Sleeping sickness, also known as Human African trypanosomiasis (HAT), is a vector-borne parasitic disease. It is restricted to areas where the tsetse fly is endemic and affects especially rural areas of sub-Saharan Africa, where a weak health system and political instability make disease surveillance and management difficult. In 2009 some 30.000 new cases were reported from 17 countries with 74% alone from the Democratic Republic of Congo. Untreated sleeping sickness is invariably fatal.
The parasites infect first the blood and lymphatic system, before they move to the central nervous system. This second stage is particularly difficult to treat. Of the two currently available drugs, one, the arsenic-based melarsoprol has fatal side effects in 1 of 20 patients, while the other, eflornithine, is costly, requires prolonged hospital treatment and is effective only against one of the two infective subspecies. The currently used combination therapy (eflornithine and nifurtimox) improved application but not their effectiveness. Increasing reports of treatment failures has led to concern that soon there may be no effective sleeping sickness treatment, unless safe, effective and inexpensive new drugs are developed.
We perform basic research to obtain detailed knowledge about the life threatening 2nd stage of the disease, including differentiation of trypanosomes and their host-independent cell density regulation. In addition we work on possible drug targets (e.g. Cathepsin B) and the development of nano-particle based drug application.
As the causative agent of African Sleeping Sickness, trypanosomes enter the brain during second stage infection, where they deregulate sleep-wake cycle by secretion of prostaglandin D2, causing the typical symptoms like insomnia at night and somnolescence at day. T. brucei was first described within the cerebrospinal fluid (csf) from sleeping sickness patients in 1903, but a final proof how trypanosomes reach the brain has not been presented till today.
Our murine model shows that the parasites first enter the brain via the choroid plexus, whereas they cannot cross the blood-brain barrier. From the plexus stroma, trypanosomes penetrate the epithelial cell layer to reach the ventricular system. Although they cannot develop in csf, they can use the liquor flow to reach the Virchow-Robin space and penetrate the glia limitans. However, we cannot find any trypanosomes within the brain parenchyma, as NMRI mice (and Wistar rats) won’t survive long enough, i.e. more than 30 days. During infection trypanosomes change their morphology repeatedly from a dividing long slender form to a cell-cycle arrested short stumpy form. Beyond this, we found not only an alternation of short (and thick) as well as intermediate length forms, but rather an increase of their length over time. Whereas until day 15 post-infection almost no trypanosome reached a length of 25 µm, there were remarkably long (i.e. up to 30 µm), extremely thin and agile parasites present after 20 days and later.We observed that brain infection and the occurrence of these long forms might correlate. This could mean that trypanosomes have to change their morphological properties before they can cross the vessels fenestrated endothelium in order to cross the blood-csf barrier. During brain infection, we can show a slight inflammation of the meninges and choroid plexus with magnet resonance imaging.
(partly out of: Brain infection by A. trypanosomes during Sleeping Sickness, NPaBR (2012), 18(2): p49-51)
African trypanosomes are unicellular parasites that cause the nagana disease in cattle and the African sleeping sickness in humans. Their life cycle involves an obligatory change between the vertebrate and the tsetse fly. Dividing and nonproliferating stages alternate within the cycle. With the bite of an infected fly metacyclic trypanosomes from the insect’s salivary glands are transferred to the bloodstream of a mammal. They then transform spontaneously to the bloodstream form that is called the long slender form. Their fast dividing rate causes an increase of parasitaemia. When the cell density reaches a certain threshold value, the slender cells differentiate to the non‐dividing short stumpy parasites. The differentiation is a transient process that involves intermediate forms whose metabolism is similar to stumpy cells, their morphology, however, is difficult to determine. Intermediate and stumpy cells possess partly activated mitochondria and parts of the respiratory chain. They are therefore pre-adapted to survive in the fly. The transduction of this cell density dependent differentiation from the long slender to the short stumpy bloodstream form is attributed to a differentiation inducing factor, that is secreted into the blood or the culture medium by the slender cells. We want to illuminate the effects of conditioned medium and its fractions on the cells in axenic culture more detailed.
Transmission electron micrographs revealed the cells that were treated with conditioned medium to show increased autophagy. In flow cytometry experiments these cells did not show signs of necrosis or enhanced apoptosis after 24 and 48 hours. However, the cells were not arrested in the G1/G0 phase of the cell cycle, which usually is typical for stumpy cells. Treated with the growth inhibitory fraction from gel filtration for 19 and 25hours, pleomorphic cells developed more intermediate morphology. A significant increase of cells with
stumpy morphology did not take place until after 40 hours. Similar to the effects observed with troglitazone, this finding could point towards a more important role of the intermediate trypanosomes during differentiation than currently assumed. The identification of the differentiation factor would be a milestone for the development of new sleeping sickness drugs. Theoretically it should be possible to isolate this molecule from the culture medium. The processing of the conditioned media in order to isolate biologically active fractions turned out to be very complex due to the small molecular weight an d the strong hydrophobicity of the factor. Isolation and the chemical characterization of this differentiation factor is one of our goals, since stumpy forms cannot survive in the mammalian host (Out of: Björn Buchholz, Doctoral thesis)