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Research on vaccines against trypanosomatids, a family of protozoa that cause neglected tropical diseases, such as Chagas disease, leishmaniasis, and sleeping sickness, is a current need. Today, according to modern vaccinology, virus-like particle (VLP) technology is involved in many vaccines, including those undergoing studies related to COVID-19. The potential use of VLPs as vaccine adjuvants opens an opportunity for the use of protozoan antigens for the development of vaccines against diseases caused by Trypanosoma cruzi, Leishmania spp., and Trypanosoma brucei. In this context, it is important to consider the evasion mechanisms of these protozoa in the host and the antigens involved in the mechanisms of the parasite–host interaction. Thus, the immunostimulatory properties of VLPs can be part of an important strategy for the development and evaluation of new vaccines. This work aims to highlight the potential of VLPs as vaccine adjuvants for the development of immunity in complex diseases, specifically in the context of tropical diseases caused by trypanosomatids.

Research on vaccines against trypanosomatids, a family of protozoa that cause neglected tropical diseases, such as Chagas disease, leishmaniasis, and sleeping sickness, is a current need. Today, according to modern vaccinology, virus-like particle (VLP) technology is involved in many vaccines, including those undergoing studies related to COVID-19. The potential use of VLPs as vaccine adjuvants opens an opportunity for the use of protozoan antigens for the development of vaccines against diseases caused by Trypanosoma cruzi, Leishmania spp., and Trypanosoma brucei. In this context, it is important to consider the evasion mechanisms of these protozoa in the host and the antigens involved in the mechanisms of the parasite-host interaction. Thus, the immunostimulatory properties of VLPs can be part of an important strategy for the development and evaluation of new vaccines. This work aims to highlight the potential of VLPs as vaccine adjuvants for the development of immunity in complex diseases, specifically in the context of tropical diseases caused by trypanosomatids.

African trypanosomiasis is infection by protozoan hemoflagellates of the Trypanosoma brucei complex, subspecies of which cause disease in humans: Trypanosoma brucei gambiense causes Gambian (chronic) trypanosomiasis and Trypanosoma brucei rhodesiense causes Rhodesian (acute) trypanosomiasis. A third economically important subspecies, Trypanosoma brucei, causes nagana, a fatal disease of animals, but does not infect humans., See also ADA545141. Chapter 3 from e-book, Topics on the Pathology of Protozoan and Invasive Arthropod Diseases.

This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural s, Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703

This thesis describes bioanalytical methods for drug determination in biological matrixes, with drugs in focus used against diseases largely affecting low-income countries. Solid-phase extraction is used for sample cleanup, and processed samples are analyzed by liquid chromatography. Developed bioanalytical methods are validated according to international guidelines. Eflornithine (DFMO) is a chiral drug, used for treating human African trypanosomiasis. A bioanalytical method for determination of DFMO enantiomers in plasma is presented. The enantiomers are detected by evaporative light-scattering detection. The method has been applied to determination of D-DFMO and L-DFMO in rats, after intravenous and oral administration of racemic DFMO. It is concluded that DFMO exhibits enantioselective absorption, with the more potent enantiomer L-DFMO being less favored. Sulfadoxine (SD) and sulfamethoxazole (SM) are sulfa-drugs used for malaria and pneumonia respectively. Two methods are described for simultaneous determination of SD and SM in capillary blood sampled on filter paper. The former method allows direct injection of extracts from dried blood spots (DBS), while for the latter method solid-phase extraction is added. Pre-analytical factors contributing to measurement uncertainty is also discussed, and it is concluded that it is of high importance that homogeneity in type of sampling paper and sampling volume is assured. Piperaquine (PQ) is an antimalarial, increasingly used in artemisinin combination therapy. A method for determination of piperaquine in DBS is presented. By using a monolithic LC column, a very short LC analysis of two minutes per sample is achieved. A method for simultaneous determination of three antiretroviral drugs i.e. lamivudine (3TC), zidovudine (AZT) and nevirapine (NVP), in DBS samples is described. The method is applied to drug determination in two subjects after receiving standard antiretroviral treatment. Conclusion is that the method is sui

The trypanolytic factor of human serum :a trojan horse.African trypanosomes, the prototype of which is Trypanosoma brucei, are protozoan parasites of huge clinical, veterinary and economical importance. They develop in the body fluids of various mammals (including humans) where they face and manipulate many different aspects of the immune system. The extent of this interplay is pivotal to both host and parasite survival, and depending on parasite virulence and host susceptibility, infection duration ranges from some months to several years. At the end, host survival is invariably compromised.Humans and few other primates provide however a striking exception to this fatal outcome. They are indeed fully protected against most trypanosome infections through the presence in their blood of a so-called trypanosome lytic factor (TLF). The TLF is known to circulate mainly in the form of a high density lipoprotein particle characterized by the simultaneous presence of two primate-specific proteins: haptoglobin-related protein (Hpr) and apolipoprotein L-I (apoL-I).We have contributed to delineate the respective roles played by Hpr and apoL-I in the lysis process.ApoL-I was shown to be the exclusive toxin of the TLF. In its absence humans get fully susceptible to any trypanosome infection. The toxin was shown to kill the parasite after endocytosis through the generation of ionic pores in the lysosomal membrane. Those pores dissipate membrane potential and trigger the influx of chloride ions from the cytoplasm into the lysosomal compartment, leading to an eventually fatal uncontrolled osmotic phenomenon. ApoL-I efficient delivery to the parasite relies on Hpr. African trypanosomes indeed fulfil their heme nutritional requirements by receptor-mediated internalization of the complex formed by haptoglobin, an evolutionary conserved acute-phase protein, and hemoglobin, resulting from physiological intravascular hemolysis. This heme uptake by the aux, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The two human pathogenic sub-species T.b.gambiense and T.b.rhodesiense can be distinguished from the morphologically identical T.b.brucei by their ability to infect humans, enabling them to cause sleeping sickness. This is because they are resistant to lysis by the lytic factor (APOL-I) present in normal human serum (NHS). In T.b.rhodesiense resistance to this lytic factor is due to a truncated VSG gene termed SRA which blocks lysis by interacting with APOL-I in the lysosome. SRA does not exist in T.b.gambiense. The search for a similar truncated VSG gene lead to the identification of a T.b.gambiense specific glycoprotein termed TGSGP. TGSGP transfected alone into the sensitive T.b.brucei is unable to confer resistance to this sub-species. This is either due to incorrect processing of this gene is this sub-species or because TGSGP requires a partner to confer resistance.In the search for a partner, the genomic locus of TGSGP was cloned and sequenced. We found that TGSGP is linked to a truncated gene homologous to the S.cerevisiae AUT1 gene, a gene implicated in autophagy and more specifically in membrane expansion. Southern blot hybridization and PCR analysis on genomic DNA from several isolates demonstrated that this feature was a specific to T.b.gambiense. In addition, we observed a correlation between the aut1 allele size and the geographical origin of the isolate.Since in trypanosomes lysis by NHS is due to an uncontrolled expansion of the lysosome, we speculated that the truncation of the aut1 allele could be implication in the resistance to human serum. We characterized the genomic organisation of the AUT1 locus. T.b.brucei possesses two native AUT1 alleles whilst T.b.gambiense possesses a truncated aut1 allele, as well as a native AUT1 allele. We showed that in the T.b.gambiense LiTAR isolate (aut1/AUT1), despite the presence of a wild-type allele this gene is no longer expressed at the mRNA and protein level. Our complimentary results by run-on t, Doctorat en sciences, Spécialisation biologie moléculaire, info:eu-repo/semantics/nonPublished

The repertory of drugs to fight protozoal diseases such as malaria, Chagas' disease, leishmaniasis, and African trypanosomiasis is woefully inadequate. Now, genome sequencing and structural genomics projects are quickly elucidating new drug targets, providing incredible opportunities for medicinal chemists. Here, we illustrate the power of structure-based drug design in this process by our efforts to selectively block trypanosomal glycolysis.

Trypanosomosis is the most widespread parasitic disease of great importance in Africa affecting human and animals. There are breeds of cattle that are trypanotolerant. Trypanotolerance is an ability of these breeds to withstand the effects of trypanosome infections. The disease impedes production in Sub-Saharan Africa. In this thesis, the main focus is on marker-assisted breeding to improve trypanotolerance and mouse is used as model organism for cattle. We have concentrated on the opportunities to exploit individual genes (QTL) that affect trypanotolerance. We have shown how the size of a breeding experiment increases with the number and the size of the QTL region to introgress. Biological factors to be considered when estimating the required size of an introgression experiment are also highlighted. Strategies to optimize designs for introgression show that selecting two lines each carrying two of the donor's QTL allele during the backcross (when introgressing three QTL) is more advantageous than monitoring simultaneously the three QTL in terms of number of animals to maintain, number of genotyping and costs involved. An experiment in mice shows that QTL introgression is feasible and successful, and that the background genotype is an important factor to be considered when analyzing the results of such an experiment. This experiment is unique. The general discussion focuses on the introgression of trypanotolerance genes in cattle with an emphasis on SubSaharan zones of Africa. The introduction of trypanotolerant cattle in the humid and subhumid zones of Africa, however, should be done with care to avoid damage to the environment and bio-diversity.

Malaria chemotherapeutic studies included a primary antimalarial blood schizontocidal test system (MM test), where 1,500 compounds were evaluated against Plasmodium berghei with 327 exhibiting activity, and a secondary antimalarial program consisting of in depth evaluation of compounds against drug-sensitive and drug-resistant lines. In a 3-dose modified MM test artelinic acid exhibited better activity than 5 analogs of artemisinin and only 2 of the 4 different 5-fluoropyrimidine analogs were active. Neither verapamil, diltiazem, nor prochloroperazine reversed chloroquine resistance in a highly chloroquine- resistant line even when mice were pretreated with phenylhydrazine. The antimalarial activity of primaquine, dapsone, and menocotone was hindered when mice received phenylhydrazine. Alloxan did not influence the antimalarial activity of several compounds. FeSO4 but not CuSO4 enhanced the activity of WR181023. Peroxidized cod-liver oil and menhaden oil had antimalarial activity.

The objective of this project is to determine the extent of antigenic variability in trypanosomes of the T. brucei group collected from man and animals in the Lambwe Valley. T. rhodesiense is endemic in the Valley causing periodic disease in man. Domestic and game animals harbor the morphologically indistinguishable T. brucei. This project is also designed to study reactions of various antisera to determine the variability of different antigenic types within the parasitic population.

Eighteen isolates of Trypanosoma brucei rhodesiense were acquired and several were selected for a preclinical evaluation of a combination of eflornithine and suramin as a treatment for African Sleeping Sickness. The sensitivity to eflornithine, suramin arsenicals and diamidines of seventeen isolates was measured in acute, non-central nervous system (CNS), disease models. The discovery of a wide variation in sensitivity of these mostly wild isolates to standard and experimental drugs has important implications for any preclinical evaluation of any anti-trypanosomal drug candidates. Seven isolates were evaluated for suitability as a CNS model; one was chosen for evaluation of the combination suramin and eflornithine as therapy for CNS disease. The combination was effective despite the fact that these two agents used singly were ineffective. In two acute infection models the combination produced cures at doses much lower than that required when the drugs were used singly. No adverse interactions were found even when high doses of suramin and eflornithine were combined.

President Roosevelt doubts if he will be able to take advantage of William Osler's hospitality, but genuinely looks forward to seeing him again. Roosevelt is not sure if his information about the sleeping sickness (African trypanosomiasis) has any value, but he trusts the observations of Major Edgar Alexander Mearns. He soon expects to receive the French report on the sleeping sickness that Osler mentioned.

Edgar Alexander Mearns will follow Caspar Whitney's suggestions for the trip. He has also received the documents on sleeping sickness and will read them with great interest.

President Roosevelt thanks Caroline Kirkland for the copy of her book, and intends to replace the one he already has in his personal library with the autographed copy she sent. He will look forward to seeing Kirkland's sister, Ethel Kirkland Ennis, if he travels to Entebbe, Uganda. Roosevelt was very interested in what Kirkland said about the sleeping sickness.

Senator Lodge returns some letters from Prescott F. Hall to President Roosevelt and reports on his findings regarding Hall's allegations. Lodge's Commision on Immigration found that appeal reversals did not increase under Secretary of Commerce and Labor Oscar S. Straus. Lodge has found no proof that Leslie M. Shaw's personal views have affected governmental operations. Lodge has visited Baltimore, Philadelphia, New York, and Boston, and can find no evidence of what Hall alleges. Lodge found evidence of immigration enforcement corruption in California, but believes it has been remedied. In Chicago, Lodge found evidence of police corruption in enforcing laws against prostitution. In the southern states, Lodge has found problems with nepotism. Lodge emphasizes that it will take a great deal of time to collect evidence and create reports of his investigations. Lodge is pleased to hear of President Roosevelt's African safari plans, but warns him to be careful of flies carrying sleeping sickness. He writes about the impressive dike engineering in the Netherlands, and of a book by Maurice Maeterlinck he believes the Roosevelts will enjoy.

In the first cartoon, two doctors look at a sleeping President Roosevelt. Caption: Doctor--"It is a very severe case of sleeping sickness. Our remedies have no effect. Mr. Roosevelt must be aroused. But how?" In the second cartoon, the doctors have an idea. Caption: "An idea!" He might respond to a severe shock. Let's try it!" In the third cartoon, one doctor reads a paper. Caption: "Ah, doctor! I see here an account of an African hunting trip, written by the--" In the fourth cartoon, Roosevelt starts to wake up. Caption: "Famous nature lover, Jack Liverpool. He says: 'I was hastening over the mountains in pursuit of the savage crocodile when suddenly I beheld--'" In the fifth cartoon, President Roosevelt has a questioning look. Caption: "A lion leap madly from the top of a tall palm tree, pursued by a frantic springbok, which was beating him terribly with her wings. The king of beasts had evidently been robbing her nest--" In the sixth cartoon, Roosevelt jumps up with a pen in his hand. The doctors shake hands, saying "Congratulations!" and "He's cured." Caption: Mr. Roosevelt--"Nature faker!!! I'll show him up!" comments and context

Sir William Osler invites President Roosevelt to use his house next year when Roosevelt comes to Oxford, England. Osler will ask Dr. Emile Roux to send Roosevelt the French Commission Report on sleeping sickness. Osler hopes Roosevelt will mention it in his speech, so the issue will generate more interest.