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[06/01/2004]
 Immunopathology : Cerebral malaria ( or pernicious malaria) |
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Valéry COMBES, Nicolas COLTEL, Samuel WASSMER and Georges E.
GRAU
Immunopathology Laboratory, Experimental Parasitology Unit, URA
3282,
Faculty of Medicine, ‘’Méditerranée’’ University
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Currently, worldwide efforts are made by large international
organizations to fight malaria, the world largest endemia that
threatens 40% of the population in some 90 countries, and claims
more than 2 millions lives each year. Among the four species of
Plasmodium that affect humans, P. falciparum is by far the most
pathogenic, causing neurological damages («pernicious malaria» or
«cerebral malaria»), hematological damages (severe anemia) and/or
complications during pregnancy («gestational malaria»).
In its non-complicated form, malaria is a fever associated with an
algetic syndrome (cephalgia, myalgia, arthralgia, abdominal pains),
and digestive disorders. Acute malaria – usually associated
with P. falciparum – occurs either unexpectedly or following
malarial signs either non acknowledged as such or the treatment of
which was ill-suited or late. Severe malaria is defined by
the detailed criteria of the WHO.
| I. Physiopathology of severe malaria
The main histopathological characteristics of P.
falciparum-induced severe malaria is the sequestration of
erythrocytes that contain mature forms of the parasite in their
deep vascular territories. This sequestration is not, however,
evenly distributed within the vital organs.
Pernicious malaria
The pathogenesis of pernicious malaria is not yet fully
understood. In this affection, sequestration is most severe
in the brain (1), which explains the high frequency of coma.
Three pathogenic theories have been suggested for human pernicious
malaria (2) : The permeability theory (3); the mechanical theory
and the immunological theory (4). It seems that a combination
of factors that depend on both the host and the parasite
contributes to the pathology of acute attacks of P. falciparum
malaria, and more specifically to the severity of cerebral lesions
(5-7).
Acute anemia
Anemia is an unavoidable consequence of the infection. Anemia
expands rapidly during the infection and, generally, the higher the
parasitemia level, the lower the hematocrit drop. In high
transmission zones, severe anemia is one of the most frequent
manifestations of complicated malaria and affects mainly children
under the age of 3.
The mechanisms of acute anemia are multifunctional and complex,
involving an hemolysis which can be mediated by the
complement. Anti-red blood cells antibodies have been
evidenced in malaria-infected patients and in patients after the
end of the parasitemia. Moreover, an inappropriate medullary
response seems indicated since dyserythropoïesis has been
described.
Kidney insufficiency
Acute renal insufficiency is a common complication of P.
falciparum-induced acute malaria and is often lethal. It
affects almost essentially adults and adolescents. The
mechanism of acute tubular necrosis in malaria is not yet
understood. Occasionally, there is cytoadherence of parasited
erythrocytes inside the capillaries of the glomeruli, but not as
pronounced as in other organs such as the brain. The clinical
and biochemical prognosis is that of ischemic nephropathy or of an
acute tubular necrosis.
Pulmonary oedema
The cause of this lethal manifestation of complicated malaria
is still poorly known. Symptomatology is similar - in
numerous aspects – to the adult respiratory distress
syndrome. The patients affected by complicated malaria are
highly susceptible to develop an acute pulmonary oedema after
hyper-hydration. This oedema can also affect patients who
have not been hyper-hydrated. The oedema can result from an
increase of the pulmonary capillary permeability, and develops
rapidly even sometimes after a chemotherapy. Hence the
physiopathology can differ from other P. falciparum-induced malaria
complications.
Among the modifications of the host’s biological parameters,
various cellular and plasmatic responses are observed, of which the
main ones are described below.
Platelets
Thrombocytopenia is often found during the infection by P.
falciparum, but its clinical relevance remains to be
defined. This thrombocytopenia can result from either a drop
in platelet production, or from an augmentation of the platelet
renewal induced by different destruction mechanisms. A central
attack seems unlikely since high numbers of megacaryocytes are
found in patients in acute phase. Conversely, some arguments
advocate for a destruction induced by immunological mechanisms,
platelet activation, elimination by the reticulo-endothelial
system, or excessive consumption during the various steps of
coagulation.
A recent study has shown that in children of Senegal, platelet
counts were lower in severe attacks than in moderate attacks, and
likewise in dead children as opposed to those who recovered
(Gérardin et al, Am J Trop Med Hyg 2002 66(6) : 686-91). A
multivariate analysis has shown that thrombocytopenia is a
predictive factor independent from death.
Monocytes
There are multiple interactions between the circulating
monocytes and the malaria agent. Namely, monocytes are
activated by the phagocytosis of parasited red blood cells, a
phenomenon that is potentialized by TNF (8). Moreover, the
inhibition of parasitic growth takes place through ADCI («antibody
- dependent cellular inhibition») which require monocytes.
The causes of leukopenia in malaria remain unknown (9,10).
Among the mechanisms mentioned, apoptosis seems to play a role
since during the acute phase of malaria the number of apoptotic
cells increases (11). On the other hand, the P.
falciparum-parasited red blood cells induce the apoptosis of the
human mononuclear cells (12).
The Duffy blood antigene is an erythrocytic chemokines receptor
which binds IL-8, the MGSA (melanoma growth-stimulating activity),
MCP-1 and RANTES. Longitudinal surveys that analyze the secretion
of IL-8 and MIP-1a in P.
falciparum-malaria infected patients have shown that the IL-8
concentration is correlated with the parasitemia and the severity
of the disease. The reason for this increase is unknown,
albeit MIP-1a is an inhibitor of
hematopoietic cell proliferation that could be responsible for
prolonged anemia in malaria (14).
Soluble molecules
Occlusive phenomena in the cerebral vessels are caused by immune
phenomena that cause a modification of the adhesive potential of
the erythrocytes, leukocytes, and platelets. Several experimental
models have insisted on the importance of the urokinase receptor,
of the CD40 and of the TNFR2. There is no data on their
impact on human pathology.
The CD40 ligand is involved in the humoral and cellular immunity
and is also a receptor capable of modulating the proliferation, the
differentiation and the cell death. Mortality drops
significantly in mice whose CD40 and CD40L genes have been
invalidated with a less severe thrombopenia despite identical
parasitemia and a diminution of the sequestration of macrophages in
the cerebral vessels (15). CD40L is a membrane receptor which,
after cleavage, can be released in the circulation. The
soluble form would preserve its activity.
TNF is involved in the pathogenesis of experimental pernicious
malaria. As to the respective role of these two receptors, we have
shown a significant increase of receptor 2 (TNFR2, p75), but not of
receptor 1 (TNFR1, p55), inside the cerebral microvessels during
the cerebral syndrome in animals susceptible to the disease. On the
other hand, TNFR2-deficient mice are protected from experimental
pernicious malaria unlike TNFR1-deficient mice. This
protection is not associated – as is the case with the
TNFR1-deficient mice – with an over-expression of ICAM-1 and with
an increase of the leukocyte sequestration specific to the vascular
lesion of mice that died from pernicious malaria (16). Furthermore,
the use of mice cerebral microvascular cells has made it possible
to show the importance of the interaction between the membrane TNF
and the TNFR2 in the development of the neurological
syndrome. Indeed, the presence of the two TNFR1 and TNFR2
receptors necessary for the over-expression of ICAM-1 by the
soluble TNF, whereas only TNFR2 is necessary for the cerebral
syndrome to occur.
The urokinase receptor (uPAR, CD87) is a key molecule of the
adhesion and monocytic spreading. Recent data have established that
CD87 is most likely involved in the pathogenesis of pernicious
malaria, since the KO mice for this molecule are protected from the
cerebral syndrome and from mortality associated with it (17).
In human being, in the case of pernicious malaria, the CD87
over-expression has been observed at the level of the Dürck
granulomas. The uPAR could be involved in the alteration of the
hemato-encephalic barrier during pernicious malaria
(18).
| II. Contribution of experimental models to the comprehension of the physiopathology
Since the study of physiopathological mechanisms in human being
is complex, we looked into an experimental model of pernicious
malaria in mice, and more recently, into an in vitro modelization
of human pernicious malaria, using human cell cultures.
Our researches into the murine experimental model of pernicious
malaria have indicated that cerebral complications depend on an
inappropriate stimulation of the immune system in the infected
host. The neurological attack and the associated mortality only
occurs in the susceptible animal that possesses intact T
lymphocytes; one of the consequences of the activation of T cells
is the production of various inflamation mediators. Among those, we
have found that a cytokine, the tumour necrosis factor (TNF), plays
an important role in the pathogenesis of pernicious malaria.
Indeed, (i) plasmatic rates of this molecule are only found, during
the acute phase, in the animals that develop pernicious malaria,
(ii) the injection of anti-TNF antibodies prevents pernicious
malaria in infected mice, (iii) the perfusion of recombining TNF
triggers cerebral lesions in genetically infected mice that resist
the neurological syndrome, and (iv) transgenic mice that express
high rates of TNF soluble receptors do not develop any neurological
syndrome. In the last few years, we focused our attention to
the mechanisms responsible for the excessive production of TNF and
to the TNF vascular toxicity effector mechanisms.
Our results indicate that the susceptibility to malaria-induced
neurovascular lesions is associated with a high production capacity
of IFN-g production as a response to
malaria antigenes. Since the production of cytokines defines the
sub-classes of T Lymphocyites CD4+, referred to as Th1 and Th2,
this data suggests that unlike any other parasitic diseases,
pernicious malaria involves a predominant expansion of Th1
lymphocytes.
The use of a panel of monoclonal antibodies as an in vivo treatment
for malaria-infected mice has shown that only the antibodies
directed against the “leukocyte function adhesion molecule 1”
(LFA-1) prevent cerebral complications and acute mortality. Since
this antibody has proven efficacious even when administered a few
minutes before the death of the mice, the known anti-LFA-1
immunosuppressive properties cannot be invoked. We had to
re-evaluate the action mechanisms of this antibody. This is why we
envisaged the possible role of the platelets in the pathogenesis of
pernicious malaria.
The key role of the platelets in the development of neurological
lesions caused by pernicious malaria has been demonstrated in mice.
The fusion of the platelets with the endothelium described in vitro
and in vivo as a physiological process that ensures the trophicity
of the endothelial cells. In the case of acute malaria, this
process could be increased and be involved in the endothelial
lesion process. These results open new perspectives as to the
mechanisms of action in vivo of the anti-LFA-1 antibody:
interference with the deleterious effects of platelets-endothelium
interactions. Besides the pernicious malaria, an in vivo treatment
using anti-integrin antibodies provides a significant
protection for various pathologies. The effector role of the
platelets in macrovascular attacks could lead to new therapeutical
approaches in the TNF-induced lesions in the broad meaning of the
term.
We have deepened the study of the implication of endothelial
adherence molecules in the pathogenesis of experimental pernicious
malaria for three reasons: first, this syndrome is characterized by
an increased adherence of the leukocytes to the vein endothelium,
leading to an endothelial lesion and focal hemorrhages; second; TNF
is a central mediator; and third, TNF increases the expression of
various endothelial adherence molecules, namely ICAM-1, one of the
LFA-1.
For these studies, we developed cultures of purified endothelial
cells of the cerebral microvessels. The morphologic, physiologic
and immunologic properties of these macrovascular endothelial cells
(MVEC) are different from those of the MVEC, such as those of the
umbilical cord usually studied. This technique enabled us to
develop a co-culture system to analyze more precisely the
interactions between the MVEC and circulating cells, namely the
platelets. Thus, we have been able to propose a sequence of
events that involve endothelial cells, parasited red blood cells
and the platelets that lead to the occlusion of the microvessel
(Fig 1 and 2). Figure 1: 
Figure 2: 
On the basis of these facts, reviewed and discussed in two recent
articles (19 - 21), our research works within Experimental
Parasitology look into the following questions:
• Which biological markers are associated with the clinical
expression of malaria, enabling a better prognosis and a better
control of the patients to be made?
• What are the molecular mechanisms of the endothelial lesion
in pernicious malaria?
• How can we define the new therapeutics for the benefit of
the patients ?
• How is it possible to better predict the complicaitons, and
thus reduce the health costs?
| Bibliographical references:
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