal submucosa (SIS), a naturally-occurring decellularized extracellular matrix, has been recognized as an ideal scaffold for tissue engineering and widely used in repairing various tissues and organs. Nowadays its application has also been gradually increased in the field of orthopedics. We reviewed laboratorial studies and clinical trails about the application of SIS in bone and joint repair, aiming to evaluate its effects on the repair of bone, cartilage, meniscus, ligament and tendon. SIS has showed promising results in repairing bone, meniscus, ligament or tendon. However, additional studies will be required to further evaluate its effects on articular cartilage and tendon-bone healing. How to optimize SIS material,is also a focused problem concerned with making SIS a potential therapeutic option with high value for orthopedic tissue repair.
Animals ; Cell- and Tissue-Based Therapy ; Humans ; Intestinal Mucosa ; cytology ; Intestine, Small ; cytology ; Joint Diseases ; physiopathology ; surgery ; therapy ; Tissue Engineering ; instrumentation ; methods ; Tissue Scaffolds ; chemistry

OBJECTIVETo study the effect of hypoxia on cofilin activation in intestinal epithelial cells and its relation with distribution of tight junction protein zonula occludens 1 (ZO-1).

METHODSThe human intestinal epithelial cell line Caco-2 was used to reproduce monolayer cells. The monolayer-cell specimens were divided into control group (no treatment), hypoxic group ( exposed to hypoxia), and normoxic group (exposed to normoxia) according to the random number table. Western blotting was used to detect the protein expressions of cofilin and phosphorylatedl cofilin (p-cofilin) of cells in normoxic group and hypoxic group exposed to normoxia or hypoxia for 1, 2, 6, 12, and 24 h and control group, with 9 samples in control group and 9 samples at each time point in the other two groups. The other monolayer-cell specimens were divided into hypoxic group (exposed to hypoxia) and control group (no treatment) according to the random number table. Cells in hypoxic group exposed to hypoxia for 1, 2, 6, 12, and 24 h and control group were obtained. Morphology and distribution of F-actin was observd with laser scanning confocal microscopy, the ratio of F-actin to G-actin was determined by fluorescence method, and distribution of ZO-l and cellular morphology were observed with laser scanning confocal microscopy. The sample number of last 3 experiments was respectively 3, 6, and 3 in both hypoxic group (at each time point) and control group. Data were processed with paired ttest, analysis of variance of repeated measurement, and LSD-t test.

RESULTSThe protein expressions of cofilin and p-cofilin of cells between normoxic group exposed to normoxia for 1 to 24 h and control group showed no significant changes (with values from -0.385 to 1.701, t(p-cofilin)values from 0. 040 to 1.538, P values above 0.05). There were no obvious differences in protein expressions of en filmn of cells between hypoxic group exposed to hypoxia for 1 to 24 h and control group ( with values from 1.032 to 2.390, P values above 0.05). Compared with that in control group, the protein expressions of p-cofilin of cells were greatly reduced in hypoxic group exposed to hypoxia for 1 to 24 h (with values from 4.563 to 22.678, P values below 0.01), especially exposed to hypoxia for 24 h. The protein expressions of cofilin of cells between normoxic group and hypoxic group at each time point were close ( with t values from -0.904 to 1.433, P values above 0.05). In hypoxic group, the protein expressions of p-cofilin of cells exposed to hypoxia for 1, 2, 6, 12, and 24 h were 0.87 +/- 08, 0.780 .05, 0.89 +/- 0.07, 0.68+0. 07, and 0.57 +/- 0.06, respectively, significantly lower than those in normoxic group (0.90 +/- 0.07, 0.97 +/- 0.06, 1.00 +/- 0.06, 1.00 +/- 0.05, and 0.99 +/- 0.05, with t values from 3.193 to 16.434, P values below 0.01). In control group, F-actin in the cytoplasm was abundant, most of it was in bunches. The trend of F-actin was disorderly in hypoxic group from being exposed to hypoxia for 1 h, shortened in length or even dissipated. The ratios of F-actin to G-actin of cells in hypoxic group exposed to hypoxia for 12 and 24 h (0.89 +/- 0.12 and 0.84 +/- 0.19) were obviously decreased as compared with that in control group (1. 00, with t values respectively 3. 622 and 3. 577, P values below 0.01). There were no obvious differences in the ratios of F-actin to G-actin of cells between hypoxic group exposed to hypoxia for 1, 2, and 6 h and control group ( with values from 0.447 to 1.526, P values above 0.05). In control group, cells were compact in arrangement, and ZO-1 was distributed continuously along the cytomnembrane. From being exposed to hypoxia for 2 h, cells became irregular in shape in hypoxic group. ZO-1 was distributed in discontinuous fashion along the cytomembrane with breakage in hypoxic group exposed to hypoxia for 24 h.

CONCLUSIONSHypoxia may cause the disorder of dynamic balance between F-actin and G-actin by inducing cofilin activation, which in turn leads to the changes in distribution of tight junction protein ZO-1 in intestinal epithelial cells.

Actin Depolymerizing Factors ; Actins ; Blotting, Western ; Caco-2 Cells ; drug effects ; physiology ; Epithelial Cells ; cytology ; drug effects ; Humans ; Hypoxia ; metabolism ; Intestinal Mucosa ; drug effects ; metabolism ; pathology ; Intestines ; Oxygen ; pharmacology ; Tight Junctions ; drug effects ; metabolism ; Zonula Occludens-1 Protein ; metabolism

Hypercoagulable state and thrombosis are major lethal causes of ulcerate colitis (UC). The aim of the present study is to explore the change and role of protein C (PC) system in UC thrombosis. 4% dextran sulfate sodium (DSS) was used to induce the UC model, and the body weight, the length of colon, and the weight of spleen were measured after intake of DSS as drinking water for 1 week. The macroscore and microscore were examined. The quantity of macrophage in colon smooth muscle was observed by immunofluorescence, and TNF-α and IL-6 levels in plasma were evaluated by ELISA. Intravital microscopy was applied to observe colonic mucosal microvascular circulation, activities of PC and protein S (PS) were determined by immunoturbidimetry, endothelial cell protein C receptor (EPCR) and thrombomodulin (TM) expressions were detected by immunohistochemistry. In vitro, TNF-α and IL-6 levels were tested in supernatant of macrophage separated from colonic tissue. After stimulation of mouse colonic mucosa microvascular endothelial cells by TNF-α and IL-6 respectively, the activities of PC, PS, activated protein C (APC) were evaluated, and the expressions of EPCR and TM were detected by Western blotting. The results revealed that compared with control, the DSS mouse showed weight loss (P < 0.05), a shortened colon (P < 0.05), and swelled spleen (P < 0.05), accompanied by higher histological score (P < 0.05), as well as infiltration of macrophages, elevated TNF-α and IL-6 levels in plasma (P < 0.01). The intravital microscopy results revealed that compared with control, DSS mice showed significantly enhanced adhesion of leukocytes and colonic mucosal microvascular endothelial cells (P < 0.01), meanwhile, decreased activity of PC and PS in plasma (P < 0.01 or P < 0.05), and down-regulated expression of EPCR (P < 0.01). The degree of inflammation was negatively correlated with the PC activity. In vitro, TNF-α and IL-6 levels were increased in the supernatant of macrophages from DSS mice colonic tissue (P < 0.05), and after incubation of TNF-α or IL-6 with colonic mucosal microvascular endothelial cells, the APC activity was decreased (P < 0.05 or P < 0.01), and expression of EPCR was down regulated (P < 0.05). These results suggest that PC system is inhibited in UC mouse. Presumably, the mechanism may be due to the secretion of cytokines from macrophages and subsequential influence on the function of endothelia cells. Furthermore, enhancement of PC system activity may serve as a new strategy for the treatment of UC.
Animals ; Blood Coagulation Factors ; metabolism ; Colitis, Ulcerative ; chemically induced ; physiopathology ; Dextran Sulfate ; Immunohistochemistry ; Inflammation ; Interleukin-6 ; blood ; Intestinal Mucosa ; pathology ; Macrophages ; cytology ; Mice ; Protein C ; metabolism ; Receptors, Cell Surface ; metabolism ; Spleen ; pathology ; Tumor Necrosis Factor-alpha ; blood

To achieve immune homeostasis in such a harsh environment as the intestinal mucosa, both active and quiescent immunity operate simultaneously. Disruption of gut immune homeostasis leads to the development of intestinal immune diseases such as colitis and food allergies. Among various intestinal innate immune cells, mast cells (MCs) play critical roles in protective immunity against pathogenic microorganisms, especially at mucosal sites. This suggests the potential for a novel MC-targeting type of vaccine adjuvant. Dysregulated activation of MCs also results in inflammatory responses in mucosal compartments. The regulation of this yin and yang function of MCs remains to be elucidated. In this review, we focus on the roles of mucosal MCs in the regulation of intestinal allergic reaction, inflammation and their potential as a new target for the development of mucosal adjuvants.
Adjuvants, Immunologic/*therapeutic use ; Animals ; Humans ; Hypersensitivity/*immunology/prevention & control ; Inflammation/immunology/metabolism/prevention & control ; Intestinal Mucosa/cytology/*immunology ; Mast Cells/*immunology

Dendritic cells (DCs) are key modulators that shape the immune system. In mucosal tissues, DCs act as surveillance systems to sense infection and also function as professional antigen-presenting cells that stimulate the differentiation of naive T and B cells. On the basis of their molecular expression, DCs can be divided into several subsets with unique functions. In this review, we focus on intestinal DC subsets and their function in bridging the innate signaling and adaptive immune systems to maintain the homeostasis of the intestinal immune environment. We also review the current strategies for manipulating mucosal DCs for the development of efficient mucosal vaccines to protect against infectious diseases.
Animals ; Dendritic Cells/*immunology/metabolism ; Humans ; Immunity, Mucosal ; Intestinal Mucosa/cytology/*immunology ; T-Lymphocytes, Helper-Inducer/immunology

Vaccination is one of the most successful applications of immunology and for a long time has depended on parenteral administration protocols. However, recent studies have pointed to the promise of mucosal vaccination because of its ease, economy and efficiency in inducing an immune response not only systemically, but also in the mucosal compartment where many pathogenic infections are initiated. However, successful mucosal vaccination requires the help of an adjuvant for the efficient delivery of vaccine material into the mucosa and the breaking of the tolerogenic environment, especially in oral mucosal immunization. Given that M cells are the main gateway to take up luminal antigens and initiate antigen-specific immune responses, understanding the role and characteristics of M cells is crucial for the development of successful mucosal vaccines. Especially, particular interest has been focused on the regulation of the tolerogenic mucosal microenvironment and the introduction of the luminal antigen into the lymphoid organ by exploiting the molecules of M cells. Here, we review the characteristics of M cells and the immune regulatory factors in mucosa that can be exploited for mucosal vaccine delivery and mucosal immune regulation.
Administration, Oral ; Animals ; Antigens, Bacterial/*immunology ; Antigens, Viral/*immunology ; Bacterial Vaccines/administration & dosage/*immunology ; Humans ; Immunity, Mucosal ; Intestinal Mucosa/cytology/*immunology ; Peyer's Patches/cytology/*immunology ; Viral Vaccines/administration & dosage/*immunology

BACKGROUND/AIMS: Doublecortin and CaM kinase-like-1 (DCAMKL1) is a marker of stem cells expressed predominantly in the crypt base in the intestine. However, DCAMKL1-positive cells have been shown to be differentiated tuft cells rather than quiescent progenitors. Tuft cells are the only epithelial cells that express cyclooxygenase 2 (COX-2) in the normal intestinal epithelium. We previously generated Cdx2-transgenic mice as model mice for intestinal metaplasia and gastric carcinoma. In the current study, we investigated the association between COX-2 and DCAMKL1 in gastric carcinoma. METHODS: We examined the association between COX-2 and DCAMKL1 expression in gastric carcinomas in clinical samples (early gastric well-differentiated adenocarcinoma) and Cdx2-transgenic mice; and the DCAMKL1-transgenic mouse stomach using immunohistochemistry and quantitative real-time polymerase chain reaction. RESULTS: The COX-2-expressing cells were scattered, not diffusely expressed, in gastric carcinomas from humans and Cdx2-transgenic mice. DCAMKL1-positive cells were also scattered in the gastric carcinomas, indicating that tuft cells could still be present in gastric carcinoma. COX-2 was expressed in DCAMKL1-positive tuft cells in Cdx2- and DCAMKL1-transgenic mouse stomachs, whereas the Sox9 transcription factor was ubiquitously expressed in gastric carcinomas, including COX-2-positive cells. CONCLUSIONS: COX-2 is expressed in DCAMKL1-expressing quiescent tuft cells in gastric carcinoma.
Adenocarcinoma/metabolism ; Animals ; Cyclooxygenase 2/genetics/*metabolism ; Epithelial Cells/metabolism ; Gastric Mucosa/metabolism ; Humans ; Intestinal Mucosa/cytology/*enzymology/metabolism ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Mice ; Mice, Transgenic ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; SOX9 Transcription Factor/genetics/metabolism ; Stomach Neoplasms/*enzymology/genetics

OBJECTIVETo study the protective effects of sacral nerve root electrostimulation on intestinal mechanical barrier in rats with spinal cord injury (SCI).

METHODSFifty six Wistar rats were divided into normal group, SCI control group and SCI group with sacral nerve root electrostimulation (8 rats in each subgroup at 24, 48, 72 h after spinal cord injury). The following experiments were performed respectively in rats from the 3 groups: bacteria culture from intestinal mesentery lymph nodes, liver, spleen, intestinal morphology observation and detection the protein expression level of ZO-1.

RESULTSThe intestinal mucosa appeared different degree of damage in SCI control group; cell-cell connections between intestinal epithelial cells were destroyed; Endotoxin levels in blood and the number of bacterial translocation increased obviously. Sacral nerve stimulation was found toimprove the intestinal mucosal, reduce the endotoxin content in the blood to normal level and the decrease the incidences of bacterial translocation of the gut origin. The expression of tight junction protein ZO-1 of rat intestinal tissue had no statistical differences among the 3 groups. On the other hand, the distribution of tight junction protein ZO-1 appeared different degrees of scattered and irregular in the control group while that in the experimental group appeared different degree of improvement as determined by the immunohistochemistry of rat intestinal tissue.

CONCLUSIONsacral nerve root electrostimulation can rehabilitate the peristalsis of denervated colon, promote defeacation and decrease bacterial amount, protection of the intestinal mechanical barrier between intestinal epithelial cells and tight junction, reducing the endotoxin content in the blood and suppressing bacterial translocation from the gut.

Animals ; Bacterial Translocation ; Electric Stimulation Therapy ; Endotoxins ; blood ; Epithelial Cells ; cytology ; Intestinal Mucosa ; physiology ; Peristalsis ; Rats ; Rats, Wistar ; Spinal Cord ; Spinal Cord Injuries ; physiopathology ; Zonula Occludens-1 Protein ; metabolism

OBJECTIVETo observe the effect of SIRT1 on intestinal barrier function of epithelial Caco-2 cells under hypoxia and investigate its mechanism.

METHODSCaco-2 cells were randomly divided into three groups: normoxia group (Nx), hypoxia group (Hx,1%O2 for 6 h) and hypoxia plus 40 μmol/L Resveratrol (agonist of SIRT1) group (Hx+Res). Transepithelial electrical resistance (TER) was determined. mRNA and protein expressions of SIRT1 and tight junctions (ZO-1, Occludin, Claudin-1) were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSBoth mRNA and protein expressions of SIRT1 were significantly reduced in Hx group as compared with Nx group (0.40±0.02 vs. 0.70±0.07, P=0.001; 0.37±0.03 vs. 0.76±0.03, P=0.001). The mRNA and protein expressions of SIRT1 were significantly increased in Hx+Res group as compared with Hx group(0.50±0.02 vs. 0.40±0.02, P=0.026; 0.54±0.02 vs. 0.37±0.03, P=0.011). The expression levels of ZO-1, Occludin and Claudin-1 in Hx group were lower than those in Nx group (P<0.05), however, pretreatment with Resveratrol could attenuate the decreased expression of above 3 molecules under hypoxia(P<0.05). TERs of Nx group, Hx group and Hx+Res group were (142±7) Ohm/cm(2), (94±3) Ohm/cm(2) and (119±7) Ohm/cm(2) respectively. Compare with the Nx group, the TER of Hx group was significantly decreased(P<0.05). TER of Hx+Res group was significantly increased compare with Hx group, but it was still significantly lower than that in Nx group(P<0.05).

CONCLUSIONSExpression of SIRT1 is significantly reduced under hypoxia. Activation of SIRT1 can maintain the epithelial barrier function through regulating the expression of tight junctions under hypoxia.

Caco-2 Cells ; Cell Hypoxia ; Claudin-1 ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Intestinal Mucosa ; cytology ; Occludin ; metabolism ; Sirtuin 1 ; metabolism ; Zonula Occludens-1 Protein ; metabolism

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.
Caco-2 Cells ; Calcium-Binding Proteins ; Calpain/genetics/metabolism ; Caspase 3/genetics/metabolism ; Caspases ; *Cell Death ; Colon/cytology ; Entamoeba histolytica/*physiology ; Epithelial Cells/cytology/parasitology ; Humans ; I-kappa B Proteins/metabolism ; Intestinal Mucosa/cytology ; NF-kappa B/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor/genetics/*metabolism ; STAT5 Transcription Factor/genetics/*metabolism ; Signal Transduction