DETECTION OF CYTOKINE mRNA IN THE BRAINS OF Trypanosoma brucei brucei -INFECTED MICE Guided by Subham and Presented by Swati

1. Centre for Biotechnology
Siksha ‘O’ Anusandhan
( Deemed to be University )
NAME- SWATI PANDA
REGD NO-1861621056
Designed by- SUBHAM PREETAM
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2.  Immunopathology of Trypanosoma brucei in the central
nervous system (CNS) was studied in infected mice with T.b
brucei were treated with dose of diaminazine aceturate
several times.
 Brains were examined using PCR to detect MRNA (cytokine).
 The infected & treated animals developed severe chronic
meningo-encephalitis characterized by inflammatory cells &
astrocyte proliferation.
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3.  African sleeping sickness is caused by Trypanozoon species,
extracellular protozoan parasites transmitted by the bite of
tsetse fly.
 Many of the clinical symptoms or disease arise as a
consequence of invasion of the central nervous system (CNS)
by the parasite.
 The pathological changes associated with this post-treatment
reactions in humans includes severe meningitis, perivascular
cuffing, encephalitis, astrocyte activation.
 The resulting inflammatory lesions in the CNS are composed
mostly of plasma cells, macrophages, activated T cell.
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4.  Recent work has pointed the production of cytokines by glial
cells within the brains of infected individuals may give rise to
the symptoms of late stage sleeping sickness & be important
in generating the inflammatory changes seen in the CNS.
 The current work was undertaken to identify which cytokines
may be involved in the pathogenesis of the post-treatment
reactions.
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5. Female CD-1 Inoculated with 2*10⁴ T.b brucei with
Mice(28-35g) PBS (ph 7.4) for 30days
(60-65 mice sacrificed)
treated with
Dose of diaminazine aceturate (Berenil) (10mg kg)
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6. Left half of the mouse brain
5% Buffered formalin
Fore brain hind brain
mid brain
Hematoxylin & Eosin
Stained with
Rehydrated in
3% H202 for 5mins
Rinsed with
PBS
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7. PBS (PH- 7.4)
incubated with
20% Swine serum (30mins)
DAKO:PBS(1:1000) (Dilution)
1% Bovine serum albumin
placed & left overnight at 4℃.
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8. right half of the brain
2ml denaturing solution( 4m guadinum
thiocyanate,25mm sodium citrate,0.5% sarcosyl,
0.1m 2-mercapto ethanol) & homogenized.
homogenized
200 μliter of 2m sodium acetate + 2ml water saturate
phenol + 400 μliter of chloroform.
Mixture left on ice for 15 mins
Centrifugation at 1000rpm for 20mins at 4℃
Ice cold isopropanol added
RNA precipitated at -70℃ for 30mins
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9. RNA pellet suspended in 300microliter of sterile water &
extracted with phenol chloroform
Aqueous layer precipitated with 30 μliter of 7m
ammonium acetate & 600μl absolute ethanol at -70℃
Washed twice in 70% ethanol, put in
200μliter sterile water.100μg of total
RNA would yield.
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10.  By using PCR, we detected a range of cytokine RNA transcripts.
Transcripts for β-actin were detected in the brains of all the infected
& uninfected controls.
 IL-1α transcripts were detected in 6 of the 10 uninfected controls,
but no transcripts for TNF- α, IFN- γ, MIP-1, IL-2, IL-4, IL-6 were
detected.
 Analysis of RNA samples from the 10 infected mice which had
developed chronic meningo encephalitis showed the presence of
RNA trascripts for IL-1 α,IL-4, TNF- α, MIP-1 in all of these mice.
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11.  IL-6 was found in only samples 4 & 8. while IFN-γ was detected
strongly in only one sample.
 Although sample 9 displayed a faint band of appropriate size IL-2
mRNA was not detected in any of these samples.
 There are several possible sources of various cytokines, activated
astrocytes in the brain of infected animals, large no. of monocytes
which are capable of synthesizing IL-1α & TNF- α. Presence of these
cytokines can initiate the inflammatory process.
CONCLUSION :
However, in view of the high level of astrocytes activation observed,
it seems likely that astrocytes contribute in some way to the
presence of cytokines & play a role in initiating the inflammatory
processes.
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12. Detection of IL-1(625bp)(A), IL-4
(399bp)(B), TNF(692bp)(C), MIP-
1(267bp)(D) in the brains of T.b.
brucei effcted mice with meningo-
encephalitis. The left most lanes
(M) contained a ladder of 123bp
fragments. A negative control &
positive control were also included.
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THANK YOU

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