Effectiveness of thin-layer preparations vs. conventional Pap smears in a blinded, split-sample study. Extended cytologic evaluation.

OBJECTIVE: To further evaluate the effectiveness of the AutoCyte PREP thin-layer slide preparation (TriPath Imaging, Inc., Burlington, North Carolina) as compared to conventional Pap smears. STUDY DESIGN: A split-sample, blinded evaluation of matched thin-layer preparations and conventional smears from 2,438 patients was performed. This material was enriched by including 260 cases of high grade squamous intraepithelial lesions (HSILs) and cancer cases from an earlier study. Many of these cases were difficult to diagnose, containing very few abnormal cells on one or both matching slides. The preparations were evaluated multiple times by both thin-layer-inexperienced and -experienced cytology professionals to better compare performance related to preparation quality alone. RESULTS: The initial evaluations of the slides by personnel with only brief training in thin-layer interpretation demonstrated equivalent performance for the two preparations. The reevaluation study by cytology professionals with several months of thin-layer experience demonstrated a statistically significant improvement in detection of both LSIL and HSIL lesions using AutoCyte PREP slides. There was also a statistically significant improvement in the number of satisfactory samples using the AutoCyte PREP method. CONCLUSION: The study demonstrated that the AutoCyte PREP thin-layer slide preparation is at least equivalent to conventional Pap smears in the detection of LSIL and HSIL, even when evaluated by cytology professionals who have been newly trained in the thin-layer method and that, with increased experience, the thin-layer AutoCyte PREP slide preparation method showed a statistically significant improvement in disease detection.

Psychophysiological effects of relaxation training in children.

OBJECTIVES: This study compares the effects of progressive muscle relaxation and an imagery-based relaxation training on childrens' physiological and subjective responses in a randomized controlled trial. DESIGN: Sixty-four children aged 9 to 13 years were randomly allocated to either one of three experimental conditions: progressive muscle relaxation, imagery-based relaxation or a control condition (neutral story). There were five training sessions in each condition. METHOD: Heart rate (HR), skin conductance level (SCL), and skin temperature (ST) were measured continuously during a 5-minute baseline period, an 8-minute relaxation training period, and a 5-minute follow-up in each session. In addition, subjective ratings of mood and physical well-being were collected intermittently. RESULTS AND CONCLUSIONS: A physiological pattern indicating relaxation was most clearly associated with the imagery-based relaxation approach (decreases in HR and SCL), although ST remained unchanged. In contrast, progressive muscle relaxation led to an increase in HR during the training. The neutral story condition showed a similar trend as the imagery-based relaxation approach (although not reaching statistical significance). Furthermore, children's ratings of positive mood and physical wellbeing increased during baseline and training periods, but there were no differences between training conditions. The results indicate psychophysiological effects of relaxation instructions which, however, are not specific for systematic relaxation training.

Study of interaction between agonists and asn293 in helix VI of human beta(2)-adrenergic receptor.

Previously, we demonstrated the involvement of Asn293 in helix VI of the human beta(2)-adrenergic receptor in stereoselective agonist recognition and activation. In the present study, we have further explored the role of this residue by synthesizing derivatives of isoproterenol and clenbuterol, two beta-adrenergic receptor agonists. We analyzed their efficacy and affinity on the wild-type and a mutant receptor (Asn293Leu). Each compound had similar efficacy (tau values) on both the wild-type and mutant receptor, although tau values varied considerably among the eight compounds studied. It appeared that one derivative of isoproterenol, but not of clenbuterol, showed a gain in affinity from the wild type to the mutant receptor. This derivative had a methyl substituent instead of the usual beta-OH group in the aliphatic side chain of isoproterenol, compatible with the more lipophilic nature of the leucine side chain. Such a "gain of function" approach through a combination of synthetic chemistry with molecular biology, may be useful to enhance our insight into the precise atomic events that govern ligand-receptor interactions.

Comparative pharmacology of human adenosine receptor subtypes - characterization of stably transfected receptors in CHO cells.

Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadeno sine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC50-value of 2.3 microM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine) as the most potent antagonist at this receptor subtype. The A3 receptor was characterized utilizing the nonselective agonist [3H]NECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A3 receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands.

Transduction of human melanoma cells with interleukin-2 gene reduces tumorigenicity and enhances host antitumor immunity: a nude mouse model.

Human melanoma tumor cells were genetically modified in vitro by transferring the interleukin-2 (IL-2) gene via a retroviral vector into established or fresh tumor cells. In addition, human melanoma cells were transduced in vivo by the direct injection of the IL-2/retroviral vector into melanoma xenografts in nude mice. The gene-modified melanoma cells expressed the IL-2 cytokine gene and secreted biologically active IL-2. Transduction of melanoma cells with the IL-2 gene did not affect the antigenic profile of the cells, but caused a strong abrogation of their tumorigenicity. One million parental cells formed subcutaneous tumors in nude mice. In contrast, various doses of up to 20 x 10(6) IL-2-transduced cells failed to form tumor in the mice. Coinjection of IL-2-producing cells with parental cells inhibited tumor formation even when highly tumorigenic doses of parental cells were used. Histochemical analysis of the injection sites of IL-2-modified cells showed an influx of host immune cells, predominantly macrophages, as early as the third day after inoculation. Neutrophils, mast cells, and eosinophils were also seen in the inflammatory exudate. Eventually, transduced cells showed signs of degeneration and necrosis and ultimately died in 4 weeks. Macrophages were seen in parental tumor sites only during the first few days after injection, and then parental tumors exhibited fast, progressive growth. The study suggests that melanoma cells transduced with the IL-2 cytokine gene may provide an effective vaccine for melanoma patients, whereas the in vivo transduction of tumors with cytokine genes is feasible and may represent a novel approach for the immunotherapy of cancer patients.

Prenatal diagnosis of Sanfilippo syndrome type A by early amniocentesis.

Early amniocentesis performed at 13 weeks gestation was utilized to obtain amniocytes for culture. Sonicates of cultured amniocytes were used to measure heparin sulfamidase activity for assessment of the status of an at risk pregnancy for Sanfilippo syndrome, type A. The heparin sulfamidase activity was not detectable in cultured amniocytes of the fetus at risk while another enzyme, N-acetylglucosamine 6-sulfatase, was comparable to that of the control. Following termination of the pregnancy, various tissue from the fetus were used for assay of both enzymes. The sulfamidase activity was not detectable in any of the fetal tissue while the 6-sulfatase activity was present in all fetal tissue but varied in activity depending on the type of tissue. Cultured fetal skin and brain contained the highest enzyme activity while skin and liver contained the lowest.

A direct comparison of procedures for the detection of mycoplasma in tissue culture.

Mycoplasma contamination of cell cultures has been shown to perturb a number of immunologic parameters. Because such contamination is almost always introduced in the laboratory, the immunologist requires a procedure to screen his cell lines frequently for mycoplasma. Two procedures recently described for the detection of mycoplasma in cell cultures, the uridine-uracil incorporation procedure and a direct fluorescent assay, were compared with the standard procedures of agar culture and transmission electron microscopy. The results with uridine-uracil incorporation were totally non-concordant with those of any of the other 3 procedures and, moreover, were inconsistent through serial assays on the same cell culture. In contrast, the direct fluorescent assay, using the fluorochrome Hoechst 33258, yielded consistent results in full agreement with the agar culture data. Since the fluorescent assay is rapid and has discriminatory capability at least equivalent to that of agar culture, it would appear to be the method of choice for routine screening of cell cultures for mycoplasma.