Vitamin D receptor expression in SLE peripheral blood CD4+T cells is associated with disease activity and cell apoptosis.

OBJECTIVES: Systemic lupus erythematosus (SLE) is characterised by accumulated cell apoptosis. Vitamin D receptor (VDR) has immunomodulatory effect and potent anti-apoptosis activities. The aim of this study was to examine the correlation between CD4+T cells VDR expression, cell apoptosis, and disease activity in patients with SLE. METHODS: Forty-five SLE patients were recruited and 50 healthy individuals served as controls. The expression of VDR in CD4+T cells and their subsets were determined by flow cytometry. The correlations between VDR expression and cell apoptosis or disease parameters in SLE patients were analysed. RESULTS: VDR expression in CD4+T cells and their subsets were upregulated in SLE patients, especially in help T (Th)1, regulatory T (Treg), and follicular helper T (Tfh) cells. Frequency of VDR-positive CD4+T cells was positively associated with SLE disease activity index (SLEDAI)-2K values and inversely correlated with serum C3 concentration. The frequency of VDR-positive CD4+T cells, Th1 cells, Th2 cells, Th17 cells, Treg cells, and Tfh cells was positively correlated with cells apoptosis. CONCLUSION: VDR expression in CD4+T cells and their subsets were increased in SLE. VDR expression was positively associated with disease activity and cell apoptosis in SLE patients.

Mesenchymal stem cells prevent podocyte injury in lupus-prone B6.MRL-Faslpr mice via polarizing macrophage into an anti-inflammatory phenotype.

BACKGROUND: Podocyte injury plays a pathogenic role in the development of lupus nephritis (LN). Mesenchymal stem cells (MSCs) have shown promising therapeutic potential for LN. However, whether MSCs can prevent podocyte injury in LN remains unknown. METHODS: Human umbilical cord-derived MSCs (UC-MSCs) were infused into lupus-prone B6.MRL-Faslpr (B6.lpr) mice to investigate the influences of UC-MSCs on podocyte injury in LN. Podocytes and macrophages were co-cultured with UC-MSCs in vitro to study the mechanism by which UC-MSC protect podocytes. We further explored the effects of UC-MSCs on macrophage polarization. RESULTS: We found that UC-MSCs promoted the expression of podocyte-specific markers, podocin and synaptopodin, in lupus-prone B6.lpr mice, along with the improvement of lupus renal pathology in terms of reduced IgG and C3 deposition in glomeruli and decreased anti-dsDNA antibody level. Besides, UC-MSC treatment decreased podocyte foot process effacement, as UC-MSCs-treated macrophages led to less podocyte injury in vitro. Interestingly, we further found that UC-MSCs-treated macrophages exhibited an anti-inflammatory phenotype with higher expression of CD206, and lower expression of tumor necrosis factor-α and interleukin-1ß. Additionally, UC-MSCs-treated lupus mice showed reduced renal macrophage infiltration and elevated CD206 expression in kidney. CONCLUSIONS: Our results demonstrated that UC-MSCs ameliorated LN by preventing podocyte injury possibly through reducing macrophage infiltration and polarizing macrophage into an anti-inflammatory phenotype.

Effects of allogeneic mesenchymal stem cell transplantation in the treatment of liver cirrhosis caused by autoimmune diseases.

AIM: There has been great interest in recent years to take advantage of mesenchymal stem cells (MSCs) to treat end-stage liver disease. This study is aimed to evaluate clinical therapeutic effects of allogeneic MSC transplantation in liver cirrhosis caused by autoimmune diseases. METHODS: The enrolled patients with liver cirrhosis were assigned to receive allogeneic MSC infusions through a peripheral vein. The primary objective of this study was to assess the safety and effectiveness of MSCT in patients with autoimmune diseases-induced cirrhosis. Secondary endpoints were to assess changes in the Models of End Stage Liver Disease (MELD) scores and liver functions after the transplantation. RESULTS: A total of 26 patients were enrolled. Of these, 23 patients received umbilical cord MSCT, two received cord blood MSCT and one received bone marrow MSCT. Three patents died of the complications caused by cirrhosis and two patients received liver transplantation after MSCT. Four patients were lost to follow-up. The mean of alanine transaminase values decreased 6 months, 1 and 2 years after the transplantation, but there were no statistical significance. The mean value of total bilirubin decreased at 6 months and 1 year follow-up. Average serum albumin levels improved at 6 months, 1 and 2 years follow-up. The mean value at 2 years increased significantly compared with the baseline value. A lowering of prothrombin time was seen at 6 months after MSCT. MELD score improved at 6 months, 1 and 2 years of follow-up. No serious adverse events were observed during or 24 h after infusions of MSCs in any of the 26 patients with liver cirrhosis. CONCLUSION: Based on this clinical trial, allogeneic MSCT through the peripheral vein probably is safe and seemingly has beneficial effect in patients with liver cirrhosis. Therefore, allogeneic MSCT is a potential option for treatment of liver cirrhosis caused by autoimmune diseases. Further studies with higher numbers of patients are warranted to better clarify the impact and mechanisms of MSCT in liver cirrhosis.

Human Umbilical Cord Mesenchymal Stem Cells Inhibit T Follicular Helper Cell Expansion Through the Activation of iNOS in Lupus-Prone B6.MRL-Faslpr Mice.

The aberrant generation or activation of T follicular helper (Tfh) cells contributes to the pathogenesis of systemic lupus erythematosus (SLE), yet little is known about how these cells are regulated. In this study, we demonstrated that the frequency of Tfh cells was increased in lupus-prone B6.MRL-Faslpr (B6.lpr) mice and positively correlated to plasma cell proportions and serum total IgG as well as anti-dsDNA antibody levels. Transplantation of mesenchymal stem cells derived from Wharton's jelly of human umbilical cords (hUC-MSCs) ameliorated lupus symptoms in B6.lpr mice, along with decreased percentages of Tfh cells. In vitro studies showed that the differentiation and proliferation of Tfh cells were markedly suppressed by hUC-MSCs. The production of inducible nitric oxide synthase (iNOS) was dramatically upregulated in hUC-MSCs when cocultured with CD4+ T cells directly, while adding the specific inhibitor of iNOS into the coculture system significantly reversed the inhibitory effect of hUC-MSCs on Tfh cell generation. Interestingly, the efficacy of hUC-MSCs in inhibiting Tfh cells was impaired in the Transwell system, with the reduction of iNOS in both mRNA and protein levels. Taken together, our findings suggest that hUC-MSCs could effectively inhibit Tfh cell expansion through the activation of iNOS in lupus-prone B6.lpr mice, which is highly dependent on cell-to-cell contacts.

Long-term safety of umbilical cord mesenchymal stem cells transplantation for systemic lupus erythematosus: a 6-year follow-up study.

The aim of this study is to assess the long-term safety of allogeneic umbilical cord mesenchymal stem cells (UC MSCs) transplantation for patients with refractory systemic lupus erythematosus (SLE). Nine SLE patients, who were refractory to steroid and immunosuppressive drugs treatment and underwent MSCs transplantation in 2009, were enrolled. One million allogeneic UC MSCs per kilogram of body weight were infused intravenously at days 0 and 7. The possible adverse events, including immediately after MSCs infusions, as well as the long-term safety profiles were observed. Blood and urine routine test, liver function, electrocardiogram, chest radiography and serum levels of tumor markers, including alpha fetal protein (AFP), cancer embryo antigen (CEA), carbohydrate antigen 155 (CA155) and CA199, were assayed before and 1, 2, 4 and 6 years after MSCs transplantation. All the patients completed two times of MSCs infusions. One patient had mild dizzy and warm sensation 5 min after MSCs infusion, and the symptoms disappeared quickly. No other adverse event, including fluster, headache, nausea or vomit, was observed. There was no change in peripheral white blood cell count, red blood cell count and platelet number in these patients after followed up for 6 years. Liver functional analysis showed that serum alanine aminotransferase, glutamic-oxalacetic transaminase, total bilirubin and direct bilirubin remained in normal range after MSCs infusions. No newly onset abnormality was detected on electrocardiogram and chest radiography. Moreover, we found no rise of serum tumor markers, including AFP, CEA, CA125 and CA199, before and 6 years after MSCs infusions. Our long-term observational study demonstrated a good safety profile of allogeneic UC MSCs in SLE patients.

Berberine Prevents Intestinal Mucosal Barrier Damage During Early Phase of Sepsis in Rat through the Toll-Like Receptors Signaling Pathway.

Our previous study has shown berberine prevents damage to the intestinal mucosal barrier during early phase of sepsis in rat through mechanisms independent of the NOD-like receptors signaling pathway. In this study, we explored the regulatory effects of berberine on Toll-like receptors during the intestinal mucosal damaging process in rats. Male Sprague-Dawlay (SD) rats were treated with berberine for 5 d before undergoing cecal ligation and puncture (CLP) to induce polymicrobial sepsis. The expression of Toll-like receptor 2 (TLR 2), TLR 4, TLR 9, the activity of nuclear factor-kappa B (NF-κB), the levels of selected cytokines and chemokines, percentage of cell death in intestinal epithelial cells, and mucosal permeability were investigated at 0, 2, 6, 12 and 24 h after CLP. Results showed that the tumor necrosis factor-α (TNF-α ) and interleukin-6 (IL-6) level were significantly lower in berberine-treated rats compared to the control animals. Conversely, the expression level of tight junction proteins, percentage of cell death in intestinal epithelial cells and the mucosal permeability were significantly higher in berberine-treated rats. The mRNA expression of TLR 2, TLR 4, and TLR 9 were significantly affected by berberine treatment. Our results indicate that pretreatment with berberine attenuates tissue injury and protects the intestinal mucosal barrier in early phase of sepsis and this may possibly have been mediated through the TLRs pathway.

Berberine prevents damage to the intestinal mucosal barrier during early phase of sepsis in rat through mechanisms independent of the NOD-like receptors signaling pathway.

NOD-like receptors play a crucial role in host defense against intestinal infection. We explored the regulatory effects of berberine on NLRs during the intestinal mucosal damaging process in rats. Male Sprague-Dawlay (SD) rats were treated with berberine for 5d before undergoing cecal ligation and puncture (CLP) to induce polymicrobiol sepsis. The expression of nucleotide-binding oligomerization domain 2 (NOD2), NLR family-pyrin domain containing 3 (NLRP3), the activity of nuclear factor-kappa B (NF-κB), the levels of selected cytokines and chemokines, percentage of cell death in intestinal epithelial cells, and mucosal permeability were investigated at 0h, 2h, 6h, 12h and 24h after CLP. Results showed that the Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) level in were significantly lower in berberine treated rats compared to the control animals. The tight junction proteins level, percentage of cell death in intestinal epithelial cells and the mucosal permeability were, on the other hand, significantly elevated in berberine treated rats. The expression of NOD and NLRP3, however, were not significantly affected by berberine treatment. Our results indicate that Pretreatment with berberine attenuates tissue injury and protects the intestinal mucosal barrier in early phase of sepsis but it is likely that the mechanisms of this preventive effect do not involve the NLR pathway.

Berberine attenuates lipopolysaccharide-induced impairments of intestinal glutamine transport and glutaminase activity in rat.

Berberine was reported to protect against the intestinal injury and improve the survival rate in sepsis, and glutamine deficiency was considered to be correlated with mortality in sepsis. We found that berberine pretreatment ameliorated lipopolysaccharide-induced direct intestinal injury and mucosal hypoplasia and attenuated impairments of intestinal glutamine transport and glutaminase activity, B(0)AT1 mRNA and protein expressions, and glutaminase protein expression. These findings showed the first time that berberine pretreatment could improve intestinal recovery and attenuate the impairment of glutamine transport and glutaminase activity in rat sepsis. This might be one of the mechanisms for the beneficial effect of berberine on sepsis.

Different alterations in rat intestinal glutamine transport during the progression of CLP- and LPS-induced sepsis.

BACKGROUND: A marked deficiency of glutamine in clinical critical illness is correlated with mortality in the intensive care unit. Though intestinal glutamine transport was reported to be impaired in late sepsis, we hypothesized that there might be a different alteration in the early stage, with differential effects on the Na(+)-dependent glutamine transporters B(0)AT1, ATB(0,+), and ATA2. MATERIALS AND METHODS: Sepsis was induced by cecal ligation and puncture or lipopolysaccharide intraperitoneal injection in Sprague Dawley rats, and the samples were collected at 0, 2, 6, 12, 24h. Small intestinal brush border glutamine transport was studied by a rapid filtration technique. The relative contributions of the three main transporter, B(0)AT1, ATB(0,+), and ATA2, were determined by competitive inhibition. The mRNA level of each transporter was analyzed by RT-PCR, and an extra immunohistochemistry analysis was performed to detect the localization of ATA2 protein in small intestine. Serum TNF-α and IL-10 concentrations were quantitated by ELISA. RESULTS: Intestinal glutamine transport showed a biphasic change with an early increase and a late decrease in both CLP and LPS group. The early increase of glutamine transport was mainly attributable to the increased contributions of ATA2 and ATB(0,+). The transport activities of B(0)AT1, ATB(0,+) altered mainly because of the number of transporters (mRNA level as an indicator), while turned to ATA2, the redistribution was also found to be involved. The plasma TNF-α and IL-10 levels, especially the former, showed similar changing profiles to glutamine transport and, thus, may have relevance to it. CONCLUSION: Rat intestinal glutamine transport showed an early increase and a late decrease in sepsis, and may provide some information for sepsis treatment.

Endothelial nitric oxide synthase as a marker for human endothelial progenitor cells.

Endothelial progenitor cells (EPCs) have been proposed as a promising tool for therapeutic neovascularization, vascular repair, tumor pathology and tissue engineering, though their identification is still a subject of much discussion. EPCs consist of two different subpopulations, termed endothelial cell (EC)-like cells and endothelial outgrowth cells (EOCs). Both types of EPCs are derived from mononuclear cells, but they have different characteristics. Our aim was to characterize and compare the two types of EPCs to find reliable biological features of EPCs that can be used for identification of EPCs. In this study, human peripheral blood mononuclear cells were isolated by density gradient centrifugation and cultured on fibronectin-coated culture plates. While adherent cells were maintained, EC-like cells appeared within 4-7 days of culture, and EOCs developed after 2-3 weeks of culture. EOCs, which were characterized by high proliferation potential, were able to form capillary tubes on Matrigel, but not EC-like cells, despite the higher concentrations of three angiogenic cytokines, vascular endothelial growth factor, granulocyte colony-stimulating factor, and interleukin 8, in the conditioned medium of EC-like cells. In contrast, endothelial nitric oxide synthase (eNOS) was expressed in both types of EPCs, and both cell types could produce nitric oxide (NO), as judged by measuring the total amounts of nitrites and nitrates in culture media. In conclusion, the expression of eNOS and the production of NO could be used as common biological features to identify EPCs. These findings provide new insights into the identification of EPCs.

[Therapeutic effects of combined enteral nutrition with Tripterygium Wilfordii poly-glycoside in remission induction of active adult Crohn's disease].

OBJECTIVE: To investigate the potential role of enteral nutrition (EN) combined with Tripterygium Wilfordii Poly-glycoside (TWP) for remission induction of active adult Crohn's disease (CD). METHODS: Clinical data of 62 adult patients with active CD treated with EN and TWP in combination (n = 42) or TWP alone (n = 20) from March 2001 to September 2008 were retrospectively analyzed. All the patients had a Crohn's Disease Activity Index (CDAI) > 150 and < 450. In TWP group, subjects received TWP tablets (1.0 - 1.5 mg x kg(-1) x d(-1)) with uncontrolled diets; while in the group of combination therapy, the patients were given total enteral nutrition (TEN) through tube feeding in addition to TWP tablets. Clinical response was defined by a decrease of at least 70 points in the CDAI from baseline after treatment, and clinical remission was defined as the absolute value of CDAI (less than 150). Patients' nutritional and disease activity index, such as CDAI score, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), were determined at 0, 4, and 12 weeks after treatment. RESULTS: The ratio of clinical response (78.6% vs. 40.0%, P = 0.003) and clinical remission (69.1% vs. 30.0%, P = 0.004) were both significantly higher in the combined treatment group than in those the TWP group at week 4. At week 12, the clinical response ratio was significantly higher in the combined treatment group (90.5% vs. 65.0%, P = 0.014); the remission ratio was also higher in the combined treatment group (76.2% vs. 55.0%, P = 0.091). The nutritional parameters improved from baseline at week 4 and 12 in the combined treatment group but not in TWP group. At week 4, blood albumin, prealbumin, and transferrin levels was higher in the combined treatment group than those in TWP group (P < 0.05); at week 12, patients in combined treatment group also had significantly higher body mass index (BMI), blood albumin, prealbumin, transferrin and hemoglobin levels (P < 0.05). CONCLUSIONS: Treatment with enteral nutrition and TWP in combination are superior to TWP alone for induction of clinical response and remission in adult Crohn's Disease. This strategy also improves patient's nutritional status and avoids the adverse effects of traditional therapy.

[Effects of perioperative combined nutritional support in Crohn disease].

OBJECTIVE: To observe the efficacy of perioperative combined nutritional support in patients with Crohn disease. METHODS: From January 2000 to June 2008, 165 patients with Crohn disease receiving perioperative nutritional support were included in this retrospective analysis. The patients were divided into three groups according to the ways of nutritional support: total enteral nutrition group, total parenteral nutrition group and combined nutrition group; there were 55 patients in each group. Each group had the same treatment except for nutritional support. The efficacy of different approaches of nutritional support was analyzed and compared among the groups. RESULTS: Compared with total enteral and total parenteral nutrition, combined nutrition supplied more sufficient energy, the nutritional status improved more significant in short time; pre-albumin, transferrin, lymphocytes and platelet count increased significantly. The disease remission rate in combined nutrition group was 80.0%, better than 76.4% in total enteral nutrition group and 74.5% in total parenteral nutrition group. The morbidity rate was 10.9% in combined nutrition group, and it was lower than that in total enteral nutrition group and total parenteral nutrition group (25.4% and 18.2%, respectively). The length of hospital stay was shorter and the treatment was more cost-effective in combined nutrition group. CONCLUSION: For patients with Crohn disease, perioperative combined nutritional support is more efficient than total enteral or parental nutrition support.

Therapeutic effects of triptolide on interleukin-10 gene-deficient mice with colitis.

BACKGROUND: Triptolide, the principal active ingredient in the extract of Chinese herb Tripterygium wilfordii Hook , has both anti-inflammatory and immunomodulatory activities. However, the potential therapeutic role of triptolide in IBD was still unknown. Interleukin-10 deficient mice, a well characterized experimental model of inflammatory bowel disease, spontaneously developed a Th1 T cell-mediated colitis with many similarities to Crohn's disease. This study was designed to investigate the therapeutic effect of triptolide on the chronic colitis in IL-10-/- mice. METHODS: Triptolide was intraperitoneally administrated every another day for 8 weeks to IL-10-/- mice. The gross and histological appearances of the colon, the level of inflammatory mediators and transcription factor activation in the colon were evaluated and compared with the control group. RESULTS: The 8-week administration of triptolide resulted in a significant decrease in the severity of colitis, together with lower production of TNF-alpha ,IFN-gamma and IL-4 in colon. The level of serum amyloid A was decreased in triptolide-treated mice. Gene expressions of IL-12 and IL-23 in colon were also downregulated after treatment. Furthermore, administration of triptolide markedly reduced NF-small ka, CyrillicB activation in colon mucosa of IL-10-/- mice. CONCLUSIONS: The efficacy of tritpolide treatment for the reduction of intestinal inflammation in IL-10-/- mice is a result of both anti-inflammatory and immunosuppressive activity. Triptolide holds significant potential for clinical applications for CD treatment.

[Study of enteral nutrients transport in intestinal hypoperfused rat model].

OBJECTIVE: To study the transport of glutamine and glucose, expression of their transporters and tissue morphology in intestinal hypoperfusion. METHODS: Sprague-Dawley rats were randomized to receive 60 min of intestinal hypoperfusion (superior mesenteric artery clamp) or serve as normoxic controls (celiotomy only). At the same time, jejunal loops were randomized to receive in situ perfusion of mannitol,glucose,or glutamine.Intestinal brush border membrane vesicles (BBMV) were prepared by calcium precipitation. Sodium-dependent uptake of glucose and glutamine into BBMV were quantitated by rapid mixing and filtration. Histologic examination and immunohistochemistry were performed by pathologists blinded to the groups. RESULTS: When compared with the control group, tissue lactate concentration of the hypoperfused group increased significantly (4.9+/-0.3 vs 3.1+/-0.2), especially in the glucose perfused groups (P<0.01). Transport and transporters of glucose in brush border, but not glutamine, decreased during hypoperfusion [(76+/-10) pmol d mg(-1) d 10 s(-1) vs (290+/-13)pmol d mg(-1) d 10 s(-1)]. Tissue structural damage was most severe in glucose perfused groups during hypoperfusion. CONCLUSION: Transport and expression of transporters of glucose and glutamine in enteral nutrition are differently regulated under conditions of trauma and stress.