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

Parasitology, Pathology

Ngure, Raphael Muchangi

University:University of Glasgow

关键词: Parasitology, Pathology;   

Others  :  http://theses.gla.ac.uk/75537/1/13832512.pdf

来源: University of Glasgow

【 摘 要 】

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.