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B-cell chronic lymphocytic leukaemia (B-CLL) originates from B lymphocytes that may differ in the activation level, maturation state or cellular subgroups in peripheral blood. Tumour progression in CLL B cells seems to result in gradual accumulation of the clone of resting B lymphocytes in the early phases (G0/G1) of the cell cycle. The G1 phase is impaired in B-CLL. We investigated the gene expression of five key cell cycle regulators: TP 53, c-Myc, cyclin D2, p21WAF1/CIP1 and p27KIP1, which primarily regulate the G1 phase of the cell cycle, or S-phase entry and ultimately control the proliferation and cell growth as well as their role in B-CLL progression. The study was conducted in peripheral blood CLL lymphocytes of 40 previously untreated patients. Statistical analysis of correlations of TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expressions in B-CLL patients with different Rai stages demonstrated that the progression of disease was accompanied by increases in p53, cyclin D2 and c-Myc mRNA expression. The expression of p27KIP1 was nearly statistically significant whereas that of p21 WAF1/CIP1 showed no such correlation. Moreover, high expression levels of TP53 and c-Myc genes were found to be closely associated with more aggressive forms of the disease requiring earlier therapy.

B-cell chronic lymphocytic leukaemia (B-CLL) originates from B lymphocytes that may differ in the activationlevel, maturation state or cellular subgroups in peripheral blood. Tumour progression in CLL B cells seems to result in gradualaccumulation of the clone of resting B lymphocytes in the early phases (G0/G1) of the cell cycle. The G1 phase isimpaired in B-CLL. We investigated the gene expression of five key cell cycle regulators: TP 53, c-Myc, cyclin D2,p21WAF1/CIP1 and p27KIP1, which primarily regulate the G1 phase of the cell cycle, or S-phase entry and ultimately controlthe proliferation and cell growth as well as their role in B-CLL progression. The study was conducted in peripheral bloodCLL lymphocytes of 40 previously untreated patients. Statistical analysis of correlations of TP53, cyclin D2, c-Myc,p21WAF1/CIP1 and p27KIP1 expressions in B-CLL patients with different Rai stages demonstrated that the progression of diseasewas accompanied by increases in p53, cyclin D2 and c-Myc mRNA expression. The expression of p27KIP1 was nearlystatistically significant whereas that of p21 WAF1/CIP1 showed no such correlation. Moreover, high expression levels of TP53and c-Myc genes were found to be closely associated with more aggressive forms of the disease requiring earlier therapy.

The molecular bases of miR-182 deregulation in epithelial ovarian cancers (EOCs) remain unknown and its diagnostic or prognostic role in EOCs is still unclear. We performed miR-182 expression analysis using a microarray approach and real-time PCR (qPCR). We also used array comparative genomic hybridization and methylated DNA immunoprecipitation to study copy number changes and methylation aberrations within coding locus/promoter sequences of miR-182 in EOC tissues, respectively. We have found that miR-182 expression is significantly increased in EOC (P < 0.00001) and that higher miR-182 expression in EOC is linked with significantly shorter overall survival (P = 0.026). The methylation of miR-182 promoter was significantly associated with lower miR-182 expression in EOC tissues (P = 0.045). miR-182 over-expression is connected with copy number (CN) gains of this miRNA coding sequences in EOC (P = 0.002), and the number of PRDM5 copies is significantly and inversely correlated with miR-182 expression evaluated by qPCR (R = -0.615, P = 0.009). We conclude that the aberrant miR-182 expression in EOC may be due to CN gains within its coding locus. The miR-182 promoter is rarely methylated in EOC, and its methylation status is associated with lower miR-182 expression. Deletion of the PRDM5 locus may play a supportive role in miR-182 overexpression in EOC. miR-182 is an unfavorable prognostic factor in EOC. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Chronic lymphocytic leukemia is characterized by the accumulation of functionally abnormal, monoclonal B lymphocytes in the peripheral blood, bone marrow, lymph nodes and spleen, resulting in a reduction count of normal immunocompetent cells and their impaired immune function. The defect in transmission of signals from various types of extracellular receptors, leading to aberrant cytokines and transcription factors gene expression, may underlie the basis of immune failure in B-CLL. The aim of the study was to assess of IL-6, IL-10, c-Jun, and STAT3 expression. In response to antigenic stimulation IL-6, IL-10, c-Jun, and STAT3 proteins induce mutual activity. The subject of the study was subpopulations of leukemic lymphocytes (CD5+ CD19+) and CD19+ B cells from healthy donors (control group). Our results provide evidence that the regulation of IL-6, IL-10, c-Jun, and STAT3 gene expression in CLL B cells is clearly different from normal B lymphocytes. In B-CLL STAT3 expression in unstimulated lymphocytes is significantly higher (p<0.0001) compared with normal subpopulation of B cell. In contrast, IL-6, IL-10, and c-Jun mRNA expressions are statistically lower in B-CLL in comparison with the control group, in all cases (p<0.0001). When analyzing the relationship between c-Jun expression and B-CLL stage according Rai we revealed decreasing c-Jun expression, both at the mRNA and protein levels, along with advancing stage of disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

TIRAP and Myd88 are adaptor proteins for Toll-like receptors-2 and -4 (TLR2/4) which are engaged in transducing the signal to downstream molecules. Several studies have shown the increased role of infection factors in pathogenesis of B cell chronic lymphocytic leukemia (B-CLL). This prompted us to test whether there is a correlation between MyD88-TIRAP dynamics before and after inflammatory stimuli. We determined the mRNA and protein expression of TIRAP and MyD88 in CD5(+)CD19(+) B-CLL cells and in a subpopulation of normal B CD19(+) lymphocytes. Additionally we determined the influence of lipopolysaccharide Escherichia coli - TLR4-ligand (LPS) and Staphylococcus aureus strain Cowan I - TLR2-ligand (SAC) on TIR-domain-containing adaptor protein, also called MyD88 adaptor-like (TIRAP) and myeloid differentiation primary response protein 88 (MyD88) expression. We have found that the mRNA and protein expression of TIRAP and MyD88 in B-CLL lymphocytes is lower compared with that in normal B lymphocytes. LPS and SAC stimulation in normal lymphocytes significantly altered neither TIRAP nor MyD88 mRNA expression, whereas TIRAP protein level substantially decreased after TLR agonist treatment. We did not observe any changes in MyD88 protein level after B lymphocyte stimulation. There was a significant increase in TIRAP mRNA expression after LPS and SAC stimulation of B-CLL cells. MyD88 mRNA expression levels in B-CLL lymphocytes slightly decreased upon treatment with either stimulator. Stimulation with TLR agonists did not cause changes in TIRAP and MyD88 expression at the protein level in B-CLL lymphocytes. The results of our study suggest that there may exist a, yet unknown, defect of TIRAP and MyD88 proteins in B-CLL lymphocytes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The mechanism of innate immunity is based on the pattern recognition receptors (PRR) that recognize molecular patterns associated with pathogens (PAMPs). Among PRR receptors Toll-like receptors (TLR) are distinguished. As a result of contact with pathogens, TLRs activate specific intracellular signaling pathways. It happens through proteins such as adaptor molecules, e.g. MyD88, TIRAP, TRIF, TRAM, and IPS-1, which participate in the cascade activation of kinases (IKK, MAP, RIP-1, TBK-1) as well as transcription factors (NF-κB, AP-1) and regulatory factor (IRF3). The result of this activation is the production of active proinflammatory cytokines, chemokines, interferons and enzymes. The PRR pathways are controlled by extra- and intracellular molecules to prevent overexpression of PRR. They include soluble receptors (sTLR), transmembrane proteins (ST2, SIGIRR, RP105, TRAIL-R) and intracellular inhibitors (SOCS-1, SOCS-3, sMyD88, TOLLIP, IRAK-M, SARM, A20, β-arrestin, CYLD, SHP). These molecules maintain the balance between activation and inhibition and ensure balancing of the beneficial and adverse effects of antigen recognition.

NOD-like proteins (NLR) are a specialized group of intracellular receptors, which constitute an essential component of the host innate immune system. They were discovered more than a decade ago, but research on this particular class of microbial detectors is still ongoing to allow for a better understanding of the mechanisms, recognition of microorganisms, transmission of signals, and carrying out the activation of inflammatory signaling pathways. In this review, we discuss the construction of NOD1 and NOD2 receptors, their functions, and significance in the pathogenesis of inflammatory diseases in humans.

B-cell receptor (BCR) and Toll-like receptor (TLR) stimulation and integration with signals from the pathogen or immune cells and their products determine the B-cell antibody response. Low expression of BCR is the hallmark of B lymphocytes in CLL, however little is known about the expression and function of TLR in B-CLL. We studied TLR2, TLR4, IL-6 and mIL-6Rα expression on mRNA and protein level in CD19(+) subpopulation of normal lymphocytes and the CD19(+)CD5(+) subpopulation from B-CLL. Experiments were performed on unstimulated and stimulated lymphocytes [Staphylococcus aureus Cowan I (SAC) and lipopolysaccharide (LPS) from Escherichia coli - TLR2- and TLR4-specific agonists, respectively]. We showed undetectable or low IL-6 expression, which seems to be specific for B-CLL lymphocytes. Induction of TLR4 mRNA upon LPS stimulation affected the expression of IL-6, but not of mIL-6Rα. Increased expression of TLR2 (MFI) after LPS and SAC stimulation did not correlate with mIL-6Rα receptor expression. B-CLL CD19(+)CD5(+) lymphocytes showed a significant increase in TLR2 expression at the protein level after stimulation with SAC and LPS compared to normal CD19(+) lymphocytes. TLR2 may influence the behaviour of the malignant clone in B-CLL., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: The alteration in the expression of some genes and proteins responsible for chosen DNA repair pathways in pterygium pathogenesis were studied. This study was focused on the examination of the expression of genes and RAD50 protein taking part in homologous recombination., Methods: Peripheral blood lymphocytes, samples of pterygium tissue, samples of conjunctiva of patients suffering from pterygium as well as peripheral blood lymphocytes and conjunctiva of patients from the control group were examined. In order to identify genes products from RNA, Ribonuclease Protection Assai method was applied. LIM15, RAD50, RAD54, RAD52, MRE11, XRCC2, XRCC3, RAD51, RAD51B, RAD51C, RAD51D genes transcripts were detected. Expression of RAD50 protein was analyzed immunohistochemically., Results: Peripheral blood lymphocytes analyses revealed lower level of RAD50 gene expression in the pterygium patients compared to the control group and the increased expression of XRCC2, XRCC3 and RAD51 genes in patients with pterygium, who declared the recurrence of the lesion in comparison to the patients with primary pterygium. Lower expression of the RAD54 gene in pterygium tissue comparing to conjunctiva from the eyes with pterygium was found. An expression of RAD50 gene in the conjunctiva originating from eyes with pterygium in comparison to the conjunctiva of control group was shown to be considerably higher. Expression of RAD50 protein in pterygium squamous epithelial cells was significantly higher than in conjunctiva from control group., Conclusion: There may exist a relationship between pterygium pathogenesis and damages of double strand DNA, however, the elucidation of its exact nature needs further study.

Numerous experimental data indicate that B-CLL development and progression are influenced by antigenic pressure. It can not be excluded that these antigens may originate from bacteria and viruses. Toll like receptors (TLRs) interact with pathogen associated molecular patterns as part of innate immunity. TLRs are currently used to target different subclasses of B-cell leukemia, and TLR agonists are being evaluated in clinical trials. It is little known regarding the repertoire and function of TLR in B-CLL. The aim of the study was to assess the CD180, CD284 and mCD14 levels in CD19+ subpopulation of B-CLL peripheral blood lymphocytes and compare them with respective levels in the normal B-cells of adult volunteers, before and after LPS stimulation. We investigated the percentage of the CD19+CD180+, CD19+CD284+, CD19+CD14+ cells and the mean fluorescence intensity (MFI) of CD180, CD284 and CD14 antigens among CD19+ B-CLL as well as in the normal B cells for comparison. MFI analysis revealed that CD180, CD284 and CD14 expression was higher on normal B cells then on CD19+ B-CLL (MFI CD180: 99.16 vs. 25.3, MFI CD284: 7.37 vs. 5.79 and MFI CD14 25.07 vs. 8.32). After 24-hour LPS activation of B-cells, CD180 MFI appeared to decrease, in both healthy and B-CLL patients. CD284 MFI in healthy controls decreased after LPS stimulation while slight increase of MFI was observed in leukemic cells. CD14 MFI in leukemic cells was moderately higher after LPS in comparison to CD14 MFI without LPS stimulation, whereas CD14 MFI in normal CD19+ cells after LPS stimulation decreased over three times. Variations observed in expression of both normal and leukemic receptors may be due to their different sensitivity to antigenic stimulation.

Recent studies have been carried out to delineate further the role of the Rb1 gene in B-cell chronic lymphocytic leukaemia (B-CLL). The suggested role of the Rb1 gene in this disease was based on cytogenetic data. CLL patients (40 in toto) were examined using cytogenetic and molecular biological methods. R-banding analysis of metaphase chromosomes revealed aberrations in only seven cases containing either the Rb1 gene or a chromosome 13 monosomy. No evident differences were found by RT-PCR analysis of Rb1 gene expression. The amounts of the RT-PCR products obtained appeared to be approximately equal in all cases, and was independent of the clinical stage, immunophenotypes and LPS or TPA stimulation.

The in vitro production and secretion of the immunoglobulins IgM, IgG, Kappa and lambda light chains by peripheral blood lymphocytes from chronic lymphocytic leukaemia patients was examined. The ELISA system was employed to quantitate the immunoglobulins secreted in the culture supernatants. Lipopolysaccharide and phorbol esters with CA++ ionophore were used as lymphocyte stimulators. The maturation of cultured lymphocytes was demonstrated by synthesis of cytoplasmic immunoglobulins and Ig secretions typical for plasma cells. In one case an isotype switch C mu for C gamma was observed.

A comparative research on lymphocyte immunological phenotypes in peripheral blood and bone marrow was carried out on 14 patients with B-cell chronic lymphocytic leukaemia. Three types of B cell markers were estimated: 1-light chains (lambda or kappa) on the surface of cells, 2-CD5 antigen, 3-capability of forming mouse erythrocyte rosettes (MER); the last two markers are mostly characteristic of leukaemic cells. The patients were divided into 3 groups according to the localisation of the main mass of tumorous cells: group I-the patients, whose clinical picture was dominated by the infiltration of lymphoid structures of the abdominal cavity and/or with massive infiltration of peripheral lymph nodes; group II-the patients, whose clinical picture was dominated by the infiltration of bone marrow; group III-the intermediate-patients with large mass of tumor in lymphoid structures and with bone marrow insufficiency symptoms. In most of the patients of group I, the percentage of CD5+ cells in peripheral blood was higher than the one in bone marrow (arithmetic means were 38.5% for blood and 26.7% for marrow). It was the reverse in group II (the means were 44.8% for blood and 64.3% for marrow, P < 0.01). The percentage in group III was similar to that of group I (the means-48% for blood and 29.25% for marrow). No correlation between the percentage of CD5+ and MER+ cells was found in respective groups of the patients although the last marker behaved similarly to CD5. The results obtained suggest that while estimating percentages of CD5+ cells in peripheral blood and bone marrow, one can differentiate between chronic lymphocytic leukaemia (with domination of CD5+ cells in bone marrow) and leukaemic phase of small lymphocyte lymphoma (with domination of CD5+ cells in peripheral blood).

Lipopolysaccharide (LPS) and phorbol esters (TPA) stimulate lymphocytes proliferation in two different ways. While LPS primary function is specific receptor binding, TPA directly activate cellular protein kinase C. The stimulation of human leukaemic lymphocytes (from chronic lymphocytic leukaemia patients) with LPS and TPA results in two different types of response: to both stimulators, and to LPS only. Therefore the supposed defect of cellular receptors can not explain all the observed effects. The existence of TPA independent second messengers and changes in signal transduction pathways downstream of PKC can be considered.

Unlabelled: In a group of 16 patients with chronic lymphocytic leukaemia and leukaemic forms of the lymphoma lymphoplasmocytoides immunophenotypes of peripheral blood and lymph node lymphocytes were studied., Conclusions: 1) immunophenotype heterogeneity observed in a minority of patients in lymph-nodes or peripheral blood seems to be connected with the co-existence of leukaemic and normal reactive B-cells, 2) SIgG+ cells seem to represent activated B lymphocytes producing and secreting autoantibodies, 3) circulating peripheral T lymphocytes do not reflect the distribution of T cell subpopulations in lymph-nodes.

This article contains the review and the critical discussion of studies of T and B lymphocytes in chronic B-cell lymphocytic leukaemia. It is not explicitly known now, which stage of differentiation of lympho-haemopoietic cells undergoes malignant transformation in CLL-B. Basing on VDJ recombination activity it is supposed that some cases derive from the stem cell, others--from B lineage cells.

The purpose of the study was evaluation of the effect of treatment on the immunological phenotype of peripheral blood lymphocytes in patients with chronic lymphocytic leukaemia or in leukaemic forms of non-Hodgkin lymphomas, with low or medium grade malignancy. The study was carried out in 13 patient. In those responding to treatment a tendency was observed for normalization of the immunological phenotype of peripheral blood lymphocytes (increased proportion of cells forming rosettes with sheep erythrocytes, reduced proportion of cells forming rosettes with murine erythrocytes), and a tendency for polyclonal pattern of B-cells with immunoglobulins on their surface (although this feature was not a rule). In non-responders these tendencies were much less pronounced or absent. Similarly, in non-responders the tendency for polyclonal B-cell pattern may be only apparent, and may be connected with proliferation of a new lymphocyte population with another type of light chain.

Physico-chemical properties and RNA synthesis in the rat liver and human lymphocytes have been compared in a nuclear system in vitro. Human lymphocytes were isolated from blood of healthy donors and of chronic lymphocytic leukaemia patients. The isolated nuclei served as the source of polymerase and template DNA. 3H-CTP was incorporated into the acid insoluble fraction linearly for 60 min. The nuclei of lymphocytes contained small amounts of RNA and protein, and the isolation procedure was complicated. Rat liver nuclei seem to be less prone to clumping at high pH values and may incorporate much more 3H-CTP. The nuclear synthesis was compared with incorporation of 3H-rU and 32P-orthophosphate into nuclear RNA of intact lymphocytes. Normal cells easily incorporated 32-P, and in contrast leukaemic cells incorporated 3H-rU to a greater extent.

In vitro transcription of stimulated T lymphocyte nuclei was studied using a previously described method for evaluating the accumulation of double labelled transcription products. Whereas RNA elongation was inhibited during the G0 and S phases of the cell cycle, the synthesis of short-living products (probably products of abortive initiation) still proceed in vitro. Release of the elongation block with concommitant decrease of short-product synthesis was observed during the G1 phase.

Peripheral blood lymphocytes from five patients with B-derived chronic lymphocytic leukaemia were stimulated by Staphylococcus aureus strain Cowan together with T cell mitogen phytohaemagglutinin in 5-9 days suspension cultures. The responses of B lymphocytes were studied on a T cell depleted subpopulation, obtained from harvested lymphocyte cultures using the sheep red blood cell rosette technique. Proliferation tests were performed using a 3H-TdR blast cell index. The maturation process of B-lymphocytes was examined with cytoplasmic Ig studied by FITC-conjugated antisera. Results analysed indicate various degrees of maturation of B cells in different patients.

Peripheral blood lymphocytes from 8 patients with B-derived chronic lymphocytic leukaemia were stimulated by Staphylococcus aureus bacteria strain Cowan 1 with T cell mitogens PHA or PWM in 5-7 day suspension cultures. For the first group of patients proliferation and maturation tests were performed on T cell enriched and T cell depleted subpopulations, obtained from harvested lymphocytes at the end of cultures by the sheep red cell rosette technique. To re-examine mutual influences of cultivated T and B cells in the second group of experiments, lymphocytes from CLL patients and from 5 healthy individuals were investigated by the use of transmembrane cocultivation system after Feldman and Basten. The proliferative responses to lectin and to bacteria were assessed by 3HTdR-blastic and mitotic indices. The maturation process of B lymphocytes was examined by cytoplasmic Ig, studied by FITC-conjugated antisera. Results obtained with cocultivation system support the view that T cell replacing factor(s) were required for inducing prolonged growth and development of maturation of more numerous B lymphocytes in response to Staphylococcus aureus, a T cell independent, B specific polyclonal stimulator. Results analysed in different patients indicate various degrees of maturation of B cells including their differentiation towards a plasmacytoid cell, accompanied by various proliferative capacities of B and T lymphocytes. This functional analysis reflects the heterogeneity of B-CLL patients group.