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parasitic dz

cerebral malaria is one of the most serious 
forms.....blah blah blah....
A. what mechanism accounts for the 
sequestration of plasmodium infected erythrocytes in deep tissues and 
organs such as the brain?
B. describe one specific receptor/ligand 
interaction that participates in the mechanism mentioned in A, and 
breifly discus the experimental evidence for this interaction and its 
role in sequestration
C. what new preventative or therapeutic modalities 
are suggested by this area of research?
D. since it isn't in the 
parasite's interest to kill the host what could be some adaptive 
advantages to the malaria parasite of the phenomenon discussed above?


redefinition of parasitism: a parasite is an organism that lives on the 
surface of, or insid,e the host, and obtains nutrients from host. the 
relationship is at the expense of the host.

the pathogenesis brought 
about by parasites can stem from several factors. first, there is a sort 
of superficial view, that pathogenesis results from competition b/w host 
and parasite for resources. ultimately, things like this, such as 
relation b/w cestode and host, will have metabolic consequences, but they 
are nonspecific, and we will not discuss them. also, we saw a number of 
instances where parasites do gross tissue damage - primary screw worm fly 
and host, for example, destroying the ear of a cow. this is also beyond 
the scope of this lecture.

many of the diseases resulting from 
parasitism in vertebrates are immune mediated in nature. today, we'll 
steer away from pathogenetic mechanisms understood strictly at the 
effector cell level...we'll focus on the things understood at the 
biochemical level.

the direct biochemical lesion - such as cachexia 
mediated by TNF in trypanosomiasis - is the subject of the lecture. 
virulence factors of parasite - how it changes self or host cells to 
increase invaseivness and proliferation, and so doing brings about 
disease.

example - peripheral to vet med in a way, but there are some 
parasitisms we see involving related protozoan parasites. 

Malaria 
parasites - much known about the biochemical mediation of malaria comes 
from the medical literature. 

Life cycle of plasmodium - mosquito 
introduces sporozoites (asexual) into body, sporozoites invade liver, 
undergo feeding trophozoite stage, giving rise to multinucleated 
schizont, rupturing cell and sending out merozooites which may infect 
other hepatocytes or go out and enter RBC for mainn phase of host life 
cycle. the whole thing happens again in RBC-  the large schizont phase 
ruptures the RBC and more merozoites go out and so forth. during schizont 
development we see signs of disease.

clinically, depending on the 
plasmodium spp (vivax, falciparum,  or malariae) there are regular 
patterns of chills and fever occuring, roughly coinciding with 
synchronous waves of RBC lysis. Some people used to call it "the shakes" 
but I like to call it shake 'n bake, myself. anyway, it results in sick 
people. 
physiologic consequences are traced to three types of patterns - 


1. disease occurs secondary to lysis of red cells - Hburia, splenomegaly, 
anemia
2. changes in Hb metabolism - iron depletion, anemia
3. changes in 
surface characteristics of host red cells - agglutination, DIC, decresed 
microcirculation, tissue anoxia

red cells are basically metabolically 
inert,right? they are anucleate, surfaces are relatively inert, but the 
malaria parasite changes these things, makes cells more active and more 
adhesive.

slide: what leads us to believe that parasitism by the malaria 
parasite may change the sequestration or adhesion patterns of RBCs? if 
you look in blood of malaria patients, you see ring stages, trophozoite 
forms. these are the feeding stages of the parasite and are what we see 
in the periphery. the big schizont stages are rarely seen inn the 
periphery. where are they? well, we find them adhering to the endothelial 
lining of small postcapillary venules. usually in deep tissues of the 
host (including brain?).
this results in small focal hemorrhages in deep 
tissues, obstruction of the venules, localized replicationn of parasites 
and destruction of tissues/cell lysis (not good when it happens in the 
brain) resulting in convulsions, coma, death, all the signs we call 
cerebral malaria.

malaria is a very severe disease but usually doesn't 
kill you. when it does kill you it is usually due to cerebral 
complications. 

slide: brain from cerebral malaria patient - focal 
hemorrhages, infiltrationn of inflammatory cells, vascular leakage. 


what's the mechanism of this sequestration in these deep tissues? what 
makes the schizont stages get hung up in these small vessels?

20 yrs 
ago, a paper in science asked - are the endothelial cells involved 
particularly sticky wrt to these schizonts? Looking at the % of RBCs 
infected with all stages of parasite that are in suspension vs attached 
to a monolayer of endothelial cells, we see that hte parasitized RBCs 
tend to stick to the endothelium. so the infected red cell is somehow 
sticky, has some kidn of adhesion factor, it seems. 

looking at the 
interaction up close- there are cells i the moolayer, and each little dot 
ont here is a parasitized RBC - you do not need quantitative data 
totellyou there is adherence. 

if you lok at the tight junctions b/w 
host endo cell and the parasitized RBC, there are weird knobs on the RBC 
- it seems that at those points the host endothelium makes contact with 
the infected RBC.

what's going on? what makes that change, which we 
think is associated with enhanced adhesiveness?

two things to enhance 
pathogenesis are going on - one, the parasite ends up aggregating inside 
the small blood vessels, and causes RBC to adhere to RBC - 
autoagglutination occurs. 
pRBC - EC 
pRBC - RBC
pRBC - pRBC

all these 
things make the pRBCs stick in the venules. that cuases the complications 
we described. what's in it for the parasite? the sequestered cells aren't 
subject to clearance by the spleen. also, these parasites are finding 
themselves in postcapillary venules, a compartment with a relatively low 
oxygen concentration. we know, from many years of trying to culture these 
parasites, that they do best at low O2 concentrations. so the key to 
finding the method for culturing them was lowering the O2 tension in the 
culture. O2 is toxic to them. so sequestering themselves in postcapillry 
venules in deep tissues is really useful for them- enhances ability to 
proliferate. good for parasite, not for host.

specific changes on 
surface of RBC that make it stick to postcap venule endothelium.

two 
proposed mechanisms: 
1. parasite actually takes its own proteins, sends 
them to the surface,and expresses them there. these enhance the 
adhesivness.
2. parasite brings about structureal modifications of host 
RBC surface proteins, making them stickier.

evidence for export of 
parasite proteins to RBC surface: 
one of the first things see was the 
knobs on the surface of the RBCs - we figured that's where the adhesion 
factors were. sure enough, looking at radiolabelled proteins, from 
strains of knob positive or knob negative parasites, we found that in the 
total protein analysis there are 2 large proteins that seem to be 
synthesized de novo in the pRBCs (pfEMP 1 and 2). only one of them, 
PfEMP1, is actually exported to the RBC surface. b/c of the 
characteristics of immune recognition by anti-knob positive sera, it 
seems to be associated with the cytoadherent strains. 

if you do it 
again with anti-knob neg serum (serum with ab to a knob neg strain) you 
still make PfEMP2 but not PfEMP1, so it looks like PfEMP1 is associated 
with knobs and cytoadherence.

what could be the receptor recognizing 
PfEMP1? this is a receptor ligand interaction. what could the receptor 
be? well, we'll see some data here....that indicates that PfEMP1 is being 
recognized by host cell CD36. CD36 is a naturally occuring receptor found 
in many cell types - monocytes, platelets, endothelial cells - under 
normal circumstances it binds collagen or thrombospondin. it is 
upregulated by IFNg and other proinflammatory cytokines associated with 
parasite infection. 

what makes you think PfEMP1 and CD36 are 
interacting in the host? experimental data from Baruch and Howard in 
handout - they took extracts of parasites, preadsorbed with different Ab 
- anti PfEMP3 (a nonadherent protein) and anti PfEMP1. then they 
subjected those cell extracts to affinity purification with different 
receptors. with PfEMP3, CD36 makes a nice band - so the parasite ligand 
is present. but if you use anti-PfEMP1, ,the CD36 binding material is no 
longer present (b/c it was adsorbed out by the antiPfEMP1 Ab). study the 
other data in the chart to see more info on how CD36 binding and 
thrombospondin binding uses different receptors. but there is evidence of 
denovo expressed parasite proteins to bind to CD36.

other evidence
p 4, 
top left
anti idiotype Ab for CD36, essentially these mimic the antigenic 
characteristics of CD36 itself. it's a way of producing the biologically 
active parts of CD36 in pure form. so using this Ab is like using CD36. 
so preadsorbing parasite extracts with nonimmune rabbit serum, these 
antiidiotype Ab bind with a band seen - if you trypsinize the extract, 
you lose the binding. i don't understand that at all.

if you run the 
same experiment, using knob + or knob - strain of parasite, you 
precipitate a band with the knob + but not knob -. so again, further 
evidence that the parasite has essentially appropriated a host receptor 
to get where it wants to be in host.

those are arguments for denovo 
expression of parasite proteins mediating this process of adherence 

the 
other avenue - parasite  modification of native surface proteins on RBC - 
crandall and sherman at UCR. focused on native anion transporter, called 
Band 3 protein, on mammalian RBC. this protein has 14 membrane spanning 
domains, and a series of 7 loops on the surface of the RBC. in the 7 
loops we look for adhesion factors or changes that enhance adhesiveness.


they made first of all synthetic peptides correspondign to 3 of the 
outward loops - 3,4 and 7. they made Ab to those peptides, so they had a 
tag to look for them. here's a photo of the IFA study - only a few of the 
cells have schizont stage parasites inside - and those cells also light 
up with the Ab - those cells  are expressing that part of the loop to the 
outside.
right hand side, p 5 diagram. 

we find that if we take those 
same Ab, and look at how they affect interactions of parasitized cells 
with host endothelium - a sort of in vitro assay - bottom left p 5 - % 
control of adherence - does monoclonal Ab do anything to change binding 
characteristics? the Ab against loop 3 interferes with binding of those 
RBCs on the surface. that Ab inhibits the binding, competitively. Ab to 
loop 4 had little effect. Ab positive in the IFA -for loop 7 - gave good 
inhibition to binding. the monoclonal which didn't light up in IFA, 
didn't recognize surface of parasitized RBC - seems to enhance binding a 
bit. hmm. so monoclonals are recognizing these peptides on the surface, 
peptides that normally are not on the surface. something about the 
parasitism causes these portions to endup on the surface. 

this is some 
of the evidence for parasite changing binding characteristics of the host 
cell.

what are the possible applications of this work, clinically/ What 
could we do with this knowledge? both of the groups doign this work are 
proposing either parasite adhesion factors, or these host protein 
segments - these molecules are proposed as the targets of clinical 
vaccines against malaria. we aren't abrogating infection - we're rather 
interfering with binding of parasite to host endothelium. so we vaccinate 
against the worst part of the disease, as opposed to against the 
infection per se.

Onward to Leishmania:

recall Dr Farrell talked about 
these in core parasit. they are apicomplexan intracellular protozan 
parasites of mphages. depending on spp, we can have either visceral form, 
which gives pronounced splenomegaly/hepatomegaly (serious form) - or we 
can see localized cutaneous lesions or more generalizing cutaneous 
lesions. in the americas, we see forms that attack cartilage - here is a 
picture of an ear totally destroyed. "Chicleros disease" - guys who go 
into forest and harvest chicle - they get this.  slide: some guy's face 
eaten off by leishmania braziliensis, which goes into the nose and soft 
palate and eats it away...

life cycle - amastigote forms taken up by 
sandfly vectors, transforming into promastigotes which proliferate in 
midgut, release hold, move into salivary glands, where they re injected 
back into host during blood meal. they turn back into amastigotes and 
parasitize host mphages.

what goes on in the sandfly? biochemical 
transformations affecting virulence and development. in gut of fly we see 
large numbers of promastigote, flagellate stages of parasite, literally 
attached to endothelial cells. at some point in the proliferative 
development something changes, they release their hold,andmove up into 
the salivary glands. if we were to take samples of those two populations 
of promastigotes - the proliferating promastigotes and the foregut, 
developed promastigotes, we would dsee differences in virulence. the 
proliferating ones raen't that infectious. the nondividing forms are 
highly virulent. so they change adhesive characteristics for fly gut, and 
also increase ability to initiate infection on host. this is fortuitous 
for researchers, b/c the transformation also occurs in simple in vitro 
culture system. during the log phase of development, we get cells that 
are not  very infective for the host (low virulence). during stationery 
phase, they are highly virulent, highly infectious. what's happening to 
the cells?

in all three panels on top L p 7, we see logphase parasites 
bring about lesions measured in terms of footpad width - these are not 
very virulent. stationery phase parasites make bigger lesions. the top 
line shows parasites from culture which are negative for peanut 
agglutinin. that's a lectin - a lectin is a CHO binding molecule from 
botanical sources, mainly - and peanut agglutinin recognizes galactose 
molecules. these have greater or lesser degrees of specificity. so you 
take the unselected culture and select out only those that do not bind to 
peanut agglutinin - those cells are highly virulent. so what changes 
virulence in our sandfly vector is a change in the surface CHO of those 
developing promastigotes. we recognize that by this shift in binding 
characteristics to peanut agglutinin, a plant lectin. 

we think the 
promastigotes have on their surfaces a large lipophosphoglycan made of a 
cap, some repeating phosphosaccharides, a saccharide core, and alkyl 
chain anchoring it into cell mmembrane.when they undergo transformation 
from uninfectious to infectious, log to stationery, we see terminal 
galactose residues become substituted by arabinoses. this accounts for 
changes in binding to peanut agglutinin. what are consequences of the 
change to the parasite? well, it makes it release from the midgut and go 
to the salivary gland, right? 

isolated sandfly midgut preps incubated 
with the parasites - in the presence of procyclic lipophophoglycan  
(multiplying type)(inhibited binding) or metacyclic lipophosphoglycan 
(infectious type)(didn't inhibit binding) - something about the procyclic 
form is essential for binding. the metacyclic form isn't. what about in 
the host? in making transition from log growth phase to stationery phase, 
they get more resistant to complement mediated lysis in the host, as 
well. bottom of p 8 - stationery phase ones survive longer, log phase 
ones do not survive well at all. so we see this also enables them to 
evade host defenses.

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