study of the enzymatic and immunological properties
of glycylpeptide N-tetradecanoyltransferase isolated
from Leishmania
By:
1. Saif Qahtan Salman
(Lecturer of Biochemistry, College of Biotechnology,
Al-Qasim Green University, Babylon, Iraq, E-mail: saif.q1988@gmail.com)
2. Karam Dawood Salman (Lecturer of Immumopathology, College of
veterinary medicine, Al-Kufa University, Kufa, Iraq, E-mail:
karam.biotech@mail.com)
3. Ghufran A. Abdulraheem (Lecturer of
Zoology, College of
Biotechnology, Al-Qasim Green University, Babylon, Iraq, E-mail:
gogo868776@yahoo.com)
Al-Qasim Green University
Iraq, 2015
Abstract
Visceral leishmaniasis caused by Leishmania donovani is the most severe systemic form of the
disease. There are still no vaccines available for humans and there are limitations associated with
the current therapeutic regimens for leishmaniasis. Recently, we reported functional importance
of glycylpeptide N-tetradecanoyltransferase enzyme from L. donovani involved in ascorbate
biosynthesis pathway. In this study, we have shown that glycylpeptide Ntetradecanoyltransferase have many immunological properties can act as enhancer for host’s
immune system and can work like leishmanial antigen after studying enzymatic enzymatic and
immunological properties of this enzyme on 4 to 6 weeks old BALB/c mice . Cytosolic antigen
was prepared by repeated freezing- thawing of leishmanial antigen, followed by ultrasonication
and ultracentrifugation. The protein concentration was estimated by Lowry method and cytosolic
protein was characterized over SDS-PAGE gel with dominant polypeptides ranging from 96 to
27 kDa . Cytosolic antigen was administered subcutaneously along with chitosan at 2 weeks
interval. 10 days postimmunization co-relates for cell mediated- and humoral- immune response
were evaluated. Successful vaccination for the control of parasite multiplication is often related
to antigen induced DTH response as an indication of activation of cell-mediated response. In the
present study, results obtained upon immunization with cytosolic antigen in association with
chitosan demonstrated induction of an appreciable DTH response suggesting the activation of
cell-mediated immunity. Activation of cell mediated immunity was further evaluated in terms of
lymphocyte proliferation. Lymphocytes from both spleen and lymph node were in vitro
restimulated with cytosolic antigen. Lymphocytes exhibited a strong in vitro recall response
where proliferation induced in response to cytosolic protein and cytosolic protein in combination
with chitosan was almost comparable . Further, induction of nitric oxide (NO) production in
splenic lymphocytes was studied as production of NO molecules is an important cytotoxic
function that macrophages used to resolve infection by several obligate intracellular protozoa and
bacterial parasites. Immunization of cytosolic protein in association with chitosan resulted in
considerable increase in NO production in comparison unimmunized, cytosolic and chitosan
alone immunized groups respectively . Through these results we saw glycylpeptide Ntetradecanoyltransferase can induce protective immunity and can provide sustained protection
against Leishmania donovani. We further conclude that the parasites attenuated in their antioxidative defence mechanism can be exploited as vaccine candidates
Key words: Leishmania donovani, glycylpeptide N-tetradecanoyltransferase, SDS-PAGE.
Introduction
Leishmania are protozoan parasites shuttling between sand fly vector where they multiply as free
promastigotes in the gut lumen, and mammalian host where they proliferate as obligatory
intracellular amastigotes in the mononuclear phagocytes
( Handmanet al., 1999 ).Leishmania
parasites
are responsible for a family of diseases, collectively known as leishmaniasis, with discrete
clinical features ranging from cutaneous lesions to a fatal systemic disease. Leishmaniasis is
prevalent in Africa, Latin America, Asia, the Mediterranean basin and the Middle East, and
recently has been identified in East Timor(Chevalier et al., 2000 ) , Thailand
kangaroos in Australia
(Rose et al., 2004)
.
(Kongkaewet al.,2007)
and in
Leishmaniasis has been classified as one of the most
neglected diseases, and the estimated disease burden places it second in mortality and fourth in
morbidity among the tropical infections. For many years, the public health impact of
leishmaniasis has been underestimated, as a substantial number of cases were never recorded.
The expansion of leishmaniasis and the sharp rise in prevalence is related to environmental
changes and migration of non-immune people to endemic areas (WHO Initiative for Vaccine Research 2008).
The former, in particular, has the potential to expand the geographic span of the vector, thus
increasing Leishmania transmission to previously unaffected areas. More recently, an increase in
the overlapping of HIV infection and visceral leishmaniasis has been observed, especially in
intravenous drug users in South-Western Europe and Brazil(Cruz et al., 2006). The situation might be
much worse in Africa and Asia where the prevalence and detection of HIV and Leishmania coinfections is still largely underestimated.
Visceral leishmaniasis (VL), also known as kala-azar, is a disseminated protozoan infection
caused by Leishmania donovani complex (Chappuiset al.,2007;Murrayet al.,2005). VL is essentially caused
by L.donovani and Leishmania infantum (synonym Leishmania chagasiin South-America).
Exceptionally, visceralization of species typically associated with cutaneous leishmaniasis has
been observed. Most commonly, this has been reported with Leishmania tropical in the Middle
East and Leishmania amazonensis in South-America. In individuals infected with human
immunodeficiency virus (HIV),visceralization of a number of dermatotropic species has been
documented as well (see section on VL-HIV coinfection.
Zoonotic visceral leishmaniasis (ZVL) caused by Leishmania infantum is a disease of humans
and domestic dogs (the reservoir) transmitted by Phlebotomine sandflies. According to the
World Health Organization
(Chaniotiset al., 1994),
ZVL was first recorded on Crete in 1907, after
which it featured prominently in medical literature as a serious public health problem. In Chania,
Crete, the annual incidence in the 1930s was 50 cases/30,000 population(Adler et al., 1938), and 21%
of 1,115 dogs were positive for ZVL by the formol-gel serologic test; 30% of those were
symptomatic
(Chaniotiset al., 1994).
After World War II, use of DDT against malaria vectors and
focal destruction of Leishmania spp.–infected dogs is thought to have reduced ZVL on Crete.
Sandflies were not found in villages systematically sprayed during 1946–1949 compared with
unsprayed villages
(Hadjinicolaou 1958).
During 1951–1975, only 33 alleged human ZVL cases were
recorded on Crete (Leger et al., 1977), and reports of canine ZVL were scanty. In 1983 a sero survey
of 72 stray dogs in Chaniaidentified only 1 infected dog
(Chaniotiset al., 1994).
By the late 1980s,
Phlebotomus neglectus, the putative vector of L. infantum on Crete, was abundant in stonewalls
inside and outside villages around Heraklion(Leger et al., 1993).During 1999–2004, P. neglectuswas
found in abundance in human dwellings and rural locations (hollows in olive trees and near
rodent burrows).
Since 1991, 38 persons who came to hospitals in Heraklion were confirmed as having cases of
ZVL. Today, canine infection is confirmed throughout the island (M. Antoniou, unpub. data),
and seroprevalences(30%–40%) are some of the highest reported in Europe. These accounts
anecdotally suggest that the incidence of ZVL has increased on Crete during the past decade,
which, if so, would be relevant to the public and veterinary health sectors.
Meterials and methods
- MICE AND PARASITES
BALB/c mice, bred in the animal care facility of the University under pathogen-free
conditions, were used at 4 to 6 weeks old for experimental purposes with prior approval from
the Animal Ethics Committee of the JamiaHamdard. An Indian strain of L. donovani AG83
was maintained by passage in BALB/c mice, amastigotes isolated from infected mice spleens
were allowed to transform to promastigotes by cultivation at 22°C in M199 medium (pH 7.4)
supplemented with 20% heat-inactivated FBS, 2 mM glutamine, 25 mM HEPES, 100 U/ml
of penicillin G-sodium and 100 µg/ml of streptomycin sulfate. Parasites were checked and
enumerated by counting in a hemocytometer on day 6 after first transformation and every 72
hrs thereafter. Designated parasites from stationary-phase cultures were diluted in fresh
medium with the same composition as that mentioned above to maintain an average density
of 2 x106 cells/ml (Afrin et al, 2002).
Determination of antigen concentration by Lowry’s method
Principle
The “Lowry Assay: Protein by Folin’sReagent”
(Lowry et al., 1951)
has been the most widely used
method to estimate the amount of protein (already in solution or easily-soluble in dilute alkali) in
biological samples. First the proteins are pretreated with copper ion in alkali solution, and then
the aromatic amino acids in the treated sample reduce the phosphomolybdatephosphotungstic
acid present in the Folin’s Reagent. The end product of this reaction has a blue color. The
amount of protein in the sample can be estimated via reading the absorbance (at 750 nm) of the
end product of the Folin reaction against a standard curve of a selected protein solution ( in our
case; Bovine Serum Albumin(BSA) solution).
The Lowry method relies on two different reactions:
1. The first reaction is the formation of a copper ion complex with amide bonds,
forming reduced copper in alkaline solutions. This is called a Biuret chromophore
and is commonly stabilized by the addition of tartarate(Gornallet al., 1949).
2. The
second
reaction
is
reduction
of
the
Folin-Ciocalteau
reagent
(phosphomolybdate and phosphotungstate), primarily by the reduced copperamide bond complex as well as by tyrosine and tryptophan residues.
PREPARING THE SOLUTION

The Lowry Solution is a mixture of the ( Na2 Tartrant.2(H2O), CuSO4.5(H2O) and Other
NaOH, Na2CO3) chemicals, except the Folin’s Reagent. The Lowry solution should be
prepared fresh, at the day of measurement. Though the individual solutions for the
Lowry solution can be prepared in advance and then mixed at the day of measurement.

Solution A is a dilute alkali solution. 2N Folin and Ciocalteau’s Phenol Reagent contain
HCl and H2PO4.
Solution A: (alkaline Solution) (for 100 ml)
0.572gm NaOH
2.862gm Na2CO3
Solution B: (for 20 ml)
0.285gm CuSO4.5(H2O)
Solution C: (for 20 ml)
0.571gm Na2Tartrant.2(H2O)
Lowry Solution: (freshly prepared, 0.7/ml sample)
Sol. A + Sol. B +Sol. C with a ratio (vol:vol) of 100:1:1
Folin Reagent: (instant fresh, 0.1 ml/sample)
5ml of 2N Folin and Ciocalteu’s Phenol Reagent + 5ml of double distilled water
this solution is light sensitive. So it should be prepared at least 5min of the first
sample incubation and kept in an amber container.
Procedure:
1. Different dilutions of BSA are prepared by mixing stock BSA (1 mg/ml) and water in the
test tube. Similarly, two dilutions of antigen are prepared in duplicate .The final volume
in each of the test tubes is 5 ml. The BSA concentration ranges from 0.05 to 1 mg/ml.
2. From these different dilutions, pipette out 0.2 ml BSA or antigen to different test tubes
and add 2 ml of alkaline copper sulphate reagent. Mix the solutions well.
3. This solution is incubated at room temperature for 10 mins.
4. Then add 0.2 ml of reagent Folin Ciocalteau to each tube and incubate for 30 min. Zero
the colorimeter with blank and take the optical density (measure the absorbance) at 660
nm.
5. Plot the absorbance against BSA concentration to get a standard calibration curve.
6. Check the absorbance of antigen and determine the concentration of the unknown sample
using the standard curve.
SDS-PAGE
Principle
Electrophoresis is the study of the movement of charged molecules in an electric field. The
generally used support medium is cellulose or thin gels made up of either polyacrylamide or
agarose. Cellulose is used as support medium for low molecular weight biochemicals such as
amino acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for
larger molecules like proteins.
The general electrophoresis techniques cannot be used to measure the molecular weight of the
biological molecules because the mobility of a substance in the gel is influenced by both charge
and size. In order to overcome this, if the biological samples are treated so that they have a
uniform charge, electrophoretic mobility then depends primarily on size. The molecular weight
of protein maybe estimated if they are subjected to electrophoresis in the presence of a detergent
sodium dodecyl sulfate (SDS) and a reducing agent mercaptoethanol ( ME). SDS disrupts the
secondary, tertiary and quaternary structure of the protein to produce a linear polypeptide chain
coated with negatively charged SDS molecules. 1.4grams of SDS binds per gram of protein.
Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds.
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Polyacrylamide gels are prepared by the free radical polymerization of acrylamide and the cross
linking agent N N’ methylene bis acrylamide.
Procedure:
Casting the gels
1. Prepare the separating gel monomer solution by combining all reagents except
ammonium persulfate (APS) and TEMED. Deaerate and mix the solution after adding
each reagent by swirling the container gently.
2. Place a comb completely into the assembled gel sandwich. With a marker pen, place a
mark on the glass
plate 1 cm below the teeth of the comb. This will be the level to
which the separating gel is poured. Remove the comb.
3. Add APS and TEMED to the monomer solution and mix well by swirling gently. Pipette
the solution to the mark.
4. Immediately overlay the monomer solution with 1 ml. of water. Use a steady, even rate of
delivery to prevent mixing with the gel.
5. Allow the gel to polymerize for 45 minutes to 1 hour. Pour the water overlaying the gel
and drain the excess water with strips of filter paper.
6. Prepare the stacking gel monomer solution. Combine all reagents except APS and
TEMED. Deaerate and mix the solution by swirling gently.
7. Place a comb in the gel sandwich.
8. Add APS and TEMED to the solution and pipette the solution down one of the spacer
until the sandwich is filled completely.
9. Allow the gel to polymerize for 15 minutes.
10. Remove the comb.
11. Gel is placed in the buffer chamber and running gel buffer is added into the chamber .
Preparation of a 10% Resolving/separating gel.
30% acrylamide + 0.8% Bisacrylamide
1.25 ml
4X Tris-HCl/SDS, pH 8.8
0.94 ml
Distilled water
1.56 ml
10% ammonium persulfate (APS)
25 l
TEMED
10 l
Preparation of a 4.5% stacking gel
30% Acrylamide + 0.8% Bis-acrylamide
0.65 ml
4X Tris-HCl/SDS, pH 6.8
1.25 ml
Distilled water
3.05 ml
Ammonium persulfate
25l
TEMED
5 l
The Leishmanial antigen (Cytosolic) from L. donovani promastigotes was subjected to sodium
dodecyl sulphate 10% polyacrylamide gel electrophoresis (SDS–10% PAGE), and the
proteinswere localized in gels, stained with Coomassie blue . (Ulrich K. Laemmli , 1970 )
Evaluation of Cell Mediated Immunity
DTH (delayed type hypersensitivity):
The delayed type hypersensitivity was the first experimental evidence of transferable
immunity carried only by the immune cells. Robert Koch, the discoverer of the tubercule bacillus
was the first to demonstrate a delayed type hypersensitivity reaction in 1882. Koch attempted to
use his killed tuberculin preparation as a prophylactic and therapeutic vaccine. Unfortunately, the
antigen did not confer protection to naive patients, and when injected intravenously in infected
patients, caused reactivation of the disease and in some cases death. Nevertheless, when the
antigen was injected intradermally, the delayed inflammatory response (tuberculin reaction)
could indicate whether or not an asymptomatic person had been exposed to Mycobacterium
tuberculosis. It was not until 1942 that Landsteiner and Chase demonstrated that the DTH
reaction could be transferred by a "cell only" fraction. The basis for the experiment was fairly
direct. Cells from guinea pigs, which had been immunized with Mycobacterium tuberculosis or
hapten, were transferred into naive guinea pigs. Later, when antigen or hapten was injected into
these guinea pigs, they elicited an immune recall response that was not present in the naive
controls. This did not happen when the serum fraction was transferred. Coombs and Gell
classified delayed type hypersensitivity as Type IV. The three other classifications of
hypersinsitivity are Type I (immediate/IgE-related) in which the cutaneous skin test reaction
peaks at 2 hours, Type II (antibody and complement related cytotoxicity) and type III (antigenantibody complex mediated).
DTH responses have been well characterized. The reaction is antigen specific and causes
erythema and induration at the site of antigen injection in immunized animals or humans.
Systemic injection of antigen results in fever, synthesis of acute phase proteins and in some
instances death. The nature of this antigen can be varied. Mycobacteria, protein, hapten, and
even grafted tissue are all capable of inducing delayed type hypersensitivity reactions. The
histology of DTH can be different for different species, but the general characteristics are an
influx of immune cells at the site of injection, either macrophages and basophils in humans and
mice or neutrophils in guinea pigs, and induration which becomes apparent within 24-72 hours.
Even though they make up only a small percentage (10-20%) of the total inflammatory infiltrate
at 48 hours, T cells (either CD4+ or CD8+ depending on the antigen) are required to initiate the
reaction.
Evaluation of DTH Response:
The response was evaluated at 24 h by measuring the difference in swelling between two hind
footpads, one injected with 50 µl of PBS alone and the other with PBS containing L. donovani
antigens (cytosolic antigen) (40 µg/50 µl PBS), thickness of foot pad swelling measured by using
a digital Vernier caliper
.Lymphocyte
proliferation by Trypan blue exclusion method
BALB/c Mice sacrificed
Isolation
.
Spleen
Lymph Nodes
Making single cell
suspension by frosted
(sterile)
Making single cell
suspension by pestle
and mortar (sterile)
RBCs lysed by lysis
buffer
Wash 2 times with
RPMI 1640 without PR
and FBS
Wash 2 times with
RPMI 1640 without PR
and FBS
Adjust cell density to 5x106 cells/ml with
complete RPMI 1640 after trypan blue
exclusion and counting under phase
contrast microscope
Adjust cell density to 2x106 cells/ml with
complete RPMI 1640 after trypan blue
exclusion and counting under phase
contrast microscope
Cultured in RPMI 1640 containing 20 mM NaHCO3, 10 mM HEPES, 100U
of penicillin per ml, 100 µg of streptomycin per ml, 2 mM L-glutamine,
and 10% fetal calf serum (complete medium [CM]), with 50 μMβmercaptoethanol.
added.
The cells were cultured in triplicate in a final volume of 200 μl/well
with cytosolic antigen (12μg/ml) at optimum concentrations, Normal
cells along with Antigen ,or with ConA (mitogen), cells from chitosan
alone , and Antigen along with chitosan immunized mice.
The cultures were incubated for 48 h at 37°C in a humidified chamber
containing 5% CO2.
The amount of proliferation was measured by counting the cells after
Trypan blue exclusion using hemocytometer
(Poulteret al.,1982)
Nitric oxide estimation
Production of NO molecules is an important cytotoxic function that macrophages use to resolve
infection by several obligate intracellular protozoa and bacterial parasites. The inducible form of
the NO Synthase enzyme (iNOS) produces large amounts of reactive nitrogen molecules by
oxidizing the terminal guanidino nitrogen of L- arginine. Since reactive nitrogen molecules can
damage host tissues as well as the invading microbes, the activation of NO production by
macrophages is a tightly regulated ligand receptor mediated process that involves two stages.
The priming stage occur when activating molecules, such as the cytokine INF-ϒ, binds to
receptors on the cell surface of macrophages, inducing alterations in cytoplasmic calcium levels
and initiating other intracellular signaling cascades. These signaling cascades ultimately alter the
expression of enzymes, including iNOS and receptor proteins to prepare the metabolic
machinery to initiate a cytotoxic response. The triggering stage is induced when another
molecule often a molecule of the intruding microbe such as bacterial LPS binds to the receptor
on macrophages initiating another series of signaling cascade that activates the previously
primed cytotoxic machinery.
Nitric Oxide Assay involves the conversion of nitrate to nitrite by the enzyme nitrate reductase.
The detection of nitrite is then determined as a coloured azo-dye product of the Griess reaction
that absorbs visible light at 540 nm. The concentration of NO can be extrapolated from a
standard curve of sodium nitrite.
The Griess reaction involves an oxidation and a nucleophilic reaction. Buffer or sample
components that interfere with the oxidation and nucleophilic reaction may interfere with color
formation. Examples of nucleophiles and antioxidants are azide, ascorbic acid, compounds
containing sulphydryl groups such as cysteine, glutathione, DTT, and β-mercaptoethanol. Nitrate
Reductase converts nitrate to nitrite. Any sample or buffer component that may interfere with
this enzyme will lower the conversion of sample nitrate to nitrite and therefore result in lower
estimates of NO. The nitrate salt content should be considered when choosing a tissue culture
media. Some media may contain relatively high levels of nitrate, which may interfere with
sensitive detections. Media that contain phenol red as a pH indicator do not interfere with the
Griess reaction as the indicator is typically yellow colored under the conditions of the Griess
reaction.
Preparation of solution:
Reagent A:- for 10ml
1 sulphanilamide (0.1gm) in 1.2 N HCl (1.08 ml).
Reagent B:- for 10ml
0.3 NEDD (1-naphthyl ethylene diaminedihydrochloride) (30mg) in milli Q water.
Reagent C:-for 10ml
0.1M NaNO2 (6.9mg) in milli Q.
Griess reagent:1 sulfanilamide and 0.1 NEDD in 50 H3PO4
Determination of nitric oxide
The spleens were removed aseptically and teased into single cell suspension in RPMI 1640
supplemented with 10% FBS, 2mM L-glutamine, Penicillin (100 U/ml), Streptomycin
(100µg/ml) and 50µM 2-mercaptoethanol. RBCs were removed by lysis with 0.14M Tris
buffered NH4Cl. The remaining cells were washed twice with culture medium and viable
mononuclear cell number was determined by counting after Trypan blue exclusion method in a
haemocytometer. The cells from the immunized and normal mice were stimulated with soluble
leishmanial antigen at a concentration of 5g/ml, cultured in complete medium at 2x105
cells/well in a 96 well plate flat bottom culture plate. ConA was taken as positive control at a
concentration 2.5µg/ml. after incubation for 48 hrs in CO2 incubator nitrite concentrations were
determined in the culture supernatants using Greiss reagent. Equal volumes (100 L) of cell
culture medium and Greiss reagent were mixed and incubated at room temperature, and
absorbance was measured 550 nm with a microplate reader. Nitrite concentration was
determined using a standard curve generated with sodium nitrite.
Results
 Determination of antigen (cytosolic) concentrations by Lowry’s method
The concentration of cytosolic antigen was determined by Lowery’s method using BSA
as a control to generate standard curve (Fig.14) and the concentration of cytosolic antigen
was found to be 3.32 mg/ml.
1.4
y = 0.012x
R² = 0.9693
1.2
OD (750 nm)
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
120
Concentration (µg/ml)
Fig.14 BSA standard graph for estimation of protein concentration
CHARACTERIZATION OF ANTIGEN (SDS-PAGE)
The protein profile was examined by SDS-PAGE. SDS-PAGE analysis revealed that cytosolic
antigen is a mixture of polypeptides with molecular mass ranging from 27 to 96 KDa. The
prominent polypeptides correspond to molecular weight 48 KDa and 38 KDa (Fig.15).
97 kD
66 kD
96kD
64kD
48kD
45 kD
38kD
31kD
29 kD
Fig.15 SDS
profiling of cytosolic proteins in comparative with ladder
Enhancement of cell-mediated
immunity
A positive DTH response towards cytosolic antigen is an indication of cell-mediated immunity.
As shown in (Fig.16), immunization with cytosolicantigen induced thehighest level of DTH. 10
days post immunization the DTH response in the group immunizedwith cytosolic antigen
increased substantially followed by levels of DTH induced by PBS control BALB/c mice. Mice
immunized with cytosolic antigen along with chitosan induced a higher DTH response when
compared to antigen alone .
DTH
Fig.16 DTH responses in terms of footpad swelling following intra dermal inoculation of the antigen
Cell proliferation assay by Trypan blue exclusion method
Vaccine-induced stimulation of the cell-mediated immune response was further investigated
through the capacity of spleen and lymph node cells to proliferate in response to concanavalin A
and cytosolic antigen after immunization. The in vitro recall responses were evaluated 10 days
after immunization. Cytoslic antigen induced strong proliferative in both cases (splenocytes and
lymphocytes). This further indicates that cytosolic antigen is a potent stimulator of cell mediated
immunity and the presence of chitosan as an adjuvant is not enhancing its immunogenicity
(Fig.17)
Fig.17 Lymphoproliferation responses after in vitro restimulation by cytosolic
antigen evaluated by Trypan blue exclusion method
NO estimation
The nitrite concentration as an indicator of NO production by activated splenocytes was
estimated in vitro for all the experimental groups. MaximumNO production was observed in
Normal + ConA group followed by Ag+ Adj. (Fig.18).
NO estimation for splenocytes
Fig.18 NO estimation for splenocytes by Griess Reaction
Conclusion
Successful vaccination for the control of parasite multiplication is often related to antigen
induced DTH response as an indication of activation of cell-mediated response. In the present
study, results obtained upon immunization with cytosolic antigen in association with chitosan
demonstrated induction of an appreciable DTH response. induction of nitric oxide (NO)
production in splenic lymphocytes was studied as production of NO molecules is an important
cytotoxic function that macrophages use to resolve infection by several obligate intracellular
protozoa and bacterial parasites. Immunization of cytosolic protein in association with chitosan
resulted in considerable increase in NO production in comparison unimmunized, cytosolic and
chitosan alone immunized groups respectively
REFERENCES








Handman E. Cell biology of Leishmania. Adv Parasitol 1999;44:1-39.
Chevalier B, Carmoi T, Sagui E, Carrette P, Petit D, De Mauleon P, et al. Report of the first cases of cutaneous
leishmaniasis in East Timor. Clin Infect Dis 2000;30:840.
Kongkaew W, Siriarayaporn P, Leelayoova S, Supparatpinyo K, Areechokchai D, Duang-ngern P, et
al. Autochthonous visceral leishmaniasis: a report of a second case in Thailand. Southeast Asian J Trop Med Public
Health 2007;38:8-12.
Rose K, Curtis J, Baldwin T, Mathis A, Kumar B, Sakthianandeswaren A, et al. Cutaneous leishmaniasis in red
kangaroos: isolation and characterisation of the causative organisms. Int J Parasitol 2004;34:655-64.
Cruz, I., M. A. Morales, et al.2002. Leishmania in discarded syringes from intravenous drug users. Lancet359:11241125
Chappuis, F., Rijal, S., Soto, A., Menten, J. & Boelaert, M. A meta-analysis of the diagnostic performance of the direct
agglutination test and rK39 dipstick for visceral leishmaniasis. BMJ 333, 723 (2006).
Chaniotis B. Leishmaniasis, sandfly fever and phlebotomine sandflies in Greece: an annotated bibliography. Geneva:
World Health Organization; 1994. WHO/LEISH/94.34.
Leger N, Pesson B, Madulo-Leblond G, Ferte H, Tselentis I, Antoniou M. The phlebotomes of Crete [in French].
Biologia Gallo- Hellenica. 1993;20:135–43.