Sputum neutrophil count after bronchial allergen challenge is related to peripheral blood neutrophil chemotaxis in asthma patients.

OBJECTIVE: The aim of this study was to estimate relations between sputum neutrophilia and the chemotactic activity of peripheral blood neutrophils after the bronchial allergen challenge in asthma patients. MATERIALS AND METHODS: Fifteen patients with allergic asthma (AA), 13 patients with allergic rhinitis (AR), all sensitized to Dermatophagoides pteronyssinus, and 8 healthy subjects (HS) underwent bronchial challenge with D. pteronyssinus. Sputum and peripheral blood collection were performed 24 h before, 7 and 24 h after the bronchial challenge. Cell counts were determined by the May-Grünwald-Giemsa method. Neutrophil chemotaxis was analyzed by a flow cytometer; IL-8 levels were measured by ELISA. RESULTS: Sputum neutrophil count and peripheral blood neutrophil chemotaxis of patients with AA were greater 7 and 24 h after the challenge compared with the baseline values and patients with AR and HS (P < 0.05). Moreover, a significant correlation was found between the neutrophil count in sputum and IL-8 levels, and the chemotactic activity of peripheral blood neutrophils 24 h after the bronchial challenge only the patients with AA (P < 0.05). CONCLUSIONS: Increased sputum neutrophil count was found to be associated with an enhanced chemotactic activity of peripheral blood neutrophils during allergen-induced late-phase airway inflammation in patients with allergic asthma.

Response of peripheral blood Th17 cells to inhaled Dermatophagoides pteronyssinus in patients with allergic rhinitis and asthma.

BACKGROUND: Recent studies have shown the importance of Th17 cells in the development of allergic airway diseases. We examined Dermatophagoides pteronyssinus-induced changes in peripheral blood Th17 cells to establish the importance of these cells in late-phase allergic inflammation in patients with allergic rhinitis (AR) and allergic asthma (AA). METHODS: Eighteen patients with mild-to-moderate/severe persistent AR, 14 patients with intermittent- or mild-to-moderate persistent AA, and 15 healthy subjects (HS) were examined. All patients had positive skin test to D. pteronyssinus. Study subjects underwent bronchial challenge with D. pteronyssinus. The peripheral blood Th1, Th2, and Th17 cells were determined by flow cytometry 24 h before and 7 and 24 h after challenge. The serum IL-17 levels were determined by ELISA. RESULTS: The percentage of Th17 cells and IL-17 levels was significantly higher in patients with AR and AA compared with HS before and after challenge. Twenty-four hours after challenge, the percentage of Th17 cells increased significantly in patients with AA compared with baseline values. The IL-17 levels rose markedly in patients with AR and AA after challenge. Moreover, 24 h after challenge, the percentage of Th17 cells and IL-17 levels were significantly higher in patients with AA than those with AR. CONCLUSIONS: Percentages of peripheral blood Th17 cells and serum IL-17 levels were found to be higher in patients with AR and AA. An increase in the percentage of Th17 cells following challenge shows that Th17 cells may have an important role in the development of late-phase allergen-induced inflammation.

Peripheral blood Th17 cells and neutrophils in Dermatophagoides pteronyssinus-induced early- and late-phase asthmatic response.

BACKGROUND AND OBJECTIVE: Biphasic cellular immune reactions, which follow allergen inhalation, are a specific feature of inflammation in allergic asthma. The aim of this study was to determine the changes in the percentage of peripheral blood Th17 cells and neutrophil functions after Dermatophagoides pteronyssinus-induced early- and late-phase asthmatic response in patients with allergic asthma. MATERIAL AND METHODS: A total of 19 patients with allergic asthma were examined. Eleven patients developed an isolated early-phase asthmatic response (EAR), whereas 8 developed both early- and late-phase (dual) asthmatic responses (DAR) after the bronchial challenge with Dermatophagoides pteronyssinus. The control group included 15 healthy subjects. Peripheral blood collection was performed 24 hours before as well as 7 and 24 hours after the bronchial challenge. The percentage of Th17 cells, and chemotaxis and apoptosis of neutrophils were analyzed by flow cytometry. The serum IL-8 and IL-17 levels were determined by ELISA. RESULTS: After the bronchial challenge, the percentage of Th17 and IL-17 levels increased considerably 7 and 24 hours after the challenge in both groups of patients. Moreover, 24 hours after the challenge, the percentage of Th17 cells and IL-17 levels were significantly higher in the patients with the DAR than those with the EAR or healthy controls. Seven and 24 hours after the challenge, neutrophil chemotaxis was greater in the patients with the DAR as compared with those with the EAR and healthy controls as well. The apoptotic activity of neutrophils was lower 24 hours after the challenge in the patients with the DAR than those with the EAR. CONCLUSIONS: Dermatophagoides pteronyssinus-induced early- and late-phase asthmatic response in patients with allergic asthma was found to be accompanied by an increased percentage of peripheral blood Th17 cells and elevated serum IL-17 levels as well as altered neutrophil functions.

The effect of induced sputum and bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease on neutrophil migration in vitro.

OBJECTIVE: The aim of study was to investigate a chemotactic effect of induced sputum and bronchoalveolar lavage fluid on blood neutrophils in patients with chronic obstructive pulmonary disease (COPD) and healthy individuals. MATERIAL AND METHODS: Forty-three smokers with COPD, 19 ex-smokers with COPD, 13 healthy smokers, and 17 healthy nonsmokers were recruited to the study. Neutrophils were isolated from peripheral blood of study individuals. For the same experimental conditions, pooled induced sputum and bronchoalveolar lavage fluid of 20 COPD patients were used. Neutrophil chemotaxis in vitro was performed in cell-transmigration chamber. Substances tested for chemoattraction (interleukin-8, induced sputum, bronchoalveolar lavage fluid directly or in addition to interleukin-8) were added to lower wells. Upper wells were filled with 2.5 x 10(6)/mL of neutrophil culture and incubated for 2 hours. Migration was analyzed by flow cytometry. RESULTS: Interleukin-8 (10-100 ng/mL) induced a dose-dependant neutrophil migration in all the groups. Only 100 ng/L of interleukin-8 induced more intensive chemotaxis of neutrophils from COPD smokers as compared to ex-smokers (P<0.05). Such difference between healthy individuals was obtained using 30 ng/mL of interleukin-8 (P<0.05). Induced sputum/interleukin-8 (10-100 ng/mL), as well as induced sputum directly, induced neutrophil migration (P<0.05). Chemotaxis of neutrophils isolated from COPD patients and healthy nonsmokers did not depend on additional interleukin-8 concentration. Bronchoalveolar lavage fluid/interleukin-8 (30-100 ng/mL) induced more intensive migration of neutrophils from COPD patients than bronchoalveolar lavage fluid (P<0.05) alone. CONCLUSIONS: Migration of neutrophils isolated from patients with COPD was more intensive compared to healthy individuals. Induced sputum and bronchoalveolar lavage fluid directly and with addition of interleukin-8 stimulated chemotaxis, and it was higher in neutrophils from COPD patients. Migration of neutrophils did not depend on smoking status.

[Pseudomonas aeruginosa bacteremia: associations with a source of infection and antibiotic resistance]. / Pseudomonas aeruginosa padermiu, sukelusiu bakteriemija, sasajos su infekcijos zidiniu ir ju atsparumas antibiotikams.

AIM OF THE STUDY: To determine the associations between the source of infection and antibiotic resistance in patients with Pseudomonas aeruginosa bacteremia. MATERIAL AND METHODS: A retrospective analysis of 50 patients with Pseudomonas aeruginosa bacteremia was carried out. If sepsis was suspected, blood culture was incubated in an automatic system BACTEC 9240. Then bacteria were identified, and their antibiotic resistance was estimated by disc diffusion method. If Pseudomonas aeruginosa strains were resistant to three or more antibiotics, they were considered as multidrug-resistant. RESULTS: The origin of bacteremia was confirmed in 33 (66%) patients. Lower respiratory tract was the predominant source of Pseudomonas aeruginosa bacteremia (81.8%, n=27) as compared with infection of wound (39.4%, n=13), urinary tract (15.2%, n=5), and drain or cerebrospinal fluid (9.1%, n=3) (P<0.05). Eighteen percent (n=9) of strains, which caused bacteremia, were resistant to ceftazidime; 38% (n=19), to piperacillin; 22% (n=11), to imipenem; 26% (n=13), to meropenem; 24% (n=12), to ciprofloxacin; 40% (n=20), to gentamicin; and only 8% (n=4), to amikacin. Multidrug-resistant Pseudomonas aeruginosa strains were more frequently isolated if a source of infection was wound comparing to a source of other localization (61.5%, n=8 and 20.0%, n=4, respectively; P<0.05). Resistance of Pseudomonas aeruginosa strains to imipenem was associated with resistance to ciprofloxacin (13.2%, n=5 and 50.0%, n=6, retrospectively; P<0.05), but resistance to meropenem--both to ciprofloxacin and amikacin. CONCLUSIONS: The predominant source of Pseudomonas aeruginosa bacteremia was lower respiratory tract, and multidrug-resistant strains caused bacteremia more frequently if a source infection was wound. Pseudomonas aeruginosa resistance to carbapenems was associated with resistance to ciprofloxacin and resistance to meropenem--also to amikacin. Resistance of strains to ceftazidime and piperacillin was associated with resistance to gentamicin.

High frequency of the c.3207C>A (p.H1069Q) mutation in ATP7B gene of Lithuanian patients with hepatic presentation of Wilson's disease.

AIM: To investigate the prevalence of the ATP7B gene mutation in patients with hepatic presentation of Wilson's disease (WD) in Lithuania. METHODS: Eleven unrelated Lithuanian families, including 13 WD patients were tested. Clinically WD diagnosis was established in accordance to the Leipzig scoring system. Genomic DNA was extracted from whole venous blood using a salt precipitation method. Firstly, the semi-nested polymerase chain reaction (PCR) technique was used to detect the c.3207C>A (p.H1069Q) mutation. Patients not homozygous for the c.3207C>A (p.H1069Q) mutation were further analyzed. The 21 exons of the WD gene were amplified in a thermal cycler (Biometra T3 Thermocycler, Gottingen, Germany). Direct sequencing of the amplified PCR products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer (Applied Biosystems, Darmstadt, Germany). RESULTS: Total of 13 WD patients (mean age 26.4 years; range 17-40; male/female 3/10) presented with hepatic disorders and 16 their first degree relatives (including 12 siblings) were studied. Some of WD patients, in addition to hepatic symptoms, have had extrahepatic disorders (hemolytic anemia 3; Fanconi syndrome 1; neurophsychiatric and behavioural disorder 2). Liver biopsy specimens were available in all of 13 WD patients (8 had cirrhosis; 1-chronic hepatitis; 3-acute liver failure, 1-liver steatosis). Twelve of 13 (92.3%) WD patients had the c.3207C>A (p.H1069Q) mutation, 6 of them in both chromosomes, 6 were presented as compound heterozygotes with additional c.3472-82delGGTTTAACCAT, c.3402delC, c.3121C>T (p.R1041W) or unknown mutations. For one patient with liver cirrhosis and psychiatric disorder (Leipzig score 6), no mutations were found. Out of 16 first degree WD relatives, 11 (68.7%) were heterozygous for the c.3207C>A (p.H1069Q) mutation. Two patients with fulminant WD died from acute liver failure and 11 are in full remission under penicillamine or zinc acetate treatment. Three women with WD successfully delivered healthy babies. CONCLUSION: The c.3207C>A (p.H1069Q) missense mutation is the most characteristic mutation for Lithuanian patients with WD. Even 92.3% of WD patients with hepatic presentation of the disease are homozygous or compound heterozygotes for the p.H1069Q mutation.

Patterns of airway inflammation and MMP-12 expression in smokers and ex-smokers with COPD.

BACKGROUND: Smoking activates and recruits inflammatory cells and proteases to the airways. Matrix metalloproteinase (MMP)-12 may be a key mediator in smoke induced emphysema. However, the influence of smoking and its cessation on airway inflammation and MMP-12 expression during COPD is still unknown. We aimed to analyse airway inflammatory cell patterns in induced sputum (IS) and bronchoalveolar lavage (BAL) from COPD patients who are active smokers and who have ceased smoking >2 years ago. METHODS: 39 COPD outpatients - smokers (n = 22) and ex-smokers (n = 17) were studied. 8 'healthy' smokers and 11 healthy never-smokers were tested as the control groups. IS and BAL samples were obtained for differential and MMP-12+-macrophages count analysis. RESULTS: The number of IS neutrophils was higher in both COPD groups compared to both controls. The amount of BAL neutrophils was higher in COPD smokers compared to healthy never-smokers. The number of BAL MMP-12+-macrophages was higher in COPD smokers (1.6 +/- 0.3 x 106/ml) compared to COPD ex-smokers, 'healthy' smokers and healthy never-smokers (0.9 +/- 0.4, 0.4 +/- 0.2, 0.2 +/- 0.1 x 106/ml respectively, p < 0.05). CONCLUSION: The lower amount of BAL neutrophils in COPD ex-smokers, compared to COPD smokers, suggests positive alterations in alveolar compartment after smoking cessation. Smoking and disease itself may stimulate MMP-12 expression in airway compartments (IS and BAL) from COPD patients.

Functional activity of peripheral blood eosinophils in allergen-induced late-phase airway inflammation in asthma patients.

OBJECTIVE: We aimed to investigate peripheral blood eosinophil chemotaxis, generation of spontaneous reactive oxygen species (ROS), and apoptosis in patients with allergic asthma after bronchial allergen challenge. MATERIAL AND METHODS: A total of 18 patients with allergic asthma (AA), 14 with allergic rhinitis (AR), and 10 healthy subjects (HS) underwent bronchial challenge with a specific allergen extract. Eosinophils from peripheral blood were isolated 24 h before as well as 7 and 24 h after bronchial allergen challenge. Chemotaxis, spontaneous ROS production in eosinophils, and apoptosis were analyzed by flow cytometry. Serum and induced sputum IL-5 levels were measured by ELISA; the cell count in sputum was analyzed by the May-Grünwald-Giemsa method. RESULTS: Before bronchial allergen challenge, peripheral blood eosinophil chemotaxis, spontaneous ROS production was enhanced and eosinophil apoptosis was reduced in the patients with AA as compared with AR patients and HS (P < 0.05). Meanwhile, eosinophil chemotaxis and ROS generation markedly increased in the patients with AA 7 h and 24 h after challenge compared with other groups and baseline values (P < 0.05). The percentage of apoptotic eosinophils in the patients with AA decreased at 7 h as well as 24 h after challenge when compared with other groups and the baseline values (P < 0.05). There was a significant correlation between the migrated peripheral blood eosinophil count and the sputum eosinophil count (Rs = 0.89, P < 0.0001) and the sputum IL-5 level (Rs = 0.68, P = 0.002) at 24 h after bronchial challenge only in the patients with AA. Furthermore, the percentage of peripheral blood apoptotic eosinophils significantly correlated with eosinophil count in sputum (Rs = -0.53, P = 0.02), and ROS production correlated with the serum IL-5 levels (Rs = 0.71, P = 0.01). CONCLUSION: During allergen-induced late-phase airway inflammation, peripheral blood eosinophils demonstrated further alterations of their functional activity manifested by enhanced spontaneous ROS production, increased chemotaxis, and diminished apoptosis in patients with AA.

Th17 response to Dermatophagoides pteronyssinus is related to late-phase airway and systemic inflammation in allergic asthma.

BACKGROUND: Th17 cells may play a role in the development of late-phase allergen-induced airway and systemic inflammation in allergic asthma, although the mechanisms involved remain to be elucidated. METHODS: A total of 36 subjects were enrolled into the study: 15 allergic asthma patients with early asthmatic reaction (n=7) or dual asthmatic reaction (n=8) developed to inhaled D. pteronyssinus, 13 patients with allergic rhinitis, and 8 healthy subjects. Peripheral blood and induced sputum were collected 24h before as well as 7h and 24h after a bronchial challenge with D. pteronyssinus. Th17 cells were analyzed by FACS; IL-17 levels were determined by ELISA. RESULTS: At baseline, the percentage of peripheral blood Th17 cells and serum and sputum IL-17 levels were significantly higher in all groups of studied patients compared with those of healthy subjects. After the bronchial challenge, there was a significant increase in the percentage of peripheral blood Th17 cells and in serum and sputum IL-17 levels in rhinitis and asthma patients compared with their baseline values, particularly in allergic asthma patients with the dual asthmatic reaction. Positive correlations were found between the percentage of Th17 cells and IL-17 levels in serum (Rs=0.649; P=0.009) as well in sputum (Rs=0.583; P=0.022) in allergic asthma patients 24h after the bronchial challenge. CONCLUSIONS: The Th17 response is associated with the development of late-phase airway and systemic inflammation after the inhalation of D. pteronyssinus in patients with allergic asthma.

Reactive oxygen species in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbation of chronic obstructive pulmonary disease.

Chronic airway inflammation can be mediated by an enhanced neutrophil oxidative burst. However, the role of bacteria in the pathogenesis of chronic obstructive pulmonary disease (COPD) exacerbations is highly controversial. The aim of this study was to evaluate the production of reactive oxygen species (ROS) in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbations of COPD (AECOPD). A total of 40 patients with AECOPD, 10 healthy nonsmokers, and 10 "healthy" smokers were enrolled into the study. Peripheral blood and sputum samples were obtained during exacerbation and after recovery. Neutrophils were isolated by high-density gradient centrifugation and magnetic separation. ROS production by neutrophils was investigated after stimulation with phorbol-myristate-acetate and Staphylococcus aureus bacteria. ROS production by neutrophils was assessed as the mean fluorescent intensity using a flow cytometer. IL-8 levels in serum and induced sputum were determinant by ELISA. Spontaneous ROS production was significantly higher in neutrophils from the patients with bacterial AECOPD as compared with nonbacterial AECOPD and stable COPD (P <0.05). ROS production stimulated with PMA and with Staphylococcus aureus was significantly higher in neutrophils isolated from the patients with bacterial AECOPD as compared with nonbacterial and stable COPD (P <0.05). The serum and induced sputum IL-8 levels were significantly increased in the patients with bacterial AECOPD than nonbacterial AECOPD, stable COPS, and "healthy" smokers and nonsmokers (P <0.05) and higher in the induced sputum as the compared with serum in all studied groups (P <0.05). Enlarge CRP level was documented during AECOPD than in all other groups (P <0.05). A markedly increased ROS production in sputum neutrophils during bacterial AECOPD shows an inflammatory response reflecting enhanced local inflammation, which can be mediated by bacterial colonization.

Peripheral blood neutrophil activity during Dermatophagoides pteronyssinus-induced late-phase airway inflammation in patients with allergic rhinitis and asthma.

Recent investigations suggest that neutrophils may play an important role in the late-phase allergen-induced inflammation in allergic airway diseases. The aim of this study was to evaluate neutrophil chemotaxis, phagocytic activity, and reactive oxygen species (ROS) production in patients with allergic rhinitis and asthma challenged with inhaled Dermatophagoides pteronyssinus. Eighteen patients with allergic rhinitis and 14 with allergic asthma, all sensitized to D. pteronyssinus, as well as 15 healthy individuals underwent bronchial challenge with D. pteronyssinus. Peripheral blood collection and neutrophil isolation were performed 24 h before as well as 7 and 24 h after bronchial challenge. Neutrophils chemotaxis, phagocytic activity, and ROS production were analyzed by flow cytometer. Neutrophil chemotaxis and ROS production were increased, while phagocytic activity was decreased 24 h before challenge in patient groups compared with healthy individuals. After challenge, neutrophil chemotaxis and phagocytic activity increased after 7 and 24 h, when ROS production, only after 24 h. Bronchial allergen challenge had no influence for neutrophils activity in healthy subjects. Activated chemotaxis, phagocytic activity, and ROS production of peripheral blood neutrophils after challenge with D. pteronyssinus reflect an enhanced systemic inflammation in allergic rhinitis and asthma patients with induced late-phase airway inflammation.