Investigation of the safety and feasibility of AAV1/SERCA2a gene transfer in patients with chronic heart failure supported with a left ventricular assist device - the SERCA-LVAD TRIAL.

The SERCA-LVAD trial was a phase 2a trial assessing the safety and feasibility of delivering an adeno-associated vector 1 carrying the cardiac isoform of the sarcoplasmic reticulum calcium ATPase (AAV1/SERCA2a) to adult chronic heart failure patients implanted with a left ventricular assist device. The SERCA-LVAD trial was one of a program of AAV1/SERCA2a cardiac gene therapy trials including CUPID1, CUPID 2 and AGENT trials. Enroled subjects were randomised to receive a single intracoronary infusion of 1 × 1013 DNase-resistant AAV1/SERCA2a particles or a placebo solution in a double-blinded design, stratified by presence of neutralising antibodies to AAV. Elective endomyocardial biopsy was performed at 6 months unless the subject had undergone cardiac transplantation, with myocardial samples assessed for the presence of exogenous viral DNA from the treatment vector. Safety assessments including ELISPOT were serially performed. Although designed as a 24 subject trial, recruitment was stopped after five subjects had been randomised and received infusion due to the neutral result from the CUPID 2 trial. Here we describe the results from the 5 patients at 3 years follow up, which confirmed that viral DNA was delivered to the failing human heart in 2 patients receiving gene therapy with vector detectable at follow up endomyocardial biopsy or cardiac transplantation. Absolute levels of detectable transgene DNA were low, and no functional benefit was observed. There were no safety concerns in this small cohort. This trial identified some of the challenges of performing gene therapy trials in this LVAD patient cohort which may help guide future trial design.

Quantification of glycolic acid in cosmetic products using reversed phase high performance liquid chromatography.

Many cosmetics contain keratolytic hydroxy acids to correct the effects of photoageing on human skin. Although methods exist for quantifying the alpha-hydroxy acid, glycolic acid in aqueous media, accurate methods for quantification in mixed hydrophobic and aqueous cosmetic creams and lotions are lacking. Glycolic acid was extracted from cosmetics using aqueous tetrahydrofuran (THF), separated with strong-anion exchange cartridges, and quantified by high performance liquid chromatography (HPLC) with UV-VIS detection without the paired-ion reagents. In a recovery experiment, the mean accuracy of the method was 100.6%. The dynamic range of the method allows for the detection of glycolic acid at concentrations used in over-the-counter cosmetics.

Treatment With tobramycin solution for inhalation in bronchiectasis patients with Pseudomonas aeruginosa.

A randomized, placebo-controlled, multicenter trial evaluated the safety and efficacy of 300 mg aerosolized tobramycin solution for inhalation (TSI) administered twice daily for 4 weeks in 74 bronchiectasis patients colonized with Pseudomonas aeruginosa (PA). Patients were evenly divided between TSI therapy and placebo. After 2 weeks of treatment, patients treated with TSI had a mean reduction in sputum PA density of 4.8 log(10.) This reduction was maintained for the duration of treatment. The placebo group showed no change in PA density during the study. Two weeks after the end of therapy, PA had been eradicated in 13 TSI-treated patients. PA was not eradicated in any placebo patients. Among those colonized with Staphylococcus aureus at baseline, 6 of 9 patients in the TSI group and 2 of 9 patients in the placebo group were culture negative for this organism 2 weeks posttreatment. Sixty-two percent of TSI-treated patients were judged by a physician as having an improved general health status, compared with 38% of placebo-treated patients. Dyspnea, wheezing, and chest tightness were reported more frequently in the TSI-treated patient group than in the placebo-treated patient group.

Basal cell proliferation in female SKH-1 mice treated with alpha- and beta-hydroxy acids.

Alpha- and beta-hydroxy acids are compounds that have been used extensively in cosmetic and dermatological formulations. Clinical and qualitative effects of alpha- and beta-hydroxy acids have been well characterized, but little is known about their mechanism of action or acute and chronic biochemical effects. In the present study, we examined the acute proliferative effects of glycolic and salicylic acids on cell proliferation in the epidermis of SKH-1 female mice, using BrdU incorporation as a marker of epidermal proliferation. In preliminary experiments, we observed an increase in the rate of proliferation after 3 days of treatment with 10% glycolic acid-containing cream and this was sustained throughout a 6.5-week (treatment 5 days/week) time course compared with untreated control animals. After each treatment with cream containing glycolic acid there was a wave of proliferation that was maximal 12 to 16 h (significant at p < 0.05) after treatment, followed by a subsequent increase in epidermal thickness at 18 to 20 h (significant at p < 0.05). The effects of the concentration and pH level of glycolic acid- and salicylic acid-containing creams on the rate of proliferation and increases in skin thickness in SKH-1 epidermis were also investigated. We observed a dose-dependent increase in epidermal proliferation of animals treated with either glycolic or salicylic acid. A similar time-dependent response was observed in the epidermal thickness in animals treated with salicylic acid, but not with glycolic acid. Differences in pH (3.5 or 4.0) had no significant effect on either epidermal proliferation or skin thickness. The data that we present here should be useful in characterizing not only the beneficial but also the adverse effects that occur following acute or chronic usage of alpha-hydroxy acids.

Azithromycin-containing regimens for treatment of Mycobacterium avium complex lung disease.

Ninety-two patients were assessable in 3 consecutive, open, noncomparative, prospective, controlled, single-center trials of the use of multidrug regimens that contain azithromycin for treating pulmonary Mycobacterium avium complex (MAC) disease. Azithromycin was provided at a dose of 300-600 mg per day with oral companion drugs administered daily (regimen A, 29 patients); 600 mg 3 times weekly (t.i.w.), with oral companion drugs administered daily (regimen B, 20 patients); and 600 mg (t.i.w.), with oral companion drugs administered t.i.w. (regimen C, 43 patients). All regimens included rifabutin (or rifampin) and ethambutol as companion drugs as well as initial streptomycin. Treatment success was defined as 12 months of negative cultures while on therapy. Treatment failure was defined as sputum culture positivity after at least 6 months of therapy. Of the patients in each regimen who reached study end points, 17 of 29 (59%) were in regimen A, 11 of 20 (55%) were in regimen B, and 28 of 43 (65%) were in regimen C met the treatment success criterion. There were no statistically significant differences in outcome between the 3 regimens. These studies demonstrate the effectiveness of daily and t.i.w. regimens containing azithromycin for treatment of MAC lung disease.

Descriptions of two new species of Eimeria (Apicomplexa: Eimeriidae) and of Eimeria chinchillae-like oocysts from the plains vizcacha Lagostomus maximus (Desmarest, 1817) (Rodentia: Chinchillidae) from Argentina.

Of 163 fecal samples collected between March 1997 and February 1999 from the plains vizcacha, Lagostomus maximus (Rodentia: Chinchillidae), 19 (12%) were found to be positive for coccidia. All species are from the genus Eimeria and 2 are described here as new. The third species is consistent with the description of Eimeria chinchillae De Vos and Van der Westhuizen, 1968. Sporulated oocysts of Eimeria lagostomi n. sp. are ellipsoidal to subspheroidal, 35.7 x 30.9 (26-36 x 30-41), with a length:width (L/W) ratio of 1.2 (1.0-1.3), without a micropyle (M) or polar granule (PG), but with an oocyst residuum (OR) comprised of a round, compact mass of many small granules. The sporocysts are lemon-shaped, 14.2 x 10.2 (9-11 x 11-16), with an L/W ratio of 1.4 (1.2-1.7) and have a Stieda body (SB) and sporocyst residuum (SR). Eimeria vizcacho n. sp. has sporulated oocysts that are subspheroidal, 26.4 x 23.4 (21-27 x 23-31), with an L/W ratio of 1.1 (1.1-1.2), lack an M and OR, but have 1-2 PGs. Sporocysts are elongate-ellipsoidal, 14.3-9.0 (8-10 x 13-15), with an L/W ratio of 1.6 (1.4-1.8), lack an SB, but have 2 SR; the first a small mass of granules lying between the sporozoites in the middle or at 1 end, and the second also of many small granules, always at the opposite end. Sporulated oocysts of the E. chinchillae-like organism are ellipsoidal to subspheroidal, 20.7 x 17.5 (15-22 x 17-24) with an L/W ratio of 1.2 (1.0-1.3), lack an M and OR, but have 0-3 PGs. Sporocysts are ellipsoidal, 10.7-6.9 (6-8 x 8-13) with an L/W ratio of 1.55 (1.3-1.8) and have an SB and SR.

Relative effectiveness of HZE iron-56 particles for the induction of cytogenetic damage in vivo.

One of the risks of prolonged manned space flight is the exposure of astronauts to radiation from galactic cosmic rays, which contain heavy ions such as (56)Fe. To study the effects of such exposures, experiments were conducted at the Brookhaven National Laboratory by exposing Wistar rats to high-mass, high-Z, high-energy (HZE) particles using the Alternating Gradient Synchrotron (AGS). The biological effectiveness of (56)Fe ions (1000 MeV/nucleon) relative to low-LET gamma rays and high-LET alpha particles for the induction of chromosome damage and micronuclei was determined. The mitotic index and the frequency of chromosome aberrations were evaluated in bone marrow cells, and the frequency of micronuclei was measured in cells isolated from the trachea and the deep lung. A marked delay in the entry of cells into mitosis was induced in the bone marrow cells that decreased as a function of time after the exposure. The frequencies of chromatid aberrations and micronuclei increased as linear functions of dose. The frequency of chromosome aberrations induced by HZE particles was about 3.2 times higher than that observed after exposure to (60)Co gamma rays. The frequency of micronuclei in rat lung fibroblasts, lung epithelial cells, and tracheal epithelial cells increased linearly, with slopes of 7 x 10(-4), 12 x 10(-4), and 11 x 10(-4) micronuclei/binucleated cell cGy(-1), respectively. When genetic damage induced by radiation from (56)Fe ions was compared to that from exposure to (60)Co gamma rays, (56)Fe-ion radiation was between 0.9 and 3.3 times more effective than (60)Co gamma rays. However, the HZE-particle exposures were only 10-20% as effective as radon in producing micronuclei in either deep lung or tracheal epithelial cells. Using microdosimetric techniques, we estimated that 32 cells were hit by delta rays for each cell that was traversed by the primary HZE (56)Fe particle. These calculations and the observed low relative effectiveness of the exposure to HZE particles suggest that at least part of the cytogenetic damage measured was caused by the delta rays. Much of the energy deposited by the primary HZE particles may result in cell killing and may therefore be "wasted" as far as production of detectable micronuclei is concerned. The role of wasted energy in studies of cancer induction may be important in risk estimates for exposure to HZE particles.

Induction and repair of HZE induced cytogenetic damage.

Wistar rats were exposed to high-mass, high energy (HZE) 56Fe particles (1000 GeV/AMU) using the Alternating Gradient Synchrotron (AGS). The animals were sacrificed at 1-5 hours or after a 30-day recovery period. The frequency of micronuclei in the tracheal and the deep lung epithelial cells were evaluated. The relative effectiveness of 56Fe, for the induction of initial chromosome damage in the form of micronuclei, was compared to damage produced in the same biological system exposed to other types of high and low-LET radiation. It was demonstrated that for animals sacrificed at short times after exposure, the tracheal and lung epithelial cells, the 56Fe particles were 3.3 and 1.3 times as effective as 60Co in production of micronuclei, respectively. The effectiveness was also compared to that for exposure to inhaled radon. With this comparison, the 56Fe exposure of the tracheal epithelial cells and the lung epithelial cells were only 0.18 and 0.20 times as effective as radon in the production of the initial cytogenetic damage. It was suggested that the low relative effectiveness was related to potential for 'wasted energy' from the core of the 56Fe particles. When the animals were sacrificed after 30 days, the slopes of the dose-response relationships, which reflect the remaining level of damage, decreased by a factor of 10 for both the tracheal and lung epithelial cells. In both cases, the slope of the dose-response lines were no longer significantly different from zero, and the r2 values were very high. Lung epithelial cells, isolated from the animals sacrificed hours after exposure, were maintained in culture, and the micronuclei frequency evaluated after 4 and 6 subcultures. These cells were harvested at 24 and 36 days after the exposure. There was no dose-response detected in these cultures and no signs of genomic instability at either sample time.

DNA adduct formation by Fusarium culture extracts: lack of role of fusarin C.

Fusarium fungi have been shown to infect corn and other crops worldwide, and have a significant impact on human health through loss of crops or contamination of food with mycotoxins. Isolates of Fusarium fungi from an area of South Africa with high incidence of esophageal cancer have been shown to induce esophageal and liver cancer in rats. Several isolates of Fusarium fungi were grown on corn to determine if genotoxic products were produced. We report the incubation of methanol extracts of Fusarium verticillioides cultures with DNA in the presence of rat liver fractions (S9) resulted in the formation of a unique DNA adduct that was detected by (32)P-postlabeling. Fusarin C was purified from cultures of Fusarium verticillioides RRC 415, and was not responsible for the formation of the DNA adduct. Treatment of the methanolic extracts with ultraviolet B radiation reduced the fusarin C content in the extract; however, this had no effect on the formation of the DNA adduct following incubation of the extract with DNA and S9. The unique DNA adduct was formed following the incubation of several Fusarium verticillioides isolates from the US and South Africa, while extracts of cultures of Fusarium graminearium and Fusarium sacchari isolates formed very little of the DNA adduct when incubated with DNA and S9. These data suggest that neither fusarin C nor any of its metabolites are responsible for formation of the DNA adduct, and that an unidentified compound is present in F. verticillioides cultures that forms a DNA adduct, and may be important in the etiology of human esophageal cancer.

Tobramycin solution for inhalation reduces sputum Pseudomonas aeruginosa density in bronchiectasis.

We conducted a placebo-controlled, double-blind, randomized study to evaluate the microbiological efficacy and safety of inhaled tobramycin for treatment of patients with bronchiectasis and Pseudomonas aeruginosa. Patients were randomly assigned to receive either tobramycin solution for inhalation (TSI) (n = 37) or placebo (n = 37), which was self-administered twice daily for 4 wk and followed by 2-wk off-drug. At Week 4, the TSI group had a mean decrease in P. aeruginosa density of 4.54 log(10) colony-forming units (cfu)/g sputum compared with no change in the placebo group (p < 0.01). At Week 6, P. aeruginosa was eradicated in 35% of TSI patients but was detected in all placebo patients. Investigators indicated that 62% of TSI patients showed an improved medical condition compared with 38% of placebo patients (odds ratio = 2.7, 95% confidence interval [CI] 1.1 to 6.9). Tobramycin-resistant P. aeruginosa strains developed in 11% of TSI patients and 3% of placebo patients (p = 0.36). The mean percent change in FEV(1) percent predicted from Week 0 to Week 4 was similar for the TSI and placebo groups (p = 0.41). More TSI-treated patients than placebo patients reported increased cough, dyspnea, wheezing, and noncardiac chest pain, but the symptoms did not limit therapy. Additional study is warranted to further evaluate TSI in bronchiectasis patients.

Fumonisin B1 induces apoptosis in cultured human keratinocytes through sphinganine accumulation and ceramide depletion.

Fumonisin B1 stimulates apoptosis in a variety of cell types and tissues. We examined the role of sphingolipid changes in fumonisin B1-stimulated apoptosis. Sphinganine accumulated rapidly, sphingosine levels remained unchanged, and ceramides decreased during fumonisin B1 exposure. Increased DNA fragmentation, decreased viability, and apoptotic morphology were observed in cells exposed to fumonisin B1, sphinganine, or N-acetylsphingosine. Co-exposure to N-acetylsphingosine or beta-chloroalanine, which blocks sphinganine accumulation, partially protected cells from fumonisin B1-induced apoptosis. These results illustrate three sphingolipid-dependent mechanisms for inducing apoptosis: accumulation of excess ceramide, accumulation of excess sphinganine, and depletion of ceramide or complex sphingolipids derived from ceramide.

Polycyclic aromatic hydrocarbons enhance terminal cell death of human ectocervical cells.

Polycyclic aromatic hydrocarbons (PAH) are a class of chemical carcinogens whose active metabolites form DNA adducts, resulting in specific mutational events. The tumor suppressor protein p53 is believed to play a pivotal role in the ability of cells to response to DNA damage, resulting in either cell cycle arrest in G1 or apoptosis under conditions of excessive damage. This growth inhibition is associated with the concomitant induction of p53 and enhanced terminal cell differentiation. In this study we evaluated the effects of PAH on cell growth, cell differentiation, xenobiotic metabolism, and DNA adduct levels in normal ectocervical epithelial cells (ECE) and compared them to cervical cells whose p53 have been inactivated either by binding to viral HPV E6 oncogene (ECE16-1) or by mutation (C33A). The PAH 3-methylcholanthrene (3MC) inhibited normal ECE and to a lesser extent ECE16-1 cell proliferation. Not only did the growth inhibition occur at lower concentrations in the normal cells but the extent of inhibition was also greater in normal as compared to immortalized cells. Benzanthracene (BA) had a minor effect on normal ECE cells with no effect on immortalized ECE16-1 cells. C33A cell growth was unaffected by 3MC and BA. Terminal cell death was enhanced only in normal ECE cells as evidenced by increased envelope formation and was paralleled by an increase in the level of p53 following 3MC treatment. The differentiation status of the 3MC-treated cells was similar to untreated cells as indicated by three independent markers of cell differentiation; transglutaminase, involucrin, keratin expression. There was no difference in the pattern or level of DNA adducts formed in normal and immortalized cells following 3MC treatment. In addition the basal level of metabolism of 14C-BaP to phenols, diols and quinnones was unaltered by pretreatment with either 3MC or BA. These results demonstrate that immortalized cervical cells are less sensitive to toxicant damage [i.e. cell proliferation and terminal differentiation], and as a result, immortalized cells proliferate in the presence of genotoxic damage and are at increased risk for mutations and cancer.

Initial (6-month) results of three-times-weekly azithromycin in treatment regimens for Mycobacterium avium complex lung disease in human immunodeficiency virus-negative patients.

Two consecutive, open, prospective trials of intermittent azithromycin (600 mg), usually given Monday, Wednesday, and Friday (TIW) for Mycobacterium avium complex (MAC) lung disease were initiated in human immunodeficiency virus-negative patients. Regimen A consisted of TIW azithromycin and daily ethambutol (15 mg/kg/day), daily rifabutin (300 mg/day), and initial twice weekly (BIW) streptomycin. Regimen B consisted of TIW azithromycin, TIW ethambutol (25 mg/kg/dose), TIW rifabutin (600 mg/dose), and initial BIW streptomycin. Of 19 patients enrolled in regimen A who completed at least 6 months of therapy, 14 (74%) had sputum samples become culture-negative. Of 39 patients enrolled in regimen B who completed at least 6 months of therapy, 24 (62%) had sputum conversion. These sputum conversion rates are comparable to previous rates at 6 months in patients receiving daily clarithromycin- or azithromycin-containing regimens. No resistance to azithromycin emerged with either regimen. This is the first study to demonstrate the efficacy of intermittent administration of medication for MAC lung disease.

Role of nitroreductases but not cytochromes P450 in the metabolic activation of 1-nitropyrene in the HepG2 human hepatoblastoma cell line.

1-Nitropyrene is an environmental contaminant that is mutagenic in many prokaryotic and eukaryotic systems, including the hypoxanthine-guanosine phosphoribosyl transferase (HGPRT) locus in the human hepatoma cell line HepG2. Metabolism and DNA adduct formation of [3H]1-nitropyrene in the HepG2 were quantified to understand the role of nitroreduction and/or cytochrome P450-mediated C-oxidation of 1-nitropyrene in DNA adduct formation and mutagenicity. In uninduced HepG2 cells, 10 microM [3H]1-nitropyrene was metabolized principally by nitroreduction to 1-aminopyrene (516 pmol/24 hr/10(6) cells), and by cytochrome P450-mediated C-oxidation to K-region trans-dihydrodiols (37 pmol/24 hr/10(6) cells), 1-nitropyren-3-ol (51 pmol/24 hr/10(6) cells), and 1-nitropyren-6-ol and 1-nitropyren-8-ol (77 pmol/24 hr/10(6) cells). Pretreatment of the HepG2 cells for 24 hr with 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a complete change in the metabolism of [3H]1-nitropyrene, with 1-nitropyren-6-ol and 1-nitropyren-8-ol formation (449 pmol/24 hr/10(6) cells) being 80-fold greater than 1-aminopyrene formation (6 pmol/24 hr/10(6) cells). This increase in C-oxidation of 1-nitropyrene was consistent with increased levels of cytochrome P450 1A. The only DNA adduct detected using the 32P-postlabeling assay in the HepG2 cells administered 1-nitropyrene was N-(2'-deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP). Induction of C-oxidative metabolism through TCDD treatment resulted in a concomitant decrease in dG-C8-AP formation. DNA adducts for oxidized 1-nitropyrene metabolites were not detected in the TCDD-treated HepG2 cells administered 1-nitropyrene, which indicates that cytochrome P450-mediated C-oxidative pathways are detoxification pathways in HepG2 cells.

A new coccidian from Acomys cahirinus Desmarest, 1819, from Evolution Canyon, Lower Nahal Oren, Mount Carmel, Israel.

In 1994, fresh fecal samples were collected and examined for coccidian parasites from 43 spiny mice (Acomys cahirinus) and from 60 wood mice (Apodemus mystacinus). The 2 genera of rodents inhabit an area in Lower Nahal Oren. Mount Carmel, Israel, known as Evolution Canyon, which consists of opposite-facing slopes that are geologically identical, but micro-climatically very different. Acomys cahirinus is found primarily on the warmer and drier south-facing slope (SFS), whereas A. mystacinus primarily inhabits the cooler and wetter north-facing slope (NFS). None of the samples from the A. mystacinus contained coccidia, but 6 of 43 (14%) A. cahirinus individuals were discharging eimerian oocysts that we describe herein as a new species. Five of the 6 positive samples were from the SFS. Sporulated oocysts are ovoidal to subspheroidal, 26.5 x 22.9 (21-29 x 19-26) microns, without a micropyle, but with an oocyst residuum of 1 to several large clear globules and a medium-sized refractile polar body; they contain lemon-shaped sporocysts, 10.4 x 8.1 (10 11 x 7-10) microns, with a sporocyst residuum and Stieda body, but no sub-/or parastieda body. Sporozoites lie side by side, completely filling oocysts; each contains a large posterior refractile body.

Identification of ceramides in human cells using liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry.

Ceramides are intermediates in the biosynthesis of membrane sphingolipids. These biomolecules are also important as second messengers in signal transduction pathways controlling cell growth. We have developed two reversed-phase high pressure liquid chromatography (RPHPLC) techniques for identification and quantification of ceramides from mammalian cells. One method was based on atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) detection of ceramides and had the advantage of requiring minimal sample preparation, yielding significant structural information, and affording high sensitivity. The second method relied on perbenzoylation of the ceramides and detection at 230 nm. The predominant ceramides detected in the human leukemic HL-60 cell were N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine. When selected ion monitoring was used with RPHPLC/APCI-MS, approximately 2.2 pmol N-(palmitoyl)-sphingosine and 1.7 pmol N-(nervonyl)-sphingosine were observed in an extract from 40,000 HL-60 cells. Perbenzoylation with benzoyl chloride permitted RPHPLC separation and 230 nm UV absorbance detection of the trisbenzoyl derivatives of sphingosine, N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine in the HL-60 cells. These results demonstrate the utility of utilizing two different methods coupled with APCI-MS for the quantification and identification of ceramides in biological samples.

The assessment of trust orientation.

The development of the Trust Inventory, a 40-item measure of trust in generalized others (Generalized Trust) and romantic partners (Partner Trust) is described. A third conceptualization of trust in friends and family members (Network Trust) is also discussed College students (N = 1,229) participated in five stages of test construction and validation. Results indicated that the Trust Inventory scales are reliable, both internally and temporally, and that the Partner Trust and Generalized Trust Scales demonstrate both concurrent and construct validity. The resulting inventory is unique in its capacity to assess these types of trust simultaneously. Evidence supporting the discriminant validity of the Network Trust was mixed, whereas factor analytic treatments of Trust Inventory items supported the distinctiveness of Network Trust as compared to Partner and Generalized Trust, the Network Trust Scale correlated to roughly the same degree as the other two scales with several variables of differential theoretical relevance. Thus, little evidence supporting the incremental validity of Network Trust was observed Discussion focuses on the evidence suggesting the validity of interpretations of Generalized and Partner Trust and considers the issue of additional research regarding Network Trust.

A survey of the coccidian parasites of reptiles from islands of the Galápagos Archipelago: 1990-1994.

From 1990 through 1994, fecal samples were collected and examined for coccidian parasites from 26 giant land tortoises Geochelone nigra, from 715 lava lizards Tropidurus spp., from 139 land iguanas Conolophus subcristatus, and from 128 marine iguanas Amblyrhynchus cristatus, all of which inhabit various islands in the Galápagos Archipelago. None of the samples from A. cristatus or from C. subcristatus was infected with coccidia. Only 1 of 26 (4%) G. nigra was infected with a single Eimeria species that we describe here as new. A total of 262 of 715 (37%) individuals representing 3 species of Tropidurus discharged oocysts of 1-3 different coccidian species; these included 2 previously described species Eimeria tropidura and Isospora insularius, and an eimerian that we describe here as new. Additionally, 104 fecal samples from Tropidurus spp. were from 51 animals recaptured in either 2 or 3 yr; 21 had no infections in any year, 15 were infected at least once, 14 were infected in 2 yr, and only 1 was infected during 3 yr. No animal was recaptured and sampled during each of the 4 yr of this study. Of the 262 infected individuals, 30 (12%) had multiple coccidial infections at the time of collection (eimerian and isosporan, or 2 eimerians). Where determination of the sexes was possible in the lava lizards, there was no difference in prevalence rates between males (39%) and females (41%). Sporulated oocysts of the new eimerian from Tropidurus are ellipsoidal, 27.1 x 15.6 (25-31 x 14-18) microns, with a polar body, but without a micropyle or oocyst residuum; they contain ellipsoidal sporocysts, 11.8 x 6.7 (10-14 x 6-8) microns, without Stieda, sub-, or parastieda bodies, but with a sporocyst residuum. Sporulated oocysts of the new eimerian from G. nigra are ellipsoidal to ovoidal, 21.6 x 18.1 (18-25 x 16-20) microns, with a large polar body, but without a micropyle or oocyst residuum; they contain ellipsoidal sporocysts 10.7 x 7.0 (8-12 x 5-8) microns, with Stieda body but no sub- or parastieda bodies. Also present is a sporocyst residuum of medium to large granules randomly distributed among the sporocysts.

Differential response of normal and HPV immortalized ectocervical epithelial cells to B[a]P.

Cigarette smoking has been established as a risk factor for the development of cervical cancer. Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P), which are present in cigarette smoke, might account for this increased risk. The effects of B[a]P on cell growth, aryl hydrocarbon hydroxylase, DNA adducts and p53 levels was measured in cervical cells. Since 90% of cervical preneoplastic lesions are positive for the human papillomavirus (HPV) we compared the effects of these chemicals in normal ectocervical epithelial cells (ECE) and human papillomavirus 16 (HPV16) immortalized ectocervical epithelial cells (ECE16-1). Exposure of normal ECE and HPV immortalized ECE16-1 cells to B[a]P inhibited cell proliferation. Inhibition occurred at 20-fold lower concentrations in the normal ECE cells compared to ECE16-1 cells. The proliferation of cervical cells which express mutated p53 was unaffected by B[a]P. Neither cervical stromal cells nor endometrial stromal cells were affected by these compounds. The effects of B[a]P on normal ECE cell proliferation correlated with increased terminal differentiation as measured by increased envelope formation. In contrast, B[a]P exposure did not induce envelope formation in immortalized ECE16-1 cells or in cervical tumor cells. Pretreatment of both ECE and ECE16-1 cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which induces P450 expression and activity, did not alter B[a]P metabolism in either normal or immortalized cells. Furthermore, equivalent levels of DNA adducts were formed by B[a]P in ECE and ECE16-1 cells. Neither the extent of adduct formation nor the rate of their removal differed in normal and immortalized cervical cells. Therefore, the diminished growth inhibition of the ECE16-1 cells as compared to normal ECE cells by B[a]P is not due to changes in cytochrome P450 of the 1A family metabolism or DNA adduct number. Furthermore, analysis of the p53 levels in both normal and ECE16-1 cells revealed that p53 levels are higher in normal versus immortalized ectocervical cells, and p53 is induced in both cell types following B[a]P treatment. Thus reduced p53 levels in ECE16-1 cells may contribute to a lack of growth suppression following B[a]P treatment. These results demonstrate that HPV16 immortalization diminishes ectocervical epithelial cell responsiveness to toxicant damage (i.e. decreased cell proliferation and increased terminal differentiation). As a result, ECE16-1 cells that sustain genotoxic damage which leads to DNA adduct formation continue to proliferate and may be at increased risk for mutations and further progression towards a fully transformed phenotype.

Isolation of chromosome-specific genes by reciprocal probing of arrayed cDNA and cosmid libraries.

We have identified and mapped 61 novel and previously described chromosome 17 and X genes, using a human placental cDNA library. These genes were isolated using a gene identification and mapping strategy based on reciprocal probing of arrayed chromosome specific cosmid and cDNA libraries. This strategy scans gridded cosmids for nuclear genes and isolates the expressed sequence by a cosmid to cDNA filter hybridization. Inherent to this approach is the advantage of identifying the corresponding genomic cosmid clone of a particular cDNA. The genomic and cDNA reagents can be used for fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) based mapping to resolve map positions of cDNAs belonging to gene families and those associated with multiple chromosomes. The downstream utility of reagents generated by the reciprocal probing methods is demonstrated in our studies.