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Please be advised that this information was generated on 2021-10-06 and may be subject to change. Murine Cerebral Malaria:

Aspects of pathogenesis & protection

Rob Hermsen Murine Cerebral Malaria:

Aspects of pathogenesis and protection

Cornelus C. (Rob) Hermsen Publication of this thesis was financially supported by: GlaxoWellcome, Zeist

Cover illustration: Electronmicroscopic photographs (magnification 1500x) of the brain of a mouse dying of experimental cerebral malaria (pathogenesis) and of the brain of a mouse protected against experimental cerebral malaria (protection). Design: Ma Donna and Rob Hermsen i Copyright 1999 Rob Hermsen. All rights reserved

ISBN 90 9012412 8 1999

Printing: PrintPartners Ipskamp, Enschede, The Netherlands Murine Cerebral Malaria:

Aspects of pathogenesis and protection

Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen.

Proefschrift

ter verkrijging van de graad van doctor aan de Katholieke Universiteit Nijmegen, volgens besluit van het College van Decanen in het openbaar te verdedigen op dinsdag 23 februari 1999 des namiddags om 3.30 uur precies

door

Cornelus Christianus Hermsen

geboren op 11 maart 1953 te Elden Promotores: Mw. Prof. Dr. J.A.A. Hoogkamp-Korstanje

Co-promotores: Dr. W.M.C. Eling Dr. R.W. Sauerwein

Manuscriptcommissie: Prof. Dr. J. van der Meer (voorzitter) Prof. Dr. W. van den Berg Prof. Dr. P. Kager (AMC)

The studies presented in this thesis were performed at the Department of Medical Microbiology, section Parasitology, University Hospital St. Radboud, Nijmegen, The Netherlands.

All published papers were printed with permission and credit to their resource Friendship is a single soul dwelling within two bodies Aristotle

ter nagedachtenis aan mijn vader en moeder Contents page

Chapter 1 General Introduction

Chapter 2 Depletion of CD4+ or CD8+ T-cells prevents Plasmodium, berghei 31 induced cerebral malaria in end-stage disease

Chapter 3 Convulsions due to increased permeability of the blood-brain-barrier 45 in experimental cerebral malaria can be prevented by splenectomy or anti-T-cell treatment

Chapter 4 Protection against Plasmodium berghei induced experimental cerebral 55 malaria by treatment with antioxidants

Chapter 5 Alloxan treatment protects Plasmodium berghei infected mice at 73 end stage disaese against experimental cerebral malaria

Chapter 6 Circulating Tumour Necrosis Factor a is not involved in the development 85 of cerebral malaria in Plasmodium berghei-inîected C57B1 mice

Chapter 7 Treatment with recombinant human tumour necrosis factor-a reduces 101 parasitaemia and prevents P. berghei K173 induced cerebral malaria in mice

Chapter 8 Thiolated recombinant human tumour necrosis factor-a enhances 121 suppression of parasitaemia and protection against P. berghei K173 induced cerebral malaria

Chapter 9 Summary and discussion 143

Chapter 10 Samenvatting 155 Publications 163 Curriculum vitae 167 Dankwoord 169 Abbreviations 171 CHAPTER

GENERAL INTRODUCTION 1.1 The malaria problem 1.1.1 Introduction 1.2 Clinical manifestations 1.2.1 Uncomplicated malaria 1.2.2 Severe malaria 1.3 Pathogenesis of human cerebral malaria 1.3.1 Introduction 1.3.2 The obstruction theory 1.3.2 The inflammation theory 1.3.2.1 Malaria and TNFa 1.4 Experimental cerebral malaria 1.4.1 Experimental models 1.4.2 Pathogenesis of experimental cerebral malaria 1.4.2.1 T cells 1.4.2.2 Tumour Necrosis Factor a 1.4.2.3 Blood-brain-barrier 1.4.2.4 Reactive Oxygen Intermediates 1.4.3 Characteristics of the Plasmodium berghei K173 cerebral malaria model 1.5 Outline of this thesis

9 1.1 THE MALARIA PROBLEM

1.1.1. Introduction Plasmodium falciparum infections are an important and increasing cause of morbidity and mortality in the tropics. Falciparum malaria is endemic in over 90 countries and it causes an estimated 300-500 million clinical cases and 1.5-2.7 million deaths per year (WHO 1996). African children carry the greatest burden of disease with an estimated 200 million episodes of clinical malaria each year. The vast majority of malaria associated deaths world-wide occurs in this group (Greenwood et al, 1991; World bank, 1993).

Malaria is caused by an unicellular parasite of the genus Plasmodium. In man four different species can cause malaria: P. vivax, P. ovale, P. malariae and P. falciparum. The first three cause severe illness, but are rarely fatal. These are the types of malaria which cause distinctive intermittent fevers, and a patient’s temperature chart can be a helpful guide for identification of the species. In Africa P. falciparum is the most common malaria parasite whereas P. vivax is rarely seen. Malaria-parasites are transmitted from man to man by a number of species of Anopheles mosquitoes which act as intermediate hosts. The Anopheles are active at night and until dawn and shelter in shaded, humid places during daytime. The parasites reproduce asexually in man, and sexually in female mosquitos. Parasites are transmitted by the bite of an infectious mosquito, migrate via the bloodstream to the liver, infect liver parenchymal cells and from there they infect erythrocytes. The parasites proliferate in erythrocytes, and the released progeny invades new red blood cells. It is at this stage that the classical symptoms of malaria occur, such as high periodic fever. Some erythrocytic parasites differentiate into sexual forms which, in turn, are infective to the mosquito.

1.2 CLINICAL MANIFESTATIONS

1.2.1 Uncomplicated malaria The classical picture of uncomplicated malaria is one of sudden cold shivers followed by a high fever lasting 6 to 8 hours. When the fever diminishes, a symptom free period of one day follows after which the fever returns. Anemia and enlargement of the spleen and liver can develop when malaria progresses. Except for the, more or less, typical fever pattern, a variety of clinical

10 manifestations can be observed that are by no means specific for malaria (Molyneux et a l. 1989). Uncomplicated falciparum malaria infection can progress rapidly and, if not properly treated, can lead to life threatening complications.

1.2.2 Severe malaria The criteria for severe falciparum malaria have been defined by the WHO (1990). One of them being cerebral malaria (CM). CM is defined as Plasmodium falciparum in the peripheral blood and unrousable coma often preceeded by fever for 1 to 3 days and convulsions. In endemic areas P. falciparum infections are especially dangerous to non-immune individuals i.e. children until the age of five in Africa, and to travellers without malaria experience. Indigenous adults are largely protected from severe disease by a form of partial immunity called premunition. Such individuals are carriers of the parasite but without minor clinical symptoms. Premunition develops slowly in response to repeated infections by the same species of Plasmodium and is lost in the absence of continuous exposure to infection (Knell, 1991).

1.3 PATHOGENESIS OF HUMAN CEREBRAL MALARIA

1.3.1 Introduction The pathogenesis of cerebral malaria (CM) in P. falciparum infected people is not completely understood. There are two theories: In the the obstruction theory it is proposed that sequestration of parasitized red blood cells (PBRCs) in the cerebral microcirculation may lead to obstruction of blood flow and subsequently anoxia (Warrell et al. 1988; Pongponratn et al. 1991; Berendt et al. 1994). Alternatively in the inflammation theory it is stated that cerebral pathology might be secondary to (an) immunological reaction(s) affecting the integrity of the vascular endothelial lining (Grau et al. 1989; Butcher et al. 1990; Clark & Rockett 1994; Eling & Kremsner 1994; Grau & Kossodo 1994; Patnaik et al. 1994; Eling & Sauerwein 1995).

11 1.3.2. The obstruction theory A distinctive feature of P. falciparum infections is selective accumulation of parasitized red blood cells (PRBCs) in the microvasculature of several organs, particularly the brains, but also heart, lung, kidney and liver (Pongponratn et al. 1991; Aikawa et al. 1992; Eling & Sauerwein 1995). A subsequent characteristic is the adherence of erythrocytes infected with mature stage parasites (trophozoites/schizonts) to endothelial cells lining the post-capillary venules. Moreover, Pongponratn et al. (1991) found a 94% PRBC sequestration rate in cerebral microvessels of patients with cerebral malaria while a 13% rate was found in noncerebral falciparum cases. Adherence in the brain microvessels is considered to potentially block blood flow resulting in confusion, lethargy, deep coma and death (Warrell 1987; MacPherson et al. 1985; Howard & Gilladoga 1989). Adherence of parasitized erythrocytes depends on the presence of a ligand on the membrane surface, P. falciparum erythrocyte major protein 1 (PfEMP1), a polymorphic (Mr 200.000-350.000) protein, that binds to receptors on the endothelial lining. PRBCs are able to bind to a number of adhesion molecules of endothelial cells, particularly of post-capillary venules (e.g. CD36, thrombospondin, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1)) and to the glycosaminoglycans chondroitin sulfate A and heparan sulfate. Moreover, Turner et al. (1994) showed co-localization of PRBC sequestration with the expression of ICAM-1, CD36 and E- selectin in cerebral vessels from fatal cases of P. falciparum malaria. Furthermore, Fried & Duffy (1996) showed adherence of P. falciparum-infected red blood cells to chondroitin sulfate A in the human placenta, and it is suggested that PfEMP1 is responsible for this binding (Rogerson & Brown 1997). Rosette formation of parasitized and unparasitized erythrocytes has been observed (Carlson et a l. 1990; Ho et al. 1991; Treutiger et al. 1992) and may contibute to obstruction of the vasculature. PfEMP1 is considered to be the rosetting ligand (Chen et al. 1998), and heparan sulfate, or a heparan sulfate-like molecule, on erythrocytes are the receptor for PfEMP1 binding. However, Rowe et al. (1995) and Newbold et al. (1997) showed that rosette formation was associated with severe malaria but not specifically with CM.

12 1.3.2 The inflammation theory The inflammation theory suggests that parasite-induced immunopathology plays an important role in the pathogenesis of CM (Grau et al. 1986; Curfs et al. 1989). Malaria parasites live and multiply in the blood vascular sytem. Therefore, infection can induce an intravascular inflammatory reaction. Inflammation is observed in organs that play an important role in the surveillance of the blood vascular compartment such as the liver and spleen but also in brains (Eling & Sauerwein 1995). Inflammatory reactions as a result of interactions between the parasite and host cells may induce changes in the permeability of the endothelial lining and cause disturbance of organ function. Moreover, expression of endothelial cell receptors used for binding by inflammatory cells is enhanced by tumour necrosis factora (TNFa; Senaldi et al. 1994) a cytokine released in increasing amounts during infection. This permeability hypothesis is supported by monkey malaria data showing the passage of labelled albumin across the blood-brain barrier in malaria-infected animals (Maegraith 1973). On the one hand this observation was not confirmed in adult Thai patients with CM (Warrell et al. 1986), whereas on the other hand enhanced capillary permeability to albumin in falciparum malaria was observed by Areekul et al. (1984, 1988). In addition, Das et al. (1991) observed increased levels of protein and lipid peroxidation products in the cerebrospinal fluid of patients with CM. The pathogenesis of fatal experimental cerebral malaria (ECM) has been studied in detail in murine models (Grau et al. 1986; Curfs et al. 1989; Grau et al. 1989a; Grau et al. 1994b). In these models, T cells and macrophages play an important role in the immunopathology of ECM. No ECM occurs in congenital T-cell deficient mice or in normal mice treated with anti-T-cell antibodies after infection with the parasite (Finley et al. 1982; Grau et al. 1986; Curfs et al. 1989). Furthermore, a timely depletion of macrophages also prevents the development of the cerebral syndrome (Grau et al. 1988; Grau et al. 1990; Curfs et al. 1993). Malaria parasites activate T cells and mononuclear phagocytes to release cytokines (Eling & Kremsner 1994). Of these cytokines, TNFa (tumour necrosis factora) has been considered important in the pathogenesis of CM and ECM.

13 1.3.2.1 Malaria and TNFa TNFa concentrations are more elevated in CM patients than in non-cerebral cases (Scuderi et al. 1986; Kern et al. 1989). In Malawi and Zaire, serum-TNFa concentrations of children infected with P. falciparum were positively correlated with disease severity (Grau et al. 1989b; Kern et al. 1989; Kwiatkowski et al. 1990). Furthermore, many of the clinical symptoms found after TNF a injection in tumour patients, i.e. fever, chills, rigor, headache, anorexia and neurological symptoms (Blick et al. 1987; Selby et al. 1987; Warren et al. 1987; Springs et al. 1988) resemble clinical symptoms observed in malaria patients with CM. Moreover, children homozygous for the TNF2 allele, a variant of the promotor region of the TNF a gene which correlates with a high production capacity of TNF a , have a higher risk for CM (McGuire et al. 1994). Effective treatment of patients with CM results in a rapid decrease of serum-TNFa levels, even before clinical recovery is observed and before fever and parasitemia have disappeared (WHO 1990). In contrast, studies of Kwiatkowski et al. (1990) and Grau et al. (1989b) showed that serum-TNF a levels of patients with uncomplicated malaria and patients with CM exhibit considerable overlap which could argue against a role for TNF a in human CM. Furthermore, studies in Gambian children with CM (Kwiatkowski et al. 1993) show that treatment with a murine monoclonal antibody against human recombinant TNF a reduces fever but does not prevent mortality. Van Hensbroek et al. (1996a) not only observed no effect after treatment with these anti-TNF a antibodies, but treatment was associated with a significant increase of cases with neurological sequelae. Another important observation made by Kern et al. (1992) was that the plasma concentration of soluble TNF a receptors correlated with parasitemia and disease severity in human malaria. In addition, Kern et al. (1992); Kwiatkowski et al. (1993) and Molyneux et al. (1993) all described the absence of bioactive TNF a in sera of P. falciparum patients despite high concentrations of immunoreactive TNF a. This finding could be explained by the presence of excess soluble TNF a receptors in the plasma (Kern et al. 1992; Molyneux et al. 1993; Deloron et al. 1994; Kremsner et al. 1995). This suggests that the risk for severe pathology by excessive release of TNFa can be controlled by shedding of TNF a receptors which forms a complex with free TNF a, thereby blocking its biological e.g. cytolytic activity ( Kremsner et al. 1995). TNF a can induce production of nitric oxide (NO) and this molecule has been implicated in malaria-associated pathology, particularly in the development of coma

14 Table 1: Cerebral malaria.

Plasmodium species Sequestration Haemorrhage Neurological Literature: PRBCs* MQs** s symptoms in the brain Human:

P. falciparum Hum an ++ ++ ++ ++ Patnaik et al. 1994 ++ -- ++ ++ Turner et al. 1994 models: Monkeys:

P. knowlesi Maccaca ++ ++ N.I. N.I. Mahdi & Ahmad 1991 m ulatta

P. coatneyi Maccaca ++ -- N.I. -- Aikawa et al.1992 m ulatta

P. falciparum Saimiri sciureus ++ -- N.I. ++ Gysin et al.1992

P. coatneyi Maccaca ++ ------Kawai et al. 1993 fuscata

P. fragile Maccaca ++ N.I. N.I. + /- & Fujioka et al. 1994 m ulatta

Rodents:

P. berghei ANKA C57L mice -- ++ ++ ++ Rest 1982 P. berghei NK65 Golden -- ++ ++ ++ Rest 1982 ham sters

P. berghei NK65 WM Rats N.I. N.I. ++ ++ Kamiyama et al. 1987

P. berghei K173 C57B1 mice -- ++ ++ ++ Polder et al. 1983 Curfs et al. 1989

P. berghei ANKA CBA mice -- ++ ++ ++ Grau et al. 1986

P. chabaudi-chabaudi Mice (N.I.) ++ ++ N.I. N.I. Dennison et al. 1993 + P. berghei ANKA

P. yoelii 17XL Swiss mice ++ -- -- N.I. Kaul et al. 1994

& : +/- monkeys became stuporous N.I.: not indicated * : PRBCs = Parasitized Red Blood Cells ** : MQs = Mononuclear cells

15 accompanying CM (Clark et al. 1992). It is hypothesized that NO released by vascular endothelial cells after stimulation with TNFa diffuses into the brain, where it disrupts the regulation of glutamate-induced neuronal NO, resulting in alteration of neurotransmission and, consequently, coma. Grau & de Kossodo (1994) have proposed that NO may mediate early changes in CM, such as neurotransmission disturbances, when the neurological syndrome is still reversible, but that NO is unlikely to be involved in the actual process causing neurovascular damage at the advanced stages of the development of CM. However, that treatment with NO inhibitors do not block development of CM in the P. berghei ANKA model, not even upon direct intracranial administration of the NO inhibitor N-Methyl-L-Arginine (Senaldi et al. 1992; Asensio et al. 1993; Kremsner et al. 1993).

1.4 EXPERIMENTAL CEREBRAL MALARIA

1.4.1 Experimental models Because of the limited availability of human specimens for the analysis of the pathogenesis of CM a number of animal models were developed (table 1). None of these models reflect exactly the human situation, although, they can mirror certain aspects. Several monkey CM models are available (table 1). Although sequestration of PRBC (P. falciparum) in cerebral microvessels, like in the human situation, is observed in most of the monkeys only 50% of the microvessels exhibit this sequestration (Gysin et al. 1992) whereas 94% of cerebral vessels in human falciparum CM cases showed sequestration (Riganti et al. 1990). Furthermore, neither haemorrhages nor adherence of monocytes/macrophages in the microvasculature of the brains of these P. falciparum infected monkeys was described. Therefore, the P. falciparum/Saimiri sciureus monkey model is considered to be more suitable to study the obstruction theory. In other monkey models, however, the proportion of cerebral microvessels with sequestration ranges from 1% to 40% (Aikawa et al. 1992; Fujioka et al. 1994). In the mice CM models mononuclear cell sequestration is followed by the development of intravascular inflammatory reactions (Grau et al. 1987; Polder et al. 1992) and haemorrhages are observed in the microvasculature of the brains but no General introduction sequestration of PRBCs (table 1). Therefore, these mice models are more suitable to investigate the inflammation theory. Rest (1982) was one of the first to describe ECM in mice. Subsequently, other investigators developed CM models with other parasites and mouse strains. The two most studied ECM models in mice (P. berghei K173 / C57Bl mice and P. berghei ANKA / CBA mice, table 1) are characterized by sequestration and adherence of mononuclear cells, often containing malaria pigment or phagocytosed erythrocytes, that interact with and migrate between the endothelial cells of post-capillary venules, causing widespread petechial haemorrhages (Rest 1983; Polder et al. 1992). Infection of C57Bl mice with P. berghei K173 parasites induces ECM in 95% of the mice, while 80% of the P. berghei ANKA infected CBA mice develop ECM. Recently, Dennison et al. (1993) and Kaul et al. (1994) described a murine model for sequestration of parasitized erythrocytes in postcapillary venules of the brain. Dennison et al. (1993) injected mice with a mixture of P. chabaudi chabaudi and P. berghei ANKA parasites. While P. c. chabaudi normally adhere to vascular lining cells in liver and spleen, but not in the brain (Cox, 1988), sequestration and adherence to brain capillary lining cells was found in the mixed infection. Dennison et al. (1993) suggest that cytoadherence in cerebral vessels of P. c. chabaudi in mixed infections, is due to “hijacking” of receptors otherwise adhered to by leukocytes in infections with P. berghei ANKA alone. Sequestration of PRBCs was also found in Swiss mice infected with P. yoelii 17XL (Kaul et al. 1994). However, neither haemorrhages nor involvement of monocytes or macrophages were found in the brains.

1.4.2 Pathogenesis of experimental cerebral malaria In mice, it is assumed that sequestration of mononuclear cells is followed by the development of intravascular inflammatory reactions, endothelial cell damage and finally petechiae, in the brains (Grau et al. 1987; Polder et al. 1992) and in the retina (Ma et al. 1996). Elimination of macrophages prevented the development of experimental cerebral malaria in the P. berghei K173 model (figure 1; Curfs et al. 1993). An interesting phenomenon parallel in both rodent ECM and human CM is the enhanced expression of adhesion molecules on endothelial cells which are involved in sequestration and adherence. Endothelial ICAM-1 can serve as a receptor for

17 Chapter 1

PfEMP1 expressed on P. falciparum-infected erythrocytes in humans (Ockenhouse et al. 1991) and for CD11a (leukocyte function antigen 1, LFA1) expressed on leukocytes in both mice and humans (Grau et a l. 1991).

Malaria may be a compartmentalised inflammation reaction: the malaria parasites live and multiply in the blood circulation and may concequently activate T cells and mononuclear phagocytes and their production of bioactive substances such as cytokines, reactive oxygen and nitrogen intermediates. These bioactive substances can activate the endothelial lining to upregulate adhesion molecules resulting in margination of inflammatory cells and through localized reactivity damage the endothelial lining. The importance of specific inflammatory cells, their interaction with PRBC and the endothelial lining and the release of inflammatory mediators varies among the available experimental CM models.

1.4.2.1 T cells Evidence for a role of T cells in the pathogenesis of rodent ECM came from experiments by Wright et al. (1968, 1971) who showed that neonatal thymectomy (hamsters) and treatment with anti-T-cell serum prevented the development of ECM in hamsters and rats. Grau et al. (1986) described an important role for CD4+, but not CD8+ T cells in the development of ECM in P. berghei ANKA infected CBA/Ca mice. Imai et al. (1994), working with WM/Ms rats infected with P. berghei NK65 parasites, observed that depletion of CD8 T cells, but not CD4 T cells prevented ECM. In our model, C57BL mice infected with P. berghei K173 parasites, Curfs et al. (1989) confirmed a role of T cells in the development of ECM by showing that nude mice, thymectomized mice and mice treated with anti-T-cell serum were protected against ECM (figure 1). The ligand of T cells, LFA-1 (leucocyte function antigen-1) can bind to ICAM-1 what is expressed on endothelial cells.

1.4.2.2 Tumour Necrosis Factor a Development of ECM closely associates with high serum concentrations of both, immunoreactive TNFa as measured by an ELISA, (Clark et al. 1990b) and bioactive TNFa as measured by a bio assay (Grau et al. 1987). In addition, in P. berghei ANKA infected CBA mice having a high concentration of bioactive TNFa , ECM

18 General introduction could be prevented by treatment with anti-TNFa antibodies (Grau et al. 1987; figure1), and transgenic mice expressing high levels of soluble fusion protein TNFa- Receptor-P55 (s-TNFaR-P55), a receptor for free TNFa, are markedly protected against development of CM (Garcia et al. 1995). Administration of high doses of recombinant TNFa to normal CBA/Ca mice (Grau et al. 1987) reproduced ECM and to P. berghei infected mice enhanced ECM (Curfs et al. 1993). In P. falciparum malaria a high concentration of immunoreactive TNFa is associated with disease severity and in some studies with CM (Grau et al. 1989; Kwiatkowski et al. 1990). On the other hand, elevated concentrations of s-TNFaR-P55 and soluble TNFa receptor P75 (s-TNFa R-P75) were observed in the sera of such patients (Kern et al. 1992; Molyneux et al. 1993; Deloron et al. 1994). Moreover, treatment of patients with an anti-TNFa mAb reduced fever but did not cure CM (Kwiatkowski et al. 1993; van Hensbroek et al. 1996a).

1.4.2.3 Blood-brain-barrier In the mouse model, ECM is associated with leukocyte adherence to the microvascular endothelium, oedema and petechiae in the brains. Using vascular or parenchymal localization of Evans blue or Monastral Blue as indicators of the integrity of the blood- brain-barrier (BBB), Thumwood et al. (1988) and Neill & Hunt (1992) described an increase in the vascular permeability at a late stage of the development of ECM in P. berghei ANKA infected CBA and DBA mice. Sequestration and adherence of P. falciparum infected erythrocytes in the cerebral vasculature (WHO, 1990) may disturb the permeability of the BBB (Das et al. 1991; Patnaik et al. 1994), permitting the entry of epileptogenic factors into the brain parenchyma which may be resposible for malaria specific epileptogenic seizures observed in patients with CM (Waruiri et al. 1996).

1.4.2.4 Reactive Oxygen Intermediates Endothelial cells (EC) are targets for interaction with neutrophils, monocytes and platelets and even PRBCs which are able to release and/or generate a variety of bioactive and noxious agents locally. These agents can cause membrane pertubation, leading to cell injury with subsequent loss of endothelial function. One group of agents, known as reactive oxygen intermediates (hydrogen peroxide, superoxide anion, hydroxyl anion), are produced primarily by activated neutrophils

19 Chapter 1 and are known to cause EC injury and damage (Halliwell, 1989; Varani et al. 1989; Ward & Varani, 1990; Ward, 1991). Oxidant-induced injury to the endothelium may play an important role in the pathophysiology of ECM. Activation of phagocytes sticking to the surface of the cerebral vasculature as observed in the mouse model (Rest,1982; Polder et al. 1983; Grau et al. 1986) can cause oxidative damage to the endothelium and disturbance of the BBB. Increased production of reactive oxygen intermediates (ROI) has been described during Plasmodium chabaudi, P vinckei and P. berghei infection (Stocker et al. 1984; Clark et al. 1987; Prasad et al. 1990) and independent of ECM. Under these conditions treatment with ROI scavengers enhanced parasitemia (Clark et al. 1987). However, Thumwood et al. (1989) showed that the antioxidants butylated hydroxyanisole (BHA) and the combination superoxide dismutase and catalase can prevent ECM in P. berghei ANKA-infected A/J mice. Moreover, a BHA diet prevented disturbance of the permeability of the BBB.

1.4.3 Characteristics of the Plasmodium berghei K173 cerebral malaria model According to table 1 and the high percentage of mice developing ECM after infection as compared to the P. berghei ANKA/CBA mouse model it appears that C57Bl mice infected with Plasmodium berghei K173 is a useful model to study the role of inflammation parameters in ECM. During the second week after a blood- induced infection with 10 parasitised red blood cells (PRBCs) the majority of animals (95%) develop cerebral malaria (Curfs et al. 1992). After 9 - 10 days at end-stage disease, these mice become progressively inert with a ruffled fur, humped back and locomotor disturbances, and show a dramatic decrease of their body temperature to values below 30° C usually within a period of 24 hours, and die shortly thereafter (Curfs et al. 1989). Histopathological examination of the brains of these mice, either post mortem or in the period of a rapidly decreasing body temperature, reveals a high frequency of perivascular edema, intravascular accumulations of mononuclear cells adhering to endothelial cells and haemorrhages. The topographic distribution of the petechiae in the brains coincides with that of major cerebral arteries and veins (Polder et al. 1983; Curfs et al. 1992; Polder et al. 1992). Mice that survive this critical period (about 5%) exhibit only a limited and transient decrease of their body temperature and die in the third week after infection or later

20 General introduction with high parasitemia, anaemia and frequently secondary infections without any cerebral pathology. Because the progressive decrease in body temperature and death during the second week strongly correlated with haemorrhages in the brains after infection with P. berghei K173 (Curfs et al. 1989), the low body temperature and death during the second week of infection are useful surrogate markers for the development of cerebral haemorrhages.

1.5 OUTLINE OF THIS THESIS

The aim of the present study was to further elucidate the role of T cells, ROI and TNFa in the pathogenesis of ECM in C57Bl/6J and C57Bl/10 mice infected with P. berghei K173 parasites. To examine the role of T cells, ROI and TNFa, in the pathogenesis of murine ECM intervention studies were carried out with depletion of specific T cells or using inhibitors of the inflammation mediators mentioned during various stages of infection.

T cells: To elucidate the role of T cells in the pathogenesis of ECM, P. berghei K173 infected C57Bl mice were treated with anti-CD4 and/or anti-CD8 antibodies during infection and at end stage disease (chapter 2). Convulsions induced by folic-acid treatment were used as a marker for increased permeability of the BBB in end-stage disease mice. Mice, having an increased permeability of the BBB, were treated with anti-CD4 or anti-CD8 antibodies (chapter 3) and recovery from ECM and restoration of function of the BBB were determined.

ROI: We analysed the effect of treatment with several antioxidants: apocynin, desferal-Zn, DMSO and ethanol. In addition SOD transgenic mice, producing high concentrations of SOD, a scavenger of ROI, were infected and observed for ECM (chapter 4). In chapter 5, we studied the effect on parasitemia and development of ECM of treatment with the ROI inducing drugs, artemether and alloxan that are also fast

21 Chapter 1 acting anti-malarials on. The effect of treatment was also determined at a very late stage of the development of ECM.

TNFa: Intervention studies with anti-TNFa antibodies (chapter 6) were carried out because of the differences between the effect of anti-TNF antibody treatment in the ECM mouse model of Grau et al. (1987) and in humans with CM (Kwiatkowski et al. 1993; van Hensbroek et al. 1996a). In chapter 7, the role of TNFa was further investigated by administration of low doses rec-h-TNFa monomer given as a bolus or by sustained release during infection. The effect of monomeric rec-h-hTNFa treatment on parasitemia and on the development and outcome of ECM was examinated. Stabilised rec-h-TNFa trimers, which structure is very important for its action (Smith & Baglioni 1987; Van Ostade et al. 1994), were prepared by a chemical reaction from rec-h-TNFa monomers. Chapter 8 describes the analysis of the therapeutic potential of stabilised rec-h-TNFa trimers during development of ECM. vessel lumen Macrophages (Curfs ‘93)

T-cells (Curfs ‘89) TNFt ^ anti-TNF (Grau ‘87) 1?

TNF-Receptor ICAM-1 t signal transduction 1

• t cell damage endothelial cell 1

H em orrhage _ brain tissue

Figure 1: Schematic representation of the possible involvement of cells, cytokines and their receptors (e.g.) TNFa and sTNFa R, and unknown reactions (black box) after signal transduction in the pathogenesis of experimental cerebral malaria.

22 General introduction

REFERENCES

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24 General introduction

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ROGERSON, S.J. & BROWN, G.V. (1997) Chondroitin sulphate A as an adherence receptor for Plasmodium falciparum-infected erythrocytes. Parasitol. Today, 13, 70-75. ROWE, A., OBEIRO, J., NEWBOLD, C. I., MARSCH, K. (1995) Plasmodium falciparum rosetting is associated with malaria severity in Kenya. Infect. Immun.. 63, 2323-2326. SCHMID, A.H. (1974) Cerebral malaria: on the nature and significance of vascular changes. Europ. Neurol. 12, 197-208. SCUDERI, P., STERLING, K.E., LAM, K.S., FINLEY, P.R., RYAN, K. J., RAY, C.G., PETERSEN, E., SYMEN, D.J., SALMON, S.E. (1986) Raised serum levels of tumor necrosis factor in parasitic infections. Lancet ii: 1364-1365. SELBY, P., HOBBS, S., VINER, C., JACKSON, E., JONES, A., NEWELL, D., CALVERT, A.H., MCELWIAN, T., FEARON, K., HUMPHREYS, J., SHIGA, T. (1987) Tumor necrosis factor in man: clinical and biological observations. Brit. J. Cancer, 56, 803-808. SENALDI, G., KREMSNER, P.G., GRAU, G.E. (1992) Nitric oxide and cerebral malaria Lancet 340, 1554-1555. SENALDI, G., VESIN, C., CHANG, R., GRAU, G.E., PIGUET, P.F. (1994) Role of polymorphonuclear neutrophil leukocytes and their integrin CD11a (LFA-1) in the pathogenesis of severe murine malaria. Infect. Immun. 62, 1144-9. SENOK, A.C., NELSON, A.A.S., LI, K., OPPENHEIMER, S.J. (1997) Thalassaemia trait, red blood cell age and oxidant stress: effects on Plasmodium falciparum and sensitivity to artemisinin. Trans. R. Soc. Trop. Med. Hyg. 91, 585-589. SMITH, R.A. & BAGLIONI, C. (1987) The active form of tumour necrosis factor is a trimer. J. Biol. Chem. 162, 6951-6954. SPRIGGS, D.R., SHERMAN, M.L., MICHIE, H., ARTHUR, K.A., IMAMURA, K., WILMORE, D., FREI, E., KEEFE, D. W. (1988) Recombinant human tumor necrosis factor administered as a 24-hour intravenous infusion. A phase1 and pharmacological study. J. Natl. Cancer I. 80, 1039-1044. STOCKER, R., HUNT, N.H., CLARK, I.A., WEIDEMANN, M.J. (1984). Production of luminol-reactive oxygen radicals during Plasmodium vinckei infection. Inf. Immun. 45, 708-12. TORO, G. & ROMAN, G. (1978) Cerebral malaria, a disseminated vasomyelinopathy. Arch. Neurol. 35, 271-275. TRACEY, K.J., BEUTLER, B., LOWRY, S.F., MERRYWEATHER, J., WOLPE, S., MILSARK, I.W., HARIRI, I.J., FAHEY III, T.J., ZENTALLA, A., ALBERT, J.D., SHIRES, G.T., CERAMI, A. (1986) Shock and tissue injury induced by recombinant human cachectin. Science 232, 470-474. TRACEY, K.J., WEI, H, H., MANOGUE, K., FONG, Y., HESSE, D.G., NGUYEN, H.T., KUO, G.C., BEUTLER, B., COTRAN, R.S., CERAMI, A., LOWRY, S.F. (1988) Cachectin/tumor necrosis factor induces cachexia, anemia and inflammation. J. Exp. Med. 167, 1211-1227. THUMWOOD, C.M., HUNT, N.H., CLARK, I.A., COWDEN, W.B . (1988) Breakdown of the blood-brain barrier in murine cerebral malaria. Parasitology 96, 579-589. THUMWOOD, C.M., HUNT, N.H., COWDEN, W.B., CLARK, I.A. (1989) Antioxidants can prevent cerebral malaria in Plasmodium berghei-infected mice. J. Exp. Pathol. 70, 293-303.

29 Chapter 1

TREUTIGER, C.J., HEDLUND, I., HELMBY, H., CARLSON, J., JEPSON, A., TWUMASI, P., KWIATKOWSKI, D., GREENWOOD, B.M., WAHLGREN, M. (1992) Rosette formation in Plasmodium falciparum isolates and anti-rosette activity of sera from gambians with cerebral or uncomplicated malaria. Am. J. Trop. Med. Hyg. 46, 503-510. TURNER, G., MORRISO, H., JONES, M., DAVIS, T., LOOAREESUWAN, S., BULEY, I., GATTER, K., NEWBOLD, C., PYKRITAYAKAMEE, S., NAGACHINTA, B., WHITE, N., BERENDT, A. (1994) An immunohistochemical study of the pathology of fatal malaria. Am. J. Pathol. 145, 1057-1069. UDOMSANGPETCH, R., CHIVAPAT, S., VIRIYAVEJAKUL, P., RIGANTI, M., WILAIRATANA, P., PONGPONRATIN, E., LOOAREESUWAN, S. (1997) Involvement of cytokines in the histopathology of cerebral malaria. Am. J. Trop. Med. Hyg. 57, 501-506. VAN OSTADE, X., TANERNIER, J., FIERS, W. (1994) Structure-activity studies of human tumour necrosis factors. Protein Eng. 7, 5-22. VARANI, J., GINSBURG, I., SCHUGER, L., GIBBS, D.F., BROMBERG, J., JOHNSON, K.J., RYAN, U.S., WARD, P.A. (1989) Endothelial cell killing by neutrophils. Am. J. Pathol. 135, 435-438. VOGELS, M.T.E., LINDLEY, IJ.D., CURFS, J.H.A.J., ELING, W.M.C., van der MEER J.W.M. (1993) The effects of interleukin-8 on non-specific resistance to interfection in neutropenic and normal mice. Antimicrob. Agents Ch. 37, 276-280. WARD, P.A. & VARANI, J. (1990) Mechanisms of neutrophil-mediated killing of endothelial cells. J.Leukocyte Biol. 48, 97-102. WARD, P.A., (1991) Mechanisms of endothelial cell injury. J Lab. Clin. Med. 118, 421­ 426. WARRELL, D.A., LOOAREESUWAN S., PHILLIPS R.E., WHITE, N.J., WARRELL, M.J., CHAPEL, H.M., AREEKUL, S., THARAVANIJ, S. (1986) Function of the blood cerebrospinal fluid barrier in human cerebral malaria: rejection of the permeability hypothesis. Am. J. Trop. Med. Hyg. 35, 882-889. WARRELL, D.A. (1987) Pathophysiology of severe falciparum malaria in man. Parasitology 94, S53-S76 WARRELL, D.A., VEAL, N., CHANTHAVANICH, P., KARBWANG, J., WHITE, N.J., LOOAREESUWAN, S., PHILIPS, R.E., PONGPAEW, P. (1988) Cerebral anaerobic glycolysis and reduced cerebral oxygen transport in human cerebral malaria. Lancet 2, 534-537. WARREN, R.S., STARNES, H.F., GABRILOVE, J.L., OETTGEN, H.F., BRENNAN, M.F. (1987) The acute metabolic effects of tumor necrosis factor administration in humans. Arch. Surg. 122, 1396-1400. WARUIRU, C.M., NEWTON, C.J.J.C., FORSTER, D., NEW, L., WINSTANLEY, P., MWANGI, I., MARSH, V., WINSTANLEY, M., SNOW, R.W., MARSH, K. (1996) Epileptic seizures and malaria in Keyan children. Trans. R. Soc. Trop. Med. Hyg. 90, 152-155. WENISCH, C., PARSCHALK, B., BURGMANN, H., LOOAREESUWAN, S., GRANINGER, W. (1995) Decreased serum levels of TGF- ß in patients with acute Plasmodium falciparum malaria. J. Clin. Immunol. 15, 69-73.

30 General introduction

WERNSDORFER, W.H. & McGREGOR, SIR IAN (1988) Malaria (2 volumes) Churchill Livingstone. WRIGHT D.H. (1968) The effect of neonatal thymectomy on the survival of golden hamsters infected with Plasmodium berghei. Brit. J. Exp. Pathol. 49, 379-384. WRIGHT, D.H., MASEMBE, R.M., BAZIRA, E.R. (1971) The effect of antithymocyte serum on golden hamsters and rats infected with Plasmodium berghei. Brit. J. Exp. Pathol. 52, 465-477 WORLD BANK. World development report 1993: investing in health. Oxford, England (1993), Oxford University Press. WORLD HEALTH ORGANISATION (1990) Severe and complicated malaria (2nd ed.) Trans. Roy. Soc. Trop. Med. Hyg. (Spl.2) 84, 1-65 WORLD HEALTH ORGANISATION (1996) The World Health Report. World Health Organisation, Geneva.

31 CHAPTER

Depletion of CD4+ or CD8+ T-cells prevents P.berghei induced cerebral malaria in end-stage disease

C. Hermsen, T. van de Wiel, E. Mommers, R. Sauerwein and W. Eling

Parasitology 1997; 114, 7-14. Chapter 2

ABSTRACT

The role of T-cells in development of experimental cerebral malaria was analysed in C57B1/6J and C57Bl/10 mice infected with Plasmodium berghei K173 or Plasmodium berghei ANKA by treatment with anti-CD4 or anti-CD8 MAbs. Mice were protected against experimental cerebral malaria (ECM) when anti-CD4 or anti-CD8 MAbs were injected before or during infection. Even in mice in end stage disease i.e. with a body- temperature below 35.5°C, treatment with anti-CD4 or anti-CD8 antibodies or the combination protected against ECM whereas chloroquine treatment was completely ineffective in inhibiting further development of the cerebral syndrome.

32 Prevention of murine ECM by anti-CD4 or anti-CD8

INTRODUCTION

There are a number of rodent models available for the study of the pathogenesis of experimental cerebral malaria. In these studies mice (Rest, 1982, Grau et al. 1986, Thumwood et al. 1988, Curfs et al. 1989), rats (Wright et al. 1971) and hamsters (Rest & Wright, 1979) were used in combination with different strains of P.berghei parasites e.g. ANKA (Grau et al. 1986) or K173 (Curfs et al. 1989). In all these models an immunopathological reaction is considered to be involved in the development of the syndrome. Studies in rodent models showed sequestration and adherence of white blood cells to the endothelial lining of post-capillary venules in the brains in association with development of petechiae (Rest et al. 1982, Polder et al. 1992). In the model described by Grau et al. (1986) (P. berghei ANKA, CBA/Ca mice), CD4+ T-cells play an important role since depletion of CD4+ T-cells prevents and transfer of CD4+ T-cells from mice developing cerebral malaria enhances development of the cerebral syndrome. In addition, depletion of CD8+ T-cells did not prevent cerebral malaria. In mice with Murine Acquired Immunodeficiency Syndrome (MAIDS), which is characterized by abnormal functioning of CD4+ T-cells the level of protection against murine ECM is significantly increased and related to duration of the viral infection and, hence, with the severity of CD4 T-cell immunodeficiency (Eckwalanga et al, 1994). In the model using WM/Ms rats and P. berghei NK65 parasites (Imai et al, 1994) cerebral malaria is prevented by CD8+ T-cell depletion, but not by CD4 T-cell depletion. In our model (P. berghei K173, C57Bl mice) Curfs et al. (1989) confirm a role of T-cells in the development of experimental cerebral malaria since nude mice, thymectomized mice and mice treated with an anti-T-cell serum were protected against cerebral malaria. Here, we describe the results of treatment with anti-CD4 and anti-CD8 antibodies in P. berghei ANKA and K173 infected C57Bl mice. The results show that treatment with anti-CD4 or anti-CD8 antibodies can prevent development of experimental cerebral malaria. Moreover, even when performed shortly before expected death, i.e. in end stage disease when body-temperature has decreased to 35.5°C or lower, depletion of CD4 or CD8 T-cells effectively prevented further development of the cerebral syndrome.

33 Chapter 2

MATERIALS AND METHODS

Mice: C57Black/6J or C57Black/10 mice, aged 6-10 weeks, were obtained from speci­ fic pathogen free colonies maintained at the Central Animal Facility of the University of Nijmegen. All mice were housed in plastic cages and received water and standard RMH food (Hope Farms, Woerden, The Netherlands) ad libitum.

Parasite: Plasmodium berghei K173 and Plasmodium berghei ANKA were maintained by weekly tranfer of parasitized erythrocytes (PE) from infected into naive mice. Experimental mice were infected intraperitoneally with 10 PE from blood of infected donor animals of the same strain. P. berghei ANKA (originally obtained from Dr. B. Mons, Laboratory of Parasitology, University of Leiden, The Netherlands) is a gametocyte-producing strain which is passaged through Anopheles gambiae mosquitoes after 4 weekly blood passages. For this purpose mosquitoes are allowed to feed on mice 3 days after infection with 10 parasites. Blood-fed mosquitoes receive an additional blood-meal containing normal erythrocytes 5 days later and are allowed to infect mice 14 days later.

Parasitaemia: thin blood films were made from tail-blood, stained with May- Grunwald and Giemsa's solutions, and the proportion of red blood cells infected with the parasite was determined.

Detection of cerebral malaria: approximately 95% of C57Bl mice infected with 103 P. berghei K173 (Curfs et al. 1992) or P.berghei ANKA parasites die early in the second week after infection. Approximately one day before death a progressive hypo­ thermia develops which is strongly correlated with development of haemorrhages in the brains as observed by histology (Curfs et al, 1989). Mice that show a transient hypothermia (in the majority of cases >32°C) survive this critical period but die in the third week or later after infection without any noticeable cerebral pathology.

Body temperature: Body temperature was measured with a digital thermometer (Technoterm 1200) introduced into the rectum and read after 10 seconds.

34 Prevention of murine ECM by anti-CD4 or anti-CD8

Histology: Mice were killed by an overdose of aether and their brains were collected or they were collected post mortem. Brain tissue was fixed in Carnoy's fluid for 4 hours. Paraffin sections (5mm) were stained with PAS/Hematoxilin or Goldner's trichroom stain. Sections were scored for the presence of petechiae.

In-vivo CD4+ or CD8+ T-cell depletion: Rat monoclonal anti-CD4 and anti-CD8 antibodies were produced by respectively hybridoma YTS 191.1.2, ECACC no 87072282 and YTS 169.4.2.1, ECACC no 87072283. Both MAbs are of the IgG2b isotype. Pristane primed nude mice were injected i.p. with 3.106 hybridoma cells and the ascites produced was collected. After ammonium sulfate (45%) precipitation and dialysis against HO , the solution was freeze-dried and stored at 4°C until used. Ali­ quots were solubilized in sterile, pyrogen-free saline and used immediately. Normal and thymectomized mice were treated once or twice (4 days apart) by i.p. injection of 0.3 mg of either anti-CD4 or anti-CD8 MAb. Treatment with an irrelevant isotype matched, ammonium sulfate precipitated rat MAb was used in comparison to treatment with normal rat serum as another control. Both control treatments did not prevent development of ECM and did not affect parasitaemia. Therefore, both control treatments were used in the CD4+/CD8+ T-cell depletion experiments. The efficacy of T-cell depletion was determined in peripheral leucocytes isolated by watershock- treated peripheral blood, or in ACT-treated spleen cell suspensions from samples collected 3 days after treatment with the anti-T-cell MAbs (Hudson & Hay, 1989). Analysis of CD4+/CD8+ T-cell depletion immediately after injection of the MAbs was complicated by the presence of the injected MAb coating the CD4+/CD8+ T-cells in the cell suspensions, a problem that was absent when the analysis was performed 3 days after the last anti-T-cell treatment. Cell suspensions were incubated in PBS containing 10% FCS, 0.05% sodium azide and 0.05 mg/ml of the anti-CD4 or anti- CD8 MAbs for 30 minutes at 4°C followed by a 30 minutes incubation of a 50 times diluted FITC-labelled rabbit anti-rat Ig (Dako) in PBS containing 40% mouse serum and 0.05% sodium azide. After washing with PBS + 0.05% sodium azide fluorescen- se was read microscopically. From the peripheral blood and the spleen respectively 80% and 92% of CD4+ and 62% and 79% of CD8+ T-cells were depleted. Treatment with either anti-CD4 or anti- CD8 MAbs did not change significantly the proportion of B-cells (data not shown).

35 Chapter 2

Protection against cerebral malaria was independent of iv. or ip. injection of the anti- T-cell MAbs (data not shown), and ip. injections were used for all experiments.

Thymectomy: thymectomy (Tx) was performed on mice anaesthetized with chloral- hydrate (4.5% solution; 10 ml per gram body weight). Mice were allowed to recover from surgery for a period of 2 weeks before they were used in experiments.

Statistical analysis: For statistical analysis the student's t-test (comparison of 2 groups) and the Kruskal-Wallis test (comparison of more than 2 groups) were used. P values <0.05 were considered significant.

RESULTS

The effect of treatment with anti-CD4 or anti-CD8 MAbs on development of experimental cerebral malaria was analysed in thymectomised and intact C57Bl/6J and C57Bl/10 mice being infected with P. berghei ANKA or P. berghei K173 parasites. Figure 1 shows that treatment with anti-CD4 or anti-CD8 MAbs protected against ECM, irrespective of thymectomy in both C57Bl/6J or C57Bl/10 mice infected with either P. berghei K 173 or P. berghei ANKA parasites. Parasitaemia in control mice that die of ECM was approximately 5-10% with a reduction of the haematocrit by approximately 20%. Neither thymectomy, nor treatment with anti-CD4 or anti-CD8 MAbs significantly changed parasitaemia and haematocrit during infection as compared to controls. All treated mice that did not die of cerebral malaria developed the same severe anaemia in the course of the infection (data not shown). Treatment with either an isotype-matched rat MAb or normal rat serum as a control for depletion of CD4+ or CD8+ T-cells by specific rat MAbs, had no effect on development of ECM (table 1) and did not change parasitaemia. The data in table 1 show that treatment with anti-CD4 or anti-CD8 MAbs 2 days before and 2 days after infection protected respectively 80% and 92% of the mice whereas 12% of the control mice were protected. Moreover, T-cell depletion during infection and even when performed on day 8 i.e. shortly before expected death as observed in non-depleted controls, effectively prevented development of the fatal syndrome (table 1).

36 Prevention of murine ECM by anti-CD4 or anti-CD8

Protection against ECM (%) anti-CD4 anti-CD8 Controls treated treated

1 0 0 ■ 7/7 • 6 / 6 T 19/20 ^ 1 1 / 1 2 1 6/7

x 6 / 8 a 3 / 4 75 ■ 3/4 " 8 / 1 1

50 • 3/6

25 ■ 5/20

' 1/10 - 2/23 0/9 * 1/16 0 Tx intact Tx intact Tx intact

Figure 1: Protection against cerebral malaria in thymectomized (Tx) versus intact mice and in relation to treatment with anti-CD4 or anti-CD8 MAbs two days before and two days after infection. C57B1/6J infected with P.berghei K173 (▼) or with ANKA (•); C57B1/10 infected with P.berghei K173 (■). The number of mice protected against ECM versus the total number of infected mice is indicated for each experimental group.

The effect on body-temperature of treatment with anti-CD4 or anti-CD8 MAbs two days before and two days after infection with P. berghei K173 in C57B1/6J mice was analysed in eight independent experiments. The results of one representative experi­ ment are depicted in figure 2. Body-temperature of untreated infected mice dramatically decreased after 9 days of infection. Anti-CD4 or anti-CD8 treated mice showed a limited decrease of their body-temperature which stabilized. The same effect on body-temperature was found when treatment with anti-CD4 or anti-CD8 MAbs was given as a single treatment up to day 8 of infection (results not shown).

37 Chapter 2

Table 1: Effect of treatment with anti-CD4 or anti-CD8 MAbs before and during infection with P. berghei K173 parasites on development of cerebral malaria in C57B1 mice.

Treatment Day(s) of Mice protected against ECM/ treatment* treated mice

n

Control 9/78° (12%)

IgG2b-controls# Day -2/2 0/5 (0%)

Day 9 1/7 (14%)

Anti-CD4: Day -2/2 39/49' (80%)

Day 4 3/3 (100%)

Day 6 3/3 (100%)

Day 8 7/8 (88%)

Day 8 + 10 5/5 (100%)

Anti-CD8: Day -2/2 33/36' (92%)

Day 4 3/3 (100%)

Day 6 2/3 (66%)

Day 8 5/8 (63%)

Day 8 + 10 5/5 (100%)

* in relation to infection at day 0 o summerized data from P. berghei K173 or P. berghei ANKA infected mice (see figure 1) # Isotype matched irrelevant MAb as a control of MAb anti-CD4 or anti-CD8 treatment

Anti-CD8 treatment 2 days before and 2 days after infection prevented development of ECM as effective as CD4+ T-cell depletion (table 1) and no significant difference in body-temperature during infection of the anti-CD4 and anti-CD8 treated mice was found too (figure 2). To further determine the latest possible moment for effective anti-CD4 or anti-CD8 treatment in the course of infection, mice were selected with a body-temperature

38 Prevention of murine ECM by anti-CD4 or anti-CD8 between 35.5 and 30°C. If left untreated such infected mice die within 24 hours as shown in figure 2. The results in table 2 show that mice with such a hypothermia could be protected against fatal cerebral malaria by treatment with anti-CD4 and/or anti-CD8 antibodies, whereas chloroquine treatment could not save any of these mice. No relation could be found between body-temperature on the moment of anti-T-cell treatment (30 - 35.5°C) and protection against fatal cerebral malaria (data not shown). Anti-CD4 or anti-CD8 treatment of mice with a body-temperature below 30°C never prevented death. Treatment with anti-CD4+ or anti-CD8+ MAbs in mice with end stage disease did not save all mice from the cerebral syndrome (table 2). These last two observations are probably due to petechiae that already developed before treatment (Polder et al. 1988). Depletions that prevented further development of the cerebral syndrome also resulted in a subsequent increase of the body-temperature (table 2). Overall in 93% (27/29) of the anti-T-cell treated mice body-temperature increased within 24 hours and prevented further development of the cerebral syndrome.

Figure 2: The effect on body-temperature of treatment with anti-CD4 or anti-CD8 MAbs 2 days before and 2 days after P. berghei K173 infection in C57B1/6J mice. (•) Control mice, (Q) anti-CD4 treated mice, ( ) anti-CD8 treated mice; (n=5).

39 Chapter 2

Table 2: Effect of treatment with anti-CD4 and/or anti-CD8 MAbs or chloroquine on development of cerebral malaria in C57B1/6J mice infected with P.berghei K173 parasites.

Treatment ECM protected/total increased T C** /toi:al

Saline 2/52 (4%) 1/2 (50%)

Chloroquine0 0/10 (0%) NS°°

anti-CD4 12/17 (71%) 11/12 (92%)

anti-CD8 10/20 (50%) 9/10 (90%)

anti-CD4 + anti-CD8 7/8 (88%) 7/7 (100%) * Treatment was given when the body-temperature was between 35.5 and 30.0°C. ** Mice with a statistically significant increase of their body-temperature at 24 hours after treatment* 0 Chloroquine treatment: 0.8 mg i.p. mouse/daily. 00 NS = no survivors

DISCUSSION These studies demonstrate the importance of CD4+ and CD8+ T-cells in the pathogenesis of murine cerebral malaria (ECM), and this was independent of the use of C57B1/6J or C57B1/10 mice, or infection with P. berghei K173 or P. berghei ANKA parasites. Thymectomized mice were used to prevent the infux of new T-cells after treatment with anti-T-cell MAbs, but no significant difference was found when data were compared to intact mice. Treatment with anti-T-cell MAbs did not completely eliminate all CD4+ or all CD8+ T-cells. Together these observations suggest that complete absence of CD4 or CD8 T-cells is not necessary to prevent development of murine ECM. Anti-CD4+ or anti-CD8+ T-cell treatment had no effect on parasitaemia and development of anemia confirming the observations made by Grau et al. (1986) in another murine P. berghei ECM model and by Imai & Kamiyama (1994) in a rat P. berghei NK 65 ECM model. The observation that both anti-CD4 and anti-CD8 T-cell depletion can prevent ECM, is in contrast to observations made previously by Grau et al. (1986) who described a role for CD4 but not CD8 T-cells in development of ECM in P. berghei ANKA infected CBA mice. In contrast, Waki et al. (1992) and Imai & Kamiyama

40 Prevention of murine ECM by anti-CD4 or anti-CD8

(1994) found a role for CD8+ but not CD4+ T-cells in preventing either early mortality or ECM respectively in P. berghei NK65 infected rodents. A role for both CD4 and CD8 T-cells in development of ECM in P. berghei ANKA infected mice was noted by Weidanz and collaborators using various types of knock-out mice (personal communication). Hancock et al (1994) found that treatment with anti-CD4 antibodies not only depletes CD4-positive T-cells but also CD4-positive mononuclear cells, but their possible role in ECM remains to be determined. In contrast to our results Grau et al (1986) found that CD8+ T-cell depletion did not prevent development of ECM in P. berghei ANKA infected CBA mice. This apparent discrepancy may relate to different mouse strains used and/or a difference in the strain of P. berghei ANKA. Anti-CD4 or anti-CD8 T-cell treatment was effective even in mice with end stage disease (body-temperature between 35,5 and 300C). Histological analysis of brains of mice developing the cerebral syndrome showed that white blood cells adhere to the endothelial lining (Curfs et al. 1989, Polder et al. 1992) suggesting that these CD4+ and CD8 T-cells are involved in disturbance of the endothelial lining and thereby in the functioning of the blood-brain barrier. Disturbance of the blood-brain barrier in mice developing ECM was also observed by Thumwood et al. (1988) and Neil & Hunt (1992). They described leakage of Evans blue from the vasculature into brain parenchyma. In addition, P. berghei K173 infected mice with end stage disease are sensitive to development of folic acid induced convulsions, while control mice are not. Treatment with anti-CD4+ or anti-CD8+ MAbs not only prevents ECM but successfully treated mice also recover from their sensitivity to folic acid induced convulsions within 4 hours after MAb treatment (Hermsen et al., unpublished observations). In human cerebral malaria convulsions are common (Waruiru et al. 1996) raising the question whether in human CM adherence of sequestered infected red blood cells and possibly the presence of leukocytes (Polder, 1989, Porta et al. 1993, Eling & Kremsner, 1994, Patnaik et al. 1994, Eling & Sauerwein, 1995) are involved in excessive activation of endothelial cells and subsequent disturbance of the function of the blood-brain barrier. Chloroquine treatment could not save mice in end-stage disease while anti-CD4 or anti-CD8 treatment was still effective. This may be explained by the fact that chloroqu- ine like most other anti-malarial drugs need more than one day to suppress

41 Chapter 2 parasitaemia effectively, whereas most of the mice in end-stage disease die within one day. Our data are compatible with the hypothesis that a localized inflammatory reaction involving both CD4 and CD8 T-cells disturbs and damages the endothelial lining of the blood-brain barrier eventually leading to petechiae. Our data suggest that both CD4 and CD8 T-cells are involved in stimulation of a downstream process of the development of the petechiae.

ACKNOWLEDGEMENTS We thank G. Poelen, T. van den Ing and Y. Brom for skilled biotechnical assistance.

REFERENCES CURFS, J.H.A.J., SCHETTERS, T.P.M., HERMSEN, C.C., JERUSALEM, C.R., VAN ZON, A.A.J.C. & ELING, W.M.C. (1989). Immunological aspects of cerebral lesions in murine malaria. Clinical Experimental Immunology 75, 136-140. CURFS, J.H.A.J., HERMSEN, C.C., MEUWISSEN, J.H.E.TH. & ELING, W.M.C. (1992). Immunization against cerebral pathology in Plasmodium berghei-infected mice. Parasitology 105, 7-14. ECKWALANGA, M., MARUSSIG, M., TAVARES, M.D., BOUANGA, J.C., HULIER, E., PAVLOVITCH, J.H., MINOPRIO, P., PORTNOI, D., RENIA, L. & MAZIER, D. (1994). Murine AIDS protects mice against experimental cerebral malaria: Down- regulation by interleukin 10 of a T-helper type 1 CD4 cell-mediated pathology. Proceedings of the National Academy of Sciences of the United States of America 91(17), 8097-8101. ELING, W.M.C. & KREMSNER, P.G. (1994). Cytokines in malaria, pathology and protection. Biotherapy 7, 211-221. ELING, W.M.C. & SAUERWEIN, R. (1995). Severe and cerebral malaria: common or distinct pathophysiology? Reviews in Medical Microbiology 6(1), 17-25. GRAU, G. E., PIGUET, P. F., ENGERS, H. D., LOUIS, J. A., VASSALLI, P. & LAMBERT, P.H. (1986). L3T4 T-lymphocytes play a major role in the pathogenesis of murine cerebral malaria. The Journal of Immunology 137, 2348-2354. HANCOCK, W. W., ADAMS, D. H., WYNER, L. R., SAYEGH, M. H. & KARNOVSKY, M. J. (1994). CD4+ Mononuclear cells induce cytokine expression, vascular smooth muscle cell proliferation, and arterial occlusion after endothelial injury. American Journal of Pathology 145(5), 1008-1014. HUDSON, L. & HAY, F (1989). Practical Immunology, Blackwell scientific publications, Oxford London. IMAI, Y & KAMIYAMA, T. (1994). T-lymphocyte-dependent develop ment of cerebral symptoms in WM/Ms rats infected with Plasmodium berghei. Annals of Tropical Medicine and Parasitology 88, 83-85. NEILL, A.L. & HUNT, N.H. (1992). Pathology of fatal and resolving Plasmodium berghei cerebral malaria in mice. Parasitology 105, 165-75.

42 Prevention of murine ECM by anti-CD4 or anti-CD8

PATNAIK, J.K., DAS, B.S., MISHRA, S.K., MOHANTY, S., SATPATHY, S.K. & MOHANTY, D. (1994). Vascular clogging, mononuclear cell migration, and enhanced vascular permeability in the pathogenesis of human cerebral malaria. American Journal of Tropical Medicine and Hygiene 51, 642-47. POLDER, T.W., ELING, W.M., KUBAT, K. & JERUSALEM, C.R. (1988). Histochemistry of cerebral lesions in mice infected with Plasmodium berghei. Tropical Medicine and Parasitology 39, 277-83. POLDER, T.W. (1989). Dissertation: Morphology of cer ebral malaria clinical and experimental study. POLDER, T.W., ELING, W.M.C., CURFS, J.H.A.J., JERUSALEM, C.R., & WIJERS- ROUW, M. (1992). Ultrastuctural changes in the blood-brain barrier of mice infected with Plasmodium berghei. Acta Leidensia 60, 31-46. PORTA, J., CAROTA, A., PIZZOLATA, G. P., WILDI, E., WIDMER, M. C., MARGAIRRAZ, C., & GRAU, G. E. (1993). Immunopathological changes in human cerebral malaria. Clinical Neuropathology 12 142-146. REST, J.R. & WRIGHT, D.H. (1979). Electron microscopy of cerebral malaria in golden hamsters (Mesocricetus auratus). Journal of Pathology 127,115-120. REST, J.R. (1982). Cerebral malaria in inbred mice. A new model and its pathology. Transactions of the Royal Society of Tropical Medicine and Hygiene 76(3), 410-415. THUMWOOD, C.M., HUNT, N.H., CLARK, I.A. & COWDEN, W.B. (19 88). Breakdown of the blood-brain barrier in murine cerebral malaria. Parasitology 96, 579-589. WAKI, S., UEHARA, K., KANBE, K., ONO, K., SUZUKI, M. & NARIU CHI, H. (1992). The role of T-cells in pathogenesis and pro tective immunity to murine malaria. Immunology 75, 646-651. WARUIRI, C.M., NEWTON, C.R.J.C., FORSTER, D., NEW, L., WINSTAN LEY, R., MWANGI, I., MARSH, V., WINSTANLEY, M., SNOW, R.W. & MARSH, K. (1996). Epileptic seizures and malaria in Kenyan children. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 152-155. WRIGHT, D. H., MASEMBLE, R. M. & BAZIRA, E.R. (1971). The effect of anti­ thymocyte serum on golden hamsters and rats infected with P. berghei. Britisch Journal of Experimental

43 CHAPTER

Convulsions due to increased permeability of the blood-brain-barrier in experimental cerebral malaria can be prevented by splenectomy or anti-T cell treatment

C.C. Hermsen, E. Mommers, T. van de Wiel, R.W. Sauerwein and W.M.C. Eling

Journal of Infectious Disaeses 1998; 178:1225-1227 Chapter 3

ABSTRACT

Experimental cerebral malaria (ECM) can be induced in C57B1 mice by infection with Plasmodium berghei K173 parasites. Behavioral changes shortly before they die of ECM may reflect disturbance of the integrity of the blood-brain-barrier (BBB). Folic acid elicits strong convulsive activity if the permeability of the BBB is increased. Administration of folic acid to mice during development of ECM induced convulsions. Interventions known to prevent fatal outcome from ECM, such as splenectomy or treatment with anti-CD4 or anti-CD8 MAbs also prevented sensitivity to folic acid- induced convulsions. In addition, infected mice with ECM and sensitive to folic acid- induced convulsions, recovered from this sensitivity alter treatment with anti-T cell antibodies within 4 hours. These data suggest that disturbance of the permeability of the BBB can be reversed and depends on the involvement of T cells.

46 Permeability of the BBB and ECM

INTRODUCTION

C57B1 mice infected with Plasmodium berghei K173 parasites die of experimental cerebral malaria (ECM) [1], Mice that develop ECM exhibit behavioral changes during the last 24 hours of life that reflect convulsive activity [2], In this model, ECM is associated with leukocyte adherence to the microvascular endothelium, edema, and petechiae in the brain [3, 4], Using Evans blue or Monastral Blue as indicators of the integrity of the blood-brain-barrier (BBB), Thumwood [5] and Neill and colleagues[6,7] reported increased vascular permeability in mice that developed ECM (P. berghei ANKA-infected CBA mice), but not in mice without cerebral malaria (CBA mice infected with P. berghei K173 parasites) exept at a very late stage of the disease [7], Convulsions are common in childhood malaria and are associated with severity of disease and mortality [8], Recent observations show that childhood convulsions are particularly associated with malaria, but not necessarily in combination with fever, suggesting that Plasmodium falciparum parasites have epileptogenic properties [10], Sequestration and adherence of P. falciparum-infected erythrocytes in the cerebral vasculature [9] may disturb the permeability of the BBB [11, 12], permitting the entry of epileptogenic factors into the brain parenchyma, Hommes et al, [13] demonstrated that strong, rapidly lethal convulsions could be evoked in rats by injection of folic acid if the BBB was disturbed, This technique of provoking strong epileptic activity by folic acid treatment if the permeability of the BBB was increased was applied in our model of ECM, In this study, we assessed the effect of folic acid treatment on mice with ECM and of interventions with anti-CD4 and anti-Cd8 monoclonal antibodies (MAbs),

MATERIALS AND METHODS

Mice: female C57Bl/6J mice, ages 6-10 weeks, were obtained from specific pathogen­ free colonies maintained at the Central Animal Facility, University of Nijmegen. All mice were housed in plastic cages and received water and standard RMH food (Hope Farms, Woerden, Netherlands) ad libitum, Parasite: P. berghei K173 (originally obtained from W, Kretschmar, Tübingen, Germany) has been maintained by weekly transfer of parasitized erythrocytes (PE)

47 Chapter 3 from infected into naive mice for >30 years. Experimental mice were infected intra- peritoneally (ip) with 10 PE from blood of infected donors.

Parasitaemia: thin blood films were made from tail-blood stained with May-Grunwald and Giemsa solutions, and the proportion of parasitized red blood cells was determined.

Detection ECM: ECM was detected as described by Curfs et al. [3]. In brief, 95 % of C57Bl mice infected with 10 P. berghei K173 parasites die early in the second week after infection. One day before death a progressive hypothermia develops that is strongly correlated with development of hemorrhages in the brains as observed by histology [1]. The parasitaemia in mice that die of ECM is between 5%-10%, and such mice have a 20% reductionin haematocrit. Mice that show a transient hypothermia (in most cases >32°C) survive this criti­ cal period but die in the third week or later after infection with signs of severe anemia and a parasitaemia of 20%-40% but without any detectable cerebral pathology as determined by light microscopy.

End-stage disease: mice infected with P. berghei K173 parasites exhibit a rapidly decreasing body temperature and usually die within 24 hours [1] about day 8/9 after infection. Mice are considered to be in end-stage disease when their body temperature drops to < 35.5°C.

Body temperature: body temperature was measured by a digital thermometer (Technoterm 1200, Thermotex, The Hague, Netherlands) introduced into the rectum and read after 10 seconds.

Splenectomy: mice were anesthetized, the splenic pedicles were ligated, and the spleen was removed. Mice were infected with P. berghei K173 parasites 7 days after splenectomy.

Anti-CD4 or anti-CD8 Mab treatment: Mice were treated with anti-CD4 or anti-CD8 MAbs [14]. In brief, rat anti-CD4 or anti-CD8 MAbs were produced by hybridoma YTS 191.1.2 and YTS 169.4.2.1, respectively. Mice received 0.3 mg of the MAb ip 2 days before and 2 days after infection. From the peripheral blood and the spleen, respectively 80% and 92% of CD4 and 62% and 79% of CD8 T cells were depleted 3 days after treatment.

48 Permeability of the BBB and ECM

Convulsions: mice received 0.2 ml of a folic acid solution (25mg/ml) intravenously. The folic acid (Genfarma, Maarsen, Netherlands) was dissolved in saline and adjusted to pH 7 with 1 M NaOH. Mice were observed after injection of 5 mg of folic acid for 90 minutes. During this period sensitive mice developed generalized convulsions and died. Phenobarbitone (0.75 mg/mouse ip) dissolved in saline and adjusted to pH 7 with 1 M HCl was used as an anticonvulsant and injected 10 minutes before folic acid.

Statistical analysis: all data are expressed as mean ± SD. Student's t-test was used for statistical analysis. P < .05 was considered significant.

RESULTS

Induction of convulsions by folic acid in P. berghei-infected mice Convulsions after folic acid administration were not observed in normal C57Bl mice. In the absence of folic acid treatment, P. berghei-infected mice did not show obvious convulsions. C57Bl mice infected with 10 P. berghei K173 PE developed convulsions after injection of 5 mg of folic acid/mouse at day 8 or 9 after infection, depending on body temperature. There was a significant difference (P < .05) in mean body temperatures of infected mice that developed (32.1 + 2.8°C) or did not develop convulsions (36.3 + 2.1°C) after folic acid treatment. Of 28 infected mice, 26 (93%) mice with body temperature < 35.5°C and 2 of 12 (16.7%) mice with a temperature >35.5°C developed generalized convulsions (P < .05). The proportion of mice that developed convulsions decreased when <5 mg folic acid was injected in infected mice with body temperatures <35.5°C (data not shown). Thus, in the standard test to determine sensitivity to induction of folic acid-dependent convulsions, mice with body temperatures < 35.5°C were given 5 mg folic acid i.v. Pretreatment of infected mice with a body temperature <35.5°C with the anti­ convulsant phenobarbitone neither affected parasitaemia nor prevented ECM (n=7), but it protected all mice (n=8) against development of convulsions induced by a subsequent injection of folic acid.

49 Chapter 3

Sensitivity to folic acid-induced convulsions in splenectomized or anti-T cell- treated infected mice. Splenectomized mice and mice depleted of CD4 or CD8 T cells before infection were protected against development of ECM and against folic acid-induced convulsions to a varying degree (table 1), whereas controls developed convulsions and ECM.

Table 1: Effect of splenectomy (day -1) or anti-CD4 or anti-CD8 T cell treatment (days - 2 and 2) on prevention of ECM and of convulsions induced by folic acid treatment in P. berghei infected mice.

Treatment Number of mice protec­ Number of mice* ted against ECM/ without convulsions/ total number total number

Control 0/5 (0%) 0/8 (0%)

Splenectomy 3/3 (100%)' 17/28 (61%)

Anti-CD4 (Day -2/2) 5/6 (83%)' 9/9 (100%)

Anti-CD8 (Day -2/2) 3/4 (75%)' 10/15 (67%)

*: In infected mice with a body temperature between 30° and 35.5°C convulsions were scored after administration of 5 mg folic acid i.v. t: Only small numbers of mice were used as controls, because protection against ECM by these treatments was shown before [1, 14],

When infected mice with body temperatures of 30-35.5°C were treated with folic acid 4 or 24 hours after treatment with anti-T cell MAbs a considerable proportion were protected against development of convulsions, whereas all untreated infected mice developed convulsions after folic acid injection (table 2). There was no significant difference in the proportion of mice protected against convulsions when folic acid was given 4 or 24 hours after the anti-T cell treatment.

50 Permeability of the BBB and ECM

Table 2: Effect of treatment with anti-CD4 or anti-CD8 MAbs of P. berghei K173 infected mice with a body temperature between 30° and 35.5°C on development of folic acid induced convulsions.

Treatment with:

Time of Anti-CD4* Anti-CD8* Saline administration

of folic acid after No convulsions / No convulsions / No convulsions / anti-T cell treatment folic acid treated folic acid treated folic acid treated mice mice mice

4 h 5/9 (56%) 11/18 (61%) 0/5 (0%)

24 h 5/9 (56%) 4/8 (50%) 0/8 (0 %)

*: 0.3 mg/0.2 ml anti-CD4 or anti-CD8 mAb was injected i.p. once.

DISCUSSION

C57Bl mice infected with P. berghei K173 develop ECM characterized by leukocyte adherence to the vascular endothelial lining, edema, and petechiae in the brain [1] suggesting a progressive loss of BBB integrity. These results show that mice during development of ECM become sensitive to the induction of convulsions by folic acid treatment and that protection against ECM by splenectomy or anti-T cell treatment, also protects against development of convulsions after folic acid treatment. Moreover, when mice in end-stage disease are treated with anti-CD4 or anti-CD8 MAbs, the evolution of ECM is disrupted [14] and they recover within 4 h from sensitivity to folic acid- induced convulsions. In another experimental murine model of ECM [2] convulsions occured in end-stage disease. In our model, behavioral changes (progressively inert with ruffled fur, humped back, and locomotor disturbances) were not explicit enough for further analysis. Therefore, we injected folic acid, which induces convulsions if the BBB is damaged [13], to analyze the hypothesis that during development of ECM the permeability of the BBB is increased and causes all or part of the accompanying behavioral changes observed. Only infected mice developing ECM were sensitive to

51 Chapter 3 folic acid could be prevented by treatment with the anti-convulsant phenobarbitone, suggesting that the permeability of the BBB is increased during development of ECM. Thumwood et al. [5] and Neill & Hunt [6] also found an increased permeability for Evans blue or Monastral blue of the BBB in late stage ECM development in P. berghei ANKA-infected mice. Neill et al. [7] observed an increased permeability only in terminally ill CBA or DBA mice infected with P. berghei K173 parasites that did not develop cerebral symptoms. The absence of ECM in their experiments may relate to their use of DBA or CBA mice (we used C57Bl/6J mice) or to the number of parasites used for infection (10/mouse in their model vs. 10 in ours). Our previous experi­ ments showed that the proportion of mice that develop ECM decreases as more parasi­ tes are used to infect mice [3], which may explain why Neill and colleages [6, 7] observed neither ECM nor increased permeability until the mice are terminally ill. Although ECM cannot readily be compared with P. falciparum-induced childhood malaria, CM it is of interest that Waruiru et al. [10] describe malaria-specific convulsions in P. falciparum- infected children. Previous work showed that splenectomy [1] or treatment with either anti-CD4 or anti- CD8 T cell MAbs [14] prevented development of ECM in P. berghei K173-infected mice. A close correlation between sensitivity to folic acid-induced convulsions and development of ECM is supported by the observation that prevention of ECM by splenectomy or anti-T cell treatment also prevented sensitivity to induction of convulsi­ ons by folic acid (table 1). These data support and complement the observation of Neill & Hunt [15] who prevented an increase in the permeability of the BBB by treatment with dexamethasone, which in our model successfully prevents ECM when given prior to day 5 of infection [1]. Treatment with anti-CD4 or anti-CD8 MAbs in our model prevented ECM [14] and prevented development of convulsions after folic acid treatment 4 or 24 hours later (table 2). Why treatment with anti-T cell MAbs is not 100% successful remains to be determined. In some mice with body temperature "35.5°C anti-T cell treatment may be ineffective because of preexisting fatal petechiae as observed by light and electron microscopy [1, 4]. This may also explain why recovery from sensitivity to folic acid- induced convulsions was not always successful and did not increase when folic acid treatment was delayed to 24 hours after anti-T cell treatment. In summary, our results suggest that mice developing ECM suffer from an increasing permeability of the BBB that may further lead to petechiae. CD4 and CD8 T cells are

52 Permeability of the BBB and ECM involved in the increase of permeability, probably by triggering reactions that are more directly involved in damage.

ACKNOWLEDGEMENTS

We thank G. Poelen, T. van den Ing, and Y. Brom for skilled biotechnical assistance.

REFERENCES

1. CURFS JHAJ, SCHETTERS TPM, HERMSEN CC, JERUSALEM CR, VAN ZON AAJC, ELING WMC. Immunological aspects of cerebral lesions in murine malaria. Clin. Exp. Immunol. 1989; 75: 136-40. 2. GRAU GE, FAJARDO LF, PIQUET PF, ALLET B, LAMBERT PH, VASSALLI P. Tumor necrosis factor (cachetin) as an essential mediator in murine cerebral malaria. Science 1987; 237:1210-2. 3. CURFS JHAJ, HERMSEN CC, MEUWISSEN JETH, ELING WMC. Immunization against cerebral pathology in Plasmodium berghei-infected mice. Parasitology 1992;105:7-14. 4. POLDER TW, ELING WMC, CURFS JHAJ, JERUSALEM CR, WIJERS-ROUW M. Ultrastructural changes in the blood-brain barrier of mice infected with Plasmodium berghei. Acta Leidensia 1992; 60:31-46. 5. THUMWOOD CM, HUNT NH, CLARK IA, COWDEN WB. Breakdown of the blood- brain-barrier in murine cerebral malaria. Parasitology 1988; 96:579-89. 6. NEILL AL, HUNT NH. Pathology of fatal and resolving murine cerebral malaria. Parasitology 1992;105: 165-75. 7. NEILL AL, CHAN-LING T, HUNT NH. Comparisons between microvascular changes in cerebral and non-cerebral malaria in mice, using the retinal whole-mount technique. Parasitology 1993;107:477-87. 8. MOLYNEUX ME, TAYLOR TE, WIRIMA JJ, BORGSTEIN J. Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q. J. Med. 1989;71:441-59. 9. WHO, World Health Organization, Division of control of Tropical Diseases,Geneve, Switzerland Severe and complicated malaria. Trans. R. Soc. Trop. Med. Hyg. 1990;84:S2,1-65. 10.WARUIRU CM, NEWTON CRJC, FORSTER D, et al. Epileptic seizures and malaria in Kenyan children. Trans. R. Soc. Trop. Med. Hyg. 1996;90:152-5. 11.DAS BS, MOHANTY S, MISHRA SK. et al. Increased cerebrospinal fluid protein and lipid peroxidation products in patients with cerebral malaria. Trans. R. Soc. Trop. Med. Hyg. 1991;85:733-4.

53 Chapter 3

12.PATNAIK JK, DAS BS, MISHRA SK, MOHANTY S, SATPATHY SK, MOHANTY D. Vascular clogging, mononuclear cell margination, and enhanced vascular permeability in the pathogenesis of human cerebral malaria. Am. J. Trop. Med. Hyg. 1994;51:642-47. 13.HOMMES OR, OBBENS EAMT, WIJFFELS CCB. Epileptogenic activity of sodium- folate and the blood-brain-barrier in the rat. J. Neurol. Sci. 1973;19:63-71. 14.HERMSEN CC, VAN DE WIEL T, MOMMERS E, SAUERWEIN R, ELING W. Depletion of CD4 or CD8 T cells prevents Plasmodium berghei induced cerebral malaria in end-stage disease. Parasitology 1997;114:7-12. 15.NEILL AL, HUNT NH. Effects of endotoxin and dexamethasone on cerebral malaria in mice. Parasitology 1995;111:443-54.

54 CHAPTER

Protection against Plasmodium berghei induced experimental cerebral malaria upon treatment with antioxidants

C.C. Hermsen1, N.S. Postma2, R.W. Sauerwein1 , Y. Groner3, J. Golenser4 and W.M.C. Eling1

1 Department of Medical Microbiology, University of Nijmegen, Nijmegen, The Netherlands. 2 Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands. 3 Department of Biochemistry, The Weizmann Institute, Jerusalem, Israel. 4 Department of Parasitology, Molecular Genetics, Rehovot, Israel.

Submitted Chapter 4

ABSTRACT

C57B1 mice infected with Plasmodium berghei K173 parasites develop experimental cerebral malaria (ECM). TNFa is considered to play an important role in the pathogenesis by damaging the endothelial lining causing haemorrhages. A role for a TNFa dependent induction of reactive oxygen intermediates (ROI) in L929 cell killing was analysed by the addition of antioxidants. TNFa mediated lysis of L929 cells was inhibited in a dose dependent way in the presence of the antioxidants apocynin, desferal-Zn (DFO-Zn), ethanol or dimethyl sulfoxide (DMSO). Addition of catalase or sodium oxide dismutase (SOD) was ineffective. Treatment of P. berghei infected mice by sustained release of ethanol or DMSO starting one day before infection or by repeated injections of DMSO starting on day 8 after infection protected mice against ECM. However, treatment of mice in end-stage disease (body temperature "35.5°C) did not. Mice treated with DMSO did not develop convulsions after injection of folic acid indicating that the permeability of the blood-brain-barrier remained intact. Treatment with ethanol or DMSO before infection significantly inhibited parasitaemia but this inhibition did not explain protection against ECM. Mice treated with apocynin or DFO-Zn or SOD transgenic mice were not protected against ECM. These data indicate that ROI are involved in the pathogenesis of ECM, and that certain antioxidants can protect against ECM while others do not.

56 ECM and protection by antioxidant treatment

INTRODUCTION

Development of (experimental) cerebral malaria ((E)CM) is associated with high plasma concentrations of tumour necrosis factor-a (TNFa). Likewise in CBA mice infected with P. berghei ANKA, development of ECM is associated with high concentrations of biologically active TNFa which can be prevented by treatment with anti-TNFa antibodies (Grau et al. 1987). TNFa is cytotoxic (Matthews & Watkins, 1978) and can stimulate upregulation of the expression of adhesion receptors (Vlassara et al. 1989; Mackay et al. 1993). Ockenhouse et al. (1992) reported that circulating TNFa and interferon-gamma, being elevated during P. falciparum infections, systematically activate postcapillary venular endothelial cells. Furthermore, the activated endothelial cells not only bind neutrophils (Varani et al. 1989; Ward & Varani 1990), monocytes (Carlos et al. 1991) and platelets (Fry et al. 1980) but also P. falciparum infected red blood cells (Aikawa 1988; Ockenhouse et al. 1991). The interactions with adherent neutrophils and monocytes cause local release of bioactive and noxious agents (Golenser et al. 1992; Chiu et al. 1997) notably cytokines (and more TNFa) and ROI (hydrogen peroxide, superoxide anions and hydroxyl anions). This can cause membrane perturbation, cell injury and loss of endothelial integrity (Bjork et al. 1980; Varani et al. 1989; Ward & Varani 1990; Ward, 1991; Sulkowska & Sulkowski 1997). Thumwood et al. (1989) found that treatment with antioxidants (butylated hydroxyanisole; BHA) or the combination of superoxide dismutase and catalase coupled to polyethylene glycol) prevented ECM in 45-60% of Plasmodium berghei ANKA infected AJ or CBA/H mice. In addition, mice on a diet containing BHA did not exhibit an increase in the permeability of the blood-brain- barrier (BBB) in the course of infection. Thus, oxidant-induced injury of the vascular endothelial lining, particularly in the brain, may play an important role in the pathophysiology of ECM (for review see Postma et al. 1996). Parallely, TNFa mediated cytotoxicity in vitro is inhibited in the presence of the antioxidants BHA and 4-hydroxymethyl-2,6-di-t-butylphenol (Brekke et al. 1992) also implicating reactive oxygen intermediates (ROI) in this damage. This study analysed the role of several antioxidants on TNFa induced cytotoxicity in the L929 in vitro, as well as the capacity of these antioxidants to prevent ECM in P. berghei infected C57Bl mice.

57 Chapter 4

MATERIALS AND METHODS

Mice: female C57B1/6J mice, aged 6-10 weeks, were obtained from specific pathogen free colonies maintained at the Central Animal Facility, University of Nijmegen. All mice were housed in plastic cages and received water and standard RMH food (Hope Farms, Woerden, The Netherlands) ad libitum.

Parasite: P. berghei K173 (originally obtained from W. Kretschmar, Tübingen, Germany) has been maintained by weekly transfer of parasitized erythrocytes (PE) from infected into naive mice >30 years. Experimental mice were infected intra- peritoneally (i.p.) with 103 PE from blood of infected donors.

Parasitaemia: thin blood films were made from tail blood on days 8, 9 and 10 after infection. They were stained with May-Grünwald and Giemsa solutions and the proportion of parasitized red blood cells was determined.

Detection of ECM: ECM was detected as described by Curfs et al. (1992). In brief, 95% of C57Bl mice infected with 103 P. berghei K173 parasites die early in the second week after infection. One day before death a progressive hypothermia develops that is strongly correlated with development of haemorrhages in the brain as observed by histology (Curfs et al. 1989). The parasitaemia in mice that die of ECM is between 5­ 10%, and such mice have a 20% reduction in haematocrit. Mice that show a transient hypothermia (in most cases >32°C) survive this critical period but die in the third week or later after infection with signs of severe anaemia, a parasitaemia of 20%-40% but without detectable cerebral pathology as determined by histology.

End-stage disease: mice infected with P. berghei K173 parasites exhibit a rapidly decreasing body temperature, and usually die within 24 hours (Curfs et al. 1989), 8 or 9 days after infection. Mice are considered to enter end-stage disease when their body temperature drops to " 35.5°C (Hermsen et al. 1998).

Body temperature: measured with a digital thermometer (Technoterm 1200, Thermotex, The Hague, Netherlands) introduced into the rectum and read after 10 seconds.

58 ECM and protection by antioxidant treatment

Tumour Necrosis Factora (TNFa): recombinant-murine-TNFa (rec-m-TNFa) was generously donated by Dr. G. Adolf, Ernst-Boehringer Institut, Vienna, Austria. The specific activity was 1.2.10 U of TNFa/mg in a TNFa dependent cytotoxicity assay using murine LM cells. The endotoxin content was <51 pg/ml.

L929 cell lysis assay: the TNFa bioassay was performed as described by Aggarwal (1985) with minor modifications. Briefly, rec-mTNFa was added to 10 L929 cells per well in the presence or absence of a concentration range of antioxidants and cultured in RPMI 1640 DM (Gibco, Life Technologies, 31870-025) containing 10% foetal calf serum (FCS; Integro, Zaandam, The Netherlands) and 8 mg/ml emetine (Sigma) and 5% CO in air at a 90% relative humidity at 37°C for 20 h. A TNF-mediated cytopathic effect on L929 cells was measured by adding to each well 20 zl EZ4U solution (Biomedica, Austria) which is taken up only by live, intact cells and converted from a yellow tetrazolium-compound to a red formazan-derivative. After 2 hrs incubation at 37°C the optical density was measured in an ELISA reader at 450 nm with 620 nm as a reference (Titertek Multiskan MCC/340; ICN, Zoetermeer, The Netherlands). The detection limit of the L929 bioassay was 0.5 pg rec-mTNFa/ml.

Apocynin: 4-hydroxy-3-methoxy-acetophenone, acetovanillone 98%, (Jansen Chimica, Belgium) was dissolved in 96% ethanol. Saline was used for further dilutions and injection. The stock solution was diluted in normal drinking water for treatment per os. Osmotic pumps (Alzet 2001, pumping rate 1 zl/hour, 7 days, or Alzet 2002, pumping rate 0.5 zl/hour, 14 days) were filled with a solution of apocynin (range 50 - 200 mM) dissolved in 96% ethanol and implanted i.p. in mice under ether anaesthesia. Osmotic pumps filled with 96% ethanol were implanted i.p. as controls. Apocynin inhibits production of hydrogen peroxide and superoxide anion (Hart & Simmons, 1992).

Catalase: ( Sigma, C6665, 1540 Units/mg solid) was dissolved in RPMI 1640 DM containing 10% FCS. Concentrations used in the L929 cell assay: 15 - 30,000 Units/ml.

Desferal-Zn (DFO-Zn): was prepared by mixing 110 mM desferal (DFO, Ciba-Geigy AG, Bazel, Swiss) and 110 mM ZnSO4.7HO (Merck, Germany; in aqua dest). Alzet

59 Chapter 4

2002 mini-osmotic pumps, pumping rate 0.5 ul/hour for 14 days, were filled with DFO-Zn solution and implanted i.p. in mice under ether anaesthesia. Mini-osmotic pumps filled with saline were used as controls. DFO-Zn is an inhibitor of the production of hydroxyl radicals (Ophir et al. 1994).

Dimethyl sulfoxide (DMSO): (Merck, Germany) was either diluted in saline and injected i.p. or mini-osmotic pumps were filled with DMSO and implanted i.p. DMSO is an inhibitor of the production of superoxide anions and hydroxyl radicals (Grankvist, 1981; Miller & Raleigh, 1983).

Ethanol: (Merck, Germany) 100% was diluted in saline and injected i.p. or mini- osmotic pumps were filled with ethanol and implanted i.p. Ethanol, a hydroxyl radical scavenger (Kumagai et al. 1991) was used in a concentration range of 0.5 - 3% in the L929 cell assay.

SOD-transgenic mice: carry the human CuZn-superoxide dismutase gene (SOD-1). Mice with the following genetic background were used: F2 = F1(J C57Bl/6J, DBA) x F1(N Balb/C, C57Bl/6J). In addition, mice homozygous for TgHS-69 or TgHS-51 and control F2 mice were included in the experiments (Tg=Transgenic; HS=Human Cu/Zn SOD-1; respectively 69 and 51=Human Cu/Zn SOD-1 gene integrated at different sites in the mouse genome; Shanley et al. 1992).

Superoxide dismutase: (Sigma, S2515, EC 1.15.1.1., 2000 Units/mg solid) was diluted in RPMI containing 10%FCS. Concentrations used in the L929 cell assay: 2 - 4.000 Units/ml.

Folic acid induced convulsions: infected mice that develop ECM and have a body temperature " 35.5°C show generalised convulsions upon i.v. injection of 5 mg folic acid (Genfarma, Maarsen, Netherlands), in 0.2 ml saline, adjusted to pH 7 with 1 M NaOH (Hermsen et al. 1998). Mice were observed for the development of convulsions after injection of folic acid for a period of 90 minutes.

Statistical analysis: all data are expressed as mean ± SD. Student's t-test was used for statistical analysis. P<0.05 was considered significant.

60 ECM and protection by antioxidant treatment

RESULTS

Effect of antioxidants on rec-m-TNFa dependent cytotoxicity in vitro Tumour necrosis factor-a (TNFa) is cytotoxic for L929 cells in a concentration dependent manner (Aggarwal 1985; starting points in fig. 1a,b,c). The presence of either apocynin, desferal-Zn (DFO-Zn) or dimethyl sulfoxide (DMSO) during incubation of L929 cells with rec-m-TNFa, inhibited TNFa-dependent cytotoxicity in a concentration dependent way (fig. 1a,b,c). Similar data were obtained with ethanol in a dose range 0.5 - 3% (data not shown). The presence of catalase or sodium oxide dismutase (SOD) in the L929 test system had no effect on TNFa-mediated cytotoxicity (data not shown).

Effect of antioxidants on the development of Plasmodium berghei K173 induced experimental cerebral malaria (ECM) The effect of several different regimens of apocynin treatment on ECM was analysed. Apocynin was administered either via the drinking water (range 8 - 600 mg/L starting one day before infection, or orally (by gastric tube, 1 mg in 0.2 ml saline) on day 8, or intravenously (0.25 mg in 0.2 ml saline/mouse) on days 7-11 after infection and none of these treatments protected against ECM (data not shown). Apocynin, dissolved in ethanol and administered via osmotic pumps (range 50 - 200 mM; release rate 1 zl/hour) implanted intra-peritoneally (i.p.) one day before infection also did not protect against ECM (table 1). Finally, i.p. injection of 0.25 mg apocynin in ethanol 96% on day 7 (n=3), day 8 (n=3), day 9 (n=3) or day 10 (n=3) after infection did not protect against ECM. Sustained release of 96% ethanol alone from 1 day before infection onwards, which was originally included as a solvent control in the apocynin studies, protected against ECM (table 1), but injection (i.p.) of 0.5 ml 10% ethanol on day 8 (n=3) or on day 9 (n=3) after infection did not protect. Desferal-Zn (55, 110 or 220 mM), administered by sustained release from mini- osmotic pumps implanted i.p. one day before infection or on day 6 after infection, did not protect against ECM (table 1). DMSO (100%) delivered via mini-osmotic pumps implanted one day before infection protected mice against ECM (table 1), but implantation 6 days after infection (n=6) did not protect. In addition, 4 out of 6 (67%) mice were protected against ECM by i.p. injection of 0.2 ml of 0.25% DMSO in saline on 3 consecutive days starting 8

61 Chapter 4

Figure 1: Effect of increasing concentrations of apocynin, desferal-Zn (DFO-Zn) or DMSO on the TNFa dependent lysis of L929 cells. • = no rec-m-TNFa, 0 = 5 pg/ml rec-m- TNFa, □ = 20 pg/ml rec-m-TNFa, V = 80 pg/ml rec-m-TNFa. Data are presented as the mean ± SD of triplicate determinations. *: P= < 0.05 as compared to TNFa control. Reduction of the optical density reflects lysis of L929 cells.

62 ECM and protection by antioxidant treatment days after infection, when mice had a normal body temperature, but no protection was observed when DMSO was administered to mice in end-stage disease (body temperature " 35.5°C; days 9 and 10 after infection, n=6).

Effect of antioxidant treatment on parasitaemia Treatment with DMSO or ethanol 96% via mini-osmotic pumps implanted one day before infection significantly inhibited parasitaemia as compared to their infected controls while sustained release of apocynin in ethanol or DFO-Zn from 1 day before infection onwards did not (table 1). Further analysis showed that the inhibition of parasitaemia by DMSO or ethanol occured independent of protection against ECM.

Sensitivity of DMSO treated, infected mice to folic acid-induced convulsions Mice in the course of development of ECM, particularly in end stage disease develop convulsions upon folic acid treatment which is indicative of an increased permeability of the BBB (Hermsen et al. 1998). Table 2 shows that all infected mice treated with DMSO released from implanted mini-osmotic pumps from 1 day before infection onwards and were protected against ECM (78%, table 1) did not develop sensitivity to the epileptogenic activity of folic acid treatment during the whole infection period.

Table 2: Effect of DMSO treatment by sustained release from mini-osmotic pumps implanted i.p. in mice one day before infection with 103 Plasmodium berghei K173 parasites on folic acid induced convulsions.

Treated* with Number of mice developing 5 mg folic acid convulsions/ total number

Saline/no 8/8

Day 9 after infection 0/3

Day 15 after infection 0/3

Day 20 after infection 0/3

*: only mice with a body temperature between 35.5° and 30°C received folic acid.

63 Table 1: Effect of treatment with apocynin, desferal-Zn, ethanol or dimethyl sulfoxide (DMSO) on parasitaemia and development of experimental cerebral malaria in C57B1/6J mice infected with Plasmodium berghei K173 parasites.

Treatment Mode of treatment ECM Parasitaemia at day 8 post infection protected/total n/n % controls treated mice treated mice CM protected CM unprotected

No treatment 1/32 3 Saline Mini-osm. pump; day -1 2/10 20 i.p. injection; day 8,9 and 10 1/10 10

Apocynin/ Mini-osm. pump; day -1 11/56 20 22.9 ± 14.9 14.3 ± 8.1 N.D. N.D. ethanol (n=17) (n=56)

Ethanol Mini-osm. pump; day -1 5/9 56* 20.2 ± 10.8 5.1 ± 3.0+ ± ± (n = ll) (n=9) (n=5) (n=4)

Desferal-Zn Mini-osm. pump; day -1 4/24 17 ± ± N.D. N.D. (n=6) (n=24)

DMSO Mini-osmotic pump; day -1 28/36 78* ± ± ± ± (n= 18) (n=36) (n=28) (n=8)

DMSO i.p. injection; day 8, 9 and 10 4/6 67* N.D. N.D.

*: P < 0.05 as compared to mice with saline containing mini-osmotic pumps implanted on day -1. ■f: P<0.05 as compared to parasitaemia of their controls. ECM development in SOD-transgenic mice infected with Plasmodium berghei K173. Homozygous SOD-transgenic TgHS-69 or TgHS-51 mice infected with P. berghei K173 developed ECM after infection just as their control F2 littermates (table 3). Furthermore, no significant difference was found between the parasitaemias observed in P. berghei infected homozygous TgHS-69 or TgHS-51 mice and their controls (data not shown).

Table 3: Development of experimental cerebral malaria in SOD transgenic (TgHS-69 and TgHS-51), control F2 and control C57B1/6J mice infected with 103 Plasmodium berghei K173 parasites.

Mice ECM protected/total n/n %

SOD-TgHS-69 0/15 0

SOD-TgHS-51 0/5 0

Control F2 1/24 4

Control Black/6J 0/6 0

DISCUSSION C57B1 mice infected with P. berghei K173 develop ECM characterized by leukocyte adherence to the vascular endothelial lining, edema, and petechiae in the brain (Curfs et al. 1989). Local production of TNFa and/or ROI by the adherent leukocytes can cause cell injury and loss of endothelial integrity (Bjork et al. 1980; Varani et al. 1989; Ward & Varani 1990; Ward, 1991; Sulkowska & Sulkowski 1997). This study shows that TNFa dependent lysis of L929 cells in vitro can be inhibited by a number of antioxidants that either inhibit the production of (apocynin, DFO-Zn and DMSO) or scavenge (ethanol) produced reactive oxygen intermediates (ROI). In the L929 cytotoxicity assay binding of TNFa to its membrane receptors triggers internal production of ROI which can lead to cell-lysis (Beyaert & Fiers, 1994). Whereas, presence of apocynin, DFO-Zn, DMSO or ethanol prevented TNFa dependent cell lysis, presence of catalase or SOD did not protect. Probably in Chapter 4 contrast to apocynin, DFO-Zn, DMSO and ethanol, catalase and SOD cannot cross the cell membrane and, therefore cannot exert an effect on internally produced ROI. TNFa plays an important role in the pathogenesis of ECM in the P. berghei-ANKA model (Grau et al. 1987) and treatment with rec-h-TNFa during infection immediately provokes an ECM like syndrome (Curfs et al. 1993). Therefore the possibility that a TNFa dependent production of ROI is involved in the damage of endothelial cells during the development of ECM was investigated. ROI secreted by activated macrophages can damage endothelial cells (DelMaestro, 1982, Wei et al. 1985) and adherent macrophages are observed in the brain pathology of all ECM models described so far (Polder et al. 1983, Grau et al. 1986; Curfs et al. 1992). Furthermore, extensively triggered endothelial cells can produce an overshoot of ROI, that are harmful for these cells (Beyaert & Fiers, 1994). Finally, Thumwood et al. (1989) observed that treatment with butylated hydroxyanisole, a free radical scavenger, or treatment with the combination of catalase and SOD coupled to polyethylene glycol protected a proportion of A/J and CBA/H mice infected with P. berghei ANKA against ECM. These observations argue for a role of ROI in the pathogenesis of ECM. Our data on the effect on antioxidants only partly confirm this hypothesis. Administration of ethanol, a hydroxyl radical scavenger (Kumagai et al. 1991) and DMSO, an inhibitor of the production of superoxide anions and hydroxyl radicals (Grankvist, 1981; Miller & Raleigh, 1983) protected mice against ECM, whereas apocynin and DFO-Zn did not protect. DMSO and ethanol treatment administered before infection inhibited parasitaemia, but this was not specific for the mice which were protected against ECM. A comparable observation was made in the same model after treatment of infected mice with rec-h-TNFa (Postma et al. 1998) and also in the P. berghei ANKA mouse model when mice were treated with the antioxidant BHA (Thumwood et al. 1989). Previous observations in our model (Curfs et al. 1992) showed that ECM developed equally well when mice were infected with a range of 10-10,000 parasites which results in a three days delay in the course of the parasitaemia. The inhibition observed after antioxidant treatment is within this range. Thus, ROI probably interferes with parasite proliferation but their role in development of ECM is mediated through other mechanisms of action.

56 ECM and protection by antioxidant treatment

In addition, it is important to note that the antioxidants DMSO and ethanol were only effective when present during infection and did not protect mice in end-stage disease (body temperature "35.5°C ). Thumwood et al. (1989) succeeded in protecting mice against ECM with the combination catalase and SOD coupled to polyethylene glycol or BHA also by treatment during infection. The observation that DMSO treatment also prevented the development of convulsions after administration of folic acid is in agreement with the observation of Thumwood et al. (1989) that damage of the BBB scored by uptake of Evans blue or radiolabeled erythrocytes was prevented by BHA treatment. Thus, according to these data ROI are involved in the pathogenesis of ECM but a direct link between ROI and the development of haemorrhages has not been established. The results may suggest that ROI are involved in modulation of the immune response to the parasite contributing to the development of ECM and that antioxidant treatment can prevent this. Depletion of either CD4 or CD8 cells protects against ECM (Hermsen et al. 1997), but DMSO treatment did not change numbers of these cells in the spleen or in the peripheral blood as compared to infected controls on day 8 after infection (unpublished data). Administration of ethanol prevented mice against ECM while apocynin dissolved in ethanol did not (table 1). Recently, Norton et al. (1998) descibed that ethanol can also act as an inducer of ROI. This could explain the failure of the combination of apocynin and ethanol to protect against ECM. In addition, recent observations in our model showed that treatment with an inducer of ROI, alloxan, could protect mice in end stage disease against ECM (Hermsen et al. Alloxan protects Plasmodium berghei infected mice in end-stage disease against experimental cerebral malaria. submitted). Antioxidants may also inhibit NFkB preventing upregulation of adhesion molecules (Weber et al. 1994), but a possible role in ECM remains to be determined. Results obtained with apocynin and DFO-Zn do not support a role for ROI in ECM and the reason is not clear. Apocynin inhibits production of hydrogen peroxide and superoxide anion (Hart & Simmons, 1992) and inhibits ROI production by activated mononuclear cells in vitro (Beukelkamp et al. 1995). In addition, 0.06 mM apocynin administered in the drinking water reduced joint swelling in collagen induced arthritis in rats (Hart et al. 1990). On the other hand, peroxidase deficient cells such as lymphocytes and mature macrophages (Welch et al. 1980), which are involved in pathology of ECM, are insensitive to apocynin (Hart et al. 1992).

57 Chapter 4

DFO-Zn is an inhibitor of the production of hydroxyl radicals (Ophir et al. 1994) and its failure to protect against ECM might relate to the use of an insufficient regimen. Our sustained release system was limited to a maximal daily dose of 2 mg/day/animal. Golenser et al. (1997) observed reduction in parasite proliferation and protection against ECM in mice that received a polymeric implant containing 140 mg DFO subcutaneously on day 6 postinfection. In addition, protection against ischemia due to reperfusion injury in the rat retina was obtained with 7.5 mg injected 5 minutes before reperfusion (Ophir et al. 1994). SOD-transgenic mice, with a 2.3 to 6 fold elevated intracellular SOD activity in various tissues including erythocytes (Shanley et al. 1992), were not protected against ECM. Despite increased SOD concentrations in erythrocytes parasitaemia was the same as in infected controls. Thumwood et al. (1989) reported protection when mice were treated with a mixture of SOD and catalase coupled to polyethylene glycol, but the mechanism e.g. prolonged circulation or cell uptake, was not established. One possible explanation for the lack of effect in SOD-trangenic mice is that an increment in SOD alone is not sufficient (Golenser et al. 1998). In summary, the data indicate that ROI are involved in the pathogenesis of ECM. ROI generated during infection contribute to the development of immunopathological reactions leading to ECM rather than causing heamorrhages.

ACKNOWLEDGEMENTS

We thank G. Poelen, T,. van de Ing and Y. Brom for biotechnical assistance and E. Bennink and M. van Lare for technical assistance.

REFERENCES

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58 ECM and protection by antioxidant treatment

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19.GRAU, G.E., L.E. FAJARDO, P.E. PIGUET, B. ALLET, P.H. LAMBERT, AND P. VASSALLI 1987. Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 237:1210-1212. 20.HART ‘T, B.A., J.M. SIMONS, S. KNAAN-SHANZER, N.P.M. BAKKER, AND R.P. LABADIE. 1990. Antiarthritic activity of the newly developed neutrophil oxidative burst antaganist apocynin. Free Radical Bio. Med. 9:127-131. 21.HART ‘T, B.A. AND J.M. SIMONS. 1992. Metabolic activation of phenols by stimulated neutrophils: a concept for a selective type of anti-inflammatory drug. Biotechnol. Therap. 3:119-135. 22.HERMSEN, C.C., T. VAN DE WIEL, E. MOMMERS, R. SAUERWEIN, AND W. ELING. 1997. Depletion of CD4 or CD8 T cells prevents Plasmodium berghei induced cerebral malaria in end-stage disease. Parasitology 114:7-12. 23.HERMSEN, C.C., E. MOMMERS, T. VAN DE WIEL, R. SAUERWEIN, AND W.M.C. ELING. 1998. Convulsions due to increased permeability of the blood-brain- barrier in experimental cerebral malaria can be prevented by splenectomy or anti-T-cell treatment. J. Infect. Dis. 178:1225-1227. 24.KUMAGAI, Y., L.Y. LIN, D.A. SCHMITZ AND A.K. CHO. 1991. Hydroxyl radical mediated demethylenation of (methylenedioxy)phenyl compounds. Chem. Res. Toxicol. 4:330-334. 25.MACKAY, F., H. LOETSCHER,D. STUEBER, G. GEHR, AND W. LESSLAUER. 1993. Tumor necrosis factor a (TNFa)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor, TNF-R55. J.Exp. Med. 177:1277-1286. 26.MATTHEWS, N. AND J.F. WATKINS. 1978. Tumour-necrosis factor from the rabbit. I. Mode of action, specificity and physicochemical properties. Br. J. Cancer 38:302-309. 27.MILLER, G.G, AND J.A. RALEIGH. 1983. Action of some hydroxyl radicals scavengers on radiation-induced haemolysis. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 43:411- 419. 28.NORTON, I.D., M.V. APTE, O. LUX, P.S. HABER, R.C. PIROLA, AND J.S. WILSON. 1998. Chronic ethanol administration causes oxidative stress in the rat pancreas. J. Lab. Clin. Med. 131:442-446. 29.OCKENHOUSE, C.F., M. HO, N.N. TANDON, G.A. VAN SEVENTER, S. SHAW, N.J. WHITE, G.A. JAMIESON, J.D. CHULAY, AND H.K. WEBSTER. 1991. Molecular basis of sequestration in severe and uncomplicated P. falciparum malaria: differential adhesion of infected erythrocytes to CD36 and ICAM-1. J. Infect. Dis. 164:163-169. 30.OCKENHOUSE, C.F., T. TEGOSHI, Y. MAENO, C. BENJAMIN, M. HO, K.E. KAN, Y. THAW, K. WIN, M. AIKAWA, AND R.R. LOBB. 1992. Human vascular endothelial cell adhesion receptors for Plasmodium falciparum-infected erythrocytes: roles for endothelial leukocyte adhesion molecule 1 and vascular adhesion molecule 1. J. Exp. Med. 176:1183-1189. 31.OPHIR, A., E. BERENSTEIN, N. KITROSSKY, AND E. AVERBUKH. 1994. Protection of the transiently ischemic cat retina by zinc-desferrioxamine. Invest. Ophthalmol. Vis. Sci. 35:1212-1222. 32.POSTMA, N.S., E.C. MOMMERS, W.M.C. ELING, AND J. ZUIDEMA. 1996. Oxidative stress in malaria; implications for prevention and therapy. Pharm.World Sci. 18:121-129.

60 ECM and protection by antioxidant treatment

33.POSTMA, N.S., C.C. HERMSEN, D.J.A. CROMMELIN, J. ZUIDEMA, AND W.M.C. ELING. 1998. Treatment with reconbinant human tumour necrosis factor-a reduces parasitaemia and prevents P. berghei K173 induced cerebral malaria in mice. Parasitology in press. 34.POLDER, T.W., C.R. JERUSALEM, AND W.M.C. ELING. 1983. Topographical distribution of the cerebral lesions in mice infected with Plasmodium berghei. Tropenmed. Parasit. 34:235-243. 35.SHANLEY, P.F., C.W. WHITE, K.B. AVRAHAM, Y. GRONER, AND T.J. BURKE. 1992. Use of transgenic animals to study disease models: Hyperoxic lung injury and ischemic acute renal failure in “high SOD” mice. Renal Failure 14: 391-394. 36.SULKOWSKA, M., AND S. SULKOWSKI. 1997. The effect of pentoxifylline on ultrastructural picture of type ii alveolar epithelial cells and generation of reactive oxygen species during cyclophosphamide-induced lung injury. J. Submicrosc. Cytol. Pathol. 29:487-496. 37.THUMWOOD, C.M, N.H. HUNT, W.B. COWDEN, AND I.A. CLARK. 1989. Antioxidants can prevent cerebral malaria in Plasmodium berghei-infected mice. Brit. J. Exp. Pathol. 70:293-303. 38.VARANI, J., I. GINSBURG, L. SCHUGER, D.F. GIBBS, J. BROMBERG, K.J. JOHNSON, U.S. PYAN, AND P.A. WARD. 1989. Endothelial cell killing by neutrophils. Am. J. Pathol. 135:435-438. 39.VLASSARA, H., L. MOLDAWER, AND B. CHAN. 1989. Macrophage/monocyte receptor for nonenzymatically glycosylated protein is upregulated by cachectin/tumor necrosis factor. J. Clin. Invest. 84:1813-1820. 40.WARD, P.A., AND J. VARANI. 1990. Mechanisms of neutrophil-mediated killing of endothelial cells. J. Leukocyte Biol. 48:97-102. 41.WARD, P.A. 1991. Mechanisms of endothelial cell injury. J. Lab. Clin. Med. 118:421­ 426. 42.WEI, E.P., C.W. CHRISTMAN, H.A. KONTOS ,AND J.T. POVLISHOCH. 1985. Effects of oxygen radicals on cerebral arterioles. Am. J. Physiol. 248:H157-162. 43.WELCH, W.D., C.W. GRAHAM, J. ZACCHARI, AND L.P. THRUPP. 1980. Analysis and comparison of the luminol-dependent chemiluminescence response of alveolar macrophages and neutrophils. J. Reticolo-endothelial Soc. 28:275. 44.WEBER, C., W. ERL, A. PIETSCH, M. STOBEL, H.W. ZIEGLER-HEITBROCK, AND P.C.WEBER. 1994. Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-kappa B mobilization and induction of vascular cell adhesion molecule-1 in endothelial cells stimulated to generate radicals. Arterioscler. Thromb. 14:1665-1673.

61 CHAPTER

Alloxan protects Plasmodium berghei infected mice in end-stage disease against experimental cerebral malaria

C.C. Hermsen, R.W. Sauerwein and W.M.C. Eling

Submitted Chapter 5

ABSTRACT

C57B1 mice infected with 103 Plasmodium berghei K173 parasites develop experimental cerebral malaria (ECM). Mice in end-stage disease, with a body temperature "35.5°C, which is associated with an increased permeability of the blood- brain-barrier, were treated with the rapidly acting antimalarials alloxan or artemether. Treatment with alloxan protected all mice in end-stage disease against ECM while none of the artemether-treated mice were protected. Treatment with alloxan resulted in a 6 fold reduction in parasitaemia at 2 hours after treatment while reduction of parasitaemia after artemether treatment was observed after one day. Pretreatment with a mixture of the anti-oxidants: desferal and apocynin reduced the alloxan dependent protection against ECM but not the antiparasitic activity. These results indicate that alloxan-induced hydroxyl radicals prevent lethality by ECM and that this activity is independent of its parasiticidal action.

74 Prevention of end-stage ECM by alloxan

INTRODUCTION

C57Bl mice infected with Plasmodium berghei K173 parasites develop experimental cerebral malaria (ECM)[1]. One day before death (approximately at day 9) a progressive hypothermia develops which strongly correlates with an increased permeability of the blood-brain-barrier (BBB) [2] and development of haemorrhages in the brains [1]. ECM is associated with leukocyte adherence to the microvascular endothelium, edema and petechiae in the brains [3, 4]. Mice in end-stage ECM can be protected by treatment with anti-CD4 or anti-CD8 mAbs [5] or anti-LFA1 [6] suggesting that lymphocytes and more specifically those adhering to brain vascular endothelial cells are involved in the pathogenesis of petechiae. A current hypothesis is that adherent immunocompetent cells [4] locally stimulated by parasites or their products are involved in destabilization of the BBB. Since elimination of parasites by fast acting antimalarials may protect against fatal ECM, the effects of treatment with alloxan or artemether was analysed on parasitaemia and late stage ECM. Alloxan is an inducer of hydroxyl radicals [7] and a rapidly acting antiparasitic drug which has been shown to reduce parasitaemia and cure P. vinckei-infected mice [8]. Artemether acts not as fast as alloxan but clears parasitaemia faster than chloroquine [9]. Treatment with chloroquine could not prevent ECM in mice with end-stage disease [5]. The antiparasitic activity of artemether is most likely caused by free-radical damage of parasite membrane systems [9]. Radical production can be inhibited by desferal which is an iron chelator blocking radical formation by the iron-catalyzed Haber-Weiss reaction [8, 10] and also an antiparasitic drug [11, 12, 13]. Apocynin is another anti-oxidant that can block an oxidative burst [14] and treatment with apocynin inhibits joint swelling in collagen induced arthritis in rats [15]. A mixture of desferal and apocynin was administered before treatment with alloxan to analyse the possibility that the alloxan mediated effect on parasitaemia and protection against ECM is mediated by the induction of hydroxyl radicals.

MATERIALS AND METHODS

Mice: female C57Bl/6J mice, ages 6-10 weeks, were obtained from specific pathogen­ free colonies maintained at the Central Animal Facility, University of Nijmegen. All

75 Chapter 5 mice were housed in plastic cages and received water and standard RMH food (Hope Farms, Woerden, The Netherlands) ad libitum.

Parasite: P. berghei K173 (originally obtained from W. Kretschmar, Tübingen, Germany) has been maintained by weekly transfer of parasitized erythrocytes (PE) from infected into naive mice >30 years. Experimental mice were infected intra- peritoneally (i.p.) with 10 PE from blood of infected donors.

Parasitaemia: thin blood films made from tail-blood on days 8, 9 and 10 after infection. They were stained with May-Grünwald and Giemsa solutions and the proportion of parasitized red blood cells was determined.

Detection of ECM: ECM was detected as described by [3]. In brief, 95% of C57Bl mice infected with 10 P. berghei K173 parasites die early in the second week after infection. One day before death a progressive hypothermia develops that is strongly correlated with development of haemorrhages in the brains as observed by histology [1]. The parasitaemia in mice that die of ECM is between 5-10%, and such mice have a 20% reduction in haematocrit. Mice that show a transient hypothermia (in most cases >32°C ) survive this critical period but die in the third week or later after infection with signs of severe anemia, a parasitaemia of 20%-40% but without detectable cere­ bral pathology as determined by histology.

End-stage disease: mice infected with P. berghei K173 parasites exhibit a rapidly decreasing body temperature, and usually die within 24 hours [1] about 8 or 9 days after infection. Mice are considered to be in end-stage disease when their body temperature drops to " 35.5°C [2].

Body temperature: was measured with a digital thermometer (Technoterm 1200, Thermotex, The Hague, Netherlands) introduced into the rectum and read after 10 seconds.

Heamatocrit: measured in heparinized 10 zl Drummond® capillaries (Drummond scientific company, Pennsylvania, USA) after centrifugation for 5 minutes in a Hawksley® haematocrit centrifuge.

76 Prevention of end-stage ECM by alloxan

Glucose levels: was measured with the digital one touch glucose meter (Lifescan, Beerse, Belgium). Glucose concentrations are expressed as mmol/L.

Artemether: 12-P-methyldihydro-quinghaosu, 100 mg/ml, (arteminh, Euro-vina, Geel, Belgium) was diluted in arachis oil and injected intraperitoneally.

Alloxan: alloxan monohydrate (Sigma A7413) was dissolved in saline and injected intra-peritoneally. To prevent damage to the pancreatic beta-cells 0.05 mg D- glucose/0.2 ml saline was injected i.p. 15 minutes before the injection of alloxan [16].

Desferal/Apocynin: desferal (Ciba, Arnhem, Netherlands) was dissolved in distilled * 0 to 220 mM and 1:1 diluted with 2 times concentrated phosphate buffered saline (PBS). Apocynin (Acetovanillone, 98%, Jansen Chimica, Belgium) was dissolved in 96% ethanol and further diluted in PBS. Equal volumes of desferal and apocynin were mixed and 0.2 ml/mouse was injected i.p. 10 minutes before alloxan treatment. Thus, mice received 125 mg/kg desferal and 12.5 mg/kg apocynin. In a dose finding study of desferal it was observed that higher concentrations were toxic (data not shown).

Statistical analysis: all data are expressed as mean ± SD. For statistical analysis the Student's V-test was used. P values < 0.05 were considered to be significant.

RESULTS

When Plasmodium berghei infected mice were treated with alloxan (250 mg/kg) on day 8 after infection, 4/5 mice were protected against experimental cerebral malaria (ECM; table 1). The therapeutic range of alloxan is rather narrow since 500 or 150 mg/kg or less showed no effect. Further analysis showed that all mice in end-stage disease (n=8; body temperature "35.5°C) were protected after treatment with 250 mg/kg alloxan (table 2), while only 42% of the mice at earlier stages of development of ECM (body temperature >35.5°C ) were protected. No protection against ECM was observed after single treatment with artemether (5 mg/kg - 20 mg/kg) 8 days after

77 Chapter 5 infection (table 2) nor after treatment with 10 mg/kg given on days 8 and 9 (data not shown).

Table 1: Effect of alloxan on protection against development of Plasmodium berghei induced experimental cerebral malaria (ECM) in C57B1/6J mice.

Dose* Number of mice protected against ECM / (mg/kg) total number of alloxan treated mice

500 0/5 250 4/5 150 0/5 75 0/5

Treatment with alloxan on day 8 after infection.

To determine whether the capacity of alloxan to induce hydroxyl radicals played a role in its protective effect, a mixture of the anti-oxidants desferal and apocynin (DFO/Apo) was administered 10 minutes before injection of alloxan. Treatment of infected mice with DFO/Apo alone did not protect against ECM (table 2). Treatment with DFO/Apo before alloxan reversed protection in mice in end-stage disease (from 100 to 47%) as compared to alloxan alone. DFO/Apo pretreatment did not affect the outcome of mice at earlier stages of development of ECM (table 2).

Figure 1 shows the effect of treatment with alloxan (250 mg/kg) or artemether (20 mg/kg) on parasitaemia. Reduction of the parasitaemia and of the number of viable parasites as judged from pycnosis of parasites in bloodsmears was already significant 2 hours after treatment with alloxan, but only 24 hours after artemether treatment. Pretreatment with DFO/Apo did not affect the antiparasitic activity of alloxan (fig. 1). Furthermore, parasitaemia was not different in mice that were protected or not against ECM after treatment with DFO/Apo and alloxan (fig. 1).

78 Prevention of end-stage ECM by alloxan

Table 2: Effect of alloxan or artemether on protection against development of Plasmodium berghei induced experimental cerebral malaria (ECM) in C57B1/6J mice.

Treatment* Dose Pretreatment+ Number of mice protected against ECM (mg/kg) / total number Body temperature early ECM end-stage ECM (>35.5°C) (<35.5°C)

Alloxan 250 Saline 3/7 8/8

Alloxan 250 DFO/Apo 2/4 7/15+

- - DFO/Apo 0/4 0/10

Artemether 5 None 0/4 0/4 10 None 0/9 0/13 20 None None 0/6

Saline 0/4 0/18 Saline/oil 0/1 0/2

*: Administration on day 8 after infection. ■f: Pretreatment was carried out 10 minutes before treatment with alloxan =(=: P < .05 as compared to alloxan treated mice with end-stage disease (body temperature <35.5°C)

Clark & Hunt [8] observed hemolysis 2 hours after alloxan injection in P. vinckei infected mice which was not confined to parasitized erythrocytes. In our study alloxan treated mice have a significantly lower haematocrit level (43% ± 2; n=5) as compared to sham-treated infected mice (48% ± 1; n=5; P < 0.05) 2 hours after treatment. Treatment with DFO/Apo before alloxan prevented the alloxan dependent decrease (Ht= 48% ± 1; 2 hours after treatment; n = 5) but a possible to protection against ECM could not be determined because of the small numbers of mice involved. D-glucose was administered to mice 15 minutes before alloxan to prevent hypoglycaemia as a result from P pancreatic cell damage [16, 8]. The glucose concentration (7.4 ± 1.2, n=5, 120 min.; 6.3 ± 0.3, n=5, 7 days after alloxan treatment) were not significantly different from those of sham-treated infected (6.8 ± 0.8, n=4) and uninfected controls (7.1 ± 0.4, n=5).

79 Chapter 5

Figure 1: Parasitaemia (mean and SD) after treatment with alloxan or artemether on day 8 after infection of C57B1/6J mice with 103 Plasmodium berghei K173 parasites. • = Alloxan treated mice, 250 mg/kg, n=6; =Artemether treated mice, 20 mg/kg, n=6; _= Alloxan+DFO/Apo treated mice which are protected against ECM, n=5; ▼ = Alloxan+DFO/Apo treated mice which are not protected against ECM, n=4. *: p < 0.05 as compared with parasitaemia at day 8 after infection and before drug treatment.

DISCUSSION

This study shows that mice can be protected against ECM by treatment with alloxan which is particularly effective in end-stage disease (body temperature is "35.5°C). Treatment with artemether late in the infection did not protect against ECM in our studies whereas treatment (5 mg/kg daily for three days) starting 5 or 6 days after infection protected P. berghei ANKA infected mice against ECM [17]. This difference in effectivity of artemether treatment may be explained by the earlier and longer time period of the antiparasitic treatment before ECM occurs in the study of Prada et al. [17] as compared to our study.

80 Prevention of end-stage ECM by alloxan

The ECM protective effect of alloxan could be mediated through one or more of its activities: a strong and rapid antiparasitic activity [8, 18], the capacity to generate hydroxyl radicals [7] or the capacity to lyse erythrocytes [8]. Alloxan treatment caused a 6-fold reduction of the parasitaemia 2 hours later, whereas reduction of parasitaemia by treatment with artemether only became apparent 24 hours later. Our data also show, however, that the antiparasitic activity of alloxan and protection against ECM are not directly linked. Alloxan treatment strongly reduced parasitaemia both in ECM protected and non-protected mice and did not prevent recrudescences one week after alloxan treatment. Finally, neutralization of the ECM protective effect of alloxan treatment by pretreatment with the anti-oxidants DFO and apocynin did not abolish the antiparasitic activity of alloxan. These results may indicate that apart from its antiparasitic activity, other activities of alloxan are involved in protection against ECM. The assumption that rapid parasite clearance by alloxan is not the critical factor in protection against ECM may be supported by results described by Hensbroek van et al. [19] who observed that although P. falciparum parasites (sequestered in brain microvasculature) in children with cerebral malaria were cleared from the circulation significantly faster by artemether than by quinine, the fatality rate was similar for both drugs. The observation that pretreatment with DFO and apocynin can abolish the alloxan dependent protection against ECM points to a role of the generation of hydroxyl radicals by alloxan. Generation of ROI, measured as hydrogen peroxide, in relation to antiparasitic treatment has been described by Prada et al. [17] though a causative relation was not described. Sensitivity of the parasite to ROI mediated damage has been shown before [20, 21] and possibly explains the antiparasitic activity of alloxan and artemether [8, 9]. In addition to parasite killing alloxan also caused lysis of uninfected erythrocytes confirming observations of Clark et al. [8] in P. vinckei infected mice. In addition, this haemolysis could be prevented by pretreatment with anti-oxidantia. Thus, hydroxyl radicals generated by alloxan are probably involved in erythrolysis. Treatment with DFO/Apo before alloxan prevented lysis of uninfected erythrocytes but not killing of parasites. This may indicate that the antioxidant effect of DFO/Apo on alloxan induced ROI is incomplete and that parasites are more sensitive to the effect of ROI than erythrocytes. The capacity of the hydrophilic DFO to enter

81 Chapter 5 parasitized red blood cells is rather low [22, 23, 24]. Inhibition of the antiparasitic effect of alloxan by pretreatment with 200 mg/kg of DFO was observed by Clark et a l. [8] in P. vinckei infections in CBA/CaH mice, but this regime was too toxic in our P. berghei model, where only 125 mg/kg could be administered maximally. The observation that alloxan causes damage of pancreatic P cells and diabetes [25] and lyses of erythrocytes and killing of parasites might indicate that alloxan also affects other as yet undefined cells (e.g. T cells) and that such cells or their products play a role in development of ECM. Mice infected with P. berghei K173 parasites exhibit a rapidly decreasing body temperature and usually die within 24 hours [1] and light- and electronmicroscopical analysis show an increasing adherence of white blood cells to the brain vascular endothelium [4]. Possibly, alloxan interferes with the activity of these adhering cells preventing damage to the endothelial lining. Previous work has shown that depletion of CD4 or CD8 T-cells in end-stage disease mice can protect against ECM [5], however, an effect of alloxan on T-cell activity has not been described. Artemether treatment did not protect against ECM, despite its capacity to generate free radicals [9, 26, 27]. Our studies suggest that artemether treatment did not generate enough or the right type of free radicals or to the appropriate cells in time to be effective against the rapidly evolving ECM. Thus, artemether treatment still requires 24 hours to show a substantial effect on parasites and if this is indicative for effects on other cells that play a role in development of ECM the artemether dependent induction of ROI might be too slow to be effective in the protection against ECM. Alloxan treatment protected against ECM more efficiently when given to mice in end stage disease ("35.5°C) than at an earlier stage (> 35.5°C ; table 2). This may reflect a narrow timewindow for effective treatment with alloxan. Such a timewindow has been described for treatment of sepsis in which administration of anti-TNF is likely to provide the most benefit in patients with severe sepsis or early shock, but not those with refractory shock [28].

In summary, these results show that alloxan treatment protects P. berghei-infected mice in end-stage disease, i.e. when the permeability of the blood-brain-barrier is already disturbed, against fatal ECM. The capacity of alloxan to generate hydroxyl radicals, rather than its fast acting antiparasitic activity is probably involved in the mechanism of protection.

82 Prevention of end-stage ECM by alloxan

ACKNOWLEDGEMENTS

We thank G. Poelen, T. van den Ing and Y. Brom for skilled biotechnical assistance.

REFERENCES

1. CURFS JHAJ, SCHETTERS TPM, HERMSEN CC, JERUSALEM CR, VAN ZON AAJC, ELING WMC. Immunological aspects of cerebral lesions in murine malaria. Clin Exp mmunol 1989; 75:136-40. 2. HERMSEN CC, MOMMERS E, WIEL VAN DE T, SAUERWEIN R, ELING WMC. Convulsions due to increased permeability of the blood-brain-barrier in experimental cerebral malaria can be prevented by splenectomy or anti-T-cell treatment. J Infect Dis 1998; 178:1225-1227. 3. CURFS JHAJ, HERMSEN CC, MEUWISSEN JHETH, ELING WMC . Immunization against cerebral pathology in Plasmodium berghei-infected mice. Parasitology 1992; 105:7-14. 4. POLDER TW, ELING WMC, CURFS JHAJ, JERUSALEM CR, WIJERS-ROUW M. Ultrastuctural changes in the blood-brain barrier of mice infected with Plasmodium berghei. Acta Leidensia 1992; 60:31-46. 5. HERMSEN C, WIEL VAN DE T, MOMMERS E, SAUERWEIN R, ELING W. Depletion of CD4+ or CD8+ T-cells prevents Plasmodium berghei induced cerebral malaria in end-stage disease. Parasitology 1997; 114:7-12. 6. GRAU GE, POITAIRE P, PIGUET PF, VESIN C, ROSEN H, STAMENKOVIC I, TAKEI F, VASSALLI P. Late administration of monoclonal antibody to leukocyte function-antigen 1 abrogates incipient murine cerebral malaria. Eur J Immunol 1991; 21: 2265-7. 7. GRANKVIST K, MARKLUND S, SEHLIN J, T äLJEDAL IB. Superoxide dismutase, catalase and scavengers of hydroxylradicals protect against the toxic action of alloxan on pancreatic islet cells in vitro. Biochem J 1979; 182:17-25 8. CLARK IA, HUNT NH. Evidence for reactive oxygen intermediates causing hemolysis and parasite death in malaria. Infect Immun 1983; 39:1-6. 9. HIEN TT, WHITE NJ. Qinghaosu. Lancet 1993; 341:603-8. 10.GUTTERIDGE KMC, RICHMOND R, HALLIWELL B. Inhibition of the iron-catalyzed formation of hydroxyl radicals from superoxide and of lipid peroxidation by desferrioxamine. Biochem J 1979; 184:469-72. 11.TSAFACK A, LOYEVSKI M, PONKA P, CABANTCHIK ZI. Mode of action of iron (III) chelators as antimalarials. IV. Potetiation of desferal action by benzoyl and isonicotinoyl hydrazone derivatives. J Lab Clin Med 1996; 127:574-82. 12.GOLENSER J, DOMB A, TEOMIM D, TSAFACK A, NISIM O, PONKA P, ELING W, CABANTCHIK ZI. The treatment of animal models of malaria with iron chelators by use of a novel polymeric device for slow drug release. J Pharmacol. Exp Ther 1997; 281:1127- 35.

83 Chapter 5

13.POSTMA NS, HERMSEN CC, ZUIDEMA J, ELING WMC. Plasmodium vinckei: Optimisation of desferrioxamine B delivery in the treatment of murne malaria. Experimental Parasitology 1998; 89:323-30. 14.HART HET BM, SIMONS JM. Metabolic activation of phenols by stimulated neutrophils: a concept for a selective type of anti-inflammatory drug. Biotechnol Therap 1992 ; 3:119­ 135. 15.HART HET BM, SIMONS JM, KNAAN-SHANZER S, BAKKER NPM, LABADIE RP. Antiarthritic activity of the newly developed neutrophil oxidative burst anatgonist apocynin. Free Rad Biol Med 1990; 9:127-31 16.MCDANIEL ML, ROTH CE, FINK CJ, LACY PE. Effect of anomers of D-glucose on alloxan inhibitions of insulin release in isolated perifused pancreatic islets. Endocr 1976; 99:535-40. 17.Prada J, Muller S, Bienzle U, Kremsner PG. Upregulation of reactive oxygen and nitrogen intermediates in Plasmodium berghei infected mice after therapy with chloroquine or artemether. J Antimicrob Chemoth 1996; 38:95-102. 18.COX FEG, MILLOTT SM. The importance of parasite load in the killing of Plasmodium vinckei in mice treated with Corynebacterium parvum or alloxan monohydrate. Parasitol 1984; 89:417-24. 19.HENSBROEK VAN BM, ONYIORAH E, JAFFER S, SCHEIDER G, PALMER A, FRENKEL J, ENWERE G, FORCK S, NUSSMEIER A, BENNETT S, GREENWOOD B, KWIATKOWSKI D. A trial of artemether and quinine in children with cerebral malaria. New Engl J Med 1996; 335:69-75. 20.OCKENHOUSE CF, SHEAR HL. Oxidative killing of the intraerythrocytic malaria parasite P. yoelli by activated macrophages. J Immunol 1984; 132:424-31. 21.POSTMA NS, MOMMERS EC, ELING WMC, ZUIDEMA J. Oxidative stress in malaria; implications for prevention and therapy. Pharm World Sci 1996; 18:121-9. 22.FRITSCH G, JUNG A. 14C-DFO Uptake into erythrocytes infected with P. falciparum. Z Parasietenkd 1986; 72:709-13. 23.SCOTT MD, RANZ A, KUYPERS FA, LUBIN BH, MESHNICK SR. Parasite uptake of desferoxamine a prerequiste for antimalarial activity. Br J Haematol 1990; 75:598-602. 24.LOYEVSKY M, LYTTON SD, MESTER B, LIBMAN J, SHANZER A, CABANTCHIK ZI. The antimalarial action of desferal involves a direct access route to erythrocytic (Plasmodium falciparum) parasites. J Clin Invest 1993; 91:218-24. 25.HEIKKILA RE, WINSTON B, COHEN G, BARDEN H. Alloxan-induced diabetes- evidence for hydroxyl radical as a cytotoxic intermediate. Biochem Pharmacol 1976; 25:1085-92. 26.KRUNGKRAI SR, YUTHAVONG Y. The antimalarial action on Plasmodium falciparum of qinghaosu and artesunate in combination with agents which modulate oxidant stress. Trans R Soc Trop Med Hyg 1987; 81:710-4. 27.MESHNICK SR. The mode of action of antimalarial endoperoxides. Trans R Soc Trop Med Hyg 1994; 88:(suppl 1),31-2. 28.GRAU GE, MAENNEL DN. TNF inhibition and sepsis sounding a cautionary note. Nature Medicine 1997; 11:1193-5.

84 CHAPTER

Circulating tumour necrosis factor a is not involved in the development of experimental cerebral malaria in Plasmodium berghei-infected C57B! mice

C.C. Hermsen, J. v d Crommert, H. Fredrix, R.W. Sauerwein and W.M.C. Eling

Parasite Immunology 1997; 19, 571-577. Chapter 6

ABSTRACT

Administation of neutralizing anti-TNFa antibodies did not prevent Plasmodium berghei induced cerebral malaria (ECM) in C57B1/6 mice, when given at different dosages and time intervals. Elevated concentrations of immunoreactive TNFa were found in the circulation but no bioactive TNFa could be detected in mice developing ECM. Furthermore, elevated TNFa receptor concentrations were measured in the plasma of mice developing ECM. Plasma of these mice neutralized bioactive recombi- nant-mouse TNFa indicating that a part of the plasma TNFa-receptors were not complexed with TNFa. The apparent absence of free TNFa probably explains why treatment with antibodies against TNFa failed to prevent development of ECM.

86 TNFa is not involved in murine ECM

INTRODUCTION

Experimental cerebral malaria (ECM) can be induced in CBA mice by experimental infection with Plasmodium berghei ANKA (reviewed Eling & Sauerwein 1995). Development of ECM is closely associated with high serum concentrations of immunoreactive Tumor Necrosis Factora (TNFa), measured by ELISA, (Clark et al. 1990) and of bioactive TNFa, measured by bio-assay, (Grau et al. 1987, Neill & Hunt 1995). In mice with high concentrations of bioactive TNFa ECM can be prevented by treatment with anti-TNFa antibodies (Grau et al. 1987), and transgenic mice expres­ sing high levels of soluble fusion protein TNFaR-P55 are markedly protected against CM (Garcia et al. 1995). In human P. falciparum malaria high concentrations of immunoreactive TNFa have been associated with both disease severity and cerebral malaria (Grau et a l. 1989, Kwiatkowski et al. 1990). Recent studies have shown that children homozygous for the TNF2 allele, a variant of the TNFa gene promotor region, which corresponds to a high production capacity of TNFa, have a higher risk for CM (McGuire et al. 1994). However, in other studies the relation between plasma concentrations of immunore- active TNFa and clinical outcome was unclear (Shaffer et al. 1991; Yamada-Tanaka et al. 1995). Whilst a number of studies (Kern et al. 1992, Molyneux et al. 1993 and Kossodo, Houba & Grau 1995) have detected high levels of immunoreactive TNFa in the plasma of patients with complicated P. falciparum malaria, very low concentrations of bioactive TNFa have been reported. Strongly elevated concentrations of soluble TNFa receptors P55 (s-TNFaR-P55) and P75 (s-TNFaR-P75) have also been observed in the sera of such patients (Kern et al. 1992; Molyneux et al. 1993; Deloron et al. 1994). Importantly treatment with anti-TNFa MAbs did not cure cerebral malaria in children (Kwiatkowski et al. 1993). It can be concluded from both murine and human studies that bioactive TNFa is the important denominator for success of anti-TNFa antibody treatment rather than immu- noreactive TNFa. We have studied the effect of anti-TNFa antibodies, that neutralize bioactive TNFa, on the development of Plasmodium berghei K173 induced cerebral malaria in C57Bl mice. The results have been related to concentrations of immunore­ active and bioactive TNFa and of soluble TNFa receptors.

87 Chapter 6

MATERIALS AND METHODS

Mice: Female C57Bl/6J mice, aged 6-10 weeks, were obtained from specific pathogen free colonies maintained at the Central Animal Facility of the University of Nijmegen. All mice were housed in plastic cages and received water and standard RMH food (Hope Farms, Woerden, The Netherlands) ad libitum.

Parasite: Plasmodium berghei K173 was maintained by weekly transfer of parasitized erythrocytes (PE) from infected into naive mice. Experimental mice were infected intraperitoneally with 10 PE from blood of infected donor animals of the same strain and sex.

Parasitaemia: Thin blood films were made from tail-blood, stained with May-Grun- wald and Giemsa's solutions, and the proportion of parasitized red blood cells was determined.

Detection of experimental cerebral malaria: The detection of ECM was performed as described by Curfs et al. (1992). Briefly, 95% of C57Bl mice infected with 10 P. berghei K173 parasites die early in the second week after infection. One day before death a progressive hypothermia develops which is strongly correlated with develop­ ment of haemorrhages in the brain as observed by histology (Curfs et al. 1989). The parasitaemia in mice that die from ECM is between 5 and 10%, and such mice have a 20% reducion of the haematocrit. Mice that show a transient hypothermia (in the majority of cases >32°C ) survive this critical period but die in the third week or later after infection with severe anaemia, a parasitaemia between 20 and 40 % but without any noticeable cerebral pathology.

Body temperature: Body temperature was measured with a digital thermometer (Technoterm 1200) introduced into the rectum and read after 10 s.

Lipopolysaccharide: LPS E. coli phenol extract was obtained from Sigma (L-3129), dissolved in pyrogen-free saline and injected intraperitoneally. Body-temperature was measured and plasma collected 1.5 hours after the LPS injection when TNFa levels were expected to be maximal (Fong et al. 1989).

88 TNFa is not involved in murine ECM

Collection of plasma: Blood was collected in endotoxin free tubes (4°C) which contained endotoxin free (LAL assay) heparin (NPBI, The Netherlands). Plasma was col lected by centrifugation within 20 min. after bleeding and stored at -20°C.

Tumour Necrosis Factor. (TNFa): Recombinant-murine-TNF a (rec-mTNFa) was generously donated by Dr. G. Adolf, Ernst-Boehringer Institut, Vienna, Austria. The specific activity was 1.2.107U/mg in murine LM cells. The endotoxin content was <51 pg/ml.

Anti-TNFa: Neutralizing (Echternacher et al. 1990) rat anti-murine TNF a MAbs produced by V1Q hybridoma cells (gift of P.H. Krammer, Heidelberg, FRG) were used. Pristane primed nude mice were injected i.p. with 3.106 hybridoma cells and the ascites produced was collected. After ammoniumsulfate (45%) precipitation and dialysis against H O , the solution was freeze-dried and stored at 4°C until used. Aliquots were solubilized in sterile, pyrogen­ free saline and used immediately. The in-vitro neutralizing capacity was determined in a TNFa dependent L929 cell-lysis assay (Aggarwal, 1985). The amount of rec-mTNF athat caused lysis of 50% of the L929 cells as determined in a standard serial twofold dilution curve was defined as 1 unit of rec-mTNF a and corresponded to 100 pg/ml. The amount of anti- TNFa MAbs neutralizing 1 unit rec-mTNF a was defined as one neutralizing unit (nU). Normal rat-Ig (Sigma) was used as a control of anti-TNF a MAbs in the in-vivo studies. The LPS content (LAL assay) of the anti-TNF a (V1Q) MAbs solution used in-vivo was less then 8.2 pg/ml.

Rabbit anti mouse-TNFa antibodies: A New Zealand rabbit was immunized with 50 mg rec- mTNF mixed with Freunds Complete AdLuvant and boosted 3 times with a four week interval with 25 mg rec-mTNF mixed with Freunds Incomplete Adjuvant. One week after the last booster, serum was collected and stored at -20°C. The antiserum was a gift from Dr.J.W.M. van der Meer, Department of Internal Medicine, Academic Hospi tal, NiLmegen, The Netherlands. The neutralizing capacity was determined in the L929 assay. Using the procedure as described above for anti-TNF a MAbs, one unit rabbit anti mouse-TNFa neu­ tralized 100 pg rec-mTNF a/ml.

Immunoreactive TNFa: The plasma concentration of immunoreactive TNFa was determined by a sandwich ELISA which recognize free unbound TNFa and TNFaR- TNFa complexes. An ammoniumsulfate precipitate of ascites containing anti-TNFa

89 Chapter 6

MAbs were purified by protein G (Pharmacia) affinity chromatography followed by dialysis. ELISA plates were coated with 100 ml of 0.01 mg anti-TNFa MAbs/ml phosphate buffered saline (PBS) overnight at 4°C. After washing with PBS plates were incubated with 250 ml 1% BSA + 0.1% Tween 20 in PBS for one hour at room temperature. After washing five times with PBS, 100 ml of serial dilutions of standard rec-mTNFa or test samples were added and incubated for 1 hour. After washing, plates were incubated with 100 ml rabbit anti rec-mTNFa antiserum (see below). 100 ml of a 1:500 dilution of a swine-anti rabbit Ig horseradish peroxidase antiserum (Dakopatts) was added to the plates followed by 100 ml of tetramethyl benzidine solution (0.25 mM 3,3',5,5'-tetramethyl benzidine, 0.7 mM H2O2 in 0.1 M sodium acetate, pH 5.5) as the substrate. The peroxidase substrate reaction was stopped by adding 100 ml of 4 M H2SO4 and the optical density was determined in an ELISA reader at 450 nm (Titertek Multiskan MCC/340;ICN, Zoetermeer, The Netherlands). The detection limit was 100 pg rec-mTNFa/ml.

Bioactive TNFa: TNFa bioassay was performed as described by Aggarwal (1985) with minor modifications. Briefly, rec-mTNFa or test samples were added to 100,000 L929 cells per well. The mixture was cultured in the presence of 8 mg/ml emetine (Sigma) and 5% C O in air at 90% relative humidity at 37°C for 20 h. TNF-mediated cytopathic effects on L929 cells were measured by discarding the supernatant, washing the adherent cells gently and fix the remaining cells. After drying, 1% crystal violet (Sigma) in PBS was added and incubated for 10 min. After removing the excessive crystal violet acetic acid was added to dissolve the colored cells. The optical density was determined in an ELISA reader at 540 nm (Titertek Multiskan MCC/340;ICN, Zoetermeer, The Netherlands). The L929 bioassay detection limit was 50 pg rec- mTNFa/ml.

TNFa neutralizing assay: L929 cells were cultured as described above. To each well 50 ml cellsuspension (2.10 cells/ml) in culture medium without fetal calf serum was added. After addition of rec-m-TNFa to mouse plasma (20 and 40 % in culture medium) 50 ml was added to the L929 cells. Serial dilutions of rec-m-TNFa in culture medium with 20% fetal calf serum (FCS) were taken as a standard. The TNFa mediated cytopathic effect on L929 cells was measured by adding 20 ml EZ4U substrate (Nuclilab b.v. Ede, The Netherlands) to each well and cells were incubated

90 TNFa is not involved in murine ECM at 5% CO in air at 90% relative humidity at 37°C for 2 h. Absorbance was determined at 450 nm with 620 nm as a reference on an ELISA reader (Titertek Mul- tiskan MCC/340;ICN, Zoetermeer, The Netherlands).

TNFa receptor P55 (TNFaR-P55) and P75 (TNFaR-P75) ELISAs: The ELISAs recognizing both free, unbound TNF receptors and TNFaR-TNFa complexes, were carried out according to the methods described by Bemelmans et al. (1994). Briefly, microtiter plates were coated with anti-TNFR-P55 or anti- TNFR-P75. Aspecific binding was blocked with 1% BSA. After washing, serum samples were added. A standard titration curve was made by recombinant murine TNFR-P55 of TNFR-P75. Subsequentely, plates were washed and incubated with biotinylated anti-TNF-receptor antibodies. After washings, plates were incubated with streptavidin peroxidase followed by enzyme reaction. The detection limit for murine sTNFR-P55 was 5 pg/ml and for sTNFR-P75 50 pg/ml.

Statistical analysis: For statistical analysis the student's t-test was used. P values < 0.05 were considered to be significant.

RESULTS

Immunoreactive TNFa concentrations in ECM In the course of developing ECM, immunoreactive TNFa concentration was determined in plasma of P. berghei K173 infected C57Bl mice (figure 1). An increa­ sing concentration of immunoreactive TNFa was measured in plasma of mice in the second week after infection, whereas the immunoreactive plasma TNFa concentration was below the detection limit before infection.

Neutralizing effect of anti-TNFa MAbs on LPS challenge in-vivo Initially, the in-vivo neutralizing capacity of anti-TNFa MAbs was tested by injecting 12,500 - 50,000 nU of anti-TNFa MAbs, intravenously in uninfected mice 3 hours before an intraperitoneal injection of 100 or 500 mg of LPS. LPS treatment induced a decrease in body-temperature , which was inhibited by treatment with anti-TNFa MAbs in a dose-dependent manner (R value = 0.75, p=<0.005, Table 1). The body-

91 Chapter 6 temperature of mice treated with either 50,000 or 25,000 nU of anti-TNF a MAbs followed by injection of LPS (100 mg/mouse) was not significantly different from untreated mice. LPS dependent mortality after injection of 500 mg LPS/mouse (4/4 mice died) was prevented by pretreatment with 50,000 nU of anti-TNF a MAbs/mouse (1/4 mice died). Plasma concen­ trations of immunoreactive TNF a and bioactive TNFa were increased 90 minutes after injection of 100 mg LPS/mouse (Table 1). Pretreatment with 50,000 nU of anti-TNF a MAbs 3 hours before injection of 100 mg LPS significantly reduced immunoreactive TNF a concentrations and reduced the bioactive TNF a concentration below the detection limit.

Figure 1: Concentration of immunoreactive TNFa in plasma of Plasmodium berghei infected mice (n=5); *:p<0.05 compared to day 7; data are depicted as mean + SD

Neutralizing effect of anti-TNKx MAbs on LPS challenge in P. berghei infected mice. To confirm the neutralizing activity of anti-TNF a MAbs during malaria infection, the concentration of immunoreactive and bioactive TNFa was determined 90 minutes after injection of 100 mg LPS on day 8 after P. berghei infection. The bioactive TNF a plasma level in infected / LPS treated mice (Table 2) was approximately 1300 times higher in compari son to uninfected-LPS treated mice. Pretreatment with 50,000 nU anti-TNF a MAbs of infected / LPS treated animals neutralized bioactive TNFa to levels below the detection limit (Table 2).

92 TNFa is not involved in murine ECM

Table 1: The effect of anti-TNFa MAbs (V1Q) on a LPS mediateci decrease of thè body- temperature and on thè concentration immunoreactive and bioactive TNFa.

Injection of Injection of LPS Body-temperature neutralizing (T = 3.0 h) (°C) TNFa units (T = 4.5 h) anti-TNFa

(T= Ohrs) immuno reactive bioactive pg/ml pg/ml

No No 37.6 ± 0.5 (n=18) <100 <50

Saline 100 mg 36.4 ± 0.2 (n=3)* 500 220

50,000 nU 100 mg 37.3 ± 0.4 (n=3) 303 <50

25,000 nU 100 mg 37.2 ± 0.3 (n=3) nd nd

12,500 nU 100 mg 36.7 ± 0.4 (n=3)* nd nd n = number of mice * = significantly different as compared to untreated mice nd = not done

Table 2: Effect of anti-TNFa MAbs (V1Q) on the concentration of immunoreactive and bioactive TNFa in LPS treated P. berghei infected mice.

P. berghei infected mice 8 days after infection

anti-TNFa LPS Immunoreactive TNFa Bioactive TNFa (T= Oh) (T= 3 h) (T= 4.5 h) (T= 4.5 h)

mg/mouse pg/ml pg/ml

No No 292 ± 180* nd

Saline 100 265,000# 294,000#

50,000 nU 100 5,00 O' < 50#

* n=5 nd = not done # pool (n=5)

93 Chapter 6

Effect of anti-TNFa treatment on the development of ECM Mice were treated with anti-TNFa MAbs before or in the course of development of ECM. Individual groups of mice received a single dose of anti-TNFa MAbs. In a second experiment infected mice received three times anti-TNFa MAbs 2 days apart (Table 3). The results show that almost all mice treated with anti-TNFa MAbs develo­ ped ECM as did untreated or saline treated controls. Similarly, mice treated with polyvalent rabbit anti-mouse recombinant TNFa antibodies also developed ECM (Table 3).

Table 3: Effect of anti-TNFa MAbs and polyvalent anti-TNFa treatment on the develop- ment of cerebral malaria in P. berghei infected mice.

Anti-TNFa treatment Number of ECM protec­ ted mice / number of treated mice

Days after infection Treatment nU

-1 anti-TNFa MAbs 17,000 0/2

2,4,6 tf 50,000/day 0/6

3 tf 17,000 0/2

5 tf 43,000 0/2

6 tf 50,000 0/5

7 tf 17.000 2/7 7 tf 50.000 0/10 7 tf 100.000 0/3

8 tf 100,000 1/3

9 tf 100,000 0/12

7 R-M-TNFa* 50,000 0/7

2,4,6 saline 0/7

7 tf 0/15

8 tf 1/2

9 tf 0/7

Controls No 2/52 * Polyvalent rabbit-anti-mouse rec TNFa antibodies ** Neutralizing Units (determined in the L929 cell-lysis assay)

94 TNFa is not involved in murine ECM

TNFa receptors during development of ECM. Measurement of the concentration bioactive TNFa in the course of the infection showed high concentrations of immunoreactive TNFa (Figure 1), concentrations of bioactive TNFa below 50 pg/ml (Table 4).

Table 4: Concentration of bioactive TNFa and soluble TNFa receptors during develop­ ment of cerebral malaria

Plasma samples of Bioactive Soluble TNFa-receptors normal and infected mice TNFa

pg/ml s-TNFa-P55 s-TNFa-P75 ng/ml ng/ml

Normal mice < 50 0.04-0.1* 3-6*

Exp 1:

day 7s < 50 0.57° 34.7°

day 10s < 50 0.79° 97.4°

Exp 2:

day 10s < 50 0.54 + 0.2400 108.5 ± 42.5- O O

$: after infection *: range °: pool (n=3) 00: individual samples (n=9)

Plasma concentrations of soluble TNFa receptors P55 and P75 were 10 and 20 times higher at day 10 after infection compared to control mice (Table 4). Furthermore, plasma of mice collected on day 10 after infection, inhibited significantly the cytopathic effect of rec-m-TNFa in the L929 cell-lysis assay in a dose-dependent manner (Figure 2) as compared to plasma of normal mice. The assay was carried out in 2 individual experiments, one with 10% and one with 20% plasma of day 10 after infection.

95 Chapter 6

Figure 2: Neutralization of bioactive recombinant-mouse TNFa induced L929 cell lysis by plasma from normal mice (Q = 10%, #= 20% , n=5) or plasma from mice of day 10 after infection with 103 Plasmodium berghei K173 parasites ( =10%, B=20% , n=9). A dose dependent lysis of L929 cells by rec-m-TNFa was taken as a control (+ = 10% fetal calf serum (FCS), +=20% FCS); *:p<0.05 compared to plasma from normal mice; data are depicted as mean + SD.

DISCUSSION

Our principle finding is that the administration of either anti-TNFa MAbs or polyva­ lent rabbit anti-TNFa antibodies could not prevent development of P. berghei K173 induced cerebral malaria in C57Bl mice. Furthermore no bioactive TNFa could be detected in plasma of mice developing ECM. Anti-TNFa antibodies were ineffective when given at different dosages and time intervals despite proven neutralization of bioactive TNFa (Table 3). These anti-TNFa antibodies effectively neutralized LPS induced bioactive TNFa and prevented LPS dependent decrease of body-temperature and mortality (Table 1,2). Moreover, anti-TNFa MAbs treatment reduced the plasma

96 TNFa is not involved in murine ECM concentration of bioactive TNFa to < 50 pg/ml, even when the plasma concentration of bioactive TNFa in P. berghei infected mice treated with LPS was a 1300-fold higher as compared to non-infected mice. The reduction of immunoreactive TNFa in anti-TNFa / LPS treated normal mice was about 50% (Table 1), while a 50-fold reduction was obtained in anti-TNFa / LPS treated and P. berghei infected mice (Table 2). In both instances bioactive TNFa was not detectable, which indicates that immunoreactive TNFa is complexed to anti-TNFa antibodies. A possible explanation of the difference in reduction of bioactive TNFa in anti-TNFa / LPS treated normal mice compared with anti-TNFa / LPS treated and P. berghei infected mice could be that the TNFa - anti-TNFa complex circulates longer in normal mice as compared to infected mice. A longer circulation period of the TNFaa / anti-TNFa complex is also found in P. falciparum infected children treated with anti-TNFa MAbs (Kwiatkowski et al. 1993). Our results are in contrast with those obtained by Grau et al. (1987) in P. berghei ANKA infected CBA/Ca mice, but in agreement with studies in P.falciparum infected children (Kwiatkowski et al. 1993, van Hensbroek et al. 1996). Another discrepancy between our results and those of Grau et a l. (1987) is the absence of bioactive TNFa in plasma of our P. berghei infected C57Bl mice that develop the cerebral syndrome (Table 4). These differences may be due to the different combinations of mouse (resp. CBA/Ca vs. C57Bl mice) and parasite strains used (resp. ANKA parasites vs. K173 parasites). The absence of bioactive TNFa despite high concentrations of immunoreactive TNFa is similar to data obtained from P. falciparum in humans (Kern et al. 1992; Kwiat­ kowski et al. 1993; Molineux et al. 1993). This finding could be explained by the presence of excess soluble TNFa receptors in the plasma (Kern et al. 1992; Molyneux et al. 1993; Deloron et al. 1994; Kremsner et al. 1995). This is in line with the observation of Garcia et a l. (1995) showing that in contrast to normal infected mice, transgenic mice, expressing high levels of soluble TNFaR-P55 fusion protein, are markedly protected against development of a P. berghei ANKA induced ECM. Our studies show increased concentrations of soluble TNFa receptors (Table 4). Not all TNFa-receptors are complexed with TNFa as plasma taken on day 10 after infection is still able to neutralize bioactive rec-mTNFa (Figure 2). Kwiatkowski et al. (1993) observed a reduction of fever after treatment with anti- TNFa MAbs in Gambian children suffering from P. falciparum induced CM. In our

97 Chapter 6 mouse model we observe an increase in body-temperature to peak values of 39°C around day 8/9 after infection, which is just before body-temperature rapidly decreases during development of the cerebral malaria syndrome (Curfs et al. 1989). Treatment with either anti-TNFa MAbs or polyvalent rabbit anti-TNFa antibodies on different days during infection (day 2 to day 8) with different doses (2.104 to 1 .1 0 nU) did not prevent this increase in body-temperature. In summary, the results suggest that in P. berghei K173 infected mice plasma levels of soluble TNFa receptors increase as a response to produced bioactive TNFa. Fur­ thermore, soluble TNFa receptors can neutralize bioactive TNFa and the resulting bio-inactive complex can be measured as an increasing concentration of immunoreacti- ve TNFa during infection. Not all TNFa receptors are complexed. The apparent absence of free TNFa probably explains why treatment with a MAbs against native TNFa or polyclonal antibodies against rec-mTNFa failed to prevent development of ECM in this model.

ACKNOWLEDGEMENTS

We thank Dr. W. Buurman, Department of General Surgery, University Limburg, the Netherlands for performing the TNF-receptor ELISAs, G. Poelen, T. van den Ing and Y. Brom for skilled biotechnical assistance and Dr. C. Drakeley for reading the manuscript.

REFERENCES

AGGARWAL B.B., (1985) Human lymphotoxin. Methods Enzymology 116, 441-444 BEMELMANS M.H., ABRAMOWICZ D., GOUMA D.J. ET AL. (1994) In vivo T cell activation by anti-CD3 monoclonal antibo dy induces soluble TNF receptor release in mice. Effects of pentoxifylline, methylpredniso lone, anti-TNF and anti-IFN-gamma antibodies. Journal of Immunology 153, 499-506 CLARK I.A., ILSCHNER S., MACMICKING J.D. ET AL. (1990) TNF and Plasmodium berghei ANKA-induced cerebral malaria. Immunology Letters 25, 195-198 CURFS J.H.A.J., SCHETTERS T.P.M., HERMSEN C.C. ET AL. (1989) Immunological aspects of cerebral lesions in murine malari a. Clinical and Experimental Immunology 75, 136-140 CURFS J.H.A.J., HERMSEN C.C., MEUWISSEN J.H.E.TH. ET AL. (1992) Immunization against cerebral pathology in Plasmodium berghei-infected mice. Parasitology 105, 7-14

98 TNFa is not involved in murine ECM

DELORON P., LOMBARD ROUX P., RINGWALD P. ET AL. (1994) Plasma levels of TNFa soluble receptors correlate with outcome in human falciparum malaria. European Cytokine Networke 5, 331-336 ECHTERNACHER B., WERNER F., MANNEL D.N. ET AL. (1990) Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis. The Journal of Immunology 145, 3762-3766 ELING, W.M.C. & SAUERWEIN, R (1995) Severe and cerebral malaria: common or distinct pathophysiology? Reviews in Medical Microbiology 6(1), 17-25 FONG Y., TRACEY K.J., MODAWER L.L. ET AL. (1989) Antibody to cachectin/TNF reduce interleukin-1-beta and interleukin-6 appearance during lethal bacteremia. Journal of Experimental Medicine 170, 1627-33 GARCIA I., MIYAZAKI Y., ARAKI K. ET AL. (1995) Transgenic mice expressing high levels of soluble TNF-R1 fusion protein are protected from lethal septic shock and cerebral malaria, and are highly sensitive to Listeria monocytogenes and Leishmania major infections. European Journal of Immunology 25, 2401-7 GRAU G.E., FAJARDO L.J., PIQUET P.F. ET AL. (1987) Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 237, 1210-1212 GRAU G.E., TAYLOR T.E., MOLYNEUX M.E. ET AL. (1989) Tumor necrosis factor and disease severity in children with Halciparum malaria. The New England Journal oH Medicine 320, 1586-91 HENSBROEK VAN M.B., PALMER A., ONYIORAH E. ET AL. (1996) The effect of a monoclonal antibody to tumor necrosis factor on survival from childhood cerebral malaria. Journal of Infectious Diseases 174, 1091-7 KERN P., HEMMER C.J., GALLATI H. ET AL. (1992) Soluble tumor necrosis factor receptors correlate with parasitemia and disease severity in human malaria. The Journal oH Infectious Diseases 166, 930-4 KOSSODO DE S., HOUBA V., GRAU G.E. (1995) Assaying tumor necrosis factor concentrations in human serum. A WHO international colaborative study. Journal of Immunological Methods 182, 107-14 KREMSNER P.G., WINKLER S., BRANDTS C. ET AL. (1995) Prediction of accelerated cure in Plasmodium Halciparum malaria by the elevated capacity of tumor necrosis factor production. American Journal of Tropical Medicine and Hygiene 53, 532-538 KWIATKOWSKI D., HILL A.V.S., SAMBOU I. ET AL. (1990) TNF concentration in fatal cerebral, non-fatal cerebral, and uncomplicated Plasmodium falciparum malaria. The Lancet 336, 1201-1204 KWIATKOW SKI D., MOLYNEUX M.E., STEPHENS S. ET AL. (1993) Anti-TNF therapy inhibits fever in cerebral malaria. Quarterly Journal of Medicine 86, 91-98 MCGUIRE W., HILL A.V., ALLSOPP C.E. ET AL. (1994) Variation on the TNF a promotor region associated with susceptibility to cerebral malaria. Nature 371, 508-10 MOLYNEUX M.E., ENGELMANN H., TAYLOR T.E., ET AL. (1993) Circulating plasma receptors for tumor necrosis factor in Malawian children with severe Halciparum malaria. Cytokine 5, 604-9 NEILL A.L. & HUNT N.H. (1995) Effects of endotoxin and dexamethasone on cerebral malaria in mice. Parasitology 111, 443-454 SHAFFER N., GRAU G.E., HEDBERG K. ET AL. (1991) Tumor necrosis factor and severe malaria. Journal of Infection and Disease 163, 96

99 Chapter 6

YAMADA-TANAKA M.S., FATIMA FERREIRA-DA-CRUZ M., DAS GRACAS ALECRIM M. ET AL. (1995) Tumor necrosis factor alpha, interferon gamma and macrophage stimulating factor in relation to the severity of Plasmodium falciparum malaria in the Brazilian Amazone. Tropical and Geographical Medicine 6, 282-285

100 CHAPTER

Treatment with recombinant human tumour necrosis factor-a reduces parasitaemia and prevents P. berghei K173-induced experimental cerebral malaria in mice

N.S. POSTMA1, C.C. HERMSEN2, D.J.A. CROMMELIN1,

J. ZUIDEMA1, w .m .c . e l i n g 2

'Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands department of Medical Microbiology, University Hospital Nijmegen, Nijmegen, The Netherlands

Parasitology, accepted juni 1998 Chapter 7

ABSTRACT

The present study shows that treatment with recombinant human tumour necrosis factor-a (rec-h-TNFa) can suppress parasitaemia and prevents development of cerebral malaria (ECM) in P. berghei K173 infected mice. Mice received rec-h- TNFa treatment either by subcutaneous injection of free or liposome-encapsulated rec-h-TNFa or sustained intraperitoneal administration of rec-h-TNFa given via mini-osmotic pumps. Low dose treatment with a subcutaneous bolus injection of rec- h-TNFa protected against ECM when treatment was started at day 5 or 6 after infection. The same protective efficacy was obtained either by subcutaneous injection of liposome-encapsulated rec-h-TNFa or by sustained release from osmotic pumps, but in the latter case a 10-fold lower daily dose of rec-h-TNFa was sufficient. Treatment with rec-h-TNFa substantially suppressed parasitaemia in ECM-protected mice, but not in mice developing ECM. Thus, the rec-h-TNFa mediated suppression of parasitaemia is directly or indirectly involved in protection against ECM. Sustained delivery of rec-h-TNFa through osmotic pumps, but not by subcutaneous injection of liposome-encapsulated rec-h-TNFa, resulted in increased concentrations of soluble mouse TNF receptor R75 (sTNFR75) in plasma at day 9 after infection when non-treated mice die of ECM. Thus, protection against ECM is not directly correlated with the sTNFR75 concentrations at day 9 after infection.

102 rec-h-TNFa reduces parasitaemia and prevents cerebral malaria

INTRODUCTION

Tumour necrosis factora (TNFa) plays a dual role in malaria, being involved both in the control of parasitaemia (Taverne et al. 1994; Kremsner et al. 1995; Jacobs et al. 1996) and in the development of malaria-pathology, like cerebral malaria (ECM) (Grau et al. 1989; McGuire et al. 1994). Administration of high doses of rec-h- TNFa to P. berghei K173 infected mice induced pathology in the brains that resembled the pathology found in the brains of non-treated mice dying of ECM (Curfs et al. 1993). However, a preliminary observation in this model showed that low dose rec-h-TNFa treatment protected against ECM, which merits further attention. A possible mechanism involved in the rec-h-TNFa mediated protection against ECM is the production of soluble TNF receptors (sTNFRs) (Engelmann, Novick & Wallach, 1990). STNFRs are derived from proteolytic cleavage of the cellular receptors TNFR55 and TNFR75. They competitively inhibit interaction of TNFa with the membrane-bound receptor (Lesslauer et al. 1991) and thus provide a possible mechanism for regulating the availability of TNFa. In another observation, repeated administration of rec-h-TNFa resulted in the development of tolerance to the lethal effects of TNFa (Takahashi, Brouckaert & Fiers, 1995). Sustained release (from osmotic pumps or liposomes) of small amounts of rec-h-TNFa might prevent development of the cerebral syndrome e.g. through induction of refractoriness of TNFa sensitive cells to subsequent stimuli (Alexander et al. 1991). The successful use of liposomes as carrier systems for sustained release of encapsulated agents in general (Kadir, Zuidema & Crommelin, 1993) and in the treatment of malaria (Peeters et al. 1989) has been described before. The aim of the present study was to analyse the effect of treatment with low doses of rec-h-TNFa on the protection against the development of ECM in P. berghei K173 infected mice. Sustained release of rec-h-TNFa from intraperitoneally (i.p.) implanted mini-osmotic pumps is compared with a subcutaneous (s.c.) injection of free or liposome-encapsulated rec-h-TNFa. The protective effect of rec-h-TNFa treatment on ECM is analysed in relation to its effect on parasitaemia and on the production of soluble TNF receptors (sTNFR55 and sTNFR75).

103 Chapter 7

MATERIALS AND METHODS

Materials: Highly purified rec-h-TNFa from Escheria coli (Wang et al. 1985) with a biological activity of 2.5 10 U/mg (Creasey et al. 1987) was a kind gift of Chiron Corporation (Emeryville, California USA). Egg phosphatidylcholine (EPC) and egg phosphatidylglycerol (EPG) were donated by Lipoid (Ludwigshafen, Germany). All other reagents used were of analytical grade.

Preparation of liposomes: A mixture of EPC and EPG (9:1, molar ratio) was dissolved in ethanol and evaporated to dryness in a rotary evaporator under reduced pressure at 35° C. After drying the lipid film with nitrogen for at least 20 min, the lipid film was hydrated with the rec-h-TNFa solution (100 zg rec-h-TNFa/ml in 20 mM Hepes, 149 mM NaCl, pH 7.4, at a lipid concentration of 40 mM). The resulting liposome dispersion (MLVs) was centrifuged three times (275000 g for 30­ 60 min at 4° C) to remove the non-liposomal rec-h-TNFa (Beckmann Optima™ LE- 80K, Beckman Instruments, Palo Alto, CA). Empty or buffer-loaded liposomes were prepared in the same way, but hydrated with Hepes buffer only (20 mM Hepes, 149 mM NaCl, pH 7.4). Liposome dispersions were used within 1 week after preparation; they were stored in the refrigerator (4- 6°C).

Liposome characterisation: Lipid phosphate was determined by the method of Rouser, Flusher & Yamamoto (1970). Approximately 2% bioactive rec-h-TNFa appeared to be encapsulated as determined in the WEHI cytotoxicity assay after solubilisation of the liposomes (see below). The liposomes used in the experimental studies were non-sized.

Determination of bioactive TNFa: The bioactivity of rec-h-TNFa, free or liposome-associated (expressed as dose of free rec-h-TNFa in the aqueous phase of the liposomes), was determined using the WEHI cytotoxicity assay essentially according to the method described previously (Espevik & Nissen-Meyer, 1986). WEHI 164 clone 13 mouse fibrosarcoma cells were cultured in Iscove's Modified Dulbecco's Medium with glutamine (IMDM; Gibco Life Technologies, Breda, The Netherlands) supplemented with 10% foetal bovine serum (FBS; Integro, Zaandam,

104 rec-h-TNFa reduces parasitaemia and prevents cerebral malaria

The Netherlands), penicillin (100 IU/ml), streptomycin (100 zg/ml) and amphotericin B (0.25 zg/ml) at 37°C in a humidified atmosphere containing 5% CO2. One day before testing, WEHI cells were transferred to new flasks and grown overnight. The next day cells were collected to a final concentration of 0.5 10 cells/ml medium of which 50 zl was pipetted into 96 wells microtiter plates (Falcon, Micronic, Lelystad, The Netherlands). Following incubation for 3 to 5 hours at 37°C and 5% CO , 50 zl of standard or test sample was added. A standard curve (up to 20000 pg/ml) was made from rec-h-TNFa (Boehringer Ingelheim, Germany) with an originally defined biological activity of 6 10 7 U/mg in a murine LM bioassay according to WHO standards. Serial dilutions of free TNF a in medium were added directly to the cells. In case of liposome-encapsulated rec-h-TNFa, liposomes were disrupted with 10% Triton X-100 in Hepes buffer (20 mM Hepes, 149 mM NaCl, 0.5 % bovine serum albumin, pH 7.4) in a volume ratio of 1:1. No effect of Triton X-100 was observed in the bioactivity assay of rec-h-TNFa with a sample dilution of 30000 times.

After incubation for 18 hours at 37°C and 5% CO 2 the viable cells were quantitated using the XTT assay (Scudiero et al. 1988). Briefly, 100 zl of N-methyldibenzopyrazine methyl sulfate (0.4 mg/ml in phosphate buffered saline) from Sigma ( Bornem, Belgium) was added to 5 ml of sodium 3-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene sulfonic acid hydrate (XTT, Sigma, Bornem, Belgium) and 1 mg/ml RPMI 1640 (Gibco, Life Technologies, Breda, The Netherlands). Fifty microliters of the XTT labelling mixture was added to the wells and incubated for 90 min at 37°C and 5% CO . The absorbance was measured using a Biorad Novapath microplate reader (Bio-Rad Laboratories, Veenendaal, The Netherlands) at a wavelength of 495 nm (reference wavelength: 655 nm). The WEHI cytotoxicity assay lower detection limit was approximately 80 pg rec-h-TNF a/ml. In the above WEHI cytotoxicity assay, the specific biological activity (U/mg) of rec-h- TNFa used for the experiments was about 8-10 times higher than for the standard rec-h- TNFa (Boehringer Ingelheim, Germany).

Mice: Female C57Bl/6J mice, 6 to 10 weeks old, were obtained from a specific pathogen free colony maintained at the Central Animal Facility of the University of NiLmegen. All mice were housed in plastic cages and received water and standard RMH food (Hope Farms, Woerden, The Netherlands) ad libitum. Parasite and development of cerebral malaria Mice were infected with the murine parasite Plasmodium berghei K173. The parasite was maintained by weekly transfer of

105 Chapter 7 parasitised erythrocytes from infected into naive mice. About 95% of C57Bl/6J mice i.p. infected with 10 parasitised erythrocytes die early in the second week after infection (day 9-12) due to the development of ECM as described by Curfs et al. (1992). One day before death a progressive hypothermia develops which is strongly correlated with development of haemorrhages in the brain as observed by histology (Polder et al. 1992). Mice that survive this critical period only show a limited, transient hypothermia and the majority dies in the third week or later after infection with severe anaemia and a parasitaemia of 20-40%, but without any noticeable cerebral pathology. Day of death after infection was used as the parameter for ECM or protection against ECM in the experiments described in this paper.

Experimental design: For each experiment mice received 10 parasitised erythrocytes i.p. on day 0. At day 5, 6, 7 or 8 after infection, mice were treated with a single s.c. injection of either free rec-h-TNFa or liposome-associated rec-h-TNFa. Sustained delivery of rec-h-TNFa through i.p. implanted mini-osmotic pumps (Alzet model 2001; release rate 1 ml/hour for 7 days, Alza, Palo Alto, CA) was started at day 5 or 7 after infection. Prior to injection, appropriate dilutions were prepared using Hepes buffer (20 mM Hepes, 149 mM NaCl, pH 7.4) containing 1% mouse plasma. The effect of treatment on parasitaemia (% infected erythrocytes) at day 8 after infection was studied from thin blood films made from tail-blood and stained with May-Grunwald and Giemsa’s solutions. A considerable variation is observed in parasitaemia at day 8 after infection among independent infected control groups (average parasitaemia + s.d.: 20 + 15%; 21 experiments and 3-5 mice per experimental group). Therefore, within each experiment the average parasitaemia in non-treated mice was determined and the parasitaemia of each individual mouse from either a control or a treatment group was divided by this average. These ratios were used to compare and summarise the results of independent repeat experiments. The effect of treatment on the development of ECM was studied by monitoring survival of mice. Death of mice within two weeks after infection was used to identify ECM-death (Curfs et al. 1992). Persistent, recurrent parasitaemia and survival for more than 2 weeks after infection indicated protection against ECM. TNF receptor R55 and R75 (TNFR55 and TNFR75) ELISAs: Mice treated with rec-h-TNFa and non-treated mice were sacrificed at day 9 after infection to study the

106 rec-h-TNFa reduces parasitaemia and prevents cerebral malaria effect of treatment on the plasma concentrations of soluble mouse TNF receptors. Blood was collected in endotoxin free tubes (4°C) which contained endotoxin free (LAL assay) heparin (NPBI, Emmer Compascuum, The Netherlands). Plasma was collected by centrifugation within 2 h after bleeding and stored at -20°C. The ELISAs recognising both free and complexed TNF receptors, were carried out with the materials and according to the methods described by Bemelmans et a l. (1994). Briefly, microtiter plates were coated with anti-TNFR55 or anti-TNFR75 monoclonal antibody. Aspecific binding was blocked with 1 % BSA. After washing, mouse plasma samples were added. A standard curve was made using recombinant murine TNFR55 and TNFR75 protein. Subsequently, plates were washed and incubated with biotinylated anti-TNF receptor antibodies. After washing, plates were incubated with streptavidin peroxidase followed by an enzyme reaction providing a coloured reaction product. The lower detection limit for murine soluble TNFR55 was 20 pg/ml and for soluble TNFR75 was 500 pg/ml.

Statistical analysis: The effect of treatment on parasitaemia was analysed by one­ way ANOVA. The effect of treatment on survival was analysed by the Chi-Square test. Differences were considered significant at a level a =0.05.

RESULTS

Subcutaneous injection of free rec-h-TNFa in P. berghei infected mice Mice received a single s.c. injection of free rec-h-TNFa at day 5, 6, 7 or 8 after infection and protection against ECM was monitored (Fig. 1). Approximately 50­ 90% of the mice were protected against ECM when treatment was started at day 5 after infection (0.5-5 zg rec-h-TNFa). The protective efficacy against ECM depended on the day of treatment (day 5-8, p < 0.0001; summarised data of all doses) and decreased when treatment was started later in infection. Protection against ECM was independent of the dose of rec-h-TNFa in the studied dose range 0.5-5 zg rec-h-TNFa (summarised data of treatment of days 5-7). Administration of 10 zg rec-h-TNFa was toxic (death within 24 h after injection) at day 5 for 7 out of 10

107 Chapter 7

Mice (%) protected against cerebral malaria 100 18/20 □ 0.5 fig rliTNF-alplia □ 1 /xg rliTNF-alplia □ 2 /xg rliTNF-alpha ■ 5 fig rliTNF-alpha 10 fig rliTNF-alplia

L 0/4 Day of rec-h-'fNF-a treatment7after infection

Figure 1: The effect of treatment with free rec-h-TNFa during infection on protection against ECM. Mice received a single subcutaneous injection of rec-h-TNFa (0.5-10 fxg rec-h-TNFa) at day 5, 6, 7 or 8 after infection with 103 P. berghei parasites and survival for more than 2 weeks after infection was used as a marker for protection against ECM. The percentage of mice protected against ECM is plotted against the day of treatment and the dose of rec-h-TNFa. Above each bar, the number of mice protected against ECM divided by the number of treated mice is indicated mice, while all mice died within 24 h when injected at day 8. No toxic effects were observed after injection of 5 zg rec-h-TNFa at day 8 after infection. The parasitaemia data are expressed as the ratio of the parasitaemia of a certain mouse divided by the mean parasitaemia of the infected but otherwise non-treated mice in the same experiment. Calculation of this ratio permitted to summarise the results of independent repeat experiments. The effect of rec-h-TNFa treatment on parasitaemia was similar when treatment was started at day 5, 6 or 7 and in the dose­ range of 0.5-5 zg rec-h-TNFa (Fig. 2). Therefore, statistical analysis was performed on the summarised data. Treatment with rec-h-TNFa significantly (p<0.0001 compared to non-treated mice) suppressed parasitaemia independent of the day of treatment (day 5-7; summarised data of doses ranging from 0.5-5 zg rec-h-TNFa) and independent of dose of rec-h-TNFa (0.5-5 zg rec-h-TNFa; summarised data of treatment on days 5-7) (Table 1). The effect of rec-h-TNFa treatment on parasitaemia was also studied in relation to protection against ECM; parasitaemia was significantly suppressed in ECM-protected mice but not in mice developing ECM (p<0.0001, Table 1).

108 rec-h-TNFa reduces parasitaemia and prevents cerebral malaria

Figure 2: The effect of treatment with rec-h-TNFa during infection on parasitaemia. Mice received a single subcutaneous injection of rec-h-TNFa (0.5-5 fig rec-h-TNFa) at day 5, 6 or 7 after infection with 103 P. berghei parasites. The average parasitaemia at day 8 after infection was determined in each control group and the parasitaemia of each individual mouse was divided by the average of the corresponding control group. The ratio is plotted against the dose of rec-h-TNFa for each mouse. When treatment has no effect on parasitaemia the ratio scatters around 1, while ratios < 1 indicate that parasitaemia is suppressed.

Table 1: Effect of treatment with a subcutaneous bolus injection of rec-h-TNFa on parasitaemia in relation to development of cerebral malaria in /5, berghei infected mice.

Treatment+ Ratio parasitaemia at day 8/ mean parasitaemia non-treated mice* All mice Mice with ECM Mice without ECM no treatment 1 ± 0.6 (n=87) 1 ± 0.6 (n=87) (n=0)

rec-h-TNFa 0.6** + 0.6 (n = 101) 0.9 ± 0.6 (n=48) 0.4*** ± 0.4 (n=53) +Mice received a subcutaneous injection of rec-h-TNFa (0.5-5 fig) at day 5, 6 or 7 after infection with 103 parasites and the effect on parasitaemia at day 8 after infection was studied in relation to protection against cerebral malaria. *The average parasitaemia at day 8 after infection was determined in each control group and the parasitaemia of each individual mouse was divided by the average of the corresponding control group. This permitted to summarise results of independent repeat experiments. Values represent averages ± s.d. of the combined data of different doses (0.5-5 ¿tg rec-h-TNRx) and days of treatment (day 5, 6 or 7). ** p <0.0001 as compared to non-treated mice. #p < 0.0001 as compared to mice developing ECM in the same treatment group.

109 Chapter 7

Sustained release of rec-h-TNFa in P. berghei infected mice Sustained release of low doses of 0.05-0.15 zg rec-h-TNFa/day, from i.p. implanted osmotic pumps (release rate 1 ml/hour during 7 days) starting at day 5 after infection protected 65-100% of the mice against the development of ECM (Table 2). The protective efficacy against ECM was independent of dose in the dose range studied. The summarised data of mice receiving saline containing pumps exhibit protection against ECM in 5 out of 16 mice. This was an unexpectedly high protection rate when considering the outcome of earlier work (unpublished data). For unknown reasons in one of the four independent experiments 3 out of 3 mice treated with the saline containing pumps were protected against ECM. Despite this, protection against ECM was significantly (p<0.01) higher by sustained release of rec-h-TNFa (0.05-0.15 zg rec-h-TNFa/day) as compared to saline treated controls. Treatment with rec-h-TNFa via osmotic pumps (0.05-0.15 zg rec-h-TNFa/day) starting at day 5 after infection significantly (p< 0.001) suppressed parasitaemia (average + s.d.: 0.4 + 0.3; n=14) (data not shown). When sustained release of rec-h-TNFa started at day 7 after infection, no effect was observed on parasitaemia and mice were not protected against ECM (data not shown).

Table 2: Effect of sustained release of rec-h-TNFa from intraperitoneally implanted osmotic pumps on protection against cerebral malaria.

Treatment+ Number of ECM protected mice / number of treated mice no treatment 2/86 (2%)

pump: saline 5/16 (31%) 0.05 fig rec-h-TNFa/day 13/20 (65%) 0.10 fig rec-h-TNFa/day 4/4 (100%) 0.15 fig rec-h-TNFa/day 8/9 (89%) + The pumps were implanted at day 5 after infection with 103 P. berghei parasites and released 1 ml/hour during 7 days. Controls received either pumps containing saline or no treatment at all.

110 rec-h-TNFa reduces parasitaemia and prevents cerebral malaria

S.c. injection of liposomal rec-h-TNFa in P. berghei infected mice Subcutaneous injection of rec-h-TNFa encapsulated into fluid type (EPC:EPG) liposomes was used as another approach for sustained release of rec-h-TNFa. The dose range of 0.5-5 zg of bioactive rec-h-TNFa as used for free rec-h-TNFa was also used for the liposomal rec-h-TNFa preparation. Protection against ECM was dose-dependent (0.5-5 zg liposomal rec-h-TNFa, p<0.001; summarised data of treatment on days 5-7) and increased to 100% protection when 5 zg liposomal rec-h- TNFa was injected at day 5 or 6 after infection (Fig. 3). The protective efficacy of liposomal rec-h-TNFa against ECM also depended on the day of treatment (day 5-8, p< 0.0001; summarised data of all doses) and decreased when treatment was started later in infection, similar to the results obtained with free rec-h-TNFa (cf Fig. 1 and Fig. 3). Injection of liposomal rec-h-TNFa at day 8 was completely ineffective, but also non-toxic.

Mice (%) protected against cerebral malaria 120 □ 0.5 fig liposomal rhTNF-alpha 10/10 □ 1 fig liposomal rhTNF-alpha 100 □ 2 fig liposomal rhTNF-alpha

80 U 5 fig liposomal rhTNF-alpha

60 2/4 4/8

40

20

0 —«------mrr ■TO* 5 6 7 8 Day of rec-h-TNF-a treatment after infection

Figure 3: The effect of treatment with liposomal rec-h-TNFa during infection on protection against ECM. Mice received a single subcutaneous injection of liposomal rec-h-TNFa (0.5-5 fig bioactive rec-h-TNFa) at day 5, 6, 7 or 8 after infection with 103 P. berghei parasites and survival for more than 2 weeks after infection was used as a parameter for protection against ECM. The percentage of

mice protected against ECM is plotted against the day of treatment and the dose of rec-h-TNFa administered. Above each bar, the number of mice protected against ECM divided by the number of treated mice is indicated.

I ll Chapter 7

The effect of treatment with liposomal rec-h-TNFa on parasitaemia as compared with non-treated mice did not reach significance (p = 0.06), which is probably linked to the remarkably high parasitaemia of a few mice treated with 2 zg liposomal rec-h- TNFa (Fig. 4 and Table 3). On the other hand, parasitaemia was significantly (p<0.001) suppressed in ECM-protected mice as compared to non-treated mice or mice developing ECM (Table 3). Subcutaneous injection of empty, buffer-loaded liposomes at day 5, 6 or 7 after infection did not protect against ECM (data not shown) and did not affect parasitaemia (Table 3).

Parasitaemia at day 8 after infection/mean parasitaemia non-treated mice 3

□ 5 a 6 o 7

2 □o o o 06 AO B □ 1 S B § ID a 0 % □ n 0 1 i i “I 2 3 4 6 Dose of rec-h-TNF-a ( u, a)

Figure 4: The effect of treatment with liposomal rec-h-TNFa during infection on parasitaemia. Mice received a single subcutaneous injection of liposomal rec-h-TNFa (1-5 fig rec-h-TNFa) at day 5, 6 or 7 after infection with 103 P. berghei parasites. The average parasitaemia at day 8 after infection was determined in each control group and the parasitaemia of each individual mouse was divided by the average of the corresponding control group. The ratio is plotted against the dose of rec-h-TNFa for each mouse. When treatment has no effect on parasitaemia the ratio scatters around 1, while ratios < 1 indicate that parasitaemia is suppressed.

112 rec-h-TNFa reduces parasitaemia and prevents cerebral malaria

Effect of sustained treatment with rec-h-TNFa on sTNFRs in plasma The effect of treatment by sustained release of rec-h-TNFa on the concentrations of sTNFRs in plasma was determined at day 9 after infection when non-treated mice normally die of ECM. Groups of mice received protective doses of 0.5 zg rec-h-TNFa/day (mini-osmotic pump) or 2 zg liposomal rec-h-TNFa s.c. at day 5 after infection. The concentrations of soluble mouse TNFR55 and TNFR75 in rec-h-TNFa treated and non-treated mice are depicted in Table 4. No differences were observed in the concentration of soluble mouse TNFR55 at day 9 after infection in plasma samples of non-treated and rec-h-TNFa treated mice. In contrast, soluble mouse TNFR75 concentrations were increased significantly (p<0.05) at day 9 after infection when rec-h-TNFa was released from osmotic pumps, but not when rec-h-TNFa containing liposomes were administered s.c. at day 5.

Table 3: Effect of treatment with a subcutaneous injection of liposomal rec-h-TNFa on parasitaemia in relation to development of cerebral malaria in /5, berghei infected mice.

Treatment Ratio parasitaemia at day 8 / mean parasitaemia non-treated mice*

All mice Mice with ECM Mice without ECM No treatment 1.0 ± 0.6 (n=86) 1.0 ± 0.6 (n=86) (n=0)

Empty 0.8 ± 0.3 (n= 15) 0.8 ± 0.3 (n= 15) (n=0) liposomes

Liposomal 0.8 ± 1.0 (n=82) 1.5* ± 1.3 (n=23) 0.5** ± 0.6 (n=59) rec-h-TNFa +Mice received a subcutaneous injection of empty liposomes or liposome-encapsulated rec-h-TNFa (1-5 ¡xg rec-h-TNFa) at day 5, 6 or 7 after infection with 103 parasites and the effect on parasitaemia at day 8 after infection was studied in relation to protection against cerebral malaria. ::The average parasitaemia at day 8 after infection was determined in each control group and the parasitaemia of each individual mouse was divided by the average of the corresponding control group. The ratios were used for further analysis. Values represent averages + s.d. of the combined data of different doses (1-5 fxg rec-h-TNFa) and days of treatment (day 5, 6 or 7). * p<0.01 as compared to non-treated mice, #p < 0.0001 as compared to mice developing ECM in the same treatment group.

113 Chapter 7

Table 4: Effect of sustained rec-h-TNFa treatment starting at day 5 after infection on the concentrations of soluble mouse TNF receptor in plasma at day 9 after infection in P. berghei infected mice.

Treatment+ Soluble mouse TNF receptor in plasma (ng/ml)

STNFR55 STNFR75 no treatment 0.3 ± 0.2 (n=5) 50 + 9 (n=5)

rec-h-TNFa infusion < 0.2 (n=3) 84* + 13 (n=3) (0.5 /¿g/day)

liposome-encapsulated rec-h-TNFa 0.3 ± 0.1 (n=5) 54 + 13 (n=5) (2 /xg) +Mice received rec-h-TNFa either from i.p. implanted mini-osmotic pumps (release rate 1 ml/h, 0.5 fig rec-h-TNFa/day for 7 days) or from a single s.c. injection of 2 ¡xg liposome-encapsulated rec-h-TNFa at day 5 after infection with 103 parasites. A third group of mice did not receive rec-h-TNFa treatment. Groups of mice were sacrificed at day 9 after injection and plasma was collected for analysis by specific ELISAs. Values represent averages + standard deviation. * p < 0.05 as compared with non-treated mice.

DISCUSSION The most important observation of this study is that low dose treatment with rec-h- TNFa protected against ECM and suppressed parasitaemia when treatment was started at day 5 or 6 after infection. The rec-h-TNFa mediated protection against ECM gradually disappeared when treatment was postponed to day 7 or 8 after infection. The same protective efficacy was obtained by either s.c. injection of free or liposome-encapsulated rec-h-TNFa or i.p. infusion, but it should be noted that a 10 times lower daily dose of rec-h-TNFa was sufficient when continuously released from an i.p. implanted osmotic pump. Sustained release of rec-h-TNFa probably results in prolonged therapeutic concentrations of rec-h-TNFa in the circulation. It has been demonstrated that rec-h-TNFa is rapidly cleared from the circulation after i.v. injection into mice with an initial half-life less than 20 min and a subsequent slower b-phase (Ferraiolo et al. 1988). The sustained effect of rec-h-TNFa released from osmotic pumps is also shown by the higher plasma concentrations of sTNFR75 as compared with non-treated or liposomal rec-h-TNFa treated mice (Table 4). Bolus injection of rec-h-TNFa resulted in increased levels of sTNFR75 4 h after

114 rec-h-TNFa reduces parasitaemia and prevents cerebral malaria injection, returning to levels seen in the infected controls within 24 h after injection (unpublished observations). The observation that doses needed for protection against ECM by treatment with liposome-encapsulated rec-h-TNFa were as high as for free rec-h-TNFa (Fig. 1 and 3) and the observation that sTNFR75 was not increased by this treatment (Table 4), suggest that the s.c. liposomal depot released protective amounts of rec-h-TNFa only for a limited period of time. The mortality observed after s.c. injection of 10 zg rec-h-TNFa at day 5 or 8 probably reflects the role of TNFa in malaria-pathology as has been described before (Curfs et al. 1993) and limits the dose that can be administered by s.c. bolus injection. A significant suppression of parasitaemia by rec-h-TNFa treatment was observed in ECM-protected mice (Table 1 and 3). Under the experimental conditions used, the reduction of parasitaemia was independent of day of treatment or treatment regimen. Suppression of parasitaemia by administration of rec-h-TNFa to mice has been observed before (Stevenson & Ghadirian, 1989). TNFa alone does not inhibit parasite-growth in vitro (Taverne et al. 1987) and activated macrophages and neutrophils are probably involved in TNFa mediated parasite-killing (Kumaratilake, Ferrante & Rzepczyk, 1990; Taverne et al. 1994). In addition, TNFa affects erythropoiesis, which may also play a role in suppression of parasitaemia (Johnson et al. 1989; Moldawer et al. 1989). The observation that parasitaemia was suppressed in ECM-protected mice, but not in mice developing ECM (Table 1 and 3), suggests that the effect of rec-h-TNFa on parasitaemia plays a role in rec-h-TNFa mediated protection against ECM. It should be noted that almost all mice infected with parasites in a range of 10 to 10000, develop ECM despite a widely varying parasitaemia after infection (Curfs et al. 1993). Moreover, parasitaemia in non-treated mice varies extensively before the mice die of ECM. Therefore, the course of an untreated infection is probably not a critical factor in development of ECM in P. berghei infected mice. Thus, the change in parasitaemia by treatment with rec-h-TNFa may interfere with the development of ECM. On the other hand, the effect of rec-h-TNFa on parasitaemia may be an associated but not causally related factor in protection against ECM. Possible other mechanisms involved in the rec-h-TNFa mediated protection are the stimulation of the production of sTNFRs and the induction of tolerance or

115 Chapter 7 refractoriness of TNFa sensitive cells. Elevated concentrations of sTNFRs have been observed during malaria infection in humans (Deloron et al. 1994) and in the murine model used in the present study (Hermsen et al. 1997). STNFRs are probably increased as a response to endogenous TNFa production. They can inhibit TNFa interaction with membrane-bound receptors (Lesslauer et al. 1991) and high levels of sTNFR55 were shown to protect against ECM (Garcia et al. 1995). Our study did not reveal an increase in the plasma concentration of sTNFR55 in relation to sustained treatment with rec-h-TNFa. The increase in sTNFR75 at day 9 after infection is only observed in mice receiving rec-h-TNFa from osmotic pumps, not in mice receiving rec-h-TNFa from a s.c. liposomal depot, while both treatment regimens protected against ECM (Table 4). This suggests that the increase in sTNFR75 is not linked to protection against ECM. However, only one time point was measured and that may not be the critical point to observe functional changes in sTNFR75 involved in the protection against ECM. Another mechanism involved in rec-h-TNFa mediated protection against ECM may be the induction of refractoriness of tolerance of TNFa sensitive cells. Several studies demonstrated that pre-treatment with TNFa induces refractoriness and therefore tolerance to its pathological effects (Fraker et al. 1988; Takahashi et al. 1995). Tolerance persists for several days and might be a “natural” regulatory mechanism to control toxicity of this cytokine (Takahashi, Brouckaert & Fiers, 1993). Our data do not permit to decide whether treatment with rec-h-TNFa induces refractoriness and therefore prevents development of ECM. In summary, low dose treatment with rec-h-TNFa at day 5 or 6 after infection protects against ECM in P. berghei K173 infected mice. The effect of rec-h-TNFa treatment on suppression of parasitaemia may play a role. No indications were found that treatment with rec-h-TNFa protects through triggering of production of sTNFRs. The mechanism by which rec-h-TNFa prevents the development of ECM remains to be elucidated.

ACKOWLEDGEMENTS

We thank Dr. M. Hora (Chiron Corporation, Emeryville, CA) for the generous gift of rec-h-TNFa. We also gratefully thank Dr. W. Buurman (Department of General

116 rec-h-TNFa reduces parasitaemia and prevents cerebral malaria

Surgery, University Limburg, The Netherlands) for supplying us with the materials used for the sTNF receptor ELISAs and Prof.dr. J. van de Meer (University Hospital Nijmegen, Nijmegen, The Netherlands) for the gift of rec-h-TNFa used as a standard in the WEHI cytotoxicity assay. We thank G. Poelen, T. van de Ing and Y. Brom for their skilled biotechnical assistance

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119 CHAPTER

Thiolated recombinant human tumour necrosis factor-a enhances suppression of parasitaemia and protection against P. berghei K173-induced experimental cerebral malaria in mice

Nancy S. Postma1, C.C. Hermsen2, Daan J.A. Crommelin1,

Wijnand M.C. Eling2, Jan Zuidema1

'Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands 2Department of Medical Microbiology, University Hospital Nijmegen, Nijmegen, The Netherlands

Submitted. ABSTRACT

The introduction of reactive thiol groups in recombinant human tumour necrosis factor-a (rec-h-TNFa) by the reagent succinimidyl-S-acetylthioacetate resulted in the formation of a chemically stabilised rec-h-TNFa trimer (rec-h-TNFa-AT, SDS- PAGE analysis). Rec-h-TNFa-AT showed a slightly reduced in vitro bioactivity, but a substantially enhanced protective efficacy against development of murine cerebral malaria (CM) after intravenous injection as compared with non-modified rec-h- TNFa. Administration of thiolated rec-h-TNFa with protected thiol groups (rec-h- TNFa-ATA, no stabilised trimers in vitro) exhibited the same protective efficacy against CM while in vitro bioactivity was reduced. Parasitaemia was significantly suppressed in CM-protected mice, but not in treated mice developing CM. Protection against CM was not related to enhanced plasma concentrations of soluble TNF receptor R55 and R75 directly after injection, nor prior to the development of CM in non-treated mice. The present study demonstrates that chemical stabilisation of rec- h-TNFa trimers increases its potential (therapeutic and protective) in vivo. INTRODUCTION Tumour necrosis factor-a (TNFa) is a pleiotropic cytokine primarily produced by stimulated macrophages and a major mediator of the inflammatory and cellular immune response. Although TNFa was originally identified by its antitumour properties and cytotoxicity to some transformed cell lines [1], it has also been implicated in the pathophysiology of septic shock, cachexia, certain inflammatory diseases (e.g. rheumatoid arthritis) and infections, like malaria [2-4]. Recent studies demonstrated that treatment with low doses of recombinant human TNFa (rec-h- TNFa) inhibited parasite proliferation and prevented the development of cerebral malaria (CM) in P. berghei K173 infected mice (unpublished observations). Momomeric rec-h-TNFa is a non-glycosylated polypeptide of 157 amino acids with a molecular mass of 17 kD [5]. Studies from analytical ultracentrifugation [6-8], cross-linking [9-12], gel filtration chromatography [13, 14] and X-ray-scattering [15] showed that rec-h-TNFa was present as a trimer in solution. X-ray crystallographic studies of rec-h-TNFa revealed that rec-h-TNFa forms a compact, bell-shaped homotrimer [16, 17]. The effects of both rec-h-TNFa and rec-h-TNFP are mediated by two receptors with a distinct molecular weight, TNFR55 and TNFR75 [18]. Studies with TNFR55 provided evidence that each rec-h-TNFa trimer can interact with three receptor molecules [19, 20]. Additional mutational analysis studies with rec-h-TNFa suggest that each trimer has three receptor interaction sites located in the intersubunit grooves near the base of the trimer and that integrity of the trimeric structure is essential for rec-h-TNFa activity [21, 22]. These studies clearly demonstrate that the trimeric structure of rec-h-TNFa is very important for its action. However, rec-h-TNFa oligomers (dimers and trimers) have been shown to be unstable at low concentrations or upon incubation in buffer resulting in the formation of inactive monomers and polymers [12, 14, 23, 24]. This suggests that when rec-h-TNFa is applied as a therapeutic agent, bioactive rec-h- TNFa may only be present at or near the site of administration [24]. Stabilisation of rec-h-TNFa trimers by chemical cross-linking prevents the dissociation into inactive monomers and may therefore enhance the therapeutic potential of rec-h-TNFa due to higher and longer-circulating concentrations of bioactive rec-h-TNFa with an enhanced access to deeper body compartments. Stabilised rec-h-TNFa trimers were prepared by reaction of the protein with the reagent succinimidyl-S-acetylthioacetate (SATA). Reactive thiol groups are introduced in the protein at the site of primary amino groups [25], which can lead to formation of disulphide bridges between adjacent rec-h-TNFa monomers. The aim of this study was to analyse the therapeutic potential of covalently stabilised rec-h-TNFa trimers in P. berghei K173 induced CM in mice. The effect on parasitaemia and development of CM was monitored after intravenous injection and compared with non-modified rec-h-TNFa. The protective effect of rec-h-TNFa treatment on CM was analysed in relation to its effect on the production of soluble TNF receptors (sTNFR55 and sTNFR75).

MATERIALS AND METHODS Materials: Rec-h-TNFa (4 mg/ml in 10 mM sodium phosphate pH 7, 200 mM NaCl) (E.coli-derived) [26] with a specific activity of 5.10 U/mg (bioassay: murine LM cells) was a gift from Dr. G.R. Adolf (Boehringer Ingelheim, Vienna, Austria). N-succinimidyl S-acetylthioacetate (SATA) was purchased from Pierce (Rockford, USA). All other reagents used were of analytical grade.

Thiolation of rec-h-TNFa by the SATA-reaction: Basically, the procedure to thiolate proteins with SATA as described by Duncan et al. [25] was used. Rec-h- TNFa (4 mg/ml in 10 mM sodium phosphate pH 7, 200 mM NaCl) was applied on a Sephadex G-25M column (PD-10 column, Pharmacia, Uppsala, Sweden) and eluted with Hepes buffer (10 mM Hepes, 135 mM NaCl, 1 mM EDTA, pH 7.5). The eluted rec-h-TNFa solution was concentrated using microsep filters with a molecular weight cut-off of 10kD (Pall Filtron, Breda, The Netherlands) at 4°C to approximately 2 mg/ml before reaction with SATA. SATA was dissolved in dimethylformamide (DMF) and mixed with the rec-h-TNFa solution in a volume ratio of DMF:buffer=1:100 and a molar ratio of SATA:rec-h-TNFa = 8:1. The mixture was incubated at room temperature under continuous rotation for at least 20 min to allow formation of rec-h-TNFa with protected thiol groups (rec-h-TNFa- ATA). After incubation, the unreacted SATA was separated from rec-h-TNFa-ATA on a PD-10 column. The protein fraction in the eluate was detected by monitoring the absorption at 280 nm, collected and stored at -20°C (maximally 3 weeks). Rec-h- TNFa-ATA was de-acetylated by adding a freshly prepared hydroxylamine-HCl solution (0.5 M Hydroxylamine-HCl, 0.5 M Hepes, 25 mM EDTA, pH 7.5). The mixture was incubated for 60 min at room temperature under continuous rotation at a volume ratio of rec-h-TNFa-ATA:hydroxylamine = l0:l, yielding rec-h-TNFa with reactive thiol groups (rec-h-TNFa-AT). This product was used in all studies.

Protein determination: To determine the concentration of rec-h-TNFa and rec-h- TNFa-ATA the method described by Wessel and Flugge [2T] was used. However, the amount of rec-h-TNFa-AT could not be determined by the same method due to interference of hydroxylamine with the analysis. Therefore, the amount of protein in the rec-h-TNFa-AT solutions was estimated from the amount of rec-h-TNFa-ATA measured and corrected for dilution upon addition of hydroxylamine, as it is not likely that addition of hydroxylamine will change the amount of protein in any other way.

Gel permeation chromatography: The molecular weights of rec-h-TNFa, rec-h- TNFa-ATA and rec-h-TNFa-AT were determined by gel permeation chromatography (GPC) with a system consisting of a Model Sl0 HPLC pump (Waters Associates, Milford, MA, USA), a thermostated (3S0C) pre-column (Biosep SEC S3000 PEEK, S0 x T.S mm, Phenomenex, Torrance CA) and a TSK-GEL G3000SW column (TS x 300 mm, particle size l0 mm, Tosohaas, Japan). The system was equilibrated with l00 mM sodium sulphate/phosphate buffer, pH 6.T. The material was eluted with the same buffer at a flow rate of l.0 ml/min and the elution profile was monitored at 280 nm with a UV-detector (Model 486, Waters Associates, Milford, MA, USA). The system was calibrated with mixtures of standards of known molecular weight (dextran blue 2,000,000, ovalbumin 43,000, and ribonuclease A l3,T000; thyroglobulin 669,000, lactoglobulin l8,400, and insulin 6,000; immunoglobulin G lS0,000, chymotrypsin 2S,000, bovine serum albumin 6T,000, and myoglobin lT,200).

SDS polyacrylamide gel electroforesis (SDS-PAGE): SDS-PAGE analysis of rec-h- TNFa, rec-h-TNFa-ATA and rec-h-TNFa-AT was performed in a modified Laemmli system [28] using a horizontal 8-l8% gradient gel (ExelGel®, Pharmacia, Uppsala, Sweden) and gels were silver stained. Gels were run with non-reduced and reduced (0.l M DTT and l0% iodocetamide) samples. Molecular weight markers (Bio-Rad Laboratories, Veenendaal, The Netherlands) were used with a molecular mass ranging from l4 to 200 kD. In vitro bioactivity: The biological activity of rec-h-TNFa, rec-h-TNFa-ATA and rec-h-TNFa-AT was determined in the WEHI cytotoxicity assay essentially according to the method described previously [29], WEHI 164 clone 13 mouse fibrosarcoma cells were cultured in Iscove's Modified Dulbecco's Medium with glutamine (IMDM; Gibco Life Technologies, Breda, The Netherlands) supplemented with 10% foetal bovine serum (FBS; Integro, Zaandam, The Netherlands), penicillin (100 IU/ml), streptomycin (100 zg/ml) and amphotericin B (0.25 zg/ml) at 37°C in a humidified atmosphere containing 5% C 02. One day before testing, WEHI cells were transferred to new flasks and grown overnight. The next day cells were collected to a final concentration of 0.5 10 cells/ml medium of which 50 zl was pipetted into 96 wells microtiter plates (Falcon, Micronic, Lelystad, The Netherlands). Following incubation for 3 to 5 hours at 37°C and 5% CO, 50 zl of standard or test sample was added. A standard curve (10 to 20,000 pg/ml) was made from rec-h-TNFa (Boehringer Ingelheim, Germany) with an originally defined biological activity of 6 10 U/mg in a murine LM bioassay according to WH0 standards. Serial dilutions of samples in medium were added directly to the cells. After incubation for 18 hours at 37°C and 5% C O the viable cells were quantitated using the XTT assay [30]. Briefly, 100 zl of N-methyldibenzopyrazine methyl sulfate (0.4 mg/ml PBS) from Sigma (Bornem, Belgium) was added to 5 ml of sodium 3-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT, Sigma, Bornem, Belgium) with a concentration of 1 mg/ml RPMI 1640 (Gibco Life Technologies, Breda, The Netherlands). Fifty microliter of the XTT labelling mixture was added to the wells and incubated for 90 min at 37°C and 5% CO . The absorbance was measured using a Biorad Novapath™ microplate reader (Bio-Rad Laboratories, Veenendaal, The Netherlands) at a wavelength of 495 nm (reference wavelength: 655 nm). The WEHI cytotoxicity assay lower detection limit was approximately 80 pg rec-h-TNFa/ml. In the above WEHI cytotoxicity assay the specific biological activity of rec-h-TNFa (U/mg) used for the experiments was about 2-4 times higher than for the standard rec-h-TNFa (Boehringer Ingelheim, Germany). Mice: Female C57Bl/6J mice, 6 to 10 weeks old, were obtained from a specific pathogen free colony maintained at the Central Animal Facility of the University of Nijmegen. All mice were housed in plastic cages and received water and standard RMH food (Hope Farms, Woerden, The Netherlands) ad libitum. Parasite and development of cerebral malaria: Mice were infected with the murine parasite Plasmodium berghei K173. The parasite was maintained by weekly transfer of parasitised erythrocytes from infected into naive mice. About 95% of C57B1/6J mice intraperitoneally (i.p.) infected with 103 parasitised erythrocytes die in the second week after infection (day 9-12) due to the development of CM [31]. One day before death a progressive hypothermia develops which is strongly correlated with development of haemorrhages in the brain as observed by histology [32]. Mice that survive this critical period only show a limited, transient hypothermia and the majority dies in the third week or later after infection with severe anaemia and a parasitaemia between 20 and 40%, but without any noticeable cerebral pathology. Time of death after infection was used as the parameter for CM or protection against CM in the experiments described in this paper. Experimental set-up: For each experiment mice received 103 parasitised erythrocytes i.p. on day 0. At day 5 mice were treated with a single intravenous (i.v.) injection of either rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa-AT. Prior to injection, appropriate dilutions were prepared using Hepes buffer (10 mM Hepes, 135 mM NaCl, 1 mM EDTA, pH 7.5) containing 1% mouse plasma. Placebo- treated mice received buffer only. Control mice did not receive any treatment (non­ treated mice). The effect of treatment on parasitaemia (% infected erythrocytes) at day 8 after infection was studied from thin blood films made from tail-blood and stained with May-Grunwald and Giemsa’s solutions. A considerable variation is observed in parasitaemia at day 8 after infection among independent infected control groups (average parasitaemia and standard deviation: 17 + 8%; n=8 experiments). Therefore, within each experiment the average parasitaemia in non-treated mice was determined and the parasitaemia of each individual mouse from either a control or a treatment group was divided by this average. These ratios were used to compare the results of independent repeat experiments. The effect of treatment on the development of CM was studied by monitoring survival of mice. Death of mice within two weeks after infection was used to identify CM-death [31]. Persistent, recurrent parasitaemia and survival for more than 2 weeks after infection indicated protection against CM.

TNF receptor R55 and R75 (TNFR55 and TNFR75) ELISAs: Mice treated with buffer, rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa-AT at day 5 after infection were sacrificed at pre-defined time points after injection (t = 4 h, 24 h, T2 h or 96 h). Blood was collected in endotoxin free tubes (40C) which contained endotoxin free (LAL assay) heparin (NPBI, Emmer Compascuum, The Netherlands). Plasma was collected by centrifugation within 2 h after bleeding and stored at -200C. The ELISAs recognising both free and complexed TNF receptors were carried out with the materials and according to the methods described by Bemelmans et al. [33]. Briefly, microtiter plates were coated with anti-TNFRSS or anti-TNFRTS monoclonal antibody. Aspecific binding was blocked with l % BSA. After washing, mouse plasma samples were added. A standard curve was made using recombinant murine TNFRSS and TNFRTS protein. Subsequently, plates were washed and incubated with biotinylated anti-TNF receptor antibodies. After washing, plates were incubated with streptavidin peroxidase followed by an enzyme reaction providing a coloured reaction product. The detection limit for murine soluble TNFRSS was 40 pg/ml and for soluble TNFRTS was lS60 pg/ml.

Statistical analysis: The effect of treatment on parasitaemia was analysed by the one-way ANOVA. The effect of treatment on survival was analysed by the Chi- Square test. Differences were considered significant at a level a=0.0S.

RESULTS

Characterisation of rec-h-TNFa, rec-h-TNFa-ATA and rec-h-TNFa-AT by GPC and SDS-PAGE. Using GPC under non-denaturing conditions, rec-h-TNFa is eluted as a single peak corresponding to a molecular mass of approximately 42 kD indicative of a compact trimer (data not shown). No other peaks were present. Chromatography of both rec- h-TNFa-ATA or rec-h-TNFa-AT showed a major peak corresponding to a molecular mass of 3T and 36 kD, respectively (dimer or compact trimer). Small fractions eluted with molecular mass of approximately 80 kD (multimers) and in the void volume (molecular weight > S00 kD). When subjected to SDS-PAGE analysis, non-reduced samples of non-modified rec-h-TNFa showed a strong monomeric band of lT kD (Fig. lA, lane l). Rec-h-TNFa-ATA exhibited two major bands around lT kD (Fig. lA, lane 2), whereas rec-h-TNFa-AT showed a major band at S2 kD (indicative of a trimer, Fig. lA, lane 3). Reduced samples yielded a single band corresponding to the rec-h-TNFa monomer of about 17 kD (Fig. 1B). No other products were observed.

Figure 1A and B: Samples of rec-h-TNFa (1), rec-h-TNFa-ATA (2) and rec-h-TNFa- AT (3) were analysed by SDS-PAGE in the absence (A) or presence (B) of reducing agents as described in the materials and methods section. Photometric scans of silver stained SDS-PAGE gels are presented. The major bands are indicated by arrows with the corresponding molecular mass. In vitro bioactivity The bioactivity of non-modified rec-h-TNFa, rec-h-TNFa-ATA and rec-h-TNFa- AT was determined in a standard WEHI cytotoxicity assay. Equal concentrations of protein were added to the cells. The cytotoxicity of rec-h-TNFa-ATA was about 8-9 fold lower compared with non-modified rec-h-TNFa (Fig. 2). The cytotoxic activity was almost completely restored (70-90%) upon de-acetylation of rec-h-TNFa-ATA to rec-h-TNFa-AT. Under the experimental conditions used, hydroxylamine was not cytotoxic and did not change the cytotoxicity of rec-h-TNFa (data not shown).

Effect of treatment with rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa-AT on CM Intravenous injection of increasing doses of rec-h-TNFa of 2-18 mg protein protected up to 30% of the mice against CM (Fig. 3). No further increase was observed when 80 mg rec-h-TNFa was administered, and no toxicity of this dose A490 nm rec-h-TNFa

Protein (pg/ml)

Figure 2: Dose-response curves of the cytotoxic activity of rec-h-TNFa, rec-h-TNFa- ATA and rec-h-TNFa-AT. Equal protein doses of rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa-AT were added to TNF-sensitive WEHI cells. Absorbance was measured at 490 nm and plotted against the logio of protein concentration. Samples were measured in triplicate and averages + standard deviation are presented was observed (data not shown). Administration of either rec-h-TNFa-ATA (1-18 mg protein) or rec-h-TNFa-AT (1­ 18 mg protein) showed a significantly (p<0.05, Chi-Square test) enhanced protection against CM compared with non-modified rec-h-TNFa (Fig. 3). The protective efficacy increased up to 80% at doses of 9 and 18 mg protein for both rec- h-TNFa-ATA and rec-h-TNFa-AT. Differences in the protective effects of rec-h- TNFa-ATA and rec-h-TNFa-AT were not significant (summarised data of doses of 1-18 mg protein). However, in the WEHI cytotoxicity assay equal protein concentrations were associated with a 8-9 fold lower bioactivity of rec-h-TNFa-ATA as compared to rec-h-TNFa and rec-h-TNFa-AT (Fig. 2). A 9-fold higher dose of rec-h-TNFa-ATA (81 mg protein corresponding to 9 mg bioactive protein in the WEHI cytotoxicity assay) killed 13 out of 15 mice within 24 h, which is in contrast with the observation that 80 mg of non-modified rec-h-TNFa Mice (%) protected against cerebral malaria 6/6 120 ■ rec-h-TNF-a □ rec-h-TNFa-ATA 13/15 5/6 5/6 5/6 100 ■ rec-h-TNFa-AT 14/20 80

60 3/8

40

20

P/4 0/9 ■ I ■

Protein dose (¡J, g)

Figure 3: Effect of treatment with rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa-AT during infection on prevention of cerebral malaria (CM). Mice received a single intravenous injection of rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa-AT at day 5 after infection with 103 P. berghei parasites and survival for more than two weeks after infection was used as a marker for protection against CM. The percentage of mice protected against CM is plotted against protein dose. Above each bar the number of mice protected against CM divided by the number of treated mice is indicated. was not toxic to the infected mice. A comparable toxicity was observed after i.v. injection of 80 mg rec-h-TNFa-AT (data not shown). Injection of buffer only did not protect against CM (data not shown).

Effect of treatment with rec-h-TNa, rec-h-TNFa-ATA or rec-h-TNFa-AT on parasitaemia The effect of treatment with rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa-AT on parasitaemia at day 8 after infection is shown for rec-h-TNFa-ATA (Fig. 4A) and rec-h-TNFa-AT (Fig. 4B). The data are expressed as the ratio of the parasitaemia of a certain mouse divided by the mean parasitaemia of the infected but otherwise non­ treated mice in the same experiment. In the studied dose range, the effect treatment on parasitaemia was independent) of protein dose rec-h-TNFa-ATA(fig. 4A) or Table 1: Effect of an intravenous injection of rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa-AT on parasitaemia in relation to development of cerebral malaria in /5, berghei infected mice.

Treatment+ Parasitaemia at day 8 after infection/mean parasitaemia of non-treated mice in the same experiment* All mice Mice with CM Mice without CM No treatment 1 + 0.4 (n=27) 1 + 0.4 (n=27) (n=0)

rec-h-TNFa 1 ± 0.5 (n=33) 1 ± 0.5 (n=27) 0.6” + 0.2 (n =6) (2-18 mg)

rec-h-TNFa-AT A 0.8 ± 0.5 (n=52) 1 ± 0.6 (n= 17) 0.6 : + 0.2 (n==35) (1-18 mg)

rec-h-TNFa-AT 0.7' + 0.4 (n=35) 0.7 ± 0.2 (n=9) 0.6' + 0.4 (n==26) (1-18 mg)

+Mice received a single intravenous injection of non-modified rec-h-TNFa, or thiolated rec-h-TNFa with either protected thiol groups (rec-h-TNFa-ATA) or reactive thiol groups (rec-h-TNFa-AT) at day 5 after infection with 103 parasites. Doses ranged from 1-18 mg protein. *The average parasitaemia at day 8 after infection was determined in each control group and the parasitaemia of each individual mouse was divided by the average of the corresponding control group. The ratios were used for further analysis. Values represent averages + standard deviation of the combined data of different doses (1-18 mg protein). p<0.05 (ANOVA) as compared to non-treated mice, ::p<0.05 (ANOVA) as compared to mice developing CM in the same treatment group. rec-h-TNFa-AT (fig 4B). The summarised parasitaemia data (l-l8 mg protein) are shown in Table l. Parasitaemia was significantly (p

The effect of treatment on changes in plasma concentrations of soluble TNFR55 and TNFR75 (sTNFR55 and sTNFR75) was analysed in comparison with buffer-treated P. berghei infected mice. Since rec-h-TNFa-ATA exhibited a 8-Q fold lower in vitro bioactivity than non-modified rec-h-TNFa and rec-h-TNFa-AT, the effect of both treatment with comparable amounts of protein (Q mg) and bioactive rec-h-TNFa- ATA (8l mg protein) was analysed. At all time points concentrations of sTNFR55 in plasma were below the detection limit (<4O pg/ml). The plasma concentration of sTNFR75 at different time points after treatment is shown in Table 2. The plasma concentration of sTNFR75 remained below the detection limit in buffer- treated mice until day 6, and increased thereafter. Treatment with either rec-h- TNFa, rec-h-TNFa-ATA (Q mg protein) or rec-h-TNFa-AT significantly (p

Protein dose (|ig)

Parasitaemia at day 8 after infection / mean parasitaemia non-treated mice

Figure 4A and B: The effect of treatment with rec-h-TNFa-ATA or rec-h-TNFa-AT during infection on parasitaemia. Mice received a bolus i.v. injection of rec-h-TNFa-ATA (A; 1-18 /ig protein) or rec-h-TNFa-AT (B; 1-18 fig protein) at day 5 after infection with 103 P. berghei parasites. The average parasitaemia at day 8 after infection was determined in each control group and the parasitaemia of each individual mouse was divided by the average of the corresponding control group. The ratio is plotted against the protein dose for each mouse. When treatment has no effect on parasitaemia the ratio scatters around 1, while ratios < 1 indicate that parasitaemia is suppressed. Table 2: Effect of treatment with non-modified of rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa- AT on mouse soluble TNFR75 in plasma in P. berghei infected mice. Time after Time after Soluble mouse TNFR75 (ng/ml) infection (days) injection (h) Buffer rec-h-TNFa rec-h-TNFa-ATA rec-h-TNFa-ATA rec-h-TNFa-AT 9 iig protein 9 iig protein 81 iig protein8 9 iig protein 5 0 < 1.6 (n=3) 5 4 < 1.6 (n=3) 62* + 6 (n=4) 85* + 25 (n=7) 96*' + 4 (n=4) 84* + 17 (n=3) 6 24 < 1.6 (n=3) < 1.6 (n=4) < 1.6 (n=7) 136, 129' < 1.6 (n=3) 8 72 22 ± 10 (n=5) 38* + 8 (n=4) 36* + 11 (n=7) 33* + 7 (n=3) 9 96 28 + 7 (n=3) 45 + 23 (n=4) 52 + 18 (n=7) 103 + 28 (n=3) Mice received an intravenous injection of buffer, rec-h-TNFa, rec-h-TNFa-ATA, or rec-h-TNFa- AT at day 5 after infection with 103 parasites. At pre-defined time points after injection, mice were sacrificed and blood samples were taken. Plasma was stored at -20°C until analysis of soluble TNFR75. Values represent averages + standard deviation. s9 fxg bioactive protein in WEHI cytotoxicity assay *p < 0.05 as compared to buffer-treated mice p < 0.05as compared to treatment with rec-h-TNFa ::13 out of 15 mice died; values represent data of the surviving mice (n=2). DISCUSSION

The present study demonstrates that the introduction of reactive thiol groups in rec- h-TNFa by the SATA-reaction results in the formation of a covalently stabilised trimer (rec-h-TNFa-AT) with a significantly enhanced protective efficacy against P. berghei induced CM. Administration of thiolated rec-h-TNFa with protected thiol groups (rec-h-TNFa-ATA, no stabilised trimers in vitro) exhibited similar protective efficacy against CM, in spite of a Q-fold reduced in vitro bioactivity. SDS-PAGE analysis under non-reducing conditions confirmed that stabilised rec-h- TNFa trimers were formed upon thiolation of rec-h-TNFa (rec-h-TNFa-AT) with SATA (Fig. l). Stabilised trimers of thiolated rec-h-TNFa probably result from the formation of disulphide bridges between monomers already locked into a trimeric position. Monomeric rec-h-TNFa contains one terminal valine and six lysyl residues. It was shown before that up to l2 residues could be acetylated in the rec-h- TNFa trimer [34], indicating that three amino groups in the monomer were not available for modification e.g., because they participate in intra- and inter-subunit interactions [l6]. The total number of thiolated residues in rec-h-TNFa was not determined in the present study. The heterogeneity of bands on the SDS gel probably reflects differences in disulphide-bridging resulting in different molecular complexes [lO]. The absence of other products besides the trimer suggests that the trimer is the actual conformational state of rec-h-TNFa in solution and is not merely a cross-linking phenomenon of rec-h-TNFa monomers. Gel permeation chromatography (GPC) analysis of rec-h-TNFa under non-denaturing conditions revealed a single peak with a molecular weight of 42 kD, indicative of a dimer or compact trimer which is in accordance to literature [6, ll, 24]. Under denaturing conditions (SDS-PAGE) only the monomeric form of rec-h-TNFa was present, demonstrating that the rec-h-TNFa oligomer is held together by non-covalent interactions. The presence of bioactive oligomers and inactive monomers and polymers of hTNFa has also been demonstrated in vivo and may represent a “natural” mechanism in regulating cytokine activity [35]. Taken together the present data suggest that the enhanced therapeutic potential of rec-h-TNFa-AT results from an increased availability of bioactive rec-h-TNFa (i.e. rec-h-TNFa trimers). Chapter 8

Interestingly, administration of rec-h-TNFa-ATA showed the same protective efficacy against CM, while the majority of the protein was present as non-covalently bound oligomers (GPC data and Fig. 1), and in vitro bioactivity was reduced 8-9 fold as compared with rec-h-TNFa and rec-h-TNFa-AT (Fig. 2). The latter may be explained by perturbation of the tertiary structure of rec-h-TNFa interfering with receptor binding or some post-binding events [34]. De-acetylation of rec-h-TNFa- ATA to rec-h-TNFa-AT restored the in vitro bioactivity probably because a functional, bioactive trimer is formed again after fomation of disulphide-bridges between the monomers in the trimer. It is hypothesised that rec-h-TNFa-ATA is transformed into a bioactive compound in vivo. This hypothesis is supported by three observations. First, treatment with 80 mg rec-h-TNFa-ATA and rec-h-TNFa-AT, but not with rec-h-TNFa, is toxic in vivo whereas in vitro cytotoxicity of rec-h-TNFa-ATA was 8-9 fold lower as compared with rec-h-TNFa and rec-h-TNFa-AT. Secondly, the observation that the increase of sTNFR75 is stronger after treatment with rec-h-TNFa-ATA as compared with rec-h-TNFa (Table 2) is also in line with the above hypothesis. Thirdly, treatment with rec-h-TNFa-ATA, like rec-h-TNFa-AT, showed enhanced protection against CM as compared with rec-h-TNFa (Fig. 3). This leads to the assumption that rec-h- TNFa-ATA is de-acetylated in vivo to rec-h-TNFa-AT which may form stabilised trimers in vivo. The mechanism by which treatment with rec-h-TNFa, rec-h-TNFa-ATA or rec-h- TNFa-AT protected against the development of CM remains to be elucidated. Our data suggest that reduction of parasitaemia might be a factor that plays a role, particularly because the reduction was observed in CM-protected mice but not in treated mice developing CM (Table 1). Reduction of parasitaemia by TNFa has been described before in the same model (unpublished observations) and in other models [36, 37] and is probably mediated through rec-h-TNFa activated cells (e.g. macrophages and neutrophils) [36, 38]. Inhibition of erythropoiesis by rec-h-TNFa may also play a role in suppression of parasitaemia [39, 40]. It should be noted that almost all mice infected with a range of 10 to 10,000 parasites develop CM despite a widely varying parasitaemia after infection [31] and parasitaemia in controls varies extensively before they die of CM. These observations suggest that the course of an untreated infection is not a critical factor in development of CM in P. berghei

2 Thiolated rec-h-TNFa in malaria treatment infected mice. Thus, other, as yet undefined processes may be involved as well in the rec-h-TNFa-ATA or rec-h-TNFa-AT mediated protection against CM. Another possible factor in protection against CM could be the timely production of sufficient sTNFRs. STNFRs have been shown to competitively inhibit interaction of TNFa with membrane bound receptor [41] and transgenic mice expressing high levels of soluble TNFR55 were protected against CM [42]. On the other hand, an increase in sTNFR results in a decreased amount of receptors on the cell membrane, which could reduce the sensitivity of cells to TNFa. Previous observations have shown that TNFR75 knock out mice are protected against CM in a murine model [43]. Our data indicate that treatment with either rec-h-TNFa, rec-h-TNFa-ATA or rec-h-TNFa-AT induced the production of sTNFR75 4 h after injection, but this was not directly related to protection against CM (15% in rec-h-TNFa treated mice and 80% in the rec-h-TNFa-ATA and rec-h-TNFa-AT treated groups; Fig. 3 and Table 2). The enhanced concentrations of sTNFR75 24 h after injection of 81 mg rec-h- TNFa-ATA may reflect prolonged circulation times of bioactive protein, and may be a proof of a strong compensating response of the body to an overdose of bioactive rec-h-TNF a-ATA. No increase of sTNFR55 was observed in our studies, probably because peak levels were reached earlier in time (< 2 h after injection) and had returned to normal levels [44]. Interestingly, treatment with rec-h-TNFa induced the production of sTNFR75, whereas rec-h-TNFa binds only to mouse TNFR55 [45]. The induction of both sTNFRs after injection of rec-h-TNFa was observed before in non-infected mice, indicating that triggering of TNFR55 is sufficient to cause shedding of both TNFR [44]. The enhanced protection against CM by treatment with rec-h-TNFa-ATA and rec-h- TNFa-AT compared with rec-h-TNFa is not reflected by a difference in sTNFR75 levels at day 8 after infection (Fig. 3 and Table 2). Mice with non-treated infections develop CM despite increasing concentrations of sTNFRs [46]. The observation that protection against CM is not correlated with sTNFR-concentrations is in agreement with the observation that no bioactive mTNFa was present in the plasma of non­ treated infected mice and the inability to protect against CM by treatment with anti- TNF antibody in this model [46]. Interestingly, sTNFR75 plasma concentrations at day 9 after injection were strongly enhanced after injection of rec-h-TNFa-AT. The enhanced plasma concentrations of sTNFR75 were not related to an enhanced

3 Chapter 8 suppression of parasitaemia (Table 1), nor an enhanced protective efficacy against CM as compared with rec-h-TNFa-ATA (Fig. 3). However, parasitaemia was only determined at day 8 after infection, one day before elevated plasma concentrations of sTNFR75 were observed. The mechanism behind the increase in sTNFR75 and potential therapeutic implications remains to be elucidated. The mechanism(s) by which rec-h-TNFa-ATA and rec-h-TNFa-AT protect against CM is also not known, yet. Other processes involved may be the development of refractoriness or tolerance after treatment with rec-h-TNFa or stimuli that provoke TNFa release [47, 48, 49]. In summary, intravenous injection of covalently stabilised trimers of rec-h-TNFa strongly enhances the bioactivity of rec-h-TNFa in vivo. This is reflected in protection against CM at low doses (2-18 mg protein). Moreover, the data suggests that activation of protected thiol groups into reactive thiol groups by de-acetylation and formation of the bioactive configuration can take place in vivo. Finally, the effect on parasitaemia could play a role in the rec-h-TNFa mediated protection against CM, whereas sTNFRs are probably not involved.

ACKNOWLEDGEMENTS

We gratefully thank Dr. G.R. Adolf (Boehringer Ingelheim, Vienna, Austria) for the generous gift of rec-h-TNFa. We also thank Drs. A. Mekking and J. Smal (Octoplus, Leiden, The Netherlands) for performing the SDS-PAGE analysis. We thank Dr. W. Buurman (Department of General Surgery, University Limburg, The Netherlands) for supplying us with the materials used for the sTNF receptor ELISAs and Prof.dr. J. van de Meer (University Hospital Nijmegen, Nijmegen, The Netherlands) for the gift of rec-h-TNFa used as a standard in the WEHI cytotoxicity assay. We thank M.J. van Steenbergen (Department of Pharmaceutics, Utrecht University,The Netherlands) for performing the GPC-analysis and we thank G. Poelen, T. van de Ing and Y. Brom for their skilled biotechnical assistance.

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4 Thiolated rec-h-TNFa in malaria treatment

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22. ZHANG X-M, WEBER I, CHEN M-J. Site-directed mutational analysis of human tumour necrosis factor-a receptor binding site and structure-functional relationship. J Biol Chem 1992;267:24069-75. 23. CORTI A, FASSINA G, MARCUCCI F, BARBANTI E, CASSANI G. Oligomeric tumour necrosis factor a slowly converts into inactive forms at bioactive levels. Biochem J 1992;284:905-10. 24. PETERSEN CM, NYKLER A, CHRISTIANSEN BS, HEICKENDORFF L, MOGENSEN SC, M0LLER B. Bioactive human recombinant tumour necrosis factor-a: an unstable dimer? Eur J Immunol 1989;19:1887-94. 25. DUNCAN RJS, WESTON PD, WRIGGLESWORTH R. A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal Biochem 1983;132:68-73. 26. PENNICA D, NEDWIN GE, HAYFLICK JS, ET AL. Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 1984;312:724-9. 27. WESSEL D, FLUGGE UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analyt Biochem 1984; 138:141-3. 28. LAEMMLI UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5. 29. ESPEVIK T, NISSEN-MEYER J. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor / tumor necrosis factor from human monocytes. J Immunol Methods 1986;95:99-105. 30. SCUDIERO DA, SHOEMAKER RH, PAULL KD, ET AL. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumour cell lines. Cancer Res 1988;48:4827-33. 31. CURFS JHAJ, HERMSEN CC, MEUWISSEN JHETH, ELING WMC. Immunisation against cerebral pathology in Plasmodium berghei infected mice. Parasitology 1992;105:7-14. 32. POLDER TW, ELING WMC, CURFS JHAJ, JERUSALEM CR, WIJERS-ROUW M. Ultrastructural changes in the blood-brain barrier of mice infected with Plasmodium berghei. Acta Leiden 1992;60:31-46. 33. BEMELMANS MH, ABRAMOWICZ D, GOUMA DJ, ET AL. In vivo T cell activation by anti-CD3 monoclonal antibody induces soluble TNF receptor release in mice. Effects of pentoxifylline, methylprednisolone, anti-TNF and anti-IFN-gamma antibodies. J Immunol 1994;153:499-506. 34. UTSUMI T, HUNG M-C, KLOSTERGAARD J. The role of amino functions in recombinant human tumour necrosis factor in expression of biological activity. Mol Immunol 1992;29:77- 81. 35. M0LLER B, MOGENSEN SC, WENDELBOE P, BENDTZEN K, PETERSEN CM. Bioactive and inactive forms of tumour necrosis factor-a in spinal fluid from patients with meningitis. J Infect Dis 1991;163:886-9. 36. TAVERNE J, SHEIKH N, DE SOUZA JB, PLAYFAIR JHL, PROBERT L, KOLLIAS G. Anaemia and resistance to malaria in transgenic mice expressing human tumour necrosis factor. Immunology 1994;82: 397-403. 37. STEVENSON MM, GHADIRIAN E. Human recombinant tumour necrosis factor alpha protects susceptible A/J mice against lethal Plasmodium chabaudi AS infection. Infect Immun 1989;57:3936-9.

6 Thiolated rec-h-TNFa in malaria treatment

38. KUMARATILAKE LM, FERRANTE A, RZEPCZYK CM. Tumor Necrosis Factor Enhances Neutrophil-Mediated Killing of Plasmodium falciparum. Infect Immun 1990;58:788-93. 39. JOHNSON RA, WADDELOW TA, CARO J, OLIFF A, ROODMAN GD. Chronic exposure to tumour necrosis factor in vivo preferentially inhibits erythropoiesis in nude mice. Blood 1989;74:130-8. 40. MOLDAWER LL, MARANO MA, WEI H, ET AL. Cachectin/tumour necrosis factor-a alters red blood cell kinetics and induces anaemia in vivo. FASEB J 1989;3:1637-43. 41. LESSLAUER W, TABUCHI H, GENTZ R, ET AL. Recombinant soluble tumour necrosis factor receptor proteins protect mice from lipopolysaccharide-induced lethality. Eur J Immunol 1991;21:2883-6. 42. GARCIA I, MIYAZAKI Y, ARAKI K, ET AL. Transgenic mice expressing high levels of soluble TNF-R1 fusion protein are protected from lethal septic shock and cerebral malaria, and are highly sensitive to Listeria monocytogenes and Leishmania major infections. Eur J Immunol 1995;25:2401-7. 43. LUCAS R, LOU J-N, JUILLARD P, MOORE M, BLUETHMANN H, GRAU GE. Respective role of TNF receptors in the development of experimental cerebral malaria. J Immunol 1997;72:143-8. 44. BEMELMANS MHA, GOUMA DJ, BUURMAN WA. LPS-induced sTNF-receptor release in vivo in a murine model. J Immunol 1993;151:5554-62. 45. LEWIS M, TARTAGLIA LA, LEE GL, RICE GC, WONG GHW, CHEN EY. Cloning and expression of cDNAs for two distinct murine tumour necrosis factor receptors demonstrate one receptor is species specific. Proc Natl Acad Sci USA 1991;88:2830-4. 46. HERMSEN CC, CROMMERT VAN DE J, FREDERIX H, SAUERWEIN RW, ELING WMC. Circulating tumour necrosis factor a is not involved in the development of cerebral malaria in Plasmodium berghei-infected C57B1 mice. Parasite Immunol 1997;19:571-7. 47. TAKAHASHI N, BROUCKAERT P, AND FIERS W. Mechanism of tolerance to tumour necrosis factor: receptor-specific pathway and selectivity. Am J Physiol 1995;269:R398-405. 48. ALEXANDER HR, SHEPPARD BC, JENSEN JC, ET AL. Treatment with recombinant human tumour necrosis factor-alpha protects rats against the lethality, hypotension, and hypothermia of gram-negative sepsis. J Clin Invest 1991;88:34-9. 49. FRAKER DL, STOVROFF MC, MERINO MJ, NORTON JA. Tolerance to tumour necrosis factor in rats and the relationship to endotoxin tolerance and toxicity. J Exp Med 1988;168:95-105.

7 CHAPTER

SUMMARY AND DISCUSSION Chapter 9

SUMMARY AND DISCUSSION

We studied the role of subsets of T cells, reactive oxygen intermediates (ROI) and TNFa in the pathogenesis of ECM in our P. berghei K173-mouse model. Data from several murine models (reviewed in Chapter 1, table 1) support the hypothesis (Chapter 1, figure 1) that the infection generates a localised inflammatory reaction activated by the parasite, resulting in damage of the endothelial lining. This reaction is mediated by T cells and macrophages that adhere to the endothelial cells.

The involvement of T cell subsets and TNFa has been the subject of debate over the last years based on results in different rodent ECM models. Grau et al. (1986) described protection against ECM after depletion of CD4, but not CD8 T cells, while Imai et al. (1994) observed protection after depletion of CD8 T cells, but not CD4 T cells. With respect to the role of TNFa, it became clear that anti-TNFa treatment protected against death from ECM in the model of Grau et al. (1987). An important question was whether this was also true in our model and if so how TNFa contributed to the development of the haemorrhages. In view of the well documented cytolytic activity of TNFa the hypothesis was developed that excessive TNFa generates an excessive amount of ROI that damages cells. If this is the case, this might open new venues for prevention of ECM and therapy in end-stage disease. Our studies surprisingly showed that depletion of CD4 and/or CD8 T cells before or during the infection or in the end-stage of the disease protected against ECM (Chapter 2 and 3). The role of CD4 T cell subset in ECM was also noted in mice with acquired immunodeficiency syndrome (MAIDS): protection against ECM increased with the severity of MAIDS and the absence of functional CD4 cells (Eckwalanga et al. 1994). The finding that both depletion of CD4 and CD8 independently protected against ECM suggest that these T cells are involved in the stimulation of a downstream process in the development of the petechiae. Possible downstream mechanisms are excessive stimulation of endothelial receptor molecules or the release of (inflammatory) mediators from adherent (T cells, macrophages; Curfs et al. 1993) or trapped WBC activated by parasitized erythrocytes or their proteins.

144 Summary and discussion

To study the possibility of a beneficial intervention late in the course of the infection, we looked for a stage in ECM with apparent disturbances of the blood brain barrier (BBB), but still permitted recovery from fatal ECM. Earlier observations showed that folic acid can only cross the BBB when it is damaged (e.g. by heat coagulation of brain tissue); under these conditions, folic acid can induce severe seizures and even death. Folic acid was administered to test the integrity of the BBB in the course of ECM. Almost all infected mice with a body temperature "35.5 °C (defined as mice in end-stage disease) developed convulsions after folic acid treatment (Chapter 3). This suggested that changes in the permeability of the BBB were demonstrable within one day before the mice died with extensive haemorrhages. Next, it was shown that depletion of CD4 and/or CD8 T cells (Chapter 3) or treatment with alloxan (Chapter 5) of mice in end-stage disease protected mice against fatal ECM. In addition, within 4 hours after anti-T cell treatment mice did not develop convulsions after folic acid treatment. This supported the hypothesis that at first the permeability of the brain vascular endothelial lining was increased by a reversible process and soon thereafter was disrupted causing the haemorrhages and death of the mice. These data are compatible with the hypothesis that a local inflammatory reaction mediated by adherent T cells contribute to the development of the haemorrhages. TNFa and ROI are possible candidates as inflammatory mediators that can directly damage the endothelial lining. Administration of anti-TNFa antibodies in end-stage disease protected against ECM in the model of Grau et al. (1987). TNFa dependent cell lysis can be mediated by generation of excessive amounts of ROI and ROI can cause membrane pertubation, leading to cell injury with subsequent loss of endothelial function (Bjork et al. 1980; Varani et al. 1989; Ward & Varani 1990; Ward, 1991; Sulkowska & Sulkowski 1997). Thus, ROI dependent cell damage can be the result of TNFa dependent cell lysis. Antioxidants included in an in vitro test for TNFa dependent lysis of L929 cells prevent cell lysis (Chapter 4) and confirm the hypothesis that biologically active TNFa exerts its lytic activity through generation of ROI. However, treatment of mice with inhibitors, scavengers or inducers of ROI before or during infection as well as in end-stage disease does not give unequivocal answers with respect to the role of ROI in ECM (Chapter 4 and 5). Only the ROI scavengers dimethyl sulfoxide (DMSO) and ethanol, but not the antioxidants apocynin and desferal-zinc (DFO-Zn),

145 Chapter 9 can protect against ECM and this is only achieved when treatment is given before or during infection but not in end-stage disease. In addition, alloxan, a reported inducer of ROI, prevents rather than induces ECM when applied in end-stage disease. Of course, differences in pharmacokinetics of the antioxidants in vivo versus in vitro may explain discrepancies but it remains unclear why mice with increased SOD expression after transfection of the human SOD gene still develop ECM after infection. Antioxidant treatment was protective when started before or in the first part of the infection, but showed no effect in end-stage disease. Therefore, ROI are modulators of the immunopathological reactions leading to ECM rather than key effector molecules. This is supported by the observation that treatment with the ROI-inducer alloxan in end-stage disease protects against rather than induces ECM and that this protection can be abolished by additional antioxidant (apocynin and DFO) treatment. Alloxan treatment also killed the majority of the parasites within 2 hours. This might imply that alloxan depletes the antigen that otherwise stimulates adherent lymphocytes and macrophages on endothelial cells in a localised inflammatory reaction. On the other hand, antioxidant treatment in combination with alloxan reduced the protective capacity of alloxan treatment, but did not prevent killing of the parasites. This could argue for an effect on adherent lymphocytes or macrophages or an additional effect of alloxan next to ROI generation. The role of ROI in the generation of immunopathological reactions during development of ECM and/or in the protection against ECM in end-stage disease is complex and needs further analysis. High plasma levels of TNFa correlated with ECM as was observed by Grau and collaborators (1987) who showed that excessive levels of biologically active TNFa were present in their P. berghei-ANKA model and that treatment of severely ill mice with antibodies to TNFa protected against fatal ECM. Our studies (Chapter 6) demonstrated also increased TNFa plasma concentrations in P. berghei-infected mice with ECM, but treatment with antibodies to TNFa did not protect against ECM. In addition, plasma TNFa was not biologically active and associated with elevated levels of sTNFRs. Treatment with rec-h-TNFa was found to trigger and enhance ECM-like pathological changes (Curfs et a l. 1993). This suggests that circulating TNFa is not directly involved in the pathogenesis of ECM in our model, but does not exclude an indirect role for TNFa. Recent experiments in

146 Summary and discussion a P. berghei ANKA-mouse model showed protection against ECM in TNFR75 but not in TNFR55 knock-out mice and an association between ECM and endothelial cell upregulation of TNFR75 but not TNFR55 (Lucas et al. 1997). It also appears that membrane bound TNFa needs TNFR75 interaction for cell signalling and upregulation of ICAM-1, which in turn is a receptor for LFA1 on lymphocytes. Thus, membrane bound TNFa in a cell-to-cell contact rather than systemic TNFa in the presence of sTNFR may be the factor that contributes to endothelial damage. This supports the observations that membrane bound TNFa is superior over sTNFa in the activation of TNFR, particularly TNFR75 of endothelial cells (Grell et a l. 1995) and that TNFa dependent cytotoxicity mediated through interaction with TNFR55 is 1000-fold enhanced by co-stimulation of TNFR75 (Weiss et al. 1997). Therefore, further studies could explore the role of various types of TNFa (membrane bound vs. free; human vs. mouse; native vs. recombinant) and the differential activation of TNFR55 and TNFR75 by TNFa, free or membrane bound, in the pathogenesis of (E)CM. Earlier observations showed that high concentrations of rec-h-TNFa (15 zg/mouse) enhanced the ECM syndrome, whereas smaller doses (0.5-1 zg/mouse) can protect (Curfs et al. 1993). We could confirm these observations in more extensive studies (Chapter 7). Bolus injection of free or liposome encapsulated TNFa and even 10 times lower daily doses achieved through sustained release from mini-osmotic pumps were effective in the protection against ECM. Further studies with TNFa coupled to the outside of the liposomes showed better protection than free TNFa and this triggered the idea that chemical stabilisation of TNFa on the outside of liposomes mimicked to some extent the formation of the functionally active trimeric complex of TNFa (Zhang et al. 1992; Van Ostade et al. 1993). Therefore, the activity of thioliated rec-h-TNFa was analysed and found superior over rec-h-TNFa in the protection against ECM (Chapter 8). In addition, thioliation produced stable TNFa trimers. The data support the notion that a stable trimer in vivo is responsible for the biological activity of TNFa (Zhang et al. 1992; Van Ostade et al. 1994) and stable trimers can now be prepared to further study of the effects of TNFa. An important question is how TNFa treatment protects against ECM. Two observations from our experiments are relevant. The first observation is that TNFa treatment was successful when given before the animals were in end-stage disease (Chapter 7 and 8). Possibly TNFa treatment can trigger TNFa release from TNFaR

147 Chapter 9 bearing cells (e.g. macrophages and lymphocytes) that subsequently enter a refractory state (Patton et al. 1987; Fraker et al. 1988; Takahashi et al. 1991). If so, this may implicate that this action has to take place before the cells become adherent and critical amounts of TNFa are released locally, or before membrane-bound TNFa interacts with endothelial TNFaR and activation of the endothelial cell takes place and the addition of more TNFa is only more harmful. The second observation is that TNFa treatment can reduce parasitaemia in mice that later appear to be protected against ECM (Chapter 7 and 8). It is known that TNFa can suppress erythropoiesis (Roodman, 1987, Johnson et al. 1990) that may explain the reduction in parasitaemia. It should be noted, however, that the reduction in parasitaemia after TNFa treatment is limited and not as complete as observed after alloxan treatment (Chapter 5). The resulting parasitaemias after TNFa treatment remain within the range observed from individual variations within and between experiments. Moreover, induction of ECM is equally effective when 100 times less parasites are used for infection delaying parasitaemia by approximately 2 days (Curfs et al. 1992). May be not the parasitaemia as such, but other mechanisms changed by treatment with rec-h-TNFa during the ongoing infection may modulate the immunopathological process. Thus, in view of the multifaceted activity of TNFa the effect on parasitaemia may be associative and a marker that TNFa treatment was effective in another process that modulates ECM.

Many animal models (Chapter 1, table 1) are used to study the pathogenesis of ECM but none of these models reflect exactly the human situation. Nevertheless they can mirror certain aspects. There are a number of similarities comparing ECM in the P. berghei K173/C57Bl mouse model with human CM. Data from P. falciparum infected patients suggest that T cells and particularly the CD4 subset become sequestered in CM patients (Hviid et al. 1991; Elhassan et al. 1994). They re-emerge rapidly after anti-malaria therapy (Hviid et al. 1997). In the ECM mouse model depletion of CD4 and CD8 T cells turned out to be effective in protection against ECM (Chapter 2 and 3). Depletion is rapidly effective in protection against ECM suggesting depletion of adherent T cells. Post mortem histopathological analysis of patients that died of CM have shown margination of

148 Summary and discussion mononuclear cells in brain microvessels in addition to, or even in the absence of sequestered parasitized erythrocytes (Toro & Roman, 1978; Porta et al. 1993; Patnaik et al. 1994).

Malaria is by far the commonest cause of seizures in children in endemic areas (Waruiru et al. 1996). This may be due to an increased permeability of the BBB like we observed in mice developing ECM (Chapter 3). Enhanced capillary permeability to albumin is also observed in patients suffering from severe P. falciparum malaria (Areekul, 1988).

Increased TNFa plasma concentrations are observed both in human CM and in our ECM model. Children with CM do not recover from coma by treatment with neutralizing MAbs against TNFa; actually more complications are found in patients treated with anti-TNFa antibodies (Kwiatkowski et al. 1993; Hensbroek et al. 1996). This resembles our findings in anti-TNFa treated mice (Chapter 6). Mice with ECM and patients with CM have high serum levels of sTNFa receptors (ECM: Chapter 6, CM: Kern et al. 1992; Molineux et al. 1993; Kossodo et al. 1995), which are supposed to block the biological activity of the elevated serum TNFa (Mohler et al. 1993). Our studies show that plasma from mice in end-stage disease is still able to neutralize bioactive rec-m-TNFa suggesting that not all TNFa-receptors are complexed with TNFa. The above described similarities suggests that the P. berghei/C57Bl mouse model is valuable for the analysis of the role of TNFa and T cells and the increased permeability of the BBB and results can be used for comparison with human CM. In conclusion (figure 1): Hemorrhages in the brain are the result of loss of the integrity of the microvascular endothelial lining possibly as a result of excessive activation of signal transduction. The damaging mechanisms, which are still unknown, are probably triggered by activation of the TNFa receptor P75 and by ICAM both induced and/or upregulated on activated endothelial cells. This activation may be the result of binding of activated T cells (CD4 or CD8) and/or white blood cells (WBC) through their ligands, e.g. membrane bound TNFa and/or LFA1. Adhered cells can also release other substances (see ?) like reactive nitrogen intermediates which could be deleterious.

149 Chapter 9

Upregulation of the expression of ICAM (see +) is due to activation of TNFa receptors on the endothelial cell by TNFa (soluble or membrane bound). T cells, granulocytes and macrophages are activated by parasites or their products or by circulating proinflammatoir cytokines during infection. Not only T cell depletion/inactivation but depletion/inactivation of all white blood cells (WBC) in mice in end stage ECM can be considered for therapeutic intervention.

Figure 1:

150 Summary and discussion

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AREEKUL, S. (1988) Transcapillary escape rate and capillary permeability to albumin in patients with Plasmodium falciparum. Ann. Trop. Med. Parasitol. 82, 135-140. BERENDT, A.R. (1993) Sequestration and its discontents: infected erythrocyte-endothelial cell interactions in Plasmodium falciparum malaria. Res. Immunol. 144, 740-5; discussion 754-62. BEAERT, R., FIERS, W. (1994) Molecular mechanisms of tumor necrosis factor-induced cytotoxicity. What we do understand and what we do not. FEBS Lett. 340, 9-16. BJORK, J., DELMAESTRO, R.F., ARFORS, K.E. (1980) Evidence for participation of hydroxyl radical in increased microvascular permeability. Agents Actions Suppl. 7, 208-13. CRAIG, A.G. & BERENDT, A.R. (1991) The role of ICAM-1 as a receptor for rhinovirus and malaria. Chem. Immunol. 50, 116-34. CURFS, J.H.A.J., HERMSEN, C.C., MEUWISSEN, J.H.E.Th., ELING, W.M.C. (1992) Immunisation against cerebral pathology in Plasmodium berghei infected mice. Parasitology 105, 7-14. CURFS, J.H.A.J., HERMSEN, C.C., KREMSNER, P., NEIFER, S., MEUWISSEN, J.H.E.Th., Van ROOIJEN, N., ELING, W.M.C. (1993) Tumor necrosis factor-a and macrophages in Plasmodium berghei-induced cerebral malaria. Parasitology 107, 125-34. ELHASSAN, I.M., HVIID, L., SATTI, G., AKERSTROM, B., JAKOBSEN, P.H., JENSEN, J.B., THEANDER, T.G. (1994) Evidence of endothelial inflammation, T cell activation, and T cell reallocation in uncomplicated Plasmodium falciparum malaria. Am. J. Trop. Med. Hyg. 51, 372-379. ECKWALANGA, M„ MARUSSIG, M., TAVARES, M.D., BOUANGA, J.C., HULIER, E., PAVLOVITCH, J.H., MINOPRIO, P., PORTNOI, D., RENIA, L., MAZIER, D. (1994) Murine AIDS protects mice against experimental cerebral malaria; down regulation by interleukin 10 of a T- helper type 1 CD4 cell-mediated pathology. Proc. Nat. Acad. Sci. USA 91, 8097-101. FALANGA, P.B., BUTCHER, E.C. (1991) Late treatment with anti-LFA-1 (CDlla) antibody prevents cerebral malaria in a mouse model. Eur. J. Immunol. 21, 2259-63. FRAKER, D.L., STOVROFF, M.C., MERINO, M.J., NORTON, J.A. (1988) Tolerance to tumour necrosis factor in rats and the relationship to endotoxin tolerance and toxicity. J. Exp. Med. 168, 95-105. GRAU, G.E., FAJARDO, L.E., PIGUET, P.E., ALLERT, B., LAMBERT, P.H., VASSALLI, P. (1987) Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 237, 1210-12. GRAU, G.E., POINTAIRE, P., PIGUET, P.F., VESIN, C., ROSEN, H., STAMENKOVIC, I., TAKEI, F., VASSALLI, P. (1991) Late administration of monoclonal antibody to leukocyte function-antigen 1 abrogates incipient murine cerebral malaria. Eur. J. Immunol. 21, 2265-2267. GRELL, M., DOUNI, E., WAJANT, H.,LOHDEN, M., CLAUSS, M., MAXEINER, B., GEORGOPOULOS, S., LESSLAUER, W., KOLLIAS, G., PFIZENMAIER, K„ SCEURICH, P. (1995) The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80kDa tumor necrosis factor receptor. Cell 83, 793-802. HENSBROEK, VAN M.B., PALMER, A., ONYIRAH, E., SCHNEIDER, G., JAFFAR, J., DOLAN, D., MEMMING, H., FRENKEL, J., ENWERE, G., BENNETT, S., KWIATKOWSKI, D., GREENWOOD, B. (1996) The effect of a monoclonal antibody to tumor necrosis factor and survival from childhood cerebral malaria. J. Infect. Dis. 174, 1091-7.

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HVIDD, L., THEANDER, T.G., ABDULHADI, N.H., ABU-ZEID, Y.A., BAYOUMI, R.A., JENSEN, J.B. (1991) Transient depletion of T cells with high LFA-1 expression from peripheral circulation during acute Plasmodium falciparum malaria. Europ. J. Immunol. 21, 1249-1253. HVIID, L., KURTZHALS, J.A., GOKA, B.Q., OLIVER-COMMEY, J.O., NKRUMAH, F.K., THEANDER, T.G. (1997) Rapid reemergence of T cells into peripheral circulation following treatment of severe and uncomplicated Plasmodium falciparum malaria. Infect. Immun. 65, 4090-93 JOHNSON, C.S., COOK, C.A., FURMANSKI, P. (1990) In vivo suppression of erythropoiesis by tumor necrosis factor-a (TNFa): reversal with exogenous erythropoietin (EPO). Exp. Hematol. 18, 109-13. KERN, P., HEMMER, C.J., GALLATI, H., NEIFER, S., KREMSNER, P., DIETRICH, M., PORZSOLT, F. (1992) Soluble tumour necrosis factor receptors correlate with parasitemia and disaese severity in human malaria. J. Infect. Dis. 166, 930-4. KOSSODE, DE S., HOUBA, V., GRAU, G.E. (1995) Assaying tumour necrosis factor concentrations in human serum. A WHO international colaborative study. J. Immunol. Meth. 182, 107-14. KWIATKOW SKI, D., MOLYNEUX, M., STEPHENS, S., CURTIS, N., KLEIN, N., POINTAIRE, P., SMIT, A.M., BREWSTER, D., GRAU, G.E., GREENWOOD, B.M. (1993) Anti-TNF therapy inhibits fever in cerebral malaria. Quart. J. Med. 86, 91-98. LUCAS, R., LOU, J.N., JUILLARD, P., MOORE, M., BLUETHMANN, H., GRAU, G.E. (1997) Respective role of TNF receptors in the development of experimental cerebral malaria. J. Immunol. 72, 143-8. MIGOT. F., OUEDRAOGO, J.B., DIALLO, J., ZAMPAN, H„ DUBOIS, B., SCOTT- FINNIGAN, T., SANOU, P.T., DELORON, P. (1996) Selected P. falciparum specific immune responses are maintained in AIDS adults in Burkina Faso. Parasite Immunol. 18, 333-9. MOHLER, K.M., TORRANCE, D.S., SMITH, C.A., GOODWIN, R.G., STREMLER, K.E., FUNG, V.P., MADANI, H., WIDMER, M.B. (1993) Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and faction simustaneously as both TNF carriers and TNF antagonists. J. Immunol. 151, 1548-61. MOLYNEUX, M.E., ENGELMANN, H., TAYLOR, T.E., WIRIMA, J.J., ADERKA, D., WALLACH, D., GRAU, G.E. (1993) Circulating plasma receptors for tumour necrosis factor in Malawian children with severe falciparum malaria. Cytokine 5, 604-9. PATNAIK, J.K., DAS, B.S., MISHRA, S.K., MOHANTY, S., SATPATHY, S.K., MOHANTHY, D. (1994). Vascular clogging, mononuclear cell migration and enhanced vascular permeability in the pathogenesis of human cerebral malaria. Am. J. Trop. Med. Hyg. 51, 642-7. PATTON, J.S., PETERS, P.M., McCABE, J., CRASE, D., HANSEN, S., CHEN, A.B., LIGITT, D. (1987) Development of partical tolerance to the gastrointestinal effects of high doses of recombinant tumour necrosis factora in rodents. J. Clin. Invest. 80, 1587-96. PORTA, J., CAROTA, A., PIZZOLATO, G.P., WILDI E., WIDMER, M.C., MARGAIRAZ, C., GRAU, G.E. (1993) Immunopathological changes in human cerebral malaria. Clin. Neuropathol. 12, 142-146. ROBAYE, B., MOSSELMANS, R., FIERS, W., DUMONT, J.E., GALAND, P. (1991) Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro. Am. J. Pathol. 138, 447-53.

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ROODMAN, G.D. (1987) Mechanisms of erythroid suppression in the anemia of chronic disease. Blood Cells 13, 171-84. SULKOWSKA, M., & SULKOWSKI, S. (1997) The effect of pentoxifylline on ultrastuctural picture of type ii alveolar epithelial cells and generation of reactive oxygen species during cyclophosphamide-induced lung injury. J. Submicrosc. Cytol. Pathol. 29, 487-96. TAKAHASHI, N., BROUCKART, P., FIERS, W. (1991) Induction of tolerance allows separation of lethal end antitumour activities of tumour factor in mice. Cancer Res. 51, 2366-72. TORO, G., ROMAN, G. (1978) Cerebral malaria, a disseminated vasculomyelinopathy. Arch. Neurology. 35, 271-275. TURNER, G., MORRISO, H„ JONES, M„ DAVIS, T., LOOAREESUWAN, S., BULEY, I., GATTER, K., NEWBOLD, C., PYKRITAYAKAMEE, S., NAGACHINTA, B., WHITE, N., BERENDT, A. (1994) An immunohistochemical study of the pathology of fatal malaria. Am. J. Pathol. 145, 1057-69. VAN OSTADE, X., TAVENIER, J., FIERS, W. (1994) Structure-activity studies of human tumour necrosis factors. Protein Eng. 7, 5-22. VARANI, J., GINSBURG, I., SCHUGER, L„ GIBBS, D.F., BROMBERG, J., JOHNSON, K.J., RYAN, U.S., WARD, P.A. (1989) Endothelial cell killing by neutriphils. Am. J. Pathol. 135, 435-38. WARD, P.A. & VARANI, J. (1990) Mechanisms of neutrophil-mediated killing of endothelial cells. J. Leukocyte Biol. 48, 97-102. WARD, P.A. (1991) Mechanisms of endothelial cell injury. J. Lab. Clin. Med. 118, 421-6. WARUIRU, C.M., NEWTON, C.R.J.C., FORSTER, D., NEW, L., WINSTANLEY, P., MWANGI, I., MARSH, V., WINSTANLEY, M., SNOW, R.W., MARSH, K. (1996) Epileptic seizures and malaria in Kenyan children. Trans. R. Soc. Trop. Med. Hyg. 90, 152-5. WEISS, T„ GRELL, M., HESSABI, B„ BOURTEELE, S., MULLER, G.,SCHEURICH, P., WAJANT, H. (1997) Enhancement of TNF receptor p60-mediated cytotoxicity by TNF receptor p80: Requirement of the TNF receptor-associated factor-2 binding site. J. Immunol. 158, 2398­ 2404. ZANG, X.M ., WEBER, I., CHEN, M.J. (1992) Site-directed mutational analysis of human tumour necrosis factor-a receptor bindidng site and stucture-functional relationship. J. Biol. Chem. 267, 24069-75.

153 CHAPTER 10

SAMENVATTING Chapter 10

INLEIDING

Malaria tropica is nog altijd een van de belangrijkste oorzaken van ziekte en sterfte in de tropen. Malaria wordt veroorzaakt door een eencellige parasiet van het geslacht Plasmodium. Er zijn 4 typen Plasmodia die de mens kunnen besmetten, waarvan er drie min of meer goedaardig zijn en zelden leiden tot levensbedreigende situaties. Dit in tegenstelling tot Plasmodium falciparum, de veroorzaker van malaria tropica, die vele dodelijke slachtoffers eist. Jaarlijks maken tussen de 250 en 500 miljoen mensen een aanval van malaria door van wie tussen de 1 en 3 miljoen de infectie niet overleven. Malaria is verantwoordelijk voor een kwart van alle sterftegevallen onder Afrikaanse kinderen tussen 1 en 4 jaar. Oudere kinderen en volwassenen in Afrika krijgen nog wel klinische verschijnselen van malaria maar hebben over het algemeen genoeg afweer opgebouwd om niet ernstig ziek te worden. Wel heeft de malaria bij hen invloed op hun gezondheid, ontwikkeling en arbeidscapaciteit. Het merendeel van de Afrikaanse kinderen krijgt gemiddeld eenmaal per jaar een malaria aanval. Deze presenteert zich meestal als een griepachtig beeld met koorts. Na het starten van een effectieve antimalariabehandeling treedt meestal snel herstel op. Een kleine groep (1-3%) ontwikkelt echter ernstige malaria, hetgeen gepaard kan gaan met convulsies (stuipen), ernstige bloedarmoede en hersenmalaria (met een diep coma). Hersenmalaria (cerebrale malaria) heeft een sterftekans die tussen de 10 en 40% ligt. De oorzaak van cerebrale malaria is onbekend maar er bestaan een aantal hypotheses.

Voor de bestudering van cerebrale malaria zijn een aantal diermodellen beschikbaar (hoofdstuk 1, tabel 1). In hoofdstuk 1 wordt uitgelegd waarom de muizenstam C57Bl/6J en de knaagdier malariaparasiet Plasmodium berghei K173 gekozen zijn. In dit model ontwikkelen ongeveer 95% van de geïnfecteerde dieren experimentele cerebrale malaria (ECM). Een van de hypotheses stelt dat ECM ontstaat, doordat in de loop van de infectie, de malaria parasieten die in het vaatsysteem leven en zich vermenigvuldigen, een ontstekingsreactie induceren waarbij geactiveerde mononucleaire cellen aanhechten aan de bekleding van de vaatwand (endotheel) o.a. in de hersenen. Als gevolg hiervan ontstaan er ter plaatse ontstekingsreacties waardoor schade ontstaat aan het endotheel en ten slotte bloedingen in de hersenen. Uit eerder wetenschappelijk onderzoek van onze groep en anderen was gebleken dat

156 Samenvatting verwijdering van T cellen bij malaria geïnfecteerde muizen de ontwikkeling van ECM voorkomt. Een van de vragen is nu of de T cellen direct verantwoordelijk zijn voor de schade aan de vaatwand en zo ja welke subset van deze T cellen en/of zijn het de door deze cellen geproduceerde bioactieve stoffen die bijdragen aan de ontwikkeling van ECM. In dit onderzoek is gekeken naar de rol van specifieke T cellen, reactieve zuurstofradicalen en tumor necrosis factor . (TNFa) in de ontwikkeling van ECM.

T cellen Resultaten van onderzoekers die de rol van T cel subsets (CD4 en CD8) bij ECM hebben onderzocht zijn niet eensluidend. Om de rol van specifieke T cellen tijdens de pathogenese van ECM in ons model te bestuderen zijn geïnfecteerde muizen behandeld met anti-CD4 of anti-CD8 antistoffen. Deze behandeling is er op gericht om de betreffende T cel populatie te verwijderen (hoofdstuk 2). Beide behandelingen gaven bescherming tegen ECM. Dit suggereert dat deze T cellen onafhankelijk van elkaar betrokken zijn bij de stimulatie van ECM. Mogelijk zijn de T cellen betrokken bij verhoogde stimulatie van endotheel-receptormoleculen en/of de produktie en vrijmaking van (ontstekings)mediatoren van aangehechte cellen of vastgelopen witte bloedcellen die geactiveerd worden door geparasiteerde erytrocyten of hun produkten. Intregriteit van de bloed-hersen-barrière is een belangrijke functie van de stabiliteit van hersenfuncties. Convulsies na behandeling met foliumzuur zijn een aanwijzing voor een gestoorde bloed-hersen-barrière. Alleen muizen in het eindstadium van ECM (met een lichaamstemperatuur < 35.5°C) reageerden met convulsies na foliumzuur, hetgeen de aantasting van de bloed-hersen-barrière illustreert (hoofdstuk 3). Behandeling van muizen met een lichaamstemperatuur lager dan 35.5°C met anti-CD4 of anti-CD8 antistoffen, gaf bescherming tegen ECM. Binnen 4 uur na de behandeling met deze anti-T cel sera vertoonden de muizen geen convulsies meer na toediening van foliumzuur, hetgeen een aanwijzing is dat de bloed-hersen-barrière zich heeft hersteld. Deze resultaten ondersteunen de hypothese dat bij de ontwikkeling van ECM als eerste de permeabiliteit van de bloed-hersen-barrière is verhoogd, wat aanvankelijk reversibel is, maar snel verder ontwikkelt tot bloedingen die sterfte van de muizen veroorzaakt.

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Reactieve zuurstofradicalen Geactiveerde neutrophielen en macrophagen die hechten aan het oppervlak van de hersenvaten, zoals in het geval van ECM al eerder was waargenomen, kunnen diverse bioactieve stoffen uitscheiden. Een groep van deze stoffen, de reactieve zuurstofradicalen, staan bekend om hun schadelijke werking op endotheelcellen. Deze stoffen kunnen membraanschade veroorzaken, dat kan leiden tot celschade en vervolgens tot een verlies van de endotheelfunctie. In hoofdstuk 4 is geanalyseerd of antioxidantia een rol kunnen spelen in de bescherming tegen ECM. Omdat TNFa cellen kan doden en mogelijk betrokken is bij de ontwikkeling van ECM werd via in vitro proeven aangetoond, dat lysis van L929 cellen, die veroorzaakt wordt door TNFa, verhindert wordt in aanwezigheid van een aantal antioxidantia: apocynine, desferal-Zn, dimethyl sulfoxide (DMSO) en ethanol. Hiermee wordt bevestigd dat TNFa zijn lytische activiteit kan ontlenen aan het generen van reactieve zuurtofradicalen. Vervolgens werden muizen die na infectie ECM ontwikkelen met de antioxidantia behandeld, maar de resultaten waren niet eenduidig. Alleen behandeling met DMSO of ethanol en dan alleen indien die behandeling voor de infectie was gestart, beschermde tegen ECM. Behandeling met apocynine of desferal-Zn voor of tijdens infectie, of bij muizen in het eindstadium van ECM beschermde niet. Omdat de antioxidant behandeling alleen profylactisch beschermd, bestaat de indruk dat reactieve zuurstofradicalen eerder modulatoren zijn van de immunopathologische reactie dan essentiële effector moleculen die leiden tot celschade bij ECM. In een andere serie experimenten werd gevonden dat muizen die een verhoogde productie van het enzym superoxide dismutase hebben, door de extra aanwezigheid van het getranfecteerde humane superoxide dismutase gen, desalniettemin niet tegen ECM beschermd waren. Uit de resultaten beschreven in hoofdstuk 4 komt dus geen eenduidige rol voor reactieve zuurstofradicalen in de pathogenese van ECM. De resultaten beschreven in hoofdstuk 5 bevestigen dit verder. In dit hoofdstuk is gekeken naar het effect van behandeling met alloxan, een inducer van reactieve zuurstofradicalen. Behandeling in het eindstadium, kort voor de muizen overlijden aan ECM, beschermde juist in plaats dat het de ontwikkeling van ECM versnelde. Bovendien werd deze beschermende werking van alloxan opgeheven door voorafgaande

158 Samenvatting behandeling met de antioxidantia apocynine en desferal. Alloxan behandeling doodt binnen 2 uur een groot gedeelte van de aanwezige parasieten. Hierdoor kan misschien de rol van de parasieten in een ontstekingreactie, waaraan lymphocyten en/of macrophagen deelnemen die zich gehecht hebben aan de vaatwand, beperkt worden. Een direct effect van alloxan op de activiteit van de ontstekingscellen behoort ook tot de mogelijkheden. Een met alloxan vergelijkbaar effect werd niet gevonden na behandeling met artemisinine, dat zijn werking dankt aan de aanwezigheid van een peroxide brug, maar als antiparasitair middel veel langzamer en mogelijk te langzaam werkt. De rol van antioxidantia in de ontwikkeling van ECM en/of in de bescherming tegen ECM is complex en moet nog verder onderzocht worden.

Tumor necrosis factora (TNFa) Uit eerder onderzoek is gebleken dat de ontwikkeling van ECM geassocieerd is met een verhoogde concentratie TNFa in het plasma van muizen. Grau en onderzoekers (1987) konden muizen beschermen tegen ECM door antistoffen in te spuiten die de werking van TNFa neutraliseerden (hoofdstuk 1, figuur 1). In een vervolg onderzoek is dit ook toegepast bij kinderen die cerebrale malaria hadden, maar met teleurstellende resultaten. Behandeling met anti-TNFa antistoffen kon bij deze kinderen de cerebrale malaria niet opheffen. Vanwege het verschil in resultaten van de behandeling met anti-TNFa antistoffen bij muizen en mensen is de rol van TNFa in de pathogenese van ECM in ons model geanalyseerd. Onze resultaten toonden verhoogde concentraties van immunologisch aantoonbaar TNFa in het plasma van P. berghei-geïnfecteerde muizen met ECM, maar behandeling met antistoffen werkte niet beschermend (hoofdstuk 6). Uit verder onderzoek bleek dat het plasma TNFa niet biologisch actief was en associeerde met verhoogde concentraties circulerende TNF-receptoren. In vervolg experimenten werd aangetoond dat plasma van muizen, die in het eindstadium van de ziekte zitten, zelfs toegevoegd rec-m-TNFa kon neutraliseren. Deze resultaten geven aan dat in de pathogenese van ECM in ons model circulerend TNFa niet direct verantwoordelijk is voor de endotheelschade en het ontstaan van de bloedingen maar sluiten niet uit dat er een indirecte rol zou kunnen zijn via membraan-gebonden TNFa in cel-cel interacties.

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In hoofdstuk 7 is de rol van TNFa in de ontwikkeling van ECM verder onderzocht door geinfecteerde muizen te behandelen met rec-h-TNFa. Eerder onderzoek liet zien dat behandeling met hogere doseringen rec-h-TNFa het ECM beeld versneld en systematisch opriep terwijl preliminaire resultaten van een voorstudie aangaven dat lage doses rec-h-TNFa de ontwikkeling van ECM juist kon voorkomen. Resultaten van onze studies lieten zien dat dit ECM beschermende effect van rec-h-TNFa verkregen kon worden via een bolusinjectie van vrij rec-h-TNFa of van rec-h-TNFa ingevangen in liposomen. Door TNFa toe te dienen via een mini-osmotische pomp (continue uitscheiding) kon de beschermende werking ook met een 10x lagere dosering nog verkregen worden. Betere resultaten werden verkregen met rec-h- TNFa dat chemisch aan het oppervlak van liposomen was gekoppeld (Postma et al. 1998). Dit suggereerde dat door chemische stabilisering het rec-h-TNFa op het oppervlak van liposomen meer gelijkenis heeft met het actieve trimeer-complex dat TNFa onder natuurlijke omstandigheden in oplossing vormt. In hoofdstuk 8 wordt beschreven dat door chemische modificatie van de monomeer vorm van rec-h-TNFa een stabiel trimeer gevormd kan worden. Behandeling met de trimeer vorm geeft een betere bescherming tegen ECM dan behandeling met de monomeer vorm. Behandeling met zowel de monomeer als de trimeer vorm van rec- h-TNFa verhoogde de plasma concentratie van de TNF receptoren, maar dit was niet gecorreleerd met bescherming tegen ECM. Mogelijk dat de trimeer vorm en de monomeer vorm in mindere mate een interactie aangaat met TNFa receptoren op cellen, die dan vervolgens een refractaire fase doormaken voor verder stimulering of voor hernieuwde afgifte van TNFa en daardoor niet meer deelnemen aan de ontwikkeling van ECM. Vervolgonderzoek is nodig om deze hypothese uit te werken, dan wel andere processen op te sporen die door de behandeling met rec-h-TNFa de pathogenese van ECM moduleren.

Tot slot De waarschijnlijke stand van zaken na de voltooiing van de in dit proefschrift beschreven experimenten is dat de beschadigingen aan het endotheel van het vaatsysteem in de hersenen verloopt via de (overmatige) activatie/opregulatie van de TNF receptoren en van ICAM op het oppervlak van deze cellen (hoofdstuk 9, figuur

160 Samenvatting

1). Deze activatie kan tot stand komen door interactie met en hechting van geactiveerde T (CD4 en CD8) cellen, monocyten en macrophagen via hun liganden (LFA1, MAC1, membraangebonden TNFa, etc.), waarbij ook stoffen vrijgemaakt kunnen worden die celschade veroorzaken. De aan het endotheel gehechte cellen kunnen de doorstroming belemmeren en ter plaatse een interaktie aangaan met aanwezige parasieten, waardoor pro-inflammatoire cytokinen kunnen worden vrijgemaakt en het ter plaatse tot een ontstekingsreaktie kan komen, die het vaatwandendotheel kan beschadigen.

REFERENTIES

GRAU, G.E., FAJARDO, L.E., PIGUET, P.E., ALLERT, B., LAMBERT, P.H., VASSALLI, P. (1987) Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 237, 1210-12. POSTMA, N .S., CROMMELIN, D.J.A., ELING, W .M .C., ZUIDEMA, J. (1998) Treatment with recombinant tumour necrosis factor-a coupled to liposomes suppresses parasitaemia and protects against P. berghei K173 induced cerebral malaria in mice. Disseration. Drug delivery approches in malaria treatment. ISBN 90-393-1895-6. Chapter 7.

161 List of publications

LIST OF PUBLICATIONS

1. ZON, A.A.J.C. VAN, W.M.C. ELING, C.C. HERMSEN, A.A.G.M. KOEK­ KOEK. (1982) Corticosterone regulation of the effector function of malaria immunity during pregnancy. Infect. Immun.36,484-491

2. ZON, A.A.J.C. VAN, W.M.C. ELING, C.C. HERMSEN. (1983) Enhanced virulence of malaria infection in pregnant mice. I.C.R.S. Med. Sci. 11,1002-1003.

3. ZON, A.A.J.C. VAN, W.M.C. ELING, C.C. HERMSEN, TH.J.J.M.VAN DE WIEL, M.E. DUIVES. (1983) Malarial immunity in pregnant mice in relation tot total and unbound plasma corticosterone. Bull. Soc. Pathol. Exot. Filiales 76,493­ 502.

4. ELING, W.M.C., C.R. JERUSALEM, C.C. HERMSEN, U. HEINE-BORRIES, M.L. WEISS, C.H.J. VAN RUN - VAN BREDA. (1983) Endomyocerdial lesion and endomyocerdial fibrosis in experimental malaria (Plasmodium berghei) in mice. Contrib. Microbiol. Immunol. 7, 218-229.

5. ZON, A.A.J.C. VAN, W.M.C. ELING, T.P.M. SCHETTERS, C.C. HERMSEN. (1985) Pregnancy induced recrudescences strenghten malarial immunity in mice infected with P. berghei. Parasitology 91, 9-17.

6. ZON A.A.J.C., W.M.C. ELING, T.P.M. SCHETTERS, C.C.HERMSEN (1985) ACTH-dependent modulation of malaria immunity in mice. Parasite Immunol. 7, 107-117.

7. SCHETTERS, T., C. HERMSEN, J. CURFS, A. VAN ZON, T. VAN DER WIEL, P. LIEM, W. ELING (1988) Parasiet-Gastheer relaties. Vakbl. Biol. 68, 158-159.

8. SCHETTERS, T., C. HERMSEN, A. VAN ZON, W. ELING. (1988) Stage specific proteins of Plasmodium berghei infected red blood cells detected by antibodies on immune mouse serum. Parasit. Res. 75, 69-72.

9. ELING, W., C. JERUSALEM, U. HEINEN-BORRIES, C. HERMSEN, J.VAN RUN VAN BREDA. (1988) Is malaria involved in the pathogenesis of endomyocardial fibrosis. Acta Leiden. 57, 41-46.

10.CURFS, J., T. SCHETTERS, C. HERMSEN, C. JERUSALEM, A. VAN ZON, W. ELING. (1989) Immunological aspects of cerebral lesions in murine malaria. Clin. Exp. Immunol. 75, 136-140.

163 List of publications

ll.SCHETTERS, T., C. HERMSEN, A. VAN ZON, W. ELING. (1989) Protective and pathological activity in serum of mice developing resistance to Plasmodium berghei infection. Parasite Immunol. 11, 413-423.

12.SCHETTERS, T., J. VAN RUN VAN BREDA, C. HERMSEN, J. CURFS, W. ELING. (1989) Impaired immune reactivity in Plasmodium berghei immune mice. Parasite Immunol. 11, 519-528.

13.SCHETTERS, T., J. CURFS, A. VAN ZON, C. HERMSEN, W. ELING. (1989) Cerebral lesions in mice infected with Plasmodium berghei are the result of an immunopathological reaction. Trans. R. Soc. Trop. Med. Hyg. 83(S), 103-104.

14.ELING, W.M.C., C. CELLUZZI, C.C. HERMSEN, TH VAN DE WIEL, J. CURFS, P. LIEM. (1991) The need for live parasites for long-term immunity in malaria. Acta Leiden. 60, 167-175.

15.CURFS J., C.C. HERMSEN, J.H.E.TH MEUWISSEN, W.M.C. ELING. (1992) Immunisation against cerebral malaria in Plasmodium berghei infected mice. Parasitology 105, 7-14.

16.CURFS, J.A.H.J., C.C. HERMSEN, P. KREMSNER, S. NEIFER, J.H.E.TH. MEUWISSEN, N. VAN ROOYEN, W.M.C. ELING. (1993) Tumour necrosis factora and macrophages in Plasmodium berghei-induced cerebral malaria. Parasitology 107,125-134.

17.VOGELS, M.T.E., C.C. HERMSEN, H.L.P.G. HUYS, W.M.C. ELING, J.W.M. VAN DER MEER. (1994) Roles of tumour necrosis factor a, granulocyte- macrophage colony-stimulating factor, platelet-activating factor, and arachidonic acid metabolites in interleukin- 1-induced resistance to infection in neutropenic mice. Infect. Immun. 62,2065-2070.

18.SMEIJSTERS, L.J.J.W., NIEUWENHUIS H., HERMSEN R.C., DORRESTEIN G.M., FRANSSEN F.F.J., OVERDULVE J.P. (1996) Antimalarial and toxic effects of the acyclic nucleoside phosphonate (S)-9-(3Hydroxy-2- phosphonylmethoxypropyl) adenine in Plasmodium berghei infected mice. Antimicob. Agents Chemother. 40,1584-1588.

19.HERMSEN, C., T. VAN DE WIEL, E. MOMMERS, R. SAUERWEIN, W. ELING. (1997) Depletion of CD4+ or CD8+ T-cells prevents P. berghei induced cerebral malaria. Parasitology 114, 7-12.

164 List of publications

20.HAZRA, S. MOENIRALAM, WILLEM A. BEMELMAN, JOHANNES A. ROMIJN, ERIK ENDERT, MARIETTE T. ACKERMANS, J, JAN B. VAN LANSCHOT, ROB C. HERMSEN, HANS P. SAUERWEIN. (1997) Origin of endotoxemia influences the matabolic response to endotoxin in dogs. J. Surg. Res. 73, 47-53.

21.HERMSEN, C.C., J. VD. CROMMERT, H. FREDERIX, R.W. SAUERWEIN, W.M.C. ELING. (1997) Circulating tumour necrosis factor a is not involved in the development of cerebral malaria in Plasmodium berghei-infected C57B1 mice. Parasite Immunol.19, 571-577.

22.POSTMA, N.S., C.C. HERMSEN, W.M.C. ELING, O. BOERMANS, J. ZUIDEMA. (1998) Optimal delivery of desferrioxamine (DFO) in the treatment of murine malaria. Future strategies for drug delivery with particulate systems. Medpharm, GmbH Scientific Publishers Stuttgart Ed. Diederichs J.E. & Müller R.H.172-176.

23.POSTMA, N.S., HERMSEN, C.C., ELING, W.M.C., ZUIDEMA, J. (1998) Plasmodium vinckei: Optimisation of desferrioxamine B delivery in the treatment of murine malaria. Exp. Parasitol. 89, 323-330.

24.MONIQUE J.J.M. JANSEN, THIJS HENDRIKS, ROB HERMSEN, JOS W.M. VAN DER MEER, R. JAN A. GORIS. (1998) A monoclonal antibody against Tumor Necrosis Factor-a improves survival in experimental multiple organ dysfunction syndrome. Cytokine accepted

25.HERMSEN, C.C., MOMMERS, E., VD WIEL, TH., SAUERWEIN, R.C., ELING, W.M.C (1998) Convulsions due to increased permeability of the blood- brain-barrier in experimental cerebral malaria can be prevented by splenectomy or anti-T-cell treatment. J. Inf. Diseases 178, 1225-1227.

26.POSTMA, N.S., HERMSEN, C.C., CROMMELIN, D.J.A., ZUIDEMA, J., ELING, W.M.C. (1998) Treatment with recombinant human tumour necrosis factor-a reduces parasitaemia and prevents P. berghei K173 induced cerebral malaria in mice. Parasitology accepted

27.POSTMA, N.S., HERMSEN, C.C., CROMMELIN, D.J.A., ELING, W.M.C. ZUIDEMA, J. (1998) Thiolated recombinant human tumour necrosis factor-a enhances suppresion of parasitaemia and protection against P. berghei K173 induced cerebral malaria. Antimicobial Agents and Chemotherapy submitted

165 List of publications

28.SNEL, J., HERMSEN, C.C., SMITS, H.J., BOS, N.A., ELING, W.M.C., CEBRA, J.J., HEIDT, P.J. (1998) Interactions between gut-associated lymphoid tissue and colonization levels of indigenous, segmented, filamentous bacteria in the small intestine of mice. Canadian J. of Microbiology accepted

29.HERMSEN, C.C., SAUERWEIN, R.W., ELING, W.M.C. (1998) Alloxan protects Plasmodium berghei infected mice in end-stage disease experimental cerebral malaria. J. Inf. Dis. submitted

30.HERMSEN, C.C., POSTMA, N.S., SAUERWEIN, R.W., GRONER, Y., GOLENSER, J., ELING, W.M.C. (1998) Protection against Plasmodium berghei induced experimental cerebral malaria after treatment with antioxidants. Antimicob. Agents Chemother. submitted

166 Curriculum vitae

CURRICULUM VITAE

Cornelus Christianus Hermsen werd geboren op 11 maart 1953 te Elden. In 1974 behaalde hij het diploma HBO-a aan de Analistenopleiding te Arnhem. In het kader van deze opleiding werd stage verricht op de afdeling Cytologie en Histologie (Dr. W. Eling) aan de Katholieke Universiteit te Nijmegen. Het HBO-b diploma werd 2 jaar later behaald aan de Analistenopleiding te Oss. In 1977 begon hij met zijn werkzaamheden op de afdeling Cytologie en Histologie (Prof. C. Jerusalem, Dr. W. Eling) aan de Katholieke Universiteit Nijmegen. In deze periode werden de diploma’s deskundigheid stralingshygiëne voor een C-laboratorium en medisch biologische laboratorium techniek behaald en de pedagogische en didactische aantekening. Na een fusie van al het malaria-onderzoek van de faculteit werd vanaf 1993 het wetenschappelijk onderzoek dat beschreven staat in dit proefschrift uitgevoerd op de afdeling Medische Microbiologie/sectie Parasitologie (Dr. R. Sauerwein/ Dr. W. Eling).

167 Dankwoord

DANKWOORD

Hoewel op de titelpagina van dit proefschrift slechts één auteursnaam vermeld staat, moge het duidelijk zijn dat vele anderen ook een bijdrage hebben geleverd tot het tot stand komen van dit proefschrift. Hierbij wil ik iedereen die op enigerlei wijze heeft bijgedragen bedanken. Graag wil ik enkele personen met name noemen: Prof. Dr. J.H.E.Th. Meuwissen die mij indertijd de gelegenheid heeft gegeven dit onderzoek te starten. Mijn co-promotoren Dr. W.M.C. Eling en Dr. R.W. Sauerwein wil ik bedanken voor hun begeleiding. Wijnand, sinds 1977 werken we als een goed koppel samen. Jij hebt me de ruimte gegeven om me wetenschappelijk te kunnen ontplooien. Je geduld, opbouwende discussies, ideeën en steun zijn hier terdege van belang geweest. Hiervoor dank ik je hartelijk. Robert, voor mij ben je niet alleen een “facilitator” zoals je het zelf noemt. Jij hebt mij niet alleen de gelegenheid gegeven om dit wetenschappelijk onderzoek af te ronden maar je kritische blik, het bekijken van het manuscript door de “ogen van een ander” en onze discussies heb ik als zeer waardevol ervaren. Mijn promotor Mw. Prof. Dr. J.A.A. Hoogkamp-Korstanje wil ik bedanken voor de begeleiding bij de afronding van ervan. Nancy, bedankt voor de gezelligheid, de vele zakken drop, de goede samenwerking en het beschrijven van de hoofdstukken 7 en 8 die we ieder hebben gebruikt in onze manuscripten. Medewerkers van het CDL, Geert Poelen, Theo van de Ing, Yvette Brom en Henny Broekman, jullie wil ik heel hartelijk bedanken voor de vakkundige biotechnische hulp en de verzorging van de muizen. Dankzij jullie hulpvaardigheid heb ik geen enkel probleem gehad van mijn allergie tegen muizen en ratten. Ellen, jou wil ik bedanken voor je vrijwillige inzet, enthousiasme en je wezenlijke bijdrage van hoofdstuk 3. Theo v.d. ®, ik bedank je voor je betrokkenheid, discussies en je filosofische benadering van de wetenschap en van het leven. Veel steun heb ik de afgelopen jaren mogen ontvangen van mijn “lotgenoten” Ton, Annette en Will. We hebben frustraties kunnen delen maar ook veel plezier en steun aan elkaar gehad. Zo’n betrokkenheid voelt goed.

169 Dankwoord

De studenten Jos, Anita, Hanny, Marloes, Erik, Laura en Mirjam wil ik hartelijk bedanken voor hun enthousiasme, inzet en bijdrage aan mijn onderzoek ook al is jullie werk niet letterlijk terug te vinden in dit proefschrift, het heeft zeker bijgedragen. En natuurlijk wil ik de andere (ex-)medewerkers van de afdeling Medische Parasitologie, Karina, Marianne, Pieter, Jan-Peter, Theo, Pascal, Marga, Geert-Jan, Arianne, Tita, en Truus bedanken. Het zou veel minder gezellig zijn geweest zeker zo rond de soms te sterke of te slappe koffie. Last but not least, het thuisfront. Bedankt voor jullie steun, betrokkenheid en ruimte. Harm en Roel, jullie hoeven niet meer te wachten want nu is er weer tijd om te klussen. El, ja, woorden .

170 Abbreviations

ABBREVIATIONS

BBB blood-brain-barrier CM cerebral malaria CQ chloroquine DFO desferrioxamine DFO-Zn desferrioxamine-zinc ECM experimental cerebral malaria ELAM endothelial leucocyte adhesion molecule ELISA enzyme-linked immunosorbent assay EPC egg phosphatidylcholine EPG egg phosphatidylglycerol LFA leucocyte function antigen FBS foetal bovine serum FCS foetal calf serum FITC fluorescein isothiocyanate i.p. intraperitoneal i.v. intravenous ICAM intercellular adhesion molecule IFNy interferon gamma RPMI RPMI 1640 medium IMDM iscove’s modified dulbeco’s medium kD kilo Dalton MAb monoclonal antibodies NFMB nuclear factor kappa B OD optical density OH hydroxyl radical O superoxide radical P plasmodium PBL peripheral blood lymphocytes PBS phosphate buffered saline PE parasitized erythrocyte PÎEMP1 plasmodium falciparum erythrocyte membrane protein-1 rec-m-TNFa recombinant mouse tumor necrosis factor alpha

171 Abbreviations rec-h-TNFa recombinant human tumor necrosis factor alpha rec-h-TNFa-AT thioacetyl rec-h-TNFa rec-h-TNFa-ATA acetylthioacethyl rec-h-TNFa RNI reactive nitrogen intermediates ROI reactive oxygen intermediates ROS reactive oxygen species s.c. subcutaneous SATA N-succunimidyl-4-S-acetyl thioacetate SOD superoxide dismutase TNFa tumor necrosis factor alpha TNFa-R TNFa receptor sTNFa soluble TNFa VCAM vascular adhesion molecule WBC white blood cells

172