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Promising data are emerging on a new anticancer agent, Ad.EGR-TNF, an adenoviral vector, which contains radio-inducible DNA sequences from the early growth response (EGR1) gene promoter and cDNA for the gene encoding human tumour necrosis factor-alpha. Ad.EGR-TNF combines the well-documented broad-spectrum anticancer activity of TNFalpha with the proven clinical usefulness of radiotherapy. Systemic delivery of the TNFalpha protein has had limited success clinically because of severe dose-limiting toxic effects. This limitation has been overcome by the use of a gene delivery approach, combined with a radiation-inducible promoter to express the TNFalpha protein in the irradiated tumour tissue. Preclinical and early phase I clinical testing indicates that effective concentrations of TNFalpha can be delivered to the tumour site without significant systemic exposure or toxic effects. The combination of radiation and TNFalpha gene delivery has produced striking antitumour effects in model systems in animals. In the clinical setting, potent anticancer activity has been observed with a high rate of complete and partial objective tumour responses. A novel mechanism of destruction of the tumour vasculature seems to be central to this distinct antitumour activity. This review summarises the rationale, mechanistic basis, preclinical data, and preliminary clinical findings for this new treatment model. [ABSTRACT FROM AUTHOR]

Background: Long-term exposure to particulate air pollution has been associated with mortality in urban cohort studies. Few studies have investigated the association between emission contributions from different particle sources and mortality in large-scale population registries, including non-urban populations., Objectives: The aim of the study was to evaluate the associations between long-term exposure to particulate air pollution from different source categories and non-accidental mortality in the Netherlands based on existing national databases., Methods: We used existing Dutch national databases on mortality, individual characteristics, residence history, neighbourhood characteristics and modelled air pollution concentrations from different sources and air pollution components: particulate matter PM10, primary particulate matter PM10 (PPM10), particulate matter PM2.5, primary particulate matter PM2.5 (PPM2.5), elemental carbon (EC), nitrogen dioxide (NO 2 ) and secondary inorganic aerosol (SIA) in PM10 (SIA10) or in PM2.5 (SIA2.5). We established a cohort of 7.5 million individuals 30 years or older. We followed the cohort for eight years (2008-2015). We applied Cox proportional hazard regression models adjusting for potential individual and area-specific confounders., Results: We found statistically significant associations between total and primary particulate matter (PM10 and PM2.5), elemental carbon and mortality. Adjustment for nitrogen dioxide did not change the associations. Secondary inorganic aerosol showed less consistent associations. All primary PM sources were associated with mortality, except agricultural emissions and, depending on the statistical model, industrial PM emissions., Conclusions: We could not identify one or more specific source categories of particulate air pollution as main determinants of the mortality effects found in this and in a previous study. This suggests that present policy measures should be focussed on the wider spectrum of air pollution sources instead of on specific sources., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Although the health effects of long term exposure to air pollution are well established, it is difficult to effectively communicate the health risks of this (largely invisible) risk factor to the public and policy makers. The purpose of this study is to develop a method that expresses the health effects of air pollution in an equivalent number of daily passively smoked cigarettes., Methods: Defined changes in PM2.5, nitrogen dioxide (NO2) and Black Carbon (BC) concentration were expressed into number of passively smoked cigarettes, based on equivalent health risks for four outcome measures: Low Birth Weight (<2500g at term), decreased lung function (FEV1), cardiovascular mortality and lung cancer. To describe the strength of the relationship with ETS and air pollutants, we summarized the epidemiological literature using published or new meta-analyses., Results: Realistic increments of 10µg/m(3) in PM2.5 and NO2 concentration and a 1µg/m(3) increment in BC concentration correspond to on average (standard error in parentheses) 5.5 (1.6), 2.5 (0.6) and 4.0 (1.2) passively smoked cigarettes per day across the four health endpoints, respectively. The uncertainty reflects differences in equivalence between the health endpoints and uncertainty in the concentration response functions. The health risk of living along a major freeway in Amsterdam is, compared to a counterfactual situation with 'clean' air, equivalent to 10 daily passively smoked cigarettes.., Conclusions: We developed a method that expresses the health risks of air pollution and the health benefits of better air quality in a simple, appealing manner. The method can be used both at the national/regional and the local level. Evaluation of the usefulness of the method as a communication tool is needed., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Long-term exposure to air pollution has been associated with mortality in urban cohort studies. Few studies have investigated this association in large-scale population registries, including non-urban populations., Objectives: The aim of the study was to evaluate the associations between long-term exposure to air pollution and nonaccidental and cause-specific mortality in the Netherlands based on existing national databases., Methods: We used existing Dutch national databases on mortality, individual characteristics, residence history, neighborhood characteristics, and national air pollution maps based on land use regression (LUR) techniques for particulates with an aerodynamic diameter ≤ 10 μm (PM10) and nitrogen dioxide (NO2). Using these databases, we established a cohort of 7.1 million individuals ≥ 30 years of age. We followed the cohort for 7 years (2004-2011). We applied Cox proportional hazard models adjusting for potential individual and area-specific confounders., Results: After adjustment for individual and area-specific confounders, for each 10-μg/m3 increase, PM10 and NO2 were associated with nonaccidental mortality [hazard ratio (HR) = 1.08; 95% CI: 1.07, 1.09 and HR = 1.03; 95% CI: 1.02, 1.03, respectively], respiratory mortality (HR = 1.13; 95% CI: 1.10, 1.17 and HR = 1.02; 95% CI: 1.01, 1.03, respectively), and lung cancer mortality (HR = 1.26; 95% CI: 1.21, 1.30 and HR = 1.10 95% CI: 1.09, 1.11, respectively). Furthermore, PM10 was associated with circulatory disease mortality (HR = 1.06; 95% CI: 1.04, 1.08), but NO2 was not (HR = 1.00; 95% CI: 0.99, 1.01). PM10 associations were robust to adjustment for NO2; NO2 associations remained for nonaccidental mortality and lung cancer mortality after adjustment for PM10., Conclusions: Long-term exposure to PM10 and NO2 was associated with nonaccidental and cause-specific mortality in the Dutch population of ≥ 30 years of age.

Background: Studies have shown associations between mortality and long-term exposure to particulate matter air pollution. Few cohort studies have estimated the effects of the elemental composition of particulate matter on mortality., Objectives: Our aim was to study the association between natural-cause mortality and long-term exposure to elemental components of particulate matter., Methods: Mortality and confounder data from 19 European cohort studies were used. Residential exposure to eight a priori-selected components of particulate matter (PM) was characterized following a strictly standardized protocol. Annual average concentrations of copper, iron, potassium, nickel, sulfur, silicon, vanadium, and zinc within PM size fractions ≤ 2.5 μm (PM2.5) and ≤ 10 μm (PM10) were estimated using land-use regression models. Cohort-specific statistical analyses of the associations between mortality and air pollution were conducted using Cox proportional hazards models using a common protocol followed by meta-analysis., Results: The total study population consisted of 291,816 participants, of whom 25,466 died from a natural cause during follow-up (average time of follow-up, 14.3 years). Hazard ratios were positive for almost all elements and statistically significant for PM2.5 sulfur (1.14; 95% CI: 1.06, 1.23 per 200 ng/m3). In a two-pollutant model, the association with PM2.5 sulfur was robust to adjustment for PM2.5 mass, whereas the association with PM2.5 mass was reduced., Conclusions: Long-term exposure to PM2.5 sulfur was associated with natural-cause mortality. This association was robust to adjustment for other pollutants and PM2.5.

Background: Air pollution has been associated with respiratory health effects. There is little direct evidence that reductions in air pollution related to abatement policies lead to actual improvement in respiratory health. We assessed whether a reduction in (traffic policy-related) air pollution concentrations was associated with changes in respiratory health., Methods: Air pollution concentrations and respiratory health were measured in 2008 and 2010 at eight busy urban streets and at four suburban background control locations. Respiratory function was assessed twice in 661 residents by spirometry and measurements of airway resistance. Nitric oxide (NO) in exhaled air was measured as a marker for airway inflammation., Results: Air pollution concentrations were lower in 2010 than in 2008. The declines in pollutants varied among locations, with the largest decline observed in a street with a large reduction in traffic intensity. In regression analyses adjusted for important covariates, reductions in concentrations of soot, NO2, NOx, Cu, and Fe were associated with increases in forced vital capacity (FVC) (∼1% increase per interquartile range [IQR] decline). Airway resistance decreased with a decline in particulate matter (PM10) and PM2.5 (9% per IQR), although these associations were somewhat less consistent. No associations were found with exhaled NO. Results were driven largely by one street where traffic-related air pollution showed the largest reduction. Forced expiratory volume and FVC improved by 3% to 6% in residents of this street compared with suburban background residents. This was accompanied by a suggestive reduction in airway resistance., Conclusions: Reductions in air pollution may lead to small improvements in respiratory function.

Outdoor air pollution has been associated with decrements in lung function and growth of lung function in school-age children. Lung function effects have not been examined in preschoolers, with the exception of one study on minute ventilation in newborns. Our goal was to assess the relationship between long- and short-term exposure to traffic-related air pollution and interrupter resistance in 4-year-old children. Lung function was measured using the interrupter resistance method in children participating in a Dutch birth cohort study. Long-term average air pollution concentrations of fine particulate matter, nitrogen dioxide and soot at the residential address at birth were assessed using land-use regression models. Daily average air pollution concentrations on the day of clinical examination were obtained from the Dutch National Air Quality Monitoring Network. Significant associations were found between long-term average air pollution concentrations and interrupter resistance. Interrupter resistance increased by 0.04 kPa·s·L(-1) (95% CI 0.01-0.07) per interquartile range increase (3.3 μg·m(-3)) in fine particle concentration. Short-term exposure was not associated with interrupter resistance. Long-term exposure to traffic-related air pollution was associated with increased interrupter resistance in 4-year-old children, supporting previous birth cohort studies reporting effects of air pollution on subjectively reported respiratory symptoms in preschool children.

Background: Evaluations of the effectiveness of air pollution policy interventions are scarce. This study investigated air pollution at street level before and after implementation of local traffic policies including low emission zones (LEZ) directed at heavy duty vehicles (trucks) in five Dutch cities., Methods: Measurements of PM(10), PM(2.5), 'soot', NO(2), NO(x), and elemental composition of PM(10) and PM(2.5) were conducted simultaneously at eight streets, six urban background locations and four suburban background locations before (2008) and two years after implementation of the policies (2010). The four suburban locations were selected as control locations to account for generic air pollution trends and weather differences., Results: All pollutant concentrations were lower in 2010 than in 2008. For traffic-related pollutants including 'soot' and NO(x) and elemental composition (Cr, Cu, Fe) the decrease did not differ significantly between the intervention locations and the suburban control locations. Only for PM(2.5) reductions were considerably larger at urban streets (30%) and urban background locations (27%) than at the matching suburban control locations (20%). In one urban street where traffic intensity was reduced with 50%, 'soot', NO(x) and NO(2) concentrations were reduced substantially more (41, 36 and 25%) than at the corresponding suburban control location (22, 14 and 7%)., Conclusion: With the exception of one urban street where traffic flows were drastically reduced, the local traffic policies including LEZ were too modest to produce significant decreases in traffic-related air pollution concentrations., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Measuring the oxidative potential of airborne particulate matter (PM) may provide a more health-based exposure measure by integrating various biologically relevant properties of PM into a single predictor of biological activity., Objectives: We aimed to assess the contrast in oxidative potential of PM collected at major urban streets and background locations, the associaton of oxidative potential with other PM characteristics, and the oxidative potential in different PM size fractions., Methods: Measurements of PM with aerodynamic diameter ≤ 10 μm (PM10), PM with aerodynamic diameter ≤ 2.5 μm (PM2.5), soot, elemental composition, and oxidative potential of PM were conducted simultaneously in samples from 8 major streets and 10 urban and suburban background locations in the Netherlands. Six 1-week measurements were performed at each location over a 6-month period in 2008. Oxidative potential was measured as the ability to generate hydroxyl radicals in the presence of hydrogen peroxide in all PM10 samples and a subset of PM2.5 samples., Results: The PM10 oxidative potential of samples from major streets was 3.6 times higher than at urban background locations, exceeding the contrast for PM mass, soot, and all measured chemical PM characteristics. The contrast between major streets and suburban background locations was even higher (factor of 6.5). Oxidative potential was highly correlated with soot, barium, chromium, copper, iron, and manganese. Oxidative potential of PM10 was 4.6 times higher than the oxidative potential of PM2.5 when expressed per volume unit and 3.1 times higher when expressed per mass unit., Conclusions: The oxidative potential of PM near major urban roads was highly elevated compared with urban and suburban background locations, and the contrast was greater than that for any other measured PM characteristic.

Background: Air pollution may promote type 2 diabetes by increasing adipose inflammation and insulin resistance. This study examined the relation between long-term exposure to traffic-related air pollution and type 2 diabetes prevalence among 50- to 75-year-old subjects living in Westfriesland, the Netherlands., Methods: Participants were recruited in a cross-sectional diabetes screening-study conducted between 1998 and 2000. Exposure to traffic-related air pollution was characterized at the participants' home-address. Indicators of exposure were land use regression modeled nitrogen dioxide (NO2) concentration, distance to the nearest main road, traffic flow at the nearest main road and traffic in a 250 m circular buffer. Crude and age-, gender- and neighborhood income adjusted associations were examined by logistic regression., Results: 8,018 participants were included, of whom 619 (8%) subjects had type 2 diabetes. Smoothed plots of exposure versus type 2 diabetes supported some association with traffic in a 250 m buffer (the highest three quartiles compared to the lowest also showed increased prevalence, though non-significant and not increasing with increasing quartile), but not with the other exposure metrics. Modeled NO2-concentration, distance to the nearest main road and traffic flow at the nearest main road were not associated with diabetes. Exposure-response relations seemed somewhat more pronounced for women than for men (non-significant)., Conclusions: We did not find consistent associations between type 2 diabetes prevalence and exposure to traffic-related air pollution, though there were some indications for a relation with traffic in a 250 m buffer.

There are currently no epidemiological studies on health effects of long-term exposure to ultrafine particles (UFP), largely because data on spatial exposure contrasts for UFP is lacking. The objective of this study was to develop a land use regression (LUR) model for UFP in the city of Amsterdam. Total particle number concentrations (PNC), PM10, PM2.5, and its soot content were measured directly outside 50 homes spread over the city of Amsterdam. Each home was measured during one week. Continuous measurements at a central urban background site were used to adjust the average concentration for temporal variation. Predictor variables (traffic, address density, land use) were obtained using geographic information systems. A model including the product of traffic intensity and the inverse distance to the nearest road squared, address density, and location near the port explained 67% of the variability in measured PNC. LUR models for PM2.5, soot, and coarse particles (PM10, PM2.5) explained 57%, 76%, and 37% of the variability in measured concentrations. Predictions from the PNC model correlated highly with predictions from LUR models for PM2.5, soot, and coarse particles. A LUR model for PNC has been developed, with similar validity as previous models for more commonly measured pollutants.

Background: Daily variations in the levels of air pollution are well known to be associated with daily variations in mortality counts. Given the large number of time-series studies, there is little need for simple replication of these results in additional locations. However, additional analyses of time-series data might be useful in elucidating remaining questions on the role of air pollution on mortality., Objectives: Because of ongoing issues related to causality, changing toxicity, the difficulty in isolating the independent effects of individual pollutants, the availability of new methods to detect effect thresholds, and questions about the extent to which effects are restricted to frail members of the population, additional analyses of time-series data might be helpful in addressing these issues. We show an example where additional time-series analyses can be helpful in elucidating specific questions in the field of air pollution epidemiology., Methods: We analysed daily mortality and air pollution data using Poisson regression in generalised additive models. Air pollution data for the overall period 1992-2006 and for four different periods were analysed to assess the overall risk estimates for the whole period and to assess variability over time for the different effect estimates., Results and Conclusion: We found some statistically significant upward trends, but this was only the case for a few associations without a consistent pattern over the cause-specific deaths. Whether these findings are consistent over time or whether our findings are merely the result of statistical chance can only be elucidated by continuation of monitoring of the relative risks over time in the future. Although these results may indicate that both photochemical and particulate matter air pollution might have become more toxic, the lack of a clear pattern in the results makes these conclusions speculative., (Copyright © 2010 Elsevier Inc. All rights reserved.)

During 2 months of the pollen season, the acute and putative adjuvant effect of traffic-related air pollution on respiratory health was investigated in children sensitised to grass pollen or house dust mite (HDM). Respiratory complaints were objectified via measurement of exhaled NO and inflammatory mediators in nasal lavage (NAL). During the study children, skin prick negative (n = 31) or positive to grass pollen (n = 22), HDM (n = 34) or grass pollen + HDM (n = 32), kept a daily diary on respiratory symptoms, and NAL and exhaled air was sampled twice a week. The level of air pollutants and pollen was monitored continuously. Like children sensitised to HDM, those sensitised to pollen reported respiratory complaints (shortness of breath, itchy eyes or blocked nose) more frequently than non-sensitised children during (but not before) the pollen season; the respiratory complaints of sensitised children were independent of the pollen level. In addition, exposure to increased levels of PM(10) induces 'shortness of breath' in pollen- and HDM-sensitised children, whereas ozone induces a blocked nose in HDM-sensitised children. Combined exposure to PM(10) + pollen and O(3) + pollen induces a blocked nose in both HDM-sensitised children and children sensitised to pollen + HDM. Significant positive associations were found between eNO and the levels of NO(2), CO, PM(2.5) and pollen in both sensitised and non-sensitised children. At the start of the pollen season, the NAL concentration of eosinophils and ECP in pollen-sensitised children was increased compared to winter, but their levels were not further affected by increased exposure to pollen or air pollution. In conclusion, during the pollen season, sensitised children continuously report a high prevalence of respiratory complaints which coincides with increased levels of upper and lower airway inflammatory markers. No additional pro-inflammatory effect of air pollution was observed, which indicates that air pollution does not facilitate allergen-induced inflammatory responses., (Copyright 2003 S. Karger AG, Basel)

Background: Exhaled nitric oxide (eNO) may serve as a non-invasive marker of airway inflammation but its relationship with other commonly used measures has not been evaluated., Methods: Levels of eNO in a sample of 450 children aged 7-12 years out of a total sample of 2504 school children living in different urban areas near motorways were determined. The aim of this cross-sectional study was to explore the relationship between eNO, impairment of lung function (PEF, FVC, FEV(1) and MMEF), bronchial hyperresponsiveness (BHR), and blood eosinophilia in children with and without atopy as assessed by skin prick testing., Results: Regression analysis showed that wheezing and nasal discharge and conjunctivitis that had occurred during the previous 12 months were positively associated with eNO levels in atopic children (relative increase of 1.48 and 1.41, respectively; p<0.05) but not in non-atopic children. Similarly, BHR and the number of blood eosinophils per ml were positively associated with eNO levels in atopic children (relative increase of 1.55 and 2.29, respectively; p<0.05) but not in non-atopic children. The lung function indices PEF, FVC, FEV(1) and MMEF were not associated with eNO levels., Conclusions: In addition to conventional lung function tests and symptom questionnaires, eNO is a suitable measure of airway inflammation and its application may reinforce the power of epidemiological surveys on respiratory health.

Background: Exhaled nitric oxide (NO) has been proposed as novel a non-invasive marker of airway inflammation., Objective: The level of exhaled NO was determined in a random sample of school children (7-12 years old) with the aim of investigating the relationship between exhaled NO and sensitization to common allergens., Results: In the 450 children tested by skin prick tests (SPT), the prevalence of sensitization was 29.5% (overall), 21.9% (sensitization to indoor allergens), and 15.0% (sensitization to outdoor allergens). Regression analysis showed that levels of exhaled nitric oxide were closely associated with various measures of sensitization to aeroallergens. Sensitization to indoor allergens was associated with higher levels of exhaled NO (eNO) than sensitization to outdoor allergens when assessed by IgE but not when assessed by SPT. Children with reported wheeze in the past 12 months had much stronger associations between sensitization and eNO than children without wheeze., Conclusion: We conclude that allergic sensitization is strongly associated with increased levels of exhaled NO, especially in children with wheeze.

Despite the important contribution of traffic sources to urban air quality, relatively few studies have evaluated the effects of traffic-related air pollution on health, such as its influence on the development of asthma and other childhood respiratory diseases. We examined the relationship between traffic-related air pollution and the development of asthmatic/allergic symptoms and respiratory infections in a birth cohort (n approximately 4,000) study in The Netherlands. A validated model was used to assign outdoor concentrations of traffic-related air pollutants (nitrogen dioxide, particulate matter less than 2.5 micro m in aerodynamic diameter, and "soot") at the home of each subject of the cohort. Questionnaire-derived data on wheezing, dry nighttime cough, ear, nose, and throat infections, skin rash, and physician-diagnosed asthma, bronchitis, influenza, and eczema at 2 years of age were analyzed in relation to air pollutants. Adjusted odds ratios for wheezing, physician-diagnosed asthma, ear/nose/throat infections, and flu/serious colds indicated positive associations with air pollutants, some of which reached borderline statistical significance. No associations were observed for the other health outcomes analyzed. Sensitivity analyses generally supported these results and suggested somewhat stronger associations with traffic, for asthma that was diagnosed before 1 year of age. These findings are subject to confirmation at older ages, when asthma can be more readily diagnosed.

Objectives: The aim of the study was to investigate whether the level of exhaled nitric oxide (eNO) provides a more sensitive measure to assess adverse pulmonary effects of air pollution than conventional lung function indices., Method: The non-selected cohort studied consisted of 68 children (aged 10-11 years) living in an urban environment. For 7 weeks respiratory complaints were reported daily by these children in a diary, and lung function measures and eNO levels were determined in the children once a week on days with various level of air pollution., Results: During the study the increase in the levels of the various air pollutants was positively associated with eNO (6% to 31% increase; P<0.05) but not with lung function measures. In contrast to the lung function measures, the prevalence of respiratory symptoms such as "sore throat", "runny nose", "having a cold", and "sick at home" were positively associated with the level of eNO measured in the following week., Conclusions: Though clinically very meaningful, functional pulmonary measures appear to be too course to detect relatively mild clinical symptoms in response to exposure to air pollution. In an epidemiological setting the increase in eNO may represent a more suitable measure to assess such adverse effects.

The authors used a longitudinal observational design, with repeated measures, to study the association between traffic-related air pollutants (i.e., nitric oxide, nitrogen dioxide, carbon monoxide, and Black Smoke) and respiratory symptoms. Subjects (N = 82) attended an elementary school in either Utrecht (i.e., urban children) or Bilthoven (i.e., suburban children). These two geographic areas differed with respect to levels of Black Smoke (means = 53 microg/m3 and 18 microg/m3, respectively). Levels of nitric oxide, nitrogen dioxide, carbon monoxide, and Black Smoke were consistently higher in Utrecht than in Bilthoven (mean daily ratios were 8, 1.5, 1.8, and 2.7, respectively). The authors compared mean levels of short-term effects of the aforementioned air pollutants on suburban and urban children. Urban children had higher mean levels (p = .05) of interleukin-8 (32%), urea (39%), uric acid (26%), albumin (15%), and nitric oxide metabolites (21%) in nasal lavage than did suburban children. Peak expiratory flow, exhaled nitric oxide levels, and nasal markers were associated with levels of particulate matter with diameters less than or equal to 10 microm, Black Smoke, nitrogen dioxide, and nitric oxide. With respect to per-unit increases in air pollution, urban children had more increased peak expiratory flow, higher levels of exhaled nitric oxide, and more increased release of uric acid, urea, and nitric oxide metabolites than suburban children. In summary, urban children had increased levels of inflammatory nasal markers, and their responses were more pronounced than were the suburban children's responses to the same increments of air pollution.

We propose a model for activation of the epidermal cell hypersensitive response (HR) in the barley/powdery mildew interaction. The model suggests that the plasma membrane proton pump (H+-ATPase) of epidermal cells is activated following penetration by an avirulent powdery mildew fungus. This will cause an acidification of the apoplast towards the mesophyll cells, thereby activating generation of H2O2 from the mesophyll, which subsequently triggers the epidermal cell to undergo HR. The model is supported by the following data: (1) the earliest HR-related H2O2 is found in the attachment zones between the epidermal cell and underlying mesophyll cells; (2) scavenger treatment reduces HR; (3) treatment of leaves with low-pH (3.5) citrate and succinate buffers causes more cells to undergo HR in the compatible interaction, while treatment with the same buffers at pH 5.5 reduces the number of HR-cells in the incompatible interaction; (4) race-specific proton extrusion is observed underneath epidermal tissue detached from leaves inoculated 15 h earlier; and (5) treatment of leaves with fusicoccin, an activator of the plasma membrane H+-ATPase, increases the number of HR-cells in the compatible interaction.

Levels of exhaled nitric oxide (NO) were determined in well-characterized atopic and nonatopic subjects on 4 days with a different level of outdoor air pollution. The two groups matched well regarding spirometric values, i.e., no difference with regard to FEV(1), FVC, and peak flow. On the 4 test days asymptomatic atopic subjects exhaled 1.5- to 2.4-fold higher levels of NO compared with nonatopic subjects. In both groups the increase in exhaled NO in response to air pollution was similar (2.5 times maximal increase, P < 0.01). In conclusion, atopic subjects exhale higher levels of NO compared with nonatopic subjects, but respond to a similar degree to increased levels of air pollution., (Copyright 1999 Academic Press.)

The aim of this study was to assess the effect of outdoor air pollution on exhaled levels of endogenously released nitric oxide. To exclude bias from exogenous NO in the recovered exhaled air (residual NO or NO in dead volume) an experimental design was used that sampled NO of endogenous origin only. The validity of the presented experimental design was established in experiments where subjects were exposed to high levels of exogenous NO (cigarette smoke or 480 microg x m(-3) synthetic NO). Subsequent 1 min breathing and a final inhalation of NO-free air proved to be sufficient to attain pre-exposure values. Using the presented method detecting only endogenous NO in exhaled air, 18 subjects were sampled on 4 separate days with different levels of outdoor air pollution (read as an ambient NO level of 4, 30, 138 and 246 microg x m(-3)). On the 2 days with highest outdoor air pollution, exhaled NO was significantly (p<0.001) increased (67-78%) above the mean baseline value assessed on 4 days with virtually no outdoor air pollution. In conclusion, the level of endogenous nitric oxide in exhaled air is increased on days with high outdoor air pollution. The physiological implications of this findings need to be investigated further.

Exacerbations of asthma have been associated with exposure to ozone or particles with a 50% cut-off aerodynamic diameter of 10 microm (PM10). We postulated in this study that the association of summertime air pollution (i.e. ozone and PM10) with acute respiratory symptoms, medication use and peak expiratory flow differs among patients grouped according to asthma severity. During the summer of 1995, effects of ambient air pollution on these parameters were studied in a panel of 60 nonsmoking patients with intermittent to severe persistent asthma. These patients were recruited from our Pulmonary Out-patient Clinic. Subgroup analysis was performed on the degree of hyperresponsiveness and lung steroid use before the start of the study, as indictors for the severity of asthma. Associations of the parameters studied with ozone, PM10, nitrogen dioxide (NO2), sulphur dioxide (SO2) and black smoke were evaluated using time series analysis. Several episodes with increased summertime air pollution occurred during the 96 day study period. Eight hour average ozone concentrations exceeded the World Health Organization (WHO) Air Quality Guidelines (120 microg x m(-3)) on 16 occasions. Daily mean levels of PM10 were moderately elevated (range 16-98 microg x m(-3)). Levels of the other measured pollutants were low. There was a consistent, positive association of the prevalence of shortness of breath (maximal relative risk (RRmax) 1.18) with ozone, PM10, black smoke and NO2. In addition, bronchodilator use was associated with both ozone and PM10 levels (RRmax 1.16). Stratification by airway hyperresponsiveness and steroid use did not affect the magnitude of the observed associations. No associations with peak expiratory flow measurements were found. We conclude that the severity of asthma is not an indicator for the sensitivity to air pollution.

Asthma is an inflammatory disease of the airways, and exacerbations of this disease have been associated with high levels of air pollution. The objective of this study was to examine whether ambient air pollution and/or allergen exposure induces inflammatory changes in the upper airways of asthmatics. Sixty patients with intermittent to severe persistent asthma visited the Hospital's Out Patient Clinic every 2 wk for a period of 3 mo, and on each visit a nasal lavage was obtained. Associations between nasal inflammatory parameters and seasonal allergens and/or air pollution exposures were analyzed using linear regression analysis. The study ran from July 3 to October 6, 1995, during which period ozone (8-h mean: 80 micrograms/m3) and PM10 (24-h mean: 40 micrograms/m3) were the major air pollutants; the major aeroallergen was mugwort pollen (24-h mean: 27 pollen grains/m3). Effects on both cellular and soluble markers in nasal lavage were demonstrated for both ozone and mugwort pollen, but not for PM10. Ambient ozone exposure was associated with an increase in neutrophils (112% per 100 micrograms/m3 increase in 8-h average ozone concentration), eosinophils (176%), epithelial cells (55%), IL-8 (22%), and eosinophil cationic protein (ECP) (19%). Increases in environmental mugwort pollen counts were associated with an increase in nasal eosinophils (107% per 100 pollen/m3) and ECP (23%), but not with neutrophils, epithelial cells, or lL-8. This study demonstrated that both ambient ozone and allergen exposure are associated with inflammatory responses in the upper airways of subjects with asthma, although the type of inflammation is qualitatively different.

The overexpression of the erbB-2 (HER-2, neu) gene has attracted significant interest as a molecular target for the rational design of cancer therapies. This review examines the design and preclinical testing phase for one such experimental therapy, recombinant toxins targeted to the erbB-2 protein, termed e23(Fv)PEs.

The performance of OLX-209 indicates it should enter phase I clinical testing. OLX-209 is a recombinant toxin targeting the erbB-2 oncoprotein. The design of OLX-209 takes advantage of improvements in immunotoxin technology to produce a molecule that is smaller and more potent than a conventional chemically linked antibody-toxin conjugate. The targeting portion of OLX-209 is a single chain antibody structure derived from the anti-erbB-2 hybridoma, e23. This antibody has unusual specificity in that it does not bind to most normal tissue including peripheral nerve or kidney tissue. Preclinical testing shows in vitro activity against breast cancer cell lines in the pM range. Efficacy testing in five models of human cancer indicates that a dose of 43 micrograms/kg causes reproducible tumor regressions. Efficacy can be achieved on a variety of schedules of administration. The effective dose results in no measurable change in serum liver enzymes when delivered to mice or primates. The LD10 is over twice the effective dose in mice. The pharmacokinetics indicate a t 1/2 of 50 minutes for both mice and cynomolgus monkeys. Serum concentrations of more than ten times those observed at the effective dose can be achieved in monkeys with no evidence of toxicity. Antigenicity of OLX-209 is surprisingly low. These results form the basis for the clinical testing phase for OLX-209.

The recombinant oncotoxin OLX-209 [e23(Fv)PE38KDEL] has been developed to target cancers with erbB-2 expression and is nearing a clinical trial. Important in clinical planning is the selection of patients on the basis of tumor expression of erbB-2. ErbB-2 gene amplification occurs in cancers of the breast, stomach, and ovary. Patients with these diseases and evident overexpression are candidates for OLX-209 therapy. In lung cancer, overexpression of erbB-2 is also frequent, but in most cases, it is not caused by gene amplification. This study demonstrates that OLX-209 has activity on lung cancer cells with varying levels of erbB-2 expression in the presence and absence of gene amplification. In vitro sensitivity of cell lines to OLX-209 is related to erbB-2 expression level. Normal bronchial epithelial cells were not sensitive. Effective treatment of lung cancer cell lines growing as xenografts in nude mice was shown with Calu-3 (a lung adenocarcinoma line with high levels of p185(erbB-2) caused by gene amplification) and three other lung adenocarcinomas (A549, NCI-H1466, and 201T) with lower levels of p185(erbB-2) and no gene amplification. The 201T cell line was isolated recently from a lung tumor with erbB-2 expression in the original tumor. The results of this study indicate that patients with erbB-2-positive, non-small cell lung cancer should be included in clinical trials of OLX-209.

Short-term ozone exposure causes lung function decrements, increased airway reactivity, airway inflammation, increased respiratory symptoms and hospital admissions. Exposure to long-term elevated ozone levels seems to be associated with reduced lung function (aging), increase of respiratory symptoms, exacerbation of asthma, and airway cell and tissue changes. Health risk caused by exposure to ozone has been evaluated mainly in a qualitative way by comparing ozone air quality data with health-based guidelines or standards. A preliminary approach to quantifying health risk from short-term exposure to oxidant air pollution has been taken by expert judgement, describing known or expected effects at specific levels of ozone. For quantitative assessment of the health impact of distinct ozone exposure conditions (acute, repeated daily, chronic) specific exposure-dose-response models are being developed which can be linked to human exposure data. Exposure-(dose-)response models using data from epidemiological, human-clinical and animal toxicity studies are presented.

OLX-209 has readily measurable activity, is safe in experimental animals, and is efficacious in model systems. These results support the concept of OLX-209 and provide groundwork for further development of this oncoprotein targeted agent.

Forty-seven patients with advanced malignancies were treated with a concurrent 72-h continuous infusion of 5-fluorouracil (FUra) and dipyridamole. The FUra dose was escalated over the dose range of 185 to 3600 mg/m2/day for 3 days. Dipyridamole was administered in a fixed dose of 7.7 mg/kg/day for 3 days. A total of 155 courses of therapy were completed of which there were 31 paired courses of the combination and FUra alone, at the same dose of FUra and in the same patient. This was for purposes of analysis of pharmacokinetics and modulation of FUra toxicity by dipyridamole. Stomatitis was the dose-limiting toxicity experienced by patients entered into this trial. Myelosuppression was not a serious problem. Increasing FUra plasma concentration was associated with greater leukopenia and stomatitis. Dipyridamole did not appear to modulate the systemic toxicity of FUra. The pharmacokinetics of FUra were altered by the concurrent administration of dipyridamole. Dipyridamole promoted the total body clearance of FUra which resulted in lower mean steady-state FUra plasma concentrations when compared with courses of FUra alone administered at the same dose level. These differences were statistically significant over the course of the trial. For courses of the combination, FUra exhibited linear pharmacokinetics over the dose range studied. Total body clearance of FUra declined slightly at the higher dose levels, but the differences were not significant. For courses of FUra alone, total body clearance was significantly decreased above the dose level of 2300 mg/m2/day. At the maximal tolerated dose of FUra, 2300 mg/m2/day x3, mean steady-state FUra plasma concentration and total body clearance were 6.6 microM and 122 liters/h/m2, respectively, for courses of the combination. The corresponding pharmacokinetic parameters were 7.4 microM and 103 liters/h/m2 for courses when FUra was given alone. Further evaluation of the utility of this regimen and basis of these pharmacokinetic observations appear warranted.

The cytotoxic effects of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) were effectively antagonized by 5'-amino-5'-deoxythymidine (5'-AdThd). The antiproliferative actions of CF3dUrd were reduced in a dose-dependent manner by 5'-AdThd in both HeLa and Vero cells. In addition, the ability of CF3dUrd to kill HeLa cells (95% at 1 microM and 99% at 3 microM), as measured by cloning efficiency, was ablated entirely by 5'-AdThd (300 microM). In contrast, the inhibition of herpes simplex virus Type 2 (HSV-2) replication in HeLa cells was not antagonized by 5'-AdThd. In Vero cells, the combination of CF3dUrd and 5'-AdThd produced a greater antiviral effect than either agent alone. The reduction in CF3dUrd cytotoxicity caused by 5'-AdThd in uninfected HeLa and Vero cells was associated with decreased intracellular levels of CF3dUrd nucleotides. In contrast, in HSV-2-infected Vero cells the intracellular levels of CF3dUrd nucleotides were slightly elevated by 5'-AdThd and, in virally infected HeLa cells, a 300-fold excess of 5'-AdThd reduced CF3dUrd uptake only marginally. Since the relative abundance of these phosphorylated derivatives of CF3dUrd was not markedly changed by 5'-AdThd, preferential inhibition of the mammalian thymidine kinase (EC 2.7.1.21) was suggested. Using CF3dUrd as the substrate, the ability of 5'-AdThd to inhibit thymidine kinase activity in extracts prepared from parallel cultures of mock-infected or HSV-2 infected HeLa cells was compared. CF3dUrd was phosphorylated to a lesser extent and the reaction was more potently inhibited by 5'-AdThd in the extracts prepared from the uninfected cells. The HeLa cell and HSV-2 thymidine kinases were purified by affinity column chromatography, and kinetic analyses were then done. Using CF3dUrd as the variable substrate, the Ki values for 5'-AdThd were 2.2 microM for the HeLa enzyme and 36 microM for the viral enzyme. Km values for CF3dUrd were 2.6 microM and 4 microM for the viral and mammalian enzymes, respectively. These data account for the ability of 5'-AdThd to inhibit preferentially the phosphorylation of CF3dUrd in uninfected host cells. The presence of 5'-AdThd substantially increased the therapeutic index of CF3dUrd, indicating that this drug combination, an example of specific inhibition, warrants investigation in vivo.

Eighteen patients with hepatic metastases primarily from colorectal carcinoma were treated on a phase I protocol employing hepatic artery infusion (HAI) of 5-fluorouracil (FUra) and 5-iodo-2'-deoxyuridine (IdUrd) via implantable infusion pump. Patients received a 14-day continuous HAI of 300 mg/day FUra. During days 8-14 of therapy, patients received IdUrd as a separate 3-h HAI daily x 7. Treatment cycles were repeated every 28 days. IdUrd was escalated from 0.1 to 2.86 mg/kg/day x 7. Myelosuppression and stomatitis were mild and not dose limiting. Hepatotoxicity was dose limiting and similar to that reported for 5-fluoro-2'deoxyuridine alone administered as a 14-day infusion every month. One patient developed a clinical picture consistent with sclerosing cholangitis and another had biopsy-proven cholestasis and triaditis. Catheter complications occurred in 7 of 18 patients. Plasma concentrations of FUra during the 7-day continuous HAI of FUra alone were consistently either undetectable or very low (less than or equal to 0.1 microM). At level 3 (1.0 mg/kg/day IdUrd) and beyond, measurable plasma concentrations of FUra, iodouracil, and IdUrd were found at the end of the daily 3-h infusion of IdUrd. The maximum tolerated dose of IdUrd as administered in this trial is 2.2 mg/kg/day x 7 and the recommended starting dose for further clinical investigation is 1.7 mg/kg/day x 7.

The phosphorylation of thymidine and iododeoxyuridine by thymidine kinase was stimulated by 5-iodo-5'-amino-2',5'-dideoxyuridine ( AIdUrd ). Antagonism of the feedback inhibition that is normally exerted by the 5'-triphosphates of thymidine and iododeoxyuridine appears to account for the stimulation. The effect of AIdUrd on thymidine kinase purified from HeLa cells by affinity column chromatography was critically dependent on the presence of these feedback inhibitors. In the presence of thymidine triphosphate or iododeoxyuridine triphosphate, AIdUrd could markedly stimulate ( deinhibit ) enzyme activity, whereas, in their absence, AIdUrd inhibited thymidine kinase with an apparent Ki of 0.7 microM. Stimulation was evident over a wide range of concentrations of both iododeoxyuridine and adenosine triphosphate. In intact HeLa and Vero cells, phosphorylation of thymidine and iodoeoxyuridine was strongly enhanced by AIdUrd . Large increases in the intracellular levels of nucleotides derived from exogenous thymidine and iododeoxyuridine were apparent. As a consequence, the cytotoxicity of both nucleosides was exacerbated by AIdUrd . The reductions in cellular replication rates and colony formation produced by iododeoxyuridine were enhanced by AIdUrd . Although the replication of HeLa cells was not inhibited by either thymidine (30 microM) or AIdUrd (300 microM), in combination they were strongly synergistic and produced a 60% inhibition of cellular growth. Under these conditions, the uptake of thymidine was increased over 300% by AIdUrd . AIdUrd represents a new regulatory antagonist of thymidine kinase which may be useful in novel chemotherapeutic strategies.

Iododeoxyuridine (IdUrd) potentiated the lethal but not the growth inhibitory properties of fluorouracil (FUra) and fluorodeoxyuridine (FdUrd) in human bladder cancer cells (T24). The rate of incorporation of IdUrd into DNA was enhanced by both fluoropyrimidines, but to a significantly greater extent by FdUrd. Both inhibition of iododeoxyridylate dehalogenation and the depletion of thymidine triphosphate pools contributed to the increased incorporation rate. Inhibition of dehalogenation accounted for 67% of the observed stimulation in the case of FUra, but only 37% of the increase produced by FdUrd. The depletion of dTTP pools, both in the presence and absence of IdUrd, was greater after FdUrd than FUra exposure. The observed increase in the rate of incorporation of IdUrd appears to account for the enhanced toxicity seen with FdUrd, but other factors may be involved in the case of FUra. Since FUra and IdUrd appear to be mutually potentiating and do not share a dependence on thymidine kinase activity, this drug combination warrants further investigation.

The effects of a variety of 5-, 5'-, and 3'-substituted deoxyuridine derivatives on the cytoplasmic thymidine kinase (EC 2.7.1.21) purified from a human colon carcinoma cell line, HCT 116, were determined. Of particular interest was elucidation of the structural features important for antagonism of the feedback inhibition of thymidine kinase exerted by thymidine triphosphate. Substitutions at the 5-position altered the potency of the 5'-modified compounds. The replacement of the 5-hydrogen with a methyl group or an iodine greatly increased the affinity of compounds for the thymidine kinase. This was evident for enzyme substrates with 5'-hydroxyl groups [2'-deoxyuridine (dUrd), 2'-deoxythymidine (dThd) and 5-iodo-2'-deoxyuridine (IdUrd)], feedback inhibitors with 5'-triphosphate substitutions (dUTP, dTTP and IdUTP), and for 5'-amino derivatives [5'-amino-2',5'-dideoxyuridine (5'-AdUrd), 5'-amino-2'-5'-dideoxythymidine (5'-AdThd) and 5-iodo-5'-amino-2',5'-dideoxyuridine (5'-AIdUrd)]. Qualitatively, however, the 5-substitutions did not affect the nature of the interactions with dThd kinase. For example, in the presence of dTTP, 5'-AdUrd stimulated dThd kinase activity as much as 5'-AdThd, but approximately a 100-fold greater concentration of 5'-AdUrd was required. Similar results were obtained using intact cells in which substitutions at the 5-position affected the potency, but not the efficacy, of the 5'-amino derivatives to stimulate dThd phosphorylation. In contrast, substitutions at the 5'-position did alter the nature of the interaction with dThd kinase. Thus, the 5'-hydroxyl compounds, dUrd, dThd and IdUrd, did not reverse the enzyme inhibition produced by dTTP nor did they stimulate dThd uptake in intact cells. 5'-Deoxy-5'-(ethylthio)thymidine, 5'-deoxy-5'-[(2-hydroxyethyl)thio]thymidine, and dTMP, but not dTDP, also antagonized the inhibition of dThd kinase produced by dTTP. In comparison to 5'-AdThd, the 3'-amino derivatives, 3'-AdThd and 3'-5'-diAdThd, were much less potent, but still efficacious, antagonists of feedback inhibition. These results indicate that a wide range of dUrd derivatives can disrupt the regulation of dThd kinase and provide leads for the development of new nucleotide analogues.

5'-Aminothymidine represents a novel class of compounds capable of antagonizing the feedback inhibition which normally regulates thymidine kinase. As a consequence, the uptake of iododeoxyuridine, a substrate of thymidine kinase, can be substantially increased by 5'-aminothymidine. in this study the phosphorylation, uptake, and cytotoxicity of iododeoxyuridine was markedly enhanced by 5'-aminothymidine in three human bladder cancer cell lines, T24, HT1197 and 647V. In contrast, neither the uptake nor the toxicity of iododeoxyuridine was increased by 5'-aminothymidine in normal human urothelial cells propagated in vitro. Although 30 microM 5'-aminothymidine increased the uptake of 3 microM iododeoxyuridine 4- to 5-fold in the HT1197 and 647V cells and 2.5-fold in the T24 cells, no stimulation was produced in the normal urothelial cells. The modulation of iododeoxyuridine uptake was associated with parallel changes in the inhibition of cellular replication. The cytotoxicity of iododeoxyuridine was strongly augmented by 5'-aminothymidine in the HT1197 and 647V cells, modestly increased in the T24 cells, and unchanged in the normal urothelial cells. The degree to which iododeoxyuridine phosphorylation was stimulated did not correlate with cellular replication rates or with intracellular thymidine triphosphate concentrations. Iododeoxyuridine uptake was markedly increased in the HT1197 (doubling time = 52 h) and 647V (doubling time = 24 h) cells, moderately in the rapidly growing cells T24 (doubling time = 20 h), and minimally in the normal urothelial cells which doubled every 32 h. In exponentially growing cells the thymidine triphosphate pools were approximately 18 microM in the normal cells and about 21, 24, and 35 microM in the HT1197, T24, and 647V cells, respectively. The use of 5'-aminothymidine and other compounds capable of antagonizing feedback inhibition may provide a new means of increasing the efficacy of cytotoxic nucleosides.

We have previously reported that 5'-aminothymidine (5'-AdThd), an antagonist of the feedback inhibition exerted by dTTP that regulates thymidine kinase, enhances the uptake and cytotoxicity of 5-iododeoxyuridine in various human bladder cancer cell lines but not in normal human urothelial cells (HU) propagated in vitro. In this work we have analyzed the factors that could potentially account for the differential effect of 5'-AdThd among various cell types: 647V (a human bladder cancer cell line); HU; SV-HU (a SV40-transformed human urothelial cell line), and C3H/10T1/2 mouse embryo fibroblasts (10T1/2) cells. 5'-AdThd enhanced the uptake of IdUrd in SV-HU cells (greater than 400%), similar to what we have observed before for 647V cells. However, in 10T1/2 and HU cells, 5'-AdThd only minimally increased the uptake of 5-iododeoxyuridine (about 160%). Thymidine kinases purified from the different sources were similarly sensitive to inhibition by dTTP or 5'-AdThd and to deinhibition of the dTTP-induced regulation of enzyme activity by 5'-AdThd. Furthermore, [3H]-5'-AdThd permeated and accumulated intracellularly in all cell types. In none of these cultures was nucleoside phosphorylase activity detected, as indicated by the inability of the cells to produce thymine or iodouracil after exposure to the appropriate nucleosides. Also, 5'-AdThd did not affect the breakdown of dTMP by crude preparations of cytosolic 5'-nucleotidase from the different cells. We found that intracellular dTTP pools in the various cell types were substantially high (15-26 microM) compared to the sensitivity of thymidine kinase to inhibition by dTTP (IC50 2-4 microM). This suggests that thymidine kinase is in a strongly inhibited state in situ. To test the sensitivity of thymidine kinase (in situ) to regulation by dTTP we investigated: (a) the effect of depleting intracellular dTTP pools with methotrexate on the uptake of thymidine (dThd); and (b) the effect of pH on the uptake of dThd and its perturbation by 5'-AdThd, since the inhibition of thymidine kinase activity by dTTP is known to be pH dependent. We found that a 47% reduction of dTTP pools by methotrexate in 10T1/2 and HU cells did not result in an increase in thymidine kinase activity, as indicated by the lack of an effect on the uptake of dThd. However, we have previously shown that, under similar conditions, 647V cells show a substantial increase in dThd uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

We have studied the effect of pH on the interactions between thymidine kinase, thymidine triphosphate, and 5'-amino-2',5'-dideoxythymidine (5'-AdThd) in purified preparations of the enzyme and in intact 647V cells, a human bladder cancer cell line. Thymidine kinase is competitively inhibited by 5'-AdThd. dTTP feedback inhibits in a noncompetitive fashion. However, 5'-AdThd partially reverses the inhibition produced by dTTP resulting in enhanced enzyme activity. We have found that dTTP (pKa = 7.5) is a much more potent inhibitor of purified preparations of thymidine kinase activity at low pH conditions. For example, 2.5 microM dTTP inhibited thymidine kinase activity by 50, 85, and 95% at pH values of 8.0, 7.5, and 6.5, respectively. The interaction of 5'-AdThd (pKa = 8.5) at either the active (competitive) or the regulatory (deinhibition) site is not altered significantly over a pH range of 6.5 to 9.5. To extend these findings to intact cells, we studied the perturbation of the uptake of thymidine by 5'-AdThd in 647V cells incubated in media buffered at various pH values. In cells exposed to media buffered at pH 8.5 or 7.5, 5'-AdThd maximally stimulated thymidine uptake about 250 and 300% at 10 and 30 microM, respectively. However, at pH 6.5, 300 microM 5'AdThd was required to produce maximal stimulation of about 500%. These observations are consistent with the greater sensitivity of thymidine kinase (in situ) to feedback inhibition by dTTP at the lower pH conditions. Intracellular dTTP pool sizes were not affected by variations in pH during the short time course of our experiments. However, after 1 h, the intracellular concentration of 5'-AdThd was twice that of the extracellular medium in conditions at pH 7.5 and 8.5 but was equimolar across the membrane at pH 6.5. This does not account for the differences in the perturbation of thymidine uptake by 5'-AdThd at various pH values. In general, our results indicate that regulation of thymidine kinase by dTTP is pH dependent, while its modulation by 5'-AdThd is not, and that regulation of thymidine kinase in situ is sensitive to alterations in pH.

5'-Amino-5'-deoxythymidine (5'-AdThd) was found to antagonize the feedback inhibition exerted by dTTP on dThd kinase (EC 2.7.1.21). This effect was demonstrable in intact cells, cellular extracts, and purified enzyme preparations. Thus, 5'-AdThd markedly stimulated the uptake of dThd in Hela and Vero cells and reduced the inhibitory effects of dTTP on the dThd kinase activity measured in extracts from both cell types. dThd kinase was purified by affinity column chromatography from Hela and Vero cells, and 5'-AdThd was again shown to reduce the inhibition caused by dTTP. The ability of 5'-AdThd to disrupt the homeostatic mechanisms normally regulating the uptake of dThd was sufficient to convert a noncytotoxic concentration of dThd (30 microM) to one that inhibited Hela cell growth by 40%. Since the activity of regulatory enzymes critically influences the pharmacological response produced by many agents, we propose the design of compounds specifically targeted at enzyme regulatory sites as an approach to drug development.

Isopycnic centrifugation in CsCl gradients was used to quantify the incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus type 1 DNA. A parallelism between the degree of incorporation into viral DNA and the inhibition of herpes simplex virus type I replication was found for both thymidine analogs. A concentration of 5-iodo-5'-amino-2',5'-dideoxyuridine approximately 100 times greater than 5-iodo-2'-deoxyuridine was required to achieve similar levels of antiviral activity. However, the inhibitory effects of these compounds are similar when compared with respect to the percent of substitution for thymidine in herpes simplex virus type I DNA. Damage to the viral DNA, as indicated by the presence of single or double-stranded breaks, was assessed by centrifugation in alkaline and neutral sucrose gradients. The incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into herpes simplex virus type I DNA produced single and, to a lesser extent, double-stranded breaks in a dose-dependent manner. 5-Iodo-2'-deoxyuridine did not, however, induced DNA breakage. These data indicate that the additional presence of a phosphoramidate bond in the DNA produced the extensive damage detected under these conditions, but that such damage is not required for antiviral activity.

Although the interaction between FUra and DP in HCT 116 cells is fairly complex, data from other investigators indicate that in cell lines in which inhibition of TS is growth limiting at relatively low concentrations of fluoropyrimidines, DP appears to augment the cytotoxicity of FUra and FdUrd by blocking the salvage of dThd (Miller et al., 1987; Schwartz et al., 1987). The previous in vitro data regarding the ability of DP to modulate the toxicity of fluoropyrimidines was obtained in exponentially growing cells. An additional observation that warrants consideration is a report that the inhibition of nucleoside incorporation by DP changed as a function of time in culture (Zhen et al., 1986). Hepatoma 3924A cells in lag and log phase were highly sensitive to DP with IC50 values for dThd incorporation of 0.2 and 0.32 microM, respectively. In contrast, stationary phase cells were insensitive to DP (IC50 = 38.9 microM). Amphotericin B, an antifungal agent which perturbs cell membranes, restored the sensitivity to DP in stationary cells. Several investigators have presented information on the effect of DP on fluoropyrimidines in normal tissues. Lee and Park (1987) examined the effect of DP on FUra and MTX toxicity in a soft-agar cloning assay against two human cancer cell lines and on pooled normal human bone marrow (CFU-C). DP (1 microM) potentiated the action of both MTX (0.1 microM) and FUra (5 microM) on Hep-2 (epidermoid carcinoma), MCF-7 (breast carcinoma) and CFU-C in medium supplemented with either non-dialyzed or dialyzed serum. Woodcock et al. (1987) incubated gallbladder mucosa, obtained from patients undergoing elective surgery for cholelithiasis, with control medium or varying concentrations of DP for 1 hr, and then exposed the mucosal cells to 2.5 microCi [3H]-FdUrd (2.5 microM). After 1 hr, the uptake of FdUrd into the tissue was inhibited to 49% and 42% of control by 0.1 microM and 1 microM, respectively.