Haematology and Cell Biology

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Open Access
The application of high-resolution melting curve analysis for the detection of mutations in the MCR-ABL kinase domain of patients with chronic myeloid leukaemia
(University of the Free State, 2011-12) Wienand, Kirsty; Viljoen, C. D.; Louw, V. J.
CML is a haematological malignancy that is characterized by the BCR-ABL fusion oncogene that encodes a constitutively active tyrosine kinase. The treatment of choice for CML is a tyrosine kinase inhibitor and molecular monitoring of patients forms an integral part of disease management. When the expected response to tyrosine kinase inhibitor is not achieved within internationally accepted time frames, acquired resistance to tyrosine kinase inhibitors is suspected. Acquired resistance to tyrosine kinase inhibitors is primarily due to mutations in the BCR-ABL kinase domain. Types of mutations include single base mutations, insertions, deletions as well as duplications. Characterization of these mutations is important for treatment, since the type and position of the mutation may have an effect on how the patient responds to treatment. Although several methods have been described for detecting mutations, DNA sequencing is mostly used. Sequencing is currently the only technique that can simultaneously detect single base mutations, insertions and deletions in the BCR-ABL kinase domain. However, sequencing is costly as some patient samples do not have mutations and the lack of response to treatment is due to non-compliance. Thus, a screening method to exclude samples without mutations would make mutational analysis more cost-effective. High resolution melting (HRM) is a relatively new technique that is being used to screen for mutations, prior to sequencing. HRM has recently been used to screen the region of BCR-ABL encoding for the kinase domain for single base mutations. However, it was unknown whether HRM could be used to identify insertions, deletions or duplications in the kinase domain. This study has shown that HRM can be used to screen for mutations including insertions, deletions and duplications the region of BCR-ABL encoding for the kinase domain, prior to sequencing. HRM was performed on 40 patient samples, 10 of which had confirmed mutations in BCR-ABL in the region of the kinase domain. Of the 10 samples with mutations, three had single base mutations, one with a previously described insertion, seven had novel deletion variants. Furthermore, HRM detected a tandem duplication of the kinase domain in two patient samples that was not previously been possible with sequencing. There was 100% congruency between the detection of mutations using HRM and sequencing results, indicating similar sensitivity. HRM proved successful to indicate the presence of deletion variants. However, the deletion variants were detected in the HRM region preceding the area affected by the deletion. It was confirmed that the detection of the deletion variants was due to the PCR extension of HRM 1 amplicon into the HRM area of the deletion. It has been suggested that the insertion, deletions and duplications detected in this study may result in acquired resistance to tyrosine kinase inhibitor. In conclusion, this was the first study to use high-resolution melting to detect insertions, deletions and duplications in the region of BCR-ABL encoding for the kinase domain, indicating the suitability of the assay for screening for mutations prior to sequencing.
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
A case study of GM maize gene flow in South Africa
(Springer, 2011) Viljoen, Chris; Chetty, Lukeshni
Background: South Africa has been growing first-generation commercial genetically modified (GM) maize since 1997. Despite a requirement for non-GM food, especially for export, there is no system for coexistence of GM and non-GM crop. Gene flow is a major contributor to commingling, and different distances of cross-pollination have been recorded for maize, using a variety of field-trial designs under different environmental conditions, with the furthest distance being 650 m. However, these trials have usually been small plots and not on the scale of commercial farming. There are also no published data regarding the extent of cross-pollination for maize in South Africa, even after a decade of commercialization of GM. Thus, the aim of this study, conducted from 2005 to 2007, was to determine the extent of GM maize cross-pollination under South African conditions in the context of commercial farming practice. Materials and methods: Field trials were planted with a central plot of yellow GM maize (0.0576 ha) surrounded by white non-GM maize (13.76 ha), in two different geographic regions over two seasons with temporal and spatial isolations to surrounding commercial maize planting. Cross-pollination from GM to non-GM maize was determined phenotypically across 16 directional transects. Pollen counts during flowering were compared to weather data as well as percentage cross-pollination. The data were transformed logarithmically, and mean percentage cross-pollination was compared to high cross-pollination. Results and discussion: Although there was a general congruency between wind data, pollen load and crosspollination, it is evident that wind data and pollen load do not solely explain the directional extent of crosspollination and that swirling winds may have contributed to this incongruence. Based on the logarithmic equations of cross-pollination over distance, 45 m is sufficient to minimize cross-pollination to between <1.0% and 0.1%, 145 m for <0.1% to 0.01% and 473 m for <0.01% to 0.001%. However, compared to this, a theoretical isolation distance of 135 m is required to ensure a minimum level of cross-pollination between <1.0% and 0.1%, 503 m for <0.1% to 0.01% and 1.8 km for <0.01% to 0.001% based on high values of cross-pollination. Conclusions: Based on the results of this study, the use of mean values of cross-pollination over distance may result in an underestimation of gene flow. Where stringent control of gene flow is required, for example, for non- GM seed production or for GM field trials under contained use, the high values of cross-pollination should be used to determine isolation distance. However, this may not be practical in terms of the isolation distance required. We therefore suggest that temporal and distance isolations be combined, taking into account the GM maize pollen sources within the radius of the most stringent isolation distance required.
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
Characterisation of apparent mismatches detected during routine short tandem repeat analysis in parentage investigations
(University of the Free State, 2023) Soldati, Afika; de Kock, André; Kloppers, Jean F.
𝗕𝗮𝗰𝗸𝗴𝗿𝗼𝘂𝗻𝗱: Short Tandem Repeat (STR) analysis has proven effective for establishing parentage and biological relatedness. There are commercially available STR kits that allow for reliable PCR amplification and genotyping of STR loci. However, one or two STR loci mismatches may be identified in non-exclusion cases. In routine analysis, these discrepancies are classified as apparent STR loci mismatches. The mismatches result from various mutational mechanisms. However, the mechanisms that drive these mutations are poorly understood. Several STR loci mismatches have previously been reported to impact parentage analysis. The alleles involved in the mismatch affect the interpretation of genetic profiles and can sometimes lead to false parentage exclusions. As such, it is essential to identify and characterise the underlying cause of STR loci mismatches for further validation of the genotypic data produced within a specific DNA profiling laboratory. 𝗠𝗲𝘁𝗵𝗼𝗱𝘀: A laboratory-based descriptive-comparative study was conducted. This study consisted of 100 parentage cases with one or two STR loci mismatches from the DNA testing facility, Universitas Academic Unit, National Health Laboratory Services (NHLS) Bloemfontein from 1 January 2021 to 31 March 2022. The following 15 STR autosomal loci were included in the analysis: CSF1PO, FGA, vWA, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248, D13S317, D16S539, D18S51, D19S433, and D21S11. Both published and designed study primers were used to optimise the PCR assay conditions for the amplification of the selected STR loci using commercially available control DNA. The optimised PCR assay conditions were used to screen the samples across the 15 STR loci. Sanger sequencing and sequence analysis was conducted for each parentage case to identify and characterise the underlying cause of the observed apparent STR mismatches. Furthermore, the sequence-based alleles were evaluated for concordance with genotypes determined by Capillary Electrophoresis-based (CE-based) STR typing previously reported by the facility. 𝗥𝗲𝘀𝘂𝗹𝘁𝘀: An average concordance of 82% was observed between STR profiling and Sanger sequencing across the 15 STR loci studied. In 11 of the loci, a 100% concordance was obtained. In contrast, no concordance was observed for the D19S433 locus. The stepwise mutations observed at the various loci were 70% more frequent than other mutation models; these were attributed to DNA polymerase slippage. In comparison, 30% of the mutations were as a result of allelic dropouts, accounted for by primer-binding site sequence variants. It was observed that there were more mutations originating from paternal (n=76) rather than maternal (n=26) lineages. 𝗖𝗼𝗻𝗰𝗹𝘂𝘀𝗶𝗼𝗻: The observation of one or two STR loci mismatches in parentage analysis should not be overlooked; all the studied allelic mismatches between the parent and child were characterised successfully. The findings revealed that most of the apparent mismatches occurred due to DNA polymerase slippage. The results of this study provide evidence that sequencing of the core STR repeat and the flanking regions can provide valuable information to characterise STR loci mutational events when inconclusive parentage or kinship results are obtained. Because of the limited sample size, the findings of this study provide evidence that STR mutations are more prevalent in males than females. Furthermore, this study demonstrates the need for DNA testing facilities to have a method in place to characterise and confirm inconclusive genotypic data obtained using the available commercial STR kits.
Open Access
Characterisation of the fibrinolytic system and the Von Willebrand factor-Adamts13 axis in the Chacma baboon
(University of the Free State, 2021-11) Joubert, Jaco; Janse van Rensburg, W. J.; Meiring, S. M.
Background: The Chacma baboon (Papio ursinus) model of acquired thrombotic thrombocytopenic purpura (aTTP) is ideally suited to investigate novel treatments with potential application in this lethal thrombotic disorder, which hinges on the dysfunction of the VWF–ADAMTS13 axis. One such modality is the thrombolytic drugs, which activate the fibrinolytic system. Our recently published pilot study demonstrated the thrombolytic drug streptokinase as ineffective in resolving aTTP in this model and highlighted the deficits in our knowledge of the Chacma baboon’s fibrinolytic system and VWFADAMTS13 axis. The present study aimed to characterise these components of the Chacma baboon’s haemostatic system to better understand the aTTP model, the effects of thrombolytics in this model, and the implications for other haemostatic disease models in this species. Materials and Methods: Forty baboons were tested using observational and experimental assays. The VWF–ADAMTS13 axis was investigated by determining ADAMTS13 antigen and activity levels, VWF:Ag, VWF:RCo, and VWF:CB levels and VWF multimer patterns. The fibrinolytic system was explored by measuring the concentrations of fibrinogen, plasminogen, tPA, PAI-1, PAP complexes, TAFI, and α2- antiplasmin, and by assessing its in vitro clot lysis ability in a modified clot lysis time assay.The plasminogen activation potentials of streptokinase and tPA were determined using concentration escalation experiments. The effect of tPA-induced plasmin activity on VWF multimer patterns when in the non-globular state was assessed in the presence of the anti-ADAMTS13 mAb 3H9. Thrombin generation, which can influence the aTTP model and its response to thrombolytics, was also evaluated, as well as the effects of sex and ABO blood group. Reference intervals and interindividual variation were calculated and compared with human values. Results and Discussion: ADAMTS13 activities were generally below (but still comparable to) human ranges. VWF:Ag and VWF:CB values tended towards the lower limit of the human reference interval, whereas VWF:RCo activities were higher. All VWF multimer patterns were essentially equivalent to normal human patterns. Fibrinogen concentrations were similar to human values, but tPA, PAP complex, PAI-1 and α2- antiplasmin concentrations all tended toward the lower human ranges. Meaningful results could not be generated for ADAMTS13 antigen, plasminogen, or TAFI, possibly due to structural differences with the human protein. Streptokinase resulted in minimal plasmin activity, but tPA led to concentration-dependent increases. All baboon samples exceeded 100% of human plasmin activity at baseline and had clot lysis times shorter than pooled normal human plasma when activated by tPA. In the absence of ADAMTS13 activity, tPA reduced the non-globular high molecular weight VWF multimers in both human and baboon samples. Baboons had greater overall endogenous thrombin potentials than humans, which was more prominent in females (p=0.0238). Except for lower fibrinogen concentrations (p=0.0134) in male baboons, and PAP complex concentrations which were higher (p=0.0188), no other sex-related differences were apparent. No baboons were typed as ABO group B or AB, and none were Rh(D) negative. Group O baboons had higher fibrinogen concentrations (p=0.0355), but all other parameters were unaffected. Fibrinogen and especially α2-antiplasmin were subject to considerable interindividual biological variation. Conclusion: The central thesis of this research is that the components of the Chacma baboon’s haemostatic system pertinent to the pathogenesis of aTTP and its treatment with thrombolytics are similar enough to their human counterparts to enable continued use of this species as a model of human haemostasis, provided quantitative results are interpreted within the context of the novel reference intervals, and the identified limitations and interspecies differences are considered. Finally, tPA should be explored further in the,Chacma baboon aTTP model in vivo to provide proof-of-concept for the use of thrombolytic drugs in the treatment of aTTP in humans.
Open Access
Characterization of a human inhibitory antibody fragment against tissue factor
(University of the Free State, 2011-11) Vermeulen, Jan-G.; Meiring, S. M.; Van Heerden, E.
English: 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.
Open Access
Clinical haematology training in South Africa
(Health and Medical Publishing Group, 2010) Coetzee, M. J.
Abstract not available
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
Comparison of ADAMTS13 and Von Willebrand factor antigen levels and activities and plasminogen levels, in the currently available plasma products for the treatment of thrombotic thrombocytopenic purpura in South Africa
(University of the Free State, 2018) Van Marle, Anneke; Joubert, Jaco
Thrombotic thrombocytopenic purpura results from a deficiency in the Von Willebrand factor cleaving protease, ADAMTS13. Treatment involves plasma exchange therapy with either fresh frozen plasma, cryosupernatant, or solvent/detergent-treated plasma (available locally as Bioplasma FDP®). The research aimed to generate in vitro data on these products, and to explore possible differences between them that may offer treatment advantages. Twenty samples per product were analysed for levels and activities of ADAMTS13 and Von Willebrand factor, and plasminogen levels (a proposed physiological backup system for ADAMTS13). Fresh frozen plasma and cryosupernatant samples were subanalysed according to ABO blood group. All samples had normal/high ADAMTS13 activity and plasminogen levels. Von Willebrand factor content was mostly normal for Bioplasma FDP®, typically deficient for cryosupernatant and mostly (unexpectedly) deficient for fresh frozen plasma. Depending on the parameter, Bioplasma FDP® was the most standardised (CV: 14.1% to 27.3%), whilst fresh frozen plasma showed great inter-individual variation (CV: 24.6% to 208.6%). Statistically significant differences were found across products (p values ≤ 0.0095) and ABO blood groups (p value: 0.0001). In conclusion, all three products can be used for treatment of thrombotic thrombocytopenic purpura. Product choice depends on the need for additional viral safety, costs, product availability and the perceived impact of within-product variations.
Open Access
Comparison of platelet receptors P2Y12, GPIIB/IIIA, GPVI, and GPIBα between the Cape chacma baboon and the human
(University of the Free State, 2015) Janse van Rensburg, Walter James; Badenhorst, Philip N.; De Kock, André
English: Background: Acute coronary syndrome is globally a major cause of morbidity and mortality. Treatment and prevention involve the use of an anti-platelet agent. The current available agents have either side-effects or are relatively ineffective. Therefore, there exists a need to develop safer and more effective agents. Platelet receptors are a target for anti-platelet agents and new generation agents function on a molecular level. The Cape chacma baboon (Papio ursinus) has been a popular model for the pre-clinical evaluation of anti-platelet agents. However, limited molecular data are available for these animals, restricting its translational value. The aim of this study was to characterize four common platelet receptors in the Cape chacma baboon and compare the results to human data. Methods: The platelet receptors P2Y12, glycoprotein (GP) VI, GPIIb/IIIa and GPIbα were selected for this study. Light transmission platelet aggregometry was performed to assess baboon platelet function; receptor number quantification was performed by flow cytometry; and Sanger sequencing was done on genomic baboon DNA. All results were compared to normal human data. Results: Baboon ADP-induced platelet aggregation results were significantly different from normal human results, even at ADP levels four times (40 μM) the highest human concentration of 10 μM. Baboon collagen-induced aggregation remained significantly different at twice (8 μg/ml) the highest human concentration of 4 μg/ml. However, the differences in collagen-induced aggregation results were not clinically relevant from the human results, because all except one result (at 8 μg/ml) fell within the normal human reference range. At double the highest human concentration for ristocetin (2.5 mg/ml) baboon platelets gave statistically similar results. At double the highest human concentration (1 mg/ml) arachidonic acid results remained significantly different between baboons and human. Baboon quantification results showed a 37% increase in GPIIb, 27% increase in GPIIIa and 25.5% increase in GPIbα. GPVI quantification failed due to non-reactive monoclonal antibodies. P2Y12 quantification was not possible, as no commercial monoclonal antibodies exist for it. The P2Y12 protein sequence was 98.8% similar. It differed by only four amino acids, none of which have been described as functionally essential. The GPVI protein sequence showed 95% similarity. It included a 14 amino acid difference and a three amino acid deletion. One change was at a region where an amino acid change has been implicated in reduced collagen-induced platelet aggregation in humans. Two differences were directly adjacent to a collagen-binding amino acid. The deletion was within the signalling region of GPVI. Exon 28 of GPIIb could not be sequenced. The GPIIb protein sequence for exon 1-27 was 98.2% similar and for exons 29-30 there was 98.3% similarity. There was an 18 amino acid difference. One amino acid change was in the ligand-binding region. The GPIIIa protein sequence was 99.6% similar, with three amino acid changes. One change was in the ligand-binding region. 54 amino acid changes were found in GPIbα. The protein sequences of the signal peptide, VWF-binding-, PEST / macroglycoprotein-, transmembrane- and cytoplasmic domains showed 93.8%, 89.4%, 57.9%, 90.5% and 95.0% similarity, respectively. 246 bases of GPIbα failed to sequence. Discussion and Conclusion: Sequentially and functionally baboon P2Y12, GPIIb/IIIa and GPIbα is comparable to humans. The higher agonist-levels needed for baboon platelet aggregation may be attributed to the increase in surface receptor numbers. However, receptor-number, optimal agonist concentrations and potentially inhibiting amino acid changes should be noted for future studies. Non-reactive antibodies and changes in critical amino acids caused the baboon GPVI to be not comparable to humans. The Cape chacma baboon (Papio ursinus) is therefore, deemed a suitable animal model for the evaluation of human-targeted anti-platelet agents directed against the receptors P2Y12, GPIIb/IIIa and GPIbα, but not for the evaluation of human-targeted anti-GPVI agents.
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
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
Development and application of a real-time PCR method to detect selected single nucleotide polymorphisms associated with hypertension in a black South African population
(University of the Free State, 2014-08) Du Toit, Egardt; Viljoen, C. D.
English: Hypertension is one of the leading causes of death and disability in the world. In 95% of individuals with hypertension, the condition arises from the interaction of multiple environmental factors with physiological systems. Environmental factors that have been found to increase blood pressure include obesity, aging, high salt and alcohol consumption, low potassium and calcium intake, stress and insulin resistance. Physiological systems that regulate blood pressure include the autonomic nervous system, the renal system, hormonal system and the cardiovascular system. Various genes in these systems including the β1-adrenergic receptor (ADRB1), α-adducin (ADD1), angiotensinogen (AGT), aldosterone synthase (CYP11B2), CYP3A5 and G protein-coupled receptor kinase 4 (GRK4) have been implicated in hypertensive blood pressure due to the presence of single nucleotide polymorphisms (SNPs). The occurrence of such SNPs in blood pressure regulatory systems is thought to result in altered gene expression or protein function. In South Africa, the prevalence of hypertension has been determined to be approximately 39.9% in males and 34.9% in females. The Assuring Health for all in the Free State (AHA-FS) study determined that the prevalence of hypertension was approximately 48.3% in the Mangaung population. The AHA-FS study also found that 37.6% and 51.2% of individuals in the study cohort were overweight or obese, respectively, and that high body mass could be an important risk factor for hypertension. The aim of this study was to determine whether genes in the sympathetic nervous system, renal system and hormonal systems could contribute to the high prevalence of hypertension in the Mangaung population. Previously identified SNPs associated with hypertension in ADRB1 (A145G and G1165C), ADD1 (G217T), AGT (G-217A, C521T and T704C), CYP11B2 (C-344T), CYP3A5 (A6986G) and GRK4 (G448T, C679T and C1711T) were genotyped in a cohort of the AHA-FS study, which comprised black individuals from Mangaung, Free State. Six of the 11 candidate SNPs did not appear to be associated with hypertension in the black population of Mangaung. These included G1165C (ADRB1), G-217A and T704C (AGT), G448T, C679T and C1711T (GRK4). None of the latter SNPs were associated with statistically significant elevations in either systolic or diastolic blood pressure. The association remained negative even after the cohort was stratified into underweight to normal weight and overweight to obese groups. The lack of association between these SNPs and hypertensive blood pressure in the black population group in Mangaung compared to other population groups could be attributed to population differences in environmental factors, ethnicity, cohort size and/or epistasis. Five SNPs were associated with hypertension in black individuals from Mangaung. The latter included SNPs in CYP3A5 (A6986G), ADRB1 (A145G), AGT (C521T), CYP11B2 (C-344T) and ADD1 (G217T). The hypertensive A allele of the A6986G SNP of CYP3A5 has been associated with systolic hypertension in homozygous individuals of the study. Similarly, an association between the A allele and hypertension in African-Americans and Swedish Caucasians has also been found. Thus, the A allele of the A6986G SNP seems to cause hypertension susceptibility in different populations, including the Mangaung population group. As for the A145G SNP of ADRB1, hypertensive systolic blood pressure was associated with individuals that were homozygous for the hypertensive A allele of the A145G SNP. A meta-analysis determined that individuals expressing the A allele of A145G had a 24% higher risk for developing hypertension. In the Mangaung population, hypertension risk associated with the A allele of A145G was especially increased in overweight to obese individuals. The presence of the hypertensive T allele of the C521T SNP of AGT was associated with hypertensive diastolic blood pressure in the Mangaung population. Similar results were found in Hutterite, Russian and Tartar population groups. Furthermore, in the Mangaung population the hypertension risk conferred by the T allele was significantly increased in overweight and obese individuals. This suggests that the T allele of C521T may be involved in particularly overweight and obesity related hypertension. The hypertensive T allele of the C-344T SNP of CYP11B2 was only associated with hypertensive systolic and diastolic blood pressure in the overweight to obese individuals of the Mangaung population. In another study conducted on black South African individuals the T allele was also associated with hypertension. The cohort of the latter study furthermore had an overweight to obese body mass index average. It therefore appears that the T allele of C-344T could primarily be a risk factor for overweight and obesity related hypertension. Interestingly the normotensive G allele of ADD1 was implicated in obesity related hypertension, instead of the hypertensive T allele. In contrast, another study on black South Africans found that the T allele was associated with hypertension. However, results from a previous study on African-Americans suggested that the T allele may be protective against hypertension. It has been proposed that other unidentified polymorphisms, which also could affect hypertension susceptibility, could be in linkage disequilibrium with the G217T SNP and that allelic variation of the other polymorphic loci could contribute to the inconsistent findings of association studies. Several individuals in the cohort from the AHA-FS study could not be genotyped for the candidate SNPs. Attempts to obtain conventional PCR product for individuals where genotyping failed did not prove entirely successful. In the cases where no PCR amplicon could be amplified even after attempts at assay optimization, it was concluded that primer mismatch, especially at the 3‟ end of the primer may be the likely cause. Where PCR amplicon was successfully amplified, sequencing proved difficult due to short amplicon size. However, partial sequence data revealed additional SNPs in some individuals in the probe binding region that would account for failed genotyping. A limitation of this study was that only selective SNPs in genes associated with hypertension were genotyped in the cohort. Furthermore, since the study did not investigate the role of potentially novel SNPs in candidate genes, it is possible that additional unidentified SNPs in these genes may also contribute to hypertension. Despite these limitations, this study is currently the most comprehensive of its kind on the Mangaung population. Future research could focus on additional genes as well as screening these genes for novel SNPs, especially in the genes where the SNPs investigated were not associated with hypertension in the Mangaung cohort. In conclusion, the A6986G SNP in CYP3A5 appears to be an independent risk factor for hypertension, whereas A145G in ADRB1, C521T in AGT, C-344T in CYP11B2 and G217T in ADD1 may be associated with hypertension related to overweight and obese body weights. To our knowledge, this is the most comprehensive study investigating a combination of several gene SNPs associated with hypertension in a black Mangaung population.
Open Access
Differential sensitivity of von Willebrand factor (VWF) 'activity' assays to large and small VWF molecular weight forms: a cross-laboratory study comparing ristocetin cofactor, collagen-binding and mAb-based assays
(International Society on Thrombosis and Haemostasis, 2012) Favaloro, E. J.; Bonar, R.; Chapman, K.; Meiring, M.; Funk, D.
Background: von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or moreVWF'activity' assays. VWFactivity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen-binding (VWF:CB) and VWF mAbbased (VWF activity [VWF:Act]) assays are used by some laboratories. Objective: To perform a cross-laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B. Methods: A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays. Results: The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, withinmethod analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers. Conclusions: We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura.
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
The effect of inflammatory cytokines and coagulation factors on Von Willebrand factor synthesis and cleavage
(University of the Free State, 2012-02) Allers, Werner Ernst; Meiring, S. M.
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.
Open Access
Evaluation of the eosin-5-maleimide flow cytometric test and other screening tests in the diagnosis of hereditary spherocytosis
(University of the Free State, 2020-06) Mothi, Hemasha; Van Marle, A.; Roodt, J. P.
Background: Hereditary spherocytosis (HS) is a genetically determined haemolytic anaemia characterised by the spherical shape of affected red blood cells. With limited confirmatory tests currently available in South Africa, the diagnosis of HS is reliant on the clinical presentation and screening tests. Objectives: The aim of this study was to compare the sensitivity and specificity of three screening tests: flow osmotic fragility test (FOFT), cryohaemolysis test (CHT) and the eosin-5-maleimide binding test (EMA-binding test) in diagnosing hereditary spherocytosis (HS). Method: All three tests were performed on 18 subjects with confirmed HS. The negative control group was comprised of 10 subjects with haemolysis and spherocytosis, and either a positive direct antiglobulin test (DAT) or normal red cell membrane studies. The tests were also performed on samples submitted for cases of suspected HS during the study period. Results: The EMA-binding test demonstrated superior sensitivity (88.9%) compared to the CHT (61.1%) and the FOFT (38.8%). The EMA-binding test specificity (90.0%) was equal to that of the FOFT and superior to the CHT (50%). Combined sensitivities and specificities for EMA-binding test and CHT, EMAbinding test and FOFT, and CHT and FOFT, were 100% and 33.3%, 94.4% and 80.0% and 88.9% and 50.0%, respectively. Conclusion: EMA-binding test is the best screening test for cases of suspected HS. If there is a high clinical index of suspicion with a negative EMA-binding test, the CHT is recommended as a second screening test.
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
Genotypic and expression analysis of CYP3A4 and CYP3A5 in patients with chronic myeloid leukaemia
(University of the Free State, 2013-01) Thompson, Gaynor Gillian; Viljoen, C. D.
Chronic myeloid leukaemia (CML) is a haematological malignancy characterised by the BCR-ABL fusion oncogene which encodes for a constitutively active tyrosine kinase. Imatinib mesylate is a tyrosine kinase inhibitor that has effectively been used in the treatment of CML. However, some individuals experience adverse drugs reactions (ADRs) to imatinib. One of the reasons for varied treatment response among individuals may be as a result of inter-individual differences in the metabolism of imatinib. Imatinib is metabolised by the drug metabolizing enzymes CYP3A4 and CYP3A5. Single nucleotide polymorphisms (SNPs) in CYP3A4 and CYP3A5 have been described, some of which have been associated with altered catalytic activity of these enzymes. SNPs in CYP3A4 and CYP3A5 may also impact the expression of these genes and result in a less favourable response to imatinib treatment. Patients with a decrease in catalytic activity of CYP3A4 and CYP3A5 may experience ADRs due to prolonged exposure to imatinib. On the other hand an increase in activity may lead to ineffective treatment as a result of increased clearance of the drug. Thus the aim of this study was to screen CYP3A4 and CYP3A5 for SNPs using high resolution melting curve analysis (HRM) and determine the impact of these SNPs on gene expression in CML patients treated with imatinib. A total of ten SNPs were detected in CYP3A4, of which two SNPs, namely A15619G and A15649T have not been previously described in literature. A15619G was a synonymous SNP while A15649T resulted in a change in amino acid from glutamine to a histidine. A total of four SNPs were detected in CYP3A5, of which one SNP namely, G7226A, had not previously been reported in literature and did not result in an amino acid change. Out of all the detected SNPs in CYP3A4, the G20338A SNP was statistically associated with the occurrence of ADRs but not with mRNA expression. The I369V SNP was statistically associated with increased CYP3A4 mRNA expression. None of the SNPs detected in CYP3A5 significantly affected mRNA expression. Expression of CYP3A4 and CYP3A5 was not dependent on ethnicity or gender, with the exception of CYP3A5 which showed a statistically significant difference between males and females. Currently a limited amount of literature exists regarding SNPs in CYP3A4 and CYP3A5 and CML treatment. Given the potential impact that SNPs can have on the CYP3A4 and CYP3A5 enzymes and therefore imatinib treatment, it is an important issue that needs to be investigated. Determining the potential impact of SNPs and differential gene expression of CYP3A4 and CYP3A5 is important as it may allow for more effective imatinib treatment.