Acute Phase Response and the Possible Involvement of an Endotoxin-Like Molecule in the Pathogenesis of Murine African Trypanosomiasis

This thesis describes a series of studies in mice infected with either Trypanosoma brucei brucei and Trypanosoma congolense, with the primary aim of identifying the pathogenic mechanism responsible for the acute phase response which occurs during African trypanosomiasis. The acute phase response was monitored by measuring the acute phase proteins, serum amyloid P-component (SAP) and haptoglobin (Hp) during a tissue invasive and non-tissue invasive infection. The possible involvement of trypanosome endotoxin in the pathogenesis of trypanosomiasis was also investigated, by measuring the endotoxin levels and acute phase proteins in plasma of infected animals during treatment with either a general, systemic antibiotic (norfloxacin) or one that is restricted to the intestinal tract and binds to and neutralises the pathogenic effects of endotoxin (polymyxin-B). In addition, the endotoxin-like molecule(s) in trypanosome lysates or enriched protein membranes were examined to determine the presence of lipopolysaccharide or the active moiety, lipid-A, using polyacrylamide gel electrophoresis and the silver stain, or Western blot utilising a lipid-A specific monoclonal antibody. Chapter I comprises an introduction and literature review on African trypanosomiasis with emphasis on endotoxin, cytokines and acute phase proteins. Chapter II describes the general materials and methods used in these studies. Chapter III describes the development of a direct antigen enzyme linked immunosorbent assay (ELISA) for the quantification of mouse serum amyloid P-component. Chapter IV describes the acute phase response during chronic murine trypanosome infection with tissue invasive and non-invasive trypanosomes, and the effect of subcurative treatment with the trypanocidal drug, diminazine aceturate. It was found that the acute phase proteins SAP and haptoglobin increased significantly after infection. Following infection with T. b. brucei, SAP increased in both infections to peak 7-10 days after infection (DAI), after which it declined to levels just above control values where it remained for the rest of the infection period. In contrast, with T. congolense infection a second peak occurred around 34 DAI. The levels of SAP were not affected by the treatment with diminazine aceturate. Haptoglobin (Hp) increased to peak at 7-10 DAI, and remained elevated throughout the infection in both experiments, but decreased significantly following treatment with diminazine aceturate in the T. congolense but not in the T. brucei infection where it remained elevated. The diminazine aceturate treatment in T. brucei infected mice resulted in severe cellular infiltration of mononuclear cells in the brains of these animals. As acute phase response occurs with both species of trypanosomes, it was concluded that tissue invasiveness is not the main means by which trypanosomes initiate tissue damage in infected hosts but that tissue pathology in the brain can cause synthesis of acute phase protein in the liver. Chapter V describes the changes in plasma endotoxin-like activity, the concentration of the acute phase proteins, SAP and Hp, and tissue pathology during chronic T. b. brucei infections in mouse, the presence or absence of an antibiotic umbrella, with norfloxacin or polymyxin-B. The concentration of acute phase proteins and endotoxin-like activity increased significantly following infection. The animals also showed varying pathological changes during the different stages of infection. The spleen showed cellular activity, livers were markedly infiltrated with inflammatory cells with occasional necrosis, while the choroid plexus of the brains was infiltrated by trypanosome. Treatment with the antibiotic polymyxin-B, had no significant effect on any of the parameters examined, whereas in the norfloxacin-treated animals there was a small but significant decrease in the haptoglobin levels in the terminal stages of infection after 21 DAI. The norfloxacin-treated animals also showed reduced liver pathology but only in the early stages of infection, i.e., before 21 DAI. Chapter VI describes the characterisation of trypanosome lysate and protein-enriched membrane proteins by the silver stain and by Western blot. The results showed that trypanosomes do not contain a gram negative bacteria-like LPS molecule. On the other hand, the lipid-A monoclonal antibody (8A1) recognised epitopes on trypanosome lysate and protein-enriched membranes on Western blots; this were destroyed by pre-treatment of the samples with proteinase K, suggesting they are protein in nature or are associated with protein. The general discussion and conclusions drawn from the study are presented in chapter VII. African trypanosomiasis causes an acute phase response and tissue damage probably by the production of pro-inflammatory cytokines which are induced probably by molecule(s) from trypanosomes with endotoxin-like activity. The molecule(s) responsible for the increased endotoxin-like activity is of trypanosome origin. Although this molecule(s) has lipid-A like activity and epitopes recognised by lipid-A monoclonal antibody, this trypanosome molecule(s), is dissimilar to the gram negative bacteria LPS

Antioxidant activity and effects of Kenyan Tea (Camellia sinensis) on the liver function and serum biochemistry in male Wistar rats

Background: Tea is a beverage that is most widely consumed worldwide. Studies have shown that oral consumption of tea has health benefits however, there is paucity of data in Kenya detailing the biochemical effects of tea in the liver and elucidation of its mechanism of action.Methods: The polyphenol composition and antioxidant capacity of tea were determined by HPLC and the Folins Ciocalteu spectrophotometric methods. Metal levels were determined using flame Atomic Absorption Spectrometer (AAS). Aqueous black and green tea extracts were administered to the rats at dosages of 400mg/kg b.w.t. The effect of tea on total blood proteins, Albumin, ZHX1, TBARS, AST, ALP and ALT were determined by spectrophotometric methods. The body weight of each rat was also determined at one week interval.Results: Total Polyphenols (TP), Total Catechins (TC) and Antioxidant Activity (AA) between the black and green teas were significantly (P0.05) effect on TP, ALB, ALT, AST, ALP, MDA and ZHX1 in the test animals compared with the controls. This data indicates that green tea is rich in catechins while black tea being rich in Theaflavins (TFs) and Thearubigins (TRs). Both tea products possess essential and non-essential metals well within the maximum permissible concentrations.Conclusions: Findings from this study indicate both green and black tea aqueous extracts have polyphenols and high antioxidant activity. Administration of the aqueous tea extracts have no toxicological effect on the liver.

Tracking the Feeding Patterns of Tsetse Flies (Glossina Genus) by Analysis of Bloodmeals Using Mitochondrial Cytochromes Genes

Tsetse flies are notoriously difficult to observe in nature, particularly when populations densities are low. It is therefore difficult to observe them on their hosts in nature; hence their vertebrate species can very often only be determined indirectly by analysis of their gut contents. This knowledge is a critical component of the information on which control tactics can be developed. The objective of this study was to determine the sources of tsetse bloodmeals, hence investigate their feeding preferences. We used mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) gene sequences for identification of tsetse fly blood meals, in order to provide a foundation for rational decisions to guide control of trypanosomiasis, and their vectors. Glossina swynnertoni were sampled from Serengeti (Tanzania) and G. pallidipes from Kenya (Nguruman and Busia), and Uganda. Sequences were used to query public databases, and the percentage identities obtained used to identify hosts. An initial assay showed that the feeds were from single sources. Hosts identified from blood fed flies collected in Serengeti ecosystem, included buffaloes (25/40), giraffes (8/40), warthogs (3/40), elephants (3/40) and one spotted hyena. In Nguruman, where G. pallidipes flies were analyzed, the feeds were from elephants (6/13) and warthogs (5/13), while buffaloes and baboons accounted for one bloodmeal each. Only cattle blood was detected in flies caught in Busia and Uganda. Out of four flies tested in Mbita Point, Suba District in western Kenya, one had fed on cattle, the other three on the Nile monitor lizard. These results demonstrate that cattle will form an integral part of a control strategy for trypanosomiasis in Busia and Uganda, while different approaches are required for Serengeti and Nguruman ecosystems, where wildlife abound and are the major component of the tsetse fly food source

Combined Antibacterial and Antifungal Activities of Eucalyptus citriodora and Syzygium aromaticum Essential Oils

Background: The increasing proportion of skin infections encountered in general practice represents a substantial level of morbidity. The emergence of multi-drug resistant strains is a formidable threat to the fight against skin diseases and hence alternative forms of treatment are essential. Syzygium aromaticum and Eucalyptus citriodora oils as single entities have demonstrated potency against some of the concerned micro-organisms and any synergistic activity between the two oils could minimise development of resistance by the microorganisms to the two oils. Objective: The aim of this study was to evaluate for synergism between Eucalyptus citriodora and Syzygium aromaticum essential oils against selected pathogenic microorganisms of the skin. Materials and methods: Eucalyptus citriodora (eucalyptus oil) and Syzygium aromaticum (clove oil) essential oils were used in this study. In-vitro antimicrobial activities of Sysygium aromaticum and Eucalyptus citriodora oils, alone and in combination were tested against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa, E. coli ATCC 25922, MRSA, Candida albicans, Trichophyton mentagrophytes, Microsporum gypseum and Cryptococcus neoformans. Results: The combination of the two oils exhibited synergistic activity against Staphylococcus aureus (FICI: 0.240), E. coli (FICI: 0.54), MRSA (FICI: 0.48), and Microsporum gypseum (FICI: 0.36) while the combination exhibited additive activity against Candida albicans (FICI: 2.04). Conclusion: The combination of clove and eucalyptus oils possesses synergistic activity against most of the test pathogens and therefore may be combined for enhanced antimicrobial activity against a wide range of skin disease-causing microorganisms. Keywords: antifungal, antibacterial, synergism, Eucalyptus citriodora, Syzygium aromaticum, essential oil

Combined Antibacterial and Antifungal Activities of Eucalyptus citriodora and Syzygium aromaticum Essential Oils - Supporting Information

Background: The increasing proportion of skin infections encountered in general practice represents a substantial level of morbidity. The emergence of multi-drug resistant strains is a formidable threat to the fight against skin diseases and hence alternative forms of treatment are essential. Syzygium aromaticum and Eucalyptus citriodora oils as single entities have demonstrated potency against some of the concerned micro-organisms and any synergistic activity between the two oils could minimise development of resistance by the microorganisms to the two oils. Objective: The aim of this study was to evaluate for synergism between Eucalyptus citriodora and Syzygium aromaticum essential oils against selected pathogenic microorganisms of the skin. Materials and methods: Eucalyptus citriodora (eucalyptus oil) and Syzygium aromaticum (clove oil) essential oils were used in this study. In-vitro antimicrobial activities of Sysygium aromaticum and Eucalyptus citriodora oils, alone and in combination were tested against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa, E. coli ATCC 25922, MRSA, Candida albicans, Trichophyton mentagrophytes, Microsporum gypseum and Cryptococcus neoformans. Results: The combination of the two oils exhibited synergistic activity against Staphylococcus aureus (FICI: 0.240), E. coli (FICI: 0.54), MRSA (FICI: 0.48), and Microsporum gypseum (FICI: 0.36) while the combination exhibited additive activity against Candida albicans (FICI: 2.04). Conclusion: The combination of clove and eucalyptus oils possesses synergistic activity against most of the test pathogens and therefore may be combined for enhanced antimicrobial activity against a wide range of skin disease-causing microorganisms. Keywords: antifungal, antibacterial, synergism, Eucalyptus citriodora, Syzygium aromaticum, essential oil

Tea (Camellia sinensis) infusions ameliorate cancer in 4TI metastatic breast cancer model

Abstract Background Tea (Camellia sinensis) infusions are widely consumed beverages with numerous health benefits. However, physiological and molecular responses mediating these activities are poorly understood. Method Three replicates of 4TI cancer cell suspension (2.0 × 105 cells/ml) were challenged in vitro with various concentrations of green, black and purple tea infusions to asseses their cytoxicity and associated differentially expressed genes in the cells. Inhibitory activity was tested by using serial dilutions of respective tea infusions in a 96 well ELISA plate. Results Green tea had the highest inhibition on 4TI cells proliferation at a concentration of IC50 = 13.12 μg/ml. Further analysis of the 4TI cancer cell line treated with tea using 454 pyrosequencing generated 425,696 reads with an input mean length of 286.54. Trimmed sequences were imported on a CLC genomic workbench v7.03 and annotated on a reference mouse genome (Mus musculus strain C57BL/6 J). Results revealed a differential expression of apoptosis related genes in the transcriptome. Casp8, Casp9, Casp3, Casp6, Casp8AP2, Aifm1, Aifm2 and Apopt1 genes were significantly upregulated indicating the process of apoptosis was initiated and executed. Conclusion These findings on caspases offer valuable information on the mechanism of tea as an anticancer agent and will contribute to further research in future novel treatments

Major acute phase response of haptoglobin and serum amyloid-P following experimental infection of mice with <i>Trypanosoma brucei brucei</i>

Investigation of the pathophysiological role of the systemic cytokines, including interleukin-1, interleukin-6 and tumour necrosis factor α, in the host response to infection with African trypanosomes is hampered by the low and transient concentrations of these cytokines in plasma. One of the actions of these cytokines is the stimulation of hepatocyte production of acute phase proteins such as serum amyloid-P and haptoglobin. These acute phase proteins are more stable in the circulation than the cytokines and can be measured as a means of assessing the systemic cytokine response in the trypanosome-infected host. The plasma concentrations of serum amyloid-P and haptoglobin were measured in an experimental mouse model of Trypanosoma brucei brucei infection. Both serum amyloid-P and haptoglobin, increased markedly following infection. Peak concentrations of serum amyloid-P at 125 μg/ml and haptoglobin at 2 g/l were attained 10 to 12 days after infection. Thereafter, serum amyloid-P concentration decreased to approximately 40 μg/ml while the haptoglobin concentration remained elevated at approximately 1.5 g/l. The reactions of the serum amyloid-P and haptoglobin following experimental Trypanosoma brucei brucei infection in mice demonstrate that a major acute phase response has occurred indicating that the systemic cytokine network has been activated. Further studies are required to identify whether the response is stimulated by the parasite or indirectly by tissue damage