To produce monoclonal antibody (mAb) specifically against human thrombomodulin (hTM), an immune-tolerizing procedure was employed to generate monoclonal antibodies specific to hTM. Female BALB/c mice were first immunized with CHO cells following at 10 min, 24 h, 48 h by intraperitoneal injection of different doses of cyclophosphamide (CP) 2 times at an interval of 2 weeks, thereby tolerizing the mice to common epitopes shared between CHO and CHO-TM5 cells. Subsequently the selected mice with the lowest titer of serum polyclonal antibody by cellular enzyme-linked immunoabsorbent assay (CELISA) were immunized with CHO-TM5 cells, which have stable high level expression of hTM, to produce antibodies specific to hTM 3 times at an interval of 2 weeks. On the third day after the third immunization, mouse with the highest titer of serum polyclonal antibody was sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96 well plates for screening with CELISA. To improve probability to obtain specific mAb, CELISA was applied twice. The first CELISA was done with polyethylene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but having CHO cells monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5 +CHO- hybridoma cells. BALB/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded mAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. Detection of CELISA showed that 100 mg/kg dose of CP could tolerize the mouse to common epitopes shared between CHO and CHO-TM5 cells. And CELISA also discovered that all hybridomas positive for CHO-TM5 cells were negative for CHO cells. Five lines of positive hybridoma cells had been obtained altogether and 2F7 was selected randomly for next investigation. The Ig subclass of the mAb 2F7 was IgG1 and the titer of ascitic mAb was 1?10-6. Furthermore, the content of ascitic mAb was 19.56 g/L and chromosome numbers is 98. Flow cytometry, CELISA and Western blotting assays demonstrated that mAb 2F7 could specifically recognize hTM expressed on CHO-TM5 and human umbilical vascular endothelial cells (HUVEC). Meanwhile, the tissue specificity of mAb 2F7 was also identified by immunohistochemical ABC staining. On the other hand, Western blotting assays indicated that mAb 2F7 could recognize the antigen protein with 105 ku molecular mass under reduction condition. Moreover, the dissociation constant of mAb 2F7, 1.22? 10-9 mol/L, indicated the affinity higher than some others. The results suggest that the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms. mAb 2F7 can specifically recognize the natural hTM expressed mainly on vascular endothelial cells, which will potentially useful for investigating the functions and clinic values of hTM.

Objective To elucidate clinical pathological features of primary mediastinal B-cell lymphoma (PMBL) and its difference compared with diffuse large B-cell lymphoma,not otherwise specified (DLBCL,NOS).Methods The clinical histories and pathological datas of 24 PMBL cases and 31 cases of DLBCL,NOS as the control group were collected.Immunohistochemical staining and a follow-up study was conducted.Results The distribution of gender showed significant difference when the age of onset of PMBL patients was obviously younger with the medial age of 30 years old (P ＜ 0.001).All cases presented as a huge mass in mediastinal site with compression symptoms.PMBL was similar to DLBCL in the morphology of tumor cells but fibrosis of various degrees was common,more than 70.8 ％ (17/24) cases had the collagen bundles split.CD23 positive rate (40.0 ％,6/15) in PMBL was significantly higher than the control group (3.2 ％,1/31)(P =0.003).Conclusion PMBL frequently occurs in young female people,mostly happens in mediastinal site and adjacent area,but rarely has distant dissemination.PMBL has the characteristics of various degrees of collagen fiber hyperplasia,and CD23 positive could be used to differentiate PMBL from DLBCL,NOS.

Objective To investigate the clinicopathological and prognosis features of enteropathy-associated T-cell lymphoma (EATL).Methods 21 cases of EATL,6 cases of peripheral T-cell lymphoma (PTCL) and 11 cases of natural-killer/T-cell lymphoma (NKTCL) were collected from January 2008 to May 2015.The immunophenotype of the tumor cell was tested by EnVision and as well as EBV-EBER for EB virus.Some patients were performed with follow-up data.Results 21 EATL patients included 14 males,7 females and the middle age was 55 years old (40-79 years old).15 patients affected the small bowel,4 cases affected colon,2 cases affected more than one site.18 cases were mono-morpholohic EATL while 3 cases were classical EATL.The expression rates of neoplastic cells for CD3ε,CD4,CD8,CD56,Granzyme B,TIA-1 were 95.24 ％ (20/21),20.00 ％ (3/15),73.68 ％ (14/19),85.71％ (18/21),64.71％ (11/17),88.89 ％ (16/18) respectively.The expression of EBER in EATL patients (0,0/21) was obviously lower than that in NKTCL patients (100 ％,11/11).17 EATL patients had follow-up data,and the middle survival time was 15 months.No different prognosis was found in the three kinds of T-NHL (P =0.697).Conclusions EATL usually occurs in elder male and jejunum.The diagnosis of EATL needs a lot of information,including clinical history,endoscopy,histomorphology,immunophenotype and EBV-EBER result.EATL has low mobidity and high malignancy,it still lacks impactful therapeutic regimen.

ObjectiveTo investigate the diagnostic significance of different pathology techniques (Immunohistochemistry and ISH)to detect EBV on bone marrow biopsy tissues of hemophagocytic syndrome. Methods Histological,immunohistochemicalandinsituhybridizationwereusedtostudythe hemophagocytic features and expression of LMP-1,EBER and other markers.Results25 out of 51 cases (49.0 ％)showed an active proliferation growth pattern while other 12 cases(23.5 ％)demonstrated a deterioration morphological character compared with their same age group. The left 14 cases (27.5 ％) showed a normal myeloproliferative pattern. 91.4 ％ (32/35) expressed CD68/KP-1 and 89.5 ％ (17/19) cases expressed CD68/PG-M1, which showed an abnormal increase of monocytes. 26 out of 51 cases (51.0 ％) were positive for EBER,while EBER and immunohistochemistry on LMP-1 were detected simultaneously in 41 cases.The EBER were positive in 19 cases(46.3 ％)but none for LMP-1(P =0.000).ConclusionHypoplasia or hemophagocytic features could be detected on bone marrow biopsy tissues of hemophagocytic syndrome, which might be slightly difficult to diagnose. So immunohistochemistry would be essential. Meanwhile, the detection of EBER is more helpful than LMP-1 for diagnosis.

Objective:To investigate pathological characteristics and prognosis of patients with Epstein-Barr virus (EBV) positive gastric diffuse large B-cell lymphoma (DLBCL). Methods:Through retrospective study, we collected 75 cases of patients with DLBCL that oc-curs in the stomach. The patients were divided into two groups consisting of 60 cases of EBV negative control group and 15 cases of EBV positive group. To analyze the pathological characteristics and prognosis of patients with EBV positive gastric DLBCL, immunohisto-chemical and Epstein-Barr encoding region (EBER) in situ hybridization methods were used to detect Bcl-2, c-myc protein expression, and EBV-encoded RNA (EBER). Results:In certain aspects of clinical manifestations, such as age, gender, and origin, the comparison be-tween EBV-positive and EBV-negative groups had no statistically significant difference. The same results were obtained for Bcl-2 and c-myc protein expression. However, a statistically significant difference (P=0.01) was observed under the R-CHOP regimen where the me-dian overall survival (OS) of the EBV-positive and EBV-negative groups were 15.1 and 31.4 months, respectively. Conclusion:In pa-tients with DLBCL of the stomach, the EBV infection had no obvious effects in terms of clinical manifestation, origin, morphology, and protein expression of tumor cells. EBV-positive DLBCL was not limited to elderly patients. Under the R-CHOP regimen, the prognosis of EBV-positive patients was worse than that of EBV-negative patients.

Objective To evaluate the effects of Alprostadil combined with Enalapril on high sensitivity C-reactive protein and Cystatin in patients with early diabetic nephropathy.Methods One hundred and fifteen cases of outpatients were randomized into 3 groups.Thirty-seven cases were assigned to Alprostadil group and treated with Alprostadil 10 μg + NS 100 ml,iv,qid;Thirty-eight cases were assigned to the Analapril group and treated with Enalapril 5 mg bid;Forty cases were assigned to the combined treatment group and treated with Alprostadil 10 μg + NS 100 ml,iv qid and Enalapril 5mg bid.All patients were observed for twelve weeks.Changes before and after treatment in the blood pressure,plasma glucose,glycolated hemoglobin (HbA1 c),24 hours urinary albumin (24 hUAE),high-sensitive C-reactive protein(hs-CRP) and Cystain C( Cys C) were observed and compared between the three groups.Results After treatment,hs-CRP,CysC and 24 h UAE were significantly improved in the three groups compared with baseline levels( P ＜ 0.05).In the combined treatment group,hs-CRP,CysC and 24 h UAER had a more significant improvement than the other two groups (P ＜0,05 ).Conclusion Alprostadil combined with Enalapril is a clinically effective strategy in the treatment of early diabetic nephropathv and can reduce the levels of CysC and hs-CRP.

ObjectiveTo observe the clinical efficacy of combined irbesartan and alprostadil treatment of early diabetic nephropathy (DN).Methods A total of 120 patients with early type 2 diabetes of hospitalization were randomly divided into 3 groups: irbesartan group of 40 patients (150 mg, once per day)(group irbesartan),40 patients treated with alprostadil (physiological saline and alprostadi 10 μg))(group alprostadi) ,40 patients treated with alprostadil combined irbesartan (dose same as the other two groups)(combined group).All cases were observed for 4 weeks.Comparison of serum creatinine (Cr), blood urea nitrogen(BUN) ,24 hour urinary albumin(24hUAE) changes after treatment.ResultsAfter treatment 4 weeks 24hUAE of the three groups were significantly decreased (t = 2.07, t = 2.01 and t = 3.15, Ps ＜ 0.05) .The decrease of 24hUAE in the combined treatment group ([252.69 ± 33.56]mg/24h) was better than that in the irbesartan group([268.75 ± 34.42)](t = 2.11, P ＜ 0.05)and in the alprostadil group ([267.95 ± 30.75])mg/24h (t = 1.998, P ＜ 0.05) .No significant difference were observed between the Irbesartan and alprostadil group.During treatment, several patients affected by swell, uncomfortable in the alprostadil group, but improved after treatment.No other adverse effect was observed.Conclusion Alprostadil combined with irbesartan treatment of early diabetic nephropathy is an effective way.

Objective To investigate the EB virus (EBV) infection and its clinical significance in angioimmunoblastic T-cell lymphoma(AITCL).Methods 62 patients diagnosed as AITCL between 2008 Jan and 2011 Dec were retrospective analyzed.All cases were re-confirmed and classified with the histology and immunology according to 2008 WHO Classification.In situ hybridization of EBV encoded RNA (EBV-EBER) was performed.Clinical characteristics and follow-up data of patients were collected.Results 42 ％ (26/62) AITCL cases were EBER-positive.EBV infection was found to be significant correlation with age over 60 years and poor response to therapy (P =0.025,P =0.049,respectively).However,EBV infection had no relationship with the overall survival,the presence of B symptom and the Ann Arbor stage.Conclusion In AITCL,EBV infection seems not to be associated with the overall survival,B symptom and Ann Arbor stage,but it may have impacts on theraputic response.

Objective To study the frequency of diffuse large B-cell lymphoma (DLBCL) with multi-genetic alteration, and its correlation with c-myc, bcl-2 and bcl-6 protein expression. Methods 50 cases diagnosed with DLBCL from January 2012 to December 2016 were collected. The expression of c-myc, bcl-2 and bcl-6 was analyzed by immunohistochemistry. Interphase fluorescence in situ hybridization (I-FISH) analysis was performed to identify the genetic alteration of c-myc, bcl-2 and bcl-6. Results In all cases, there were 27 males and 23 females with a median age of 50 years (range: 3-85 years). 23 (46.00 %) cases were defined as primary nodal DLBCL and 27 (54.00 %) cases were primary extra-nodal DLBCL, with gastrointestinal tract (48.15 %, 13/27) being the most common site of involvement. c-myc protein expression was detected in 94.00 % (47/50) cases, in which 82.00 % (41/47) cases exhibited high levels of c-myc expression with positive nuclear staining observed in over 40.00 % of tumor cells. The positive rate of bcl-2 protein was 84.00 % (42/50), 76 % (38/50) cases presented with high-level bcl-2 expression. Concurrent high expression of c-myc and bcl-2 were presented in 18 cases (36.00%). FISH analysis demonstrated c-myc gene rearrangement in 7 cases (14.00 %) and amplification in 2 cases (4.00 %). bcl-2 gene rearrangement was detected in 6 cases (12.00 %) and 4 cases (8.00 %) exhibited gene amplification. bcl-6 gene rearrangement was identified in 8 cases (16.00%), amplification in 3 cases (6.00%), and 1 case concomitantly harbored the rearrangement and amplification of bcl-6. Multi-genetic alterations were defined in 4 cases with 3 cases fulfilling the criteria for double-hit lymphoma (DHL) and 1 case for triple-hit lymphoma (THL). For the cases with concomitant high-level expression of c-myc and bcl-2 proteins, 3 cases (16.67 %) was detected with multi-genetic alterations, including 2 cases for DHL and 1 case for THL. Conclusions The proportion of DLBCL with multi-genetic alterations is 8.00 % in this study. The genetic alterations are not consistently correlated with the protein expression. The molecular genetic testing is reliable for the identification of DHL.

Objective To study the clinicopathologic features and explore the potential prognostic factors of extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma).Methods 86 patients diagnosed with MALT lymphoma were retrieved and divided into 2 subgroups according to the location of tumor,namely gastric (32 cases) and non-gastric (54 cases) subgroup.Histological characteristics were reassessed on hematoxylin-eosin staining slides,and immunophenotypic features were determined by immunohistochemical staining.Interphase fluorescence in-situ hybridization (FISH) was carried out to detect the cytogenetic abnormalities.Results There were no significant difference of clinical behavior,histology,and immunophenotype between gastric and non-gastric subgroups.In addition,FISH detected t(14;18) in 9 ％ (4/45) and t(11;18) in 12 ％ cases (6/50) respectively.Among the 20 cases treated with H pylori (HP) eradication,a significantly lower remission rate was observed in cases with bcl-10 (20 ％) nuclear expression or those harboring t(11;18) (33 ％) compared with those in the negative group (73 ％,91％)(x2 =3.842 and 4.639,P =0.035 and 0.031,respectively).For the 35 non-gastric patients with follow-up data,males (35 ％) and patients older than 60 yrs (25 ％) tended to have a lower remission rate as compared to females (60 ％) and those younger than 60 yrs (63 ％) (x2 =3.905 and 7.373,P =0.048 and 0.007,respectively).Moreover,progression-free survival rate was significantly lower in patients with higher stage (ⅢⅣ) (25 ％) and without t(14;18) (50 ％) than that in patients with stage Ⅰ-Ⅱ (52 ％) and with t(14;18)(75 ％).The differences had statistical significance (x2 =4.207 and 4.363,P =0.040 and 0.037,respectively).Conclusion MALT lymphoma in general is an indolent B-cell non-Hodgkin’ s lymphoma which more frequently occurs in the elderly people.Differences in the response to treatment or the prognosis are observed between gastric and non-gastric MALT lymphoma patients.