~~Ir
EFF'Fr.-:'-:--S
8F
C~""C':Lr:":3'!?C'RIr'l
AN ANTI~IA ANTIBODY
ANG
An Honors Thesis (:G499)
lJy
p'
. 1 .
Dana Mc.rshall
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By ~~~~S va~i0us
measuri~g
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5. :. _ "_
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disease
treatment prot
progr~5s~on,
r t·
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it
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CsA,
possi.ble ther..., because
t.he
co~~ectioil
,'
ce ......
ir:hib:' t:.. 0::,
a2~
activat.iorl
that
CsA
esA
ex~r~s~lo~.
profo~.:.nd,
has
p~ocess
ability to
fronl
Ia
betwse
protected
direct
antigen
anirr.a~ 3
e=fects
:Jll
macrop~:ag-!?s
and
tr-_t::il.:-
(19)
'::X.2reS2
Eea~~ y
n01T:"l':":
( '7 -. 9) .
decrease :"n
~acrophage
activat~o~
i~
due
~r
r
'-
C'sA
P L ev i 0'-'."
_... 3. __ '
pror,hylactic
t~~atment wit~
lesion
32.Z2,
esA (£-3:
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i
-~.
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p
-- 1
corr_pa l.-e
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r .t: .:::.
\J ::...
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O~,
,
the
J:.sease
a~~~als
was
~
cause of disease progresslon OL an
:_:.3.t
\' :... 'i] ':]
3:":25
::: :?
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t_
3all~/,::.
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OF
CY~LOSPCR:~~
0::2.:
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'reatment schedJle aGJ dOSE
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tClat
OF CYCLOSPORIN.
S~2-C~:A~EO~S
~OSES
phY5io:og~~~I
sa:ine per ml cf
of
Seve:.:.::::.:
SO:~ti02,
inves:i';}a-!. ':"d
5ub-c~taneo;s t~~at~e~~
..' u
a
dosage
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-l
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days
day:;
+ 2 , -L 3 , + 4 , 5 , '"'- 6 ,
.j...
+l,-f-2,+3,+-";,T"
+€.
F
p
,+
ANTIBODY.
ANT:-ra
,.~
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-
';.)
:; . ("
_ l ..
( ._1
~
an(~
,
'..t..
produce
:
Asci~es
fluid was
2.1
ge
arnoun ts
'C' _ _
of
;-'
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the D313 7
~_
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ordel."
to
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,,,\.Le,'
,, -
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,
: h·.= :: I ",. ; 1
0_
comp 1 et. f"';
30
-c, ,'
concentratio~
of the dialysate wa:
at a waveleng:h of
w:..th
3~tibody
T::e
has beerl
d~~e~r:~r_e,!
280~~.
physiological
fQ~nd
sallEe.
to
sp2cif:"~al~~
__ ...;
ci.L t::..
-
.J...
~~
~.~.
protei=-~
3rect~o3i:02Y
r
I,:
f-<
an t i bod ...-'
8.5%
cont.a::..:-~=
lOmg/:-r:.:
::.. i"l
a
rnediccrn.
~abb:i..t
ver:
1!71.9
-'- J.
,.
A
...,
rabbit
1
1" ;.:i .:.
,-.
<•
detect2~
by
ar~,-
~~=ro3,:oPi
_,
,_,
( 9)
its o!:iginal
~he~
~sed
~n
tL~
'-U~
'"
The
co~ce~tration
Ia
stain~~lg
o~
9mg/r:
p~oced~res.
J:dLb::..t
!-.1-_
. _. _" .
Pa~a~o~~aldehyd~
fixative:
:a
p:rOr~edur~s
paraformaldehi~~-
whi
• .j.. ~ ,.,...
-~-'-'~~I
,::.~
,-
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<,_,' -,t-=- _,
cr
":'
__' J . -• .
-~
:-='.J __ _
~he
~eritonea:
2acrcphage3
~~
mic2 were retrieved .. Jj
,
- , '7 :!i. 1 ::e:':::-ievals
~
'......J
.! .. '
A •• '1
~ / • •"
,
....
• ;"1
_J
- .. ::..~
(Be~lco
.
int.o one
Labs ;,
\--iF-" ,-:,
mo')se to
cover sl
to
t~~
p~ov~ue dupl~cate
ip~._
s:ips ::..n 5% CO- at
-? -..1. '
1_1_
rC3ult3
~~!
a::owed to adhere
ho1.:trs.
r,
,
CE'.:. .... :...'
'1.
wa3hed agai:-.
witL
was'·
anti-Ia
:.:.::i,-1-
r ..
.:, . -,-'" ''::
:"E:p:aced
-''';
::31ip
.c ,. _
c~.
s~line.
~r.l.ey
of the
were
the~:
plac,:"·1
sol ~-.:~::'on of
we:!::-:- se:
:ce
0:::'
l.. L' ....
Again,
-.
30
,
the
s~ips
.'
were
the!"'.
refr:geI:ated
f 0::
a
'_'.:.,,:01'1:::-' •
_ C
as
tho
u;...
as
ml-,
.:;.
.l. ... _
.'
,,
b,:.:l ow.
:::e:ri.eved
f ro:-.
''f'Jer':.'
-J.
centrifuge a:' 4 3egrees
~PM!
wa3 added to a
4
50~!
C~:Sih~
:640
c~lture
me~~um
f:ask.
~it~
~he
5% FES
flas!: was
cent~ifuged
at
:ma c :.: 0
hou~.3
on
f l.-or:-:
washec:
'-
sid,:; of
r~ ~-:..a9::'-.s
(:-?2-5)
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The suspensions were
~
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t ir:,~"
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began
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i t
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-~..:.
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1.. \
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Footpad 1 esic,:!'-;.
normal
sma:le~
~evels
iF;
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lympt
node
tha~:
weights
3igr:.i f i caL t~ Ii'
,;,: 1-
33
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'
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seventh
wee~:
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:=0 __
-,,:'::1
t:: ea ted
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Le
r' t_' - (' en. t
r
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r
ag c
c
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c
2-
.. c
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,
Dose
..Lrng
Days of Treatment
-.J..
---;>
Disease Progression
Exacerbation
~47
_
,-mg
MG5~
-.~
Necrosis
Day +49
,
, '
T..J...'1c
Ext~eme
S:~gi:
EXaCelL&:io~
+44
Zxacerbatic~
• ""'1,-,
,
"'--
,44
""
::":::g
-49
:nfected,
U~treateci
ALima:s
:'lone
+19->+56
Table 1.
Several treatment protocols were investlgated uSlng
the D3137 anti-Ia antibody in Balb/c mice infected with
LeiEhma~ia m3]2r.
Eariy treatme~ts seemed to ~ave ~he gre~tes~
_~~~~~ S~ ~~~?~se ?~);~~S3~Q~,
=ausing t~e disease proceS3 to
progress quicker and to a greater degree compare,i to infecte~,
untreated ~~-:~.
~isease progression was followed by ~easur~~g
foctpad lesion sizes a~d the ~xtent of ulceration of footpad
l~sions.
disease
~at>::--:
t~ea-::-_:l;r:.-~_t_:'
progressi0~
.:;et:- __ -:1_",:
:-La·:;:::
,:._t.L~e-
e-:'i:0cT_
-.)::-1
I
1-', "Zqn
'•./ ~ ,-I '.,' 1-'
i
1.I
,
!
,
Q_ooo··~l--------+-------~-­
o
Fig.l
CsA treated animals and infected controls were infected
with lxla promastigotes on day 0. CsA treated animals were
treated "lith oral 150mg/kg/bid doses of CsA on days 1,0,+J.,+2,+3,+4,+5. The left, hind footpads f.rom two
representative mice were measured and averaged for each data
pOint.
Lyrn~,rl
C'j
ni
NOfj€ Weights of CS,A, Trt.:!cted tJI'I,j
Untreated Mke otter infection 'with Leis!'f'nanio !nd lor
(-! , - -_ _ _~. _ _ _ ._ _ •__ ~~~~_T' __ ~'-'_~~~ ..... ~ .......... _... ~ ... __~_'_._~ __ ."._"'~-----'-'~~---'--""'l
· ..•. , '~ij.........,:t.
I~'_ J,...J.
J; i"'Sil
v ,,-,.
O~060
T'" "t"'d
1 ()--() lnf~cte,j
I'
. . .- ' - .
~!
Onl\-'
r-·,!(',:'-r ..."'''.':,
··! ..
1_
'l'_~'
i!
r~WI.~
!
\r"I;~'lfF;,. . ~':t.;;,ci
, ..
! __" _ ' . ' _ _
()
!
f)·· ..f.',...
'! '1-;\
" .'I +-I
'~-,
.-
~J
Q,COC! +----- !
o
Doy ,AJter infection
Fig.2
CsA treated animals and infected controls were infected
with lxl(Y promastigotes on day 0. CsA treated animals were
treated with oral 150mg!kg!bid doses of CsA on days
-1,0,+1,+2,+3,+4,+5. The popliteal lymph nodes from the left
hind legs of two representative mice wer" weighed and averaged
for each data point.
Spleen Weiqht3 (,f CaA Treate,j mtcl
IJntreoted 1',,1ice otter- Inftlction yvith LeishrrI0n!(J rnd jor
"
i
C"""
(\. °1 ()() "1"'
i
i
i
(',
'-'1 " r i .L,
L'. ,...... ~.J \.. !
I
i
!
OJJOO +\-----+-
o
Do)'
,~fter
l!')fectfon
Fig.3
esA treated animals and infected controls were infected
with lxla promastigotes on clay 0. esA treated animals were
treated with oral 150mg/kg/bid doses of esA on days
-1,0,+1,+2,+3,+4,+5. The spleens of two representative mice were
weighed and averaged for each data point.
Per-centoge of kJ+ PeritoneGi ~,'I':JCmphIJqes in i~sA Treated ond
Untreoted Mice oftet' Infection \'llth Leishrrl,.onl(] rno[o!!
!::,) ..- , ( )
ir'I";ecte:j On!\:
!' •• :r,
;;.:, f."..1'
;~L"-·t
~,,~
' L •• l
• "'"._ ' ...
25+,
C.l..
r-. ,-.
£~u
!
t-,
i
+
i
it=: -.I.Jv J
j
...,
~
~,
+
i
!
0 +----+-1---+---It----+--,---+---,---,
(',
I)
"i
!
i')
,>-,
:j t.:;
I _,)
-::. (,-j
,,/:.)
Fig.4
esA treated animals and infected controls were infected
wi th 1x1cY promastigotes on day 0.
CsI, treated animals 'I'lere
treated with oral 150mg!kg!bid doses of esA on days
-1,0,+1,+2,+3,+4,+5. The percentages of Ia+ peritoneal
macrophages of two representative mice wers assessed by
observation under the fluorescent microscope and averaged for
each data point,.
...c
~)
.....
Fig.S
Anti-Ia treated animals and infected controls were
infected with 1x16 p.romastigotes on day 0. Anti-Ia treated
animals were treated i.p. with 2mg!day doses of D3137 Anti-Ia
antibody on days -1,0,+1,+2. The left, hind footpads from four
representative mice were measured and averaged for each data
point.
..
FootPCd ::;
-- ",
Untreate1j i\,1L:::e Gtv~(
~.
L;
\
ir/2C:ti.~:;rl
..
:
'lhitr';
i
:;J:r"':]r-"i, ~.... "':,J I:.
J'
."-,~---".--- ---.---,-~
~}-J' .!
\(~
,.J
1,. .. \
..
c
' ,'
0.4(10
+
.--_._- e--------e--------e
0_200 +1--------+----·
'J 0
Do','
, AJter infection
Fig.6
Anti-Ia treated animals and infected controls were
infected with lx16 promastigotes on day O. Anti-Ia treated
animals were treated i.p. with 1mg/day doses of D3137 Anti-Ia
antibody on days +18,+19,+20,+21,+22,+23,+24. The left, hind
footpads from six representative mice were measured and averaged
for each data pOint.
Fig.7
Three populations of peritoneal macrophages were stained
wi th fluorescent antibody for analysis by flow cytometry. 'rhe
peritoneal macrophages of the D3137 Anti-Ia treated animals
(shown here in blue) were retrieved 62 days after infection.
These mice were treated with Img!day wit.!"! D3137 Anti-·la antibody
on days -1,0,+1,+2,+3,+4,+5, with day 0 being the day of
infection with 1x1eJ promastigotes. The peritoneal macrophages
of infected, untreated mice (shown here in red) were retrieved 62
days after infection. 'rhe peritoneal macrophages shown here in
green are from uninfected, normal animals.
This graph shows the macrophage population size (Y) vs.mean
fluorescent intensity of each macrophage population (X).
.... 1- - _
~-~.
-
,-
-'-
,
'
.:.-,
J.. ,_
'---I:"
".At
- -'- -'
._'-'--'--____ 2,
__ , J.
~'xa( ~_-::':i ~
~efleral,
peritoneal
para::e:
appea:::-a.dce
macrop~ages
~~e
:~~3c::":(~"phag,=.s
the
I
In
ed
cisease
no~
0:
infected/
nor-tr~ated
animals
3~e2:5
deve:opnent of severe disea3e iL tl~ese anima!~
~,
+-
_ .:: 3.
~o
Sil-.:.ce
t:-.-.
(:he,:=-3
c. __
::.. __ 1--
Indeed,
~his
particl.~l
ar
an t i
did seem to differ
accc~d~~g
infec:ti:J:;
bod~/
was
to the timing of thp
gr.-eat 1 ";j
bea:.:in1j"
the
'-"~")
tLeat~e~~
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wi
tr~
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SL:.ppressior:
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to this parasite
,
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disease to
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et
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