The technical performance and clinical feasibility of telecolposcopy.

BACKGROUND: The purpose of our study was to demonstrate the technical performance and clinical feasibility of a telecolposcopic system through assessment of image transmission veracity, ease of office system implementation, and the patient's acceptance of the electronic image transmission. METHODS: We used a telecolposcopic system incorporating a custom software package that integrated patient history, current gynecologic status, epidemiologic risk factors, and colposcopic images for local medical documentation and transmission. Satisfaction questionnaires were developed to measure ease of implementation at the remote sites and the patients' acceptance of telecolposcopy. RESULTS: Seventy-nine women participated in our trial. From 3 to 20 images were captured for each woman, documenting cervical squamous intraepithelial lesions and vaginal and vulvar diseases. All images were received without distortions in color, size, or orientation. With complete visualization of the squamocolumnar junction there was an 86% agreement between the remote and review sites (kappa=.533, P=.019). The interobserver agreement for colposcopic impressions was 86% (kappa=.684, P <.001), and for colposcopic impressions with histology within one level of disease severity, 86% (kappa=.78, P <.001). Colposcopists' and patients' satisfaction with telecolposcopy was excellent. More than 95% of the women stated that they would rather have their colposcopy locally with electronic transmission if an experienced colposcopist were more than 25 miles away. CONCLUSIONS: The telecolposcopic system described in our study is technically feasible, can be implemented in an office system with limited technical support, and is preferred by women who have to travel many miles to receive referral health care.

Granulocyte/macrophage-colony-stimulating factor released by adenovirally transduced CT26 cells leads to the local expression of macrophage inflammatory protein 1alpha and accumulation of dendritic cells at vaccination sites in vivo.

Antigen presenting cells (APC) play an essential role in the generation of tumor-specific immune responses. Dendritic cells are the most potent of APC, capable of activating both antigen-specific CD4+ and CD8+ T cells. Previously, we have described how vaccination of mice with irradiated tumor cells producing granulocyte/macrophage-colony-stimulating factor (GM-CSF) induces tumor-specific immunity capable of protecting mice from a subsequent tumor challenge. The present study extends these findings to examine the types of APC infiltrating vaccination sites and the chemokines responsible for their recruitment. GM-CSF released from genetically engineered tumor cells led to the local accumulation of dendritic cells in and around the vaccination site. Quantification revealed a significant ten-fold increase in the number of dendritic cells infiltrating GM-CSF-producing as opposed to beta-galactosidase-producing (control) vaccination sites. Reverse transcription/polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis of vaccination sites revealed that MIP-1alpha may be responsible for dendritic cell infiltration into GM-CSF-producing tissues. These findings suggest that GM-CSF may indirectly recruit dendritic cells into vaccination sites through the local production of MIP-1alpha.

Trophoblastic cell lines generated from tumour necrosis factor receptor-deficient mice reveal specific functions for the two tumour necrosis factor receptors.

In mice and humans, expression of the tumour necrosis factor receptor-1 (TNF-R1) gene in placental trophoblast cells is constitutive whereas expression of the TNF-R2 gene is developmentally programmed. In order to study the individual functions of TNF-R1 and -R2 in this lineage, cell lines were generated from placental explants of homozygous matings of gestation day 10 outbred mice (Swiss-Webster), TNF-R1-deficient (TNF-R1-/-) and TNF-R2-/- transgenic mice as well as the background strain for the TNF-R2-/- mice (WT, C57BL/6x129). All of the cells exhibited trophoblast markers; they contained cytokeratin intermediate filaments, expressed alkaline phosphatase activity and displayed transferrin receptors, but were negative for vimentin filaments and the macrophage marker, F4/80. Analysis of DNA by polymerase chain reaction demonstrated the expected TNF-R genotype in each line. In experiments testing the effects of recombinant mouse TNF-alpha (rmTNF-alpha) on viability and proliferation of the cell lines, rmTNF-alpha modestly but dose-dependently inhibited the growth of WT and TNF-R2-/- cells while having no effect on TNF-R1-/- cells. Actinomycin D-treated WT and, to a lesser extent, TNF-R2-/- cells, were more sensitive to growth inhibition than untreated cells whereas TNF-R1-/- cell responses remained unchanged. These data indicated that rmTNF-alpha inhibits growth of trophoblastic cells through TNF-R1 and that newly synthesized protein(s) provide partial protection against toxicity. In contrast to the receptor species-specific effects on cell growth exerted by rmTNF-alpha, both TNF-R mediated inhibition of alkaline phosphatase activity. Collectively, the observations support the postulate that receptor expression is the key factor which determines the nature and extent of TNF-alpha effects on trophoblast cell growth and function.

Coronary sinus pacing prevents induction of atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is due to reentry, and its incidence has been shown to decrease after dual-site atrial or biatrial pacing. We investigated whether a simpler pacing approach via the distal coronary sinus (CSd) could eliminate AF inducibility by high right atrial (HRA) extrastimuli (APDs). We based our hypothesis on our previous observation that AF inducibility by HRA APDs was associated with conduction delays to the posterior triangle of Koch, whereas AF was never induced with CSd APDs, which were associated with minimal intra-atrial conduction delays. METHODS AND RESULTS: Programmed electrical stimulation was performed from the high right atrium and CSd, and bipolar recordings were obtained from the high right atrium, His bundle, posterior triangle of Koch, and coronary sinus. In 13 patients (age, 44+/-18 years), AF was reproducibly induced with a critically timed HRA APD (220+/-22 ms) delivered during HRA pacing. AF was not induced in any of the patients when HRA APDs were delivered during CSd pacing at the same critical coupling intervals. Coronary sinus APDs delivered during HRA pacing also were not associated with AF induction. The APD coupling interval measured at the posterior triangle of Koch during CSd pacing was significantly prolonged compared with the one measured during HRA pacing and AF induction (381+/-58 versus 263+/-37 ms; P<.0001). CONCLUSIONS: We propose that CSd pacing suppresses the propensity of HRA APDs to induce AF by limiting their prematurity at the posterior triangle of Koch and not allowing local conduction delay and local reentry to occur.

Decidual/trophoblast prolactin-related protein: characterization of gene structure and cell-specific expression.

Decidual/trophoblast PRL-related protein (d/tPRP) is a member of the PRL gene family and is dually expressed in uterine and placental tissues in a highly coordinated pattern during pregnancy. In the present study, we describe the isolation and characterization of the d/tPRP gene. A lambda DASH II Wistar-Kyoto rat genomic library was screened with a labeled d/tPRP complementary DNA, resulting in the isolation of two phage clones, RGLd-41 [17.7 kilobases (kb)] and RGLd-42 (15.8 kb). RGLd-41 alone was found to contain the full-length d/tPRP gene and was used for subsequent analyses. The d/tPRP gene possesses a six-exon, five-intron organization. Relative to other highly conserved members of the PRL gene family, d/tPRP contains a single small additional exon (exon 3) situated between exons 2 and 3 of the prototypical PRL gene. The region corresponding to exon 3 of d/tPRP encodes for a unique amino acid region found in a subset of PRL family members. A reverse transcription-PCR (RT-PCR) tissue survey for d/tPRP messenger RNA revealed that d/tPRP expression was restricted to decidual and trophoblast tissues. A single transcription start site 65 bp upstream of the initiation codon was identified in decidual tissue, whereas multiple transcription start sites ranging from 61-66 bp upstream of the initiation codon were detected in placental tissue. Various tissue culture systems (primary cultures and cell lines) were evaluated for d/tPRP expression and activation of a 3.96-kb d/tPRP promoter-luciferase reporter construct. Decidual, spongiotrophoblast, and trophoblast giant cell populations expressed d/tPRP and were capable of activating the d/tPRP promoter-reporter construct, whereas other cell types were ineffective. Limited d/tPRP promoter activation was noted in uterine stromal cell lines. In summary, d/tPRP possesses a unique six-exon, five-intron gene structure and exhibits cell-specific expression that is regulated at least in part by a 3.96-kb 5'-flanking region.

Dual expression of prolactin-related protein in decidua and trophoblast tissues during pregnancy in rats.

Decidual prolactin-related protein (dPRP) is a member of the prolactin gene family and is abundantly expressed in the rat deciduum. Previously, dPRP was shown to associate with heparin-containing molecules and was found to reside, at least in part, within the decidual extracellular matrix, where it was postulated to influence decidual cells and other cell types. The purpose of this investigation was to identify the cellular origin and the temporal and regional characteristics of dPRP expression in the rat uterus during pregnancy. Protein expression was evaluated by Western blot analysis, immunoprecipitation, and immunocytochemistry; dPRP mRNA expression was assessed by reverse transcriptase polymerase chain reaction and in situ hybridization. Decidual PRP was first detected at Day 6 of pregnancy or pseudopregnancy. Expression increased with the growth of the deciduum and then declined coincident with regression of decidual tissue. Throughout the first half of pregnancy or pseudo-pregnancy, dPRP and mRNA were predominantly localized to the antimesometrial deciduum of the developing conceptus. During the second half of gestation, expression also appeared in the chorioallantoic placenta. Trophoblast giant cells and spongiotrophoblast cells within the junctional zone of the chorioallantoic placenta expressed dPRP, as did the Rcho-1 trophoblast cell line. In conclusion, dPRP production is elevated from implantation until parturition through the participation of decidual (early pregnancy) and trophoblastic (late pregnancy) tissues.

Decidual prolactin-related protein: heterologous expression and characterization.

As a first step in understanding the role of decidual PRL-related protein (dPRP) during pregnancy, we have generated recombinant dPRP protein. In this report, we present data on the generation, purification, and characterization of recombinant dPRP protein. The dPRP complementary DNA was subcloned into the pMSXND vector, and the vector was transfected into Chinese hamster ovary (CHO) cells by electroporation. After appropriate selection, amplification, and induction procedures, recombinant dPRP was purified from conditioned medium of the CHO-dPRP cells using ultrafiltration, size-exclusion chromatography, and reverse phase HPLC. Recombinant dPRP was found to possess electrophoretic mobility, immunoreactivity, and N-terminal amino acid sequence identical to those of dPRP isolated from decidual tissue. Polyclonal antibodies were generated to the recombinant dPRP and used for Western blot analysis. dPRP is capable of binding heparin, and a significant fraction of synthesized dPRP resides within the decidual extracellular matrix. Recombinant dPRP failed to bind to PRL receptors and showed no stimulatory activity in the PRL-dependent rat Nb2 lymphoma cell proliferation assay. Additional studies have shown that heterologous expression of dPRP in CHO cells significantly increased the ability of CHO cells to form tumors in athymic mice. In conclusion, recombinant dPRP possesses characteristics similar to those of dPRP of decidual origin and is a heparin-binding protein that may facilitate the establishment of pregnancy.

The relationship of death anxiety with age and psychosocial maturity.

For this study, 194 respondents completed a biographical data sheet, the Templer (1970) Death Anxiety Scale and the Constantinople (1973) Inventory of Psychosocial Development to help assess the relationship among death anxiety, age, and psychosocial maturity. Findings showed that psychosocial maturity was a better predictor of death anxiety than age was. However, both variables were significantly negatively correlated with death anxiety, revealing that as psychosocial maturity and age increase, death anxiety decreases.

Morning peak in ventricular tachyarrhythmias detected by time of implantable cardioverter/defibrillator therapy. The CPI Investigators.

BACKGROUND: A morning peak in occurrence of sudden cardiac death has been identified in epidemiological studies, but the studies are subject to selection bias, with the exclusion of unwitnessed deaths, which are more likely to occur at night. The recent availability of implantable cardioverter/defibrillators that record the time of ventricular tachyarrhythmias requiring either pacing or shock therapy provides an opportunity to clarify the timing of ventricular tachyarrhythmias predisposing to sudden cardiac death. Analysis of the timing of arrhythmias in different patient subgroups, such as patients with poor left ventricular function, may provide further insight into the mechanism of onset of sudden cardiac death. METHODS AND RESULTS: We studied patients in whom a cardioverter/defibrillator (Ventak PRx) was implanted between September 1990 and September 1993 in US centers. Events that could be timed occurred in 483 patients. With an RR cycle length of 240 ms as a cutoff, corresponding to a heart rate of 250 beats per minute, episodes were categorized as rapid (n = 1217) or less rapid (n = 9266) ventricular tachyarrhythmias. A higher proportion of both rapid and less rapid ventricular tachyarrhythmias began in the late morning compared with other times of the day. The subgroup of patients with ejection fraction < 20% at the time of implantation demonstrated a more uniform 24-hour distribution of tachycardias < or = 250 beats per minute than patients with higher left ventricular ejection fraction. CONCLUSIONS: Further investigation of the late morning peak and of precipitants of ventricular tachyarrhythmias by use of data from the implantable cardioverter/defibrillator may provide insight into the pathophysiological mechanisms causing sudden cardiac death.

Immunocytochemical localization of desferrioxamine in the kidney, liver and brain of the developing and adult mouse: implications for drug processing and therapeutic mechanisms.

The iron chelator, desferrioxamine, has been shown to interfere with hydroxyl radical formation, which mediates tissue damage in several disease states. In this study, young and adult mice were given intraperitoneal injections of desferrioxamine and sacrificed after 1, 24 or 72 hr, and adult rats were given intraventricular injections and sacrificed after 24 or 72 hr. Immunohistochemical experiments, that utilized an anti-desferrioxamine antiserum, were performed to localize the cellular distribution of the drug in the kidney, liver and brain. In the kidney, the findings were as follows: 1) at 0 days, there was heterogeneous distribution of desferrioxamine, which suggests that there are functional differences between nephrons in the immature kidney; 2) with increasing age, the degree of staining decreased, which suggests that the efficiency of renal clearance of desferrioxamine increases with maturity; 3) punctate staining was superimposed onto diffuse staining, which suggests that the drug can get taken up in pinocytotic vesicles during tubular reabsorption; and 4) staining was present at 72 hr, which suggests a prolonged clearance period. In the liver, there was an absence of staining at 24 and 72 hr and only diffuse staining at 1 hr, which suggests a rapid processing of the drug by the liver. In the normal mouse brain, the drug was found localized to the choroid plexus and ependymal cells at all ages, and the staining decreased with increasing time following injection. These results indicate that there are differences in the way that desferrioxamine is processed by cells in different tissues and indicate possible therapeutic mechanisms.

Role of changes in [Ca2+]i in energy deprivation contracture.

Mechanisms of energy deprivation contracture were investigated in cultured chick embryo ventricular cells. In the presence of zero-extracellular-Na+, (choline chloride substitution)-nominal-zero-Ca2+ [( Ca2+] approximately 5 microM), exposure of ventricular cells to 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG)-zero-glucose solution resulted in the development of a contracture (video motion detector) in 5.9 +/- 0.5 minutes. Early after contracture development, the resupply of extracellular Na+, in the continued presence of CN + 2-DG, resulted in a rapid partial relaxation (t1/2 = 1.9 +/- 0.3 seconds), associated with an increase in 45Ca efflux, presumably due to transsarcolemmal Ca2+ extrusion due to Na+-Ca2+ exchange. Resupply of glucose and removal of CN + 2-DG, in the continued absence of Na+, resulted in an initially slower (t1/2 = 11.6 +/- 2.5 seconds), but more complete relaxation of contracture, which was not associated with increased Ca2+ efflux. Pretreatment with 20 mM caffeine delayed the onset of contracture (9.2 +/- 1.1 minutes) and resulted in a contracture that could not be relaxed by resupply of external Na+ only. Studies using the fluorescent Ca2+ probe indo 1 demonstrated that in zero-Na+-zero-Ca2+ solutions, contracture due to CN + 2-DG was associated with an initial rise in [Ca2+]i but that this did not account for all of contracture force development. In cells exposed to CN + 2-DG in the presence of normal extracellular Na+ and Ca2+ concentrations, a small rise in [Ca2+]i was associated with initial contracture development, consistently preceding the development of a larger accelerated contracture presumably due to ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ryanodine and caffeine on contractility, membrane voltage, and calcium exchange in cultured heart cells.

To investigate the mechanisms of action of ryanodine and caffeine, changes in mechanical and electrical activity caused by these agents were correlated with alterations in 45Ca fluxes and cell Ca contents in chick embryo ventricular cell monolayer cultures. Ryanodine (10(-10)-10(-5) M) irreversibly decreased contraction amplitude by 10-70% relative to control in a concentration-dependent manner with minimal effects on electrical activity. Ryanodine caused a slight decrease in rapid 45Ca uptake, but no change in total exchangeable calcium content or rapid 45Ca efflux. Caffeine (1-20 mM) caused a transient (less than 10 seconds) 5-12% increase in contraction amplitude followed by a sustained 9-76% decrease in contraction amplitude and a 10 mV decrease in diastolic membrane voltage. Caffeine caused a decrease in rapid 45Ca uptake, a decrease in total exchangeable calcium content, and an increase in rapid 45Ca efflux. These results suggest that caffeine produces a decrease in sarcoplasmic reticulum (SR) Ca2+ uptake, and/or an increase in SR Ca2+ release that eventually depletes the SR of Ca2+, presumably accounting for the negative inotropic effect. The ryanodine effects on contraction are more difficult to account for solely in terms of alterations of transsarcolemmal Ca2+ fluxes and Ca2+ contents. Our data indicate an important role for the SR in excitation-contraction coupling in cultured chick embryo ventricular cells and suggest that SR Ca2+ is part of the rapidly exchanging Ca2+ compartment noted in 45Ca flux studies.

External Na-independent Ca extrusion in cultured ventricular cells. Magnitude and functional significance.

The relative magnitudes and functional significance of Ca extrusion by Na-Ca exchange and by an Nao-independent mechanism were investigated in monolayer cultures of chick embryo ventricular cells. Abrupt exposure of cells in 0-Nao, nominally 0-Cao solution to 20 mM caffeine produced a large contracture (3.94 +/- 0.90 micron of cell shortening) that relaxed with a t1/2 of 8.60 +/- 1.22 s. An abrupt exposure to caffeine plus 140 mM Na resulted in a contracture that was smaller in amplitude (1.53 +/- 0.50 micron) and relaxed much more rapidly (t1/2 = 0.77 +/- 0.09 s). An abrupt exposure to caffeine in 0-Nao solutions produced an increase in 45Ca efflux that persisted for 20 s, and a net loss of Ca content, determined by atomic absorption spectroscopy (AAS), of approximately 4 nmol/mg protein, within 35 s. A comparable net loss of Ca was demonstrated in the presence of 100 microM [Ca]o. The abrupt exposure of cultured cells to 0 Nao in 1.8 mM Ca produced a Ca uptake, estimated with 45Ca, of 3.2 nmol/mg protein X 15 s, but produced no increase in cell Ca content (AAS). In cells in which a 30% increase in Nai was produced by 5 min exposure to 10(-6) M ouabain, the abrupt exposure to 0 Nao produced a Ca uptake of 6 nmol/mg protein X 15 s and an increase in Ca content (AAS) of 4 nmol/mg protein. We conclude that there is an Nao-independent mechanism for Ca extrusion in these cells, presumably a Ca-ATPase Ca pump, with a limited Ca transport capacity of no more than 2 nmol/mg protein X 15 s. This is five times smaller than the demonstrated maximum capacity of the Na-Ca exchanger in these cells. The relaxation of twitch tension in these cells seems to be dependent primarily on sarcoplasmic reticulum uptake of Ca, with a secondary role provided by the Na-Ca exchanger. The Ca pump appears to contribute little to beat-to-beat relaxation.