Neutrophil activation during acute human anaphylaxis: analysis of MPO and sCD62L.

BACKGROUND: The mechanisms involved in the amplification of the mast cell response during anaphylaxis are unclear. Mouse models of anaphylaxis demonstrate the critical involvement of neutrophils. These innate immune cells are highly abundant in peripheral blood and can be rapidly activated to trigger both local and systemic inflammation. OBJECTIVE: To investigate neutrophil activation in peripheral blood during acute human anaphylaxis. METHODS: Patients presenting to the emergency department with anaphylaxis underwent blood sampling upon enrolment and at up to three subsequent time-points. Traditional anaphylaxis biomarkers, histamine and mast cell tryptase, were measured by ELISA and ImmunoCAP, respectively. Plasma myeloperoxidase concentrations were measured by ELISA, serum soluble CD62L concentrations by cytometric bead array, and both compared to healthy controls. RESULTS: In 72 patients, 37 (51%) had severe anaphylaxis, 33 (60%) were histamine positive, and 47 (70%) were mast cell tryptase positive. At enrolment, myeloperoxidase concentrations were 2.9- (95% CI: 1.3, 6.5) and 5.0- (95% CI: 2.4, 10.5) fold higher in moderate and severe patients, respectively, compared with healthy controls, and remained stable over the first 5 h following symptom onset. At enrolment, soluble CD62L was 29% (95% CI: 19, 38) and 31% (95% CI: 22, 40) lower in moderate and severe patients, respectively, than healthy controls, and was stable over the first 5 h. There were no associations between myeloperoxidase or soluble CD62L concentrations and either histamine or mast cell tryptase concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: These results provide compelling evidence for the involvement of neutrophils during acute human anaphylaxis, suggesting they are activated early in the reaction, regardless of mast cell activation. This important finding increases our understanding of the basic mechanisms of anaphylaxis, a necessary precursor to improving treatment and prevention.

Factors influencing the quality of Myrmecia pilosula (Jack Jumper) ant venom for use in in vitro and in vivo diagnoses of allergen sensitization and in allergen immunotherapy.

BACKGROUND: Allergen immunotherapy uses pharmaceutical preparations derived from naturally occurring source materials, which contain water-soluble allergenic components responsible for allergic reactions. The success of in vivo and in vitro diagnoses in allergen sensitization and allergen immunotherapy largely depends on the quality, composition and uniformity of allergenic materials used to produce the active ingredients, and the formulation employed to prepare finished products. OBJECTIVES: We aimed to examine the factors influencing batch-to-batch consistency of Jack Jumper (Myrmecia pilosula) ant venom (JJAV) in the form of active pharmaceutical ingredient (AI) and informed whether factors such as temperature, artificial light and container materials influence the quality of JJAV AIs. We also aimed to establish handling and storage requirements of JJAV AIs to ensure preservation of allergenic activities during usage in the diagnosis of allergen sensitization and in allergen immunotherapy. METHODS: The quality and consistency of JJAV AIs were analysed using a combination of bicinchoninic acid assay for total protein quantification, HPLC-UV for JJAV allergen peptides quantification, ELISA inhibition for total allergenic potency, SDS-PAGE, AU-PAGE and immunoblot for qualitative assessment of JJAV components, and Limulus Amebocyte Lysate assay for the quantification of endotoxin concentration. API-ZYM and Zymogram assays were used to probe the presence of enzymatic activities in JJAV. RESULTS: Pharmaceutical-grade JJAV for allergen immunotherapy has good batch-to-batch consistency. Temporary storage at 4°C and light exposure do not affect the quality of JJAV. Exposure to temperature above 40°C degrades high MW allergens in JJAV. Vials containing JJAV must be stored frozen and in upright position during long-term storage. CONCLUSIONS AND CLINICAL RELEVANCE: We have identified factors, which can influence the quality and consistency of JJAV AIs, and provided a framework for appropriate handling, transporting and storage of JJAV to be used for the diagnosis of allergen sensitization and in AIT.

Spontaneous pneumothorax; a multicentre retrospective analysis of emergency treatment, complications and outcomes.

BACKGROUND: Spontaneous pneumothorax can be managed initially by observation, aspiration or chest drain insertion. AIMS: To determine the clinical features of spontaneous pneumothorax in patients presenting to the emergency department (ED), interventions, outcomes and potential risk factors for poor outcomes after treatment. METHODS: Retrospective chart review from ED of three major referral and two general hospitals in Australia of presentations with primary spontaneous pneumothorax (PSP) or secondary spontaneous pneumothorax (SSP). Main outcomes were prolonged air leak (>5 days) and pneumothorax recurrence within 1 year. RESULTS: We identified 225 people with PSP and 98 with SSP. There were no clinical tension pneumothoraces with hypotension. Hypoxaemia (haemoglobin oxygen saturation measured by pulse oximetry ≤92%) occurred only in SSP and in older patients (age >50 years) with PSP. Drainage was performed in 150 (67%) PSP and 82 (84%) SSP. Prolonged air leak occurred in 16% (95% confidence interval 10-23%) of PSP and 31% (21-42%) of SSP. Independent risk factors for prolonged drainage were non-asthma SSP and pneumothorax size >50%. Complications were recorded in 11% (7.5-16%) of those having drains inserted. Recurrences occurred in 5/91 (5%, 1.8-12%) of those treated without drainage versus 40/232 (17%, 13-23%) of those treated by drainage, of which half occurred in the first month after drainage. CONCLUSION: Pneumothorax drainage is associated with substantial morbidity including prolonged air leak. As PSP appears to be well tolerated in younger people even with large pneumothoraces, conservative treatment in this subgroup may be a viable option to improve patient outcomes, but this needs to be confirmed in a clinical trial.

Filtration effectiveness of HVAC systems at near-roadway schools.

UNLABELLED: Concern for the exposure of children attending schools located near busy roadways to toxic, traffic-related air pollutants has raised questions regarding the environmental benefits of advanced heating, ventilation, and air-conditioning (HVAC) filtration systems for near-road pollution. Levels of black carbon and gaseous pollutants were measured at three indoor classroom sites and at seven outdoor monitoring sites at Las Vegas schools. Initial HVAC filtration systems effected a 31-66% reduction in black carbon particle concentrations inside three schools compared with ambient air concentrations. After improved filtration systems were installed, black carbon particle concentrations were reduced by 74-97% inside three classrooms relative to ambient air concentrations. Average black carbon particle concentrations inside the schools with improved filtration systems were lower than typical ambient Las Vegas concentrations by 49-96%. Gaseous pollutants were higher indoors than outdoors. The higher indoor concentrations most likely originated at least partially from indoor sources, which were not targeted as part of this intervention. PRACTICAL IMPLICATIONS: Recent literature has demonstrated adverse health effects in subjects exposed to ambient air near major roadways. Current smart growth planning and infill development often require that buildings such as schools are built near major roadways. Improving the filtration systems of a school's HVAC system was shown to decrease children's exposure to near-roadway diesel particulate matter. However, reducing exposure to the gas-phase air toxics, which primarily originated from indoor sources, may require multiple filter passes on recirculated air.

Can an observational method of assessing hand preference be used to predict language lateralisation?

The current study investigated (a) whether or not the WatHand Cabinet Test (WHCT, Bryden, Roy, & Spence, 2007) could be used as accurately as the Waterloo Handedness Questionnaire (WHQ) to classify individuals into language lateralisation groups based on their hand preference, and (b) the relationship between direction and degree of hand preference and language lateralisation. A total of 142 participants (82 right-handers and 60 left-handers) completed the WHQ and the WHCT, and performed a fused-words dichotic listening test. Findings indicated that the WHCT was robust alternative to the WHQ in providing a measure of hand preference as there was a high correlation between the WHCT and the WHQ, and individuals were divided into similar language lateralisation groups when using either the WHCT or the WHQ as the classifying variable. More specifically, there existed a predictable pattern of language lateralisation into which members of different handedness groups fell. The same pattern exists whether handedness is defined using subjective questionnaires or more objective observational measures of hand preference.

Severe hypoglycaemia and cognitive impairment in older patients with diabetes: the Fremantle Diabetes Study.

AIMS/HYPOTHESIS: The aim was to investigate the relationship between severe hypoglycaemia and cognitive impairment in older patients with diabetes. METHODS: A sample of 302 diabetic patients aged >/=70 years was assessed for dementia or cognitive impairment without dementia in 2001-2002 and a subsample of non-demented patients (n = 205) was followed to assess cognitive decline. A history of severe hypoglycaemia was determined from self-reports, physician assessments and records of health service use for hypoglycaemia (HSH). Prospective HSH was determined up to 2006. Data analysis, including multiple logistic and Cox regression models, was used to determine whether: (1) there were cross-sectional associations between hypoglycaemia and cognitive status, (2) historical hypoglycaemia predicted cognitive decline, and (3) baseline cognitive status predicted subsequent HSH. RESULTS: There were significant cross-sectional associations between both cognitive impairment and dementia and hypoglycaemia. Independent risk factors for future HSH included dementia (hazard ratio 3.00, 95% CI 1.06-8.48) and inability to self-manage medications (hazard ratio 4.17, 95% CI 1.43-12.13). However, there were no significant associations between historical hypoglycaemia, incident HSH and cognitive decline. CONCLUSIONS/INTERPRETATION: Dementia is an important risk factor for hypoglycaemia requiring health service utilisation. We found no evidence that hypoglycaemia contributes to cognitive impairment in older patients with diabetes.

Cation currents in human airway epithelial cells induced by infection with influenza A virus.

Influenza A viruses cause lung disease via an incompletely understood mechanism that involves the accumulation of liquid within the lungs. The accumulation of lung liquid is normally prevented by epithelial Na(+) absorption, a transport process regulated via several pathways including phosphoinositide-3-kinase (PI3K). Since the influenza A virus encodes a non-structural protein (NS1) that can activate this kinase, we now explore the effects of NS1 upon the biophysical properties of human airway epithelial cells. Transient expression of NS1 depolarized electrically isolated cells maintained in glucocorticoid-free medium by activating a cation conductance identical to the glucocorticoid-induced conductance seen in single cells. This response involved PI3K-independent and PI3K-dependent mechanisms. Infecting glucocorticoid-deprived cells with influenza A virus disrupted the normal electrical coupling between neighbouring cells, but also activated a conductance identical to that induced by NS1. This response to virus infection was only partially dependent upon NS1-mediated activation of PI3K. The presence of NS1 allows influenza A to modify the biophysical properties of infected cells by activating a Na(+)-permeable conductance. Whilst the activation of Na(+)-permeable channels may be expected to increase the rate of Na(+) absorption and thus reduce the volume of liquid in the lung, liquid does normally accumulate in influenza A-infected lungs. The overall effect of influenza A on lung liquid volume may therefore reflect a balance between the activation and inhibition of Na(+)-permeable channels.

SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr346/356/366 phosphorylation.

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr(346/356/366)) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1-12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr(346/356/366) in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na(+) conductance (G (Na)) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr(346/356/366) phosphorylation. Although G (Na) is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 muM) acutely activated SGK1 and the peak of this response (2-3 h) coincided with the induction of G (Na), and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G (Na), transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G (Na) but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na(+) absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G (Na), its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.

Adrenaline for the treatment of anaphylaxis: cochrane systematic review.

BACKGROUND: Anaphylaxis is a serious allergic reaction that is rapid in onset and may cause death. Adrenaline is recommended as the initial treatment of choice for anaphylaxis. OBJECTIVES: To assess the benefits and harms of adrenaline in the treatment of anaphylaxis. METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2007, Issue 1), MEDLINE (1966 to March 2007), EMBASE (1966 to March 2007), CINAHL (1982 to March 2007), BIOSIS (to March 2007), ISI Web of Knowledge (to March 2007) and LILACS (to March 2007). We also searched websites listing ongoing trials: http://www.clinicaltrials.gov/, http://www.controlledtrials.com and http://www.actr.org.au/ and contacted pharmaceutical companies and international experts in anaphylaxis in an attempt to locate unpublished material. Randomized and quasi-randomized controlled trials comparing adrenaline with no intervention, placebo or other adrenergic agonists were eligible for inclusion. Two authors independently assessed articles for inclusion. RESULTS: We found no studies that satisfied the inclusion criteria. CONCLUSIONS: On the basis of this review, we are unable to make any new recommendations on the use of adrenaline for the treatment of anaphylaxis. In the absence of appropriate trials, we recommend, albeit on the basis of less than optimal evidence, that adrenaline administration by intramuscular injection should still be regarded as first-line treatment for the management of anaphylaxis.

Urea and its formation in coelacanth liver.

Urea occurs in liver of the coelacanth Latimeria chalumnae to the extent of about 1.7 percent by weight. It was determined quantitatively by reaction with 1-phenyl-1,2-propanedione-2-oxime (Archibald reagent) and by measurement of ammonia released upon treatment with urease. Arginase and ornithine carbamoyltransferase, enzymes instrumental in the formation of urea in typical ureotelic vertebrates, occur in homogenates of coelacanth liver. Formed in part by the ornithine-urea cycle, urea may have an osmoregulatory function in the coelacanth as it has in elasmobranchs.

Uricolytic enzymes in liver of the Dipnoan Protopterus aethiopicus.

The enzymes uricase, allantoinase, and allantoicase have been measured in liver preparations of the African lungfish Protopterus aethiopicus. The levels for these enzymes in lungfish liver suggest that the amount of urea formed in vivo in Protopterus via a uricolytic pathway may be greater than that derived via the Ornithine-urea cycle. The operation of a "purine cycle" in lungfish liver is proposed.

H1-antihistamines for the treatment of anaphylaxis with and without shock.

BACKGROUND: Anaphylaxis is an acute systemic allergic reaction, which can be life-threatening. H1-antihistamines are commonly used as an adjuvant therapy in the treatment of anaphylaxis. OBJECTIVES: To assess the benefits and harm of H1-antihistamines in the treatment of anaphylaxis. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), (The Cochrane Library), MEDLINE (1966 to June 2006);EMBASE (1966 to June 2006); CINAHL (1982 to June 2006) and ISI Web of Science (1945 to July 2006). We also contacted pharmaceutical companies and international experts in anaphylaxis in an attempt to locate unpublished material. SELECTION CRITERIA: Randomized and quasi-randomized controlled trials comparing H1-antihistamines with placebo or no intervention. DATA COLLECTION AND ANALYSIS: Two authors independently assessed articles for inclusion. MAIN RESULTS: We found no studies that satisfied the inclusion criteria. AUTHORS' CONCLUSIONS: Based on this review, we are unable to make any recommendations for clinical practice. Randomized controlled trials are needed, although these are likely to prove challenging to design and execute.

Thrombotic microangiopathy from Australian brown snake (Pseudonaja) envenoming.

BACKGROUND: Australian brown snake (genus Pseudonaja) envenoming causes a venom-induced consumptive coagulopathy (VICC). A proportion of cases go on to develop thrombotic microangiopathy characterized by thrombocytopenia, microangiopathic haemolytic anaemia (MAHA) and acute renal failure (ARF). AIM: The aim of the study was to define better the natural history and empirical treatments for thrombotic microangiopathy in brown snake envenoming. METHODS: A review of brown snake bites recruited to the Australian Snakebite Project (ASP), a national multicentre study of snake envenoming was undertaken. Serial data are recorded on clinical effects and laboratory results, including measurement of venom concentrations. We describe cases of thrombotic microangiopathy and compare these to cases without thrombotic microangiopathy. RESULTS: From 32 cases of brown snake envenoming with severe VICC, four (13%) developed thrombotic microangiopathy, we also included two cases of thrombotic microangiopathy from prior to ASP. All six developed severe thrombocytopenia (<20 x 10(-9)/L), worst 3 days after the bite and resolving over a week, MAHA with fragmented red blood cells on the blood film and five developed anuric ARF requiring dialysis and lasting 2-8 weeks. All six received antivenom, which was delayed compared with other brown snake-envenoming cases. Four were treated with plasmapheresis. The severity and recovery of the thrombocytopenia, anaemia and renal function were similar with and without plasmapheresis. The median length of stay for MAHA cases was 14 days (interquartile range (IQR) 12-14) compared to 1.8 days (IQR 1.3-2) for all other cases. CONCLUSION: Thrombotic microangiopathy resulting from brown snake bite appears to have a good prognosis and management should focus on early antivenom therapy and supportive care including dialysis. The role of plasmapheresis is yet to be defined.

Microenvironment around the essential cysteine residues in chicken liver fructose-1,6-bisphosphatase as analyzed by ESR spin labelling.

Chemical modification and electron spin resonance spectroscopy (ESR) spin-labelling techniques have been employed to investigate the local environment of the essential sulfhydryl groups of chicken liver fructose-1,6-bisphosphatase. The results demonstrate the presence of two distinct classes of sulfhydryl groups in this enzyme. The first class react preferentially with iodoacetate and its spin-labelled derivative, and this results in an increase in catalytic activity, while the second class react preferentially with N-ethylmaleimide and its spin-labelled derivative, and this leads to a decrease in catalytic activity. The ESR spectral data strongly suggest that the first class of sulfhydryl groups are located in a deep cleft of the enzyme molecule, while the second class of sulfhydryl groups are located in a shallow crevice. The environment of the second class of the sulfhydryl groups appears to undergo a significant change after the modification of the first class of sulfhydryl groups by iodoacetate.

Sequential changes in serum iron and ferritin in patients undergoing high-dose chemotherapy and radiation with autologous bone marrow transplantation: possible implications for treatment related toxicity.

In an effort to define the pattern of iron flux during high-dose chemotherapy or chemo/radiotherapy, we prospectively measured serum iron, iron binding capacity, and ferritin in patients undergoing autologous bone marrow transplantation for various malignancies. Sequential measurement of serum iron from days -7 to +12 was carried out in 88 evaluable patients, and simultaneous measurement of iron, ferritin, and total iron binding capacity was carried out in 32 patients. We found that there was a predictable rise in serum iron on day -2 or -3, and that this was accompanied by an increase in the saturation of transferrin. In addition, there was a similar increase in serum ferritin levels, which peaked by day +2. We suggest that the timing of this change in serum iron and saturation of transferrin may be important in mediating endothelial cell damage and, hence, organ toxicity in the setting of AuBMT. Based on these findings, we suggest that large clinical studies could be a source of patient samples to measure surrogate endpoints such as lipid peroxidation products (malondialdehyde or isoprostanes), or protein oxidation products following high-dose chemo/radiotherapy to determine the role of iron in cellular injury. It is possible that pharmacological manipulations to reduce free radical production or to chelate iron during the days prior to bone marrow reinfusion might help to reduce tissue injury in the setting of bone marrow transplantation.

Structure-activity relationships of pyridoxal phosphate derivatives as potent and selective antagonists of P2X1 receptors.

Novel analogues of the P2 receptor antagonist pyridoxal-5'-phosphate 6-azophenyl-2',5'-disulfonate (2) were synthesized and studied as antagonists in functional assays at recombinant rat P2X1, P2X2, and P2X3 receptors expressed in Xenopus oocytes (ion flux stimulation) and at turkey erythrocyte P2Y1 receptors (phospholipase C activation). Selected compounds were also evaluated as antagonists of ion flux and the opening of a large pore at the recombinant human P2X7 receptor. Modifications were made in the 4-aldehyde and 5'-phosphate groups of the pyridoxal moiety: i.e. a CH2OH group at the 4-position in pyridoxine was either condensed as a cyclic phosphate or phosphorylated separately to form a bisphosphate, which reduced potency at P2 receptors. 5-Methylphosphonate substitution, anticipated to increase stability to hydrolysis, preserved P2 receptor potency. At the 6-position, halo, carboxylate, sulfonate, and phosphonate variations made on the phenylazo ring modulated potency at P2 receptors. The p-carboxyphenylazo analogue, 4, of phosphate 2 displayed an IC50 value of 9 nM at recombinant P2X1 receptors and was 1300-, 16-, and > 10,000-fold selective for P2X1 versus P2X2, P2X3, and P2Y1 subtypes, respectively. The corresponding 5-methylphosphonate was equipotent at P2X1 receptors. The 5-methylphosphonate analogue containing a 6-[3,5-bis(methylphosphonate)]phenylazo moiety, 9, had IC50 values of 11 and 25 nM at recombinant P2X1 and P2X3 receptors, respectively. The analogue containing a phenylazo 4-phosphonate group, 11, was also very potent at both P2X1 and P2X3 receptors. However, the corresponding 2,5-disulfonate analogue, 10, was 28-fold selective for P2X1 versus P2X3 receptors. None of the analogues were more potent at P2X7 and P2Y1 receptors than 2, which acted in the micromolar range at these two subtypes.

Rapid, inexpensive quality control of fluorine-18 2-deoxy-2-fluoro-D-glucose preparations using the hexokinase reaction in vitro.

A rapid enzymatic method for determining the purity of 2FDG preparations has been devised. A small aliquot of the preparation is incubated with a hexokinase/adenosine triphosphate/Mg+2 mixture and passed through a Dowex 1 ion-exchange column, which retains the 2FDG-6-phosphate. Another aliquot, without prior incubation, is passed through an identical column and the 2FDG radioactivity is found in the eluant. The criteria for purity are quantitative retention of the 2FDG-6-phosphate on the column and no retention of 2FDG. Comparison of the HK method with thin layer and high performance liquid chromatography assays indicate that the HK method can serve as a rapid, simple and inexpensive alternative to these other methods. It can be used in a routine quality control program and may be easily adaptable to automated 2FDG synthetic methods.