ALBERT

Description: Introduction. B cell chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease as shown by differential expression of a variety of surface and cytoplasmic markers. In a search for markers that could define biological activity of different B-CLL subsets, we have studied the surface expression of the Toll-like receptor (TLR) family member CD180 in relation to other surface markers and mutation status of IgVh genes. Methods. Seventy eight B-CLL patients (68 untreated and 10 treated) and 15 age-matched controls were studied in three different clinics. CD19+ B cells were stained using indirect immuno fluorescence for CD180, surface IgM (sIgM), CD79b and CD38, analysed by flow cytometry and data expressed as the relative antibody binding sites (RBS)/cell for each marker. Monoclonal anti-CD5 antibodies were also used with anti CD180 to determine levels of expression of CD180 in control CD5+ cells. IgVh mutation was determined for 47 patients. Results B-CLL cells had variable levels of CD180 expression, but this was always less (1036 ± 935 RBS/cell) than that expressed by normal blood B cells (5548 ± 2271 RBS/cell) and was stable for up to 18 months. Significantly higher levels of CD180 were expressed by B-CLL cells with mutated (M) compared with those using unmutated (UM) IgVh genes. This was in contrast to the higher levels of expression of sIgM by B-CLL cells using UM than M IgVh genes (Figure). Conclusions. CD180 is expressed at higher levels on B-CLL cells using M than those using UM IgVh genes and is in contrast to the level of expression of sIgM which is higher on B-CLL cells using UM versus M genes. This differential expression of CD180 supports the notion that B-CLL cells using UM IgVh genes represent a population of cells actively responding to signals (perhaps to self antigens) via their surface IgM. Figure Figure

Description: Chronic lymphocytic leukemia is characterized by the accumulation of monoclonal malignant CD5+ B cells that show resistance to apoptosis in vivo. Berg 36 (ZFP36L1, TIS11b, BRF1, cMG1, ERF1) is a zinc finger containing early response gene that was cloned from PMA-stimulated B chronic lymphocytic leukemia cells. Induced expression of this gene has been linked to calcium ionophore and anti-CD20-induced apoptosis of human B lymphoma cells. Berg36 protein has, in common with two other members of the small gene family (TIS11, TIS11d), been reported to function as an mRNA-binding protein that may promote instability of cytokine mRNAs. We have therefore studied the regulation of Berg36 expression in B-CLL cells in vitro, in response to cytokines and other signals that regulate apoptosis. B-CLL cells from 12 patients were purified and incubated with the following agents either alone or in combinations for a number of hours; IL-4, CD40 ligand, PMA or anti-CD20 antibody (rituximab). Apoptosis was measured after 24 hours by Annexin/PI staining and Berg-36 expression by Northern blot analysis. Spontaneous apoptosis in unstimulated B-CLL cells was 20.9±5.1%. Co-incubation of B-CLL cells with IL-4 reduced the percentage of apoptotic cells to 6.2±1.2%, but had no effect on Berg-36 expression. In contrast, both PMA and CD40 stimulation reduced the percentage of apoptotic cells (to 14.8±3.4% and 10.2±4.2% respectively), and markedly induced Berg36 expression. On the other hand, anti-CD20 antibody induced apoptosis (36.2±6.6%), but also induced Berg36 expression. Specific inhibitors of various intracellular signalling molecules confirmed that induction of Berg36 by different agents was mediated through different signalling pathways. In conclusion, expression of Berg36 can be induced in B-CLL cells by stimuli that induce either survival or apoptosis in these cells.

Description: Introduction: We have previously shown that Toll-like receptor RP105 (CD180) is heterogeneously expressed on B-CLL cells and that the ligation of CD180 by monoclonal antibodies (mAb) on CD180+ B-CLL cells resulted in delineation of responder and non-responder B-CLL clones [1]. In this study we have examined the role of IL-4 together with CD180 and CD40 as activation signals. Methods: Blood mononuclear cells were separated from 7 responder B-CLL patients with both mutated and unmutated Ig Vh genes and 11 controls and were cultured for 72 hours in optimum concentrations of anti-CD180 (G28-8) or anti-CD40 mAb or both in presence and absence of 15 ng/ml of IL-4. CD19+ B cells were stained with mAb to the activation marker CD86 or cell cycle protein Ki-67, measured by flow cytometry and expressed as Mean Fluorescence Intensity (MFI) or % of Ki-67+ cells. Results: B-CLL cells and normal control B cells responded to CD180-ligation by activation and proliferation (Table). Higher levels of CD86 and Ki67 were detected when both anti CD40 mAb and anti CD180 mAb were added (p

Description: Introduction B cell Chronic lymphocytic leukemia (BCLL) is characterized by the accumulation of monoclonal malignant CD5+ B cells that show resistance to apoptosis in vivo, but are susceptible to apoptosis when incubated in vitro. A number of cytokines have been reported to protect against apoptosis in vitro, but it is not clear whether the effects of these cytokines are mediated in similar ways. We have therefore studied the regulation of apoptosis in B-CLL cells in vitro, in response to various cytokines. Methods B-CLL cells were purified and incubated with the following agents; Interferon-gamma, IL-4, IL-10, IL-13, TNF-alpha and GM-CSF, each at 3 different concentrations. Apoptosis was measured after 70 hours by Annexin/PI staining and analysed for early apoptosis (Annexin V positive), late apoptotic (Annexin V and PI positive), and necrotic (PI positive). Results The percentage of live cells in unstimulated B-CLL cells was 42.4±2.3%. Co-incubation of B-CLL cells with IL-4 at 100ng/ml increased the percentage of live cells to 63.2±8.4%, while in the presence of interferon-gamma the percentage of live cells was 48.7±5.9%. In contrast, co-incubation with TNF-alpha reduced the percentage of live cells to 30.8±6.8%. IL-10, IL-13 and GM-CSF had no effect on B-CLL cells survival at any of the concentrations used.. No significant differences in the pattern of early/late apoptosis or necrosis were seen between the different cytokines used. Figure Figure

Description: Despite advances in modern chemotherapy, CLL remains incurable. CLL is an indolent disease. It expresses a panel of Cancer-Testis (CT) antigens. CLL leukemia cells are susceptible to the cytotoxicity of T cells. CLL is, therefore, an ideal disease for immunotherapeutic approaches. Immunotherapy, in addition to being less toxic and more specific than chemotherapy, provides a different mode of cytotoxicity that may synergize with that induced by chemotherapeutic agents. Immunotherapy also offers the prospect of inducing immune memory that may be important for long term disease-free survival of patients with CLL. However, there are obstacles that may prevent successful immunotherapy. CLL patients are generally immunosuppressed even before any therapy is given and the immunosuppression increases as the disease progresses. Therefore, any immunotherapeutic approaches for CLL should be aplied in early disease when immunosuppression is least encountered. We previously demonstrated the expression of a CT antigen, SEMG 1, in 3/9 patients with CLL. Furthermore, we also demonstrated that the presence of high titer IgG in the serum of patients expressing SEMG 1, suggesting the in vivo immunogenicity of SEMG 1 in the cancer-bearing autologous host. We have also recently used SEMG 1 as the bait in a yeast two-hybrid system of testicular cDNA library and identified that Protamine 1 is the interacting ligand of SEMG 1 and that Protamine 1 is also a novel CT antigen, suggesting that both SEMG 1 and Protamine 1 may be suitable antigens for tumor vaccine development. However, the expression of SEMG 1 and Protamine 1 in early CLL is unknown. We have in this study set out to determine whether or not SEMG 1 and/or Protamine 1 could be used for the design of tumor vaccine for the targeting of patients with early CLL, in particular, those with poor risk disease, as predicted by Zap 70 expression. Using pairs of sequence-specific primers in RT-PCR on a cohort of CLL (41 Stage 0/I and 6 Stage II/III), we found that SEMG 1 gene is expressed in 24/47 (51%) and Protamine 1 in 16/47 (34%) of CLL patients. Gene expression in most cases was associated with the detection by immunocytochemistry of SEMG 1 and/or Protamine 1 in the CLL cells. The expression frequency of SEMG1 and Protamine 1 in CLL did not appear to differ between early and late stage disease. 19/41 of patients with early stage disease and 5/6 of patients with late disease expressed SEMG 1; 12/41 of patients with early stage disease and 4/6 patients with late disease expressed Protamine 1. Furthermore, the expression of these antigens was equally distributed between Zap 70+ and Zap 70− CLL. SEMG 1 was expressed in 4/6 of Zap 70+ CLL (all 6 had early disease) and 2/9 of Zap 70− CLL (1/8 early disease and 1/1 late disease). Interestingly, although Protamine 1 expression in CLL predicted for SEMG 1 co-expression, only 67% of SEMG 1+ CLL expressed Protamine 1. Our results, therefore, suggest that both SEMG 1 and Protamine 1 are suitable targets for tumor vaccine development for some patients with early CLL, especially those with high risk disease, as predicted by Zap 70 expression.

Description: Cancer patients frequently suffer with anxiety, fatigue, loss of well-being and functionality. In myeloma patients this is compounded by the effects of lytic bone disease, causing chronic pain and impaired mobility. The result is a decrease in physical fitness and loss of confidence in carrying out day-to-day activities, contributing to a reduced QoL. The development of novel therapies has extended the survival of these patients, hence such issues are of increasing importance and effective rehabilitation programmes are urgently needed. We carried out a pilot study of a tailored exercise training programme in patients in stable plateau phase. The primary aims were to determine the feasibility, adherence rate, and the effects on QoL, physiological and cardiorespiratory functions. Eligible patients underwent radiological and cardiac screening prior to study entry. There was a 25% screening failure rate due to disease progression or fracture risk, these patients proceeding to prophylactic surgery or radiotherapy. Twenty-five patients were given a programme based on their current exercise capacity, level of functioning and individual rehabilitative needs. Patients undertook exercise training 3 times a week for 6 months, with 1 supervised exercise session each week in the hospital outpatient gym. Each session comprised stretching and mobility, aerobic (treadmill, cycle ergometer or walking to 50–60% of heart rate reserve and 15–30 minutes duration) and resistance training (theraband, ankle, hand weights and body-weight) in order to improve flexibility, cardiorespiratory fitness and muscle strength. QoL and physiological outcomes were assessed at baseline, 4-weekly for 3 months, then 6 weekly for 3 months during the 6-month study period. A preliminary analysis of 17 patients who completed 3 months on the programme has been performed. Average attendance at the weekly exercise class was 84%. Adherence to the exercise programme, as assessed by inspection of a log-book was 〉50% in all patients; 35% achieved 〉90% adherence. Significant improvements were found in the FACT G (baseline: median 85; range 62 – 104, 3 months: 90; range 70 – 108, p

Description: Rituximab is a chimeric anti-CD20 monoclonal antibody that has been used successfully in the treatment of Non Hodgkin’s Lymphoma or patients with Chronic Lymphocytic Leukaemia (CLL). The mechanisms of action of Rituximab are not fully understood although antibody dependent cell mediated cytotoxicity and complement dependent cytoxicity have been shown to be important. An alternative mechanism is the induction of apoptosis through activation of pathways mediated through CD20. CD20 is involved in many cellular processes including proliferation, activation, differentiation and apoptosis. We have found that treatment of CLL cells with 20 μ g/ml Rituximab cross-linked with a secondary antibody reduced cell viability from 84±8% (in unstimulated cells) to 51.50±10% after 48h of cultivation by the Annexin/PI method. Using inhibitors specific for p38, JNK and ERK pathways, we found that inhibition of p38 inhibits the induction of apoptosis by crosslinked Rituximab. Rituximab has been reported to inhibit this pathway andlead to down regulation of bcl-2 expression in AIDS related lymphoma cells. However the mechanism is unclear. One mechanism by which many genes involved in apoptosis are regulated is through induction of mRNA instability through induction of Tis11 family genes. The Tis11 family (Tis11, Tis11b/Berg36 and Tis11d) bind to AU Rich elements present in several mRNA (eg bcl-2, TNF) and cause their degradation. We found that Tis11b/Berg36 is strongly induced by crosslinked Rituximab. Tis11d was weakly induced while Tis11 remained unchanged after treatment. Furthermore we found that induction of Tis11b/Berg36 by Rituximab is partly regulated through the p38 pathway since inhibition of this pathway resulted partial or complete inhibition of Tis11b/Berg36 induction. This suggests that Tis11b/Berg36 may mediate the induction of apoptosis by Rituximab through the degradation of proteins involved in apoptosis that contain AU Rich elements, disrupting autocrine cytokine feedback mechanisms and down regulating bcl-2.

Description: Tis11b/Berg36 is a member of a family of proteins involved in post-transcriptional gene regulation. Members of this family bind to AU rich elements in certain mRNA species and increase mRNA stability. mRNA that contain AU rich elements and can be regulated in this way include molecules involved in the regulation of apoptosis eg bcl-2 and several cytokines eg TNF, IL-2. We have recently shown that Tis11b/Berg36 is involved in the regulation of apoptosis following Rituximab treatment of CLL cells suggesting a pro-apoptotic role of this gene in CLL cells. Thus B-CLL were stimulated with IL-4, anti-CD40, anti-CD40+IL-4 and PMA. It was found that IL-4, CD40 and their combination significantly inhibited spontaneous apoptosis while PMA was able to inhibit spontaneous apoptosis in some but not all patients tested and the overall effect did not reached statistical significance. Unexpectedly Tis11b/Berg36 mRNA remained unchanged following IL-4 treatment, but expression was induced following anti-CD40 or PMA treatment. Because it was found that regulation of Tis11b/Berg36 mRNA following the latter two treatments was under the control of NF-κB pathway and not p38 (as found for Rituximab treatment) it was hypothesized that this gene may be involved in the regulation of cell cycle or differentiation of B-CLL cells. Indeed it was found that stimulation of B-CLL cells with either PMA or anti-CD40 resulted in increase in sIgM as a marker of B-CLL differentiation. CLL cells were also found to express high basal levels of two other members of this family, Tis11 and Tis11d. From these stimuli, IL-4 or anti-CD40 were found to downregulate the basal expression of Tis11 mRNA. These data suggest that members of the Tis11b/Berg36 family are involved in the regulation of apoptosis and differentiation in B-CLL cells.

Description: Chronic Lymphocytic Leukaemia (CLL) remains largely incurable despite recent advances in therapy, and therefore alternative strategies are of interest in treating this disease. One such alternative is the use of gene therapy, but this relies on developing efficient gene transfer technologies. We have compared several viral vectors coding for green fluorescent protein (GFP) for their ability to transduce CLL cells. Three serotypes of adeno-associated virus (AAV) were used, AAV-2, AAV-5 and a relatively new isolate AAV-8, an EI-EIII deleted adenoviral 5 based vector, AV-5, all with GFP regulated by the CMV promoter, and a VSVG pseudotyped lentiviral vector in which GFP expression is controlled by EF1a promotor/enhancer complex. AV-5 resulted in variable GFP expression, 24.1±3.4%, n=10 but caused cell death at high multiplicities of infection (MOI). The lentiviral vector resulted in GFP expression of 23.5±2.6%, n=12, at the highest titre used, and expression declined in a distinct dose-dependent manner as titres were reduced. Of the AAV vectors, AAV-8 was the most efficient with GFP expression at 41.3±1.0% n=14. We conclude that AAV-8 is a promising viral vector for efficient transduction of CLL cells. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions.