SUMMARY While activation of beige thermogenesis is a promising approach for treatment of obesity-associated diseases, there are currently no known pharmacological means to induce beiging in humans. Intermittent fasting is an effective and natural strategy for weight control, but the mechanism for its efficacy is poorly understood. Here, we show that an every other day fasting (EODF) regimen selectively stimulates beige fat development within white adipose tissue, and dramatically ameliorates obesity, insulin resistance and hepatic steatosis. EODF treatment results in a shift in the gut microbiota composition leading to the elevation of the fermentation products acetate and lactate, and the selective upregulation of monocarboxylate transporter 1 expression in beige cells. Microbiota-depleted mice are resistance to EODF-induced beiging, while transplantation of the microbiota from EODF-treated mice to microbiota-depleted mice activates beiging and improves metabolic homeostasis. These findings provide a new gut microbiota-driven mechanism for activating adipose tissue browning and treating metabolic diseases.

Atrial fibrillation (AF) is the most common sustained arrhythmia and is associated with significant morbidity and mortality. Timely diagnosis of the arrhythmia, particularly transient episodes, can be difficult since patients may be asymptomatic. In this study, we describe a robust algorithm for automatic detection of AF based on the randomness, variability and complexity of the heart beat interval (RR) time series. Specifically, we employ a new statistic, the Turning Points Ratio, in combination with the Root Mean Square of Successive RR Differences and Shannon Entropy to characterize this arrhythmia. The detection algorithm was tested on two databases, namely the MIT-BIH Atrial Fibrillation Database and the MIT-BIH Arrhythmia Database. These databases contain several long RR interval series from a multitude of patients with and without AF and some of the data contain various forms of ectopic beats. Using thresholds and data segment lengths determined by Receiver Operating Characteristic (ROC) curves we achieved a high sensitivity and specificity (94.4% and 95.1%, respectively, for the MIT-BIH Atrial Fibrillation Database). The algorithm performed well even when tested against AF mixed with several other potentially confounding arrhythmias in the MIT-BIH Arrhythmia Database (Sensitivity = 90.2%, Specificity = 91.2%).

SummaryHeparanase, a heparan sulfate (HS)-specific endoglycosidase, plays an important role in inflammation and mediates acute pulmonary and renal injuries during sepsis. To explore its role in septic intestinal injury, a non-anticoagulant heparanase inhibitor, N-desulfated/re-N-acetylated heparin (NAH), was administrated to a mouse sepsis model induced by cecal ligation and puncture (CLP). Immunohistochemical staining revealed massive shedding of HS from the intestinal mucosal surfaces after CLP, and effective inhibition of heparanase by NAH was confirmed by markedly reduced HS shedding. Following CLP, intestinal expression of heparanase was increased, whereas pretreatment with NAH reduced the sepsisinduced upregulation of heparanase expression. Meanwhile, CLP led to shedding of syndecan-1 and upregulated expression of proteases such as matrix metalloprotease-9 and urokinase-type plasminogen activator in the intestine, whereas NAH markedly suppressed syndecan-1 shedding and protease upregulation following CLP. In addition, pretreatment with NAH attenuated intestinal injury, inhibited neutrophil infiltration and suppressed the production of inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6) in the intestine during sepsis, and it also significantly reduced the elevation of inflammatory cytokines in the serum 24 hr after CLP. Our findings demonstrate that the activation of intestinal heparanase contributes to intestinal injury during early sepsis by facilitating the destruction of mucosal epithelial glycocalyx and promoting inflammatory responses. (J Histochem Cytochem 65:241-249, 2017)

Endometrial regenerative cells (ERCs) are mesenchymal-like stromal cells, and their therapeutic potential has been tested in the prevention of renal ischemic reperfusion injury, acute liver injury, ulcerative colitis, and immunosuppression. However, their potential in the induction of transplant tolerance has not been investigated. The present study was undertaken to investigate the efficacy of ERCs in inducing cardiac allograft tolerance and the function of stromal cell-derived factor-1 (SDF-1) in the ERC-mediated immunoregulation. The inhibitory efficacy of human ERCs in the presence or absence of rapamycin was examined in both mouse cardiac allograft models between BALB/c (H-2 d ) donors and C57BL/6 (H-2 b ) recipients and in vitro cocultured splenocytes. AMD3100 was used to inhibit the function of SDF-1. Intragraft antibody (IgG and IgM) deposition and immune cell (CD4 1 and CD8 1 ) infiltration were measured by immunohistochemical staining, and splenocyte phenotypes were determined by fluorescence-activated cell sorting analysis. The results showed that ERC-based therapy induced donor-specific allograft tolerance, and functionally inhibiting SDF-1 resulted in severe allograft rejection. The negative effects of inhibiting SDF-1 on allograft survival were correlated with increased levels of intragraft antibodies and infiltrating immune cells, and also with reduced levels of regulatory immune cells including MHC class II low CD86 low CD40 lowdendritic cells, CD68 1 CD206 1 macrophages, CD4 1 CD25 1 Foxp3 1 T cells, and CD1d high CD5 high CD83 low IL-10 high B cells both in vivo and in vitro. These data showed that human ERC-based therapy induces cardiac allograft tolerance in mice, which is associated with SDF-1 activity, suggesting that SDF-1 mediates the immunosuppression of ERC-based therapy for the induction of transplant tolerance. STEM CELLS TRANSLATIONAL MEDICINE 2017;6:1997 SIGNIFICANCE STATEMENTEndometrial regenerative cells (ERCs) are newly identified stromal cells with advantages of noninvasively obtained method, abundant resources, highly proliferative rate, immunoregulatory function, and absence of tumorigenesis. Stromal cell-derived factor-1 (SDF-1) is involved in many different physiological and pathological processes. This study demonstrates that human ERC-based therapy can effectively suppress immune response and further induce allograft tolerance in a mouse cardiac transplantation model, and SDF-1 secreted by ERCs plays an essential role in ERC-mediated graft protection. Simultaneously, as xenograft, human ERCs are proven to be safe and effective in mice, which hold significant promise for therapeutic use in future clinical transplantation.

BackgroundThe endometrial regenerative cell (ERC) is a novel type of adult mesenchymal stem cell isolated from menstrual blood. Previous studies demonstrated that ERCs possess unique immunoregulatory properties in vitro and in vivo, as well as the ability to differentiate into functional hepatocyte-like cells. For these reasons, the present study was undertaken to explore the effects of ERCs on carbon tetrachloride (CCl4)–induced acute liver injury (ALI).MethodsAn ALI model in C57BL/6 mice was induced by administration of intraperitoneal injection of CCl4. Transplanted ERCs were intravenously injected (1 million/mouse) into mice 30 min after ALI induction. Liver function, pathological and immunohistological changes, cell tracking, immune cell populations and cytokine profiles were assessed 24 h after the CCl4 induction.ResultsERC treatment effectively decreased the CCl4-induced elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and improved hepatic histopathological abnormalities compared to the untreated ALI group. Immunohistochemical staining showed that over-expression of lymphocyte antigen 6 complex, locus G (Ly6G) was markedly inhibited, whereas expression of proliferating cell nuclear antigen (PCNA) was increased after ERC treatment. Furthermore, the frequency of CD4+ and CD8+ T cell populations in the spleen was significantly down-regulated, while the percentage of splenic CD4+CD25+FOXP3+ regulatory T cells (Tregs) was obviously up-regulated after ERC treatment. Moreover, splenic dendritic cells in ERC-treated mice exhibited dramatically decreased MHC-II expression. Cell tracking studies showed that transplanted PKH26-labeled ERCs engrafted to lung, spleen and injured liver. Compared to untreated controls, mice treated with ERCs had lower levels of IL-1β, IL-6, and TNF-α but higher level of IL-10 in both serum and liver.ConclusionsHuman ERCs protect the liver from acute injury in mice through hepatocyte proliferation promotion, as well as through anti-inflammatory and immunoregulatory effects.

BackgroundEndometrial regenerative cells (ERCs), a novel type of mesenchymal-like stem cell derived from menstrual blood, have been recently evaluated as an attractive candidate source in ulcerative colitis (UC); however, the mechanism is not fully understood. The present study was designed to investigate the effects of ERCs, especially on B-cell responses in UC.MethodsIn this study, colitis was induced by administering 3% dextran sodium sulfate (DSS) via free drinking water for 7 days to BALB/c mice. In the treated group, mice were injected intravenously with 1 × 106 ERCs on days 2, 5, and 8 after DSS induction. Therapeutic effects were assessed by monitoring body weight, disease activity, and pathological changes. Subpopulations of lymphocytes were determined by flow cytometry. IgG deposition in the colon was examined by immunohistochemistry staining. Cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA), Western blot, or polymerase chain reaction (PCR) analysis. Adoptive transfer of regulatory B cells (Bregs) into colitis mice was performed.ResultsHere, we demonstrated that ERC treatment prolonged the survival of colitis mice and attenuated disease activity with fewer pathological changes in colon tissue. ERCs decreased the proportion of immature plasma cells in the spleen and IgG deposition in the colon. On the other hand, ERCs increased the production of Bregs and the interleukin (IL)-10 level. Additionally, adoptive transferred Bregs exhibited significant therapeutic effects on colitis mice.ConclusionsIn conclusion, our results unravel the therapeutic role of ERCs on experimental colitis through regulating the B-lymphocyte responses.

Endometrial regenerative cells (ERCs) have been recently evaluated as an attractive novel type of stem cell therapy. Previous studies have demonstrated that most ERCs accumulated in the lung after injection and are successfully used to treat diseases such as cardiac fibrosis. However, relevant studies of ERCs in idiopathic pulmonary fibrosis (IPF) have not been reported. The present study was designed to examine the effects of ERCs on bleomycin-induced pulmonary fibrosis. All IPF models in C57BL/6 mice were induced by administrating 5 mg/kg bleomycin in PBS intratracheally. ERCs were isolated from healthy female menstrual blood and were injected (1 million/mouse, i.v.) 24 hours after induction. Wet/dry weight ratio assay, hydroxyproline content, pathological and immunohistological changes, MDA content, T-SOD activity, cytokine profiles, and RT-qPCR analysis were assessed 2 weeks after disease induction. The results showed that ERC treatment significantly decreased the wet/dry ratio and reduced collagen deposition. Histological analyses, Masson staining, and hydroxyproline content analysis indicated that ERCs could reduce collagen fiber production. Immunohistochemical staining revealed lower expression of TGF-β after ERC treatment. Furthermore, mice treated with ERCs had lower levels of IL-1β and TNF-α, but a higher level of IL-10 in both the lung and serum. Gene expression analysis demonstrated that ERCs potently suppressed the proapoptotic gene Bax, while increasing the antiapoptotic gene Bcl-2 and antifibrosis genes HGF and MMP-9. Our results indicate that human ERCs protected the lung from pulmonary fibrosis in mice through immunosuppressive and antifibrosis effects. Moreover, these findings formed a foundation for the further use of ERCs in clinical treatment.

BackgroundUlcerative colitis (UC) is a type of inflammatory bowel disease (IBD) characterized by chronic inflammation of colon. It is commonly believed that the imbalance of immune system and overwhelming production of cytokines are involved in the pathogenesis of UC. Recent studies demonstrated that interleukin-35 (IL-35), a key player in the regulation of inflammation, has been identified as potential therapeutic target to treat UC. However, conventional intravenous administration is costly and inconvenient. The present study was designed to establish a novel IL-35 delivery system and investigate its therapeutic effects on dextran sulfate sodium (DSS)-induced experimental colitis in mice for the first time.MethodsAn engineered Escherichia coli (E. coli/IL-35) expressing IL-35 was constructed. Adult male BALB/c mice randomly got the oral administration of E. coli/IL-35, empty plasmid-transformed E. coli (E. coli0) or PBS for treatment following ingestion of 3% DSS solution for 5 days. Normal mice were used as control group. Colonic and splenic tissues were collected on day 10 post-DSS-induction. Clinical signs, disease activity index (DAI), pathological and immunohistological changes, cytokine profiles and cell populations were evaluated.ResultsIntragastric administration of E. coli/IL-35 effectively protected the colitis mice from DSS assimilation including weight loss and colon shortening. Pathological analysis showed significantly lower DAI score and much less intra-colon infiltration of neutrophils and CD3+ cells in the IL-35 treated group. Moreover, E. coli/IL-35-treated mice demonstrated much less CD4+ IL-17A+ Th17 cells and a higher level of CD4+CD25+Foxp3+ Tregs in spleen and mesenteric lymph nodes, as well as increased colon and serum level of IL-10 and IL-35 and decreased levels of IL-6.ConclusionsOur study showed that E. coli/IL-35 as a novel oral IL-35 delivery system alleviated inflammatory damage of colonic tissue in the colitic mice. Genetic therapeutic strategies using engineered E. coli encoding immunoregulatory cytokines may provide a potential approach for the treatment of IBD.