[Effect of adipose tissue-derived mesenchymal stem cell transplantation in treatment of liver fibrosis and possible mechanism].

Objective: To investigate the effect of adipose tissue-derived mesenchymal stem cell (ADSC) transplantation in the treatment of liver fibrosis rats and possible mechanism. Methods: Subcutaneous adipose tissue in the inguinal region of rats was collected to isolate ADSCs. The rats with liver fibrosis induced by intraperitoneally injected carbon tetrachloride were divided into cell transplantation group and phosphate buffer saline (PBS) injection group, and the rats which were fed normally were enrolled as negative control group. The rats in the cell transplantation group were given tail vein injection of ADSCs, and those in the PBS injection group were given injection of 0.5 ml PBS. At 7 days after transplantation, blood samples were collected from the inferior vena cava to evaluate liver function; liver tissue was collected to measure the protein expression of hepatocyte growth factor (HGF) and alpha-smooth muscle actin (α-SMA); Masson trichrome staining was used to evaluate intrahepatic collagen deposition. Hepatic stellate cells (HSCs) were collected from the rats with liver fibrosis, and indirect co-culture of HSCs and ADSCs was performed in vitro to analyze the influence of ADSCs on the proliferation and apoptosis of HSCs. The independent samples t-test was used for comparison between groups, and an analysis of variance was used for comparison of means between multiple samples. Results: ADSCs were found in liver tissue in the transplantation group, and compared with the PBS injection group, the transplantation group had significant alleviation in hepatocyte necrosis, vacuolization, and area of fibrosis and significant reductions in the serum levels of aminotransferases, while there was no significant difference in the level of albumin between the two groups. Compared with the PBS injection group, the transplantation group had significant upregulation in the protein expression of HGF and significant downregulation in the protein expression of α-SMA (both P < 0.05). In vitro co-culture for 72 hours showed that ADSCs inhibited the proliferation of HSCs, and there was a significant difference between the co-culture group and the control group with HSCs cultured alone. Caspase-3 immunostaining showed that after co-culture for 72 hours, there was a significant difference in the apoptosis rate of HSCs between the co-culture group and the control group with HSCs cultured alone (23.42% ± 3.02% vs 14.82% ± 3.93%). Conclusion: ADSC transplantation can upregulate the expression of HGF in the liver, promote the apoptosis of HSCs, and thus alleviate liver fibrosis.

Experimental study on co-culturing adipose-derived stem cells with osteoblasts under different conditions.

OBJECTIVE: To observe whether adipose-derived stem cells (ADSCS), co-cultured with osteoblasts, can differentiate into osteoblasts and, if so, to study the best-induced conditions, with an ultimate goal of repairing bone defects. MATERIALS AND METHODS: Adipose-derived stem cells and osteoblasts were isolated from New Zealand white rabbits, and co-cultured in media with either 5% or 10% fetal bovine serum, for up to 4 weeks. The morphology of collected cells was examined under a microscope, and histological staining with alkaline phosphatase and alizarin red was carried out after induction for 1, 2, 3 and 4 weeks. Osteogenesis identification, including mRNA expression of type I collagen and osteocalcin, and alkaline phosphatase, was also performed using RT-PCR. RESULTS: After 7 days of co-culture, some adipose-derived stem cells became round in both groups. After 14 days of co-culture, adipose-derived stem cells were found highly-differentiated, and stained positively with alkaline phosphatase and alizarin red, similar to mature osteoblasts. The mRNA expression of type I collagen and osteocalcin increased in both groups, especially in the 10% fetal bovine serum group. CONCLUSIONS: Our findings indicate that adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts when induced by a high concentration of serum culture.

[A comparative study of diagnostic efficacy of different diagnostic/ classification criteria in Takayasu arteritis].

OBJECTIVE: This study aimed to analyze and evaluate the diagnostic efficacy of Ishikawa's, the modified Ishikawa's criteria, 1990 American College of Rheumatology (ACR ) classification criteria and the diagnostic model based on Chinese population in Chinese TA patients. METHODS: One hundred and forty-nine patients with Takayasu arteritis and 126 patients with other vascular disorders which involved aorta or its branches were recruited in this study.All the patients were admitted to the Department of rheumatism and Immunology clinic or inpatient department of Zhongshan Hospital affiliated to Fudan University from January 1(st), 2008 to June 31(st), 2015.General characteristics, clinical manifestations, laboratory results and imaging data of all the patients were collected.Sensitivity, specificity, accuracy and area under receiver operating characteristics (ROC) curve of different criteria were analyzed. RESULTS: Sensitivity, specificity, accuracy and area under ROC curve of Chinese diagnostic model were 90.60%, 80.95%, 86.18%, and 85.80%, respectively, while those of Ishikawa criteria were 34.23%, 99.21%, 64.00%, and 66.70%, respectively.These four indicators of the modified Ishikawa criteria were 84.13%, 79.87%, 81.82%, and 82.00%, respectively and that of ACR criteria were 83.89%, 83.33%, 83.64%, and 83.60%, respectively.No significant difference was found between any two of Chinese diagnostic model, the modified Ishikawa criteria and ACR criteria in all the indicators.Sensitivity of Chinese diagnostic model was highest, while specificity of Ishikawa criteria was the highest.Among these four criteria, the diagnostic efficacy of Chinese model was the best and that of Ishikawa criteria was the worst. CONCLUSION: Chinese diagnostic model, which is based on Chinese population and adopts advanced imaging modality, has better diagnostic efficacy.

Dopamine receptor DR2 expression in B cells is negatively correlated with disease activity in rheumatoid arthritis patients.

OBJECTIVE: Dopamine receptor (DR) signaling is involved in the pathogenesis of autoimmune diseases. We aimed to measure the expression levels of DR1-5 on B cells from patients with rheumatoid arthritis (RA) and to analyze the relationship between DRs and clinical manifestations, inflammatory biomarkers, functional status and disease activity. METHODS: A total of 29 patients with RA, 12 healthy donors and 12 patients with osteoarthritis (OA) were recruited in this study. Flow cytometry was used to measure the levels of DR1-5 expressed on B cells. The relationships between B cell DR expressions and clinical features in RA patients were analyzed using the Spearman correlation test. RESULTS: The expression levels of B cell DR1-5 in both the RA and OA groups were lower than those in healthy controls. After 3 months of medication, all five receptors were elevated in RA patients, with DR2 and DR3 being significantly increased from the baseline. DR2 expression on B cells was negatively correlated with inflammatory biomarkers and disease activity. CONCLUSION: RA patients had lower expression level of DR2 on B cells compared to the healthy controls, and the level of DR2 negatively correlated with the disease activity. DR2 and DR3 might be novel predictors of patient responses to disease modifying antirheumatic drug therapy.

Direct detection of chloramphenicol in honey by neutral desorption-extractive electrospray ionization mass spectrometry.

Herein, we constructed a platform of neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) for direct and rapid detection of chloramphenicol (CAP) in honey samples diluted with methanol. Under the optimized working conditions, the quantitative information of CAP residues was acquired effectively by EESI-Ion Trap MS (n) . Using heated methanol-N2 as spray reagent, we reduced the limit of determination (LOD) from 73.3 ng/mL to 0.3 ng/mL, and the CAP detection is linear in the range of 1-5000 ng/mL (R = 0.9947). For the honey samples with CAP of 10, 100, and 1000 ng/mL, the recoveries were 133.0, 80.6, and 101.1%, and the relative standard deviations were 5.96, 8.82, and 8.71%, respectively. The reproducibility assays showed the stability of this method. Therefore, this ND-EESI-MS method is powerful for direct, rapid, and quantitative CAP analysis in honey samples with high sensitivity, precision, and specificity.

Association of ELMO1 gene polymorphisms with diabetic nephropathy in Chinese population.

BACKGROUND: Genetic variations in the engulfment and cell motility 1 (ELMO1) gene have recently been identified to be associated with nephropathy attributed to Type 2 diabetes mellitus (T2DM). Since T2DM-associated complications are proved to be more common among Asians than Western individuals, and Chinese people have a high incidence of diabetic nephropathy (DN), this study sought to analyze the association of ELMO1 gene polymorphisms with DN to probe into the effects of ELMO1 gene on susceptibility of DN in Chinese population. METHODS: We genotyped 6 polymorphism sites of ELMO1 gene in 200 unrelated Chinese subjects (123 T2DM with DN case subjects and 77 T2DM without DN control subjects). Genotyping was detected by a Sequenom MassARRAY genotyping system. RESULTS: The strongest associations in ELMO1 gene with DN occurred at rs741301 [odds ratio (OR) 1.89; p=0.004] and rs10951509 (OR 1.76; p=0.02). Unconditional logistic regression analysis identified that the rs741301 polymorphism (presence of A allele, adjusted OR 3.27; p=0.03) and duration of T2DM (adjusted OR 1.15; p=0.04) were independent predictors for DN. The marker rs741301 located in intron 18 of ELMO1 gene was in strong linkage disequilibrium (LD) with rs11769038 (D'=0.91). Furthermore, haplotype analysis identified that haplotype 1 [CAAAGA] (OR 1.95; p=0.01), haplotype 2 [CAAAGG] (OR 0.50; p=0.01), and haplotype 9 [TGCGGG] (OR 0.17; p=0.007) of ELMO1 were significantly associated with DN. CONCLUSIONS: This study first investigated the association of ELMO1 gene polymorphisms with DN in a Chinese population, supporting its key role as a candidate gene in the susceptibility of DN.

Rescue of the colony-stimulating factor 1 (CSF-1)-nullizygous mouse (Csf1(op)/Csf1(op)) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis.

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1-null Csf1(op)/Csf1(op) mice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects of Csf1(op)/Csf1(op) mice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed beta-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.

Natural isolates of simian virus 40 from immunocompromised monkeys display extensive genetic heterogeneity: new implications for polyomavirus disease.

Simian virus 40 (SV40) DNAs in brain tissue and peripheral blood mononuclear cells (PBMCs) of eight simian immunodeficiency virus-infected rhesus monkeys with SV40 brain disease were analyzed. We report the detection, cloning, and identification of five new SV40 strains following a quadruple testing-verification strategy. SV40 genomes with archetypal regulatory regions (containing a duplication within the G/C-rich regulatory region segment and a single 72-bp enhancer element) were recovered from seven animal brains, two tissues of which also contained viral genomes with nonarchetypal regulatory regions (containing a duplication within the G/C-rich regulatory region segment as well as a variable duplication within the enhancer region). In contrast, PBMC DNAs from five of six animals had viral genomes with both regulatory region types. It appeared, based on T-antigen variable-region sequences, that nonarchetypal virus variants arose de novo within each animal. The eighth animal exclusively yielded a new type of SV40 strain (SV40-K661), containing a protoarchetypal regulatory region (lacking a duplication within the G/C-rich segment of the regulatory region and containing one 72-bp element in the enhancer region), from both brain tissue and PBMCs. The presence of SV40 in PBMCs suggests that hematogenous spread of viral infection may occur. An archetypal version of a virus similar to SV40 reference strain 776 (a kidney isolate) was recovered from one brain, substantiating the idea that SV40 is neurotropic as well as kidney-tropic. Indirect evidence suggests that maternal-infant transmission of SV40 may have occurred in one animal. These findings provide new insights for human polyomavirus disease.

Outbreaks of gastroenteritis in elderly nursing homes and retirement facilities associated with human caliciviruses.

Eleven outbreaks of acute gastroenteritis, eight of which were in nursing homes or retirement facilities, were reported in virginia during the winter of 1993-1994. Serum samples (four outbreaks) and stool samples (two outbreaks) from involved people were tested for human calicivirus (HuCV) infection by enzyme immune assays (EIAs) using recombinant Norwalk virus (rNV) and Mexico virus (rMX) capsid antigens and reverse transcription-polymerase chain reaction (RT-PCR). Of the 31 pairs of acute and convalescent serum specimens tested, 24 had a fourfold or more titer increase to rMX and 4 responded to rNV. In all four outbreaks, the geometric mean titers (GMTs) against rMX were significantly higher than those against rNV in the convalescent, but not in the acute phase of illness. The antibody response to rMX among these patients was also higher than to rNV (summary mean 32-fold increase vs. 0.7-fold increase, respectively, P < .001). Antigen was detected in 5 of 21 stool specimens tested by the rMX EIA, RNA in 12 of 17 stool specimens tested by RT-PCR, and small round structured virus (SRSV) particles in 12 of 21 by electron microscopy (EM); none were positive by the rNV EIA. Sequence analysis of the RT-PCR-amplified products from the viral RNA polymerase region revealed 92-93% amino acid identity with Snow Mountain agent (SMA), 86% with MX, 58-59% with NV, and 31-32% with Sapporo HuCV, suggesting that these viruses belong to the SMA HuCV genogroup.

Partial characterization of the genome of nine animal caliciviruses.

Caliciviruses (CVs) include at least 42 distinct serotypes. Seventeen CV serotypes have been isolated from marine sources and are called San Miguel sea lion caliciviruses (SMSVs). CVs also have been isolated from reptiles, primates, and other terrestrial animals. Nucleotide sequences from portions of genome of prototype strains for six SMSV serotypes, the reptile CV, Cro-1, the cetacean CV, Tur-1, and the primate CV, Pan-1, are presented. cDNA products of the polymerase (all strains characterized) and capsid (SMSV-17) regions were produced by reverse transcription-polymerase chain reaction using Pan-1 primers. Comparisons of nucleotide and amino acid identity among these and published CV sequences indicated that the nine characterized CVs fall into a phylogenetic group that includes SMSV-1 and SMSV-4 and that is more closely related to other characterized animal CVs than to most human CVs. The phylogenetic analysis also indicated that distinct genera exist among the Caliciviridae. SMSV-17 and SMSV-4 are predicted to be closer to each other than other caliciviruses of known serotype; 574 (82%) of the 704 amino acids in the SMSV-17 and SMSV-4 capsid genes were identical.