Piotr Kopinski

Endobronchial ultrasound-guided needle aspiration in the non-small cell lung cancer staging.

OBJECTIVE The aim of the study was to assess the diagnostic yield of the endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-NA) in the mediastinal staging in non-small cell lung cancer (NSCLC) patients. METHODS Consecutive NSCLC patients with enlarged or normal mediastinal nodes on CT scans underwent EBUS-NA. All patients with negative EBUS-NA subsequently underwent the transcervical extended bilateral mediastinal lymphadenectomy (TEMLA) as a confirmatory test. RESULTS Two hundred and twenty-six patients underwent EBUS-NA between 1.02.07 and 30.04.08. There were 320 mediastinal lymph nodes biopsied (stations: 2R - 8, 4R - 83, 2L - 1, 4L - 61, 7 - 167). EBUS-NA revealed metastatic lymph node involvement in 129/226 patients (57.1%) and in 171/320 biopsies (53.4%). In 97 patients with negative EBUS-NA, who underwent subsequent TEMLA, metastatic nodes were diagnosed in 16 patients (7.1%) - in 12 (5.3%) in stations accessible for EBUS-NA (stations: 4R - 3, 4L - 2, 7 - 8) and in 4 (1.8%) in stations not accessible for EBUS-NA (stations: 5 - 4, 6 - 1). All positive N2 nodes diagnosed by the TEMLA contained only small metastatic deposits. A diagnostic sensitivity, specificity, accuracy, PPV and NPV of EBUS-NA were 89.0%, 100%, 92.9%, 100% and 83.5%, respectively. No complications of EBUS-NA were observed. CONCLUSIONS (1) EBUS-NA is an effective and safe technique for mediastinal staging in NSCLC patients. (2) In patients with negative results of EBUS-NA, surgical exploration of the mediastinum should be performed.

Aptamers in Diagnostics and Treatment of Viral Infections

Aptamers are in vitro selected DNA or RNA molecules that are capable of binding a wide range of nucleic and non-nucleic acid molecules with high affinity and specificity. They have been conducted through the process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). It serves to reach specificity and considerable affinity to target molecules, including those of viral origin, both proteins and nucleic acids. Properties of aptamers allow detecting virus infected cells or viruses themselves and make them competitive to monoclonal antibodies. Specific aptamers can be used to interfere in each stage of the viral replication cycle and also inhibit its penetration into cells. Many current studies have reported possible application of aptamers as a treatment or diagnostic tool in viral infections, e.g., HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), HCV (Hepatitis C Virus), SARS (Severe Acute Respiratory Syndrome), H5N1 avian influenza and recently spread Ebola. This review presents current developments of using aptamers in the diagnostics and treatment of viral diseases.

Interleukin-27: Biological Properties and Clinical Application

Interleukin (IL)-27 is a novel cytokine secreted by stimulated antigen-presenting cells. Initial studies on the biology of IL-27 provided evidence for its role in the initiation of TH1 responses; however, subsequent work has indicated that IL-27 has broad inhibitory effects on TH1, TH2, and TH17 subsets of T cells as well as the expansion of inducible regulatory T cells. The involvement of IL-27 in the regulation of angiogenesis and antiviral response has also recently been reported. The aim of this review is to highlight the potential areas of IL-27 clinical application, especially the management of neoplastic and viral diseases as well as autoimmune disorders, including rheumatoid arthritis and multiple sclerosis. The review will also serve to elaborate on the molecular mechanisms involved in the expression of this cytokine and signaling from the IL-27 receptor.

Real-time PCR quantification of plasma DNA in non-small cell lung cancer patients and healthy controls

IntroductionFree-circulating DNA is present in minute amounts in plasma of healthy individuals, whereas increased levels are found in a number of malignant pathologies including non-small cell lung cancer (NSCLC). The objective of this research was the evaluation of the plasma DNA quantification capacity to distinguish between healthy subjects and non-small cell lung cancer (NSCLC) patients.Materials and methodsPlasma samples were collected prospectively from 16 healthy volunteers and 30 untreated NSCLC patients (I-IIIA). Subsequently, free-circulating DNA extraction and quantitative real-time PCR analysis were performed.ResultsThe values of plasma DNA concentration ranged from 0.9 up to 7.0 ng/ml in healthy individuals and from 1.5 up to 50 ng/ml in NSCLC patients before treatment. Cancer group showed several-fold higher mean free-circulating DNA concentration than that present in healthy subjects (mean 12.00 vs. 2.65 ng/ml; P < 0.001). A greater variability of plasma DNA concentrations was observed in NSCLC patients than in controls (SD 14.50 vs. 2.02, respectively). The area under the ROC curve was 0.87 (95% CI, 0.744 to 0.954, P < 0.001).ConclusionNon-small cell lung cancer is associated with elevated levels of cell-free DNA in plasma with respect to healthy controls. Real-time PCR method proved its utility in effective free-circulating DNA detection and quantification.

Merkel cell carcinoma: is this a true carcinoma?

Recent years have brought an enhanced understanding of Merkel cell carcinoma (MCC) biology, especially with regard to the Merkel cell polyoma virus as a causative agent. Differences between Merkel cell polyomavirus‐positive and Merkel cell polyomavirus‐negative MCC in morphology,, gene expression, miRNA profiles and prognosis have been reported. Origin of MCC is controversial. Presence of neurosecretory granules has suggested that these carcinomas originate from one of the neurocrest derivatives, most probably Merkel cells; the name Merkel cell carcinoma is now widely accepted. Expression of PGP 9.5, chromogranin A and several neuropeptides, initially regarded as specific markers for neural and neuroendocrine cells, has recently been shown in a subset of lymphomas. MCC commonly expresses terminal deoxynucleotidyl transferase and PAX5. Their co‐expression under physiologic circumstances is restricted to pro/pre‐B cells and pre‐B cells. These findings lead to the hypothesis by zur Hausen et al. that MCC originates from early B cells. This review was intended to critically appraise zur Hausens hypothesis and discuss the possibility that MCC is a heterogenous entity with distinct subtypes.

Challenges and Prospects for Alpha-1 Antitrypsin Deficiency Gene Therapy

Alpha-1 antitrypsin (AAT) is a protease inhibitor belonging to the serpin family. A number of identified mutations in the SERPINA1 gene encoding this protein result in alpha-1 antitrypsin deficiency (AATD). A decrease in AAT serum concentration or reduced biological activity causes considerable risk of chronic respiratory and liver disorders. As a monogenic disease, AATD appears to be an attractive target for gene therapy, particularly for patients with pulmonary dysfunction, where augmentation of functional AAT levels in plasma might slow down respiratory disease development. The short AAT coding sequence and its activity in the extracellular matrix would enable an increase in systemic serum AAT production by cellular secretion. In vitro and in vivo experimental AAT gene transfer with gamma-retroviral, lentiviral, adenoviral, and adeno-associated viral (AAV) vectors has resulted in enhanced AAT serum levels and a promising safety profile. Human clinical trials using intramuscular viral transfer with AAV1 and AAV2 vectors of the AAT gene demonstrated its safety, but did not achieve a protective level of AAT >11 μM in serum. This review provides an in-depth critical analysis of current progress in AATD gene therapy based on viral gene transfer. The factors affecting transgene expression levels, such as site of administration, dose and type of vector, and activity of the immune system, are discussed further as crucial variables for optimizing the clinical effectiveness of gene therapy in AATD subjects.

Enhanced expression of Fas Ligand (FasL) in the lower airways of patients with fibrotic interstitial lung diseases (ILDs).

The exact role of FasL, and particularly its soluble and membrane-bound forms, in the development of chronic ILDs and lung fibrosis has not been extensively explored. We aimed at analyzing membrane-bound FasL expression on alveolar macrophages (AM) and lymphocytes (AL) as well as soluble FasL (sFasL) levels in bronchoalveolar lavage (BAL) from ILDs patients, incl. pulmonary sarcoidosis (PS), hypersensitivity pneumonitis (HP), silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), and healthy subjects (n = 89, 12, 7, 8, 23, 6, 17, respectively). In IPF, significantly increased percentage of AM FasL(+) and CD8(+)FasL(+) cells as well as sFasL levels in BAL were found. Increased sFasL levels were also observed in HP. NSIP and asbestosis were characterized by higher AM FasL(+) relative number; CD8(+)FasL(+) population was expanded in asbestosis only. There was a significant decline in AL FasL(+) percentage in PS and HP. Vital capacity was negatively correlated with sFasL levels, AM FasL(+) and CD8(+)FasL(+) cell relative count. CD4(+)FasL(+) and CD8(+)FasL(+) percentage strongly correlated with BAL neutrophilia, an unfavorable prognostic factor in lung fibrosis. The concurrent comparative BAL analysis of FasL expression indicates that FasL(+) AM and AL (mainly Tc cells) comprise an important element of the fibrotic process, mostly in IPF. FasL might play a crucial role in other fibrosis-complicated ILDs, like NSIP and asbestosis.

Apoptosis of alveolar lymphocytes. Part 1: pathways of lymphocyte apoptosis

Apoptosis is a form of programmed cell death essential for maintaining homeostasis, including onset, progress and resolution of immune reactions. Two major apoptosis pathways: extrinsic (mediated by death receptors) and intrinsic (mitochondrial), were distinguished. Lymphocytes with cytotoxic activity may also initiate apoptosis of target cells by granzyme/perforin (pseudoreceptor) pathway. The specific apoptotic processes, i.e. activation induced cell death (AICD) and neglect induced death (NID), are types of extrinsic and intrinsic pathways, respectively. They both seem to be crucial in apoptosis of antigen-primed T cells during the contraction phase of inflammation. Alveolar lymphocytes (AL) are almost exclusively T effector cells, engaged in interstitial lung disease (ILD) pathophysiologies. The AL numbers in lower airways depends on recruitment to the lung, proliferation and local apoptosis. According to the references, it should be noted that AL usually do not proliferate in alveoli; their apoptosis rate accounts, on average, for 1% of cells in healthy subjects, and this is significantly decreased in disorders with lymphocytic alveolitis such as sarcoidosis and extrinsic allergic alveolitis (EAA). The mechanisms of AL apoptosis have not been completely explained. However, it is the NID process that is probably critical for the culling of T-cell response, as in EAA or sarcoidosis remission, with AICD as an auxiliary and/or modulating mechanism only. It should be emphasised that many ILDs are chronic disorders with no remission or improvement, and it is difficult to describe the AL response in terms of immune expansion/contraction.