Clinical Efficacy of Kuijietong Against Mild to Moderate Active Ulcerative Colitis / 中国实验方剂学杂志

Objective:To re-evaluate the intervention effect of Kuijietong（KJT） on ulcerative colitis（UC）. Method:Sixty patients with mild-to-moderate UC in the active stage were enrolled and randomized into a KJT group （<italic>n</italic>=30） and a sulfasalazine （SASP） group （<italic>n</italic>=30）. Patients in the KJT group were treated with KJT granules， one bag divided in two daily doses， once in the morning and once in the evening， while those in the SASP group received SASP， 1 g per time， four times per day. Then the clinical efficacy was evaluated. Result:According to the modified Mayo score，the clinical remission rates of the KJT group and SASP group were determined to be 46.7% （14/30）and 40% （12/30），exhibiting no significant difference between the two groups （<italic>P</italic>>0.05）. The clinical effective rate of the KJT group was 83.3% （25/30），which was better than 60% （18/30） of the SASP group （<italic>P</italic><0.05）. The mucosal healing rate in the KJT group was 36.7% （11/30）， not significantly different from 30% （9/30） in the SASP group. In the alleviation of UC symptoms，the score of large intestine dampness heat syndrome in the KJT group was remarkably better than that in the SASP group （<italic>P</italic><0.05），but there was no significant difference in inflammatory bowel disease questionnaire （IBDQ） score between the two groups. In terms of physical and chemical indexes，serum erythrocyte sedimentation rate （ESR） in the KJT group after intervention was lower than that in the SASP group （<italic>P</italic><0.05），whereas the interleukin-10 （IL-10） level was higher（<italic>P</italic><0.05）. The comparison between the two groups revealed no significant difference in C-reactive protein （CRP）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， CD4⁺ T cells and regulatory T （Treg） cells after intervention. During the intervention，no obvious adverse reactions were found in the two groups，indicating good safety. Conclusion:KJT is not inferior to SASP in relieving mild-to-moderate UC in the active stage.

Classification study of Coptis chinensis based on quantitative physical property characteristics of appearance and internal quality evaluation / 中国中药杂志

<p><b>OBJECTIVE</b>Combining the quantitative physical property characteristics of the appearance with the internal quality evaluation index, its aims to provide experimental basis for the classification and quality evaluation of Coptis chinensis.</p><p><b>METHOD</b>Fourteen batches of C. chinensis from different areas were respectively measured in size (total length, total width, root length, taproot diameter, branch number, branch length, branch diameter, length of the bridge, weight), color (external color, internal color), content (epiberberine, coptisine, palmatine, berberine). Then the determination data were evaluated by spss principal component analysis and cluster analysis.</p><p><b>RESULT</b>Three principal components were extracted from the original data. The principal component analysis results showed that the characteristic elements might be the total length, main root length, taproot diameter, branch length, weight, the total color value of the appearance and content of epiberberine and berberine. The results of cluster analysis showed that 14 batches of samples could be clustered reasonably into two groups. In terms of the appearance and quality, there were some differences between in the geo-authentic and non-authentic producing areas of C. chinensis.</p><p><b>CONCLUSION</b>The method which was combining the quantitative physical property characteristics of the appearance with the internal quality evaluation index, and through the processing of mathematical statistics, could be used for the the classification of C. chinensis.</p>

Rapid identification of UCA1 as a very sensitive and specific unique marker for human bladder carcinoma.

PURPOSE: The most common genitourinary malignancy in China is bladder transitional cell carcinoma (TCC). Early diagnosis of new and recurrent bladder cancers, followed by timely treatment, will help decrease mortality. There are currently no satisfactory markers for bladder cancer available in clinics. Better diagnostic methods are highly demanded. EXPERIMENTAL DESIGN: In this research, we have used comprehensive expressed sequence tag analysis, serial analysis of gene expression, and microarray analysis and quickly discovered a candidate marker, urothelial carcinoma associated 1 (UCA1). The UCA1 gene was characterized and its performance as a urine marker was analyzed by reverse transcription-PCR with urine sediments. A total of 212 individuals were included in this study, 94 having bladder cancers, 33 ureter/pelvic cancers, and 85 normal and other urinary tract disease controls. RESULTS: UCA1 was identified as a novel noncoding RNA gene dramatically up-regulated in TCC and it is the most TCC-specific gene yet identified. The full-length cDNA was 1,439 bp, and sequence analysis showed that it belonged to the human endogenous retrovirus H family. Clinical tests showed that UCA1 assay was highly specific (91.8%, 78 of 85) and very sensitive (80.9%, 76 of 94) in the diagnosis of bladder cancer and was especially valuable for superficial G2-G3 patients (sensitivity 91.1%, 41 of 45). It showed excellent differential diagnostic performance in various urinary tract diseases without TCC. CONCLUSIONS: UCA1 is a very sensitive and specific unique marker for bladder cancer. It could have important implications in postoperative noninvasive follow-up. This research also highlights a shortcut to new cancer diagnostic assays through integration of in silico isolation methods with translational clinical tests based on RNA detection protocols.

[Expression of melanoma antigen gene in urinary transitional cell carcinomas].

OBJECTIVE: To evaluate the significance of melanoma antigen (MAGE) gene expression in bladder transitional cell carcinoma (TCC). METHODS: MAGE-A1, A2, A3, A4 mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR) in 3 clusters bladder TCC cells and 20 samples of bladder TCC patients (T(1) 7 samples, T(2) 5 samples, T(3) 6 samples, T(4) 2 samples, G(1) 1 samples, G(2) 11 samples, G(3) 8 samples). MAGE-A4 protein was detected by immunohistochemistry in 105 samples of bladder TCC patients (T(1) 35 samples, T(2) 12 samples, T(3) 26 samples, T(4) 13 samples, G(1) 13 samples, G(2) 44 samples, G(3) 48 samples). RESULTS: Three clusters bladder TCC cells had MAGE gene mRNA expression. In detection of MAGE mRNA of 20 samples of bladder TCC patients, 12 samples (60%) expressed MAGE-A1, 16 samples (80%) expressed MAGE-A2, 11 samples (55%) expressed MAGE-A3, 18 samples (90%) expressed MAGE-A4, 8 samples (40%) expressed all of MAGE-A1--A4. In 105 bladder TCC samples, 53 samples (50%) expressed MAGE-A4 protein. Strong expression (++ or +++) was significant higher in higher grade (13 samples/48 samples) or stage (14 samples/51 samples) than in lower grade (2 samples/57 samples) or stage (0 samples/35 samples). CONCLUSION: MAGE gene highly expresses in bladder TCC. Bladder TCC of high grade or high stage has higher MAGE-A4 protein strong expression.

[Expression of SSX2 gene in human urologic neoplasms].

OBJECTIVE: To investigate the expression of SSX(2)gene in human renal cell carcinoma and urinary transitional cell carcinoma. METHODS: Reverse-transcription polymerase chain reaction (RT-PCR) was used for detecting SSX(2) gene in the specimens from renal cell carcinoma (n = 26), urinary transitional cell carcinoma (n = 27) and in 15 specimens taken from the tumor surrounding tissues. RESULTS: Positive expression of SSX(2) gene at mRNA was detected in 69% renal cell carcinomas (18/26), in 81% urinary transitional cell carcinomas (22/27). The mRNA of SSX(2) was not detected in the 15 specimens from tumor surrounding tissues. CONCLUSION: The SSX(2) gene is highly expressed in human renal cell carcinoma and urinary transitional cell carcinoma.

Preparation of arsenic trioxide-loaded albuminutes immuno-nanospheres and its specific killing effect on bladder cancer cell in vitro.

BACKGROUND: Recently, arsenic trioxide (As2O3) was considered as a novel anti-tumor agent. However, it showed severe toxicity effect on normal tissue at the same time. To improve its therapeutic efficacy and decrease its toxicity,we prepared arsenic trioxide-loaded albuminutes immuno-nanospheres [As2O3-(HAS-NS)-BDI-1] targeted with monoclonal antibody (McAb) BDI-1 and tested its specific killing effect against bladder cancer cell. METHODS: As2O3-HAS-NS was prepared by chemical cross-linking method. Monoclonal antibody BDI-1 was purified with ammonium sulphate saltingout and chromatography. Albuminutes microspheres were conjugated with McAb by SPDP cross-linking method. Concentration of As in As2O3-(HAS-NS)-BDI-1 and As2O3-HAS-NS was measured by atomic fluometry method. As2O3-(HAS-NS)-BDI-1 and its activity were detected by SDS-PAGE reduction electrophoresis, indirect immunofluorescence test, light microscope and scanning electron microscope observation. Acridine orange staining and tritiated thymidine (3H-TdR) incorporation tests were used to indicate specific killing activity of As2O3-(HAS-NS)-BDI-1 in vitro. RESULTS: In As2O3-(HAS-NS)-BDI-1 groups, we saw two protein bands in SDS-PAGE reduction electrophoresis. Albuminutes immuno-nanospheres were rounded with clear green fluorescence by immunofluorescence test. Under microscope, we observed that BIU-87 cells were covered with the As2O3-(HAS-NS)-BDI-1 and that As2O3-(HAS-NS)-BDI-1 moved with the BIU-87 cells. The albuminutes immuno-nanospheres were tightly junctioned with the BIU-87 cells. Specific killing activity of As2O3-(HAS-NS)-BDI-1 on bladder tumor cells was observed by acridine orange staining and 3H-TdR incorporation assays. CONCLUSIONS: As2O3-(HAS-NS)-BDI-1 might bind specifically against BIU-87 cells, thus leading to high activity of killing bladder tumor cells.

[Expression of MAGE genes and MAGE gene products in human renal and urinary bladder tumor].

OBJECTIVE: To observe the expression of MAGE-1 MAGE-3 genes and MAGE-3 gene product in renal and urinary bladder tumor, and to explore the possibility of MAGE-1 and MAGE-3 genes encoding proteins or MAGE-3 gene product used as a target for immunotherapy in renal and urinary bladder tumor patients. METHODS: Reverse transcriptase polymerase chain reaction for MAGE-1 and MAGE-3 genes was performed using 39 renal and urinary bladder tumor specimens. Immunohistochemical technique for MAGE-3 antigen was performed using formal infixed paraffin embedded section of 121 renal and urinary bladder tumor specimens. RESULTS: MAGE-1 and MAGE-3 mRNAs were detected in 23(59.0%) and 22(56.4%) of 39 patients with renal and urinary bladder tumor without expression in 10 tumor surrounding tissues.MAGE-3 antigen was detected in 56(46.3%) of 121 patients with renal and urinary bladder tumor without expression in 10 tumor surrounding tissues. The expression rates of MAGE-1 and MAGE-3 mRNA and MAGE-3 gene product were significantly higher in renal and urinary bladder tumors tissues than in tumor surrounding tissues. The frequency of MAGE-3 gene product expression was examined according to clinical stage and differentiation of histopathology. The results revealed no significant differences in MAGE gene product expression among the clinical stage and the grade of differentiation of histopathology according to Logistic Regression test(P>0.05). CONCLUSION: The tumor-specific antigens might be used as molecular markers and targets for human renal and urinary bladder tumor.

[Expression of human-mouse chimeric antibody ch-BD1 and its affinity to human bladder cancer in vitro and in vivo].

OBJECTIVE: To investigate the expression of human-mouse chimeric antibody ch-BD1 against human bladder cancer and its affinity to human bladder cancer in vitro and in vivo. METHODS: Three kinds of mutated fragments of dihydrofolate reductase (DHFR) gene were created by techniques of molecular biology to decrease the transcriptional activities and then cloned into pDHL-BD1 so as to construct the vectors pWSD1-BD1, pWSD2-BD1, and pWSD3-BD1 expressing the human-mouse chimeric antibody ch-BD1 against human bladder cancer with decreased expression of DHFR gene. These vectors and pDHL-BD1 were transfected into Chinese hamster ovary cell (CHO)/DHFR- cell respectively. 72 hours later Northern blotting was used to examine their DHFR gene expression. Methotrexate (MTX) of increasing concentrations was added into the culture of transfected CHO/DHFR-cells. The ch-BD1 levels in the supernatants were measured. Purified ch-BD1 was labeled by (99m)TcO(4)(-) and added into the serially diluted solutions of human bladder cancer EJ cells to examine their radioactivities and calculate their affinity constants. EJ cells were injected into the roots of hind limb of 3 Balb/C mice. Four weeks later, (99m)TcO(4)(-)-labeled antibody ch-BD1 were injected into the rats' caudal veins. Radioimmunoimaging was conducted to examine the distribution of the antibody. RESULTS: The sequence of DHFR gene expression levels from strong to weak in the constructed vectors was as follows: pDHL-BD1 > pWS1-BD1 > pWS3-BD1 > pWS2-BD1. When the concentration of MTX was 10(-6) mol/L the expression level of ch-BD1 was significantly correlated to the expression level of DHFR gene, the lower the baseline expression level of DHFR gene the higher the expression level of ch-BD1. After the serially diluted EJ cells were co-incubated with the (99m)TcO(4)(-)-labeled antibody ch-BD1 the immunoactivity ratio of ch-BD1 was 76%, and that of murine monoclonal antibody was 81%; the affinity constant of ch-BD1 was 3.56 x 10(9) M(-1), and that of murine monoclonal antibody BD1 was 1.22 x 10(9) M(-1). 6 hours after injection of the (99m)TcO(4)(-)-labeled antibody ch-BD1 into the mice body it was mainly distributed in the tumor, 22 hours later, it was specifically distributed in tumor, and 24 hours later it was still concentrated here. CONCLUSION: Decrease of the baseline expression level of DHFR gene effectively increases the amplification of MTX to exogenous gene. The human-mouse chimeric antibody ch-BD1 shows an ideal affinity activity to human bladder cancer in vivo and in vitro and has a certain clinical prospect.

[Prevention of postoperative recurrence of human bladder carcinoma by intravesical instillation of immunotoxin, a clinical study].

OBJECTIVE: To observe the effects of intravesical instillation of immunotoxin (IT) on the prevention of recurrence of bladder carcinoma. METHODS: 128 patients with superficial bladder carcinoma or bladder carcinoma of T2 stage underwent operation, and then were randomly divided into 3 groups 2 - 3 weeks after operation to be instilled intravesically with solution of mitomycin (n = 53), Calmette-Guerin vaccine (n = 30), or IT (n = 45) once a week for 8 weeks and then once every month for 8 months respectively. Cystoscopy, blood routine examination, routine urine examination, and liver function test were conducted every 3 months. Immunohistochemistry was used to examine 30 samples of resected carcinoma. The effects and side effects were observed. Recurrence was verified by cystoscopy and/or ultrasonography, CT, and surgery. RESULTS: Out of the 30 samples of resected bladder carcinoma, 27 were staining positive (+ - + + +) with a positive rate of 90%. The binding activity was significantly different between the G(1) carcinoma of and the G(3) carcinoma (P < 0.05) and not significantly different between the G(1) and G(2) carcinomas and between the G(2) and G(3) carcinomas (both P > 0.05). The higher the grade of carcinoma, the stronger the binding activity of IT (P < 0.05). The recurrence rate in the first year were 11.1%, 10.0%, and 15.1% in the IT, BCG, and MMC groups respectively without significant difference between any 2 groups (all P > 0.05); and the recurrence rate in the 2nd year were 24.4%, 30.0%, and 28.3% in the 3 groups respectively without significant difference between any 2 groups (all P > 0.05). The side effect rate were 17.8% on the IT group, significantly lower than those in the other 2 groups (70.0% and 56.6% respectively, both P < 0.01). However, the side effect rates between the BCG group and MMC group was not significantly different (P > 0.05). CONCLUSION: IT is more effective on the prevention of recurrence of carcinomas with higher malignancy and has fewer side effects. The short-term effect of IT is similar to those of BCG and MMC. The activity of IT is associated with the grade and not the stage of carcinoma.

[In vitro and in vivo functional evaluation of anti-human bladder tumor human-mouse chimeric antibody ch-BDI].

OBJECTIVE: To evaluate the in vitro and in vivo function of anti-human bladder tumor human-mouse chimeric antibody ch-BDI and its future clinical application. METHODS: With ch-BDI in high-expression cell-line medium, affinity chromatography was used for the purification. Labeled with (99m)Tc through reduction method, its immunoreactive fraction and association constant were measured. The constant was injected into nude mice with xenografted human bladder tumor. The biodistribution of the labeled ch-BDI was studied with radioimmunoimaging. RESULTS: ch-BDI showed desirable immunoreactive fraction (76%) and association constant (3.56 x 10(9) M(-1)) in vitro and a terrific specific targeting effect in vivo. CONCLUSION: ch-BDI has fairly good function against human bladder tumor both in vitro and in vivo, and is promising in clinical use.

[Significantly improvement of antibody expression level in CHO cells through downregulation of the DHFR gene in expression vector].

AIM: To increase the expressed level of a human-mouse chimeric antibody against human bladder tumor in dihydrofolate reductase (DHFR) defective CHO cells(CHO/DHFR) via weakening the transcription of DHFR gene in the vector. METHODS: A series of chimeric antibody expression vectors with different deletions and mutations in the modulator sequence of DHFR gene were constructed to downregulate the DHFR gene expression. The vectors were used to transfect CHO/DHFR cells and the transfected cells were subjected to gene amplification in medium containing gradually increasing methotrexate (MTX). The expressed chimeric antibody was quantitated by ELISA. RESULTS: The downregulation of vector-produced DHFR gene introduced by mutation of the modulator sequence could significantly improve the gene amplification effect and the increased antibody production correlated to the reduction of DHFR gene expression. From the best group, a clone with antibody production of 55 microg/(10(6) cells.24 h) was obtained by subcloning. More than 100 microg/(10(6) cells.24 h) was achieved by zinic ion induction. CONCLUSION: MTX induced-increase of recombinant antibody production in CHO cells can be increased by weakening the expression of DHFR gene.

[Induction of apoptosis in prostate cancer cell line PC-3 by BBSKE, a novel organoselenium compound, and its effect in vivo].

OBJECTIVE: To investigate the effects of BBSKE (1,2-[bis (1,2-benzisoselenazolone-3 (2H)-ketone)]ethane), a novel organoselenium compound, on the proliferation and apoptosis of the prostate cancer cell line PC-3, and to study its effect on the growth of prostate cancer in vivo. METHODS: Prostate cancer cells of the cell line PC-3 was cultivated in media with different concentrations of BBSKE and cisplatin. The inhibition of proliferation was measured by colorimetric MTT assay. The morphologic changes were observed by fluorescence microscopy, DNA fragmentation was visualized by agarose gel electrophoresis, and the DNA degradation was determined by flow cytometry. Western blot analysis was used to identify the expression of bcl-2 and bax. The activity of caspase-3 was determined by a micro-ELISA reader. Mouse prostate cancer cells of the TRAMP-C2 line were cultured and then injected subcutaneously into 2 male C57BL/6 mice to establish the animal model. Then the 2 mice were killed to collect the cancer cells. Twenty-four mice were injected intraperitoneally with single cell suspension of TRAMP-C2 cell and then divided into 3 groups of 8 mice undergoing intraperitoneal injection for 7 days: BBSKE group (BBSKE was administered at the dosage of 25mg/kg/day), cisplatin group (cisplatin 2mg/kg/d was injected), and control group (pure solvent was injected). Three weeks after the mice were killed and the tumors were taken out to calculate the inhibition rate. RESULTS: BBSKE inhibited the growth of the PC-3 cells dosage-dependently with a value of IC(50) of 17.90 micro mol/L after a 48 h exposure, higher than that in the case of cisplatin (15.00 micro mol/L). After exposure of PC-3 cells to BBSKE at the dosage of 20 micro mol/L for 48 hours the apoptosis rate was 26.32%, significantly higher than that of the control group (1.75%, P < 0.01). The expression of bcl-2 was decreased and the expression of bax remained almost unchanged along with the increase of BBSKE concentration. The activity of caspase 3 in the subgroup of BBSKE of the concentration of 5 micro mol/L remained almost unchanged, and was increased to 3.65 +/- 0.57 and 4.39 +/- 1.01 respectively in the BBSKE 10 micro mol/L and 20 micro mol/L subgroups, both significantly higher than that of the control group (both P < 0.05). In the in vivo experiment, the growth of tumor was significantly inhibited by BBSKE with an inhibition rate of 40% and the inhibition rate of the cisplatin group was 48%. CONCLUSION: The novel organoselenium BBSKE inhibits the proliferation of PC-3 cell and promote its apoptosis, probably through downregulating the expression of bcl-2 and the activity of caspase-3. BBSKE also inhibits the growth of prostate cancer in vivo.

[Detection of cell apoptosis induced with anti-Fas antibody in bladder carcinoma cell line by single cell gel electrophoresis].

BACKGROUND & OBJECTIVE: Fas, a death factor, can induce apoptotic death of cells. Many malignant tumor cells expressed Fas can not develop apoptotic death, this may be because of low Fas expression. Therefore we investigate the relationship between expression level of Fas and anti-Fas biological responsiveness. METHODS: With direct immunofluorescence flow cytometry, the expression of Fas was detected in bladder carcinoma cell line EJ, TNF alpha treated EJ, and Fas gene transfected EJ. The three kinds of EJ cells died by apoptosis were induced with anti-Fas antibody DX2 IgG1K and determined by SCGE and flow cytometry (FCM). RESULTS: Expression of Fas in bladder carcinoma cell line EJ, TNF alpha treated EJ, and Fas gene transfected EJ was 19.18%, 28.03%, and 68.69%, respectively. Cellular apoptosis induced with anti-Fas antibody DX2 IgG1K in bladder carcinoma cell line EJ was least, in TNF alpha treated EJ moderate, and in Fas gene transfected EJ the most. CONCLUSIONS: Fas gene transfection is an effective method to increase Fas expression; the sensitivity of EJ cell line to Fas-mediated apoptosis is mainly dependent upon the expression of Fas.

[Expression of Fas in six human urogenital malignant cell lines].

BACKGROUND & OBJECTIVE: Expression of Fas in tumors is an important basis for the treatment of the patients with tumor and determination of the prognosis. This study was designed to investigate the expression of Fas in urogenital tumor cell lines. METHODS: Using direct immunofluorescence flow cytometry, Western blot, we detected and Northern blot, the expression of Fas in six urinary malignant cell lines [bladder carcinoma(T24, EJ, BIU-87), renal carcinoma (GRC-1, RCC-949), prostatic carcinoma (PC-3M)] and one primary in vitro cultured renal fibroblast. RESULTS: Expression of Fas in 6 urinary tumor cell lines was commonly presented but with limited positive cell percentage (range 16.51%-49.13%) and relatively lower fluorescence intensity, compared with expression of Fas in normal control of primary in vitro cultured renal fibroblast (the positive rate of Fas reached 77.98%) measured by flow cytometry with direct immunofluorescence; In detection of expression of Fas in 6 urogenital cell lines, a 48 kDa clear band was found with the strongest intensity in T24 cell line and the weakest in BIU-87. Fas mRNA from all tumor cell lines but BIU-87 was detected. CONCLUSIONS: Fas was extensively expressed in urogenital malignant cells and normal cell. The lower expression of Fas in urinary malignant cell lines than that in normal cells might be the reason for occurrence and progression of urinary malignant tumors.