Potential regulatory role of the Nrf2/HMGB1/TLR4/NF-κB signaling pathway in lupus nephritis.

OBJECTIVES: Systemic lupus erythematosus is an autoimmune disease that involves multiple organ systems. One of its major complications, lupus nephritis (LN), is associated with a high mortality rate, and children-onset LN have a more severe course and worse prognosis than adults. Oxidative stress and inflammatory responses are involved in LN development and pathogenesis. Thus, this study aimed to explore the role of signaling regulation of the Nrf2/HMGB1/TLR/NF-κB pathway in LN pathogenesis and unravel the expression of TLR4+CXCR4+ plasma cells subset (PCs) in LN. METHODS: C57BL/6 and MRL/lpr mice were divided into four groups: control, model, vector control, and Nrf2 overexpression groups. The vector control and Nrf2 overexpression groups were injected with adenoviral vectors into the kidney in situ. Pathological changes in kidney tissues were observed by hematoxylin-eosin staining. The expression of Nrf2, HMGB1, TLR4, NF-κB, and downstream inflammatory factors in kidney samples was analyzed by quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The ratios of TLR4+CXCR4+ PC subsets in the blood and kidneys of mice were determined by flow cytometry. RESULTS: In MRL/lpr mice, Nrf2 was downregulated while HMGB1/TLR4/NF-κB pathway proteins were upregulated. Nrf2 overexpression decreased the expression of HMGB1, TLR4, NF-κB, and its downstream inflammatory cytokines (IL-1ß and TNFα). These cytokines were negatively correlated with an increase in Nrf2 content. PC and TLR4 + CXCR4 + PCs in the blood and kidney samples were significantly increased in MRL/lpr mice; however, they were decreased upon Nrf2 overexpression. CONCLUSION: This study showed severe kidney injury in an LN mouse model and an increased ratio of TLR4 + CXCR4 + PCs. Furthermore, we observed that Nrf2 regulates LN immune response through the Nrf2/HMGB1/TLR4/NF-κB pathway, which can be considered an important target for LN treatment. The clinical value of the findings of our study requires further investigation.

Influence of prostate size on the perioperative and postoperative outcome of transurethral plasmakinetic enucleation of the prostate: Results of 892 patients with 3 years of follow-up.

To explore the influence of prostate size on the outcome of Plasmakinetic enucleation of the prostate (PkEP) for the treatment of benign prostate hyperplasia (BPH), The data of 892 patients with symptomatic BPH who underwent PkEP were retrospectively reviewed. Among them, 199 (22.31%) had the prostate size smaller than 40 g (Group 1), 409 (45.85%) between 40 and 79 g (Group 2), 197 (22.09%) between 80 and 120 g (Group 3), and 87 (9.75%) larger than 120 g (Group 4). Perioperative variables, perioperative and postoperative complications were recorded. Patients were followed up for 36 months postoperatively. The efficiency of the surgery increased as the prostate size increased. Greater decreases in hemoglobin were noted in groups with larger prostates, while the duration of catheterization after the operation was similar across all groups. During the 3-year follow-up, the postoperative improvement in International Prostate Symptom Score (IPSS), Quality of Life (QOL), maximal flow rate (Qmax) and post-void residual urine volume (PVR), as well as longterm complications including urethral stricture and bladder-neck contracture were comparable across the 4 groups. These findings revealed that PkEP is more efficient for large prostate and can treat all prostates regardless of the size with equivalent symptom relief and micturition improvement.

Overexpression of ULK1 Represents a Potential Diagnostic Marker for Clear Cell Renal Carcinoma and the Antitumor Effects of SBI-0206965.

BACKGROUND: Uncoordinated 51-like kinase 1 (ULK1) plays a vital role in autophagy. ULK1 dysregulation has recently been found in several human cancers. METHODS: mRNA expression levels of ULK1 and clinical information were analysed from The Cancer Genome Atlas data. ULK1 expression levels were verified in 36 paired fresh ccRCC tissue specimens by western blot analysis. Expression of ULK1 was knockdown by shRNA lentivirus. ULK1 activity was inhibited by SBI-0206965. The effect of inhibition of ULK1 was measured by detecting the apoptotic rate, autophagy, and the ratio of ROS and NADPH. The efficacy of SBI-0206965 in vivo was assessed by the murine xenograft model. FINDINGS: ULK1 mRNA expression was significantly upregulated in clear cell renal cell carcinoma (ccRCC) and overexpression of ULK1 correlated with poor outcomes. We found that ULK1 was highly expressed in 66.7% of ccRCC tumours (p < 0·05). Knockdown of ULK1 and selective inhibition of ULK1 by SBI-0206965 induced cell apoptosis in ccRCC cells. We demonstrated that SBI-0206965 triggered apoptosis by preventing autophagy and pentose phosphate pathway (PPP) flux. Furthermore, blocking the kinase activity of ULK1 with SBI-0206965 resulted in a level of anticancer effect in vivo. INTERPRETATION: Taken together, our results suggested that ULK1 was upregulated in ccRCC tumours and may be a potential therapeutic target. Therefore, SBI-0206965 should be further considered as an anti-ccRCC agent. FUND: This work was supported in part by The National Natural Science Foundation of China (No. 81570748) and Natural Science Foundation of Fujian Province (No. 2018J01345, 2017XQ1194).

Apolipoprotein C1 promotes prostate cancer cell proliferation in vitro.

Here, we aimed to investigate the carcinogenic effects of apolipoprotein C1 (APOC1) in prostate cancer (PCa). APOC1 expression was evaluated in PCa and normal prostate specimens, and lentivirus-mediated RNA interference was used to knockdown APOC1 in DU145 cells. The effects of APOC1 silencing on cell proliferation, cell cycle arrest, and apoptosis were assessed. APOC1 expression was much higher in PCa tissues than in normal tissues. Moreover, APOC1 silencing inhibited cell proliferation and colony formation, arrested cell cycle progression, and enhanced apoptosis in DU145 cells. Additionally, APOC1 silencing decreased survivin, phospho-Rb, and p21 levels and increased cleaved caspase-3 expression. These data supported the procarcinogenic effects of APOC1 in the pathogenesis of PCa and suggested that targeting APOC1 may have applications in the treatment of PCa.

Chemotherapy response evaluation in a mouse model of gastric cancer using intravoxel incoherent motion diffusion-weighted MRI and histopathology.

AIM: To determine the role of intravoxel incoherent motion (IVIM) diffusion-weighted (DW) magnetic resonance imaging (MRI) using a bi-exponential model in chemotherapy response evaluation in a gastric cancer mouse model. METHODS: Mice bearing MKN-45 human gastric adenocarcinoma xenografts were divided into four treated groups (TG1, 2, 3 and 4, n = 5 in each group) which received Fluorouracil and Calcium Folinate and a control group (CG, n = 7). DW-MRI scans with 14 b-values (0-1500 s/mm2) were performed before and after treatment on days 3, 7, 14 and 21. Fast diffusion component (presumably pseudo-perfusion) parameters including the fast diffusion coefficient (D*) and fraction volume (fp), slow diffusion coefficient (D) and the conventional apparent diffusion coefficients (ADC) were calculated by fitting the IVIM model to the measured DW signals. The median changes from the baseline to each post-treatment time point for each measurement (ΔADC, ΔD* and Δfp) were calculated. The differences in the median changes between the two groups were compared using the mixed linear regression model by the restricted maximum likelihood method shown as z values. Histopathological analyses including Ki-67, CD31, TUNEL and H&E were conducted in conjunction with the MRI scans. The median percentage changes were compared with the histopathological analyses between the pre- and post-treatment for each measurement. RESULTS: Compared with the control group, D* in the treated group decreased significantly (ΔD*treated% = -30%, -34% and -20%, with z = -5.40, -4.18 and -1.95. P = 0.0001, 0.0001 and 0.0244) and fp increased significantly (Δfptreated% = 93%, 113% and 181%, with z = 4.63, 5.52, and 2.12, P = 0.001, 0.0001 and 0.0336) on day 3, 7 and 14, respectively. Increases in ADC in the treated group were higher than those in the control group on days 3 and 14 (z = 2.44 and 2.40, P = 0.0147 and P = 0.0164). CONCLUSION: Fast diffusion measurements derived from the bi-exponential IVIM model may be more sensitive imaging biomarkers than ADC to assess chemotherapy response in gastric adenocarcinoma.

Apple Polyphenols Decrease Atherosclerosis and Hepatic Steatosis in ApoE-/- Mice through the ROS/MAPK/NF-κB Pathway.

In this study, we examined the effects of apple polyphenols (APs) on hyperlipidemia, atherosclerosis, hepatic steatosis and endothelial function and investigated the potential mechanisms. ApoE(-/-) mice were fed a western-type diet and orally treated with APs (100 mg/kg) or atorvastatin (10 mg/kg) for 12 weeks. Hyperlipidemia and atherosclerosis in the aortic sinuses and, and hepatic lipidosis were measured. The treatment with APs or atorvastatin induced a remarkable reduction in the atherosclerotic lesions and hepatic steatosis and decreased the levels of low-density lipoprotein, triglyceride, CCL-2 and VCAM-1 levels in the plasma. Conversely, the APs significantly increased the plasma levels of high-density lipoprotein (HDL) cholesterol and markedly up-regulated the glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) levels in liver tissues. Moreover, the APs treatment modulated lipid metabolism by up-regulating the transcription of associated hepatic genes including PPARα, while down-regulating the transcription of SCAP and its downstream genes associated with lipid synthesis in the liver. Histological assessment showed that the APs treatment also reduced the macrophage infiltration in the aortic root plaque and the inflammatory cells infiltrations to the liver tissues. Moreover, we confirmed that the APs treatment greatly reduced the ox-LDL-induced endothelial dysfunction and monocyte adhesion to rat aortic endothelial cells (RAECs). Mechanistically, the APs treatment suppressed the ROS/MAPK/NF-κB signaling pathway, and consequently, reduced CCL-2, ICAM-1 and VCAM-1 expression. Our results suggest that the APs are a beneficial nutritional supplement for the attenuation of atherosclerosis.

Adjuvant dendritic cells vaccine combined with cytokine-induced-killer cell therapy after renal cell carcinoma surgery.

PURPOSE: To observe the efficacy and side effects of adjuvant dendritic cells' (DCs) vaccine combined with cytokine-induced killer cell (CIK) therapy after renal cell carcinoma (RCC) surgery (RCCS). METHODS: DCs vaccine and CIK that loaded the autologous tumor cell lysate were prepared in vitro. Four hundred and ten RCC patients were recruited, and the study group was given DCs-CIK immunotherapy, while the control group was given IFN-α therapy. RESULTS: Disease progression (recurrence, metastasis or death) showed significant differences between the two groups in clinical stage I and II patients, as well as in highly and moderately differentiated disease (p<0.05), while there was no significant difference between the two groups in patients with poorly differentiated disease (p>0.05). The 3- and 5-year overall survival rates of the DCs-CIK group (96% and 96%, respectively) exhibited significant difference compared to the IFN-α group (83% and 74%, respectively (p<0.01). Progression-free survival (PFS) between the two groups was significantly different (p<0.01). Tumor stage and DCs-CIK treatment were independent factors concerning prognosis of RCC (p<0.05). There was no severe toxicity observed in the DCs-CIK treatment group. CONCLUSIONS: Adjuvant post-RCCS DCs-CIK treatment prolonged PFS and reduced mortality, showing better overall activity compared to interferon treatment.

Characterization of a water-soluble polysaccharide from Boletus edulis and its antitumor and immunomodulatory activities on renal cancer in mice.

A polysaccharide (BEP, Mw=113,432Da) was purified from Boletus edulis, which had a backbone consisting of (1→6)-linked-α-d-glucopyranosyl, (1→2,6)-linked-α-d-galactopyranosyl, (1→6)-linked-α-d-galactopyranosyl, and (1→3)-linked-α-d-rhamnopyranosyl residues, which were branched at O-2 position of (1→2,6)-linked-α-d-galactopyranosyl residue with a single terminal (1→)-linked-α-l-arabinofuranosyl residue. After 32 days' BEP administration to Renca tumor bearing mice, the tumor mass of Renca transplanted in mice was significantly repressed. Furthermore, BEP could significantly increase the spleen and thymus indices, stimulate splenocytes proliferation, augment NK cell and CTL activities in spleen, and promote the secretion of the cytokines IL-2 and TNF-α in Renca tumor bearing mice. Meanwhile oral administration of BEP (100 and 400mg/kg) restored all the altered hematological and biochemical parameters of tumor-bearing mice to normal levels. Thus, these data demonstrate that BEP possesses potential immunomodulatory activity and might be employed as effective therapeutic agents for the prevention of renal caner.

Prognostic factors for renal allograft survival in patients with immunoglobulin A nephropathy: a case control study.

The renal allograft survival rates of patients with immunoglobulin A nephropathy (IgAN), and patients with or without other glomerular diseases, have yet to be fully elucidated. In this study, the clinicopathological factors associated with long-term allograft survival for the prognosis of renal allograft recipients with IgAN were examined. All patients enrolled in this study were diagnosed with IgAN following clinical and pathological examinations. Patients underwent renal graft biopsy and were hospitalized at the Fuzhou General Hospital between June, 2004 and December, 2010. Common demographic and clinical indicators were recorded in patients who had graft loss and in those who had functional renal grafts. Forty-two of the 202 biopsy specimens (20.8%) met the diagnostic criteria for IgAN and were divided into two groups, the graft loss group (n=17) and the functional graft group (n=25). Patients were followed up for 1-257 months after kidney transplantation. The mean patient age was 40.6 ± 9.3 years at the time of renal graft biopsy. Examination results indicated concomitant proteinuria and hematuria in 25 patients (59.5%) and proteinuria alone in six patients (14.3%). Graft loss occurred in 17 patients during the follow-up period. Comparison of the graft loss and the functional graft groups indicated that patients in the graft loss group were more likely to have proteinuria (P=0.047), high creatinine levels at the time of biopsy (P=0.009), low glomerular filtration rates (P=0.013), low serum total protein (P=0.01), a high Banff score (P=0.001), extensive glomerulosclerosis (P=0.002), a greater likelihood of crescent formation (P=0.01), severe tubular atrophy (P=0.013) and more extensive interstitial fibrosis (P=0.033). However, the two groups showed no significant differences in blood pressure, hematuria, BUN, UA, Hb, TG and CHO levels. The allograft survival rate of patients with IgAN was identified to be similar to that of patients with and without other glomerular diseases.

Suppression of SCIN inhibits human prostate cancer cell proliferation and induces G0/G1 phase arrest.

SCIN is a calcium regulated actin severing and capping protein. Its homologue in zebrafish is found to be related with cell death. In the present study, we found that SCIN is highly expressed in human prostate cancer specimens. However, the functions of SCIN in human prostate carcinoma cells are largely unknown. To address the function of SCIN in prostate carcinoma cells, we used lentivirus-mediated RNAi to knock down SCIN expression in PC3 cells, a prostate carcinoma cell line. We found that in vitro silencing of SCIN could inhibit the proliferation and colony formation ability of PC3 cells. Furthermore, cell cycle analysis showed that reduced SCIN expression lead to G0/G1 cell cycle arrest through the regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin-dependent kinase inhibitor 2A (CDKN2A, p16Ink4A) and cyclin A2. These results suggest that SCIN plays an important role in the proliferation of prostate cancer cells and lentivirus-mediated inhibition of SCIN expression may be a potential therapeutic method for the treatment of prostate cancer.

BMSCs reduce rat granulosa cell apoptosis induced by cisplatin and perimenopause.

BACKGROUND: The objective of this study was to evaluate the effect of bone marrow mesenchymal stem cells (BMSCs) on the apoptosis of granulosa cells (GCs) in rats. RESULTS: Cisplatin increased GC apoptosis from 0.59% to 13.04% in the control and cisplatin treatment groups, respectively, which was significantly reduced upon co-culture with BMSCs to 4.84%. Cisplatin treatment increased p21 and bax and decreased c-myc mRNA expression, which was reversed upon co-culture with BMSCs. As compared to young rats, increased apoptosis was observed in the perimenopausal rats (P < 0.001). After 3 months, the apoptosis rate in the BMSC group was significantly lower than that of the control group (P = 0.007). CONCLUSIONS: BMSC therapy may protect against GC apoptosis induced by cisplatin and perimenopause. Further studies are necessary to evaluate therapeutic efficacy of BMSCs.

Successful treatment of patients with paraquat intoxication: three case reports and review of the literature.

OBJECTIVE: To report on three patients with paraquat (PQ) intoxication surviving after combined therapy with hemoperfusion (HP), cyclophosphamide (CTX), and glucocorticoid. METHODS: Three patients suffered acute renal failure in a few days after ingesting a lethal amount of PQ. Chest computed tomography (CT) scans revealed obvious pulmonary inflammation, pleural effusion, and fibrous lesions several days after ingestion. HP was performed immediately, followed by large doses of glucocorticoid (methylprednisolone, 500 g/d) and CTX (approximately 4 g). RESULTS: After 50 d of treatments, all three patients were discharged in healthy condition, with chest CT showing small fibrous lesions, exudation, and both lungs clear of auscultation. CONCLUSIONS: The protective effect of the lungs may have been due to timely treatment at adequate doses.

Alemtuzumab induction therapy in highly sensitized kidney transplant recipients.

BACKGROUND: Immunosuppression for immunologically high-risk kidney transplant patients usually involves antithymocyte globulin induction with triple drug maintenance therapy. Alemtuzumab, a humanized anti-CD52 antibody, was expected to be a promising induction therapy agent for kidney transplantation. However, currently no consensus is available about its efficacy and safety. This study aimed to evaluate the efficacy and safety of alemtuzumab as immune induction therapy in highly sensitized kidney transplant recipients. METHODS: In this prospective, open-label, randomized, controlled trial, we enrolled 23 highly immunological risk patients (panel reactive antibody > 20%). They were divided into two groups: alemtuzumab group (trial group) and anti-thymocyte globulin (ATG) group (control group). Patients in the alemtuzumab group received intravenous alemtuzumab (15 mg) as a single dose before reperfusion. At the 24th hour post-operation, another dosage of alemtuzumab (15 mg) was given. The control group received a bolus of rabbit ATG (9 mg/kg), which was given 2 hours before kidney transplantation and lasted until the removal of vascular clamps when the anastomoses were completed. Maintenance immunosuppression in both groups comprised standard triple therapy consisting of tacrolimus, prednisone, and mycophenolate mofetil (MMF). Acute rejection (AR) and infection episodes were recorded, and kidney function was monitored during a 2-year follow-up. χ(2) test, t test and Kaplan-Meier analysis were performed with SPSS17.0 software. RESULTS: Median follow-up was 338 days. In both the alemtuzumab group and ATG group, creatinine and blood urea nitrogen values in surviving recipients were similar (P > 0.05). White blood cell counts were significantly reduced in the alemtuzumab group for the most time points up to 6 months (P < 0.05). One patient receiving alemtuzumab died for acute myocardial infarction at the 65th day post-operation. Two ATG patients died for severe pulmonary infection or cardiac and pulmonary failure. Cumulative 2-year graft survival rate was 90.9% in the alemtuzumab group and 81.8% in ATG group (P > 0.05) respectively. There was one graft failure in the alemtuzumab group and two graft failures in ATG group, with all graft failures at tributed to rejection episodes. The alemtuzumab group had a 2-year cumulative freedom from rejection rate of 81.8%, compared with 72.7% for the ATG group (P > 0.05). CONCLUSION: Alemtuzumab induction therapy for highly sensitized kidney transplant recipients is an effective and safe protocol yielding an acceptable acute rejection rate.

Pre-transplant soluble CD30 level as a predictor of not only acute rejection and graft loss but pneumonia in renal transplant recipients.

Pre-transplant sera of 586 renal graft recipients were tested to investigate whether soluble CD30 (sCD30) is a useful predictor of some severe clinical episodes post-transplant. Correlation analysis showed sCD30 level was significantly correlated with acute rejection (AR) (r=0.242, P<0.001), graft loss (r=0.162, P<0.001), and pneumonia (r=-0.147, P<0.001). Higher sCD30 levels were observed in patients with AR than the others (180.0+/-89.1 vs. 135.3+/-72.7U/ml, P<0.001). And patients with pneumonia had significantly lower pre-transplant sCD30 level than the others (123.2+/-75.5 vs. 150.7+/-79.6U/ml, P=0.003). Based on statistical results, 120 and 240U/ml were selected as the optimal couple of cut-off value to divide patients into three groups: Group High (H), Group Intermedial (I) and Group Low (L). The lowest AR rate of 17.4% was observed in Group L (P<0.001). Significant difference of AR rate was also observed between Group I (29.2%) and H (42.9%) (P<0.001). There were much more patients suffering pneumonia in Group L (P=0.001). Significantly lower 5-year patient survival rate (79.4%) was observed in Group H (P=0.016). These data showed that elevated pre-transplant sCD30 level of renal allograft recipients may reflect an immune state detrimental for renal allograft survival. But sCD30 level lower than <120U/ml may be associated with a high risk of pneumonia. Pre-transplant sCD30 level is an independent predictor of acute rejection, lung infection, even graft survival. Suitable immunosuppression protocol should be selected according to pre-transplant sCD30 level in an attempt to promote patient and graft survival.

Pre- and post-transplant monitoring of soluble CD30 levels as predictor of acute renal allograft rejection.

Identification of renal graft candidates at high risk of impending acute rejection (AR) and graft loss may be helpful for patient-tailored immunosuppressive regimens and renal graft survival. To investigate the feasibility with soluble CD30 (sCD30) as predictor of AR, sCD30 levels of 70 patients were detected on day 0 pre-transplant and day 1, 3, 5, 7, 10, 14, 21, and 30 post-transplant. AR episodes in 6 months were recorded and then patients were divided into Group AR (n=11) and Group UC (n=59). Results showed that the patients had higher pre-transplant sCD30 levels than healthy people. A significant decrease of sCD30 was observed on the first day post-transplant and continued until day 14 post-transplant. Soluble CD30 presented a stable level from day 14 to 30 post-transplant. Pre-transplant sCD30 levels of Group AR were much higher than those of Group UC (P<0.001). Patients of Group AR also had higher sCD30 levels than those of Group UC on day 1, 3, 5, 7, 10 and 14 (P<0.001). The sCD30 level presented a significantly delayed decrease in the patients of Group AR. Statistical results showed that the highest value of area under ROC curve (0.95) was obtained on day 5 post-transplant, suggesting that sCD30 levels on day 5 are of high predictive value. Therefore, sCD30 level may be a good marker of increased alloreactivity and of significant predictive value. It's necessary to monitor the variation of sCD30 in the early period post-transplant.

[Comparison of biologic activity of two anti-PSA/anti-CD3 bispecific single-chain antibodies].

OBJECTIVE: To study and compare the biologic activity of two anti-PSA/anti-CD3 bispecific single-chain antibodies. METHODS: Flow cytometry (FCM) was used to detect the binding activity of two antibodies to CD3-positive cell line Jurkat and prostate carcinoma cell line LNCaP. The effect of the two antibodies in mediating tumor cell lysis in vitro was determined by using the 51Cr-release test. For in vivo evaluation of the two antibodies activity, a nude mouse model was used. The mice were inoculated with LNCaP prostate cancer cells. RESULTS: FCM showed that both the antibodies could bind Jurkat and LNCaP cells with high specificity. The percentages of the cells bond by the bispecific single-chain antibodies were 56.3% and 55.4%, and those by the multivalent antibodies were 74.0% and 83.0% respectively. Both the antibodies mediated a specific lysis of LNCaP cells in vitro, with activated CTLs as effector cells, and significantly reduced tumor growth of nude mice in vivo as compared with the untreated controls and the group treated with CTLs only (P <0.05). The experiment also showed that the multivalent antibody had a better activity than the bispecific antibody in binding antigens, mediating lysis of LNCaP cells and reducing tumor growth (P < 0.05). CONCLUSION: Both the anti-PSA/anti-CD3 bispecific single-chain antibody and multivalent antibody have good biologic activity, and the formation of the tetramerization of single-chain antibody can improve its biologic activity.

[Biologic activity of bispecific single chain antibody against human gamma-seminoprotein and CD3 molecule].

AIM: To construct a recombinant vector which expresses bispecific single chain antibody (BsscFv) against human gamma-seminoprotein and CD3 molecule and evaluate its biologic activity. METHODS: The BsscFv gene was constructed by the splicing overlap extensive (SOE) PCR and then a flexible peptide linker was inserted between anti-human gamma-seminoprotein single chain Fv gene and anti-CD3 single chain Fv gene. The fusion gene was subcloned into the pSectag2-B plasmid and was expressed in HeLa cell lines. After being analyzed by SDS-PAGE and Western blot, the expressed product was purified through a Ni(2+)-NTA superflow affinity chromatography column. Flow cytometry (FCM) was used to detect the binding activity of BsscFv to CD3(+) cell line Jurkat cells and prostate carcinoma cells LNCaP. In vitro killing effect on target cells (LNCaP) mediated by BsscFv was determined by chromium(51)-release test. The effect of CTLs mediated by BsscFv on inhibiting tumor growth was observed by utilizing nude mice bearing prostate cancer cells. RESULTS: DNA sequencing indicated that BsscFv gene consisted of 1,500 bp, encoding 500 amino acids. SDS-PAGE and Western blot analysis showed that the expressed product with relative molecular mass of 61,000 existed in culture supernatant of Hela cells. Flow cytometry analysis demonstrated that the binding rate of BsscFv to LNCaP cells and Jurkat cells was 54.1% and 53.7%, respectively. In vitro, BsscFv mediated cytotoxicity of CTLs to LNCaP cells as confirmed by chromium(51)-release assay. In prostate cancer nude mouse model, BsscFv inhibited tumor's growth as compared with control group. CONCLUSION: The BsscFv against human gamma-seminoprotein and CD3 molecule possesses certain biologic activity, and in vitro and in vivo it can mediate cytotoxicity of CTLs to prostate cancer cells.

[Construction and expression of anti-CD3/anti-prostate-cancer bispecific single-chain antibody].

OBJECTIVE: To construct a recombinant vector that expresses anti-CD 3/anti-prostate-cancer bispecific single-chain antibody (BsAb), and study its biological activities and clinical significance. METHODS: An anti-CD 3/anti-prostate-cancer BsAb was constructed by PCR and molecular biological technique of DNA cloning. The fusion gene, confirmed by sequencing, was subcloned into the pSectag2-B plasmid from the pUC18 vector by digestion with EcoR I and Hind III restriction endonucleases, whose sites exist in both the vectors. Then the recombinant plasmid was transfected into HeLa cells. The expression products in the supernatant were analyzed by both SDS-PAGE and Western blot technique, then were purified with Ni(2+)-NTA superflow affinity chromatography. Its biological activities were examined by flow cytometry (FCM). RESULTS: A fragment of 1.5 kb was inserted into the pUC18 vector, which was sequenced and verified to be identical with that designed. The expression of anti-CD 3 x anti-prostate-cancer BsAb yielded a soluble protein with an apparent molecular mass of 61 KD. The purification rate of the expressed BsAb was up to 90.537% and the yield of purified BsAb from this procedure was 0.09 mg/ml. The positive binding rates of BsAb to PBMC and to PC-3 cell were 54.1% and 53.7% respectively. CONCLUSION: The anti-CD 3 x anti-prostate-cancer BsAb thus constructed exhibits beneficial biological activities and may play an important role in the treatment of prostate cancer.