Cell-free DNA levels in plasma of patients with non-small-cell lung cancer and inflammatory lung disease.

BACKGROUND: The analysis of plasma cell-free DNA (cfDNA) is expected to provide useful biomarkers for early diagnosis of non-small-cell lung cancer (NSCLC). However, it remains unclear whether the intense release of cfDNA into the bloodstream of NSCLC patients results from malignancy or chronic inflammatory response. Consequently, the current diagnostic utility of plasma cfDNA quantification has not been thoroughly validated in subjects with chronic respiratory inflammation. Here we assess the effect of chronic respiratory inflammation on plasma cfDNA levels and evaluate the potential clinical value of this phenomenon as an early lung cancer diagnostic tool. METHODS: We measured plasma cfDNA concentrations in 50 resectable NSCLC patients, 101 patients with chronic respiratory inflammation (chronic obstructive pulmonary disease, sarcoidosis, or asthma) and 40 healthy volunteers using real-time PCR. RESULTS: We found significantly higher plasma cfDNA levels in NSCLC patients than in subjects with chronic respiratory inflammation and healthy individuals (P<0.0001). There were no significant differences in plasma cfDNA levels between patients with chronic respiratory inflammation and healthy volunteers. The cutoff point of >2.8 ng ml(-1) provided 90% sensitivity and 80.5% specificity in discriminating NSCLC from healthy individuals (area under the curve (AUC)=0.90). The receiver-operating characteristics curve distinguishing NSCLC patients from subjects with chronic respiratory inflammation indicated 56% sensitivity and 91% specificity at the >5.25-ng ml(-1) cutoff (AUC=0.76). CONCLUSIONS: We demonstrated that elevated plasma cfDNA levels in NSCLC resulted primarily from tumour development rather than inflammatory response, raising the potential clinical implications for lung cancer screening and early diagnosis. Further research is necessary to better characterise and identify factors and processes regulating cfDNA levels in the blood under normal and pathological conditions.

TNFalpha and INFgamma inducing capacity of sera from patients with interstitial lung disease in relation to its angiogenesis activity.

Sera from interstitial lung diseases (ILD) constitute a source of mediators participating in angiogenesis. The nature of these mediators is unknown. The aim of our study was to asses whether preincubation with sera from ILD patients could influence TNFalpha and INFgamma production by normal mononuclear cells (MNC) challenged with LPS (for TNFalpha) or PHA (for INFgamma), and to correlate the cytokine levels with angiogenic properties of sera. The study population consisted of 53 patients with ILD, 16 with sarcoidosis (SAR), 11 with avian fanciers' lung (AFL), 10 with scleroderma with pulmonary manifestations (SCL), 9 with Wegener's granulomatosis (WG), and 7 with pulmonary Langerhans' cell histiocytosis (PLH). As a control, sera from 10 healthy volunteers were used. Neovascularization was measured by a leukocyte-induced angiogenesis assay according to Sidky and Auerbach. TNFalpha and INFgamma production was estimated by a one-step culture immunoassay CytoTraptrade mark TNFalpha DIA (Biosource Europe S.A.) after 3 h of incubation with LPS (TNFalpha) and 24 h incubation with PHA (INFgamma). Sera from sarcoidosis patients, WG patients, and AFL patients significantly stimulated angiogenesis in comparison with sera from healthy donors (P<0.001). Sera from PLH and SCL patients presented anti-angiogenic properties in comparison with sera from healthy donors and from each examined group (P<0.001). Comparing with other groups, preincubation with sera from AFL and WG patients led to a significant increase in TNFalpha production by normal MNC. Highly significant correlation between serum angiogenic activity and TNFalpha production by MNC was observed in SCL, WG, and AFL (r=0.74, P<0.01). we conclude that TNFalpha may play an important role in neovascularization in ILD.

Inflammatory markers in the exhaled breath condensate of patients with pulmonary sarcoidosis.

Pulmonary sarcoidosis may progress to fibrosis in some patients, so that close monitoring of its activity is essential for recommending clinical strategy. Examination of airway inflammatory markers in bronchoalveolar lavage (BAL) is one of the methods applied to assess the disease severity. Recently, the expired breath condensate (EBC) has become another source of cytokines and mediators. In sarcoidosis, except for NO and oxidative stress markers, no other mediators have yet been estimated in the exhaled air. In the present study we attempted to answer the question of whether airway inflammatory markers in pulmonary sarcoidosis patients might be assessable in EBC and to what extend these markers might reflect the disease activity in the lungs IL-6, TNF-alpha, PAI-1, and IGF-1 were measured by Elisa method in EBC and BALF samples from 9 patients with newly-diagnosed pulmonary sarcoidosis. TNF-alpha, IGF-1, and PAI-1 levels in EBC and BAL samples were comparable and closely positively correlated [TNF-alpha (r=0.79, P<0.001), IGF-1 (r=0.94, P<0.001), and PAI-1 (r=0.81, P<0.001)]. In contrast, IL-6 concentration in EBC was significantly lower compared with that in BALF, while the correlation between both materials was negative (r=-0.47, P<0.05). An important distinction in IL-6 performance, which might explain this inconsistency, is its tendency to form more complex molecular forms of a higher weight than that of other cytokines. Our study shows that EBC reflects cytokine production in the lung as effectively as BALF, providing that the characteristics of proteins evaluated allow their easy transfer into the exhaled air. Further studies are required before accepting EBC samples as an equivalent to BALF.

Human leukocyte antigen-DRB1 position 11 residues are a common protective marker for sarcoidosis.

Genetic factors, in particular human leukocyte antigens (HLAs) are important determinants of susceptibility to sarcoidosis, a chronic granulomatous disease of undetermined etiology. To clarify the role of HLA in sarcoidosis we determined HLA-DR and -DQ alleles in case-control samples from three European populations (United Kingdom, Czech, and Polish) and compared these results with those published for three additional populations (Italian, Japanese, and Scandinavian) to determine whether the HLA-DR and/or -DQ alleles act as ethnic-dependent, or ethnic-independent modifiers of disease risk. Although variations were apparent in the alleles associated with susceptibility, reductions in the frequency of alleles associated with protection were remarkably consistent in the six populations. Previously detected associations between single-nucleotide polymorphisms at the TAP2 locus and sarcoidosis were shown to be due to linkage disequilibrium with the HLA-DR locus. The protective HLA-DR alleles, which encode the DR1 and DR4 antigens, were found to share characteristic small hydrophobic residues at position 11, which were replaced by small hydrophilic residues in the remaining, nonprotective, HLA-DR alleles. This residue position is within a pocket of the HLA-DR complex antigen binding groove (designated P6), where it is the only variable amino acid and therefore determines the peptide binding preferences of this pocket. A highly significant reduction in the frequency of individuals carrying HLA-DR alleles with a hydrophobic residue at position 11 was observed in the sarcoidosis cases in the three populations we examined. This suggests this HLA-DR residue is an important protective marker in sarcoidosis.

[Predictors of success in smoking cessation among participants of spirometric screening for COPD]. / Czynniki okreslajace sukces w porzucaniu palenia u osób uczestniczacych w srodowiskowych badaniach spirometrycznych.

The aim of the study was to evaluate factors that could predict smoking cessation after a minimal antismoking counseling during spirometric screening for COPD. Every subject filled-in a simple questionnaire on clinical signs of COPD and tobacco habit, had a spirometry performed according to ATS standards and received a short antitobacco counseling together with a booklet on how to quit smoking. Out of 800 smokers over 40 years of age, smoking history of more than 10 packyears, screened for COPD in 1999, four hundred were invited a year later for a follow-up spirometry and evaluation of anti-smoking intervention. Of 383 patients, who responded to the invitation (208 M and 175 F, mean age 56.6 +/- 10.7 yrs), 52 (13.6%) quit smoking for one year and another 48 (12.5%) quit smoking temporarily and than resumed smoking. Smokers who permanently succeeded in quitting smoking were older (60.5 vs 55.9 years p < 0.01), started smoking later (age at starting smoking 22 vs 19.5 years p < 0.001), had a shorter tobacco exposition (28.8 vs 34.3 packyears p < 0.05), had lower lung function (FEV1%pred 80.5 vs 89.2% p < 0.05) and were less nicotine dependent (FTQ score 1 vs 4.8 p < 0.00001).

[Comparison of HLA class I and DR class II antigen frequency in Polish patients, in relation to different stages of the disease]. / Porównanie czestosci wystepowania swoistosci serologicznych antygenów klasy I i grup alleli DR klasy II ukladu HLA, wsród chorych na sarkoidoze w populacji polskiej, w odniesieniu do stadium choroby.

The aim of this study was to analyze the association between HLA class I and class II DR frequency in the different stages of sarcoidosis in Polish population. 88 patients and 30 healthy controls have been typed. Patients were divided into three groups depending on radiological findings. In the first group were 28 cases presenting the regression of the disease. In the second were 33 patients in stable stage II or III and in the third group 27 patients with pulmonary fibrosis (stage IV). The typing was performed by NIH method using commercial sera. There were no statistically significant differences between studied group in HLA-A class I. The frequency of HLA B-18 was statistically more frequent in patients with sarcoidosis compared to healthy controls. HLA-DR1 was not present in third group of patients and the difference was significant compared to healthy controls.

[Familial sarcoidosis in Poland: analysis of clinical characteristics and environmental aspects]. / Sarkoidoza rodzinna w Polsce: analiza obrazu klinicznego choroby oraz uwarunkowan srodowiskowych.

Sarcoidosis is a systemic disease of undetermined etiology although it can be related to genetic or environmental factors or both. We investigated 19 polish caucasian families with at least two affected relatives and healthy families members in order to evaluate the clinical aspects of familial sarcoidosis and the influence of environmental factors. We have found three types of familial sarcoidosis: 10 sib pairs, 1 sib triplet, 5 parent offspring pairs and one case of sarcoidosis in cousins. Most of sarcoidosis cases were histologically proved. A high frequency of chronic onset of the disease, chronic form with extrapulmonary sarcoidosis and fibrosis were observed. In only three families the course of the disease was similar in both affected relatives. There was no specific environmental agent found that could be related to the development of the disease. We concluded that it may be possible that familial sarcoidosis can have poorer prognosis that non-familial form, but there is no epidemiological data available about the clinical aspects of sarcoidosis in Poland. The clinical aspects of familial sarcoidosis can suggest the inherited susceptibility to the disease. The etiologic extrinsic factor has not been identified, but it doesn't exclude its role in the pathogenesis.

[Histocompatibility class I antigens loci A and B in familial sarcoidosis in Poland]. / Antygeny zgodnosci tkankowej klasy I loci A i B w sarkoidozie rodzinnej w Polsce.

Thirty three affected members from 17 Polish sarcoidosis families and their healthy relatives (61 subjects) have been typed for HLA class I (A and B). Controls consisted of 101 healthy Polish subjects. The HLA typing was performed by the serological method using the standard microcytotoxicity test. No significant differences were observed between group 1 (33 affected family members), group 2 (61 healthy relatives) and group 3 (healthy controls) considering the HLA class I locus A antigens. The frequencies of HLA B8, HLA B16 and HLA B40 were significantly higher in affected sarcoidosis families members compared to control subjects (p < 0.05). When comparing all families members (affected and non-affected as a one group) to the control group we found a significant overrepresentation of HLA B12 and B35 in the control group. We concluded that HLA antigens locus B: B12 and B35 may have a protective function against the disease; the role of HLA class I antigens in the pathogenesis of sarcoidosis needs further evaluation.

[Polymorphism of histocompatibility class II antigens coded with the DRB gene in in familial sarcoidosis in Poland]. / Polimorfizm ukladu antygenów zgodnosci tkankowej klasy II kodowanych genem DRB w sarkoidozie rodzinnej w Polsce.

Several studies suggested association between some human leukocyte antigen (HLA) alleles and sarcoidosis, but none has been conclusive. To confirm possible association of sarcoidosis with HLA-DRB1, -DRB3,- DRB4, -DRB5 associated alleles HLA-DR genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in 17 polish families with familial sarcoidosis and in 101 healthy controls. The families with sarcoidosis consisted of 31 affected first-degree relatives from 16 families, 2 affected cousins from 1 family and 78 healthy relatives of those patients. We found 3 varieties of familial sarcoidosis: a) in parent and offspring (5 pairs), b) in siblings (10 sib pairs and 1 sib triplet) and in cousins (1 family). Genotyping for HLA-DRB1,-DRB3,-DRB4,-DRB5 revealed an over-representation of HLA-DR5(12) and DRw52 among antigens shared by affected relatives comparing to the control group. A significant increase in the frequency of HLA-DR7 and HLA-DRw53 antigens (p < 0.05) was found in subjects from the control group. Comparing the group of family members (affected and healthy relatives taken together, n = 111) with the control group (n = 101) we found a significant differences in the distribution of HLA-DR2(15), HLA-DR5(12), HLA-DR6, HLA-DR9 and HLA-DRw52. Those antigens were more frequent (p < 0.05) in members from families with sarcoidosis. The frequencies of HLA-DR1, HLA-DR2(16), HLA-DR7 and HLA-DRw53 were significantly higher (p < 0.05) in the control group. Presented results suggest that HLA-DRB alleles contribute to the susceptibility to sarcoidosis in the Polish population.

[A probe for evaluating the role of HLA class II antigens coded with the DRB gene in etiopathogenesis of familial sarcoidosis in Poland using the K-nearest neighbor experimental statistical method]. / Próba oceny roli antygenów ukladu HLA klasy II kodowanych genem DRB w etiopatogenezie sarkoidozy rodzinnej w Polsce eksperymentalna metoda statystyczna K-najblizszych sasiadów.

The aim of this study was evaluation of the significance of HLA-DRB1,-DRB3,-DRB4,-DRB5 associated alleles in the genetic susceptibility to sarcoidosis. We investigated 17 Polish 'families with' familial occurrence of the disease. Thirty three affected family members and 78 healthy relatives and 101 healthy individuals (control group) have been typed for HLA class II DR antigens. Assuming that relatives from families with two or more affected members are more susceptible to develop sarcoidosis we considered two classes: affected and healthy family members taken together (class 1, N = 111) and healthy control group (class 2, N = 101). HLA antigens from both classes were compared using a statistical pattern recognition method. This method, called k-NN method, assumes that objects (individuals) are described by a certain number of variables called features. Selected features that played an important role in the decision to which class an individual person would be classified were: HLA-DR7, HLA-DR2(16), HLA-DR1, HLA-DR5(12), HLA-DR6(14), HLA-DR6(13), HLA-DR9, HLA-DR5(11) and HLA-DRw52. K-NN method allowed to classify properly 76% of studied subjects to healthy or disease susceptible group. However, 24 out of 100 individuals would be misclassified which gives the total error rate of 0.24. We concluded that using HLA-DR antigens as features characterising every individual we can predict with high probability to which class ("high risk" or "healthy") individual would belong.

Analysis of MHC encoded antigen-processing genes TAP1 and TAP2 polymorphisms in sarcoidosis.

Sarcoidosis is a chronic granulomatous disease of unknown etiology. Several studies have suggested involvement of human leukocyte antigen (HLA) genes in sarcoidosis susceptibility. HLA associations described have not been consistent, possibly because of additional susceptibility genes adjacent to or within the major histocompatibility complex (MHC) such as genes for the transporter associated with antigen processing (TAP). The aim of this study was to analyze TAP gene polymorphisms in patients with sarcoidosis using the amplificatory refraction mutation system (ARMS) PCR. To determine whether any association between TAP gene variation and sarcoidosis was ethnic-independent we examined two European populations: 117 unrelated UK Caucasoid patients with sarcoidosis and 290 healthy UK control subjects, and 87 unrelated Polish Slavonic patients with sarcoidosis and 158 healthy Polish control subjects. We detected significant differences in TAP2 between the UK control and patient groups, and in TAP2 between the Polish control and patient groups. Comparing the UK and Polish control groups, we observed a difference in TAP1. Examination of HLA-DPB1 in our UK population showed no associations with disease or between variants at the TAP gene loci and HLA-DPB1 variants. These results suggest associations at the TAP loci occur independently of HLA-DPB1 associations, that TAP associations seen may be involved in determining sarcoidosis susceptibility, and that such susceptibilities differ between UK and Polish populations. This first study of TAP genes in UK and Polish sarcoid populations has demonstrated the importance of using multiple defined ethnic populations in defining the role genetic factors play in sarcoidosis susceptibility and the importance of candidate gene studies.

[Early, targeted population based screening for COPD. Preliminary study]. / Wczesne, srodowiskowe, celowane wykrywanie POCHP. Doniesienie wstepne.

COPD is the fourth leading cause of death in Poland, unfortunately diagnosed not early enough. The aim of the study was to establish prevalence of COPD in chronic smokers. Therefore, using daily press and TV, smokers with at least 10 year history of smoking, over 40 years of age, were invited for a free spirometry. 263 subjects (177 M and 86 F) mean age 54 +/- 0.6 years were examined. Most of them (97.7%) were smokers with a history of 32.2 +/- 0.9 pack-years, 6 persons (2.3%) were passive smokers. 110 persons (41.8%) presented bronchial obstruction, the remaining (58.2%) had normal spirometric values. Following recommendations of the Polish Society of Physio-pneumonology bronchial obstruction was classified as mild in 25.1%,- moderate in 12.1% and severe in 4.6% subjects. Majority of examined subjects presented with COPD symptoms, cough (62.7%), expectoration (68.8%) and dyspnoea (50.2%). The presence of those symptoms did not differ among groups with different severity of bronchial obstruction. However, there were significant differences in age (p < 0.05) and years of smoking habit (p < 0.01). The great efficacy of targeted screening for COPD (40%) should be an incentive to perform routine spirometric examination in smokers with more than 20 years of smoking history.

[Evaluation of effects of antituberculous treatment on the course of sarcoidosis]. / Ocena wplywów leczenia przeciwpratkowego na przebieg sarkoidozy.

From 1960 to 1994 2150 sarcoid patients were observed in our Sarcoidosis Clinic. Before the diagnosis of sarcoidosis was confirmed, 52 of those patients were treated with tuberculostatics because of the radiological changes in the lung, diagnosed as tuberculosis. In no case any conventional method-smear examination for acid bacilli or culture identification was positive. There was no radiological improvement after treatment in any patient. Subsequently in all those patients sarcoidosis was diagnosed, by typical histology (48 cases) or on clinical grounds (4 patients). In 15 cases spontaneous remission was observed. 37 patients were treated with steroids and in 31 of them radiological improvement was found. In six cases stabilization of the disease was noted. The observation time after steroids treatment was from one year to 27 years. In only one case tuberculosis of the lung has developed.

[Co-stimulating effect of extracellular matrix proteins on T lymphocyte proliferation in patients with sarcoidosis. Preliminary tests]. / Ko-stymulacyjny efekt bialek ECM na proliferacje limfocytów T u chorych na sarkoidoze. Badania wstepne.

Although very little is known about the aetiology of sarcoidosis, its immunopathogenesis is now better known. The interaction of alveolar macrophages and T cells may play a role in the pathomechanism of the disease. To infiltrate the tissue, lymphocytes have to migrate through the subendothelial basement membrane and interstitium, rich in extracellular matrix (ECM). The interaction of lymphocytes with proteins of the ECM may play an important role in the migration, accumulation and activation of these cells. The aim of our study was to estimate the ability of the ECM components (collagen I, collagen IV and fibronectin) to co-stimulate T-cells in patients with sarcoidosis. The peripheral blood was obtained from 14 sarcoid patients. The disease was confirmed histologically in 9 cases and in 5 patients on clinical grounds. In radiological findings 4 persons were at the first stage of the disease, 4 at the second, in three cases interstitial changes were found and in three patients, the fibrosis on the X-ray was noticed. No one of those patients were treated with steroids during last 2 months. Normal peripheral blood T cells are strongly co-stimulated by ECM proteins. In contrast, lymphocytes from patients with sarcoidosis were inhibited by the ECM proteins. The mean co-stimulation ratios (OKT3 + ECM proteins:OKT3 alone) were significantly lower for all ECM proteins (collagen I: p < 0.00009; collagen IV: p < 0.02; fibronectin: p < 0.04). Our data shed a new light on the nature of sarcoidosis associated immunodeficiency and suggest that disturbed T cells: ECM interactions may play a role in the pathogenesis.

[Recurrence of pulmonary sarcoidosis]. / Nawroty sarkoidozy pluc.

From September 1960 to December 1992, 2021 patients with pulmonary sarcoidosis were registered in our Sarcoidosis Clinic. 462 patients were treated with steroids. Treatment was initiated either because of radiological evidence of disease progression after 6 months observation, lack of radiological improvement together with abnormalities of pulmonary function, presence of extra-pulmonary lesions. In 42 cases (9% of treated group); 19 men and 23 women, mean age 35 years, relapse of disease was observed from 6 months to 6 years after completion of treatment. At the entry to the first treatment 11 patients were assessed as having stage I disease, 30 as stage II and one as stage III. In 16 subjects of this group (38%), extra-pulmonary lesions were presents; skin lesions in 8 cases and enlarged peripheral lymph nodes in 6. After initial treatment, radiological and clinical improvement was observed in 32 patients. The second treatment results in radiological improvement in 38 patients, 10 are being still treated. In 4 cases a second relapse was noted. In three patients regression of pulmonary changes was achieved, in one case stabilization of the disease was observed.

[Effect of intravenous ambroxol hydrochloride on lung function and exercise capacity in patients with severe chronic obstructive pulmonary disease]. / Wplyw chlorowodorku ambroksolu podawanego dozylnie na czynnosc pluc i wydolnosc wysilkowa u chorych na ciezka, przewlekla obturacyjna chorobe pluc.

The effect of Ambroxol hydrochloride on spirometry, pulmonary gas exchange and exercise capacity in patients with COPD were evaluated. Twenty patients mean age 59 +/- 9 yr presenting with severe airways obstruction were divided by random number to two groups: treated (L) and control (K). L patients received iv infusion of one gram of Ambroxol in 500 ml of 0.9% saline daily, for 5 days. K patients received infusions of 500 ml 0.9% saline at the same intervals. Blood gases, capnography and incremental exercise test on cycloergometer were performed before treatment and after the fifth dose of the drug or placebo. Before treatment spirometry, blood gases, capnometric++ indices and exercise tolerance were similar in both groups. After treatment in patients from L group an increase in VC from 1.78 +/- 0.61 L to 2.04 +/- 0.58 L (p < 0.01), FEV1 from 0.96 +/- 0.43 L to 1.11 +/- 0.49 L (p < 0.01), MEF25 from 1.36 +/- 0.93 L/sec to 1.61 +/- 0.96 L/sec (p < 0.01) was found. End-tidal carbon dioxide in 60 sec hyperventilation test decreased from 3.7% to 2.9% (p < 0.001). There were no differences in exercise tolerance and blood gases after treatment. In the control group all studied variables remained unchanged. We conclude that Ambroxol improves airways patency in patients with severe COPD.

[Evaluation of the effectiveness of medical consultation regarding smoking cessation]. / Ocena skutecznosci porady lekarskiej na porzucenie nalogu palenia.

Smoking habits were assessed by a questionnaire in workers of a small factory during their prophylactic medical check-up. In all subjects spirometry and CO levels in expired air were determined. In the studied group 43.8% were smokers. Respiratory indices were decreased in smokers in comparison to ex- and non-smokers. In all smokers higher (3x) levels of CO were found. After a year 5 subjects ceased to smoke (15.6% of the smokers).