Platelet Aggregation in Apparently Healthy Elderly Populations: Reference Ranges and Influence of Hematology Parameters.

BACKGROUND: Reference intervals based on younger populations may not apply to elderly populations. The aim of the current study was to calculate reference ranges for platelet aggregation in apparently healthy Chinese elderly populations and analyze the impact of gender, age, and hematological parameters on platelet aggregation in vitro. METHODS: A total of 138 males and 161 females were enrolled based on stringent inclusion and exclusion criteria. Platelet aggregation was measured with sodium citrate anticoagulation whole blood samples using a PL12 analyzer, triggered by adenosine-5'-diphosphate (ADP) and arachidonic acid (AA) agonists. Hematological parameters were measured using a Sysmex XE2100 instrument. RESULTS: In the total sample tested in this study, the platelet aggregation induced by AA and ADP was not dependent on age (p > 0.05) but gender. Platelet aggregation triggered by ADP and AA was more enhanced in females than males (p = 0.024 for AA, p = 0.036 for ADP). The recommended reference values of AA-induced platelet aggregation were 48.42% - 85.93% for males and 43.62% - 87.80% for females. The recommended reference values of ADP-induced platelet aggregation were 38.12% - 80.21% for males and 40.12% - 83.05% for females. Furthermore, for all agonists, a positive correlation was found between platelet aggregation and platelet count. The ADP-triggered aggregation showed a significant positive correlation with white blood cell (WBC) count and a negative correlation with red blood cell (RBC) count. Moreover, platelet aggregation showed no significant correlation with mean platelet volume or hemoglobin concentration. CONCLUSIONS: Combined and gender-specific reference ranges for platelet aggregation in healthy elderly populations were established in whole blood measured by a sequential platelet counting method.

The impact of prophylactic ascending aorta replacement on the long-term prognosis of zone 0 hybrid arch repair.

OBJECTIVES: To investigate the impact of prophylactic zone 0 replacement with prosthetic grafts on the long-term prognosis and perioperative safety of zone 0 hybrid arch repair (HAR) when zone 0 is neither dilated nor pathologic. METHODS: We retrospectively reviewed 115 patients whose zone 0 aorta was neither dilated nor pathologic and who underwent zone 0 HAR from January 2009 to December 2020 and divided then into two groups depending on whether zone 0 was replaced, with 46 patients in the no-replacement group and 69 patients in the replacement group. Inverse probability of treatment weighting (IPTW) was used to balance the baseline difference, and outcomes were compared after IPTW adjustment. The primary end points were overall survival and adverse aortic events (AAEs). The secondary end points were early composite adverse events and other perioperative complications. Subgroup analysis was performed by age, diagnosis, zone 0 maximum diameter and risk stratification. RESULTS: The 5-year IPTW-adjusted overall survival rate was 84% in the no-replacement group 90% in the replacement group (P = .61). With death as a competing risk, the IPTW-adjusted cumulative incidence of AAEs at 5 and 10 years was 23% and 41% in the no-replacement group, and 14% and 25% in the replacement group, respectively (subdistribution hazard ratio [sHR], 0.56; 95% confidence interval [CI], 0.23-1.39; P = .23). Considering proximal complications alone, the replacement group exhibited lower 5-year (3% vs 18%) and 10-year (6% vs 36%) cumulative incidences of proximal complications (sHR, 0.11; 95% CI, 0.01-0.91; P = .04) after IPTW adjustment. A subgroup analysis demonstrated that the benefits of zone 0 replacement in decreasing AAEs were observed in those aged 60 years or less (sHR, 0.15; 95% CI, 0.03-0.75; P = .02) and those with type B aortic dissection (sHR, 0.24; 95% CI, 0.07-0.82; P = .02). Additionally, zone 0 replacement did not increase early composite adverse event morbidity (9% vs 21%; P = .08) or early mortality (7% vs 6%; P = .87). CONCLUSIONS: Although zone 0 was neither dilated nor pathologic, prophylactic zone 0 replacement in zone 0 HAR significantly decreased the incidence of proximal complications, without impairing perioperative safety. Additionally, this strategy was associated with benefits in reducing AAEs in younger patients and patients with type B aortic dissection. Thus, prophylactic zone 0 replacement should be considered for reconstructing a stable proximal landing zone in zone 0 HAR.

Cannulation strategy in frozen elephant trunk for type A aortic dissection: double arterial cannulation approach.

OBJECTIVES: The goal of this study was to compare clinical outcomes of double arterial cannulation (DAC), axillary cannulation and femoral cannulation in patients undergoing frozen elephant trunk for type A aortic dissection. METHODS: Between 2015 and 2020, the study included 488 patients and was divided into 3 groups: 171 in the DAC group, 217 in the axillary group and 100 in the femoral group. Overall survival was the primary end point and clinical outcomes were analysed after inverse probability weighting. RESULTS: A total of 43 patients died during the follow-up period. DAC group presented higher percentages of coeliac trunk, renal and iliac artery malperfusion, but early outcomes and overall survival did not differ among groups. Subgroup analyses suggested that in patients requiring cardiopulmonary bypass duration ≥180 min, DAC approach was associated with a tendency to improved overall survival compared with axillary [hazard ratio (HR): 0.35, 95% confidence interval (CI): 0.14-0.90, P = 0.029) and femoral cannulation (HR: 0.38, 95% CI: 0.14-1.03, P = 0.058). Inverse probability weighting adjustment (axillary as reference: HR: 0.34, 95% CI: 0.13-0.86, P = 0.022; femoral as reference: HR: 0.33, 95% CI: 0.11-0.90, P = 0.030) and multivariable Cox proportional hazards model (covariates including age, gender, acute dissection, any organ malperfusion and deep hypothermic circulatory arrest) confirmed this result. CONCLUSIONS: DAC approach was commonly used in patients with branch artery malperfusion and clinical outcomes did not differ compared with axillary and femoral cannulation. It provides a flexible and effective option with adequate perfusion for cases with various dissection-involved statuses and prolonged cardiopulmonary bypass duration.

Impact of frozen elephant trunk on the outcomes of thoracoabdominal aortic repair with normothermic iliac perfusion.

Background: Frozen elephant trunk technique (FET) has been proven to provide an excellent landing zone for second-stage thoracoabdominal (TA) aortic repair. The aim of this study was to evaluate the impact of FET in TA aortic repair with normothermic iliac perfusion. Methods: From January 2008 to December 2019, 144 patients undergoing TA repair with normothermic iliac perfusion were enrolled in this study. Early and mid-term outcomes of patients with previous FET implantation (group A, n = 62) were compared with patients without previous FET implantation (group B, n = 82). The logistic regression analysis was performed to investigate the risk factors for adverse events, which were defined as early death, permanent stroke, permanent paraplegia, or permanent renal failure necessitating dialysis. Results: The proximal aortic clamp time and operating time was 14.26 ± 5.57 min and 357.40 ± 94.51 respectively in group A, which were both significantly shorter than that in group B (18.67 ± 5.24 min and 18.67 ± 5.24 min). The incidence of adverse event was significantly lower in group A than that in group B (9.7% vs. 25.6%, P = 0.027). There was no significant difference between two groups with regard to other complications or late outcomes. In addition, age >50 years, a Ccr < 90 ml/min/1.73 m2 and the operating time were identified as significant risk factors through logistic regression analysis for adverse events of TA repair. Conclusions: The FET technique simplifies the operative technique of proximal anastomosis, decreases the operating time and improves the early outcomes in TA repair, whereas does not provide a significant benefit with regard to late outcomes. Long-term follow-up and studies with larger sample sizes are necessary for further confirmation.

Improvement of Clinical Outcomes of Total Aortic Arch Replacement and Frozen Elephant Trunk Surgery With Aortic Balloon Occlusion.

Background: Total aortic arch replacement (TAR) with frozen elephant trunk (FET) surgery provides improved long-term results, but the surgery itself is associated with higher risks compared with isolated proximal reconstructions. We applied an aortic balloon occlusion (ABO) technique to reduce the circulatory arrest (CA) time and improve other clinical outcomes. Methods: All patients who underwent TAR with FET surgery (130 with ABO technique, 230 with the conventional approach) in Fuwai Hospital from August 2017 to February 2019 were reviewed in this retrospective observational cohort study. Intra- and early-postoperative results and clinical characteristics were analyzed. Results: After 1:1 propensity score matching (130 cases in each group), the 30-day mortality of the ABO group and the conventional group were 4.6% and 10.8% (p = 0.063), respectively. Although the reduction in complications was not statistically significant, the complication rate in the ABO group was relatively low, having fewer cases of postoperative renal (23.1 vs. 38.5%, p = 0.007) and hepatic (12.3 vs. 30.0%, p < 0.001) injury, lower postoperative wake-up time (15.2 ± 23.6 h vs. 20.1 ± 26.5 h, respectively, p < 0.001), reduced chest tube output (176.03 ± 143.73 ml vs. 213.29 ± 130.12 ml, respectively, p = 0.003), lower red blood cell transfusion volume (4.98 ± 6.53 u vs. 7.28 ± 10.41 u, respectively, p = 0.008), and no fatal events. Conclusions: The ABO technique is a simple method that can reduce the CA time and improve the recovery stage following TAR with FET surgery. The technique represents a practical strategy to treat patients with high operative risks due to its lower complication rate compared with the conventional approach.

Impact of body mass index on early and mid-term outcomes after surgery for acute Stanford type A aortic dissection.

BACKGROUND: Obesity is dramatically increasing worldwide, and more obese patients may develop aortic dissection and present for surgical repair. The study aims to analyse the impact of body mass index (BMI) on surgical outcomes in patients with acute Stanford type A aortic dissection (ATAAD). METHODS: From January 2017 to June 2019, the clinical data of 268 ATAAD patients in a single centre were retrospectively reviewed. They were divided into three groups based on the BMI: normal weight (BMI 18.5 to < 25 kg/m2, n = 110), overweight (BMI 25 to < 30 kg/m2, n = 114) and obese (BMI ≥30 kg/m2, n = 44). RESULTS: There was no statistical difference among the three groups in terms of the composite adverse events including 30-day mortality, stroke, paraplegia, renal failure, hepatic failure, reintubation or tracheotomy and low cardiac output syndrome (20.9% vs 21.9% vs 18.2% for normal, overweight and obese, respectively; P = 0.882). No significant difference was found in the mid-term survival among the three groups. The proportion of prolonged ventilation was highest in the obese group followed by the overweight and normal groups (59.1% vs 45.6% vs 34.5%, respectively; P = 0.017). Multivariable logistic regression analysis suggested that BMI was not associated with the composite adverse events, while BMI ≥30 kg/m2 was an independent risk factor for prolonged ventilation (OR 2.261; 95% CI 1.056-4.838; P = 0.036). CONCLUSIONS: BMI had no effect on the early major adverse outcomes and mid-term survival after surgery for ATAAD. Satisfactory surgical outcomes can be obtained in patients with ATAAD at all weights.

Early-to-midterm outcomes of aortic balloon occlusion technique versus hybrid procedure for aortic arch diseases.

OBJECTIVES: The goal of this study was to compare the early-to-midterm outcomes of patients treated with the frozen elephant trunk procedure with aortic balloon occlusion (FET-ABO) versus hybrid repair for aortic arch diseases. METHODS: Patients who underwent the FET-ABO (n = 134) and the hybrid procedure (n = 220) from 2017 to 2020 at our institution were analysed retrospectively. Early-to-midterm outcomes were compared using inverse probability weighting. Low-risk and high-risk subgroup analyses were performed according to the cut-off of the additive European System for Cardiac Operative Evaluation value of 6. RESULTS: The present study demonstrated similar 30-day mortality (3.7% vs 8.6%; P = 0.118) and adverse events between the FET-ABO and the hybrid groups. Fewer intraoperative red blood cell transfusions (0.54 ± 1.45 vs 1.26 ± 2.47 U; P = 0.001), decreased total hospital costs (P < 0.001) and considerable early-to-midterm survival [crude: hazard ratio (HR) 0.40, 95% confidence interval (CI) 0.17-0.91; P = 0.030; adjusted: HR 0.35, 95% CI 0.13-0.91; P = 0.032) were obtained with the FET-ABO compared to the hybrid procedure. The inverse probability weighting method substantiated the foregoing results. Adjusted subgroup analyses suggested that the FET-ABO procedure had a trend towards improved survival in low-risk patients (HR 0.17, 95% CI 0.03-0.93; P = 0.041) and achieved outcomes comparable to those of the hybrid procedure in high-risk patients (HR 0.46, 95% CI 0.15-1.42; P = 0.176). CONCLUSIONS: The FET-ABO technique could be better promoted in hospitals lacking experience and equipment and could be more viable and cost-effective for selected patients compared with the hybrid procedure.

Type II hybrid arch repair versus total arch replacement with frozen elephant trunk: a propensity score-matched analysis.

OBJECTIVES: The aim of this study was to evaluate the objective outcomes of type II hybrid arch repair (HAR) and total arch replacement with frozen elephant trunk (TAR with FET). METHODS: Data from 528 patients who underwent aortic arch repair from January 2017 to June 2019 were collected, which consisted of 175 type II HAR and 353 TAR with FET. The propensity score-matched analysis identified a subgroup of 90 pairs. Perioperative data and mid-term follow-up results were assessed. RESULTS: There was no significant difference in the composite adverse events (type II HAR, 20.6%, 36/175 vs TAR with FET, 17.8%, 63/353, P = 0.450). Multivariable logistic analysis of the 528 patients showed that the procedure type (type II HAR or TAR with FET) was not associated with composite adverse events, 30-day mortality or stroke. The 3-year survival rates were 84.8% in the type II HAR group and 90.1% in the TAR with FET group (P = 0.12). The 3-year reintervention-free rates in the type II HAR and TAR with FET groups were 98.7% and 96.5% (P = 0.22), respectively. After matching, no significant difference was found in the incidence of composite adverse events or the 3-year survival and reintervention-free rates. CONCLUSIONS: No significant clinical differences were found in the early and mid-term outcomes of type II HAR and TAR with FET. The long-term outcomes remain to be investigated. Careful patient selection for individualized approaches is the key to taking full advantage of the 2 surgical procedures.

Safety and durability of single-stage type I hybrid total aortic arch repair for extensive aortic arch disease: early- and long-term clinical outcomes from a single center and our 10-year of experience.

BACKGROUND: Single-stage type I hybrid total aortic arch repair is a surgical treatment for extensive aortic arch disease, but the clinical outcomes were distinguishing. The purposes of this study were to share our experience and evaluate the perioperative safety and long-term durability. METHODS: Thirty-six patients who underwent single-stage type I hybrid total aortic arch repair in Fuwai Hospital between January 2010 and June 2020 were respectively reviewed. Early primary endpoint was defined as early composite adverse events, including mortality, multiple organ dysfunction syndrome (MODS), unplanned reoperation, stroke, paraplegia, acute renal failure (ARF) necessitating continuous renal replacement therapy (CRRT), respiratory failure and stents related complications. Long-term endpoints included late mortality, late aortic related reintervention and late adverse aortic events. When evaluating the early- and long-term outcomes, all patients were stratified into two subgroups by age (65 years). RESULTS: All patients acquired technical success. Early composite adverse events rate was 11.1% (4/36), in-hospital mortality was 8.3% (3/36). Average follow-up period was 48.0±35.3 months. Overall survival rate was 83.3% and 51.9% at 5 and 10 years respectively. Late aortic related reintervention occurred at one (3.0%, 1/33) patient and this patient died after reintervention. Overall freedom from adverse aortic events was 79.2% and 47.5% at 5 and 10 years respectively. Significant difference was not observed between the elderly and young subgroups, no matter in early- and long-term outcomes. CONCLUSIONS: Single-stage type I hybrid total aortic arch repair has achieved desirable outcomes in our center, which does not increase perioperative risk in the elderly patients, meanwhile, also acquire acceptable durability in the young patients. In conclusion, this surgery is a practical mini-invasive treatment for extensive aortic arch disease with strict and limited indications.

A Comparison of Frozen Elephant Trunk, Aortic Balloon Occlusion, and Hybrid Repair for Total Arch Replacement.

This study aimed to compare clinical outcomes of patients treated by total arch replacement (TAR) with frozen elephant trunk (FET), aortic balloon occlusion (ABO) technique and hybrid arch repair (HAR). Between January 2017 and July 2019, 643 consecutive patients with aortic arch diseases were eligible for TAR, including 356 in conventional FET, 112 in ABO based on FET, and 175 in HAR. A retrospective cohort analysis of perioperative results was undertaken, performed with inverse probability weighting. The primary endpoint was composite endpoints included 30-day mortality, stroke, paraplegia, hemodialysis, reintubation, and intra-aortic balloon pump or extracorporeal membrane oxygenation support, and visceral dysfunction was secondary endpoint. Overall in-hospital mortality was 2.2% (FET = 2.5% vs ABO = 0 vs HAR = 2.9%, P= 0.210). Parallel early outcomes were demonstrated among three groups. ABO group was associated with significantly shorter circulatory arrest time (5, IQR 3-7 vs 16, IQR 14-18 minutes, P < 0.001), and a lower incidence of visceral dysfunction compared with FET group (25.1% vs 47.3%, P= 0.003). Patients receiving ABO suffered a significantly lower rate of prolonged ventilation (more than 72 hours; P= 0.014). Furthermore, a tendency toward decreasing composite endpoints was suggested in ABO (7.2%) compared with FET (15.5%, P= 0.061) and HAR (19.8%, P= 0.032). ABO technique obtains considerable early clinical outcomes for TAR compared with conventional FET and HAR, which could be a feasible and effective approach for patients with aortic arch diseases.

Early outcomes of hybrid type II arch repair versus total arch replacement with frozen elephant trunk in acute DeBakey type I aortic dissection: a propensity score-matched analysis.

OBJECTIVES: The aim was to evaluate the short-term outcomes of hybrid type II arch repair (HAR) and total arch replacement with frozen elephant trunk (TAR with FET) for acute DeBakey type I aortic dissection patients. METHODS: From January 2017 to June 2019, the clinical data of acute DeBakey type I aortic dissection patients in a single centre were retrospectively reviewed; there were 92 cases of HAR and 268 cases of TAR with FET, with 56 pairs by propensity score matching. RESULTS: After matching, the composite end points including 30-day mortality, stroke, paraplegia, renal failure, hepatic failure, reintubation or tracheotomy and low cardiac output syndrome were comparable (21.4%, 12/56 in the HAR group vs 21.4%, 12/56 in the TAR with FET group, P = 1.000). The rate of acute kidney injury (AKI) was significantly lower in the HAR group (58.9%, 33/56 vs 80.4%, 45/56, P = 0.031). The distribution of AKI stage according to the Kidney Disease Improving Global Outcomes criteria was different (P = 0.039), with more patients suffering from high-grade AKI in the TAR with FET group. Multivariable logistic analysis showed that the procedure type (HAR or TAR with FET) was not an independent predictor of composite adverse events or stroke. HAR was identified as a protective factor against AKI (odds ratio 0.485, 95% confidence interval 0.287-0.822; P = 0.007). CONCLUSIONS: In the treatment of acute DeBakey type I aortic dissection, no significant differences were found in early outcomes between the 2 groups, but HAR was associated with a significantly lower incidence of AKI.

Renal protective effect of the aortic balloon occlusion technique in total arch replacement with frozen elephant trunk.

BACKGROUND: Organ dysfunction caused by hypothermic circulatory arrest continues to concern surgeons. The aortic balloon occlusion (ABO) technique can significantly shorten the circulatory arrest time in total arch replacement with frozen elephant trunk (TAR with FET). This study aims to analyze the renal protective effect of the ABO technique and to analyze the predictors of acute kidney injury (AKI) and continuous renal replacement therapy (CRRT) after TAR with FET. METHODS: Between August 2017 and September 2018, 247 patients who underwent TAR with FET were divided into ABO and moderate hypothermic circulatory arrest (MHCA) groups. The primary endpoint was postoperative AKI defined by the Kidney Disease Improving Global Outcomes (KDIGO) criteria. Multivariable logistic analysis was used to identify the predictors of AKI and CRRT after TAR with FET. RESULTS: With the application of the ABO technique, the circulatory arrest time was significantly shortened (ABO 4, IQR: 3-6 vs. MHCA 18, IQR: 16-20, P<0.001). Meanwhile, surgeons safely set the lowest nasopharyngeal temperature at a higher grade (ABO 28.1, IQR: 27.4-28.5 vs. MHCA 24.7, IQR: 24.1-25.1, P<0.001). The peak serum creatinine (SCr) values within 48 hours after the surgery was lower in the ABO group than in the MHCA group (ABO 124, IQR: 97-173 vs. MHCA 146, IQR: 108-221, P=0.008). The distribution of AKI grade according to the KDIGO criteria differed between the two groups (P=0.04): more patients in the ABO group were free from AKI (Grade 0) than patients in the MHCA group (33% vs. 23.1%), and the proportion of patients with high-grade AKI (Grades 2 and 3) in the ABO group was lower than that in the MHCA group (21% vs. 32%). The ABO technique was associated with reduced potential for AKI, but was not protective for CRRT. CONCLUSIONS: The ABO technique significantly shortened the circulatory arrest time and safely elevated temperature, and provided better renal protection in patients undergoing TAR with FET. The ABO technique did not reverse the need for CRRT, nor did it reduce mortality or major adverse events.

Application of Aortic Balloon Occlusion in Total Aortic Arch Replacement with Frozen Elephant Trunk on Clinical Endpoints for Aortic Dissection Patients.

PURPOSE: Total aortic arch replacement (TAR) with frozen elephant trunk (FET) is the standard operation for treating aortic dissection (AD) patients involving aortic arch with high operative risk due to long circulatory arrest (CA). We used aortic balloon occlusion technique that safely reduced the CA time to 5 min in average and investigated whether it can improve the clinical endpoints. METHODS: All patients diagnosed with AD and underwent TAR with FET operation (123 with aortic balloon occlusion and 221 with conventional method) in Fuwai Hospital during August 2017 and February 2019 was reviewed in this retrospective observational study. RESULTS: After propensity score matching, the 30-day mortality of aortic balloon occlusion group and conventional group was 4.88% and 11.38% (P = 0.062), respectively. In multivariate analysis, aortic balloon occlusion is one of the factors that reduced the risk for renal and hepatic injury, shortened postoperative conscious revival time, and reduced red blood cell (RBC) transfusion during operation. CONCLUSIONS: The aortic balloon occlusion technique, as a perfusion strategy during operation, could alleviate postoperative complication. This method deserves further attention in future clinical practice for its value in treating patients with higher operative risks.

A synthetic multi-epitope antigen enhances hepatitis C virus-specific B- and T-cell responses.

Combining results from previous studies, a multi-epitope antigen PCXZ against the hepatitis C virus was synthesized in this study. The antigenic specificity of PCXZ was determined by recognizing antibodies in serum samples from hepatitis C virus patients, but not from healthy subjects or subjects who had the hepatitis B virus. The characteristics of PCXZ immunogenicity were evaluated in BALB/c mice. Strong antibody responses were generated in mice immunized with either naked PCXZ or PCXZ in Freund's adjuvant. As for the T-cell responses, Freund's adjuvant significantly increased interferon-γ secretion and enhanced the lytic activity of cytotoxic T lymphocytes. The epitope Pa, one component of PCXZ, made the most significant contribution to specific CTL lysis; this epitope was also a B-cell epitope and was able to induce high IgG titers. In summary, PCXZ was found to be highly immunogenic, and elicited both humoral and cellular immune responses in mice.

Tumor antigen delivered by Salmonella III secretion protein fused with heat shock protein 70 induces protection and eradication against murine melanoma.

Attenuated Salmonella typhimurium possess the ability to stimulate innate immune responses and preferentially allocate within the solid tumor. These two main characteristics make attenuated Salmonella one of the most attractive vehicles for development of vaccine and also targeted cancer therapies. However, location of Salmonella prevents the process of antigen presentation. Salmonella Type III secretion system can be utilized to circumvent this problem because this system secretes the protein it encoded outside the cells. Heat shock protein 70 (Hsp70) is referred to as an "immunochaperone" for its capacity to elicit tumor-specific adaptive immune responses in the form of Hsp70-TAA (tumor associated antigen) complex. Hsp70 facilitates the cross-presentation of exogenous antigens through its receptor on antigen-presenting cells and therefore activates an antigen-specific cytotoxic T lymphocyte (CTL) response, which can directly contribute to potent anti-tumor immunity. Here, we designed a novel therapeutic vaccine utilizing the type III secretion system and Hsp70 to deliver and present the tumor-specific antigen. This live recombinant bacteria vaccine, when administrated orally, successfully broke the immune tolerance, induced a specific CTL response against tumor cells, and therefore revealed protective and therapeutic effects against generation and growth of B16F10 melanoma in C57BL/6J mice.

The intrabody targeting of hTERT attenuates the immortality of cancer cells.

hTERT (human telomerase reverse transcriptase) plays a key role in the process of cell immortalization. Overexpression of hTERT has been implicated in 85% of malignant tumors and offers a specific target for cancer therapy. In this paper, we describe an effective approach using a single-chain variable fragment (scFv) intrabody derived from monoclonal hybridoma directed against hTERT to attenuate the immortalization of human uterine cervix and hepatoma cells. The scFv we constructed had a high affinity to hTERT, and specifically neutralized over 70% of telomere synthesis activity, thereby inhibiting the viability and proliferation of the cancer cells. Our results indicate that this anti-hTERT intrabody is a promising tool to target hTERT and intervene in the immortalization process of cancer cells.

Suicide gene/prodrug therapy using salmonella-mediated delivery of Escherichia coli purine nucleoside phosphorylase gene and 6-methoxypurine 2'-deoxyriboside in murine mammary carcinoma 4T1 model.

Attenuated salmonella have been reported to be capable of both selectively growing in tumors and expressing exogenous genes for tumor-targeted therapy. As 6-methoxypurine 2'-deoxyriboside (MoPdR) is similar to 6-methylpurine 2'-deoxyriboside in structure, we aimed to evaluate the antitumoral effect of the Escherichia coli purine nucleoside phosphorylase (ePNP) gene, using an attenuated salmonella-mediated delivery system, in combination with MoPdR. A novel mutant serovar Typhimurium (SC36) was used to carry the pEGFP-C1-ePNP vector that contains an enhanced green fluorescent protein and an ePNP gene under the control of the cytomegalovirus promoter. The function of the ePNP expression vector was confirmed in vitro using the enzymic conversion of MoPdR into methoxypurine. We also observed a high bystander effect induced by the ePNP/MoPdR system with a very low proportion (1%) of ePNP-positive cells and 5 microg/mL MoPdR, although the growth of parental cells was affected appreciably by MoPdR. The killing effect and increased apoptosis induced by SC36 carrying the ePNP expression vector (SC/ePNP) were detected by cytotoxicity assay and propidium iodide staining flow cytometry analysis, in combination with MoPdR. SC/ePNP was given orally to mice bearing mammary carcinomas, and its antitumor effect was evaluated. SC/ePNP plus MoPdR significantly inhibited tumor growth by approximately 86.6-88.7% and prolonged the survival of tumor-hosting mice. Our data support the view that MoPdR combined with the ePNP gene could be used in gene-directed enzyme prodrug therapy. Attenuated salmonella could be a promising strategy to improve ePNP/MoPdR bystander killing due to its preferential accumulation and anticancer activity in tumors.