Gene delivery corrects N-acetylglutamate synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-acetylglutamate synthase.

The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-acetylglutamate synthase (NAGS) catalyzes formation of N-acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags-/- mice, established the dose of the vector needed to rescue Nags-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.

FBXL4 defects are common in patients with congenital lactic acidemia and encephalomyopathic mitochondrial DNA depletion syndrome.

Mutations in FBXL4 have recently been recognized to cause a mitochondrial disorder, with clinical features including early onset lactic acidosis, hypotonia, and developmental delay. FBXL4 sequence analysis was performed in 808 subjects suspected to have a mitochondrial disorder. In addition, 28 samples from patients with early onset of lactic acidosis, but without identifiable mutations in 192 genes known to cause mitochondrial diseases, were examined for FBXL4 mutations. Definitive diagnosis was made in 10 new subjects with a total of 7 novel deleterious variants; 5 null and 2 missense substitutions. All patients exhibited congenital lactic acidemia, most of them with severe encephalopathic presentation, and global developmental delay. Overall, FBXL4 defects account for at least 0.7% (6 out of 808) of subjects suspected to have a mitochondrial disorder, and as high as 14.3% (4 out of 28) in young children with congenital lactic acidosis and clinical features of mitochondrial disease. Including FBLX4 in the mitochondrial diseases panel should be particularly important for patients with congenital lactic acidosis.

Localization of TEIF in the centrosome and its functional association with centrosome amplification in DNA damage, telomere dysfunction and human cancers.

Centrosome amplification and telomere shortening, which are commonly detected in human cancers, have been implicated in the induction of chromosome instability in tumorigenesis. The functions of these two structures are closely related to DNA damage repair machinery, and some factors that operate in the maintenance of telomeres also take part in the regulation of centrosome status, suggesting they are functionally linked. We report that TEIF (telomerase transcriptional elements-interacting factor), a transactivator of the hTERT (human telomerase reverse transcriptase subunit) gene, is distributed in the centrosome throughout the cell cycle, but its transport into the centrosome is increased under some conditions, and its distribution is dependent on its C-terminal domain. Experimental modulation of TEIF expression through overexpression, polypeptide expression or depletion affected centrosome status and increased abnormalities of cell mitosis. Localization of TEIF to the centrosome was also stimulated by treatment with genotoxic agents and experimental telomere dysfunction, accompanying centrosome amplification. Moreover, we demonstrated that the expression level of TEIF is not only closely correlated with centrosome amplification in soft tissue sarcomas but it is also significantly related to tumor histologic grade. Our data confirmed TEIF functions as a centrosome regulator. Its participation in DNA damage response, including telomere dysfunction and tumorigenesis, indicates TEIF is likely to be a factor involved in linking centrosome amplification and telomere dysfunction in cancer development.

Distinctly different expression of cytokines and chemokines in the lungs of two H5N1 avian influenza patients.

The pathogenesis of human H5N1 influenza remains poorly understood and controversial. 'Cytokine storm' has been hypothesized to be the main cause of the severity of this disease. However, the significance of this hypothesis has been called into question by a recent report, which demonstrates that inhibition of the cytokine response did not protect against lethal H5N1 influenza infection in mice. Here we showed discrepant findings in two adult H5N1 autopsies and a fetus obtained at autopsy which also raise doubt about this hypothesis. Antigens of 10 cytokines/chemokines which were found to be significantly elevated in previous H5N1-infected patients and in vitro experiments, and mRNA of eight of these, were absent from the lungs of a pregnant woman and her fetus. In contrast, antigens of seven cytokines/chemokines and mRNA of six of these were found to be increased in the lungs of a male autopsy. The cells expressing these cytokines and chemokines were identified as type II pneumocytes, bronchial epithelial cells, macrophages and vascular endothelial cells. Levels of cytokines and chemokines in the serum of the male case were also significantly higher than those of infectious (infection other than by H5N1) and non-infectious controls. In comparison with results from our previous study, it appeared that the male case had increased cytokine/chemokine expression but reduced viral load, while the pregnant female had diminished cytokine/chemokine expression but a significantly increased viral load in the lungs. These disparate findings in these two cases suggest that 'cytokine storm' alone could not be a sufficient explanation for the severe lung injury of this newly emerging disease.

Pharmacologic profiling of transcriptional targets deciphers promoter logic.

The blueprint for cellular diversity and response to environmental change is encoded in the cis-acting regulatory sequences of most genes. Deciphering this 'cis-regulatory code' requires multivariate data sets that examine how these regions coordinate transcription in response to diverse environmental stimuli and therapeutic treatments. We describe a transcriptional approach that profiles the activation of multiple transcriptional targets against combinatorial arrays of therapeutic and signal transducing agents. Application of this approach demonstrates how cis-element composition and promoter context combine to influence transcription downstream of mitogen-induced signaling networks. Computational dissection of these transcriptional profiles in activated T cells uncovers a novel regulatory synergy between IGF-1 and CD28 costimulation that modulates NF-kappaB and AP1 pathways through signaling cascades sensitive to cyclosporin A and wortmannin. This approach provides a broader view of the hierarchical signal integration governing gene expression and will facilitate a practical design of combinatorial therapeutic strategies for exploiting critical control points in transcriptional regulation.

Assessing the Saskatchewan database for outcomes research studies of depression and its treatment.

This study was conducted to evaluate the validity of using the Saskatchewan Health administrative claims databases for conducting depression research. To develop a claims-based definition of depression, we identified a cohort of individuals who began a "new" period of antidepressant use (no use 180 days prior) from which we selected a stratified random sample (n = 600) for medical record abstraction. The medical record diagnosis was used as the gold standard for judging our database definitions of depression. After defining a primary database definition of depression, we tried to refine it using medically probable scenarios and assessed refinement by agreement statistics. Defining depression with ICD9 codes 296 (affective disorders), 309 (adjustment reaction), and 311 (depressive disorders), the sensitivity (Se), specificity (Sp), positive (PV+) and negative predictive (PV-) values were: 71%, 85%, 86%, and 70%, respectively. Algorithms that limited the number of false-negatives resulted in: Se = 84% and PV- = 77% whereas those that limited false-positives resulted in: Sp = 90% and PV+ = 86%. Although our depression definition requires treatment with antidepressants, this definition will allow us to conduct future studies of depression and its treatment using the Saskatchewan Health databases.

Nested case-control study of the effects of non-steroidal anti-inflammatory drugs on breast cancer risk and stage.

We carried out a nested case-control study to measure the rate ratio (RR) for invasive female breast cancer in relation to non-steroidal anti-inflammatory drug (NSAID) use. The source population consisted of the female beneficiaries of the Saskatchewan Prescription Drug Plan from 1981 to 1995 with no history of cancer since 1970. Four controls/case, matched on age and sampling time, were randomly selected. Dispensing rates during successive time periods characterized NSAID exposure. RRs associated with exposure during each period were adjusted for exposure during the others. Confounding by other determinants was studied in analyses adjusted with data obtained by interviewing samples of subjects accrued from mid-1991 to mid-1995. We accrued 5882 cases and 23,517 controls. Increasing NSAID exposure 2-5 years preceding diagnosis was associated with a trend towards a decreasing RR (P-trend = 0.003); for the highest exposure level RR = 0.76, 95% confidence interval 0.63-0.92. This protective effect could not be attributed to confounding by other determinants. In analyses involving only the cases, NSAID exposure 2-5 and 6-10 years preceding diagnosis was associated with significantly reduced risks of presenting with a large tumour (> 5 cm diameter) or distant metastasis, but not regional lymph node metastasis. The use of NSAIDs may retard the growth of breast cancers and prevent distant metastasis.

Importance of surgical resection in the successful management of soft tissue sarcoma.

HYPOTHESIS: A systemic disease-free state necessitates a local disease-free state. This cannot be accomplished without a properly performed resection by an experienced surgical team. Successful local management of soft tissue sarcoma (STS) may lead to improved disease-free survival. An STS treatment protocol using wide local excision followed by radiation therapy is effective in achieving local tumor control and survival similar to that of multiple-modality regimens, but with lower morbidity. DESIGN: Retrospective cohort review (August 1, 1987, to May 6, 1998). SETTING: Referral to a single musculoskeletal oncologic surgeon, with surgery performed at a tertiary care medical center in a large urban area. PATIENTS: Ninety patients with STS of the trunk or extremities. INTERVENTIONS: Preoperative evaluation included surveillance computed tomographic scan of the chest, magnetic resonance imaging of primary site to assess tumor extent and to plan the surgical approach, and angiography if vascular bypass was proposed. Wide local excision of tumor was performed, with concomitant vascular bypass and/or complex plastic reconstruction as needed. Postoperative radiation therapy was given in most patients. Adjuvant chemotherapy was used selectively. MAIN OUTCOME MEASURES: Morbidity, local recurrence rates, and survival. RESULTS: Histologically negative margins were obtained in 89 (99%) of 90 patients; 86 (96%) remained free of local disease at follow-up. Five patients died of systemic metastatic disease. CONCLUSION: Excellent local control obtained with aggressive, appropriate surgery followed by radiation therapy in most patients and chemotherapy in only selective high-risk patients leads to excellent survival, with low morbidity and good functional outcome.

Head and neck osteosarcoma at the University of Washington.

BACKGROUND: Head and neck osteosarcoma is a comparatively rare and aggressive malignancy. Our goal was to examine the experience of head and neck osteosarcoma patients seen over a 15-year period at the University of Washington Medical Center and compare this with the published experience of other centers in terms of demographics, histology, treatment, and survival rate. METHODS: We reviewed surgical pathology slides and clinical treatment records of 13 patients who were treated at the University of Washington Medical Center between 1981 and 1996. A total of 17 cases from 13 patients (13 primary tumors and 4 recurrences) were studied. RESULTS: There was a slight male predominance, with a male:female ratio of 1.6:1, and median age at diagnosis of 40.9 years (range 22 to 75 years), both slightly higher than has been generally reported. Three of 13 patients had recognized risk factors for the development of osteosarcoma: 2 with a history of prior radiotherapy and 1 with Paget's disease. All surgical pathology specimens were examined independently by two pathologists for histologic grading and typing. At initial presentation, 9/13 (69%) cases had conventional (osteoblastic) histology; 2/13 (15%) were fibroblastic, 1 chondroblastic (8%) and 1 parosteal (8%). Eight of 13 (62%) cases were high grade at initial presentation. Four of 13 (30%) of the primary tumors were low grade 2, of which did not recur over a median follow-up period of 24 months. The other 2 low-grade tumors later recurred locally, as high-grade osteosarcomas, after disease-free intervals of 1 year and 14 years, respectively. One patient had an intermediate-grade tumor which has not recurred as of last follow-up. Combined-modality treatment, including surgery with or without radiotherapy and/or chemotherapy, was given depending on the histologic grade, surgical margins, and recurrence. Some patients with low-grade tumors had surgery only. There were 5 local recurrences, 1 of these following a disease-free interval of 14 years. One patient had 3 separate recurrences at the same site. Ten of 13 (77%) are alive and disease-free. Of the 3 deaths, 1 was related to radiation-induced brain necrosis, without evidence of recurrent tumor. The project 5-year overall survival in this series is 72%, with a mean follow-up of 58 months (median, 36 months). Of those receiving neoadjuvant chemotherapy, 6/7 have survived to the present. CONCLUSION: Given the limitations of a small patient population, our data suggest that neoadjuvant chemotherapy may provide benefit in terms of survival. Longer follow-up will be necessary to support this conclusion. Our data also show that our population has a higher-than-average age of onset, low presence of risk factors, and better survival rate in comparison with the published series from other institutions.

Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunocytochemical and biochemical study of myogenic regulatory protein expression.

There has been persistent controversy regarding the nature of cell differentiation in alveolar soft-part sarcoma (ASPS) since its first description in 1952. Some studies suggest that ASPS might represent an unusual variant of skeletal muscle tumor. Given the availability of new monoclonal antibodies to probe for skeletal muscle differentiation and the rapid advance in immunocytochemical techniques for deparaffinized, formalin-fixed tissue sections, we wished to test the proposed hypothesis that ASPS might represent a new type of rhabdomyosarcoma. Twelve archival samples of ASPS were retrieved, and we investigated the expression of two myogenic regulatory proteins, MyoD1 and myogenin, as well as other muscle-associated proteins, using sensitive immunocytochemical techniques. Despite the presence of desmin immunostaining in six ASPSs, no tumors were positive for either muscle actin or myoglobin. Most importantly, no specimen showed nuclear expression of MyoD1 or myogenin. In 11 tumors, however, there was considerable granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 monoclonal antibody 5.8A, a phenomenon observed in various nonmuscle normal and neoplastic tissues with this antibody. To analyze the exact nature of immunostaining of MyoD1 and desmin in ASPS, biochemical analyses using available fresh frozen tumor tissue were performed. Although a 53-kDa band was noted with antidesmin antibody on Western blot analysis, no specific protein band that corresponds to the 45-kDa MyoD1 was detected with antibody 5.8A. These results confirm the presence of desmin in ASPS but argue against authentic expression of MyoD1. They also suggest that the cytoplasmic immunostaining observed with anti-MyoD1 antibody 5.8A most likely represents a nonspecific cross-reaction with an unknown cytoplasmic antigen. Considering the master role that MyoD1 and myogenin play in skeletal muscle commitment and differentiation and the lack of expression of these two proteins in ASPS as determined immunocytochemically and biochemically, we think that the histogenesis of ASPS remains unknown.

Forces contributing to the conformational stability of proteins.

For 35 years, the prevailing view has been that the hydrophobic effect is the dominant force in protein folding. The importance of hydrogen bonding was always clear, but whether it made a net favorable contribution to protein stability was not. Studies of mutant proteins have improved our understanding of the forces stabilizing proteins. They suggest that hydrogen bonding and the hydrophobic effect make large but comparable contributions to the stability of globular proteins.

Expression of myogenic regulatory proteins (myogenin and MyoD1) in small blue round cell tumors of childhood.

The distinction of rhabdomyosarcoma (RMS) from other small blue round cell tumors of childhood, such as Ewing's sarcoma/peripheral primitive neuroectodermal tumor (pPNET) and neuroblastoma, continues to present a diagnostic challenge to pathologists. The recent recognition of the master role of myogenic regulatory proteins in skeletal muscle commitment and differentiation, and the availability of monoclonal antibodies to two of them (myogenin and MyoD1), has prompted us to test their diagnostic utility in routinely processed, formalin-fixed, and deparaffinized tissue. Preliminary studies had demonstrated that, with the use of heat-induced epitope retrieval techniques, expression of myogenin and MyoD1 could be documented specifically in nuclei of fetal skeletal muscle by the respective antibodies. We performed a retrospective immunohistochemical analysis on 72 cases of small blue round cell tumors, including 33 RMSs, 1 metastatic myogenous Wilms' tumor, 26 Ewing's sarcomas/pPNETs, and 12 neuroblastomas. Nuclear expression of myogenin and MyoD1 were both found in 30/33 non-overlapping cases of RMS, with no significant differences in the sensitivity with respect to histological subtypes, and in 1/1 case of myogenous Wilms' tumor. None of the neuroblastomas or Ewing's sarcomas/pPNETs demonstrated positive nuclear staining with either antibody. However, most of the neuroblastomas, and occasional Ewing's sarcomas/pPNETs, showed variable fibrillary, cytoplasmic immunoreactivity with antibody to MyoD1. We conclude that, with the use of microwave-based epitope retrieval, antibodies to myogenin and MyoD1 are both useful markers for the identification of RMS among other small blue round cell tumors of childhood, but antibodies to myogenin have technical advantages over those to MyoD1, as the latter may cross-react with an unknown cytoplasmic antigen in non-muscle cells and tumors.

Recent trends in the use of inhaled beta 2-adrenergic agonists and inhaled corticosteroids in Saskatchewan.

OBJECTIVE: To examine recent trends in the use of inhaled beta 2-adrenergic agonists and inhaled corticosteroids for the treatment of asthma among Saskatchewan residents and to determine whether these trends are in keeping with widely publicized guidelines recommending a reduction in the use of agents to treat symptoms (i.e., inhaled beta 2-adrenergic agonists) and increased use of prophylactic agents (i.e., inhaled corticosteroids). DESIGN: Descriptive pharmacoepidemiologic study conducted with the use of data from the computerized database of the Saskatchewan Prescription Drug Plan. SETTING: Saskatchewan. PATIENTS: Saskatchewan residents 5 to 54 years of age who received one or more outpatient prescriptions for drugs to treat asthma (inhaled drugs, ingested beta 2-adrenergic agonists and ingested methylxanthines) from 1989 to 1993. OUTCOME MEASURES: Epidemiologic trends, calculated for each year: number of prescriptions per 1,000 persons; number of patients who received prescriptions for inhaled corticosteroids, inhaled beta 2-adrenergic agonists and any type of drug to treat asthma; mean number of such prescriptions per patient; and weighted mean amount of salbutamol, fenoterol and beclomethasone dispensed per patient. RESULTS: There has been an increase in the proportion of the population receiving prescriptions for drugs to treat asthma. The number of patients receiving these drugs per 1,000 people rose during the study period from 33.38 to 46.59 for any drug to treat asthma, from 24.70 to 33.77 for inhaled beta 2-adrenergic agonists and from 6.1 to 19.9 for inhaled corticosteroids. The mean number of prescriptions per patient decreased steadily for all drugs to treat asthma (from 5.34 in 1989 to 3.88 in 1993), for inhaled beta 2-adrenergic agonists (from 4.35 to 3.09) and for inhaled corticosteroids (from 2.98 to 2.25). The weighted mean amount of inhaled salbutamol dispensed per patient per year decreased by 40%, from 178.08 mg in 1989 to 109.14 mg in 1993. The weighted amount of fenoterol dispensed per patient per year declined even more, by 58%, from 387.91 mg in 1989 to 164.00 mg in 1993. Conversely, the weighted amount of inhaled beclomethasone dispensed per patient per year increased by 35% from 46.95 mg in 1989 to 63.50 mg in 1992, then dropped to 56.17 mg per year in 1993. CONCLUSION: These data demonstrate a substantial change in Saskatchewan in the prescribing of drugs to treat asthma; they suggest that many physicians responded to current guidelines advocating increased attention to prevention of airway inflammation in the treatment of asthma.

Cohort study of incidence of sudden unexplained death in persons with seizure disorder treated with antiepileptic drugs in Saskatchewan, Canada.

To measure the incidence of sudden unexplained death in treated persons with epilepsy (SUDEP) and to identify risk factors for SUDEP, a cohort of 6,044 persons aged 15-49 years with more than four prescriptions for antiepileptic drugs (AEDs) was identified from the Saskatchewan Health prscription drug file. To exclude subjects whose sudden deaths (SUDs) might be misattributed to another chronic underlying disease, subjects with hospitalizations for cancer or heart problems were excluded. To exclude subjects without epilepsy, subjects with > 2-year AED treatment followed by AED-free time and subjects receiving < 1 U/day were excluded. The final cohort consisted of 3,688 subjects. Follow-up was started at the first AED prescription listed in the prescription drug file and ended at the earliest of the following: age 50 years, death, or last registration in the Saskatchewan Health. For 153 of 163 deaths occurring in the cohort, copies of anonymized death certificates were obtained and copies of anonymized autopsy reports of potential SUDEP cases were examined. There were 18 definite/probable SUDs and 21 possible SUDEPs, yielding a minimum incidence of 0.54 SUDEP per 1,000 person-years and a maximum of 1.35 SUDEP per 1,000 person-years. SUDEP incidence increased with male sex, number of AEDs ever prescribed, and prescription of psychotropic drugs and was highest in males with a history of treatment with three or more AEDs and four or more psychotropic drug prescriptions. Poisson regression showed a 1.7-fold increase in risk of SUDEP for each increment in maximum number of AEDs administered, a likely surrogate for severity and persistence of seizures.

A cohort analysis of excess mortality in asthma and the use of inhaled beta-agonists.

The association between the use of inhaled beta-agonists and the risk of death and near-death from asthma has previously been reported. It was based on a nested case-control study of 129 cases and 655 control subjects selected from a cohort of 12,301 users of asthma drugs followed during the period 1980 through 1987. In this paper we examine the question of asthma and non-asthma mortality using data from the entire cohort of 12,301 asthmatics. There were 46 asthma and 134 non-asthma deaths in this cohort, for which there were 47,842 person-years of follow-up. The overall rate of asthma death was 9.6 per 10,000 asthmatics per year. This rate varied significantly according to the use of fenoterol, albuterol, or oral corticosteroids in the prior 12 months and the number of asthma hospitalizations in the prior 2 years. The rate decreased significantly, by 0.6 asthma deaths per 10,000 asthmatics per year over the study period, after controlling for the effect of the four other risk factors. It also increased significantly with the use of all beta-agonists, and more so for fenoterol than for albuterol, although this difference was partly explained by the dose inequivalence of the two drugs. Change-point dose-response curves showed that the risk of asthma death began to escalate drastically at about 1.4 canisters (of 20,000 micrograms each) per month of inhaled beta-agonist, the recommended limit. For non-asthma death, the overall rate of 28 deaths per 10,000 asthmatics per year was not related to the use of inhaled beta-agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Is the association between inhaled beta-agonist use and life-threatening asthma because of confounding by severity?

We have previously reported an increasing dose-response relationship between the regular use of beta-agonist inhalers and the risk of asthma death and near death among a cohort of 12,301 subjects who had been dispensed 10 or more prescriptions of asthma drugs from January 1980 to April 1987. That analysis was based solely on information obtained from linkable computerized data bases. Such an association might be explained in part by the tendency of patients with more severe asthma, that is, those at greatest risk for an adverse outcome, to use more beta-agonist medication. To further examine this potential confounding by severity, we gathered clinical information independently from the field on the 129 case patients and their 655 control patients from the matched case-control analysis of 12,301 subjects. In 68% of the control patients with a life-threatening episode and 75% of the matched control subjects, we obtained a valid questionnaire from at least one physician who had seen the patient during the previous 2 yr. Acceptable information on hospitalizations because of asthma was obtained in 87% of those hospitalized. Clinical features associated with an increased risk of fatal and near-fatal asthma were: a history of loss of consciousness or seizures during a previous asthma attack (odds ratio, 10.2; 95% CI, 3.9 to 26.7), a history of attacks of asthma precipitated by eating certain foods (odds ratio, 5.1; 95% CI, 2.4 to 11.1), a clinical score designed to reflect the severity of prior attacks of asthma leading to hospitalization, and prior respiratory acidosis among those in whom a blood gas determination was recorded.(ABSTRACT TRUNCATED AT 250 WORDS)

Gravity Field over Northern Eurasia and Variations in the Strength of the Upper Mantle.

The correlation of long-wavelength gravity anomalies in northern Eurasia with seismic velocity anomalies in the upper mantle reverses in sign between western and eastern Eurasia. The difference between western and eastern Eurasia can be explained by the presence of a low-viscosity zone in the uppermost mantle beneath eastern Eurasia that is absent to the west. The location of the lateral change in viscosity corresponds with the geologic boundary between the older shields and platforms of the Baltics, Russia, and Siberia and the younger, geologically active mountain belts of eastern Asia. This relation provides evidence that differences in the strength of the upper mantle control the locus of intracontinental deformation.

Risk of fatal and near-fatal asthma in relation to inhaled corticosteroid use.

OBJECTIVE: To examine the relationship between patterns of use of inhaled beclomethasone dipropionate and the risk of fatal and near-fatal asthma. DESIGN: Nested case-control analysis of a historical cohort; a further analysis. SETTING: The 12,301 residents of Saskatchewan aged 5 to 54 years who were dispensed 10 or more asthma drugs from 1978 to 1987. PATIENTS: The 129 persons who experienced asthma death (n = 44) and near-death (n = 85) and their 655 controls matched as to age and date of entry into the cohort, with the additional matching criteria of at least one hospitalization for asthma in the prior 2 years, region of residence, and having received social assistance. MAIN OUTCOME: Life-threatening attacks of asthma defined as death due to asthma or the occurrence of hypercarbia, intubation, and mechanical ventilation during an acute attack of asthma. RESULTS: After accounting for the risk associated with use of other medications and adjustment for markers of risk of adverse events related to asthma, subjects who had been dispensed, on average, one or more metered-dose inhalers of beclomethasone per month over a 1-year period had a significantly lower risk of fatal and near-fatal asthma (odds ratio, 0.1; 95% confidence interval, 0.02 to 0.6). CONCLUSION: These data support recent guidelines from several countries that recommend the use of inhaled corticosteroids in moderate and severe asthma.