Giorgos Margaritopoulos

Investigation of Toll-like receptors in the pathogenesis of fibrotic and granulomatous disorders: a bronchoalveolar lavage study

Background and aimToll-like receptors (TLRs), a key component of innate immunity, have recently been implicated in the pathogenesis of interstitial lung diseases (ILDs). As the involvement of TLRs has not yet been fully elucidated, the aim of the current study was to examine the expression of various TLRs in the bronchoalveolar lavage fluid (BALF) of patients with ILDs.Patients and MethodsWe studied prospectively three groups of patients: (1) one group of 35 patients with fibrotic disorders, 16 with idiopathic pulmonary fibrosis (IPF) and 19 with fibrotic interstitial pneumonias associated with collagen tissue disorders (CTD-IPs); (2) one group of 14 patients with pulmonary sarcoidosis; and (3) 11 normal subjects. We evaluated TLR expression with flow cytometry and mRNA expression with real-time PCR.ResultsAn overexpression of TLR-3 mRNA was found in fibrotic disorders (CTD-IPs/IPF) in comparison with sarcoidosis (mean ± SD, 1.104 ± 1.087 versus 0.038 ± 0.03; P = 0.04). Additionally, TLR-3 mRNA was increased in CTD-IPs in comparison with IPF (P = 0.001), sarcoidosis (P = 0.002) and controls (P = 0.05). An upregulation in TLR-7 and -9 mRNA expression was detected in IPF (P = 0.05) and sarcoidosis (P = 0.05), respectively, when compared to controls. A higher percentage of TLR-9-expressing cells was found in BALF of CTD-IPs when compared to IPF (mean ± SD, 36.7 ± 7.06 versus 14.85 ± 3.82; P = 0.025).ConclusionWe observed distinct profiles of TLR expression in fibrotic and granulomatous disorders. It is likely that they could play a key role in the pathogenesis of these diseases and represent future therapeutic targets.

Differential telomerase expression in idiopathic pulmonary fibrosis and non-small cell lung cancer

Telomerase is a reverse transcriptase ribonucleo-protein (h-TERT) that synthesizes telomeric repeats using its RNA component (h-TERC) as a template. Telomerase dysfunction has been associated with both fibrogenesis and carcinogenesis. In this study, we aimed to evaluate the telomerase mRNA expression levels of both subunits (h-TERT and h-TERC) in lung tissue and bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) and non-small cell lung cancer (NSCLC), since there are indications of common pathogenetic pathways in these diseases. We prospectively examined lung tissue samples from 29 patients with IPF, 10 patients with NSCLC and 21 controls. Furthermore, we examined BALF samples from 31 patients with NSCLC, 23 patients with IPF and 12 control subjects. The mRNA expression for both h-TERT and h-TERC was measured by real-time RT-PCR. In the lung tissue samples, both h-TERT and h-TERC mRNA expression levels varied among the 3 groups (p=0.036 and p=0.002, respectively). h-TERT mRNA levels in the patients with IPF were lower compared with those in the controls (p=0.009) and patients with NSCLC (p=0.004). h-TERC mRNA levels in the patients with IPF were lower compared with those in the controls (p=0.0005) and patients with NSCLC (p=0.0004). In the BALF samples, h-TERT mRNA expression levels varied among the groups (p=0.012). More specifically, h-TERT mRNA levels in the patients with IPF were higher compared with those in the controls (p=0.03) and patients with NSCLC (p=0.007). The attenuation of telomerase gene expression in IPF in comparison to lung cancer suggests a differential role of this regulatory gene in fibrogenesis and carcinogenesis. Further functional studies are required in order to further elucidate the role of telomerase in these devastating diseases.

Yin Yang-1(YY-1) expression in idiopathic pulmonary fibrosis

Context: Yin Yang-1 (YY-1) is implicated in the pathogenesis of lung cancer which can be complicated with idiopathic pulmonary fibrosis (IPF). Objective: The aim of the study was to investigate whether YY-1 is involved in the pathogenesis of IPF and whether represents a common pathogenetic pathway which could explain the coexistence of these disorders. Materials and methods: Lung tissue from 52 patients (37 with IPF and 15 controls) and bronchoalveolar lavage fluid (BALF) from 34 patients (25 with IPF and 9 controls) were studied and YY-1 mRNA expression was evaluated by real-time PCR. Results: YY-1 was expressed in 8% (3/37) of IPF patients and in 6% (1/15) of healthy controls in tissue samples. In addition, 12% (3/25) of IPF patients and 33% (3/9) of healthy controls have expressed YY-1 gene in BALF samples. However, no statistical significant difference in mRNA expression between patients and controls has been detected in both tissue and BAL fluid samples. Discussion and conclusion: Our results do not support the hypothesis of YY-1 involvement in IPF. However, similar expression of YY-1 gene in two biological samples cannot exclude a possible role of this polymorphic gene in the pathway of IPF. Further studies in a larger scale of patients are needed.

Upregulation of stromal cell derived factor-1α in collagen vascular diseases-associated interstitial pneumonias (CVDs-IPs)

OBJECTIVE We speculated that distinct angiogenic profiles are involved in idiopathic interstitial pneumonias (IIPs) in comparison with interstitial pneumonias associated with collagen vascular disease (CVD-IPs). This hypothesis was investigated by measuring the expression of a cardinal biologic axis, the vascular endothelial growth factor (VEGF)-stromal derived growth factor [SDF-1alpha, transcripts 1 and 2 (TR1 and TR2)] and receptor, CXCR4 and the angiogenetic receptors CXCR2 and CXCR3 in bronchoalveolar lavage fluid (BALF) in both conditions. METHODS We studied prospectively 25 patients with fibrotic IIPs (f-IIPs) [20 with idiopathic pulmonary fibrosis (IPF) and 5 with idiopathic non-specific interstitial pneumonia (NSIP)] and 16 patients with CVD-IPs. mRNA expression was measured by Real-Time RT-PCR and protein was evaluated by Western Blotting. RESULTS A significantly greater value has been detected in SDF-1alpha-TR1 mRNA expression levels of CVD-IPs (p=0.05) in comparison with IPF group. A similar trend has been also detected in protein expression in favor of CVD-IP group. In addition, VEGF mRNA levels have been found significantly increased in CVD-IPs in comparison with the NSIP group (p=0.05). No significant difference has been found in SDF-1alpha-TR2-CXCR4 mRNA and CXCR2-CXCR3 between the two groups. CONCLUSION These results showed increased expression of SDF-1alpha in CVD-IPs, suggesting different angiogenic procedures. Further studies are needed in order to better explore the angiogenetic pathway in these disorders.

Immunopathogenesis of Sarcoidosis

Sarcoidosis is a multisystemic disease in which inflammatory cells gather and form nodules known as non caseating epithelioid granulomas. The most commonly affected organs are the lungs, the eyes and the skin whereas all the organs can be potentially affected. The disease can develop when genetically susceptible individuals are exposed to environmental agents with antigenic properties. These can be either exogenous agents (infections, antigenic structures) or endogenous agents produced by damaged cells. Usually the immune system is able to eliminate the granulomas over a few years but if this is not the case, a progression to fibrosis and permanent organ damage is observed. It is commonly accepted that the pathogenesis of the disease is mediated by an interplay of cells of both innate and adaptive immunity as well as by their products. Interestingly, the pathogenetic process is compartmentalized and there is an exuberant immune response occurring in the affected tissues such as increase of lymphocytes in the bronchoalveolar lavage fluid in contrast to the peripheral blood lymphocytopenia and cutaneous anergy to tuberculin and other skin tests (Daniele& Rowlands, 1976; Hunninghake,1979,1981; Siltzbach et al,1974; Winterbauer et al,1993; Yeager et al 1977). The role of the immune cells and cytokines involved in the pathogenesis of sarcoidosis will be discussed in this chapter.

A 35 year old smoker with shortness of breath

A previously well 35 year old man who currently smoked cigarettes (40 pack years) was referred because of a history of dyspnoea on exertion that had gradually got worse during the two months before presentation and a cough that was initially productive but then dry. He regularly smoked crack cocaine and took cocaine intranasally. At presentation he had hypoxaemia (partial pressure of oxygen 57 mm Hg), with a respiratory rate of 20 breaths/min. Physical examination showed only bilateral fine inspiratory crackles at the middle and lower zones of the chest. Initial blood tests were unremarkable. He had no signs of collagen tissue disorder, his autoantibody profile was negative, and HIV screening was negative. Chest radiography showed a bilateral reticular pattern at the middle and lower zones and bilateral infiltrates in the lower zones of the lung. Pulmonary function testing showed a restrictive pattern (total lung capacity 49%, forced expiratory volume in one second 48%, forced vital capacity 47%, diffusing capacity of the lung for carbon monoxide 36%). A 12 lead resting electrocardiogram showed sinus rhythm and an echocardiogram showed no abnormalities and no indirect evidence of pulmonary hypertension. Baseline saturation was 96% with a heart rate of 100 beats/min. During the six minute walking distance test he desaturated to 83% after three minutes and having completed 110 m. He underwent high resolution computed tomography of the chest (figs 1 and 2⇓ ⇓). Fig 1 High resolution computed tomography of the chest: upper lung zones Fig 2 High resolution computed tomography of the chest: lower lung zones