The -2549 -2567 del18 Polymorphism in VEGF and Irreversible Bronchoconstriction in Asthmatics.

BACKGROUND: While the importance of vascular endothelial growth factor (VEGF) in the pathogenesis of several diseases (eg, neoplasms) has been proven, its role in asthma, especially in terms of the potential associations between genetic variants of VEGF and airway remodeling, has received relatively little attention. Objectives: This study aimed to evaluate the possible connection between a genetic factor, ie, the polymorphism del/ins in the VEGF promoter region, and airway remodeling potential in asthmatics with and without irreversible bronchoconstriction. MATERIAL AND METHODS: The study population comprised 82 patients with asthma (of whom 42 had irreversible bronchoconstriction) and a group of 40 controls. DNA was isolated from peripheral blood leukocytes. Polymerase chain reaction was used to type the VEGF (18-bp deletion/insertion) gene polymorphism at loci -2549 -2567. Other factors (ie, smoking, disease duration) were also taken into consideration. RESULTS: The del/del genotype was found in 74.39% of patients with asthma (P=.031; OR=2.38), 80.95% of patients with irreversible bronchoconstriction (P=.012; OR=3.48), and 67.5% patients with reversible bronchoconstriction (P=.251; OR=1.70). The proportion of smokers to nonsmokers was higher (P=.032) and disease duration was longer (P=.041) in patients with irreversible bronchoconstriction than in those with reversible bronchoconstriction. CONCLUSIONS: Our results showed that the risk of irreversible bronchoconstriction in asthmatics was associated with the presence of the del18 genotype at the -2549 -2567 position in the promoter region of the VEGF gene, as were disease duration and other factors such as smoking.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome: a multicenter study. II. Basophil activation by nonsteroidal anti-inflammatory drugs and its impact on pathogenesis.

BACKGROUND: Patients who are clinically hypersensitive to nonsteroidal anti-inflammatory drugs (NSAIDs) sometimes present basophil activation in vitro, and in 50% of cases a parallel response to release of sulfidoleukotrienes (cellular allergen stimulation test) is observed. These phenomena occur not only in clinically hypersensitive patients, but also in some healthy controls who tolerate NSAIDs. MATERIAL AND METHODS: We studied 16 clinically hypersensitive patients, 22 controls tolerating NSAIDs, and 29 healthy blood donors (clinical NSAID status unknown) using 2 different basophil isolation techniques (buffy coat or plasma leukocytes). RESULTS: In a population of 13 aspirin-tolerant healthy controls and 29 healthy blood donors, basophil activation with aspirin, diclofenac, and naproxen was analyzed at 4 different concentrations. The results in the 2 groups were quite similar in qualitative terms. Choosing a cutoff of 5% and a stimulation index >2, the proportion of positive results increased with the concentration. There were more positive results at all concentrations using the plasma leukocyte technique. CONCLUSIONS: The most important finding of this study is that basophil activation by NSAIDs occurs not only in clinically hypersensitive patients but also, to a very variable extent and on an individual basis, in apparently normal healthy individuals who tolerate NSAIDs. The phenomenon is clearly dose-related, and hypersensitive patients seem to react to lower NSAID concentrations.

A new variant of the basophil activation test for allergen-induced basophil CD63 upregulation. The effect of cetirizine.

OBJECTIVE: The aim of our study was to determine the diagnostic usefulness of a newly developed basophil activation test (BAT) in patients allergic to Dermatophagoides pteronyssinus and pollens. We also analyzed the influence of cetirizine on CD63 upregulation. This popular antihistamine strongly inhibits skin tests, but its impact on BAT sensitivity remains unknown and deserves at least preliminary determination. METHODS: The study sample comprised 22 patients allergic to house dust mite and pollens and 19 healthy controls. All participants underwent skin prick testing and the newly developed flow-cytometric basophil activation test. The protocol for allergen-induced basophil CD63 upregulation consisted of whole blood samples that were processed and stained with anti-CCR3/CD63 antibodies added to the buffer at the beginning of stimulation. Skin prick tests and BAT were performed twice--before and 2 hours after ingestion of 10 mg of cetirizine. RESULTS: The new BAT is characterized by its short processing time, easy basophil gating, and strong CD63 upregulation with very high sensitivity and excellent specificity. Our results suggest that allergen-induced CD63 upregulation by higher doses of allergens is not inhibited 2 hours after administration of cetirizine (unlike skin prick tests). CONCLUSION: The BAT is a very useful and precise method for the diagnosis of allergy to aeroallergens. It is not influenced by cetirizine.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome. A multicenter study. I. Clinical findings and in vitro diagnosis.

BACKGROUND: We present the results obtained from the largest series of in vitro diagnostic tests ever reported in patients with clinically validated hypersensitivity to acetylsalicylic acid (ASA)/nonsteroidal anti-inflammatory drugs (NSAID) compared with various categories of controls tolerating ASA/NSAIDs. This multicenter study, which was performed within the framework of the European Network for Drug Allergy (ENDA) group, showed that the basophil activation test (BAT), particularly when used with the 3 NSAIDs aspirin (ASA), diclofenac (DIC), and naproxen (NAP), allows us to confirm the diagnosis of NSAID hypersensitivity syndrome. The results of the cellular allergen stimulation test (CAST) frequently correlate with those of the BAT, although not always. An unexpected finding was that basophil activation by NSAIDs is not an all-or-nothing phenomenon restricted to clinically hypersensitive patients, but that it also occurs in a dose-related manner in some NSAID-tolerant control individuals.Therefore, NSAID hypersensitivity appears as a shift in the normal pharmacological response to NSAIDs. These findings allow us to formulate a new rational hypothesis about the mechanism of NSAID hypersensitivity syndrome, a mechanism that most authors continue to describe as "unknown." METHODS: We enrolled 152 patients with a history of hypersensitivity to NSAIDs and 136 control participants in 11 different centers between spring 2003 and spring 2006. Flowcytometric BAT was performed. RESULTS: The most noteworthy results of our study were that 57% of 140 patients presented very clear-cut positive BAT results to multiple NSAIDs, and 16% were entirely negative. In about 27% of cases, positive results were obtained with 1 or 2 concentrations of a single NSAID. There is clearly a correlation between the results of BAT and CAST. CONCLUSIONS: BAT seems particularly indicated in patients with a clinical history of NSAID intolerance, and in whom a provocation test is not advisable for ethical, clinical, or other reasons. Clear-cut positive results can be considered as confirming a history of NSAID hypersensitivity, although negative results may not exclude it.

Food-dependent exercise-induced anaphylaxis: possible impact of increased basophil histamine releasability in hyperosmolar conditions.

We present a case of anaphylactic shock induced by exercise following celery ingestion. The possible mechanism of food-dependent exercise-induced anaphylaxis (FDEIA) and the laboratory tests for its diagnosis are discussed. We evaluated spontaneous, celery-allergen-induced, and anti-FcepsilonRI-antibody-induced histamine release from basophils obtained from the patient, 2 celery-allergic controls, and 3 healthy controls. Buffers of increasing osmolarity were used to mimic conditions of vigorous physical exercise. Only the patient's basophils showed an increase in spontaneous, anti-FcepsilonRI antibody-induced and allergen-induced histamine release under physiological conditions and with slightly increased medium osmolarity. To our knowledge, this is the first report on the possible role of increased histamine releasability in the pathogenic mechanism of FDEIA. We suggest that FDEIA results from increased histamine releasability triggered by physical effort after exposure to a sensitizing food allergen.

Immunosuppressive Treatment and Its Effect on the Occurrence of Pneumocystis jiroveci, Mycoplasma pneumoniae, Chlamydophila pnemoniae, and Legionella pneumophila Infections/Colonizations Among Lung Transplant Recipients.

BACKGROUND: The aim of the study was to assess the frequency of infections caused by Pneumocystis jiroveci, Chlamydophila pneumoniae, Legionella pneumophila, and Mycoplasma pneumoniae among lung transplant recipients in the context of immunosuppression. METHODS: The study group consisted of 94 patients (37 women and 57 men; mean age 42.03 years) transplanted between 2009 and 2016 at the Silesia Center for Heart Diseases (SCCS). Immunosuppressive treatment (induction and maintenance therapy) was assessed. The immunofluorescence methods were used to detect the P. jiroveci, L. pneumophila, C. pneumoniae, and M. pneumoniae antigens in samples obtained from the respiratory tract. RESULTS: Thirty-two of 94 graft recipients developed atypical or opportunistic infection. The median time of its occurrence was 178 days after transplantation. P. jiroveci was responsible for 84.38% of first infections. Five patients developed infection with P. jiroveci and C. pneumoniae. None of the infections occurred during induction of immunosuppression. An opportunistic or atypical infection developed in 19.35% of the patients treated with a tacrolimus-based regimen, and in 43.33% of patients on a cyclosporine-based regimen. CONCLUSION: Infection with P. jiroveci is a recognized problem after lung transplantation and should be monitored. The percentage of infected patients is higher in patients treated with a cyclosporine-based regimen in comparison to those treated with tacrolimus.

Bacterial Infections During Hospital Stay and Their Impact on Mortality After Lung Transplantation: A Single-Center Study.

INTRODUCTION: The aim of the study was to assess the impact of bacterial infection during hospital stay on long-term follow-up. MATERIALS AND METHODS: This was a retrospective single-center study of 97 recipients of lung transplantations performed between December 2004 and June 2016 at a single center. Information about age, sex, underlying lung disease, and date and type of procedure was gathered from patients' charts. Immunosuppressive treatment has been analyzed individually among the cohort. Microbiological evaluation included the presence of infection, bacterial species in recipients and donors, as well as type of biological material. RESULTS: During a mean hospitalization time of 57 days (range 4-398 days), 67 patients (69%) were diagnosed with bacterial infection. There were 120 episodes of infection caused by 32 species of bacteria. The most common were Pseudomonas aeruginosa (27%), Acinetobacter baumanii (21%), Klebsiella pneumoniae (10%) and Stenotrophomonas maltophilia (11%). Analysis revealed that 39 patients developed bronchiolitis obliterans syndrome (43%). Patients with A baumanii had a lower probability of survival than the rest of the population (P < .05). Patients treated with mammalian target of rapamycin inhibitors had a higher probability of survival. CONCLUSIONS: Infection with A baumanii affects lung transplant recipients' survival. Incorporating sirolimus could be beneficial for the lung transplant recipients' survival.

Production of leukotriene C4 by peripheral blood leukocytes stimulated with anti-fcepsilonRI antibody, PMA, and fMLP does not correlate with irreversible airway obstruction in asthmatics.

BACKGROUND: Airway remodeling has recently emerged as a major problem in an increasing percentage of patients with asthma. Reasons for great diversity in the progression of irreversible bronchoconstriction among asthmatics remain unclear. OBJECTIVE: The aim of this study was to assess whether the potential ability of leukocytes to produce cysteinyl leukotrienes in response to various stimuli is correlated with magnitude of irreversible airway obstruction in asthmatics. MATERIALS AND METHODS: The study sample comprised 76 asthmatics (34 males, mean +/- SD age 52 +/- 13 years), and 35 healthy controls (18 males, 38.2 +/- 15 years). Each subject underwent 2 pulmonary function tests: before and after bronchodilator administration. In addition, approximate annual decline in forced expiratory volume in 1 second (FEV1) (% of predicted) was calculated. Leukotriene C4 (LTC4) production was assessed combining a cellular antigen stimulation test and enzyme-linked immunosorbent assay using Bulhmann Laboratories AG kits. Leukocytes isolated from peripheral blood were stimulated with anti-FcepsilonRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). In separate tubes each subject's leukocytes were tested for spontaneous LTC4 production. Finally, stimulated LTC4 production was expressed in pg/mL after subtraction of values of spontaneous production. RESULTS: In asthmatics, baseline FVC% and FEV% values ranged from 24.4% to 122.4% (mean, 75.5%) and from 23.4% to 126.6% (mean, 74.4%), respectively. There were no significant differences between asthmatics and controls in LTC4 production stimulated by anti-FcepsilonRI antibody (P = .79), fMLP (P = .33) or PMA (P = .86). We found no correlation between stimulated LTC4 production and spirometric parameters at baseline or after bronchodilator administration or annual decline in FEV1%. CONCLUSION: Our results do not confirm the hypothesis that airway remodeling in asthma might be related to enhanced ability of leukocytes to produce cysteinyl leukotrienes in response to various stimuli.

Eosinophil cationic protein in serum of corticosteroid-sensitive and resistant bronchial asthma.

Theories for the inflammatory basis of bronchial asthma are presented. The phenomenon of corticosteroid resistance (CR) in bronchial asthma is also discussed. Resistance to corticosteroids, which occurs in about 5% patients with moderate and severe asthma, presents still an important diagnostic and therapeutical problem. In addition, present opinions on the role of eosinophils in the allergic bronchial inflammation were analyzed. The aim of this study was to monitor serum eosinophil cationic protein (ECP) level in asthma patients, sensitive and resistant to glucocorticosteroids (GCS), before and after prednisolone treatment. The resistance to steroids was determined, based on the oral prednisolone test according to Carmichael and vasoconstriction assay according to Stoughton and McKenzie. In the group of corticosteroid-sensitive (CS) asthmatic patients a statistically significant decrease of ECP level was observed, after 10 day administration of prednisolone in a daily dose of 20 mg, which was associated with a meaningful increase of FEV1 value. On the other hand, the level of ECP in the serum of patients resistant to corticosteroids, although also decreased under influence of prednisolone, was not correlated with the increase of FEV1 value.

Effect of recombinant IFN-gamma on igE-dependent leukotriene generation by peripheral blood leukocytes in patients with pollinosis and asthma.

Interferon gamma (IFN-gamma) is considered one of the causative and intensifying factors in inflammation. The reaction to allergens releases IFN-gamma, an immunomodulatory cytokine known to inhibit IgE synthesis and Th cell proliferation. The aim of the study was to evaluate the influence of IFN-gamma on leukotriene (LT) release in vitro, from human leukocytes of atopic patients with pollinosis and asthma. Thirty-eight patients were enrolled in the study: 15 with pollinosis and 23 asthmatics. In the presence of IL-3, leukocytes were stimulated with specific allergens. Other samples of leukocytes were preincubated with different concentrations of IFN-gamma for 15 min before allergen stimulation. The concentration of LT in supernatants was measured according to the CAST-ELISA procedure. We stated that IFN-gamma had significantly diminished LT release in a dose-dependent mode from the leukocytes of pollinotics. IFN-gamma did not change LT release in the asthmatic group, although, in leukocytes the small and medium basic production of LT, IFN-gamma caused a statistically significant fall in LT generation.

Effect of hydrocortisone treatment on oxygen metabolism and phagocytosis of peripheral blood neutrophils in asthmatic patients, sensitive and resistant to glucocorticosteroid therapy.

The inflammatory activity of peripheral blood neutrophils of patients suffering from atopic asthma and treated with glucocorticosteroids was investigated. Two tests were performed: 1. chemiluminescence test of neutrophils, and 2. phagocytosis test of latex and bacteria (Staphylococcus aureus 209 P) by neutrophils before and after incubation of the cells with hydrocortisone. The tests were carried out in 3 selected groups of (1) asthmatic steroid sensitive patients, (2) asthmatic steroid resistant patients, and (3) healthy individuals. The neutrophil chemiluminescence stimulation index was found to be significantly lower in the asthmatic steroid sensitive group as compared to the other groups. The index increased in this group after neutrophil incubation with hydrocortisone. The results of phagocytosis of latex by neutrophils before and after their incubation with hydrocortisone were similar in all the 3 groups. The results of phagocytosis of bacteria by neutrophils in the asthmatic steroid sensitive group were significantly lower after incubation of neutrophils with hydrocortisone only.

Bacterial lipopolysaccharide-induced sulfidoleukotriene release from peripheral blood leukocytes in patients with asthma and chronic obstructive pulmonary disease.

Bacterial endotoxins are seen to possess strong proinflammatory activities. These substances may intensify inflammation in the airways of patients with chronic obstructive pulmonary disease (COPD) and asthma by facilitating release of various mediators from different types of cells. Sulfidoleukotrienes (sLT) cause bronchoconstriction, increase vascular permeability and stimulate mucous secretion. The aim of our study was to evaluate sLT release from peripheral blood leukocytes stimulated by Klebsiella pneumoniae lipopolysaccharide (LPS) and obtained from COPD and asthma patients. Nineteen subjects with mild or moderate stable bronchial asthma, nine patients with COPD and 10 healthy controls entered the study. Cellular allergen stimulation test (CAST)-ELISA test was performed using Bühlmann Laboratories AG kits to determine sLT production. The differences between atopic (462.57 SD = 215.89 pg/ml) and nonatopic (474.25 SD = 158.02 pg/ml) asthmatics in comparison to healthy controls (191.55 SD = 53.2 pg/ml) were statistically significant (p < 0.005) upon LPS stimulation at the concentration of 10 micrograms/ml. At lower LPS concentration (1 microgram/ml) the difference was statistically significant only between nonatopic asthmatics and healthy subjects (p < 0.02). In the COPD group the sLT production in either LPS concentration was higher than in the controls but the difference was not significant. We suppose that leukocytes obtained from asthmatics and COPD patients are more susceptible to LPS than these cells from healthy individuals. An increased sLT production upon LPS stimulation during respiratory bacterial infection may intensify inflammation, bronchoconstriction and increase nonspecific bronchial hyperreactivity.

Comparative study between skin prick tests and TOP-CAST allergen leukocyte stimulation in diagnosis of allergic status.

A recently developed CAST-ELISA method was applied to determine allergen-induced leukocyte stimulation. This method is based on the measurement of sulfidoleukotriene secretion by peripheral blood leukocytes previously stimulated with specific allergen in the presence of interleukin 3. 24 patients allergic to different aeroallergens and some food allergens were included in this study. Subjects received no medication 2 weeks before the testing. Leukocytes were isolated by dextran sedimentation. After incubation with allergen at two concentrations, the cells were centrifuged and the concentration of sulfidoleukotrienes in supernatant was determined. The results were expressed in pg/ml after subtraction of values of spontaneous sulfidoleukotriene production in portions incubated without allergen. We observed a wide range of sulfidoleukotriene secretion upon allergen stimulation. Concentrations of leukotrienes ranged from 0 to 5780 pg/ml at lower allergen concentration and from 210 to 5680 at higher allergen concentration. On the basis of observed results, we conclude that the better allergen concentration is the higher one because there was no appearance of negative cell stimulation. In the lower allergen concentration we observed negative results in two cases. Sixteen healthy control subjects were also included in this study. Eleven subjects had negative skin prick tests (SPT) as well as CAST results. In 5 healthy subjects with positive SPT, we also observed positive CAST results in 4 persons. In 1 healthy person with positive SPT results, CAST results were negative. We conclude that TOP-CAST allergen is a valuable mixture of different aero and food allergens for determining the allergic status in patients with suspicious allergic status, although it cannot differentiate between health and disease states.

CAST-ELISA test in diagnosis of mite allergy.

The recently developed CAST-ELISA method was applied to determine allergen-induced leukocyte stimulation. This method is based on the measurement of sulfidoleukotriene secretion by peripheral blood leukocytes stimulated with specific allergen in the presence of interleukin 3.23 patients allergic to Dermatophagoides pteronyssinus were included in this study. All subjects received no medication 2 weeks before the testing. Leukocytes were isolated by dextran sedimentation. After incubation with allergen the cells were centrifuged and the concentration of sulfidoleukotrienes in supernatant was determined. The results were expressed in pg/ml after subtracting the values of spontaneous sulfidoleukotriene production in portions incubated without allergen. We observed a wide range of sulfidoleukotriene secretion upon allergen stimulation. Concentrations of leukotrienes ranged from 0 to 3440 pg/ml. The mean level in the whole group was 1521.3 pg/ml at the optimal allergen concentration. Positive results were observed in 21 subjects. In 2 subjects we could not demonstrate leukotriene secretion by stimulation with allergen. 15 healthy control subjects were also included in this study. 12 subjects had negative skin prick tests (SPT) as well as CAST results. In 2 healthy subjects with positive SPT, we also observed positive CAST results. However, leukotriene secretion was lower than 300 pg/ml in these subjects. We conclude that CAST-ELISA is a valuable method for determining the allergic status in patients with mite allergy.

Is mast cell activation during asthmatic reaction reflected in the circulation?

Allergic asthmatic patients were challenged with specific allergen that resulted in early asthmatic reaction (EAR). Serum tryptase concentration (STC) and neutrophil chemotactic activity (NCA) were measured before and during EAR. A significant increase in neutrophil chemotactic activity was noticed in the 60th min, without an accompanying increase in serum tryptase concentration. The rise in neutrophil chemotactic activity in our study confirms previous observations in this field. However, because of the numerous cellular sources for neutrophil chemotactic activity, it does not seem to be a gold standard for measurement of mast cell activation. Undetectable levels of serum tryptase concentration during EAR in our study do not exclude the role of the mast cell in its pathogenesis. To our knowledge, only massive mast cell degranulation is reflected in the circulation as an increased tryptase concentration. In the case of upper respiratory allergic reactions, mast cell degranulation definitely takes place, but does not result in evident changes in serum tryptase concentration. We conclude that mast cell activation during allergen-induced EAR is poorly represented in the circulation.

CAST-ELISA test--a new diagnostic tool in pollen allergy.

We have applied a recently developed method called CAST-ELISA to evaluate the degree of leukocyte stimulation by specific allergen. This method is based on the measurement of sulfidoleukotriene levels in supernatants taken from previously stimulated peripheral blood leukocytes by specific allergen in the presence of interleukin 3.23 patients with pollinosis entered the study. All of them received no medication during 2 weeks before the test. Leukocytes were isolated by dextran sedimentation followed by single centrifugation. After removal of platelet rich plasma the cells were suspended in stimulation buffer and divided into portions incubated with or without specific allergen for 40 minutes at 37 degrees C. After the incubation, the cells were centrifuged and the evaluation of sulfidoleukotrienes in supernatant was performed. The results were expressed in pg/ml after subtraction of the value of spontaneous sulfidoleukotriene production in portions incubated without allergen. The concentration higher than 200 pg/ml of sulfidoleukotrienes above spontaneous production was regarded as a positive result. We have observed a large spectrum of the leukocyte response upon allergen stimulation. In the initial part of our study we established the optimal allergen concentration. The concentration of sulfidoleukotrienes in supernatants ranged from 10 to 5130 pg/ml. The mean sulfidoleukotriene concentration in the whole group was 1671.69. Positive results were observed in 20 persons. In 3 persons the results of allergen stimulation were negative. We conclude that CAST-ELISA is a reliable method to determine the allergic status in persons with pollinosis.

[The evaluation of concentration myeloperoxidase and interleukin-8 in blood serum of pregnant women with or without pregnancy induced hypertension]. / Ocena stezen mieloperoksydazy i interleukiny-8 w surowicy krwi ciezarnych z nadcisnieniem indukowanym ciaza oraz w ciazach fizjologicznych.

In the serum of woman during the pregnancy with and without PIH the activity of interleukin-8 and myeloperoxydase was measured. 40 pregnant woman with PIH and 16 normal pregnant woman were included in the study. We have not found significative difference between examined populations.

[Pathogenesis of exercise induced asthma]. / Patogeneza astmy wysilkowej.

Exercise induced asthma is an exaggerated airway response to airway dehydration and the following mediators release from the inflammatory cells. The airway narrowing is primarily caused by bronchial smooth muscle contraction, but in milder form mucus production, airway edema and cough can be observed. In this study we have described some previously and currently proposed hypotheses which may explain pathomechanism of this form of bronchial hyperreactivity.