Magister Pharmaceuticae - MPharm / The declining efficacy of antimalarial drugs against resistant Plasmodium falciparum strains in several endemic regions has amplified the world’s burden of neglected diseases. This has highlighted the need for alternate strategies for chemotherapy and chemoprophylaxis. Since malaria is prevalent primarily in third world countries, it is critical for novel therapies to be affordable. Previous research has found that some antihistamines possess inherent antimalarial activity and cause a marked reversal of chloroquine resistance in vitro and in vivo. Promising results have been demonstrated when chlorpheniramine was combined with chloroquine to reverse chloroquine resistance in two African studies (Sowunmi et al, 1997; Abok., 1997).Recently, astemizole and its principle human metabolite desmethylastemizole were identified as potent inhibitors of Plasmodium falciparum at sub-micromolar concentrations in both chloroquine sensitive and chloroquine resistant parasites, showing efficacy in vitro and in two mouse models. The promising results observed with these studies warrant a more comprehensive understanding of how antihistamines interact with the malaria parasite. Additionally, analysing the different structural and mechanistic characteristics of antihistamines may lead to the design and development of effective and affordable antimalarial agents or chloroquine resistance modulators.This thesis describes the antimalarial activity of mainly off-patent (generic) antihistamines by comparing the efficacy of a total of 24 antihistamines, representing histamine1, histamine2, and histamine3 receptor antagonists, against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. Cyproheptadine, ketotifen, loratadine, desloratadine, 3-(1HImidazol-4-yl) propyldi (p-fluorophenyl) methyl ether hydrochloride and ciproxifan display IC50 values less than 4μg/ml. There was no significant difference in the sensitivity to antihistamines among the chloroquine sensitive and resistant parasites tested. A tricyclic nucleus appears to be an important structural scaffold for antihistamines which exhibit low IC50 values. Synergistic studies indicate that enhancement of the antimalarial effect of chloroquine on P.falciparum was observed with the ethanolamines against the chloroquine sensitive parasites.Cyproheptadine, ketotifen and desloratadine exerted a marked synergistic action with chloroquine against chloroquine sensitive and resistant parasites. Chlorpheniramine exhibited synergism with chloroquine against resistant parasites only.Microscopic studies illustrate the effect of antihistamines on parasite morphology when compared to control. Using immunofluorescence microscopy, it was seen that ketotifen decreases haemoglobin localization while cyproheptadine increases haemoglobin localization in the parasite’s food vacuole. Western blots have confirmed these results, in addition to indicating that chlorpheniramine decreases the haemoglobin content in the parasite. The results confirm that certain antihistamines do indeed cause a reduction in the growth of malaria parasites. Furthermore, the histamine1 and histamine3 receptor antagonists are most active while histamine2 receptor antagonists have no antimalarial activity. Microscopic studies suggest that antihistamines do not exert their antimalarial effect via a single mechanism of action.I wish to express my sincere appreciation to the following people and institutions whose supervision and assistance made the presentation of this thesis possible:My supervisor, Prof. Henry Leng. Thank for always believing in me. Your encouragement, kindness and calm temperament has given me the strength to complete this thesis even when times were tough. Your wisdom and understanding will always be remembered.My co-supervisor, Prof. Pete Smith. I sincerely thank you for allowing me the opportunity to work in your laboratory and for welcoming me into the department. Your kindness and welcoming attitude will forever be appreciated. Thank you for always being patient and understanding.Dr. Uschi Wiehart. Thank you for all the help in the laboratory and always being there for me. I truly value and appreciate your contribution to this thesis. Your friendship has added so much positive energy to my life. Thank you for your wisdom, inspirational advice and unfaltering encouragement Sumaya and Ntokosi, your help, advice and company in tissue culture, are truly appreciated.The UCT, Pharmacology students. Thank for all your assistance.My dearest Pharmaceutical Chemistry colleagues, Jaques Joubert, for your friendship and support and for always listening and Prof. Peter Eagles, your kindness, support and wise advice has given me strength when I needed it most. To my other School of Pharmacy colleagues. Prof. Sarel Malan and team, for your support and motivation.To my family for all your support and wisdom and to my baby brothers; Omar and Uzair for all the joy that you bring to my life.And finally to my dearest husband, Zaheer for all your love and support throughout my studies and for taking me to UCT to culture parasites every weekend

Objective: The objective of this retrospective cohort study is to assess whether demographic and anthropometric parameters, laboratory tests, co-morbidity, co-infection, treatment regimen, IRIS and adherence predict response to HAART as measured by CD4 count, weight gain and functional status in a cohort of patients in Roma, the Kingdom of Lesotho. Method: Data were collected from a computerised database of the Antiretroviral Centre of the hospital. A cohort of 300 subjects was identified from hospital records from January 2007. Each of these subjects was followed up over a period of 12 months with data obtained for at least two visits within the 12-month span. Data were obtained on weight and CD4 at baseline, three months and also at six and 12 months, and data for haemoglobin were obtained only at 12 months. Variables that may be potential confounders were identified and univariate and multivariate logistic regression analyses were carried out to establish differences independent of confounding factors for the combined endpoints, as well as for each endpoint separately. Results: Three-hundred patient records were analysed. Approximately 70% of the patients had a CD4 increase of at least 150 cells over baseline values at the end of the review period and in 52.3% of the patients an increase in weight of 10% over baseline measurements was seen. Seventy-nine patients (26.3%) had a haemoglobin level of at least 14g/dL at 12 months, regardless of baseline values or gender. The inclusion of Zidovudine (AZT) in treatment regimens was found in 73% of the patients and in multivariate analysis AZT was associated with not having anaemia at the end of the review period. However there was a slight reduction in haemoglobin level in the first two to three months of therapy in comparison with both Stavudine (d4T) and Tenofovir (TDF) but not significant enough to result in clinical anaemia. Baseline CD4 values were similar for all treatments options but dissimilar in other outcome variables and continued to vary significantly throughout the review period. The outcomes of multivariate analyses suggest that the male gender appears to have better response to HAART as seen in each of the multivariate models. The most important determinant of haemoglobin response was baseline haemoglobin values. In the haemoglobin-associated multivariate model, HAART is associated with an increase in haemoglobin over baseline values. A history of TB prior to HAART was a major factor in weight response and it is thought to be as a result of IRIS, which is the unmasking of latent infections as the immune system reconstitutes. CD4 values have no direct influence on weight however, but an increase in weight was observed in all therapy groups. Conclusion: Clinical and immunological parameters can be used to monitor response to HAART and predict treatment outcomes. These parameters can be organised into monitoring tools that will be useful in resource-limited areas. This study suggests that AZT-containing regimens appear not to result in anaemia and that symptomatic anaemia might need additional investigation. Treatment with TDF appeared to have shown the best possible response pattern more but patients on TDF therapy will have to be included in the study to justify this observation. / Dissertation (MSc)--University of Pretoria, 2012. / Clinical Epidemiology / unrestricted

Many organisms have geographical distributions extending from the tropics to near polar regions or can experience up to 30°C temperature variation within the lifespan of an individual. Two forms of evolutionary adaptation to such wide ranges in ambient temperatures are frequently discussed: local adaptation and phenotypic plasticity. The freshwater planktonic crustaceanDaphnia magna, whose range extends from South Africa to near arctic sites, shows strong phenotypic and genotypic variation in response to temperature. In this study, we use D. magna clones from 22 populations (one clone per population) ranging from latitude 0° (Kenya) to 66° North (White Sea) to explore the contributions of phenotypic plasticity and local adaptation to high temperature tolerance. Temperature tolerance was studied as knockout time (time until immobilization, Timm) at 37°C in clones acclimatized to either 20°C or 28°C. Acclimatization to 28°C strongly increased Timm, testifying to adaptive phenotypic plasticity. At the same time, Timm significantly correlated with average high temperature at the clones' sites of origin, suggesting local adaptation. As earlier studies have found that haemoglobin expression contributes to temperature tolerance, we also quantified haemoglobin concentration in experimental animals and found that both acclimatization temperature (AccT) and temperature at the site of origin are positively correlated with haemoglobin concentration. Furthermore, Daphnia from warmer climates upregulate haemoglobin much more strongly in response to AccT, suggesting local adaptation for plasticity in haemoglobin expression. Our results show that both local adaptation and phenotypic plasticity contribute to temperature tolerance, and elucidate a possible role of haemoglobin in mediating these effects that differs along a cold-warm gradient.

Many organisms have geographical distributions extending from the tropics to near polar regions or can experience up to 30°C temperature variation within the lifespan of an individual. Two forms of evolutionary adaptation to such wide ranges in ambient temperatures are frequently discussed: local adaptation and phenotypic plasticity. The freshwater planktonic crustaceanDaphnia magna, whose range extends from South Africa to near arctic sites, shows strong phenotypic and genotypic variation in response to temperature. In this study, we use D. magna clones from 22 populations (one clone per population) ranging from latitude 0° (Kenya) to 66° North (White Sea) to explore the contributions of phenotypic plasticity and local adaptation to high temperature tolerance. Temperature tolerance was studied as knockout time (time until immobilization, Timm) at 37°C in clones acclimatized to either 20°C or 28°C. Acclimatization to 28°C strongly increased Timm, testifying to adaptive phenotypic plasticity. At the same time, Timm significantly correlated with average high temperature at the clones' sites of origin, suggesting local adaptation. As earlier studies have found that haemoglobin expression contributes to temperature tolerance, we also quantified haemoglobin concentration in experimental animals and found that both acclimatization temperature (AccT) and temperature at the site of origin are positively correlated with haemoglobin concentration. Furthermore, Daphnia from warmer climates upregulate haemoglobin much more strongly in response to AccT, suggesting local adaptation for plasticity in haemoglobin expression. Our results show that both local adaptation and phenotypic plasticity contribute to temperature tolerance, and elucidate a possible role of haemoglobin in mediating these effects that differs along a cold-warm gradient.

Daphnia is a widespread freshwater zooplankton species, which is both a classic and emerging new model for research in ecological physiology, ecotoxicology and evolutionary biology of adaptation to novel environments. Heat tolerance in Daphnia is known to depend both upon evolutionary history of a genotype and on individuals' acclimation to elevated temperature and to correlate with the level of haemoglobin expression. We demonstrate the existence of north-south gradient of heat tolerance in North American D. pulex, which is not associated with any parallel changes in haemoglobin expression. Geographically distinct clones differ in the way their haemoglobin expression changes due to acclimation to a sub-stressful (28°C) temperature, but these changes are not correlated with the latitude of clones' origin. Likewise, the effect of acclimation to sub-stressful temperature is independent from, and cannot be fully explained by, haemoglobin expression changes during acclimation. The degree of oxidative damage to haemoglobin, measured as the ratio of absorbance at 540:576 nm at the acclimation temperature, is a strong predictor of 28°C-acclimated Daphnia survival during an acute heat exposure. The comparison of haemoglobin expression in resistant and tolerant clones acclimated to different temperatures indicates that tolerant clones exhibit canalization of haemoglobin expression, possessing a high level of haemoglobin even at non-stressful temperatures. We discuss the evolutionary biology of adaptation and acclimation to elevated temperatures in an ecologically important component of freshwater ecosystems in the context of global climate change.

Magister Artium (Human Ecology) - MA(HE) / Iron deficiency anaemia is the most common abnormality of the blood in childhood (Karabus 1987). If the quantity of iron lost by the body exceeds iron intake, the body will draw on its iron reserves to counterbalance this deficit. However, the continuance of an iron imbalance will eventually lead to a reduction in body iron stores. Because iron forms an integral component of the oxygen transport mechanism of the body, it is understandable that the functional capacity of this system will be compromised under conditions of iron deficiency. A deficit in oxygen transport capacity will presumably indicate a decreased capacity to persevere in the face of continuing strenuous physical activity. The decrement in physical aerobic working capacity (maximal oxygen consumption) will largely be indicative of the decrease in oxygen transport capacity. Routine haemoglobin determinations carried out in the outpatient department of the Red Cross War Memorial Children's Hospital in Cape Town showed that many Coloured and African pre-schoolgoing children had abnormally low haemoglobin levels which occurred as a manifestation of iron deficiency anaemia (Lanzkowsky 1961). In another similar but more recent study also in the Cape Peninsula, Lamparelli et al. (1988) showed that the prevalence of iron deficiency anaemia in Coloured and African children was 15.5 % and 36.0 %, respectively . In this study, the condition of iron deficiency anaemia was particularly pronounced in urban Coloured children. In both these studies done in the Western Cape, the majority of Coloured children were classified as coming from the lowest socioeconomic income group in the community. In the majority of studies concerning the relationship between socioeconomic status and iron deficiency anaemia, it is often stated that low socioeconomic circumstances are significantly correlated to low blood haemoglobin levels (Expert Scientific Working Group 1985; Lanzkowsky 1959; Lanzkowsky 1961; World Health Organization 1972; World Health Organization 1975).

ABSTRACTBackground: Determination of blood loss can be a crucial factor at surgical procedures, especially when the amount of blood is small and mixed with other fluids. The existing methods to measure this are still not supported with evidence enough.Aim: To validate the accuracy of the HemoCue® system (HemoCue, Ängelholm, Sweden) at estimation of blood loss in a setup where blood is mixed with saline and saliva.Materials and methods: The haemoglobin concentration of defibrinated horse blood was measured using the haematology analysers Hemocue® 201+ and Hemocue Plasma/Low Hb Photometer. Series of non-diluted blood (control) and blood diluted with saline and saliva (test) were conducted to mimic a clinical situation at oral and maxillofacial surgical treatment. Following each dilution, a new measurement of the haemoglobin concentration was performed using the appropriate haematology analyser to measure the blood loss.Results: There were no statistically significant differences regarding the measured Hb concentration in relation to the degree of dilution. The accuracy of measured blood volume in the diluted and non-diluted blood (control) was within a level of ± 11,5%.Conclusion: The measurement of haemoglobin concentrations in a mixture of blood, saline, and saliva, proved to be accurate when compared to non-diluted blood. It is suggested that the HemoCue® system can be applied in the field of oral and maxillofacial surgery to improve the accuracy of the blood loss measurement.

Objetivos: Este Estudo randomizado duplo cego placebo controlado comparou, clínica, sitemica e imunologicamente um protocolo de Terapia fotodinâmica antimicrobiana (TFA) de aplicações múltiplas com um protocolo já consagrada com o uso da Doxiciclina sistêmica no tratamento da doença periodontal em pacientes diabéticos tipo 2 descontrolados. Materiais e Métodos: Vinte seis pacientes com HbA1c> 7% foram selecionados e randomicamente alocados em dois grupos que receberam raspagem e alisamento radicular. Um dos grupos recebeu a aplicação adjunta de aplicações múltiplas de TFA (n=12) e o outro utilizando a doxiciclina sistêmica na dose de 100mg (n=14). Os parâmetros monitorados foram índice de placa, Sangramento à sondagem, Profundidade de sondagem, Supuração, Recessão gingival e nível clinico de inserção relativo, o parâmetro sistêmico avaliado foi a HbA1c, medida antes e 3 meses pós tratamento. Os níveis de IL1-β, TNF-α e TGF-β foram medidos antes, 1 e 3 meses pós tratamento através da coleta de fluido crevicular gengival. Resultados: Não houveram diferenças significantes em nenhum dos parâmetros clínicos avaliados e nos níveis de HbA1c. O uso do antibiótico sistêmico demonstrou ser superior na redução de IL1-β até o período de 1 mês pós tratamento, porém, em 3 meses a TFA se mostrou superior. Não houve diferença na redução dos níveis de TNF-α e TGF-β entre os grupos. Conclusões: Ambos tratamentos foram eficientes nas melhoras dos parâmetros clínicos e sistêmicos. A TFA parece possuir maior estabilidade na redução dos níveis de citocinas inflamatórias. / Aim: This randomized, double-blind, placebo-controlled, clinical study compared a multiple application Antimicrobial Photodynamic Therapy (aPDT) treatment protocol with systemic doxycycline as adjuvant to scaling and root planning, to treat chronic periodontitis on type 2 diabetic patients on clinical, systemic and immune-inflammatory outcomes. Materials and Methods: Twenty six patients with Hba1c >7% were randomically allocated in two groups, SRP+Doxy (n=14) using systemic doxycycline 100 mg and SRP+aPDT (n=12) with multiple applications (0, 3, 7 and 14 days). Monitored parameters of plaque score (PS), bleeding on probe (BOP), probing depth (PD), suppuration (S), gingival recession, and relative clinical attachment level (RCAL), glycated haemoglobin (HbA1c) were measured at baseline and 3 months after therapy, the cytokine profile was assessed at 0, 1 and 3 month to measure IL1-β, TNF-α and TGF-β on Gingival Crevicular Fluid. Results: There were no statistically significant differences on intergroup on clinical parameters and HbA1c levels. Systemic doxycycline shoed difference in reduction of IL1-β at 1 month, but aPDT better results at 3 months IL1-β levels. There were no differences between TNF-α and TGF-β trough experimental times Conclusions: Both treatments were effective to improve clinical and systemic outcomes and aPDT seems to have a great stability on of IL1-β reductions. (Clinicaltrials.gov Identifier: NCT01175720).

Objetivos: Este Estudo randomizado duplo cego placebo controlado comparou, clínica, sitemica e imunologicamente um protocolo de Terapia fotodinâmica antimicrobiana (TFA) de aplicações múltiplas com um protocolo já consagrada com o uso da Doxiciclina sistêmica no tratamento da doença periodontal em pacientes diabéticos tipo 2 descontrolados. Materiais e Métodos: Vinte seis pacientes com HbA1c> 7% foram selecionados e randomicamente alocados em dois grupos que receberam raspagem e alisamento radicular. Um dos grupos recebeu a aplicação adjunta de aplicações múltiplas de TFA (n=12) e o outro utilizando a doxiciclina sistêmica na dose de 100mg (n=14). Os parâmetros monitorados foram índice de placa, Sangramento à sondagem, Profundidade de sondagem, Supuração, Recessão gingival e nível clinico de inserção relativo, o parâmetro sistêmico avaliado foi a HbA1c, medida antes e 3 meses pós tratamento. Os níveis de IL1-β, TNF-α e TGF-β foram medidos antes, 1 e 3 meses pós tratamento através da coleta de fluido crevicular gengival. Resultados: Não houveram diferenças significantes em nenhum dos parâmetros clínicos avaliados e nos níveis de HbA1c. O uso do antibiótico sistêmico demonstrou ser superior na redução de IL1-β até o período de 1 mês pós tratamento, porém, em 3 meses a TFA se mostrou superior. Não houve diferença na redução dos níveis de TNF-α e TGF-β entre os grupos. Conclusões: Ambos tratamentos foram eficientes nas melhoras dos parâmetros clínicos e sistêmicos. A TFA parece possuir maior estabilidade na redução dos níveis de citocinas inflamatórias. / Aim: This randomized, double-blind, placebo-controlled, clinical study compared a multiple application Antimicrobial Photodynamic Therapy (aPDT) treatment protocol with systemic doxycycline as adjuvant to scaling and root planning, to treat chronic periodontitis on type 2 diabetic patients on clinical, systemic and immune-inflammatory outcomes. Materials and Methods: Twenty six patients with Hba1c >7% were randomically allocated in two groups, SRP+Doxy (n=14) using systemic doxycycline 100 mg and SRP+aPDT (n=12) with multiple applications (0, 3, 7 and 14 days). Monitored parameters of plaque score (PS), bleeding on probe (BOP), probing depth (PD), suppuration (S), gingival recession, and relative clinical attachment level (RCAL), glycated haemoglobin (HbA1c) were measured at baseline and 3 months after therapy, the cytokine profile was assessed at 0, 1 and 3 month to measure IL1-β, TNF-α and TGF-β on Gingival Crevicular Fluid. Results: There were no statistically significant differences on intergroup on clinical parameters and HbA1c levels. Systemic doxycycline shoed difference in reduction of IL1-β at 1 month, but aPDT better results at 3 months IL1-β levels. There were no differences between TNF-α and TGF-β trough experimental times Conclusions: Both treatments were effective to improve clinical and systemic outcomes and aPDT seems to have a great stability on of IL1-β reductions. (Clinicaltrials.gov Identifier: NCT01175720).

Previous data have shown that the most prevalent, indirect plasma volume (PV) measurement technique, which utilises changes in haematocrit (Hct) and haemoglobin concentration ([Hb]), underestimates actual PV changes during immersion, when compared to a direct tracer-dilution method. An increase in the F-cell ratio (whole-body haematocrit (Hctw) to large-vessel haematocrit (Hctv) ratio) has been purported as a possible explanation, probably due to hydrostatic and thermally-mediated changes during water immersion. Previous investigators have not quantified the F-cell ratio during immersion. Therefore, this study sought to determine the effect of the F-cell ratio on the indirect method during both, thermoneutral and cold-water immersions. Seven healthy males were tested three times, seated upright in air (control: 21.2°C SD ±1.1), and during thermoneutral (34.5oC SD ±0.2) and cold-water immersion (18.6oC SD ±0.2), immersed to the third intercostal space for 60 min. Measurements during the immersion tests included PV (Evans blue dye column elution, Evans blue dye computer programme, and Hct [Hb]), red cell volume (RCV; sodium radiochromate), cardiac frequency (fc) and rectal temperature (Tre). Plasma volume during the control trial remained stable, and equivalent across the three tests. There was a hydrostatically-induced increase in PV during thermoneutral immersion, when determined by the Evans blue dye method (16.2%). However, the Hct/[Hb] calculation did not adequately reflect this change, and underestimated the relative PV change by 43%. In contrast, PV decreased during cold immersion when determined using the Evans blue dye method by 17.9% and the Hct/[Hb] calculation by 8.0%, respectively, representing a 52% underestimation by the latter method. There was a non-significant decline in RCV during both immersions. Furthermore, an increase (8.6%) and decrease (-14.4%) in blood volume (BV) was observed during thermoneutral and cold-water immersions, respectively. The decline in RCV during thermoneutral immersion attenuated the BV expansion. Despite the disparity between the PV methods, there was no increase in the F-cell ratio during either immersion. In contrast, there was a significant decline in the F-cell ratio during the control: air and thermoneutral immersion, which may indicate that other, undefined variables may impact on the stability of the red cell compartment. The current study is the first to show that the Hct/[Hb] method clearly underestimates PV changes during both thermoneutral and cold-water immersion. Furthermore, RCV was shown, for the first time, to decline during both immersions. However, the changes in the F-cell ratio during this study, did not account for the underestimation of PV change using the Hct/[Hb] method.