Jacek Bartkowiak

The positive correlation between gene expression of the two angiogenic factors: VEGF and BMP-2 in lung cancer patients

Lung cancer is a particular challenge in oncology. More than 1 million new cases occur worldwide every year and despite many clinical trials and modern diagnostic techniques, long-term survival rate has only marginally improved. The aim of the current research is to explore new molecular prognostic factors and identify new targets for anticancer therapy. Current evidence shows that angiogenesis is controlled by several angiogenic factors including VEGF and BMP-2. It has been also demonstrated that VEGF plays a key role in this process that is essential in carcinogenesis. Our study has shown that the expressions of the VEGF, BMP-2 and BMP-4 mRNAs were significantly higher (7.1-fold, 25.6-fold and 2.3-fold, respectively) in lung cancer samples than in adjacent normal lung tissues (real-time RT-PCR). Analysis based on the Pearsons correlation coefficient indicated the positive correlation between VEGF and BMP-2 gene expression, whereas no significant correlation between VEGF and BMP-4 gene expression was found. The mean+/-standard deviation serum level of VEGF was 423+/-136 pg/ml. Significant differences in the serum levels of VEGF between patients with T1 tumors and patients with T2, T3 or T4 tumors were observed. Patients with T2, T3 and T4 tumors, respectively, had 1.6-fold, 1.8-fold and 2.3-fold greater serum levels of VEGF than their peers with T1 tumors. In current study patients homozygous for the 936T allele of the +936C/T VEGF gene polymorphism had 12-fold lower VEGF gene expression and 1.3-fold lower VEGF serum level than patients homozygous for the 936C allele. In conclusion, our findings underline the importance of the two angiogenic factors namely VEGF and BMP-2 as well as +936C/T VEGF gene polymorphism in the evaluation of lung cancer patients.

Lymphocyctes Tgammadelta in clinically normal skin and peripheral blood of patients with systemic lupus erythematosus and their correlation with disease activity.

Human Tgammadelta lymphocytes constitute from 1 to 15% of all peripheral blood lymphocytes. Recent work has demonstrated that this population plays a major role in the pathogenesis of infectious and immune diseases. Increased numbers of gammadelta T cells have been found in affected skin from systemic sclerosis and chronic cutaneous lupus erythematosus patients. In our study, we have determined the numbers of Tgammadelta lymphocytes and their subpopulations in peripheral blood from 29 patients with systemic lupus erythematosus (SLE) and in 19 healthy volunteers using flow cytometry and specific monoclonal antibodies. The same cells in uninvolved skin from SLE patients and human controls using immunohistochemical analysis were estimated. T-Cell receptor (TCR) delta chain gene rearrangement was identified with primers for Vdelta1, Vdelta2 and Vdelta3 by the polymerase chain reaction. Statistical analysis showed a significantly decreased number of gammadelta T cells in SLE patients (26.4+/-16.9/microl) compared with the control group (55.3+/-20.6/microl (p < 0.001). The number of Vdelta2 TCR+ and Vgamma9 TCR+ subpopulations was also lower in SLE patients than in healthy persons. No statistical correlation between disease activity and the number of gammadelta T cells was demonstrated. The percentage of Tgammadelta lymphocytes in clinically normal skin from SLE patients was twice (22.0+/-9.4%) that found in the skin from healthy persons (11.1+/-5.5%) (p < 0.002). Higher percentages of the Vdelta2 TCR+ and Vgamma9 TCR+ subpopulation of lymphocytes were found in the skin from SLE patients. We have also found positive correlation between the percentage of Tgammadelta lymphocytes in skin and the activity of SLE (r=0.594, p < 0.001), and between subpopulation Vdelta3 TCR+ and disease activity (r=0.659, p< 0.001). In conclusion, the results of our studies demonstrate that, in patients with SLE, accumulation of Tgammadelta lymphocytes can be seen in clinically normal skin, and the percentage of these cells correlates with the activity of the disease.

Immunohistochemical profile of invasive lobular carcinoma of the breast: Predominantly vimentin and p53 protein negative, cathepsin D and oestrogen receptor positive

Vimentin, p53 protein and cathepsin D positivity were assessed by immunohistochemistry, and oestrogen receptor (ER) by an enzyme immunoassay, in invasive lobular carcinomas (LC) of the breast. While vimentin was positive in only 5% (3/57) and p53 protein was positive only in 3% (2/63), cathepsin D was expressed in 86% (48/56) and ER in 78% (25/32). Classical LC were negative for p53 protein and all except one were cathepsin D positive. These results are in contrast to invasive ductal breast carcinomas (DC), where the reported average incidence of vimentin and p53 protein is much higher (19% and 33% respectively) and that of cathepsin D and ER lower (63% and 67% respectively). Thus lack of expression of vimentin and lack of p53 positivity together with high incidence of expression of cathepsin D and ER are more often associated with lobular than with ductal differentiation of invasive breast cancer. The results show that LC, distinguished morphologically, can further be defined by its immunohistochemical profile. This in turn may point to underlying biological differences between LC and DC.

A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant - An in-vivo study

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAK-RGD-K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir. The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg. There were no observable changes in plasma fibrinogen concentration. In conclusion, our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats. Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGD-K2-Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAK-RGD-K2-Hir bears a risk of bleeding, but this may be true for higher doses.

Hodgkin's type of Richter's syndrome in familial chronic lymphocytic leukemia treated with cladribine and cyclophosphamide.

Second malignancies are frequent complications in patients with chronic lymphocytic leukemia (CLL). Hodgkins disease (HD) has been observed in approximately 0.5% of the patients with CLL and is known as Hodgkins type Richters syndrome (H-RS). We present a 64-year-old male patient with a familial history of CLL who developed H-RS in abdominal lymph nodes 6 years after CLL diagnosis and 18 months after treatment with cladribine (2-CdA) and cyclophosphamide. HD was diagnosed by fine needle aspiration. The disease was refractory to treatment with two courses of CHOP and three courses of ABVD chemotherapy. In the current literature we found case reports of only 6 patients with H-RS who were treated with fludarabine (FA) before transformation, and, to our knowledge the presented patient is the first to develop H-RS after treatment with 2-CdA combined with cyclophosphamide. He is also the first published patient with familial CLL in whom this complication developed.

Coordinated increase of miRNA-155 and miRNA-196b expression correlates with the detection of the antigenomic strand of hepatitis C virus in peripheral blood mononuclear cells

A tight relationship has been revealed between cellular microRNAs (miRNAs) and the course of hepatitis C virus (HCV) replication in human hepatoma cells. Although the detection of the antigenomic HCV RNA strand in peripheral blood mononuclear cells (PBMCs) has provided evidence for viral replication in PBMCs, no reports have shown how miRNAs are affected upon HCV RNA synthesis in PBMCs. The aim of the present study was to assess if and how the expression levels of miRNA-155 and miRNA-196b in PBMCs are related to HCV replication in PBMCs of chronic hepatitis C (CHC) patients. Supporting analyses were performed to evaluate the expression of precursor pri-miR-155 (BIC) and Dicer protein. The genomic and antigenomic HCV RNA strands in PBMCs were detected by strand-specific qRT-PCR. The expression levels of miRNAs, BIC RNA and Dicer protein were assayed on PBMCs by qRT-PCR and Western blotting, respectively. miRNA-155 and miRNA-196b were detected in all studied PBMC samples, but their levels varied according to the presence of the antigenomic HCV RNA strand in PBMCs. Increased expression levels of miRNA-155 and miRNA-196b were associated with the presence of the antigenomic HCV RNA strand in PBMCs. In this group of patients higher frequency of BIC RNA and Dicer protein detection was also found. This study demonstrates that HCV RNA replication in PBMCs of CHC patients is connected with the increased and coordinated expression of miRNA-155 and miRNA-196b.

Effect of oxidative stress on the expression of t-PA, u-PA, u-PAR, and PAI-1 in endothelial cells.

In this study we examined the effects of exogenous nitric oxide (sodium nitroprusside, SNP) and hydrogen peroxide (H2O2) on the expression level of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVEC). The expression of selected genes involved in fibrynolysis under the influence of oxidative stress was analyzed at the levels of mRNA, protein, and promoter activity. The results of the conducted studies revealed that oxidative stress in endothelial cells causes a significant increase in PAI-1 and u-PAR expression and a moderate increase in t-PA and u-PA expression at all of the investigated levels. We attempted to elucidate the molecular signaling mechanisms by which SNP and H2O2 regulate expression of the respective fibrinolytic factors. Therefore, we tested the protein levels of AP-1, NF-kappaB, and HIF-1 and their DNA-binding activity in endothelial cells subjected to oxidative stress. We found strong correlation between AP-1, NF-kappaB, and HIF-1 in the contribution of regulation of selected genes. In addition, we also found that the inhibition of PAI-1 synthesis by antisense oligonucleotide to PAI-1 mRNA results in markedly increased u-PAR expression and that NF-kappaB and AP-1 are involved in this regulation.

Atypical molecular background of glioblastoma and meningioma developed in a patient with Li–Fraumeni syndrome

We observed three neoplasms with completely different histologies: malignant fibrous histiocytoma (MFH), atypical meningioma (AM), and glioblastoma (GB), developing in a patient with Li–Fraumeni syndrome. By using a combined molecular approach we performed molecular characterization of all three tumours. Data obtained showed an interesting molecular background of the AM and GB. AM showed TP53mutations and a 22q loss of heterozygosity (LOH). GB showed epidermal growth factor receptor (EGFR) amplification and TP53 mutations, whereas P16, PTEN, Rbwere intact in terms of LOH and/or multiplex PCR (polymerase chain reaction) analysis. Additionally, GB has a 1q LOH, which is an extremely rare alteration in glioblastomas. Identical 1q LOH was also observed in MFH.

Richter's Syndrome Following Cladribine Therapy for Chronic Lymphocytic Leukemia First Manifested as Pathologic Fracture of the Femur

Richters syndrome (RS) refers to the development of aggressive non-Hodgkins lymphoma (NHL) during the course of chronic lymphocytic leukaemia (CCL). It occurs in approximately 3% of patients with CLL. The isolated form of this complication in bone is extremely rare and, so far, has not been described in a patient treated with cladribine (2-CdA). We report a case of CLL treated successfully with 2-CdA, where isolated diffuse large B-cell lymphoma (LBCL) developed 2 years after the diagnosis of CLL Rai II and one year after the completion of 2-CdA treatment. RS was first manifested as a pathologic fracture of the left femur. The LBCL was clonally distinct from the original CLL cells. The patient was successfully treated with CHOP and radiotherapy and obtained complete response of the LBCL.

Circulating TCR gammadelta cells in the patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a disorder with a wide range of immunological abnormalities. The results of the studies undertaken in the last decade indicated that SLE pathogenesis was mainly connected with the breakdown of the activation control of B and T cells, generating humoral or cell-mediated responses against several self-antigens of affected cells. The last studies demonstrate that the role of gammadelta T lymphocytes in autoimmune diseases can be especially important. Flow cytometry techniques were used to investigate the number and percentage of TCR gammadelta T cells and their most frequent subtypes in peripheral blood of 32 patients with SLE and 16 healthy volunteers. We also correlated TCR gammadelta cells number with the level of T CD3+, T CD4+, T CD8+, and NK (CD16) cells (cytometric measurements) and SLE activity (on the basis of clinical investigations). Our studies were preliminary attempts to evaluate the role of that minor T cell subpopulation in SLE. Absolute numbers of cells expressing gammadelta TCR in most SLE blood specimens were significantly lower than in the control group (P<0.006). However, since the level of total T cell population was also decreased in the case of SLE, the mean values of the percentage gammadelta T cells of pan T lymphocytes were almost the same in both analysed populations (7.1% vs 6.3%, respectively). In contrast to Vdelta2+ and Vgamma9+ subtypes of pan gammadelta T cells, Vdelta3+ T cells number was higher in SLE patients (20 x 10 cells/microl) than in healthy control group (2 x 2 cells/microl) (P=0.001). However, we found no differences between the numbers of pan gammadelta T lymphocytes and studied their subtypes in the patients with active and inactive disease. These cell subpopulations were doubled in the treated patients with immunosuppressive agents in comparison with untreated ones; however, data were not statistically significant. Our study indicated that Vdelta3+ subtype of gammadelta T cells seems to be involved in SLE pathogenesis; however, we accept the idea that the autoimmunity does not develop from a single abnormality, but rather from a number of different events.