Comprehensive identification of genes driven by ERV9-LTRs reveals TNFRSF10B as a re-activatable mediator of testicular cancer cell death.

The long terminal repeat (LTR) of human endogenous retrovirus type 9 (ERV9) acts as a germline-specific promoter that induces the expression of a proapoptotic isoform of the tumor suppressor homologue p63, GTAp63, in male germline cells. Testicular cancer cells silence this promoter, but inhibitors of histone deacetylases (HDACs) restore GTAp63 expression and give rise to apoptosis. We show here that numerous additional transcripts throughout the genome are driven by related ERV9-LTRs. 3' Rapid amplification of cDNA ends (3'RACE) was combined with next-generation sequencing to establish a large set of such mRNAs. HDAC inhibitors induce these ERV9-LTR-driven genes but not the LTRs from other ERVs. In particular, a transcript encoding the death receptor DR5 originates from an ERV9-LTR inserted upstream of the protein coding regions of the TNFRSF10B gene, and it shows an expression pattern similar to GTAp63. When treating testicular cancer cells with HDAC inhibitors as well as the death ligand TNF-related apoptosis-inducing ligand (TRAIL), rapid cell death was observed, which depended on TNFRSF10B expression. HDAC inhibitors also cooperate with cisplatin (cDDP) to promote apoptosis in testicular cancer cells. ERV9-LTRs not only drive a large set of human transcripts, but a subset of them acts in a proapoptotic manner. We propose that this avoids the survival of damaged germ cells. HDAC inhibition represents a strategy of restoring the expression of a class of ERV9-LTR-mediated genes in testicular cancer cells, thereby re-enabling tumor suppression.

A novel and selective sodium-glucose cotransporter-2 inhibitor, tofogliflozin, improves glycaemic control and lowers body weight in patients with type 2 diabetes mellitus.

AIM: To assess the efficacy, safety and tolerability of different doses of tofogliflozin, a novel, highly selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, in patients with type 2 diabetes mellitus (T2DM). METHODS: In a 12-week, multicentre, multinational, randomized, double-blind, parallel-group, placebo-controlled, dose-finding study, patients with inadequate glycaemic control from diet and exercise alone, or from diet and exercise plus a stable dose of metformin, were randomized to one of five doses of tofogliflozin (2.5, 5, 10, 20, or 40 mg) or placebo. The primary efficacy endpoint was absolute change at week 12 from baseline in glycated haemoglobin (HbA1c), minus the change in the placebo group. RESULTS: Statistically significant dose-dependent reductions in HbA1c were shown in all treated groups except the 2.5-mg dose group, with a maximum reduction of 0.56% (placebo-subtracted) at the 40-mg dose, along with increased urinary glucose excretion. Metformin treatment had no substantial influence on tofogliflozin efficacy. Dose-dependent reductions in fasting plasma glucose and body weight were observed, and glucose intolerance was improved, with a trend towards blood pressure reduction. Slight increases were observed for mean ketone bodies with no abnormal change in ketone body ratio. No deaths or treatment-related serious adverse events were reported. The incidence of adverse events was similar in the placebo (37.9%) to that in the tofogliflozin group (35.9-46.3%). Withdrawal because of adverse events was rare (≤2 patients per treatment group), with similar rates of withdrawal in the placebo and tofogliflozin groups. CONCLUSIONS: A once-daily dose of tofogliflozin for 12 weeks was an effective, safe and well-tolerated treatment for T2DM.

C.E.R.A. safety profile: a pooled analysis in patients with chronic kidney disease.

BACKGROUND: C.E.R.A., a continuous erythropoietin receptor activator, is a long-acting erythropoiesis-stimulating agent (ESA) that is approved for the treatment of renal anemia. This analysis evaluated the safety profile of C.E.R.A. in comparison to that of other ESAs in patients with chronic kidney disease (CKD). METHODS: Safety parameters were analyzed in a pooled population comprising all patients with CKD on dialysis and not on dialysis from the completed Phase II and Phase III studies in the C.E.R.A. clinical program (Phase II/III population); patients were treated with either C.E.R.A. (n = 1,789) or comparator ESA (n = 948). Differences between treatment groups in safety parameters were identified by either a 2% difference in incidence between groups, or a statistically significant difference between groups (p < or = 0.05 with the Fisher's exact test, which was used as a conservative screening tool). To assess changes in safety findings over time, long-term safety data were analyzed from patients who were given the option to enter long-term safety studies upon completing their initial Phase II/III study (safety extension population). RESULTS: Compared with the C.E.R.A. group, the incidence of adverse events (AEs) was higher in the comparator ESA group in the Phase II/III population (C.E.R.A. vs. comparator ESA, 89.5% vs. 91.8%, p = 0.067), and significantly so in the safety extension population (93.0% vs. 95.8%, p = 0.003). The incidence of serious AEs was significantly higher in the comparator ESA group than in the C.E.R.A. group in both analysis populations (Phase II/III population, 37.8% vs. 42.4%, p = 0.021; safety extension population, 53.3% vs. 59.7%, p = 0.001). However, there was no consistent pattern of clinical events that could explain these differences between the treatment groups. CONCLUSION: Analysis of safety events in patients with renal anemia receiving long-term treatment with C.E.R.A. shows a safety profile comparable to that of other ESAs.

The alpha/beta carboxy-terminal domains of p63 are required for skin and limb development. New insights from the Brdm2 mouse which is not a complete p63 knockout but expresses p63 gamma-like proteins.

p63, an ancestral transcription factor of the p53 family, has three C-terminal isoforms whose relative in vivo functions are elusive. The p63 gene is essential for skin and limb development, as vividly shown by two independent global knockout mouse models. Both strains, although constructed differently, have identical and severe phenotypes, characterized by absent epidermis and hindlimbs and only rudimentary forelimbs at birth. Here we show that mice from one model, Brdm2, express normal levels of truncated p63 proteins that contain the DNA binding and oligomerization domain but lack the long carboxy-terminal SAM (sterile alpha-motif) and post-SAM domains that are specific for the alpha and beta isoforms. As such, transcriptionally active p63 proteins from Brdm2 mice resemble the naturally occurring p63gamma isoforms, which of all the p63 isoforms most closely resemble p53. Thus, Brdm2 mice are p63alpha/beta isoform-specific knockout mice, gaining unexpected new importance. Our studies identify that p63alpha/beta but not p63gamma are absolutely required for proper skin and limb development.

Calibration of the viscometric glucose sensor before its use in physiological liquids--compensation for the colloid-osmotic effect.

The function of the recently described viscometric affinity sensor (VAS), which measures glucose due to its strong effect on the viscosity of a sensitive liquid containing Concanavalin A (ConA) and dextran, was analysed for osmotic and colloid-osmotic effects on the glucose reading. The suction of low- and high-molecular weight osmotica on the membrane of the microdialysis fibre was measured using a membrane osmometer built from the microdialysis probe of the VAS. The reduction of the sensor read-out in blood plasma can be completely explained by a change in small osmotic volume fluxes through the dialysis membrane, which affect the ConA concentration and the viscosity after the flow of the sensitive liquid through the dialysis probe. The measuring error could be prevented by the presence of the polyethylene glycol 6000 at an isotonic concentration in the glucose standard solutions used for sensor calibration.

Ring-shaped vortex domain in type-II superconductors.

We present results of experiments in superconducting niobium and numerical simulations showing the creation of a metastable ring-shaped vortex domain by heating. Such vortex rings, if pinned by structural defects, can exist forever.

Recording of subcutaneous glucose dynamics by a viscometric affinity sensor.

AIMS/HYPOTHESIS: To provide a nonenzymatic sensor for glucose monitoring in subcutaneous tissue. METHODS: A continuously working affinity sensor based on the glucose-dependent viscosity of a sensitive liquid containing dextran and concanavalin A has been designed by arranging a microdialysis probe, two flow-resisting capillaries and two pressure transducers in a linear flow system. It allows synchronous processing of the viscosity of the sensitive liquid at the standard glucose concentration and the glucose concentration to be measured. In preliminary human trials the sensor was implanted into the subcutaneous tissue of the forearm and its read-out was compared with capillary blood concentrations. RESULTS: In vitro, the viscometric sensor shows a linear and long-term stable dependence on the glucose concentration without detectable drift. At the applied flow rate of the sensitive liquid (about 5 microliters/h) the technical delay is 5 to 10 min. The read-out of the implanted sensor followed the dynamics of the capillary blood glucose concentrations with a time-shift of 10 to 15 min but showed a systematic error when based on precalibration with polymer-free glucose solutions. After appropriate in vivo calibration, the read-out was in good or acceptable coincidence with capillary blood concentrations according to the error grid method and did not show any detectable reduction of sensitivity during the periods of measurement (up to 44 h). CONCLUSION/INTERPRETION: The viscometric-affinity sensor is an efficient tool for current research on glucose monitoring in the subcutaneous tissue and can potentially be further developed for routine clinical use.

Differences in the intracellular distribution of acid-sensitive doxorubicin-protein conjugates in comparison to free and liposomal formulated doxorubicin as shown by confocal microscopy.

PURPOSE: To investigate differences in the cellular uptake and intracellular distribution of protein-bound doxorubicin in comparison to free doxorubicin and a liposomal formulation (CAELYX) METHODS: LXFL 529 lung carcinoma cells were incubated with an acid-sensitive transferrin and albumin conjugate of doxorubicin, a stable albumin doxorubicin conjugate, and free and liposomal doxorubicin for up to 24 h. The uptake of doxorubicin was detected with confocal laser scanning microscopy (CLSM). To investigate the intracellular localization of the anticancer drug, lysosomes, Golgi apparatus, and mitochondria were also stained by various organelle-specific fluorescent markers. In vitro efficacy of the doxorubicin derivatives was examined with the BrdU incorporation assay. RESULTS: The acid-sensitive albumin and transferrin doxorubicin conjugates showed enhanced cytotoxicity in comparison to liposomal doxorubicin, whereas the stable albumin-doxorubicin conjugate showed only marginal activity. Of all compounds tested, doxorubicin showed the highest cytotoxicity. CLSM studies with specific markers for lysosomes, mitochondria, and the Golgi apparatus demonstrated that protein-bound doxorubicin or liberated doxorubicin was accumulated in the mitochondria and Golgi compartments, but not in the lysosomes after 24 h. Free doxorubicin showed a time-dependent intracellular shift from the nucleus to the mitochondria and Golgi apparatus. Fluorescence resulting from incubation with CAELYX was primarily detected in the nucleus. CONCLUSIONS: Our results indicate that other organelles in addition to the cell nucleus are important sites of accumulation and interaction for protein-bound doxorubicin or intracellularly released doxorubicin as well as for free doxorubicin.

Compensation of temperature and concanavalin A concentratration effects for glucose determination by the viscometric affinity assay.

A viscometer suitable for rapid measurements in small volumes of highly viscous liquids is described. Using this device the viscometric affinity assay for glucose was studied under variable conditions in order to obtain basic information for the design of a viscometric glucose sensor. The viscosity of the dextran/Concanavalin A (ConA) solution is sensitive to glucose in a broad range of the shear stress. However, for measuring the glucose concentration with this sensitive liquid the strong dependence of the absolute viscosity on temperature and ConA concentration has to be taken into account. For the purpose of calibration a parameter more suitable than the absolute viscosity is the relative fluidity (F(r)) that is defined by the actual measured viscosity at a given glucose concentration, the reference viscosity at a standard glucose concentration, and a constant linearization coefficient. F(r) shows a linear dependence on the glucose concentration in the therapeutically interesting range up to 30 mM and is not significantly changed by moderate variations of the ConA concentration or temperature.

Drug-polymer conjugates containing acid-cleavable bonds.

Drug-polymer conjugates are potential candidates for the selective delivery of anticancer agents to tumor tissue. Incorporating acid-sensitive bonds between the drug and the polymer is an attractive approach because it ensures effective release of the polymer-bound drug at the tumor site. This release is either extracellular, resulting from the slightly acidic pH in tumor tissue, or intracellular, in acidic endosomes or lysosomes after cellular uptake of the drug-polymer conjugate. This paper reviews acid-sensitive drug-polymer conjugates developed during the past 20 years and outlines aspects for further development in this research field.

Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and intracellular distribution.

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to alpha-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), alpha,omega-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or alpha-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02-1.5 microm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.

Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil.

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.

Systemic Fusarium oxysporum infection in an immunocompetent patient with an adult respiratory distress syndrome (ARDS) and extracorporal membrane oxygenation (ECMO).

We report a case of disseminated fusariosis in a 42-year-old patient with adult respiratory distress syndrome (ARDS) and extracorporal membrane oxygenation (ECMO), but without definite immunosuppression. Fusarium oxysporum was isolated from a bronchial lavage taken 6 days ante mortem. Despite antifungal treatment with amphotericin B and flucytosine the patient died in septic multiorgan failure. A post-mortem examination was performed. The patient's liver was found to contain fungus cells and F. oxysporum could be cultured from ascites.

Transferrin conjugates of doxorubicin: synthesis, characterization, cellular uptake, and in vitro efficacy.

One strategy for improving the antitumor selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing suitable carrier proteins. Thus, thiolated human serum transferrin was conjugated with four maleimide derivatives of doxorubicin that differed in the stability of the chemical link between drug and spacer. Of the maleimide derivatives, 3-maleimidobenzoic or 4-maleimidophenylacetic acid was bound to the 3'-amino position of doxorubicin through a benzoyl or phenylacetyl amide bond, and 3-maleimidobenzoic acid hydrazide or 4-maleimidophenylacetic acid hydrazide was bound to the 13-keto position through a benzoyl hydrazone or phenylacetyl hydrazone bond. The acid-sensitive transferrin conjugates prepared with the carboxylic hydrazone doxorubicin derivatives exhibited an inhibitory efficacy in the MDA-MB-468 breast cancer cell line and U937 leukemia cell line comparable to that of the free drug (employing the BrdU (5-bromo-2'-deoxyuridine) incorporation assay and tritiated thymidine incorporation assay, respectively, IC50 approximately 0.1-1 mM), whereas conjugates with the amide derivatives showed no activity. Furthermore, antiproliferative activity of the most active transferrin conjugate (i.e. the conjugate containing a benzoyl hydrazone link) was demonstrated in the LXFL 529 lung carcinoma cell line employing a sulforhodamine B assay. In contrast to in vitro studies in tumor cells, cell culture experiments performed with human endothelial cells (HUVEC) showed that the acid-sensitive transferrin conjugates of doxorubicin were significantly less active than free doxorubicin (IC50 values approximately 10-40 higher by the BrdU incorporation assay), indicating selectivity of the doxorubicin-transferrin conjugates for tumor cells. Fluorescence microscopy studies in the MDA-MB-468 breast cancer cell showed that free doxorubicin accumulates in the cell nucleus, whereas doxorubicin of the transferrin conjugates is found localized primarily in the cytoplasm. The differences in the intracellular distribution between transferrin-doxorubicin conjugates and doxorubicin were confirmed by laser scanning confocal microscopy in LXFL 529 cells after a 24 h incubation that revealed an uptake and mode of action other than intercalation with DNA. The relationship between stability, cellular uptake, and cytotoxicity of the conjugates is discussed.

Albumin conjugates of the anticancer drug chlorambucil: synthesis, characterization, and in vitro efficacy.

In our efforts to improve the selectivity and toxicity profile of antitumor agents, four maleimide derivatives of chlorambucil (1-4) were bound to thiolated human serum albumin which differ in the stability of the chemical link between drug and spacer. 1 is an aliphatic maleimide ester derivative of chlorambucil, whereas 2-4 are acetaldehyde, acetophenone, and benzaldehyde carboxylic hydrazone derivatives. HPLC stability studies at pH 5.0 with the related model compounds 5, 7, 8, and 9, in which chlorambucil was substituted by 4-phenylbutyric acid, demonstrated that the carboxylic hydrazone derivatives have acid-sensitive properties; the acid lability of 7 was particular prominent with a half-life of only a few hours. The alkylating activity of albumin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)-pyridine (NBP), demonstrating that on average three equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective albumin conjugates in the MCF7 mamma carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugate in which chlorambucil was bound to albumin through an ester bond was not as active as chlorambucil. In contrast, the conjugates in which chlorambucil was bound to albumin through carboxylic hydrazone bonds were as or more active than chlorambucil in both cell lines. In particular, the conjugate in which chlorambucil was bound to albumin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 4-fold (MCF7) to 13-fold (MOLT4) lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil.

Preparation, characterization and in vitro efficacy of albumin conjugates of doxorubicin.

One strategy for improving the antitumor selectivity and toxicity profile of antitumor agents is to design drug carrier systems with suitable transport proteins. Thus, four maleimide derivatives of doxorubicin were bound to thiolated human serum albumin which differed in the stability of the chemical link between drug and spacer. In the maleimide derivatives, 3-maleimidobenzoic or 4-maleimidophenylacetic acid was bound to the 3'-amino position of doxorubicin through a benzoyl or phenylacetyl amide bond and 3-maleimidobenzoic acid hydrazide or 4-maleimidophenylacetic acid hydrazide was bound to the 13-keto position through a benzoyl hydrazone or phenylacetyl hydrazone bond. The acid-sensitive albumin conjugates prepared with the carboxylic hydrazone doxorubicin derivatives exhibited an inhibitory efficacy in the MDA-MB-468 breast cancer cell line and U937 leukemia cell line comparable with that of the free drug (using the BrdU-(5-bromo-2'-deoxyuridine)-incorporation assay and tritiated thymidine incorporation assay respectively, IC50 approximately 0.1-1 microM) whereas conjugates with the amide derivatives showed no or only marginal activity. These results demonstrate that antiproliferative activity depends on the nature of the chemical bond between doxorubicin and carrier protein. Acid-sensitive albumin conjugates are suitable candidates for further in vitro and in vivo assessment.

Serum proteins as drug carriers of anticancer agents: a review.

Targeted delivery of anticancer drugs is one of the most actively pursued goals in anticancer chemotherapy. Serum proteins such as transferrin, albumin, and low-density lipoprotein (LDL) offer promise for the selective delivery of antineoplastic agents due to their accumulation in tumor tissue. Uptake of these proteins in solid tumors is mediated by a number of factors, including an increased metabolic activity of tumors, an enhanced vascular permeability of tumor blood vessels for circulating macromolecules, and a lack of a functional lymphatic drainage system in tumor tissue. At the tumor site, transferrin, low-density lipoprotein, and albumin are taken up by the tumor cell through receptor-mediated and fluid phase endocytosis, respectively. Serum protein conjugates can be designed to release the bound antitumor drug after cellular uptake of the drug conjugate. This review covers the diagnostic evidence for tumor accumulation of serum proteins and the design, development, and biological evaluation of drug conjugates with transferrin, albumin, and low-density lipoprotein.

Large increase in cardiac output in a patient with ARDS and acute right heart failure during inhalation of nitric oxide.

BACKGROUND: Inhaled nitric oxide (NO), a selective pulmonary vasodilator, reduces pulmonary artery pressure in patients with acute respiratory distress syndrome (ARDS). In spite of the reduction of right ventricular afterload, the effect of NO on cardiac output remains unclear. METHODS: A patient with ARDS and echocardiographically determined severe acute right heart failure was treated with increasing concentrations of inhaled nitric oxide (NO). Haemodynamic and gas exchange variables were determined for each concentration of NO. NO treatment was continued for 3 days. RESULTS: During initial right heart failure, administration of NO resulted in a large increase (32%) in cardiac output in a dose-dependent manner. When right ventricular function had improved, inhalation of NO did not increase cardiac output. CONCLUSION: Our observations suggest that inhalation of NO is likely to increase cardiac output in ARDS when severe acute right heart failure is present.

Acute health effects of exposure to high levels of air pollution in eastern Europe.

Acute effects of winter-type air pollution characterized by high levels of SO2, moderate levels of particles, and low acidity were studied. A panel of 155 asthmatic children and 102 adults with a history of asthma from the former German Democratic Republic cities of Erfurt and Welmar and from the Czech Republic city of Sokolov participated from September 1990 through June 1992. The panelists recorded daily symptoms, medication intake, and peak expiratory flow (PEF). Statistical analysis was based on linear regression of population-averaged time series controlling for trend, meteorology, and autocorrelation. A temporospatial time series approach was also applied to the data to eliminate possible confounding by some known or unknown variables that occurred simultaneously in two of the study areas. Weak same-day effects and a stronger cumulative effect of air pollution on children was observed both for PEF and for symptoms. PEF decreased -0.90% (-1.35 to -0.46%), and a symptom score increased 14.7% (0.8-28.6%) in association with an average increase of 128 micrograms/m3 SO2 over the previous 5 days. Effects on adults were smaller and less consistent. Morbidity of children was best predicted by SO2 and sulfate concentrations. The authors conclude that prolonged, high exposure to winter-type pollution was associated with small adverse health effects in asthmatics.