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1
Schlaak, Max Simon. "Differentielles Genexpressionsmuster in chronischen lymphatischen Leukämiezellen vom B-Zell-Typ (B-CLL-Zellen)." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=966320069.
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Schlaak, Max Simon. "Differentielles Genexpressionsmuster in chronischen lymphatischen Leukämiezellen vom B-Zell-Typ (B-CLL-Zellen)." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2003. http://dx.doi.org/10.18452/14817.
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Die B-CLL ist eine niedrigmaligne Erkrankung, die im höheren Lebensalter auftritt und für die es weiterhin keine dauerhaft kurative Therapie gibt. Die Erforschung der genetischen Grundlagen dieser Krankheit könnte Erkenntnisse über die Funktionsmechanismen und neue Diagnose- und Therapieansätze erbringen. Ziel dieser Arbeit war es, eine Genbank von CLL-Patienten bzw. gesunden Spendern zu erstellen, um Gene, die für die Entstehung der Erkrankung mitverantwortlich sein könnten, zu identifizieren. Die subtraktive suppressive Hybridisierung (SSH) wurde als Methode eingesetzt, um cDNA-Mischungen zu generieren, in denen differentielle Gene angereichert wurden. In dieser Arbeit wurden drei differentielle Gene identifiziert. Eines dieser Gene kodiert den Oberflächenmarker CD5, der bei CLL-Erkrankten stärker exprimiert wurde. Da CD5 ein für die B-CLL relativ spezifischer Marker ist, bestätigte dieses Ergebnis die Qualität der Genbank. Mit Hilfe der SSH wurde ein Verlust der Expression der cDNA für den Oberflächenmarker CD20 bei CLL-Erkrankten nachgewiesen. CD20 ist möglicherweise ein Kalziumkanal und wird als Ziel des anti-CD20-Antikörpers Rituximab verwendet. Der Verlust von CD20 im Verlauf der Erkrankung konnte in dieser Arbeit angedeutet werden. Weiterhin wurde eine verringerte Expression von cDNA für IkappaBa (Mad-3) bei CLL-Patienten entdeckt. Der Transkriptionsfaktor IkBa spielt im zellulären Ablauf der Apoptose eine wichtige Rolle. Phosphoryliertes IkBa inhibiert die Wirkung von NF-kB. Eine Reduktion von IkBa-cDNA könnte ebenfalls die Funktion von NF-kB beeinflussen. Dieses Ergebnis könnte auch für zukünftige immuntherapeutische Ansätze von Bedeutung sein. Weiterhin sind die nachgewiesenen Gene interessant für die Technik des "DNA-Microarrays". Diese schnelle Methode ermöglicht kostengünstige Diagnosen und könnte daher eine zukunftsweisende Alternative zu herkömmlichen Ansätzen bieten. In der vorliegenden Arbeit konnten differentielle Transkripte auf mRNA-Ebene bei B-CLL-Erkrankten und gesunden Kontrollen festgestellt und untersucht werden. Die Ergebnisse geben neue Einsichten über Onkogene und Tumorsurpressorgene in der B-CLL und eröffnen zukünftige immuntherapeutische Ansätze.<br>The B-CLL is a malignancy that appears in the higher age and for which it gives further no durably curative therapy. The investigation of the genetic bases of this illness could furnish understandings of the function mechanisms and new diagnosis and therapy extensions. It was the goal of this work of generating a gene bank of CLL-patients and healthy donors, in order to identify genes, that could be responsible for the origin of the disease. The subtractive suppressive hybridisation (SSH) was inserted as a method in order to generate cDNA-mixtures, in which differential genes were enriched. In this work, three differential genes were identified. One of these genes codes for CD5 that in CLL-patients was stronger expressed. Because CD5 is a marker relatively specific for the B-CLL, this result confirmed the quality of the gene bank. By means of the SSH, a loss of the Expression of the cDNA was proved for CD20 in CLL-patients. Possibly CD20 is a calcium canal and is used as a goal of the anti-CD20-antibody Rituximab. The loss of CD20 in the progress of the disease could be indicated in this work. Further a diminished expression was discovered by cDNA for IkBa (Mad-3) in CLL-patients. The transcription factor IkBa plays an important role in the cellular system of apoptosis. Phosphorylated IkBa is inhibiting the effect of NF-kB. A reduction of IkBa-cDNA could influence also the function of NF-kB. This result could be interesting for future immune therapeutic extension. Further the proved genes are interesting for the technology of the "DNA-Microarrays". This fast method enables favorable diagnoses and could offer therefore a leading-edge alternative to conventional extensions. In the existing work, differential transcripts could be assessed on mRNA-levels in B-CLL-patients and healthy donors. The results give new insights over oncogenes and tumor surpressing genes in the B-CLL and open future immune therapeutic extensions.
3
Kurzeder, Christian. "Gentransfer in primäre B-CLL-Zellen mittels EBV abgeleiteter Genvektoren." [S.l.] : [s.n.], 2004. http://edoc.ub.uni-muenchen.de/archive/00004955.
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Kurzeder, Christian. "Gentransfer in primäre B-CLL-Zellen mittels EBV abgeleiteter Genvektoren." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-49552.
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Bund, Dagmar. "hTERT, CD23 und CD229 als Tumorantigene bei der B-CLL." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-145282.
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Almond, Jason Baron. "Mechanisms of proteasome inhibitor-induced apoptosis of B-cell chronic lymphocytic leukaemia (B-CLL) cells." Thesis, University of Leicester, 2002. http://hdl.handle.net/2381/30761.
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Proteasome inhibitors, including lactacystin, LLnL (N-acetyl-N-leucinyl-L-leucinyl-L- norleucinal) and MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), potently induce apoptosis in leukaemic B-cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL). This pro-apoptotic effect occurs in cells from patients at all stages of the disease, including those resistant to conventional chemotherapy, suggesting that proteasome inhibitors may be useful for treatment of B-CLL. Following initial inhibition of proteasomal activity and an increase in ubiquitinated proteins, these agents induce mitochondrial cytochrome c release and caspase-dependent apoptosis, involving cleavage/activation of caspases -2, -3, -7, -8 and - 9. Pre-treatment with the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe)fluoromethyl ketone (Z-VAD.fmk), does not prevent the release of cytochrome c or partial processing of caspase-9 but prevents activation of effector caspases and induction of apoptosis. These results suggest that the release of cytochrome c is caspase independent and that caspase-9 is the initiator caspase in proteasome inhibitor-induced apoptosis of B-CLL cells. Activation of B-CLL lysates with dATP results in the formation of an -700 kDa caspase-activating apoptosome complex containing Apaf-1. A similar complex is formed in B-CLL cells induced to undergo apoptosis by proteasome inhibitors. Mechanisms of proteasome inhibitor-induced apoptosis vary between cell types, but often involve accumulation of short-lived proteins such as p53, p27 and pro-apoptotic Bcl-2 family members, activation of the stress kinase JNK, or inhibition of NF-kB transcriptional activity. Proteasome inhibitor-induced apoptosis in B-CLL cells is not triggered by alterations in the Bcl-2:Bax ratio, increase in pro-apoptotic t-Bid, or alterations in p27, XIAP, cIAPl or cIAP2. There is also no accumulation of IkB proteins that would inhibit NFkB survival signalling. Although proteasome inhibitors cause an accumulation of p53 and p21, this is not sufficient to induce apoptosis, as etoposide causes more pronounced increases in these molecules but is a less potent inducer of apoptosis. Activation of the stress kinases c-Jun N- terminal kinase (JNK) and p38 also does not appear to be involved. Therefore proteasome inhibitors induce mitochondrial perturbation in B-CLL cells by an apparently novel mechanism.
7
Walton, Alexander James. "CD4+PF+ T Cells in B-CLL Patients & Normal Controls." Thesis, University of Westminster, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.502405.
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Our group has previously shown that CD4+Perforin (PFt T cells with cytotoxic potential are expanded in patients with B-CLL, accounting for up to 50% of CD4+ T cells in this disease. This study confinns the finding of increased percentages of CD4+PF+ T cells in a new cohort of B-CLL patients. However, the significance of this subset in B-CLL remains unclear. Evidence has accumulated for the potential role of CD4+ cytotoxic T cells in controlling cytomegalovirus (CMV). Therefore, a potential relationship between chronic CMV infection and CD4+PF+ T cell expansion in B-CLL was investigated. CMV seropositivity was found to be strongly associated with CD4+PF+ T cell expansion in both B-CLL patients and controls. This suggested that CD4+PF+ T cells from CMV seropositive (SP) patients and controls might contain clonally expanded populations of CMV-specific cells. To test this hypothesis, the CMV reactivity of CD4+PF+ and CD4+PF- T cells from B-CLL patients and controls was detennined using an intracellular cytokine staining flow cytometric assay. CD4+PF+ cells from untreated patients were enriched for CMV-reactive cells, as measured by . IFN-y production, compared to the CD4+PF- subset. In addition, the data indicated that CD4+ T cell immunity to CMV is dysregulated in B-CLL patients. CD4+PF+T cells from B-CLL patients are characterised by a highly differentiated CD2S-CD57+ phenotype. To further characterise the differentiation phenotype of these cells, the distribution of CD45RA and CCR7 was detennined on CD4+PF+ T cells from B-CLL patients and controls. CD4+PF+ T cells from B-CLL patients were both characterised by a highly differentiated T-effector memory TEM (CCRT) phenotype, but differed in the proportion of CD45RA expressing cells, which might reflect chronic T cell activation in the fonner group. ~inally, flowFISH analysis revealed that CD4+PF+ T cells had shorter telomeres compared CD4+PF- T cells in B-CLL patients, indicative of a more extensive replicative history. The results indicate that low, but persistent, antigenic exposure in CMVinfected individuals leads to the emergence of a CD4+PF+ T cell subset, characterised by a highly differentiated cell surface phenotype and shortened telomeres.
8
Munoz-Ritchie, Varinia Graciela. "P-glycoprotein-associated anthracycline resistance in B-CLL : potential for cytokine modulation." Thesis, University of Plymouth, 2001. http://hdl.handle.net/10026.1/2809.
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The phenomenon of multidrug resistance (MDR) in cancer cells is generally associated with P-glycoprotein (P-gp) expression and presents an obstacle to successful chemotherapy. Attempts to overcome P-gp-associated MDR using P-gp modulators, such as verapamil, have been hindered by their intrinsic in vivo toxicity. In 1991, however, Scala et al. demonstrated the alteration of P-gp function by interferon-alpha (IFN-α) in vitro at non-toxic in vivo concentrations, suggesting a basis for the use of IFN-α clinically in patients exhibiting P-gp-associated MDR. Drug resistance in B-CLL has been linked to the phenomenon of MDR, however, publications regarding this have been conflicting. The contrasting results prompted further investigation of the role of P-gp-associated anthracycline resistance and, using isolated β-lymphocytes from B-CLL patients, this investigation examined P-gp expression, function and IFN-α modulation in vitro. Optimum conditions for in vitro analysis of P-gp-associated anthracycline resistance were determined by examining the stability of the anthracycline, daunorubicin, in varying cell culture conditions. The resulting system balanced conditions affecting drug stability with those affecting cell survival. While other investigations have neglected the issue of drug stability, this study demonstrates that the instability of daunorubicin may be a critical variable determining the outcome of drug sensitivity studies. In RPMI + 2mM L-glutamine and 10% (v/v) FBS, loss of drug concentration is due to both adsorption and degradation and these experiments show that the presumed availability of drug may be over-estimated in in vitro studies. Furthermore, the degradation products might interfere with P-gp function and modulation. MDRl gene mRNA was detected in the B-cells of forty-three out of fifty B-CLL patients analysed, whereas P-gp expression, as measured by flow cytometry, resulted in only sixteen patients out of fifty-five being classed as positive (> 10% increase in staining as compared to the control). P-gp functionality and modulation studies on the B-cells of eleven patients confirmed the existence of an efflux mechanism with identical characteristics to P-gp using verapamil, the dye rhodamine 123 (rho123) and daunorubicin. Four patients were classed as functional low expressers (functional P-gp with low P-gp expression (7-10% increase in staining)), six were classed as functional high expressors (functional P-gp with high P-gp expression (20-57% increase in staining)) and one as a non-functional high expressor (non-functional P-gp with high P-gp expression (13.4% increase in staining)). Verapamil modulated rho123 efflux in all ten patients classed as P-gp functional expressors, and daunorubicin efflux in eight of these patients. However, IFN-α modulated rho123 and daunorubicin efflux in only two and one patients, respectively, even at concentrations higher than 500I.U./ml. In contrast to Scala et al. (1991), this finding suggests that at a well tolerated concentration IFN-α may not be suitable for use as a P-gp modulating agent in vivo in B-CLL, although conclusive evidence would require a larger study.
9
Hellqvist, Eva. "Antigen interaction with B cells in two proliferative disorders : CLL and MGUS." Doctoral thesis, Linköping : Department of Clinical and Experimental Medicine, Linköping University, 2010. http://www2.bibl.liu.se/liupubl/disp/disp2010/med1158s.pdf.
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Gorgone, Ausilia Giuseppa. "Correlazione tra dati biologici e prognosi in pazieti affetti da B-CLL." Doctoral thesis, Università di Catania, 2012. http://hdl.handle.net/10761/1138.
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Gli studi di questi anni hanno dimostrato che i fattori biologici giocano un ruolo fondamentale per la stratificazione del rischio come elementi predittivi del treatment-free survival e dell' overall survival nei pazienti affetti da CLL in stadio iniziale.La maggioranza di questi markers prognostici ,anche se ormai in uso quotidiano nella pratica clinica, non è ancora incluso nelle linee guida internazionali che si basano ancora su criteri esclusivamente clinici e individuano la valutazione biologica da usare in combinazione ai parametri clinici.Il loro impiego può certamente contribuire al miglioramento del clinical management dei pazienti affetti da CLL .
11
Memon, Azka. "The function of CD180 toll like receptor(TLR) on control B cells and B cell chronic lymphocytic leukaemia (B-CLL) cells." Thesis, University of Westminster, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507859.
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Hellebrand, Eva. "Entwicklung von GM-CSF-produzierenden EBV-Vektoren für die Immuntherapie der B-CLL." Diss., lmu, 2003. http://nbn-resolving.de/urn:nbn:de:bvb:19-12575.
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Niu, Suli. "Quantitative Determination of Surface Markers on B-cell Chronic Lymphocytic Leukemia (CLL) Cells." Thèse, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/30982.
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To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.
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McWilliams, Emily Mary. "Restoring Innate NK-cell Immunity with Antibody Therapeutics in CLL B-Cell Malignancy." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1479863842166353.
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Kiaii, Shahryar. "T cells in patients with B-cell chronic lymphocytic leukemia (B-CLL) and multiple myeloma (MM) : an immunological study /." Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-050-3/.
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Abdulmajed, Hind Abdulrazak Yassin. "Identification of tumour associated antigens as potential targets for the immunotherapy of B-cell chronic lymphocytic leukaemia (B-CLL)." Thesis, University of Leicester, 2008. http://hdl.handle.net/2381/9157.
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B-CLL, the most common adult B-cell malignancy in USA and the western world, is characterized by the accumulation of CD5+ mature B-cells in blood, bone marrow and lymphoid tissues. Many cases require no treatment because of an indolent course, while other patients become symptomatic or develop signs of rapid progression. Treatment is usually non-curative and is directed at reducing the symptoms. However, new therapies are currently under investigation aiming to achieve a curative therapy for B-CLL. The increasing understanding of how peptide antigens are processed, transported and presented by HLA, the identification of tumour antigens recognised by cytotoxic T lymphocytes and the identification of T-cell epitopes on human tumor cells, have opened the routes for the development of more efficient strategies for tumour immunotherapy. Several antigens have been characterized as tumour associated antigens (TAA) in B-CLL, with the potential to elicit specific anti-tumour responses. This thesis addresses the role of survivin, a member of the family of inhibitor of apoptosis proteins, as a potential target for the immunotherapy of B-CLL. Data on survivin expression showed no/ little expression of survivin in resting B-CLL, which was upregulated by in vitro activation with CD40 ligation, CpG-oligodeoxynucleotides (ODN), and with both stimulators together. CD40 ligation resulted in greater B-CLL cell activation, survivin expression, upregulation of activation markers (CD54, CD80 and CD86), and in enhanced activity of B-CLL cells to stimulate allogeneic T cell proliferation, compared to the other two ways of stimulation. Studying the induction of autologous tumour specific T cell responses in vitro, CD40L activated B-CLL cells enhanced CTL responses compared to unactivated cells. No significant difference in response of B-CLL patients T cells to survivin peptide pulsed T2 cells was found between HLA-A2+ and HLA-A2- patients, and no clear peptide specific responses were seen in either group. After investigating survivin peptides for their potential to induce cytotoxic T cell responses, generating survivin specific CTL line from a healthy donor was attempted using peptide pulsed autologous monocyte derived dendritic cells, but was unsuccessful suggesting that no T cells in the culture were able to recognise survivin peptides, or that the culture conditions used were inappropriate for the generation and maintenance of such responses. Finally, in investigation of other TAA expression in B-CLL cells, no expression of MAGE-A1, MAGE-A3, Proteinase-3, WT-1 was detected pre- and post CD40 activation. Positive PRAME expression was detected in 8 out of 20 CD40L activated CLL cells whilst NY-ESO-1 showed an upregulation in one sample. Overall results indicate that much work is still needed to study the potential of immunotherapy in the treatment of B-CLL.
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Osorio, Fernández Lyda María. "Regulation of T-cell proliferation and B-CLL apoptosis by CD6 and FAS/FASL /." Stockholm, 1998. http://diss.kib.ki.se/search/diss.se.cfm?19980605osor.
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Ferrario, A. "THE CLINICAL AND BIOLOGICAL FEATURES OF A SERIES OF IMMUNOPHENOTYPIC VARIANT OF B-CLL." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/203768.
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We described the clinical and biological features of 63 cases of immunophenotypic variant of B-CLL (v-CLL) characterised by intermediate RMH score, in absence of t(11;14)(q13;q32) in FISH analysis in comparison with 130 cases of typical CLL.
We observed significant differences in terms of age <70 yrs (p <.001), lymphocytosis <20 x 109/l (p <.001), lymphocyte doubling time <12 months (p = .02), high serum beta2-microglobulin levels (p <.001) and splenomegaly (p = .002); CD38, CD49d, CD1c were more expressed in v-CLL, CD43 in CLL (p <.001). IgVH mutation and trisomy 12 were more frequent in v-CLL group (p = .001; p<.001); del13q14 in CLL (p=.008). Gene expression profiling of nine v-CLL and 60 CLL indicated that the atypical group presented a specific molecular pattern. After a median follow-up of respectively 55 (4-196) and 60 months (6-180), 25/42 v-CLL (48%) and 55/93 CLL patients (59%) were treated. Time to treatment was significantly shorter in v-CLL when IgVH mutational status was considered (p= .006). The median overall survival was worse in v-CLL mutated cases (p= 0.062).
In conclusion, v-CLL should be identified and dealt with separately from classic CLL. In particular, the prognostic markers that are routinely used to characterise classical B-CLL should not be interpreted as having the same meaning.
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Jordanova, Maya. "Veränderungen von B-Zellantigenen unter Rituximab-Therapie." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2004. http://dx.doi.org/10.18452/15121.
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Das FMC7-Antigen, eine unbekannte B-Zell-Membranstruktur, dient als eines der immunphänotypischen Grundkriterien zur Diagnose der typischen B-CLL. Die unterschiedliche CD20-Expressionsintensität ist auch ein charakteristisches Merkmal bei der Subtypisierung der Lymphomentitäten. In der vorliegenden Arbeit wurde die Modulation des CD20- und des FMC7-Antigens während einer Therapie mit CD20-Antikörper (Rituximab) bei Patienten mit indolenten B-Zell-Lymphomen untersucht. Bei der durchflusszytometrischen Untersuchung quantitativer und qualitativer Charakteristika der monoklonalen B-Zell-Population bei CLL- (n=12) und Non-CLL-Patienten (n=10) unmittelbar nach der Antikörperinfusion und bis zur 8. Woche nach der Therapie wurden parallele Veränderungen von FMC7 und CD20 festgestellt. Die Anzahl und die Fluoreszenzintensitäten der für die beiden Antigene positiven Zellen korrelierten signifikant sowohl bei der malignen Zell-Population (Kurzzeitbeobachtung: n=89; r=0,9; p<br>The FMC7, although being an unknown structure of the B-cell membrane, represents one of the basic immunophenotypic criteria for the diagnosis of the typical B-CLL. A different CD20 expression is a characteristic sign for the sub-typing of lymphomas also. The underlying study investigated the qualitative and quantitative modulation of the CD20 and FMC7 antigens in patients with indolent B-cell lymphomas (CLL, n=12 and non-CLL, n=10) during the therapy with a CD20 antibody (rituximab). Concomitant changes of FMC7 and CD20 expression were found immediately after rituximab infusion and up to 8 weeks thereafter. A correlation was seen for the number of positive malignant cells and for the corresponding fluorescence intensity (short-time observation n=89; r=0.9; p
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Gu, Baijun. "TWO PATHWAYS OF SHEDDING OF L-SELECTIN AND CD23 FROM HUMAN B-LYMPHOCYTES." University of Sydney, 2000. http://hdl.handle.net/2123/821.
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Lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) express large numbers of P2X7 receptors for extracellular adenosine triphosphate (ATP). Activation of P2X7 receptors induces multiple downstream effects, of which the best documented is the opening of an ionic channel that is selective for divalent cations. Another effect of ATP is to induce the shedding of L-selectin (CD62L), a molecule which is involved in the adhesive interactions of lymphocytes on endothelial cells. High levels of soluble L-selectin and CD23 are found in the serum of patients with B-CLL, although the mechanisms involved in their production are poorly characterized. Because extracellular ATP causes shedding of L-selectin, we studied the effect of ATP on shedding of CD23, an adhesion molecule expressed on the surface of B-CLL lymphocytes. ATP induced the shedding of CD23 at an initial rate of 12% of that for L-selectin, while the EC50 of ATP (35 uM) and BzATP (10 uM) was identical for shedding of both molecules. Inactivation of the P2X7 receptor by pre-incubation with OxATP, an irreversible inhibitor of P2X7 purinoceptor, abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2X7 receptor inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nM). Ro 31-9790, a membrane permeant zinc chelator which inhibits the phorbol-ester stimulated shedding of L-selectin also inhibited shedding of CD23 from B-CLL lymphocytes, but the IC50 was different for the two shed molecules (25 versus 1 ug/ml respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester no CD23 was lost under these conditions. Also, Ca2+ inhibits ATP-induced CD23 shedding but not L-selectin shedding. Since soluble CD23 and L-selectin are found in the serum of normal subjects and B-CLL patients, the expression of these two adhesion molecules on lymphocytes before and after transendothelial migration was studied in an in vitro model of this process. In normal and B-CLL subjects, 71�b5% of L-selectin from both T and B cells and 90% of CD23 from B cells was lost following transmigration, while the expression of a range of other adhesion molecules such as VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with OxATP retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Ro 31-9790, which can protect ATP-induced both L-selectin and CD23 shedding, had no effect on inhibiting L-selectin and CD23 lost during transmigration. These data show the presence of a second pathway for the downregulation of L-selectin and CD23 from the lymphocyte surface. Data in vivo from 'knock-out' mice show that L-selectin is essential for the emigration of lymphocytes through high endothelial venules into lymph nodes. The migration of normal and B-CLL lymphocytes across confluent human umbilical vein endothelial monolayers was studied in an in vitro model of this process. Lymphocytes treated with ATP or BzATP showed 56�b25% or 67�b16% loss of L-selectin on the surface and 36�b24% or 64�b19% decrease of transmigration, respectively, while OxATP, which does not alter the L-selectin level, had no effect on lymphocyte transmigration. Further experiments examined this correlation between L-selectin expression and lymphocyte transendothelial migration in this model system. A quantitative assay for cell surface L-selectin showed that expression of L-selectin was lower on B-CLL lymphocytes (8,880�b5,700 molecules/cell) than on normal lymphocytes (29,500�b7,500 molecules/cell, p less than 0.001). Also the rate of transmigration of B-CLL lymphocytes (1.5�b0.9 migrated cells/HUVEC) was lower than normal peripheral lymphocytes (2.4�b0.9 migrated cells/HUVEC, p=0.04). Incubation of lymphocytes in complete medium for 24 hrs increased the expression of L-selectin on B-CLL lymphocytes by 1.5 to 2 fold while the normal lymphocyte L-selectin remained at the initial level. This upregulation of B-CLL L-selectin correlated with a 2 fold increased rate of transendothelial migration. A correlation was found between L-selectin expression on lymphocytes and their ability for transendothelial migration (r^2=0.6). This study shows that the adhesion molecules L-selectin and CD23 can be lost from lymphocytes by two different physiological pathways. One is by P2X7 receptor activation by extracellular ATP while the second is activated by transendothelial migration of these cells. A second finding is that B-CLL lymphocytes have lower level of L-selectin expression and an impaired ability for transendothelial migration compared with normal peripheral blood lymphocytes. Do these results explain the high serum levels of soluble L-selectin and CD23 observed in B-CLL? Although B-CLL lymphocytes do not recirculate as rapidly as normal peripheral blood lymphocytes, the greatly increased number of leukaemic cells in B-CLL ensures that much more soluble L-selectin and CD23 is generated during the recirculation of these cells through the body.
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Rezvany, Mohammad Reza. "Natural specific T cell immunity in patients with B-cell chronic lymphocytic leukaemia (B-CLL) : (a clinical and immunological study) /." Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4841-0/.
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Trimarco, Valentina. "Role of Nocodazole on the survival of chronic lymphocytic leukemia B cells." Doctoral thesis, Università degli studi di Padova, 2012. http://hdl.handle.net/11577/3422967.
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B-cell Chronic Lymphocytic Leukemia (B-CLL) is the most common leukemia in adults and is characterized by the accumulation of clonal CD19+/CD5+/CD23+ B lymphocytes, due to uncontrolled growth and resistance to apoptosis. Leukemic cells from B-CLL show reduced crosslink with specific molecules and high susceptibility to microtubule disrupting drugs, which suggest cytoskeletal alterations.
Microtubules play a crucial role in the vital functions of neoplastic cells, including mitosis, motility and cell-cell contact, and for this reason they became an important target in cancer therapies. In particular, tubulin, a cytoskeletal member, is the target of specific drugs, named microtubule inhibitors. Among these inhibitors, nocodazole induces tubulin depolimerization, mitotic process blocking and shows an apoptotic effect in B leukemic cells.
The aim of this study was to define the effects of nocodazole on B-CLL cells.
First of all, we verified nocodazole capability to favour the depolymerization of tubulin cytoskeleton in different cell types. In addition, we tested nocodazole-induced apoptosis in normal and leukemic B cells, in cell lines (Jurkat, Raji, and K562), in mesenchymal stromal cells (MSCs), and in T lymphocytes of B-CLL patients. Our data pointed out the high specificity of nocodazole for B-CLL cell apoptosis (leukemic cells: 57±25% vs normal B cells: 98±6%, p<0.0001; data are expressed as mean±standard deviation (SD) of percentage of viable cells after treatment with nocodazole) and the absence of toxicity to others cell types.
Growing evidence suggests that the marrow microenvironment, where MSCs are present, protects B-CLL cells from conventional anti-neoplastic drugs. The cultures of neoplastic B cells with MSCs and nocodazole demonstrated that nocodazole is able to overcome MSC protective effect, even after survival signal supplemental, such as CD40L or plasma from the same patients.
The action mechanism of nocodazole in B-CLL cells is still under investigation. However, we observed that nocodazole is able to turn off the increased basal tyrosine phosphorylation of leukemic cells mediated by Src-kinase Lyn through the down-modulation of Lyn active site. Since the specific inhibition of Lyn induces B-CLL cells apoptosis, this linking will be further investigated.
The results obtained in this study suggest a future role of nocodazole as a possible agent for treatment of B-CLL, for its extreme selectivity, the absence of toxicity and its ability to counteract the protective effect provided by marrow microenvironment.<br>La Leucemia Linfatica Cronica di tipo B (LLC-B) è la forma più comune di leucemia nell’adulto ed è caratterizzata dall’accumulo clonale di piccoli linfociti B CD19+/CD5+/CD23+, dovuto sia ad una crescita incontrollata che ad una resistenza all’apoptosi. Le cellule leucemiche di LLC-B presentano inoltre alcune anomalie, come ridotta capacità di legare specifiche molecole e suscettibilità a farmaci che distruggono i microtubuli, che indicano la presenza di alterazioni a livello citoscheletrico.
Il ruolo cruciale che i microtubuli rivestono nelle funzioni vitali delle cellule neoplastiche, quali mitosi, motilità e contatti cellula-cellula, li ha resi un importante target nelle terapie anti-tumorali. In particolar modo la tubulina, componente dei microtubuli, è il bersaglio di una categoria specifica di farmaci anti-tumorali, gli inibitori dei microtubuli; di questa famiglia fa parte anche il nocodazolo, un agente sintetico che induce la depolimerizzazione della tubulina, arresta il processo mitotico ed ha una peculiare specificità nell’indurre l’apoptosi nelle cellule B di LLC-B.
Sulla base di queste considerazioni, abbiamo voluto approfondire gli effetti ed il meccanismo d’azione del nocodazolo sulle cellule di LLC-B.
Dopo aver verificato che il nocodazolo sia effettivamente responsabile della depolimerizzazione dei filamenti di tubulina citoscheletrica in numerosi tipi cellulari, abbiamo valutato l’effetto apoptotico indotto dal nocodazolo in cellule B normali e di LLC-B, in linee cellulari (Jurkat, Raji e K562), in cellule stromali mesenchimali (MSC) e nei linfociti T residui di pazienti affetti da LLC-B. I risultati ottenuti evidenziano l’estrema selettività del nocodazolo nell’indurre l’apoptosi nelle sole cellule B di LLC-B (linfociti B di LLC-B: 57±25% vs B normali: 98±6%, p<0,0001; dati espressi come media±deviazione standard (DS) della percentuale di cellule vive dopo trattamento con nocodazolo) e l’assenza di tossicità nei confronti delle altre popolazioni cellulari prese in esame.
Studi recenti suggeriscono che il microambiente midollare, in cui si trovano anche le MSC, sia in grado di proteggere le cellule leucemiche dall’azione dei farmaci chemioterapici convenzionali. La co-coltura di MSC e cellule B di LLC-B in presenza di nocodazolo ha dimostrato che tale inibitore è in grado di annullare l'effetto protettivo esercitato dalle MSC, nonostante la presenza di segnali di sopravvivenza quali CD40L o plasma ricavato dagli stessi pazienti.
I meccanismi d’azione del nocodazolo rimangono ancora da chiarire, tuttavia abbiamo osservato come nelle cellule leucemiche di LLC-B il nocodazolo sia in grado di ridurre l’aumentata fosforilazione tirosinica basale mediata dalla Src-chinasi Lyn, mediante down-regolazione del sito attivatorio di Lyn. Dal momento che abbiamo dimostrato che l’inibizione specifica di Lyn induce apoptosi nelle cellule di LLC-B, questi primi risultati diventano rilevanti e dovranno essere ulteriormente indagati.
In conclusione, i risultati ottenuti in questo studio hanno evidenziato l’estrema selettività del nocodazolo nell’indurre apoptosi nei linfociti B leucemici, l’assenza di tossicità in vitro e la capacità di contrastare l’effetto protettivo fornito dal microambiente midollare, suggerendo un futuro ruolo di questa sostanza quale possibile agente terapeutico per la cura della LLC-B.
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Rossmann, Eva D. "Targets for immune mediated killing of tumor cells and T cell functions in B-CLL /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-622-7/.
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Bund, Dagmar [Verfasser], and Michael [Akademischer Betreuer] Hallek. "hTERT, CD23 und CD229 als Tumorantigene bei der B-CLL / Dagmar Bund. Betreuer: Michael Hallek." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1024243443/34.
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Dein, Thomas [Verfasser], and Martin [Akademischer Betreuer] Trepel. "Untersuchungen zur Interaktion von CLL B-Zell-Rezeptoren mit Autoantigenen / Thomas Dein. Betreuer: Martin Trepel." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2014. http://d-nb.info/1048626407/34.
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Nothing, Maria Grazia [Verfasser]. "Lenalidomid in der B-CLL: direkte Effekte auf die Tumorzelle in vitro / Maria Grazia Nothing." Ulm : Universität Ulm, 2016. http://d-nb.info/1112603077/34.
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Dai, Yuntao. "CHARACTERIZATION OF TCL1-MURINE B-1A CELL TRANSCRIPTOME DYNAMICS REVEALS NOVEL INSIGHTS INTO CHRONIC LYMPHOCYTIC LEUKEMIA ONSET." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1434632288.
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Schlaak, Max Simon [Verfasser], F. [Gutachter] Schriever, J. [Gutachter] Klose, and U. [Gutachter] Keilholz. "Differentielles Genexpressionsmuster in chronischen lymphatischen Leukämiezellen vom B-Zell-Typ (B-CLL-Zellen) / Max Simon Schlaak ; Gutachter: F. Schriever, J. Klose, U. Keilholz." Berlin : Humboldt-Universität zu Berlin, 2003. http://d-nb.info/1207658294/34.
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Nardini, Elena. "Characterization of genetic events involving IgH switch regions in gastric low grade MALT lymphomas and B CLL." Thesis, Open University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251405.
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Mohamed, Ahmed. "Deciphering the ontogeny of unmutated and mutated subsets of Chronic Lymphocytic Leukemia." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17286.
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Chronic Lymphocytic Leukemia (CLL) is a type of cancer that affects the B cells of the immune system causing problems in the process of producing antibodies. It can be sorted into mutated and unmutated CLL based on the percentage of somatic mutations in the Immunoglobulin Heavy chain Variable region (IgHV). The B cells of healthy individuals can be sorted into three groups; CD27dull memory B cells (MBCs), CD27bright MBCs and naïve B cells. The hypothesis for the project was that the unmutated CLL subset originates from CD27dull MBCs and the mutated CLL subset originates from CD27bright MBCs. RNA-sequencing data from healthy individuals were acquired from a collaboration partner in Rome and CLL-patients were collected from public datasets available online. Several bioinformatic tools were used to analyze the data. First, the quality of the data files was checked, then adapter sequence from the sequencing process and low-quality bases were removed (trimming). Good quality of the files was confirmed after the trimming. Secondly, these files were mapped against the human reference genome (GRCh38/hg38) for alignment, then the resulted data was used to check for genes that showed differential expression between the different groups. Results were analyzed and visualized using Venn diagrams, Principal Component Analysis (PCA) and heatmap plots and random forest. A list of 85 genes was generated based on the different comparisons and was used in one PCA plot that showed clear separation between the different groups. The SWAP70 gene was analyzed for single nucleotide polymorphisms (SNPs). The study concluded five genes that could be used as biomarkers for CLL and the diagnosis of its subtypes where some of them were discussed in previous studies. Also, the mutated CLL subset showed a similar behavior to the healthy individuals and this could validate the original hypothesis and justifies the better disease prognosis for this subtype.
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Blanco, Ares Gonzalo 1989. "Molecular characterization of the microenvironment in CLL-like monoclonal B cell lymphocytosis and early-stage chronic lymphocytic leukemia." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/664506.
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The analysis of the microenvironment in CLL-like monoclonal B cell lymphocytosis (MBL) and early-stage chronic lymphocytic leukemia (CLL) is relevant for understanding the natural history of CLL. To this end, a total of 58 MBL, 54 early-stage CLL and 31 healthy subjects were extensively characterized by various immunological and molecular methods. Purified CD4+ and CD8+ mononuclear cells from peripheral blood were subjected to gene expression studies and T cell receptor (TR) repertoire analysis, whereas cytokine immunoassays were performed in serum samples. Gene expression studies in CD4+ cells revealed increased cytotoxic and inflammatory pathways, which were higher in MBL than in early-stage CLL. Gene dysregulation was not remarkable in CD8+ cells. Increased serum levels of cytokines such as IL8, IFNγ and TNFα were also observed in MBL, while early-stage CLL generally displayed lower cytokine levels, especially amongst cases bearing somatically hypermutated IGHV genes. TR analysis demonstrated oligoclonality in both entities with persisting T cell clones over time and increasing clonality within CD4+ T cells concurrently with the expansion of neoplastic B cells. Besides, identical T cell clonotypes were identified in different MBL/CLL cases. All these findings implicate inflammatory processes and antigenic elements in the immune background of CLL, whose effects are significantly altered during progression from MBL to CLL.<br>El estudio del microambiente en la linfocitosis B monoclonal (LBM) de tipo LLC y en estadios iniciales de la leucemia linfática crónica (LLC) tiene gran relevancia para entender la historia natural de la enfermedad. Con este objetivo se caracterizaron 58 casos de LBM, 54 de LLC en fases iniciales y 31 sujetos sanos. Se analizó la expresión génica y el repertorio del receptor de células T (TR) en fracciones de células mononucleares CD4+ y CD8+ purificadas a partir de sangre periférica. Además, se realizaron inmunoensayos para medir niveles de citoquinas en suero. Los estudios de expresión génica revelaron patrones citotóxicos e inflamatorios aumentados en células CD4+, superiores en LBM con respecto a LLC en fases iniciales. En las células CD8+ no se observó ninguna disfunción remarcable en la expresión génica. Se detectaron niveles aumentados de citoquinas como IL8, IFNγ y TNFα en sueros de sujetos con LBM, mientras que en LLC en fases iniciales los niveles de citoquinas fueron generalmente inferiores, principalmente debido a los casos con hipermutaciones del gen IGHV. El análisis del TR mostró la existencia de oligoclonalidad en ambas entidades y de clones T persistentes en el tiempo, así como niveles de clonalidad en la fracción T CD4+ que aumentan conjuntamente con la expansión de las células B malignas. Asimismo, se identificaron clonotipos comunes en diferentes casos con LBM/LLC. Todos estos hallazgos implican un papel clave de los procesos inflamatorios y de los elementos antigénicos desde las etapas más tempranas de la enfermedad, cuyos efectos varían notablemente durante la progresión desde LBM a LLC.
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Rawluk, Justyna. "Untersuchungen zur Signaltransduktion des CXCR4-(CD-184)-Chemokinrezeptors im Hinblick auf Vitalität und Migration humaner B-CLL-Zellen." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972879765.
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Talab, Fatima. "Identification of LCK as a key mediator of B cell receptor (BCR) signalling in chronic lymphocytic leukaemia (CLL)." Thesis, University of Liverpool, 2013. http://livrepository.liverpool.ac.uk/14973/.
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Chronic lymphocytic leukaemia (CLL) is a common type of adult leukaemia that accounts for approximately 30% of all mature B-lymphocyte malignancies. An important contributor to CLL pathogenesis is B-cell receptor (BCR) signalling which promotes survival of the malignant clone. BCR engagement on CLL cells provides survival signals by activating the NFκB pathway. Previous work to this thesis showed that CLL cells overexpress PKC-betaII and c-Abl, kinases which have been implicated in mediating NF-kappaB pathway activation in normal B cells. In particular, PKC-beta mediates activation of the CARMA1-Bcl10-MALT1 (CBM) complex leading to eventual activation of I-κB kinases (IKKs) in normal B cells responding to BCR engagement. Considering the importance of the BCR in providing pro-survival signals to CLL cells, the initial aim of this thesis was to characterise any potential role of PKCII and c-Abl in BCR-mediated activation of the NFκB pathway. We addressed this question in Chapter 3 and showed that inhibition of PKC-beta had no effect on BCR-induced activation of IKK, likely because Bcl10 is expressed at low levels in CLL cells. Investigation of the role of c-Abl using the inhibitor imatinib showed that the presence of this compound partially inhibited IKK phosphorylation in BCR-stimulated CLL cells, but the observed effect was variable between CLL patients, and this variability was unrelated to c-Abl expression. Imatinib can also inhibit Lck, a T cell-specific Src-family tyrosine kinase involved in antigen receptor signalling that is also expressed by CLL cells. A further aim of this thesis, answered in Chapter 4, is to define a possible role for Lck in BCR signalling in CLL cells. We showed that inhibition of this Src family kinase (SFK) with the specific inhibitor [4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2d] pyrimidin -7-yl-cyclopentane (Lck-i)], or reduction of its expression with siRNA blocked BCR-stimulated induction of CD79a, Syk, IKK, Akt and ERK phosphorylation in CLL cells. Furthermore, we demonstrated that CLL cells with high levels of Lck expression had higher levels of BCR-mediated IKK, Akt and ERK phosphorylation as well as cell survival than did CLL cells with low levels of Lck expression. These data demonstrated a major role for Lck in proximal and distal BCR signalling in CLL cells. Importantly, these data suggested that Lck expression levels may be linked to disease prognosis. In Chapter 5 we investigated this possibility and showed that high Lck expression was associated with good disease outcome. We hypothesized that this may be because of a dual role played by Lck in promoting and suppressing BCR-induced signalling. We provided data to support this hypothesis and showed that CLL cells bearing low levels of Lck expressed a significantly higher proportion of mannosylated BCR than did CLL cells bearing high levels of Lck, a finding which was consistent with the presence of in vivo constitutive BCR signals. Furthermore, we found that Lck mediated the phosphorylation of ITIMs within CD22 during BCR stimulation of CLL cells. Taken together, these data suggest that high levels of Lck expression within CLL cells may function to interact with phosphatases and set an activation threshold to limit in vivo BCR signalling.
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Müller, David Johannes [Verfasser]. "Charakterisierung des B-Zell-Rezeptors (BCR) auf NFAT2-deletierten CLL-Zellen im Eµ-TCL1-Mausmodell / David Johannes Müller." Tübingen : Universitätsbibliothek Tübingen, 2020. http://d-nb.info/1217249230/34.
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Abbaci, Amazigh. "Caractérisation fonctionnelle du récepteur de type 2 de la neurotensine dans la résistance à la mort cellulaire des lymphocytes B au cours de la Leucémie Lymphoïde Chronique." Thesis, Limoges, 2017. http://www.theses.fr/2017LIMO0029/document.
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La leucémie lymphoïde chronique (LLC) est caractérisée par une accumulation anormale de lymphocytes B matures. Les thérapies actuelles reposent sur l'utilisation d'inhibiteurs ciblant les kinases impliquées dans la voie du récepteur des cellules B (BCR), mais elles sont limitées par le niveau élevé de résistance à l’apoptose des cellules leucémiques. En effet, celles-ci échouent à éradiquer les cellules résistantes à l'apoptose, il est donc essentiel d'identifier d'autres voies de survie comme nouvelles cibles pour les thérapies anticancéreuses. La surexpression des récepteurs de surface couplés aux protéines G (RCPGs) entraîne une transformation cellulaire et joue ainsi un rôle essentiel dans les tumeurs malignes. Dans cette étude, nous montrons que le récepteur de la neurotensine de type 2 (NTSR2), un récepteur couplé aux protéines G, est un acteur essentiel dans les mécanismes de résistance à l'apoptose dans les cellules leucémiques. Le récepteur NTSR2 est surexprimé et constitutivement actif dans les cellules leucémiques, son activation dépend de son interaction avec le récepteur TrkB (Tropomyosin-related kinase B) et du recrutement des protéines Giα à la place de son interaction avec son ligand naturel, la neurotensine (NTS). L'interaction NTSR2-TrkB agit comme un oncogène conditionnel nécessitant le BDNF (Brain-Derived Neurotrophic Factor), le ligand de TrkB, qui est fortement exprimé dans les cellules B leucémiques, contrairement à son ligand naturel la NTS. L'interaction NTSR2-TrkB active les voies de signalisation de survie, y compris les voies de Src et Akt, ainsi que l'expression des protéines anti-apoptotiques Bcl-2 (B-cell lymphoma-2) et Bcl-xL (B-cell lymphoma-extra large). Néanmoins le récepteur TrkB seul ne protège pas les cellules B leucémiques d'une diminution drastique de la viabilité par apoptose lorsque NTSR2 est inactivé. L’ensemble de ces résultats suggèrent que l'interaction NTSR2-TrKB et l'activation soutenue des voies de signalisation dépendante de cette interaction constituent un mécanisme essentiel d’échappement à l'apoptose des cellules B leucémiques. Le ciblage du récepteur NTSR2 représente une stratégie prometteuse pour le traitement de cette pathologie<br>Chronic lymphocytic leukemia (CLL) is characterized by the abnormal accumulation of mature B lymphocytes. Current therapies for CLL rely on using kinase inhibitors targeting B-cell receptor (BCR) pathways, but they are limited by the high level of apoptosis-resistant B-CLL cells, which results in a high frequency of patient relapse. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Overexpression of cell-surface G protein-coupled receptors (GPCRs) drives cell transformation, and thus plays a critical role in malignancies. In this study, we show that neurotensin receptor 2 (NTSR2), a G-protein-coupled receptor, is an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed and constitutively active in B-CLL cells, and its activation depended on its interaction with the tropomyosin-related kinase B receptor (TrkB) and the recruitment of Gi proteins, instead of its interaction with its natural ligand, neurotensin (NTS). The NTSR2-TrkB interaction acted as a conditional oncogenic driver requiring the TrkB ligand BDNF (Brain-Derived Neurotrophic Factor), which is highly expressed in B-CLL cells, unlike its natural ligand NTS. The NTSR2-TrkB interaction activates survival signaling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 (B-cell lymphoma-2) and Bcl-xL (B-cell lymphoma-extra large). TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death when NTSR2 was down-regulated. Taken together, the results suggest that the NTSR2-TrKB interaction and the sustained activation of signaling pathways reliant on this interaction constitute an essential driving force for apoptosis evasion of B-CLL cells. Targeting NTSR2 could represent a promising strategy for treating B-CLL malignancy
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Auchter, Morgan. "Implication de la topoisomérase IIIa dans la stabilité chromosomique au cours de la recombinaison télomérique des cellules cancéreuses." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4008.
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Dans les cellules somatiques, les télomères s'érodent à chaque division cellulaire. Ce processus appelé « Sénescence Réplicative» est contrebalancé de manière basale chez la levure bourgeonnante S. cerevisiae par l'action de la télomérase qui, alors qu'elle est inactive dans les cellules somatiques des eucaryotes supérieures, est activée dans 85% des cancers. Un autre mécanisme impliqué dans les 15% des cas de cancer restants et est appelé Alternative Lengthening of Telomere (ALT). Dans ce processus, le maintien des télomères est assuré par des mécanismes de recombinaison télomérique induisant des échanges de séquences télomériques de chromatides sœurs (T-SCE).Nous avons évalué l'existence d'ALT dans la LLC-B connue pour rarement exprimer la télomérase. Nous avons montrer que 90% des patients LLC-B présentent une diminution de l'expression de TopoIIIα corrélée à une méthylation plus importante des îlots CpG de la région promotrice du gène suggérant que dans les LLC-B le maintien des télomères est défectueux.Nous avons étudié l'implication de la SUMOylation de TopoIIIα/Top3 dans les mécanismes de régulation du ALT. Nous avons montré que TopoIIIα était SUMOylée in vitro et in vivo au sein des cellules U2-OS ALT. Nous avons aussi observé chez S. cerevisiae que Top3 ne serait SUMOylée qu'en absence d'une activité télomérase. Nos résultats suggèrent que la SUMOylation de TopoIIIα augmenterait son activité in vitro et in vivo en diminuant son affinité pour les télomères une fois la recombinaison achevée et qu'elle serait requise pour son accumulation dans les APBs mais pas pour leur formation<br>In somatic cells, telomeres erode with each cell division. This process named « Replicative Senescence » is basically counterbalanced in the budding yeast Saccharomyces cerevisiae by the action of telomerase which, while it is inactive in somatic cells of higher eukaryotes is activated in 85 % of cancer cases. Another process of telomere maintenance is involved in 15% of remaining cancer cases and is called Alternative Lengthening of Telomere (ALT). In this process, telomere maintenance is provided by telomeric recombination mechanisms inducing exchange of telomeric sister chromatid (T-SCE).We assessed the existence of an ALT mechanism in B-CLL known to rarely express telomerase. We have shown that 90% of B-CLL patients have a decreased expression of TopoIIIα correlated with largest methylation of CpG islands of the gene promoter region. Our results suggest that in B-CLL, telomere maintenance is defective either by telomerase or ALT mechanism.We investigated the involvement of post- SUMOylation of TopoIIIα/Top3 in mechanisms regulating ALT phenomenon. We have shown that TopoIIIα was SUMOylated in vitro and in vivo in U2-OS ALT cells. We also observed in S. cerevisiae that Top3p might be SUMOylated in absence of telomerase activity. Our results suggested that the SUMOylation of TopoIIIα increased its activity in vitro and in vivo by reducing its affinity for telomeres once recombination occurred and would be required for its accumulation in APBs but not for their formation
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Schönsteiner, Stefan [Verfasser]. "Analyse des B-Zell-Rezeptor-Rearrangements an Patienten der CLL-8 Studie: Assoziation mit Charakteristika und Überlebenszeiten / Stefan Schönsteiner." Ulm : Universität Ulm. Medizinische Fakultät, 2015. http://d-nb.info/1071629328/34.
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Stadler, Maike. "Die Wirkung des Chemokins "Pulmonary and activation-regulated chemokine" (PARC)auf B-CLL-Zellen und B-Zell-Linien, sowie Untersuchungen zur Expression und Signaltransduktion eines potentiellen PARC-Rezeptors." Doctoral thesis, Universitätsbibliothek Leipzig, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:15-20100121-100623-7.
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Bis heute sind über 50 Chemokine und fast 20 Chemokinrezeptoren identifiziert. Dennoch gibt es Chemokine und Chemokinrezeptoren, deren zugehörige Rezeptoren bzw. Liganden noch nicht bekannt sind. PARC (=CCL18) ist ein ausschließlich in Primaten nachgewiesenes, bisher nur wenig charakterisiertes, im Organismus jedoch weit verbreitetes Chemokin, für das bisher noch kein Rezeptor beschrieben wurde. Die Wirkung dieses Chemokins wurde bisher vor allem an T Lymphozyten nachgewiesen. Die vorliegende Arbeit untersucht die Wirkung von PARC auf B-Lymphozyten und das Vorkommen des putativen PARC-Rezeptors DRY12. Dabei wurden B-Zellen von CLL Patienten sowie mehrere standardisierte B-Zelllinien als Untersuchungsgut verwendet. In funktionellen Assays (Kalziummobilisation, Aktinpolymerisation und Chemotaxis) wurde die Wirkung von PARC auf diese Zellen charakterisiert. Untersuchungen zu beteiligten Signalkaskaden wurden durch Einsatz von spezifischen Inhibitoren (Pertussis-Toxin) und mittels Western Blot durchgeführt. Weiterhin wurde das Vorkommen des putativen PARC-Rezeptors DRY12 bei den verschiedenen B Lymphozyten mittels Antikörperfärbung und RT-PCR sowohl auf Protein- als auch auf mRNA-Ebene nachgewiesen. Der Nachweis der genauen Lokalisation des Rezeptors in der Zelle erfolgte mittels Immunfluoreszenzcytologie. Abschließend wurde vergleichend das Vorkommen des DRY12 im Lymphknoten von CLL-Patienten und gesunden Spendern untersucht. PARC löst bei den B-Zellen der CLL-Patienten die Polymerisation von Aktin aus. Es induziert jedoch keine gerichtete Migration der Zellen. PARC wirkt auf die in dieser Arbeit untersuchten B-Zellen also nicht als Chemokin im klassischen Sinne. Seine Wirkung besteht möglicherweise in einem synergistischen Effekt, indem es im Zusammenspiel mit anderen Faktoren die Migration der Zellen beeinflusst. Weiterhin wäre denkbar, dass PARC das Verhalten von hämatopoetischen Stamm- und Vorläuferzellen beeinflusst. Die beteiligte Signalkaskade beinhaltet ein Pertussis-Toxin-sensitives Gi-Protein und die Aktivierung der p42/44-MAP Kinase. Ein intrazellulärer Einstrom von Ca2+ spielt bei der Wirkungsvermittlung von PARC keine Rolle. Der putative PARC-Rezeptor DRY12 konnte bei verschiedenen B-Zellen in unterschiedlicher Intensität nachgewiesen werden. Die Expression des DRY12 scheint sowohl auf Ebene der mRNA als auch auf Proteinebene durch multiple Faktoren reguliert zu sein. Dazu gehören z.B. der Reifungs- und Aktivierungszustand der Zellen oder die Kultivierungsdauer nach dem Auftauen der Zellen bis zur Durchführung des Versuchs. Bisher konnten jedoch keine entsprechenden Zusammenhänge nachgewiesen werden. Der DRY12 ist demnach kein konstitutiv exprimierter Rezeptor. Durch Immunfluoreszenzcytologie konnte die Lokalisation des Rezeptormoleküls auf der Zelloberfläche gezeigt werden. Im Lymphknoten wird DRY12 v.a. von Lymphozyten exprimiert. Bei Makrophagen konnte das Rezeptorprotein nicht nachgewiesen werden. In den Lymphknoten von CLL-Patienten exprimieren die Lymphozyten deutlich mehr DRY12 als Lymphozyten im Gewebe gesunder Individuen. Ein direkter Zusammenhang zwischen Rezeptorexpression und Reaktion auf PARC konnte nicht sicher aufgezeigt werden. Die Ergebnisse dieser Arbeit schließen aber auch nicht aus, dass PARC ein möglicher Bindungspartner von DRY12 ist. Bei der Wirkungsvermittlung spielen vermutlich auch andere Botenstoffe und weitere Faktoren eine Rolle, indem sie die Reaktionsfähigkeit der Zellen gegenüber PARC bzw. die Rezeptorexpression des DRY12 beeinflussen. Hinsichtlich der Frage, ob es sich bei DRY12 um einen Rezeptor für PARC handelt, kann diese Untersuchung zu keinem abschließenden Ergebnis gelangen, so dass dieser Aspekt in weiterführenden Analysen eingehender betrachtet werden sollte<br>Today there are more than 50 chemokines and almost 20 chemokine receptors described. Despite growing knowledge, the ligands for some orphan chemokine receptors have not been identified and for several chemokines the receptor has not been discovered. PARC (=CCL18) is one of these chemokines for which the receptor has not been recognized. It has been detected in primates only and, despite being widely spread in the organism, it is still poorly characterized. Up to now, the effects of PARC were mainly shown on T-lymphocytes. Therefore, the objective of this study was to investigate the function of PARC and the expression of the putative PARC-receptor DRY12 in B-lymphocytes. For the purpose of the present study, B-CLL-cells and several lymphocytic B-cell-lines served as models to cover different stages of B-cell maturation. In order to characterize the effect of PARC, several functional assays (calciummobilisation, actinpolymerisation and chemotaxis), specific inhibitors (pertussis toxin) and Western Blotting were used. Expression analyses of the DRY12-receptor were performed by FACS-analysis, RT-PCR and immunofluorescence cytochemistry. In addition, lymph nodes from patients with CLL and healthy donors were stained immunohistochemically. In B-CLL-cells, PARC stimulation leads to phosphorylation of p42/44-MAP-Kinase and polymerization of actin, which can be inhibited by pertussis toxin, but does not induce calcium signaling or chemotactic migration. In this case, PARC is no classical chemokine but may act as synergist to potentiate the effect of other chemokines or may influence the behavior of hematopoetic stemm-cells. The results of the study show expression of the putative PARC-receptor DRY12 present on several subsets of B lymphocytes. As they showed different intensity of expression, DRY12 may be regulated by different factors in translation as well as transduction. Among these factors might be their current state of maturation and activation and the time period from revitalization to the start of the experiments. The reasons for these differences are still unknown. According to these findings, the receptor is not constitutively expressed, but may be itself regulated by several chemokines and other factors. DRY12 is located at the surface of the cell, as shown by immunocytochemistry. In lymph nodes, particularly lymphocytes but not macrophages express DRY12. In lymph nodes of CLL-patients lymphocytes express much more DRY12 than in healthy samples. However, it could not be proved that DRY12 is the agonistic receptor for PARC, as the expression of DRY12 did not completely correlate with the effects on PARC stimulation. But results of this study do not exclude this possibility either, as different factors are considered to influence the effect of PARC and the expression of DRY12 in B-cells. Although there are hints to it, from this study we can not conclude that DRY12 is the agonistic receptor for PARC. Therefore, further investigation is necessary to find the answer to this question
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Rajakaruna, A. V. Nadeeka P. "Functional interaction between CD180 Toll-like receptor (TLR) and B cell receptor (BCR) in the biology of Chronic Lymphocytic Leukaemia (CLL)." Thesis, University of Westminster, 2017. https://westminsterresearch.westminster.ac.uk/item/q254x/functional-interaction-between-cd180-toll-like-receptor-tlr-and-b-cell-receptor-bcr-in-the-biology-of-chronic-lymphocytic-leukaemia-cll.
Full textAbstract:
Chronic Lymphocytic Leukaemia (CLL) is the most common leukaemia in the western world and remains incurable. It is driven by as yet unknown (auto)antigens via the B cell receptor (BCR) and growth, survival and expansion signals it receives from the microenvironment through a range of cytokines and receptors, including Toll-like receptors (TLR). The role of the microenvironment in the development and progression of CLL is currently of major interest. Pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively) represent exogenous and endogenous microenvironmental factors acting via a range of receptors, including TLR. CD180/RP105 is a membrane-associated orphan receptor of the TLR family which is expressed on various cells of the immune system including macrophages, peripheral blood monocytes, dendritic cells, naive and mature B cells and marginal zone/mantle zone B cells driving normal B-cell activation and proliferation. However, it is not expressed on germinal centre (GC) B cells. CD180 is expressed heterogeneously on CLL cells, and predominantly on CLL with mutated IGVH genes (M-CLL). Although approximately 60% of CLL clones expressed CD180, only half responded to ligation with anti-CD180 monoclonal antibody (mAb) by activation, cycling, and reduced basal apoptosis. They were termed responders (R) and CD180+ CLL samples that failed to respond to anti-CD180 mAb despite expressing a high density of CD180 receptors, were termed non-responders (NR). Our group has previously demonstrated that CD180 ligation significantly induced phosphorylation of ZAP70/Syk, ERK, p38MAPK, and AKT in R-CLL cells whilst CD180-mediated signalling in NR CLL cells did not progress downstream from ZAP70/Syk phosphorylation indicating a block in activation of downstream protein kinases, and possible anergy. However, the responses were quite heterogeneous and to further understand the CD180-mediated signalling pathways in CLL; downstream signal transduction was studied by defining CLL samples into R and NR through their proximal ability to activate AKT. R-CLL cells could be divided into two categories as AKT signallers (AKT-S) and AKT non-signallers (AKT-NS) based on the ability to increase level of phosphorylation of AKT (Ser 473) compared with the basal levels. AKT-NS showed a significant increase in p38MAPK-P and this group was, therefore, re-categorised as p38MAPK signallers (p38MAPK-S). A small cohort of patients showed phosphorylation of both AKT and p38MAPK and they were categorised as double signallers (DS) whereas the remaining CLL samples which showed a decrease in percentages of both AKT-P and p38MAPK-P expressing cells and were categorised as non-signallers (NS). Ligation of CD180 with mAb on CLL cells can activate two alternative signalling pathways, one being pro-survival and operating through activation of protein kinases (PK) BTK-AKT (AKT-signallers); and the second - predominantly pro-apoptotic, operating through activation of p38MAPK (p38MAPK-signalers) but not through BTK. This may have implications for CLL therapy where BTK inhibitors are being used. To assess cross-talk between CD180 and BCR (sIgM) signalling pathways in CLL, the effect of pre-engagement of CD180 on sIgM-mediated signalling was investigated. While sIgM ligation with goat anti-human IgM F(ab)2 alone led to a significant activation of pro-survival BTK-AKT pathway, pre-treatment with anti-CD180 mAb redirected pro-survival signalling mediated through BCR towards pro-apoptotic p38MAPK pathway. Application of specific inhibitors of AKT and p38MAPK signalling pathways confirmed that, in many of the CLL samples, activation of AKT and p38MAPK pathways is exclusive. This dichotomy appears to be a feature of CLL cells, and not of normal B cells that responded to CD180 ligation as double AKT/p38MAPK signallers. It is becoming apparent that the clinical outcome of patients with CLL is significantly affected by intraclonal diversity. CLL clones which can respond more vigorously to external stimuli may gain selective advantage for growth and survival and perhaps promote clonal evolution. That CD180 expression modulates in some categories of CD180+ CLL cells suggests a possible link between dynamic expression of CD180 and CD180-mediated intracellular signalling, survival or apoptosis. Finding on rewiring of signalling pathways from BTK/AKT pro-survival circuit to p38MAPK pro-apoptotic pathway in AKT-S CLL cells may suggest a possible cross-talk between CD180 and BCR in AKT-S is consistent with that CD180+sIgM+ CLL cells may receive simultaneous signals through both receptors in vivo and microenvironment plays a major role in the survival of CLL cells. Hence, this study helped defineify mechanisms leading to the expansion of the leukaemic cells and thus contribute to development of novel therapies of CLL.
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Culpin, Rachel Emily. "Expression of the MiR-17-92 cluster in DLBCL and B-CLL and its relationship with disease outcome and established prognostic factors." Thesis, University of Newcastle Upon Tyne, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.582656.
Full textAbstract:
Diffuse large B-cell lymphoma is an aggressive malignancy of large transformed B- lymphocytes. The disease is biologically and clinically highly heterogeneous and this nature has impacted on patients response to treatment. Gene profiling studies have identified two biologically diverse sub-groups, that exhibit different prognoses, namely; the germinal centre (GC)- and activated B-cell (ABC)- like DLBCL. B-cell chronic lymphocytic leukemia is also a heterogeneous disease. Patients may follow an indolent clinical course and survive for >20 years, but others have a rapidly progressive disease requiring aggressive treatment, with some dying a few years after diagnosis. It is possible to identify some of the high-risk patients with either DLBCL or B-CLL on the basis of clinical parameters. However, these do not always reflect the molecular heterogeneity of the diseases and have not impacted on the routine treatment or clinical management. Therefore, there is a need to identify molecular factors that represent these diseases more accurately and provide better prognostic information. Recent studies implicate microRNAs in the regulation of gene expression. The microRNAs are endogenous, non-coding RNAs that play key regulatory roles in a diverse range of pathways, including development, cell proliferation, differentiation and apoptosis. These 18-24 nucleotide single-stranded RNAs are involved in post- transcriptional gene regulation. They bind to complimentary sites in the 3' untranslated region (UTR) of messenger RNAs to induce gene silencing. In 2002, Calin et al directly associated deregulated expression of miR -15 and miR -16 in the pathogenesis of B-CLL. Since this time, a number of microRNAs have been implicated in haematological neoplasms, including members of the oncogenic miR-17-92 cluster, encoded by the CJ3orj25 gene located on chromosome 13q31. In the present study; i) microRNA microarray profiling was employed to identify microRNA signatures that are accurately assigned to the DLBCL prognostic sub-groups and ii) qRT-PCR technology was used to examine the role of expression of the mature microRNAs of the miR-17-92 cluster in the prognostication of DLBCL and B-CLL. Using a number of well characterised DLBCL cell lines, a 9 microRNA signature was identified that discriminated between GC-like and ABC-like prognostic sub-groups. This included 4 microRNAs of the miR-17-92 cluster and 3 newly identified microRNAs, not previously associated with DLBCL and predicted to target genes that are deregulated in lymphoma. A 4- and 18- microRNA signature was identified that differentiated between DLBCL and FL cell lines and between lymphoma and normal B-cells, respectively. These signatures have yet to be assessed in clinical material. Due to the emerging role of the miR-17-92 cluster in B-cell malignancy, using robust qRT-PCR technology the expression levels in B-cell lymphoma cell lines, DLBCL and B-CLL clinical samples were investigated. It was observed that, despite being transcribed from the same parental cluster, the expression levels of mature microRNAs varied, suggesting the existence of additional mechanisms of regulation in the processing of these molecules from their transcription to the mature form. In DLBCL clinical samples, high expression of miR-17-5p or miR-92 were identified as significant high risk factors when analysed as continuous variables (OS: miR-17-5p p = 0.025 and miR-92 p = 0.014, PFS: miR-92 p = 0.017). Similarly, as dichotomous variables, high expression of miRs -17-5p (p = 0.001), -18 (p = 0.051),- 19a (p = 0.011), -20 (p = 0.010) or -92 (p = 0.050) predicted for shorter OS and in addition, high expression of miRs -17-5p (p = 0.006) or -20 (p = 0.038) for shorter PFS. Expression of microRNAs of the cluster further risk stratified immunophenotypic- or Ann Arbor disease stage-based prognostic sub-groups. In multivariate analysis, miR-17- 5p (p = 0.012) and IPI (p = 0.009) were identified as independent prognostic factors. In B-CLL, when samples were analysed as dichotomous variables, high expression ofmiR-18 (p = 0.013) and miR-19a (p = 0.009) predicted for shorter TFS in the entire cohort and miR-19a (p = 0.008) retained prognostic significance in Binet stage A group of patients. Conversely, high expression of miR-17-5p (p = 0.044) or miR-92 (p = 0.052) predicted for longer TFS and remained predictive in Binet stage A (miR-17-5p: p = 0.022, miR-92: p = 0.014). In multivariate analysis CD38 (p < 0.001) and Binet disease stage (p = 0.002) were identified as independent prognostic factors but in the Binet stage A group, only CD38 expression (p = 0.011) retained prognostic significance. In conclusion: i) microRNA array-based signatures provide a useful mechanism to classify lymphoma sub-groups. Investigation into the role of these microRNAs may provide further insights into disease pathophysiology, ii) varying expression levels of mature miR-17-92 microRNAs in B-cell lymphoma and B-CLL suggest additional regulatory mechanisms in the processing of this cluster and iii) mature miR-17-92 microRNAs may act as either high, or low risk factors, depending upon the cellular context and provide useful prognostic information for patients with DLBCL or B-CLL. Given the recent identification of the involvement of miR-17-92 oncomiR-1 in the activation of anti-apoptotic and pro-proliferative pathways, the role of this cluster in DLBCL and B-CLL pathophysiology warrants further investigation.
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Ghosh, Anil Kumar. "The role of heat shock proteins, amyloid precursor protein and B-cell CLL/lymphoma 3 and their co-chaperones in colorectal cancer." Thesis, Queen Mary, University of London, 2013. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8392.
Full textAbstract:
The elevated expression of Heat shock proteins (HSPs) has been implicated in CRC prognosis and tumour cells may require the expression of HSPs and BCL2-associated athanogene (BAG1), the HSP co-chaperone, to survive. BAG1 is a regulator of amyloid precursor protein (APP); both are important in cellular proliferation and cancer. B-cell CLL/lymphoma 3 (BCL3) is an agonist in the tumour-associated NF-κB pathway. This study aims to establish the mutational status of TP53, KRAS and PIK3CA, and the activation status of AKT, in primary CRCs and then investigate the role of HSPs, BAG1, APP, and BCL3. Oncogene induced senescence (OIS) is a potential roadblock to CRC. Recent cell culture studies suggest that OIS mediated by PI3K/AKT activation can be circumvented by high expression of HSPs in the absence of TP53 mutation. While PI3K/AKT activation and KRAS mutations are independent inducers of OIS, PI3K/AKT activation can suppress KRAS-induced OIS when both are present in cultured cells. KRAS mutations, PI3K/AKT activation and TP53 mutations are all common features of CRCs. For HSP to inhibit OIS in CRC may be dependent on the tumour’s mutation spectrum. CRCs with oncogenic activation of the AKT pathway were associated with increased HSP27 expression, which may represent an important mechanism in suppressing p53 dependent senescence. No association was found between HSP27 or HSP72 expression with TP53 mutation status, but HSP27 expression was strongly associated with the co-presence of wildtype KRAS and activated PI3K/AKT, indicating a possible 4 role of HSP27 in overcoming PI3K/AKT induced OIS in tumours. There was no correlation between BAG1 and APP co-expression in CRCs. BCL3 expression was heterogeneous, and elevated at the invasive edge. The expression of HSPs, APP, BCL3 was not correlated with any CRC clinicopathological features. Our studies suggest a role for using archival tissues in validating hypotheses generated from cell culture based investigations.
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Lindqvist, Camilla. "T Regulatory Cells – Friends or Foes?" Doctoral thesis, Uppsala universitet, Enheten för klinisk immunologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-128837.
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T regulatory cells (Tregs) have been extensively studied in patients with cancer or autoimmunity. These cells hamper the immune system’s ability to clear tumor cells in cancer patients. In autoimmune diseases, on the other hand, they are not able to restrain autoreactive immune responses. If we manage to understand Tregs and their role in health and diseases we may be able to develop better immunomodulatory therapies. Early studies demonstrated that tolerance was maintained by a subset of CD25+ T-cells. CD25 was the earliest marker for Tregs and is still often used to define these cells. Several Treg-associated markers have been suggested throughout the years. However, these markers can be upregulated by activated T-cells as well. The most specific marker for Tregs is currently the transcription factor forkhead box P3 (FoxP3). In this thesis, we investigated the presence of CD25- Tregs in patients with B-cell malignancies and in patients with autoimmunity. These cells were identified in both patient groups. Further, patients with B-cell malignancies often have high levels of soluble CD25 (sCD25) in the periphery. In our patient cohorts, the level of peripheral Tregs correlated with the level of sCD25 in patients with lymphoma. Tregs were shown to release sCD25 in vitro and sCD25 had a suppressive effect on T-cell proliferation. These data show that Tregs may release CD25 to hamper T-cell proliferation and that this may be an immune escape mechanism in cancer patients. Previous studies have demonstrated that an increased infiltration of FoxP3+ cells into lymphoma-affected lymph nodes is associated with a better patient outcome. This is in contrast to studies from non-hematological cancers where an increased presence of Tregs is associated with a poor prognosis. Since previous studies have shown that Tregs are able to kill B-cells, we wanted to investigate if Tregs are cytotoxic in patients with B-cell tumors. In the subsequent studies, Tregs from patients with B-cell lymphoma and B-cell chronic lymphocytic leukemia (CLL) were phenotyped to investigate the presence of cytotoxic markers on these cells. FoxP3-expressing T-cells from both patients with CLL and B-cell lymphoma displayed signs of cytotoxicity by upregulation of FasL and the degranulation marker CD107a. Tregs from CLL patients could further kill their autologous B-cells in in vitro cultures. Taken together the studies in this thesis have demonstrated two possible new functions of Tregs in patients with B-cell malignancies and the presence of CD25- Tregs in both cancer and autoimmunity.
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CASTELLI, MONICA. "INCREASED SURVIVAL OF CLL B CELLS IN THE PRESENCE OF MARROW MESENCHYMAL STROMAL CELLS: A NOVEL MODEL TO DEFINE NEW TARGETS FOR THERAPY." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424459.
Full textAbstract:
Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western World, accounting for about 30% of adult leukemia, and it is characterized by the clonal expansion and accumulation of mature CD19+/CD5+/CD23+ B lymphocytes in the peripheral blood, bone marrow and secondary lymphoid organs. Despite their apparent longevity in patients, in vitro CLL leukemic B cells rapidly undergo spontaneous apoptosis. The selective survival advantage is due both to intrinsic defects on apoptosis mechanism and to signals delivered by accessory cells at the active site of the disease. Previous studies demonstrated that mesenchymal stromal cells (MSCs), derived from bone marrow, and CD68+ nurse-like cells, derived from peripheral blood, are involved in CLL clone longevity and migration, suggesting a crucial role of MSCs on favouring disease progression.
Therefore, in this thesis we evaluated the effect of MSCs, the main stromal population in the bone marrow of CLL patients, on the survival of leukemic B cells and their role in drug resistance.
MSCs were isolated from the bone marrow of 46 CLL patients; their immunophenotypic characterization was based on the expression of CD105, CD73 and CD90 and the negativity of CD14, CD34, CD45 and CD31. Co-culturing MSCs and CLL B cells, we confirmed that MSCs are able to support malignant B cell survival, providing an in vitro culture system that closely approximates CLL microenvironment in vivo. We observed that different leukemic clones demonstrated a large variety in the pro-survival effect. Evaluating the cleavage pattern of PARP, we revealed two subsets of CLL clones with different sensitivity to MSCs pro-survival signals. Our results indicate that both cell-cell contact and soluble molecules are actors in the relationship between malignant B cells and the MSCs, promoting CLL B cell survival and migration.
Later, we evaluated the role of the MSCs on CLL B cells during the most common cytotoxic therapy used in clinical practice. Our data demonstrate that MSCs are able to protect leukemic B cells from apoptosis during Fludarabine and Cyclophosphamide treatment, both in vitro and in vivo. We tested MSCs protective role also during CLL B cells treatment with Ibrutinib, a novel inhibitor of Btk involved in the BCR signaling pathway, and we found that the treatment counteracts the MSC pro-survival effect. To better understand the effect of Ibrutinib on the cross-talk between CLL B cells and MSCs, we evaluated its role on leukemic B cell migration, also analyzing the expression levels of CCR7 and CXCR4, two chemokine receptors that are central in the homing of the neoplastic clone. We demonstrated that malignant B cell migration is not significantly affected by the Btk inhibitor; since cell-cell contact with MSC is crucial for CLL B cell survival, we analyzed the adhesion of leukemic B cells to MSCs after treatment with Ibrutinib. We found a significant reduction in leukemic B cells and MSCs interactions mediated by the CD49d integrin.
In this thesis, we demonstrate that MSCs enhance the survival of leukemic B cells through the release of soluble factors and cell-cell direct contact and that each CLL clone reveals a peculiar response to the anti-apoptotic signals delivered by MSCs. These observations could be relevant to identify patients more responsive to druggable targets on marrow microenvironment and also to find putative new strategies for CLL therapy. A better understanding on the complexity of the cross-talk between CLL cells and their microenvironment during CLL therapy could also help to define mechanisms of drug resistance and treatment failure, as well to plan randomized clinical trials comparing new compounds and their combinations with standard chemo-immunotherapy.<br>La Leucemia Linfatica Cronica (LLC) è considerate la più comune leucemia del mondo occidentale, rappresentando circa il 30% delle leucemie dell’adulto, ed è caratterizzata dalla proliferazione clonale e dall’accumulo nel sangue periferico, nel midollo osseo e negli organi linfatici secondari di linfociti B maturi CD19+/CD5+/CD23+. Nonostante i linfociti B leucemici mostrino un’aumentata sopravvivenza nei pazienti affetti da LLC, in vitro vanno rapidamente incontro ad apoptosi. Il vantaggio sulla sopravvivenza è legato sia a difetti intrinseci del meccanismo di apoptosi sia a segnali forniti da cellule accessorie, presenti nel sito attivo della malattia. Precedenti studi hanno dimostrato che le cellule mesenchimali stromali (MSC) e le cellule accessorie (“nurse-like”) derivate rispettivamente dal midollo osseo e dal sangue periferico, sono coinvolte nell’aumentata longevità e mobilità del clone leucemico, suggerendo un ruolo cruciale delle MSC nel favorire la progressione della malattia.
In questa tesi abbiamo valutato l’effetto delle MSC, la principale popolazione stromale nel midollo osseo dei pazienti affetti da LLC, sulla sopravvivenza dei linfociti B neoplastici e il loro ruolo sulla resistenza ai farmaci.
Le MSC sono state isolate da campioni di sangue midollare provenienti da 46 pazienti affetti da LLC; la loro caratterizzazione immunofenotipica è stata effettuata sulla base dell’espressione di CD105, CD73 e CD90 e sulla negatività di CD14, CD34, CD45 e CD31. Allestendo co-colture di MSC e linfociti B leucemici, abbiamo confermato la capacità delle MSC di incrementare la sopravvivenza delle cellule B neoplastiche, fornendo un sistema di coltura in vitro che mima profondamente il microambiente della LLC in vivo. Abbiamo osservato una grande varietà sulla vitalità dimostrata dai diversi cloni leucemici e, mediante la valutazione del frammento clivato della proteina PARP, abbiamo identificato due differenti gruppi di cloni di LLC, con una diversa sensibilità ai segnali di stimolo provenienti dalle MSC. I nostri risultati indicano che sia il diretto contatto cellula-cellula che la presenza di molecole solubili sono coinvolte nell’interazione tra le cellule B leucemiche e le MSC, promuovendo la sopravvivenza e la migrazione della cellula B leucemica.
Successivamente, abbiamo valutato l’effetto delle MSC sui linfociti B neoplastici durante un trattamento chemioterapico di uso comune nella pratica clinica. I nostri dati hanno dimostrato che le MSC sono in grado di proteggere le cellule B leucemiche dall’apoptosi durante il trattamento con Fludarabina e Ciclofosfamide, sia in vitro che in vivo. Abbiamo esaminato il ruolo protettivo delle MSC anche durante il trattamento dei linfociti neoplastici con Ibrutinib, un nuovo inibitore della Btk, una chinasi coinvolta nella cascata del segnale del BCR, e abbiamo dimostrato che il trattamento delle cellule B con Ibrutinib è in grado di contrastare l’effetto anti-apoptotico delle MSC. Per meglio definire l’azione di Ibrutinib nell’interazione tra le cellule B di LLC e le MSC, abbiamo valutato il suo ruolo sulla mobilità dei linfociti B leucemici, analizzando inoltre i livelli di espressione di CCR7 e CXCR4, due recettori chemiochinici fondamentali nella migrazione del clone neoplastico. Abbiamo dimostrato che la migrazione delle cellule B neoplastiche non è significativamente influenzata dall’inibitore del Btk; inoltre, considerando che il diretto contatto cellula-cellula con le MSC è di notevole importanza per la sopravvivenza dei linfociti B leucemici, abbiamo analizzato l’adesione delle cellule B alle MSC dopo il trattamento con Ibrutinib, evidenziando che la loro adesione era significativamente ridotta.
In questa tesi abbiamo dimostrato che le MSC incrementano la sopravvivenza delle cellule B neoplastiche attraverso il rilascio di fattori solubili e mediante il diretto contatto cellula-cellula, e che ogni clone leucemico rivela una peculiare risposta ai segnali anti-apoptotici rilasciati dalle MSC. Queste osservazioni potrebbero essere determinanti al fine di identificare i pazienti più sensibili a trattamenti mirati a colpire il microambiente midollare ed a trovare potenziali nuove strategie terapeutiche per la LLC. Una migliore comprensione della complessità delle interazioni tra i linfociti leucemici e il loro microambiente nel corso del trattamento potrà inoltre aiutare a chiarire i meccanismi di chemioresistenza e refrattarietà, così come a pianificare studi clinici randomizzati che confrontino nuovi farmaci e la loro combinazione con i trattamenti chemio-immunoterapici già in uso.
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Rehman, Haroon, Asha Chepkorir Segie, Kanishka Chakraborty, and Devapiran Jaishankar. "Bone Marrow Wars: Attack of the Clones." Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/asrf/2020/presentations/33.
Full textAbstract:
Multiple myeloma is characterized by the malignant proliferation of clonal plasma cells producing monoclonal paraproteins, leading to multi-organ damage. On the other hand monoclonal B-cell lymphocytosis (MBCL) is characterized by the malignant proliferation of clonal B-lymphocytes, with potential to develop into chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). CLL/SLL can result in visceromegaly, anemia, thrombocytopenia, fevers, night sweats and unintentional weight loss. Literature review demonstrates these two malignant clonal bone marrow disorders are most frequently seen independently in patients; however, we report one rare diagnostic challenge where both clonal disorders were identified in a single patient concurrently. A 64-year-old man initially presented with worsening back pain. Thoracic spine x-ray revealed a T11 compression fracture, confirmed by magnetic resonance imaging. Complete blood count revealed a white blood cell count of 7.3 K/uL with 54% lymphocyte predominance and peripheral smear demonstrated a population of small lymphocytes with round nuclei and an atypical chromatin pattern suggestive of CLL/MBCL. Flow cytometry revealed a monoclonal B-cell CD5 positive, CD23 positive, CD10 negative population with an absolute count of 1.6 K/uL. Due to the instability and pain associated with the spinal fracture, patient had kyphoplasty performed and intraoperative bone biopsies were taken from both T11 and T12 vertebrae. Interestingly each bone biopsy revealed involvement by both a kappa-light chain restricted plasma cell neoplasm, ranging from 15% to 30% cellularity, as well as a CD5-positive B-cell lymphocyte population. It suggested two concurrent but pathologically distinct pathologies including plasma cell myeloma and a separate B-cell lymphoproliferative disorder with immunophenotypic features suggestive of CLL/MBCL. Bone marrow biopsy was performed for definitive evaluation and confirmed multiple myeloma with 15-20% kappa-restricted plasma cells identified, and also confirmed concurrent MBCL with CD5 and CD23-positive, kappa-restricted B-cells identified on bone marrow flow cytometry. Adding an additional layer of complexity, bone marrow molecular genetics revealed presence of a MYD88 mutation, raising concern for possible lymphoplasmacytic lymphoma (LPL). However, secondary pathologic review ruled out LPL, as the immunophenotypic pattern of the clonal B-cells was not consistent with that of LPL, and although the MYD88 mutation is predominantly seen in LPL, it has also been seen in a small percentage of CLL/SLL cases and exceedingly rarely described in MM as well. Serum protein electrophoresis with immunofixation, serum quantitative immunoglobulins and serum quantitative free light chain assay revealed findings consistent with IgG kappa multiple myeloma and systemic CT imaging was negative for any lymphadenopathy, confirming MBCL. Patient was started on first-line multiple myeloma systemic therapy for transplant eligible patients and has demonstrated an excellent response to treatment thus far. This patient case serves to demonstrate the importance of maintaining a broad differential when approaching hematological problems; It also underlines the necessity for a complete diagnostic evaluation to identify rare clinical conundrums such as with our patient, allowing for proper and timely treatment. While we use “Occam’s razor” to explain multiple problems with a single unifying diagnosis the rare possibility of divergent diagnosis is to be always entertained.
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Chiodin, Giorgia. "Chronic Lymphocytic Leukemia: analysis of microenvironmental influence on neoplastic clone survival and IgM signaling during Ibrutinib therapy." Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3422771.
Full textAbstract:
Chronic Lymphocytic Leukemia (CLL) is characterized by the monoclonal expansion of mature CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow and lymphoid tissues. Surface IgM (sIgM) signaling is key to CLL behavior and is a therapeutic target of the BTK-inhibitor Ibrutinib. SIgM levels and signaling capacity are variable in CLL and correlate with the behavior of the disease. In CLL, the microenvironment also plays an important role in disease support and progression. In this thesis two projects are presented: the analysis of the microenvironmental influence on neoplastic clone survival in different in vitro culture conditions, and the study of the effects that Ibrutinib in vivo therapy exerts on sIgM in CLL patients.
Mesenchymal Stromal Cells (MSCs), which represent the major component of the stromal microenvironment, were isolated from the marrow aspirate of CLL patients and co-cultured with leukemic cells. After 7 days, we observed a relevant extended survival of leukemic cells in respect to the B cells cultured alone, and the behavior of the neoplastic clones could be differently dependent on the signals coming from the stromal cells. MSCs were able to counteract the cytotoxic effect of Fludarabine/Cyclophosphamide in vivo administration, confirming the important role played by the microenvironment during therapy. However, the kinase inhibitors Ibrutinib and Bafetinib could induce apoptosis of leukemic cells co-cultured with MSCs, and inhibited CLL B cell CD49d-mediated adhesion and pseudoemperipolesis, suggesting that the new kinase inhibitors are effective in targeting the pro-survival cross-talk between leukemic lymphocytes and stromal cells.
In patients, Ibrutinib treatment induces a rapid redistribution of CLL cells into the blood. In this study, the expression and function of sIgM was analyzed in 12 CLL patients after 1 week of Ibrutinib therapy. At this time point, the expression of sIgM increased significantly (P=0.001), accompanied by full N-glycan maturation of sIgM heavy-chain, indicating recovery from antigen engagement. In addition, the sIgM levels correlated with increased sIgM-mediated SYK phosphorylation. The data suggest that Ibrutinib could prevent antigen encounter, thus favoring sIgM expression and maturation.<br>La leucemia linfatica cronica (LLC) e’ caratterizzata dall’accumulo di linfociti B maturi con fenotipo CD19+/CD5+/CD23+ nel sangue periferico, nel midollo osseo e nei tessuti linfatici. I segnali mediati dalle immunoglobuline M di superficie (sIgM) sono fondamentali per il comportamento dei linfociti di LLC, e sono divenuti target di inibitori chinasici come Ibrutinib. I livelli di sIgM e la capacita’ di mediare segnali intracellulari sono variabili nei cloni tumorali e si associano al comportamento della malattia. Inoltre, anche il microambiente tumorale ricopre un ruolo importante nel supporto e nella progressione della LLC. In questa tesi sono presentati due progetti: l’analisi dell’influenza del microambiente sulla sopravvivenza del clone neoplastico in diverse condizioni di coltura in vitro, e lo studio degli effetti della terapia con Ibrutinib su signalling e funzionalita’ delle sIgM in pazienti di LLC.
Nella prima parte dello studio, le cellule mesenchimali stromali (MSCs) sono state isolate da aspirati midollari da pazienti affetti da LLC e sono state poste in co-coltura con cellule B neoplastiche. Dopo 7 giorni di incubazione, abbiamo osservato un rilevante incremento della sopravvivenza delle cellule leucemiche poste in co-coltura con MSCs rispetto alle cellule poste in coltura singola; abbiamo osservato che cloni diversi mostrano comportamento diverso in termini di sopravvivenza, in base alle caratteristiche intrinseche dei cloni stessi. Le MSCs, inoltre, sono in grado di contrastare l’effetto citotossico della terapia Fludarabina/Ciclofosfamide quando somministrata in vivo in pazienti con LLC, a conferma dell’importante ruolo svolto dal microambiente. Tuttavia, i risultati ottenuti hanno mostrato che gli inibitori chinasici Ibrutinib e Bafetinib sono invece in grado di indurre apoptosi nelle cellule tumorali anche in presenza di MSCs e di inibirne l’adesione mediata da CD49d e la pseudemperipolesi, suggerendo che gli inibitori del signalling del BCR sono efficaci nel bloccare il cross-talk tra linfociti neoplastici e cellule stromali.
Nei pazienti affetti da LLC il trattamento con Ibrutinib induce una rapida ridistribuzione delle cellule tumorali nel sangue. In questo studio, l’espressione e la funzione delle sIgM e’ stata analizzata in 12 pazienti con LLC dopo 1 settimana di terapia con Ibrutinib. A questo time point, l’espressione di IgM sulla superficie delle cellule neoplastiche e’ risultata significativamente aumentata (P=0.001); allo stesso tempo abbiamo anche osservato un aumento della forma matura della catena pesante delle sIgM, indicativa di un mancato incontro con l’antigene. Inoltre, i risultati ottenuti hanno mostrato una correlazione tra l’incremento dei livelli di sIgM e l’aumentata fosforilazione di SYK mediata da IgM. I dati suggeriscono che Ibrutinib potrebbe prevenire l’incontro con l’antigene, favorendo quindi espressione e maturazione delle sIgM nelle cellule di LLC.
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Pötzsch, Birgit [Verfasser]. "Die Bedeutung des Ang2-Tie2-Signalweges für die Interaktion von malignen B-Lymphozyten mit ihrer Mikroumgebung bei der chronischen lymphatischen Leukämie (CLL) / Birgit Pötzsch." Köln : Deutsche Zentralbibliothek für Medizin, 2014. http://d-nb.info/1056004169/34.
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Veuillen, Caroline. "Caractérisation des mécanismes d'échappement tumoral à la lyse NK dans la LLC-B et le cancer de la prostate." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX20708.
Full textAbstract:
De nombreuses données expérimentales et cliniques ont montré l'importance des cellules Natural Killer (NK) dans l'immunosurveillance antitumorale. Les stratégies thérapeutiques basées sur les cellules NK pourraient donc être une alternative de choix dans le traitement de certains types de cancers. Nous avons focalisé notre étude sur deux types de cancer incurables malgré les récents progrès thérapeutiques : la leucémie lymphoïde chronique B (LLC-B) et le cancer de la prostate. Le but de notre étude est une meilleure compréhension des mécanismes mis en place par les cellules B leucémiques et les cellules tumorales prostatiques pour échapper à la réponse antitumorale des cellules NK. La connaissance de ces mécanismes d'échappement est un pré-requis indispensable à l'utilisation des cellules NK dans les thérapies antitumorales. Concernant la LLC-B, nos résultats suggèrent que les cellules NK de patients, fonctionnellement compétentes, ne peuvent pas initier une réaction immunitaire appropriée envers les cellules B leucémiques due au manque de reconnaissance de ces dernières. Concernant le cancer de la prostate, nos données préliminaires montrent que les cellules NK circulantes de patients sont également fonctionnellement compétentes, quelque soit le stade de la maladie, malgré la diminution significative de l'expression du récepteur NKp30. Ainsi, le degré d’immunogénicité des cellules B leucémiques et celui des cellules tumorales prostatiques devra être autant pris en compte que la fonctionnalité des cellules NK dans les stratégies visant à optimiser l'activité antitumorale de ces dernières<br>Many experimental and clinical data have enlightened the importance of Natural Killer (NK) cells in tumor immunosurveillance. Therapeutic strategies based on NK cells could be an alternative in the treatment of certain cancers. We focused our study on two types of incurable cancers despite recent advances in treatment: B chronic lymphocytic leukemia (B-CLL) and prostate cancer. The aim of our study is a better understanding of the mechanisms set up by leukemic B cells and prostate cancer cells to escape from NK antitumor response. The knowledge of these escape mechanisms is an essential prerequisite to the use of NK cells in antitumor therapies. Regarding B-CLL, our results suggest that NK cells, although functionally competent, can not initiate an appropriate immune response against leukemic B cells due to a lack of recognition of the latter. Concerning the prostate cancer, our preliminary data show that circulating NK cells are functionally competent, whatever the stage of disease, despite the significant decrease in expression of the receptor NKp30. Thus, the degree of immunogenicity of leukemic B cells and of the prostate cancer cells must be taken into account as well as the functionality of NK cells in strategies aiming at improving NK antitumor activity
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Hadife, Nader. "Interleukine-24 : rôle immunologique et mécanismes d'induction de mort cellulaire dans les lymphocytes B." Thesis, Université de Lorraine, 2013. http://www.theses.fr/2013LORR0018/document.
Full textAbstract:
Notre équipe a précédemment démontré que l'Interleukine (IL)-24 une cytokine de la famille de l'IL-10 a un effet cytostatique voire cytotoxique sur des cellules B normales ou leucémiques mises préalablement en cycle mais non sur des cellules quiescentes. L'IL-24 inhibe également la différenciation plasmocytaire des cellules B humaines du centre germinatif dans un modèle de différentiation in vitro. Nous avons utilisé ce modèle pour analyser pour la première fois le transcriptome de cellules B stimulées ou non par IL-24 au bout de 6 et 36 h. Plusieurs transcrits impliqués dans le métabolisme et la réplication de l'ADN sont inhibés précocement à l'exception d'IGF-1 qui est stimulé. L'IGF1 ayant été décrit comme une molécule de survie des cellules B ou pré-B, nous avons analysé son effet biologique et démontré qu'il a au contraire un rôle proapoptotique à doses physiologiques. En revanche, l'IL-24 induit l'expression plus tardive des gènes de la voie mitochondriale de la mort cellulaire programmée (MCP). Cet effet est également retrouvé dans des LLC « IgVH mutées » ou non mais avec une cinétique distincte des cellules B normales. Au total, dans des cellules activées au préalable, l'IL-24 induit séquentiellement un blocage précoce du cycle cellulaire suivi d'une apoptose. D'autres gènes potentiellement importants dans la synapse immune ainsi que dans la régulation de l'immunité innée sont décrits et suggèrent que l'IL-24 a un rôle immunologique particulier au-delà de son effet cytostatique et potentiellement anti-tumoral<br>We have previously shown that Interleukin(IL)-24 a class-II cytokine of the IL-10 family has cytostatic and cytotoxic properties on normal and malignant human B-cells previously engaged into the cell cycle, but not on quiescent B-cells. IL-24 also inhibits the differentiation of germinal center B-cells in plasma cells in an in vitro model; the later was used to compare for the first time the transcriptome of B-cells cultured or not with IL-24 for 6 and 36h. Several "early" transcripts involved in DNA metabolism and replication were inhibited whereas that of Igf1 a molecule described as a B-cell growth factor was induced. We show herein that IgF1 has instead a proapoptotic role on B-cells at physiological concentrations. In contrast, several genes of the intrinsic apoptotic pathway were stimulated after 36h. This expression pattern was also found in CLL cells whether they were "IgVH mutated" or "unmutated", albeit with distinct kinetics from normal B-cells. In addition several genes belonging to the immune synapse and innate immunity were regulated by IL-24. These results disclose additional, possibly immunoregulatory properties, for IL-24 than its already described cytostatic and potentially anti-tumoral effects
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Orlandi, Veronica. "Effects of p66shc expression on bioenergetics and cell viability of b-cell chronic lymphocytic leukemia." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3423953.
Full textAbstract:
During tumorigenesis many oncogenic pathways are reprogrammed and modulate mitochondrial metabolism, decreasing oxidative phosphorylation (OXPHOS) activity. They inhibit ATP synthesis and ROS production from OXPHOS complexes favoring metabolic switching toward a more glycolytic metabolism and tumor growth. This metabolic rewiring is well established in solid tumor but very little is known about this event in non-solid tumors such as leukemias. Indeed, it has been demonstrated that in chronic lymphocytic leukemia (B-CLL), cells show a high OXPHOS activity and elevated ROS levels. Notably, expression of the p66Shc protein, whose mitochondrial fraction modulates cell bioenergetics and increases ROS generation, is lost during B-CLL tumorigenesis. I investigate the effects of the expression of p66Shc on bioenergetics and viability of B-CLL cells. After p66Shc expression in MEC-1 cells, a B-CLL cell model, a fraction of it is in the intermembrane space of mitochondria. p66Shc expression decreases both basal and maximal respiration, decreases mitochondrial membrane potential, total ATP levels and renders cells less dependent from OXPHOS to ATP supply without changes mitochondrial mass. I propose that decreased mitochondrial respiration is due to decreased activity of respiratory complex I and II. In more detail, there is a decrease in complex I assembly with a consequent inhibition of its activity, and a decrease in complex II activity caused by a multi-protein complex in which TRAP1 and active ERK are involved. p66Shc expression in MEC-1 cells increases mitochondrial ERK activation. The mitochondrial fraction of ERK contribute to tumor cell survival by inhibiting opening of the permeability transition pore (PTP), a mitochondrial channel whose opening irreversibly commits cells to death. I check the effect of p66Shc expression on cell viability. Although its pro-apoptotic function well described in many cell models, in MEC-1 cells p66shc protects cells from death induced by mitochondrial-derived oxidative stress derived from starvation, EM20-25 and CisPlatin treatments. In depletion conditions, p66Shc expression correlates with more active mitochondrial ERK and the inhibition of ERK decreases cell viability. Inhibition of mitochondrial chaperones activity with Cyclosporine A, an inhibitor of CyP-D, or 17AAG, an inhibitor of TRAP1, also affects cell viability under depletion condition, thus supporting the hypothesis that mitochondrial ERK is involved in survival mechanism induced by p66Shc expression.
Taken together these data indicate that regulation of RC complex activity can be linked to regulation of cell survival. We hypothesize that the mitochondrial branch of the Ras-ERK signaling pathway regulates mitochondrial bioenergetics by affecting SDH activity and resistance to apoptosis of B-CLL cells. Through the activation of mitochondrial ERK and inhibition of SDH activity, p66Shc can decrease OXPHOS activity and ROS production, a condition that is necessary in the early stages of tumorigenesis. It can also support tumour cell growth making mPTP less sensitive to opening by activating mitochondrial ERK.<br>Durante lo sviluppo tumorale, molte vie di segnale delle cellule vengono riprogrammate cambiando il metabolismo mitocondriale e diminuendo l’attività della fosforilazione ossidativa. In questa maniera viene diminuita la produzione di ATP e di ROS da parte della fosforilazione ossidativa favorendo un metabolismo glicolitico e la crescita tumorale. I meccanismi alla base dello sviluppo di questo fenotipo sono ben caratterizzati nelle cellule di tumori solidi, ma poco si sa a riguardo delle leucemie. Nella leucemia linfocitica cronica, le cellule presentano elevata attività OXPHOS e alti livelli di ROS. È noto che la proteina p66Shc, di cui una frazione si trova nello spazio intermembrana del mitocondrio, modula la bioenergetica cellulare e induce la produzione di ROS. Durante il processo di tumorigenesi della B-CLL l’espressione di p66Shc viene persa. Quindi, sono andata ad analizzare l’effetto della espressione di p66Shc sulla bioenergetica e la vitalità cellulare della leucemia linfocitica cronica. Dopo l’espressione di p66Shc nel modello cellulare di leucemia linfocitica cronica di tipo B, MEC-1, una frazione della proteina è stata trovata nello spazio intermembrana dei mitocondri.
L’espressione di p66Shc nelle cellule diminuisce la respirazione mitocondriale, massima e basale, ma anche il potenziale di membrana e i livelli totali di ATP. Ho proposto che queste differenze sono dovute alla diminuzione dell’attività dei complessi respiratori: complesso I e complesso II indotta dalla espressione di p66Shc nelle cellule MEC-1. In particolare, il complesso I è meno assemblato con la conseguente diminuzione della sua attività mentre il complesso II diminuisce la sua attività a causa di un complesso multi proteico in cui sono coinvolti la proteina chinasi ERK e lo sciaperone mitocondriale TRAP1.
L’espressione di p66Shc nelle MEC-1 aumenta l’attivazione della frazione mitocondriale di ERK. Questa frazione, contribuisce alla sopravvivenza delle cellule tumorali inibendo l’apertura del poro di transizione di permeabilità (mPTP), un canale mitocondriale la cui apertura porta alla morte cellulare. Ho quindi analizzato l’effetto dell’espressione di p66Shc sulla vitalità cellulare delle MEC-1. Sebbene, p66Shc ha una attività pro-apoptotica, ho osservato che la sua espressione nelle MEC-1 protegge le cellule dalla morte cellulare indotta dallo stress ossidativo prodotto dal mitocondrio da trattamenti come EM20-25, CisPlatino e assenza di glucosio. In assenza di glucosio, l’espressione di p66Shc correla con una maggiore attivazione di ERK nel mitocondrio. L’inibizione di ERK diminuisce la vitalità cellulare. In assenza di glucosio, anche l’inibizione degli sciaperoni mitocondriali CyP-D e TRAP1 influenzano la vitalità cellulare. Quindi, la frazione mitocondriale di ERK è coinvolta nella regolazione delle vie di segnale che proteggono le cellule dalla morte cellulare dopo l’espressione di p66SHhc.
Questi dati, indicano che la regolazione dell’attività dei complessi respiratori è legata alla regolazione della sopravvivenza cellulare. Abbiamo ipotizzato che nella leucemia linfocitica cronica la parte mitocondriale della via di segnale RAS-ERK può regolare la bioenergetica influenzando l’attività enzimatica del complesso II e la resistenza alla morte cellulare. Inoltre, abbiamo ipotizzato anche una nuova funzione per p66Shc in cui, attraverso l’attivazione di ERK e l’inibizione dell’attività del complesso II può diminuire l’attività della fosforilazione ossidativa e i livelli di ROS, una condizione necessaria nelle prime fasi della tumori genesi. p66Shc può anche sostenere la crescita tumorale rendendo mPTP meno sensibile all’apertura attraverso l’attivazione della frazione mitochondrial di ERK.
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Lopes, Ana Alexandra Festas. "Estudo funcional das células T de gânglio normal/reativo e com doença linfoproliferativa crónica de células B." Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/16768.
Full textAbstract:
Mestrado em Bioquímica - Bioquímica Clínica<br>As doenças linfoproliferativas crónicas de células B são um grupo
heterogéneo de entidades que representam cerca de 80-90% de todas as
síndromes linfoproliferativas crónicas, nas quais podem ser observadas uma
proliferação clonal de linfócitos B. Amostras de biópsia de gânglio linfático
podem ser usadas para o diagnóstico destas doenças. Os gânglios linfáticos são
o local adequado para as interações entre as células B e as células T e para
ocorrer uma resposta imune. Neste sentido, é esperado que, na presença de
células B malignas, com uma capacidade diferente para interagir com as células
T, estas irão responder de uma forma diferente.
O objetivo do presente trabalho consistiu na análise numérica e funcional
das células T, nomeadamente de células T ativadas, células T reguladoras,
Th/c17 e Thc/1, bem como, das células NK e quantificação de monócitos e
células dendríticas.
Foram analisadas dezanove amostras de biópsia de gânglio linfático, sete
normais/reativas, cinco com linfoma linfocítico/LLC-B e sete com linfomas Não-
Hodgkin B. Quantificou-se as subpopulações de células T CD4 e CD8; Th/c17;
Th/c1; Tregs; células T ativadas através da citometria de fluxo, e após
purificação das células TCD4 e TCD8, procedeu-se à quantificação da
expressão de mRNA para os genes IL2 e IL10.
Os nossos resultados mostraram um aumento das células Treg bem
como, um decréscimo das células Th/c17 e Th/c1 no grupo com linfomas
linfocíticos/LLC-B e no grupo com LNH-B. Relativamente à expressão génica,
verificou-se uma diminuição de IL2 e um aumento de IL10 tanto para as células
T CD4, como para as células T CD8 no grupo com linfomas linfocíticos/LLC-B e
no grupo com LNH-B.
Os resultados, embora preliminares, devido ao reduzido números de
amostras estudadas, apontam para alterações significativas fenotípicas e
funcionais nas células T e NK dos gânglios linfáticos com infiltração por células
B patológicas, o que sugere diferenças ao nível da resposta imune anti-tumoral,
que podem contribuir para o prognóstico destas entidades.<br>B cells Chronic lymphoproliferative diseases are a heterogeneous group of
entities representing about 80-90% of all chronic lymphoproliferative syndromes,
in which can be observed a clonal proliferation of B lymphocytes. Lymph node
biopsy samples may be used to diagnosis of these diseases. Lymph nodes are
suitable location for the interaction between B cells and T cells and an immune
response to occur. Therefore, it is expected that in the presence of malignant B
cells with a different capacity to interact with T cells, they will respond in a
different way.
The objective of this study was to numerical and functional analysis of T
cells, particularly of activated T cells, regulatory T cells, Th/c17 and Th/c 1, as
well as quantitation of NK cells and monocytes and dendritic cells.
Nineteen lymph node biopsy samples were analyzed, seven
normal/reactive, five with lymphocytic lymphoma/CLL-B and seven non-
Hodgkin's lymphomas. Subpopulations of CD4 and CD8 T cells; Th/c17; Th/c1
Tregs; and T cells activated by flow cytometry were quantitated. After purification
of CD4 and CD8 T cells, we proceeded to quantify the expression of mRNA for
IL-2 and IL-10 genes.
Our results showed an increase of Treg cells as well as a decrease in Th/
c17 cells and Th/c1 in the group with lymphocytic lymphoma/B-CLL and NHL-B
group. For the gene expression, there was a decrease in IL2 and an increase of
IL10 both CD4 T cells, as for CD8 T cells in the group with lymphocytic
lymphomas/B-CLL and NHL-B group.
The results, although preliminary, due to the small number of samples
analyzed, indicate phenotypic and functional significant changes in the T and NK
cells from lymph nodes with infiltration by pathological B-cells, suggesting
differences in anti-tumor immune response, which may contribute to the
prognosis of these entities.