Medical Haematology Immunocytochemical Staining of a Chronic Myeloid Leukaemia Cell Line - Assignment Example

MEDICAL HAEMATOLOGY IMMUNOCYTOCHEMICAL STAINING OF A CHRONIC MYELOID LEUKAEMIA CELL LINE Customer inserts His/her name Customer inserts grade course Customer inserts Tutor’s name 21st February 2012 Outline Introduction Results Discussion Conclusion References Introduction Chronic myeloid leukemia is a myeloprofifwerative disease under which leukemia cells thus exhibits the reciprocal translocation t (9; 22) and the bcr/abl oncogene. This report was conducted in order to understand how several different mechanisms have been implicated in BCR/ABL dependent growth and accumulation of leukemia cells in CML. The report was also conducted in order to understand the role that BCR/ABL in the expression of MCL-1 in leukemia cells and as analyzed underlining signaling pathways. The results showed that primary CML cells expressed MCL-1 in a constitutive manner and that BCR/ABL thus promotes the expressions of MSL-1 through the activation of ARS/ RAF/MAP kinase pathways. This method was designed so as to understand how chronic myeloid leukemia inhibits the signaling pathways through the association with variety of tyrosine kinase proteins. There was the need to develop the methods so as to analyze mutation and expression of the gene in 112, chronic myeloid leukemia. The results conducted demonstrated that the SOCSI gene silencing is caused by methylation of CPG islands in CML and is further reversed by a methylated status in molecular remission over time. In order to arrive at the cause of the disease the methods used expressed reliability and accuracy for the experiment. During the practical I found out that it is not very easy to determine the actual cause of chronic myeloid leukemia unless time and advanced technology equipments are used. Aims and objectives The aims of doing the experiment were for the purpose of detecting and enumerate basophils in born marrow sections in patients with CML and other MPD. More so, thrombocythemia being a chronic myeloproliferative disorder with an abnormal platelet production, being a member of BCL-2 of proteins that inhibits apoptosis. Results During performing of the experiment in the first step, it was reconfirmed that the specificity of using ficoll separated peripheral blood leucocytes that are obtained from those patients with CML showing basophilia and out of the cells provided 4 emerged to be positive and accurate. The cells showed negative fraction when they were further subjected to cytometry flow obtained from CD203c and thus they showed a negative fraction as was obtained from less than 1% basophilis. In other aspects, the monoclonal antibody 2D7 was found to react with an enriched CML basophilis and refused to react with bone marrow cells depleted of basophilis. However, identical results were obtained when two CML basophilis CML donors were put together. The data suggested that 2D7 is thus a basophilis specific antigen in patients with CML but is not expressed in other leucocytes confirming the results with normal blood basophilis. In addition, direct sequencing did not classify the nonspecific mutation or crossing out of SOCSI gene coding areas within the CML patient’s samples and the five cell lines. In trying to analyze the methylation status of the promoting area of the gene through the use of MSP method showed that none of the methylation was thus observed in the normal individuals. However, in contrast to the normal individual the hypermethylation was then noted 52% of the CML samples which included 67% and 46% blastic and chronic phase samples respectively. The results showed that in those patients with series phase showed a major gene in the acute blastic phase always more hypermethylated than in the chronic phase. In respect to the case, MSP results demonstrated a co-existence of both SOCSI-M and SOCSI-U in the chronic phase and they were positive in SOCSI-M but negative in SOCSI-U within a blastic crisis phase. SOCSI protein expression was then investigated using an immunocytochemistry, majority of the white cells of the normal controls were thus seen to be positively stained by the presence of SOCSI antibody. In contrast it was found out that heterogeneous positive staining was in the CML cases in the chronic phase and majority of the cells in the blastic phase were thus negatively stained. These results inveterate the examination that was observed and obtained from MSP and in real time qualitative RT-PCR. As the reversibility shown in methylation which was induced epigeneric transcriptional repression was a hall mark of the gene silencing, demethylation studies as were carried out. In other cases, the hypermethylated cell line mostly K562 was then treated by the use of a 5-aza- 2’ deoxycytidine so as it could be demethylated by SOCS1 locus over a period of 24h exposure period incubation in micromol/. The MSP results also showed a time dependent an increase in intensity of the SOCSI-U PCR products. However, the immunocytochemical staining also showed no significance by Galm et al (2003). Also, the loss in cytokine regulations further causes the constitution activation of the STAT/JAK pathways which has been reported among several hematopoietic malignancies that includes CML. The t (9, 22) chromosomal translocation results wiythi the fusion of ABL and BCR genes and the synthesis of the chimaeric BCR-ABL p210 protein with deregulated event in CML. The activated BCR –ABL tyrosine kinase actively induces anti-apoptotic and mitogenic stimuli and further reduces cytokine dependency through the GRB2-Ras mitogen activated protein kinase and JAK/STAT pathways. The final results showed that, hypermethylation of the SOCSI1 gene with the expression down regulation did demonstrate that the loss of the negative regulation of cytokine signaling by epigenetic silencing of the SOCSI proteins played a role in the CML pathogenesis. In the event, SOCSI expression provided a novel and a very useful marker for clinical follow-up of CML and thus provided a new thepeutic strategy for design of the effective target therapies in CML. Discussion Hairy cell leukemia is usually uncommon hematological malignancy which is characterized by accumulations of abnormal B lymphocytes. In most cases it is classified as a group of a sub-type of a chronic lymphoid leukemia. It constitutes of around 2% of all leukemia’s with fewer than 2000 cases that are diagnosed in Western Europe and North America. The infection of human B cell line JOK-1 with herpes simplex virus type 1 persists over a period of 12 months in the body cells. However, limited cytopathic effects are seen to be in the form of viral infection which does not lead to an extinction of the culture. In the present study, HSN replication was checked in JOK-1 cells where a human B cell line was derived from the patient with hairly cell leukemia. In contrast, majority of the systems that were described in JOK-1 cell cultures tdid survived an initial infection despite the aspects of producing high tires of HSV and thus became persistently infected. In the experiment it was found out that, in order to analyze the mechanism that led to the establishment of persistent replication which provides evidence that in strict cell property which restricts the production of infectious particles. In this aspect, JOK-1 cells were thus cultured in RPM1 1640 medium which are supplemented with L-glutamine (2mm), sodium pyruvate (1mm), and heat-inactivated foetal calf serum. In order to understand the experiment, subcultures cells were thus diluted in culture medium for about 3 to 4 day intervals. Viability of the cells was determined by trypan blue dye exclusion. The infection cells were washed twice with hanks balanced salt solution and incubated with HV at m.o.i.’s ranging ferom 0.05 to 100 p.f.u./cell. The virus was then allowed to adsorb for a period of 30 min. after two major washeds with Hanks balanced salt solution to remove residual non-adsorbed virus particles, the cells were cultured at a density of 2x10 cells/ml. the samples for the virus titration were then removed at an indicated times and then stored at -70centrigade until caused for an assay virus yield. In the event virus titres were then determined by a plaque assay through the use of RITA cells of African green monkey kidney origin (RC-37 RITA Italdiagnostics). Read More