[Clinical application of split liver transplantation: a single center report of 203 cases].

Objective: To investigate the safety and therapeutic effect of split liver transplantation (SLT) in clinical application. Methods: This is a retrospective case-series study. The clinical data of 203 consecutive SLT, 79 living donor liver transplantation (LDLT) and 1 298 whole liver transplantation (WLT) performed at the Third Affiliated Hospital of Sun Yat-sen University from July 2014 to July 2023 were retrospectively analyzed. Two hundred and three SLT liver grafts were obtained from 109 donors. One hundred and twenty-seven grafts were generated by in vitro splitting and 76 grafts were generated by in vivo splitting. There were 90 adult recipients and 113 pediatric recipients. According to time, SLT patients were divided into two groups: the early SLT group (40 cases, from July 2014 to December 2017) and the mature SLT technology group (163 cases, from January 2018 to July 2023). The survival of each group was analyzed and the main factors affecting the survival rate of SLT were analyzed. The Kaplan-Meier method and Log-rank test were used for survival analysis. Results: The cumulative survival rates at 1-, 3-, and 5-year were 74.58%, 71.47%, and 71.47% in the early SLT group, and 88.03%, 87.23%, and 87.23% in the mature SLT group, respectively. Survival rates in the mature SLT group were significantly higher than those in the early SLT group (χ2=5.560,P=0.018). The cumulative survival rates at 1-, 3- and 5-year were 93.41%, 93.41%, 89.95% in the LDLT group and 87.38%, 81.98%, 77.04% in the WLT group, respectively. There was no significant difference among the mature SLT group, the LDLT group and the WLT group (χ2=4.016, P=0.134). Abdominal hemorrhage, infection, primary liver graft nonfunction,and portal vein thrombosis were the main causes of early postoperative death. Conclusion: SLT can achieve results comparable to those of WLT and LDLT in mature technology liver transplant centers, but it needs to go through a certain time learning curve.

[Effect of renal function on sarcopenia in elderly male patients with chronic kidney disease].

Objective: To evaluate the effect of renal function on sarcopenia in elderly male patients with chronic kidney disease (CKD). Methods: A total of 105 male CKD patients aged ≥65 years who were admitted to the Chinese PLA General Hospital between October 1, 2018 and January 30, 2019 were included in this study. Using two different equations to estimate glomerular filtration rate (GFR), respectively. According to the sarcopenia criteria, the participants were categorized as the non-sarcopenia group (n=72) and the sarcopenia group (n=33), respectively. The association of estimated GFR (eGFR) and the sarcopenia in the male CKD patients was analyzed using the model of multivariate logistic regression. Results: Among the 105 patients, the median age was 74 (68, 77) years old. The prevalence of sarcopenia was 31.4% (33/105). According to the multivariate logistic regression analysis, eGFR based on serum creatinine and Cys-C (eGFRscr-cys) lower than 45 ml·min(-1)·(1.73 m(2))(-1) (OR=4.17, 95%CI:1.08-16.02, P=0.038) and eGFR based on Cys-C (eGFRcys) lower than 45 ml·min(-1)·(1.73 m(2))(-1) (OR=3.99, 95%CI:1.08-14.75, P=0.038) were independent risk factors for underlying sarcopenic, respectively. The area under the receiver operating characteristics curve (AUC) revealed that eGFRscr-cys (AUC=0.67) was more suitable than eGFRcys (AUC=0.64) to predict the sarcopenia in elderly male patients with CKD. Conclusion: The increased incidence of sarcopenia in elderly men with CKD is accompanied with deterioration of renal function.

[Influence of different timing of Saccharomyces boulardii combined with bismuth quadruple therapy for Helicobacter pylori eradication].

Objective: To assess the efficacy and safety of different opportunity of Saccharomyces boulardii (S. Boulardii) Sachets combined with bismuth quadruple therapy for Helicobacter pylori(H. pylori)eradication. Methods: This experiment was a prospective study. A total of 300 H. pylori-infected patients were enrolled and randomized assigned into three groups. Quadruple therapy group received pantoprazole 40 mg+bismuth potassium citrate capsule 220 mg+amoxicillin 1 000 mg+furazolidone 100 mg, bid, oral for 14 days. The simultaneous probiotic group received pantoprazole 40 mg+bismuth potassium citrate capsule 220 mg+amoxicillin 1 000 mg+furazolidone 100 mg+S. Boulardii Sachets 500 mg,bid,oral for 14 days.There after probiotic group:S.Boulardii Sachets was added on the first day after the end of 14 days in the quadruple group, for 500 mg, bid 14 days. The eradication rates and adverse reactions of the three groups were compared. Results: The eradication rates of H.pylori were 89.0%,90.4% and 91.3% in the quadruple therapy group, the simultaneous probiotic group and the there after probiotic group according to Per-protocol(PP) analysis,respectively,with no statistical difference (P=0.870). According to intention-to-treat(ITT)analysis, 81.0%, 85.0% and 84.0%, respectively, the difference was not statistically significant (P=0.732).The overall incidence of adverse reactions and the incidence of diarrhea and nausea in the simultaneous probiotic group, and the there after probiotic group were lower than those in the quadruple group(P<0.05),and the difference was statistically significant.The overall incidence of adverse reactions and diarrhea in the simultaneous probiotic were lower than those in the there after probiotic group (P=0.021, P=0.007), and the difference was statistically significant. Conclusions: S. Boulardii Sachets combined with quadruple therapy at the same time or after eradication treatment can not improve the H.pylori eradication rate,but can reduce the overall incidence of adverse reactions and the incidence of diarrhea and nausea. It is safer to add S. Boulardii Sachets at the same time than after eradication therapy.

Sildenafil might impair maternal-fetal immunotolerance by suppressing myeloid-derived suppressor cells in mice.

Myeloid-derived suppressor cells (MDSCs) as an important population of immune cells were found to restrain T cell function, polarize T-helper cells (Th) 1/Th2 toward Th2 response and induce regulatory T cells (Tregs), therefore enhancing the immunotolerance during pregnancy. Sildenafil has been applied for poor endometrial quality in implantation failure patients. Nevertheless, investigations have shown that sildenafil could reduce MDSCs-dependent immunosuppression. Whether sildenafil affects embryo implantation by suppressing MDSCs? To address this question, using the mice model, we investigated the amounts of immune cells in peripheral blood and endometrial cells from control group (CG), sildenafil low-dose group (LDG) and high-dose group (HDG). We found that both treatment groups displayed a marked deficiency in polymorphonuclear (PMN)-MDSCs and Th2 from mice blood and endometrium as compared to these from CG. The frequency of Tregs in endometrium from HDG was lower than those from CG. Th1/Th2 ratio in both periphery and uterus from study groups showed a significant increase as compared to those from CG. By relevance analysis, we found that the level of Tregs positively correlated with the level of PMN-MDSCs, whereas the Th1/Th2 ratio negatively correlated with the frequency of PMN-MDSCs in uterus. Moreover, there was a positive relationship between the amount of blood PMN-MDSCs and endometrial PMN-MDSCs. These results suggest that we should carefully weigh the pros and cons of using sildenafil when applied to patients with poor endometrial receptivity.

Simvastatin alleviates inflammation and oxidative stress in rats with cerebral hemorrhage through Nrf2-ARE signaling pathway.

OBJECTIVE: To investigate the regulatory effects of simvastatin on the inflammation and oxidative stress in rats with cerebral hemorrhage through the nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE) signaling pathway. MATERIALS AND METHODS: A total of 120 healthy male rats weighing 280-300 g and 7-8 weeks old were selected to establish the traumatic brain injury (TBI) model. Rats were divided into group A (trauma operation, n=30), group B (no treatment, n=30), group C (drug administration after trauma operation, n=30), and group D (no trauma operation, drug administration, n=30). Cerebral edema content in brain tissues was measured by calculating the dry and wet weight. Neurological dysfunction was scored using the Garcia method. Positive levels of the Toll-like receptor 4 (TLR4) and interleukin-1ß (IL-1ß) were qualitatively analyzed via immunohistochemistry. Protein levels of TLR4 and IL-1ß were quantitatively analyzed via Western blotting. Moreover, the brain injury volume and neuronal apoptosis were evaluated via Nissl staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. At 48 h after injury, activities of superoxide dismutase (SOD), reduced glutathione (GSH), and oxidized glutathione (GSSG) in brain tissues were detected, and levels of malondialdehyde (MDA) and nitric oxide (NO) were detected using the enzyme activity assay kits. Finally, relative levels of the Nrf2-ARE signaling pathway and its downstream molecules heme oxygenase-1 (HO-1) and NAD (P)H dehydrogenase, quinone 1 (NQO1) were detected via reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting. RESULTS: Compared with those in group B, cerebral edema content in brain tissues significantly increased (p<0.05), the neurological dysfunction score significantly declined (p<0.05), and protein levels of TLR4 and IL-1ß were significantly upregulated in group A (p<0.05). In group C, relative levels of TLR4 and IL-1ß were down-regulated, cerebral edema content decreased, and the neurological dysfunction score significantly increased (p<0.05). After 48 h, activities of SOD, reduced GSH and GSSG and levels of MDA and NO all increased, and levels of MDA and NO declined in group C (p<0.05). Western blotting and RT-PCR showed that simvastatin could increase the transcriptional level of Nrf2. After simvastatin intervention, expression levels of downstream molecules HO-1 and NQO1 were upregulated. CONCLUSIONS: Simvastatin alleviates TLR4-mediated inflammatory injury, promotes neurological recovery and resists oxidative stress through the Nrf2-ARE signaling pathway, thus exerting a neuroprotective effect in TBI.

Effect of miR-130a on neuronal injury in rats with intracranial hemorrhage through PTEN/PI3K/AKT signaling pathway.

OBJECTIVE: The aim of this study was to investigate the effect of micro-ribonucleic acid (miR)-130a on neuronal injury in rats with intracerebral hemorrhage (ICH) through the phosphatase and tensin homolog deleted on chromosome ten/phosphatidylinositol 3-hydroxy kinase/protein kinase B (PTEN/PI3K/AKT) signaling pathway. MATERIALS AND METHODS: A total of 30 healthy male rats were randomly divided into three groups, including the blank control group, ICH model group (ICH group) and ICH model + miR-130a treatment group (miR-130a treatment group). The differences in neurological injury, the number of apoptotic cells in brain tissues, the activity of Caspase-9 and protein expressions of PTEN/PI3K/AKT were analyzed among the three groups, respectively. RESULTS: Neurological function was normal without injury in the control group. However, the neurological injury was severe in the ICH group and mild in the miR-130a treatment group. There were statistically significant differences in neurological function in the control group relative to those of the ICH group and miR-130a treatment group (p<0.05). Meanwhile, the neurological injury was markedly milder in the miR-130a treatment group than that of the ICH group, showing a statistically significant difference (p<0.05). The number of apoptotic cells was remarkably smaller in the control group when compared with the ICH group and miR-130a treatment group. However, it was markedly larger in the ICH group than that of the miR-130a treatment group, showing significant differences (p<0.05). The activity of Caspase-9 was significantly lower in the control group than ICH group and miR-130a treatment group (p<0.05). However, it increased remarkably in the ICH group compared with that of the miR-130a treatment group (p<0.05). Moreover, the protein level of PTEN in the ICH group was significantly higher than control group and miR-130a treatment group, displaying statistically significant differences (p<0.05). However, no marked difference in the protein level of PTEN was observed between the control group and miR-130a treatment group (p>0.05). The protein levels of the phosphorylated 3-hydroxy kinase (p-PI3K) and phosphorylated protein kinase B (p-AKT) were remarkably lower in the ICH group than those of the control group and miR-130a treatment group, displaying statistically significant differences (p<0.05). However, they were remarkably higher in the miR-130a treatment group than that of the control group (p<0.05). CONCLUSIONS: MiR-130a promotes neuronal growth in brain tissues in ICH rats and alleviates neuronal injury after ICH through the PTEN/PI3K/AKT signaling pathway. Our findings suggest that miR-130a exerts important clinical significance in the treatment of ICH.

[The role of Th1/Th2 cells imbalance in the pathogenesis of secretory otitis media].

Objective:The aim of this study is to explore the role of Th1/Th2 cells imbalance in the pathogenesis of secretory otitis media. Method:Ninety secretory otitis media patients were enrolled in observation group. According to medical history, they were divided into acute and chronic group. In addition, 90 healthy volunteers during the same period were selected as the control group. The levels of Th1-type cytokines IFN-γ, Th2-type cytokines interleukin-4 (IL-4) and IFN-γ/IL-4 in peripheral blood were compared between observation group and control group. Compare with acute and chronic secretory otitis media patients IFN-γ, IL-4, IFN-γ/IL-4 levels as well as the compare with middle ear effusion and peripheral blood sIFN-γ, IL-4, IFN-γ/IL-4 levels in observation group. Result:The level of IFN-γ, IL-4 and IFN-γ/IL-4 in the peripheral blood in the observation group were higher than those of the control group (P<0.05). The levels of IFN-γ, IL-4 and IFN-γ/IL-4 in the peripheral blood of patients in the chronic group were higher than those in the acute group. There was no significant difference in IL4, IFN-γ/IL-4 levels between the observation group and the middle ear effusion (P>0.05), IFN-γ levels in peripheral blood were lower than those of the middle ear effusion IFN-γ (P<0.05). Conclusion:Abnormal IFN-γ, IL-4 levels of the peripheral blood and the middle ear effusion have some relationship with secretory otitis media, and Th1/Th2 imbalance may be a risk factor for secretory otitis media.

Laparoscopic primary choledochorrhaphy over endonasobiliary drainage tubes.

BACKGROUND: The T-tube is widely used in laparoscopic choledochotomy to decompress the biliary tree. However, there are high morbidity rates related to the T-tube. This study reviewed the results of laparoscopic primary choledochorrhaphy over endonasobiliary drainage (ENBD) tubes to find an effective alternative to the T-tube for the performance of laparoscopic choledochotomy. METHODS: From March 2003 to September 2005, 23 patients (9 men and 14 women) with choledocholithiasis underwent laparoscopic choledochotomy over ENBD tubes. The mean age of these patients was 47 years (range, 32-73 years). At admission, six patients had cholangitis. All the patients had ENBD tubes placed preoperatively after the failure of endoscopic sphincterotomy. RESULTS: There was no conversion to open surgery. The mean operative time was 90 min (range, 70-150 min). There were no biliary complications such as bile leaks, biliary peritonitis, or pancreatitis. No residual stones were found by postoperative cholangiograms. The ENBD tubes were removed between postoperative days 7 and 9. The hospital stay ranged from 8 to 14 days, with 16 patients (70%) discharged on postoperative day 8. The complications were limited to one umbilical infection and one case of pneumonia. The median follow-up period was 24 months (range, 8-36 months), and none of the patients were readmitted with biliary symptoms. CONCLUSION: Laparoscopic choledochotomy over ENBD tubes proved to be technically feasible and safe. The ENBD tube decompresses the biliary tree and allows for cholangiography after surgery. Its removal does not need to wait for tract maturation, which allows an earlier removal of the tube and a shorter postoperative hospital stay. Laparoscopic choledochotomy over ENBD tubes is an effective alternative to the T-tube in laparoscopic choledochotomy.

Reoperation of biliary tract by laparoscopy: a consecutive series of 26 cases.

BACKGROUND: In the past, previous operation of biliary tract represented a contraindication to perform reoperation by laparoscopy. As experience with laparoscopic techniques and instrumentation has expanded, reoperation of biliary tract with laparoscope has become an accepted procedure in the management of cholelithiasis. We present our interesting experience with regard to reoperation of biliary tract by laparoscopy. MATERIAL AND METHODS: Laparoscopic operation of biliary tract was performed on 3,674 consecutive patients from April, 1992 till June, 2005. Among these patients, 26 had a previous open operation of biliary tract and their clinical data were retrospectively analyzed as follows: seven cases had complicated intrahepatic bile duct stones (restricted at hepatic duct of the first and second order). Diameter of common bile duct in patients with common duct stones was above 1.2 cm, the number of stones for each patient was more than 3 and all the biggest stones exceeded 1 cm. In the 26 patients, preoperatively, stenosis of bile duct and malignant tumour were excluded by both radiological examination and detection of serological tumour markers. RESULTS: The mean operative time was 125 min (75-190 min). Reoperations of biliary tract by laparoscope were successfully accomplished in 25 patients. One patient was converted to open operation and the common duct stones were removed by right angle forceps through short incision. None of the patients developed any severe complication, all of them recovered and were successfully discharged. Three cases with retained calculuses were successfully cured by removing these through the sinus tract of T tube. CONCLUSIONS: Laparoscopic procedure is minimally invasive, safe and feasible for laparoscopic experts in case of reoperation of biliary tract. It is also a first method for patients for whom endoscopic sphincterotomy is contraindicated.

Intraepithelial Attack Rather than Intratumorally Infiltration of CD8+T Lymphocytes is a Favorable Prognostic Indicator in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) are one of the major participants in the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC). However, the mechanism of interaction between TILs and tumors is complex and remains unclear. OBJECTIVE: To evaluate the state of immunoreactions in PDAC tissues, and explore the prognostic value of these markers in a large sample, to provide a new theoretical basis for PDAC immunotherapy. METHOD: Immunohistochemical staining of CD4+ and CD8+T cells was performed in a tissue microarray (TMA) of 143 cases of PDAC. Two major variables for the spatial distributions of CD4+T and CD8+T cells in PDAC tissues, intraepithelial attack and intratumoral infiltration, were used to evaluate the state of immunoreactions, and the interrelationships with the clinicopathological variables were analyzed. RESULTS: Our data showed that both the intraepithelial CD4+T and CD8+T attack were less frequent than the intratumoral infiltration. CD8+T intraepithelial attack and intratumoral infiltration were more intense than CD4+T. CD8+T intraepithelial attack was an independent favorable prognostic factor for overall survival, correlating negatively with vascular invasion and positively with CD4+T and CD8+T high intratumoral infiltration. CD8+T high intratumoral infiltration without CD8+T intraepithelial attack was a poor prognostic factor. CD8+T high intratumoral infiltration was accompanied by T stage progression. Conclusively, in PDAC progression, imbalances of T cells occurred in CD4+ and CD8+ immunoreactions. The CD8+T intraepithelial attack was an independent favorable prognostic indicator, however the intraepithelial attack of CD4+T and the both intratumoral infiltration of CD8+T and CD4+T played an ambiguous role. CONCLUSION: Our data suggested that it is a potential approach to increasing the number of intraepithelial attacking CD8+T cells for tumor immunotherapy, and exploring a new mechanism for immunosuppression in a tumor microenvironment with high T cell infiltration without attack.

Enhanced dissipation of phenanthrene and pyrene in spiked soils by combined plants cultivation.

Polycyclic aromatic hydrocarbons (PAHs), a class of POPs, are widely distributed in the environment. Phytoremediation has long been recognized as a cost-effective method for removal of PAHs pollutants from soil. This study was conducted to investigate the capability of three plant species separately and their combination to promote the degradation of phenanthrene and pyrene in soil. The performance of three plant species, maize, ryegrass and white clover for phenanthrene and pyrene removal was also compared. The result showed that the presence of vegetation significantly enhances the dissipation of phenanthrene and pyrene in the soil environment. This effect was especially marked with maize. At the end of 60 days treatment, phenanthrene and pyrene concentrations in treated soils declined from an initial 52.52 mg kg-1 and 58.19 mg kg-1 to 4.15 mg kg-1 and 6.77 mg kg-1, respectively, indicating that phenanthrene and pyrene was successfully removed by maize. Around 92.10% of phenanthrene and 88.36% of pyrene were removed from soils planted with maize. Within approximately two months experimental period, the dissipation extent showed that the 4-ring pyrene was more recalcitrant than 3-ring phenanthrene. Although the extents did not differ significantly among three tested species, the rates of degradation were different. The maize treatment had the highest rate of contaminant removal after two months, followed by white clover and annual ryegrass. As compare to single plant cultivation, combined plants cultivation significantly enhanced the destruction rate and extent of phenanthrene and pyrene in soils. Around 98.22% of phenanthrene and 95.81% of pyrene were removed from soils planted with maize and ryegrass. This research indicates the potential for phenanthrene and pyrene mineralization in combined plants cultivation, which may be especially useful for phytoremediation of soils contaminated with PAHs.

CCR6 overexpression predicted advanced biological behaviors and poor prognosis in patients with gastric cancer.

BACKGROUND: CCR6 expression is deregulated in some human malignancies and may be involved in the tumor progression. The aim of the present study was to determine the CCR6 expression in gastric cancer (GC) and to clarify its clinical significance. METHODS: We used western blotting to examine CCR6 protein expression in GC tissues and matched adjacent non-tumor tissues. Immunohistochemistry was performed on a large cohort of 372 postoperative GC samples. Chi-square test, Kaplan-Meier analysis and Cox regression model were used to analyze the data. RESULTS: Upregulated CCR6 protein expression was observed in the GC tissues by western blotting compared with the adjacent non-cancerous gastric tissues. High CCR6 expression was detected in 56.5 % (210/372) samples and significantly associated with the extracapsular extension of the tumor, tumor relapse and poor overall survival in GC (P < 0.001). Further analysis demonstrated that the CCR6 expression level stratified the patient outcome in stage II, stage III, T3/4, N positive and poorly differentiated/undifferentiated tumor subgroups. The Cox regression analysis showed that high expression of CCR6 was an independent prognostic factor for GC patients. CONCLUSIONS: CCR6 expression may be a novel biomarker for predicting clinical outcomes for GC patients.

Epidemiology of prostate cancer: current status.

Prostate cancer is one of the most common cancers affecting men with > 1,100,000 new cases and 300,000 deaths worldwide each year. The disease is more common among older men, with a median age at diagnosis around age above 60 years. Prostate cancer is a major medical problem that needs immediate attention as the disease is indolent, shows prolonged latency in association with high morbidity and mortality. Administration of diagnostic tests including PSA test and biopsies and the advances in other diagnostic procedures have led to early detection of the disease with therapeutic steps being taken early on, there has been a steady decline in the disease-specific mortality. Global incidence and mortality rates show that the disease is more prevalent among black people, even though the differences cannot be attributed entirely to race, as the influence of socioeconomic situation and the resultant limited access to medical technologies and treatment could not be ruled out completely. Several genes have been identified that when mutated confer high risk for the disease. Besides the genetic factors, family history and nutritional factors such as lack of enough vitamin D, high intake of calcium, obesity and high fat diets have been implicated as risk factors for prostate cancer. Therapeutic measures for prostate cancer involve mostly radical prostatectomy followed by radiotherapy in combination with hormonal treatment as needed.

Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid:pDNA complexes.

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-dependent inflammatory response that is characterized by an influx of leukocytes and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). We have examined the contribution of plasmid DNA (pDNA) to this observed toxicity, specifically the role of unmethylated CpG dinucleotides, which have been previously shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of BALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-gamma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contrast, BALF of animals administered either GL-67 alone or GL-67 complexed with SssI-methylated pDNA contained low levels of these cytokines. Similar results were observed using a plasmid (pCF1-null) that does not express a transgene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response observed was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokines. Concomitant with this increase in cytokine levels were also elevated numbers of neutrophils in the BALF, suggesting a possible cause- and-effect relationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1alpha and LFA-1 also diminished cytokine levels. This reduction in cytokine levels in the BALF was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY, shown to be most immunostimulatory, were selectively mutagenized. However, instillation of a plasmid in which 14 of the 17 CpG sites were altered into BALF/c mice did not reduce the levels of cytokines in the BALF compared with the unmodified vector. This suggests that other unmethylated motifs, in addition to RRCGYY, may also contribute to the inflammatory response. Together, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines associated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic diseases.

Efficiency of cationic lipid-mediated transfection of polarized and differentiated airway epithelial cells in vitro and in vivo.

Systematic analysis of a large number of different cationic lipids has led to the identification of novel structures (GL-67) and formulations of cationic lipid:plasmid DNA (pDNA) complexes that facilitate high levels of gene expression in lungs of mice. However, despite significant improvement in gene transfer activity, we show here that the efficiency of GL-67-mediated gene transduction of intact airway epithelia is still relatively low. Administration of GL-67:pCF1-CFTR (encoding the cystic fibrosis transmembrane conductance regulator) complexes into the nasal epithelium of cystic fibrosis (CF) transgenic mice resulted only in marginal correction of the ion transport defects. Measurements of nasal potential differences (PD) showed no correction of the sodium (Na+) transport defect, and only partial restitution of the chloride (Cl-) transport defect was achieved in a small proportion of the animals after perfusion of the nasal epithelium with the complexes. Furthermore, in contrast to results obtained following instillation of GL-67:pDNA complexes into the lungs of mice, perfusion of GL-67:pDNA into the nasal epithelium resulted only in a moderate enhancement of gene transduction activity relative to that attained with naked pDNA alone. To determine the basis for this low efficiency of transfection, a series of studies was conducted to identify some of the barriers governing cationic lipid-mediated gene transfer to the airway epithelium. We show here that the transfection activity of GL-67 was affected by the polarization, differentiation, and proliferative state of the cells. Diminished transfection activity was observed with nonmitotic, highly polarized and differentiated airway epithelial cells. This observed reduction in gene expression with nonmitotic cells was determined to be due in part to inefficient nuclear translocation of the pDNA from the cytoplasm. Together these data indicate that much improvement in the ability of cationic lipids to transfect polarized and differentiated airway epithelial cells is a necessary prerequisite for effective cationic lipid-mediated gene therapy of airway diseases such as CF.

Optimization of plasmid vectors for high-level expression in lung epithelial cells.

Nonviral gene therapy approaches use a plasmid vector to express the desired transgene. We have systematically examined several regulatory elements within plasmid vectors that govern gene expression, e.g., the promoter, enhancer, intron, and polyadenylation signal, by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoters and polyadenylation signal sequences that were tested, the human cytomegalovirus (CMV) immediate early gene promoter and the addition of polyadenylation signal sequences from the bovine growth hormone (BGH) gene or rabbit beta-globin gene produced the highest levels of expression in vitro. The inclusion of a hybrid intron 3 to the promoter further increased expression 1.6-fold. The addition of a region of the CMV enhancer 5' to several weak promoters increased expression 8- to 67-fold, and co-transfection with a second plasmid encoding a chimeric transcription factor also enhanced expression. On the basis of these results, the CMV promoter, the hybrid intron, and the BGH polyadenylation signal were selected for consistent high level expression in vitro and in the mouse lung. However, expression was transient, with greater than 60% loss of activity in the first 7 days. This transient expression was not specific to CMV promoter-containing plasmids, because plasmids containing other heterologous promoters showed a similar profile of transient expression in vivo. These comparative analyses begin to provide a basis for the development of optimized expression plasmids for gene therapy of lung diseases.

Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung.

Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.

Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung.

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.

Acoustic pulse propagation and localization in bubbly water.

Acoustic pulse propagation in bubbly water is studied using a self-consistent method. The acoustic transmission and backscattering are evaluated numerically. Under proper conditions, the localization of acoustic waves is identified within a range of frequency. The results show that when a short pulse is transmitted the waves of frequencies within the localization regime will be trapped in the system and reveal a coherent behavior. A phase diagram approach is used to describe the localization behavior.

Advanced analytical determination of volatile organic compounds (VOC) and other major contaminants in water samples using GC-ion trap MS.

The GC-Ion Trap MS is recently one of the most efficient instrumental analysis recommended for understanding the chemistry of volatile organic compounds, not only in water but even in the food chain and other environmental media (air and soil). Results of the experiment conducted on water samples from Kuguri and Yatsutani sampling stations showed considerably higher levels of organic enrichment (COD = 10 mg/L and 11 mg/L respectively). Total concentrations of Pb (0.072 mg/L and 0.093 mg/L) and Cd (0.004 mg/L and 0.011 mg/L) on the other hand, invariably exceeded the maximum allowable concentrations for human health and the living environment (Pb = 0.005 mg/L; Cd = 0.001 mg/L respectively). And the toxicity levels for these contaminants at LC50 showed critical impact on rainbow trout (hypersensitive species) at 0.14 mg/L for Pb and 0.007 mg/L for Cd in 96 hours respectively. Although these major contaminants including phenol and 3-, 4-cresol, showed relatively higher toxicity impact in the experimental media, it would remain contentious to justify any associated potential dangers without regular routine water monitoring, at least for a period of one year. Nevertheless, the data could serve as a benchmark through which other phenomena can easily be investigated.