Orientadores: Adriana Maria Alves De Tommaso, Gabriel Hessel / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T20:45:32Z (GMT). No. of bitstreams: 1 Rosa_MarianaNogueiradePaula_M.pdf: 1246789 bytes, checksum: 4d75875746a0c9dddc300904721c7941 (MD5) Previous issue date: 2007 / Resumo: Anualmente ocorrem, no Brasil, cerca de 130 casos novos de hepatite A por 100000 habitantes e o país é considerado área de risco para a doença. A soroprevalência vai aumentando com a idade. Estima-se que, aproximadamente, 15% da população já entrou em contato com o vírus da hepatite B e que 1% apresenta doença crônica. A resposta à vacina da hepatite A está diminuída em pacientes com doença hepática crônica descompensada e a taxa de resposta à vacinação rotineira contra o vírus B em indivíduos imunossuprimidos é menor que em indivíduos saudáveis. A hipertensão portal pode determinar esplenomegalia e levar ao hiperesplenismo com redução nos números absoluto e percentual de células T, podendo determinar, dessa forma, uma diminuição da resposta à vacinação. Este estudo tem por objetivo avaliar a resposta à vacinação contra as hepatites A e B, em pacientes com hipertensão portal secundária a hepatopatia crônica ou trombose de veia porta. Foram avaliados 36 pacientes, de 2 a IS anos, com hipertensão portal, atendidos no Ambulatório de Hepatologia Pediátrica do Hospital de Clínicas da Universidade Estadual de Campinas, no período de 1994 a 2006. As sorologias para hepatites A e B foram realizadas nos pacientes já vacinados e nos pacientes que nunca receberam as vacinas. Os pacientes não vacinados receberam as vacinas durante o período do estudo. Os pacientes com títulos negativos de anti-HBs foram submetidos a dose de reforço e a sorologia foi repetida após o reforço. Avaliações hematimétricas dos pacientes foram realizadas, juntamente com as sorologias, para avaliar as alterações decorrentes da imunossupressão secundária à doença de base e ao hiperesplenismo. Dezenove pacientes receberam a vacina contra hepatite A e todos apresentaram resposta à vacina. Todos os pacientes receberam 3 doses da vacina contra hepatite B e vinte e sete pacientes (75%) apresentavam anti-HBs positivo. Nove pacientes foram submetidos à dose de reforço e nove apresentaram anti-HBs positivo após o reforço. A análise estatística realizada, por meio do teste de Mann-Whitney, para a comparação dos pacientes anti-HBs positivo e anti-HBs negativo com relação às variáveis idade, leucócitos, linfócitos e tempo entre a vacinação e a realização da sorologia não demonstrou diferença estatisticamente significativa. Os resultados permitem concluir que: 1. a vacina contra hepatite A apresentou 100% de resposta em pacientes com hipertensão portal e 2. a vacina contra hepatite B parece conferir proteção e induzir à resposta anamnéstica nesses pacientes. / Abstract: Anually, about 130 new cases of hepatitis A per 100000 inhabitants occur in Brazil, and the country is considered a risk area for the disease. About 15% of brazilian population has been in contact with hepatitis B virus and 1% has chronic disease. Hepatitis A vaccine response is diminished in patients with decompensated chronic disease, and response to routine vaccination against VHB in imunossupressed subjects is lower than in healthy subjects. Portal hypertension lead to hypersplenysm and decrease of absolute and percentual numbers of T cells, and a lower response to vaccination could occur. The purpose of this study was to evaluate response to hepatitis A e B vaccination in patients with portal hypertension secondary to chronic liver disease or portal vein thrombosis. Thirty six patients (2 to 18 years) with portal hypertension were evaluated, assisted at Pediatric Hepatology Service of the Hospital das Clínicas da Universidade Estadual de Campinas, from 1994 to 2006. Hepatitis A and B serologies were done in all patients. Patients who weren't vaccinated received vaccines during the study period and patients with negative anti-HBs received a booster dose, and serology was repeated after booster. Hematimetric evaluation of patients was done as the same time as serologies, for evaluation of immunosupression secondary to chronic liver disease and to hypersplenism. Nineteen patients received hepatitis A vaccine and all presented positive anti-VHA. All patients received hepatitis B vaccine, and 27 patients (75%) presented positive anti-HBs. Nine patients received a booster dose, and nine presented positive anti-HBs after booster. Statistic analysis was done by Mann-Whitney test to compare positive anti-HBs group and negative anti-HBs group. Variables age, leucocytes, limphocytes and time between vaccination and serology were analyzed, and there wasn't statistically significative diference between the groups. These results demonstrate that vaccine against hepatitis A presented a 100% response in patients with portal hypertension, and vaccine against hepatitis B seems to confer protection and to induce an anamnestic response in these patients. / Mestrado / Pediatria / Mestre em Saude da Criança e do Adolescente

Orientador: Maria Marluce dos Santos Vilela / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T01:20:12Z (GMT). No. of bitstreams: 1 Zorzeto_TatianeQueiroz_M.pdf: 1695884 bytes, checksum: 267f0534bc256440762ad9d78bc6402d (MD5) Previous issue date: 2008 / Resumo: A associação temporal de eventos adversos de variada gravidade à imunização com pertussis celular total (DTP) tem estimulado o desenvolvimento de vacinas antipertussis menos reatogênicas. Este ensaio clínico fase I visou à avaliação da imunogenicidade da vacina pertussis celular modificada pela extração do lipopolissacarídeo (DTPm) em comparação com a vacina convencional (DTP). Um total de 234 lactentes foi imunizado aos dois, quatro e seis meses de idade com DTPm ou DTP. Os títulos de anticorpos para os componentes pertussis, tétano, difteria e hepatite B foram determinados um mês após a terceira dose de vacina. A proliferação de células T CD3+ foi avaliada por citometria de fluxo após seis dias de cultivo de células mononucleares de sangue periférico estimuladas com células inativadas de B. pertussis ou com fitohemaglutinina (PHA). Células CD4+, CD8+ e TCR ?d+ foram identificadas no gate de blastos. Os níveis de IFN-?, TNF-a, IL-4 e IL-10 no sobrenadante de cultura foram quantificados por ensaio imunoenzimático (ELISA). A vacina modificada DTPm mostrou-se inferior à DTP quanto ao título de anticorpos antipertussis, mas não houve diferença de resposta aos outros componentes vacinais avaliados. A porcentagem líquida de blastos sob estímulo da B. pertussis foi menor no grupo que recebeu três doses de DTPm (mediana de 3,9% para DTPm e 6,2% para DTP, p=0,029), mas as freqüências de células CD4+, CD8+ e ?d+ em proliferação e as concentrações de citocinas não diferiram entre os grupos. A vacina DTPm não apresentou, portanto, imunogenicidade similar à da vacina DTP convencional nos ensaios laboratoriais / Abstract: Concerns about systemic reactions after immunization with whole-cell pertussis (wP) have stimulated efforts to produce less reactogenic vaccines. This phase I comparative trial aimed the efficacy evaluation of a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wPlow) in comparison to conventional wP vaccine. A total of 234 infants was vaccinated at 2, 4, and 6 months with conventional wP or wPlow. Serum antibody titers to pertussis, diphtheria, tetanus and hepatitis B were measured 1 month after the third dose of vaccine. Proliferation of CD3+ T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cells culture, with heat-killed B. pertussis or phytohemagglutinin (PHA) stimulation. CD4+, CD8+ and TCR ?d+ cells were identified in the gate of blast lymphocytes. IFN-?, TNF-a, IL-4 and IL-10 levels in supernatants were determined by ELISA. wPlow was inferior to wP in terms of anti-pertussis titers, but there was no diference in other serum antibody evaluations. Net percent blasts in cultures with B. pertussis was lower in the group vaccinated with wPlow (medians of 3.9% and for wPlow and 6.2% for wP; p=0.029), but the frequency of proliferating CD4+, CD8+ and ?d+ cells and the cytokine concentrations in supernatants were similar between vaccination groups. Therefore, wPlow wasn't as imunogenic as conventional wP in experimental evaluations / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente

Little is known about the effect of different modes of expression of an antigen in rBCG on immune response. An appropriate wing of the immune system, with different degrees, is activated upon encounter with a foreign antigen. Knowledge of these responses is vital to the development of future recombinant vaccine. Various E. coli-mycobacterial species shuttle vector constructs were made using a combination of mycobacterial promoters and signal sequences. Thus enabling foreign antigens to be expressed cytoplasmically or secreted outside rBCG as native proteins or membrane-associated lipoproteins. A pivotal study using an E. coli beta-lactamase as a reporter gene is described for the evaluation of the strength of promoter and signal sequence constructs both in vitro and most importantly in vivo using the mouse macrophage cell line J-774. Expression of the diphtheria toxin fragment B as a foreign antigen was detected in vitro with all constructed plasmid vectors in rBCG using a western blot as a means of detection. It was observed that all hsp60 promoter-based constructs exhibited a high frequency with variable degree of plasmid DNA deletions Using three different rBCG substrains, the BCG Tokyo was found to be more stable (P < 0.01) and exhibited less degree of deletion (P < 0.001) compared to either BCG Moreau or BCG Pasteur. Sequence analysis of deleted plasmid DNA revealed a specific region common with nearly all plasmid deletions. Such a region of the DNA was found to correspond to the first transcriptional starting site of the hsp60 promoter. Furthermore no differences were observed in the level of expression among the three-rBCG substrains, retaining plasmid DNA, when detected by immunoblotting.

Master of Science in Biomedical Sciences / Department of Clinical Sciences / Elizabeth G. Davis / To protect horses from disease, equine practitioners typically prescribe a protocol of an initial primary vaccination followed by a booster vaccination 3-4 weeks later. Subsequent boosters are given every 6-12 months depending on the pathogen of concern. Each vaccination incurs an additional cost and increased chance for adverse reactions. Despite wide-spread protocol acceptance, duration of effectiveness of vaccines in protecting horses from disease is not well documented. It was hypothesized that horses vaccinated annually since birth have increased antibody production that remains consistent and sufficient for long-term protection from common diseases. This work resulted in the development of a novel, multiplex-magnetic bead-based indirect immunoassay to screen sera from vaccinated adult horses to measure antibody levels in response to vaccine administration. Antigens tested included West Nile Virus, Eastern Equine Encephalitis, Western Equine Encephalitis, Equine Influenza Virus, Equine Herpes Virus 1 and 4, Tetanus, and 7 different Rabies antigens (3 lab and 4 wild strains). The developed assay was a 7-plex capture antibody, which quantified equine IgG (Immunoglobulin G) that binds viral antigens derived from different rabies virus strains along with pure vaccine samples of the 7 different antigens. A 7-point standard curve was developed to quantify the viral-antigen reactive IgG concentration in vaccinated horse serum. Vaccinated horses increased serum antibody concentration for each antigen post-vaccination with the percent increase ranging between 34.0% for Equine Herpes Virus 4 and 257.3% for Equine Influenza Virus. Use of the novel assay will provide equine veterinarians with an economical method to measure immune activation toward common pathogens of concern. This methodology will provide foundation level information regarding antigen specific IgG concentrations that ultimately may be extrapolated to establish protective levels of immunity resulting in establishment of vaccine protocols.

African horsesickness virus (AHSV) belongs to the Orbivirus genus in the family Reoviridae. This non-enveloped virus consists of an outer capsid formed by two structural proteins, VP2 and VP5, and an inner core formed by structural proteins VP7 and VP3. Three additional structural proteins associated with viral replication, as well as ten dsRNA molecules responsible for replication, are found inside the core. VP7 is the smallest of the structural proteins and each monomer consist of two domains, a hydrophilic top and hydrophobic bottom domain. Upon expression of VP7, the protein spontaneously assembles into trimers. Recombinant expression of the core protein VP7 results in large hexagonal structures formed by a double layer of these VP7 trimers with the hydrophobic bottom domains on the inside and hydrophilic top domains on the outside. The use of these crystal structures as a general display system for the display of foreign peptides/epitopes is being investigated in our group. In this regard, sites for the insertion of foreign peptides/epitopes were constructed at amino acid positions 177, 144 and 200 of the top domain of the VP7 protein and the resultant proteins named vectors AHSV-9 VP7-177, AHSV-9 VP7-144 and AHSV-9 VP7-200. Various inserts ranging from the HIV-1 ELDKWA epitope and FMDV VP1 epitopes to the eGFP peptide were inserted and subsequently analysed for immunogenicity. Results showed that a significant immune response was only elicited if the soluble trimer component of a chimeric VP7 protein was used for inoculation purposes. The crystal particles initially investigated as a display system did not result in any immune response. These results emphasized the importance of protein solubility for eliciting a significant immune response. The importance of solubility prompted an investigation into the use of the Bluetongue virus (BTV) VP7 protein as a vaccine display system. This protein is inherently more soluble than AHSV VP7 and does not result in crystal hexagonal structures if recombinantly expressed. An insertion site analogous to that of the AHSV-9 VP7-177 vector, located at amino acid 177 within an RGD loop in the top domain of VP7 was constructed. This new BTV VP7 vector, BTV-10 VP7-169, was characterised with regard to solubility and the ability to form trimers. In order to investigate the effect on solubility and trimerisation, FMDV VP1 epitope and eGFP were inserted into the BTV-10 VP7-169 vector. Results showed that following the construction of the insertion site, the vector was largely insoluble compared to the AHSV VP7 vectors and that insertion of the abovementioned peptides/epitopes did not have a significant effect on solubility. Although trimers were present, the yield was low compared to that of the AHSV chimeric VP7 proteins. Methods of improving the solubility of the chimeric VP7 proteins were investigated by treatment with solubilisation agents, sarkosyl and L-arginine. The results indicated that a strong denaturant such as sarkosyl can solubilise the particulate component of all chimeric VP7 proteins whereas Larginine had limited effect. The effect of these agents on the folding of the proteins were evaluated using fluorescence, since the ability to fluoresce is regarded as an indicator of correct folding. A comparison of the different VP7-eGFP proteins treated with these solubilisation agents showed that the sarkosyl solubilised proteins were not necessarily correctly folded. These results combined with the previously performed solubility assays indicated that a large proportion of correctly folded chimeric VP7 proteins associate with the particulate fraction. Investigation showed that expression of a large amount of correctly folded chimeric proteins results in the aggregation of these proteins within the expressing host cell. Once harvested these proteins remain associated with the insoluble fraction but can be solubilised by arginine treatment, or in some cases mere resuspension in a low-salt buffer, and used for vaccination purposes. In conclusion, the comparative analyses of solubility and trimerisation for the display vectors indicated that AHSV-9 VP7-144 vector may be most suitable for the display of foreign epitopes/peptides as it consistently yielded the largest component of correctly folded proteins. Furthermore, considering that large amounts of correctly folded chimeric VP7 proteins occurred in the insoluble component of each the VP7 display proteins, this study emphasize that use of solubility assays alone does not provide adequate information regarding the potential of a display vector for vaccination purposes. / Dissertation (MSc)--University of Pretoria, 2013. / Genetics / unrestricted

DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Vaccination is one of the major strategies available for combating viral infections in humans. However, currently available vaccines are not without pitfalls; they are laborious to produce, could potentially be unsafe, and in the case of the highly variable influenza virus need to be reformulated each season. The use of synthetic peptides thus represents an exciting alternative to traditional vaccines. However, these synthetic peptides are not highly immunogenic without the use of potent adjuvants. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of branched peptides, lipidated peptides, or designs combining both of these elements might also enhance the immunogenicity, as they might target Toll-like receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. In this study, a recognized T-B peptide based on the hemagglutinin protein of the A/Puerto Rico/8/34 influenza virus was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties leads to a greatly enhanced activity as compared to other similar T-B diepitope constructs.

The overarching objective of this thesis was to investigate the phenomenon of vaccine hesitancy in Canada and examine relationships among vaccine beliefs, socio-demographic characteristics, and seasonal influenza immunization. Quantitative findings were derived from a national health risk perception survey administered to adults across Canada (N = 1,125). Respondents were asked to provide their level of agreement (1 = do not agree at all to 5 = agree completely) with 2 vaccine-related behaviour statements and 21 vaccine-related beliefs statements. A principal components analysis was performed to reduce the number of belief statements into meaningful components. Two components were retained and reflected negative beliefs about ‘vaccine safety’ and positive beliefs about ‘vaccine regulation and benefits’. Descriptive results presented in the first study indicated a heightened uncertainty about the long-term side effects of vaccination, particularly with respect to the purported link between the Measles-Mumps-Rubella vaccine and the development of autism, among survey respondents. Multivariate analyses identified differences in the endorsement of numerous vaccine beliefs according to age and educational attainment. Findings revealed that older respondents and respondents without a university education demonstrated more negative attitudes towards vaccination, whereas younger respondents and respondents with a university education demonstrated more positive vaccine attitudes, respectively. Finally, both components of vaccine beliefs were significant predictors of vaccine-related behaviours, including discussing information about vaccines with others and reported receipt of the seasonal influenza vaccine. The second study investigated interrelationships among components of vaccine beliefs, socio-demographic characteristics, and reported receipt of the seasonal influenza vaccine. A mediation analysis revealed that higher levels of agreement with the statement ‘I usually get the seasonal flu vaccine’ among older adults was associated with lower levels of agreement with negative beliefs about vaccine safety in conjunction with higher levels of agreement with positive beliefs about the regulation and benefits of vaccines, whereas the opposite was true for younger adults. Also, a significant moderation analysis revealed that among respondents with greater concern about vaccine safety, those with higher educational attainment reported lower levels of agreement with the statement ‘I usually get the seasonal flu vaccine’ compared to those with lower educational attainment. Recognizing the limitations of quantitative findings, a qualitative investigation was undertaken to provide more in-depth insight on the factors driving influenza immunization among healthy adults. A thematic analysis was performed on transcripts from 6 semi-structured focus group discussions with a total of 18 participants residing in Ottawa, Ontario. Findings identified 7 themes and 8 sub-themes related to contextual, vaccine specific, and individual determinants of vaccine hesitancy. Participants predominantly discussed themes related to individual determinants of vaccine hesitancy (perceived severity, susceptibility, and likelihood of contracting the influenza virus; personal interests; interactions with healthcare professionals). The perceived novelty, severity and effectiveness of the influenza vaccine, as well as a lack of information and discontent with communication by government health authorities and the media were also discussed. Overall, findings identified salient themes informing vaccine decision-making and behaviours among a sample of educated adults, which can inform subsequent studies investigating influenza immunization in a more representative sample of Canadian adults.

The antigenic variability of the Influenza virus hinders our ability to develop new therapeutic and vaccine strategies which provide a broad protection against all influenza strains. It has been previously suggested that a means to approach this challenge is to identify conserved sequences within viral proteins and use these for future therapeutic targets. Although such conserved sequences are plentiful amongst the internal viral proteins, their lack of exposure to the host immune system makes mounting an immune response against these regions difficult. Alternatively, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) have been shown to provide host protection against a limited number of influenza strains when used as vaccine targets; however conserved regions within these proteins which are also antibody accessible are extremely rare. My Ph.D. thesis project is focused on investigating the functional role of a conserved region within the NA protein and to further determine the protection afforded by a monoclonal antibody to this region. In a comprehensive bioinformatics analysis, the only universally conserved sequence amongst all influenza A and B viral NA has been previously identified as being located between amino acids (a.a.) 222-230 (dubbed the HCA-2 region). However, the potential role of this region remains largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics and growth kinetics, I have found that substitutions in this sequence significantly affect viral replication by impairing the catalytic activity, substrate-binding and thermostability of NA. These findings prompted me to further investigate if antibody to this region may provide protection against influenza infection. Indeed, universal monoclonal antibody (HCA-2 MAb) against this peptide provided broad inhibition against all nine subtypes of NA in vitro and heterosubtypic protection in mice challenged with lethal doses of mouse-adapted viruses. I further demonstrated that residues within this peptide that are exposed on the surface of NA and located in close proximity to the active site, I222 and E227, are indispensable for antibody-mediated inhibition. These data are the first to demonstrate a monoclonal antibody against the NA protein which provides heterosubtypic protection. Since I observed that the HCA-2 antibody provided a broad inhibition against all nine subtypes of influenza A NA, I decided to investigate whether this inhibitory effect could be extended against Influenza B. Here, I have further reported that HCA-2 MAb provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. I also demonstrate that the growth and NA enzymatic activity of two drug resistant influenza B strains are also inhibited by the HCA-2 antibody. The findings of my Ph.D. thesis project have thus demonstrated that the HCA-2 region is paramount to optimal viral function. Additionally, my data show that antibodies generated against this region provide heterosubtypic protection both in vitro and in vivo and against drug resistant strains. These results indicate that this universally conserved epitope should be further explored as a potential target for future antiviral intervention and vaccine-induced immune responses.

Malaria remains the most serious and deadly parasitic disease, affecting millions of people mostly in the poorest countries in the world. With no vaccine likely in the foreseeable future, drugs remain the best means of controlling the disease. Plants have provided most of the antimalarial drugs so far and it is likely that more antimalarial drugs will be discovered in this way. A previous study on South African plants yielded very good results on the extract level. In this study Croton steenkampianus leaf extract was selected for isolation of active principles. Bio-guided fractionation of the extract was done on silica column chromatography and Sephadex column chromatography. Five compounds, two favonoids, a triterpene and two new diterpenes, with a novel skeleton were isolated. Compounds were identified with NMR, MS and X-ray crystallography. Antiplasmodial activity of the compounds varied from moderate to excellent, with crotrene A having excellent activity. Further studies on the antimalarial potential of this compound are planned. Cytotoxicity of compounds and extract were determined against human lymphocytes. Results obtained had an ID50 between >16,61 µg/ml. The therapeutic indexes were between 2.75 and 55.18, showing poor to moderate selectivity towards Plasmodium. Crotrene A had the best therapeutic index and more detailed studies on its cytotoxicity are necessary. Resistance to antimalarial drugs is a major problem in effective treatment of the disease. One way of overcoming this problem is combination drugs working synergistically. Chloroquine the most affordable antimalarial drug was combined with the isolated compounds. Two compounds showed synergistic activity with crotrene A having excellent activity, completely reversing chloroquine resistance. This combination of drugs showed no synergistic cytotoxic effects and its potential as a drug will be further investigated. The mode of action of antimalarial drugs can provide useful information about the long term potential and the likelihood of resistance development. Crotrene A was subjected to a basic test to determine a possible mode of action. Results showed a marked effect in the early phase of development (rings). The results suggest a very potent mode of action able to reduce the amount of parasites quickly and this holds promise for further development of this compound. / Thesis (DPhil)--University of Pretoria, 2008. / Plant Science / unrestricted