ABSTRACT
Metagenomic studies show that diverse resident viruses inhabit the healthy gut; however, little is known about the role of these viruses in the maintenance of gut homeostasis. We found that mice treated with antiviral cocktail displayed more severe dextran sulfate sodium (DSS)-induced colitis compared with untreated mice. DSS-induced colitis was associated with altered enteric viral abundance and composition. When wild-type mice were reconstituted with Toll-like receptor 3 (TLR3) or TLR7 agonists or inactivated rotavirus, colitis symptoms were significantly ameliorated. Mice deficient in both TLR3 and TLR7 were more susceptible to DSS-induced experimental colitis. In humans, combined TLR3 and TLR7 genetic variations significantly influenced the severity of ulcerative colitis. Plasmacytoid dendritic cells isolated from inflamed mouse colon produced interferon-ß in a TLR3 and TLR7-dependent manner. These results imply that recognition of resident viruses by TLR3 and TLR7 is required for protective immunity during gut inflammation.
Subject(s)
Colitis/immunology , Gastrointestinal Tract/virology , Interferon-beta/immunology , Membrane Glycoproteins/immunology , Rotavirus/immunology , Toll-Like Receptor 3/immunology , Toll-Like Receptor 7/immunology , Animals , Antiviral Agents/pharmacology , Colitis/chemically induced , Dendritic Cells/immunology , Dextran Sulfate , Gastrointestinal Microbiome , Gastrointestinal Tract/immunology , Humans , Inflammation/immunology , Interferon-beta/biosynthesis , Membrane Glycoproteins/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , RNA, Ribosomal, 16S/genetics , Toll-Like Receptor 3/genetics , Toll-Like Receptor 7/geneticsABSTRACT
A polarization-independent multilayer dielectric diffraction grating with a low aspect ratio and high diffraction efficiency was designed and fabricated. The diffraction grating designed with a grating density of 1200 lines/mm had an aspect ratio of 0.59, mean polarization-independent diffraction efficiency in the Littrow angle of ±2.5∘, and 1030-1080 nm wavelength range of 97.2%. The designed grating was fabricated using ion assisted deposition and reactive ion etching techniques. The mean polarization-independent diffraction efficiency of the fabricated grating was 96.1%, and its standard deviation was 0.68%. The fabricated diffraction grating was irradiated with a 1064 nm cw laser, with a power density of 30kW/cm2, for 1 min to measure the temperature change before and after the laser application. It was verified that the temperature variation of the diffraction grating without heat treatment was 8.8°C, and the temperature variation after heat treatment at 400°C decreased to 2.3°C.
ABSTRACT
PURPOSE: To evaluate the effects of diabetes and corticosteroid injected in the joints on the shoulder motion, gait, and joint capsular properties in a rat stiffness model. METHODS: A total of 27 rats were randomly distributed into 3 groups-nondiabetes group (group A), diabetes group (group B), and diabetes plus steroid injection group (group C). The diabetes model was developed by inducing hyperglycemia with a submaximal dose of streptozotocin and the stiffness model by completely immobilizing the right shoulder of each animal in all groups with sutures passed between the scapula and humeral shaft. The left shoulder was used as an untreated control in all groups. Three weeks after immobilization, the sutures were removed in all groups, and a single dose of triamcinolone acetonide (0.5 mg/kg) was injected into the glenohumeral joint in group C. After 3 weeks of free activity, range of motion (ROM) evaluation, gait analysis by stride length, and capsular area measurement were performed in all rats. RESULTS: Hyperglycemia was successfully induced with a mean blood glucose level of 448.9±55.9 mg/dL in group B and 431.6±17.8 mg/dL in group C, which were significantly higher than 136.5±13.4 mg/dL in group A (P < .001). A significantly smaller ROM and stride length were found in the right (stiffness-induced) shoulder than that in the left (control) shoulder only in group B, and significantly larger capsular area in the right shoulder than that in the left shoulder in groups A and B (all P < .05). However, in group C, there were no differences between the right and left shoulders in all measurements (all P > .05). In case of the right shoulders in each group, group C showed significantly larger ROM (68° ± 11° vs. 42° ± 7°) and smaller capsular area (3934.4 ± 537.1 pixels vs. 7402.3 ± 1840.3 pixels) than group B (all P < .0167). CONCLUSIONS: The diabetic model had a detrimental effect on the development of stiffness by thickening the joint capsule, and an intra-articular steroid injection resolved the thickened joint capsule and restored shoulder motion.
Subject(s)
Diabetes Mellitus , Shoulder Joint , Adrenal Cortex Hormones , Animals , Joint Capsule , Range of Motion, Articular , RatsABSTRACT
BACKGROUND: Fatty infiltration (FI) is a key prognostic factor that affects outcomes after rotator cuff repair and is radiologically evaluated using the Goutallier classification. The purpose of this study was to assess alterations in gene and protein expression according to the Goutallier classification in the supraspinatus muscle and any relationships among various gene expression profiles. METHODS: Twenty-four samples of the supraspinatus muscle from 12 patients with a high FI grade (grade 3 or 4) and 12 patients with a low FI grade (grade 1 or 2) with medium-sized tears were acquired during arthroscopic surgery. Alterations in the expression of genes and proteins associated with adipogenesis, fibrosis, inflammation, and muscle atrophy were compared between the high- and low-FI groups using reverse-transcription quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. RESULTS: mRNA expression of not only the adipogenic genes (peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α; P < .001 and P = .020) but also the fibrosis-related gene (α-smooth muscle actin; P < .001), inflammation-related genes (interleukin [IL]-1ß and tumor necrosis factor α; P = .041 and P = .039), and muscle atrophy-related genes (atrogin 1 and myostatin; P = .006 and P < .001) was higher in the high-FI group compared with that in the low-FI group. In addition, adipogenic gene expression was significantly correlated with the expression of other categories of genes (all P < .05, except atrogin 1). A correlation of gene and protein expression was observed for IL-1ß (P = .027) and myostatin (P = .029). CONCLUSIONS: The radiologic grading of FI was associated with the expression of various genes, including adipogenic, fibrotic, inflammatory, and atrophy-related genes, and these genes were closely correlated with each other in terms of expression. This information could be helpful in patient counseling.
Subject(s)
Rotator Cuff Injuries , Rotator Cuff , Adipose Tissue , Arthroscopy , Gene Expression , Humans , Magnetic Resonance Imaging , Rotator Cuff Injuries/diagnostic imaging , Rotator Cuff Injuries/genetics , Rotator Cuff Injuries/surgeryABSTRACT
Despite the presence of vitamin D receptor (VDR) in skeletal muscle cells, the relationship between VDR expressions and muscle mass or function has not been well studied. The purpose of this study was to compare VDR gene and protein expression in the forearm muscle between sarcopenic and non-sarcopenic individuals who have sustained distal radius fractures. Twenty samples of muscle tissue from sarcopenic patients (mean age 63.4 ± 8.1 years) and 20 age- and sex-matched control tissues (62.1 ± 7.9 years) were acquired from the edge of dissected pronator quadratus muscle during surgery for distal radius fractures. The mRNA expression levels of VDR as well as the myokines of interest that may be associated with muscle mass change (myogenin and myostatin) were analyzed with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, Western blot assay and immunohistochemistry for VDR were performed. Sarcopenic patients showed a significantly lower level of gene expression for VDR and myogenin, but a greater level of gene expression for myostatin than the controls according to qRT-PCR analysis. The density of VDR protein expressions was 2.1 times greater, while that of myostatin was 2.6 times lower, in the control group than in the sarcopenic group according to Western blot analysis. On immunohistochemical analysis, the density of the cells expressing VDR was significantly decreased in the sarcopenic patients. Sarcopenic patients who sustained distal radius fractures presented lower vitamin D receptor gene and protein expression in skeletal muscles compared to non-sarcopenic individuals.
Subject(s)
Gene Expression Regulation , Muscle, Skeletal/metabolism , Radius Fractures/genetics , Receptors, Calcitriol/genetics , Sarcopenia/genetics , Female , Forearm , Humans , Male , Middle Aged , Muscle, Skeletal/pathology , Myogenin/genetics , Myogenin/metabolism , Myostatin/genetics , Myostatin/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Radius Fractures/pathology , Receptors, Calcitriol/metabolism , Sarcopenia/complications , Sarcopenia/pathologyABSTRACT
PURPOSE: To evaluate the altered gene and protein expression patterns in the rotator cuff muscles of smokers and non-smokers with rotator cuff tears and to identify the smoking-associated key genetic factor(s) involved in rotator cuff muscle physiology. METHODS: Twenty-four samples of rotator cuff muscle from 12 current heavy smokers (mean age 61.8 ± 5.1 years) and age- and sex-matched 12 non-smokers (mean age 61.8 ± 6.9 years) with medium-sized tears were acquired during arthroscopic surgery. As a statistical method, the propensity score matching technique was used to select control group by 1:1 matching for age and sex. Inclusion criteria were patients who underwent arthroscopic repair for medium-sized full-thickness rotator cuff tears and those that were current smokers with a smoking history >20 packs/year. Patients lacking medium-sized tears, those with recent steroid injection history, isolated subscapularis tear, preoperative stiff shoulder, acute traumatic tear, or previous surgery on the same shoulder, or those that declined to participate were excluded. Alterations in the expression of genes and proteins associated with myogenesis, inflammation, adipogenesis, and muscle fibrosis were compared between smokers and non-smokers with reverse-transcription quantitative polymerase chain reaction, western blotting, and immunohistochemistry. RESULTS: Histologic analysis revealed increased inflammation and remarkable fat accumulation and fibrogenesis in the rotator cuff muscle from smokers compared with that from non-smokers. The mRNA expression levels of inflammatory high mobility group box 1 (HMGB1; P = .043), adipogenic CCAAT/enhancer-binding protein alpha (P = .046) and peroxisome proliferator-activated receptor gamma (PPARγ; P = .048), myogenic differentiation 1 (P = .032), fibrogenic alpha-smooth muscle actin (α-SMA; P = .033), and metalloproteinase 9 (P = .036) were significantly greater in samples from smokers than from non-smokers. A correlation was observed between gene and protein expression of HMGB1 (P = .034), PPARγ (P = .021), and α-SMA (P = .021). CONCLUSIONS: Smokers with rotator cuff tears showed high inflammation, large fat infiltration, and fibrosis in rotator cuff muscle that is associated with the increased expression of HMGB1, PPARγ, and α-SMA, respectively. LEVEL OF EVIDENCE: Case control study (Prognostic level III).
Subject(s)
Adipose Tissue/pathology , Fibrosis/pathology , Inflammation/pathology , Proteins/metabolism , Rotator Cuff Injuries/pathology , Rotator Cuff/metabolism , Smoking/adverse effects , Aged , Case-Control Studies , Female , Gene Expression Profiling , Humans , Joint Diseases/pathology , Male , Middle Aged , Pilot Projects , RNA, Messenger/metabolism , Shoulder Joint/pathologyABSTRACT
Diode-pumped alkali-atom laser (DPAL) has attracted intense attention due to its inherently high quantum efficiency, a good beam quality, and a high potential in the power scaling. However, most of DPAL research has been confined to the continuous wave and only a few pulsed operations have been attempted with limited performances. Here, we proposed and experimentally demonstrated a new scheme using a fast mode-hopping in the pump laser diode (LD), which enabled the quasi-continuous-wave (QCW) pulse modulation in a cesium (Cs) DPAL to control both the pulse width and the repetition rate. The pump wavelength was efficiently modulated in a fast cycle within discrete spectral ranges provided by the mode-hopping in the pump LD. The spectral range was successfully adjusted to include the resonant D2 absorption line of Cs atom to result in an effective gain modulation. Using this proposed scheme, we successfully achieved Cs-DPAL QCW modulation, whose pulse width was varied from tens of microseconds to a few milliseconds and the repetition rate was also variable in a wide frequency range from 10 Hz to 7.0 kHz. Detailed pump modulation method and the corresponding laser characteristics are discussed. The proposed method can be readily applied to pulse modulation of other types of alkali vapor lasers overcoming the previous limitations of DPAL to further expand applications in various light-matter interactions.
ABSTRACT
Upon liver intoxication with malnutrition or high-fat diet feeding, fibrinogen is synthesized by hepatocytes and secreted into the blood in human and mouse. Its primary function is to occlude blood vessels upon damage and thereby stop excessive bleeding. High fibrinogen levels may contribute to the development of pathological thrombosis, which is one mechanism linking fatty liver disease with cardiovascular disease. Our previous results present ERRγ as key regulator of hepatocytic fibrinogen gene expression in human. In a therapeutic approach, we now tested ERRγ inverse agonist GSK5182 as regulator of fibrinogen levels in mouse hyperfibrinogenemia caused by diet-induced obesity and in mouse hepatocytes. ACEA, a CB1R agonist, up-regulated transcription of mouse fibrinogen via induction of ERRγ, whereas knockdown of ERRγ attenuated the effect of ACEA (10 µM) on fibrinogen expression in AML12 mouse hepatocytes. Deletion analyses of the mouse fibrinogen γ (FGG) gene promoter and ChIP assays revealed binding sites for ERRγ on the mouse FGG promoter. ACEA or adenovirus ERRγ injection induced FGA, FGB and FGG mRNA and protein expression in mouse liver, while ERRγ knockdown with Ad-shERRγ attenuated ACEA-mediated induction of fibrinogen gene expression. Moreover, mice maintained on a high-fat diet (HFD) expressed higher levels of fibrinogen, whereas cannabinoid receptor type 1 (CB1R)-KO mice fed an HFD had nearly normal fibrinogen levels. Finally, GSK5182 (40 mg/kg) strongly inhibits the ACEA (10 mg/kg) or HFD-mediated induction of fibrinogen level in mice. Taken together, targeting ERRγ with its inverse agonist GSK5182 represents a promising therapeutic strategy for ameliorating hyperfibrinogenemia.
Subject(s)
Fibrinogen/biosynthesis , Liver/metabolism , Receptor, Cannabinoid, CB1/metabolism , Receptors, Estrogen/metabolism , Tamoxifen/analogs & derivatives , Animals , Cells, Cultured , Diet, High-Fat/adverse effects , Fibrinogen/genetics , Gene Expression Regulation/drug effects , Hepatocytes/drug effects , Hepatocytes/metabolism , Liver/drug effects , Male , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , Receptor, Cannabinoid, CB1/genetics , Receptors, Estrogen/genetics , Tamoxifen/pharmacologyABSTRACT
PURPOSE: To analyze and compare the gene and protein expression characteristics in torn rotator cuff tendon tissues between diabetic and nondiabetic patients. METHODS: This was a pilot study. Twelve samples of rotator cuff tendon tissue from diabetic patients (mean age, 62.3 ± 9.9 years) and 12 age- and sex-matched nondiabetic tendon tissues (62.3 ± 9.9 years) were acquired from the torn tendon end of medium rotator cuff tears during arthroscopic surgery, after applying the same inclusion and exclusion criteria. Expressions of various genes of interest, including collagens I and III, matrix metalloprotease (MMP)-2, MMP-3, MMP-9, MMP-13, interleukin (IL)-1, IL-6, insulin-like growth factor-1, vascular endothelial growth factor, tenomodulin, tumor necrosis factor-α, and p53, were analyzed with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, immunohistochemistry and western blot assay were performed for the genes that revealed significantly different expressions in real-time qRT-PCR between groups. RESULTS: Gene expression levels of MMP-9, MMP-13, IL-6, and tenomodulin were significantly higher in the diabetic than in the nondiabetic group by real-time qRT-PCR analyses (P = .011, .004, .009, and .010, respectively). The density of cells expressing MMP-9 and IL-6 was significantly increased in the torn tendons of the diabetic patients on immunohistochemical analysis, and the density of MMP-9 and IL-6 protein expressions was significantly higher in the diabetic group on western blot (P = .018 and .044, respectively). CONCLUSIONS: Diabetic torn cuff tendon tissues showed MMP-9 and IL-6 overexpressions compared with controls. CLINICAL RELEVANCE: The overexpressions of MMP-9 and IL-6 may be one of the explanations for the high healing failure rate after rotator cuff repair in the diabetic patients.
Subject(s)
Diabetes Mellitus/metabolism , Rotator Cuff/metabolism , Case-Control Studies , Female , Gene Expression , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Male , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Middle Aged , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Rotator Cuff Injuries/metabolismABSTRACT
Attaching and effacing pathogens, including enterohemorrhagic Escherichia coli in humans and Citrobacter rodentium in mice, raise serious public health concerns. Here we demonstrate that interleukin-1 receptor (IL-1R) signaling is indispensable for protection against C. rodentium infection in mice. Four days after infection with C. rodentium, there were significantly fewer neutrophils (CD11b+ Ly6C+ Ly6G+) in the colons of IL-1R−/− mice than in wild-type mice. Levels of mRNA and protein of KC/CXCL1 were also significantly reduced in colon homogenates of infected IL-1R−/− mice relative to wild-type mice. Of note, infiltrated CD11b+ Ly6C+ Ly6G+ neutrophils were the main source of IL-22 secretion after C. rodentium infection. Interestingly, intestinal stromal cells isolated from IL-1R−/− mice secreted lower levels of KC/CXCL1 than stromal cells from wild-type mice during C. rodentium infection. Similar effects were found when mouse intestinal stromal cells and human nasal polyp stromal cells were treated with IL-1R antagonists (i.e., anakinra) in vitro. These results suggest that IL-1 signaling plays a pivotal role in activating mucosal stromal cells to secrete KC/CXCL1, which is essential for infiltration of IL-22-secreting neutrophils upon bacterial infection.
Subject(s)
Chemokine CXCL1/metabolism , Citrobacter rodentium/physiology , Enterobacteriaceae Infections/metabolism , Interleukin-1/metabolism , Interleukins/metabolism , Intestinal Mucosa/metabolism , Neutrophils/metabolism , Stromal Cells/metabolism , Animals , Cells, Cultured , Chemokine CXCL1/genetics , Enterobacteriaceae Infections/genetics , Enterobacteriaceae Infections/microbiology , Female , Humans , Interleukin-1/genetics , Interleukins/genetics , Intestines/microbiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/immunology , Signal Transduction , Interleukin-22ABSTRACT
Both the thymus (T) and bone (B) are necessary hematopoietic niches in adult humans. We previously showed that co-transplantation of human fetal T and B tissues into neonatal immunodeficient NOD/SCID IL2Rγ(null) (NSG, N) mice facilitated hematopoiesis. However, transplantation into neonatal mice resulted in high frequency of early death, making it unrealistic for repetitive experiments. In this study, young adult N mice were pre-engrafted with T and B, T alone, B alone or no tissues. The animals were irradiated and injected with autologous fetal liver (FL)-derived CD34(+) cells (34). The resultant mice were TB34N, T34N, B34N and 34N, respectively, and challenged with T cell dependent antigens (Ags). The humanized TB34N mice showed best performance of these mouse models in many aspects resembling the adult human Ag-experienced spleen. The TB34N mice exhibited better hematopoietic reconstitution; balanced development of T- and B-cell, and common progenitor cells; follicular lymphoid structures with a functional germinal center (GC) enriched with follicular dendritic cells (FDCs) and plasma cells (PCs); secretion of hIgG in the sera in response to Ags at comparable levels to those of human; derivations of hIgG mAb-secreting hybridoma clones. Collectively, the humanized TB34N mice could develop an adaptive immunity that was capable of producing Ag-specific hIgG at a significant level via class switching. This unprecedented TB34N platform in humanized mice would be useful in dissecting human immunity, for generating human Abs and clinical applications.
Subject(s)
Adaptive Immunity/immunology , Antibodies/immunology , Antigens, CD34/metabolism , Bone Transplantation , Fetal Tissue Transplantation , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Spleen/immunology , Thymus Gland/transplantation , Animals , Antibody Formation , Hematopoiesis , Heterografts , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Interleukin Receptor Common gamma Subunit/genetics , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Spleen/pathologyABSTRACT
We propose a novel photonic crystal fiber (PCF) design with high birefringence and low confinement loss based on a diamond unit cell. Unit cell is a degree of freedom of PCF design, and highly asymmetric diamond unit cell can deliver both high birefringence and low confinement loss. In our fiber design, each diamond unit cell consists of two different sizes of circular air holes in a conventional hexagonal lattice, which can enhance the confinement loss with increased birefringence and can overcome the previous fabrication challenges of elliptic air hole PCFs due to the all-circular air hole structure. The optimized design shows high birefringence of 6×10(-3) over S, C, and L bands while maintaining low confinement loss.
ABSTRACT
AIMS/HYPOTHESIS: Obesity is associated with ageing and increased energy intake, while restriction of energy intake improves health and longevity in multiple organisms; the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1) is implicated in this process. Pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in the arcuate nucleus (ARC) of the hypothalamus are critical for energy balance regulation, and the level of SIRT1 protein decreases with age in the ARC. In the current study we tested whether conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevents age-associated weight gain and diet-induced obesity. METHODS: We targeted Sirt1 cDNA sequence into the Rosa26 locus and generated conditional Sirt1 knock-in mice. These mice were crossed with mice harbouring either Pomc-Cre or Agrp-Cre and the metabolic variables, food intake, energy expenditure and sympathetic activity in adipose tissue of the resultant mice were analysed. We also used a hypothalamic cell line to investigate the molecular mechanism by which Sirt1 overexpression modulates leptin signalling. RESULTS: Conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevented age-associated weight gain; overexpression in POMC neurons stimulated energy expenditure via increased sympathetic activity in adipose tissue, whereas overexpression in AgRP neurons suppressed food intake. SIRT1 improved leptin sensitivity in hypothalamic neurons in vitro and in vivo by downregulating protein-tyrosine phosphatase 1B, T cell protein-tyrosine phosphatase and suppressor of cytokine signalling 3. However, these phenotypes were absent in mice consuming a high-fat, high-sucrose diet due to decreases in ARC SIRT1 protein and hypothalamic NAD(+) levels. CONCLUSIONS/INTERPRETATION: ARC SIRT1 is a negative regulator of energy balance, and decline in ARC SIRT1 function contributes to disruption of energy homeostasis by ageing and diet-induced obesity.
Subject(s)
Hypothalamus/metabolism , Leptin/pharmacology , Sirtuin 1/metabolism , Weight Gain/physiology , Animals , Calorimetry, Indirect , Genotype , Hypothalamus/drug effects , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , Sirtuin 1/genetics , Weight Gain/geneticsABSTRACT
OBJECTIVE: Allogeneic transplantation of human embryonic stem cell (hESC) derivatives has the potential to elicit the patient's immune response and lead to graft rejection. Although hESCs and their derivatives have been shown to have advantageous immune properties in vitro, such observations could not be determined experimentally in vivo because of ethical and technical constraints. However, the generation of humanized mice (hu-mice) harboring a human immune system has provided a tool to perform in vivo immunologic studies of human cells and tissues. Using this model, we sought to examine the therapeutic potential of hESC-derived endothelial cells, human embryonic fibroblasts, and cord blood-derived endothelial progenitor cells in a human immune system environment. APPROACH AND RESULTS: All cell types transplanted in hu-mice showed significantly reduced cell survival during the first 14 days post-transplantation compared with that observed in immunodeficient mice. During this period, no observable therapeutic effects were detected in the hindlimb ischemic mouse models. After this point, the cells demonstrated improved survival and contributed to a long-term improvement in blood perfusion. All cell types showed reduced therapeutic efficacy in hu-mice compared with NOD scid IL2 receptor gamma chain knockout mice. Interestingly, the eventual improvement in blood flow caused by the hESC-derived endothelial cells in hu-mice was not much lower than that observed in NOD scid IL2 receptor gamma chain knockout mice. CONCLUSIONS: These findings suggest that hESC derivatives may be considered a good source for cell therapy and that hu-mice could be used as a preclinical in vivo animal model for the evaluation of therapeutic efficacy to predict the outcomes of human clinical trials.
Subject(s)
Cord Blood Stem Cell Transplantation , Embryonic Stem Cells/transplantation , Endothelial Cells/transplantation , Fetal Blood/immunology , Ischemia/surgery , Muscle, Skeletal/blood supply , Animals , Biomarkers/blood , Cell Line , Cell Survival , Disease Models, Animal , Embryonic Stem Cells/immunology , Endothelial Cells/immunology , Fibroblasts/immunology , Fibroblasts/transplantation , Graft Survival , Hindlimb , Humans , Immunocompetence , Immunocompromised Host , Interleukin Receptor Common gamma Subunit/deficiency , Interleukin Receptor Common gamma Subunit/genetics , Ischemia/immunology , Ischemia/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Neovascularization, Physiologic , Recovery of Function , Regional Blood Flow , Species Specificity , Time Factors , Transplantation, HeterologousABSTRACT
The pancreas is critical for maintaining glucose homeostasis. Activating transcription factor 3 (ATF3) is an adaptive response transcription factor. There are major discrepancies in previous reports on pancreatic ATF3; therefore, its role in the pancreas is unclear. To better elucidate the role of ATF3 in the pancreas, we conducted in vitro studies using pancreatic α and ß cell lines, and also evaluated the use of ATF3 antibodies for immunohistochemistry. We determined ATF3 expression was increased by low glucose and decreased by high glucose in both αTC-1.6 and ßTC3 cells. We also showed that adenovirus-mediated ATF3 overexpression increased glucagon promoter activity and glucagon mRNA levels in αTC-1.6 cells; whereas, it had no effect on insulin promoter activity and insulin mRNA levels in ßTC3 cells. Although immunostaining with the C-19 ATF3 antibody demonstrated predominant expression in α cells rather than ß cells, ATF3 staining was still detected in ATF3 knockout mice as clearly as in control mice. On the other hand, another ATF3 antibody (H-90) detected ATF3 in both α cells and ß cells, and was clearly diminished in ATF3 knockout mice. These results indicate that previous discrepancies in ATF3 expression patterns in the pancreas were caused by the varying specificities of the ATF3 antibodies used, and that ATF3 is actually expressed in both α cells and ß cells.
Subject(s)
Activating Transcription Factor 3/genetics , Gene Expression/drug effects , Glucagon/genetics , Glucose/administration & dosage , Insulin/genetics , Islets of Langerhans/metabolism , Activating Transcription Factor 3/analysis , Animals , Cell Line , Glucagon-Secreting Cells/chemistry , Glucagon-Secreting Cells/metabolism , Insulin-Secreting Cells/chemistry , Insulin-Secreting Cells/metabolism , Mice , Mice, Knockout , Promoter Regions, Genetic/genetics , RNA, Messenger/analysisABSTRACT
OBJECTIVE: To overcome the therapeutic limitations of malignant fibrous histiocytoma (MFH), we evaluated human adipose tissue-derived mesenchymal stromal cells (MSCs) that secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on metastatic MFH. BACKGROUND: MFH is a highly malignant and metastatic type of sarcoma but surgical removal is the only effective method for treating MFH. MSCs are easily transduced to express a high level of transgene and can migrate toward cancer. For this reason, MSCs are a promising candidate for metastatic MFH therapies. METHODS: In vitro sustainability of MSC-TRAIL against MFH-ino was analyzed by apoptosis assay. For preclinical study, anti-MFH effects of MSC-TRAILs were validated in murine models for local tumorigenesis and metastasis. Furthermore, a time-interval metastasis model of MFH was applied to confirm antimetastatic ability of MSC-TRAIL for preestablished metastatic MFH. RESULTS: We found that MFH-ino is highly susceptible to recombinant TRAIL and MSC-TRAIL, which selectively induce apoptosis via caspase-8 activation in vitro. Moreover, not only MFH-ino but xenograft explants were also significantly inhibited by MSC-TRAIL in local tumorigenesis. In particular, the metastatic ability of MFH-ino was considerably reduced by MSC-TRAIL in metastasis murine model, particularly for preestablished metastatic MFH. CONCLUSIONS: These results suggest that MSC-TRAIL is sufficiently effective in inhibiting MFH-ino metastasis and the application using MSC-TRAIL could be extended to other sarcomas and recurrent metastatic cancers for cell-mediated cancer therapy.
Subject(s)
Adipose Tissue, White/cytology , Antineoplastic Agents/therapeutic use , Genetic Therapy/methods , Histiocytoma, Malignant Fibrous/therapy , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , TNF-Related Apoptosis-Inducing Ligand/therapeutic use , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Flow Cytometry , Histiocytoma, Malignant Fibrous/pathology , Humans , Mice , Mice, Inbred NOD , Neoplasm Metastasis , TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Transduction, Genetic , Transgenes , Tumor Cells, CulturedABSTRACT
Diabetes is characterized by an absolute or relative deficiency of pancreatic ß-cells. New strategies to accelerate ß-cell neogenesis or maintain existing ß-cells are desired for future therapies against diabetes. We previously reported that forkhead box O1 (FoxO1) inhibits ß-cell growth through a Pdx1-mediated mechanism. However, we also reported that FoxO1 protects against ß-cell failure via the induction of NeuroD and MafA. Here, we investigate the physiological roles of FoxO1 in the pancreas by generating the mice with deletion of FoxO1 in the domains of the Pdx1 promoter (P-FoxO1-KO) or the insulin 2 promoter (ß-FoxO1-KO) and analyzing the metabolic parameters and pancreatic morphology under two different conditions of increased metabolic demand: high-fat high-sucrose diet (HFHSD) and db/db background. P-FoxO1-KO, but not ß-FoxO1-KO, showed improved glucose tolerance with HFHSD. Immunohistochemical analysis revealed that P-FoxO1-KO had increased ß-cell mass due to increased islet number rather than islet size, indicating accelerated ß-cell neogenesis. Furthermore, insulin-positive pancreatic duct cells were increased in P-FoxO1-KO but not ß-FoxO1-KO. In contrast, db/db mice crossed with P-FoxO1-KO or ß-FoxO1-KO showed more severe glucose intolerance than control db/db mice due to decreased glucose-responsive insulin secretion. Electron microscope analysis revealed fewer insulin granules in FoxO1 knockout db/db mice. We conclude that FoxO1 functions as a double-edged sword in the pancreas; FoxO1 essentially inhibits ß-cell neogenesis from pancreatic duct cells but is required for the maintenance of insulin secretion under metabolic stress.
Subject(s)
Diabetes Complications/metabolism , Disease Models, Animal , Forkhead Transcription Factors/physiology , Insulin-Secreting Cells/metabolism , Obesity/metabolism , Pancreas/metabolism , Animals , Cell Count , Cell Differentiation , Crosses, Genetic , Diabetes Complications/pathology , Diet, High-Fat/adverse effects , Dietary Sucrose/adverse effects , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Gene Expression Regulation , Glucose Intolerance/complications , Glucose Intolerance/etiology , Glucose Intolerance/prevention & control , Insulin/blood , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/ultrastructure , Mice , Mice, Knockout , Mice, Mutant Strains , Obesity/complications , Obesity/pathology , Pancreas/pathology , Pancreatic Ducts/metabolism , Pancreatic Ducts/pathology , RNA, Messenger/metabolism , RatsABSTRACT
Mesenchymal stem cells (MSCs) have known to induce immunosuppressive properties by preventing T cell proliferation. However, it is remains unclear how MSCs inhibit T cell proliferation. To identify the factor that inhibits T cell proliferation, we conducted a cytokine array analysis of culture medium from a co-culture of MSCs and T cells and found that the chemokines, CXCL1, 2 and 3, were induced in T cells. MSCs also induced the expression of the CXCR2 receptor on T cell surface. Particularly, CXCL3 inhibited proliferation and increased apoptosis in T cells, which were reversed by CXCR2 inhibitor treatment. Moreover, CXCL3 decreased JAK2, STAT3, and AKT phosphorylation and these responses were also abolished by CXCR2 inhibitor treatment. MSCs suppressed the proliferation of T cells into tumor tissue. Collectively, these data demonstrate that MSCs directly regulate T cell proliferation by induction of CXCL3 chemokine and its receptor, CXCR2 on the surface in T cells.
Subject(s)
Cell Proliferation , Mesenchymal Stem Cells/immunology , Receptors, Interleukin-8B/immunology , T-Lymphocytes/immunology , Animals , Animals, Newborn , Apoptosis/drug effects , Apoptosis/immunology , Cells, Cultured , Chemokine CXCL1/immunology , Chemokine CXCL1/metabolism , Chemokine CXCL2/immunology , Chemokine CXCL2/metabolism , Chemokines, CXC/immunology , Chemokines, CXC/metabolism , Chemokines, CXC/pharmacology , Cord Blood Stem Cell Transplantation , Flow Cytometry , HeLa Cells , Humans , Janus Kinase 2/immunology , Janus Kinase 2/metabolism , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Microscopy, Fluorescence , Phosphorylation , Receptors, Interleukin-8B/metabolism , STAT3 Transcription Factor/immunology , STAT3 Transcription Factor/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
The goal of this study was to evaluate the impact of thermal and chemical aging processes on high-density polyethylene (HDPE), low-density polyethylene (LDPE), unplasticized polyvinyl chloride (U-PVC), and high-impact polyvinyl chloride (Hi-PVC) pipes. The materials were exposed to 1-10 ppm chemical disinfectants [chlorine dioxide (ClO2) and hypochlorite (HOCl)] at 40-80 °C for 1200 h. The diffusion properties of the materials were systematically analyzed based on the change in their sorption characteristics and activation energies according to the Arrhenius model. Moreover, the structural changes were analyzed with scanning electron microscopy (SEM), Fourier transform infrared (FTIR) radiation, and thermogravimetric analysis (TGA). The results show that the materials have Fickian characteristics in the aging environment. Specifically, the water sorption rates of HDPE and LDPE increase first and then decrease after reaching saturation (Ms); those of U-PVC and Hi-PVC its increasing continuously with different rate. This behavior of materials was prominent for ClO2 at high temperature and disinfectant dose because of polymeric chains crosslinking and rearrangement, extraction of monomers, and stable compounds removal during aging under exposed conditions. The deleterious effects decreased the activation energies of the materials and increased the concentrations of carbonyl groups [CO] via the formation of ketones, aldehydes, and carboxylic acids. The decomposition temperature increased with the changes in the material morphology and elemental contents under the investigated conditions. Moreover, LDPE and Hi-PVC were more severely affected in the thermal aging process with 10 mg.L-1 ClO2 at 80 °C.