A comparative study on basophil activation test, histamine release assay, and passive sensitization histamine release assay in the diagnosis of peanut allergy.

BACKGROUND: Allergy can be diagnosed using basophil tests. Several methods measuring basophil activation are available. This study aimed at comparing basophil activation test (BAT), histamine release assay (HR), and passive sensitization histamine release assay (passive HR) in the diagnosis of peanut allergy. METHODS: BAT, HR, and passive HR were performed on 11 peanut-allergic and 14 nonallergic subjects. Blood was incubated with peanut extract or anti-IgE and tests were performed as follows: BAT-CD63 upregulation was assessed by flow cytometry; HR-released histamine was quantified by a glass fiber-based fluorometric method; passive HR-IgE-stripped donor basophils were incubated with participants' serum and histamine release was quantified as HR. RESULTS: CDsens, a measure of basophil allergen sensitivity, was significantly higher for BAT (80.1±17.4) compared to HR (23.4±10.31) and passive HR (11.1±2.0). BAT, HR, and passive HR had a clinical sensitivity of 100%, 100%, and 82% and specificity of 100%, 100%, and 100%, respectively, when excluding inconclusive results. BAT identified 11 of 11 allergic patients, HR 10, and passive HR 9. Likewise, BAT recognized 12 of 14 nonallergic subjects, HR 10, and passive HR 13. However, the tests' diagnostic performances were not statistically different. Interestingly, nonreleasers in HR but not in BAT had lower basophil count compared to releasers (249 vs 630 counts/min). CONCLUSION: BAT displayed a significantly higher CDsens compared to HR and passive HR. The basophil tests' diagnostic performances were not significantly different. Still, BAT could diagnose subjects with low basophil number in contrast to HR.

Dynamics of plasma levels of specific IgE in chlorhexidine allergic patients with and without accidental re-exposure.

BACKGROUND: Chlorhexidine is an effective disinfectant, which may cause severe allergic reactions. Plasma level of specific IgE to chlorhexidine (ImmunoCAP(®) ) has high estimated sensitivity and specificity when measured within 6 months of allergic reaction, but knowledge of the dynamics over longer time periods is lacking and it is unknown whether levels fall below <0.35 kUA/L in patients with previously elevated levels. It is also unclear whether re-exposure influences levels of specific IgE. OBJECTIVE: To investigate the dynamics of specific IgE in chlorhexidine allergic patients with and without re-exposure. METHODS: All patients diagnosed with chlorhexidine allergy in the Danish Anaesthesia Allergy Centre January 1999 to March 2015 were invited to participate. The study included blood samples from the time of reaction and time of investigation and blood samples drawn prospectively over several years. RESULTS: Overall, 23 patients were included. Specific IgE within hours of reaction was available in eight patients and was >0.35 kUA/L in six of these. During allergy investigations, usually 2-4 months later, specific IgE was >0.35 kUA/L in 22 of 23 patients. In the following months/years specific IgE declined <0.35 kUA/L in 17 of 23 patients (most rapidly within 4 months). Nine re-exposures in the healthcare setting were reported by seven patients (35%). Most re-exposures caused symptoms and were followed by an increase in specific IgE. Two patients with specific IgE <0.35 kUA/L reacted upon re-exposure. CONCLUSIONS & CLINICAL RELEVANCE: Time from reaction should be considered when interpreting specific IgE results. Specific IgE is >0.35 kUA/L in most patients at time of reaction but should be repeated after a few weeks/months if negative. The optimal sampling time seems to be >1 month and <4 months. A value <0.35 kUA/L neither excludes allergy nor implies loss of reactivity in previously sensitized patients. Re-exposures are common, often iatrogenic, and can cause a rebound in specific IgE.

IgE-mediated basophil tumour necrosis factor alpha induces matrix metalloproteinase-9 from monocytes.

BACKGROUND: IgE-mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF-α), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase-9 (MMP-9). While mast cells and basophils have been shown to express complementary and partially overlapping roles, it is not clear whether a similar IgE/TNF-α/MMP-9 axis exists in the human basophil. The purpose of this study was thus to investigate whether IgE-mediated activation of human basophils induces TNF-α and MMP-9 release. METHODS: Human peripheral blood mononuclear cells (PBMC), isolated basophils and monocytes were stimulated up to 21 h with anti-IgE. Mediator releases were assessed by ELISA, and surface expressions of mediators were detected by flow cytometry. Upregulation of cytokine production was detected by Western blot and polymerase chain reaction (PCR). RESULTS: IgE-mediated activation of basophils induced the synthesis and release of both TNF-α and MMP-9 from PBMC. In contrast, IgE-mediated activation of purified basophils induced the release and cellular expression of TNF-α but not MMP-9. Isolated monocytes did not release MMP-9 upon anti-IgE stimulation, but MMP-9 release was induced by stimulating monocytes with supernatants from activated basophils, and this release was inhibited by anti-TNF-α neutralizing antibodies. CONCLUSION: Our results strongly indicate that human basophils release TNF-α following IgE-dependent activation and that this cytokine subsequently stimulates MMP-9 release from monocytes. These findings support a direct involvement of basophils in inflammation as well as suggesting a role for the basophil in tissue remodelling.

Authentication of food allergen quality by physicochemical and immunological methods.

Purified allergens are required to detect cross-contamination with other allergenic foods and to understand allergen interaction with other components of the food matrix. Pure allergens are also used for the diagnosis and treatment of food allergies. For example, serological methods are being developed to improve the quality of diagnosis, and to reduce the need for food challenge tests. In addition, recombinant allergens are being evaluated as candidate vaccines for safe and efficacious specific immunotherapy. Pure allergens are indispensable as reference materials for the calibration and standardization of methods between different laboratories and operators for risk assessment in the food industry. Therefore, there is a need for well-defined purified food allergens. In this context, a panel of 46 food allergens from plant and animal sources has been purified, from either the food sources or as recombinant forms, within the EU-funded EuroPrevall project. These allergens have been characterized by a battery of diagnostic tests demonstrating that they constitute an authentic, well-defined library of comparable quality. The review summarizes the applications, potentials and limitations of key techniques used for the characterization and authentication of these allergen preparations, with a special emphasis on protein purity and identity, folding, post-translational modifications and immunochemical properties. One key area identified is the development of powerful analytical techniques, such as mass spectrometry and nuclear magnetic resonance, to improve the authentication of allergens for routine applications in allergy management.

Pharmacological targeting of the KIT growth factor receptor: a therapeutic consideration for mast cell disorders.

KIT is a member of the tyrosine kinase family of growth factor receptors which is expressed on a variety of haematopoietic cells including mast cells. Stem cell factor (SCF)-dependent activation of KIT is critical for mast cell homeostasis and function. However, when KIT is inappropriately activated, accumulation of mast cells in tissues results in mastocytosis. Such dysregulated KIT activation is a manifestation of specific activating point mutations within KIT, with the human D816V mutation considered as a hallmark of human systemic mastocytosis. A number of other activating mutations in KIT have recently been identified and these mutations may also contribute to aberrant mast cell growth. In addition to its role in mast cell growth, differentiation and survival, localized concentration gradients of SCF may control the targeting of mast cells to specific tissues and, once resident within these tissues, mast cell activation by antigen may also be amplified by SCF. Thus, KIT inhibitors may have potential application in multiple conditions linked to mast cells including systemic mastocytosis, anaphylaxis, and asthma. In this review, we discuss the role of KIT in the context of mast cells in these disease states and how recent advances in the development of inhibitors of KIT activity and function may offer novel therapies for the treatment of these disorders.

Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

BACKGROUND: Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells. However, a relationship between the AS status and FcepsilonRI has not been investigated. We aimed to characterize basophils from AS by looking at histamine release (HR) (sensitivity and reactivity) and the FcepsilonRI molecule, and compare it with nonatopic (NA) or allergic (A) persons. METHODS: Blood was obtained from NA (n = 14), grass and/or birch A persons (n = 17) and mono-sensitized grass or birch pollen AS (n = 12). The basophil sensitivity and reactivity were examined by anti-IgE triggered HR. Surface expression of FcepsilonRI and IgE were measured by flow cytometry, FcepsilonRIalpha protein was identified using a radioimmunoassay and Western blot. mRNA coding for the classic FcepsilonRIbeta-chain and the truncated form (FcepsilonRIbetaT) were determined by real-time PCR. RESULTS: The AS group was less reactive than NA or A persons when triggered by anti-IgE and had a significant higher number of nonresponders. However, there was no difference in sensitivity among the three groups and furthermore; the groups did not vary in FcepsilonRI- and IgE-surface expression, FcepsilonRIalpha-protein level or beta/betaT ratio. CONCLUSION: Basophils from AS persons are less reactive and include more nonresponders than basophils from NA and A persons, but do not differ regarding the FcepsilonRI molecule.

A comparative study of the FcepsilonRI molecule on human mast cell and basophil cell lines.

BACKGROUND: Mast cells and basophils express the high-affinity IgE receptor FcepsilonRI. We have analysed the human mast cell line LAD2 and four subclones of the basophil cell line KU812 in order to reveal possible differences concerning the FcepsilonRI surface regulation, anti-IgE-triggered activation, FcepsilonRIalpha protein stability and the mRNA level of FcepsilonRIalpha-, beta- and the truncated beta-chain (beta(T)), and thereby determine the utility of these cell lines in investigations of the FcepsilonRI biology. METHODS: The surface expression of FcepsilonRI was assessed by flow cytometry, using the monoclonal antibody CRA1. The FcepsilonRI-induced cellular activation (i.e. cross-linking of FcepsilonRI) was determined by changes in the intracellular level of Ca2+, which was measured by fluorescence of Fura-2. The level of the FcepsilonRIalpha protein was determined by a Western blot technique and by a radioimmunoassay. The mRNA level of FcepsilonRIalpha, beta- and beta(T)-chain was analysed using real-time PCR. RESULTS: Two KU812 subclones and especially LAD2 had FcepsilonRI surface expression which was capable of inducing cellular activation. Both the FcepsilonRI expression and stability of the FcepsilonRIalpha protein were increased when IgE was present. All the cell lines expressed mRNA of FcepsilonRIalpha-, beta- and beta(T), with LAD2 tending to have the highest expression. However, a determination of the beta/beta(T) ratio demonstrated no difference between any of the cell clones. CONCLUSION: These cell lines are important tools in the investigation of both the FcepsilonRI molecule and the effects induced by its activation.

Monomeric immunoglobulin E stabilizes FcepsilonRIalpha from the human basophil cell line KU812 by protecting it from natural turnover.

BACKGROUND: The high affinity IgE receptor (FcepsilonRI) on mast cells and basophils is up-regulated by its own ligand IgE; however, the mechanism is unknown. OBJECTIVE: To study the IgE-mediated effect on FcepsilonRI on basophils by using the human basophilic cell line KU812. METHODS: Expression of cell surface FcepsilonRI was assessed by flow cytometry. Western blot technique was used to illustrate tyrosine-phosphorylation and the Ca2+ level in KU812 was measured by fluorescence of Fura-2. Soluble specimens of the alpha-chain from FcepsilonRI (FcepsilonRIalpha) were obtained by lysing 107 KU812 pr. mL. FcepsilonRIalpha was detected by a sandwich immunoradiometric assay employing the IgE-binding capacity of FcepsilonRIalpha in conjunction with a monoclonal antibody. Polyclonal rabbit anti-FcepsilonRIalpha was used for detection of FcepsilonRIalpha by Western blotting. RESULTS: We found that monomeric IgE did not induce tyrosine-phosphorylation in KU812, which was the case when stimulating with IgE cross-linked by anti-IgE binding. Further, only cross-linking of IgE, but not monomeric IgE, increased the Ca2+ level. Using the immunoradiometric assay, we found a temperature dependent reduction in the amount of FcepsilonRIalpha. Samples incubated at 37 degrees C for 5 h displayed a 16-fold decrease in the FcepsilonRIalpha level compared with samples incubated at 4 degrees C. In the presence of IgE the reduction at 37 degrees C was only threefold. CONCLUSION: These results indicate that IgE does not induce intracellular signals in KU812, i.e., tyrosine-phosphorylation or Ca2+ release. Instead it appears that FcepsilonRIalpha is an unstable protein that IgE stabilizes and thereby protects from a temperature dependent turnover.

[Study of selected methods of self-assessment of health, quality of life and satisfaction with treatment. Use among patients four weeks after discharge from a psychiatric ward]. / Undersøgelse af udvalgte metoder til selvvurdering af helbred, livskvalitet og tilfredshed med behandling. Anvendelse til patienter fire uger efter udskrivning fra psykiatrisk indlaeggelse.

INTRODUCTION: The purpose of the investigation was to test selected methods for self-evaluation of health, the quality of life, and satisfaction with treatment. As part of this, to test their fitness as quality estimates in a psychiatric ward. MATERIAL AND METHODS: Four weeks after discharge questionnaires were sent to all patients and they were asked to participate. The methods used were SF-36 (short-form-36 questions), SIP (sickness impact profile), WHO-5, and a scale measuring patient satisfaction with treatment and care. RESULTS: The response rate was almost 50%. Seventy-five per cent were feeling better or the same as they did one year before admission. Compared with average values in a healthy Danish population, the psychic health of the patients was much lower, as evaluated by the SF-36 and WHO-5. The functional level measured by the SIP was reduced 12-32% from the optimum. About 75% of the patients rated the treatment and care positively. DISCUSSION: The applied methods of self-evaluation of health, quality of life, and satisfaction with treatment were found to be appropriate. For one year, it was possible to obtain and register important data from participating patients without using too many resources. Nevertheless, only 50% answered the questionnaire. It would be desirable to simplify the necessary questionnaires, thus encouraging more patients to respond. From the agreement between the methods, the use of the SF-36, WHO-5, and the satisfaction scale would be sufficient to obtain a qualified measure of the patients' health, quality of life, and satisfaction with treatment and care. Time spent on answering could be reduced to about 20 minutes. Resources spent on registration would be so small that the methods could be used for on-line quality surveillance.

Chronic wound assessment.

Chronic wounds are a drain on health care resources, and as such, continue to challenge health care providers to define and create more effective intervention strategies. Wound assessment is the foundation for maintaining and evaluating a therapeutic plan of care. Without adequate baseline wound assessment and valid interpretation of the assessment data, the plan of care for the wound may be inappropriate or ineffective. This article discusses comprehensive assessment of the total patient as well as assessment of wound severity and wound status.

[Surgical treatment of cancer of the esophagus and cardia]. / Kirurgisk behandling af cancer esophagi og cancer cardiae.

The purpose of the present study was to evaluate the results following surgical resection for cancer of the gastro-oesophageal junction. From 1. january 1988 to 1. april 1996 radical resection was intended in 107 patients at the Department of Thoracic and Cardiovascular Surgery at Odense University Hospital. Resection was possible in 75 patients. The operative mortality was 6.7% insufficiency of the gastro-oesophageal anastomosis was found in 6.7%. Five year survival was 24.1%. However in 52 patients where the resection was found to be radical the five-year survival was 35.3%. The results show that oesophago-gastrectomy could be performed with low mortality and morbidity. Long term survival is still low. To improve the results efforts should be directed toward earlier diagnosis, better selection and minimising post-operative complications.

Multiple anomalies, hypokalaemic paralysis and partial symptomatic relief by terbutaline.

In this paper a follow-up is presented of a case report initially described by Andersen in 1971. The patient presented with a syndrome including elements of familial periodic paralysis with hypokalaemia, long QT syndrome, ventricular ectopy, myopathy with fibre-type disproportion and dysmorphic features resembling Treacher Collins' syndrome. The main symptom was hypokalaemic paralysis. The episodes were accompanied by a lowered intracellular potassium content and an increase in intracellular sodium. Treatment with terbutaline, a Na/K-ATPase-stimulating drug, resulted in attack-free periods of approximately 9 months, after which the attacks reoccurred. The patient suffered severe attacks whenever treatment with terbutaline was stopped. The patient experienced two attacks of respiratory arrest, the second being fatal.

The Pressure Sore Status Tool a few thousand assessments later.

Although the Pressure Sore Status Tool (PSST) was developed using expert panelists and its reliability has been previously reported, the patterns that seem to be forming based on the first few thousand PSST assessments have not been reported yet. This paper will focus on analyses of a subset of PSST assessments that were collected as a by-product of testing a computerized assessment and decision support system. Changes in wound characteristics that appear to serve as "lead indicators" of healing, preliminary factor analysis data, and a correlation of total PSST scores with recorded stage will be discussed. Due to the sample size and variability within the data, it is premature to draw firm conclusions from the data. However, analyses of the first few thousand assessments and over 100 healed pressure ulcers show promise in confirming some prior speculations about wound healing. The data also appear to suggest the possibility of surprises that are not necessarily part of current understanding of wound healing in pressure ulcers.

Magnesium substitution and postoperative arrhythmias in patients undergoing coronary artery bypass grafting.

Sixty coronary artery bypass grafting patients were randomized to receive either magnesium sulphate or placebo for 4 days postoperatively. The magnesium substitution reduced the duration of atrial fibrillation or flutter (p < 0.05), but not the number of patients developing these arrhythmias. The number of ventricular ectopic beats was also reduced among patients receiving magnesium sulphate compared to placebo (p < 0.05). To evaluate whether the anti-arrhythmic effect of magnesium sulphate was explained by a faster resumption of cellular potassium postoperatively, skeletal muscle electrolyte concentrations were measured pre-operatively and on the third day postoperatively. No significant difference was found in skeletal muscle potassium or magnesium contents on the third day postoperatively when comparing the two groups. The serum magnesium level declined postoperatively in the placebo group, whereas an increase was found in patients receiving magnesium sulphate. We suggest magnesium substitution as a routine postoperatively, because this treatment seems to reduce the severity of postoperative arrhythmias.

Wound care nurses' judgements on healing time in chronic wounds.

With the current push to develop critical pathways for chronic wound patients, information on "normal" versus problematic wound healing is important. This descriptive study begins to define chronic wound healing time markers by examining clinicians' own healing expectations with pressure ulcers. A 30-item questionnaire concerning partial and full thickness pressure ulcers on the sacrum, trochanter and heel was sent to 272 wound/ET nurses. Total response rate was 32.3% with 74 analyzable questionnaires returned. Data was analyzed using descriptive statistics and correlations where appropriate. Results were: (1) The longer respondents thought it would take for wounds to heal, the more variable their responses. (2) Respondents viewed heel wounds as taking the longest to heal and were more certain about time to healing in sacral wounds. (3) Responses indicated longer healing times than those shown in recent studies looking at change in wound surface area. (4) Results on time of 3.2 weeks to autolytic debridement of wounds with black eschar does not differ greatly from published time for enzymatic debridement, in spite of guidelines stating that autolysis may take longer than other methods. This study demonstrates that there remains variability in wound nurses' perceptions of time to healing and, therefore, recent attempts at defining critical pathways may be premature.

Postoperative arrhythmias and myocardial electrolytes in patients undergoing coronary artery bypass grafting.

Electrolyte changes in right atrial and skeletal muscle pre- intra- and postoperatively, and their relationship to the development of postoperative atrial fibrillation or flutter were evaluated in 31 patients with coronary artery bypass grafting (CABG). Such postoperative arrhythmias occurred in 14 patients (45%). Before CABG the skeletal muscle potassium concentration was lower in these patients than in the others: median 261.4 (range 148.2-329.5) vs 298.6 (167.1-416.4) mumol/g dry weight, p = 0.017. The right atrial potassium concentration was normal, but sodium levels were higher in the patients with, than in those without postoperative arrhythmias: median 340.3 (263.7-454.9) vs 296.3 (203.9-355.0) mumol/g dry weight, p = 0.008, indicating disturbed transmembrane electrolyte transfer. During CABG the potassium levels fell and sodium increased in both right atrium and skeletal muscle, and on postoperative day 2 the potassium content in skeletal muscle was not yet restored. Magnesium levels showed no changes in right atrium or skeletal muscle, but serum magnesium declined postoperatively. As the observed electrolyte derangements may be important in the development of postoperative arrhythmias, concomitant potassium and magnesium supplement postoperatively may be beneficial in restoring cellular potassium concentration.

Toward an intelligent wound assessment system.

There is general agreement regarding the need for pressure ulcer assessment methodology which more discretely reflects relevant aspects of wound status than does the commonly used staging system. The Pressure Sore Status Tool (PSST) is one such instrument which was developed with consensual expert input. While the psychometric properties of the PSST have been reported in the literature, the instrument was validated using ET nurses, highly trained wound care specialists, and existed only in manual form. This paper reports results from attempts to establish reliability estimates for healthcare practitioners without extraordinary wound care training or experience. The paper further describes the automation of the PSST and provides examples of pressure ulcer profiles tracked over time. Results indicate that inter-rater reliability with general healthcare practitioners was .78 and intra-rater reliability was .89. The practitioners were able to use the PSST for over six months and the automated system allowed analysis of wound healing profiles that would have been difficult using a manual system. These results imply that movement toward an automated system which makes discriminations regarding the effects of various treatment and intervention strategies is possible and practical.

Glucose content in human skin: relationship with blood glucose levels.

In order to ascertain the dynamic relationship between the extracellular glucose in upper skin layers and blood glucose, skin suction blisters were raised in six Type 1 diabetic patients during a three-step glucose clamp. Blister glucose closely paralleled venous glucose (mean of r = 0.998). However, in three patients blister glucose was constantly lower than plasma glucose and this appeared to be related to their slower formation of skin blisters. A substantial difference in skin blister suction time was noted among patients and it was found that suction time was linearly correlated to glycosylated haemoglobin (HbA1C) (n = 6, r = 0.865, p = 0.026). It is concluded that a non-invasive blood glucose monitoring system could be successfully based on measurement of alterations in skin glucose contents.