European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia

The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no "gold standard" reference test. Hence, a Task Force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia

A portal to validated websites on cosmetic surgery: the design of an archetype.

There has recently been an increase in the usage of the Internet as a source of patient information. It is very difficult for laypersons to establish the accuracy and validity of these medical websites. Although many website assessment tools exist, most of these are not practical.A combination of consumer- and clinician-based website assessment tools was applied to 200 websites on cosmetic surgery. The top-scoring websites were used as links from a portal website that was designed using Microsoft Macromedia Suite.Seventy-one (35.5%) websites were excluded. One hundred fifteen websites (89%) failed to reach an acceptable standard.The provision of new websites has proceeded without quality controls. Patients need to be better educated on the limitations of the Internet. This paper suggests an archetypal model, which makes efficient use of existing resources, validates them, and is easily transferable to different health settings.

Mechanisms of inhibition of aldehyde dehydrogenase by nitroxyl, the active metabolite of the alcohol deterrent agent cyanamide.

Nitroxyl, produced in the bioactivation of the alcohol deterrent agent cyanamide, is a potent inhibitor of aldehyde dehydrogenase (AIDH); however, the mechanism of inhibition of AlDH by nitroxyl has not been described previously. Nitroxyl is also generated from Angeli's salt (Na2N2O3) at physiological pH, and, indeed, Angeli's salt inhibited yeast AlDH in a time- and concentration-dependent manner, with IC50 values under anaerobic conditions with and without NAD+ of 1.3 and 1.8 microM, respectively. Benzaldehyde, a substrate for AlDH, competitively blocked the inhibition of this enzyme by nitroxyl in the presence of NAD+, but not in its absence, in accord with the ordered mechanism of this reaction. The sulfhydryl reagents dithiothreitol (5 mM) and reduced glutathione (10 mM) completely blocked the inhibition of AlDH by Angeli's salt. These thiols were also able to partially restore activity to the nitroxyl-inhibited enzyme, the extent of reactivation being dependent on the pH at which the inactivation occurred. This pH dependency indicates the formation of two inhibited forms of the enzyme, with an irreversible form predominant at pH 7.5 and below, and a reversible form predominant at pH 8.5 and above. The reversible form of the inhibited enzyme is postulated to be an intra-subunit disulfide, while the irreversible form is postulated to be a sulfinamide. Both forms of the inhibited enzyme are derived via a common N-hydroxysulfenamide intermediate produced by the addition of nitroxyl to active site cysteine thiol(s).

Differential expression of c-jun and junD in end-stage human cardiomyopathy.

The proto-oncogenes c-jun and junD are closely related transcriptional factors with opposing actions on cell growth and division. Expression of c-jun rapidly increases as cells enter the cell cycle. Levels of c-jun are also increased in the early stages of experimental cardiac hypertrophy and failure but expression decreases with time. In contrast, junD accumulates in quiescent cells. Expression in end-stage cardiomyopathy has not been studied. Steady-state levels of c-jun and junD mRNA were determined in failing human myocardium (obtained at the time of cardiac transplantation) and in control myocardium from patients who died of noncardiac causes. Relative expression was normalized for glyceraldehyde-3-phosphate dehydrogenase expression. Levels of junD were almost four-fold depressed in myocardium from myopathic hearts (2.1 +/- 0.27, x +/- SE; n = 20) vs. the controls (7.7 +/- 1.1; n = 3). Levels of c-jun were similar in both myopathic and control hearts. Relative expression of beta-myosin heavy chain was the same in both myopathic and control hearts. Levels of junD were still found to be depressed in the myopathic hearts after normalization for myosin heavy chain gene expression. We conclude that c-jun and junD are differentially regulated in end-stage human cardiomyopathy with expression of junD being decreased while relative levels of c-jun mRNA remain unchanged. Further studies are needed to determine the role of junD down-regulation in the development and/or maintenance of the abnormalities present in end-stage heart disease.

Mechanism for the inhibition of aldehyde dehydrogenase by nitric oxide.

The inhibition of Saccharomyces cerevisiae aldehyde dehydrogenase (AlDH) by gaseous nitric oxide (NO) in solution and by NO generated from diethylamine nonoate was time and concentration dependent. The presence of oxygen significantly reduced the extent of inhibition by NO, indicating that NO itself rather than an oxidation product of NO such as N2O3 is the inhibitory species under physiological conditions. A cysteine residue at the active site of the enzyme was implicated in this inhibition based on the following observations: a) NAD+ and NADP+, but not reduced cofactors, significantly enhanced inhibition of AlDH by NO; b) the aldehyde substrate, benzaldehyde, blocked inhibition; and c) inhibition was accompanied by loss of free sulfhydryl groups on the enzyme. Activity of the NO-inactivated enzyme was readily restored by treatment with dithiothreitol (DTT), but not with GSH. This difference was attributed, in part, to a redox process leading to the formation of a cyclic DTT disulfide. Based on the chemistry deduced from model systems, the reaction of NO with AlDH sulfhydryls was shown to produce intramolecular disulfides and N2O. These disulfides were shown to be intrasubunit disulfides by nonreducing SDS-PAGE analysis of the NO- inhibited enzyme. Following complete inhibition of AlDH by NO, four of the eight titratable (Ellman's reagent) sulfhydryl groups of AlDH were found to be oxidized to disulfides. These results suggest that a) the sulfhydryl group of active site Cys-302 and a proximal cysteine are oxidized to form an intrasubunit disulfide by NO; b) only two of the four subunits of AlDH are catalytically active; and c) NO preferentially oxidizes sulfhydryl groups of the catalytically active subunits. A detailed mechanism for the inhibition of AlDH by NO is presented.

Reaction of nitric oxide with the free sulfhydryl group of human serum albumin yields a sulfenic acid and nitrous oxide.

Nitric oxide (NO) generated by diethylamine nonoate (DEA/NO), an NO donor, readily oxidized the free sulfhydryl group of human serum albumin (HSA) as well as the sulfhydryl groups of reduced glutathione (GSH) and dithiothreitol (DTT) at pH 7.4 and 37 degrees C. Under anaerobic conditions, the major products of the oxidation of HSA thiol by NO were the sulfenic acid (RSOH) of HSA and nitrous oxide (N2O). The stoichiometry for this reaction, viz., 1 mol of HSA sulfhydryl oxidized to 1 mol of N2O produced, is consistent with a net two-electron oxidation of the protein thiol to a sulfenic acid. The sulfenic acid product of HSA was shown to react with dimedone and GSH, two known reactions of sulfenic acids. In contrast, anaerobic oxidation of GSH and DTT by NO gave a stoichiometry close to the expected ratio of 2:1 (sulfhydryl oxidized to N2O produced) for the oxidation of these thiols to their disulfides and N2O. Under aerobic conditions, significant fractions of the sulfhydryl groups of HSA, GSH, and DTT were oxidized to their respective thionitrites, presumably by N2O3. Thionitrite formation was not observed in the absence of oxygen. The production of HSA-sulfenic acid by NO, as well as by other oxidizing agents such as H2O2 and peroxynitrite, followed by its reaction with circulating GSH or L-Cys may account for the mixed disulfides of HSA observed in plasma.

Comparative oxidation of 2-propyn-1-ol with other low molecular weight unsaturated and saturated primary alcohols by bovine liver catalase in vitro.

The oxidative metabolism of low molecular weight, saturated and unsaturated, primary alcohols, which include ethanol, allyl alcohol (2-propen-1-ol), and propargyl alcohol (2-propyn-1-ol), is generally accepted to occur via alcohol dehydrogenase; however, compared to other short-chain alcohols, 2-propyn-1-ol is a poor substrate for this enzyme. Accordingly, we have examined liver catalase as an alternative pathway for the oxidation or bioactivation of 2-propyn-1-ol to 2-propyn-1-al, a highly reactive alpha,beta-unsaturated aldehyde. The rates of oxidation for a series of low molecular weight, saturated, primary alcohols and selected unsaturated alcohols were determined for the bovine liver catalase-catalyzed reaction by measuring aldehyde production over time employing a GC procedure. A negative correlation was found for log rates of oxidation versus molecular size (volume) of the substrates (p < 0.01); however, the rate of oxidation for 2-propyn-1-ol was higher than predicted by this relation and was 30% greater than the oxidation rate determined for ethanol. In addition, 2-propyn-1-ol-derived 2-propyn-1-al inhibited the peroxidatic and catalytic activities of catalase, whereas 2-propen-1-ol-derived 2-propen-1-al had no effect on these activities of catalase. Inhibition was blocked by GSH; and the activity was not restored to the inhibited enzyme by GSH treatment or dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic activation of n-butyraldoxime by rat liver microsomal cytochrome P450. A requirement for the inhibition of aldehyde dehydrogenase.

n-Butyraldoxime (n-BO) is known to cause a disulfiram/ethanol-like reaction in humans, a manifestation of the inhibition of hepatic aldehyde dehydrogenase (AIDH). As with a number of other in vivo inhibitors of AIDH, n-BO does not inhibit purified AIDH in vitro, suggesting that a metabolite of n-BO is the actual inhibitor of this enzyme. In re-examination of the effect of n-BO on blood acetaldehyde levels following ethanol in the Sprague-Dawley rat, we found that pretreatment with substrates and/or inhibitors of cytochrome P450 blocked the n-BO-induced rise in blood acetaldehyde in the following order of decreasing potency: 1-benzylimidazole (0.1 mmol/kg) > 3-amino-1,2,4-triazole (1.0 g/kg) > ethanol (3.0 g/kg) > phenobarbital (0.1% in the drinking water, 7 days) > SKF-525A (40 mg/kg). Rat liver microsomes were shown to catalyze the conversion of n-BO to an active metabolite that inhibited yeast AIDH. This reaction was dependent on NADPH and molecular oxygen and was inhibited by CO and 1-benzylimidazole. Hydroxylamine, postulated by others to be a metabolite of n-BO, inhibited AIDH via a catalase-mediated reaction and not through an NADPH-supported microsome-catalyzed reaction. Using GLC-mass spectrometry, 1-nitrobutane (an N-oxidation product) and butyronitrile (a dehydration product) were identified as metabolites from microsomal incubations of n-BO. However, neither of these metabolic products inhibited AIDH directly or in the presence of liver microsomes and NADPH. We conclude that another NADPH-dependent, cytochrome P450-catalyzed metabolic product of n-BO is responsible for the inhibition of AIDH by n-BO.

Differential inhibition of rat tissue catalase by cyanamide.

The relative sensitivity of rat tissue catalase to inhibition by intraperitoneally administered cyanamide was liver greater than kidney greater than heart greater than brain, whereas the activity of the erythrocyte enzyme was affected minimally. The measured ED50 values for cyanamide in these tissues were 31, 44, 107 and 680 mumoles/kg body weight for liver, kidney, heart and brain respectively. On a molar basis, cyanamide was approximately twenty times more potent than 3-amino-1,2,4-triazole (3-AT) in inhibiting hepatic catalase in vivo in the rat. Like 3-AT, cyanamide inhibited erythrocyte catalase activity in vitro in the presence of hydrogen peroxide. The apparent similarities between the inhibition of hepatic catalase by cyanamide and 3-AT in vivo suggest that cyanamide belongs to the family of 3-AT-like catalase inhibitors.

Analysis of hepatic reduced glutathione, cysteine and homocysteine by cation-exchange high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method employing a mercury-based electrochemical detector and a cation-exchange column is described for the simultaneous measurement of reduced glutathione, cysteine, and homocysteine in liver homogenates. Sample preparation involves precipitation of protein with perchloric acid, removal of perchlorate by precipitation as its potassium salt and dilution with mobile phase. Mercaptoethylglycine is used as the internal standard. Using this procedure, the sum of the individual hepatic thiols agreed well with the total thiols determined with Ellman's reagent. Comparisons were made with (a) control rats, (b) rats depleted of hepatic thiols by pargyline pretreatment, and (c) rats administered L-cysteine.

Elimination of artifactual acetaldehyde in the measurement of human blood acetaldehyde by the use of polyethylene glycol and sodium azide: normal blood acetaldehyde levels in the dog and human after ethanol.

A new procedure is described for the preparation of human blood samples for analysis of acetaldehyde and ethanol by head space gas chromatography. High concentrations of polyethylene glycol were used to remove the hemoglobin and approximately 50% of the plasma protein. Artifactual formation of acetaldehyde from ethanol was inhibited by sodium azide. Using this method, no artifactual acetaldehyde was detectable in human, dog, sheep, and rat blood when spiked with ethanol in final concentrations of 65 mM. The recovery of added acetaldehyde was approximately 80% for human dog, and sheep blood, whereas it was only 30% for rat blood. Following ethanol administration, acetaldehyde levels were determined in blood taken from the pulmonary artery and descending aorta of the dog and human, and also from the hepatic vein of the latter. The relative blood acetaldehyde concentrations at these sites were hepatic vein greater than pulmonary artery greater than descending aorta.

Calcium metabolism in acromegaly.

A total of 78 acromegalic patients were studied before and after treatment by yttrium-90 needle implantation.Among the untreated patients 16% had a borderline or raised serum calcium. In half of these patients the serum calcium fell to normal after remission of their acromegaly. In the others the hypercalcaemia was due to associated proved or probable hyperparathyroidism. A downward trend of the serum calcium was noted even in the normocalcaemic patients with remission of their disease.Only 20% of untreated patients had a raised serum phosphate, and follow-up showed this measurement to be a poor index of disease activity.Net calcium absorption and calcium balances in five patients in this series and 12 others from the literature were essentially normal for their given level of calcium intake. No patient showed definite radiological evidence of osteoporosis and vertebral fractures.Bone uptake rate of calcium-47 and stable strontium was raised in the untreated state in all nine patients studied. The 24-hour strontium space was raised in 73% of untreated patients and fell to normal after treatment in all the retested patients in whom it was high initially.