A step-wise approach to developing indicators to compare the performance of maternity units using hospital administrative data.

Hospital administrative data are attractive for comparing performance of maternity units because of their often large sample sizes, lack of selection bias and the relatively low costs of accessing these data compared with conducting primary data collection. However, using administrative data to develop indicators can also present challenges including varying data quality, the limited detail on clinical risk factors and a lack of structural and user experience measures. This review illustrates how to develop performance indicators for maternity units using hospital administrative data, including methods to address the challenges that administrative data pose. TWEETABLE ABSTRACT: How to develop maternity indicators from administrative data.

Painful sex (dyspareunia) in women: prevalence and associated factors in a British population probability survey.

OBJECTIVE: To estimate the prevalence of painful sex among women in Britain, and to explore associated sexual, relationship and health factors that should be considered in assessment. DESIGN: Multi-stage, clustered and stratified population probability sample survey, using computer-assisted self-interview. Sample frame was the British Postcode Address File. SETTING: Participants interviewed at home between 2010 and 2012. SAMPLE: A total of 15 162 adults aged 16-74 years (8869 women). Data reported from 6669 sexually active women. METHODS: Age-adjusted logistic regressions to examine associations between painful sex and indicators of sexual, relational, mental and physical health. MAIN OUTCOME MEASURE: Physical pain as a result of sex for ≥3 months in the past year, plus measures of symptom severity. RESULTS: Painful sex was reported by 7.5% (95% CI 6.7-8.3) of sexually active women, of whom one-quarter experienced symptoms very often or always, for ≥6 months, and causing distress. Reporting painful sex was strongly associated with other sexual function problems, notably vaginal dryness (age adjusted odds ratio 7.9; 6.17-10.12), anxiety about sex (6.34; 4.76-8.46) and lacking enjoyment in sex (6.12; 4.81-7.79). It was associated with sexual relationship factors [such as not sharing same level of interest in sex (2.56; 1.97-3.33)], as well as with adverse experiences such as non-volitional sex (2.17; 1.68-2.80). Associations were also found with measures of psychological and physical health, including depressive symptoms (1.68; 1.28-2.21). CONCLUSION: Painful sex is reported by a sizeable minority of women in Britain. Health professionals should be supported to undertake holistic assessment and treatment which takes account of the sexual, relationship and health context of symptoms. TWEETABLE ABSTRACT: Painful sex-reported by 7.5% of women in Britain-is linked to poorer sexual, physical, relational and mental health.

Trends in the use of emergency contraception in Britain: evidence from the second and third National Surveys of Sexual Attitudes and Lifestyles.

OBJECTIVE: To examine the changes in the prevalence of, and the factors associated with, the use of emergency contraception (EC) in Britain between 2000 and 2010, spanning the period of deregulation and increase in pharmacy supply. DESIGN: Cross-sectional probability sample surveys. SETTING AND POPULATION: British general population. METHODS: Data were analysed from the second and third British National Surveys of Sexual Attitudes and Lifestyles (Natsal), undertaken in 1999-2001 and 2010-12. Univariate and logistic regression analyses were used to measure change in EC use amongst sexually active women aged 16-44 years not intending pregnancy. MAIN OUTCOME MEASURES: Prevalence of EC use and factors associated with use. RESULTS: Of the 5430 women surveyed in 1999-2001 and the 4825 women surveyed in 2010-12, 2.3 and 3.6%, respectively, reported using EC in the year prior to interview (P = 0.0019 for change over time). The prevalence of EC use increased amongst single women and those with higher educational attainment (adjusted odds ratio, aOR 1.51; 95% confidence interval, 95% CI 1.04-2.20; P = 0.0308). Increases in EC use were generally greater among women without behavioural risk factors, such as those with no history of abortion within 5 years (aOR 1.57; 95% CI 1.17-2.12; P = 0.0029), or those whose first heterosexual intercourse occurred after the age of 16 years (aOR 1.68; 95% CI 1.21-2.35; P = 0.0021). The increase in EC use was also more marked among women usually accessing contraception from retail sources than among those doing so from healthcare sources, which may reflect a use of condoms amongst EC users. CONCLUSION: The increase in EC use among women in Britain in the first decade of the 21st century was associated with some, but not all, risk factors for unplanned pregnancy. Advice and provision may need to be targeted at those at highest risk of unplanned pregnancy. TWEETABLE ABSTRACT: Despite pharmacy access, only a small rise in emergency contraception use has been seen in Britain over 10 years.

Immunogenetic characterization of a captive colony of sooty mangabeys (Cercocebus atys) used for SIV research.

BACKGROUND: African non-human primates are SIV natural hosts and do not develop disease following infection. Understanding disease avoidance mechanisms in these species is important for HIV vaccine development. The largest captive population of sooty mangabeys, a SIV natural host species, resides at the Yerkes National Primate Research Center. METHODS: Thirteen primer sets that amplify polymorphic microsatellite loci within the MHC region were used to genotype 144 animals. Immunogenetic Management Software (IMS) was used to identify MHC haplotypes and organize data. RESULTS: Seventy-three haplotypes were identified. Limited haplotype diversity was observed in this population with 88.2% of included animals carrying one of 18 haplotypes. Differences in haplotype frequency were observed between SIV (+) and SIV (-) populations. CONCLUSIONS: We have developed a novel tool for others to use in the analysis of the role of the MHC in a natural host non-human primate model species used for SIV research.

Uptake and outcomes of robotic gynaecological surgery in England (2006-2018): an account of Hospital Episodes Statistics (HES).

This was a retrospective study to review the uptake and outcomes of robotic gynaecological surgery in England between 1st April 2006 and 31st March 2018, analysing Hospital Episode Statistics form National Health Service hospitals in England. Women aged 18 years and above who had elective gynaecological surgery were included and those who had undergone robotic gynaecology surgery were included. Robotic gynaecological procedures were defined as procedures that used a robotic minimal access approach for hysterectomy, adnexal surgery and urogynaecological surgery (sacrocolpopexy, sacrohysteropexy and colposuspension). Numbers of procedures were reviewed by year and mapped to the 44 NHS healthcare regions. Length of stay (nights in hospital), laparotomy (conversion during primary procedure or after return to theatre for management of complication), and 30-day emergency readmission rates were calculated by year and procedure type. Overall 527,217 elective gynaecological procedures were performed in the English NHS (1st April 2006 and 31st March 2018), of which 4384 (0.83%) were performed with robotic assistance (3864 (88%) hysterectomy, 706 (16%) adnexal surgery, 192 (4%) urogynaecological surgery). There was gradual rise in the uptake of robotic surgery but there was a marked geographical variation. Median (IQR) length of stay (LOS) was 1(1-2) night, laparotomy rate was 0.3% and 30-day emergency readmission rate was 4.7%. LOS was statistically, but not clinically, different across time. Other outcomes did not differ by year. Robotic gynaecological procedures are increasingly being used in the English NHS, predominantly for hysterectomy, although in small proportions (2.6% in the most recent study year). There was wide geographical variation in robotic uptake across England and overall, outcomes were comparable to those reported in other countries.

Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain.

RNA interference (RNAi) for spinocerebellar ataxia type 1 can prevent and reverse behavioral deficits and neuropathological readouts in mouse models, with safety and benefit lasting over many months. The RNAi trigger, expressed from adeno-associated virus vectors (AAV.miS1), also corrected misregulated microRNAs (miRNA) such as miR150. Subsequently, we showed that the delivery method was scalable, and that AAV.miS1 was safe in short-term pilot nonhuman primate (NHP) studies. To advance the technology to patients, investigational new drug (IND)-enabling studies in NHPs were initiated. After AAV.miS1 delivery to deep cerebellar nuclei, we unexpectedly observed cerebellar toxicity. Both small-RNA-seq and studies using AAVs devoid of miRNAs showed that this was not a result of saturation of the endogenous miRNA processing machinery. RNA-seq together with sequencing of the AAV product showed that, despite limited amounts of cross-packaged material, there was substantial inverted terminal repeat (ITR) promoter activity that correlated with neuropathologies. ITR promoter activity was reduced by altering the miS1 expression context. The surprising contrast between our rodent and NHP findings highlight the need for extended safety studies in multiple species when assessing new therapeutics for human application.

Osteosarcoma and second malignant neoplasms: a case series.

BACKGROUND: The overall survival of patients with localised osteosarcoma has dramatically improved with the introduction of multidrug chemotherapeutic regimens into the treatment paradigm. However, despite optimal treatment, all-cause mortality remains higher among osteosarcoma survivors than in the general population. The development of second malignant neoplasms contributes to this higher mortality rate. CASE SERIES: We present three cases of patients definitively treated for osteosarcoma who subsequently developed a second malignant neoplasm. The first case describes a 17-year-old female with osteosarcoma of her right femur treated with surgical resection and perioperative chemotherapy. Ten years later, she was diagnosed with metastatic HER2-positive breast cancer. Genetic testing identified a germline TP53 mutation, confirming the presence of Li-Fraumeni syndrome. The second case details an 18-year-old male with osteosarcoma of his right humerus treated with definitive resection and perioperative chemotherapy. He was diagnosed with appendiceal adenocarcinoma after presenting with acute abdominal pain 17 years later. The third case reviewed is of a 36-year-old male with osteosarcoma of his right femur treated with definitive resection and adjuvant chemotherapy. A diagnosis of leiomyosarcoma was made 7 years later following surveillance imaging. DISCUSSION: The risk of second malignant neoplasms in osteosarcoma may relate to previous oncological treatment, an inherited cancer predisposition syndrome or a spontaneous new neoplasm. Although screening for a second malignancy is not routinely recommended for osteosarcoma survivors, a high degree of clinical suspicion should be maintained during surveillance.

Bioavailability and therapeutic activity of alicaforsen (ISIS 2302) administered as a rectal retention enema to subjects with active ulcerative colitis.

BACKGROUND: Alicaforsen is a phosphorothioate-modified antisense oligodeoxynucleotide designed to sequence-specifically reduce intercellular adhesion molecule 1 messenger RNA levels. AIMS: To determine the systemic and local bioavailability of alicaforsen, and its activity when administered as a once daily enema in subjects with active ulcerative colitis. METHODS An open-label study was conducted to assess the relative absorption (local and systemic pharmacokinetics) and pharmacologic activity of alicaforsen enema in subjects with active ulcerative colitis. Fifteen subjects received nightly enemas of alicaforsen (240 mg) for a treatment period of 6 weeks. Alicaforsen concentrations in plasma and colonic tissue biopsies were determined. Disease activity index and multiple measurements including endoscopy were used to assess alicaforsen activity in these subjects. RESULTS: Plasma concentrations of parent alicaforsen represented < 0.6% mean bioavailability when compared with historical intravenous area under the plasma concentration-time curves. Concentrations of the intact oligonucleotide in mucosal colonic tissue biopsies were orders of magnitude higher than those observed in plasma. A 46% reduction in mean Disease Activity Index and 33% rate of remission as defined by complete mucosal healing were observed at the end of treatment. Conclusion These data confirm that alicaforsen enema provides local treatment for a local disease with little meaningful systemic exposure.

A comparison of aorta and vena cava medial message expression by cDNA array analysis identifies a set of 68 consistently differentially expressed genes, all in aortic media.

We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles of macaque aorta and vena cava media using a cDNA array containing 4048 known human genes, approximately 35% of currently named human genes (approximately 11,000). The data show extensive differences in RNA expression in artery versus vein media. Sixty-eight genes had consistent elevation in message expression by the aorta, but none were elevated in the vena cava. The most differentially expressed gene was regulator of G-protein signaling (RGS) 5, at an expression ratio of 46.5+/-12.6 (mean+/-SEM). The data set also contained 2 genes already known to be expressed in the aorta, elastin at 5.0+/-1.4, and the aortic preferentially expressed gene 1 (APEG-1) at 2.3+/-0.6. We chose to analyze RGS5 expression further because of its high level of differential expression in the aorta. Levels of RGS5 mRNA were confirmed by Northern analysis and in situ hybridization. A human tissue RNA dot blot showed that RGS5 message is highest in aorta, followed by small intestine, stomach, and then heart. Northern analysis confirmed that RGS5 expression in human aorta is higher than in any region of the heart. RGS5 is a G-protein signaling regulator of unknown specificity most homologous to RGS4, an inhibitory regulator of pressure-induced cardiac hypertrophy. The expression pattern of the 68 differential genes as a whole is a start toward identifying the molecular phenotypes of arteries and veins on a systematic basis.

Hyaluronan enhances contraction of collagen by smooth muscle cells and adventitial fibroblasts: Role of CD44 and implications for constrictive remodeling.

Remodeling contributes to restenosis when cells shrink the artery wall at sites of injury. This may be analogous to wound healing, where tissue remodeling achieves wound contraction. Hyaluronan (HA) is prominent in wound matrix and inhibits fetal scarring. HA is also produced in the artery wall after angioplasty, where it may inhibit constrictive remodeling. This hypothesis was tested in vitro using a model of matrix contraction. Primate aortic smooth muscle cells and adventitial fibroblasts were seeded into collagen I gels containing increasing amounts of HA (0% to 50%, wt/wt). Both cell types reduced the diameter of collagen alone approximately 65% at 18 hours. HA significantly increased gel contraction (diameter in mm: 0% HA, 7. 7+/-0.9; 2%, 7.1+/-0.7; 10%, 6.7+/-0.5; 50%, 5.6+/-0.9; P<0.05 for >/=10%), cell spreading and telopodia, and pericellular accumulation of collagen fibrils. These effects were mediated in part by cellular HA binding, because an antibody against CD44 receptors blocked pericellular collagen accumulation and enhanced gel contraction without altering cell shape. The role of CD44 was specific, because inhibiting receptor for hyaluronic acid-mediated motility (RHAMM) had no effect. Blocking ss(1)-integrins completely inhibited contraction of collagen, but gels containing HA required CD44 and ss(1)-integrin blockade for complete inhibition. Enhanced collagen reorganization and contraction were not attributable to increased collagenase activity, because the metalloproteinase inhibitor batimastat had no effect. In summary, HA enhanced collagen reorganization by the cell types most likely to mediate constrictive remodeling after angioplasty. These effects were CD44-dependent, thus providing a potential target for therapies to prevent constrictive remodeling and restenosis.

Self-reported Sleep Improvement in Buprenorphine MAT (Medication Assisted Treatment) Population.

This is a prospective, naturalistic study to evaluate patient's report on sleep and depression in early recovery while receiving buprenorphine in Medication Assisted Treatment (MAT). 40 Subjects entering into MAT with buprenorphine/naloxonefor opioid dependence disorder were recruited. No change of concurrent treatment was made. Subjects were administered Sleep Scale from the Medical Outcomes Study (MOS-Sleep), a 5-item Supplemental Sleep Scale (SSS), and the Beck Depression Inventory II (BDI-II). The measures were administered at day 0 (baseline), 30, 60 and 90 days. The result showed that patients reported significant progressive improvements in three MOS-Sleep subscales: sleep disturbance, sleep indices I and II. The mean scores of SLPD4 (Sleep disturbance) at day 0, 30, 60, 90 were 62.4, 53.2, 53.3, and 48.4 respectively (p=0.0029). Similarly, subscores of SLP6 (Sleep Problem Index I) and SLP 9 (Sleep Problem Index II) were also significantly decreased over time (P=0.038 for SLP6 and p=0.007 for SLP9). BDI-II depression scores improved from "Moderate depression" at baseline to "Mild depression". The mean BDI score decreased from 24.2 to 17.0 after 90 days of treatment. Findings suggest that subjects reported improvement in both sleep and depression after initiating MAT with buprenorphine/naloxone.

Phase I evaluation of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C-alpha, in patients with advanced cancer.

PURPOSE: To determine the maximum-tolerated dose (MTD) and pharmacologic behavior of ISIS 3521 (ISI 641A), an antisense phosphorothioate oligonucleotide to protein kinase C-alpha. PATIENTS AND METHODS: Thirty-six patients with advanced cancer received 99 cycles of ISIS 3521 (0.15 to 6.0 mg/kg/d) as a 2-hour intravenous infusion administered three times per week for 3 consecutive weeks and repeated every 4 weeks. Plasma and urine sampling was performed during the first week of treatment and subjected to capillary gel electrophoresis to determine full-length antisense oligonucleotide in addition to chain-shortened metabolites. RESULTS: Drug-related toxicities included mild to moderate nausea, vomiting, fever, chills, and fatigue. Hematologic toxicity was limited to thrombocytopenia (grade 1, four patients; grade 2, one patient; grade 3, one patient). There was no relationship between dose, maximum concentration of the drug (C(max)), or area under the plasma concentration versus time curve (AUC) and coagulation times or complement levels. Dose escalation was discontinued because of the attainment of peak plasma concentrations, which approached that associated with complement activation in primates. Two patients with non-Hodgkin's lymphoma who completed 17 and nine cycles of therapy achieved complete responses. The pharmacokinetic profile of ISIS 3521 revealed a short elimination half-life (18 to 92 minutes), as well as a dose-dependent decrease in clearance and dose-dependent increases in C(max), AUC, and elimination half-life. CONCLUSION: No dose-limiting toxicity of ISIS 3521 was identified, and clinical activity was observed. A short elimination half-life was identified, which suggests that alternate schedules with prolonged administration may be necessary for further clinical development.

Conjugated equine estrogens inhibit progression of atherosclerosis but have no effect on intimal hyperplasia or arterial remodeling induced by balloon catheter injury in monkeys.

OBJECTIVES: This study sought to determine the effects of estrogen treatment on atherosclerosis progression and the proliferative and structural responses of the atherosclerotic arteries to injury. BACKGROUND: Estrogen treatment suppresses the intimal response to arterial injury in nonatherosclerotic rodents and rabbits and inhibits the in vitro proliferation of smooth muscle cells. However, the effect of estrogen on the response of atherosclerotic arteries to transmural injury, as occurs in balloon catheter angioplasty in humans, is unknown. METHODS: Forty-six ovariectomized cynomolgus monkeys were fed an atherogenic diet for 30 months; 25 received 175 microg/day of conjugated equine estrogens, and 21 served as untreated control animals. All animals underwent balloon catheter injury of the left iliac artery. Subsets of animals underwent a necropsy study at 4, 7, 14 and 28 days after injury; injured and contralateral (uninjured) arteries were pressure-fixed and evaluated morphometrically. RESULTS: Estrogen treatment resulted in a 37% decrease (p < 0.05) in atherosclerosis (plaque area) in the uninjured artery. In response to injury, arterial cell proliferation increased at days 4 and 7, and intimal area was increased two- to threefold at day 28 (p < 0.05). Although estrogen treatment resulted in a trend toward decreased arterial cell proliferation at day 4, there was evidence of increased cell proliferation in both media and intima at day 7 (p < 0.05). However, there was no effect of estrogen treatment on intimal area or indexes of arterial remodeling in the injured artery at day 28 (p > 0.4). CONCLUSIONS. In contrast to previous studies of nonatherosclerotic animals, the results indicate that in the circumstance of transmural injury to arteries of primates with preexisting atherosclerosis, estrogen does not suppress arterial neointimal or structural responses to injury.

A phase I trial of c-Raf kinase antisense oligonucleotide ISIS 5132 administered as a continuous intravenous infusion in patients with advanced cancer.

Raf proteins play a central role in the mitogen-activated protein kinase signaling pathway and hence are involved in oncogenic transformation and tumor cell proliferation. ISIS 5132 is a 20-base antisense phosphorothioate oligodeoxyribonucleotide that specifically down-regulates c-raf expression. We report here an initial study of the safety and tolerability of an i.v. infusion of ISIS 5132 in patients with advanced cancer. A continuous i.v. infusion of ISIS 5132 was administered for 21 days every 4 weeks to 34 patients with a variety of solid tumors refractory to standard therapy. The dose of ISIS 5132 was increased in sequential cohorts of patients, as toxicity allowed, until a final dose of 5.0 mg/kg body weight was reached. Toxicity was scored by common toxicity criteria, and tumor response was monitored. Pharmacokinetic studies were performed for 30 patients treated at doses of < or =4.0 mg/kg/day. The initial dose of ISIS 5132 was 0.5 mg/kg body weight and was successfully increased incrementally to 5.0 mg/kg body weight. Toxicities through the 4.0 mg/kg dose level were not dose limiting. Side effects were minimal and could not be specifically related to ISIS 5132. Two patients had prolonged stabilization of their disease, and one patient with ovarian carcinoma had a significant response with a 97% reduction in CA-125 levels. ISIS 5132, an antisense oligonucleotide against c-raf, was well tolerated at doses up to and including 4.0 mg/kg/day by 21-day continuous i.v. infusion and demonstrated antitumor activity at the doses tested.

Phase I study of an antisense oligonucleotide to protein kinase C-alpha (ISIS 3521/CGP 64128A) in patients with cancer.

Protein kinase C (PKC) is an attractive target in cancer therapy. It is overexpressed in a variety of cancers, and nonspecific inhibitors of PKC have demonstrated antitumor activity. Antisense oligonucleotides targeted against PKC-alpha, which have high specificity, can inhibit mRNA and protein expression as well as the growth of tumors in vitro and in vivo. This Phase I study sought to characterize the safety profile and to determine the maximum tolerated dose of antisense to PKC-alpha when administered by continuous infusion in patients. Patients with incurable malignancies received ISIS 3521, a 20-length phosphorothioate oligodeoxynucleotide specific for PKC-alpha. Treatment was delivered over a period of 21 days by continuous i.v. infusion followed by a 7-day rest period. Doses were increased from 0.5 to 3.0 mg/kg/day. Patients continued on the study until evidence of disease progression or unacceptable toxicity was detected. Between August 1996 and September 1997, 21 patients were treated in five patient cohorts. The maximum tolerated dose was 2.0 mg/kg/day. The dose-limiting toxicities were thrombocytopenia and fatigue at a dose of 3.0 mg/kg/day. Pharmacokinetic measurements showed rapid plasma clearance and dose-dependent steady-state concentrations of ISIS 3521. Evidence of tumor response lasting up to 11 months was observed in three of four patients with ovarian cancer. The recommended dose of ISIS 3521 for Phase II studies is 2.0 mg/kg/day when given over a period of 21 days. Side effects are modest and consist of thrombocytopenia and fatigue. Evidence of antitumor activity provides the rationale for Phase II studies in ovarian cancer and other malignancies.

Phase I Trial of ISIS 5132, an antisense oligonucleotide inhibitor of c-raf-1, administered by 24-hour weekly infusion to patients with advanced cancer.

Raf-1 is a serine/threonine kinase that functions as a critical effector of Ras-mediated signal transduction via the mitogen-activated protein kinase pathway. Constitutive activation of this pathway directly contributes to malignant transformation in many human tumors. A 20-base phosphorothioate oligonucleotide complementary to c-raf-1 mRNA (ISIS 5132; CGP 69846A) has been shown to specifically suppress Raf-1 expression both in vitro and in vivo. This Phase I trial, involving 22 patients with advanced cancer, was designed to evaluate the safety, feasibility, and maximum tolerated dose of ISIS 5132 administration as a weekly 24-h i.v. infusion. Pharmacokinetic analysis was performed, and c-raf-1 mRNA levels in peripheral blood mononuclear cells were assessed using quantitative reverse transcription-PCR. This trial defined a maximum tolerated dose of 24 mg/kg/week on this schedule. Two of four patients treated at 30 mg/kg/week had serious adverse events after the first dose of ISIS 5132, including acute hemolytic anemia and acute renal failure and anasarca. There were no major responses documented. Dose-dependent complement activation was demonstrated on this schedule, but not on previously evaluated schedules, of ISIS 5132 administration. In contrast to other trials of ISIS 5132, there appeared to be no consistent suppression of peripheral blood mononuclear cell c-raf-1 mRNA level on this schedule at any of the dose levels analyzed. These data suggest that the efficacy and toxicity profiles of antisense oligonucleotides may be highly dependent on the schedule of administration and support the analysis of the putative molecular target in the evaluation of novel therapeutics.

Adenoviral gene transfer in arteries of hypercholesterolemic nonhuman primates.

Arterial gene transfer with adenoviral vectors is a promising approach for the treatment and prevention of vascular disorders. However, in small animals such as rats and rabbits adenoviral vectors can have deleterious effects on the artery wall. The effects of adenovirus in primate arteries have not been studied. AdRSVn-LacZ, a replication-defective adenoviral vector, was delivered to the left brachial arteries of six hypercholesterolemic cynomolgus monkeys; right brachial arteries received vehicle only. Serum was collected before gene transfer and at vessel harvest 9 or 10 days later. Recombinant gene expression was present in occasional endothelial cells of transduced arteries, and all animals generated neutralizing antibodies. In transduced arteries, immunostaining revealed a fourfold increase in intimal and medial macrophage accumulation (p < 0.05); intimal cellularity was also significantly increased (twofold; p < 0.05). T cell density and total cellular proliferation (determined by bromodeoxyuridine labeling) were unaffected. In hypercholesterolemic nonhuman primates, adenoviral vectors increase vessel wall inflammation and promote the progression of early atherosclerotic lesions. The long-term consequences of these observations remain unclear; however, a better understanding of host responses to specific vector systems appears necessary for the development of safe and effective approaches to human vascular gene therapy.

Gene transfer in baboons using prosthetic vascular grafts seeded with retrovirally transduced smooth muscle cells: a model for local and systemic gene therapy.

Prosthetic vascular grafts containing retrovirally transduced autologous vascular smooth muscle cells were studied as a model for introduction of human genes into baboons. Retroviral vectors encoding beta-galactosidase (beta-Gal) (LNPoZ) or human purine nucleoside phosphorylase (LPNSN-2), a control gene, were used for ex vivo transduction of autologous baboon smooth muscle cells obtained from vein biopsies. Transduced cells were placed into a collagen solution and seeded into the interstices of polytetrafluoroethylene vascular grafts. Endothelial cells were then seeded onto the luminal surface of the grafts to reduce thrombus formation. One LNPoZ-seeded graft and one LPNSN-2-seeded control graft were implanted bilaterally into the aorto-iliac circulation of each of 4 animals. All grafts remained patent until they were removed after 3-5 weeks and examined histochemically for vector-expressing cells. All histological cross-sections from the beta-Gal vector seeded grafts contained cells staining blue with the X-Gal chromogen. For the four grafts, the mean fraction of LNPoZ expressing cells was 10%, with a range of 2-20%, while no sections from the control grafts contained stainable cells. Smooth muscle cells expressing the reporter gene were localized within the graft wall but not in the newly forming intima or outer capsule of fibrous tissue. Implantation of transduced cells within this type of vascular graft may provide a useful approach for long-term local and systemic gene therapy.

Angiotensin-converting enzyme expression in human carotid artery atherosclerosis.

Angiotensin-converting enzyme (ACE) inhibitors reduce the progression of atherosclerosis in animal models and reinfarction rates after myocardial infarction in humans. Although expression of components of the renin-angiotensin system has been reported in human coronary arteries, no data regarding their presence in carotid arteries, a frequent site for the occurrence of atherosclerosis plaques, are available. The following study sought to determine whether ACE mRNA and protein can be detected in human carotid atheromatous lesions. Twenty-four intact endarterectomy specimens were obtained from patients with severe carotid occlusive disease (17 males and 7 females, aged 68+/-1 years) and fixed within 30 minutes. Carotid artery specimens contained advanced Stary type V and VI lesions, and human ACE mRNA expression and protein were localized in cross sections by the combination of in situ hybridization and immunohistochemistry. Cell type-specific antibodies were used to colocalize ACE to smooth muscle cells, endothelial cells, macrophages, or lymphocytes. ACE protein was localized in the intima, whereas the overlying media was largely free of ACE staining. In less complicated lesions, ACE staining was modest and could be visualized in scattered clusters of macrophages and on the luminal side of carotid artery vascular endothelium. Smooth muscle cells were largely negative. ACE staining increased as lesions became more complex and was most prominent in macrophage-rich regions. The shoulder regions of plaques contained numerous ACE-positive macrophage foam cells and lymphocytes. In these areas, microvessels were positive for endothelial cell and smooth muscle cell ACE expression. However, microvessels in plaques free of inflammatory cells were stained only faintly for ACE expression. Labeling for ACE mRNA mirrored the pattern of protein expression, localizing ACE mRNA to macrophages and microvessels within the intima. In conclusion, atherosclerosis alters carotid artery ACE production, increasing transcription and translation within regions of plaque inflammation. These data provide another important mechanism by which inflammation associated with increased ACE expression may contribute to the progression of atherosclerosis.

Pharmacokinetics, metabolism, and elimination of a 20-mer phosphorothioate oligodeoxynucleotide (CGP 69846A) after intravenous and subcutaneous administration.

The pharmacokinetics, tissue distribution and metabolism of CGP 69846A, a 20-mer phosphorothioate oligodeoxynucleotide targeted against the 3'-untranslated region of human c-raf-1 kinase mRNA, were investigated in vivo in rats after intravenous and subcutaneous administration. Intravenous disposition studies with [3H]CGP 69846A were supported with analysis by capillary gel electrophoresis and electrospray mass spectrometry. In combination, these techniques provide a detailed account of the pharmacokinetic and metabolic profile for this compound. The elimination of CGP 69846A after a single intravenous dose was studied over extended periods in mice using whole-body autoradiography and capillary gel electrophoresis. Subcutaneous administration to rats resulted in a significant bioavailability with peak plasma levels 4.5-fold lower than after intravenous dosing. This dose route resulted in low interanimal variability and only slightly greater metabolism of the oligonucleotide compared to the intravenous administration.