Lymphocytes In Chronic Leukemia Research Articles

Infectious Diseases and Secondary Antibody Deficiency in Patients from a Mesoregion of São Paulo State, Brazil.

Our aim was to determine the secondary antibody deficiency (SAD) profiles of patients in a mesoregion of São Paulo state, Brazil, focusing on infectious diseases. Demographic characteristics, and clinical and laboratory data were obtained from electronic files; infections were classified as organ-specific and graded as mild, moderate, life-threatening, and fatal. Non-Hodgkin's lymphoma (NHL) accounted for 30% of patients, nephrotic syndrome (NS) 25%, chronic lymphocyte leukemia 20%, and multiple myeloma 15%. Patients with NS were younger than those in other groups, and hypo-γ-globulinemia was detected in 94.1%, IgG < 400 mg/dL in 60.0%, IgA < 40 mg/dL in 55.0%, and CD19 < 20 cells/mm3 in 30.0%. One hundred and one infections were found; 82.1% were classified as mild or moderate, 7.9% as life-threatening, and 3.0% as fatal. Respiratory tract infections were more prevalent (41.5%), and pneumonia accounted for 19.8%. Lower levels of infections were found in patients with NS compared with NHL (p = 0.0001). Most patients progressed to hypo-γ-globulinemia and SAD after treatment with immunosuppressants, and mild and moderate infections were predominant. These therapies are increasing in patients with different diseases; therefore, monitoring hypo-γ-globulinemia and infections may help to identify patients at high risk for severe complications, antibiotic prophylaxis or treatment, and immunoglobulin replacement.

Generation of iPSC derived CD19 CAR iNK cells for treatment of autoimmune disorders

Abstract Treatment of autoimmune diseases (ADs) relies on broad immunosuppressive agents that are often not curative. Autologous CAR T cell approaches demonstrated disease remission in patients with Systemic lupus erythematosus (SLE). Among cellular abnormalities typical for ADs, NK cells dysfunction was reported. We believe that using iPSC derived CD19 CAR NK cells (iNK cells) to eliminate disease-causing circulating plasma cells could be a novel therapeutic approach for ADs mediated by B cells. Evotec’s validated GMP iPSC line was genetically modified by a knock-in of a CD19-CAR and differentiated into iNK cells using a feeder-free 3D differentiation process. Successful knock-in was confirmed by flow cytometry. Expression of the CAR did not negatively impact the differentiation of the iNK cells. The CD19 CAR iNK cell phenotype and scRNA sequencing profiles are comparable to blood-derived NK cells. To evaluate the ability of CD19 CAR iNK cells to selectively eliminate primary B cells, patient-derived B cell chronic lymphatic leukemia (CLL) cells and primary B cells from healthy donors were co-cultured with CAR iNK cells. Compared to wildtype (WT) iNK cells, CD19 CAR iNK showed specific killing of CD19-positive CLL cells and primary B cells. iPSC-derived CD19-CAR NK cells show increased cytotoxicity and specific targeting of CD19 positive primary patient and healthy subject derived B cells, demonstrating their potential suitability for the treatment of ADs such as SLE.

Structure of BTK kinase domain with the second-generation inhibitors acalabrutinib and tirabrutinib.

Bruton's tyrosine kinase (BTK) is the target of the therapeutic agent, Ibrutinib, that treats chronic lymphocyte leukemia (CLL), mantle cell lymphoma (MCL) and other B cell malignancies. Ibrutinib is a first in class, covalent BTK inhibitor that limits B-cell survival and proliferation. Designing new inhibitors of BTK has been an important objective for advancing development of improved therapeutic agents against cancer and autoimmune disorders. Based on the success of Ibrutinib, several second-generation irreversible BTK inhibitors have been developed that exhibit fewer off-target effects. However, the binding-mode and their interaction with Btk have not been experimentally determined and evaluated at atomic resolution. Here we determined the first crystal structure of the BTK kinase domain in complex with acalabrutinib. In addition, we report a structure of the BTK/tirabrutinib complex and compare these structures with previously solved structures. The structures provide insight in the superior selectivity reported for acalabrutinb and guide future BTK inhibitor development.

Retrospective Study of T Cell Leukaemia (Large Granular Lymphocyte Variant) in Dogs Associated with Suspected Immune-Mediated Cytopaenia(s) in the Absence of Peripheral Lymphocytosis.

Canine chronic large granular lymphocyte (LGL) leukaemia is commonly characterised by moderate to marked lymphocytosis but not neutropaenia. In humans, LGL leukaemia is often associated with autoimmune disorders, including immune-mediated cytopaenias (mainly neutropaenia). This presentation is rare in dogs. The aim of this retrospective study was to describe the clinical characteristics, treatments, and outcomes of dogs with chronic LGL leukaemia with suspected immune-mediated cytopaenia. Six dogs with a median age of 4.5 years (range 2-8 years) were included in the study. The most common presenting signs were pyrexia and lethargy. All dogs had severe neutropaenia (median neutrophil count 0.07 × 109/L), three had thrombocytopaenia (median platelet count 66 × 109/L), and one had anaemia (HCT 0.32 L/L). In all dogs, bone marrow cytology revealed infiltration of granular T lymphocytes; PARR analysis confirmed clonality in four, and bone marrow flow cytometry identified CD3+ CD8+ neoplastic cells in two cases. All patients received systemic chemotherapy, and the cytopaenias resolved after 1-19 weeks. Two dogs were euthanised 133 and 322 days after diagnosis, two were lost to follow-up after 224 and 357 days, and two were alive at 546 and 721 days. A subset of LGL leukaemia in dogs is associated with immune-mediated cytopaenia and has a unique clinical presentation.

CD229 (Ly9) a Novel Biomarker for B-Cell Malignancies and Multiple Myeloma.

Simple SummaryCD229 is a cell-surface molecule predominantly expressed on lymphocytes. Its expression in B-cell malignancies is poorly known. We tested the presence of this immunoreceptor on a large number of malignancies and normal tissue using a new monoclonal antibody and tissue microarrays. Our data show that CD229 expression is restricted to hematopoietic cells. It was strongly expressed in myeloma and marginal-zone lymphomas. Because of the high expression on multiple myeloma cells, we also analyze the presence of soluble CD229 in the sera of these patients. We showed that serum levels of soluble CD229 in myeloma patients, at the time of diagnosis, could be useful as a prognostic biomarker. Altogether, our results indicate that CD229 represents not only a useful disease biomarker but also an attractive therapeutic target.CD229 (Ly9) homophilic receptor, which belongs to the SLAM family of cell-surface molecules, is predominantly expressed on B and T cells. It acts as a signaling molecule, regulating lymphocyte homoeostasis and activation. Studies of CD229 function indicate that this receptor functions as a regulator of the development of marginal-zone B cells and other innate-like T and B lymphocytes. The expression on leukemias and lymphomas remains poorly understood due to the lack of CD229 monoclonal antibodies (mAb) for immunohistochemistry application (IHC). In this study, we used a new mAb against the cytoplasmic region of CD229 to study the expression of CD229 on normal tissues and B-cell malignancies, including multiple myeloma (MM), using tissue microarrays. We showed CD229 to be restricted to hematopoietic cells. It was strongly expressed in all cases of MM and in most marginal-zone lymphomas (MZL). Moderate CD229 expression was also found in chronic lymphocyte leukemia (CLL), follicular (FL), classic mantle-cell (MCL) and diffuse large B-cell lymphoma. Given the high expression on myeloma cells, we also analyzed for the presence of soluble CD229 in the sera of these patients. Serum levels of soluble CD229 (sCD229) at the time of diagnosis in MM patients could be useful as a prognostic biomarker. In conclusion, our results indicate that CD229 represents not only a useful biomarker but also an attractive therapeutic target.

MYD88 Gene Polimorphism in Patients With Chronic Lenfoid Leukemia

Leukemia is a type of cancer that manifests itself with the excessive proliferation of blood cells, especially white blood cells, and affects the blood production system in the body. Leukemias are classified as acute or chronic and according to the extent and development of the tumor. In chronic lymphocyte leukemia (CLL), cancer cells are found in the bone marrow, blood, and lymph nodes. Therefore, in this study, the effects of polymorphisms in the MYD88 gene of CLL patients were investigated. For this purpose, 100 patients diagnosed with CLL and 70 healthy individuals without cancer history were included in the study as the control group. DNA was isolated from CLL patients and control groups. Polymorphisms in the MYD88 (L265p T/C) promoter regions were studied by RT-PCR. Genotype and allele frequencies were calculated directly. Statistical significance of genotypic distributions between CLL patients and control groups was evaluated with Z Ratios test and Fisher's Exact test.For the MYD88 L265p T/C region in 100 CLL patients, 95 wild type TT, 3 TC and 5 mutant type CC genotypes were detected. No statistically significant difference was found between CLL patients and control groups in the MYD88 L265p T/C region (p 0.05). According to the allele frequencies of the region, it was determined as 0.965 and 0.065 for T and C, respectively. According to Fisher's Exact results, it was calculated as 0.078 for TT, 0.078 for TC and 0.269 for Mutant, respectively. The MYD88 genotype frequencies of the patients included in the study do not appear to be statistically correlated with CLL patients. The research we have done as a result of our study will shed light on the studies with different SNPs to be made with the increase in the number of CLL patients..

Ibrutinib’s off-target mechanism: cause for dose optimization

ABSTRACT Ibrutinib (Imbruvica®, 2013) is a Bruton’s tyrosine kinase (BTK) inhibitor approved for multiple B-cell malignancies and cGVHD. Its treatment is associated with increased risk of cardiac adverse events. Atrial fibrillation is a common cause of therapy discontinuation and interruptions, which have been correlated with shorter progression-free survival in chronic lymphocyte leukemia (CLL) patients. Recently, Xiao et al. identified that ibrutinib-mediated atrial fibrillation is likely due to off-target CSK inhibition. Given promising in vitro and in vivo evidence of maintained biological activity in CLL at lower-than-labeled ibrutinib doses, this elucidated mechanism substantiates the case to investigate alternative dosing schedules. The potential to minimize ibrutinib’s off-target effects while conserving response warrants further discussion and investigation of optimal ibrutinib dosing.

Association of BTLA rs1982809 polymorphism with lung cancer risk in Tunisian population.

B and T lymphocyte attenuator (BTLA) is an immune-inhibitory receptor that negatively regulates the lymphocyte activation. A few studies have been devoted to the relationship between BTLA gene variations and cancer's risk. It has been essentially demonstrated to be involved in increasing cancer risk in chronic lymphocyte leukaemia, renal cell carcinoma, breast and colorectal cancer predispositions in Asian population. The aim of this study was to evaluate the association between BTLA gene polymorphisms and the risk of lung cancer in the Tunisian population. In a case-control study, three BTLA single-nucleotide polymorphism (SNP): rs1982809 (A>G), rs9288952 (G>A) and rs9288953(C>T) were genotyped with the use of TaqMan probes in 169 lung cancer patients and in 300 controls. The rs1982809 SNP was significantly associated with an increased risk of lung cancer compared with controls in codominant and dominant models. The heterozygous rs1982809-AG genotype carriers had a higher risk of developing lung cancer when compared to AA genotype carriers in Tunisian population (OR (95%CI)=1.63 (1.09-2.42), p=.01]. The AG genotype is an important risk factor associated with lymphatic invasion (OR=3.71) and large-sized lung tumour (OR=1.80). It is also a risk factor for the development of an adenocarcinoma subtype (OR=2.08). However, the BTLA rs9288953 and rs9288952 SNPs were not associated with susceptibility for lung cancer (p>.05). Haplotype comparison did not show any significant association in our research. For the survival analysis, there was no impact of BTLA SNPs on the mortality risk associated to lung cancer in Tunisian patients. The current study is the first to demonstrate an association between BTLA rs1982809 polymorphism and an increased lung cancer risk in the Tunisian population.

Evaluating the mechanism underlying antitumor effect of interleukin 27 on B cells of chronic lymphocytic leukemia patients.

Chronic lymphocyte leukemia (CLL) is a B-cell malignancy resisted to apoptosis. Recently, some studies indicated that cytokines such as interleukin 27 (IL-27) can reduce B-cell proliferation. The aim of this study is to evaluate the mechanism underlying the proapoptotic effect of IL-27 on B cells of patients with CLL in comparison with B cells of normal subjects. The effect of IL-27 on the antitumor activity of natural killer (NK) and T cells was also evaluated. Peripheral blood mononuclear cells (PBMCs) were isolated from 35 patients with CLL and 15 normal subjects. B cells and PBMCs were cocultured with IL-27 and B cells apoptosis to evaluate proliferation. Both messenger RNA and protein expression of IL-27 and IL-27 receptor were determined using flow cytometry and real-time polymerase chain reaction analysis. To evaluate the apoptotic effect of IL-27 on B cells of patients with CLL, Annexin V-FITC and 7-AAD (BioLegend) fluorescent dyes were used. In addition, the IL-27 effect on activation of T cell and NK cell was determined by determining CD96 molecule expression. IL-27 and IL-27 receptor expression in patients with CLL was significantly lower than that of normal subjects (p < .05). IL-27 enhanced apoptosis of B cells in patients with CLL (p < .05) but this effect was not significantly observed in B cells of normal subjects (p > .05). Consequently, IL-27 reduced the proliferation of B cells and enhanced NK cell activity (p < .05). IL-27, through inducing apoptosis, can exert an inhibitory effect on cancer B cells of CLL patients with minimal effect on normal B cells.

The importance of pharmacological modulation of the enzymatic activity of CD38 and intracellular NAD levels

CD38 may play important roles in the pathophysiology of different human diseases, like obesity, diabetes, chronic lymphocyte leukemia and other disease either through the enzymatic or receptorial functions. Also, it has a role in energy homeostasis, signal transduction, and aging. Interestingly, CD38 is a major regulator of intracellular nicotinamide adinin dinucleotide (NAD+) levels. It is the main NADase enzyme, it uses NAD to produce the main calcium mobilizing messengers. On the other hand, NAD is a key molecule that is involved in multiple signaling pathways and metabolic conditions. Thus, it is of highly importance to regulate of CD38 catalytic activity. Therefore, it is of interest to many investigators to find inhibitors of CD38 function to verify CD38 as a new potential therapeutic target. Here we draw attention to the importance of CD38 catalytic function and its involvement in several disease. In addition to the importance of intracellular NAD, the main NAD-consuming enzymes, and biosynthesis enzymes. The main regulators of CD38 activity (inhibitors or activators) are also reported to provide a theoretical basis for further drug design.This review is aimed at giving a brief insight into the progress made to date about CD38/NAD relationship. CD38/NAD link plays important roles in signaling pathways that may contribute to human pathologies, it is a potential target for drug development.

Expression of Cytokine of Chronic Lymphocyte Leukemia from Baghdad / Iraq Population

Aim of the current study is to investigate correlation proinflammatory cytokines (Interleukin 6, 8, 10, and TNF α) with Chronic Lymphocyte Leukemia. In this study, serum was collected from (100) sample, a representative of 80 samples of chronic lymphocyte leukemia who were newly diagnosed by the consultant medical staff at the National Center of Hematology and 20 healthy sample on the other hand. IL-6, IL8, IL10 and TNF α were studied using the enzyme linked immunosorbent assay methods from (R and D system/USA). This study have shown that IL6 levels increased about two fold compared with healthy groups and TNF α levels was three times higher in CLL patient compared with healthy and IL10 levels was 3.5 fold that healthy. The conclusion IL 6, 10, and TNF α levels have significantly increased in patients compared with healthy control. However, IL8 was the same production in CLL compared with control.

Role of T Lymphocytes In Chronic Leukemia

Functional immune cells are required for the prevention or resolution ofimmune-mediated disease , in particular T cells, including helper (CD4+; Th) or cytotoxic (CD8+; Tc) T cells. Th cells differentiate into Th1 or Th2 type cells, through production of inflammatory (IL-2, IFN-a) or anti-inflammatory (IL-4, TGF-ß) cytokines, respectively. Th cells also can differentiate into a third type of Th cells termeds Th17 type cells that produces IL-17 thereby mimicing the effects of Th1 cells. Th type cells can also differentiate under certain conditions into a regulatory (T-reg) type cell capable of secreting immunosuppressive cytokines such as TGF-ß and IL-10. This mini-review discusses the importance of T cell plasticity in regulating the nature of the immune cell responses seen in inflammatory diseases or cancer.Bangladesh Journal of Medical Science Vol.16(2) 2017 p.207-211

Class I histone deacetylases regulate p53/NF-κB crosstalk in cancer cells

The transcription factors NF-κB and p53 as well as their crosstalk determine the fate of tumor cells upon therapeutic interventions. Replicative stress and cytokines promote signaling cascades that lead to the co-regulation of p53 and NF-κB. Consequently, nuclear p53/NF-κB signaling complexes activate NF-κB-dependent survival genes. The 18 histone deacetylases (HDACs) are epigenetic modulators that fall into four classes (I-IV). Inhibitors of histone deacetylases (HDACi) become increasingly appreciated as anti-cancer agents. Based on their effects on p53 and NF-κB, we addressed whether clinically relevant HDACi affect the NF-κB/p53 crosstalk. The chemotherapeutics hydroxyurea, etoposide, and fludarabine halt cell cycle progression, induce DNA damage, and lead to DNA fragmentation. These agents co-induce p53 and NF-κB-dependent gene expression in cell lines from breast and colon cancer and in primary chronic lymphatic leukemia (CLL) cells. Using specific HDACi, we find that the class I subgroup of HDACs, but not the class IIb deacetylase HDAC6, are required for the hydroxyurea-induced crosstalk between p53 and NF-κB. HDACi decrease the basal and stress-induced expression of p53 and block NF-κB-regulated gene expression. We further show that class I HDACi induce senescence in pancreatic cancer cells with mutant p53.

Hypomethylation coordinates antagonistically with hypermethylation in cancer development: a case study of leukemia.

BackgroundMethylation changes are frequent in cancers, but understanding how hyper- and hypomethylated region changes coordinate, associate with genomic features, and affect gene expression is needed to better understand their biological significance. The functional significance of hypermethylation is well studied, but that of hypomethylation remains limited. Here, with paired expression and methylation samples gathered from a patient/control cohort, we attempt to better characterize the gene expression and methylation changes that take place in cancer from B cell chronic lymphocyte leukemia (B-CLL) samples.ResultsAcross the dataset, we found that consistent differentially hypomethylated regions (C-DMRs) across samples were relatively few compared to the many poorly consistent hypo- and highly conserved hyper-DMRs. However, genes in the hypo-C-DMRs tended to be associated with functions antagonistic to those in the hyper-C-DMRs, like differentiation, cell-cycle regulation and proliferation, suggesting coordinated regulation of methylation changes. Hypo-C-DMRs in B-CLL were found enriched in key signaling pathways like B cell receptor and p53 pathways and genes/motifs essential for B lymphopoiesis. Hypo-C-DMRs tended to be proximal to genes with elevated expression in contrast to the transcription silencing-mechanism imposed by hypermethylation. Hypo-C-DMRs tended to be enriched in the regions of activating H4K4me1/2/3, H3K79me2, and H3K27ac histone modifications. In comparison, the polycomb repressive complex 2 (PRC2) signature, marked by EZH2, SUZ12, CTCF binding-sites, repressive H3K27me3 marks, and “repressed/poised promoter” states were associated with hyper-C-DMRs. Most hypo-C-DMRs were found in introns (36 %), 3′ untranslated regions (29 %), and intergenic regions (24 %). Many of these genic regions also overlapped with enhancers. The methylation of CpGs from 3′UTR exons was found to have weak but positive correlation with gene expression. In contrast, methylation in the 5′UTR was negatively correlated with expression. To better characterize the overlap between methylation and expression changes, we identified correlation modules that associate with “apoptosis” and “leukocyte activation”.ConclusionsDespite clinical heterogeneity in disease presentation, a number of methylation changes, both hypo and hyper, appear to be common in B-CLL. Hypomethylation appears to play an active, targeted, and complementary role in cancer progression, and it interplays with hypermethylation in a coordinated fashion in the cancer process.Electronic supplementary materialThe online version of this article (doi:10.1186/s40246-016-0071-5) contains supplementary material, which is available to authorized users.

Dishevelled proteins are significantly upregulated in chronic lymphocytic leukaemia.

Dishevelled (DVL) proteins are components of the Wnt signalling pathways, and increased expression is associated with various malignancies. Information on DVLs in chronic lymphatic leukaemia (CLL) is limited. The aim of the present study was to investigate the role of DVLs in CLL cells and association with Wnt pathways downstream of ROR1. DVL1, 2 and 3 were exclusively expressed in CLL cells as compared to normal peripheral blood mononuclear cells (PBMCs). The expression of DVL1 and DVL3 proteins was significantly more pronounced in progressive than in non-progressive disease (p < 0.01), whereas the level of DVL2 was significantly higher in non-progressive as compared to progressive disease (p < 0.001). Treatment of CLL cells with anti-ROR1 specific monoclonal antibodies induced dephosphorylation of ROR1 as well as of tyrosine and serine residues of both DVL2 and DVL3. However, gene silencing of DVLs in the CLL cell line (EHEB) did not induce detectable apoptosis. Non-progressive CLL patients had a different protein activity pattern with regard to Wnt signalling pathway proteins as GSK-3β, β-catenin and AKT as compared to progressive disease. The DVL2 protein may play a role in the activation of signalling pathways in CLL during early stages of the disease, while DVL1 and 3 may have a role in later phases of the leukaemia.

Application Of Raman Microscopy For The Diagnosis Of The Chronic Lymphocytic Leukemia (Cll)

Introduction/ Background In hematology, actual diagnosis of B chronic lymphocyte-leukemia (CLL) is based on the microscopic analysis of cell morphology from patient blood smear. However, new photonic technologies appear promising to facilitate and improve the early diagnosis, prognostic and monitoring of personalized therapy. The development of automated diagnostic approaches could assist clinicians in improving the efficiency and quality of health services, but also reduce medical costs. Aims The M3S project aims at improving the diagnosis and prognosis of the CLL pathology by developing a multimodal microscopy platform, including Raman spectrometry, dedicated to the automatic analysis of lymphocytes. Methods Blood smears were prepared on glass slides commonly used in pathology laboratories for microscopy. Two types of sample per patient were prepared: a conventional blood smear and a deposit of “pure” lymphocyte subtypes (i.e. normal B, CLL B, T and NK), sorted out in flow cytometry by using the negative double labelling technique. The second sample is used for the construction of a database of spectral markers specific of these different cell types. The preparations were analyzed with the multimodal machine which combines i) a Raman micro-spectrometer, equipped with a 532nm diode laser excitation source; ii) a micro- scope equipped with 40x and 150x lenses and a high precision xyz motorized stage for scanning the blood smear, and localizing x-y coordinates of representative series (~100 for each patient) of lymphocyte cells before registering three Raman spectra; these cells of interest being previously localized by an original method based on the morphology analysis. After the Raman acquisitions, the conventional blood smears were submitted immunolabelling using specific antibodies. For the establishment of the Raman classifiers, this post-acquisition treatment was used as reference to distinguish the different lymphocyte sub-populations. Raman data were then analyzed using chemometric processing and supervised statistical classifiers in order to construct a spectral library of markers highly specific of the lymphocyte type and status (normal or pathological). Results Currently, a total of 60 patients (CLL and healthy) were in- cluded in the study. Various classification methods such as LDA (Linear Discriminant Analysis), PLS-DA (Partial Least Square Discriminant Analysis), RF (RandomForest) and SVM (Support Vector Machine), were tested in the purpose to distinguish tumoral B lymphocytes from other cell types. These classification algorithms were combined with feature selection approaches. The best performances were around 70% of correct identification when a three-class model (B-CLL vs B-normal vs T and NK lymphocytes) was considered, and 80% in case of a two-class model (B-CLL vs B-normal lymphocytes). These encouraging results demonstrate the potential of Raman micro-spectroscopy coupled to supervised classification algorithms for leukemic cell classification. The approach can find interest more generally in the field of cyto-hematology. Further developments will concern the integration of additional modality such as Quantitative Phase Imaging on one hand to speed the exploration process of cells of interest to be probed, and on the other hand to extract additional characteristics likely to be informative for CLL diagnosis. In addition, the identification of prognostic markers will be investigated by confronting the photonic data to clinical patient information.

Diagnosis Of The Chronic Lymphocytic Leukemia (CLL) Using A Raman-Based Scanner Optimized For Blood Smear Analysis (M3s Project)

Introduction/ Background In hematology, actual diagnosis of B chronic lymphocyte-leukemia (CLL) is based on the microscopic analysis of cell morphology from patient blood smear. However, new photonic technologies appear promising to facilitate and improve the early diagnosis, prognostic and monitoring of personalized therapy. The development of automated diagnostic approaches could assist clinicians in improving the efficiency and quality of health services, but also reduce medical costs. Aims The M3S project aims at improving the diagnosis and prognosis of the CLL pathology by developing a multimodal microscopy platform, including Raman spectrometry, dedicated to the automatic analysis of lymphocytes. Methods Blood smears were prepared on glass slides commonly used in pathology laboratories for microscopy. Two types of sample per patient were prepared: a conventional blood smear and a deposit of “pure” lymphocyte subtypes (i.e. normal B, CLL B, T and NK), sorted out in flow cytometry by using the negative double labeling technique. The second sample is used for the construction of a database of spectral markers specific of these different cell types. The preparations were analyzed with the multimodal machine which combines i) a Raman micro-spectrometer, equipped with a 532nm diode laser excitation source; ii) a microscope equipped with 40x and 150x lenses and a high precision xyz motorized stage for scanning the blood smear, and localizing x-y coordinates of representative series (~100 for each patient) of lymphocyte cells before registering three Raman spectra; these cells of interest being previously localized by an original method based on the morphology analysis. After the Raman acquisitions, the conventional blood smears were submitted to immunolabelling using specific antibodies. For the establishment of the Raman classifiers, this post-acquisition treatment was used as reference to distinguish the different lymphocyte sub-populations. Raman data were then analyzed using chemometric processing and supervised statistical classifiers in order to construct a spectral library of markers highly specific of the lymphocyte type and status (normal or pathological). Results Currently, a total of 60 patients (CLL and healthy) were included in the study. Various classification methods such as LDA (Linear Discriminant Analysis), PLS-DA (Partial Least Square Discriminant Analysis), RF (Random Forest) and SVM (Support Vector Machine), were tested in the purpose to distinguish tumoral B lymphocytes from other cell types. These classification algorithms were combined with feature selection approaches. The best performances were around 70% of correct identification when a three-class model (B-CLL vs B-normal vs T and NK lymphocytes) was considered, and 80% in case of a two-class model (B-CLL vs B-normal lymphocytes). These encouraging results demonstrate the potential of Raman micro-spectroscopy coupled to supervised classification algorithms for leukemic cell classification. The approach can find interest more generally in the field of cyto-hematology. Further developments will concern the integration of additional modality such as Quantitative Phase Imaging on one hand to speed the exploration process of cells of interest to be probed, and on the other hand to extract additional characteristics likely to be informative for CLL diagnosis. In addition, the identification of prognostic markers will be investigated by confronting the photonic data to clinical patient information.

Homoharringtonine binds to and increases myosin-9 in myeloid leukaemia.

Homoharringtonine (HHT) is a natural alkaloid isolated from various Cephalotaxus species. HHT has been used to treat acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML), chronic lymphocyte leukaemia and myelodysplastic syndromes. Although HHT inhibits protein synthesis and promotes apoptosis of leukaemia cells in preclinical studies, its molecular target proteins remain unknown. The aim of this study was to identify target proteins of HHT. We have synthesized a biotinylated affinity column and used it to identify targets of HHT and confirmed the results by MS and Western blots. We also examined the effects of HHT on the target protein and determined roles of the target protein in anti-leukaemia activities of HHT through Western blots, flow cytometry and retrovirus transfection. Myosin-9, a member of the myosin super-family, was identified as a direct interactor of HHT. Furthermore, HHT up-regulated the expression level of myosin-9 in both AML and CML cell lines in a time-dependent manner. Thus, HHT-induced apoptosis of leukaemia cells begins in 6 h and continues to increase for 24 h. There is a positive correlation between up-regulated myosin-9 expression level and increased percentage of apoptotic cells mediated by HHT. Overexpression of myosin-9 could increase the sensitivity of the leukaemia cells to the cytotoxicity of HHT and arrest cells in S and G2/M phases. Our results indicated that myosin-9 was the target protein of HHT and played an important role in the HHT-induced apoptosis of leukaemia cells.

Reference intervals and its verification for leukocyte differential count in peripheral blood by CytoDiff flow cytometry

To establish and verify the reference intervals for leukocyte differential count in peripheral blood by CytoDiff flow cytometry. Leukocyte differentiation count was analyzed by using hematology analyzer, CytoDiff flow cytometry and morphology differential count in 278 healthy controls, 550 flagged and 102 unflagged samples. The reference intervals of leukocyte parameters were established and verified according to the healthy controls. Then the correlations of five leukocytes were analyzed among hematology analyzer, CytoDiff flow cytometry and morphology differential count. Lymphocyte subsets were analyzed and compared between healthy controls and patients with different diseases. The CytoDiff flow cytometric blast counts were explored to analyze the clinical diagnostic efficiency compared to morphology differential count as a reference method. CytoDiff flow cytometry can differentiate the leukocyte into 16 parameters, including percentage and absolute count, therefore 32 parameters in total. Among these parameters, 18 parameters had significant difference between male and female (P < 0. 05), and the others had no difference (P >0. 05). Except the CD16pos monocyte, there were no difference among ages in other parameters. The correlation between hematology analyzer, CytoDiff flow cytometry and morphology differential count were good for neutrophils, lymphocytes, monocytes, basophils and eosinophils in healthy controls and clinical samples. When the cutoff value of the ratio of T + NK and B lymphocyte was set at 1. 0 by ROC, the sensitivity was 90. 9% and specificity was 99. 8% for diagnosing the chronic lymphocyte leukemia (CLL). When the cutoff value of blast count by CytoDiff flow cytometry was set at 1%, the sensitivity was 100%, specificity was 96. 1% and accuracy was 96. 2% by morphology differential blast count for the reference method. Establish and verify the reference interval of leukocyte differential count in peripheral blood by CytoDiff flow cytometry in the adults of Beijing region. CytoDiff flow cytometry provide more parameters than hematology analyzer and morphology differential count, and therefore have excellent clinical diagnostic efficiency, especially for CLL and blasts from acute leukemia.

Association between Leukemia and Exon 2 CD19 Gene Variants in a sample of Iraqi Patients

Background: The human CD19 (Cluster Differentiation) antigen is a 95 kd transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 gene located on the short arm of chromosome 16p11.2 (P: petit). CD19 is a member of the Ig immunoglobulin superfamily expressed on the surface of B lymphocytes, and may play a pivotal role in B-cell differentiation and activation. Research suggests that mutations in a gene CD19 leads to a lack of expression of CD19 membrane and result in an antibody deficiency syndrome.Objective: The aim of this work is to study the mutations in Exon 2 CD19gene in leukemia patients in Baghdad/Iraq.Patients and Methods: This cross sectional study was performed in the National Center of Hematological Diseases/Al Mustanisyria University. Blood samples were collected from 50 leukemia patients including (25 acute lymphocyte leukemia ALL and 25 chronic lymphocyte leukemia CLL) and 50 samples of apparently healthy individuals. DNA was isolated and the CD19 gene was amplified by using specific primers for exon2 of this gene. The nucleotide sequences of CD19 gene was according to Macro gene company, USA. Analysis was done using BLAST program which is available online at (http:// www.ncbi.nlm.nih.gov) and BioEdit program.Results: The DNA sequencing results of flank sense of CD19 gene from healthy individual was found to be compatible 100% with wild type of Homo sapiens from the Gene Bank, while 99% compatibility was found for that gene of all ALL and CLL patients with wild type of gene. The difference was attributed to insertion of 1900 C nucleotide in position +48 of exon 2 of CD19 gene resulted in the replacement of a serine (TCC) residue into isoluseine (ATC), and deletion 1904 C in position + 49 of exon 2. This mutation resulted in change of codon from GCA to GCC but there was no changes in the amino acid (Alanine to Alanine). The second mutation (Deletion nucleotide) amended the first mutation effect (Insertion nucleotide) and did notlead to a change of all the amino acid sequence (framshift). The results showed that the incidence of insertion and deletion mutation at position +48 and +49 in exon 2 of CD19 gene and leukemia was highly significant (X2 = 15.75, P&lt;0.01).Conclusion: This study suggests that the ploymrphism in exon 2 of the CD19 gene is strongly associated with leukemia patient of Baghdad population.