Physicochemical and mineralogical characterization of test materials used in 28-day and 90-day intratracheal instillation toxicology studies in rats.

Two recent intratracheal instillation toxicology studies in rats clearly show that a naturally occurring quartz, with occluded crystal surfaces (quartz isolate), produced significantly less inflammatory response than a crushed reference quartz (DQ12). Respirable-size quartz isolate was isolated from bentonite parent rock, without crushing or the use of chemicals, to ensure that the surface properties of the quartz particles were unaltered. The isolation technique utilized gentle mechanical dispersion followed by sedimentation in an aqueous medium. Extensive mineralogical and chemical characterizations were undertaken to define the physicochemical properties of the test materials. The characterizations showed significant, measurable physicochemical differences between the two quartz types. These differences may help to explain the difference in toxicological response associated with these materials. The evaluation methods and resulting data that characterized the chemical and physical properties of the instillation test materials are discussed. The data presented show that such characterizations are essential if meaningful correlations are to be made between test materials and their toxicological profiles.

Establishment of a human in vitro model of the outer blood-retinal barrier.

The outer blood-retinal barrier is composed of a monolayer of retinal pigment epithelium, Bruch's membrane and the choriocapillaris which is fenestrated. Endothelial proliferation and breaching of Bruch's membrane leads to the neovascular form of age-related macula degeneration (ARMD). The aim of this study was to generate an in vitro model that mimics more faithfully the phenotype of the choriocapillaris and the trilayer architecture in vitro. A trilayer culture model was generated with retinal pigment epithelium (ARPE-19) cell cultures on the epithelial surface of amniotic membrane and with human umbilical vein-derived endothelial cells on the other surface. A control model for the effect of retinal pigment epithelium on endothelial changes was generated with corneal epithelial cells replacing the ARPE-19. Both human umbilical vein-derived endothelial and ARPE-19 cells formed confluent monolayers on respective surfaces of the amnion. The human umbilical vein-derived endothelial cells in the trilayer became fenestrated when co-cultured with the ARPE-19 cells, but not with corneal epithelial cells, or when grown as monolayers on the amnion, showing a loss of fidelity of origin in the presence of ARPE-19 cells. These cells also revealed VE-cadherin and ZO-1 at cell-cell contacts from 24 h in the trilayer. The tight junctional molecules, occludin and ZO-1, were localized to cell-cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. Permeability of the trilayer was tested by using fluorescein and fluorescein-conjugated tracers under flow. At 72 h the trilayer severely restricted transfer of sodium fluorescein (NaF) (ten-fold reduction) whilst transfer of a 4 kDa FITC-conjugated dextran was virtually occluded, confirming a restrictive barrier. Ultrastructural studies showed the retinal pigment epithelium monolayer was polarized with microvilli present on the apical surface. Paracellular clefts showed numerous tight junctional-like appositions, similar to that seen on amnion alone. This study demonstrates that ARPE-19 and human umbilical vein-derived endothelial cells can be co-cultured on the amniotic membrane and that the resultant cross-talk leads to formation of a fenestrated endothelium, whilst maintaining a polarized restrictive epithelial layer. The fenestrated endothelial phenotype achieved in this human in vitro trilayer model is a first and offers an outer-retinal barrier which approaches the in vivo state and has potential for studies into induced junctional disruption, endothelial proliferation and migration: features of ARMD.

First Report of White Rust Caused by Albugo bliti on Seabeach Amaranth in the United States.

Seabeach amaranth (Amaranthus pumilus Raf.), a threatened annual marine plant, is a primary colonizer of the windward side of Atlantic coastal dunes. It serves an important ecological role in dune accumulation and stabilization. Because Hurricane Floyd eliminated all native seabeach amaranth in South Carolina in 1999, experimental reestablishment plantings have been attempted. In August 2000, seabeach amaranth on Dewees and Cape Island in Charleston County, Huntington Beach in Georgetown County, and Otter Island in Colleton County, South Carolina were stunted and senesced prematurely. Leaves on affected plants were only one-half of the normal size and internodes were shortened. Most plants (>90%) at each location were affected. Diseased leaves had small, pale green-to-tan spots above hypophyllous pustules that contained numerous, dry, hyaline, subglobose conidia. Conidia measured 13.5 (10 to 17) × 15.0 (11 to 18) µm. Based on morphological characters and the host, the pathogen was identified as Albugo bliti (Biv.-Bern.) Kuntze (1,2). No oospores were observed. Diseased plants were collected from Dewees and Otter Islands and kept frozen for use as a source of inoculum. Six A. pumilus plants each of six Plant Introductions (PI), 553080 through 553085, that had been grown from seed were sprayed with a suspension of 4.7 × 105 conidia per ml. One plant of each PI was sprayed with sterile distilled water as a noninoculated control. All plants were placed in a humidity chamber for 48 h and then moved to a greenhouse bench. Thirteen days after inoculation, all inoculated plants had pustules of white rust. Diseased plants had a mean of 42 pustules per plant and PI's did not differ in susceptibility. Five of six noninoculated plants also had white rust pustules, but only a mean of 2.3 (range 1 to 5) pustules each. White rust likely appeared on noninoculated plants because plants were spaced closely together in the chamber. Pustules and conidia on inoculated plants were identical to those on plants collected originally. Albugo bliti has been reported on 19 other Amaranthus species (1), but to our knowledge, this is the first report of white rust on seabeach amaranth in the United States. White rust reduced the biomass of infected plants and, hence, their ability to trap sand. White rust was not observed on subsequent plantings in 2001 and 2002 at any location. References: (1) D. F. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory, On-line publication. ARS USDA, 2002. (2) G. W. Wilson. Bull. Torrey Bot. Club 34:61, 1907.

The performance impact of context in TQM implementation: the nursing facility industry.

This study develops a model and empirically assesses how# organizational context mediates the impact of total quality management (TQM) implementation on perceived performance in the nursing facility industry. Outcomes are analysed for financial, human resources and resident-care performance. Contextual factors related to TQM implementation include managerial control, reward systems, organizational structure and the extent of implementation. Duration of TQM implementation is included as a control variable. Benchmarking has a positive impact on financial outcomes, and the extent of TQM implementation and required reporting of quality improvement activity results have a positive impact on both financial and human resources performance. The presence of a Quality Steering Council has a positive impact on financial performance, but only among larger facilities.

The importance of fiber biopersistence and lung dose in determining the chronic inhalation effects of X607, RCF1, and chrysotile asbestos in rats.

The chronic inhalation effects in rats of X607 (a rapidly dissolving synthetic vitreous fiber) were compared with those previously reported for RCF1 (a refractory ceramic synthetic vitreous fiber) and chrysotile asbestos. Of primary concern was the importance of biopersistence as a mechanism of fiber toxicity. Fischer rats were exposed to fiber aerosol by nose-only inhalation for 6 h/day, 5 days/week for 2 years. X607 and RCF1 aerosols were similar in concentration (approximately 200 fibers/cc) and average dimensions (approximately 20 x 1 microns). Chrysotile aerosol was higher in concentration (10,600 fibers/cc) and an order of magnitude smaller in average dimensions. Lung fiber deposition after 6 h inhalation was greater for X607 than for RCF1. However, at later time points, fibers/lung (especially long fibers) were much lower for X607 than for RCF1, suggesting less biopersistence for X607. X607 was neither fibrogenic nor tumorigenic and induced only minimal lung cellularity that reversed after exposure was terminated. In contrast, RCF1 and chrysotile asbestos induced pulmonary fibrosis and thoracic neoplasms (chrysotile induced 32% more pulmonary neoplasms than RCF1). Lung deposition and fiber lengths did not explain the toxicologic differences between the three fibers. Fiber biodurability, including chemical and physical parameters, appears to be a major toxicologic determinant here. Chemical analysis of lung fibers revealed rapid leaching of X607 compared to RCF1. In in vitro dissolution tests, X607 underwent rapid dissolution (kdis = 990 ng/cm2/h) and transverse fragmentation, RCF1 dissolved slowly (kdis = 6 ng/cm2/h) and did not fragment, and chrysotile dissolution was negligible (< 0.1 ng/cm2/h).

Biopersistence of man-made vitreous fibers and crocidolite asbestos in the rat lung following inhalation.

This study investigated possible relationships between fiber bio-persistence in the lung and previously observed differences in pulmonary toxicity between asbestos and man-made vitreous fibers (MMVF) following inhalation exposure. Fischer 344/N rats were exposed nose only, 6 hr/day for 5 days to 30 mg/m3 MMVF (two fiberglass compositions, rock wool, or slag wool) or to 10 mg/m3 crocidolite asbestos. At eight time points up to 1 year postexposure, lung fiber burdens were analyzed for number/lung and bivariate dimensions using scanning electron microscopy (SEM) and for chemical composition using SEM energy dispersive spectroscopy. After 365 days, > 95% of long (> 20 microns) MMVFs had disappeared from the lung compared to only 17% of long crocidolite fibers. Longer MMVFs disappeared more rapidly than short MMVFs, suggesting that long fibers were dissolving or breaking. Mean diameters and lengths of the MMVFs decreased with time, while the mean diameter of crocidolite remained unchanged and its mean length showed an apparent increase, probably related to macrophage-mediated clearance of short fibers. Leaching of oxides occurred in the fibrous glasses and slag wool and correlated with morphological changes in the fibers over time. No chemical or morphological changes were observed in crocidolite fibers. These changes in MMVF number, chemistry, and morphology over time in lung tissue compared to crocidolite asbestos demonstrate the relatively low biological persistence of some MMVFs in the lung and may explain why these MMVFs are not tumorigenic in rats, even after chronic exposure at high concentrations.

Central and reflex neural control of genioglossus in subjects who underwent laryngectomy.

Inspiratory activation of the genioglossus (GG) may occur by central drive or as a reflex to negative airway pressure. To distinguish between these, we studied seven laryngectomy patients who breathe via tracheal stomas. Negative pressure stimuli (-15 and -25 cmH2O for 500 ms) were applied 1) at functional residual capacity and 2) during early inspiration via (i) the upper airway (UA) and (ii) the tracheal stoma. Intraoral surface GG electromyogram was quantified, as described previously (R. L. Horner, J. A. Innes, K. Murphy, and A. Guz, J. Physiol. Lond. 436: 15-29, 1991). Phasic GG activity was also measured from an integrated electromyogram during spontaneous and inspiratory loaded breathing. Reflex GG activation occurred with negative UA pressure both at functional residual capacity and during inspiration (P < 0.001), but pressure stimuli at the stoma caused no significant activation (P = 0.07). Phasic inspiratory activation occurred in four patients at rest and in all seven patients during inspiratory loading (P < 0.02). These patients demonstrate 1) reflex activation of the GG by negative UA pressure without airflow or respiratory effort and 2) central inspiratory GG activation that is not mediated by negative airway pressure.

Characterization of exposure and dose of man made vitreous fiber in experimental studies.

The use of fibrous test materials in in vivo experiments introduces a number of significant problems not associated with nonfibrous particulates. The key to all aspects of the experiment is the accurate characterization of the test material in terms of fiber length, diameter, particulate content, and chemistry. All data related to fiber properties must be collected in a statistically sound manner to eliminate potential bias. Procedures similar to those outlined by the National Institute of Occupational Safety and Health (NIOSH) or the World Health Organization (WHO) must be the basis of any fiber characterization. The test material to which the animal is exposed must be processed to maximize the amount of respirable fiber and to minimize particulate content. The complex relationship among the characteristics of the test material, the properties of the delivery system, and the actual dose that reaches the target tissue in the lung makes verification of dose essential. In the case of man-made vitreous fibers (MMVF), dose verification through recovery of fiber from exposed animals is a complex task. The potential for high fiber solubility makes many of the conventional techniques for tissue preservation and digestion inappropriate. Processes based on the minimum use of aggressive chemicals, such as cold storage and low temperature ashing, are potentially useful for a wide range of inorganic fibers. Any processes used to assess fiber exposure and dose must be carefully validated to establish that the chemical and physical characteristics of the fibers have not been changed and that the dose to the target tissue is completely and accurately described.

Breathing patterns of monozygous twins during behavioural tasks.

To better understand behavioural and genetic influences upon breathing, the breathing patterns of 8 pairs of monozygous (MZ) twins were measured under 4 behavioural conditions; relaxed without standardisation; eyes closed; eyes open; and reading. Breathing was quantified by inspiratory and expiratory durations (TI, TE), tidal volume (VT) and derived variables. Airflow shape was normalised and quantified using 8 dimensions. Reading caused breathing to increase by > 500 ml/min compared to the other four conditions. Differences in breathing between combinations of two conditions were compared by testing whether the differences within an individual were smaller than the differences between random pairs of individuals from the same 16 subjects. For almost all respiratory variables, and whatever the behavioural condition, there were highly significant similarities within an individual (p < 0.00025 on 32/80 comparisons). Under each condition, the differences within MZ twin-pairs were compared to the differences within random-pairs from the same subject population. There were highly significant similarities within twin-pairs for the airflow shape across all conditions. However, TI, TE and VT failed to consistently show significant similarities within twin pairs. Hence, an individual's airflow shape appears to be a fundamental characteristic which is conserved when behavioural condition and level of ventilation changes. Further, MZ twins have similar airflow shapes--whatever the behavioural situation. Hence, behavioural influences upon airflow shape act upon monozygous twin pairs in similar ways, or such influences were negligible under the conditions of the present study.

Prevention of tilorone developmental toxicity with progesterone.

The immunomodulator tilorone hydrochloride was administered (gastric intubation) once to time-pregnant Upj:TUC(SD)spf (Sprague-Dawley) rats in four experiments. In experiment 1, tilorone (250 or 500 mg/kg) was administered on day 10 of gestation. The dams were killed 4 or 72 hr after dosing. Interferon-like activity and drug levels were determined in maternal blood, spleen, and thymus, as well as in the embryos. In experiment 2, the test groups received progesterone (2 mg/kg), or tilorone (200 or 400 mg/kg), or progesterone and tilorone. The dams from each group were killed 24 or 48 hr after receiving tilorone. Experiment 3 was similar to experiment 2, except that the dams were killed on gestation day 20. In experiment 4, tilorone (400 mg/kg) was administered on gestation day 17, 18, or 19, and the dams were killed 24 hr after dosing or on gestation day 20. In all four experiments, tilorone-related maternal toxicity (regardless of whether progesterone also was administered) was observed, as characterized by marked decreases in weight gain, the occurrence of clinical signs, and in experiment 1 by decreased thymus weights, 72 hr post-dosing. Dose-related increases in the mean number of dead embryos and in serum interferon titers occurred 72 hr postdosing. In experiment 2, there was an increase in the number of dams in the 400-mg/kg (tilorone only) group with dead embryos only, 24 hr postdosing; similar results occurred in both the 200- and 400-mg/kg groups, 48 hr postdosing. However, in the groups that also received progesterone, a partial prevention of such embryolethality was evident. In experiment 3, embryotoxicity again was observed in both tilorone-treated groups, whereas several of the dams that were also given progesterone through day 19 of gestation experienced at least a partial prevention of the embryolethal effects of tilorone. In experiment 4, no fetotoxicity was observed despite the severe maternal toxicity evident.

Volume detection during voluntary and passive breathing.

The ability to detect small changes in tidal volume (VT) during either volitional or passive breathing was compared in seven normal subjects. Passive breathing was achieved with positive pressure applied at the mouth by a ventilator. Although baseline breathing pattern was similar for each subject during the two types of breathing, the ability of the subjects to detect changes in VT was at least as good, and in general better, during passive as compared to volitional breathing. This suggests that the generation of a motor cortical command to inspire and the resultant respiratory muscle contraction are not essential to the perception of a change in lung volume. An increase in information from receptors in the mouth, pharynx and extrathoracic airways sensitive to positive pressure may be responsible for the increased ability of most subjects to detect changes in VT during passive breathing.

Effect on breathing of raising end-expiratory lung volume in sleeping laryngectomized man.

In animals, tonic vagal activity from lung receptors provides a means by which changes in end-expiratory lung volume can influence respiratory timing. We wished to examine whether increasing the end-expiratory lung volume within the tidal volume range had a similar effect in man. In order to minimize behavioral influences on breathing, the study was performed in subjects during deep non-rapid eye movement sleep. Five laryngectomized subjects were chosen for the study since their permanent tracheal stomata allow simple, airtight connection to respiratory apparatus and avoided problems with glottic closure. During EEG-documented sleep, end-expiratory volume was increased by up to 350 ml with the addition of expiratory threshold loads of 1 to 10 cm H2O. End-expiratory volume increased linearly with expiratory pressure. Inspiratory and expiratory times (TI and TE) were not affected by increases in end-expiratory volume. Tidal volume (VT) was decreased such that end-inspiratory volume remained unchanged. The decrease in VT may result from a reduction in inspiratory muscle efficiency at a higher lung volume. The results of the study provide no evidence that tonic vagal afferent information from the lungs is important in controlling respiratory rhythm within the tidal volume range in man when behavioral control of breathing is minimized.

Effects of cadmium on a microbial food chain, Chlamydomonas reinhardii and Tetrahymena vorax.

A steady-state microbial food chain consisting of the green alga Chlamydomonas reinhardii and the ciliated protozoan Tetrahymena vorax was established in a two-stage, nitrogen-limited chemostat. The lowest concentration of cadmium which produced a toxic effect at the population level was between 7.5 and 10 micrograms l-1. The algal population acclimated to the presence of cadmium up to 40 micrograms l-1 added in increments over time, but demonstrated lowered cell numbers and reduced cell weights. Protozoan populations acclimated to 40 micrograms Cd l-1 added incrementally if the rate of dilution was lowered. Abrupt elevation of the cadmium concentration to 40 micrograms l-1 resulted in extreme fluctuations in the specific growth rates of both populations and the incipient loss of all cells from the system (washout).

Cardiopulmonary response to dynamic exercise after heart and combined heart-lung transplantation.

The exercise capacity and cardiopulmonary response to progressive dynamic exercise of eight healthy recipients of heart-lung transplants were compared with those of matched recipients of orthotopic cardiac transplants and normal controls. In both transplant groups the maximum workloads were lower than that in the normal group. The transplant recipients had higher pre-exercise heart rates and lower maximum heart rates than the normal controls. Ventilation during submaximal exercise was similar in the heart transplant group and the controls. The heart-lung group had an increased ventilatory response associated with lower end tidal carbon dioxide concentrations. Exercise capacity after combined heart-lung transplantation is similar to that after cardiac transplantation. Transplant recipients have an abnormal heart rate response during exercise related to cardiac denervation. The altered ventilatory response in heart-lung recipients may be the result of pulmonary denervation.

The breathing patterns of identical twins.

The resting breathing patterns of healthy adult identical twins were compared to see if there was any possible genetic component in the determination of this pattern. From breath-by-breath analysis of airflow, measured with a pneumotachometer (9 pairs of twins), the pattern of breathing was quantified in terms of individual respiratory variables; inspiratory time (TI), expiratory time (TE), total breath duration (TTOT), VT/TI, TI/TTOT, and by taking TI, TE and VT all together (TRIAD). Also, the airflow shape was quantified by harmonic analysis (ASTER). A second study was performed under more strictly defined conditions of rest and where the respiratory variables were estimated with respiratory inductance plethysmography to eliminate the possible effect of a facemask (5 pairs of twins). In each study, for each variable, the differences within twin-pairs were compared to the differences within random-pairs from the same subject population. In both studies, there were highly significant similarities within twin-pairs in the pattern of breathing, being best demonstrated when the entire 'shape' of the pneumotachogram (ASTER) or the spirogram (TRIAD) was considered.

Phase I clinical trial of a combination of dipyridamole and acivicin based upon inhibition of nucleoside salvage.

A Phase I clinical trial of simultaneous 72-h infusions of dipyridamole and acivicin was carried out in patients with advanced malignancies. The objective of this trial was to determine the maximum tolerated dose of dipyridamole when administered as a 72-h infusion in combination with acivicin. The development of this combination is of interest because of in vitro observations which demonstrate that dipyridamole potentiates the cytotoxic action of acivicin by blocking nucleoside salvage. Patients were treated with concomitant i.v. infusions of dipyridamole and acivicin for 72 h. The acivicin dose infused remained constant during the trial at 60 mg/m2/72 h. The maximum tolerated dose (MTD) of dipyridamole was 23.1 mg/kg/72 h. Limiting toxicities at the MTD of dipyridamole with acivicin were severe gastrointestinal and constitutional symptoms which appeared to be caused by the high doses of dipyridamole administered. Escalation of dipyridamole did not potentiate the mild myelosuppression or the neurotoxicity which occurs with acivicin alone. At a dose of dipyridamole which was well below the MTD, one patient experienced symptomatic orthostatic hypotension, and another patient with coronary artery disease developed dizziness and transient electrocardiogram abnormalities. However, no other hypotensive or cardiovascular events occurred as dipyridamole was escalated to the MTD. Phlebitis occurred at the site of infusion when the dose of dipyridamole exceeded 13.5 mg/kg/72 h. Because of this local toxicity, it was necessary to administer dipyridamole through a central venous catheter to achieve maximum plasma levels. At the MTD of dipyridamole, steady-state total and free plasma levels of 11.9 microM and 27.8 nM, respectively, were attained by 24 h. These are free dipyridamole levels which in vitro were sufficient to block cytidine salvage and to potentiate the biochemical and cytotoxic effects of acivicin against human colon cancer cells (P.H. Fischer et al., Cancer Res., 44:3355-3359, 1984).

The effect of lung inflation on breathing in man during wakefulness and sleep.

To determine whether the Hering-Breuer inflation reflex could be demonstrated in the presence and absence of behavioral influences on breathing, lung inflation was performed via the tracheal stoma in eleven laryngectomized subjects: seven seated at rest with their eyes closed and six recumbent during their deepest non-rapid eye movement sleep. Laryngectomized subjects were chosen for study since their permanent tracheal stoma and absence of a glottis abolished the sensation of inflation in the upper airways, avoided problems with glottic closure and allowed simple, airtight connection to respiratory apparatus. At rest awake, inflation volumes of 500-3050 ml caused no reproducible apnea. During EEG-documented sleep, the inflation reflex was tested on 75 occasions using inflation volumes of 540-2100 ml. On the 66 occasions not associated with subject arousal inflations exceeding approximately 1 L produced apnea terminated by inspiration; greater volumes gave longer apneas. At the end of the study the subjects were woken and retested; no reproducible apnea was found. We conclude that the Hering-Breuer inflation reflex can be demonstrated above the resting tidal volume range in adult man in the absence of the behavioral control of breathing.

Ventilation and breathlessness on maximal exercise in patients with interstitial lung disease after local anaesthetic aerosol inhalation.

1. The ventilatory response to maximal incremental exercise and the accompanying sensation of breathlessness were studied after the inhalation of 0.9% sodium chloride (saline) and 5% bupivacaine aerosols in six patients with interstitial lung disease. 2. The adequacy of airway anaesthesia induced by bupivacaine aerosol was confirmed by the absence of the cough reflex to 5% citric acid aerosol on completion of exercise. 3. All subjects first performed a trial exercise test to familiarize them with the procedure and to assess the degree of arterial oxygen desaturation on exercise. In subsequent tests, supplementary oxygen was given to maintain the saturation at 95% or above. 4. Airway anaesthesia had no effect on the ability to perform exercise as assessed by maximum workload, CO2 production or heart rate. No significant changes were seen on the pattern of breathing, minute ventilation or end-tidal PCO2 on exercise. There was, however, a small but statistically significant increase in ventilation related to CO2 production (VE/VCO2) at the end of exercise. 5. There was a tendency for breathlessness to be increased by airway anaesthesia but this did not reach statistical significance. 6. These results provide no evidence that vagal afferent activity is responsible for the abnormal ventilatory response to exercise in patients with interstitial lung disease. The perception of breathlessness in these patients was not diminished by anaesthesia of the airway.