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PubMed1999

The seven amino acids (547-553) of rat glucocorticoid receptor required for steroid and hsp90 binding contain a functionally independent LXXLL motif that is critical for steroid binding.

Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547-553) overlapping the amino-terminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in full-length GR and glutathione S-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg(651). The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550-554 acting as a hydrophobic clasp.

Amino Acid Substitution↗
PubMed2003

A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes.

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Delta(1)-dehydrogenase and 3-ketosteroid-Delta(4)(5alpha)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9alpha-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.

Amino Acid Sequence↗
PubMed2007

Sciatica: treatment with intradiscal and intraforaminal injections of steroid and oxygen-ozone versus steroid only.

PURPOSE: To prospectively compare the clinical effectiveness of intraforaminal and intradiscal injections of a mixture of a steroid, a local anesthetic, and oxygen-ozone (O(2)-O(3)) (chemodiscolysis) versus intraforaminal and intradiscal injections of a steroid and an anesthetic in the management of radicular pain related to acute lumbar disk herniation. MATERIALS AND METHODS: Medical Ethical Committee approval and informed consent were obtained. One hundred fifty-nine patients (86 men, 73 women; age range, 18-71 years) were included and were randomly assigned to two groups. Seventy-seven patients (group A) underwent intradiscal and intraforaminal injections of a steroid and an anesthetic, and 82 patients (group B) underwent the same treatment with the addition of an O(2)-O(3) mixture. Procedures were performed with computed tomographic guidance. An Oswestry Low Back Pain Disability Questionnaire was administered before treatment and at intervals, the last at 6-month follow-up. Patients and clinicians were blinded as to which treatment was performed. Results were compared with the chi(2) test. RESULTS: After 6 months, treatment was successful in 36 (47%) patients in group A and in 61 (74%) patients in group B. The difference was significant (P < .01). CONCLUSION: Intraforaminal and intradiscal injections of a steroid, an anesthetic, and O(2)-O(3) are more effective at 6 months than injections of only a steroid and an anesthetic in the same sites.

Adolescent↗
PubMed1985

Fat tissue: a steroid reservoir and site of steroid metabolism.

Sex steroid concentrations and 17 beta-hydroxy-steroid dehydrogenase and aromatase activities were determined in fat tissue removed at surgery or, in order to allow comparisons in different sites, postmortem. Except for dehydroepiandrosterone (DHEA) sulfate (DHEAS), there existed a positive tissue/plasma gradient for all steroids studied (testosterone, androstenedione, DHEA, androstenediol, estrone, and estradiol), suggesting androgen uptake and estrogen synthesis in situ. Androgen concentrations did not vary according to site of origin of fat tissue, except that the DHEAS concentration was significantly lower in abdominal sc and omental fat than in breast, pericardial, or sc pubic fat. Tissue androgen concentrations were positively correlated with their plasma concentrations, but tissue and plasma estrogen concentrations were not correlated. All tissue steroid concentrations, with the exception of estradiol in men, decreased with age. Aromatase activity [androstenedione----estrone; mean maximum velocity, 7.4 +/- 3.7 (+/- SD) fmol estrone/mg protein . h] did not vary between sexes or with site of origin of fat tissue. 17 beta-Hydroxysteroid dehydrogenase activity (estradiol----estrone, mean maximum velocity 9.8 +/- 5.4 pmol/mg protein . h) was higher in fat from women than in that from men, higher in premenopausal than in postmenopausal women, and higher in omental than in sc fat. Its activity was noncompetitively inhibited in vitro by DHEA and DHEAS in near-physiological concentrations, and the enzyme activity was inversely correlated (P less than 0.001) with the tissue DHEA and DHEAS concentrations. We conclude that fat tissue is an important steroid hormone reservoir, that it is the site of active aromatase and 17 beta-hydroxysteroid dehydrogenase, and that tissue DHEA(S) may have a modulating effect on tissue estrogen production.

17-Hydroxysteroid Dehydrogenases↗
PubMed1997

Differential sex steroid negative feedback regulation of pulsatile follicle-stimulating hormone secretion in healthy older men: deconvolution analysis and steady-state sex-steroid hormone infusions in frequently sampled healthy older individuals.

The healthy aging male reproductive axis tends to exhibit a progressive decline in serum concentrations of biologically available testosterone with gradual concomitant reciprocal increases in both LH and FSH concentrations. However, relatively little is known about the sex steroid-mediated negative feedback regulation of physiologically pulsatile gonadotropin release in general, and episodic FSH release in particular, in older males. To examine the steroid hormone negative feedback control of pulsatile FSH secretion in healthy older men, we applied multiparameter deconvolution analysis to serum FSH (immunoradiometric assay) profiles obtained by sampling every 10 min over 24 h during steady state (4.5-day) infusions of estradiol (E2; 48 micrograms/day), 5 alpha-dihydrotestosterone (DHT; 7.0 mg/day), or 5% dextrose in water in five healthy older men, aged 60-73 yr. We observed the following principal responses: 1) both E2 and DHT significantly suppressed mean and 24-h integrated serum FSH concentrations (P < 0.032); 2) the calculated daily secretion rate of FSH fell significantly in all five individuals during DHT infusion; 3) the apparent half-life of FSH decreased during E2 (but not DHT) infusion; 4) DHT infusion reduced the mass and frequency of FSH secretory bursts significantly; 5) neither E2 nor DHT treatment significantly attenuated the release of FSH stimulated by consecutive iv injections of GnRH (10 and 100 micrograms); and 6) integrated 24-h serum LH (immunoradiometric assay) concentrations decreased significantly during both DHT and E2 infusions, whereas mean LH release after the serial GnRH injections was not altered. Compared to younger men studied earlier in an identical fashion, older men had significantly reduced FSH intersecretory burst intervals, reflecting a higher FSH pulse frequency at baseline and during the steroid infusions and a significantly lower mass of FSH secreted per burst during E2 infusion. We conclude that healthy older men maintain intact negative feedback responsiveness of the hypothalamo-pituitary gonadotroph unit to exogenously delivered sex steroid hormones, and that individual sex steroid hormones differentially regulate specific features of pulsatile FSH release and half-life in older men.

Aged↗
PubMed2001

Changes in follicular fluid concentrations of steroids, the pattern of steroid secretion and aromatization capability of incubated preovulatory follicular wall of rats.

Female Wistar rats exhibiting a regular 4-day oestrous cycle were included in this study. They were killed on the day of pro-oestrus at 11.00, 14.00, 16.00, 2.00, 22.00 and 24.00 h. The isolated preovulatory follicles were placed individually on a small triple disc of filter paper. They were cut in half and the flowing out follicular fluid (FF) was allowed to soak into the underlying filter paper disc, which was subsequently placed at the bottom of incubation chamber. It was rinsed with the incubation medium and this medium, containing FF, was collected. The remaining follicular wall was incubated for 3h. Some of the incubation media were supplemented with testosterone (T). The steroid content was estimated by radioimmunoassay. The secretion of estradiol (E) by follicular wall was high until 20.00 h while that of androgens until 22.00 h, and both declined thereafter. Relatively low progesterone (P) release rose sharply at 22.00 h and this hormone became the main steroid secreted at 24.00 h. The addition of T always enhanced the release of estradiol. Follicular fluid contained very little estradiol, while prevailing steroids were androgens. Decrease of androgen concentrations was found only at 24.00 h. Progesterone level, much lower than that of androgens, started to rise at 20.00 h and P was the main steroid present in FF at 24.00 h. The present results suggest an existence of selective passage of steroids into FF and show a presence of androgenic milieu surrounding cumulus oophorus in preovulatory FF. The results also indicate that the aromatase activity is sustained until ovulation and stress the role of the P rise observed in the secretion rate of follicular wall and FF content just before ovulation.

Androgens↗
PubMed1987

Epidermal growth factor activates steroid biosynthesis in cultured Leydig tumor cells without affecting the levels of cAMP and potentiates the activation of steroid biosynthesis by choriogonadotropin and cAMP.

The studies presented herein were designed to investigate the effects of mouse epidermal growth factor (mEGF) on steroid biosynthesis in a clonal strain of cultured murine Leydig tumor cells (designated MA-10). We show that in short-term incubations (up to 8 h), mEGF activates steroid biosynthesis without affecting cAMP levels. The maximal activation of steroid biosynthesis by mEGF (about 10-fold) is, however, much lower than the maximal activation detected with human choriogonadotropin (hCG) or cAMP analogues (about 1000-fold). We also show that mEGF has two (opposing) effects on the activation of steroidogenesis by hCG. Initially, it transiently attenuates the increase in intracellular cAMP and steroid biosynthesis provoked by submaximal concentrations of hCG. At later times, however, it potentiates the stimulatory effects of submaximal concentrations of hCG on steroid biosynthesis in a synergistic fashion. Last, we show that mEGF and submaximal concentrations of cAMP analogues also activate steroidogenesis in a synergistic fashion and that the degree of synergism attained with cAMP analogues plus mEGF is much higher than that attained with hCG plus mEGF. Taken together, our results show that mEGF (i) activates steroidogenesis without affecting cAMP levels and (ii) modulates the activation of steroidogenesis by the cAMP second messenger system.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Steroid-sparing effect of ketotifen in steroid-dependent asthmatics: a long period evaluation in 12 patients.

An open pilot study was carried out over a period of more than 2 years to assess the steroid-sparing effect of ketotifen (1 mg twice daily) in 12 steroid-dependent asthmatic patients. Daily steroid consumption (prednisolone equivalent) was calculated for the year before and for 1 and 2 years of ketotifen treatment from the total of all doses given throughout the year. Results showed that in 6 of the 12 patients who responded with reduced steroid consumption the mean daily dose decreased from 5.7 mg to 4.1 mg/day (p less than 0.05) in the first year of ketotifen: the final mean reduction rate at the end of the 2-year trail was 83.6% (p less than 0.005). Furthermore, the mean daily consumption of beta 2-stimulants (salbutamol equivalent) decreased from 8.7 to 6.2 mg/day (p less than 0.025) in the responders. In the 6 non-responders, there was a slight increase in mean daily steroid consumption from 2.4 mg to 3.0 mg/day. The most frequently reported side-effect of ketotifen was dizziness, which occurred in one-third of the patients; all patients, however, were able to continue with treatment.

Adrenocorticotropic Hormone↗
PubMed1983

Steroids excreted by human skin. I. C19-steroids in axillary hair.

Seven free androgen steroids and four C19-steroid sulphates were isolated and determined from the axillary hair of sexually mature healthy males and females. In 1 g axillary hair the free 4-androstene-3 beta, 17 beta-diol, testosterone, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, androsterone, 4-androstene-3,17-dione and 5 alpha-androstane-3,17-dione occurred in nanomol amounts, whereas dehydroepiandrosterone sulphate, androsterone sulphate and 5-androstene-3 beta,17 beta-diol-3-sulphate were found in approximately 1000 times these amounts. The quantity of testosterone sulphate lies among the values for the free steroids. The free steroids present in hair are regarded as products of the sebaceous glands, and the sulphate ester steroids as products of the sweat glands.

Adult↗
PubMed1980

Steroid diabetes--a sign of overtreatment with steroids in the renal graft recipient?

The development of steroid diabetes was studied in a consecutive series of 114 patients receiving a cadaveric renal graft. All were first-time transplantations. For acceptance in the series the graft should have functioned for at least 2 weeks after the transplant operation. The median age of the patients was 52 years. The follow-up period was at least one year. The criterion for a diagnosis of steroid diabetes was an elevated fasting blood glucose concentration of more than 8 mmoles/l. Fifty-two of the patients (46 per cent) developed steroid diabetes. Thirty-four (65 per cent) of them required insulin therapy or peroral anti-diabetic drugs. The mean age of these patients was higher than for those not afflicted by this complication. The first-year mortality was significantly higher, 42 against 13 per cent, respectively (p less than 0.01). The level of significance was not notably affected by correction for age at transplantation. Severe infection was the cause of a large proportion of the deaths in the diabetes group. The findings suggest that the development of steroid diabetes can serve as a warning signal for excessive dosage of corticosteroids, and help to identify patients that are particularly sensitive to immunosuppressive therapy with large doses of steroids.

Adolescent↗
PubMed1985

Azathioprine and steroids are not more effective in decreasing multiple sclerosis intra-blood-brain-barrier IgG synthesis than steroids alone.

Intra-blood-brain-barrier IgG synthesis rates and oligoclonal IgG banding patterns were examined in 9 patients with multiple sclerosis who were treated with azathioprine and steroids for 2 to 4.5 years. The IgG synthesis rates of 5 patients were significantly decreased from the pretreatment mean values 1 month after treatment, and their synthesis rates remained at the decreased levels throughout treatment. However, among the remaining 4 patients, the rates exceeded the pretreatment means. This continuous suppressive effect of the combined azathioprine and steroids upon the IgG synthesis rate was similar to that of steroids, suggesting that azathioprine and steroids in combination were not more effective in reducing intra-blood-brain-barrier IgG synthesis than steroids alone. Oligoclonal IgG patterns in all cerebrospinal fluid samples were not significantly altered during the study.

Adrenocorticotropic Hormone↗
PubMed2000

Cyclosporine-A plus steroids versus steroids alone in the 12-month treatment of systemic lupus erythematosus.

The positive results obtained with cyclosporine-A both in an experimental model and in selected patients with advanced systemic lupus erythematosus support the hypothesis that the drug could be used as a steroid sparer in the earliest stages of active disease. To determine the 12-month clinical efficacy (disease control and steroid sparing), safety, and tolerability of low-dose cyclosporine-A plus steroids versus steroids alone, we designed a multicenter, open, prospective, randomized, pilot study, controlled for parallel groups. The patients were then followed up to month 24. A total of 18 consenting patients with recently diagnosed systemic lupus erythematosus of moderate severity indicated for the use of steroids in acute boluses and subsequently per os were enrolled at two university hospital medical centers. The protocol was based on three 1-g boluses of 6-methylprednisolone followed by cyclosporine-A (<5 mg/kg per day) plus prednisone 0.5-1 mg/kg per day per os, reduced by 5 mg/day every 2 weeks following clinical remission, versus the same doses of oral prednisone alone. The efficacy evaluation was based on a four-point scale (from absent/none to severe) for signs and symptoms of systemic lupus erythematosus and immunoserological parameters. The disease activity index and cumulative prednisone dose per patient were analyzed. Any adverse events were reported. All patients showed a reduction in disease activity index within the 1st month. The results were significantly better in the group with cyclosporine-A plus prednisone throughout month 12 (baseline and 12-month disease activity indexes: 21.3+/-8.6 and 5.0+/-2.5 versus 20.4+/-7.1 and 8.8+/-6.0 in the prednisone group, P<0.05). The 12-month cumulative mean dose of prednisone was significantly lower in the group with both cyclosporine-A plus prednisone (179.4+/-40.1 versus 231.8+/-97.1 mg/kg, P<0.005). No unusual adverse events related to the study drugs have been reported. In particular, renal function and blood pressure monitoring revealed no significant changes from mean baseline values in either group. No disease flares were reported in the group treated with cyclosporine-A plus prednisone during the 12- to 24-month period. Thus cyclosporine-A represents a useful corticosteroid sparer in the maintenance of clinical remission in patients with an early-stage, active systemic lupus erythematosus.

Adolescent↗
PubMed1985

Effect of dexamethasone treatment on sex steroid-binding protein, corticosteroid-binding globulin, and steroid hormones in cycling rhesus macaques.

We tested the hypothesis that dexamethasone lowers sex steroid-binding protein levels and observed the effect of dexamethasone on corticosteroid-binding globulin and specific steroid hormones in plasma. Four cycling rhesus macaques were studied during three consecutive menstrual cycles (first and third cycles served as controls). In the second cycle, each animal received 0.5 mg of dexamethasone intramuscularly at 8:00 AM and 8:00 PM daily for 21 consecutive days. Blood samples were taken at 1- to 3-day intervals during each cycle. Concentrations of sex steroid-binding protein, corticosteroid-binding globulin, cortisol, testosterone, progesterone, and estradiol were measured in plasma. The percentage and plasma concentration of free testosterone were also determined. Within 2 days of treatment, dexamethasone suppressed cortisol to 5% of baseline values, which returned in the third cycle. All cycles were ovulatory. Dexamethasone significantly lowered plasma levels of all the compounds except progesterone: sex steroid-binding protein, -30%; corticosteroid-binding globulin, -14%; testosterone, -36%; and estradiol, -45%. The percentage of free testosterone was significantly elevated, but free testosterone concentrations were unchanged. Although our data conclusively show that dexamethasone suppresses plasma sex steroid-binding protein levels in the rhesus macaque, it remains to be established whether this suppressive effect leads to an increase in the metabolic clearance rate of testosterone.

Animals↗
PubMed1989

Partial characterization of steroid sulfohydrolase and steroid sulfotransferase activities in purified porcine Leydig cells.

Subcellular fractions of purified pig Leydig cells from 7 different animals have been investigated with respect to their abilities to catalyze the sulfation of several steroids and the hydrolysis of the sulfated forms of these same steroids. Considerable estrone sulfate sulfohydrolase of pH optimum 7.5 and high apparent Km was found to be concentrated in the 105,000 g pellet but no evidence was obtained, in any subcellular fraction, for the presence of any activity toward the 3-sulfate of pregnenolone, dehydroepiandrosterone (DHA) or delta 5-androstene-3 beta,17 beta-diol (androstenediol). Cytosolic sulfotransferase activity toward estrone, pregnenolone, DHA and androstenediol was present in each animal. The activity toward these 4 substrates was eluted from a gel filtration column as a single peak of apparent molecular weight 43 KDa. Upon chromatofocusing, a sharp estrogen sulfotransferase peak of apparent pI 6.1 and pH optimum 9.5, was clearly separated from the neutral steroid sulfotransferase which eluted over a more acidic pH range in a manner suggestive of the presence of several isozymes. This latter, which exhibited a wide pH optimum range between 6 and 8.5, was most active toward androstenediol, and least active toward pregnenolone. The estrogen sulfotransferase exhibited Michaelis-Menten kinetics (apparent Km = 4 microM). The neutral steroid sulfotransferase activity increased in velocity with increasing androstenediol or DHA concentration up to 1 microM beyond which considerable substrate inhibition occurred. It appears from these data that neutral steroid sulfates synthesized in the pig Leydig cell are not subject to enzymic desulfation in the same cells.

Androstenediols↗
PubMed1989

The influence of various steroids on the binding of methyltrienolone (R1881) to human placenta: evidence for a second steroid-binding site which stabilizes the R1881 binding site.

Specificity studies of the binding of R1881 to crude placental homogenate gave surprising results in that certain steroids increased the binding of [3H]R1881 rather than displacing it. While data for 'competing', i.e. displacing, steroids were similar to those reported by other authors, there have been no previous reports of increased binding due to added steroids. This increased binding was due mainly to an increase in capacity (about 10-fold). These data suggest that the placental steroid-binding protein is unusual in that there is a second steroid-binding site whose occupancy increases the stability of the protein, thereby increasing its capacity to bind R1881.

Androstane-3,17-diol↗
PubMed2003

Sex and season influence gonadal steroid biosynthetic pathways, end-product production and steroid conjugation in blotched blue-tongued lizards (Tiliqua nigrolutea).

We examined differences in gonadal steroid production and biosynthetic pathway activity with changing reproductive condition and between sexes in the scincid lizard, Tiliqua nigrolutea. We observed clear seasonal and sexual variation in the production of androgens and steroid conjugates, but detected no 17beta-estradiol or 5alpha-dihydrotestosterone produced by the gonads. An alternative steroid, more polar than estradiol, was detected: an investigation of this steroid is reported separately [Gen. Comp. Endocrinol. 129 (2002) 114]. There were seasonal and sex-related differences in steroid biosynthetic pathway activity. The Delta5 pathway metabolite, dehydroepiandrosterone, was detected only in males, and only from incubations using regressed testicular tissue. There was also a seasonal difference between the sexes in rates of progesterone accumulation, although the absence of corresponding elevated plasma concentrations suggests that the role of progesterone switches from a directly acting hormone to a precursor for others during the reproductive cycle in females. These results suggest that within the traditional view that vertebrate biosynthetic pathway activity and end-products are phylogenetically conserved, there is likely to be considerably species- and/or genus-specific variation.

Animals↗
PubMed1989

Mesangial IgA nephropathy with steroid-responsive nephrotic syndrome: disappearance of mesangial IgA deposits following steroid-induced remission.

This report describes the clinical features and renal biopsy pathology in two patients with immunoglobulin A (IgA) nephropathy and nephrotic syndrome before and after steroid-induced remission. Apart from confirming the frequently relapsing course and mild glomerular changes observed in patients with IgA nephropathy and steroid-responsive nephrotic syndrome (SRNS), we were able to show that mesangial expansion and mesangial IgA deposits disappeared or were greatly reduced in repeat renal biopsies following steroid-induced remission. Because mesangial IgA deposits usually persist in repeat biopsies obtained from patients with typical IgA nephropathy, their resolution in our patients following steroid remission would support the proposal that the association of IgA nephropathy and SRNS may represent a distinct clinical syndrome. It is postulated that the presence of mesangial IgA deposits during nephrotic presentation and their disappearance following steroid-induced remission may result from increased mesangial sequestration of IgA circulating immune complexes (CIC) during the period of enhanced glomerular permeability and that the increased load of IgA CICs may reflect a common defect in mucosal immunity or immunoregulation in these patients.

Adult↗
PubMed1992

High dose nebulized steroid in the treatment of chronic steroid-dependent asthma.

Despite the dramatic success of inhaled steroids in controlling asthma symptoms there remains a small number of patients in whom asthma can only be treated with continuous oral steroids. Eighteen such patients, aged 19-62 years (seven males, 11 females) were followed in an open trial of nebulized budesonide over 12-18 months. All had required at least 7.5 mg or more daily prednisolone to control their symptoms over the preceeding 2 or more years and were taking 1200 micrograms beclomethasone dipropionate or 1600 micrograms budesonide daily. With a daily dose ranging between 4 and 8 mg nebulized budesonide, 14 patients successfully stopped oral steroids while in three the dose was reduced; only one patient failed to benefit. There was an increase in the mean FEV1 from 1.9 (+/- 0.9) to 2.2 (+/- 0.9) l, and in the mean morning PEFR, from 238 (+/- 119) to 286 (+/- 130) l min-1. There was also a significant decrease in the mean number of hospital admissions for acute severe asthma, from 1.5 (+/- 1.8) to 0.9 (+/- 1.1) per year. These findings should encourage a careful and controlled evaluation of nebulized steroids as a substitute for oral steroids in this difficult group of asthmatics.

Adult↗