Barbara Cebula-Obrzut

Expression of Toll-Like Receptors 3, 7, and 9 in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology. The results of experimental studies point to the involvement of innate immunity receptors—toll-like receptors (TLR)—in the pathogenesis of the disease. The aim of the study was to assess the expression of TLR3, 7, and 9 in the population of peripheral blood mononuclear cells (PBMC) and in B lymphocytes (CD19+), T lymphocytes (CD4+ and CD8+) using flow cytometry. The study group included 35 patients with SLE and 15 healthy controls. The patient group presented a significantly higher percentage of TLR3- and TLR9-positive cells among all PBMCs and their subpopulations (CD3+, CD4+, CD8+, and CD19+ lymphocytes) as well as TLR7 in CD19+ B-lymphocytes, compared to the control group. There was no correlation between the expression of all studied TLRs and the disease activity according to the SLAM scale, and the degree of organ damage according to the SLICC/ACR Damage Index. However, a correlation was observed between the percentage of various TLR-positive cells and some clinical (joint lesions) and laboratory (lymphopenia, hypogammaglobulinemia, anaemia, and higher ESR) features and menopause in women. The results of the study suggest that TLR3, 7, and 9 play a role in the pathogenesis of SLE and have an impact on organ involvement in SLE.

Polymorphisms of TNF and IL‐10 genes and clinical outcome of patients with chronic lymphocytic leukemia

Genetic variations in tumor necrosis factor (TNF) and interleukin‐10 (IL‐10) were reported to influence susceptibility to and outcome of patients with non‐Hodgkin lymphoma. Therefore, we investigated whether single nucleotide polymorphisms in TNF and IL‐10 may play a role in the clinical course of patients with chronic lymphocytic leukemia (CLL). TNF‐308G>A, IL‐10‐3575T>A, and IL‐10‐1082A>G seem to be functionally relevant, were genotyped in 292 previously untreated patients with CLL. The control group consisted of 192 randomly selected blood donors. The patients carrying TNF‐308GG and IL‐10‐1082AA genotypes presented a higher 3‐year treatment‐free survival (56.6 vs. 40.6%, P = 0.05) as well as a 10‐year overall survival (OS) rates (92.3 vs. 57.6%, P = 0.005) than those with other TNF‐308 and IL‐10‐1082 genotype combinations. Multivariate analysis demonstrated the Rai stage (P = 0.0002), IGHV mutation status (P = 0.01), TNF‐308G>A (P = 0.03), and TNF/IL‐10 polymorphism‐based risk groups (P = 0.05) to be independent factors predicting OS. When the mutated IGHV patients were analyzed, the homozygotes TNF‐308GG and IL‐10‐1082AA presented a higher 10‐year OS rate than those carrying other TNF‐308 and IL‐10‐1082 genotypes (100 vs. 67.7%, P = 0.01). In the unmutated IGHV patients, only the TNF‐308G>A polymorphism influenced OS. The genetic variations in TNF and IL‐10 genes work as independent predictors of survival and may play a role in the clinical course of CLL. It suggests inherited ability of the host to shift the balance between the Th1 and Th2 response, which in turn might contribute to the pathogenesis and prognosis of B‐cell malignancies.

Influence of high expression of Smac/DIABLO protein on the clinical outcome in acute myeloid leukemia patients

The role of the Smac/DIABLO protein, a novel apoptosis agonist, in acute myeloid leukemia (AML) is not clearly determined. The expression of Smac/DIABLO protein in AML leukemic cells and its relationship with clinical outcome was evaluated in this study. The intracellular expression of Smac/DIABLO protein was assessed using multi-color flow cytometry in 71 newly diagnosed AML patients treated with conventional chemotherapy. It was found that the high expression of Smac/DIABLO protein was an independent prognostic factor in terms of higher complete remission rate (p<0.001) and longer overall survival (p=0.003). Moreover the low expression of Smac/DIABLO protein was associated with poor karyotype.

Sulforaphane Alone and in Combination with Clofarabine Epigenetically Regulates the Expression of DNA Methylation-Silenced Tumour Suppressor Genes in Human Breast Cancer Cells.

Background/Aim: Sporadic breast cancer is frequently associated with aberrant DNA methylation patterns that are reversible and responsive to environmental factors, including diet. In the present study, we investigated the effects of sulforaphane (SFN), a phytochemical from cruciferous vegetables, on the methylation and expression of PTEN and RARbeta2 tumour suppressor genes as well as on the expression of regulators of DNA methylation reaction, DNMT1, p53, and p21, in MCF-7 and MDA-MB-231 human breast cancer cells with different invasive potential. We also evaluate the role of SFN epigenetic effects in support of therapy with clofarabine (ClF) that was recently shown to modulate the epigenome as well. Methods: Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis and real-time PCR, respectively. Results: In both MCF-7 and MDA-MB-231 cells, SFN at IC50 (22 and 46 µM, respectively) and a physiologically relevant 10 µM concentration lead to hypomethylation of PTEN and RARbeta2 promoters with concomitant gene up-regulation. The combination of SFN and ClF enhances these effects, resulting in an increase in cell growth arrest and apoptosis at a non-invasive breast cancer stage. Conclusions: Our findings provide evidence that SFN activates DNA methylation-silenced tumour suppressor genes in breast cancer cells and may contribute to SFN-mediated support of therapy with an anti-cancer drug, ClF, increasing its applications in solid tumours.

Intravenous immunoglobulin replacement therapy in the treatment of patients with common variable immunodeficiency disease: an open-label prospective study.

BACKGROUND Common variable immunodeficiency (CVID) is characterized by humoral immunodeficiency resulting in increased susceptibility to infections and diminished responses to protein and polysaccharide vaccines. Intravenous immunoglobulins (IVIgs) constitute a replacement therapeutic regimen for CVID and other primary and selected secondary immunodeficiencies but their mode of action is still not fully understood. OBJECTIVE The purpose of this study was to assess the effect of IVIg replacement therapy on the population of regulatory T cells (cells expressing CD4, CD25 and low levels of CD127) [T(regs)]), plasma levels of interleukin (IL)-2 and IL-10, and expression of fragment, crystallizable γ receptor IIb (Fc γ RIIb) [CD32b] on CD19+ B cells in CVID patients. METHODS This was an open-label prospective trial that included 17 CVID patients and seven healthy subjects as case controls. The diagnosis of CVID was primarily established by clinical criteria designed by the European Society for Immunodeficiencies (ESID) and was confirmed by low serum levels of two out of three subclasses of immunoglobulins (IgG, IgA or IgM). All CVID patients were treated with the IVIg preparation Flebogamma® 5%, a highly purified, pasteurized normal human IgG extracted from the serum of healthy individuals, administered at a dose of 300 mg/kg by slow 2-hour intravenous infusion. Blood samples were collected 30 minutes before the infusion and 30 minutes and 2 weeks after the termination of the infusion. We examined: (i) the plasma levels of IL-2 and IL-10; (ii) the percentage of CD4+ T cells and T(regs); and (iii) the expression of Fc γ RIIb on the surface of CD19+ B cells. RESULTS CVID patients had higher plasma levels of IL-2 (p = 0.045) and IL-10 (p = 0.002) as well as a higher expression of Fc γ RIIb on CD19+ B cells (p =  0.0119) before IVIg compared with healthy controls. The infusion of IVIg led to further increases in the plasma levels of these cytokines 30 minutes after the termination of the infusion versus baseline (IL-2: p =  0.0004; IL-10: p = 0.0003). IVIg did not affect the expression of Fc γ RIIb. Finally, IVIg infusion resulted in elevation of the percentages of CD4+ T cells (p = 0.028) and T(regs) (p = 0.006) in the blood 30 minutes after the infusion. CONCLUSION Flebogamma® 5% as replacement therapy not only supplies immunoglobulins but also modulates the immune response, and in this way may provide additional benefits to patients with CVID.

Intravenous Immunoglobulin Replacement Therapy in the Treatment of Patients with Common Variable Immunodeficiency Disease

AbstractBackground: Common variable immunodeficiency (CVID) is characterized by humoral immunodeficiency resulting in increased susceptibility to infections and diminished responses to protein and polysaccharide vaccines. Intravenous immunoglobulins (IVIgs) constitute a replacement therapeutic regimen for CVID and other primary and selected secondary immunodeficiencies but their mode of action is still not fully understood. Objective: The purpose of this study was to assess the effect of IVIg replacement therapy on the population of regulatory T cells (cells expressing CD4, CD25 and low levels of CD127) [Tregs]), plasma levels of interleukin (IL)-2 and IL-10, and expression of fragment, crystallizable γ receptor IIb (Fc γ RIIb) [CD32b] on CD19+ B cells in CVID patients. Methods: This was an open-label prospective trial that included 17 CVID patients and seven healthy subjects as case controls. The diagnosis of CVID was primarily established by clinical criteria designed by the European Society for Immunodeficiencies (ESID) and was confirmed by low serum levels of two out of three subclasses of immunoglobulins (IgG, IgA or IgM). All CVID patients were treated with the IVIg preparation Flebogamma® 5%, a highly purified, pasteurized normal human IgG extracted from the serum of healthy individuals, administered at a dose of 300 mg/kg by slow 2-hour intravenous infusion. Blood samples were collected 30 minutes before the infusion and 30 minutes and 2 weeks after the termination of the infusion. We examined: (i) the plasma levels of IL-2 and IL-10; (ii) the percentage of CD4+ T cells and Tregs; and (iii) the expression of Fc γ RIIb on the surface of CD19+ B cells. Results: CVID patients had higher plasma levels of IL-2 (p = 0.045) and IL-10 (p = 0.002) as well as a higher expression of Fc γ RIIb on CD19+ B cells (p = 0.0119) before IVIg compared with healthy controls. The infusion of IVIg led to further increases in the plasma levels of these cytokines 30 minutes after the termination of the infusion versus baseline (IL-2: p = 0.0004; IL-10: p = 0.0003). IVIg did not affect the expression of Fc γ RIIb. Finally, IVIg infusion resulted in elevation of the percentages of CD4+ T cells (p = 0.028) and Tregs (p = 0.006) in the blood 30 minutes after the infusion. Conclusion: Flebogamma® 5% as replacement therapy not only supplies immunoglobulins but also modulates the immune response, and in this way may provide additional benefits to patients with CVID.

Pro-apoptotic effect of an anti-CD37 scFv-Fc fusion protein, in combination with the anti-CD20 antibody, ofatumumab, on tumour cells from B-cell malignancies

SMIP-016, a new anti-tumour agent, is a mouse/human chimeric fusion protein built on the ADAPTIR™ (modular protein therapeutic) platform targeting human CD37. In this study, for the first time, we examined pro-apoptotic activity of SMIP-016 in combination with monoclonal anti-CD20 antibody, ofatumumab (HuMax-CD20) in de novo chronic lymphocytic leukaemia (CLL) cells and in different B-cell neoplasm-derived lines. In CLL cells SMIP-016 exerted significant cytotoxicity (versus control - p=0.01). In the in vitro models, SMIP-016 was also distinctly active against Raji line (Burkitt lymphoma; BL) (versus control - p=0.007), Riva-1 line (diffuse large B-cell lymphoma; DLBCL) (versus control - p=0.002) and RPMI 8226 line (multiple myeloma cells; MM) (versus control - p=0.03). In studies combining SMIP-016 and ofatumumab, the cytotoxicity against CLL cells was significantly higher than the agents used alone (p<0.03). Remarkably enhanced cytotoxic activity of SMIP-016 and ofatumumab in combination was also observed in Raji and Riva-1 cell lines (p<0.01 and p<0.003, respectively). Importantly, both agents induced cytotoxicity at very low concentrations which suggests that potential side-effects may be decreased in clinical practice. The mechanism responsible for cytotoxicity of SMIP-016 in all the examined models was connected with caspase-dependent apoptosis. In majority of cell types SMIP-016 induced overexpression of Bax protein, as well as downregulation of Bcl-2, cIAP1 (p<0.03) and Smac/DIABLO (p<0.003) apoptosis-regulating proteins. In conclusion, our study demonstrated high pro-apoptotic activity of SMIP-016, especially in combination with ofatumumab, against ex vivo CLL cells, and BL or DLBCL in vitro cell lines. Thus, further preclinical studies in in vivo models are warranted, as this combination may be a promising therapeutic concept for treatment of those malignancies.

Effects of Toll-like receptor 7 and Toll-like receptor 9 signaling stimulators and inhibitors on chronic lymphocytic leukemia cells ex vivo and their interactions with cladribine

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant B lymphocytes in the peripheral blood that are regarded as poorly antigenic to the host immune system. Nonetheless, they are thought to be able to undergo stimulation and become antigen-presenting cells possibly through Toll-like receptors (TLRs). Several studies have examined the effect of TLR7 and TLR9 stimulation on the biology of CLL cells, revealing contradictory results in terms of cell proliferation and apoptosis. On the other hand, suppression of TLRs has not been studied in CLL so far, and the rationale for this may be increasing evidence of the supportive role of TLR signaling in CLL. In our study we assessed the effect of a synthetic oligodeoxynucleotide with immunoregulatory sequences (IRS 954) on peripheral blood cells from patients with untreated CLL, in terms of expression of costimulatory molecules, production of cytokines and cell viability ex vivo. Agonists of TLR7 (imiquimod, IMI) and TLR9 (oligodeoxynucleotide ODN 2006) acted as positive internal controls. ODN 2006 most markedly induced CD86 expression compared to IMI and IRS 954. Both oligodeoxynucleotides – IRS 954 and ODN 2006 – caused 1.5- and 5-fold increases of CD40 on CLL cells, respectively. Immunostimulatory ODN 2006 induced CD95 expression 1.5-fold. Changes in costimulatory molecule expression were accompanied by a moderate response from CD4 + and CD8 + T lymphocytes. TLR7 and TLR9 agonists led to significantly higher production of interleukin 6 (IL-6) and IL-10. IRS 954 and ODN 2006 markedly increased the concentration of tumor necrosis factor α (TNFα). IL-17A was significantly decreased by 50% after IMI. IRS 954 and IMI induced significant necrosis at all concentrations, and the effect was augmented by the addition of cladribine (2CdA). ODN 2006 presented a dual effect on cell viability, which was related to disease stage and baseline IL-17A concentration. The addition of 2CdA had little effect in a group where ODN 2006 supported cell survival, and further enhanced cytotoxicity of ODN 2006 in the second group. Inhibitory oligodeoxynucleotides seem to exert promising antileukemic effects regardless of sample background, and thus may become a new modality in CLL. The response of leukemic cells to ODN 2006 varies between samples and cannot yet be predicted.

Mechanisms of action of the anti-VEGF monoclonal antibody bevacizumab on chronic lymphocytic leukemia cells.

INTRODUCTION Chronic lymphocytic leukemia (CLL) remains incurable; therefore searching for new therapeutic strategies in this disease is necessary. An important mechanism of tumor development is neoangiogenesis. A potent antiangiogenic factor, bevacizumab (Avastin, AVA), has been poorly explored in CLL so far. In the current study we assessed cytotoxic activity of AVA alone or in combinations with drugs routinely used in this disease. MATERIALS AND METHODS Cells isolated from 60 CLL patients were treated with AVA alone or in combination with anti-CD20 monoclonal antibody (MoAb), rituximab (RIT), anti-CD52 MoAb, alemtuzumab (ALT), 2-CdA (2-chlorodeoxyadenosine), FA (fludarabine), MAF (mafosfamide) or RAPA (rapamycin). Cytotoxicity was assessed by propidium iodide staining. Apoptosis was evaluated using annexin-V and TUNEL assays. Additionally, a drop of mitochondrial potential (DYm) as well as expression of apoptosis-regulating proteins Bax, Bak, Bid, Bad, Bcl-2, Mcl-2, XIAP, FLIP, Akt and Bcl-2-A1 were determined by flow cytometry. RESULTS At the dose of 40 μg/ml, after 48 hours of incubation, AVA induced significant cytotoxicity against CLL cells. The drug triggered apoptosis, with activation of caspase-3 and -9, but not caspase-8, along with a drop of DYm. Incubation with AVA induced significant overexpression of proapoptotic Bak and Bad as well as downregulation of antiapoptotic Mcl-2 and Akt proteins. Combination of AVA with RIT, ALT or RAPA significantly increased cytotoxicity when compared with the effects of single drugs. DISCUSSION In conclusion, this is the first report showing proapoptotic activity of AVA against CLL cells. Combination of AVA with RIT, ALT or RAPA may be a promising therapeutic strategy, which requires confirmation in further studies.

Cytotoxic activity of the amphibian ribonucleases onconase and r-amphinase on tumor cells from B cell lymphoproliferative disorders

Although major advancements in antitumor treatment have been observed, several B cell-derived malignancies still remain incurable. A promising approach that involves targeting RNA either by the use of specific antisense oligonucleotides or cytostatic/cytotoxic ribonucleases (RNases) is being promoted. Two amphibian RNases, onconase (ONC; ranpirnase) and, more recently, r-amphinase (r-Amph), have already been tested, but thus far, mostly on solid tumors. In this study, for the first time we provide comprehensive data on ex vivo and in vivo cytotoxic activity of ONC or r-Amph against cancer cells from different B cell lymphoid malignancies, together with their detailed mode of antitumor action. Our data revealed strong pro-apoptotic activity of ONC and r-Amph in both chronic lymphocytic leukemia and aggressive B cell lymphomas, with less impact on acute lymphoblastic leukemia or multiple myeloma cells. Moreover, the antitumor action of ONC and r-Amph was markedly selective against neoplastic cells sparing normal, healthy control‑derived lymphocytes.