Background Mesenchymal stem cells (MSCs) have been applied in basic and clinical researches of organ transplantation.Our previous study showed that intravenous injection of MSCs prolonged corneal allograft survival in rat.However,the effect of local administration of MSCs on corneal allograft rejection remains unclear.Objective The aim of this study was to investigate the effect of subconjunctival injection of MSCs on corneal allograft rejection in rat model of keratoplasty.Methods MSCs were isolated and cultured from femur and tibia bone marrow of clean Wistar rats,and then the cells were identified by induced differentiation of osteoblast and adipocyte.The third generation of MSCs was used in subsequent experiment.Allogenic penetrating keratoplasty was performed with the bilateral corneas of 20 Wistar rats as donor grafts and the right eyes of 40 Lewis rats as recipients.PBS 0.1 ml containing 2 × 106 MSCs and 0.1 ml PBS only was subconjunctivally injected immediately and postoperative 3 days respectively in randomized two groups,and another 6 normal Lewis rats served as the normal control group.Corneal rejection response was evaluated under the slit lamp after surgery based corneal opacity,edema and neovascularization,and the grafts were scored according to the criteria of Larkin.The corneal samples were extracted from 12 rats of the PBS control group and the MSCs group separately 10 days after surgery.The relative expressions of Th1 cytokines (interferon-γ [IFN-γ] mRNA and interleukin-2 [IL-2] mRNA) and Th2 cytokines (IL-4 mRNA and IL-10 mRNA) were detected by real-time quantitative PCR.Protein levels of IL-4 and IL-10 proteins in the corneas were assayed by ELISA.All experimental protocols involving rats were approved by the laboratory animal care and use committee of the Tianjin Medical University and treated with the ARVO statement for the use of animals in ophthalmic and vision research.Results The cells grew well with the orange stain for alizarin red in differentiated the osteoblasts and red stain for Oil red O in differentiated adipocytes.The survival time of corneal graft in the MSCs group was (11.8±1.6) days,it was significantly longer than (9.6±1.4) days in the PBS control group (P=0.004).The levels of IL-4 mRNA and IL-10 mRNA in the MSCs group were significantly higher than those in the PBS control group (both at P =0.00);while the levels of IFN-γ mRNA and IL-2 mRNA were not significantly different between the groups (both at P＞0.05).The IL-10 protein contents were (22.74 ±7.06),(68.40±12.83) and (215.41 ±44.66)pg/ml in the normal control group,PBS control group and MSCs group,showing significant difference among the three groups (F =55.06,P =0.00) and a significant increase in the MSCs group compared with the PBS control group and the normal control group (both at P ＜ 0.05).Conclusions Subconjunctival injection of MSCs prolongs the survival time of cornea allograft in penetrating keratoplasty probably by modulating the balance between Th1 and Th2 cytokines,especially by up-regulating Th2 cytokines.

Objective To investigate distribution and drug resistance of pathogenic bacteria in lower respitatory tract infection. Methods Distribution and drug resistance of pathogenic bacteria in lower respitatory tract infection of patients in ICU and non-ICU of our hospital during 2013 were retrospectivly analyzed. The pathogens were identified by manual methods routinely and those difficult to be identified were analyzed by using the VITEK-2-COMPACT instrument. Antimicrobial susceptibility of these isolates were tested by Kirby-Bauey methods routinely. Results In total, 956 strains were isolated from lower respitatory tract infection of patients in ICU, including 231 strains of gram-positive cocci (24. 2%), 680 strains of gram-negative bacteria (71. 1%), 45 strains of fungi (4. 7%). In patients of non-ICU, 4 464 strains were isolated, including 1 090 strains of gram-positive cocci (24. 4%), 3 226 strains of gram-negative bacteria (72. 3%), and 148 strains of fungi (3. 3%). Staphylococcus aureus, acinetobacter baumannii and pseudomonas aeruginosa were the most frequent isolates in patients of ICU and non-ICU. The overall prevalence of methicillin resistant staphylococcus aureus (MRSA) in staphylococcus aureus was 87. 0%in ICU and 74. 0% in non-ICU. MSSA was sensitive to the most antibiotics ( more than 80. 0% of the strains were sensitive to common antibiotics) except penicillin, erythromycin and clindamycin. MRSA was sensitive to trimethoprim-sulfamethoxazole and fosfomycin (more than 75. 0% of the strains were sensitive to the antibiotics) except for vancomycin, teicoplanin and linezolid. Acinetobacter baumannii was more resistant to the antibiotics (less than 40. 0% of the strains were susceptible to the antibiotics). Pseudomonas aeruginosa from ICU was more resistant to the antibiotics ( less than 50. 0% of the strains were sensitive to the antibiotics) than that from non-ICU. Stenotrophomonas maltophilia was sensitive to trimethoprim-sulfamethoxazole, levofloxacin and minocycline (more than 80. 0% of the strains were sensitive to the antibiotics). Escherichia coli and Klebsiella pneumoniae were sensitive to Piperacillin-tazobactam and Amikacin except for meropenem and imipenem ( more than 80. 0% of the strains were sensitive to the antibiotics) . Conclusion Gram-negative bacteria was the most frequent isolate in lower respitatory tract infection of our hospital during 2013. Staphylococcus aureus, acinetobacter baumannii and pseudomonas aeruginosa were the most frequent isolates in ICU and non-ICU. Resistance to the antibiotics was more common in ICU than in non-ICU. Antibiotics should be prescribed according to bacterial resistance results reasonably in order to prevent the spread of drug-resistant strains.

Objective To investigate the antimicrobial resistance in the clinical strains isolated from Tongji Hospital to the antimicrobial agents commonly used in 2012.Methods Antimicrobial susceptibility was tested by Kirby-Bauer method.The minimum inhibitory concentration (MIC) of penicillin and ceftriaxone for Streptococcus pneumoniae and vancomycin for Staphylococcus spp.were determined by E-test.All data were analyzed by WHONET 5.6 software.Results A total of 8 191 strains were isolated in 2012,including gram-positive bacteria (2 815,34.4%)and gram-negative bacteria (5 376,65.6%). The top five pathogenic bacteria isolated from outpatients were Escheria coli,Pseudomonas aeruginosa,coagulase-negative Staphylococcus (CNS),Klebsiella spp.and Staphylococcus aureus.For the isolates from non-ICU inpatients,the top five were E.coli,S.aureus,Acinetobacter spp.,Klebsiella spp.and P.aeruginosa.For those isolated from ICU patients,the top five were Acinetobacter spp.,S.aureus,P.aeruginosa,Enterococcus spp.and E.coli.The prevalence of MRSA and MRCNS was 58.1% and 64.3%,respectively.Seventeen strains of vancomycin resistant Enterococcus were identified, including 13 strains of E.faecium with VanA and 4 strains of E.gallinarum with VanA and VanC.The percentage of antimicrobial resistance in E.faecium was significantly higher than that in E.faecalis (P<0.05).A total of 94 strains of carbapenem-resistant Enterobacteriaceae were detected.The prevalence of penicillin-non-susceptible S.pneumoniae in children was much higher than that in adults. The prevalence of carbapenem-resistant P.aerugonosa and Acinetobacter spp. was 28.1% and 56.2% respectively.Beta-lactamase was produced in 41.8% of the H.influenzae and 98.6% of the M. catarrhalis isolates. Conclusions The prevalence of multidrug resistant strains has been increasing, especially vancomycin-resistant Enterococcus and carbapenem-resistant Enterobacteriaceae.

Objective To investigate the distribution,variability and antibiotic resistance of the pathogen sisolated from bloodstream infections.Methods The bacterial strains were routinely isolated from blood samples and identified.Brucella trains were identified by PCR and gene sequencing.Antimicrobial susceptibility were routinely tested for these isolates.Results A total of 2 152 strains,excluding coagulase negative Staphylococcus,were isolated from blood specimens during the 5-year period from 2009 through 2013.Gram-positive cocci,gram-negative bacilli and fungi accounted for 35.3% (761/2 152), 57.7% (1 242/2 152)and 6.9% (149/2 152),respectively.The top three gram-positive bacteria were S.aureus,E.faecium and E.faecalis.E.coli,K.pneumoniae,P .aeruginosa,A.baumannii,and Brucella were the most frequently isolated gram-negative bacteria.In 2009,only one Brucella melitensis was isolated,but increased to 50 in 2013 (7.1%),including one strain of Brucella suis .The prevalence of MRSA was 54.1% in S.aureus.No staphylococcal isolate was found resistant to vancomycin or linezolid.More than 60% of the E.faecalis isolates still showed low resistance (< 30%)to ampicillin and levofloxacin.But E.faecium strains were highly resistant to most antibiotics (>70%)except glycopeptides and linezolid.E. coli and K.pneumoniae isolates displayed low resistance rate (< 15%)to piperacillin-tazobactam,amikacin,cefoxitin and
were still the most common pathogens in blood stream infections.Attention should be paid to the increasing prevalence of Brucella in blood stream infections.Various levels of antibiotic resistance are found in the pathogens of blood stream infections.Antibiotics should be prescribed reasonably according to local susceptibility testing data.

Objective To analyze the influence of hepatosteatosis on pancreatic β-cell function in type 2 diabetes mellitus (T2DM).Methods A total of 213 subjects with T2DM from Metabolic Disease Hospital,Tianjin Medical University from January 2013 to December 2013 were included in the study.Non-alcoholic fatty liver disease (NAFLD) was diagnosed with abdominal ultrasonography.Patients were divided into two groups:T2DM with NAFLD and T2DM without NAFLD.ALT,AST,γ-glutamyltransferase,serum lipid,glycosylated hemoglobin A1 c (HbA1c),fructosamine,fasting glucose,insulin and 2 hours plasma glucose,insulin after 75g glucoseload were detected.The insulin resistance and β-cell function were assessed by HOMA-IR and HOMA-β.Results Among the 213 T2DM subjects,51％ (108 cases) were with NAFLD.The HOMA-IR [4.76 (2.83,7.21) vs 2.79 (1.76,4.37),P ＜ 0.05] and HOMA-β [49.18 (37.78,85.09) vs 29.50 (18.09,45.54),P ＜ 0.05] were significantly higher in T2DM with NAFLD than those in T2DM alone.Within subjects with T2DM and NAFLD,the HOMA-IR [6.28 (2.87,8.17) vs 2.95 (2.07,3.66) P ＜ 0.05] and HOMA-β [59.18 (37.78,85.09) vs 30.59 (28.56,34.49),P ＜ 0.05] levels were higher in subjects with normal liver function than those with abnormal liver function.Conclusions T2DM patients with NAFLD have severer insulin resistance than those without NAFLD.The β-cell function of those patients was compensatory increased,which was decreased in subjects with abnormal liver function.

BACKGROUND:cAMP response element binding protein (CREB) is a key protein of memory, which is closely related to long-term memory. It wil provide a new way for the treatment of hypoxic ischemic brain damage (HIBD) to study the effects of dental pulp stem cel s transplantation on the long-term behavior and CREB protein via the lateral ventricle in neonatal HIBD rats. OBJECTIVE:To observe the changes in long-term behavior and CREB protein expression in neonatal HIBD rats after human dental pulp stem cel transplantation, thereby providing scientific evidence for clinical treatment of neonatal HIBD. METHODS:Thirty-six healthy 7-day-old Sprague-Dawley rats were randomly divided into normal, HIBD and cel transplantation group. The hypoxic ischemic brain damage models were established in the brain damage and cel transplantation groups. Twenty-four hours after HIBD, human dental pulp stem cel s were injected into the left lateral cerebral ventricle of rats in the cel transplantation group, total y 3×106 living cel s. Equal volume of normal saline was injected into the left lateral cerebral ventricle of rats in the normal control and HIBD groups. RESULTS AND CONCLUSION:The average time to seek water, the average escape latency and escape distance of the human dental pulp stem cel s group were significantly shorter than those of hypoxic ischemic brain injury group (P<0.01), but longer than those in the normal group (P<0.01). Nissl staining showed that the cel s in the hippocampal CA1 region in human dental pulp stem cel s group were more regular, the number of cel s was significantly higher than that of hypoxic ischemic brain injury group, but stil significantly less than that in the normal group (P<0.05). Immunohistochemical staining results showed that the number of CREB positive cel s in human dental pulp stem cel s group was significantly higher than those in HIBD group, but stil significantly less than those in the normal group (P<0.01). It is suggested that human dental pulp stem cel s transplantation could promote the expression of CREB protein in the hippocampal CA1 region, to improve the long-term learning and memory ability of hypoxic ischemic neonatal rats, and thus repair HIBD.

Objective To observe the difference between Radix AstragaLi(RA) and Radix Fici Hirtae(RFH) in immune regulation and improvement of anaemia.Methods Fatigue resistance,brain hypoxia and normal hypoxia tolerance were observed to measure the anti-stress function.Bloodletting-induced anemia model and cyclophosphamid-induced anemia model were used to observe the effect of blood enrichment.Phagocytic function of macrophage,serum hemolysin level and delayed type hypersensitivity(DTH) reaction were also detected.Results RA and RFH could improve the fatigue resistance,normal hypoxia tolerance and brain hypoxia tolerance,and increase the red blood cell count and hemoglobin content,enhance phagocytic function of macrophage and serum hemolysin level,and increase the DTH reaction.Conclusion Both RA and RFH have an effect on improving anti-stress ability,immune function and blood enrichment,and there are no significant difference between RA and RFH.

Background Reasearches showed that α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation and ameliorates the ocular surface abnormalities in a scopolamine-induced dry eye rat model,and the managing effect of sodium carboxymethylcellulose (CMC) on dry eyes also has been determined.However,whether α-MSH can enhance the therapeutic effects of CMC remains to be investigated.Objective This study was to investigate the protective effects of α-MSH combined with CMC on ocular surface in a scopolamine-induced dry eye rat model.Methods Sixty clean female Wistar rats were randomly divided into normal control group,model control group,NaCl group,CMC group,α-MSH group and α-MSH+CMC group,and 10 rats for each group.The dry eye models were established by subcutaneous injection of scopolamine hydrobromide at 9:00,12:00,15:00 and 18:00 per day for 28 days.0.9％ NaCl solution,1×10 3 mg/ml α-MSH solution,0.5％ CMC eye drop,and 1 ×10-3 mg/ml α-MSH+0.5％ CMC solution were topically administered twice a day (8:00,17:00) since the initial day of modeling according to grouping.Shirmer Ⅰ test (S Ⅰ t),breakup time of tear film (BUT) and corneal fluorescence staining were performed before and 7,14,21,28 days after the application of drugs.At 28 days following the administration of drugs,the eyeballs of the rats were collected.Hemotoxylin and eosin staining was employed to examine the morphology of corneas,and periodic acid schiff (PAS) staining was used to count the conjunctival goblet cells.This study protocol was approved by Experimental Animal Ethic Committee of Tianjin Medical University (SYXK 2009-0001),and the use and care of the rats complied with ARVO Statement.Results The S Ⅰ t and BUT values were significantly reduced,and the corneal fluorescence staining scores were significantly increased over time following modeling in the model control group (all at P＜0.01).No significant differences were found in the S Ⅰ t,BUT and corneal fluorescence staining scores between model control group and NaCl group at various time points (all at P＞0.05).At 7,14 and 21 days after intervention,the S Ⅰ t values were (4.800±0.789),(4.100±0.516) and (4.300±0.856) mm in the α-MSH+CMC group,which were considerably higher than (2.875 ±0.719),(2.375 ±0.619) and (2.532±0.957)mm in the NaCl group (all at P＜0.01).At 7 days after intervention,the BUT values were (4.938± 1.843) seconds and (5.000±1.491) seconds in the α-MSH group and α-MSH+CMC group,which were significantly higher than (3.250±1.000) seconds in the NaCl group (both at P＜0.01).The corneal fluorescence staining scores in the CMC group,α-MSH group and α-MSH+CMC group were significantly lower than that in the NaCl group,with the lowest score in the α-MSH +CMC group (all at P＜0.05).The thickening of corneal epithelial layer,corneal edema and arrangement disorder of corneal stroma were found in the model control group and NaCl group;while slight corneal edema and epithelial cell proliferation were exhibited in the α-MSH+CMC group by hemotoxylin and eosin staining.PAS staining showed that the number of goblet cells was much more in the CMC group,α-MSH group and α-MSH+ CMC group than that in the model control group and NaCl group (all at P ＜ 0.01).Conclusions The sole application of α-MSH or CMC alleviates ocular surface damage and morphological abnormality to certain extent,and the combination of α-MSH and CMC generates more effective protection in comparison with sole administration of α-MSH or CMC.The early application of the drugs plays an improvement role in tear secretion and tear film stability in dry eyes.

Objective Based on middle cerebral artery occlusion (MCAO) model,to investigate the effects of melatonin (MT) on DWI and expression of Fas,FasL and cleaved Caspase-3 proteins in rat model with focal cerebral ischemia.Methods Eighty SD rats were randomly divided into Sham group (n=16),MCAO group (n=32) and MT group (n=32).The rats in sham group were treated with sham-operation.And the rats in MCAO and MT groups were peritoneally injected with saline and MT respectively.The behavioral scores were assessed in the three groups.The rats in MCAO and MT group with the behavioral scores of 1 3 points were selected in the study.The DWI relative signal intensity (rDWI-SI),Fas,FasL and cleaved Caspase-3 proteins were respectively examined by MR scaning and immunohistochemical staining in all rats of each group at 6 h,24 h,72 h and 7 days after ischemia reperfusion (IR) or sham-operation.And the DWI and immunohistochemical results for each group were compared.Results At last,there were 16 rats in sham group,29 rats in MCAO group and 30 rats in MT group,respectively.There was significant difference of the behavioral scores among the three groups (x2 =50.125,P＜0.01).The behavioral scores of MT and MCAO groups were higher than those of sham group (all P ＜0.05).And the behavior scores of the MT group were lower compared with MCAO group after IR.Compared with the rDWI-SI values measured at 6 h,24 h and 72 h,7 days in sham group,the rDWLSI values of MT and MCAO groups were significantly higher (all P＜0.01).And the rDWI-SI was higher in MCAO group than those in MT group at 6 h,24 h and 72 h after IR (all P＜0.01).And there was no significant difference of rDWI-SI at 7 days after IR between MT and MCAO groups (P＞0.05).The immunohistochemical staining results showed that the number of Fas,FasL and cleaved Caspase-3 positive cells in MCAO and MT groups were significantly higher than those in sham group (all P＜0.01).And there were less Fas,FasL and cleaved Caspase-3 positive cells in MT groups compared with MCAO group (all P＜0.05) at 6 h,24 h and 72 h after IR.There was no significant difference of Fas,FasL and cleaved Caspase-3 positive cells among the three groups at 7 days after IR (P＞0.05).Conclusion MT can effectively alleviate the rDWI-SI value and inhibit the expression of Fas,FasL and cleaved Caspase-3 proteins in rats of focal cerebral ischemia.

Objective:To retrospectively analyze the clinical characteristics and drug resistance among COVID-19 patients with bacterial and fungal infections.Methods:Clinical data of COVID-19 patients whose blood, urine, sputum and alveolar lavage fluid samples were positive for bacteria and fungi were collected in Tongji Hospital from February 10 to March 31, 2020. WHONET5.6 software was used to analyze drug susceptibility test results.Results:A total of 95 COVID-19 patients positive for pathogenic bacteria were enrolled and among them, 23 were non-critical patients and 72 were critical patients. The main symptoms in these patients included fever, cough with sputum, fatigue and dyspnea. Male and female critical patients accounted for 63.89% and 36.11%, respectively. Most of the patients with bacterial and fungal infections were critical type, accounting for 23.61%. The mortality rates of non-critical and critical patients were 13.04% and 61.11%, respectively. A total of 179 strains of pathogenic bacteria were isolated. The positive rate of Escherichia coli in non-critical patients was 37.50%, which was higher than that in critical patients. However, the positive rates of Acinetobacter baumannii and Klebsiella pneumoniae in critical patients were both 29.87%, higher than those in non-critical patients. There was no significant difference in the positive rate of gram-positive bacteria or fungi between non-critical and critical patients. It was noteworthy that the positive rate of Candida parapsilosis in blood samples of critical patients was relatively high, reaching 36.40%. Drug susceptibility test results showed that no carbapenem-resistant Escherichia coli stains were detected and 60.87% of Klebsiella pneumoniae strains were resistant to carbapenems. Acinetobacter baumannii strains were 100% resistant to three antimicrobial drugs. Methicillin-resistant Staphylococcus aureus strains accounted for 71.43%, but no vancomycin-resistant gram-positive cocci were found. Conclusions:Critical COVID-19 patients were mostly male and prone to multiple bacterial and fungal infections. The mortality of critical patients was higher than that of non-critical patients. Critical COVID-19 was often complicated by hospital acquired infections caused by bacteria including Acinetobacter baumannii and Klebsiella pneumoniae with high drug resistance.