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Immune Monitoring and Adoptive Immunotherapy FollowingAllogeneic Hematopoietic Stem Cell Transplantation
Hejretová, Lenka ; Lysák, Daniel (advisor) ; Frič, Jan (referee) ; Raida, Luděk (referee)
Immune monitoring and adoptive immunotherapy following allogeneic hematopoietic stem cell transplantation - ABSTRACT Allogeneic hematopoietic stem cell transplantation (aloHSCT) is a specialized therapeutic method that is potentially curative for a number of hematological and non-hematological diseases. Despite continuous advances in transplant medicine, the prognosis of patients is still limited by a number of post-transplant complications, dominated by relapse and graft-versus- host disease (GvHD). These complications affect the survival of patients, but also reduce their quality of life. So, we are looking for new treatment modalities that would improve their prognosis. One of the promising procedures in the treatment of GVHD is the administration of cells with immunomodulatory potential (immunotherapy). In a number of European centers, including ours, mesenchymal stem cells (MSCs) are administered to patients after alloHSCT in the steroid-refractory GvHD (SR-GvHD) indication. Invariant NKT lymphocytes are a new cell population with a promising potential to suppress GvHD and at the same time they potentiate the graft-versus-leukemia (GvL) effect. The introduction of these cell populations into clinical practice must be preceded by a number of preclinical studies and continuous optimization of...
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Human Cytomegalovirus Infection in Patients after Allogeneic Hematopoietic Stem Cell Transplantation
Vejražková, Eva ; Žák, Pavel (advisor) ; Faber, Edgar (referee) ; Lysák, Daniel (referee)
Eva Vejražková: Human Cytomegalovirus Infection in Patients after Allogeneic Hematopoietic Stem Cell Transplantation - Summary Introduction: Specific anti-cytomegalovirus (CMV) treatment reduces CMV-related morbidity and mortality in patients after allogeneic hematopoietic stem cell transplantation (HSCT) but carries a risk of viral resistance. The viral resistance after HSCT treatment is still a relatively unexplored phenomenon. The aim of the study was to determine the incidence of CMV infection in patients within one year after HSCT in our centre. The goal was also to analyse CMV infection data, incidence of graft-versus-host disease and other clinical endpoints by comparing two patients' cohorts that were administered two different Thymoglobuline Genzyme (TG) doses as part of the HSCT conditioning regimen. Incidence of treatment failure cases and viral CMV drug resistance were also to be determined. Two methods of sequence analysis (Sanger and Next-Generation Sequencing, NGS) were compared in cases of proven viral resistance. Patients cohort and methods: The study included 101 adult patients after allogeneic HSCT for haematological diseases treated between July 2012 and December 2014 at the Hradec Kralove University Hospital, Czech Republic. CMV DNAemia was determined by quantitative real-time PCR, the...
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The use of novel technologies in the identification of unique molecular markers for minimal residual disease assessment in acute leukemia patients
Jančušková, Tereza ; Peková, Soňa (advisor) ; Jarošová, Marie (referee) ; Lysák, Daniel (referee)
Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include immunoglobulin heavy chain or T-cell receptor gene rearrangements, recurrent cytogenetic abnormalities and mutations in important hematological genes. Whereas in the majority of adult acute lymphoblastic leukemia patients a suitable MRD target can be identified, in adult acute myeloid leukemia patients well-characterized targets are found in only half of cases. The identification of new specific molecular markers of leukemic blasts for MRD assessment, particularly in AML patients, is therefore highly desirable. Our aim was to develop a flexible strategy for mapping of cytogenetically identified unique clone-specific abnormalities down to the single nucleotide level and, based on the sequence, design a specific real-time PCR assay for MRD assessment in AL patients without any previously described MRD marker. Using a combination of cytogenetic (chromosome banding, chromosome microdissection), molecular cytogenetic (mFISH, mBAND) and molecular biological (next- generation sequencing, long-range...
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New biological markers in lymphoproliferative diseases
Špaček, Martin ; Mandys, Václav (advisor) ; Smolej, Lukáš (referee) ; Lysák, Daniel (referee)
New biological markers in lymphoproliferative diseases Summary The prognosis of patients with lymphoid neoplasms is extremely variable. Reliable prognostic markers could allow the identification of patient subsets that may benefit from alternate approaches. Epstein-Barr virus (EBV) is associated with approximately one third of Hodgkin lymphoma (HL) cases. EBV-DNA is often present in the plasma and whole blood of EBV-associated HL patients. However, the significance of EBV-DNA monitoring is debated. In a cohort of 165 adult HL patients EBV-DNA viral load was prospectively monitored. Our results suggest that assessment of plasma EBV-DNA viral load might be of value for estimation of prognosis and follow-up of patients with EBV-positive HL. The role of the TP53 gene's R72P polymorphism in NHL has been analyzed in several studies but it has not been studied in HL. We have evaluated the role of R72P in 340 NHL and 298 HL patients. Our results support the evidence that R72P is not a prognostic factor in Caucasian NHL patients, and they indicate its irrelevance for HL development or prognosis. In a study of 73 patients with chronic lymphocytic leukemia we have assessed IgVH mutational status, presence of mutation in TP53 gene and expression of CD38 and ZAP-70. Overall concordance between ZAP-70 expression and IgVH...
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Mesenchymal stem cells and their regenerative and immunomodulatory potential
Brychtová, Michaela ; Lysák, Daniel (advisor) ; Horák, Vratislav (referee) ; Raška, Milan (referee)
Mesenchymal stem cells and their regenerative and immunomodulatory potential Abstract Mesenchymal stem cells (MSCs) possess multidirectional regenerative ability, which, together with their immunomodulatory potential, makes them promising cell type for therapy of wide variety of diseases. Despite ongoing research, which proved MSCs application to be safe, reported effect of MSCs administration on patients is not convincingly beneficial yet. In our work we focused on elucidation of MSCs role in regeneration of vital organs, heart and liver, where a large damage is life threatening for patients and any improvement in therapy would save many lives. Similar situation is in Graft versus host disease (GVHD), where MSCs immunomodulatory properties could be beneficial. Role of MSCs in heart regeneration was examined in vitro. Primary adult swine cardiomyocytes (CMCs) were co-cultured with or without swine MSCs for 3 days and morphological and functional parameters (contractions, current, respiration) of CMCs were measured. MSCs showed supportive effect on CMCs survival, especially at day 3 of the experiment, where in co-culture was significantly higher number of viable CMCs with physiological morphology and maintained function. Effect of MSCs on liver regeneration was observed in swine model of chronic liver...
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Specific antiviral immunity in immunocompromised patients, not only after bone marrow transplantation
Pelák, Ondřej ; Kalina, Tomáš (advisor) ; Lysák, Daniel (referee) ; Němečková, Šárka (referee)
-3- SUMMARY Viral reactivations after hematopoietic cell transplantation contribute to significant morbidity and mortality. Timely immune reconstitution of functional T cell immunity is crucial in controlling these viral reactivations. In this thesis we were able to identify several functional T cell populations, which are responsible for fast resolution of viral reactivation. Appearance of some of these populations may be even used for prediction of the occurrence of viral reactivation. On the other hand, the administration of corticoids due to the treatment of graft versus host disease contributes as significant negative predictor to viral reactivation incidence. We are also offering an option of adoptively transferred virus specific T cells for patients suffering from prolonged virus complications, through identification of suitable donors by two different methods. Viral reactivations cause complication also in patients who underwent the solid organ transplantation. In this thesis we have found the connection between allo and virus specific T cells. We have successfully identified several cross-reactive T cell clones which have responded to both allo and viral stimulation. Further it seems that these clones may play an important role in rejection of transplanted kidney if there is also present an ongoing...
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Human Cytomegalovirus Infection in Patients after Allogeneic Hematopoietic Stem Cell Transplantation
Vejražková, Eva ; Žák, Pavel (advisor) ; Faber, Edgar (referee) ; Lysák, Daniel (referee)
Eva Vejražková: Human Cytomegalovirus Infection in Patients after Allogeneic Hematopoietic Stem Cell Transplantation - Summary Introduction: Specific anti-cytomegalovirus (CMV) treatment reduces CMV-related morbidity and mortality in patients after allogeneic hematopoietic stem cell transplantation (HSCT) but carries a risk of viral resistance. The viral resistance after HSCT treatment is still a relatively unexplored phenomenon. The aim of the study was to determine the incidence of CMV infection in patients within one year after HSCT in our centre. The goal was also to analyse CMV infection data, incidence of graft-versus-host disease and other clinical endpoints by comparing two patients' cohorts that were administered two different Thymoglobuline Genzyme (TG) doses as part of the HSCT conditioning regimen. Incidence of treatment failure cases and viral CMV drug resistance were also to be determined. Two methods of sequence analysis (Sanger and Next-Generation Sequencing, NGS) were compared in cases of proven viral resistance. Patients cohort and methods: The study included 101 adult patients after allogeneic HSCT for haematological diseases treated between July 2012 and December 2014 at the Hradec Kralove University Hospital, Czech Republic. CMV DNAemia was determined by quantitative real-time PCR, the...
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Mesenchymal stem cells and their regenerative and immunomodulatory potential
Brychtová, Michaela ; Lysák, Daniel (advisor) ; Horák, Vratislav (referee) ; Raška, Milan (referee)
Mesenchymal stem cells and their regenerative and immunomodulatory potential Abstract Mesenchymal stem cells (MSCs) possess multidirectional regenerative ability, which, together with their immunomodulatory potential, makes them promising cell type for therapy of wide variety of diseases. Despite ongoing research, which proved MSCs application to be safe, reported effect of MSCs administration on patients is not convincingly beneficial yet. In our work we focused on elucidation of MSCs role in regeneration of vital organs, heart and liver, where a large damage is life threatening for patients and any improvement in therapy would save many lives. Similar situation is in Graft versus host disease (GVHD), where MSCs immunomodulatory properties could be beneficial. Role of MSCs in heart regeneration was examined in vitro. Primary adult swine cardiomyocytes (CMCs) were co-cultured with or without swine MSCs for 3 days and morphological and functional parameters (contractions, current, respiration) of CMCs were measured. MSCs showed supportive effect on CMCs survival, especially at day 3 of the experiment, where in co-culture was significantly higher number of viable CMCs with physiological morphology and maintained function. Effect of MSCs on liver regeneration was observed in swine model of chronic liver...
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The use of novel technologies in the identification of unique molecular markers for minimal residual disease assessment in acute leukemia patients
Jančušková, Tereza ; Peková, Soňa (advisor) ; Jarošová, Marie (referee) ; Lysák, Daniel (referee)
Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include immunoglobulin heavy chain or T-cell receptor gene rearrangements, recurrent cytogenetic abnormalities and mutations in important hematological genes. Whereas in the majority of adult acute lymphoblastic leukemia patients a suitable MRD target can be identified, in adult acute myeloid leukemia patients well-characterized targets are found in only half of cases. The identification of new specific molecular markers of leukemic blasts for MRD assessment, particularly in AML patients, is therefore highly desirable. Our aim was to develop a flexible strategy for mapping of cytogenetically identified unique clone-specific abnormalities down to the single nucleotide level and, based on the sequence, design a specific real-time PCR assay for MRD assessment in AL patients without any previously described MRD marker. Using a combination of cytogenetic (chromosome banding, chromosome microdissection), molecular cytogenetic (mFISH, mBAND) and molecular biological (next- generation sequencing, long-range...
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