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CHARACTERIZATION OF A HUMAN INHIBITORY ANTIBODY FRAGMENT AGAINST TISSUE FACTORVermeulen, Jan-G 23 August 2012 (has links)
Tissue factor is a transmembrane glycoprotein that functions as the primary initiator
of coagulation in response to mechanical or chemical damage. Due to its key
position within the coagulation cascade it also plays an important role in the
pathology of thrombosis and thrombotic complications associated with
cardiovascular disease as well as in non-thrombotic disorders and diseases such as
obesity, diabetes, cancer and HIV-AIDS. Recognising the potential in tissue factor
inhibition as a novel approach to antithrombotic therapy, our laboratory utilized
phage display technology in a previous study, in order to identify a 26 kDa single
chain antibody fragment which functionally inhibits human tissue factor.
In the current study, the tissue factor inhibitory single chain variable fragment (TFIscFv)
was expressed by means of the pIT2 plasmid vector by Escherichia coli
HB2151. This expression system was utilised in an up-scale setting in an attempt to
improve the TFI-scFv yield. Although functional TFI-scFv was successfully purified
from the culture media by means of Protein A affinity chromatography, the process
was hampered by large sample volumes, low levels of expression as well as the high
cost involved in Protein A purification.
Due to an initial focus on improving TFI-scFv yield through the processing of larger
sample volumes rather than the improvement of the expression system, immobilised
nickel affinity chromatography was investigated as a more cost effective alternative
to Protein A affinity chromatography. It was found that the original expression system
was incompatible with immobilised nickel affinity chromatography as the protein was
expressed into the culture media. The culture media contained nickel chelating
elements that stripped the nickel from the column and consequently prevented TFIscFv
purification.
Subsequently the TFI-scFv gene was isolated, cloned into an over-expression
system and modified to redirect the expression to the bacterial cytoplasm. Although
TFI-scFv was successfully redirected to the bacterial cytoplasm and purified by
means of nickel affinity chromatography, it was found that expression was hampered
due to the presence of rare codons. The detrimental effect of rare codons on TFI scFv yield was addressed through the modification of the expression host by the coexpression
of the pRARE plasmid as well as by the rare codon optimization of the
TFI-scFv gene sequence for expression in E. coli. Although the co-expression of the
pRARE plasmid only slightly improved TFI-scFv yield, a sufficient amount of TFIscFv
was generated for functional testing. The modified TFI-scFv displayed a similar
inhibition effect with reference to the original construct. The rare codon optimisation
resulted in a substantial increase in TFI-scFv yield but consequently resulted in the
loss of solubility and the production of inclusion bodies. Although the loss of TFIscFv
solubility is unwanted, the high level of expression achieved provides an ideal
platform for the further development and characterization TFI-scFv in animal
thrombosis models.
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MUTATIONAL ANALYSIS OF THE JANUS KINASE 2 GENE IN PATIENTS WITH POLYCYTHAEMIA VERA, ESSENTIAL THROMBOCYTHAEMIA AND PRIMARY MYELOFIBROSISGoodyear, Quintin Clive 23 August 2012 (has links)
All the cells of blood arise from two lineages, the myeloid and the lymphoid lineage.
The various cells of blood perform vital functions in the body. These cell counts are
closely regulated by regulatory pathways. Mutations within genes that encode for the
proteins involved in these pathways can occur. These mutations can cause
uncontrolled proliferation of the cells. Myeloproliferative neoplasms are malignancies
where there is an uncontrolled increase in the formation of the myeloid cells. The four
classical neoplasms are polycythaemia vera, essential thrombocythaemia, primary
myelofibrosis and chronic myeloid leukaemia.
A mutation (V617F) in the tyrosine kinase, Janus kinase 2, has been found to be the
cause of at least three of the classical MPNs. The mutation lies in the domain of the
protein that controls its tyrosine kinase activity. The tyrosine kinase thus is
constitutively active and causes proliferation of the myeloid cells. The V617F mutation
lies in exon 14 and more recently several mutations have been described in the
neighbouring exons encoding for the regulatory domain of the gene. Very few studies
have been done on the other exons of the JAK 2 gene.
In the study it was attempted to screen 15 MPN patients for mutations in the JAK 2
gene. Two different cell populations (lymphocytes and granulocytes) of each patient
were screened. It was found that the cell purity was not sufficient in the study and
better separation techniques are required for future studies. Only the granulocytes were
used for the remainder of the study. High resolution melting curve analysis was used to
screen the patients for mutations, however the data did not correlate with the
sequencing results and it was decided to proceed with sequencing of all the samples. Seven of the 25 exons of the JAK 2 gene were successfully sequenced. The remaining
exons could not be screened due to time constraints and complications such as multiple
amplicon formation. Two previously reported single nucleotide polymorphisms were
found in exons 11 and 15 in two patients. The clinical significance thereof is uncertain
however, the patient whom had the SNP in exon 15 was negative for the V617F
mutation and had a MPN. In exon 14 the V617F mutation was identified and the
prevalence thereof correlates to that reported in literature. A novel SNP was found in
exon 13 of a PV patient negative for the V617F mutation and the significance thereof is
also uncertain. Additionally a novel inverse duplication consisting of at least of exon 13
was also identified. No mutations were identified in exons 10, 12, 16 and 17 of the JAK
2 gene.
This was, to our knowledge, the first report in South Africa that found the prevalence of
the V617F mutation in MPN patients correlating to the prevalence reported in literature.
A novel SNP was identified in exon 13 and further studies are needed on the possible
effect thereof. The previously reported SNPs found in exons 11 and 15 might be the
cause of the formation of a MPN, however further research is needed. A novel
duplication variant was also identified and this might be a possible splice variant. The
study showed that the region between exons 10 and 15 in the JAK 2 gene is a
mutational hotspot and further studies are needed to elucidate the effect thereof.
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THE EFFECT OF INFLAMMATORY CYTOKINES AND COAGULATION FACTORS ON VON WILLEBRAND FACTOR SYNTHESIS AND CLEAVAGEAllers, Werner Ernst 27 June 2014 (has links)
When injured, endothelial cells secrete inflammatory cytokines such as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumour necrosis factor-α (TNF-α). These inflammatory cytokines stimulate the endothelial release of ultra large Von Willebrand factor (ULVWF) multimers that bind platelets to form thrombi in small vessels. The interaction between thrombosis and inflammation is not fully elucidated. A disintegrin-like metalloprotease with thrombospondin type I repeats-13 (ADAMTS-13) is freshly released from Weibel-Palade bodies of endothelial cells into the plasma and it cleaves the ultra large and hyperactive VWF multimers into smaller and less active forms. These VWF multimers mediate the initial adhesion of activated platelets, the first step in both inflammation and thrombosis. This process may be affected by the amount of ULVWF released and the processing capacity of ADAMTS-13. Little is known about the initial onset of HIV-associated TTP, a fatal thrombotic disease that is characterised by the absence of ADAMTS-13. The mechanisms underlying the initial onset and/or burst of TTP episodes still remain poorly understood. Interrelated components, such as coagulation factors and inflammatory cytokines, all contribute to the development of TTP, since increased levels of cytokines interleukin-6 and tumour necrosis factor and the coagulation factor, tissue factor is measured in these patients. Therefore, we hypothesised that certain inflammatory cytokines and coagulation factors released during inflammation may stimulate the release of VWF simultaneously while inhibiting the synthesis of ADAMTS-13, which results in an acquired deficiency of plasma ADAMTS-13 and ultimately in a TTP episode. Our aim was to examine the effects of inflammatory cytokines and coagulation factors such as tissue factor and thrombin as well as combinations thereof on the release and cleavage of ULVWF by cultured human umbilical vein endothelial cells (HUVECs). HUVECs were treated with cytokines, IL-6, IL-8, and TNF-α and coagulation factors, tissue factor and thrombin, and their combinations, for 24 hours under static conditions. The cells were then exposed to a shear stress of 2.5 dyne/cm2 to expose the VWF cleaving sites. The VWF, VWF propeptide and ADAMTS-13 secretion were measured by an ELISA technique. ADAMTS-13 content was measured using Western blot technology with densitometry. All treatments and their combinations, excluding IL-6, significantly stimulated the release of VWF and VWF propeptide from HUVECs. The VWF propeptide levels were constantly higher than the major VWF protein levels suggesting that the measurement of the VWF propeptide levels may be a better representation of the amount of VWF secreted from endothelial cells. Tissue factor alone and in combination with inflammatory cytokines increase the amount of VWF release from endothelial cells substantially. This correlates with the situation in thrombotic patients with inflammation where extremely high VWF levels are measured. Densitometric analysis of the Western blots indicated that lower levels of ADAMTS-13 secretion were found with all treatments. These results suggest that inflammatory cytokines such as IL-8 and TNF-α, coagulation factors such as thrombin and tissue factor, as well as combinations thereof, stimulate the release of ULVWF and inhibit the release of ADAMTS-13 in HUVECs, resulting in the accumulation of hyperreactive ULVWF in plasma and on the surface of endothelial cells to induce platelet aggregation and adhesion on the vascular endothelium. Our study may offer a logical explanation of how systemic inflammation and thrombosis might trigger the onset and/or burst of TTP in patients with HIV-associated TTP.
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Collaboration of Ezh2 and Runx1 inactivating mutations in malignant haematopoiesisBooth, Christopher January 2017 (has links)
Extensive efforts have shed light on the identity and biology of cancer stem cells, required and sufficient for the propagation of hematological malignancies and solid tumours. Much less is understood about the closely related issue as to the identity and properties of the normal stem and progenitor cells targeted by oncogenic lesions, and how the nature of the targeted cell might impact on the biology and clinical picture of the resulting cancer. To address this, we developed a mouse model allowing targeted inactivation of Ezh2 and Runx1 to different haematopoietic compartments. Inactivating mutations of EZH2 and RUNX1 frequently co-occur in haematological malignancies with markedly different phenotypes including myelodysplastic syndrome (MDS) and early thymic progenitor (ETP) leukaemia. Inactivation of Ezh2 and Runx1 in adult haematopoietic stem cells (HSCs) resulted in perturbed haematopoiesis leading to development of an MDS-like disease. Unexpectedly, this MDS phenotype could be fully reproduced when Ezh2 and Runx1 inactivation was targeted to multipotent progenitors (MPPs) using Flt3-Cre. Furthermore, the disease was transplantable by MPPs, but not more committed progenitor populations, demonstrating that MDS tumour propagating potential is not exclusive to intrinsically self-renewing HSCs. Targeting Ezh2 and Runx1 inactivation to early lympho-myeloid progenitors did not result in an MDS phenotype. These mice showed a marked expansion of ETPs within the thymus, combined with a block in thymocyte differentiation. These expanded ETPs displayed transcriptional features characteristic of ETP leukaemia, a treatment-resistant acute leukaemia subtype hypothesised to originate from ETPs. Combination of inactivation of Ezh2 and Runx1 in ETPs with the constitutively activating Flt3-ITD signalling mutation resulted in an aggressive lympho-myeloid acute leukaemia, which could be propagated by the expanded ETP population. These findings demonstrate the potential of lympho-myeloid progenitors such as ETPs to become leukaemia stem cells which propagate a disease retaining lympho-myeloid features. We used this novel ETP leukaemia model to explore therapeutic targeting of Ezh2-inactivated ETP leukaemias using inhibitors of the bromodomain and extra terminal (BET) proteins. Aberrant transcription resulting from epigenetic changes induced by Ezh2 loss could be reversed by BET inhibitors, and these compounds showed therapeutic efficacy against both mouse and human ETP leukaemias in vitro and in vivo.
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Possible effects of HIV infection on overall survival of patients diagnosed with acute myeloid leukaemiaDyer, Greg Bryan January 2019 (has links)
Background
The effects of Human Immunodeficiency Virus (HIV) on the Overall Survival (OS) in patients diagnosed with Acute Myeloid Leukaemia (AML) are not well documented. All studies to date have been with small sample sizes and based on collections of case studies from different facilities with different treatment protocols, as a result it has been difficult to draw definitive conclusions.
Method
This retrospective record review of a cohort of AML patients (n=304) treated at a single site between 2000 and 2017 was conducted. Age (16-93 years), gender (Male: n=157 ; Female: n=138), ECOG PS (Eastern Co-Operative Oncology Group Performance Status), FAB (French-American-British) staging, blast count, CD4 count, HIV viral load, financial status, response to treatment as measured on bone marrow biopsy and OS were measured. The OS was compared for HIV status. Further comparisons were conducted in a sub-group where age, ECOG PS and FAB staging were controlled.
Results
210 (69.07%) were HIV negative, 31 (10.1%) were HIV positive, 63 (20.7%) had an unknown HIV status. A statistically significant difference was found between HIV negative and HIV positive groups’ OS (563 vs. 121 days ; P<0.01)(HR 2.02 ; 95% CI 1.36 - 2.99) in the main analysis. This difference was also noted when patients who were not treated for AML were excluded from the comparison (OS, 740 vs 194 days, P<0.01)(HR, 2.10 ; 95% CI 1.26-3.50). In the main analysis mean ECOG PS was better in the HIV negative population compared to the positive population (1.80 vs. 2.06). In the controlled group sub-study, where Age, ECOG PS and FAB staging were controlled, the OS between HIV positive and HIV negative patients was not statistically significant (141 days vs. 121 days) (P=0.17; 95% CI). CD4 counts ranged from 29 – 1416, with a mean CD4 of 432 on presentation. No statistically significant difference could be found between CD4 and OS (HR, 1.0 ; 95% CI 0.99-1.00), possibly due to very few patients presenting with a low CD4 count. HIV Viral Loads ranged from <100 – 106640. Similarly, no statistically significant difference was found between HIV Viral Load and OS (HR 0.99 ; 95% CI 0.99-1.00).
Conclusion
HIV has a negative impact upon the OS of patients with AML. HIV appears to impact on OS as a chronic comorbidity by affecting ECOG PS on presentation, reducing their chance of being treated as well as possibly reducing a patients’ functional reserve. This impact does not appear to be as a result of a direct interaction between the HIV and AML disease processes, as when controlling for other factors that may influence OS there is no statistically significant difference in OS between HIV positive and negative patients. / Dissertation (MSc (Medical Oncology))--University of Pretoria, 2019. / This thesis/dissertation is under embargo until September 2023. / Medical Oncology / MSc (Medical Oncology) / Restricted
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Red cell membrane abnormalities in hereditary spherocytosis patients of KwaZulu-Natal.Bridgemohan, Roshini. January 2006 (has links)
Hereditary Spherocytosis (HS) is a common inherited haemolytic anaemia with variable clinical expression. Fifty subjects with HS from KwaZulu-Natal were studied with the aim of providing further information on the protein abnormalities of the red blood cell (RBC) membrane and their relationship with clinical presentations. Haematological and biochemical tests were performed by routine procedures. Mean Corpuscular Haemoglobin Concentration ( MCHC) in the HS group was 35.1g /dl. This was significantly higher than in normal control subjects (33.6g /dl) (p value < 0.001); indicating its usefulness for the screening of HS. Mean Red Cell Distribution Width (RDW) was also significantly higher in subjects with HS (p<0.001); thus providing an additional screening tool. Erythrocyte membrane proteins from 21 subjects were analysed by SDS - polyacrylamide gel electrophoresis (SDS-PAGE) using the Laemmli and Fairbanks methods. The most common abnormality was a deficiency of band 3 (10 subjects), followed by a combined spectrin and ankyrin deficiency in five subjects. One subject had increased band 6 and in five cases no abnormality was detected. A decreased ratio of protein 4.1a / 4.1b on the Laemmli SDS PAGE correlated with an increased reticulocyte count. The degree of haemolysis and clinical findings did not correlate with the type of red cell membrane protein defect. In this study red cell membrane analysis did not contribute further to the initial laboratory diagnosis. In addition it did not influence clinical management. The presence of red cell membrane abnormalities, either single or multiple, did not correlate with disease severity. Red cell membrane analysis, however, will play an important role for future management such as gene therapy. Red cell membrane analysis is also useful as a research tool to determine the underlying molecular defect and to assess racial or ethnic differences. It is also of value as a differential diagnostic tool in cases where the clinical and laboratory findings are not conclusive for HS. / Thesis (M.Med.Sci)-University of KwaZulu-Natal, Durban, 2006.
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The application of flow cytometeric and fluorescent microscopic techniques to the study of multiple myelomaAlSaeed, Abbas Habeeb January 1995 (has links)
No description available.
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The gut mucosal barrier following bone marrow transplantationFegan, C. D. January 1992 (has links)
No description available.
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The evaluation of a new haematological cell counter, the CELL-DYN 3500, on canine leukocyte differential countsPrinsloo, T. 23 March 2006 (has links)
Please read the abstract in the section 00 front of this document / Dissertation (M Med Vet (Clinical Laboratory Diagnostics))--University of Pretoria, 2001. / Companion Animal Clinical Studies / unrestricted
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William Bosworth Castle: Pioneer of Haematological Clinical InvestigationElrod, Jeffrey M., Karnad, Anand B. 01 May 2003 (has links)
No description available.
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