Sandwich ELISA for hemoglobin A<Subscript>2</Subscript> quantification and identification of β-thalassemia carriers

Hemoglobin (Hb) A₂ (α₂δ₂) is a minor hemoglobin in human red blood cells. An abnormal increase in the level of HbA₂ is the most significant parameter in the diagnosis of β-thalassemia carriers. In this study, we produced two monoclonal antibodies (mAbs) that specifically react to the δ-globin chain of HbA₂. A sandwich type ELISA was developed employing the produced anti-HbA₂ mAbs. HbA₂ levels quantified by the developed sandwich ELISA were highly correlated with those obtained from the standard HPLC method (r = 0.934, p < 0.001). HbA₂ levels determined by the ELISA were 4.4 ± 1.9% in β-thalassemia heterozygotes compared to 1.4 ± 0.8, 1.9 ± 0.8, 1.5 ± 0.8 and 1.5 ± 0.6% in normal subjects, HbE heterozygotes, suspected α-thalassemia heterozygotes and HbE homozygotes, respectively. Using a cut-off value of 2.5%, β-thalassemia heterozygotes could be separated from non-β-thalassemia heterozygotes with the same accuracy as obtained using the standard HPLC method. More importantly, the developed ELISA was able to determine HbA₂ levels in HbE-bearing individuals which could not be done by the HPLC method. Our results suggest that this sandwich ELISA can be applied for mass screening for β-thalassemia heterozygotes, especially in resource-limited countries, where β-thalassemia is highly prevalent.     

Incidence of ischemic stroke and systemic embolism in patients with hypertrophic cardiomyopathy,nonvalvular atrial fibrillation,CHA<Subscript>2</Subscript>DS<Subscript>2</Subscript>-VASc score of ≤1 and without anticoagulant therapy

Data on the risk of ischemic stroke and systemic embolism (iSSE) events in patients with nonvalvular atrial fibrillation (NVAF), a CHA₂DS₂-VASc score of ≤1, hypertrophic cardiomyopathy (HCM), and without anticoagulant therapy are still lacking. The aim of this study was to investigate the incidence of iSSE events in these patients. We consecutively screened medical records of patients with HCM and NVAF referred to Fuwai Hospital between January 1994 and March 2014. The primary end point was iSSE events, defined as a composite of ischemic stroke and systemic embolism. Follow-up was carried out to ascertain end point status. Medical records of 522 patients with NVAF and HCM were screened. A total of 108 patients (20.7 %) with a CHA₂DS₂-VASc score of ≤1 and without anticoagulant therapy were enrolled and constituted our study population. After a median follow-up of 2.4 years (range 0.6–14.1 years; 376.2 patient-years), ischemic stroke occurred in 2 patients, resulting in death of 1 patient in the first year and paralysis of the other patient in the fourth year. No other iSSE events occurred. The incidence of iSSE was 0.9 % [95 % confidence interval (CI) 0.0–5.0 %] in the first year, and 0.5 % per 100 patient-years (95 % CI 0.1–1.9 %). The risk of iSSE events seems low in patients with NVAF, a CHA₂DS₂-VASc score of ≤1, HCM, and without anticoagulant therapy. Multicenter studies with sizeable study populations are needed to validate the risk of iSSE events in these patients.     

The roles of Transforming growth Factor Type β<Subscript>3</Subscript>(TGF-β<Subscript>3</Subscript>) and mast cells in the pathogenesis of scleroderma

Scleroderma is a connective tissue disorder characterised by excessive accumulation of collagen in the skin and internal organs. The most likely explanation for this process is local activation of collagen synthesis from fibroblasts. Our intention was to elucidate whether TGF-₃ and mast cells play a pathogenic role in abnormal connective tissue formation in scleroderma. In this study, skin biopsies from 20 patients with scleroderma and five from healthy individuals were studied by an indirect immunoperoxidase technique to determine the immunoreactivity of TGF-₃ in the dermis. In addition, skin samples were stained with toluidine blue to count the number of mast cells in scleroderma, and tissues were examined under the electron microscope to evaluate the ultrastructural changes. Increased TGF-₃ immunoreactivities were detected in the dermis in the patient's skin, suggesting the presence of a subpopulation responsible for the increased collagen production. Mast cell counts in the skin of patients with scleroderma were significantly greater (19.2 ± 4.1/unit) than those of normal controls (4.4 ± 1.2/unit). Ultrastructural observations indicated that there is a close relationship between the mast cells and fibroblasts. These results suggest that fibrosis in scleroderma could evolve through the activation of fibroblasts and the regulatory mechanisms that appear to modulate the behavior of these cells with respect to collagen production.AcknowledgementWe are grateful for the support of Professor M. Kaya in our work with the electron microscope and thank Professor E. Erten for supplying tissue samples.     

Prevalence of Anticardiolipin and Anti-β<Subscript>2</Subscript>-Glycoprotein I Antibodies in Celiac Disease

The aim of this study was to evaluate the prevalence of anticardiolipin antibodies (aCL) and anti-β₂-glycoprotein I antibodies (aβ2GPI) in patients with celiac disease and to analyze the clinical features of antiphospholipid syndrome in these patients. We conducted a prospective case-control study based on the evaluation of IgG, IgM and IgA aCL, and IgG and IgA aβ2GPI in celiac disease patients and in controls. All patients were asked about any occurrence of thrombotic manifestations. In addition, women were asked about pregnancy morbidity. Fifty celiac disease patients and 50 healthy controls were studied. IgM aCL were not detected in study group or in controls. IgG aCL were found in two patients and in one control. IgA aCL were significantly more frequent in celiac disease patients compared with controls (13/50 (26%) vs. 2/50 (4%), p=0.004, OR [95% CI]=9.09 [1.81–50]). There was no statistically significant difference for the prevalence of IgG and IgA aβ2GPI between patients and controls. Clinical features of antiphospholipid syndrome were noted in two patients with negative antibodies. Prevalence of IgM and IgG aCL and of aβ2GPI were not increased in celiac disease. IgA aCL were more frequently detected in celiac disease. However, no clinical features of antiphospholipid syndrome were noted.     

Pancreatic β-cell K<Subscript>ATP</Subscript> channels: Hypoglycaemia and hyperglycaemia

The pancreatic β-cell ATP-sensitive K⁺ channel (K_ATP channel) plays a critical role in glucose homeostasis by linking glucose metabolism to electrical excitability and insulin secretion. Changes in the intracellular ratio of ATP/ADP mediate the metabolic regulation of channel activity. The β-cell K_ATP channel is a hetero-octameric complex composed of two types of subunits: four inward-rectifying potassium channel pore-forming (Kir6.2) subunits and four high-affinity sulfonylurea receptor 1 (SUR1) subunits. Kir6.2 and SUR1 are encoded by the genes KCNJ11 and ABCC8, respectively. Mutations in these genes can result in congenital hyperinsulinism and permanent neonatal diabetes. This review highlights the important role of the β-cell K_ATP channel in glucose physiology and provides an introduction to some of the other review articles in this special edition of the Reviews in Endocrine and Metabolic Disorders.     

Pharmacological Stimulation of β <Subscript>2</Subscript>-adrenergic Receptors (β <Subscript>2</Subscript>AR) Enhances Therapeutic Effectiveness of β <Subscript>1</Subscript>AR Blockade in Rodent Dilated Ischemic Cardiomyopathy

Background. We have reported that β ₂ adrenoreceptor (β ₂AR) stimulation is anti-apoptotic, and has strong beneficial effect on cardiac remodeling in an experimental model of post myocardial infarction chronic heart failure (CHF) in rats. Here we investigate whether the addition of chronic pharmacological β ₂AR stimulation enhances the therapeutic effects of β ₁AR blockade on cardiac remodeling in the same model. Methods and Results. Metoprolol, a β ₁AR blocker, given alone (β ₁) or in combination with β ₂AR agonist, fenoterol (β ₁β ₂) were administered to rats via drinking water for 6 weeks, beginning 2 weeks following permanent coronary ligation. Progressive left ventricular (LV) remodeling of untreated animals, assessed by repeated echocardiography, occurred during the observation time, i.e., 42% and 25% increases in end-systolic and end-diastolic LV volumes respectively, 27% fall in ejection fraction, and 35% infarct expansion. Pressure-volume loop analyses at 2d and 8th post infarction weeks showed continuous deterioration of systolic and diastolic functions and arterio-ventricular mismatch. Histological evaluation at the end of 8 weeks revealed the MI expansion and hypertrophy of cardiomyocytes. β ₁β ₂ prevented LV remodeling, MI expansion and cardiomyocytes hypertrophy to a greater degree than β ₁, due, in large part, to a vasodilatory effect of β ₂AR stimulation and thus improvement of arterio-ventricular mismatch. The abnormal diastolic performance improved only in β ₁β ₂. β ₁β ₂ treatment reduced myocardial apoptosis throughout myocardium, but β ₁ reduced apoptopsis only in the areas remote from MI. Conclusion. The therapeutic effects of chronic β ₁AR blockade on cardiac remodeling of heart failure are enhanced and extended when supplemented with β ₂AR stimulation. This research was supported by the Intramural Research Program of the National Institute on Aging, NIH.     

Non-inferiority multicenter prospective randomized controlled study of rectal cancer T<Subscript>2</Subscript>–T<Subscript>3s</Subscript> (superficial) N<Subscript>0</Subscript>, M<Subscript>0</Subscript> undergoing neoadjuvant treatment and local excision (TEM) vs total mesorectal excision (TME)

Purpose

The standard treatment of rectal adenocarcinoma is total mesorectal excision (TME), in many cases requires a temporary or permanent stoma. TME is associated with high morbidity and genitourinary alterations. Transanal endoscopic microsurgery (TEM) allows access to tumors up to 20 cm from the anal verge, achieves minimal postoperative morbidity and mortality rates, and does not require an ostomy. The treatment of T2, N0, and M0 cancers remains controversial. Preoperative chemoradiotherapy (CRT) in association with TEM reduces local recurrence and increases survival. The TAU-TEM study aims to demonstrate the non-inferiority of the oncological outcomes and the improvement in morbidity and quality of life achieved with TEM compared with TME.

Methods

Prospective, multicenter, randomized controlled non-inferiority trial includes patients with rectal adenocarcinoma less than 10 cm from the anal verge and up to 4 cm in size, staged as T2 or T3-superficial N0-M0. Patients will be randomized to two areas: CRT plus TEM or radical surgery (TME). Postoperative morbidity and mortality will be recorded and patients will complete the quality of life questionnaires before the start of treatment, after CRT in the CRT/TEM arm, and 6 months after surgery in both arms. The estimated sample size for the study is 173 patients. Patients will attend follow-up controls for local and systemic relapse.

Conclusions

This study aims to demonstrate the preservation of the rectum after preoperative CRT and TEM in rectal cancer stages T2–3s, N0, M0 and to determine the ability of this strategy to avoid the need for radical surgery (TME).

Trial registration

ClinicalTrials.gov identifier: NCT01308190. Número de registro del Comité de Etica e Investigación Clínica (CEIC) del Hospital universitario Parc Taulí: TAU-TEM-2009-01.

     

Lack of Association Between the TGF-β<Subscript>1</Subscript> Gene and Development of COPD in Asians: A Case–Control Study and Meta-analysis

Abnormalities in the transforming growth factor-β₁ (TGF-β₁) gene are thought to be linked to chronic obstructive pulmonary disease (COPD). We investigated the association between the single nuclear polymorphisms (SNPs) of TGF-β₁ and the risk of COPD in a case–control study and meta-analysis. We genotyped 160 cases and 177 control subjects in a local hospital using the Mass-Array^TM Technology Platform and then tested the association of four SNPs in TGF-β₁ (rs6957, rs1800469, rs2241712, and rs2241718) with COPD. Plasma TGF-β₁ level measurement was performed later. A database covering all papers published up to October 30, 2010, was then reviewed. Statistical analysis was performed using Revman 5.0 and STATA 11.0 software. No association was found between TGF-β₁ gene SNPs and an increased risk of COPD in Asians. By meta-analysis, the link of two polymorphisms, rs1800469 and rs1982073, was investigated in seven and eight studies, respectively, involving 1508 COPD patients and 2608 control subjects. The results showed that there was no significant association between an increased risk of COPD in carriers of the T allele (TT+TC) versus the CC genotype in rs1800469 and rs1982073. In ethnic subgroup analysis, the risk of COPD associated with the rs1800469 T allele was not significantly elevated among Asians. TGF-β₁ gene polymorphisms are not associated with an increased risk of COPD in the Asian population.     

Contribution of <Emphasis Type="Italic">I</Emphasis><Subscript>Kr</Subscript> and <Emphasis Type="Italic">I</Emphasis><Subscript>K1</Subscript> to ventricular repolarization in canine and human myocytes: is there any influence of action potential duration?

Background  The aim of the present work was to study the profile of the rapid delayed rectifier potassium current (I _Kr) and the inward rectifier potassium current (I _K1) during ventricular repolarization as a function of action potential duration and rate of repolarization. Methods  Whole cell configuration of the patch clamp technique was used to monitor I _Kr and I _K1 during the action potential plateau and terminal repolarization. Action potentials recorded at various cycle lengths (0.4–5 s) and repolarizing voltage ramps having various slopes (0.5–3 V/s) were used as command signals. I _Kr and I _K1 were identified as difference currents dissected by E-4031 and BaCl₂, respectively. Results  Neither peak amplitudes nor mean values of I _Kr and I _K1 recorded during the plateau of canine action potentials were influenced by action potential duration. The membrane potential where I _Kr and I _K1 peaked during the terminal repolarization was also independent of action potential duration. Similar results were obtained in undiseased human ventricular myocytes, and also in canine cells when I _Kr and I _K1 were evoked using repolarizing voltage ramps of various slopes. Action potential voltage clamp experiments revealed that the peak values of I _Kr, I _K1, and net outward current during the terminal repolarization were independent of the pacing cycle length within the range of 0.4 and 5 s. Conclusions  The results indicate that action potential configuration fails to influence the amplitude of I _Kr and I _K1 during the ventricular action potential in dogs and humans, suggesting that rate-dependent changes in action potential duration are not likely related to rate-dependent alterations in I _Kr or I _K1 kinetics in these species. Returned for 1. Revision: 5 February 2008 1. Revision received: 11 April 2008 Returned for 2. Revision: 14 May 2008 2. Revision received: 15 May 2008     

Loss of β<Subscript>1</Subscript>D-integrin function in human ischemic cardiomyopathy

Integrins play a pivotal role in cardiomyocyte survival and function, with integrin loss leading to myocyte apoptosis and heart failure. The aim of this study was to characterize whether regulation of integrins may contribute to cardiac remodeling in human ischemic cardiomyopathy (ICM). Myocardial tissues of the left ventricle were obtained from patients with ICM (n = 8) undergoing cardiac transplantation and from unused donor hearts (NF, n = 8). In addition, tissue samples from patients with dilated cardiomyopathy (DCM, n = 5) were analyzed. Expression of integrins β₁D and β₃, the effector proteins focal adhesion kinase (FAK) and melusin, and FAK phosphorylation were examined by Western blotting, real-time-PCR and immunofluorescence analysis, respectively. β₁D-integrin protein was decreased in ICM vs. NF by 36%. β₁D-integrin mRNA levels and β₁D-integrin shedding were unchanged. Corresponding to β₁D-integrin regulation, FAK and phosphorylated FAK were decreased in ICM vs. NF by 54% and 49%, respectively. β₃-integrin and melusin were not altered in ICM. As a mediator of integrin effects, AKT kinase activity was examined. In parallel to β₁D-integrin and FAK, AKT activity was decreased in ICM by 44%. In contrast, none of the proteins were significantly altered in DCM compared to NF. Integrins and integrin signaling are regulated differentially in ICM and DCM with a decrease of β₁D-integrin and FAK in ICM. The loss of the β₁Dintegrin-FAK-complex in ICM was paralleled by a reduced AKT activity supporting in vitro data which demonstrate the pivotal role of intact integrin function in anti-apoptotic signaling and cell survival.     

Tumor-derived prostaglandin E<Subscript>2</Subscript> and transforming growth factor-β stimulate endothelial cell motility through inhibition of protein phosphatase-2A and involvement of PTEN and phosphatidy linositide 3-kinase

Tumor vascularization is a complex process that requires structural reorganization and increased motility by endothelial cells. Studies were conducted to identify the tumor-derived mediators and signaling pathways that lead to this increased endothelial cell motility. Using the Lewis lung carcinoma (LLC) tumor model, these studies showed that prostaglandin E₂ (PGE₂) and transforming growth factor- (TGF) were the mediators that were responsible for the migration-stimulatory activity produced by the tumor cells. The response of endothelial cells to these tumor-derived motility-stimulatory factors involved a decline in the activity of the serine/threonine phosphatase PP-2A. Inhibition PP-2A either pharmacologically or genetically increased endothelial cell migration. Concurrent with the decline in PP-2A activity as a result of exposure to PGE₂/TGF was a loss of PP-2A co-precipitation with the inositol phosphatase PTEN and an increase in the PTEN serine phosphorylation level. Since hyperphosphorylation has been shown to inhibit the ability of PTEN to act as an antagonist to phosphatidylinositide 3-kinase (PI3K), the role of PI3K in PGE₂/TGF-stimulated migration was examined. These studies showed that the increased endothelial cell motility that resulted from PGE₂/TGF inhibition of PP-2A was dependent on PI3K.     

Effect of Interferon-α<Subscript>2a</Subscript> on Neutrophil Adhesion and Phagocytosis in Chronic Myeloid Leukemia and Behçet’s Disease

Alpha-interferon (alpha-IFN) is implicated in a Beh?et's disease (BD)-like syndrome observed in a small number of chronic myeloid leukemia (CML) patients. The effect of alpha-IFN on neutrophil adhesion and phagocytosis in CML patients, BD patients and healthy volunteers was investigated to clarify the reason for this observation. Ten subjects were studied for each group by incubating neutrophils with various doses of alpha-IFN. Basal neutrophil adhesions for CML patients, BD patients and healthy volunteers were similar. However, BD patients had greater basal phagocytosis than CML patients, and both groups had greater basal phagocytosis than healthy volunteers. Neutrophil adhesion and phagocytosis of CML patients increased following incubation with higher doses of alpha-IFN, and phagocytosis approached the high levels observed with BD neutrophils. This study provides evidence that alpha-IFN activates neutrophils in CML patients in a dose-dependent manner, and leads to a neutrophil function profile that resembles BD.     

α<Subscript>1</Subscript>-Antitrypsinmangel

α₁-antitrypsin deficiency is characterized by a pathologic reduction of the serum concentration of α₁-antitrypsin, the most important antiprotease in man. It is one of the most common hereditary diseases in Caucasians. Approximately 2% of obstructive airway diseases are caused by α₁-antitrypsin deficiency. Patients above 35 years may develop lung emphysema, especially in the lower lobes. Symptoms are those of chronic obstructive pulmonary disease such as cough, sputum expectoration, and progressive dyspnoea. Patients with homozygous defect often develop cholestatic hepatitis in the neonatal period. However, only few adult patients develop chronic liver disease up to liver cirrhosis with an elevated risk for malignant liver tumors. The diagnostic hallmark is the reduced serum concentration of α₁-antitrypsin while genetic testing proves the defect. An early recognition of the disease is decisive for prophylactic and therapeutic measures. Smoking should be stopped immediately. Treatment of lung disease includes physiotherapy, antiobstructive and antiinflammatory medication, augmentation with human α₁-antitrypsin and lung surgery including lung transplantation. Liver toxins should be avoided. Besides experimental therapeutic approaches, liver disease can only be treated by liver transplantation.     

Diastolic dysfunction and arrhythmias caused by overexpression of CaMKIIδ<Subscript>C</Subscript> can be reversed by inhibition of late Na<Superscript>+</Superscript> current

Transgenic (TG) Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) δ_C mice develop systolic heart failure (HF). CaMKII regulates intracellular Ca²⁺ handling proteins as well as sarcolemmal Na⁺ channels. We hypothesized that CaMKII also contributes to diastolic dysfunction and arrhythmias via augmentation of the late Na⁺ current (late I _Na) in early HF (8-week-old TG mice). Echocardiography revealed severe diastolic dysfunction in addition to decreased systolic ejection fraction. Premature arrhythmogenic contractions (PACs) in isolated isometrically twitching papillary muscles only occurred in TG preparations (5 vs. 0, P < 0.05) which could be completely terminated when treated with the late I _Na inhibitor ranolazine (Ran, 5 μmol/L). Force–frequency relationships revealed significantly reduced twitch force amplitudes in TG papillary muscles. Most importantly, diastolic tension increased with raising frequencies to a greater extent in TG papillary muscles compared to WT specimen (at 10 Hz: 3.7 ± 0.4 vs. 2.5 ± 0.3 mN/mm²; P < 0.05). Addition of Ran improved diastolic dysfunction to 2.1 ± 0.2 mN/mm² (at 10 Hz; P < 0.05) without negative inotropic effects. Mechanistically, the late I _Na was markedly elevated in myocytes isolated from TG mice and could be completely reversed by Ran. In conclusion, our results show for the first time that TG CaMKIIδ_C overexpression induces diastolic dysfunction and arrhythmogenic triggers possibly via an enhanced late I _Na. Inhibition of elevated late I _Na had beneficial effects on arrhythmias as well as diastolic function in papillary muscles from CaMKIIδ_C TG mice. Thus, late I _Na inhibition appears to be a promising option for diastolic dysfunction and arrhythmias in HF where CaMKII is found to be increased.     

CHA<Subscript>2</Subscript>DS<Subscript>2</Subscript>–VASc score predicts short- and long-term outcomes in patients with acute ischemic stroke treated with intravenous thrombolysis

The CHA₂DS₂–VASc score is a validated tool to assess the thromboembolic risk in patients with atrial fibrillation. Pre-stroke CHA₂DS₂–VASc score may predict outcome in patients with acute ischemic stroke (AIS) without atrial fibrillation. The aim of this study was to investigate if the pre-stroke CHA₂DS₂–VASc score is able to predict short- and long-term outcomes in AIS patients treated with intravenous thrombolysis (IVT). The study group consisted of 256 consecutive patients admitted to the Udine University Hospital with AIS and underwent IVT between January 2015 to March 2017. The pre-stroke CHA₂DS₂–VASc score for each patient was calculated from the collected baseline data. Patients were classified into three groups according to their pre-stroke CHA₂DS₂–VASc score: a score of 0 of 1, a score of 2 or 3 and a score above 3. Primary outcome measures were: rate of favorable outcome at 90-days and at 1-year, and mortality at 90-days and at 1-year. Data on functional outcome and mortality 1 year after stroke were collected in 165 patients (65% of the entire sample). Favorable outcome was defined as a modified Rankin Scale score?≤?2. Compared with the CHA₂DS₂–VASc score 0–1 group, patients with higher CHA₂DS₂–VASc scores had a worse outcome and a higher mortality 3 months and 1 year after stroke. The diagnostic performance of the CHA₂DS₂–VASc score as judged with AUC-ROC was 0.70 (95% CI, 0.64–0.76; p?<?0.001) for favorable outcome at 90-days, 0.78 (95% CI, 0.71–0.85; p?<?0.001) for favorable outcome at 1-year, 0.71 (95% CI 0.61–0.79) for mortality at 90-days, 0.73 (95% CI 0.64–0.80; p?<?0.001) for mortality at 1-year. Pre-stroke CHA₂DS_2–VASc score represents a good predictor for short- and long-term outcomes in AIS patients treated with IVT.     

rAAV2-TGF-β<Subscript>3</Subscript> Decreases Collagen Synthesis and Deposition in the Liver of Experimental Hepatic Fibrosis Rat

Background/Aims  

Hepatic fibrosis is one kind of common wound-healing response to chronic liver injury. Transforming growth factor (TGF)-β₃ performs an anti-fibrosis function under certain conditions such as pancreatic fibrosis and wound healing. This study aimed at investigating the effect of TGF-β₃ on the histology in the liver of rat with liver fibrosis.     

Effects of the inhibition of cytosolic phospholipase A<Subscript>2</Subscript>α in non-small cell lung cancer cells

Purpose  

The aim of this study was to investigate the expression of cPLA₂α in non-small lung cancer cell lines and tissues, and we sought to determine the in vitro effects of the pyrrolidine-2 inhibitor on cPLA₂α sensitivity in three different non-small lung cancer cell lines.     

β<Subscript>2</Subscript>-Agonist Eutomers

β₂-adrenoceptor agonists (β₂-agonists) such as albuterol (salbutamol) and terbutaline and their long-acting analogs salmeterol and formoterol are widely used as bronchodilators in the treatment of asthma. They are chiral drugs historically marketed as racemic mixtures of an active (eutomer) and essentially inactive (distomer) stereoisomer. Despite their obvious therapeutic value and widespread use, β₂-agonists have been implicated, somewhat controversially, in causing an increase in asthma mortality and a deterioration of asthma control by a mechanism that remains elusive. Inherent toxicity of the distomers has been widely touted as an explanation and has given rise to pressure for the replacement of the racemates with pure eutomer formulations (the so-called chiral or racemic switch). This has culminated in the recent introduction into clinical practice of the single active stereoisomer of albuterol (levalbuterol) and the promise of other pure β₂-agonist eutomer formulations to follow. This article examines the evidence on which these chiral switches are based.Clinical studies designed to reveal negative effects of β₂-agonists have searched for reductions in lung function, increases in airway responsiveness to bronchoconstrictor mediators and worsening of asthma control. Crossover studies administering the pure stereoisomers and racemate of albuterol have not shown a clear superiority of the pure eutomer formulation over the racemate in terms of either bronchial hyperresponsiveness, tachyphylaxis to bronchoprotective effects or improvements in lung function. Clinical toxicity of β₂-agonist distomers on any aspect of asthmatic lung function has also not been demonstrated in the relatively short-term inhalational studies (single dose or repeated dose studies <1 week) that have been carried out.In animal studies, the administration of β₂-agonist racemates and distomers has been shown to enhance bronchial hyperresponsiveness but only in ovalbumin-sensitized animals where the relevance to humans is questionable.The pharmacokinetics and metabolism of β₂-agonist stereoisomers appear to be essentially similar whether administered as single stereoisomers or as racemates. Levalbuterol may be slightly more potent than an equivalent dose given as racemate, but there is some evidence that it forms a small amount of the distomer in vivo which detracts somewhat from its purported benefits over use of the racemate.Whilst there remains a clear need for studies of longer duration with sensitive clinical endpoints to evaluate the benefits of β₂-agonist eutomers and to investigate distomer toxicity, the chiral switch for β₂-agonists in general, and for albuterol in particular, does not appear to be justified on the basis of the evidence available to date.