Suppression of CXCL-1 Could Restore Necroptotic Pathway in Chronic Lymphocytic Leukemia

The defective necroptotic pathway and cytokines were important in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). On the other hand, selenite could induce necroptosis in various sold malignancies by generating reactive oxygen species. However, the relationship between necroptosis and cytokines still remains unclear. Here, we managed to clarify the role of different cytokines and selenite in the defective necroptotic pathway of CLL. We first screened the expression of different cytokines related to malignancies between CLL patients and controls using Realtime RT-PCR. Only the expression of CXCL-1, MCP-1, IL-6 and GM-CSF was significantly higher in CLL patients than that of controls. (Figure A, B) In normal B lymphocytes, TNF-α and z-VAD could induce necroptosis and also downregulated the expression of CXCL-1 and MCP-1, which indicated that CXCL-1 and MCP-1 might have correlation with necroptosis. (Figure C) After knockdown of CXCL-1 rather than MCP-1 by siRNA, CLL cells restored the TNF-α/z-VAD induced necroptosis measured by flow cytometry. (Figure D, E) To assess the association between CXCL-1 and lymphoid enhancer-binding factor 1 (LEF-1), which has already been confirmed as the key protein in the necroptotic pathway of CLL, we first performed flow cytometry to verify that CLL cells restored TNF-α/z-VAD induced necroptosis after inhibition of either CXCL-1 or LEF-1. (Figure F) Then, we used Realtime RT-PCR, Western Blot and ELISA to confirm that the expression of LEF-1 was downregulated after inhibiting the expression of CXCL-1 by siRNA, however, the expression of CXCL-1 did not change significantly after knockdown of LEF-1. (Figure G, H, I) This results demonstrated that CXCL-1 located the upstream of LEF-1. To figure out the relationship between cytokines, necroptosis and selenite, we first construct the selenite concentration gradient and selenite with different concentrations was added into CLL cells together with TNF-α and z-VAD. We found that selenite could downregulate the expression of CXCL-1 but had little influence on LEF-1 measured by Realtime RT-PCR. (Figure J) Then, flow cytometry was performed to calculate the percentage of survival CLL cells and only 3.2μM selenite could significantly induce necroptosis of CLL cells. (p = 0.0102, Figure K) Finally, both Western Blot and ELISA showed the similar result that 3.2μM sodium selenite downregulated the translational expression of CXCL-1 but had little impact on LEF-1. (Figure L) Figure Disclosures No relevant conflicts of interest to declare.

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Heat stress combined with lipopolysaccharide alter the activity and superficial molecules of peripheral monocytes

International Journal of Immunopathology and Pharmacology ◽

10.1177/2058738419828891 ◽

2019 ◽

Vol 33 ◽

pp. 205873841982889

Author(s):

Jiajing Luo ◽

Yi Chen ◽

Chengjia Ding ◽

Jialing Qiu ◽

Yulan Chen ◽

...

Keyword(s):

Flow Cytometry ◽

Heat Stress ◽

Western Blot ◽

Inflammatory Mediators ◽

Peripheral Blood ◽

Heat Stroke ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Rt Pcr ◽

Tnf Α

The purpose of this study was to focus on the underlying relationship between the hyperactivity for the peripheral monocytes and heat stroke by investigating the inflammatory oxidative activity of and the expression of superficial molecules. Peripheral blood samples were collected from 10 healthy adult volunteers. Human blood monocytes were isolated by density gradient centrifugation and sequent adherent culture. The objectives were divided into four groups: 43°C heat stress combined with lipopolysaccharide (LPS) group, 43°C heat stress group, LPS group, and control group. There were 10 cases in each group. An enzyme-linked immunosorbent assay (ELISA) test was used to measure the concentrations of supernatant inflammatory mediators (tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-10 (IL-10)). After loaded by 2,7-Dichlorodi-hydrofluorescein-diacetate (DCFHDA) fluorescent probe, intracellular reactive oxygen species (ROS) levels were determined by a flow cytometry. After fluorescent microspheres incubation, the phagocytosis of monocytes was observed under a fluorescent microscope. Respectively, the flow cytometry and Western blot were used to evaluate the level of triggering receptor expressed on myeloid cells-1 (TREM-1) and Toll-like receptor-4 (TLR-4) on the monocytes. Furthermore, the mRNA expression of TREM-1 and TLR-4 was detected by real-time polymerase chain reaction (RT-PCR). The heat stress combined with LPS stimulation promoted the peripheral monocytes to produce inflammatory mediators (TNF-α, IL-1β, and IL-10) and release ROS. Otherwise, such complex strike significantly suppressed the phagocytic activity of monocytes in peripheral blood. Moreover, the expression of TREM-1, TLR-4 and CD86 was measured by the flow cytometry on peripheral monocytes which were respectively promoted by the union of heat stress and LPS. The results of Western blot and RT-PCR demonstrated the similar kinetics on these superficial molecules (TREM-1, TLR-4, and CD86) stimulated by the combination of heat stress and LPS. The underlying mechanism of the dysfunction for the peripheral monocytes may be related to the abnormal expression of superficial molecules TREM-1, TLR-4, and CD86 on the monocytes induced by heat stress and LPS.

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Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38+CD49d+ Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v114.22.2350.2350 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2350-2350

Author(s):

Antonella Zucchetto ◽

Dania Benedetti ◽

Claudio Tripodo ◽

Riccardo Bomben ◽

Fleur Bossi ◽

...

Keyword(s):

Endothelial Cells ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Conflicts Of Interest ◽

Cell Types ◽

Lymphocytic Leukemia ◽

Bone Marrow Biopsies ◽

Tnf Α ◽

Significant Difference

Abstract Abstract 2350 Poster Board II-327 Introduction: CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells. Methods: CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays. Results: IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells. Conclusions: CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study. Disclosures: No relevant conflicts of interest to declare.

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CD23 Receptor Positivity Has No Prognostic Implication in Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.4397.4397 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4397-4397

Author(s):

Jahan Aghalar ◽

Charles Chu ◽

Rajendra N Damle ◽

Che-Kai Tsao ◽

Nina Kohn ◽

...

Keyword(s):

Flow Cytometry ◽

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Prognostic Markers ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Lymphocytic Leukemia ◽

Mutation Status ◽

Percent Positivity ◽

Cd23 Expression

Abstract Abstract 4397 BACKGROUND Chronic Lymphocytic Leukemia (CLL) phenotypically expresses CD23, although the percentage of positive cells measured by flow cytometry is variable. We sought to analyze whether the percent of CD23 positive cells in the CLL clone correlates with time to treat (TTT), overall survival (OS) and prognostic markers CD38, ZAP-70, and IGHV mutation status. METHODS We retrospectively analyzed the flow cytometry data of 332 CLL patients on the gated population of cells that were CD5 and CD19 positive. Percentage positivity for CD23, CD38, and ZAP-70 was noted. CD38 and ZAP-70 were considered positive at cut-offs of >= 30% and >=20%, respectively. CD23 was considered negative at <30% and positive at >= 30%. IGHV sequence was determined from cDNA and then compared to germline to assess mutation status using IMGT/V-QUEST. The distributions of time from diagnosis until start of treatment and overall survival were stratified by CD23 positivity, estimated using the product limit method, and compared using the log rank test. Those who had expired without treatment or were alive and not treated at this time point were censored in the TTT analysis. Those who were still alive were censored in the OS analysis. Associations of CD23 positivity with IGHV mutation status, ZAP-70, and CD38 positivity were examined using the chi-square test. RESULTS Out of 332 patients, 25 had diminished CD23 expression (<30%) whereas 307 had normal CD23 expression (>30%). There was no difference in time until start of treatment or overall survival based on CD23 %positivity. CD23 %positivity showed no associations with IGHV mutation status, ZAP-70 or CD38 positivity. CONCLUSION CD23 percent positivity has no prognostic significance in CLL. There is no correlation between CD23 percent positivity and poor prognostic markers such as CD38, ZAP-70, or IGHV mutation status. Disclosures: No relevant conflicts of interest to declare.

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Phenotypic Characteristics of Tumor Cells in Patients with Chronic Lymphocytic Leukemia into Different Prognostic Groups

Blood ◽

10.1182/blood.v126.23.5276.5276 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5276-5276

Author(s):

Aleksei Kuvshinov ◽

Sergei Voloshin ◽

Irina Martynkevich ◽

Ludmila Martynenko ◽

Andrei Garifullin ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Tumor Cells ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Phenotypic Characteristics ◽

Hla Dr ◽

Number Of Patients ◽

Genetic Abnormalities ◽

Prognostic Groups ◽

The Relationship

Abstract Background. The presence or absence of certain cluster of differentiation on the tumor cells of chronic lymphocytic leukemia may affect the course of the disease. Influence of genetic abnormalities on the prognosis of the disease was also proved. Aim. To determine the relationship of the phenotype of tumor cells with genetic prognostic groups of patients with chronic lymphocytic leukemia (CLL). Methods. Thirty-five adult pts (median age 61 year, range 44 - 82; male 24, female 11) with diagnosed CLL were included in the study. The CLL was diagnosed according to the standard basic examination (complete blood count with differential, multicolor flow cytometry (MFC) of blood and bone marrow (BM), lymph node and BM immunohistochemistry (IHC), computered tomography). Cytogenetic studies were performed on blood samples using standard GTG-method. Interphase FISH analyses were performed according to the manufacturer's protocol using DNA probes: LSI 13(RB1)13q14, LSI ATM (11q22), CEP12, LSI TP53 (17p13.1) (Abbott). Immunophenotype (IFT) of CLL cells assessed with combinations: CD3/CD19, CD19/CD5, CD19/CD11c, CD19/CD20, CD19/CD22, CD19/CD23, CD19/CD25, CD19/CD38, CD19/CD43, CD19/CD81, CD19/HLA-DR, and CD19/CD5/CD23. Results. Stratification of patients into prognostic groups was performed based on identified GA. Favorable prognosis - patients with del(13q) (n = 9); neutral prognosis - normal karyotype (n = 14) or trisomy of chromosome 12 (n = 4); unfavorable prognosis - del(17p) (n = 3), del(11q) (n = 3) and the complex karyotype (n = 2). Expression of CD20 was lower, and CD38 - higher in adverse group (51.0±16.31 % and 36.02±10.35 %, respectively) versus neutral or favorable groups (CD20+ - 83.17±5.52 % and 84.41±4.7 %; CD38 - 10.46±4.8 %, and 12.44±4.1 %, respectively, p <0.05). The expression level of CD20 and CD38 did not differ between the neutral and favorable groups. The number of patients with CD38 expression more than 10% was higher in the unfavorable group (7/8) versus favorable (4/8) (p <0.05). At the same time overexpression of CD38 was observed more frequently in patients with a lack of expression of CD23 on CD5 tumor cells (CD5+CD23-) (p<0.05). Expression of HLA-DR was higher in patients with MRD-negative remissions (3/4) versus patients with MRD-positive remissions (1/8) (p<0.05). Conclusions. The study of the influence of various factors on the prognosis and course of CLL requires a comprehensive approach. Further researches are needed to determine the relationship between CLL affecting factors like genetic abnormalities, phenotypic characteristics of the tumor cells and MRD. Disclosures No relevant conflicts of interest to declare.

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Association of GRP78, HIF-1α and BAG3 Expression with the Severity of Chronic Lymphocytic Leukemia

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666191211101357 ◽

2020 ◽

Vol 20 (4) ◽

pp. 429-436

Author(s):

Roghayeh Ijabi ◽

Parisa Roozehdar ◽

Reza Afrisham ◽

Hemen Moradi-Sardareh ◽

Saeed Kaviani ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Western Blot ◽

Disease Progression ◽

Blot Analysis ◽

Western Blot Analysis ◽

Strong Association ◽

Hypoxia Inducible Factor ◽

Lymphocytic Leukemia ◽

Rt Pcr ◽

Metabolic Factors

Introduction: Parallel with the progression of Chronic Lymphocytic Leukemia (CLL), the levels of 78KDa Glucose-Regulated Protein (GRP78) and Hypoxia-Inducible Factor 1 alpha (HIF-1α) are increased as they may activate the induction of anti-apoptotic proteins such as BCL2 Associated Athanogene 3 (BAG3). Previous studies have indicated that there is a positive correlation among GRP78, HIF-1α and BAG3. Objective: This study aimed to evaluate the effect of metabolic factors involved in invasive CLL on apoptotic factors. Methods: A case-control study was conducted on 77 patients diagnosed with CLL along with 100 healthy individuals. Cell blood count was performed for all participants. According to Binet's classification, CLL patients were divided into different groups. B cells were isolated from the peripheral blood of CLL patients by binding to anti-CD19 beads. The expression of BAG3, GRP78 and HIF-1α genes was analyzed using the RT-PCR method. To confirm the results of RT-PCR, western blot analysis was carried out. Results: The results showed that there was a strong association among the expression of BAG3, GRP78 and HIF-1α. The stage of CLL in patients was highly correlated with the expression rate of each gene (p<0.001). Accordingly, the western blot analysis indicated that the concentrations of GRP78 and HIF-1α were significantly higher than the expression of BAG3, considering the stage of CLL. Conclusion: It was shown that increased expression of GRP78 and HIF-1α could result in the elevation of BAG3, as well as the disease progression. Therefore, the role of these metabolic factors might be more pronounced compared with the anti-apoptotic agents to monitor disease progression in CLL patients.

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The Earliest Activation Marker CD69 Is An Independent and Strong Progression Indicator in B-Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.2359.2359 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2359-2359

Author(s):

Giovanni Del Poeta ◽

Maria Ilaria Del Principe ◽

Cristina Simotti ◽

Francesco Buccisano ◽

Luca Maurillo ◽

...

Keyword(s):

Flow Cytometry ◽

Multivariate Analysis ◽

Prognostic Factor ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Significant Prognostic Factor ◽

Therapeutic Decisions ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abstract 2359 Poster Board II-336 B-cell chronic lymphocytic leukemia (B-CLL) exhibits features of activated and antigen-experienced B-lymphocytes and CD69 overexpression resembles B cells at an earlier and greater state of activation (Damle, 2002 and 2007). Moreover, the recent in vitro generation of anti-CD69 monoclonal antibodies able to inhibit either tumor growth or enhance NK cell function (Esplugues, 2005) prompt us to extensively evaluate CD69 antigen in our B-CLL cases. The primary endpoints of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD69; 2) to assess the additive prognostic value of CD69 and ZAP-70 and finally 3) to confirm CD69 as an independent prognostic factor. We investigated 401 patients (pts), median age 65 years, 219 males and 182 females. With regard to modified Rai stages, 120 pts had a low stage, 263 an intermediate stage and 18 a high stage. ZAP-70 and CD69 were determined by multicolor flow cytometry, fixing the cut-off values > 20% and >30%, respectively. ZAP-70+ and CD69+ pts were 166/401 (41%) and 108/401 (27%), respectively. CD69 lower than 30% was significantly associated with low Rai stage (105/120; P<0.0001), lymphocyte doubling time >12 months (258/332; P=0.00001), beta-2 microglobulin (B-2M) <2.2 mg/dl (181/223; P=0.00004) and soluble CD23 <70 U/ml (196/240; P<0.00001). There were significant correlations between lower CD69 and IgVH gene mutated status (283 total cases, 150/210; P=0.0001) or low risk (normal or 13q-) FISH cytogenetics (296 total cases, 149/213; P<0.00001). Equally, a strict association was found between lower CD69 and lower ZAP-70 (185/293; P=0.0003). With regard to clinical outcome, both a shorter PFS and OS were observed in ZAP-70+ pts (6% vs 56% at 12 years and 30% vs 95% at 16 years; P<0.00001) as well as in CD69+ pts (9% vs 49% at 14 years, P<0.00001 and 49% vs 75% at 16 years; P=0.00002). Noteworthy, ZAP-70 and CD69 showed additive prognostic properties, since ZAP-70 <20% plus CD69 <30% identified a B-CLL subset at better prognosis with regard to PFS (65% vs 2% at 12 years; P<0.00001, Figure) and OS (97% vs 35% at 14 years; P<0.00001). The two discordant subsets (CD69+ZAP-70 negative and CD69-ZAP-70+) showed an intermediate outcome (Figure). In multivariate analysis of PFS, CD69 (P=0.001) and ZAP-70 (P=0.005) together with cytogenetics and B-2M were confirmed to be independent prognostic factors. On the other hand, with regard to multivariate analysis of OS, age > or < 60 years resulted to be the most significant prognostic factor (P=0.004) followed by CD38 (P=0.01), IgVH status (P=0.02) and ZAP-70 (P=0.04). In conclusion, CD69 antigen, determined by flow cytometry, should be considered a novel important prognostic parameter in B-CLL and its easy and rapid laboratory determination may allow us to identify early progressive pts in order to take timely therapeutic decisions. Disclosures: No relevant conflicts of interest to declare.

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The Sesquiterpene Oil α-Bisabolol Induces Apoptosis of B-Chronic Lymphocytic Leukemia Primary Cells

Blood ◽

10.1182/blood.v118.21.1319.1319 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1319-1319

Author(s):

Massimiliano Bonifacio ◽

Antonella Rigo ◽

Angela Bonalumi ◽

Emanuele Guardalben ◽

Ilaria Nichele ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Lipid Rafts ◽

Conflicts Of Interest ◽

Membrane Integrity ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Primary Cells

Abstract Abstract 1319 We have recently demonstrated that the sesquiterpene oil α-bisabolol is cytotoxic against primary acute leukemia cells ex vivo and in chronic myeloid leukemia cell lines. It enters cells via lipid rafts and activates the mitochondrial-dependent intrinsic pathway of apoptosis, exerting a preferential toxicity against malignant vs normal cells probably due to their higher content in lipid rafts. Here we investigated the in vitro activity of α-bisabolol in primary cells from patients with B-Chronic Lymphocytic Leukemia (B-CLL). Twenty-six patients with newly diagnosed B-CLL gave their informed consent to the study. Cells were collected before any treatment, purified and cultured for 24 hours with serial dilutions of α-bisabolol. Citotoxicity was quantified in flow cytometry by the BD Trucount™ technology to allow comparison between neoplastic and normal residual lymphocytes. B-CLL cells (IC50 42±15 μM) were significantly more sensitive towards α-bisabolol than normal B- (IC50 82±34 μM, p=.005) and T-cells (IC50 120±35 μM, p<.001). Citotoxicity was similar between the IgVH mutated (n=11) and the IgVH unmutated samples (n=7), as well as between the Binet stage A (n=20) and B-C (n=6) patients. To investigate the mechanisms of α-bisabolol-induced toxicity we treated B-CLL cells with 40 μM α-bisabolol for up to 3 hours. We observed a time-dependent increase in fluorescence of cells treated with the membrane-impermeant nucleic acid stain TO-PRO-3, already detactable after 30 minutes. When cells were loaded with the Ca2+ indicator Fluo-4 AM, an increase of Ca2+ influx was revealed already after 15 minutes. These early events indicate that α-bisabolol induces the loss of cellular membrane integrity, so triggering the apoptotic cascade. Then we assessed the mitochondrial transmembrane potential (ΔΨm) with the fluorochrome JC-1 to confirm that a mitochondrial damage is a concurrent mechanism in the apoptotic process induced by α-bisabolol. By flow cytometry we demonstrated that, after 3-hour incubation with 40 μM α-bisabolol, ΔΨm dissipation was already detectable in leukemic cells, while T-lymphocytes, evaluated as internal control in the same samples, stayed vital. To investigate the mitochondrial target of α-bisabolol we examined the function of the mitochondrial permeability transition pore (mPTP). After 5-hour incubation with 40 μM α-bisabolol we loaded cells with the calcein AM dye and added CoCl2 to distinguish between intact and damaged mitochondria, confirming that the function of mPTP was compromised in B-CLL cells but not in normal controls. Finally, to determine whether α-bisabolol affects the oxydative state of treated cells, we evaluated the intracellular concentration of reactive oxygen species (ROS) by measuring the fluorescent signal of CM-H2DCFDA loaded cells. When B-CLL cells were exposed to 40 μM a-bisabolol for 3 hours, they exhibited a clear fluorescence increase, indicating the striking generation of ROS: this was completely abrogated by the addition of N-acetylcysteine, a scavenger of intracellular ROS. Clues about the molecular mechanistics of α-bisabolol have also emerged from in vitro models based on treating cells previously transfected with BH3-only molecules. In this setting, α-bisabolol exposed cells seem to undergo detrimental, non-selective autophagy-like phenomena. Our data indicate that α-bisabolol exerts a level of cytotoxicity against B-CLL cells at concentrations that only partially affect normal B- and T-cells. Moreover, a brief exposure (3–5 hours) to α-bisabolol is sufficient to elicit multiple pro-apoptotic signals independently of the patients' mutational status. Disclosures: No relevant conflicts of interest to declare.

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The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells

Blood ◽

10.1182/blood-2007-04-084756 ◽

2008 ◽

Vol 112 (3) ◽

pp. 711-720 ◽

Cited By ~ 79

Author(s):

Mohammad Luqman ◽

Sha Klabunde ◽

Karen Lin ◽

Georgios V. Georgakis ◽

Anu Cherukuri ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Surface Expression ◽

Lymphocytic Leukemia ◽

The Novel ◽

Antibody Dependent Cellular Cytotoxicity ◽

Gm Csf ◽

Tnf Α ◽

Cd40 Activation ◽

Induced Apoptosis

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-α, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis.

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The Alemtuzumab Treatment of Refractory to Fludarabine Chronic Lymphocytic Leukemia Patients.

Blood ◽

10.1182/blood.v114.22.4415.4415 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4415-4415 ◽

Cited By ~ 1

Author(s):

Elena Kataeva ◽

Anatoly Golenkov ◽

Elena Triphonova ◽

Iolanta Buravtsova

Keyword(s):

Flow Cytometry ◽

Chronic Lymphocytic Leukemia ◽

Dose Escalation ◽

Conflicts Of Interest ◽

Residual Disease ◽

Stage Iv ◽

Median Number ◽

Lymphocytic Leukemia ◽

Level 1 ◽

High Level

Abstract Abstract 4415 The aim of this study was to explore the efficiency of treatment with Alemtuzumab (A.) of chronic lymphocytic leukemia (CLL) patients (pts) refractory to Fludarabine(F.). Meterials and methods We observed 6 pts (3 male and 3 female). The median age was 56 years old(52-69). 4 of the pts had Rai stage I desease, 1 patient had Rai stage III, 1 had Rai stage IV. We revealed that 4 pts had 17p deletion with high level, 1 patient did not have del 17p, 1 - was not assessed. High level of the expression of CD38+ was determined, the median number was 37,3%(15-82). All pts were pretreated with Chlorambucil, COP, CHOP, FC for 18 months(7-48). All pts were refractory to F. 5 pts received A. monotherapy during median 9 weeks (2-18). Subcutaneous(SQ) A. dose escalation: 3mg-10mg-30mg on days 1,3,5, followed by 30mg Monday, Wednesday and Friday for 17 weeks. 1 patient received 5 courses FluCam(A. 30 mg 1,2,3 days IV after dose escalation 3mg-10mg-30mg, F. 25mg/m2 1,2,3 days). All pts received PCP, herpes and cytomegalovirus(CMV) and fungal prophylaxis as well as CMV viral DNA monitoring. Responses were based on NCI-WG 1996 criteria. Minimal residual disease (MRD) was measured in peripheral blood and bone marrow aspirate using flow cytometry for CD19+/CD5+/CD23+ lymphocytes. Results The efficiency of the treatment of CLL pts was following: 2 pts (33,3%) displayed disease progression(PD) in 2 and 4 weeks of A. monotherapy, 2 pts achived (33,3%) CR in 14 and 16 weeks, 2 pts (33,3%), achived PR, among them 1 patient showed PR after 5 FluCam courses, the other — after 14 weeks of A. monotherapy. At the completion of the study 4 pts (66,6%) had no evidence of MRD by flow cytometry(<0,01%). Conclusion Obtained results showed high effectiveness of A. and acceptable toxity, among resistant to F. CLL pts (66,6% responded), the majoority of them had unfavourable del 17p. Disclosures: No relevant conflicts of interest to declare.

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Preliminary Study of the Autoantigens on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb’s Test(+) Hemocytopenia

Blood ◽

10.1182/blood.v118.21.4382.4382 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4382-4382

Author(s):

Rong Fu ◽

Hui Liu ◽

Zonghong Shao ◽

Jun Wang ◽

Lijuan Li

Keyword(s):

Western Blot ◽

Conflicts Of Interest ◽

Healthy Controls ◽

Newly Diagnosed ◽

Rt Pcr ◽

Glycine Buffer ◽

Preliminary Study ◽

The Relationship ◽

Epor Expression ◽

Normal Controls

Abstract Abstract 4382 Objective To observe the relationship between EPO receptor(EPOR) and autoantibodies-IgG/IgM(auto-Ab) on the membrane of erythropoietic cells of the patients with IRP and then explore the probable autoantigens of auto-Ab in IRP. Methods 46 newly diagnosed IRP patients(15 with auto-Ab on erythropoietic cells and 31 without auto-Ab on erythropoietic cells) and 18 healthy controls were enrolled in this study. EPOR expression on their nuclear erythrocytes were tested with FCM to observe the relationship between EPOR and auto-Ab; EPOR mRNA were tested by RT-PCR; Stat5 and P-Stat5 proteins in nucleared erythrocytes were measured by Western blot; EPOR expression on the nucleared erythrocytes membrane were tested again after stripping autoantibodies with glycine buffer. Results (1)EPOR of auto-Ab(+) arm(1.59±0.87)% was significantly lower than that of auto-Ab (−) arm(4.58±4.09)%(P<0.01)and the latter was significantly higher than that of normal controls(2.27±1.76)%(P<0.05); GEPOR of IRP patients was inversely correlated with their auto-Ab (r=−0.543,P=0.000).(2) EPOR mRNA of auto-Ab(+) arm(0.685±0.136)was significiantly higher than that of auto-Ab (−) arm(0.554±0.116)(P<0.01)and normal controls(0.580±0.119)(P<0.05);(3) Protein Stat5 of auto-Ab(+) arm(1.45±0.94) was significantly higher than that of normal controls(0.54±0.36)(P<0.05). While P-Stat5 of auto-Ab(+) arm(0.42±0.18) was significantly lower than that of normal controls(0.85±0.38)(P<0.05). (4) EPOR expression increased significantly after auto-Ab stripped from nucleared erythrocytes with glycine buffer. Conclusion The auto-Ab of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR might be one of autoantigens in IRP. Disclosures: No relevant conflicts of interest to declare.

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