Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity.

Biosensors with high sensitivity and short time-to-result that are capable of detecting biomarkers in body fluids such as serum are an important prerequisite for early diagnostics in modern healthcare provision. Here, we report the development of an electrochemical impedance-based sensor for the detection in serum of human interleukin-8 (IL-8), a pro-angiogenic chemokine implicated in a wide range of inflammatory diseases. The sensor employs a small and robust synthetic non-antibody capture protein based on a cystatin scaffold that displays high affinity for human IL-8 with a KD of 35 ± 10 nM and excellent ligand specificity. The change in the phase of the electrochemical impedance from the serum baseline, ∆θ(ƒ), measured at 0.1 Hz, was used as the measure for quantifying IL-8 concentration in the fluid. Optimal sensor signal was observed after 15 min incubation, and the sensor exhibited a linear response versus logarithm of IL-8 concentration from 900 fg/ml to 900 ng/ml. A detection limit of around 90 fg/ml, which is significantly lower than the basal clinical levels of 5-10 pg/ml, was observed. Our results are significant for the development of point-of-care and early diagnostics where high sensitivity and short time-to-results are essential.

Recent advances in designs, approaches and materials in total knee replacement: literature review and evidence today.

Ever since Themistocles Gluck described the use of an ivory cup as a tibial hemiarthroplasty in 1894, knee arthoplasty has continued to evolve. Both human ingenuity and intensive clinical research has led to an improved understanding of biomaterials and knee kinematics, resulting in the modern total knee replacement which has enjoyed such a clinical and commercial success. As it increases in popularity, attempts to improve knee arthroplasty have been driven by demands for improved function and implant survival, particularly in younger, more demanding patients. Research continues to see if advances in implant instrumentation, materials and design will translate into improved clinical outcomes and longevity.

Results of contralateral total knee arthroplasty in patients with a history of stiff total knee arthroplasty.

This study seeks to evaluate the clinical outcomes of a second primary total knee arthroplasty in patients whose initial (contralateral) primary total knee arthroplasty was complicated by stiffness. We retrospectively compared the preoperative and postoperative range of motion and Knee Society Scores from a study group of 15 patients with an age-matched control group. Statistical analysis did not reveal a significant difference in final postoperative range of motion or Knee Society Scores between the 2 groups. However, there was a statistically significant higher rate of closed manipulation in the study group. Therefore, although the study group did show a higher rate of early stiffness, eventual functional outcome was comparable with a nonstiffness control group.

Changes in cecal microbial metabolism of rats induced by individual and a mixture of drinking water disinfection by-products.

Disinfection of drinking water has been one of the greatest public health successes. Numerous halogenated disinfection by-products (DBPs) occur and chronic ingestion has been associated with an increased risk for colorectal cancer in human populations. Because the intestinal microbiota can bioactivate xenobiotics, studies have been performed to examine the effects of individual DBPs on intestinal microbial metabolism. No studies have been conducted on a defined mixture of DBPs to determine if there is an enhancement of response to a mixture. Ten-week-old male Long-Evans rats were treated in their drinking water for 17 weeks with 0.4 g/l potassium bromate, 1.8 g/l chloroform, 0.7 g/l bromodichloromethane (BDCM), 0.07 g/l 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), or a mixture of the four chemicals or distilled water. Cecal nitroreductase (NR), azoreductase (AR), dechlorinase (DC), beta-glucuronidase (GLR), beta-galactosidase (GAL), and beta-glucosidase (GLU) were assayed. No change in GLU or GLR activity was detected after treatment. BDCM treatment reduced DC and GAL activities and elevated NR and AR activity. GAL, AR, and NR activities were significantly different after treatment with bromate, chloroform, BDCM, and MX, but not the mixture. DC activity after chloroform-, MX-, or BDCM-treatment was significantly below control levels. The present study shows that changes in intestinal microbial metabolism do occur after treatment with individual and a mixture of DBPs but the changes were not additive in the mixture group.

Standardized mortality ratios and fatal pulmonary embolism rates following total knee replacement: a cohort of 936 consecutive cases.

Mortality and fatal pulmonary embolism rates in 936 consecutive primary total knee replacements (TKR) were determined during a 3-month postoperative period. Postmortem examinations verified the cause of death in all but 3 patients, and follow-up was performed on all but 1 patient. All patients had elastic stockings as mechanical prophylaxis. No deaths occurred from pulmonary embolism confirmed by postmortem examinations. At worst, the fatal pulmonary embolism rate was 0.43% (4/936; confidence interval [CI]=0.14%-1.17%). The all-cause mortality rate was 0.64% (6/936; CI=0.26%-1.46%). The patient mortality was compared with the population mortality of England and Wales using standardized mortality ratios. The standardized mortality ratios for both sexes combined was 0.74 (CI=0.29-1.52). A lower mortality was observed in women (0.67) than in men (0.84) during the first 3 postoperative months compared to the general population. Fatal pulmonary embolism after TKR with the routine use of graded elastic stockings and early mobilization is rare. In this series, the death rate in patients undergoing TKR appears to be lower than that in the general population.

Nitric oxide synthase (nNOS) gene transfer modifies venous bypass graft remodeling: effects on vascular smooth muscle cell differentiation and superoxide production.

BACKGROUND: Pathological vascular remodeling in venous bypass grafts (VGs) results in smooth muscle cell (SMC) intimal hyperplasia and provides the substrate for progressive atherosclerosis, the principal cause of late VG failure. Nitric oxide (NO) bioactivity is reduced in VGs, in association with increased vascular superoxide production, but how these features relate to pathological VG remodeling remains unclear. We used gene transfer of the neuronal isoform of nitric oxide synthase (nNOS) to investigate how increased NO production modulates vascular remodeling in VGs and determined the effects on late VG phenotype. METHODS AND RESULTS: New Zealand White rabbits (n=60) underwent jugular-carotid interposition bypass graft surgery with intraoperative adenoviral gene transfer of nNOS or beta-galactosidase. Vessels were analyzed after 3 days (early, to investigate acute injury/inflammation) or 28 days (late, to investigate SMC intimal hyperplasia). In early VGs, nNOS gene transfer significantly increased NOS activity and substantially reduced adhesion molecule expression and inflammatory cell infiltration. In late VGs, recombinant nNOS protein was no longer evident, but there were sustained effects on VG remodeling, resulting in a striking reduction in SMC intimal hyperplasia, a more differentiated intimal SMC phenotype, and reduced vascular superoxide production. CONCLUSIONS: Intraoperative nNOS gene transfer has sustained favorable effects on VG remodeling and on the vascular phenotype of mature VGs. These findings suggest that early, transient modification of the response to vascular injury is a powerful approach to modulate VG biology and highlight the potential utility of NOS gene transfer as a therapeutic strategy in VGs.

Oral treatment of Fischer 344 rats with weathered crude oil and a dispersant influences intestinal metabolism and microbiota.

When oil is spilled into aquatic systems, chemical dispersants frequently are applied to enhance emulsification and biological availability. In this study, a mammalian model system was used to determine the effect of Bonnie Light Nigerian crude oil, weathered for 2 d with continuous spraying and recirculation, and a widely used dispersant, Corexit (Cx) 9527, on intestinal microbial metabolism and associated populations. To determine the subchronic dose, concentrated or diluted (1:2, 1:5, 1:10, 1:20) Cx9527 or oil was administered by gavage to Fischer 344 rats and the effect on body weight was determined. Next, rats were treated for 5 wk with oil, dispersant, or dispersant + oil. Body and tissue weights, urine mutagenicity, and the impact on the intestinal microflora and three microbial intestinal enzymes linked to bioactivation were determined in the small and large intestines and cecum. Two tested dispersants, Cx9527 and Cx9500, were toxic in vitro (1:1,000 dilution), and oil was not mutagenic in strains TA98 and TA100(+/-S9). None of the treated rats produced urine mutagens detected by TA98 or TA100. Undiluted dispersant was lethal to rats, and weight changes were observed depending on the dilution, whereas oil generally was not toxic. In the 5-wk study, body and tissue weights were unaffected at the doses administered. Small-intestinal levels of azoreductase (AR), beta-glucuronidase (BG), and nitroreductase (NR) were considerably lower than cecal and large-intestinal activities at the same time point. A temporal increase in AR activity was observed in control animals in the 3 tissues examined, and large-intestinal BG activity was elevated in 3-wk controls. No significant changes in cecal BG activity were observed. Oil- or dispersant-treated rats had mixed results with reduced activity at 3 wk and elevated activity at 5 wk compared to controls. However, when the dispersant was combined with oil at 3 wk, a reduction in activity was observed that was similar to that of dispersant alone. One-week nitroreductase activity in the small intestine and cecum was unaffected in the three treatment groups, but elevated activity was observed in the large intestines of animals treated with oil or dispersant. The effect of the combination dose was not significantly different from the control value. Due to experimental error, no 3- or 5-wk NR data were available. By 5 wk of treatment, enterobacteria and enterococci were eliminated from ceca of oil-treated rats. When oil was administered in combination with dispersant, an apparent protective effect was observed on the enterococci and lactose-fermenting and nonfermenting enterobacteria. A more detailed analysis at the species level revealed qualitative differences dependent on the treatment. This study suggests that prolonged exposure of mammals to oil, dispersant, or in combination impacts intestinal metabolism, which ultimately could lead to altered detoxification of oil constituents and coexposed toxicants.

Improved adenoviral vector for vascular gene therapy : beneficial effects on vascular function and inflammation.

First-generation, E1-deleted adenoviral vectors (E1-AV) can transduce the vascular endothelium with high efficiency, but their use is limited by the resulting acute endothelial injury and the long-term development of intimal hyperplasia. To reduce the impact of viral proteins on the gene-modified cells, a second-generation adenoviral vector with an additional pair of deletions in the E4 region was developed. To determine whether this E1/E4-AV vector would be useful for vascular gene transfer, we directly compared the efficiency of gene transfer to uninjured rabbit carotid arteries using either an E1/E4-AV or an E1-AV vector encoding beta-galactosidase. Both vectors efficiently transduced vascular endothelium; however, the E1/E4-AV vector gene-modified vessels showed higher beta-galactosidase expression 10 days after gene transfer. Importantly, the E1/E4-AV vector produced substantially less endothelial cell activation, less inflammation, and reduced neointimal hyperplasia compared with the E1-AV vector-treated vessels. The E1-AV vector-transduced vessels also demonstrated significantly impaired endothelium-dependent relaxation whereas the E1/E4-AV vector did not impact vasomotor function, even at doses of virus in 5-fold excess of the amount required for >90% transduction of the endothelium. We conclude that the E1/E4-AV vector is superior to the E1-AV vector for vascular gene therapy because of the prolonged transgene expression, reduced vascular inflammation, reduced intimal hyperplasia, and maintenance of normal vasomotor function.

Impacts of aging on in vivo and in vitro measurements of soil-bound polycyclic aromatic hydrocarbon availability.

Ingestion of contaminated soil is an exposure pathway at approximately one-half of the Superfund sites in the United States. This study was designed to evaluate the impacts of aging in soil on the availability of polycyclic aromatic hydrocarbons (PAHs). Two coal tar (CT)-amended soils were prepared. One was aged for 270 days and the other was not aged. Both of these treatments were incorporated into pellets and fed to male Fischer 344 rats. Excretion of 1-hydroxypyrene (1-OHP) in urine and PAH concentrations in the liver were monitored as end points. Additionally, soil:water partitioning and desorption were measured as comparisons to the in vivo results. After 5 days of ingesting their respective treatments, rats in the aged soil group excreted 4.41 +/- 1.67 ppm 1-OHP/mg of pyrene ingested while rats in the unaged soil group excreted 5.27 +/- 1.37 ppm/mg of pyrene ingested. Animals fed aged CT soil had 0.051 +/- 0.011 ppm carcinogenic PAHs in livers/mg ingested while rats fed unaged CT soil had 0.063 +/- 0.037 ppm carcinogenic PAHs in livers/mg ingested. Partitioning and desorption results revealed a similar results. These results indicate that, at high application rates, soil contact time may not play as significant a role in determining availability as simple dispersion and sorption on soil.

Pentachlorophenol potentiates benzo[a]pyrene DNA adduct formation in adult but not infant B6C3F1 male mice.

The objective of this study is to determine whether pentachlorophenol (PCP) alters benzo[a]pyrene (B[a]P)-induced DNA adduct formation in infant and adult B6C3F1 male mice. Mice were exposed intraperitoneally to 55 microg B[a]P/g body weight (BW) alone and in combination with several doses of PCP in DMSO. The 32P-postlabeling assay was used to analyze for (+/-) anti-7,8-diol-9,10-epoxide-B[a]P-N(2)deoxyguanosine (BPDE-N(2)G) adducts formed in liver and lung DNA. Hepatic DNA also was analyzed for 8-hydroxy-2'-deoxyguanosine (8-OHdG) base damage in mice exposed to PCP. 8-OHdG was not detected at any dose of PCP in infant or adult mice. PCP exhibited an antagonistic effect on BPDE-N(2)G accumulation in infant mice exposed to B[a]P in combination with 50 microg PCP/g BW at both 12 and 24 hr. Comparatively, BPDE-N(2)G adducts were increased in adult mice exposed to binary mixtures at 24 hr in both hepatic and lung DNA (P < 0.05). Multiple comparison analysis between infant and adult mice revealed that adduct levels in infants exposed to B[a]P alone or in combination with PCP were not different from those observed in adult mice exposed to B[a]P. However, a significant increase in adducts was observed in adult mice exposed to a combination of B[a]P and PCP compared to that in all other treatment groups (P < 0.05). These results suggest that PCP alters the metabolism of B[a]P in both infant and adult mice through different mechanisms, and that infants are not susceptible to the potentiating effects of PCP observed in adult mice.

Metabolism, microflora effects, and genotoxicity in haloacetic acid-treated cultures of rat cecal microbiota.

Haloacetic acids are by-products of drinking water disinfection. Several compounds in this class are genotoxic and have been identified as rodent hepatocarcinogens. Enzymes produced by the normal intestinal bacteria can transform some promutagens and procarcinogens to their biologically active forms. The present study was designed to investigate the influence of the cecal microbiota on the mutagenicity of haloacetic acids, and to look at changes in the microbiota populations and enzyme activities associated with exposure to haloacetic acids. PYG medium containing 1 mg/ml of monochloroacetic (MCA), monobromoacetic (MBA), dichloroacetic (DCA), dibromoacetic (DBA), trichloroacetic (TCA), tribromoacetic (TBA), or bromochloroacetic (BCA) acid was inoculated with rat cecal homogenate and incubated anaerobically at 37 degrees C. Growth curves were performed with enumeration of the microflora populations on selective media. Mutagenicity in a Salmonella microsuspension bioassay was determined after incubation for various lengths of time, with or without the cecal microbiota. At 15 h of incubation, enzyme assays determined the activities for beta-glucuronidase, beta-galactosidase, beta-glucosidase, azoreductase, nitroreductase, dechlorinase, and dehydrochlorinase. The haloacetic acids, with the exception of BCA, were toxic to the cecal microbiota, and especially to the enterococci. DBA, TBA, and BCA were mutagenic in the microsuspension assay, but the presence of the intestinal flora did not significantly alter the mutagenicity. BCA increased the activities of several enzymes, and therefore has the potential to affect the biotransformation of co-exposed compounds.

p27-p16 Chimera: a superior antiproliferative for the prevention of neointimal hyperplasia.

Cyclin-dependent kinase inhibitors (CDKi's) may be useful to treat hyperproliferative vascular disorders, such as restenosis induced following angioplasty or vein engraftment. We have shown that a novel fusion protein of the CDKi's p27 and p16, named W9, significantly reduces proliferation of human coronary smooth muscle cells in vitro, by blocking cell proliferation without inducing apoptosis. We have now evaluated the efficacy of adenovirus-mediated gene transfer of W9 (AV-W9) in a balloon-injury model, in the carotid arteries of cholesterol-fed rabbits. We observed that intravascular delivery of 2 x 10(11) viral particles of AV-W9 3 days following balloon injury inhibited intimal hyperplasia by 60% compared to a control virus (P > 0.001). PCNA expression in the AV-W9-treated vessels, a marker of injury-induced cell proliferation, was also reduced compared to the control virus-treated vessels. Direct comparison of the efficacy of AV-W9 and AV-p16 and AV-p27 in this model indicated that delivery of either of the parental genes was significantly less effective in inhibiting intimal thickening compared to the AV-W9 treatment. We conclude that combining the activities of multiple cell cycle regulatory proteins greatly increases the potency of cytostatic gene therapy in the treatment of balloon injury-induced intimal hyperplasia and represents a promising potential approach to preventing postangioplasty restenosis.

Use of a Salmonella microsuspension bioassay to detect the mutagenicity of munitions compounds at low concentrations.

Past production and handling of munitions has resulted in soil contamination at various military facilities. Depending on the concentrations present, these soils pose both a reactivity and toxicity hazard and the potential for groundwater contamination. Many munitions-related chemicals have been examined for mutagenicity in the Ames test, but because the metabolites may be present in low environmental concentrations, a more sensitive method is needed to elucidate the associated mutagenicity. RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), TNT (2,4,6-trinitrotoluene), tetryl (N-methyl-N-2,4,6-tetranitroaniline), TNB (1,3,5-trinitrobenzene) and metabolites were examined for mutagenicity in a microsuspension modification of the Salmonella histidine reversion assay with and without metabolic activation. TNB and tetryl were positive in TA98 (32.5, 5.2revertants/nmole) and TA100 (7.4, 9.5revertants/nmole) without metabolic activation and were more potent than TNT (TA98, 0.3revertants/nmole; TA100, 2.4revertants/nmole). With the exception of the tetranitroazoxytoluene derivatives, TNT metabolites were less mutagenic than TNT. RDX and two metabolites were negative in both strains, however, hexahydro-1,3,5-trinitroso-1,3,5-triazine was positive in TA100 with and without S9. Microsuspension bioassay results tend to correlate well with published Ames test data, however, there are discrepancies among the published data sets and the microsuspension assay results.

A high-throughput glow-type aequorin assay for measuring receptor-mediated changes in intracellular calcium levels.

A glow-type aequorin luminescence assay for measuring receptor-mediated stimulation of intracellular calcium levels is described and characterized. The human 5-hydroxytryptamine(2A) receptor stably coexpressed in human embryonic kidney cells with apoaequorin was used to characterize the system and showed that following the flash reaction, a stable luminescence signal could be measured using a microplate scintillation counter for between 3 and 7 h after the addition of receptor agonist. Furthermore, this luminescence was dependent on the concentration of agonist used and gave potency values that were stable over this time period. Testing a range of 5-hydroxytryptamine(2A) receptor agonists gave the expected rank order of potency for this receptor. The glow luminescence could also be inhibited by 5-hydroxytryptamine(2A) receptor antagonists, generating affinity values that directly correlated with those determined for inhibition of the flash reaction carried out under the same buffer conditions. The assay therefore gave pharmacologically relevant data and allows a significant improvement of throughput over the traditional flash-type measurements made using an injecting luminometer.

Identification and characterization of a truncated variant of the 5-hydroxytryptamine(2A) receptor produced by alternative splicing.

We have identified an alternatively spliced 5-hydroxytryptamine 2A receptor (5-HT(2A)-R) transcript by PCR of human brain cDNA using degenerate oligonucleotide primers to transmembrane (TM) domains 3 and 7 of the 5-HT(2)-R subfamily. The variant contains a 118-bp insertion at the exon II/III boundary of the 5-HT(2A)-R, which produces a frame shift in the coding sequence and a premature stop codon. PCR analysis showed that the truncated receptor (5-HT(2A-tr)) and native 5-HT(2A)-R were co-expressed in most brain tissues, with the highest levels being found in hippocampus, corpus collosum, amygdala and caudate nucleus. Western blot analysis of HEK-293 cells transfected transiently with a 5-HT(2A-tr) construct showed that a 30-kDa protein was expressed on cell membranes. Co-transfection studies showed no effect of the 5-HT(2A-tr) variant on 3H-ketanserin binding to the native 5-HT(2A)-R or on functional coupling of the 5-HT(2A)-R to 5-HT-stimulated Ca(2+) mobilization. The functional significance of the 5-HT(2A-tr) variant and other truncated receptors remains to be established.

Statistical modelling of general practice medicine for computer assisted data entry in electronic medical record systems.

Electronic medical record (EMR) systems have much potential, however, there are still a number of issues that need to be resolved before EMRs are widely accepted. One of these issues is the data input task, a potentially serious practical barrier to on-line medical computer usage. This paper reports the empirical modelling of data input requirements for physicians who use a problem-orientated medical record system. Three statistical models (Bayesian conditional probability, multiple linear regression and discriminant analysis) to predict drug treatment given problem diagnoses are derived from EMRs of 2500 general Practice encounters. Two metrics are used to measure the predictive power of the models considering both the number of drugs correctly predicted and the strength with which the models predict them. The models are tested on 500 unseen records from the same patient-physician population and the data used to build the models. The Bayesian model produces the best predictions on unseen data and is also the easiest model to compute. A prototype interface that enables new patient cases to be entered is constructed to demonstrate how the predictive power of the model can translate into benefits in the data entry task.

Nitric oxide synthase in atherosclerosis and vascular injury: insights from experimental gene therapy.

Gene therapy aims to intervene in a disease process by transfer and expression of specific genes in a target tissue or organ. Cardiovascular gene therapy in humans remains in its infancy, but in the last decade, experimental gene transfer has emerged as a powerful biological tool to investigate the function of specific genes in vascular disease pathobiology. Nitric oxide synthases, the enzymes that produce nitric oxide, have received considerable attention as potential candidates for vascular gene therapy because nitric oxide has pleiotropic antiatherogenic actions in the vessel wall, and abnormalities in nitric oxide biology are apparent very early in the atherogenic process. In this article, we review the use of nitric oxide synthases in experimental vascular gene therapy and assess the utility of these approaches for investigating the role of nitric oxide in atherosclerosis and their potential for human gene therapy.

The disinfection by-products dichloro-, dibromo-, and bromochloroacetic acid impact intestinal microflora and metabolism in Fischer 344 rats upon exposure in drinking water.

Human consumption of chlorinated drinking water has been linked epidemiologically to bladder, kidney, and rectal cancers. The disinfection by-product (DBP) dichloroacetic acid is a hepatocarcinogen in Fischer 344 rats and B6C3F1 mice. The objective of this study is to determine the effect of the DBPs dichloro-, bromochloro-, and dibromoacetic acids (DCA, BCA, DBA) on intestinal microbial populations and their metabolism, with emphasis on enzymes involved in the bioactivation of procarcinogens and promutagens. One-month-old male Fischer 344 rats were provided water ad libitum containing 1 g/l DCA, BCA, or DBA for up to 5 weeks. At 1, 3, and 5 weeks of treatment, beta-glucuronidase (GLR), beta-galactosidase (GAL), beta-glucosidase (GLU), nitroreductase (NR), azoreductase (AR), and dechlorinase (DC) activities were determined in cecal and small and large intestinal homogenates. After 5 weeks of treatment, intestinal populations were enumerated on selective media. Cecal GAL (DCA, BCA, DBA) and GLR (DCA, DBA) activities were reduced after 1 and 3 weeks of treatment and GAL activity was elevated at 5 weeks (BCA). Large intestinal GAL (DCA, BCA) and GLU (DCA, BCA, DBA) activities were elevated after 5 weeks of treatment. Week 5 cecal AR (DCA, BCA, DBA), NR (DCA), and DC (DCA, DBA) activities were reduced. Even though some significant changes in intestinal populations were observed, use of selective media was not sensitive enough to explain fluctuations in enzyme activity. Haloacetic acids in the drinking water alter intestinal metabolism, which could influence bioactivation of promutagens and procarcinogens in the drinking water.

Bioavailability of the genotoxic components in coal tar contaminated soils in Fischer 344 rats.

The effect of chemical aging on the bioavailability and subsequent genotoxicity of coal tar (CT)-contaminated soils was evaluated in a 17-day feeding study using Fischer 344 male rats. Rats consumed a control diet or diets amended with soil, 0.35% CT, or soil freshly prepared or aged for 9 months with 0.35% CT. Mild treatment-related microscopic lesions in liver tissue and elevated enzyme levels in serum were detected in all CT treatment groups. The (32)P-postlabeling assay was employed to determine DNA adduct formation in treated animals. All CT treatment groups induced DNA adducts in both the liver and lung. Adduct levels were 3-fold higher in lung DNA compared to hepatic DNA. After correcting adduct levels for total ingested polycyclic aromatic hydrocarbons (PAHs), a significant decrease (p < 0.05) in adduct levels was observed in both CT/soil treatment groups compared to CT control in liver and lung DNA. Adduct profiles of (32)P-postlabeled hepatic and lung DNA displayed several nonpolar DNA adducts that comigrated with PAH-adducted calf thymus DNA standards as determined through both thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). These results suggest that soil, but not aging of contaminants in soil, decreases the bioavailability of genotoxic components in CT, as evidenced by DNA adduct analysis.

Gene transfer of the neuronal NO synthase isoform to cirrhotic rat liver ameliorates portal hypertension.

Reduced production of nitric oxide (NO) in the cirrhotic liver results from a defect in hepatic endothelial cell nitric oxide synthase (ecNOS) and appears to contribute to the high intrahepatic resistance and portal hypertension typical of cirrhosis. Therefore, we postulated that targeting a heterologous NOS isoform to sinusoidal endothelial cells or other perisinusoidal cells, such as hepatic stellate cells, would counter the defect in NO production and reduce resistance to blood flow. Recombinant adenovirus (Ad) carrying the neuronal NOS gene (nNOS) targeted liver sinusoidal endothelial cells, stellate cells, and hepatocytes more efficiently than the corresponding cells in cirrhotic livers, but transduction rates were substantial even in cirrhotic animals. Expression of nNOS in each liver cell type, whether from normal or injured liver, caused increased NO production and inhibited endothelin-1-induced contractility of perisinusoidal stellate cells. Finally, in 2 different in vivo models of cirrhosis and portal hypertension, transduction of livers with recombinant Ad.nNOS significantly reduced intrahepatic resistance and portal pressure. The data highlight the feasibility of gene transfer to diseased liver and hepatic cells and demonstrate the potential of a novel therapy for portal hypertension caused by cirrhosis.