Postoperative pulmonary complications in older patients undergoing elective surgery with a supraglottic airway device or tracheal intubation.

The two most commonly used airway management techniques during general anaesthesia are supraglottic airway devices and tracheal tubes. In older patients undergoing elective non-cardiothoracic surgery under general anaesthesia with positive pressure ventilation, we hypothesised that a composite measure of in-hospital postoperative pulmonary complications would be less frequent when a supraglottic airway device was used compared with a tracheal tube. We studied patients aged ≥ 70 years in 17 clinical centres. Patients were allocated randomly to airway management with a supraglottic airway device or a tracheal tube. Between August 2016 and April 2020, 2900 patients were studied, of whom 2751 were included in the primary analysis (1387 with supraglottic airway device and 1364 with a tracheal tube). Pre-operatively, 2431 (88.4%) patients were estimated to have a postoperative pulmonary complication risk index of 1-2. Postoperative pulmonary complications, mostly coughing, occurred in 270 of 1387 patients (19.5%) allocated to a supraglottic airway device and 342 of 1364 patients (25.1%) assigned to a tracheal tube (absolute difference -5.6% (95%CI -8.7 to -2.5), risk ratio 0.78 (95%CI 0.67-0.89); p < 0.001). Among otherwise healthy older patients undergoing elective surgery under general anaesthesia with intra-operative positive pressure ventilation of their lungs, there were fewer postoperative pulmonary complications when the airway was managed with a supraglottic airway device compared with a tracheal tube.

[Effects of remimazolam versus propofol on postoperative recovery quality in elderly patients undergoing thoracoscopic laparoscopic radical esophagectomy].

Objective: To investigate the effects of remimazolam versus propofol on postoperative recovery quality in elderly patients undergoing thoracoscopic laparoscopic radical esophagectomy. Methods: A total of 108 elderly patients undergoing thoracoscopic laparoscopic radical esophagectomy under general anesthesia in the Affiliated Cancer Hospital of Zhengzhou University from May to October 2022 were prospectively included. The participants were divided into two groups by the random number table method: remimazolam group (R group, n=54) and propofol group (P group, n=54). There were 54 cases in the R group, with 35males and 19 females, and aged (65.4±3.1) years. Meanwhile, there were 54 cases in the P group, with 33males and 21 females, and aged (64.5±3.0) years. Anesthesia was induced as follows: remimazolam 0.2-0.3 mg/kg and remifentanil 0.5-1.0 µg/kg were intravenously injected in R group, while propofol 1-2 mg/kg and remifentanil 0.5-1.0 µg/kg were intravenously injected in P group. Subsequently, anesthesia was maintained as follows: remimazolam 0.4-1.0 mg·kg-1·h-1 and remifentanil 0.05-0.2 µg·kg-1·min-1 were intravenously infused in group R, while propofol 4-10 mg·kg-1·h-1 and remifentanil 0.05-0.2 µg·kg-1·min-1 were intravenously infused in group P. Bispectral index (BIS) was maintained at 45-60 during operation. The main outcome measures were the 15-item quality of recovery (QoR-15) scores 1 day before surgery, 1 day and 3 days after surgery. Secondary outcome measures included mean arterial pressure (MAP), heart rate and pulse oxygen saturation (SpO2) recorded 5 min before anesthesia induction (T0), 1 min after induction (T1), 1 min after endotracheal intubation (T2), immediately after skin incision (T3) and tracheal extubation (T4). The incidence of bradycardia and hypotension and the frequency of application of vasoactive drugs during anesthesia were recorded. Restlessness score (RS) and Ramsay sedation scale during the awakening period were recorded. Emergence time, tracheal extubation time, duration of postanesthesia care unit (PACU) stay and postoperative length of hospital stay were recorded. The incidence of postoperative pulmonary infection and other complications were also recorded. Results: The QoR-15 scale scores [M (Q1, Q3)] of R group 1 day and 3 days after surgery were 114.0 (109.0, 118.3) and 131.0 (127.8, 133.0), which were higher than those of P group [106.0 (101.0, 112.0) and 127.0 (125.0, 129.3)] (both P<0.001). The incidence of bradycardia, hypotension and the frequency of application of vasoactive drugs of R group during anesthesia were 5.6% (3/54), 35.2% (19/54) and 27.8% (15/54), which were lower than those in P group [33.3% (18/54), 63.0% (34/54) and 55.6% (30/54), respectively] (all P<0.05). RS score during the awakening period in R group was 0.9±0.5, which was lower than that of P group (1.1±0.6) (P=0.046). Emergence time, tracheal extubation time and postoperative length of hospital stay of R group were (15.4±4.9) min, (16.6±4.7) min and (11.6±1.4) d, which were shorter than those of P group [(26.2±6.4) min, (27.8±5.8) min and (12.6±1.3) d] (all P<0.05). There were no statistically significant differences in Ramsay scores during the awakening period, duration of PACU stay and the incidence of postoperative complications (all P>0.05). Conclusions: Both remimazolam and propofol can achieve satisfactory postoperative recovery quality in elderly patients undergoing thoracoscopic laparoscopic radical esophagectomy. Remimazolam has more stable hemodynamics and lower incidence of adverse reactions.

[Effects of patient-controlled intravenous analgesia with butorphanol versus sufentanil on early postoperative rehabilitation following radical laparoscopic nephrectomy].

Objective: To investigate the effects of patient-controlled intravenous analgesia with butorphanol versus sufentanil on early postoperative rehabilitation following radical laparoscopic nephrectomy. Methods: One hundred patients undergoing radical laparoscopic nephrectomy in Affiliated Cancer Hospital of Zhengzhou University from September 2018 to February 2020 were divided into two groups (n=50) using a random number table: butorphanol patient-controlled intravenous analgesia group (group A) and sufentanil patient-controlled intravenous analgesia group (group B). Patient-controlled intravenous analgesia (PCIA) was performed at the end of surgery. The formulation of group A was butorphanol (0.15 mg/kg) and ketorolac tromethamine (180 mg) using the physiological saline at a dilution of 100 ml. The formulation of group B was sufentanil (1.5 µg/kg) and ketorolac tromethamine (180 mg) using the physiological saline at a dilution of 100 ml. At the time points of 4, 8, 24, 48 h after operation (T(1), T(2), T(3), T(4)), VAS scores at rest and cough were recorded. The incidence of remedial analgesia, the number of pressings during 48 h after the operation, the postoperative anal exhaust recovery time of the patients were recorded. Quality of recovery-40(QoR-40) scores were recorded at T(3) and T(4). Adverse reactions were recorded. Results: There was no significant difference in VAS scores at rest and cough at T(1), T(2), T(3) and T(4) between two groups (all P>0.05). There was no significant difference in the incidence of remedial analgesia and the number of pressings during 48 h after the operation between two groups (all P>0.05). The postoperative anal exhaust recovery time of the patients in group A was (32±6) h, which was lower than that in group B with statistically significant difference [(40±5) h, t=7.937, P<0.01]. The QoR-40 total scores in group A were higher than those in group B at T(3) and T(4), which were (185.8±2.5) vs (170.7±2.7), (194.8±1.9) vs (183.6±2.6), and the differences were statistically significant (t=28.878, 25.025, all P<0.01). The incidence of nausea, retching/vomiting, respiratory depression and itch during 48 h after the operation in group A were 10%, 6%, 2%, 2%, which were lower than that in group B (32%, 20%, 14%, 18%), with statistically significant difference (χ(2)=7.294, 4.322, 4.891, 5.983, all P<0.05). Conclusion: PCIA with butorphanol or sufentanil can provide satisfactory analgesia for patients undergoing radical laparoscopic nephrectomy, but butorphanol can promote postoperative rehabilitation with fewer adverse reactions.

[Effects of desflurane and sevoflurane anesthesia on postoperative recovery after long-term tumor surgery].

Objective: To analyze the effects of desflurane and sevoflurane anesthesia on postoperative recovery after long lasting tumor surgery. Methods: One hundred and sixty patients undergoing endoscopic radical esophagectomy and gastrectomy (80 cases of each surgical type) from November 2019 to March 2020 at Henan Cancer Hospital, were randomized into 4 groups(n=40): group CS (esophageal cancer+sevoflurane anesthesia), group DS (esophageal cancer+desflurane anesthesia),group CW (stomach cancer+sevoflurane anesthesia) and group DW (gastric cancer+desflurane anesthesia). General anesthesia was induced by intravenous agents in all four groups, which were maintained by inhaled anesthetic during the operation. The mean arterial pressure (MAP), heart rate (HR), and surplus pulse O(2) (SpO(2)) immediately before induction (T(1)), the moment of operation begin (T(2)), operation end (T(3)) and extubation (T(4)) were recorded. Also, the duration required for inhalation anesthetic alveolar concentration reaching 0.5 minimum alveolar concentration (MAC) during induction, the alveolar anesthetic concentration at the beginning of the operation, the duration required for XMAC (patients specific alveolar concentration) declining to 0.5 MAC on recovery period, and the duration of alveolar concentration of 0.5 MAC declining to 0.2 MAC were determined. Additionally, the durations of spontaneous breathing recovery, eyes opening, extubation and recovery of consciousness were recorded. Finally, restlessness score (RS) during recovery period was used to evaluate postoperative agitation. Results: Compared with group CS and group CW, no significant differences in MAP, HR, SpO(2) in group DS and group DW at T(1) to T(4) were found (all P>0.05). The durations required for inhalation anesthetic alveolar concentration reaching 0.5 MAC were (5.6±1.3), (5.8±2.1), (3.5±1.5) and (3.8±1.0) min in group CS, group CW, group DS and group DW, where the durations in group DS and group DW were significantly shorter than those in group CS and group CW (F=32.538, P<0.05). The durations of alveolar concentration of 0.5 MAC declining to 0.2 MAC were (6.4±2.2), (7.0±1.5), (4.2±2.2) and (4.1±1.5) min in group CS, group CW, group DS and group DW, and the durations in group DS and group DW were significantly shortened as compared with group CS and group CW (F=42.113, P<0.05). Compared with group CS and group CW, group DS and group DW required significantly shorter time for spontaneous breathing recovery, eye opening,extubation, and directional force recovery after operation (all P<0.05). Conclusions: Both desflurane and sevoflurane anesthesia can achieve satisfactory anesthesia depth during long lasting tumor surgery. Desflurane can shorten the recovery time and early extubation, and improve the quality of recovery.

Randomized clinical trial of continuous transversus abdominis plane block, epidural or patient-controlled analgesia for patients undergoing laparoscopic colorectal cancer surgery.

BACKGROUND: The optimal analgesia regimen after laparoscopic colorectal cancer surgery is unclear. The aim of the study was to characterize the beneficial effects of continuous transversus abdominis plane (TAP) blocks initiated before operation on outcomes following laparoscopic colorectal cancer surgery. METHODS: Patients undergoing surgery for colorectal cancer were divided randomly into three groups: combined general-TAP anaesthesia (TAP group), combined general-thoracic epidural anaesthesia (TEA group) and standard general anaesthesia (GA group). The primary endpoint was duration of hospital stay. Secondary endpoints included gastrointestinal motility, pain scores and plasma levels of cytokines. RESULTS: In total, 180 patients were randomized and 165 completed the trial. The intention-to-treat analysis showed that duration of hospital stay was significantly longer in the TEA group than in the TAP and GA groups (median 4·1 (95 per cent c.i. 3·8 to 4·3) versus 3·1 (3·0 to 3·3) and versus 3·3 (3·2 to 3·6) days respectively; both P < 0·001). Time to first flatus was earlier in the TAP group (P < 0·001). Visual analogue scale (VAS) scores during coughing were lower in the TAP and TEA groups than the GA group (P < 0·001). Raised plasma levels of vascular endothelial growth factor C, interleukin 6, adrenaline and cortisol were attenuated significantly by continuous TAP block. CONCLUSION: Continuous TAP analgesia not only improved gastrointestinal motility but also shortened duration of hospital stay. A decreased opioid requirement and attenuating surgical stress response may be potential mechanisms. Registration number: ChiCTR-TRC-1800015535 ( http://www.chictr.org.cn).

ANTECEDENTES: El régimen analgésico óptimo para los pacientes tras cirugía laparoscópica del cáncer colorrectal se desconoce. El objetivo de este estudio fue caracterizar los efectos beneficiosos del bloqueo continuo del plano transverso abdominal (transversus abdominis plane, TAP) iniciado preoperatoriamente sobre los resultados después de cirugía laparoscópica del cáncer colorrectal. MÉTODOS: Los pacientes sometidos a cirugía del cáncer colorrectal fueron divididos aleatoriamente en tres grupos: anestesia combinada general-TAP (grupo TAP), anestesia epidural combinada general-torácica (grupo TEA) y anestesia general estándar (grupo GA). El resultado primario fue la duración de la estancia hospitalaria. Los resultados secundarios incluyeron la motilidad gastrointestinal, puntuaciones de dolor y niveles plasmáticos de citocinas. RESULTADOS: En total, 180 pacientes fueron aleatorizados y 165 completaron el ensayo. El análisis por intención de tratar mostró que la duración de la estancia hospitalaria en el grupo TEA fue significativamente más larga que en el grupo TAP y GA respectivamente (4,1 (3,8-4,3) versus 3,1 (3,0-3,3) días, P < 0,001; 4,1 (3,8-4,3) versus 3,3 (3,2-3,6) días, P < 0,001). El tiempo hasta la primera eliminación de gases fue más precoz en el grupo TAP (P < 0,001). Las puntuaciones de la escala analógica visual (visual analogue scale, VAS) durante la tos en el grupo TAP y TEA fueron inferiores (P < 0,01). Los niveles elevados en plasma del factor de crecimiento endotelial C (VEGF-C), interleucina (IL)-6, epinefrina y cortisol fueron atenuados significativamente por el bloque TAP continuo. CONCLUSIÓN: La analgesia TAP continua no solo mejora la motilidad gastrointestinal, sino que también acorta la estancia hospitalaria. Una disminución en los requerimientos de opiáceos y la atenuación de la respuesta al estrés quirúrgico podrían ser mecanismos potenciales de la acción de TAP.

Effects of anaesthesia on proliferation, invasion and apoptosis of LoVo colon cancer cells in vitro.

Tumour cell proliferation, invasion and apoptosis are crucial steps in tumour metastasis. We evaluated the effect of serum from patients undergoing colon cancer surgery receiving thoracic epidural and propofol anaesthesia on colon cancer cell biology. Patients were randomly assigned to receive propofol anaesthesia with a concomitant thoracic epidural (PEA, n = 20) or sevoflurane anaesthesia with opioid analgesia (SGA, n = 20). Venous blood was obtained before induction of anaesthesia and 24 hours postoperatively. The LoVo colon cancer cells were cultured with patient serum from both groups and the effects on proliferation, invasion and apoptosis were measured. Twenty-four hours after surgery, the absorbance value of LoVo cells at 10% serum concentration from PEA was decreased when compared with SGA (0.302 (0.026) vs 0.391 (0.066), p = 0.005). The inhibitory rate of LoVo cells at 10% serum concentration from PEA was higher than that from SGA (p = 0.004) 24 h after surgery. The number of invasive LoVo cells at 10% serum concentration from PEA was reduced when compared with SGA (44 (4) vs 62 (4), p < 0.001). Exposure of LoVo cells to postoperative serum from patients receiving PEA led to a higher luminescence ratio (apoptosis) than those receiving SGA (0.36 (0.04) vs 0.27 (0.05), p < 0.001). Serum from patients receiving PEA for colon cancer surgery inhibited proliferation and invasion of LoVo cells and induced apoptosis in vitro more than that from patients receiving SGA. Anaesthetic technique might influence the serum milieu in a way that affects cancer cell biology and, thereby, tumour metastastasis.

Systemic or intra-amygdala infusion of an endocannabinoid CB1 receptor antagonist AM251 blocked propofol-induced anterograde amnesia.

Propofol is well-known for its anterograde amnesic actions. However, a recent experiment showed that propofol can also produce retrograde memory enhancement effects via an interaction with the endocannabinoid CB1 system. Therefore, the authors hypothesized that the regulating effect of propofol on the endocannabinoid CB1 system might also decrease the anterograde amnesic effect of propofol under some conditions, which might be a risk factor for intraoperative awareness. Since, the basolateral amygdala (BLA) has been confirmed to mediate propofol-induced anterograde amnesia and the BLA contains a high concentration of CB1 receptors, the authors investigated whether and how the endocannabinoid system, particularly the CB1 receptor within BLA, influences propofol-induced anterograde amnesia. Male Sprague-Dawley rats trained with inhibitory avoidance (IA) were systematically pre-trained using a memory-impairing dose of propofol (25 mg/kg). Before propofol administration, rats received an intraperitoneal injection of a CB1 receptor antagonist AM251 (1 mg/kg or 2 mg/kg) or a bilateral intra-BLA injection of AM251 (0.6 ng or 6 ng per 0.5 µl). Twenty-four hours after IA training, the IA retention latency was tested. It was found that systemic or intra-BLA injection of a non-regulating dose of AM251 (2 mg/kg or 6 ng per 0.5 µl, respectively) blocked the memory-impairing effect of propofol. These results indicate that the anterograde amnesic effect of propofol is mediated, in part, by activation of the CB1 cannabinoid receptors in the BLA.

Pooled analysis of association between a genetic variant in the 3'-untranslated region of Toll-like receptor 4 and cancer risk.

Many epidemiological studies have shown the association between certain genetic variations in the Toll-like receptor 4 (TLR4) gene (for example, rs4986790 and rs4986791) and cancer risk. However, the results from investigations into the association between rs11536889, a genetic variant in the 3'-untranslated region of TLR4, and cancer risk lack consensus. We performed a meta-analysis to investigate the effect of rs11536889 on cancer risk. A total of 12 relevant case-control studies were included in this analysis (6222 cases and 7948 controls). The pooled ORs with their corresponding 95%CIs were estimated. We did not detect any association between rs11536889 and overall cancer risk (P = 0.13). However, stratification analysis by cancer type revealed a borderline statistically significant increased risk in both genotype comparison (OR for the variant genotype in the dominant model = 1.17; 95%CI = 0.98-1.40; P = 0.08) and allele comparison (OR for variant allele = 1.14; 95%CI = 0.99-1.32; P = 0.07) for prostate cancer. In contrast, no statistically significant or borderline result was found for gastric cancer. These findings indicate that rs11536889 in TLR4 might be specifically associated with prostate cancer. However, owing to the modest and underpowered results, and the limitations of the original studies included for analysis, further prospective studies with larger sample sizes are required to confirm our findings.

Effect of thoracic epidural anaesthesia on serum vascular endothelial growth factor C and cytokines in patients undergoing anaesthesia and surgery for colon cancer.

BACKGROUND: Serum vascular endothelial growth factor-C (VEGF-C), transforming growth factor-ß (TGF-ß), and interleukin (IL)-6 promote angiogenesis and metastases in colon cancer. We hypothesized that patients who received propofol-epidural anaesthesia (PEA) would exhibit decreases in VEGF-C, TGF-ß, and IL-6 and an increase in IL-10 compared with patients who received general anaesthesia (GA). METHODS: Colon cancer surgery patients were randomly assigned to the PEA (n=20) or GA (n=20) group. Serum VEGF-C, TGF-ß, IL-6, and IL-10 levels before surgery and 24 h after surgery were measured. RESULTS: Patients who received PEA showed decreases in VEGF-C [526 (261) vs 834 (304) pg ml(-1), P=0.001], TGF-ß (P=0.027), and IL-6 (P=0.007) and an increase in IL-10 (P=0.001) 24 h after surgery compared with patients subjected to GA. The visual analogue scale scores at rest and during coughing at 2 and 24 h after operation were significantly lower in PEA patients (P<0.05). CONCLUSIONS: PEA reduces serum concentrations of factors associated with angiogenesis during colon cancer surgery. CLINICAL TRIAL REGISTRATION: ChiCTR-TRC-13003146 (www.chictr.org).

Long-term tolerance to factor VIII is achieved by administration of interleukin-2/interleukin-2 monoclonal antibody complexes and low dosages of factor VIII.

BACKGROUND: Regulatory T cells (Tregs) play a pivotal role in regulating anti-factor VIII (FVIII) immune responses. Interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb; JES6-1) can induce the selective expansion of Tregs in vivo. METHODS: In the prevention experiments, we treated mice with hemophilia A with IL-2/IL-2mAb complexes (three times per week) and concurrently with FVIII protein (80 U kg(-1) per week) for 4 weeks. Generation of anti-FVIII immune responses was examined afterward. Next, to induce long-term tolerance to FVIII, a series of treatment dosages and schedules for administering IL-2/IL-2mAb complexes and FVIII protein in mice with hemophilia A was evaluated. RESULTS: Compared to control mice that were treated with only FVIII, which produced high-titer anti-FVIII antibodies, mice treated with IL-2/IL-2mAb complexes plus FVIII produced no antibodies. A marked seven-fold increase in CD4(+) CD25(+) Foxp3(+) Helios(+) natural Tregs was maintained for 4 weeks in blood, spleen, and lymph nodes and then dropped to normal levels within the next 10 days. The suppressive functions of expanded Tregs were demonstrated with suppressive, proliferative, and cytokine assays. The administration of anti-CD25 mAb (PC-61) blocked this protective effect, confirming the involvement of Tregs in suppressing anti-FVIII immune responses. Importantly, administration of IL-2/IL-2mAb complexes (three times per week for 8 weeks) combined with contiguous weekly injections of low-dosage FVIII protein (20 U kg(-1) per week for 18 weeks) not only abrogated the formation of anti-FVIII antibodies but also induced long-term tolerance to FVIII. CONCLUSIONS: Treatment with IL-2/IL-2mAb complexes is highly promising for the induction and maintenance of FVIII-specific tolerance after FVIII protein replacement therapy.

Explorations of high-intensity therapeutic ultrasound and microbubble-mediated gene delivery in mouse liver.

Ultrasound (US) combined with microbubbles (MBs) is a promising technology for non-viral gene delivery. Significant enhancements of gene expression have been obtained in our previous studies. To optimize and prepare for application to larger animal models, the luciferase reporter gene transfer efficacy of lipid-based Definity MBs of various concentrations, pressure amplitudes and a novel unfocused high-intensity therapeutic US (HITU) system were explored. Luciferase expression exhibited a dependence on MB dose over the range of 0-25 vol%, and a strong dependence on acoustic peak negative pressure at over the range of 0-3.2 MPa. Gene expression reached an apparent plateau at MB concentration ≥2.5 vol% or at negative pressures >1.8 MPa. Maximum gene expression in treated animals was 700-fold greater than in negative controls. Pulse train US exposure protocols produced an upward trend of gene expression with increasing quiescent time. The hyperbolic correlation of gene expression and transaminase levels suggested that an optimum gene delivery effect can be achieved by maximizing acoustic cavitation-induced enhancement of DNA uptake and minimizing unproductive tissue damage. This study validated the new HITU system equipped with an unfocused transducer with a larger footprint capable of scanning large tissue areas to effectively enhance gene transfer efficiencies.

Generation of a novel factor IX with augmented clotting activities in vitro and in vivo.

BACKGROUND: Hemophilia B is an X-linked inherited disorder caused by the lack of functional factor IX (FIX). Currently, treatment of hemophilia B is performed by intravenous infusion of plasma-derived or recombinant FIX. OBJECTIVE: In an effort to reduce factor usage and cost, we investigated the potential use of FIX variants with enhanced specific clotting activity. METHODS: Seven recombinant FIX variants using alanine replacement were generated and assayed for their activity in vitro and in vivo. RESULTS: One variant containing three substitutions (V86A/E277A/R338A, FIX-Triple) exhibited 13-fold higher specific clotting activity and a 10-fold increased affinity for human FVIIIa compared with FIX-wild-type (FIX-WT) and was thus investigated systematically in vivo. Liver-specific FIX-Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5-fold higher specific clotting activity compared with FIX-WT. Human FIX-Triple and FIX-WT knock-in mice were generated and it was confirmed that FIX-Triple has 7-fold higher specific clotting activity than FIX-WT under normal physiological conditions. Protein infusion of FIX-Triple into hemophilia B mice resulted in greater improvement of hemostasis than that achieved with FIX-WT. Moreover, tail-vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple in hemophilia B mice demonstrated a 7-fold higher specific clotting activity of FIX-Triple than FIX-WT. CONCLUSIONS: Our results indicate that the FIX-Triple variant exhibits significantly enhanced clotting activity relative to FIX-WT due to tighter binding to FVIIIa, as demonstrated both in vitro and in vivo. Therefore, FIX-Triple is a good candidate for further evaluation in protein replacement therapy as well as gene-based therapeutic strategies.

Ultrasound with microbubbles enhances gene expression of plasmid DNA in the liver via intraportal delivery.

Current ultrasound (US)-mediated gene delivery methods are inefficient due, in part, to a lack of US optimization. We systematically explored the use of microbubbles (MBs), US parameters and plasmid delivery routes to improve gene transfer into the mouse liver. Co-presentation of plasmid DNA (pDNA), 10% Optison MBs and pulsed 1-MHz US at a peak negative pressure of 4.3 MPa significantly increased luciferase gene expression with pDNA delivered by intrahepatic injection to the left liver lobe. Intraportal injection delivered pDNA and MBs to the whole liver; with insonation, all lobes expressed the transgene, thus increasing total gene expression. Gene expression was also dependent on acoustic pressure over the range of 0-4.3 MPa, with a peak effect at 3 MPa. An average of 85-fold enhancement in gene delivery was achieved. No enhancement was observed below 0.25 MPa. Increasing pulse length while decreasing pulse repetition frequency and exposure time to maintain a constant total energy during exposure did not further improve transfection efficiency, nor did extend the US exposure pre- or postinjection of pDNA. The results indicate that coupled with MBs, US can more efficiently and dose-dependently enhance gene expression from pDNA delivered via portal vein injection by an acoustic mechanism of inertial cavitation.

Expression of foreign genes in chicks by hydrodynamics-based naked plasmid transfer in vivo.

The study of gene function in vivo is considered one of the top achievements of modern biology, inasmuch as it provides tools to study gene function in the context of the whole animal. In chickens, techniques of DNA-mediated gene transfer are less advanced than in other animal or livestock models, and remain a significant challenge. The study presented here is the first to show that a hydrodynamics-based gene-transfer technique, originally developed for naked DNA transfer in mice, can be applied to chickens. Rapid injection of naked plasmids containing expression cassettes into the jugular vein of 6- to 10-day-old chicks resulted in specific expression of the transgenes. A CMV promoter-driven luciferase reporter gene was expressed at significant levels in the liver during the first 3 days post-injection with lower levels also detected in the kidney. Significantly, all injected birds showed detectable levels of luciferase expression. Similarly, injection of a plasmid containing the secreted human coagulation factor IX (hFIX) gene under the control of human alpha-1-anti-trypsin promoter resulted in detectable levels of the hFIX in the plasma during the first 2 days post-injection. The method described herein has the potential for a quick and simple route for gain and loss-of function experiments in chicken liver and kidney, as well as for studying systemic effects of secreted proteins and hormones.

Complete and sustained phenotypic correction of hemophilia B in mice following hepatic gene transfer of a high-expressing human factor IX plasmid.

Therapeutic correction of hemophilia B was achieved by rapid infusion of a large-volume solution containing a high-expressing human factor IX (hFIX) plasmid into the tail vein of hemophilia B mice. hFIX circulated at therapeutic levels (1-5 micro g mL-1) in all animals for more than 1 year as determined by both species-specific antigen assay and an activated partial thromboplastin time (APTT)-based clotting assay. There was acute, transient hepatic tissue damage by the infusion procedure and no significant inhibitory anti-hFIX antibodies developed. No bleeding episode was observed during or after treatment. Immunohistochemical studies indicated that the hFIX gene was exclusively expressed in hepatocytes, and that transduced cells had readily detectable hFIX protein at 4 h postinfusion, and stainable protein persisted for up to 1 year. Repeated infusions of hFIX plasmids boosted the hFIX expression to higher levels. These results demonstrate that hemophilia B can be treated by gene transfer of naked hFIX plasmids.

Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo.

Naked DNA transfer of a high-expressing human factor IX (hFIX) plasmid yielded long-term (over 1 1/2 years) and therapeutic-level (0.5-2 microg/ml) gene expression of hFIX from mouse livers. The expression cassette contained a hepatic locus control region from the ApoE gene locus, an alpha1-anti-trypsin promoter, hFIX cDNA, a portion of the hFIX first intron, and a bovine growth hormone polyadenylation signal. In contrast, a hFIX plasmid containing the expression cassette without effective regulatory elements produced initially low-level gene expression that rapidly declined to undetectable levels. Southern analyses of the cellular DNA indicated that the majority of the input genome from either vector persisted as episomal forms of the original plasmids. Together with RT-PCR analyses of the transcripts, these data indicated that at least two processes are critical for sustained gene expression: persistence of vector DNA and transcriptional/posttranscriptional activation. Liver regeneration after partial hepatectomy resulted in a significant decline in transgene expression, further suggestive of decreased episomal plasmid maintenance rather than transgene integration. Transaminase levels and liver histology showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage (< 5% of hepatocytes), which was rapidly repaired within 3 to 10 days and resulted thereafter in histologically normal tissue. No significant differences were observed between rapid injection of plasmid and saline control solutions. Transient, very low level antibodies directed against hFIX did not prevent the circulation of therapeutic levels of the protein. Gene transfer of hFIX plasmid DNA into liver elicited neither transgene-specific cytotoxic effect nor long-term toxicity. These results demonstrate that long-term expression of hFIX can be achieved by nonviral plasmid transfer and suggest that this occurs independent of integration.

Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro.

We systematically compared human factor IX gene expression from a variety of plasmids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection into the livers of mice. Although there was a 1.5- to 2.0-fold variation in gene expression from cultured cells, a 65-fold variation was observed in the in vivo studies. We found that a plasmid containing the apolipoprotein E locus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX minigene (hFIXmg) sequence including a portion of the first intron (intron A), 3'-untranslated region (3'-UTR), and a bovine growth hormone polyadenylation signal (bpA) produced the highest serum level of human factor IX, reaching 18 microg/ml (normal = 5 microg/ml) 1 day after injection. Although most of the plasmid DNAs resulted in transient gene expression, inclusion of an intron, a polyadenylation signal from either the 1.7-kb 3'-UTR or the 0.3-kb bpA, and the HCR resulted in persistent and therapeutic levels of hFIX gene expression, ranging from 0.5 to 2 microg/ml (10 to 40% of normal) for 225 days (length of experiment). These data underscore the importance of cis sequences for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.

Effect of long-term selection for early postnatal growth rate on survival and prenatal development of transferred mouse embryos.

Reciprocal embryo transfer procedures were performed among mouse selection lines to examine prenatal maternal effects on survival and development of transferred embryos. Mice were from generations 28 and 29 of an experiment to select for (i) increased body weight again from 0 to 10 days (E+); (ii) decreased body weight gain from 0 to 10 days (E-); or (iii) a randomly bred control line (C). A total of 118 embryo transfer procedures performed 12 h after conception resulted in 983 progeny born to 89 litters. There was a 39% overall embryo survival rate and 75% overall pregnancy success rate. Response to superovulation and oestrous synchronization was significantly lower (P < 0.01) in the E+ line. E+ individuals that did superovulate produced an average of 37 oocytes per flush, which was significantly higher than in the control line mice (29 oocytes per flush; P < 0.01). The ability to complete pregnancy successfully was not influenced by uterine environment or embryo-uterine interaction. In contrast, embryo survival in successful pregnancies was significantly affected by uterine environment. There were large maternal effects for body weight and tail length at birth; E+ recipients produced pups that were significantly larger than E- recipient pups (P < 0.01), which in turn were significantly larger than pups gestated by control recipients (P < 0.01).