Gene delivery to human sweat glands: a model for cystic fibrosis gene therapy.

Gene therapy vectors are mostly studied in cultured cells, rodents, and sometimes in non-human primates, but it is useful to test them in human tissue prior to clinical trials. In this study, we investigated the possibility of using human sweat glands as a model for testing cystic fibrosis (CF) gene therapy vectors. Human sweat glands are relatively easy to obtain from skin biopsy, and can be tested for CFTR function. Using patients' sweat glands could provide a safe model to study the efficacy of CF gene therapy. As the first step to explore using sweat glands as a model for CF gene therapy, we examined various ex vivo gene delivery methods for a helper-dependent adenovirus (HD-Ad) vector. Gene delivery to sweat glands in skin organ culture was studied by topical application, intradermal injection or submerged culture. We found that transduction efficiency can be enhanced by pretreating isolated sweat glands with dispase, which suggests that the basement membrane is a critical barrier to gene delivery by adenoviral vectors. Using this approach, we showed that Cftr could be efficiently delivered to and expressed by the epithelial cells of sweat glands with our helper-dependent adenoviral vector containing cytokeratin 18 regulatory elements. Based on this study we propose that sweat glands might be used as an alternative model to study CF gene therapy in humans.

Effect of nicotine on vasoconstrictor and vasodilator responses in human skin vasculature.

Our objective was to test the hypothesis that acute exposure of human skin vasculature to nicotine may have deleterious effects on endothelial function. Vasoconstriction and vasorelaxation in isolated perfused human skin flaps (approximately 8 x 18 cm) derived from dermolipectomy specimens were assessed by studying changes in skin perfusion pressure measured by a pressure transducer, and skin perfusion was assessed by a dermofluorometry technique (n = 4 or 5). It was observed that nicotine (10(-7) M) amplified (P < 0.05) the norepinephrine (NE)-induced concentration-dependent (10(-7)-10(-5) M) increase in skin vasoconstriction compared with the control. This amplification effect of nicotine in NE-induced skin vasoconstriction was not blocked by the nicotine-receptor antagonist hexamethonium (10(-6) M) or the cyclooxygenase inhibitor indomethacin (10(-5) M). It was also observed that ACh and nitroglycerin (NTG) elicited a concentration-dependent (10(-8)-10(-5) M) vasorelaxation in skin flaps preconstricted with 8 x 10(-7) M of NE. The vasorelaxation induced by ACh was attenuated (P < 0.05) in the presence of nicotine (10(-7) M) compared with the control. However, skin vasorelaxation induced by NTG was not affected by nicotine (10(-7) M). ACh and NTG are known to induce endothelium-dependent and -independent vasorelaxation, respectively. The present findings were interpreted to indicate that acute exposure of human skin vasculature to nicotine was associated with 1) amplification of NE-induced skin vasoconstriction and 2) impairment of endothelium-dependent skin vasorelaxation. Cyclooxygenase products and nicotine receptors blocked by hexamethonium were not involved in the amplification of NE-induced skin vasoconstriction by nicotine. These findings may provide further insight into the pathogenesis of skin vasospasm in skin flap surgery and skin ischemic disease associated with cigarette smoking or use of smokeless tobacco.

Pharmacological characterization of vasomotor activity of human musculocutaneous perforator artery and vein.

Vasospasm is one of the main causes of skin ischemic necrosis in cutaneous and musculocutaneous flap surgery, but the pathogenic mechanism is unclear. We planned to test the hypothesis derived from clinical impression that veins are more susceptible to vasospasm than arteries in flap surgery and, once established, that venous vasospasm is difficult to resolve and more detrimental than arterial vasospasm. To this end, we investigated the differences in sensitivity to vasoconstrictors and vasodilators between the human musculocutaneous perforator (MCP) artery and vein by measuring the isometric tension of arterial and venous rings suspended in organ chambers. Vascular contraction was expressed as a percentage of the tension induced by 50 mM KCl. Relaxation was expressed as a percentage of contraction induced by a submaximal concentration (3 x 10(-9) M) of endothelin-1 (ET-1). We observed that the vasoconstrictor potency of norepinephrine was significantly higher in the MCP vein than in the MCP artery. The vasoconstrictor potency of ET-1 and the thromboxane A(2) mimetic U-46619 were similar in the MCP vein and artery, but the maximal contraction induced by ET-1 and U-46619 was significantly higher in the MCP vein than in the MCP artery. On the other hand, the MCP vein was less sensitive than the MCP artery to the relaxation effect of nitroglycerin, nifedipine, and lidocaine. These differences between the human MCP artery and vein in response to vasoactive agents lend support to the clinical impression in flap surgery that veins appear to be more susceptible to vasospasm than arteries and venous vasospasm seems to be more difficult to resolve than arterial vasospasm in cutaneous and musculocutaneous flap surgery.

Vasoconstrictor effect of endothelin-1 in human skin: role of ETA and ETB receptors.

The aim of this project was to investigate the role of ETA and ETB receptors in the mediation of endothelin (ET)-1-induced vasoconstriction in human skin. This information should provide important insights into the design of pharmacological intervention against skin vasospasm induced by ET-1 in peripheral vascular disease or surgical trauma. Vasoconstriction in response to intra-arterial drug infusion in isolated perfused human skin flaps (8 x 18 cm) derived from dermolipectomy specimens was assessed by studying changes in skin perfusion and perfusion pressure under constant flow rate in each drug treatment (n = 4). It was observed that ET-1 (10(-10) to 10(-8) M) and norepinephrine (NE, 10(-8) to 10(-5) M) caused skin vasoconstriction in a concentration-dependent manner, with the vasoconstrictor potency of ET-1 approximately 200-fold higher than NE. The ETA-receptor antagonist BQ-123 but not the ETB-receptor antagonist BQ-788 blocked the vasoconstrictor effect of ET-1. This observation was confirmed by studying skin perfusion using the dermofluorometry technique. In addition, ETB-receptor agonists BQ-3020 and sarafotoxin S6c (10(-9) to 10(-6) M) did not evoke skin vasoconstriction. BQ-3020 also did not elicit skin vasoconstriction even in the presence of 10(-5) M of Nomega-nitro-L-arginine methyl ester and indomethacin. Furthermore, results from saturable and competitive ET-1 radioligand membrane receptor binding assays revealed that high-affinity and capacity binding sites are predominantly the ETA receptor subtype in endothelium-denuded skin arteries and veins of 0.5-1.5 mm diameter, with an ETA-to-ETB receptor ratio of 83:17 in arteries (n = 5) and 78:22 in veins (n = 7). Results from the present functional and radioligand receptor binding studies clearly indicate that ET-1 is a very potent vasoconstrictor in human skin and its vasoconstrictor effect is primarily mediated by ETA receptors, with no significant participation from ETB receptors.

Comparison of the enzyme activities between the thickest and the thinnest parts of the abdominal pannus.

OBJECTIVE: According to some reports, there are variations in metabolism in adipocytes from different areas of the body. The purpose of this study was to determine if there are differences in some of the lipid assimilating enzyme activities between the thickest (overhang) and the thinnest (upper margin) parts of the abdominal pannus. METHODS: The abdominal panniculectomy activities of sn-glycerol-3-phosphate dehydrogenase (31 subjects, spectrophotometric method), fatty acid synthetase (14 subjects, spectrophotometric method) and lipoprotein lipase (18 subjects, radioactive method) were determined in the thickest and the thinnest parts of the pannus of lipectomy patients. RESULTS: The enzyme activities were as follows: sn-glycerol-3-phosphate dehydrogenase: thickest: 2083 +/- 227.7 nm/mg/min; thinnest: 2084 +/- 208.3 nm/mg/min (p < 0.098, T = 0.02). Fatty acid synthetase: thickest: 22.0 +/- 3.9 microns/mg/min; thinnest 25.9 +/- 6.9 microns/mg/min (p < 0.36, T = 0.94). Lipoprotein lipase: thickest: 0.70 +/- 0.11% of control; thinnest: 0.61 +/- 0.14% of control (p < 0.47, T = 0.75). Thus no differences in specific enzyme activities were found between the two sites studied. CONCLUSIONS: There was no difference in the activity of the enzymes studied at the thickest and the thinnest part of the pannus.

Serial fluorometric assessments of skin perfusion in isolated perfused human skin flaps.

The applicability of serial skin surface fluorometry for repeated assessments of skin flap perfusion was investigated using the isolated perfused human transverse paraumbilical (TP) skin flap model. The flow rate, perfusion pressure and skin surface temperature were kept constant in seven human TP skin flaps and a low dose of fluorescein (3 x 10(-5) M) was used for each assessment. It was observed that the mean values for total dye fluorescence measured by a fluorometer and the maximum distance of perfusion estimated by dye fluorescein index remained consistent in five repeated assessments at 15 min interval. The variation in the maximum distance of perfusion within each TP skin flap over 5 repeated assessments was also relatively small, as judged by the mean coefficient of variation (6.1%; SEM 0.4%). Furthermore, a highly significant correlation between microsphere (15 microns) radioactivity index and dye fluorescence index was observed at corresponding locations in these seven TP skin flaps (r = 0.81; p < 0.001, n = 75). Taken together, these observations indicate that serial skin surface fluorometry provided consistent repeated assessments of skin perfusion in human skin flaps in vitro and the dye fluorescence index provided a consistent assessment of skin perfusion distance along the length of the TP skin flap. These observations lead us to speculate that critical (threshold) dye fluorescence index determined at various postoperative time points should be useful for prediction of skin viability in clinical skin flaps; thus, a clinical investigation is recommended.

Physician reimbursement in Medicare risk contracting.

The Medicare population remains largely unmanaged despite its increasing cost burden. With a few notable exceptions, health maintenance organizations (HMOs) have historically avoided serving this expensive segment. In many cases, the inability to control costs results from failing to understand the importance of specialist reimbursement mechanisms. This article examines the importance of these mechanisms and describes how to successfully capitate specialty physicians in a Medicare risk program. The article also includes a case study on a successful capitation agreement between an HMO and a specialty group.

Quantitative electroencephalographic subtyping of obsessive-compulsive disorder.

Current neuropsychological, electrophysiological, and other imaging data strongly suggest the existence of a neurobiological basis for obsessive-compulsive disorder (OCD), which was long considered to be exclusively of psychogenic origin. The positive response of some OCD patients to neurosurgery, as well as the efficacy of agents that selectively block serotonin reuptake, lends further support to a biological involvement. However, a survey of the treatment literature reveals that only 45-62% of OCD patients improve with these specific medications. In a pilot study using a quantitative electroencephalographic (QEEG) method known as neurometrics, in which QEEG data from OCD patients were compared statistically with those from an age-appropriate normative population, we previously reported the existence of two subtypes of OCD patients within a clinically homogeneous group of patients who met DSM-III-R criteria for OCD. Following pharmacological treatment, a clear relationship was found between treatment response and neurometric cluster membership. In this study, we have expanded the OCD population, adding patients from a second site, and have replicated the existence of two clusters of patients in an enlarged, statistically more robust population. Cluster 1 was characterized by excess relative power in theta, especially in the frontal and frontotemporal regions; cluster 2 was characterized by increased relative power in alpha. Further, 80.0% of the members of cluster 1 were found to be nonresponders to drug treatment, while 82.4% of the members of cluster 2 were found to be treatment responders.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoembryonic antigen assay in fine needle aspirate of pancreas: a diagnostic adjunct to cytology.

Carcinoembryonic antigen (CEA) was measured in 21 consecutive fine needle aspirates (FNA) of solid pancreatic lesions from 20 patients to determine whether elevated levels would predict the presence of pancreatic carcinoma in cytologically negative aspirates. Final diagnoses were correlated with clinical, radiologic, and pathologic (four patients) findings and follow-up. Twenty aspirates had malignancy, and one was benign. FNAs were performed under radiologic guidance with a 22-gauge Chiba needle and a 20-ml syringe. Cytologic examination was rendered on Papanicolaou-stained slides and, when available, hematoxylin and eosin-stained cell blocks. CEA was measured by enzyme immunoassay (Abbott Laboratories). Sensitivity of cytologic diagnosis was 80%; specificity was 100%. With 5 ng/ml as cutoff, the sensitivity of CEA for malignancy was 70% and for adenocarcinoma of pancreas, 78%; the specificity was 100%. The mean CEA in pancreatic carcinoma was 152.1 ng/ml (range 1.4 to greater than 880 ng/ml). The mean CEA for lymphoma, metastatic lung carcinoma, and benign aspirate was 1.0 ng/ml. Elevated CEA was diagnostic of pancreatic carcinoma in three cytologically negative aspirates. Combined sensitivity of CEA and cytology was 95%. Elevated CEA in FNA of pancreas increases the sensitivity of cytologic diagnosis and may suggest carcinoma in cytologically negative aspirates.