The trypanosome project

Human African trypanosomiasis (HAT), or sleeping sickness, is a parasitic infection caused by the extracellular flagellate Trypanosoma brucei (Tb). The parasite is transmitted by the tsetse fly or Glossina which is located to the tropical belt on the African continent. The disease is in late stages characterized by a severely disturbed sleep pattern and chronic pain. Arsenic compounds are used for treatment, with severe side-effects as a result. In a model of experimental T.b. brucei infection, rats infected with trypanosomes show disturbances in the locomotor activity pattern, alterations in body temperature rhythms and fragmentation of the slow wave synchronized sleep. In the experimental infection model, the parasites are located early to regions in the nervous system that lack the blood-brain barrier (BBB). Since trypanosomes are extracellular parasites, the dysfunctions in the nervous system are probably caused by molecules released either directly from the parasites or from host cells that respond to the infection.

Trypanosomes and the blood brain barrier

The overall objective of the present research project on “trypanosomes and the BBB” is to unravel basic cellular and molecular processes that underlie the passage of the parasites across the BBB into the brain parenchyma. Thereby, we hope to discover: i) molecules that may be considered as markers for an effective staging of HAT, and ii) design new therapies by using drugs, which have already passed clinical trials in humans for other indications. These drugs could impede trypanosome neuroinvasion and/or eliminate trypanosomes after they have invaded the brain; an invasion that causes the most debilitating and invalidating complication of HAT.

The specific objectives of the project are to: (a) Identify molecules involved in trypanosome neuroinvasion that could be developed into marker-based diagnostic tools for therapeutic decision and cure assessment; and (b) Investigate the therapeutic potential of molecules that interfere with trypanosome neuroinvasion and/or eliminate trypanosomes from the brain parenchyma.

(Supported by grants from the United Nations Development Programme (UNDP)/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR); the Swedish Research Council (4480); Swedish International Development Cooperation; the National Heart, Lung, and Blood Institutet (i-R01-H1-71510-01); and the EC-FP6-2004-DEV-3032324)

Relevant publications

1. Masocha W, Rottenberg ME, Kristensson K. Minocycline impedes African trypanosome invasion of the brain in a murine model. Antimicrob Agents Chemother. 2006 (accepted)
2. Rottenberg ME, Masocha W, Ferella M, Petitto-Assis F, Goto H, Kristensson K, McCaffrey R, Wigzell H. Treatment of African trypanosomiasis with cordycepin and adenosine deaminase inhibitors in a mouse model. J Infect Dis. 2005, 192:1658-65.
3. Masocha W, Robertson B, Rottenberg ME, Mhlanga J, Sorokin L, Kristensson K. Cerebral vessel laminins and IFN-gamma define Trypanosoma brucei brucei penetration of the blood-brain barrier. J Clin Invest. 2004, 114:689-94.
4. Mulenga C, Mhlanga JD, Kristensson K, Robertson B. Trypanosoma brucei brucei crosses the blood-brain barrier while tight junction proteins are preserved in a rat chronic disease model. Neuropathol Appl Neurobiol. 2001, 27:77-85.

Alterations in the biological clock

There are strong indications that alterations in circadian rhythms during experimental infection with trypanosomes occur, which correlate with reported clinical symptoms in humans with the disease. We are studying physiological and molecular alterations in the mammalian pacemaker for circadian rhythms, the hypothalamic suprachiasmatic nuclei (SCN), and investigating if these alterations correlate with the dysfunctions in endogenous rhythms in African trypanosomiasis. New information in this specific field could lead to a better understanding of how circadian rhythms are regulated in the nervous system and a deeper knowledge of how the neuronal symptoms occur in trypanosomiasis, and therefore provide an increased chance to a more efficient treatment of the disease.

The SCN governs sleep, body temperature, hormonal and locomotor activity rhythms and therefore functions as a "biological clock". The nuclei are synchronized by light through the retinohypothalamic tract, where glutamate is considered to be the main transmitter, and by non-photic stimuli such as melatonin released from the pineal gland. The SCN has its own endogenous rhythm in spontaneous neuronal firing, which peaks in activity during the light hours of the day. By performing extracellular and intracellular electrophysiological studies on brain slices containing the SCN we have shown that spontaneous impulse and synaptic activities are altered after infection with trypanosomes. Also, there are indications of a down-regulation of glutamate receptors. In collaboration with Professor Gene Block, University of Virginia, Charlottesville, USA, we are currently investigating the impact of sleeping sickness on clock gene expression and locomotor activity in rats infected with trypanosomes (supported by NHLBI 117541).

Rhythm alterations may be mediated by pro-inflammatory cytokines

Pro-inflammatory cytokines may play a role in mediating sleep and rhythm disturbances in sleeping sickness. We have previously demonstrated that trypanosomes show immunopositivity to monoclonal antibodies directed against the interferon-g (IFN-g), a pro-inflammatory cytokine released during early stages of the infection. Recombinant IFN-g binds to the trypanosomes and promotes their growth and proliferation, and Tb brucei induces production of IFN-g from CD8⁺ T-cells. In the project we investigate the impact of pro-inflammatory cytokines, such as IFN-g, on circadian rhythm function with electrophysiology, immunohistochemistry, Western blotting and molecular biology techniques. We have found that IFN-g modulates impulse frequency and postsynaptic activity in SCN brain slices, and the receptor of this cytokine is selectively located in the ventrolateral part of the rat SCN. The receptor protein shows a daily variation in its expression when the rats are kept in a 12:12 hr light:dark schedule. Furthermore, in collaboration with Dr Yongho Kwak and Professor Gene Block, University of Virginia, Charlottesville, USA, we are investigating the impact of cytokines on clock gene expression and electrical activity in SCN neurons with luciferase-reporter technology and multi-electrode array recordings (supported by NHLBI 117541).

Relevant publications

1.      Lundkvist G. B., Kristensson. K. and Bentivoglio M (2004). Why trypanosomes cause sleeping sickness. Physiology 19:198-206.

2.      Lundkvist G. B., Christenson J., ElTayeb R. A. K., Peng Z-C., Grillner P., Mhlanga J., Bentivoglio M. and Kristensson K. (1998), Altered neuronal activity rhythm and glutamate receptor expression in the suprachiasmatic nuclei of Trypanosoma brucei- infected rats, J. Neuropath. Exp. Neurol., 57: 21-29.

3.      Lundkvist G. B., Robertson, B., Mhlanga J. D. M., Rottenberg M. E. and Kristensson K. (1998), Expression of an oscillating interferon-g receptor in the suprachiasmatic nuclei, NeuroReport, 9:1059-1063.

4.      Lundkvist G. B., Robertson B., Andersson A., Rottenberg M. E., and Kristensson K. (1999), Light-dependent regulation and postnatal expression of the IFN-g receptor in the rat suprachiasmatic nuclei, Brain Res. 849 (1-2):231-4.

 The work with trypanosomes is currently performed by

Willias Masocha (Blood-brain barrier)

Gabriella Lundkvist (Alterations in circadian rhythms)

Mikael Nygård (Ageing and inflammation)