High burden of clonal mast cell disorders and hereditary α-tryptasemia in patients who need Hymenoptera venom immunotherapy.

BACKGROUND: In patients who require venom immunotherapy (VIT), there is a need to identify underlying mast cell (MC) disorders since these may affect the risk and severity of future sting reactions and the long-term effectiveness of VIT. METHODS: 1319 individuals with Hymenoptera venom allergy (HVA) who needed VIT from referral centers in Slovenia, Austria, Croatia, and Poland underwent examination for KIT p.D816V in peripheral blood leukocytes (PBL) using a highly sensitive PCR test and tryptase genotyping by digital droplet PCR. We also included 183 control individuals with large local reactions (LLRs) to Hymenoptera stings and with asymptomatic sensitization to Hymenoptera venoms. RESULTS: 285 of 1319 individuals recommended for VIT (21.6%) were positive for KIT p.D816V in PBL, preferably those who present with severe reaction (33.9% [n = 207 of 610] with Ring-Messmer grade 3-4 vs. 11% [n = 78 of 709] with Grade 1-2; p < .0001), whereas only 1.3% (n = 2 of 152) of controls with LLR and none with asymptomatic sensitization (n = 31) had KIT p.D816V. KIT p.D816V allelic burden was higher in those with severe reaction (median 0.018% [n = 207] in Grade 3-4 vs. 0.001% [n = 78] in Grade 1-2; p < .0001), and the majority had normal baseline serum tryptase levels (69% [n = 196 of 285]). All KIT p.D816V-positive individuals (n = 41) who underwent bone marrow (BM) biopsy were found to have underlying clonal diseases, principally BM mastocytosis. HαT was also associated with severe HVA and symptoms (p < .01), and remarkably, 31.0% (n = 31 of 100) were found to have concomitant KIT p.D816V. Concomitant HαT and KIT p.D816V showed an additive effect, and having both was associated with the highest risk for severe HVA, even higher than having either HαT or KIT p.D816V alone (OR = 3.8; p < .01). CONCLUSIONS: By employing prospective universal tryptase genotyping and examination for KIT p.D816V in PBL in large HVA populations, we have demonstrated a high burden of clonal MC disorders and HαT in patients who require VIT.

Fatal Hymenoptera Venom-Triggered Anaphylaxis in Patients with Unrecognized Clonal Mast Cell Disorder-Is Mastocytosis to Blame?

Hymenoptera venom-triggered anaphylaxis (HVA) affects up to 8.9% of the general population and is the most frequent cause of anaphylaxis in adults, accounting for approximately 20% of all fatal anaphylaxis cases. Quite often, a fatal reaction is a victim's first manifestation of HVA. Mastocytosis represents one of the most important risk factors for severe HVA. We analyzed patients with documented fatal HVA for the presence of underlying clonal mast cell disorder (cMCD). Here, we report three cases of fatal HVA, with undiagnosed underlying cMCD identified by the presence of the peripheral blood and/or bone marrow KIT p.D816V missense variant postmortem. In the first case, anaphylaxis was the initial episode and was fatal. In the other two cases, both patients were treated with specific venom immunotherapy (VIT), nevertheless, one died of HVA after VIT discontinuation, and the other during VIT; both patients had cardiovascular comorbidities and were taking beta-blockers and/or ACE inhibitors. Our results point to the importance of screening all high-risk individuals for underlying cMCD using highly sensitive molecular methods for peripheral blood KIT p.D816V variant detection, including severe HVA and possibly beekeepers, for proper management and the need for lifelong VIT to prevent unnecessary deaths. Patients at the highest risk of fatal HVA, with concomitant cardiovascular and cMCD comorbidities, might not be protected from field stings even during regular VIT. Therefore, two adrenaline autoinjectors and lifelong VIT, and possibly cotreatment with omalizumab, should be considered for high-risk patients to prevent fatal HVA episodes.

Combination of Experimental and Bioinformatic Approaches for Identification of Immunologically Relevant Protein-Peptide Interactions.

Protein-peptide interactions are an essential player in cellular processes and, thus, of great interest as potential therapeutic agents. However, identifying the protein's interacting surface has been shown to be a challenging task. Here, we present a methodology for protein-peptide interaction identification, implementing phage panning, next-generation sequencing and bioinformatic analysis. One of the uses of this methodology is identification of allergen epitopes, especially suitable for globular inhaled and venom allergens, where their binding capability is determined by the allergen's conformation, meaning their interaction cannot be properly studied when denatured. A Ph.D. commercial system based on the M13 phage vector was used for the panning process. Utilization of various bioinformatic tools, such as PuLSE, SAROTUP, MEME, Hammock and Pepitope, allowed us to evaluate a large amount of obtained data. Using the described methodology, we identified three peptide clusters representing potential epitopes on the major wasp venom allergen Ves v 5.

Patch testing with the European baseline series and 10 added allergens: Single-centre study of 748 patients.

BACKGROUND: The European baseline series (EBS) of contact allergens is subject to change. An allergen is considered for inclusion when routine patch testing of patients with suspected contact dermatitis results in ≥0.5% prevalence rate. OBJECTIVES: We aimed to determine the frequency of sensitizations to 30 EBS allergens and 10 locally added allergens. Additionally, we assessed the strength and evolution of reactions to all tested allergens and co-reactivity of additional allergens. METHODS: Patch testing with our baseline series of 40 allergens was done in 748 consecutive adults. Tests were applied to the upper back and removed by patients after 48 h. Readings were done on Day 3 (D3) and D6 or D7 (D6/7). Positive reactions fulfilled the criteria of at least one plus (+) reaction. A retrospective analysis was done. RESULTS: Eight allergens not listed in the EBS had ≥0.5% prevalence rate (i.e., cocamidopropyl betaine, thiomersal, disperse blue mix 106/124, 2-bromo-2-nitropropane-1,3-diol, diazolidinyl urea, propylene glycol, Compositae mix II and dexamethasone-21-phosphate), and 16.6% of positive reactions would have been missed without D6/7 readings. CONCLUSION: We propose further studies to evaluate whether cocamidopropyl betaine, disperse blue mix 106/124, 2-bromo-2-nitropropane-1,3-diol, diazolidinyl urea and Compositae mix II need to be added to the EBS.

Routine KIT p.D816V screening identifies clonal mast cell disease in patients with Hymenoptera allergy regularly missed using baseline tryptase levels alone.

BACKGROUND: Clonal mast cell disorders and elevated basal serum tryptase (BST) levels with unknown cause(s) are associated with severe Hymenoptera venom-triggered anaphylaxis (HVA). However, some individuals with clonal disease have a normal BST level (<11.4 ng/mL). OBJECTIVE: Our aim was to evaluate whether screening for KIT p.D816V in the blood is a useful clinical tool to risk-stratify patients with venom allergy. METHODS: We prospectively recruited 374 patients with Hymenoptera allergy and no overt signs of mastocytosis who were referred to our center during the years 2018 and 2019. KIT p.D816V was determined in their peripheral blood by quantitative PCR, and tryptase genotyping was performed by droplet digital PCR. RESULTS: In all, 351 patients (93.9%) had normal levels of BST, and KIT p.D816V was detected in 8% of patients (28 of 351), predominantly in patients with the most severe Mueller grade IV anaphylaxis (18.2% [24 of 132] vs 1.8% in patients with lower grades [4 of 88 with grade III and 0 of 131 with other grades]; P < .001). In grade IV patients with a normal BST level, KIT p.D816V was associated with more severe symptoms, including a significantly higher frequency of loss of consciousness (58.3% [14 of 24] vs 34.3% [37 of 108]; P = .03) and absence of skin symptoms (41.7% [10 of 24] vs 15.7% [17 of 108]; P = .004). Among patients with a normal BST level, KIT p.D816V (OR = 10.25 [95% CI = 3.75-36.14]; P < .0001) was the major risk factor associated with severe HVA. Hereditary α-tryptasemia (HαT) due to increased germline copies of TPSAB1 encoding α-tryptase was the most common cause (65.2% [15 of 23]) of elevated BST level in patients with HVA, and together with KIT p.D816V, it accounted for 90% of BST level elevations (20 of 23) in patients with HVA. CONCLUSION: These results indicate that routine KIT p.D816V screening identifies clonal disease in high-risk patients with HVA who are regularly missed when BST level is used alone.

Down-regulation of FcεRI-mediated CD63 basophil response during short-term VIT determined venom-nonspecific desensitization.

BACKGROUND: We recently showed a desensitization of FcεRI-mediated basophil response after short-term VIT. Our aim was to evaluate the allergen specificity of this desensitization. METHODS: In 11 Hymenoptera-venom double positive subjects, basophil threshold sensitivity (CD-sens) to anti-FcεRI, honeybee, and Vespula venom was assessed at the beginning and just before the first maintenance dose (MD) of single ultra-rush VIT. In some patients we also monitored CD-sens to rApi m 1 and/or rVes v 5 or other co-sensitizations (i.e., grass pollen). In additional 7 patients, basophils were stripped and sensitized with house dust mite (HDM) IgEs at the same time points. RESULTS: We demonstrated a marked reduction of CD-sens to anti-FcεRI and VIT-specific venom before the first MD in all 18 subjects included. Furthermore, in 10 out of 11 double positive subjects, a significant and comparable decrease before the first MD was also evident for non-VIT venom; this nonspecific decrease was further supported by the opposite recombinant species-specific major allergen. In one subject with additional grass pollen allergy, a decrease of CD-sens to grass allergen was also demonstrated. Similarly, in 7 cases of patients with passively HDM-sensitized basophils, a significant reduction of CD-sens was also evident to de novo sensitized HDM allergen. CONCLUSIONS: Short-term VIT induced basophil desensitization to VIT-specific as well as to VIT-nonspecific venom. As opposed to long-term VIT, which induces venom-specific changes, the effect of short-term VIT seems to be venom-nonspecific.

Discharge coordinator intervention prevents hospitalizations in patients with COPD: a randomized controlled trial.

OBJECTIVES: To test whether coordination of discharge from hospital reduces hospitalizations in patients with chronic obstructive pulmonary disease (COPD). DESIGN: Randomized controlled clinical trial. SETTING: Specialized pulmonary hospital. PARTICIPANTS: Patients hospitalized for an acute exacerbation of COPD. INTERVENTION: Care as usual included routine patient education, supervised inhaler use, respiratory physiotherapy, and disease-related communication. The discharge coordinator intervention added assessment of patient situation and homecare needs. Patients and caregivers were actively involved and empowered in the discharge planning process, which was communicated with community medical professionals to provide continuity of care at home. MEASUREMENTS: The primary end-point of the study was the number of patients hospitalized because of worsening COPD. Key secondary end-points were time-to-COPD hospitalization, all-cause mortality, all-cause hospitalization, days alive and out of hospital, and health-related quality of life. RESULTS: Of 253 eligible patients (71 ± 9 years, 72% men, 87% GOLD III/IV), 118 were assigned to intervention and 135 to usual care. During a follow-up of 180 days, fewer patients receiving intervention were hospitalized for COPD (14% versus 31%, P = .002) or for any cause (31% versus 44%, P = .033). In time-to-event analysis, intervention was associated with lower rates of COPD hospitalizations (P = .001). A Cox model of proportional hazards, adjusted for sex, age, GOLD stage, heart failure, malignant disease, and long-term oxygen treatment, demonstrated that intervention reduced the risk of COPD hospitalization (hazard ratio 0.43, 95% confidence interval 0.24-0.77, P = .002). CONCLUSION: Among patients hospitalized for acute COPD exacerbation, discharge coordinator intervention reduced both COPD hospitalizations and all-cause hospitalizations.

let-7b and miR-126 are down-regulated in tumor tissue and correlate with microvessel density and survival outcomes in non--small--cell lung cancer.

Angiogenesis is a critical event in the development, progression, and spread of various human cancers, including lung cancer. Molecular mechanisms that underlie the complex regulation of angiogenic processes are poorly understood. However, an increasing body of evidence indicates miRNAs as important regulators of tumor angiogenesis. Forceps biopsies were collected from tumor tissue, surrounding tissue, and non-tumor tissue from 50 NSCLC patients. Lung tissue samples from individuals with no clinical evidence of a cancerous disease served as controls. Immunohistochemical staining for Factor VIII was used to evaluate microvessel density (MVD). TaqMan® primer-probe sets were used in quantitative real-time RT-PCR reactions to determine expression levels of let-7b, miR-126, miR-9, and miR-19a. We demonstrated significantly higher MVD and decreased expression levels of let-7b and miR-126 in tumor tissue and surrounding tissue in comparison to corresponding non-tumor tissue or lung tissue from the control group. In addition, no differences in MVD and expression levels of both miRNAs between tumor tissue and surrounding tissue from NSCLC patients were observed. Low expression of both miRNAs correlated with high MVD and worse progression-free survival and overall survival. These observations strongly suggest similar molecular alternations within tumor tissue and surrounding tissue that comprise a specific microenvironment. Low expression of let-7b and miR-126 seems to have a possible anti-angiogenic role in lung tumor tissue and significantly correlates with worse survival outcomes for lung cancer patients. Moreover, the regulation of let-7b and miR-126 expression could have therapeutic potential because it could reduce tumor angiogenesis and therefore suppress tumor growth in lung cancer patients.

House dust mite-specific immunotherapy alters the basal expression of T regulatory and FcεRI pathway genes.

BACKGROUND: Regulatory T (Treg) cells and IgE-mediated signaling pathways could play important roles in the induction of allergen tolerance during house dust mite-specific subcutaneous immunotherapy (HDM-SCIT). Our aim was to compare the basal expression levels of Treg, T helper 1 (Th1) and Th2 transcription factors and components involved in IgE-mediated signaling in healthy subjects with those in HDM-allergic patients both untreated and successfully treated with HDM-SCIT. METHODS: Thirty-nine HDM-allergic patients who completed a 3- to 5-year course of mite extract SCIT, 20 mite-allergic controls and 25 healthy controls participated in this study. The efficacy of SCIT was monitored using skin-prick tests (SPTs), total immunoglobulin E (tIgE), specific IgE (sIgE), sIgG(4), nasal challenge and visual analog scale (VAS) scores at several time points. The mRNA levels of forkhead box protein 3 (FOXP3), T-BET, GATA-3, FcεRI, spleen tyrosine kinase (Syk), phosphatidylinositol 3 kinase (PI3K) and SH2 domain-containing inositol phosphatase (SHIP) were quantified by real-time RT-PCR using nonstimulated whole blood samples. RESULTS: Decreased wheal sizes and VAS scores, negative challenges and increased sIgG(4) levels indicated that SCIT was effective in the treated patients. Basal expression levels of FOXP3 and GATA-3 decreased and T-BET levels increased in both treated patients and in healthy controls compared to untreated patients. The IgE-mediated pathway kinases Syk and PI3K exhibited reduced expression, whereas SHIP phosphatase levels were elevated in both treated patients and healthy controls relative to untreated patients. The expression levels of FcεRI were not significantly altered. CONCLUSIONS: Immunotherapy using HDM extracts results in a modification of the basal expression levels of several IgE-related signaling factors and induces a highly significant upregulation of Th1-response and downregulation of Th2-response transcription factors. Interestingly, this therapy also appears to reduce the basal expression of FOXP3.

Asthma treatment outcome in children is associated with vascular endothelial growth factor A (VEGFA) polymorphisms.

BACKGROUND: Asthma is a common chronic disease characterized by airway inflammation and structural remodeling. Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis, is elevated in asthma patients. VEGF contributes to airway responsiveness and remodeling. It has been shown that treatment of asthma patients decreases VEGF levels, and inhibition of VEGF diminishes asthma symptoms in mice. Therefore, polymorphisms in the vascular endothelial growth factor A (VEGFA) gene might be associated with asthma treatment response. METHODS: This study enrolled 131 children with asthma treated with different therapies - specifically, the inhaled corticosteroid (ICS) fluticasone propionate or the leukotriene receptor antagonist (LTRA) montelukast. We performed an association analysis between improvement of lung function - assessed by measurement of the percentage of the predicted forced expiratory volume in 1 second (%predicted FEV(1)), the ratio between the FEV(1) and the forced vital capacity (FEV(1)/FVC) after 6 and 12 months of treatment, and asthma control after 12 months of treatment - and two polymorphisms, rs2146323 and rs833058, in the VEGFA gene. RESULTS: Polymorphism rs2146323 A>C in VEGFA was associated with response to ICS therapy. Asthma patients with the AA genotype had a greater improvement in the %predicted FEV(1) than those with the AC or CC genotype (p = 0.018). Conversely, the AA genotype in rs2146323 was associated with uncontrolled asthma in patients regularly receiving LTRA therapy (p = 0.020) and a worse FEV(1)/FVC ratio in patients who episodically used LTRA therapy (p = 0.044). Furthermore, polymorphism rs833058 C>T was associated with treatment response to episodically used LTRA therapy. A subgroup of patients with the TT genotype had an improvement in the %predicted FEV(1), compared with no improvement in patients with the CT or CC genotype (p = 0.029). CONCLUSIONS: Our results showed that treatment response to commonly used asthma therapies (ICS or LTRA) is associated with polymorphisms rs2146323 and rs833058 in VEGFA. With additional replication of this preliminary study, our findings could contribute to the development of individualized asthma therapy.

Angiogenin and vascular endothelial growth factor expression in lungs of lung cancer patients.

BACKGROUND.: Lung cancer is the leading cause of cancer deaths. Angiogenesis is crucial process in cancer growth and progression. This prospective study evaluated expression of two central regulatory molecules: angiogenin and vascular endothelial growth factor (VEGF) in patients with lung cancer. PATIENTS AND METHODS.: Clinical data, blood samples and broncho-alveolar lavage (BAL) from 23 patients with primary lung carcinoma were collected. BAL fluid was taken from part of the lung with malignancy, and from corresponding healthy side of the lung. VEGF and angiogenin concentrations were analysed by an enzyme-linked immunosorbent assay. Dilution of bronchial secretions in the BAL fluid was calculated from urea concentration ratio between serum and BAL fluid. RESULTS.: We found no statistical correlation between angiogenin concentrations in serum and in bronchial secretions from both parts of the lung. VEGF concentrations were greater in bronchial secretions in the affected side of the lung than on healthy side. Both concentrations were greater than serum VEGF concentration. VEGF concentration in serum was in positive correlation with tumour size (p = 0,003) and with metastatic stage of disease (p = 0,041). There was correlation between VEGF and angiogenin concentrations in bronchial secretions from healthy side of the lung and between VEGF and angiogenin concentrations in bronchial secretions from part of the lung with malignancy. CONCLUSION.: Angiogenin and VEGF concentrations in systemic, background and local samples of patients with lung cancer are affected by different mechanisms. Pro-angiogenic activity of lung cancer has an important influence on the levels of angiogenin and VEGF.

Natural killer T cells in pulmonary disorders.

Natural killer T (NKT) cells, a unique subgroup of lymphocytes with features of both T and natural killer (NK) cells, represent a bridge between innate and adaptive immunity. They have the ability to either promote or suppress immune responses. With these immunoregulatory functions, NKT cells have emerged as an important subset of lymphocytes with a protective role in some disorders, such as infections, cancer, and possibly sarcoidosis, and a pathogenic role in others, such as asthma, chronic obstructive pulmonary disease and hypersensitivity pneumonitis. Immunotherapeutic interventions to modulate the immune response by targeting iNKT cell functions has become a challenging field and has shown promising results for the development of new therapies.

Body mass index and prognosis in patients hospitalized with acute exacerbation of chronic obstructive pulmonary disease.

BACKGROUND: Nutritional status, weight loss and cachexia have important prognostic implications in patients with chronic obstructive pulmonary disease (COPD). Body mass index (BMI) has been implicated in COPD risk assessment, but information is mostly limited to composite scores or to patients with stable disease. We aimed to analyse the association between BMI and mortality in acute exacerbation of COPD. METHODS: This retrospective survey included 968 patients hospitalized due to acute exacerbation of COPD at the University Clinic Golnik from February 2002 to June 2007. Vital status was ascertained with Central Population Registry, and database was censored on November 1, 2008. RESULTS: Median BMI was 25.08 kg/m(2) (interquartile range, 21.55-29.05 kg/m(2)) and 210 patients (22%) had BMI < 21 kg/m(2). During median follow-up of 3.26 years (1.79-4.76 years), 430 patients (44%) died. Lowest mortality was found for BMI 25.09-29.05 kg/m(2). When divided per BMI decile, mortality was lowest for BMI 25.09-26.56 kg/m(2) (33%). In univariate analysis, BMI per quartile and BMI per unit increase were predictive for all-cause mortality. In an adjusted model, BMI per 1 kg/m(2) unit increase was associated with 5% less chance of death (hazard ratio 0.95, 95% confidence interval 0.93-0.97). CONCLUSIONS: Low BMI < 21 kg/m(2) is frequent in patients hospitalized due to acute exacerbation of COPD. Higher BMI was independently predictive of better long-term survival. A better outcome in obese patients compared to normal weight is in contrast to primary prevention data but concurs with observations of an obesity paradox in other cardiovascular diseases.

Expressions of topoisomerase IIα and BCRP in metastatic cells are associated with overall survival in small cell lung cancer patients.

The aim of this study was to investigate the mRNA expression levels of multidrug resistance-associated proteins in chemo-naïve metastatic lung cancer cells and to determine the correlation with response to chemotherapy and overall survival. Metastatic cells were obtained by transbronchial fine needle aspiration biopsy of enlarged mediastinal lymph nodes in 14 patients with small cell lung cancer (SCLC) and 7 patients with non-small cell lung cancer (NSCLC). After cytological confirmation of lung cancer type, total RNA was extracted from biopsy samples and reverse transcribed to cDNA, and real-time PCR for the genes of interest [P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), breast cancer resistance protein (BCRP), lung resistance protein (LRP) and topoisomerase IIα (TOPIIα)], was performed. We observed significantly decreased expression of BCRP and significantly increased expression of TOPIIα in metastatic SCLC cells compared to NSCLC. Furthermore, in SCLC high topoisomerase IIα and low BCRP expression levels positively correlated with longer overall survival. Our results showed higher expression levels of BCRP as well as lower levels of topoisomerase IIα in chemo-naïve metastatic cells in NSCLC than in SCLC. These results correlate with previous observations that metastatic SCLC cells at the beginning of chemotherapy are potentially more sensitive to chemotherapeutic agents while in metastatic NSCLC cells resistance is usually inherent. We also showed that altered levels of topoisomerase IIα and BCRP in SCLC are important factors that contribute to resistance to chemotherapeutics that interfere with the enzyme and/or DNA and are highly associated with overall survival.

Predictive value of multidrug resistance proteins, topoisomerases II and ERCC1 in small cell lung cancer: a systematic review.

In small-cell lung cancer (SCLC), resistance to cancer drugs presents a major problem, limiting the effectiveness of chemotherapy. A better understanding of the molecular biology is essential to improve currently available cytotoxic therapy. Herein, a systematic review of studies evaluating the predictive value of multidrug resistance-associated proteins (MDR1, MRP1, MRP2 and MVP), topoisomerase II and ERCC1 for chemotherapy outcomes is presented. The role of MDR1, MRP1 and MRP2 as predictive markers in SCLC has not yet been elucidated. The majority of studies reported an association between protein or gene expression and response to chemotherapy; however, the evidence is limited to univariate analyses performed in the frame of small retrospective trials. In addition, the largest trial did not confirm an independent predictive value for response rates or survival. Genetic variability may be overseen as a more promising marker. Available data on the predictive value of topoisomerase II are scarce and in contrast to the general idea that higher protein or gene expression correlate with greater chemo-sensitivity. The data on a possible predictive value of ERCC1 are also quite limited; in two retrospective studies, ERCC1 turned out to be a significant predictive marker for survival, but only for limited disease patients. In conclusion, a continuous research, with standardized and validated methodology of markers' determination, should be aspired at all times; a better understanding of the biology of SCLC is of utmost importance to enable personalized therapy and to improve survival rates in this, so far, poorly controlled disease.

Basophil responsiveness and clinical picture of acetylsalicylic acid intolerance.

BACKGROUND: Exposure to acetylsalicylic acid (ASA) may exacerbate respiratory or skin diseases or induce anaphylactoid reactions in apparently healthy individuals. We wanted to evaluate specific responsiveness of basophils to ASA in correlation with the clinical picture. METHODS: We performed a prospective single-blind study of 59 subjects involved in clinical evaluation and/or ASA provocation testing. Whole blood basophils were stained with anti-CD63/CD123/HLA-DR mAbs after stimulation with 0.25 or 1 mg/ml ASA. RESULTS: We found that 40 subjects were ASA tolerant and 19 were ASA intolerant. Both groups had comparable manifestations of asthma and/or rhinitis (13 in the tolerant and 9 in the intolerant group). Intolerant subjects showed significantly higher basophil responsiveness to ASA in comparison to tolerant subjects, which was concentration-dependent in both groups. The ratio between responses at 1 mg/ml of ASA and at baseline (activation index) was analyzed according to the clinical picture. We demonstrate that the activation index was higher only in the intolerant subjects with anaphylactoid reactions, but not in a subgroup of subjects with asthma/rhinitis. The ROC calculations show that the optimal threshold activation index was more than 2.18. The sensitivity was 80% and the specificity was 83% in the subgroup with anaphylactoid reactions. In the asthma/rhinitis subgroup, the sensitivity was 78% and the specificity was 50%. CONCLUSIONS: Our study demonstrates that there is a significantly higher in vitro basophil response to ASA in intolerant as compared to tolerant subjects. ROC analyses suggest that this measurement might only have a diagnostic value in subjects without asthma and/or rhinitis.

Local CD4+, CD8+ and CD56+ reactions to lung cancer in regard to pathohistological type and clinical stage.

AIM: To determinate the difference of abundance of CD4+, CD8+ and CD56+ bronchoalveolar fluid's lymphocytes and their subpopulations between non- and small cell lung cancer. Also, the differences of abundance of examined lymphocytes were compared between main clinical stages of lung cancer. METHODS: Mini-bronchoalveolar lavate was taken from lungs of 55 patients with cancer. After laboratory processing and adding CD3, CD4, CD8, CD27, CD28 and CD56 antibody, the material was analysed by flow cytometer. Results of Mini-BAL for non- and small cell lung cancer were compared, as well as the different clinical stages of the disease. RESULTS: Immature and regulatory forms of lymphocytes are more activated, while mature and activated forms are less activated in small cell lung cancer compared to non small type. With an increase of the clinical stage of disease, immunological reaction of T lymphocytes is better expressed because of increasing of abundance of immature and regulatory forms of different subpopulations of lymphocytes. CONCLUSION: All components of local CD4+ and CD8+ T lymphocyte, as well as NK and NKT cells response were more activated in lungs with small cell lung cancer, and these reactions were more expressed with an increase in the clinical stage.