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Evaluarea comparativa a markerilor serologici in sarcoidoza pulmonara

Articol publicat in revista CHEST decembrie 2010

Sarcoidoza este o boala inflamatorie multiorganica de etiologie necunoscuta, caracterizata prin granuloamele epiteloide necazeificate si alveolita limfocitara. Este o boala cu evolutie si prognostic imprevizibil, de aceea monitorizarea evolutiei bolii are o importanta deosebita. Markerii serologici cunoscuti care se pot obtine usor si in mod repetitiv sunt SAA (Amiloidul seric A), receptorul solubil pentru interleukina 2(sIL-2R), Lizozimul, Enzima de conversie a angiotensinei (ACE) si glicoproteina KL 6. In sarcoidosis, several cytokines, including IL-1and IL-6, are produced by alveolar macrophages that are activated by unknown immunological processes.9-12 IL-1 and IL-6 stimulate production of SAA and IL-2. Amiloidul seric A este o proteina de faza acuta mai putin sensibila la medicamentele imunosupresive cum ar fi corticosteroizii, din aceasta cauza este foarte utila in monitorizarea evolutiei bolii la pacientii tratati cu corticosteroizi. IL-2 production leads to T-cell activation. Activated T-cells express the IL-2 receptor (55kDa/75-kDa heterodimer) on their surfaces and release a soluble form of the 55-kDa chain. Nivelul seric al receptorului solubil pentru interleukina 2 creste la pacientii cu boala activa, datorita activarii limfocitelor T.

Lizozimul este o enzima bacteriolitica cu greutate moleculara mica prezenta in numeroase tesuturi si celule. In sarcoidoza lizozimul a fost identificat in macrofagi si in celulele epiteloide ale granuloamelor noi, nu si in leziunile mai vechi. Enzima de conversie a angiotensinei, cel mai cunoscut marker al activitatii bolii este produsa si de celulele epiteloide si reflecta cantitatea de granuloame. KL-6 este o glicoproteina asemanatoare mucinei, asociata cu pneumocitele de tip II si celulele epiteliale bronsice. Nivelul KL 6 este crescut in sarcoidoza, dar si in alte boli cum ar fi fibroza pulmonara idiopatica si pneumonita de iradiere. Previous studies reported that sIL-2R levels, but not ACE and lysozyme, were reflective of lymphocytic alveolitis.4 However, there have been no reports that compared the usefulness of serum markers as parameters reflecting alveolitis. Although it was demonstrated that sIL-2R and KL-6 levels, but not ACE and lysozyme, were suitable parameters for predicting disease progression in sarcoidosis patients,20-22 there have been no reports that compared the predictive values of these markers for increased parenchymal infiltration. Scopul studiului prezent este evaluarea utilitatii clinice a acestor markeri si de a determina daca nivelul lor poate fi corelat cu un risc crescut de infiltrate pulmonare in

cursul evolutiei. The aim of the present study was to evaluate the clinical usefulness of serum markers, including SAA, sIL-2R, lysozyme, ACE, and KL-6, to reflect lymphocytic alveolitis, and to assess if these serum markers levels at diagnosis reflect increased pulmonary infiltration during follow-up. Material si metoda
S-au colectat datele de la pacientii nou diagnosticati cu sarcoidoza la Ehime University Hospital in perioada 1990 2006. S-au selectionat numai cazurile confirmate histopatologic, fara comorbiditati: in total 43 de cazuri, 18 barbati si 25 femei. Parametrii urmariti: nivelul seric de Amiloidul seric A, receptorul solubil pentru IL2, Lizozim, glicoproteina KL 6. Radiografia toracica: pe baza radiografiei toracice pacientii au fost impartiti in 2 grupe: fara infiltrat parenchimal (stadiile radiografice 0 si 1) si cu infiltrat parenchimal (stadiile II, III, IV) Probele functionale respiratorii (VEMS) Capacitatea de difuziune a monoxidului de carbon Lichidul de lavaj bronhoalveolar.

stage 0, normal chest radiograph; stage I, bilateral hilar lymphadenopathy without parenchymal infiltration; stage II, bilateral lymphadenopathy with parenchymal infiltration; stage III, parenchymal infiltration without lymphadenopathy; stage IV, advanced fibrosis with evidence of honeycombing, hilar retraction, bullae, cysts and emphysema. In addition to this staging, patients were classified as follows: without parenchymal infiltration (stages 0 and I) and with parenchymal infiltration (stages II, III and IV).

Study population Clinical data for sarcoidosis patients, who were newly diagnosed at Ehime University Hospital (a referral center for sarcoidosis) between 1990 and 2006, were retrospectively analyzed. The diagnosis of sarcoidosis was established on the basis of clinical findings and histological evidence of non-caseating epithelioid cell granulomas, after excluding known causes of granulomatous diseases, according to the American Thoracic Society (ATS)/European Respiratory Society (ERS)/World Association of Sarcoidosis and other Granulomatous Disorders (WASOG) guidelines.1 No co-morbidity was present in any of these patients. At time of presentation, no patient was receiving any medication. The study protocol was approved by the Ethical Committee of the hospital. Informed consent was obtained from each patient prior to taking a blood sample. Serum Measurements Blood samples were taken at diagnosis, and were stored at -80 C until analysis. SAA was quantified by latex agglutination assay (EIKEN CHEMICAL CO, Tokyo, Japan). Serum sIL-2R was measured by a two-site chemiluminescent enzyme immunometric assay (Siemens Medical Solutions Diagnostics, Tokyo, Japan). Lysozyme activity was assayed by a turbidimetric method (Sigma-Aldrich Co, Tokyo, Japan). ACE was measured by a

colorimetric method using phydroxyhippuryl- L-histidyl-L-leucine as the substrate (FUJIREBIO Inc, Tokyo, Japan). KL-6 was measured by sandwich-type electrochemiluminescence immunoassay using a Picolumi 8220 Analyzer (Sanko Junyaku Co, Tokyo, Japan). Chest Radiography All chest radiographs, including follow-up study data, were available for review. They were evaluated based on a consensus by 2 experienced chest radiologists who were blinded to any clinical information, except for patients age, sex and diagnosis. These were classified according to standard radiographic stages: stage 0, normal chest radiograph; stage I, bilateral hilar lymphadenopathy without parenchymal infiltration; stage II, bilateral lymphadenopathy with parenchymal infiltration; stage III, parenchymal infiltration without lymphadenopathy; stage IV, advanced fibrosis with evidence of honeycombing, hilar retraction, bullae, cysts and emphysema.1 In addition to this staging, patients were classified as follows: without parenchymal infiltration (stages 0 and I) and with parenchymal infiltration (stages II, III and IV). Pulmonary Function Testing 1 second (FEV1), were measured by spirometry. Diffusing capacity for carbon monoxide (DLCO) was measured by the single-breath method. These indices were calculated as percentages of predicted normal values. Bronchoalveolar lavage Bronchoalveolar lavage (BAL) was performed using a fiberoptic bronchoscope (Olympus, Tokyo, Japan) at the time of diagnosis. In brief, 150 ml sterile saline was instilled into the middle-lobe or lingula segment in 50 ml aliquots. Each aliquot was immediately aspirated and pooled. Bronchoalveolar lavage fluid (BALF) was centrifuged and separated into cells and supernatant. The cell pellet was suspended in RPMI 1640 medium (Nikken Bio Medical Laboratory, Kyoto, Japan) and counted by hemacytometer. Cytospin slides were made from cell suspensions and stained with May-Grunwald-Giemsa (Wako Pure Chemical Industries, Osaka, Japan) for cell differentiation. We used flow cytometry to determine T-cell populations that were stained by monoclonal antibodies: anti-CD4-fluorescein isothiocyanate and anti-CD8-phycoerythrin from Becton Dickinson (San Jose, CA, USA). Flow cytometric analysis used a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). Follow-up Study All patients underwent repeat chest radiography at least every 6 months during the 2 years after diagnosis. This provided the opportunity to evaluate each serum marker as a predictive parameter for increased parenchymal infiltration with regard to radiologic outcomes. Changes in radiographic outcomes during follow-up were classified as follows: grade 1, normalized chest radiographic findings, defined as stage 0 or decreased parenchymal infiltration; grade 2, unchanged parenchymal infiltration at the end of the follow-up period; and grade 3, increased parenchymal infiltration at the end of the follow-up period. Based on these results, patients were categorized into 2 groups: increased parenchymal infiltration (grade 3) or no increase (grades 1 and 2). We analyzed patients with and without administration of corticosteroids separately, as treatment with corticosteroids may influence radiologic outcomes. The decision to treat was based on the following criteria: 1) progressive deterioration of pulmonary function, 2) progressive change on chest radiographs or extensive pulmonary involvement, 3) impairment of

organs other than the lung and 4) persistent symptoms in combination with parameters of disease activity. Statistical Analysis Results were expressed as median values and ranges. Mann-Whitney U test was used to compare groups. Correlations between variables were determined by Spearmans rank correlation coefficient. To find an optimal cut-off level that could discriminate between patients with and without increased parenchymal infiltration, receiver operating characteristics (ROC) curves were used. Cut-off levels for parameters were set as the closest points to 100% sensitivity and 100% specificity. Univariate analysis by logistic regression was used to identify parameters predictive of increased parenchymal infiltration. Parameters found to be significant by univariate analysis were taken as potential predictors of increased parenchymal infiltration and were also used as covariates in multivariate logistic regression analysis to identify independent predictors of increased parenchymal infiltration. Odds ratios and 95% confidence intervals were computed for variables. The Kaplan-Meier method was used to evaluate the predictive value for increased parenchymal infiltration, and comparisons were made using a log rank test. All tests were twotailed, and p < 0.05 was considered statistically significant. Statistical analysis used SPSS for Windows (Chicago, IL, USA). Rezultate Serum marker levels and BALF analyses in patients with and without parenchymal infiltration Serum levels of sIL-2R, lysozyme and KL-6 in patients with parenchymal infiltration were significantly higher than those in patients without parenchymal infiltration. The numbers of total cells and lymphocytes in BALF were significantly higher in patients with parenchymal infiltration compared to patients without parenchymal infiltration (Table 2). Nivelul seric de receptor solubil pentru IL 2, Lizozim si KL 6 a fost mai crescut la pacientii cu infiltrate parenchimatoase comparativ cu cei fara infiltrate. Celularitatea totala precum si numarul de limfocite din lichidul de lavaj bronhoalveolar a fost mai crescut la pacientii cu infiltrate.