Jeremy M. Sternberg

Human African trypanosomiasis: clinical presentation and immune response

Human African trypanosomiasis or sleeping sickness is caused by infection with two subspecies of the tsetse‐fly‐vectored haemoflagellate parasite Trypanosoma brucei. Historically, epidemic sleeping sickness has caused massive loss of life, and related animal diseases have had a crucial impact on development in sub‐Saharan Africa. After a period of moderately successful control during the mid‐part of the 20th century, sleeping sickness incidence is currently rising, and control is hampered by a combination of factors, including civil unrest and the possible development of drug resistance by the parasites. The prevailing view is that the disease is invariably fatal without anti‐trypanosomal drug treatment. However, there have also been intriguing reports of wide variations in disease severity as well as evidence of asymptomatic carriers of trypanosomes. These differences in the presentation of the disease will be discussed in the context of our knowledge of the immunology of trypanosomiasis. The impact of dysregulated inflammatory responses in both systemic and CNS pathology will be examined and the potential for host genotype variation in disease severity and control will be discussed.

Meningoencephalitic African trypanosomiasis: Brain IL-10 and IL-6 are associated with protection from neuro-inflammatory pathology

The relationship of neuropathology to CNS inflammatory and counter-inflammatory cytokine production in African trypanosome-infected mice was studied using an infection model with a defined disease progression. The initial phase of CNS infection by trypanosomes, where only mild neuropathology is evident, was characterised by high levels of IL-10 and IL-6. In the later phase of CNS infection and in a post-drug treatment model, moderate to severe neuropathology was associated with high levels of IFN-gamma and TNF-alpha. The relationship of these cytokines to neuropathological grade suggests that IL-10 and IL-6 protect the CNS from inflammatory pathology when parasites first enter the brain and the data reconcile previously contradictory clinical measurements of CSF cytokines in meningoencephalitic patients with post-mortem histopathology observations.

Spatially and Genetically Distinct African Trypanosome Virulence Variants Defined by Host Interferon-γ Response

We describe 2 spatially distinct foci of human African trypanosomiasis in eastern Uganda. The Tororo and Soroti foci of Trypanosoma brucei rhodesiense infection were genetically distinct as characterized by 6 microsatellite and 1 minisatellite polymorphic markers and were characterized by differences in disease progression and host-immune response. In particular, infections with the Tororo genotype exhibited an increased frequency of progression to and severity of the meningoencephalitic stage and higher plasma interferon (IFN)- gamma concentration, compared with those with the Soroti genotype. We propose that the magnitude of the systemic IFN- gamma response determines the time at which infected individuals develop central nervous system infection and that this is consistent with the recently described role of IFN- gamma in facilitating blood-brain barrier transmigration of trypanosomes in an experimental model of infection. The identification of trypanosome isolates with differing disease progression phenotypes provides the first field-based genetic evidence for virulence variants in T. brucei rhodesiense.

SUPPRESSOR MACROPHAGES IN TRYPANOSOMA BRUCEI INFECTION : NITRIC OXIDE IS RELATED TO BOTH SUPPRESSIVE ACTIVITY AND LIFESPAN IN VIVO

African trypanosome infections cause immunosuppression in both experimental rodent and natural hosts. One characteristic of this is an eliciting of suppressor macrophages which results in an unresponsiveness in lymphocytes. Macrophages from Trypanosoma brucei‐infected mice have previously been shown to produce high levels of nitric oxide (NO). Using model systems based on in vivo macrophage transfer and drug cure, we have sought to determine the relationship between NO and suppressed lymphocyte responses. Peritoneal macrophages from T. brucei‐infected mice inhibited the Concanavalin A (Con‐A) response of spleen cells from syngeneic recipients 3–4 days after transfer in vivo due to the activity of suppressor macrophages. When macrophage NO synthesis was inhibited either in vitro or in vivo the suppressive effects were partially abrogated. These data provide evidence of a role for NO in mediating immunosuppression during murine T. brucei infection. Suppression in spleens of mice receiving suppressor macrophages was transient, with total recovery of spleen cell mitogen responses six days after transfer. Suppression and recovery was found to coincide with the presence or absence (respectively) of donor macrophages in recipient spleens. When T. brucei‐infected mice were treated with a curative dose of a trypanocide there followed a recovery of lymphocyte responsiveness after a period of 4–5 days, and this directly correlated with a reduction of macrophage NO synthesis to control levels both in vivo and in vitro. The apparent loss of suppressor macrophage activity after 4–6 days in both drug cured animals and recipients of macrophage transfer was shown to be due to NO‐mediated apoptosis of these cells.

Focus-Specific Clinical Profiles in Human African Trypanosomiasis Caused by Trypanosoma brucei rhodesiense

Background Diverse clinical features have been reported in human African trypanosomiasis (HAT) foci caused by Trypanosoma brucei rhodesiense (T.b.rhodesiense) giving rise to the hypothesis that HAT manifests as a chronic disease in South-East African countries and increased in virulence towards the North. Such variation in disease severity suggests there are differences in host susceptibility to trypanosome infection and/or genetic variation in trypanosome virulence. Our molecular tools allow us to study the role of host and parasite genotypes, but obtaining matched extensive clinical data from a large cohort of HAT patients has previously proved problematic. Methods/Principal Findings We present a retrospective cohort study providing detailed clinical profiles of 275 HAT patients recruited in two northern foci (Uganda) and one southern focus (Malawi) in East Africa. Characteristic clinical signs and symptoms of T.b.rhodesiense infection were recorded and the degree of neurological dysfunction determined on admission. Clinical observations were mapped by patient estimated post-infection time. We have identified common presenting symptoms in T.b.rhodesiense infection; however, marked differences in disease progression and severity were identified between foci. HAT was characterised as a chronic haemo-lymphatic stage infection in Malawi, and as an acute disease with marked neurological impairment in Uganda. Within Uganda, a more rapid progression to meningo-encephaltic stage of infection was observed in one focus (Soroti) where HAT was characterised by early onset neurodysfunction; however, severe neuropathology was more frequently observed in patients in a second focus (Tororo). Conclusions/Significance We have established focus-specific HAT clinical phenotypes showing dramatic variations in disease severity and rate of stage progression both between northern and southern East African foci and between Ugandan foci. Understanding the contribution of host and parasite factors in causing such clinical diversity in T.b.rhodesiense HAT has much relevance for both improvement of disease management and the identification of new drug therapy.

Characterization of the B‐cell inhibitory protein factor in Ixodes ricinus tick saliva: a potential role in enhanced Borrelia burgdoferi transmission

We recently described the inhibition of host B lymphocytes by Ixodes ricinus tick saliva. In this study, we characterized the factor responsible for this activity and examined the modulation of lipopolysaccharide (LPS)‐ and Borrelia burgdorferi outer surface protein (Osp)‐induced proliferation of naive murine B lymphocytes by an enriched fraction of this factor. The B‐lymphocyte inhibitory activity was destroyed by trypsin treatment, indicating that a proteinaceous factor was responsible for this activity. The removal of glutathione‐S‐transferase (GST) from tick salivary glands extracts (SGE) showed that this B‐cell inhibitory protein (BIP) was not a GST. Gel filtration liquid chromatography indicated that BIP has a native molecular weight of ≈ 18 000. An enrichment protocol, using a combination of anion‐exchange and reverse‐phase liquid chromatography, was established. BIP‐enriched fractions did not suppress T‐cell proliferation. Delayed addition of BIP‐enriched fractions, up to 7 hr after LPS addition, inhibited the proliferation of isolated B cells. BIP‐enriched fractions dramatically inhibited both OspA‐ and OspC‐induced proliferation of isolated B cells. These results strongly suggest that BIP may facilitate B. burgdorferi transmission by preventing B‐cell activation, and also highlights the potential of BIP as a therapeutic agent in B‐cell maladies.

Ixodes ricinus tick salivary gland extract inhibits IL‐10 secretion and CD69 expression by mitogen‐stimulated murine splenocytes and induces hyporesponsiveness in B lymphocytes

Tick saliva contains immunosuppressive factors allowing this blood‐feeding ectoparasite to remain on hosts and enhancing pathogen transmission. In this study, we examined the modulation of mitogen‐induced activation of naive murine splenocytes by the saliva and salivary gland extract (SGE) of I. ricinus ticks. We found that saliva‐specific factors reduced IL‐10 production by both concanavalin A (ConA) and lipopolysaccharide (LPS) stimulated splenocytes. The LPS‐induced IL‐10 production is 10 times more sensitive to SGE than the ConA‐induced IL‐10 production. Flow cytometric analysis determined that SGE particularly inhibited B (B220+) cell IL‐10 production in mitogen‐stimulated splenocyte preparations. Moreover, SGE reduced the early activation marker CD69 expression on ConA‐activated T cells and also on B cells in presence of ConA or LPS. Annexin V and Via‐probe staining demonstrated that SGE did not increase cell death in activated splenocytes and slightly decreased apoptosis in B lymphocytes. By employing assays with isolated B cells, we further showed that SGE had a direct effect on B cells and inhibited LPS‐induced B cell proliferation. Taken together, our results indicate that salivary immunomodulators induce hyporesponsiveness to mitogen in both T and B cells, and that a direct B‐cell inhibitory activity is present in tick saliva.

A spectrum of disease in Human African trypanosomiasis: the host and parasite genetics of virulence.

For over 50 years it has been known that there are considerable differences in the severity and rate of progression of both Trypanosoma brucei rhodesiense and T. b. gambiense infection between individuals. Yet research into the factors, whether parasite or host, which control virulence in Human African trypanosomiasis is in its infancy. In this paper we review the clinical evidence for virulence variation and the epidemiological and experimental data that give clues as to the mechanisms involved. Evidence will be presented for both asymptomatic forms of T. b. gambiense infection and low virulence forms of T. b. rhodesiense infection in humans. While in both cases the mechanisms remain to be elucidated, the overall infection virulence phenotype is determined by both parasite and host genotype.

Trypanosoma brucei is protected from the cytostatic effects of nitric oxide under in vivo conditions

In mice infected withTrypanosoma brucei, splenic and peritoneal macrophages release substantial amounts of nitric oxide (NO). The production of NO by activated macrophages has been reported to be a nonspecific immune-effector mechansism against several parasites, and in this work we investigate the role of NO in killingT. brucei. Addition of bloodstream trypanosomes to peritoneal macrophages activated in vitro resulted in an NO-dependent inhibition of parasite growth. This effect was totally abrogated when dilutions of whole blood were included in the cultures, suggesting that bloodstream parasites such asT. brucei are not susceptible to NO-mediated killing in vivo.

African trypanosome infections in mice that lack the interferon-gamma receptor gene: nitric oxide-dependent and -independent suppression of T-cell proliferative responses and the development of anaemia.

Infection of mice with African trypanosomes leads to a severe immunosuppression, mediated by suppressor macrophages. Using ex vivo macrophage culture and in vivo cell transfer, it has been shown that nitric oxide (NO) is a potent effector product of these cells and causes both lymphocyte unresponsiveness and dyserythropoiesis. We explored the role of NO in vivo during trypanosome infection using mice with a disrupted interferon‐γ‐receptor gene, which were unable to respond with macrophage activation and NO synthesis. These mice were less effective at controlling parasitaemia than the wild types, but showed an improved splenic T‐cell responsiveness and reduced anaemia during the early stages of infection. The data indicate that, in the mouse, NO is a significant mediator of immunosuppression only in early infection. Beyond day 10 of infection, NO‐independent mechanisms are of primary significance and the control of parasitaemia and T‐cell responsiveness are not directly related.