Zeta-associated protein of 70 kDa (ZAP-70) is a tyrosine kinase that plays a role in signal transduction from the T-cell receptor. ZAP-70 is expressed in normal T-cells and NK-cells. Increased expression of ZAP-70 has been identified in chronic lymphocytic leukemia (CLL). CLL patients with increased ZAP-70 expression have significantly worse prognosis in terms of both progression-free survival and overall survival. There are several methods to quantify ZAP-70: polymerase chain reaction (PCR), immunoblotting, immunohistochemistry, and flow cytometry. Use of flow cytometry for ZAP-70 detection seems to be advantageous as this technique enables us to assess the presence of ZAP-70 separately on CLL clone, T-cells, and NK-cells. On the other hand, detection of ZAP-70 by flow cytometry is substantially influenced by many variables. The principal drawback of flow cytometry is the absence of consensus regarding selection of optimal anti-ZAP-70 antibody, fluorochrome conjugate, the most reliable staining technique, and optimal positivity threshold. This article summarizes pitfalls of flow cytometric analysis of ZAP-70 in CLL.

OBJECTIVE:To compare plasma VEGF (vascular endothelial growth factor) levels in ovarian cancer patients, in patients with benign ovarian tumors and healthy women.DESIGN:Prospective study.SETTING:Department of Gynecology and Obstetrics, Medical Faculty Charles University, Prague and University Hospital, Hradec Králové. Department of Immunology and Alergology, Medical Faculty Charles University, Prague and University Hospital, Hradec Králové.METHODS:VEGF was estimated by ELISA (R&D Systems).RESULTS:We found that plasma VEGF levels were associated with the International Federation of Gynecology and Obstetrics (FIGO) stage (FIGO I+II, n=8) Med = 425,53 pg/ml (range 142,30-982,40 pg/ml), (FIGO III+IV, n=29) Med = 941,48 pg/ml (range202,10-2857,80 pg/ml) (p=0,03). Patients with primary ovarian cancer (n=37) had a significantly higher plasma VEGF level Med = 829,93 pg/ml (range142,30-2857,80 pg/ml), compared with patients with benign ovarian tumors (n=15) Med = 426,28 pg/ml (range 32,00-922,20 pg/ml) and healthy women (n=21) Med = 283,13 pg/ml (range 80,50-735,20 pg/ml) (p=0,0003). VEGF levels were lower in plasma (n=79) Med = 575,49 pg/ml (range 55,80-2185,00 pg/ml) compared with VEGF levels in ascitic fluid (n=37) Med = 745,74 pg/ml (range 142,30-2185,00 pg/ml) (p=0,04) in ovarian cancer patients.CONCLUSION:Plasma VEGF assay before primary treatment and the changes during the other treatment should contribute to better understanding of angiogenesis in ovarian cancer patients. Plasma VEGF correlates with the stage of primary ovarian cancer.

Chronic lymphocytic leukemia is the most common leukemia type in Western countries. Even incidence of chronic lymphocytic leukemia is high, this disease remained beyond interest for a very long time. However, in the last few years the view of this disease fundamentally changed and due to intensive study, new knowledge especially on pathogenesis, prognostic factors and therapy based on intensive therapeutic procedures were made. Today we know that usage of classical prognostic factors is insufficient for prognosis evaluation in the individuals. However modern (IgVH mutation status, cytogenetic abberations) and new markers (LPL/ADAM29 ratio, microRNA, markers of angiogenesis etc) have potential to distinguish patients in early stages to groups with significantly different prognosis and predict clinical course of the disease.

Expression of ZAP-70 measured by flow cytometry belongs to the most powerful prognostic parameters in chronic lymphocytic leukemia (CLL).However, many technical factors such as setting of the positivity threshold may significantly influence results.. Quantification using mean fluorescent intensity (MFI) may eliminate the subjective error which is inevitable in the isotype control method.The aim of the present project was therefore to assess the prognostic significance of ZAP-70 using three different methods.Between 2005 and 2010 we measured ZAP-70 expression in 157 patients with CLL (108 males, 49 females, median age 60 years [range, 31-82]; low/intermediate/high Rai risk in 41/48/11%).Expression of ZAP-70 was determined by flow cytometry using phycoerythrin (PE)-conjugated monoclonal antibody, clone 1E7.2.Evaluation was performed by 1) percentage of positive cells compared to isotype control (cut-off 20%), 2) MFI ratio of Tcells/CLL cells (cut-off 3.0); 3) MFI ratio of ZAP-70/isotype control on CLL cells (cut-off 2.5).MFI method with T-cells/CLL cells ratio was the best in the identification of patients with unfavourable outcome: ZAP-70 positive patients had significantly shorter time to treatment (TTT, median 24 vs. 55 months, p=0.0001) and overall survival (OS, median 97 vs 174 months, p=0.0074).The differences in TTT a OS were not significant with the use of isotype percentage and MFI isotype methods.Combined analysis of ZAP-70 with CD38 expression or IgVH mutation status lead to identification of a subgroup with the longest TTT and OS (ZAP-70 and CD38 negative, p<0.0001 and p=0.012;ZAP-70 negative and mutated IgVH genes, p<0.0001 and p=0.0019).In conclusion, our results suggest that measurement of ZAP-70 expression in CLL by MFI using T-cells/CLL cells ratio might be the optimal method for accurate prediction of clinical course.Combined analysis of ZAP-70 with CD38 or IgVH mutation status further refined individual patient´s prognosis.

Several studies have demonstrated the potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiopoietin-2 (Ang-2), an angiogenic cytokine, was recently reported in CLL. However, data regarding prognostic significance of Ang-2 in CLL are limited. Therefore, we quantitated Ang-2 mRNA in purified mononuclear cells of 33 untreated CLL patients and compared the transcript levels to traditional as well as modern prognostic factors in patients with CLL (clinical stage, disease course, IgVH mutation status, CD38, and ZAP-70 expression). Elevated Ang-2 mRNA concentrations were detected in 12 cases; 21 patients had very low or undetectable levels of Ang-2 transcript. There was significant association between high Ang-2 mRNA levels and unmutated IgVH genes (n=27, P=0.010) and with CD38 expression (n=32, P=0.011), but not with ZAP-70 expression (n=32, P=0.784), Rai stage (n=33, P=0.305) or stable versus progressive clinical course (n=33, P=0.443). There was a trend towards shorter progression-free survival in patients with high Ang-2 expression; however, it did not reach statistical significance (P=0.090). Our pilot data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features. Further studies are needed to confirm the results in a larger patient cohort.

Fas ligand (FasL) causes apoptosis of epidermal keratinocytes and triggers the appearance of spongiosis in eczematous dermatitis. We demonstrate here that FasL also aggravates inflammation by triggering the expression of proinflammatory cytokines, chemokines, and adhesion molecules in keratinocytes. In HaCaT cells and in reconstructed human epidermis (RHE), FasL triggered a NF-κB-dependent mRNA accumulation of inflammatory cytokines (tumor necrosis factor-α, IL-6, and IL-1β), chemokines (CCL2/MCP-1, CXCL1/GROα, CXCL3/GROγ, and CXCL8/IL-8), and the adhesion molecule ICAM-1. Oligomerization of Fas was required both for apoptosis and for gene expression. Inhibition of caspase activity abolished FasL-dependent apoptosis; however, it failed to suppress the expression of FasL-induced genes. Additionally, in the presence of caspase inhibitors, but not in their absence, FasL triggered the accumulation of CCL5/RANTES (regulated on activation normal T cell expressed and secreted) mRNA. Our findings identify a novel proinflammatory role of FasL in keratinocytes that is independent of caspase activity and is separable from apoptosis. Thus, in addition to causing spongiosis, FasL may play a direct role in triggering and/or sustaining inflammation in eczemas.

Using the protein array method we determined the serum levels of a number of angiogenic factors. We identified serum levels of angiogenin, PDGF and MCP-1 (CCL2 chemokine) in serum of 32 patients with RCC, and 14 healthy volunteers by means of antibody array analysis. The patients were divided into three groups according to their disease stages (I+II, III, and IV). We found significant differences between the controls and patients with RCC both pre-operatively and post-operatively in angiogenin, PDGF and MCP-1 serum levels. The increase in angiogenin, PDGF and MCP-1 lasted in patients with RCC stages I-III even without metastases eight weeks post-operatively. The patients with stage IV RCC showed disturbed production of PDGF and MCP-1. Protein array analysis is a powerful tool for the identification of large numbers of trace proteins. Multiplex antibody array is able to provide data more precisely reflecting the nature of pathological processes.

INTRODUCTION When checking tumour growth, a number of observations indicate that the immune system plays a significant role in patients with renal cell carcinoma (,,RCC"). Infiltration by lymphocytes (tumour infiltrating lymphocytes, "TILs") is more prevalent in RCC than any other tumours. T lymphocytes are the dominant population of TIL cells. Views concerning the role ofT lymphocytic subpopulations, B lymphocytes and NK cells in an anti-tumour response are not established. AIM The aim is to determine the phenotype and activation of lymphocytic cells and to compare their representation in tumour stroma (TIL), peripheral blood (PBL) and renal vein blood in patients with RCC. PATIENTS AND METHODS The samples of peripheral blood taken from the cubital and renal veins and tumour stroma cells were obtained from 60 patients in the course of their surgeries carried out due to primary RCC. TILs were isolated from mechanically disintegrated tumour tissue. Immunophenotype multiparametric analysis of PBL and TILs was carried out. Their surface and activation characteristics were determined by means of flow cytometer. RESULTS CD3+ T lymphocytes (70.4%) were the main population of TILs. The number of CD3+/CD8+ T lymphocytes was significantly higher in TILs, 39.7% (p < 0.01), while CD4+ T lymphocytes were the majority population in peripheral blood, 41.35% (p < 0.001). The representation of CD3+/69+ T lymphocytes was significantly higher in TILs, 32.05%, compared to PBL (p < 0.001). On the contrary, the numbers of CD3+/CD25+, CD8+/57+ and CD4+/RA+ (naive CD4+ T lymphocytes) were higher in PBL (p < 0.001). The differences in representation of (CD3+/16+ 56+) NK cells and CD3+/DR+ T cells in TILs and PBL were not significant. CONCLUSION The above-mentioned results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (tumour/PBL). CD3+/CD8+ T lymphocytes are the dominant lymphocytic population of TILs. The knowledge of phenotype and functions ofeffector cells, which are responsible for anti-tumour response, are the basic precondition for understanding the anti-tumour immune response and the cause of its failure.

Le cancer du rein représente actuellement 3 % de l’ensemble des cancers. L’utilisation de cytokines dans le stade métastatique de la maladie a été le standard jusqu’à ces dernières années, produisant des réponses partielles et de courtes durées. La recherche sur l’angiogenèse dans le cancer du rein a entraîné des avancées considérables pour le développement de nouvelles thérapies antiangiogéniques. Le sunitinib (SUTENT®), le sorafenib (NEXAVAR®), et le bevacizumab (AVASTIN®) sont trois des quatre molécules acceptées à ce jour pour leurs utilisations dans le cancer du rein métastatique qui présentent une bonne tolérance démontrée au cours des différentes études. L’évidence clinique a fait du sunitinib le standard de référence en première ligne de traitement du cancer du rein métastatique, l’association bevacizumab + interféron alpha (IFN alpha) peut-être considérée comme une option. Le sorafenib est quant à lui recommandé en seconde ligne de traitement chez les patients réfractaires aux cytokines, le sunitinib étant aussi accepté dans cette indication. Des schémas d’associations de ces molécules sont en phase d’évaluation, leurs utilisations en phase néoadjuvante et adjuvante sont également étudiées et devraient changer, à moyen terme, la prise en charge de cette maladie.Renal cell carcinoma accounts for approximately 3% of all human malignancies. The use of cytokines in metastatic stage of disease has been the standard until last decades, presenting partial and short duration responses. Research on angiogenesis in renal carcinoma has brought important advances to understand tumor biology and to allow us development of new antiangiogenic drugs. Sunitinib (SUTENT®), sorafenib (NEXAVAR®) and bevacizumab (AVASTIN®) are actually three molecules accepted to use in metastatic renal cell carcinoma (mRCC), with a good tolerability demonstrated in different studies. Clinical evidence shows sunitinib to be reference standard of care for the first-line treatment of mRCC. The use of bevacizumab in combination with interferon alfa (IFN alfa) can also be considered in this setting. Sorafenib is recommended for second-line treatment in cytokine-refractory patients, sunitinib being also accepted in this situation. Other combination of these molecules and their use as neo-adjuvant and adjuvant therapy is being evaluated and should change in the short term the management of the disease.

Inflammation plays a key role in diseases such as diabetes, asthma, cardiovascular diseases and cancer. Diet can influence different stages of inflammation and can have an important impact on several inflammatory diseases. Increasing scientific evidence has shown that polyphenolic compounds, such as flavonoids, which are found in fruits, vegetables, legumes, or cocoa, can have anti-inflammatory properties. Recent studies have demonstrated that flavonoids can inhibit regulatory enzymes or transcription factors important for controlling mediators involved in inflammation. Flavonoids are also known as potent antioxidants with the potential to attenuate tissue damage or fibrosis. Consequently, numerous studies in vitro and in animal models have found that flavonoids have the potential to inhibit the onset and development of inflammatory diseases. In the present review, we focused in flavonoids, the most abundant polyphenols in the diet, to give an overview of the most recent scientific knowledge about their impact on different inflammatory diseases.

Common variable immunodeficiency (CVID) is the most frequent serious humoral deficiency manifested in adulthood in the form of acute and chronic respiratory infections which in most patients lead to respiratory failure. Retrospective analysis of 28 CVID patients was made. Mean age at time of diagnosis was 38.6 +/- 18.6 years. The time which elapsed from the first symptoms to determining the diagnosis was three times as long as that indicated in other studies, i.e., 14.1 +/- 10.2 years. Twenty-three patients (82.1%) had respiratory complications. Chronic obstructive pulmonary diseases, n=16 (57.1%), and bronchiectasis, n=10 (35.7%), were the most frequent types of lung damage. In addition, two patients (7.1%) displayed evidence of interstitial lung process. Morbidity associated with CVID can be reduced by early diagnosis and adequate dosage of immunoglobulins to minimise the occurrence and progression of lung damage.