Haematology and Cell Biology

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Open Access
A yeast-based assay for detection of mutations in the human p16 gene
(University of the Free State, 1999-05) Botha, Chantal; Pretorius, G. H. J.
Cyclin-dependent kinases (CDKs) are crucial regulators of the cell cyele. CDKs themselves are subject to control by both cyelins and CDK inhibitors. Among the inhibitors, p 16 is very prominent, since it has been found to be mutated or lost in a variety óf tumours, We are interested in mutations involved in the progression of leukemia from the chronic to the acute phase. The p 16 gene has been implicated in this progression, therefore we needed an assay for p 16 status that could be applied to screen patients in chronic phase regularly. Traditional mutation screening makes use of physical methods such as Single Stranded Conformational Polymorphism (SSCP) analysis. These methods are generally labour intensive and are not always informative. If tests for the actual function for the gene products could be devised, it could be used to screen tumour samples for the status of these genes. We have decided to develop a yeast-based test that would directly assay for activity rather than just nucleotide changes. The assay is based on the yeast two-hybrid system, where protein-protein contact is reflected in colony colour. We have designed a primer set to amplify the p16 reading frame by RT-PCR from small amounts of leukocyte mRNA. This cDNA is then cotransformed with a gapped plasmid containing terminal p 16 overlaps, allowing homologous recombination to splice the reading frame into the plasmid. The host strain also contains a CDK4- expressing plasmid and if the amplified p16 can still bind to CDK4, the colonies would turn blue. We have successfully constructed and tested the system and found it to be very sensitive, being able to assay p 16 from as little as 300 microliters of whole blood.
Open Access
Co-expression and functional assay of human Rb and E2F1 proteins in yeast
(University of the Free State, 1999-09) Wollenschlaeger, Alex; Pretorius, G. H. J
The mammalian cell cycle is composed of a myriad interactions occurring in a defined sequence dictated by the flow of entropy. The decision to study the cell cycle requires entry into a world where events take place, not because they want to, but because they have to. Cells do not 'decide' to perform certain actions, but are driven by the laws of nature, the same laws that are responsible for the existence of the universe. Study of this maelstrom of reactions is truly analogous to opening Pandora's proverbial box. A peek inside and all the inner workings of the cell start to spill out. Unfortunately, this is where things start to get complicated. Serendipitously, simpler alternatives are available. The yeast cell cycle is remarkably similar to that of higher animals. The Rb and E2F 1 proteins are integral components of the mammalian cell cycle, and as such, they are found in aberrant forms in numerous malignancies. This necessitates an adequate means for the determination of the cellular status of these proteins, as prognosis and diagnosis of several neoplastic disorders are dependentthereon. This study aimed to develop a yeast-based strategy for functional analysis of the human tumour suppressor protein Rb. This goal was impeded by one factor; the yeast cell cycle is too similar to that of humans. Several yeast strains, derived from W303-lA, were constructed that each contain a reporter gene -the lacZ gene of E. coli- regulated by E2F recognition elements introduced within the upstream CyC1 promoter. The theory was that in the absence of Rb, ectopically expressed human E2F 1 protein would be able to bind the RE and activate transcription of the reporter gene, ultimately resulting in a readily observable product. In the presence of functional Rb, E2Fl would be bound by the tumour suppressor protein, and thus be incapable of activating the reporter gene. This would provide an assay of Rb status, based not on tedious sequencing analysis of the genetic material, but on the actual functional activity of the protein. When dealing with Mother Nature, though, we are oftentimes reminded that she has thought of everything. S. cerevisiae contains an E2F-like activity, capable of binding the exact RE introduced into the reporter gene promoter. This was confirmed by experiments in this study, where the reporter gene was activated in the absence of ectopically expressed E2F1 protein. The endogenous yeast E2F-like protein is thus able to activate transcription of the reporter gene, negating the effect that would be observed by ectopic expression of human E2F 1, and thus, all Rb-expression was performed in the absence of eo-expressed human E2F 1. Since it is impossible to distinguish between the yeast and human E2F activities, it is impossible to create a functional assay for Rb activity in the W303-1Aderived chimeras constructed. As mentioned previously, it could be possible to overcome this problem by knocking out the yeast-borne E2F activity, but this approach is restricted by two barriers. Firstly, the yeast equivalent of the E2F-family is yet to be cloned. This problem can be approached with a transposon-based strategy. The endogenous E2F activity is capable of activating transcription of the lacZ reporter gene, and in so doing, provides a convenient assay for YE2F integrity. Through the use of a plasmid containing an inducible transposase it should be possible to disrupt the YE2F -encoding gene through integration of a transposon. This would be accompanied by an inability of the yeast to activate the reporter gene. The transposon, containing flanking genomic DNA, could then be retrieved and provide the basis for cloning the gene coding for YE2F. Since the reporter gene is specific for E2F-like binding, retrieval of non-specific factors should be negated. A second problem is that, since the E2F proteins play an important role in the progression of the cell cycle in higher animals, it is possible that the yeast would not survive knocking out its homologue. Relegation of YE2F to the role of bystander could possibly wreak havoc with the delicate mechanism that is the cell cycle. Still, it would be interesting to further pursue this idea. Ectopic expression of human Rb in the various strains used in this study provided some interesting results. Transformation of pAWl, followed by galactose-based induction of Rb expression resulted in observable differences in growth characteristics of certain strains. Those most affected were W-lf and W-1r, which each contain a single repeat of the E2F RE within the upstream promoter of the reporter gene, albeit in forward and reverse orientations, respectively. The effect was particularly evident in W-1r, where expression of Rb resulted in complete cessation of growth, probably due to binding of yeast E2F-like activity. These results could not, however, be reconciled with those obtained from cell cycle analysis with the aid of flow cytometry. These experiments did not show any significant effect of Rb expression on the cell cycle of the examined strains. This is possibly due to an insufficient period of observation, but this could, unfortunately, neither be confirmed nor dismissed. From the results obtained in this study it appears that construction of an apposite reporter system for the functional assay of Rb is perhaps more tricky than would be expected. The interference of endogenous proteins is a cause for concern in a development strategy such as this, and serves as a caveat for future studies in this field.
Open Access
Recombinant production and evaluation of a multifunctional haemostatic fusion protein
(University of the Free State, 1999-10) Van Zyl, Walda Brenda; Pretorius, G. H. J.; Kotzé, H. F.
Platelets and coagulation both play a pivotal role in thrombosis, one of the major life-threatening diseases in our society. We have thus experienced a drastic increase in the development of potent and secure antithrombotic, antiplatelet and fibrinolytic agents during the past decade. Recently, much research has been devoted to the development of chimeric proteins, where haemostasis is simultaneously targeted at different levels. For the purpose of this study, such a chimera, named PLATSAK, was designed. A 29 amino acid antithrombotic and antiplatelet peptide, comprising three inhibitory regions, was linked to staphylokinase via a cleavable factor Xa recognition sequence. The overall activities of PLATSAK should include inhibition of thrombin, prevention of platelet aggregation and activation of fibrinolyis. The gene encoding PLATSAK was expressed in E. coli cells under controlled conditions. PLATSAK was produced as a strongly expressed protein of 18 kDa and was purified form native E. coli proteins using metal affinity chromatography. In vitro analysis of PLATSAK activity revealed strong inhibition of thrombin and potent fibrin degradation. However, no effect on platelet aggregation could be observed. Several attempts at producing more potent antiplatelet variants were unsuccessful. According to its in vitro activity, PLATSAK appeared to be a potent novel haemostatic agent and was prone to be evaluated in an in vivo system. The in vivo activity of PLATSAK was evaluated by assessing its effect on platelet deposition in a baboon model of arterial and venous thrombosis. Dacron vascular graft segments and expansion chambers, inserted as extensions into permanent femoral arteriovenous shunts, were used to simulate arterial and venous thrombosis, respectively. PLATSAK (3.68 mg/kg) was administered as a bolus. Platelet deposition onto the graft surface and in the expansion chamber was imaged in real time with a scintillation camera as the deposition of 111ln-labelled platelets. After two hours, platelet deposition in the graft segments and expansion chambers was inhibited by 50% and 85% respectively when compared to control studies. The aPTT was lengthened to >120 seconds. Interestingly, the level of FOP in plasma did not increase after administration of PLATSAK. These results demonstrate that PLATSAK effectively inhibited platelet deposition in both arterial- and venous-type thrombosis an animal model. This is in contrast to the lack of the antiplatelet activity of PLATSAK in vitro. This illustrates that in vitro platelet aggregation results can not be directly applied to an in vivo situation. In summary, the recombinant production of a multifunctional haemostatic fusion protein, PLATSAK, was successful. In vitro PLATSAK showed significant antithrombin and fibrinolytic activity, but trivial antiplatelet activity. In vivo studies revealed that PLATSAK is a potent antithrombin and also prevented platelet deposition on thrombogenic material. The strong immuun response of PLATSAK however needs to be investigated and a variant with a weak immunogenic nature needs to be produced.
Open Access
Telomeres and telomerase in cancer of esophagus
(University of the Free State, 2001-11) Van den Heever, Wilhelmina Maria Jacoba; Pretorius, G. H. J.
English: Squamous cell carcinoma of the esophagus is a cancer with a high incidence in South Africa. We have investigated the prognostic value of telomerase activity in tumors as well as in nearby normal tissue. Biopsies from 98 patients were analyzed using an adaptation of the TRAP assay. We found all tumor biopsies to have moderate to high telomerase activity, while one third of biopsies from normal mucosa were negative. The telomerase activity level of the tumors had no prognostic value (P=0.95) as determined by the log rank test. A P-value of 0.02 was found when the telomerase-negative and moderately positive normal biopsies were grouped together and compared to those with high activity. Our results show that telomerase activity of normal mucosa in the vicinity of the tumor can identify a population of patients with significantly worse prognosis, even in late stage patients. Telomerase has attracted intense interest as a possible target for cancer therapeutics. Previous attempts at inhibiting telomerase activity utilized antisense oligonucleotides targeted at the template region of the RNA subunit of the enzyme. Although it worked well in cell extracts, getting the oligonucleotides into intact cells are difficult. We attempted a peptide-based approach to overcome the transport problem. A phage-display selection strategy was designed to isolate peptides (7 and 12 amino acids) capable of binding to the RNA template with high affinity and in so doing preventing the enzyme from elongating existing telomeres. Both libraries showed no high affinity binding to the RNA. The most probable explanation is that such short peptides are incapable of binding sequence-specifically to nucleic acids. Many studies have indicated the importance of telomerase activity as an independent prognostic indicator in a variety of cancers, but recent results have shown that telomere length is actually a better indicator, as it shows the final result oftelomerase activity. We thus decided to develop a flow cytrometric method that would allow us to analyze telomere length in small tissue samples. After the initial technique had been established on lymphocytes, it was tested on the SNO cell line before we switched to solid tissue. The background of cellular debris and cell clusters was much worse than for lymphocytes, but differences in signal strength could be seen. From the results obtained it is clear that this method is not optimized yet. The results also indicate that at this stage of development, flow-FISH is inferior to telomerase activity as prognostic indicator. The best way to validate the current method is to analyze the same samples by Southern hybridization and flow-FISH, preferably well defined tumor and normal tissue.
Open Access
Phage display selection of peptide inhibitors of FVIIa and their functional characterisation
(University of the Free State, 2002-06) Roets, Catharina Elizabeth; Meiring, S. M.
English: The importance of FVlla and the FVlla/TF complex for the initiation of not only hemostasis but also thrombosis is now generally accepted. It was shown that the blockade of coagulation at the level of FVlla provided full anti thrombotic protection without abnormal bleeding (Harker et aI, 1996), therefore FVlla is a suitable candidate for the development of novel antithrombotics. We selected inhibitors to FVlla using the technique of phage display. A repeated selection of phages from a cyclic heptapeptide and a linear 12-mere phage display library resulted in the enrichment of phages that bind to human FVlla. We selected twelve colonies (6 from each library) that showed the strongest binding to FVlla. The colonies from the cyclic 7-mere library showed a higher affinity binding for FVlla than the colonies from the linear 12- mere library. TF also prevents the binding of one of the cyclic colonies to FVlla. This colony as well as one colony from the linear library showed lengthening of the prothrombin time (PT) as well as the thrombin time (TT) in a dose-dependent manner. A cyclic heptapeptide was synthesised with the corresponding sequence as the sequence displayed on the cyclic 7-mere colony. The peptide showed lengthening of the PT and TT in a dose-dependent manner with a more pronounced effect on the PT than the TI. We also studied the effect of this peptide on platelet adhesion on human vascular endothelial cell matrix, collagen and TF under both venous and arterial shear stresses. The peptide inhibits platelet adhesion to HMEC-1 under both shear stresses. The effect on arterial shear is however more pronounced. It does not inhibit platelet adhesion to collagen, but has a dose-dependent inhibitory effect on platelet adhesion TF at arterial shear. Kinetic analysis of the peptide showed that this peptide is a competitive inhibitor of FVlla by altering the Km-values but not the Vmax-values. The Lineweaver-Burk plot also indicates a competitive inhibition, because the slope of the graphs increased with increasing inhibitor concentrations. The Ki-value was determined at 0.1232 mM. In summary, this peptide inhibits thrombus formation by preventing FVlla from binding to TF and therefore preventing the activation of FX by the FVlla/TF complex. This study suggests that inhibitors to FVlla provide a novel therapeutic approach to prevent thrombosis.
Open Access
Selection and characterization of a novel factor XI inhibiting peptide by using phage display technology
(University of the Free State, 2002-11) Motloi, Nthabiseng Cecilia; Meiring, S. M.
English: The role of factor XI in hemostasis can be seen as a combination of a procoagulant action (the formation of fibrin) and an antifibrinolytic action (the protection of fibrin). High levels of factor XI lead to a prolonged down regulation of fibrinolysis and therefore a risk of thrombosis (Meijers et ai, 2000). Under disease conditions associated with Disseminated Intravascular Coagulation (DIC), the continuous exposure to excess TF typically exhaust the available tissue factor pathway inhibitor (TFPI), leading to rampant thrombin generation by factor XI feedback and therefore also a risk of thrombosis (0sterud and Bjerlid, 2001). I selected possible .inhibitors of factor XI using phage display technology. I started the phage display selection by biopanning in immuno-tubes and eluted the factor XI binding phages non-specifically from the immuno-tube. I did four selection rounds, to enrich the factor XI binding phages. I found only two strong factor XI binding phage clones from a linear 12-mer phage library. Both phage clones bound dose dependently and with a high affinity to factor XI. Both clones also lengthened the partial thromboplastin time (aPTT) dose dependently. The amino acid sequences of the peptides displayed on these two clones indicate that both peptides contain three amino acid sequences of HMWK and thrombin. One clone also contains a three amino acid sequence of factor XII. None of them contains a three amino acid sequence of factor IX. I synthesize a linear peptide with the corresponding sequence as the peptide displayed on the clone that was prevented from binding to factor XI by both factor IX and thrombin. I characterized the peptide by studying its effect on the aPTT. This peptide lengthens the aPTT dose dependently. The lengthening in aPTT of our peptide however indicates that I have selected an inhibitor of the contact system factors of coagulation. In summary, this study shows that the phage display can be used to select novel factor XI inhibitors from random peptide libraries. With further studies, this peptide may be developed as an antithrombotic.
Open Access
DNA characterization of the FGA locus in the human genome
(University of the Free State, 2002-11) Asfaw, Estifanos Kebede; De Kock, André; Pretorious, G. H. J.
English: The human alpha fibrinogen (FGA) short tandem repeat locus is found in the long arm of chromosome 4. It is located in the third intron of the alpha fibrinogen gene. This complex highly polymorphic tetranucleotide repeat locus together with other STR DNA markers is extensively used in personal identification in medical and forensic sciences. STRs are also used to study genetic variation in distinct ethnic groups and in disease diagnosis. More than 80 alleles have been reported for this locus from various population frequency studies. A few sequence studies have also reported 11 sequence variants to date. The FGA locus was found to have high heterozygosity and power of discrimination. The aim of this study was to characterise the sequence of microvariant and off-ladder complete and microvariant alleles of the FGA locus that were observed during routine paternity analyses. The characterization of the sequence of as many as possible of alleles observed in our study population would also be attempted. A total of 62 DNA specimens were selected and sequence characterized either for one or both alleles. The DNA specimens were 52 from Negroid, 5 mixed ancestry, 4 Caucasian and 1 SAN origin. The PCR reaction was used to amplify the selected alleles. The band of interest was cut from the gel and purified in consecutive PCR and purification steps till separate single bands were obtained. The purified single bands were sequenced using a BigDye terminator ready reaction kit in both forward and reverse reactions separately. These products were precipitated with ethanol acetate and subjected to capillary electrophoresis on an ABIPrism 310 Genetic Analyser using POP6 polymer. The results were analysed using the "Sequence Analysis Software version 2.1". The data obtained were checked, printed and compared with STR analysis results and FGA sequence reports. From the selected 62 specimens a total of 76 complete and microvariant alleles, the size of which ranged between 16.1 (224bp) to 44.2 (337bp) were found. These represent 27 different alleles (13 complete and 14 interalleles). In this study 2 novel (previously undescribed) alleles (40.2 and 41.2) were found. Three sequence variants (26, 28 and 43.2) with two variants each were observed. Two alleles 43.2 and 44.2 that had reported sequence variants were found to have different sequence structures from the published sequences. Forty-nine of the 76 sequenced alleles were within ladder and the remaining 29 were off-ladder. Only 8.41% (4/49) of the within ladder alleles had been wrongly assigned allelic numbers with routine STR analysis. The difference between the routine assignment and the sequencing of these alleles was only 1 or 2 bp. In contrast, all of the 29 off-ladder alleles were wrongly assigned. In this instance the difference was 2 or more base pairs. Although this study was conducted on conveniently selected DNA samples, it had significant results. Three sequence variants, 2 newalleles and, 1 allele, which had been reported, but sequence had not been described was found. Additionally, two other alleles with reported sequences were found, but their sequence structure differed from the published sequences. The samples in this investigation were not representative of the population groups that are found in the Free State province and we suggest further population-based studies of STR loci that are commonly used in paternity and forensic investigation. The information obtained from such studies will disclose the frequency of sequence variant alleles.
Open Access
Investigating the genetic profile of the E-cadherin gene in squamous carcinoma of the esophagus
(University of the Free State, 2003-06) Mabina, Boitumelo Desiree; Pretorius, G. H. J.; Van der Merwe, N. C.
Open Access
Construction of cDNA libraries, and the selection and expression of proteins and peptides involved in haemostasis
(University of the Free State, 2004-06) De Bruin, Karen; Meiring, S. M.; Deckmyn, H.
English: The need to find new manners in which to combat cardiovascular disease and associated thrombotic complications, remains a high priority in industrialised countries. Even in third-world countries the implications and associated risks of these diseases are being felt more and more. The advent of the biotechnology era and employment of recombinant DNA techniques has brought about exponential advances in understanding the complex mechanisms of haemostasis, and is employed to find new ways to combat pathological thrombotic complications. The challenge is to harness the many tools and techniques produced by the ongoing biotechnology explosion, and apply them to elucidate questions still unanswered and explore areas still unknown. In this study it was illustrated that modern molecular biology techniques can be applied in many areas of thrombosis and haemostasis research. The display of cDNA libraries on the surfaces of filamentous bacteriophages was used in the search for novel antithrombotic compounds from a haematophagous insect Hippobosca rufipes. Phages displaying the cDNA libraries were panned against human a-thrombin and selected according to their binding affinity and inhibition ability. To illustrate the use of a Escherichia coli expression system, a domain of a enzyme was cloned, expressed, and the recombinant peptide isolated and refolded. ADAMTS-13 was recently identified as an important role player in the realm of von Willebrand factor activity, including primary haemostasis and pathological disorders. The second carboxy-terminal CUB domain of ADAMTS-13 was amplified from full-length cDNA, cloned into a expression vector system, and expressed as insoluble inclusion bodies in the cytoplasm of E. coli, from where it was isolated and refolded. In this study, molecular techniques were used in different phases of research into the specific activity and interactions of a particular component of the haemostatic system. This illustrated the marriage of biotechnology with fundamental medical research in an era of interdisciplinary sciences.
Open Access
"Killer-cell immunoglobulin-like receptor haplotype diversity in three Free State population groups"
(University of the Free State, 2006-11) Louw, Marius; De Kock, André; Louw, Vernon; Coetzee, Marius
English: In the foregoing project, an investigation was made into the relative KIR gene frequencies of three South African cohorts. Playing an important part in innate immunity, KIR fill a vital gap between viral onsets and cell mediated humeral immunity. Being able to sense when cells are abnormal, NK cells possess the ability to destroy cells which show altered HLA molecules during KIR/HLA interaction. Ethnic cohorts that were investigated included African black, mixed ancestry and the Caucasian populations. From these individuals DNA material was extracted using a “salting out” method before SSP-PCR genotyping. Seventeen primer pairs were used in the identification of individual KIR genes. PCR products were electrophoresed against a molecular weight marker in order to verify the correct fragment size. Products were viewed on a UV light where observations were noted, and indicated as present or absent. Data was recorded onto a spreadsheet indicating the absence or presence of each particular gene. Tabulated results were used in the construction of graphs as well as χ2 calculations. These graphs were used in the critical analysis of linkage disequilibrium as well as comparative analysis between the ethnic cohorts. Findings indicate that all framework genes are present in all cohorts. The Black African and mixed ancestry cohorts have not been genotyped for the KIR genes before. Investigation within non-framework genes revealed the identification of several new haplotypes, with the majority observed within the mixed ancestry cohort. Positive linkage disequilibrium was detected between 2DL2-2DS2 and 2DL5B-2DS5 for both the black African and Caucasian cohorts while 2DL1-2DL2 and 2DL5B- 2DS5 linkages were found in the mixed ancestry population. No negative linkages were observed for any of the three cohorts.
Open Access
The use of topical crushed tranexamic acid tablets to control bleeding after dental surgery and from skin ulcers in haemophilia
(Wiley, 2007) Coetzee, M. J.
Open Access
Prevalence of helicobacter pylori and its relation to cytotoxin-associated gene a status in HIV positive and negative haematology patients
(University of the Free State, 2007-10) Abbott, Tanya Claire; Louw, V. J.; Badenhorst, P. N.; Meiring, S. M.
Abstract not available
Open Access
Chakalaka-induced vasodilatation in patients with chronic myeloid leukemia on tyrosine kinase inhibitors
(Health and Medical Publishing Group, 2009) Coetzee, J.; Louw, Vernon J.; Gartrell, Kevin; Viljoen, Chris D.
Open Access
Sequensing of exon 28 of Von Willebrand factor in five patients with type 2 Von Willebrand disease
(University of the Free State, 2009-05) Mothabeng, Maliengoane Sylvia; Meiring, S. M.; De Kock, A.
English: Von Willebrand disease (VWD) is a common bleeding disorder caused by either quantitative (type1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF). The diagnosis of VWD usually requires a panel of tests. Several analyses therefore are required to diagnose VWD. These tests are also subjected to pitfalls and it is important to take the pitfalls in to consideration when diagnosing VWD. Despite all these tests, the diagnosis and classification of VWD often remains a challenge. Identification of mutations that cause functional defects of VWF (type 2 VWD) is needed to improve the diagnosis of the disease. Mutations that cause functional abnormalities of VWF occur mostly in exon 28 of the VWF gene. Exon 28 primarily encodes the platelet GPIb and collagen binding domains of VWF (A1 domains) and the ADAMTS13 cleavage domain (A2 domains). Recently, studies in industrialised countries have been conducted on finding mutations on exon 28 but none have been done on South African populations. In this study we searched for mutations in exon 28 of the VWF gene in 5 patients with functional defects of VWF in order to set up the method for genetic analysis of VWD. We used two patients with type 2M, two with type 2B and one with type 2A VWD in this study. The whole exon 28 was analysed in four specific fragments, using PCR with primers that mismatch the pseudogene. The mutations were identified by automatic sequencing of the different fragments. The following polymorphisms were detected. A silent SNP 4641T/C in all five patients, the SNP 4141A/G in three patients, a silent SNP 3795G/A in one patient and a novel silent SNP 4923G/A in another patient. It is important to note that we found a novel SNP in an African patient with type 2B VWD, since no polymorphisms reported in exon 28 were from African populations. Several studies have proven the importance of mutational analysis is solving laboratory diagnosis paradox. The mutations found in the patients with type 2 VWD confirm the diagnosis and validates the importance of molecular diagnosis in VWD. With this study, we have successfully implemented a method to detect mutations in exon 28 of the VWF gene.
Open Access
The screening for single nucleotide polymorphisms of CYP3A4 in chronic myelogenous leukemia patients receiving imatinib
(University of the Free State, 2009-05) Lamprecht, G. A.; Viljoen, C. D.
English: Chronic myelogenous leukaemia (CML) is a malignant clonal disorder that results in the uncontrolled production of white blood cells. This disease is a result of a reciprocal translocation of the long arms of chromosome 9 and 22 resulting in a shortened chromosome 22, harbouring the BCR-ABL fusion gene, known as the Philadelphia chromosome. The BCR-ABL oncogene encodes for a constitutively activated tyrosine kinase that interferes with normal cell differentiation and apoptosis. CML can be effectively treated with tyrosine kinase inhibitors, such as imatinib mesylate (GleevecÒ). However, some CML patients experience adverse drugs reactions (ADRs) to imatinib and cannot be treated at the recommended dose. There is a concern that lowering the dose of imatinib to reduce the side effects can result in the development of resistant cancer cells, and thus a cessation in treatment is rather recommended. Imatinib is metabolized by the cytochrome P450 enzyme, CYP3A4. However, if the ADRs were a result of decreased metabolic effect of CYP3A4, it would be possible to reduce the dose of imatinib without effecting efficacy. It is hypothesised that single nucleotide polymorphisms (SNPs) can alter the catalytic activity of the CYP3A4 enzyme. Thus a decrease in metabolic rate can result in ADRs due an increased exposure to the drug. Therefore, the aim of this study was to determine whether SNPs in the CYP3A4 gene are associated with ADRs from imatinib treatment. In this study, the DNA sequence of the CYP3A4 gene from 25 CML patients treated with imatinib were compared to a reference DNA sequence obtained from Genbank. The SNPs identified during this study was statistically analysed, and their association with the presence of ADRs was determined using the online statistics package, SNPator. A total of six SNPs were detected, I193I, T15871G, CYP3A4*1G, C23187T, I369V and G73239A. Of these, I369V and G73239A are novel and not described previously in literature. It was found that I369V resulted in an amino acid change, involving a substitution of isoleucine with valine. The remaining SNPs identified in this study were located in intron regions, with the exception of I193I which is a synonymous SNP. There is little information available on the frequency of SNPs located in introns, since these SNPs are generally regarded to have no impact on the expression or activity of a protein. However, in this study an SNP located in intron 10 was significantly associated with the presence ADRs. Current hypothesises suggest that intron SNPs could affect the expression levels of a protein by influencing the splicing efficiency of mRNA and subsequently translation efficacy. Future research needs to elaborate on the role of CYP3A4*1G on CYP3A4 expression as well as on the prevalence of other alleles identified in this study in South African populations.
Open Access
The application of real-time quantitative PCR in the diagnostics of chronic myeloid leukaemia
(University of the Free State, 2009-05) Van Deventer, Jacob Jacobus; Viljoen, C. D.; Louw, V. J.
English: CML is a cancer of the white blood cells and it effects on average one individual in every 100,000. Since it was first described in 1845 by John Hughes Bennett and the subsequent discovery of the Philadelphia chromosome by Nowell and Hungerford in 1960, this hematopoietic malignancy has received much attention in terms of scientific study. Elucidating the pathogenic pathway has lead to the development of targeted therapy. In 2001 imatinib mesylate was introduced as first line therapy for CML. The success of imatinb was illustrated during the IRIS trial by Real-time quantification of BCR-ABL mRNA. BCR-ABL expression levels are correlated to disease stage and progression. BCR-ABL mRNA quantification is therefore the most accurate and sensitive prognostic marker to monitor CML patients. Hence, Real-time PCR for BCRABL has been introduced in many international laboratories to allow for accurate and reliable monitoring to improve and manage patient treatment. Standardization became problematic due to the ease of method development and robustness for Real-time quantification of BCR-ABL mRNA by different laboratories. As a result a plethora of methods for Real-time quantification of BCR-ABL mRNA have been published. This is especially problematic for laboratories with limited means undertaking to develop and implement such a method. Since there are no standardized guidelines, in-house development is required. Furthermore, availability of commercial copy number standards for control and target genes makes it difficult to implement any one method from the literature especially since there is criticism for the genes where standards are commercially available. From a thorough analysis of the literature, problem areas considering RNA extraction, the choice of priming for cDNA synthesis, primers and probes for Real-time PCR as well as a specific control gene together with copy number standards and reference material were clearly defined. Based on this information, best laboratory practice regarding common methodology from literature was established. Only recently through an initiative known as Europe Against Cancer (EAC) has there been a concerted effort to facilitate regional standardization of Real-time quantification of BCR-ABL mRNA. During this study a modified EAC method for Real-time quantification of BCRABL mRNA was developed and validated with the emphasis to improve reproducibility. Instead of ABL or BCR, GUS was used as control gene based on recommendations from literature. Based on statistical analysis it was concluded that the modifications did not bias the percentage BCR-ABL result. It cannot be emphasised enough that standardization for Real-time monitoring of BCR-ABL is most crucial as it will ultimately facilitate molecular laboratories to develop this diagnostic with much greater ease. In order for standardization to be realized, copy number standards as well as reference material for quality control purposes needs to become more readily available. In addition to that, specific guidelines for assay criteria such as appropriate Ct values and analysis of data must also be developed. By streamlining Real-time quantification of BCR-ABL the treatment and monitoring of CML patients can be improved on a global scale.
Open Access
The evaluation of tirofiban hydrochloride in a high shear rate arterial thrombosis model in baboons
(University of the Free State, 2009-11) Janse van Rensburg, Walter James; Meiring, S. M.; Roodt, J. P.
English: Background: Acute coronary syndrome (ACS) is a major cause of mortality and morbidity world-wide, and is responsible for roughly 2.5 million hospital admissions world-wide annually. ACS is commonly associated with platelet thrombus formation on disrupted atherosclerotic plaques, therefore effective and safe anti-platelet drugs are needed to help treat and prevent ACS. The current most popular anti-platelet drugs are associated with increased bleeding risk and reduced efficacy, thus drugs with a wider therapeutic window (more efficacy with less bleeding) need to be developed. Tirofiban hydrochloride is a small, short half-life molecule that inhibits platelet aggregation by antagonising the glycoprotein IIb/IIIa receptor on platelets preventing fibrinogen and von Willebrand factor to cross-link platelets, thereby inhibiting the final pathway of platelet aggregation. Tirofiban hydrochloride was believed to be a very promising drug due to its short half-life, as an antidote strategy is not needed to reverse adverse bleeding events, but it soon fell out of favour when it was found not to be as effective as for example abciximab in preventing ischaemic events. This was possibly due to the recommended dose being suboptimal. Methods and Results: We studied the efficacy of tirofiban hydrochloride to inhibit platelet thrombus formation on an injured and partially occluded artery by evaluating the effect of escalating doses on cyclic flow reduction (CFR) formation in a high shear arterial thrombosis model in baboons, and also evaluated its safety in two different bleeding models. We then compared our results to results found in the same model using clopidogrel. A significant effect on the number of CFRs was only observed after injection of three times (30 μg/kg bolus plus 0.45 μg/kg/min infusion) the therapeutic dose tirofiban, but it was a weak inhibitor at this dose. Only after injection of nine times (90 μg/kg bolus plus 1.35 μg/kg/min infusion) the recommended therapeutic dose, a strong complete inhibition was observed. A further dose of 27 times (270 μg/kg bolus plus 4.05 μg/kg/min infusion) the recommended therapeutic dose was given to evaluate the effect of an overdose on the bleeding tendency. A significant prolongation in bleeding time (3.05 minutes to 11.90 minutes) was observed after injection of nine times the therapeutic dose, an average 2.7 ± 2.44 fold increase in blood loss was also observed at this dose. A maximum increase in blood loss of an average of 3.4 ± 1.77 fold was seen after injection of 27 times the therapeutic dose. The efficacy of tirofiban hydrochloride was comparable to that of clopidogrel found in earlier studies, but the blood loss was much less when compared to the average 4.3 ± 2.6 fold increase with clopidogrel at 2.5 mg/kg and 8.0 ± 5.0 fold increase at 5 mg/kg. Conclusion: Tirofiban hydrochloride is an effective anti-platelet drug, but only offers adequate protection against arterial thrombosis at a dose between three and nine times the recommended therapeutic dose. However, it still remains safer in terms of bleeding than the most common anti-platelet drugs used today. We recommend that further in vivo studies be done to determine the optimal dose for tirofiban hydrochloride treatment, and that new clinical trials be done with higher dose tirofiban hydrochloride.
Open Access
Clinical haematology training in South Africa
(Health and Medical Publishing Group, 2010) Coetzee, M. J.
Abstract not available
Open Access
Cucurbitacin B inhibits growth, arrests the cell cycle, and potentiates antiproliferative efficacy of cisplatin in cutaneous squamous cell carcinoma cell lines
(Spandidos Publications, 2010) Chen, Weikai; Leiter, Amanda; Yin, Dong; Meiring, Muriel; Louw, Vernon J.; Koeffler, H. Phillip
Cutaneous squamous cell carcinoma (CSCC) is the second most common skin cancer with a substantial risk of metastasis which causes clinical treatment failure. This study investigated the anti-CSCC effects of a triterpenoid compound, Cucurbitacin B (CuB). Dose-response studies showed that CuB inhibited 50% growth (ED50) of the CSCC cell lines (SRB1, SRB12, SCC13, COLO16) in liquid culture at 4x10-7 -10-5 M. Soft-agar assays demonstrated that nearly all of the CSCC clonogenic cells were inhibited at 10-7 M CuB. FACS analysis found that the compound (10-7 M, 48 h) caused G2/M arrest. The CSCC cells underwent profound morphologic changes within 60 min after exposure to CuB (10-7 M), rounding up and losing their pseudopodia. CuB (10-7 M) caused prominent multinucleation of the cells after they were pulse-exposed (24 h) to the drug, washed and cultured in normal medium for an additional 24 h. The drug (10-8-10-6 M, 3-24 h) decreased levels of CDC2 and cyclin B1 in SRB1 and SRB12 cell lines as seen by Western blot analysis. Migration of SRB1 and SRB12 cells was inhibited by 10-7 M CuB. Interestingly, CuB synergistically potentiated the anti-proliferative effect of cisplatin in CSCC. In summary, CuB has a prominent anti-proliferative activity on CSCC cells. In vivo studies and clinical trials of this drug should be pursued in CSCC.
Open Access
Monitoring of genetically modified food products in South Africa
(University of the Free State, 2010-12) Marx, Gertruida M.; Viljoen, C. D.
English: Globally, South Africa is the eighth largest producer of GM crops and also imports GM food. In addition to the promise of increased agricultural production, the introduction of GM crops is also having an impact on society in terms of consumer acceptance and trade. As a result, most countries manage GMOs in terms of development, use and application as well as require mandatory GM labelling for consumer preference. With an increase in GM developments, monitoring the food chain in terms of GM labelling and unapproved GM events will continue to pose a regulatory challenge. The aims of this thesis were the following: 1. To determine the uptake of GM food into the food chain; 2. To study the application of voluntary GM labelling; 3. To investigate the impact of mandatory GM labelling; and 4. To establish a monitoring system to detect illegal GMOs in South Africa. Until 2005 it was assumed that there were only low levels of GM crop in the food chain, based on production volumes. However, results from this thesis have shown that 76% of food products tested positive for the presence of GM in 2005. There was also no consideration of mandatory GM labelling as it was thought that voluntary GM labelling was successfully being applied in South Africa. Despite this, 31% of products labelled to indicate an absence of GM, such as “GMO free”, “non-GM” and “organic”, contained genetic modification above 1%, and 20% of these contained more than 5% genetic modification. These results demonstrated the extent of GM in the food chain in South Africa and highlighted the fact that voluntary GM labelling does not protect consumers against misleading claims. In 2008, the Consumer Protection Act mandated the labelling of GM in food products and ingredients. However, there was a lot of uncertainty as to how this would impact the food industry. The subsequent research on the impact of mandatory GM labelling in South Africa determined that 67% of maize and 54% of soybean products will have to be labelled for GM content. In addition to this, GM was also detected in 50% of products labelled to indicate an absence of GM. Furthermore, results indicated that the use of either a 1% or 5% threshold does not make a considerable difference in terms of the number of products implicated. The use of the term “may contain genetic modification” as suggested by draft regulations to the Consumer Protection Act may provide a cost effective manner in which GM labelling can be applied in a developing country similar to South Africa, as it would reduce costs in terms of GM detection. The draft regulations for the Consumer Protection Act also make provision to indicate the absence of GM below a threshold that does not included terminology such as ”GMO free” or “non-GM”. Furthermore, the draft regulations do not require third party verification and compliance will mainly be self-regulating. The implication of this is that consumers or consumer groups will become responsible for policing the application of GM labelling in South Africa. Finally, this thesis presents a GM monitoring scheme for unapproved GMOs, that have not been proven safe for human health and/or the environment. The scheme has the advantage of being cost effective and can be applied to the regulatory situation in any country, taking approved GM events into consideration. The scheme was applied to off-the-shelf food products in South Africa to determine the presence of illegal GMOs. Even though no unapproved GM events were detected, a potential illegal import of GM soybean event A2704-12 was found. It was also found that an approved GM soybean event was comingled with rice and wheat products, although not indicated in the ingredients. The research emanating from this thesis has contributed to inform discussions that have resulted in the inclusion of mandatory GM labelling in the Consumer Protection Act 68 of 2008. It is hoped that the research on the application of mandatory GM labelling and the monitoring for unapproved GM events in the food chain will have a similar impact on the regulatory system in South Africa.