The discovery of limbal stem cells is the key advancement in ophthalmology in the 20th century.Many clinical researches have proved that limbal stem cells transplantation is an effective method to treat severe ocular surface diseases.However, some important issues associated with the identification,isolation,standardized culture in vitro and differentiation after transplantation are still not clear and need to be paid more attention.Strengthening the underlaying research in this field will play important role in treating corneal blindness.

Vibrio parahaemolyticus has been considered as one of the most important foodborne bacterial pathogens.The loop-mediated isothermal amplification(LAMP) that amplifies DNA with high specificity and rapidity under an isothermal condition was applied for rapid detection of this pathogen for the first time.A set of four primers,two outer and two inner primers,was designed specifically to recognize the thermolabile hemolysin gene(tlh) of V.parahaemolyticus.The LAMP reaction mix was optimized.The most optimal reaction temperature and time of the LAMP assay for the tlh gene were 60℃ and 60min,respectively.Genomic DNAs from 28 bacterial strains including 14 V.parahaemolyticus strains were amplified using LAMP,and no amplicon was observed in other bacterial strains.The detection limit of this LAMP assay was around 90 fg of V.parahaemolyticus genomic DNA and 24 colonies forming units for pure cultures.In addition,this method was applied to detect artificially contaminated food samples,and the detection limit was 89 cfu/g for non-cultured artificially contaminated food samples.These results suggested that detection of V.parahaemolyticus by LAMP is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipment.This assay is expected to become a valuable tool for rapid detection and identification of V.parahaemolyticus.

Objective: To observe the clinical efficacy of electroacupuncture (EA) with different frequencies in treating migraine.Methods: Ninety patients with migraine were divided into a low frequency electroacupuncture (LF-EA) group, a high frequency electroacupuncture (HF-EA) group and a variable frequency electroacupuncture (VF-EA) group by the random number table method. Shuaigu (GB 8), Hegu (LI 4), Waiguan (TE 5), Taichong (LR 3), Taiyang (EX-HN 5), Fengchi (GB 20) and Ashi points were selected for all three groups. After achieving needling sensation (Deqi), the LF-EA group received 2 Hz continuous wave EA stimulation; the HF-EA group received 100 Hz continuous wave EA stimulation, and the VF-EA group received 2 Hz/100 Hz sparse-dense wave EA stimulation. The EA stimulation lasted for 30 min in all the three groups, once a day, 10 times as a course, and the clinical efficacy was evaluated after 2 consecutive courses of treatment. Results: After treatment, the total effective rates in the LF-EA group, HF-EA group and VF-EA group were 77.7％, 83.3％ and 93.3％, respectively. The difference in total effective rate between the LF-EA and HF-EA groups was not statistically significant (P>0.05), but the total effective rate was significantly higher in the VF-EA group than in the LF-EA and HF-EA groups (both P<0.01). After treatment, the headache scores significantly decreased in all three groups (all P<0.01). The difference in headache score between the LF-EA and HF-EA groups was not statistically significant (P>0.05), while the score was significantly lower in the VF-EA group than in the LF-EA and HF-EA groups (both P<0.01). Conclusion: VF-EA (2 Hz/100 Hz), compared with LF-EA (2 Hz) and HF-EA (100 Hz), shows superior clinical efficacy in treating migraine.

ObjectiveTo establish the reference intervals for ALT,AST,GGT and LDH among the Han nationality in Beijing.MethodsThe document C28-P3 issued by CLSI was a guideline about how to define,establish,and verify reference intervals in the clinical laboratory.IFCC had established multicenter enzymes reference intervals based on the guideline.Exclusion criteria were designed for screening candidate reference individual according to the document C28-P3 and the multicenter study's experience.Blood specimens were collected from 315 healthy individuals aged 20 to 60 years old,including 132 males and 183 females.Reference materials were used to ensure the accuracy of the test results of the four liver enzymes.The methods which used to test the four liver enzymes could be traced to the IFCC enzymes reference measure procedure,the reagent of ALT and AST included pyridoxal phosphate.Results There was statistically difference between males and females of the referenceranges forALT, ASTand GGT.Therefore,gender-specific reference intervals were established as ALT:8.2 -50.8 U/L (F),12.7 -71.8 U/L (M) ; AST:15.0 -36.7 U/L ( F),16.6 -51.1 U/L (M) ;GGT:9.0 -37.3 U/L (F),12.0 -50.9 U/L (M).For LDH,the reference interval was 127 -224 U/L,as no significant gender difference was found.ConclusionsThe reference intervals for the four liver enzymes based on the population of the Han nationality in Beijing are established.The upper reference limit for ALT in Beijing Han population is higher than that from other similar studies.

objective To explore the role of cytokines and dendritic cells (DCs) in rat high-risk corneal allograft survival pro- longed by superantigen Staphylococcal Enterotoxin B (SEB) and to compare the different effects between SEB and glucocorticoid. Design Experimental study.Participants Fisher 344 and Lewis rats.Methods The Fisher 344 and Lewis inbred rats were used for high-risk penetrating keratoplasty model.All of the Lewis rat recipients were divided into three groups by blinded fashion.GroupⅠand GroupⅡrats were injected intraperitoneally with 0.2ml saline buffer or SEB (75?g/ml) respectively at 4-day intervals on three occasions before transplantation.GroupⅢrats were injected subconjunctivally with 0.1ml dexamethasone (1mg/ml) daily from the first day after surgery for 2 weeks.The allograft survival was examined under slit-lamp.The concentration of interleukin IL-1?,IL-2,IL-4,IL-5,IL-6, IL-10,TNF-?in rat aqueous humor and peripheral blood were measured by liquichip and the cytokine and CD11c,CD80,MHC-Ⅱex- pression in corneal grafts were examined by immunohistochemestry staining.Main Octcome Measures Mean survival time of the allo- grafts,the level of cytokines in aqueous humor,peripheral blood and corneal grafts.Results Compared with group control,the grafts mean survival time was delayed about 3.8d in group SEB (P=0.00) and 7.1d in group dexamethasone(P=0.01).But there was no signifi- cant difference between group SEB and dexamethone (P=0.26).Liquichip test showed that the level of IL-1?in aqueous humor was re- duced and IL-4,IL-6 and IFN-?,ascended.Only IFN-?,and IL-6 could be found in peripheral blood,and the changing shift of them was similar to that in aqueous humor.The immunohistochemistry staining showed that the expression of IL-2 in rat corneal grafts was signif- icantly decreased but IL-4,IL-6 and IL-10 elevated.The expression of DCs in group SEB was similar to that in group control,which was elevated after keratoplasty,but the phenotype of DCs was not the same.There were predominantly mature DCs in group control while immature DCs in group SEB.Conclusions The immunological inhibiting effect of SEB is same to glucocorticoid,but the mecha- nism is different.SEB can modulate immune response,which might induce immune inhibition via the local production of cytokines and effect on DCs maturation involving corneal graft rejection prevention.

OBJECTIVETo master the distribution of male and female soldiers' body composition with bioelectrical impedance analysis (BIA) method in order to provide data for building up the standards in comparison with those of ordinary residents.

METHODSA cluster stratified sample of 5968 graduated soldiers among different armed services colleges was measured by BIA. Five percent-95% distributions with in the upper and lower limits were established on basis of the above result. The crosswise comparison was also performed.

RESULTS(1) Five percent-95% reference ranges of people in the graduating class of armed services colleges were body fat percentage (BF%): 10.30%-20.70% (male) and 19.20%-30.10% (female), body mass index (BMI): 19.30-25.70 (male) and 18.00-23.99 (female), lean body mass percentage (LBM%): 79.27%-86.69% (male) and 69.89%-80.69% (female), muscle percentage (M%): 74.24%-83.96% (male) and 65.23%-75.27% (female), bone percentage (B%): 5.01% 5.77% (male) and 4.65%-5.51% (female). (2) Soldiers in the graduating class of armed services colleges have less BF%, more B% and M% than those of ordinary residents in the same age and the same sex.

CONCLUSION(1) Soldiers in the graduating class of armed services colleges have better body composition than that of ordinary residents in the same age and the same sex; (2) Standard of body composition for soldiers in the graduating class of armed services colleges should be different from that of ordinary residents, a new standard should be built up.

Objective To investigate the activation state of and expressions of surface co-stimulatory molecules on peripheral CD4+ T cells from patients with pemphigus and healthy human controls.Methods Ninety patients with pemphigus including 24 patients with first-onset pemphigus,51 with quiescent pemphigus and 15 with recurrent pemphigus,as well as 30 healthy human controls were enrolled in this study.Peripheral blood samples were obtained from these subjects followed by lymphocyte isolation.Flow cytometry was performed to detect the expressions of CD69,intercellular adhesion molecule-1 (ICAM-1),inducible co-stimulatory molecule (ICOS),CD40 ligand (CD40L) and OX40 on CD4+ T cells.Statistical analysis was done by Mann-Whitney test using Graphpad 5.0 software.Results The expression rate of CD69 on peripheral CD4+ T cells from the healthy human controls was significantly lower than that from patients with pemphigus,patients with first-onset pemphigus,patients with quiescent pemphigus,and patients with recurrent pemphigus ((1.26 ± 0.19)％ vs.(2.46 ± 0.19)％,(2.77 ± 0.40)％,(2.15 ± 0.25)％ and (2.36 ± 0.35)％,all P ＜ 0.05).The patients with pemphigus also showed a significant increase in the expression rates of ICAM-1,CD40L and OX40 compared with the healthy human controls ((55.88 ± 1.67)％ vs.(47.75 ± 2.52)％,P＜ 0.05; (2.23 ± 0.22)％ vs.(0.73 ± 0.07)％,P＜ 0.01; (2.55 ± 0.29)％vs.(0.62 ± 0.17)％,P ＜ 0.01).No significant differences were observed between patients with different stages of pemphigus in the expression rates of CD69,ICAM-1,CD40L or OX40 (all P ＞ 0.05).The percentage of ICOS-expressing CD4+ T cells was significantly up-regulated in only patients with first-onset pemphigus as compared to the healthy controls ((3.73 ± 0.60)％ vs.(2.39 ± 0.16)％,P ＜ 0.05).Conclusions The peripheral blood CD4+ T cells from patients with pemphigus are in a relatively active state with up-regulated surface expressions of many costimulatory molecules,suggesting that CD4+ T cells are involved in the initiation and progression of pemphigus by interacting with B cells through co-stimulatory molecules.

Objective To study the changes of antigen-specific T helper type 1 (Th1) cells and Th2 cells in patients with pemphigus vulgaris (PV) at different stages,so as to better understand the roles of autoreactive T cells in PV.Methods The DG3 (96-112) peptide was synthesized.Twenty-four patients with PV and 10 health checkup examinees were included in this study.Peripheral blood mononuclear cells (PBMCs) were obtained from the health checkup examinees and all the patients before treatment and seven patients about one month after treatment,and stimulated with the DG3 peptide of 10 mg/L for different durations.Then,enzyme-linked immunospot (ELISPOT) assay was performed to count the number of DG3-specific IFN-γ+ Th1 cells,IL-4+ Th2 cells and memory B cells.One-way analysis of variance (ANOVA) and t test were done to compare the number of cells between different groups,and Pearson correlation coefficient was used to evaluate the correlation among Th cells,memory B cells and anti-desmoglein 3 (Dsg3) antibody titers.Results In these patients,the male to female ratio was 1.67 ∶ 1,and the average age was (56.59 ± 14.66) years.Compared with the health check-up examinees,the patients with PV showed a higher absolute number of DG3-specific IFN-γ+ Th1 cells (420.18 ± 350.29 vs.145.12 ± 86.56,t =3.25,P ＜ 0.05),but a similar absolute number of specific IL-4+ Th2 cells (366.76 ± 192.44 vs.335.88 ± 164.96,P＞ 0.05) per 5 × 105 PBMCs.The percentage of DG3-specific IL-4+ Th2 cells in Th2 cells was 37.03％ ± 23.44％,and the percentage of IFN-γ+ Th1 cells was 23.62％ ± 16.77％ in peripheral blood of patients with PV.The number of DG3-specific IL-4+ Th2 cells per 5 × 105 PBMCs significantly decreased from 452.82 ± 199.29 before treatment to 241.68 ± 160.60 after treatment in seven patients (t =2.48,P ＜ 0.05).There was no significant correlation between specific Th cells,memory B cells and anti-Dsg3 antibody titers (all P ＞ 0.05).Conclusions The peptide DG3 (96-112) has pathogenic epitopes which could be recognized by specific Th cells of patients with PV.Both antigen-specific IFN-γ+ Th1 cells and IL-4+ Th2 cells play certain roles in the pathogenesis of pemphigns vulgaris,and IL-4+ Th2 cells appear to be more important.

Objective To explore the role of Beclin 1 and PTEN in gastric caicinoma genesis and the effects on prognosis.Methods The expression of Beclin 1 and PTEN in 199 gastric caicinoma specimens and corresponding adjacent noncancerous tissues were examined by tissue microarrays and immunohistochemistary,and the relation with gastric cancer was analyzed.The rate of Beclin 1 and PTEN expression in 15 fresh gastric carcinoma samples and corresponding adjacent noncancerous tissues were detected by Western blot.All the samples were from Changzheng Hospital.Results The Results of immunohistochemistary showed that the rates of Beclin 1 and PTEN positive expression in cancinoma tissues were 47.2％ (94/199) and 55.8％ (111/199),both were lower than that of adjacent noncancinoma tissues (94.5％,188/199 and 92.5％,184/199; P ＜ 0.01).The lower expression of Beclin 1 and PTEN in gastric carcinoma were associated with gender,differentiation degree,depth of tumor invasion,lymph node metastases and disease stage(P＜0.05).There was a positive correlation between Beclin 1 and PTEN expression in gastric carcinoma tissues (r =0.680,P＜0.01). The survival analysis indicated that Beclin 1 and PTEN were independent factors in determining the prognosis of gastric cancinoma patients.The 5-year survival rate of Beclin 1 positive patients was 67.0％ (63/94),and of negative patients was 33.3％ (35/105).The 5-year survival rate of PTEN positive patients was 71.2％ (79/111),and of negative patients was 21.6％ (19/88) ( all P＜0.001).The Results of Western blot indicated that the expression of Beclin 1 and PTEN in gastric carcinoma tissues were significantly lower than that in the adjacent noncarcinoma tissues ( all P＜0.001).Conclusion The abnormal expression of Beclin 1 and PTEN may be related to carcinogenesis and the development of gastric carcinoma.

Objective To study the efficacy of rituximab in the treatment of pemphigus vulgaris (PV) and its underlying mechanism. Methods A 38-year-old female with PV presented with refractory, painful oral ulcers and erosions. Since she was poorly responsive to prednisone 80 mg daily, intravenous ritu-ximab of 500 mg once a week was given for successive 3 weeks followed by 5 successive days of intra-venous gamma globulin at a dose of 400 mg/kg per day, and a total of two treatment sessions were conducted. ELISA was used to detect the serum titer of anti-Dsg3 antibodies and their IgG subtypes (IgG1 and IgG4) as well as serum level of interferon-gamma (IFN-gamma), interleukin-4 (IL-4), interleukin-10 (IL-10). Results Three months after the end of two treatment sessions, the symptom was obviously improved, and lesions subsided; alter another 1 month, clinical symptom fully disappeared. During the 1-year follow-up, no lesions recurred. The anti-Dsg3 antibody titer was 253.33 U/mL before treatment, plateaued at 250 U/mL within 4 weeks after the initial infusion, decreased till 3 months after the withdrawal, and reached 26.06 U/mL 7 months after the withdrawal, and remained within normal range till the time of this writing. The serum titer of IgGl subclass of anti-Dsg3 antibodies dropped dramatically fight after the first infusion, but that of IgG4 subclass remained at a high level at early stage of medication, began to decline until 3 months after the with-drawal, and finally reached the normal range following clinical remission. Also, serum level of IFN-garnma and IL-10 correlated with the severity of PV. Conclusions Combined rituximab is effective for the control of PV, likely by eliminating Dsg3-specific antibodies, especially IgG4 subclass of them.