Redox Biomarkers and Matrix Remodeling Molecules in Ovarian Cancer.

Ovarian cancer (OC) has emerged as the leading cause of death due to gynecological malignancies among women. Oxidative stress and metalloproteinases (MMPs) have been shown to influence signaling pathways and afflict the progression of carcinogenesis. Therefore, the assessment of matrix-remodeling and oxidative stress intensity can determine the degree of cellular injury and often the severity of redox-mediated chemoresistance. The study group comprised 27 patients with serous OC of which 18% were classified as Federation of Gynecology and Obstetrics (FIGO) stages I/II, while the rest were diagnosed grades III/IV. The control group comprised of 15 ovarian tissue samples. The results were compared with genetic data from The Cancer Genome Atlas. Nitro-oxidative stress, inflammation and apoptosis biomarkers were measured colorimetrically/fluorometrically or via real-time PCR in the primary ovarian tumor and healthy tissue. Stratification of patients according to FIGO stages revealed that high-grade carcinoma exhibited substantial alterations in redox balance, including the accumulation of protein glycoxidation and lipid peroxidation products. TCGA data demonstrated only limited prognostic usefulness of the studied genes. In conclusion, high-grade serous OC is associated with enhanced tissue oxidative/nitrosative stress and macromolecule damage that could not be overridden by the simultaneously augmented measures of antioxidant defense. Therefore, it can be assumed that tumor cells acquire adaptive mechanisms that enable them to withstand the potential toxic effects of elevated reactive oxygen species.

Lipid peroxidation and sphingolipid alterations in the cerebral cortex and hypothalamus of rats fed a high-protein diet.

OBJECTIVES: High-protein diets (HPDs) are widely accepted to enhance satiety and energy expenditure and thus have become a popular strategy to lose weight and facilitate muscle protein synthesis. However, long-term high-protein consumption could be linked with metabolic and clinical problems such as renal and liver dysfunctions. This study verified the effects of 8-wk high-protein ingestion on lipid peroxidation and sphingolipid metabolism in the plasma, cerebral cortex, and hypothalamus in rats. METHODS: Immunoenzymatic and spectrophotometric methods were applied to assess oxidation-reduction (redox) biomarkers and neutral sphingomyelinase activity, whereas gas-liquid chromatography and high-performance liquid chromatography were used to examine sphingolipid levels. RESULTS: The vast majority of HPD-related alterations was restricted to the hypothalamus. Specifically, an increased rate of lipid peroxidation (increased lipid hydroperoxides, 8-isoprostanes, and thiobarbituric acid reactive substances) associated with ceramide accumulation via the activation of de novo synthesis (decreased sphinganine), salvage pathway (decreased sphingosine), and sphingomyelin hydrolysis (decreased sphingomyelin and increased neutral sphingomyelinase activity) was noted. CONCLUSIONS: This study showed that HPD substantially affected hypothalamic metabolic pathways, which potentially alter cerebral output signals to the peripheral tissues.

Branched chain amino acids-friend or foe in the control of energy substrate turnover and insulin sensitivity?

Branched chain amino acids (BCAA) and their derivatives are bioactive molecules with pleiotropic functions in the human body. Elevated fasting blood BCAA concentrations are considered as a metabolic hallmark of obesity, insulin resistance, dyslipidaemia, nonalcoholic fatty liver disease, type 2 diabetes and cardiovascular disease. However, since increased BCAA amount is observed both in metabolically healthy and obese subjects, a question whether BCAA are mechanistic drivers of insulin resistance and its morbidities or only markers of metabolic dysregulation, still remains open. The beneficial effects of BCAA on body weight and composition, aerobic capacity, insulin secretion and sensitivity demand high catabolic potential toward amino acids and/or adequate BCAA intake. On the opposite, BCAA-related inhibition of lipogenesis and lipolysis enhancement may preclude impairment in insulin sensitivity. Thereby, the following review addresses various strategies pertaining to the modulation of BCAA catabolism and the possible roles of BCAA in energy homeostasis. We also aim to elucidate mechanisms behind the heterogeneity of ramifications associated with BCAA modulation.

The Effect of α-Lipoic Acid on Oxidative Stress in Adipose Tissue of Rats with Obesity-Induced Insulin Resistance.

BACKGROUND/AIMS: Correlation between type 2 diabetes and other abnormalities such as obesity with redox balance disturbance was analyzed in many reports. Nonetheless, antioxidants impact on parameters accompanying these conditions is still unknown. Currently the role of redox imbalance in the adipose tissue has gained a lot of attention. METHODS: We investigated the impact of α-lipoic acid (ALA) on plasma glucose and insulin concentrations, oxidative stress and inflammation parameters in the subcutaneous (SAT) and visceral (VAT) adipose tissue of high fat diet-fed (HFD) rats. Male Wistar rats were randomly divided into three groups (n = 6) - control diet (CTRL), HFD and HFD with α-lipoic acid (HFD+ALA). RESULTS: HFD increased body weight, plasma insulin and glucose as well as leads to oxidative stress parameters in the adipose tissue. ALA supplementation reduced body weight and oxidative stress parameters more effectively in the visceral than subcutaneous adipose tissue of insulin resistant rats. CONCLUSION: Insulin resistance led to increased enzymatic and non-enzymatic antioxidant systems, protein and lipid glycoxidation, nitrosative stress, and selected inflammatory parameters more in VAT than in SAT of insulin resistant rats. Moreover, ALA inhibited HFD consequences mainly in VAT mostly through glutathione (GSH) biosynthesis.

Diverse impact of N-acetylcysteine or alpha-lipoic acid supplementation during high-fat diet regime on fatty acid transporters in visceral and subcutaneous adipose tissue.

PURPOSE: Adipose tissue's (AT) structural changes accompanying obesity may alter lipid transport protein expression and, thus, the fatty acids (FAs) transport and lipid balance of the body. Metabolic abnormalities within AT contribute to the elevated production of reactive oxygen species and increased oxidative/nitrosative stress. Although compounds such as N-acetylcysteine (NAC) and α-lipoic acid (ALA), which restore redox homeostasis, may improve lipid metabolism in AT, the mechanism of action of these antioxidants on lipid metabolism in AT is still unknown. This study aimed to examine the impact of NAC and ALA on the level and FA composition of the lipid fractions, and the expression of FA transporters in the visceral and subcutaneous AT of high-fat diet-fed rats. MATERIALS AND METHODS: Male Wistar rats were randomly divided into four groups. The mRNA levels and protein expression of FA transporters were assessed using real-time PCR and Western Blot analyses. The collected samples were subjected to histological evaluation. The level of lipids (FFA, DAG, and TAG) was measured using gas-liquid chromatography. RESULTS: We found that antioxidants affect FA transporter expressions at both the transcript and protein levels, and, therefore, they promote changes in AT's lipid pools. One of the most remarkable findings of our research is that different antioxidant molecules may have a varying impact on AT phenotype. CONCLUSION: NAC and ALA exert different influences on AT, which is reflected in histopathological images, FA transport proteins expression patterns, or even the lipid storage capacity of adipocytes.

α-Lipoic Acid Reduces Ceramide Synthesis and Neuroinflammation in the Hypothalamus of Insulin-Resistant Rats, While in the Cerebral Cortex Diminishes the ß-Amyloid Accumulation.

Background: Oxidative stress underlies metabolic diseases and cognitive impairment; thus, the use of antioxidants may improve brain function in insulin-resistant conditions. We are the first to evaluate the effects of α-lipoic acid (ALA) on redox homeostasis, sphingolipid metabolism, neuroinflammation, apoptosis, and ß-amyloid accumulation in the cerebral cortex and hypothalamus of insulin-resistant rats. Methods: The experiment was conducted on male cmdb/outbred Wistar rats fed a high-fat diet (HFD) for 10 weeks with intragastric administration of ALA (30 mg/kg body weight) for 4 weeks. Pro-oxidant and pro-inflammatory enzymes, oxidative stress, sphingolipid metabolism, neuroinflammation, apoptosis, and ß-amyloid level were assessed in the hypothalamus and cerebral cortex using colorimetric, fluorimetric, ELISA, and HPLC methods. Statistical analysis was performed using three-way ANOVA followed by the Tukey HSD test. Results: ALA normalizes body weight, food intake, glycemia, insulinemia, and systemic insulin sensitivity in HFD-fed rats. ALA treatment reduces nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidase activity, increases ferric-reducing antioxidant power (FRAP) and thiol levels in the hypothalamus of insulin-resistant rats. In addition, it decreases myeloperoxidase, glucuronidase, and metalloproteinase-2 activity and pro-inflammatory cytokines (IL-1ß, IL-6) levels, while in the cerebral cortex ALA reduces ß-amyloid accumulation. In both brain structures, ALA diminishes ceramide synthesis and caspase-3 activity. ALA improves systemic oxidative status and reduces insulin-resistant rats' serum cytokines, chemokines, and growth factors. Conclusion: ALA normalizes lipid and carbohydrate metabolism in insulin-resistant rats. At the brain level, ALA primarily affects hypothalamic metabolism. ALA improves redox homeostasis by decreasing the activity of pro-oxidant enzymes, enhancing total antioxidant potential, and reducing protein and lipid oxidative damage in the hypothalamus of HFD-fed rats. ALA also reduces hypothalamic inflammation and metalloproteinases activity, and cortical ß-amyloid accumulation. In both brain structures, ALA diminishes ceramide synthesis and neuronal apoptosis. Although further study is needed, ALA may be a potential treatment for patients with cerebral complications of insulin resistance.

α-Lipoic Acid Strengthens the Antioxidant Barrier and Reduces Oxidative, Nitrosative, and Glycative Damage, as well as Inhibits Inflammation and Apoptosis in the Hypothalamus but Not in the Cerebral Cortex of Insulin-Resistant Rats.

The research determined the role of α-lipoic acid (ALA) in reducing the brain manifestations of insulin resistance. The mechanism of ALA action is mainly based on its ability to "scavenge" oxygen free radicals and stimulate biosynthesis of reduced glutathione (GSH), considered the most critical brain antioxidant. Although the protective effect of ALA is widely documented in various diseases, there are still no studies assessing the influence of ALA on brain metabolism in the context of insulin resistance and type 2 diabetes. The experiment was conducted on male Wistar rats fed a high-fat diet for ten weeks with intragastric administration of ALA for four weeks. We are the first to demonstrate that ALA improves the function of enzymatic and nonenzymatic brain antioxidant systems, but the protective effects of ALA were mainly observed in the hypothalamus of insulin-resistant rats. Indeed, ALA caused a significant increase in superoxide dismutase, catalase, peroxidase, and glutathione reductase activities, as well as GSH concentration and redox potential ([GSH]2/[GSSG]) in the hypothalamus of HFD-fed rats. A consequence of antioxidant barrier enhancement by ALA is the reduction of oxidation, glycation, and nitration of brain proteins, lipids, and DNA. The protective effects of ALA result from hypothalamic activation of the transcription factor Nrf2 and inhibition of NF-κB. In the hypothalamus of insulin-resistant rats, we demonstrated reduced levels of oxidation (AOPP) and glycation (AGE) protein products, 4-hydroxynoneal, 8-isoprostanes, and 3-nitrotyrosine and, in the cerebral cortex, lower levels of 8-hydroxydeoxyguanosine and peroxynitrite. In addition, we demonstrated that ALA decreases levels of proinflammatory TNF-α but also increases the synthesis of anti-inflammatory IL-10 in the hypothalamus of insulin-resistant rats. ALA also prevents neuronal apoptosis, confirming its multidirectional effects within the brain. Interestingly, we have shown no correlation between brain and serum/plasma oxidative stress biomarkers, indicating the different nature of redox imbalance at the central and systemic levels. To summarize, ALA improves antioxidant balance and diminishes oxidative/glycative stress, protein nitrosative damage, inflammation, and apoptosis, mainly in the hypothalamus of insulin-resistant rats. Further studies are needed to determine the molecular mechanism of ALA action within the brain.

High-Sugar Diet Disrupts Hypothalamic but Not Cerebral Cortex Redox Homeostasis.

Despite several reports on the relationship between metabolic and neurodegenerative diseases, the effect of a high-sugar diet (HSD) on brain function is still unknown. Given the crucial role of oxidative stress in the pathogenesis of these disorders, this study was the first to compare the effect of an HSD on the activity of prooxidative enzymes, enzymatic and non-enzymatic antioxidants, and protein oxidative damage in the brain structures regulating energy metabolism (hypothalamus) and cognitive functions (cerebral cortex). Male Wistar rats were randomly divided into two groups (n = 10)-control diet (CD) and high-sugar diet (HSD)-for 8 weeks. We showed a decrease in glutathione peroxidase and superoxide dismutase activity and an increase in catalase activity in the hypothalamus of HSD rats compared to controls. The activity of xanthine oxidase and NADPH oxidase and the contents of oxidation (protein carbonyls), glycoxidation (dityrosine, kynurenine and N-formylkynurenine) and protein glycation products (advanced glycation end products and Amadori products) were significantly higher only in the hypothalamus of the study group. The HSD was also responsible for the disruption of antioxidant systems and oxidative damage to blood proteins, but we did not show any correlation between systemic redox homeostasis and the brain levels. In summary, HSD is responsible for disorders of enzymatic antioxidant defenses only at the central (plasma/serum) and hypothalamic levels but does not affect the cerebral cortex. The hypothalamus is much more sensitive to oxidative damage caused by an HSD than the cerebral cortex.

Altered Sphingolipid Metabolism Is Associated With Asthma Phenotype in House Dust Mite-Allergic Patients.

PURPOSE: Sphingolipids play an important role in cell growth, survival, inflammation and tissue remodeling. House dust mite (HDM) allergy is a major risk factor for asthma. The aim of the study was to evaluate if allergic asthma phenotype is associated with altered sphingolipid metabolism. METHODS: Twenty-two HDM-allergic asthmatic patients and 11 HDM-allergic rhinitis patients were challenged intrabronchially with biologically standardized Dermatophagoides pteronyssinus extract. Whole blood and platelet-poor plasma samples were collected before, during early asthmatic response (EAR), late asthmatic response (LAR) and 24 hours after the challenge. Concentrations of sphinganine (SFA), sphinganine-1-phosphate (SFA1P), ceramide, sphingosine (SFO) and sphingosine-1-phosphate (S1P) were measured using high performance liquid chromatography. RESULTS: In all house dust mite-allergic patients (HDM-APs), baseline lung function and severity of airway hyperreactivity (AHR) correlated significantly with plasma S1P and SFA1P concentrations. Exhaled nitric oxide concentration, however, correlated with SFA and ceramide, but not with S1P or SFA1P concentration. Allergen challenge increased plasma S1P concentration during EAR, but only in patients who developed both EAR and LAR. The magnitude of the increase determined during EAR correlated with the severity of subsequently developed LAR. Platelet and eosinophil counts were independent predictors of plasma S1P concentration. A significant increase in plasma SFA concentration in response to allergen challenge was seen only in patients who did not develop asthmatic response. CONCLUSIONS: Altered sphingolipid metabolism, with augmented synthesis of S1P and impaired de novo sphingolipid synthesis in response to allergen challenge, may participate in the development of asthma phenotype in HDM-APs.

High Protein Diet Induces Oxidative Stress in Rat Cerebral Cortex and Hypothalamus.

This is the first study to analyze the impact of high protein diet (HPD) on antioxidant defense, redox status, as well as oxidative damage on both a local and systemic level. Male Wistar rats were divided into two equal groups (n = 9): HPD (44% protein) and standard diet (CON; 24.2% protein). After eight weeks, glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase-1 (SOD-1), reduced glutathione (GSH), uric acid (UA), total antioxidant (TAC)/oxidant status (TOS) as well as advanced glycation end products (AGE), 4-hydroxynonenal (4-HNE), and malondialdehyde (MDA) were analyzed in the serum/plasma, cerebral cortex, and hypothalamus of HPD and CON rats. HPD resulted in higher UA concentration and activity of GPx and CAT in the hypothalamus, whereas in the cerebral cortex these parameters remained unchanged. A significantly lower GSH content was demonstrated in the plasma and hypothalamus of HPD rats when compared to CON rats. Both brain structures expressed higher content of 4-HNE and MDA, whereas AGE was increased only in the hypothalamus of HPD animals. Despite the enhancement in antioxidant defense in the hypothalamus, this mechanism does not protect the hypothalamus from oxidative damage in rats. Hypothalamus is more susceptible to oxidative stress caused by HPD.

Insulin Resistance and Oxidative Stress in the Brain: What's New?

The latest studies have indicated a strong relationship between systemic insulin resistance (IR) and higher incidence of neurodegeneration, dementia, and mild cognitive impairment. Although some of these abnormalities could be explained by chronic hyperglycaemia, hyperinsulinemia, dyslipidaemia, and/or prolonged whole-body inflammation, the key role is attributed to the neuronal redox imbalance and oxidative damage. In this mini review, we provide a schematic overview of intracellular oxidative stress and mitochondrial abnormalities in the IR brain. We highlight important correlations found so far between brain oxidative stress, ceramide generation, ß-amyloid accumulation, as well as neuronal apoptosis in the IR conditions.

Redox Balance, Antioxidant Defense, and Oxidative Damage in the Hypothalamus and Cerebral Cortex of Rats with High Fat Diet-Induced Insulin Resistance.

Oxidative stress is a key pathogenic factor in both neurogenerative and metabolic diseases. However, its contribution in the brain complications of insulin resistance is still not well understood. Therefore, the aim of this study was the evaluation of redox homeostasis and oxidative damage in the hypothalamus and cerebral cortex of insulin-resistant and control rats. 16 male Wistar rats were divided into two equal groups (n = 8): the control and high fat diet group (HFD). Prooxidant enzymes (xanthine oxidase and NADPH oxidase); enzymatic and nonenzymatic antioxidants [glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase-1 (SOD-1), and uric acid (UA)]; and oxidative damage products [advanced glycation end products (AGE), 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] as well as the total antioxidant capacity (TAC), total oxidant status (TOS), oxidative stress index (OSI), and total ferric reducing ability of sample (FRAP) were evaluated in the hypothalamus and cerebral cortex as well as serum/plasma of HFD-fed and control rats. The activity of prooxidant enzymes was significantly increased in the cerebral cortex and hypothalamus of HFD-fed rats vs. control rats. Additionally, we have showed enhanced antioxidant efficiency in the hypothalamus (↑CAT, ↑UA, ↑TAC, and ↑FRAP) and cerebral cortex (↑GPx, ↑CAT, ↑SOD-1, ↑UA, ↑TAC, and ↑FRAP) of HFD-fed rats. All of the oxidative damage markers (AGE, 4-HNE, MDA, 8-OHdG, and OSI) were significantly increased in the cerebral cortex of insulin-resistant rats, while only 4-HNE and MDA were markedly higher in the hypothalamus of the HFD group. Summarizing, the results of our study indicate an adaptive brain response to the increased production of free radicals under insulin resistance conditions. Despite the increase in antioxidative defense systems, this mechanism does not protect both brain structures from oxidative damages. However, the cerebral cortex is more susceptible to oxidative stress caused by HFD.

Mechanisms of oat (Avena sativa L.) acclimation to phosphate deficiency.

BACKGROUND: Deficiency of available forms of phosphorus is common in most soils and causes reduction of crop plants growth and yield. Recently, model plants responses to phosphate (Pi) deficiency have been intensively studied. However, acclimation mechanisms of cereals like oat (Avena sativa L.), to low Pi stress remains not fully understood. Oat plants have been usually cultured on poor soils, with a low nutrient content, but their responses to such conditions are not well known, therefore the main goal of the study was to investigate the mechanisms that enable oat plants to grow under low Pi conditions. METHODS: Four oat cultivars (A. sativa, cv. Arab, Krezus, Rajtar and Szakal) were grown for three weeks in a nutrient media with various P sources: inorganic-KH2PO4 (control), organic-phytate (PA) and with no phosphate (-P). The effects of Pi deficiency on the level of P, oat growth parameters, intensity of photosynthesis, plant productivity, root exudation ability, localization, activity and isoforms of acid phosphatases, enzymes involved in Pi mobilization, were estimated. In addition, the effect of mycorrhization on plant growth was also observed. RESULTS: All studied oat cultivars grown on Pi-deficient media had significantly decreased Pi content in the tissues. Pi deficiency caused inhibition of shoot growth, but generally it did not affect root elongation; root diameter was decreased, root/shoot ratios increased, whereas PA plants showed a similar growth to control. Photosynthesis rate and productivity parameters decreased under low Pi nutrition, however, sugar content generally increased. Studied oat cultivars did not respond to low Pi via increased exudation of carboxylates from the roots, as pH changes in the growth media were not observed. Pi starvation significantly increased the activity of extracellular and intracellular acid phosphatases (APases) in comparison to the control plants. Three major APase isoforms were detected in oat tissues and the isoform pattern was similar in all studied conditions, usually with a higher level of one of the isoforms under Pi starvation. Generally no significant effects of mycorrhizal colonization on growth of oat cultivars were observed. DISCUSSION: We postulated that acid phosphatases played the most important role in oat cultivars acclimation to Pi deficiency, especially extracellular enzymes involved in Pi acquisition from soil organic P esters. These APases are mainly located in the epidermis of young roots, and may be released to the rhizosphere. On the other hand, intracellular APases could be involved in fast Pi remobilization from internal sources. Our study showed that oat, in contrast to other plants, can use phytates as the sole source of P. The studied oat cultivars demonstrated similar acclimation mechanisms to Pi deficiency, however, depending on stress level, they can use different pools of acid phosphatases.

Interleukin-18 and NGAL in assessment of ESWL treatment safety in children with urolithiasis.

Urolithiasis is recurrent chronic disease and a complex nephro-urological problem. Currently it is diagnosed in very young children, even infants in the first quarter of life. Until recently the main method of treatment for stones, which for various reasons did not pass spontaneously, was open surgery. At present, the main method replacing open surgery is extracorporeal shock wave lithotripsy (ESWL). Usefulness of common known indicators of the renal function to assess the safety of ESWL procedure is evaluated and verified. The basic markers are serum creatinine, cystatin C, urea, glomerular filtration rate and albuminuria assessment. Unfortunately all these methods show little sensitivity in the case of acute injury processes. There are efforts to use new biomarkers of renal tubular activity, which include among others interleukin 18 (IL-18) and neutrophil gelatinase-associated lipocalin (NGAL). The aim of the study was to assess the safety of ESWL by means of albumin to creatinine ratio, serum cystatin C levels and concentration of two new markers: IL -18 and NGAL. Albumin to creatinine ratio (p = 0.28) and serum cystatin C (p = 0.63) collected before and 48 hours after ESWL did not show statistically significant differences. Similarly, both new markers (IL -18 and NGAL) showed no significant differences (urine IL -18 p = 0.31; serum NGAL p = 0.11; urine NGAL p = 0.29). In conclusion, serum cystatin C tests, urine albumin to creatinine ratio and new early markers of renal tubular injury confirmed the safety of the extracorporeal shock wave lithotripsy (ESWL) and show that the procedure does not cause any episode of acute renal injury.

[Kidney tumor in 12-year-old girl--case study of the rare histopatologic form of neoplasm in children]. / Nowotwór nerki u 12-letniej dziewczynki--przypadek rzadko wystepujacej postaci histopatologicznej u dzieci.

The authors present an atypical case of MEST uncovered in 12-year-old girl diagnosed with recurrent pyuria and persistent albuminuria with concomitant hypertension. Urine changes persisted despite the antibiotics therapy and they were accompanied with aseptic urine cultures. While investigating these changes, USG, urography, CT and MRI were performed and these manifested tumor-like change in the right kidney. Right-side nefrectomy was performed. Microscopically MEST was found while infection was found in parenchyma. Mixed epithelial and stromal tumor is a very rare kidney tumor that occurs almost exclusively in perimenopausal women or women after a long-term estrogen replacement. There are only few cases of this tumor in premenarcheal girls described in literature.

[Spirometry evaluation of montelukast treatment in children with bronchial asthma]. / Spirometryczna ocena wyników leczenia montelukastem u dzieci chorych na astme oskrzelowa.

Difficulties associated with the management of asthma were the cause of using the leukotriene antagonists. There was performed a randomised group of 31 children aged 6-18 y (mean 12 yrs., 10 girls and 21 boys) with persistent asthma and poor asthma control. Montelukast was administered once daily, 5-10 mg depended of the age of the patients. This treatment was conducted for three months. Montelukast significantly improved pulmonary lung function (as measured by spirometry). Another effect of montelukast treatment was possibility the reduction beta 2-agonist use and the required dose of inhaled corticosteroids.