EFFECTS OF THREE NIGERIAN PLANTS ON THE HISTOPATHOLOGY OF CUTANEOUS LEISHMANIASIS (Leishmania major) AND IMMUNOTHERAPY OF LEISHMANIASIS USING DNA/PROTEIN VACCINE IN BALB/c MICE By JUDITH NKIRUKA
EFFECTS OF THREE NIGERIAN PLANTS ON THE HISTOPATHOLOGY OF CUTANEOUS LEISHMANIASIS (Leishmania major) AND IMMUNOTHERAPY OF LEISHMANIASIS USING DNA/PROTEIN VACCINE IN BALB/c MICE By JUDITH NKIRUKA
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Date
2008-06
Authors
ALAWA, JUDITH NKIRUKA
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Abstract
Three Nigerian plants (Annona senegalensis, Azadirachta indica and Vernonia
amygdalina) were evaluated for antileishmanial effects in vitro and in vivo using the
Leishmania major parasites. It involved the preliminary testing of the hexane,
methanol and aqueous extracts of these plants on infected murine macrophages
and other cell lines. This was to determine the leishmanicidal activity and
cytotoxicity on normal cell lines (L929 fibroblasts and macrophages), cancer cell
lines (Jurkat and SH-5YSY) and Trypanosome brucei brucei. The leishmanicidal
activity was determined using the microscopic counting method while the
cytotoxicity involved the Alamar Blue colorimetric technique. Furthermore, doseresponse
sensitivity tests were carried out on bone marrow macrophages infected
with Leishmania major parasites and only extracts with positive indications from
the preliminary tests were used. Also, the efficacy of the extracts which gave
positive indications was tested in vivo in BALB/c mice treated orally for five days at
a dose of 50 mg/kg per day. The aqueous extract of Vernonia amygdalina
significantly (p<0.001) suppressed infection rate of intracellular amastigotes
(>60%) and significantly (p<0.001) reduced parasite levels to almost zero in the
infected macrophages. In the dose-response sensitivity test, the methanol extract
of Vernonia amygdalina suppressed infection rates (>50%) at a lower
concentration (50 g/ml) than that which caused toxicity to the macrophages. It
also significantly reduced the lesion sizes by about 50% as well as minimised
histopathological changes in the skin of infected mice. The hexane extract of
Annona senegalensis, showed good trypanocidal effect against Trypanosome
brucei brucei at a concentration of 3.13 g/ml. This was over 15 fold more potent
than the reference drug, cylemersan used at 50 g/ml.
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Also, the potentials of a DNA based vaccine and its recombinant protein were
evaluated as vaccine candidates. Leishmania donovani gene encoding gamma
glutamylcysteine synthetase and its soluble recombinant GCS antigen in the
presence of a non-ionic vesicular surfactant (NIVS) as an adjuvant were tested.
Susceptible BALB/c mice were immunised with the plasmid encoding the full
sequence for GCS (pVAXGCS), recombinant protein in presence of adjuvant
(rGCS-NIV) or plasmid alone (pVAX control) prior to challenge with a high dose of
Leishmania major promastigotes. Mice immunised with the recombinant protein
(rGCS-NIV), demonstrated an enhanced production of specific IgG1 and IgG2a
antibodies indicating that it was immunogenic. Also, it resulted in significantly lower
lesion sizes (p<0.05) as compared to the controls. This partial protection
corresponded to a significant increase (p<0.05) in gamma interferon production
suggesting an enhanced Th1 response. These results showed that the protein
vaccine from Leishmania donovani induced a strong immuno protection against
cutaneous leishmaniasis, suggesting that it represents a very good candidate for
use as a vaccine against several leishmania species.
Description
A DISSERTATION SUBMITTED TO THE POSTGRADUATE
SCHOOL IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY IN HUMAN ANATOMY
DEPARTMENT OF HUMAN ANATOMY
FACULTY OF MEDICINE
AHMADU BELLO UNIVERSITY
ZARIA
NIGERIA.
JUNE, 2008
Keywords
EFFECTS, THREE, NIGERIAN, PLANTS, HISTOPATHOLOGY, CUTANEOUS LEISHMANIASIS (Leishmania major), IMMUNOTHERAPY, LEISHMANIASIS, DNA/PROTEIN, VACCINE, BALB/c MICE