Recombinant adeno-associated virus-mediated in utero gene transfer gives therapeutic transgene expression in the sheep

Somatic in utero gene therapy aims to treat congenital diseases where pathology develops in perinatal life, thereby preventing permanent damage. The aim of this study was to determine whether delivery of self-complementary (sc) adeno-associated virus (AAV) vector in utero would provide therapeutic long-term transgene expression in a large animal model. We performed ultrasound-guided intraperitoneal injection of scAAV2/8-LP1-human Factor IX (hFIX)co (1 × 10(12) vector genomes/kg) in early (n = 4) or late (n = 2) gestation fetal sheep. The highest mean hFIX levels were detected 3 weeks after injection in late gestation (2,055 and 1,687.5 ng/ml, n = 2) and 3 days after injection in early gestation (435 ng/ml, n = 1). Plasma hFIX levels then dropped as fetal liver and lamb weights increased, although low levels were detected 6 months after late gestation injection (75 and 52.5 ng/ml, n = 2). The highest vector levels were detected in the fetal liver and other peritoneal organs; no vector was present in fetal gonads. hFIX mRNA was detectable only in hepatic tissues after early and late gestation injection. Liver function tests and bile acid levels were normal up to a year postnatal; there was no evidence of liver pathology. No functional antibodies to hFIX protein or AAV vector were detectable, although lambs mounted an antibody response after injection of hFIX protein and Freund's adjuvant. In conclusion, hFIX expression is detectable up to 6 months after delivery of scAAV vector to the fetal sheep using a clinically applicable method. This is the first study to show therapeutic long-term hFIX transgene expression after in utero gene transfer in a large animal model.     

重组AAV2/hFⅨ病毒制备及其基因治疗血友病B的实验研究

目的　制备携带人凝血因子Ⅸ (hFⅨ )基因的重组AAV2病毒 (rAAV2 /hFⅨ ) ,并对用rAAV2 /hFⅨ肌肉注射治疗血友病B模型小鼠的疗效进行评价。方法　通过“一株载体细胞 /一株辅助病毒”的双因素包装策略制备出rAAV2 /hFⅨ ,体外转导BHK 2 1、C2C12细胞后 ,检测细胞培养上清中hFⅨ的表达量 ;肌肉直接注射血友病B模型小鼠后 ,检测其血浆中hFⅨ的抗原水平和凝血活性等指标。结果　转导 2 4h后在细胞上清中即可检测到hFⅨ ,连续检测 12 0h都有表达 ,BHK 2 1、C2C12细胞 2 4h最高表达量分别达到 (5 1.0± 6 .5 )ng/ 10 5细胞和 (6 8.0± 7.2 )ng/ 10 5细胞。rAAV2 /hFⅨ经肌肉直接注射后 ,高、中、低三个剂量组均能检测到小鼠体内高效表达hFⅨ ,在给药后第 3周达到高峰 ,小鼠血浆中hFⅨ的表达量与对照组比较差异有显著性 (P <0 .0 1) ,之后缓慢下降 ,到第 10周仍可检测到低水平hFⅨ表达 ;取第 3周小鼠血浆样品检测凝血功能 ,高、中、低剂量组FⅨ活性均得到明显改善 ,小鼠的割尾实验出血时间明显缩短 ,5min失血量也相应显著减少 ,其中高剂量组hFⅨ最高表达量达到 (387.0± 12 .5 )ng/ml血浆 ,FⅨ活性达到正常水平的 (30 .0± 5 .5 ) % ;给药后第 10周 ,除在注射点外 ,其它主要脏器均未检测到AAV载体DNA。结论     

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 α1-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 μg/ml (normal = 5 μg/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 μg/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.     

High-level factor VIII gene expression in vivo achieved by nonviral liver-specific gene therapy vectors

Two liver-specific nonviral gene transfer vectors have been developed to accommodate heterologous genes. The expression cassettes contain (1) a hepatic locus control region from the apolipoprotein E (ApoE) gene (HCR), (2) a liver-specific alpha(1)-antitrypsin promoter (HP), (3) a 1.4-kb truncated factor IX first intron (I) or a synthetic minx intron (mI), (4) a multiple cloning site (MCS) for inserting cDNA sequences, and (5) a bovine growth hormone polyadenylation signal (bpA) to make pBS-HCRHPI-A or pBS-HCRHPmI-A. These vectors were first evaluated with reporter genes encoding human factor IX (hFIX) and green fluorescent protein (GFP). hFIX constructs, pBS-HCRHPI-FIXA and control pBS-HCRHP-FIXIA with the hFIX intron in its native position, produced comparable hFIX gene expression levels (0.5-5 microg/ml) 6 months after naked DNA transfer to mice, whereas the factor IX level from pBS-HCRHPmI-FIXA averaged about 50% lower. RT-PCR analysis of the mRNA indicated that introns inserted upstream from the cDNA were correctly processed and spliced. GFP expression was detected in 15-30% of the hepatocytes in pBS-HCRHPI-GFPA-treated mice. Next, a B domain-deleted human factor VIII (hFVIII) cDNA was inserted into the modified vectors. High-level hFVIII expression (up to 750 ng/ml) was achieved initially in both C57BL/6 mice and Rag2 mice. Moreover, therapeutic levels of hFVIII (20-310 ng/ml) circulated in Rag2 mice 6 months after treatment. These liver-specific gene expression cassettes can deliver a large, heterologous gene such as hFVIII cDNA to achieve high-level, persistent transgene expression after in vivo hepatic gene therapy.     

Induction of Immune Tolerance to FIX Following Muscular AAV Gene Transfer Is AAV-dose/FIX-level Dependent

Direct intramuscular injection (IM) of adeno-associated virus (AAV) has been proven a safe and potentially efficient procedure for gene therapy of many genetic diseases including hemophilia B. It is, however, contentious whether high antigen level induces tolerance or immunity to coagulation factor IX (FIX) following IM of AAV. We recently reported induction of FIX-specific immune tolerance by IM of AAV serotype one (AAV1) vector in mice. We hypothesize that the expression of high levels of FIX is critical to induction of FIX tolerance. In this study, we investigated the correlation among AAV dose, FIX expression, and tolerance induction. We observed that induction of immune tolerance or immunity to FIX was dependent on the dose of AAV1–human FIX (hFIX) given and the level of FIX antigen expressed in both normal and hemophilia mice. We then defined the minimum AAV1–hFIX dose and the lowest level of FIX needed for FIX tolerance. Different from hepatic AAV–hFIX gene transfer, we found that FIX tolerance induced by IM of AAV1 was not driven by regulatory T cells. These results provided further insight into the mechanism(s) of FIX tolerance, contributing to development of hemophilia gene therapy, and optimization of FIX tolerance induction protocols.     

In vitro activity of a new semisynthetic echinocandin, LY-303366, against systemic isolates of Candida species, Cryptococcus neoformans, Blastomyces dermatitidis, and Aspergillus species.

The in vitro activities of LY-303366, a new semisynthetic echinocandin, and comparators amphotericin B, 5-fluorocytosine, fluconazole, and ketoconazole against 205 systemic isolates of Candida species, Cryptococcus neoformans, Blastomyces dermatitidis, and Aspergillus species were determined. LY-303366 had MICs of < or = 0.32 microg/ml for all Candida albicans (n = 99), Candida glabrata (n = 18), and Candida tropicalis (n = 10) isolates tested. LY-303366 was also active against Aspergillus species (minimum effective concentration at which 90% of the isolates are inhibited, 0.02 microg/ml) (n = 20), was less active against Candida parapsilosis (MIC at which 90% of the isolates are inhibited [MIC90], 5.12 microg/ml) (n = 10), and was inactive against C. neoformans (MIC90, >10.24 microg/ml) (n = 15) and B. dermatitidis (MIC90, 16 microg/ml) (n = 29).     

Plasma and intracellular (peripheral blood mononuclear cells) pharmacokinetics of once-daily raltegravir (800 milligrams) in HIV-infected patients

The aim of this study was to evaluate the plasma and intracellular pharmacokinetics of raltegravir in HIV-infected patients receiving once-daily raltegravir. Five HIV-infected patients on stable therapy with lopinavir-ritonavir monotherapy whose HIV-1 RNA load was <50 copies/ml were included in this open-label, pilot study. Raltegravir was added to the antiretroviral regimen at a dose of 800 mg once daily from days 0 to 10. On day 10, a full pharmacokinetic profile was obtained for each participant. Raltegravir concentrations in plasma and peripheral blood mononuclear cells (PBMCs) were determined by high-performance liquid chromatography with a fluorescence detector and by liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. The values of the raltegravir pharmacokinetic parameters in plasma and PBMCs were calculated by noncompartmental analysis. Raltegravir was well tolerated, and all participants completed the study. No differences in the times to the maximum concentration of raltegravir in plasma or the raltegravir half-lives were observed between plasma and PBMCs. The geometric mean raltegravir maximum concentration, the concentration at the end of the dosing interval, and the area under the concentration-time curve during the dose interval in plasma versus PBMCs were 2,640 ng/ml (range, 887 to 10,605 ng/ml) versus 199 ng/ml (range, 82 to 857 ng/ml) (geometric mean ratio [GMR], 13.30; 95% confidence interval [CI], 3.11 to 56.89; P = 0.003); 89 ng/ml (range, 51 to 200 ng/ml) versus 7 ng/ml (range, 2 to 15 ng/ml) (GMR, 13.21; 95% CI, 3.94 to 44.26; P = 0.001); and 12,200 ng·h/ml (range, 5,152 to 30,130 ng·h/ml) versus 909 ng·h/ml (range, 499 to 2,189 ng·h/ml) (GMR, 13.43; 95% CI, 5.13 to 35.16; P < 0.001), respectively. Raltegravir does not accumulate in PBMCs, with intracellular concentrations being about 1/10 of the concentrations in plasma. Despite once-daily dosing, mean raltegravir concentrations at the end of the dosing interval in plasma and PBMCs exceeded the reported protein-binding-adjusted 95% inhibitory concentration (IC(95)) and IC(50) for wild-type viral strains, respectively.     

Pharmacokinetics of indinavir and nelfinavir in treatment-naive, human immunodeficiency virus-infected subjects

AIDS Clinical Trials Group protocol 388 was designed to compare a three-drug regimen (indinavir with dual nucleosides) to a four-drug regimen (indinavir plus nelfinavir or indinavir plus efavirenz with dual nucleosides). Blood samples from patients taking indinavir and nelfinavir were collected over 8 to 12 h following a specified dose and were analyzed with high-performance liquid chromatography. Pharmacokinetic data were derived by using noncompartmental analysis. Following administration of indinavir every 8 h in the absence of nelfinavir (n = 8), the median predose indinavir concentration (C(0)) was 369 ng/ml (range, <10 to 949 ng/ml; one subject had a concentration of <10 ng/ml), and the concentration 8 h after administration of the study dose was 159 ng/ml (range, 85 to 506 ng/ml). In the group receiving 1000 mg of indinavir every 12 h with nelfinavir (n = 10), the median indinavir C(0) was <10 ng/ml (range, <10 to 3740 ng/ml; six subjects had a value of <10 ng/ml), and the C(12 h) was 44 ng/ml (range, <10 to 4236 ng/ml; five subjects had a value of <10 ng/ml), while the subjects who received 1200 mg of indinavir every 12 h with nelfinavir (n = 7) had a C(0) of 146 ng/ml (range, 58 to 5215 ng/ml) and a C(12 h) of 95 ng/ml (range, 12 to 954 ng/ml). Indinavir clearance was significantly lower in the presence of nelfinavir (median [interquartile range], 34.1 liters/h [range, 22.6 to 45.8 liters/h] versus 47.9 liters/h [range, 42.7 to 70.3 liters/h]; P < 0.017). For subjects receiving 1,000 mg of indinavir every 12 h, the median C(0) value for nelfinavir (n = 9) was 1,779 ng/ml (range, <187.5 to 4579 ng/ml), and the C(12 h) was 1554 ng/ml (range, <187.5 to 5,540 ng/ml). Due to the unacceptable number of undetectable indinavir trough concentrations, 1200 mg of indinavir appears to be the preferred dose in a twice-daily regimen that includes nelfinavir.     

Eliminating bacteria backbone of naked DNA enhanced hFIX expression and reduced inflammatory response in mice

To test the hypothesis that the persistent high level of transgene expression of linear DNA eliminating bacterial backbone (LDEBB) results from less cytokine induction in vivo. We systematically investigated the effect of circular DNA (C DNA), linear DNA (L DNA) and LDEBB on gene expression in mice by hydrodynamics-based plasmid administration, and then determined serum cytokine levels in mice by enzyme linked immunosorbent assay (ELISA). The expression of human clotting factor IX (hFIX) gene in mice treated with LDEBB, L DNA or C DNA reached a maximum 1-day after injection (9809, 6447, 2368 ng/mL), respectively. Thirty days after injection, hFIX concentrations dropped to baseline in mice treated with C DNA group, while L DNA group and LDEBB group decreased to 207 and 377 ng/mL, respectively, at the same time-point. Mice receiving LDEBB encoding hFIX expressed approximately 1.5 to 20-fold more serum hFIX than mice injected with L DNA and C DNA for a period of 8 months, respectively. However, mice receiving LDEBB are much less inflammatory than L DNA and C DNA as shown by a 4-fold reduction in serum levels of both TNF-α and IL-12. These results demonstrate that LDEBB is not silenced and is capable of expressing persistently high levels of transgene in vivo, which result from less cytokine induction in vivo. LDEBB provides a promising approach and useful therapeutic strategy to improve naked DNA delivery.     

Cathelicidin peptides inhibit multiply antibiotic-resistant pathogens from patients with cystic fibrosis

Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease. The bactericidal activities of five cathelicidin peptides (LL37 [human], CAP18 [rabbit], mCRAMP [mouse], rCRAMP [rat], and SMAP29 [sheep]), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays. Their MICs were determined for multiply antibiotic-resistant Pseudomonas aeruginosa (n = 24), Burkholderia cepacia (n = 5), Achromobacter xylosoxidans (n = 5), and Stenotrophomonas maltophilia (n = 5) strains isolated from CF patients. SMAP29 was most active and inhibited mucoid and nonmucoid P. aeruginosa strains (MIC, 0.06 to 8 microg/ml). OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 microg/ml), but CAP18 (MIC, 1.0 to 32 microg/ml), CAP18-18 (MIC, 1.0 to >32 microg/ml), and CAP18-22 (MIC, 0.5 to 32 microg/ml) had variable activities. LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 microg/ml). Peptides had modest activities against S. maltophilia and A. xylosoxidans (MIC range, 1.0 to > 32 microg/ml), but none inhibited B. cepacia. However, CF sputum inhibited the activity of SMAP29 substantially. The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively. SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed P. aeruginosa within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h. The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.     

Efficient induction of immune tolerance to coagulation factor IX following direct intramuscular gene transfer

BACKGROUND: The formation of inhibitory anti-factor IX (anti-FIX) antibodies is a major complication of FIX protein replacement-based treatment for hemophilia B. It is difficult to treat patients with anti-FIX antibodies. Gene therapy is emerging as a potentially effective treatment for hemophilia. Direct i.m. injection of adeno-associated virus (AAV) is a safe and efficient procedure for hemophilia B gene therapy. However, the development of anti-FIX antibodies following i.m. of AAV may impede its application to patients. OBJECTIVE: We aimed to investigate induction of immune tolerance to human FIX (hFIX) by i.m. of AAV1, further validating i.m. of AAV1 for hemophilia B gene therapy. METHODS AND RESULTS: Cohorts of hemostatically normal and hemophilia B mice with diverse genetic and MHC backgrounds received i.m. of AAV-hFIX. Human FIX antigen and anti-hFIX antibodies were examined. I.m. of 1 x 10(11) vector genomes (VG) of AAV2 elicits formation of anti-hFIX antibodies comparable to those by hFIX protein replacement. I.m. of 1 x 10(11) VG of AAV1 results in expression of therapeutic levels of hFIX (up to 950 ng mL(-1), mean = 772 ng mL(-1), SEM +/- 35.7) and hFIX-specific immune tolerance in C57BL/6 mice. CONCLUSIONS: A single i.m. of AAV1 can result in efficient expression of therapeutic levels of hFIX and induction of hFIX tolerance in hemostatically normal and hemophilic B mice. Our results substantiate the prospect of i.m. of AAV1 for hemophilia B gene therapy and FIX tolerance induction.     

Clinically applicable procedure for gene delivery to fetal gut by ultrasound-guided gastric injection: toward prenatal prevention of early-onset intestinal diseases

Targeting gene therapy vectors to the fetal intestinal tract could provide a novel means toward prevention of the early postnatal intestinal pathology of cystic fibrosis and other conditions, such as congenital enteropathy, that cause intestinal failure. Among these conditions, cystic fibrosis is by far the most common lethal genetic disease. It is caused by a functional absence or deficiency of the cystic fibrosis transmembrane conductance regulator and manifests in the gut as meconium ileus. Prenatal treatment of genetic disease may avoid early-onset tissue damage and immune sensitization, and may target cells that are less accessible in the adult. We investigated gene transfer to the fetal gut, using a minimally invasive injection technique. First-generation replication-deficient adenoviral vectors encoding the beta-galactosidase gene and transduction-enhancing agents were injected into the stomach of early-gestation fetal sheep (n = 8, 60 days of gestation; term, 145 days) under ultrasound guidance. Reporter gene expression was observed 2 days after injection in the villi of the gastrointestinal epithelia after 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining and beta-galactosidase immunohistochemistry of fetal tissues. Expression of beta-galactosidase, as measured by enzyme-linked immunosorbent assay, was enhanced after pretreatment of the fetal gut with sodium caprate, which opens tight junctions, and after adenovirus complexation with DEAE-dextran, which confers a positive charge to the virus. Instillation of the fluorocarbon perflubron after virus delivery resulted in tissue transduction from the fetal stomach to the colon. Using a clinically relevant technique, we have demonstrated widespread gene transfer to the fetal gastrointestinal epithelia.     

Simple in vitro assay for determining the sensitivity of Plasmodium vivax isolates from fresh human blood to antimalarials in areas where P. vivax is endemic

The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P. falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB(+) serum. We successfully carried out 34 in vitro drug assays on 39 P. vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean percentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% +/- 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P. vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC(50)] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5 ng/ml, IC(50) = 0.47 ng/ml). The poor response of P. vivax to both tafenoquine (MIC = 14,000 ng/ml, IC(50) = 9,739 ng/ml) and sulfadoxine (MIC = 500,000 ng/ml, IC(50) = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P. vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assessing the antimalarial sensitivity of P. vivax populations and for screening new antimalarials in the absence of long-term P. vivax cultures.     

Antiherpesvirus activity of 9-(4-hydroxy-3-hydroxy-methylbut-1-yl)guanine (BRL 39123) in cell culture.

The activity of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) against several herpesviruses was compared with that of acyclovir (ACV). In plaque reduction tests with clinical isolates of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus, mean 50% inhibitory concentrations (IC50S) (n = number tested) for BRL 39123 were 0.4 (n = 17), 1.5 (n = 13), and 3.1 (n = 5) micrograms/ml, respectively. Corresponding IC50S for ACV were 0.2, 0.6, and 3.8 micrograms/ml. Cytomegalovirus was relatively resistant to BRL 39123 (IC50, 51 micrograms/ml), but equid herpesvirus 1, bovid herpesvirus 2, and felid herpesvirus 1 were susceptible (IC50S, 1.6, 1.2, and 0.9 micrograms/ml, respectively). BRL 39123 was inactive against an HSV-1 strain which does not express thymidine kinase activity, but a DNA polymerase mutant selected for resistance to ACV was sensitive to BRL 39123 (IC50, 1.5 micrograms/ml). In contrast to the results from plaque reduction tests, BRL 39123 was more active than ACV against HSV-1 and of equal activity against HSV-2 in virus yield reduction assays in MRC-5 cells. After treatment of HSV-infected cultures for short periods, BRL 39123 was considerably more effective than ACV at reducing virus replication, and furthermore, after removal of extracellular BRL 39123, virus replication remained depressed for long periods, whereas such persistent activity was not observed with ACV. Neither compound significantly affected MRC-5 cell replication at 100 micrograms/ml, but at 300 micrograms/ml BRL 39123 was more inhibitory than ACV.     

High-level mupirocin resistance in Staphylococcus aureus: evidence for two distinct isoleucyl-tRNA synthetases.

Mupirocin resistance in Staphylococcus aureus results from changes in the target enzyme, isoleucyl-tRNA synthetase (IRS). Twelve strains of S. aureus comprising four susceptible (MICs < or = 4 micrograms/ml), four intermediate level-resistant (MICs between 8 and 256 micrograms/ml), and four highly resistant (MICs > or = 512 micrograms/ml) isolates were examined for their IRS content and the presence of a gene known to encode high-level mupirocin resistance. Ion-exchange chromatography of cell extracts showed a single IRS active peak in mupirocin-susceptible strains, with 50% inhibitory concentrations (IC50s) of 0.7 to 3.0 ng of mupirocin per ml. In strains showing intermediate mupirocin resistance, similar single IRS activity peaks were observed, but these were less sensitive to inhibition, and the mupirocin IC50s for them were 19 to 43 ng/ml. Strains that were highly resistant to mupirocin displayed two distinct peaks; one was similar to that found with susceptible strains (IC50, 0.9 to 2.5 ng/ml), but an additional peak with an IC50 of 7,000 to 10,000 ng/ml was also observed. A strain cured of the plasmid encoding high-level mupirocin resistance lacked the resistant IRS peak. Restriction digests, produced by endonuclease NcoI, of total bacterial DNA isolated from the highly resistant strains hybridized with a mupirocin resistance gene probe, whereas DNA isolated from the intermediate level-resistant and susceptible strains did not. These results demonstrate that two different IRS enzymes were present in highly mupirocin-resistant S. aureus strains. In strains expressing intermediate levels of resistance, only a chromosomally encoded IRS which was inhibited less by mupirocin than IRS from fully susceptible strains was detected.     

Local delivery of VEGF adenovirus to the uterine artery increases vasorelaxation and uterine blood flow in the pregnant sheep

Impaired materno-placental perfusion causes two important obstetric complications, fetal growth restriction and preeclampsia. This study investigated whether adenoviral vector-mediated overexpression of vascular endothelial growth factor (VEGF) in the uterine arteries (UtAs) increases uterine artery blood flow (UBF). First-generation adenovirus vectors (5 x 10(11) particles) containing the VEGF gene (Ad.VEGF-A or -D) or the beta-galactosidase reporter gene (Ad.lacZ) were injected into the UtAs of pregnant sheep (n=6) at 88-102 days of gestation (term=145 days). UBF was measured using Doppler sonography before, and 4-7 days after injection. Mean UBF increased significantly from 233+/-156 (s.d.) ml min(-1) to 753+/-415 ml min(-1) following Ad.VEGF-A injection (P=0.005, n=5); Ad.lacZ infection had no significant effect. Organ bath experiments on uterine arterial sections 4-7 days after injection showed that, compared with Ad.lacZ vessels, Ad.VEGF-A-transduced vessels had a reduced contractile response to phenylephrine (E max 148+/-10.9 vs E max 228.2+/-27.5, P<0.05) but increased relaxation with bradykinin (pD2 (-log EC50) values 9.11+/-0.01 vs 8.65+/-0.11, P<0.05). Injection of Ad.VEGF-A into the UtAs increases UBF by enhancing vasodilatation. This may provide the basis for therapy in pregnancies complicated by uteroplacental insufficiency.     

Vitreous levels of vascular cell adhesion molecule and vascular endothelial growth factor in patients with proliferative diabetic retinopathy: a case-control study

OBJECTIVE: To evaluate the intravitreous concentration of vascular cell adhesion molecule (VCAM)-1 in diabetic patients with proliferative diabetic retinopathy (PDR) and the relationship of VCAM-1 with vascular endothelial growth factor (VEGF). RESEARCH DESIGN AND METHODS: Serum and vitreous fluid samples were obtained simultaneously at the onset of vitrectomy from 20 diabetic patients with PDR and 20 nondiabetic control subjects with nonproliferative ocular disease. Both groups were matched by serum levels of VCGM-1 and VEGF. VCAM-1 and VEGF were determined by enzyme-linked immunosorbent assay. Statistics were determined using the Mann-Whitney U test and Spearman's rank correlation test. RESULTS: The intravitreous concentration of VCAM-1 was signifcantly elevated in diabetic patients with PDR compared with control subjects (26 ng/ml [19-118] vs. 22 ng/ml [20-47], P < 0.05). A direct correlation between VCAM-1 and total vitreous proteins was detected in diabetic patients (r = 0.64, P = 0.003), but not in control subjects. After adjusting for total intravitreous proteins, VCAM-1 was significantly lower in diabetic patients with PDR than in control subjects (8.2 ng/ml [4-31.4] vs. 43.1 ng/ml [9.7-100], P < 0.001). Intravitreous VEGF concentrations were higher in patients with PDR than in control subjects in absolute terms (1.34 ng/ml [0.16-6.22] vs. 0.009 ng/ml [0.009-0.044], P < 0.0001) and after correcting for total vitreal proteins (0.33 ng/ml [0.01-2.3] vs. 0.013 ng/ml [0.003-0.035], P = 0.0001). Finally, the vitreous ratio of VCAM-1 to proteins correlated with the vitreous ratio of VEGF to proteins in both diabetic patients (r = 0.74, P = 0.001) and control subjects (r = 0.84, P = 0.005). CONCLUSIONS: The low proportion of VCAM-1 in relation to total vitreal proteins observed in diabetic patients with PDR suggests that VCAM-1 is quenched by diabetic retina. In addition, the direct correlation detected between VCAM-1 and VEGF suggests that cellular adhesion and neovascularization may be linked processes.     

单孔胸腔镜与两孔胸腔镜手术治疗非小细胞肺癌的手术相关指标和实验室指标比较

目的比较单孔胸腔镜与两孔胸腔镜手术治疗非小细胞肺癌的临床效果,并比较两种手术的手术相关指标和实验室指标。方法采用回顾性研究方法选取2017年1月至2019年1月广西医科大学第一临床医学院心胸外科收治的105例非小细胞肺癌(NSCLC)患者作为研究对象,根据手术方式不同将患者分为两组:观察组(n=48)和对照组(n=57)。观察组患者行单孔胸腔镜手术,对照组患者行两孔胸腔镜手术。观察并比较两组患者的手术相关指标(手术成功率、术中中转开胸率、术中淋巴结清扫数量、手术时间、术中出血量、术后拔管时间、下床活动时间、术后3 d VAS评分)、实验室指标[血清癌胚抗原(CEA)、糖类抗原125(CA125)、鳞状细胞癌抗原(SCC-Ag)、白细胞介素-1(IL-1)、降钙素原(PCT)、C反应蛋白(CRP)、P物质(SP)、前列腺素E2、去甲肾上腺素(NE)]和术中并发症发生情况。结果对照组与观察组患者的手术成功率(96.49%vs.97.92%)、术中中转开胸率(3.51%vs.2.08%)、术中淋巴结清扫数量[(16.02±6.54)个vs.(17.52±5.24)个]比较,差异均无统计学意义(P>0.05);观察组患者的手术时间[(194.62±40.58)min vs.(157.62±33.62)min]、术中出血量[(114.65±25.94)ml vs.(89.57±22.41)ml]、术后拔管时间[(5.38±1.93)d vs.(3.47±1.07)d]、下床活动时间[(6.22±1.57)d vs.(4.99±1.72)d]、术后3 d VAS评分[(4.86±1.37)分vs.(3.04±0.57)分]显著优于对照组,差异具有统计学意义(P<0.05);两组患者的血清癌胚抗原(CEA)[(6.24±0.75)ng/ml vs.(6.16±0.71)ng/ml]、糖类抗原125(CA125)[(31.25±4.21)U/ml vs.(30.27±4.92)U/ml]、鳞状细胞癌抗原(SCC-Ag)[(1.25±0.42)ng/ml vs.(1.24±0.39)ng/ml]比较,差异均无统计学意义(P>0.05);观察组患者的血清白细胞介素-1(IL-1)[(24.58±5.23)ng/L vs.(10.37±1.24)ng/L]、降钙素原(PCT)[(0.88±0.24)ng/ml vs.(0.41±0.12)ng/ml]、C反应蛋白(CRP)[(19.65±2.73)mg/L vs.(11.25±2.04)mg/L]显著低于对照组,差异均具有统计学意义(P<0.05);观察组患者的P物质(SP)[(7.63±1.29)μg/ml vs.(18.24±3.24)μg/ml]、前列腺素E2(PGE2)[(146.7±24.93)pg/ml vs.(259.37±34.62)pg/ml]、去甲肾上腺素(NE)[(154.27±32.55)ng/L vs.(268.55±40.93)ng/L]显著低于对照组,差异均具有统计学意义(P<0.05);两组患者的肺不张、肺部感染、持续液气胸、房颤发生率比较,差异均无统计学意义(P>0.05)。结论单孔胸腔镜与两孔胸腔镜手术治疗非小细胞肺癌的效果相似但前者对患者的创伤程度更小,疼痛应激和炎症反应更轻,更有利于患者的术后康复。