Virulence of clinical Mycobacterium tuberculosis strains in Lodz, Poland.

SETTING: The transmission of tuberculosis (TB) in the population and the development of disease are determined not only by the patient's immunological status, but also by the virulence of Mycobacterium tuberculosis strains. OBJECTIVE: To examine the virulence of M. tuberculosis clinical isolates with recognised transmission collected from 2006 to 2007 in a population in Lodz, Poland. METHODS: A total of 36 isolates were studied to determine their sensitivity to human neutrophil peptide 1 (HNP-1) and intracellular growth within THP-1 cells. Bacterial strains were cultured using HNP-1 at different concentrations. After incubation, the number of colony-forming units (cfu) was determined by bacteria plating. The intracellular survival was examined on days 3, 6 and 8 post-THP-1 infection by cfu enumeration. RESULTS: Overall, 69% of the isolates showed greater resistance to the highest HNP-1 concentration (15 g/ml) than the virulent H37Rv strain, and the growth of 10 strains was totally inhibited. On day 8, 56% of the strains displayed higher cfu numbers than the virulent H37Rv strain. CONCLUSION: The results suggest that isolates from our urban population represent highly virulent phenotypes. We could not find any significant difference in virulence between strains with unique genotypes and those in clusters.

Enhanced exhalation of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in patients with COPD exacerbation: a prospective study.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) and its inhibitor tissue inhibitor of metalloproteinase-1 (TIMP-1) are involved in the pathogenesis of airway inflammation in patients with chronic obstructive pulmonary disease (COPD). However, no study so far has addressed their value as noninvasive biomarkers of airways inflammation. OBJECTIVE: To evaluate MMP-9 and TIMP-1 concentrations in the exhaled breath condensate (EBC) of patients with stable COPD and also during the exacerbation episode. METHODS: EBC and serum samples were collected in 17 stable-phase COPD patients who were current smokers as well as during their first exacerbation episode, and in 22 asymptomatic smokers. EBC and serum levels of MMP-9 and TIMP-1 were measured with ELISA kit. RESULTS: Mean EBC MMP-9 and TIMP-1 levels were higher in patients with stable COPD than in asymptomatic smokers. Exacerbation of COPD increased 2-fold the exhalation of MMP-9 (18.5 ± 10.1 ng/ml vs. 8.9 ± 6.2 ng/ml, p = 0.01) and TIMP-1 (to 41.1 ± 20.4 ng/ml vs. 16.4 ± 6.8 ng/ml, p < 0.001). Both, MMP-9 and TIMP-1 in EBC correlated negatively with FEV(1) (% predicted) at baseline (r = -0.78, p < 0.001 and r = -0.73, p < 0.001) and during the exacerbation episode (r = -0.57, p = 0.02 and r = -0.65, p = 0.005). Similar negative correlations were noted with FVC (% predicted), except for MMP-9 in EBC at exacerbation. Exhaled MMP-9 and TIMP-1 did not correlate with serum concentrations in COPD patients, either at baseline or during exacerbation. CONCLUSION: Exhaled MMP-9 and TIMP-1 increased during COPD exacerbation and was negatively correlated with spirometric variables, which suggests the usefulness of their measurement in EBC for the monitoring of airways inflammation. However, to better assess their diagnostic or prognostic value larger studies are necessary.

Elevated exhalation of hydrogen peroxide and circulating IL-18 in patients with pulmonary tuberculosis.

Mycobacteria are the strong stimulators of respiratory burst, resulting in production of reactive oxygen species and nitrogen intermediates. The aim of our study was to assess the concentration of hydrogen peroxide (H(2)O(2)) in expired breath condensate (EBC) and the serum level of interleukin-18 (IL-18) in patients with active pulmonary tuberculosis (TB) before introduction of chemotherapy and after 2 months of treatment. Sixteen patients, current cigarette smokers, with advanced pulmonary TB were enrolled into the study. As a control served two groups: I group--16 asymptomatic cigarette smokers, II group--17 healthy never smoked subjects. The level of H(2)O(2) in EBC was significantly higher in patients with TB (1.3+/-0.7 microM) as compared to cigarette are healthy nonsmoker subjects (0.4+/-0.1 and 0.2+/-0.1 microM, respectively, P<0.05). Two months of treatment significantly decreased the level of H(2)O(2) exhalation in TB patients (0.5+/-0.3 microM) to the value that was not different from that in asymptomatic smokers but was still higher than in never smoked subjects. Serum concentration of IL-18 in TB patients was higher than that found in both control groups either before and after antituberculous treatment (P<0.05). Exhaled H(2)O(2) did not correlate with circulating IL-18 in TB patients before or after treatment. These results demonstrated the occurrence of oxidative stress in the airways of TB patients completely attenuating after 2 months of successful antituberculous treatment.

Circulation level of matrix metalloproteinase-9 is correlated with disease severity in tuberculosis patients.

SETTING: Parenchymal lung destruction accompanied by active tuberculosis is, at least in part, caused by host as well as bacillus metalloproteinases. Mycobacterium tuberculosis has been shown to stimulate MMP-9 expression in the lung of infected organisms. DESIGN: We have used quantitative zymography and computer-assisted image analysis to measure the levels of type IV collagenases in 20 serum samples of patients with active tuberculosis and in 23 serum samples of healthy volunteers. RESULTS: Mean levels of the serum MMP-9 were over three-fold higher in tuberculous samples compared with normal serum (P < 0.0001), whereas the MMP-2 levels did not differ in these two groups. The levels of MMP-9 were significantly higher in subjects with advanced disease than in those with only limited disease changes (P < 0.05). CONCLUSIONS: We suppose that the elevation of serum MMP-9 levels in patients with tuberculosis is affected by the augmentation of synthesis and/or secretion of this enzyme by inflammatory cells in response to M. tuberculosis infection. The observed association between the serum MMP-9 level and the extent of radiological change suggests that the quantification of the serum level of this enzyme may constitute a supplementary test in pulmonary tuberculosis diagnostics.

Enhanced lipid peroxidation in cancer tissue homogenates in non-small cell lung cancer.

The oxidative modification of nucleic acids by reactive oxygen species may lead to malignant conversion, but its exact role in lung cancer biology is still not clear. Lipid peroxidation, a well-known index of free radicals activity, is a process of oxidative polyunsaturated acids destruction. Our study was aimed to investigate the level of lipid peroxidation ex vivo in tumor tissue and lung parenchyma obtained from patients with non-small cell lung cancer. Thirty-two patients with lung cancer (including 19 with squamous cell lung cancer) were enrolled in the study. During a surgical resection, tumor tissue and lung parenchyma were obtained and the concentration of lipid peroxidation products, i.e. conjugated dienes and lipid hydroperoxides, measured. In the whole group of patients the concentrations of conjugated dienes and lipid hydroperoxides in the tumor tissue were higher than those in lung parenchyma (1.008 +/- 0.503 A233 nm vs. 0.717 +/- 0.283 A233 nm; p < 0.05 and 0.109 +/- 0.062 A532 nm vs. 0.102 +/- 0.087 A532 nm; p < 0.05, respectively). Similar results were obtained in squamous cell carcinoma patients (0.975 +/- 0.348 A233 nm vs. 0.708 +/- 0.300 A233 nm; p > 0.02 and 0.094 +/- 0.029 A532 nm vs. 0.080 +/- 0.071 A532 nm; p < 0.05, respectively). In both groups of patients, a positive correlation between concentration of conjugated dienes in tumor tissue and clinical stage (R = 0.45; R = 0.52; p < 0.05, respectively) was found. Our results confirm the enhanced lipid peroxidation in cancer tissue as compared with matched lung parenchyma. Additionally, a higher level of oxidative stress, expressed as the concentration of conjugated dienes in tumor tissue, was associated with clinical progression of the tumor.

[Atopy with recurrent wheezy bronchitis in children]. / Atopia w nawrotowym obturacyjnym zapaleniu oskrzeli u dzieci.

Childhood asthma and wheezy bronchitis are the most frequent chronic diseases of childhood. Unfortunately their clinical symptoms are similar--which makes it difficult to distinguish between the two, and therefore decide on proper treatment of patients. The aim of the study was to establish the parameters leading to right diagnosis. The study was performed in 50 children aged 3-7 years with recurrent wheezy bronchitis. All patients underwent allergological examinations (skin tests with inhaled allergens, personal and family history and serum total and specific IgE levels). 42 of them were tested for ventilatory parameters with bronchodilatation test. Three features of atopy were found in 21 (42%) patients, two features in 7 (14%) patients. In 31 (62%) children at least one feature of atopy was shown. 7 (17%) of the examined children had significant bronchodilatation after salbutamol inhalation. Finally in 24 (48%) of children suffering from wheezy bronchitis, bronchial asthma was diagnosed. The diagnosis was confirmed by antiasthma tic treatment with cromones or inhaled corticosteroids. In great majority (88%) of patients bronchial asthma was atopic. In 23% wheezy bronchitis children not diagnosed with bronchial asthma features of atopy were observed. They are of bronchial asthma risk group.

[The evaluation of efficacy and tolerability of salbutamol administered by nebulization in patients with stable severe bronchial asthma]. / Ocena skutecznosci i tolerancji Salbutamolu w nebulizacji u chorych na stabilna ciezka astme oskrzelowa.

The aim of the study was the assessment of the effect of salbutamol in nebulization on ventilatory parameters and heart action in patients with stable severe well controlled bronchial asthma. The study was performed in 30 asthmatics (19 females and 11 men) with incomplete reversibility of airflow obstruction after salbutamol inhalation administered via MDI device. The mean age was 47 years, the duration of asthma--18 years. Mean FVC value was 2.46 L (66.6%), FEV1--1.76 L (56.8%), and MEF50--1.74 L/s (40.4% predicted). The study was performed according to the double-blind crossover method with placebo used. On the first day the reversibility test with 400 mg salbutamol was performed. On the two consecutive days salbutamol (Steri-Neb Salamol 2.5 mg) and placebo in nebulization were randomly administered. Ventilatory parameters were measured before and in 20, 40, 60, 120, 180 and 240 minutes after the nebulization. At the same time points the heart action was assessed by physical examination. The changes in FVC, FEV1 and MEF50 were expressed in absolute values and as a relative increase in relation to predictive value. The significant improvement of measured ventilatory parameters was observed as early as 20 minute after the nebulization. This increase in MEF50 lasted 2 hours, in FEV1--3 hours and in FVC 4 hours. The relative increase in MEF50 was significant higher than the remaining parameters. The significant increase in heart rate was noted after salbutamol nebulization.

Comparison of hydrogen peroxide generation and the content of lipid peroxidation products in lung cancer tissue and pulmonary parenchyma.

Lipid peroxidation, as a well-known index of reactive oxygen species activity, not only in lung biochemistry, is an oxidative process associated with membrane lipid destruction. Also, the oxidative modification of nucleic acids by reactive oxygen species is of remarkable biological importance as it may contribute to malignant conversion, but its exact role in lung cancer biology is still not clear. Our study aimed to investigate the level of lipid peroxidation ex vivo in tumour tissue and lung parenchyma obtained from patients with lung cancer. Forty-two patients with lung cancer were enrolled into the study. During a surgical resection, tumour tissue and lung parenchyma were obtained and concentration of lipid peroxidation products, thiobarbituric acid-reactive substances and Schiff bases, and spontaneous generation of hydrogen peroxide, were measured. The concentration of thiobarbituric acid-reactive substances (P<0.001) in the tumour tissue was higher than that in lung parenchyma. In small cell lung cancer as well as in squamous cell carcinoma patients, a positive correlation between spontaneous generation of hydrogen peroxide in tumour tissue and clinical stage (r = 0.43; r = 0.46; respectively) was found. Our results prove enhanced lipid peroxidation in cancer tissue as compared with matched-lung parenchyma. In small cell lung cancer and squamous cell carcinoma patients, the high level of oxidative stress, expressed as a spontaneous generation of hydrogen peroxide in tumour tissue, was associated with clinical progression of tumour's stage.

[Evaluation of the relationship between the clinical course of severe asthma and ventilatory parameters]. / Ocena zwiazków miedzy klinicznym przebiegiem ciezkiej astmy oskrzelowej a wskaznikiem wentylacji.

The aim of the study was the assessement of relations between the clinical symptoms of asthma, ventilatory parameters and nonspecific bronchial hyperresponsiveness. 22 patients with severe asthma, 12 females and 10 men, aged 43-68 years (mean 56) were observed for two years. The duration of asthma ranged from 6 to 38 years (mean 18). All the patients were treated with inhalatory steroids in dose 800-1200 mg. Three months before entering the study the patients had no exacerbation or respiratory tract infection. Throughout the study diary cards were filled and the symptoms were recorded on 0-4 scale. PEF was measured two times a day, the highest value noted. Spirometry and reversibility tests were performed. On the last day patients underwent histamine challenge test. Data from four weeks were analyzed statistically. The significant correlation was established between PEF variability and clinical symptoms scores, both mean and measured in the last day of the study. The relationship between mean PEF variability and bronchial hyperresponsiveness was also observed.

[Bacteriologically unconfirmed pulmonary tuberculosis: minimal changes problem]. / Gruzlica pluc niepotwierdzona bakteriologicznie--problem zmian minimalnych.

The correct diagnosis in patients with small radiographic changes sputum smear-negative is usually time consuming and highly problematic. In this report the sensitivity of the new direct amplification tests was evaluated in these clinical situations and the immunological criteria of active disease were discussed. Patients with small radiographic changes were evaluated in epidemic terms and different kinds of therapeutic regimens were assessed.

Increased serum concentrations of conjugated diens and malondialdehyde in patients with pulmonary tuberculosis.

During pulmonary inflammation increased amounts of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) are produced as a consequence of phagocyte respiratory burst. One of the manifestation of these free radical-mediated processes is lipid peroxidation (LP). The aim of our study was to assess the concentration of lipid peroxidation products (LPPs), conjugated diens (CD) and malondialdehyde (MDA), in patients with active TB. Forty-two patients were enrolled into the study. Half (group I) had advanced TB and were sputum smear-positive. The remainder (group II) had only small radiographical changes and were sputum smear-negative. Serum concentrations of CD and MDA were measured at days 0, 7, 14 and 28 in group I and day 0 in group II. We found that in all patients with active TB CCD (1.0 +/- 0.05A233) and CMDA (2.01 +/- 0.16 nmol dl-1) were significantly elevated compared to healthy controls (0.67 +/- 0.03A233 and 1.36 +/- 0.08 nmol dl-1, respectively) (P < 0.001). The highest levels of LPPs were in patients with advanced TB. These concentrations were stable during the first month of anti-tuberculous therapy. Our data indicated that, as in bacterial pneumonia, LPPs were enhanced in active TB. The levels of LPPs depended on the form of the disease as they were higher in subjects with advanced disease than in those with only small radiographical changes. Further studies are needed to assess the role of antioxidants as adjuvant therapy in patients with pulmonary TB.

Clinical utility of a commercial ligase chain reaction kit for the diagnosis of smear-negative pulmonary tuberculosis.

SETTING: In paucibacillary forms of smear-negative tuberculosis it is very difficult to establish a correct and rapid diagnosis, as several weeks are usually required to obtain positive results from culture. In the last few years new rapid techniques based on molecular biology for the detection of Mycobacterium tuberculosis have been introduced. OBJECTIVE: The aim of this study was to evaluate the utility of the ligase chain reaction method (LCx, Abbott) for the diagnosis of smear-negative pulmonary tuberculosis. DESIGN: Thirty smear-negative patients with radiographic changes and clinical signs consistent with TB participated in the study. Sputum and bronchial aspirate were assessed according to traditional methods on Löwenstein-Jensen medium, and bronchoalveolar lavage (BAL) was assessed by the LCx test and the Bactec 460 system. Another 30 patients with non-tuberculous infections were included in the study as controls. RESULTS: Of the 30 patients suspected of tuberculosis, 19 had active disease on clinical, bacteriological and radiographic grounds, nine inactive tuberculosis and two had lung cancer. Bacteriological confirmation was obtained in 12 of the 19 (63.2%) patients with active tuberculosis. The sensitivity of sputum culture was 42.1% and bronchial aspirate culture 47.4%. BAL fluid revealed positive results in 57.9% using both LCx and Bactec. The results of the LCx assay can be obtained in 5 hours as opposed to several weeks using other methods. CONCLUSION: The LCx test may be useful in the diagnosis of smear-negative pulmonary tuberculosis and may be recommended in these clinical situations.

[Concentration of selected cytokines associated with monocytes/macrophages in serum of patients with different clinical stages of pulmonary tuberculosis]. / Stezenie wybranych cytokin zwiazanych z ukladem monocytarno/makrofagowym w surowicy chorych na gruzlice pluc o róznym przebiegu klinicznym.

Monocytes/macrophages are the main effector cells as well as the essential elements in granuloma formation in tuberculosis. Serum concentration of some cytokines connected with monocytes/macrophages like IL-12, MCP-1, TNF-alpha i sTNFRI was assessed in patients with advanced bacteriologically confirmed pulmonary tuberculosis (I group) and in those with minimal tb changes, usually without clinical signs of the disease, and sputum smear-negative (II group). We found that patients from group I had significantly higher concentration of IL-12 and sTNFRI and lower level of MCP-1 in comparison to group II. Significant difference between groups of patients with tuberculosis and the control group was found only while assessing serum concentration of TNFRI.

[The influence of salmeterol and salbutamol on bronchial constriction in patients with bronchial asthma]. / Wplyw salmeterolu i salbutamolu na zwezenie oskrzeli u chorych na astme oskrzelowa.

The aim of the study was to compare bronchodilatating effect of inhaled salmeterol, salbutamol and both drugs administered simultaneously. 14 subjects (7 females, 7 men), aged 30-65 years (mean 49 yrs), suffering from moderate to severe asthma were examined. The improvement of FEV1 greater than 15% within 15 minutes of inhaling 200 micrograms of salbutamol was demanded. The study was performed in randomized, double-blinded, placebo controlled design. Throughout the study, all patients kept a daily score of symptoms and treatment. Each day of the trial, FEV1 in 30, 60, 120, 180 and 240 minute was measured. The statistically significant increase of FEV1 was noted only in 30 minute after salbutamol and in 180 minute after inhaling of salmeterol in comparison to salbutamol alone. No significant advantage of adding salbutamol to the patients previously treated with salmeterol was observed.

[Immunological approach to the evaluation of the activity of the tuberculous process]. / Immunologiczne podejscie do oceny czynnosci procesu gruzliczego.

The aim of the study was to determine the immunological criteria of the activity of the lung tuberculosis. Some immunological tests based on cellular and humoral immunity were assessed in 3 groups: I--patients with active, bacteriologically confirmed tuberculosis, II--individuals with inactive minimal changes in the lung, III--healthy subjects. We found that among patients with active tuberculosis cellular immunity was depressed: both percentage of T-lymphocytes and their function, usually associated with cutaneous anergy to tuberculin. But the humoral immunity was enhanced. The authors have shown that the people with inactive minimal changes in the lung take place between the patients with active tuberculosis and healthy subjects.