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Human African trypanosomiasis, chemotherapy and CNS disease
Journal of Neuroimmunology 211 (2009) 16–22
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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
Review article
Human African trypanosomiasis, chemotherapy and CNS disease Jean Rodgers ⁎ Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, United Kingdom
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Article history: Received 17 December 2008 Accepted 5 February 2009 Keywords: Human African trypanosomiasis CNS Neuroinflammation Mouse model Chemotherapy
a b s t r a c t Trypanosomes have been recognised as human pathogens for over a century. Human African trypanosomiasis is endemic in an area sustaining 60 million people and is fatal without chemotherapeutic intervention. Available trypanocidal drugs require parenteral administration and are associated with adverse reactions including the development of a severe post-treatment reactive encephalopathy (PTRE). Following infection the parasites proliferate in the systemic compartment before invading the CNS where a cascade of events results in neuroinflammation. This review summarises the clinical manifestations of the infection and chemotherapeutic regimens as well as the current research findings and hypotheses regarding the neuropathogenesis of the disease. © 2009 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . Clinical manifestation of HAT . . . . . . . . Chemotherapy . . . . . . . . . . . . . . . 3.1. Early-stage treatments . . . . . . . . 3.2. Late-stage treatments . . . . . . . . 3.3. Disease staging . . . . . . . . . . . 4. Neuropathological features . . . . . . . . . 4.1. Post-mortem examinations . . . . . . 4.2. Animal models . . . . . . . . . . . 5. Parasites in the CNS . . . . . . . . . . . . 5.1. Trypanosome invasion of the brain . . 5.2. Parasites and inflammatory mediators. 6. Conclusions . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
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1. Introduction Human African trypanosomiasis (HAT), or sleeping sickness, is prevalent in 36 Sub-Saharan African countries situated between the latitudes 14o North and 29o South. The disease results from infection with protozoan parasites of the genus Trypanosoma. The infection is transmitted by the bite of the tsetse fly which is the insect vector of HAT. Untreated the disease is always fatal. There are many species of this parasite however, only two sub-species of the Trypanosoma brucei
⁎ Tel.: +44 141 330 3797; fax: +44 141 330 5603. E-mail address: [email protected]. 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.02.007
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group are infectious in man. Trypanosoma brucei (T.b.) gambiense causes HAT in West and Central Africa while T.b. rhodesiense causes disease in East and Southern Africa. Approximately 60 million people are at risk of infection and the most recent epidemiological figures report 17,500 new cases of HAT diagnosed in 2004 with a cumulative total of around 50–70,000 people infected (WHO, 2006). However, WHO estimates that less than 4 million of the population are under medical surveillance and only 10% of new infections are actually diagnosed (WHO, 2006, 1998).While there seems to be no doubt that the incidence of HAT has declined considerably over the last five years, precise figures for current HAT incidence and prevalence remain somewhat uncertain. The situation with HAT is subject to exacerbation by wars and civil unrest which result in a deterioration in health
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surveillance and disease control which increase the likelihood of disease outbreaks and higher mortality rates. 2. Clinical manifestation of HAT The clinical manifestations of sleeping sickness are both diverse and variable and have been described in depth previously (Apted, 1970; Atouguia and Kennedy, 2000). In brief, the two different forms of HAT show different clinical courses; West African sleeping sickness is a chronic disease lasting from months to years whereas East African sleeping sickness follows an acute course with a duration of weeks to months. After initial infection the trypanosomes are localised to a nonsuppurative lesion or chancre that develops at the site of the infected tsetse bite. This leads to the early, or haemolymphatic, stage of the disease in which the parasites proliferate in the blood, and lymphatic system. Clinically the patient experiences febrile episodes with headaches and enlarged lymph nodes may be present at this stage. An erythematous skin rash may also appear. From here the parasites invade a variety of tissues including spleen, liver and heart and ultimately enter the central nervous system (CNS) to cause the late or encephalitic stage of the disease. In rhodesiense disease CNS involvement occurs early after infection, usually within 3 to 4 weeks of the tsetse bite, and there may be no clear clinical distinction between early and late stage disease since it is not possible to distinguish reliably between the early and late stages on the basis of the clinical features. In gambiense disease, CNS invasion can be delayed until many months or even years after infection (Apted, 1970). Signs of neurological involvement include mental disturbances such as irritability, lack of concentration and personality changes; motor system involvement with the development of seizures, muscle fasciculations and in some cases paralysis; sensory involvement with the presence of hyperaesthesia, anaesthesia or intense itching; pout or palmo-mental reflexes; a variety of characteristic sleep disturbances such as the development of daytime somnolence accompanied by night-time insomnia and alterations of sleep structure. As the disease progresses to the final stages the patient becomes indifferent to their surroundings and more difficult to rouse. Untreated, the patient will progress to coma and death (Atouguia and Kennedy, 2000). 3. Chemotherapy Both forms of HAT are invariably fatal without chemotherapeutic intervention. To date only four drugs, suramin, pentamidine, melarsoprol and eflornithine, are licensed for treatment of HAT (Barrett et al., 2007). Their efficacy is dependent on whether the infection has reached the CNS-stage or remains in the haemolymphatic early-stage. 3.1. Early-stage treatments Suramin, first used in the early 1920s, is used to treat T.b. rhodesiense infections. It is generally administered as a series of five slow intravenous injections given over a three week period (Kennedy, 2008c). Adverse effects, including cutaneous lesions, anaphylactic shock, renal failure and neurotoxicity, are associated with its administration (Legros et al., 2002). T.b. gambiense disease can be treated with intramuscular injections of pentamidine administered over 7–10 days. This drug has been in use since 1940. Although generally well tolerated pentamidine treatment can result in either hypo- or hyperglycemia and low blood pressure (Atouguia and Kennedy, 2000). Neither suramin nor pentamidine crosses the blood-brain barrier (BBB) efficiently and therefore they cannot be used to treat CNS-stage disease.
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(Pepin and Milord, 1994). Eflornithine is only effective against CNSstage T.b. gambiense infections since T.b. rhodesiense has shown innate resistance to the drug (Iten et al., 1995). The drug is expensive and its administration protracted involving a 14 day course of intravenous infusions of 100 mg/kg body weight of eflornithine given at 6 hourly intervals (Burri and Brun, 2003). Despite this, the use of eflornithine as a first line therapy in the treatment of T.b. gambiense infections has been widely advocated (Checchi and Barrett, 2008; Balasegaram et al., 2006a; Chappuis et al., 2005; Priotto et al., 2008). Adverse effects of eflornithine treatment include pancytopenia, gastrointestinal upset, convulsions or hemiparesis, which reverse spontaneously on withdrawal of the drug (Pepin and Milord, 1994; Atouguia and Kennedy, 2000; Legros et al., 2002). The only drug that can be used to treat both T.b. rhodesiense and T.b. gambiense infections once they have reached the CNS is the trivalent arsenical melarsoprol. This has been in use since 1949. Until recently the most commonly implemented treatment regimen comprised three to four series of three to four daily intravenous injections, each round of treatment separated by a rest period of 7 days (WHO, 1998; Legros et al., 2002). However a more concise treatment protocol consisting of 10 daily intravenous injections has been introduced for T.b. gambiense infections (Burri et al., 2000). Melarsoprol is a highly toxic drug and side effects of the regimen are common. Minor problems such as fever, gastric upset, and malaise occur quickly after initiating the treatment protocol. More serious side effects including tachycardia, subconjunctival haemorrhages and retrosternal pain can also develop (Atouguia and Kennedy, 2000). However, by far the most serious adverse effect of melarsoprol treatment is the development of a severe post-treatment reactive encephalopathy (PTRE). This occurs in approximately 10% of treated patients and can be fatal in half of the affected individuals. Therefore, administration of melarsoprol results in the death of up to 5% of all patients receiving treatment (Pepin and Milord, 1994). The mechanisms leading to the development of the PTRE remain unclear however several hypotheses have been muted (Hunter and Kennedy, 1992). These include; an immune reaction to parasite antigens released within the CNS following chemotherapy (Pepin and Milord, 1991), immune complex deposition (Lambert et al., 1981), toxicity of the arsenical moiety of the melarsoprol molecule (Hurst, 1959) autoimmune reactions (Poltera, 1980; Hunter et al., 1992c) or subcurative chemotherapy (Hunter et al., 1992a). Administration of corticosteroids, as an adjunct to melarsoprol treatment, has shown some degree of success in preventing the development of the PTRE but the beneficial effects of this remain controversial (Pepin et al., 1989a; Pepin and Milord, 1994). Since the mid-1970s nifurtimox has been used to treat Chagas' disease, the South American form of trypanosomiasis caused by infection with Trypanosoma cruzi. The drug is not well tolerated and can produce serious side effects ranging from gastro-intestinal upset to neurological reactions. Although not registered for the treatment of HAT, nifurtimox has been administered in patients with melarsoprol refractory T.b. gambiense-infections. The efficacy of the treatment varied substantially. A relapse rate of 63% was seen in patients given 12–17 mg/ kg nifurtimox daily for a period of 60 days (Pepin et al., 1989b). This high failure rate was reduced to 36% by raising the dosage of nifurtimox to between 24 and 37 mg/kg daily for a 30 day period, however the higher dose rate produced significant toxicity in the patients and severe neurological reactions were not uncommon (Pepin et al., 1992). More recently, a regimen comprising a combination of nifurtimox and eflornithine has been employed and this combination chemotherapy approach has shown promising results (Priotto et al., 2007). 3.3. Disease staging
3.2. Late-stage treatments Eflornithine or D,L-α-difluoromethylornithine, first used in 1981, is the most recently registered drug for the treatment of CNS-stage HAT
The criteria used for diagnosing CNS-stage HAT remain equivocal even though the correct staging of the disease is critical in establishing the appropriate chemotherapeutic regimen (Kennedy, 2008b). The
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parameters are based on microscopic examination of a sample of cerebrospinal fluid (CSF) to determine white blood cell count and the presence of trypanosomes. World Health Organisation (WHO) guidelines stipulate that any samples showing the presence of more than 5 white blood cells (WBC)/μl and/or the presence of trypanosomes in the CSF indicates that the infection has progressed to the CNS-stage and should therefore be treated with late-stage drugs (WHO, 1998). However, in some T.b. gambiense infected patients clinicians are using the much higher figure of N20 WBC/μl before proceeding to melarsoprol therapy (Lejon et al., 2003b; Kennedy, 2006). This higher figure evolved following successful pentamidine chemotherapy of patients presenting with trypanosomes in their CSF sample and a WBC of V20, (Pepin and Milord, 1994) and has led to the notion of an intermediate classification of “early late-stage” disease (Doua et al., 1996; Lejon et al., 2003a,b; Kennedy, 2004). The safety of implementing this increased threshold is still in debate and uncertain. A retrospective analysis of data acquired from the Republic of Congo, where an intermediate WBC count of N10 cells/μl was used to diagnosed CNS-stage disease, showed that implementation of this higher threshold led to an increased risk of relapse infection following pentamidine chemotherapy compared to the lower figure of 5 WBC/μl CSF (Balasegaram et al., 2006b). Additional criteria such as intrathecal IgM synthesis, CSF end-titres in LATEX/IgM and LATEX/T.b. gambiense positive CSF have been shown to be associated with the occurrence of treatment failures and could be useful markers to help determine the correct chemotherapeutic approach (Lejon et al., 2003a,b, 2008; Bisser et al., 2002). 4. Neuropathological features 4.1. Post-mortem examinations The neuropathological features of HAT have been determined from examination of a limited number of post-mortem samples. An acute meningoencephalitis develops as the disease progresses with the presence of macrophages, lymphocytes and an abundance of plasma cells in the meninges, perivascular spaces and eventually infiltrating the neuropil. Mott cells, or morular cells, can also be found, however these are not pathognomonic of the disease (Atouguia and Kennedy, 2000; Adams and Graham, 1998). The inflammatory cell infiltration is accompanied by the activation of both astrocytes and microglia (Adams et al., 1986). These changes are most obvious in the white matter of the cerebral hemispheres but are also apparent in circumventricular areas including the thalamus, hypothalamus and supraoptic nuclei. Despite this highly inflamed picture only nominal demyelination occurs and neuronal damage is minimal (Atouguia and Kennedy, 2000). The PTRE is characterised by a conspicuous increase in the severity of this inflammatory process and can, in some instances, take the form of an acute haemorrhagic leucoencephalopathy (Adams et al., 1986). 4.2. Animal models Due to the paucity of human samples most information regarding the neuropathogenesis of the disease has been derived from animal models of HAT. These show a similar neuropathological picture to that described above. Infiltration of the brain cerebral vessels by lymphocytes, plasma cells and macrophages has been described following T.b. rhodesiense-infections in vervet monkeys (Schmidt, 1983). Proliferation of the microglial cells in immediate proximity to the inflammatory infiltrates was also detected. However, a fully developed meningoencephalitis, with the presence of inflammatory cells in the neuropil was only rarely identified (Schmidt, 1983). This was more commonly reported following ineffectual drug treatment (Schmidt, 1983; Poltera et al., 1985) and may equate to the situation found in the PTRE associated with HAT treatment.
In a murine model of T.b. rhodesiense-infection mild inflammatory cell infiltration was detected in the brain connective tissue early after infection. The severity of the reaction increased as the disease progressed with a meningoencephalitis present in animals killed 4 weeks after infection (Fink and Schmidt, 1979). In T.b. bruceiinfections of mice inflammatory changes were detected in the choroid plexus and meninges from approximately 4 weeks after infection and in advanced cases perivascular cuffing was observed in meningeal and cerebral vessels (Poltera, 1980). An analogous pattern of inflammatory changes was present in T.b. gambiense infections although this produced a more chronic disease, in some cases lasting for a 22 month period (Poltera et al., 1982). In our murine model of HAT, only mild inflammatory changes are seen when the infection is allowed to progress naturally. However, treatment with a subcurative dose of diminazene aceturate, precipitates the development of encephalitic lesions and large perivascular cuffs consisting mainly of lymphocytes and macrophages (Jennings et al., 1989; Hunter and Kennedy, 1992). The reaction can be further exacerbated by administration of a second subcurative drug treatment following relapse to parasitaemia (Hunter et al., 1992a; Jennings et al., 1997). A wide spread astrocyte activation that becomes more pronounced as the disease severity increases is also a conspicuous feature (Hunter et al., 1992b; Jennings et al., 1997). This model is highly reproducible and can be easily manipulated to mirror the progressive neuroinflammatory reactions found in each stage of HAT. We have therefore used the model to study both the disease pathogenesis and the efficacy and safety of novel chemotherapy approaches (Sternberg et al., 2005; Kennedy et al., 1997, 2003; Rodgers et al., 2007; Eckersall et al., 2001; Kennedy, 1999; Jennings et al., 1997; Jennings et al., 2002). Fig. 1 demonstrates the differing levels if neuroinflammation that can be achieved through the use of this mouse model of HAT. The overall picture portrayed by the various animal models closely simulates the reaction described in the post-mortem material from human infections. A prominent link between subcurative chemotherapy and the development of a severe CNS inflammatory reaction is also apparent. Furthermore, several of these studies make note of the relative lack of neuronal damage that results from the inflammatory cell infiltration of the brain parenchyma (Morrison et al., 1983; Fink and Schmidt, 1979). 5. Parasites in the CNS 5.1. Trypanosome invasion of the brain It has been known for many years that chronic trypanosome infection leads to CNS invasion by the parasite and results in an inflammatory reaction within the brain. Nevertheless, the demonstration of trypanosomes within the human brain has been reported only rarely (Mott, 1907). This could be the consequence of a number of factors such as trypanocidal drug treatment prior to death or deterioration of the parasites due to the time lapse between patient death and tissue fixation (Calwell, 1937). At present the mechanisms and pathways used by the trypanosomes to gain entry and become established within the CNS are not fully elucidated. In animal models, parasites have been localised in the choroid plexus, circumventricular organs and peripheral ganglia, areas, where the BBB is weak (Schultzberg et al., 1988). However, this has yet to be definitively established as the route of parasite invasion into the CNS since some investigations employing rodent models suggest that the BBB becomes progressively damaged as the disease advances (Pentreath et al., 1994; Philip et al., 1994). Using a rat model of trypanosome infection Philip and colleagues demonstrated that by day 35 postinfection with T.b. brucei, fluorescent dye injected into the jugular vein could be detected in several brain areas, including, the thalamus and hypothalamus, indicating impairment of the BBB. By 40 days postinfection, this BBB damage had become extensive and the dye could be
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Fig. 1. H&E stained sections through the hippocampal brain region prepared from T.b. brucei infected mice. The sections show the progression of the neuroinflammation throughout the disease course; early stage (A), early-CNS stage (B), late-CNS stage (C), and PTRE (D). The increasing development of the perivascular cuffs (→) and encephalitis is demonstrated. Original magnification ×100.
found throughout both the grey and the white matter of the cortex (Philip et al., 1994). In contrast, a study of trypanosome infection in rats showed no indiscriminate loss of tight junction proteins in the BBB following invasion of the CNS by trypanosome parasites and no indication of plasma protein leakage into the parenchyma (Mulenga et al., 2001) indicating that the parasites actively enter the brain rather than diffuse into the tissue as a result of barrier impairment. More recent studies have shown a link between the laminin composition of the BBB endothelial basement membrane and the ability of the trypanosome to enter the brain tissue with laminin α4 being permissive to parasite transmigration and laminin α5 being inhibitory (Masocha et al., 2007, 2004). These studies also suggest that trypanosomes may exploit transmigratory mechanisms similar to those utilised by leucocytes since the parasites could not penetrate the brain in recombinant activating gene (RAG)-1 deficient mice which lack both T and B-cells. Furthermore, the pro-inflammatory cytokine IFN-γ appears to play a prominent role in the process since the absence of this cytokine or its receptor led to reduced brain invasion by the parasites with trypanosomes becoming ‘trapped’ between the endothelial basement membrane and the parenchymal basement membrane (Masocha et al., 2007, 2004). The importance of leucocytes in trypanosome penetration of the brain parenchyma was further highlighted in a study investigating the effects of minocycline treatment in T.b. brucei infected mice. Minocycline is known to reduce leucocyte penetration of the CNS, and this inhibition of leucocyte transmigration was accompanied by a reduction in parasite CNS invasion and a marked amelioration of the CNS inflammation (Masocha et al., 2006). Retention of the functionality of the BBB until the late possibly terminal stages of trypanosome infection was also found in a study utilising an in situ BBB perfusion model (Sanderson et al., 2008). In this study T.b. brucei infected mice were perfused with radiolabelled eflornithine at a variety of time points throughout the course of the infection and the amounts of eflornithine crossing the BBB assessed. Eflornithine was shown to penetrate the normal BBB relatively poorly, however the concentration of the drug
reaching the brain increased as the infection progressed with BBB dysfunction detectable by day 28 post-infection. The integrity of the barrier was never regained (Sanderson et al., 2008). In vitro systems have also been employed to investigate trypanosome interactions with the BBB. Cultures of human brain microvascular endothelial cells (HBMEC's), can be grown in a transwell based system to mimic the BBB. Trypanosomes have been shown to cross from one side of this in vitro barrier to the other via a paracellular route with only a transient reduction in transendothelial electrical resistance (TEER) indicating that the parasites traverse the BBB without causing permanent damage to its integrity (Grab et al., 2004). Further studies utilising this model in conjunction with confocal and electron microscopy have demonstrated the presence of trypanosomes within endothelial cells, although the significance of this intracellular location and the viability of these parasites is not yet known (Nikolskaia et al., 2006b). The most recent finding from this in vitro model indicates that parasite derived cysteine protease enzymes are important in enabling the trypanosome to migrate across the BBB (Nikolskaia et al., 2006a). In a complex series of experiments its was shown that both living parasites and parasite conditioned medium could induce transient increases in intracellular calcium concentrations ([Ca2+]i) in the HMBEC's. These alterations in [Ca2+]i were abrogated when a specific irreversible cathepsin L-like cysteine protease inhibitor (K11777) was included in the culture system. The cathepsin L-like cysteine protease present in T.b. brucei is known as brucipain. Furthermore, the addition of brucipain enriched culture medium to the transwell system enhanced traversal of the BBB by the trypanosomes implicating this enzyme as an important factor in the ability of the parasites to invade the brain (Nikolskaia et al., 2006a). 5.2. Parasites and inflammatory mediators As previously stated, the precise mechanisms used by the parasite to gain entry to the brain and induce the associated inflammatory
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reaction remain unclear. However, mouse model studies indicate that the expression of inflammatory mediators including cytokines, chemokines and adhesion molecules change during the course of the infection and that the balance between pro- and anti-inflammatory mediators is central to the outcome of the disease (Kennedy, 2004, 2008a; Sharafeldin et al., 2000; Mulenga et al., 2001). Following parasite CNS invasion in a murine model of HAT, expression of IL-1, IL6, TNF-α, IFN-γ and MIP-1α was found to be upregulated within the CNS by comparative RT-PCR analysis (Hunter et al., 1991, 1992b). In a more recent study examining protein levels, increased CNS TNF-α and IFN-γ concentrations were shown to be associated with severe neuroinflammatory responses while high levels of IL-6 and IL-10 were found in mice exhibiting only mild CNS inflammation (Sternberg et al., 2005). No significant associations between concentration and neuropathology could be detected when CNS levels of IL-1β or nitrate were measured. However, a significant increase in brain IL-1β was
detected by 7 days post-infection and this remained high throughout the early and late-stages of the disease as well as the PTRE (Sternberg et al., 2005). Furthermore, intraventricular infusion of IL-1 receptor antagonist in a rat model of trypanosome infection resulted in a restoration of body weight in the animals but had no effect on neurodegeneration whereas infusion of soluble type-1 TNF-α receptor reduced the neuropathological reaction (Quan et al., 2003) strengthening the link between TNF-α and the severity of the CNS reaction and suggesting an alternative role for IL-1 in the disease pathogenesis. The association between high levels of IFN-γ and severe neuroinflammatory reactions (Sternberg et al., 2005; MacLean et al., 2007) is an interesting finding due to the likely critical role for this cytokine in parasite traversal of the BBB as described above (Masocha et al., 2004). There is also evidence for early CNS production of the chemokines MIP-2, RANTES, MIP-1α and to a lesser extent MCP-1 following T.b. brucei infection in rats (Sharafeldin et al., 2000). Using double
Fig. 2. Schematic representation of the possible pathways and cell types involved in the generation of the neuroinflammatory response associated with trypanosome infection and drug treatment. The balance of pro- and counter inflammatory regulators is crucial to the outcome of the infection since these mediators control vital components of the inflammatory process within the brain including cellular activation, adhesion molecule expression, and MHC expression. In addition recent evidence suggest a central role for IFN-γ, T-lymphocyte transmigration pathways and the presence of particular laminins in the BBB basement membranes, in the entry of the parasites to the brain parenchyma.
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labelling immunocytochemistry the initial source of the MIP-2, RANTES and MIP-1α was shown to be astrocytes and microglia with production switching to macrophages and T-cells later in the course of the disease (Sharafeldin et al., 2000). These results suggest that astrocytes and microglia become activated very quickly following infection and initiate the inflammatory reaction within the brain which is later enhanced by the infiltrating lymphocytes. Early astrocyte activation in conjunction with cytokine expression was also a prominent feature in a murine model of HAT (Hunter et al., 1992b). Recently a study of murine T.b. brucei infections has shown the influence of host genetics on CNS cytokine production with concomitant effects on parasite invasion of the CNS and host mortality. In this study C57BL/6 mice expressed higher mRNA levels of IL-1β, IL-6, IL-10, TNF-α, IFN-γ, ICAM-1 and E-selectin but lower levels of TGF-β when compared with MHC-matched 129 Sv/Ev mice. The C57BL/6 animals had higher brain parasitaemia and increased CD4+ and CD8+ T-cell infiltration as well as higher cumulative mortality when compared to the 129 Sv/Ev strain (Masocha et al., 2008). Such structured studies on brain material from human patients are not possible therefore most data regarding cytokines within the CNS comes from analysis of CSF samples. These studies again indicate the importance of pro- and anti-inflammatory mediators in the outcome of the disease. In T.b. gambiense infections in patients in the Democratic Republic of Congo elevated levels of IL-6, IL-8 and IL10 were found in the CSF of late-stage patients (Lejon et al., 2002). The concentration of these cytokines reduced following treatment. In contrast to the data gained from rodent studies neither IFN-γ nor TNF-α was detected in the CSF of these patients. A significant increase in CSF IL-10 levels was also noted in T.b. rhodesiense infections in Uganda (MacLean et al., 2001). Further studies by this group showed elevated IL-6 and IL-10 concentrations in the CSF samples taken from 91 patients with the highest levels found in latestage patients and evidence of CNS synthesis of IL-10 in a quarter of these individuals (MacLean et al., 2006). Alterations in chemokine concentrations in the CSF have been shown in T.b. gambiense infected patients from Gabon and Angola (Courtioux et al., 2006). In this study elevated levels of the chemokines MIP-1α, MCP-1 and IL-8 as well as the cytokine IL-1β were found in the CSF of late-stage patients. Moreover, the levels of these inflammatory mediators correlated with the presence of neurological signs in the infected patients. A schematic representation of the cell types and inflammatory mediators known to be involved in HAT neuropathogenesis is shown in Fig. 2. 6. Conclusions The significance of trypanosomes as human pathogens has been known for over a century however insights into the pathogenesis of the disease are relatively recent and are still largely undiscovered. This disease affects vast populations but available chemotherapy is unsatisfactory due to poor efficacy, difficulty of administration and severe adverse reactions. Despite the obvious importance in differentiating early-stage disease from late-stage infections there is no consensus on the criteria defining this critical issue. However, regardless of this bleak picture advances in each of these areas are now being made. Animal models of HAT are unveiling the neuropathological progression of the infection (Kennedy, 1999; AntoineMoussiaux et al., 2008) while both animal and in vitro BBB models help define the mechanisms and pathways used by the parasite to gain entry to the brain (Masocha et al., 2004; Grab and Kennedy, 2008). Sensitive analysis methods that can extrapolate the intrathecal immune response pattern may discover new markers that can be used to accurately diagnose the stage of the infection (Lejon et al., 2003b; MacLean et al., 2006). Furthermore, many studies, both human (Priotto et al., 2007) and animal are being carried out in an effort to
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improve chemotherapy either through the use of a combination of trypanocidal drugs (Jennings et al., 2002) or by the inclusion of adjunct therapies (Rodgers et al., 2007; Masocha et al., 2006) to prevent the development of the PTRE. So long as this thrust of interest in the disease continues hope remains for significant improvements in the management of this ancient scourge of Africa. Acknowledgement I wish to thank Professor Peter Kennedy for his continued support, dedication to HAT research and critical review of this manuscript. Our research is funded by grants from the MRC [G0601059] and the Wellcome Trust [082786]. References Adams, J.H., Graham, D.I., 1998. Virus and other infections. In: Adams, J.H., Graham, D.I. (Eds.), An Introduction to Neuropathology. Churchhill Livingstone, Edinburgh, pp. 94–117. Adams, J.H., Haller, L., Boa, F.Y., Doua, F., Dago, A., Konian, K., 1986. Human African trypanosomiasis (T.b. gambiense): a study of 16 fatal cases of sleeping sickness with some observations on acute reactive arsenical encephalopathy. Neuropathol. Appl. Neurobiol. 12, 81–94. Antoine-Moussiaux, N., Magez, S., Desmecht, D., 2008. Contributions of experimental mouse models to the understanding of African trypanosomiasis. Trends Parasitol. 24, 411–418. Apted, F.I.C., 1970. Clinical manifestations and diagnosis of sleeping sickness. In: Mulligan, H.W. (Ed.), The African Trypanosomiases. Allen & Urwin, London, pp. 661–683. Atouguia, J.L.M., Kennedy, P.G.E., 2000. Neurological aspects of human African trypanosomiasis. In: Davies, L.E., Kennedy, P.G.E. (Eds.), Infectious Diseases of the Nervous System. Butterworth-Heinemann, Oxford, pp. 321–372. Balasegaram, M., Harris, S., Checchi, F., Ghorashian, S., Hamel, C., Karunakara, U., 2006a. Melarsoprol versus eflornithine for treating late-stage Gambian trypanosomiasis in the Republic of the Congo. Bull. World Health Organ. 84, 783–791. Balasegaram, M., Harris, S., Checchi, F., Hamel, C., Karunakara, U., 2006b. Treatment outcomes and risk factors for relapse in patients with early-stage human African trypanosomiasis (HAT) in the Republic of the Congo. Bull. World Health Organ. 84, 777–782. Barrett, M.P., Boykin, D.W., Brun, R., Tidwell, R.R., 2007. Human African trypanosomiasis: pharmacological re-engagement with a neglected disease. Br. J. Pharmacol. 152, 1155–1171. Bisser, S., Lejon, V., Preux, P.M., Bouteille, B., Stanghellini, A., Jauberteau, M.O., Buscher, P., Dumas, M., 2002. Blood-cerebrospinal fluid barrier and intrathecal immunoglobulins compared to field diagnosis of central nervous system involvement in sleeping sickness. J. Neurol. Sci. 193, 127–135. Burri, C., Brun, R., 2003. Eflornithine for the treatment of human African trypanosomiasis. Parasitol. Res. 90 (Supp 1), S49–S52. Burri, C., Nkunku, S., Merolle, A., Smith, T., Blum, J., Brun, R., 2000. 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The efficacy of pentamidine in the treatment of early–late stage Trypanosoma brucei gambiense trypanosomiasis. Am. J. Trop. Med. Hyg. 55, 586–588. Eckersall, P.D., Gow, J.W., McComb, C., Bradley, B., Rodgers, J., Murray, M., Kennedy, P.G., 2001. Cytokines and the acute phase response in post-treatment reactive encephalopathy of Trypanosoma brucei brucei infected mice. Parasitol. Int. 50, 15–26. Fink, E., Schmidt, H., 1979. Meningoencephalitis in chronic Trypanosoma brucei rhodesiense infection of the white mouse. Tropenmed. Parasitol. 30, 206–211. Grab, D.J., Kennedy, P.G., 2008. Traversal of human and animal trypanosomes across the blood-brain barrier. J. Neurovirology 14, 344–351. Grab, D.J., Nikolskaia, O., Kim, Y.V., Lonsdale-Eccles, J.D., Ito, S., Hara, T., Fukuma, T., Nyarko, E., Kim, K.J., Stins, M.F., Delannoy, M.J., Rodgers, J., Kim, K.S., 2004. African trypanosome interactions with an in vitro model of the human blood-brain barrier. J. Parasitol. 90, 970–979. Hunter, C.A., Kennedy, P.G., 1992. Immunopathology in central nervous system human African trypanosomiasis. J. Neuroimmunol. 36, 91–95. Hunter, C.A., Gow, J.W., Kennedy, P.G., Jennings, F.W., Murray, M., 1991. Immunopathology of experimental African sleeping sickness: detection of cytokine mRNA in the brains of Trypanosoma brucei brucei-infected mice. Infect. Immun. 59, 4636–4640. Hunter, C.A., Jennings, F.W., Adams, J.H., Murray, M., Kennedy, P.G., 1992a. Subcurative chemotherapy and fatal post-treatment reactive encephalopathies in African trypanosomiasis. Lancet 339, 956–958.
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Jennings, F.W., Gichuki, C.W., Kennedy, P.G., Rodgers, J., Hunter, C.A., Murray, M., Burke, J.M., 1997. The role of the polyamine inhibitor eflornithine in the neuropathogenesis of experimental murine African trypanosomiasis. Neuropathol. Appl. Neurobiol. 23, 225–234. Jennings, F.W., Rodgers, J., Bradley, B., Gettinby, G., Kennedy, P.G., Murray, M., 2002. Human African trypanosomiasis: potential therapeutic benefits of an alternative suramin and melarsoprol regimen. Parasitol. Int. 51, 381–388. Kennedy, P.G., 1999. The pathogenesis and modulation of the post-treatment reactive encephalopathy in a mouse model of Human African Trypanosomiasis. J. Neuroimmunol. 100, 36–41. Kennedy, P.G., 2004. Human African trypanosomiasis of the CNS: current issues and challenges. J. Clin. Invest. 113, 496–504. Kennedy, P.G., 2006. Diagnostic and neuropathogenesis issues in human African trypanosomiasis. Int. J. Parasitol. 36, 505–512. Kennedy, P.G., 2008a. Cytokines in central nervous system trypanosomiasis: cause, effect or both? Trans. R. Soc. Trop. Med. Hyg. 103, 213–214. Kennedy, P.G., 2008b. Diagnosing central nervous system trypanosomiasis: two stage or not to stage? Trans. R. Soc. Trop. Med. Hyg. 102, 306–307. Kennedy, P.G., 2008c. The continuing problem of human African trypanosomiasis (sleeping sickness). Ann. Neurol. 64, 116–126. Kennedy, P.G., Rodgers, J., Jennings, F.W., Murray, M., Leeman, S.E., Burke, J.M., 1997. A substance P antagonist, RP-67, 580, ameliorates a mouse meningoencephalitic response to Trypanosoma brucei brucei. Proc. Natl. Acad. Sci. U. S. A. 94, 4167–4170. Kennedy, P.G., Rodgers, J., Bradley, B., Hunt, S.P., Gettinby, G., Leeman, S.E., de Felipe, C., Murray, M., 2003. Clinical and neuroinflammatory responses to meningoencephalitis in substance P receptor knockout mice. Brain 126, 1683–1690. Lambert, P.H., Berney, M., Kazyumba, G., 1981. Immune complexes in serum and in cerebrospinal fluid in African trypanosomiasis. Correlation with polyclonal B cell activation and with intracerebral immunoglobulin synthesis. J. Clin. Invest. 67, 77–85. Legros, D., Ollivier, G., Gastellu-Etchegorry, M., Paquet, C., Burri, C., Jannin, J., Buscher, P., 2002. Treatment of human African trypanosomiasis–present situation and needs for research and development. Lancet Infect. Dis. 2, 437–440. Lejon, V., Lardon, J., Kenis, G., Pinoges, L., Legros, D., Bisser, S., N'Siesi, X., Bosmans, E., Buscher, P., 2002. Interleukin (IL)-6, IL-8 and IL-10 in serum and CSF of Trypanosoma brucei gambiense sleeping sickness patients before and after treatment. Trans. R. Soc. Trop. Med. Hyg. 96, 329–333. Lejon, V., Legros, D., Savignoni, A., Etchegorry, M.G., Mbulamberi, D., Buscher, P., 2003a. Neuro-inflammatory risk factors for treatment failure in “early second stage” sleeping sickness patients treated with pentamidine. J. Neuroimmunol. 144, 132–138. Lejon, V., Reiber, H., Legros, D., Dje, N., Magnus, E., Wouters, I., Sindic, C.J., Buscher, P., 2003b. Intrathecal immune response pattern for improved diagnosis of central nervous system involvement in trypanosomiasis. J. Infect. Dis. 187, 1475–1483. Lejon, V., Roger, I., Mumba, N.D., Menten, J., Robays, J., N'Siesi, F.X., Bisser, S., Boelaert, M., Buscher, P., 2008. Novel markers for treatment outcome in late-stage Trypanosoma brucei gambiense trypanosomiasis. Clin. Infect. Dis. 47, 15–22. MacLean, L., Odiit, M., Sternberg, J.M., 2001. Nitric oxide and cytokine synthesis in human African trypanosomiasis. J. Infect. Dis. 184, 1086–1090. MacLean, L., Odiit, M., Sternberg, J.M., 2006. Intrathecal cytokine responses in Trypanosoma brucei rhodesiense sleeping sickness patients. Trans. R. Soc. Trop. Med. Hyg. 100, 270–275. MacLean, L., Odiit, M., MacLeod, A., Morrison, L., Sweeney, L., Cooper, A., Kennedy, P.G., Sternberg, J.M., 2007. Spatially and genetically distinct African Trypanosome virulence variants defined by host interferon-gamma response. J. Infect. Dis. 196, 1620–1628. Masocha, W., Robertson, B., Rottenberg, M.E., Mhlanga, J., Sorokin, L., Kristensson, K., 2004. Cerebral vessel laminins and IFN-gamma define Trypanosoma brucei brucei penetration of the blood-brain barrier. J. Clin. Invest. 114, 689–694. Masocha, W., Rottenberg, M.E., Kristensson, K., 2006. Minocycline impedes African trypanosome invasion of the brain in a murine model. Antimicrob. Agents Chemother. 50, 1798–1804. Masocha, W., Rottenberg, M.E., Kristensson, K., 2007. Migration of African trypanosomes across the blood-brain barrier. Physiol. Behav. 92, 110–114. Masocha, W., Amin, D.N., Kristensson, K., Rottenberg, M.E., 2008. Differential invasion of Trypanosoma brucei brucei and lymphocytes into the brain of C57BL/6 and 129Sv/ Ev mice. Scand. J. Immunol. 68, 484–491.
Morrison, W.I., Murray, M., Whitelaw, D.D., Sayer, P.D., 1983. Pathology of infection with Trypanosoma brucei: disease syndromes in dogs and cattle resulting from severe tissue damage. Contrib. Microbiol. Immunol. 7, 103–119. Mott, F.W., 1907. Histological observations on the changes in the nervous system in trypanosome infections especially sleeping sickness and their relation to syphilitic lesions of the nervous system. Arch. Neurol. 3, 581–646. Mulenga, C., Mhlanga, J.D., Kristensson, K., Robertson, B., 2001. Trypanosoma brucei brucei crosses the blood-brain barrier while tight junction proteins are preserved in a rat chronic disease model. Neuropathol. Appl. Neurobiol. 27, 77–85. Nikolskaia, O.V., de, A.L.A., Kim, Y.V., Lonsdale-Eccles, J.D., Fukuma, T., Scharfstein, J., Grab, D.J., 2006a. Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease. J. Clin. Invest. 116, 2739–2747. Nikolskaia, O.V., Kim, Y.V., Kovbasnjuk, O., Kim, K.J., Grab, D.J., 2006b. Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human bloodbrain barrier. Int. J. Parasitol. 36, 513–519. Pentreath, V.W., Baugh, P.J., Lavin, D.R., 1994. Sleeping sickness and the central nervous system. Onderstepoort J. Vet. Res. 6, 369–377. Pepin, J., Milord, F., 1991. African trypanosomiasis and drug-induced encephalopathy: risk factors and pathogenesis. Trans. R. Soc. Trop. Med. Hyg. 85, 222–224. Pepin, J., Milord, F., 1994. The treatment of human African trypanosomiasis. Adv. Parasitol. 33, 1–47. Pepin, J., Milord, F., Guern, C., Mpia, B., Ethier, L., Mansinsa, D., 1989a. Trial of prednisolone for prevention of melarsoprol-induced encephalopathy in gambiense sleeping sickness. Lancet 1, 1246–1250. Pepin, J., Milord, F., Mpia, B., Meurice, F., Ethier, L., DeGroof, D., Bruneel, H., 1989b. An open clinical trial of nifurtimox for arseno-resistant Trypanosoma brucei gambiense sleeping sickness in central Zaire. Trans. R. Soc. Trop. Med. Hyg. 83, 514–517. Pepin, J., Milord, F., Meurice, F., Ethier, L., Loko, L., Mpia, B., 1992. High-dose nifurtimox for arseno-resistant Trypanosoma brucei gambiense sleeping sickness: an open trial in central Zaire. Trans. R. Soc. Trop. Med. Hyg. 86, 254–256. Philip, K.A., Dascombe, M.J., Fraser, P.A., Pentreath, V.W., 1994. Blood-brain barrier damage in experimental African trypanosomiasis. Ann. Trop. Med. Parasit. 88, 607–616. Poltera, A.A., 1980. Immunopathological and chemotherapeutic studies in experimental trypanosomiaisis with special reference to the heart and brain. Trans. R. Soc. Trop. Med. Hyg. 74, 706–715. Poltera, A.A., Hochmann, A., Lambert, P.H., 1982. Trypanosoma brucei gambiense: cerebral immunopathology in mice. Acta Trop. 39, 205–218. Poltera, A.A., Sayer, P.D., Brighouse, G., Bovell, D., Rudin, W., 1985. 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Contents lists available at ScienceDirect
Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
Review article
Human African trypanosomiasis, chemotherapy and CNS disease Jean Rodgers ⁎ Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, United Kingdom
a r t i c l e
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Article history: Received 17 December 2008 Accepted 5 February 2009 Keywords: Human African trypanosomiasis CNS Neuroinflammation Mouse model Chemotherapy
a b s t r a c t Trypanosomes have been recognised as human pathogens for over a century. Human African trypanosomiasis is endemic in an area sustaining 60 million people and is fatal without chemotherapeutic intervention. Available trypanocidal drugs require parenteral administration and are associated with adverse reactions including the development of a severe post-treatment reactive encephalopathy (PTRE). Following infection the parasites proliferate in the systemic compartment before invading the CNS where a cascade of events results in neuroinflammation. This review summarises the clinical manifestations of the infection and chemotherapeutic regimens as well as the current research findings and hypotheses regarding the neuropathogenesis of the disease. © 2009 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . Clinical manifestation of HAT . . . . . . . . Chemotherapy . . . . . . . . . . . . . . . 3.1. Early-stage treatments . . . . . . . . 3.2. Late-stage treatments . . . . . . . . 3.3. Disease staging . . . . . . . . . . . 4. Neuropathological features . . . . . . . . . 4.1. Post-mortem examinations . . . . . . 4.2. Animal models . . . . . . . . . . . 5. Parasites in the CNS . . . . . . . . . . . . 5.1. Trypanosome invasion of the brain . . 5.2. Parasites and inflammatory mediators. 6. Conclusions . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
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1. Introduction Human African trypanosomiasis (HAT), or sleeping sickness, is prevalent in 36 Sub-Saharan African countries situated between the latitudes 14o North and 29o South. The disease results from infection with protozoan parasites of the genus Trypanosoma. The infection is transmitted by the bite of the tsetse fly which is the insect vector of HAT. Untreated the disease is always fatal. There are many species of this parasite however, only two sub-species of the Trypanosoma brucei
⁎ Tel.: +44 141 330 3797; fax: +44 141 330 5603. E-mail address: [email protected]. 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.02.007
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group are infectious in man. Trypanosoma brucei (T.b.) gambiense causes HAT in West and Central Africa while T.b. rhodesiense causes disease in East and Southern Africa. Approximately 60 million people are at risk of infection and the most recent epidemiological figures report 17,500 new cases of HAT diagnosed in 2004 with a cumulative total of around 50–70,000 people infected (WHO, 2006). However, WHO estimates that less than 4 million of the population are under medical surveillance and only 10% of new infections are actually diagnosed (WHO, 2006, 1998).While there seems to be no doubt that the incidence of HAT has declined considerably over the last five years, precise figures for current HAT incidence and prevalence remain somewhat uncertain. The situation with HAT is subject to exacerbation by wars and civil unrest which result in a deterioration in health
J. Rodgers / Journal of Neuroimmunology 211 (2009) 16–22
surveillance and disease control which increase the likelihood of disease outbreaks and higher mortality rates. 2. Clinical manifestation of HAT The clinical manifestations of sleeping sickness are both diverse and variable and have been described in depth previously (Apted, 1970; Atouguia and Kennedy, 2000). In brief, the two different forms of HAT show different clinical courses; West African sleeping sickness is a chronic disease lasting from months to years whereas East African sleeping sickness follows an acute course with a duration of weeks to months. After initial infection the trypanosomes are localised to a nonsuppurative lesion or chancre that develops at the site of the infected tsetse bite. This leads to the early, or haemolymphatic, stage of the disease in which the parasites proliferate in the blood, and lymphatic system. Clinically the patient experiences febrile episodes with headaches and enlarged lymph nodes may be present at this stage. An erythematous skin rash may also appear. From here the parasites invade a variety of tissues including spleen, liver and heart and ultimately enter the central nervous system (CNS) to cause the late or encephalitic stage of the disease. In rhodesiense disease CNS involvement occurs early after infection, usually within 3 to 4 weeks of the tsetse bite, and there may be no clear clinical distinction between early and late stage disease since it is not possible to distinguish reliably between the early and late stages on the basis of the clinical features. In gambiense disease, CNS invasion can be delayed until many months or even years after infection (Apted, 1970). Signs of neurological involvement include mental disturbances such as irritability, lack of concentration and personality changes; motor system involvement with the development of seizures, muscle fasciculations and in some cases paralysis; sensory involvement with the presence of hyperaesthesia, anaesthesia or intense itching; pout or palmo-mental reflexes; a variety of characteristic sleep disturbances such as the development of daytime somnolence accompanied by night-time insomnia and alterations of sleep structure. As the disease progresses to the final stages the patient becomes indifferent to their surroundings and more difficult to rouse. Untreated, the patient will progress to coma and death (Atouguia and Kennedy, 2000). 3. Chemotherapy Both forms of HAT are invariably fatal without chemotherapeutic intervention. To date only four drugs, suramin, pentamidine, melarsoprol and eflornithine, are licensed for treatment of HAT (Barrett et al., 2007). Their efficacy is dependent on whether the infection has reached the CNS-stage or remains in the haemolymphatic early-stage. 3.1. Early-stage treatments Suramin, first used in the early 1920s, is used to treat T.b. rhodesiense infections. It is generally administered as a series of five slow intravenous injections given over a three week period (Kennedy, 2008c). Adverse effects, including cutaneous lesions, anaphylactic shock, renal failure and neurotoxicity, are associated with its administration (Legros et al., 2002). T.b. gambiense disease can be treated with intramuscular injections of pentamidine administered over 7–10 days. This drug has been in use since 1940. Although generally well tolerated pentamidine treatment can result in either hypo- or hyperglycemia and low blood pressure (Atouguia and Kennedy, 2000). Neither suramin nor pentamidine crosses the blood-brain barrier (BBB) efficiently and therefore they cannot be used to treat CNS-stage disease.
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(Pepin and Milord, 1994). Eflornithine is only effective against CNSstage T.b. gambiense infections since T.b. rhodesiense has shown innate resistance to the drug (Iten et al., 1995). The drug is expensive and its administration protracted involving a 14 day course of intravenous infusions of 100 mg/kg body weight of eflornithine given at 6 hourly intervals (Burri and Brun, 2003). Despite this, the use of eflornithine as a first line therapy in the treatment of T.b. gambiense infections has been widely advocated (Checchi and Barrett, 2008; Balasegaram et al., 2006a; Chappuis et al., 2005; Priotto et al., 2008). Adverse effects of eflornithine treatment include pancytopenia, gastrointestinal upset, convulsions or hemiparesis, which reverse spontaneously on withdrawal of the drug (Pepin and Milord, 1994; Atouguia and Kennedy, 2000; Legros et al., 2002). The only drug that can be used to treat both T.b. rhodesiense and T.b. gambiense infections once they have reached the CNS is the trivalent arsenical melarsoprol. This has been in use since 1949. Until recently the most commonly implemented treatment regimen comprised three to four series of three to four daily intravenous injections, each round of treatment separated by a rest period of 7 days (WHO, 1998; Legros et al., 2002). However a more concise treatment protocol consisting of 10 daily intravenous injections has been introduced for T.b. gambiense infections (Burri et al., 2000). Melarsoprol is a highly toxic drug and side effects of the regimen are common. Minor problems such as fever, gastric upset, and malaise occur quickly after initiating the treatment protocol. More serious side effects including tachycardia, subconjunctival haemorrhages and retrosternal pain can also develop (Atouguia and Kennedy, 2000). However, by far the most serious adverse effect of melarsoprol treatment is the development of a severe post-treatment reactive encephalopathy (PTRE). This occurs in approximately 10% of treated patients and can be fatal in half of the affected individuals. Therefore, administration of melarsoprol results in the death of up to 5% of all patients receiving treatment (Pepin and Milord, 1994). The mechanisms leading to the development of the PTRE remain unclear however several hypotheses have been muted (Hunter and Kennedy, 1992). These include; an immune reaction to parasite antigens released within the CNS following chemotherapy (Pepin and Milord, 1991), immune complex deposition (Lambert et al., 1981), toxicity of the arsenical moiety of the melarsoprol molecule (Hurst, 1959) autoimmune reactions (Poltera, 1980; Hunter et al., 1992c) or subcurative chemotherapy (Hunter et al., 1992a). Administration of corticosteroids, as an adjunct to melarsoprol treatment, has shown some degree of success in preventing the development of the PTRE but the beneficial effects of this remain controversial (Pepin et al., 1989a; Pepin and Milord, 1994). Since the mid-1970s nifurtimox has been used to treat Chagas' disease, the South American form of trypanosomiasis caused by infection with Trypanosoma cruzi. The drug is not well tolerated and can produce serious side effects ranging from gastro-intestinal upset to neurological reactions. Although not registered for the treatment of HAT, nifurtimox has been administered in patients with melarsoprol refractory T.b. gambiense-infections. The efficacy of the treatment varied substantially. A relapse rate of 63% was seen in patients given 12–17 mg/ kg nifurtimox daily for a period of 60 days (Pepin et al., 1989b). This high failure rate was reduced to 36% by raising the dosage of nifurtimox to between 24 and 37 mg/kg daily for a 30 day period, however the higher dose rate produced significant toxicity in the patients and severe neurological reactions were not uncommon (Pepin et al., 1992). More recently, a regimen comprising a combination of nifurtimox and eflornithine has been employed and this combination chemotherapy approach has shown promising results (Priotto et al., 2007). 3.3. Disease staging
3.2. Late-stage treatments Eflornithine or D,L-α-difluoromethylornithine, first used in 1981, is the most recently registered drug for the treatment of CNS-stage HAT
The criteria used for diagnosing CNS-stage HAT remain equivocal even though the correct staging of the disease is critical in establishing the appropriate chemotherapeutic regimen (Kennedy, 2008b). The
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parameters are based on microscopic examination of a sample of cerebrospinal fluid (CSF) to determine white blood cell count and the presence of trypanosomes. World Health Organisation (WHO) guidelines stipulate that any samples showing the presence of more than 5 white blood cells (WBC)/μl and/or the presence of trypanosomes in the CSF indicates that the infection has progressed to the CNS-stage and should therefore be treated with late-stage drugs (WHO, 1998). However, in some T.b. gambiense infected patients clinicians are using the much higher figure of N20 WBC/μl before proceeding to melarsoprol therapy (Lejon et al., 2003b; Kennedy, 2006). This higher figure evolved following successful pentamidine chemotherapy of patients presenting with trypanosomes in their CSF sample and a WBC of V20, (Pepin and Milord, 1994) and has led to the notion of an intermediate classification of “early late-stage” disease (Doua et al., 1996; Lejon et al., 2003a,b; Kennedy, 2004). The safety of implementing this increased threshold is still in debate and uncertain. A retrospective analysis of data acquired from the Republic of Congo, where an intermediate WBC count of N10 cells/μl was used to diagnosed CNS-stage disease, showed that implementation of this higher threshold led to an increased risk of relapse infection following pentamidine chemotherapy compared to the lower figure of 5 WBC/μl CSF (Balasegaram et al., 2006b). Additional criteria such as intrathecal IgM synthesis, CSF end-titres in LATEX/IgM and LATEX/T.b. gambiense positive CSF have been shown to be associated with the occurrence of treatment failures and could be useful markers to help determine the correct chemotherapeutic approach (Lejon et al., 2003a,b, 2008; Bisser et al., 2002). 4. Neuropathological features 4.1. Post-mortem examinations The neuropathological features of HAT have been determined from examination of a limited number of post-mortem samples. An acute meningoencephalitis develops as the disease progresses with the presence of macrophages, lymphocytes and an abundance of plasma cells in the meninges, perivascular spaces and eventually infiltrating the neuropil. Mott cells, or morular cells, can also be found, however these are not pathognomonic of the disease (Atouguia and Kennedy, 2000; Adams and Graham, 1998). The inflammatory cell infiltration is accompanied by the activation of both astrocytes and microglia (Adams et al., 1986). These changes are most obvious in the white matter of the cerebral hemispheres but are also apparent in circumventricular areas including the thalamus, hypothalamus and supraoptic nuclei. Despite this highly inflamed picture only nominal demyelination occurs and neuronal damage is minimal (Atouguia and Kennedy, 2000). The PTRE is characterised by a conspicuous increase in the severity of this inflammatory process and can, in some instances, take the form of an acute haemorrhagic leucoencephalopathy (Adams et al., 1986). 4.2. Animal models Due to the paucity of human samples most information regarding the neuropathogenesis of the disease has been derived from animal models of HAT. These show a similar neuropathological picture to that described above. Infiltration of the brain cerebral vessels by lymphocytes, plasma cells and macrophages has been described following T.b. rhodesiense-infections in vervet monkeys (Schmidt, 1983). Proliferation of the microglial cells in immediate proximity to the inflammatory infiltrates was also detected. However, a fully developed meningoencephalitis, with the presence of inflammatory cells in the neuropil was only rarely identified (Schmidt, 1983). This was more commonly reported following ineffectual drug treatment (Schmidt, 1983; Poltera et al., 1985) and may equate to the situation found in the PTRE associated with HAT treatment.
In a murine model of T.b. rhodesiense-infection mild inflammatory cell infiltration was detected in the brain connective tissue early after infection. The severity of the reaction increased as the disease progressed with a meningoencephalitis present in animals killed 4 weeks after infection (Fink and Schmidt, 1979). In T.b. bruceiinfections of mice inflammatory changes were detected in the choroid plexus and meninges from approximately 4 weeks after infection and in advanced cases perivascular cuffing was observed in meningeal and cerebral vessels (Poltera, 1980). An analogous pattern of inflammatory changes was present in T.b. gambiense infections although this produced a more chronic disease, in some cases lasting for a 22 month period (Poltera et al., 1982). In our murine model of HAT, only mild inflammatory changes are seen when the infection is allowed to progress naturally. However, treatment with a subcurative dose of diminazene aceturate, precipitates the development of encephalitic lesions and large perivascular cuffs consisting mainly of lymphocytes and macrophages (Jennings et al., 1989; Hunter and Kennedy, 1992). The reaction can be further exacerbated by administration of a second subcurative drug treatment following relapse to parasitaemia (Hunter et al., 1992a; Jennings et al., 1997). A wide spread astrocyte activation that becomes more pronounced as the disease severity increases is also a conspicuous feature (Hunter et al., 1992b; Jennings et al., 1997). This model is highly reproducible and can be easily manipulated to mirror the progressive neuroinflammatory reactions found in each stage of HAT. We have therefore used the model to study both the disease pathogenesis and the efficacy and safety of novel chemotherapy approaches (Sternberg et al., 2005; Kennedy et al., 1997, 2003; Rodgers et al., 2007; Eckersall et al., 2001; Kennedy, 1999; Jennings et al., 1997; Jennings et al., 2002). Fig. 1 demonstrates the differing levels if neuroinflammation that can be achieved through the use of this mouse model of HAT. The overall picture portrayed by the various animal models closely simulates the reaction described in the post-mortem material from human infections. A prominent link between subcurative chemotherapy and the development of a severe CNS inflammatory reaction is also apparent. Furthermore, several of these studies make note of the relative lack of neuronal damage that results from the inflammatory cell infiltration of the brain parenchyma (Morrison et al., 1983; Fink and Schmidt, 1979). 5. Parasites in the CNS 5.1. Trypanosome invasion of the brain It has been known for many years that chronic trypanosome infection leads to CNS invasion by the parasite and results in an inflammatory reaction within the brain. Nevertheless, the demonstration of trypanosomes within the human brain has been reported only rarely (Mott, 1907). This could be the consequence of a number of factors such as trypanocidal drug treatment prior to death or deterioration of the parasites due to the time lapse between patient death and tissue fixation (Calwell, 1937). At present the mechanisms and pathways used by the trypanosomes to gain entry and become established within the CNS are not fully elucidated. In animal models, parasites have been localised in the choroid plexus, circumventricular organs and peripheral ganglia, areas, where the BBB is weak (Schultzberg et al., 1988). However, this has yet to be definitively established as the route of parasite invasion into the CNS since some investigations employing rodent models suggest that the BBB becomes progressively damaged as the disease advances (Pentreath et al., 1994; Philip et al., 1994). Using a rat model of trypanosome infection Philip and colleagues demonstrated that by day 35 postinfection with T.b. brucei, fluorescent dye injected into the jugular vein could be detected in several brain areas, including, the thalamus and hypothalamus, indicating impairment of the BBB. By 40 days postinfection, this BBB damage had become extensive and the dye could be
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Fig. 1. H&E stained sections through the hippocampal brain region prepared from T.b. brucei infected mice. The sections show the progression of the neuroinflammation throughout the disease course; early stage (A), early-CNS stage (B), late-CNS stage (C), and PTRE (D). The increasing development of the perivascular cuffs (→) and encephalitis is demonstrated. Original magnification ×100.
found throughout both the grey and the white matter of the cortex (Philip et al., 1994). In contrast, a study of trypanosome infection in rats showed no indiscriminate loss of tight junction proteins in the BBB following invasion of the CNS by trypanosome parasites and no indication of plasma protein leakage into the parenchyma (Mulenga et al., 2001) indicating that the parasites actively enter the brain rather than diffuse into the tissue as a result of barrier impairment. More recent studies have shown a link between the laminin composition of the BBB endothelial basement membrane and the ability of the trypanosome to enter the brain tissue with laminin α4 being permissive to parasite transmigration and laminin α5 being inhibitory (Masocha et al., 2007, 2004). These studies also suggest that trypanosomes may exploit transmigratory mechanisms similar to those utilised by leucocytes since the parasites could not penetrate the brain in recombinant activating gene (RAG)-1 deficient mice which lack both T and B-cells. Furthermore, the pro-inflammatory cytokine IFN-γ appears to play a prominent role in the process since the absence of this cytokine or its receptor led to reduced brain invasion by the parasites with trypanosomes becoming ‘trapped’ between the endothelial basement membrane and the parenchymal basement membrane (Masocha et al., 2007, 2004). The importance of leucocytes in trypanosome penetration of the brain parenchyma was further highlighted in a study investigating the effects of minocycline treatment in T.b. brucei infected mice. Minocycline is known to reduce leucocyte penetration of the CNS, and this inhibition of leucocyte transmigration was accompanied by a reduction in parasite CNS invasion and a marked amelioration of the CNS inflammation (Masocha et al., 2006). Retention of the functionality of the BBB until the late possibly terminal stages of trypanosome infection was also found in a study utilising an in situ BBB perfusion model (Sanderson et al., 2008). In this study T.b. brucei infected mice were perfused with radiolabelled eflornithine at a variety of time points throughout the course of the infection and the amounts of eflornithine crossing the BBB assessed. Eflornithine was shown to penetrate the normal BBB relatively poorly, however the concentration of the drug
reaching the brain increased as the infection progressed with BBB dysfunction detectable by day 28 post-infection. The integrity of the barrier was never regained (Sanderson et al., 2008). In vitro systems have also been employed to investigate trypanosome interactions with the BBB. Cultures of human brain microvascular endothelial cells (HBMEC's), can be grown in a transwell based system to mimic the BBB. Trypanosomes have been shown to cross from one side of this in vitro barrier to the other via a paracellular route with only a transient reduction in transendothelial electrical resistance (TEER) indicating that the parasites traverse the BBB without causing permanent damage to its integrity (Grab et al., 2004). Further studies utilising this model in conjunction with confocal and electron microscopy have demonstrated the presence of trypanosomes within endothelial cells, although the significance of this intracellular location and the viability of these parasites is not yet known (Nikolskaia et al., 2006b). The most recent finding from this in vitro model indicates that parasite derived cysteine protease enzymes are important in enabling the trypanosome to migrate across the BBB (Nikolskaia et al., 2006a). In a complex series of experiments its was shown that both living parasites and parasite conditioned medium could induce transient increases in intracellular calcium concentrations ([Ca2+]i) in the HMBEC's. These alterations in [Ca2+]i were abrogated when a specific irreversible cathepsin L-like cysteine protease inhibitor (K11777) was included in the culture system. The cathepsin L-like cysteine protease present in T.b. brucei is known as brucipain. Furthermore, the addition of brucipain enriched culture medium to the transwell system enhanced traversal of the BBB by the trypanosomes implicating this enzyme as an important factor in the ability of the parasites to invade the brain (Nikolskaia et al., 2006a). 5.2. Parasites and inflammatory mediators As previously stated, the precise mechanisms used by the parasite to gain entry to the brain and induce the associated inflammatory
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reaction remain unclear. However, mouse model studies indicate that the expression of inflammatory mediators including cytokines, chemokines and adhesion molecules change during the course of the infection and that the balance between pro- and anti-inflammatory mediators is central to the outcome of the disease (Kennedy, 2004, 2008a; Sharafeldin et al., 2000; Mulenga et al., 2001). Following parasite CNS invasion in a murine model of HAT, expression of IL-1, IL6, TNF-α, IFN-γ and MIP-1α was found to be upregulated within the CNS by comparative RT-PCR analysis (Hunter et al., 1991, 1992b). In a more recent study examining protein levels, increased CNS TNF-α and IFN-γ concentrations were shown to be associated with severe neuroinflammatory responses while high levels of IL-6 and IL-10 were found in mice exhibiting only mild CNS inflammation (Sternberg et al., 2005). No significant associations between concentration and neuropathology could be detected when CNS levels of IL-1β or nitrate were measured. However, a significant increase in brain IL-1β was
detected by 7 days post-infection and this remained high throughout the early and late-stages of the disease as well as the PTRE (Sternberg et al., 2005). Furthermore, intraventricular infusion of IL-1 receptor antagonist in a rat model of trypanosome infection resulted in a restoration of body weight in the animals but had no effect on neurodegeneration whereas infusion of soluble type-1 TNF-α receptor reduced the neuropathological reaction (Quan et al., 2003) strengthening the link between TNF-α and the severity of the CNS reaction and suggesting an alternative role for IL-1 in the disease pathogenesis. The association between high levels of IFN-γ and severe neuroinflammatory reactions (Sternberg et al., 2005; MacLean et al., 2007) is an interesting finding due to the likely critical role for this cytokine in parasite traversal of the BBB as described above (Masocha et al., 2004). There is also evidence for early CNS production of the chemokines MIP-2, RANTES, MIP-1α and to a lesser extent MCP-1 following T.b. brucei infection in rats (Sharafeldin et al., 2000). Using double
Fig. 2. Schematic representation of the possible pathways and cell types involved in the generation of the neuroinflammatory response associated with trypanosome infection and drug treatment. The balance of pro- and counter inflammatory regulators is crucial to the outcome of the infection since these mediators control vital components of the inflammatory process within the brain including cellular activation, adhesion molecule expression, and MHC expression. In addition recent evidence suggest a central role for IFN-γ, T-lymphocyte transmigration pathways and the presence of particular laminins in the BBB basement membranes, in the entry of the parasites to the brain parenchyma.
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labelling immunocytochemistry the initial source of the MIP-2, RANTES and MIP-1α was shown to be astrocytes and microglia with production switching to macrophages and T-cells later in the course of the disease (Sharafeldin et al., 2000). These results suggest that astrocytes and microglia become activated very quickly following infection and initiate the inflammatory reaction within the brain which is later enhanced by the infiltrating lymphocytes. Early astrocyte activation in conjunction with cytokine expression was also a prominent feature in a murine model of HAT (Hunter et al., 1992b). Recently a study of murine T.b. brucei infections has shown the influence of host genetics on CNS cytokine production with concomitant effects on parasite invasion of the CNS and host mortality. In this study C57BL/6 mice expressed higher mRNA levels of IL-1β, IL-6, IL-10, TNF-α, IFN-γ, ICAM-1 and E-selectin but lower levels of TGF-β when compared with MHC-matched 129 Sv/Ev mice. The C57BL/6 animals had higher brain parasitaemia and increased CD4+ and CD8+ T-cell infiltration as well as higher cumulative mortality when compared to the 129 Sv/Ev strain (Masocha et al., 2008). Such structured studies on brain material from human patients are not possible therefore most data regarding cytokines within the CNS comes from analysis of CSF samples. These studies again indicate the importance of pro- and anti-inflammatory mediators in the outcome of the disease. In T.b. gambiense infections in patients in the Democratic Republic of Congo elevated levels of IL-6, IL-8 and IL10 were found in the CSF of late-stage patients (Lejon et al., 2002). The concentration of these cytokines reduced following treatment. In contrast to the data gained from rodent studies neither IFN-γ nor TNF-α was detected in the CSF of these patients. A significant increase in CSF IL-10 levels was also noted in T.b. rhodesiense infections in Uganda (MacLean et al., 2001). Further studies by this group showed elevated IL-6 and IL-10 concentrations in the CSF samples taken from 91 patients with the highest levels found in latestage patients and evidence of CNS synthesis of IL-10 in a quarter of these individuals (MacLean et al., 2006). Alterations in chemokine concentrations in the CSF have been shown in T.b. gambiense infected patients from Gabon and Angola (Courtioux et al., 2006). In this study elevated levels of the chemokines MIP-1α, MCP-1 and IL-8 as well as the cytokine IL-1β were found in the CSF of late-stage patients. Moreover, the levels of these inflammatory mediators correlated with the presence of neurological signs in the infected patients. A schematic representation of the cell types and inflammatory mediators known to be involved in HAT neuropathogenesis is shown in Fig. 2. 6. Conclusions The significance of trypanosomes as human pathogens has been known for over a century however insights into the pathogenesis of the disease are relatively recent and are still largely undiscovered. This disease affects vast populations but available chemotherapy is unsatisfactory due to poor efficacy, difficulty of administration and severe adverse reactions. Despite the obvious importance in differentiating early-stage disease from late-stage infections there is no consensus on the criteria defining this critical issue. However, regardless of this bleak picture advances in each of these areas are now being made. Animal models of HAT are unveiling the neuropathological progression of the infection (Kennedy, 1999; AntoineMoussiaux et al., 2008) while both animal and in vitro BBB models help define the mechanisms and pathways used by the parasite to gain entry to the brain (Masocha et al., 2004; Grab and Kennedy, 2008). Sensitive analysis methods that can extrapolate the intrathecal immune response pattern may discover new markers that can be used to accurately diagnose the stage of the infection (Lejon et al., 2003b; MacLean et al., 2006). Furthermore, many studies, both human (Priotto et al., 2007) and animal are being carried out in an effort to
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improve chemotherapy either through the use of a combination of trypanocidal drugs (Jennings et al., 2002) or by the inclusion of adjunct therapies (Rodgers et al., 2007; Masocha et al., 2006) to prevent the development of the PTRE. So long as this thrust of interest in the disease continues hope remains for significant improvements in the management of this ancient scourge of Africa. Acknowledgement I wish to thank Professor Peter Kennedy for his continued support, dedication to HAT research and critical review of this manuscript. Our research is funded by grants from the MRC [G0601059] and the Wellcome Trust [082786]. References Adams, J.H., Graham, D.I., 1998. Virus and other infections. In: Adams, J.H., Graham, D.I. (Eds.), An Introduction to Neuropathology. Churchhill Livingstone, Edinburgh, pp. 94–117. Adams, J.H., Haller, L., Boa, F.Y., Doua, F., Dago, A., Konian, K., 1986. 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Hunter, C.A., Kennedy, P.G., 1992. Immunopathology in central nervous system human African trypanosomiasis. J. Neuroimmunol. 36, 91–95. Hunter, C.A., Gow, J.W., Kennedy, P.G., Jennings, F.W., Murray, M., 1991. Immunopathology of experimental African sleeping sickness: detection of cytokine mRNA in the brains of Trypanosoma brucei brucei-infected mice. Infect. Immun. 59, 4636–4640. Hunter, C.A., Jennings, F.W., Adams, J.H., Murray, M., Kennedy, P.G., 1992a. Subcurative chemotherapy and fatal post-treatment reactive encephalopathies in African trypanosomiasis. Lancet 339, 956–958.
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