1Noyes, HA: 3Arana, FE; 3Rizzo, N; 1Kemp, SJ; 2Hutchinson, IV; 2Pravica V; 1Daly, D; 3Arana, BA;

1
1Noyes, HA 3Arana, FE 3Rizzo, N 1Kemp, SJ
2Hutchinson, IV 2Pravica V 1Daly, D 3Arana,
BA 1School of Biological Sciences, University of
Liverpool, L69 7ZB, UK 2School of Biological
Sciences, University of Manchester, M13 9PT,
UK 3Medical Entomology Research and Training
Unit, University del Valle, Guatemala.
Sample Collection and Processing Samples were
collected in the Department of Peten in
Guatemala. The Peten is an agricultural frontier
area where forest has been cleared for
subsistence agriculture over the last forty
years. Cutaneous leishmaniasis (CL) due to
Leishmania mexicana and L. braziliensis is
endemic in this region. Samples were collected
from patients at the regular clinics run by the
University del Valle (Guatemala) in the town of
Poptun in Peten. These clinics are announced on
the local radio and provide a free service to all
who attend. Lesion scrapings for parasite
identification and a 10ml blood sample are
obtained from all patients who have lesions
consistent with cutaneous leishmaniasis. A
Montenegro skin test (MST) is applied to all
patients. Parasites are identified by nested PCR
(Noyes et al. 1998). Patients with a clinical
diagnosis of CL were treated with the WHO
recommended dose of Glucantime. Samples were
collected controls by establishing clinics in
communities which were known to be endemic for
CL. All members of the community were offered a
test for anaemia and an MST skin test. 10ml blood
samples were taken from all volunteers over the
age of 15. Samples were also collected from any
cases of CL that were discovered. DNA was
prepared from blood by ammonium chloride lysis of
red cells and salt precipitation of DNA. Cytokine
alleles were identified in IL-6, IL-10, TNF-ß,
TNF-a, IFN-?, IL4, IL15, TGF-beta by ARMS-PCR
(Perrey et al 1998). Additional SNP in GranzymeB
and CCL5 were genotyped by SnapShot on and ABI
Prism 3100. A Chi squared test was used to
identify significant differences between observed
and expected allele frequencies and genotype
frequencies of individual loci.

• Abstract
• Differences in expression of cytokines involved
  with the Th1 and Th2 responses are associated
  with single nucleotide polymorphisms (SNP) in the
  genes that encode them.. We have genotyped SNP in
  a panel of genes for cytokines that are known to
  be important in controlling the immune response.
• Samples were collected from the following groups
  
• Patients with chronic cutaneous leishmaniasis of
  more than four months.
• Subjects with a positive Montenegro skin test but
  no history of disease. These individuals were
  expected to be resistant to the disease but not
  infection.
• Individuals from the same endemic area with no
  history of disease and a negative skin test. This
  is expected to be a heterogeneous group who may
  or may not have been challenged
• Significant differences were found in allele
  frequencies in the TNF-beta and IL4 genes.

Although microarrays are best known for gene
expression analyses they are also a powerful
resource for identifying clusters of samples and
have been widely used for classifyin cancer
cases. PBMC were taken from 30 MSTve subjects
with a history of disease and 30 MSTve subjects
with no history of disease. PBMC were stimulated
with conA and harvested at 18 and 24 hours post
stimulation. RNA was prepared from each sample
and combined into pools of five RNA samples. Each
pool was labelled with cy3 and cy5 dyes to
provide a dye flip replicate and hybridised to
oligonucleotide microarrays representing
approximately 20,000 human genes (HGMP) using a
common reference. The normalised data was
clustered using Single Value Decomposition in
the MaxD microarray data analysis package. After
clustering the slides four groups could be
identifying corresponding to the cells from the
two groups of patients that had been stimulated
for two lengths of time. The two groups of
patients were separated on the first principal
component and the two times were separated on the
second principal component. Although the two
groups of subjects overlapped a Student T-test
indicated that the difference between them was
highly significant (p0.00059). The subjects had
been selected at random irrespective of ethnic
group, a Pearson correlation coefficient
indicated that there was no correlation between
ethnic group and co-0rdinates on the first
principal component (r2 PCA1 0.027 (r2 PCA2
0.008).
Significant differences were found at the IL4-590
and TNF-b loci between MSTve subjects with no
history of disease and both subjects with a
history of active disease and subjects with no
history of disease. In the case of IL4 this
difference was only observed in the Ladino
population of immigrant descent, and there was no
significant difference in allele frequencies
among the different patient groups of Indigenous
descent. In the case of TNF-beta the two ethnic
groups had opposite trends. If confirmed these
results would indicate that these cytokines might
play a role in conversion on the skin test but
have no effect on the development of disease.
Plt0.001
Plt0.05
Discussion Genetic risk factors for
susceptibility to cutaneous leishmaniasis There
is no evidence of any difference in
susceptibility to leishmaniasis between ethnic
groups in Guatemala, with a prevalence of history
of leishmaniasis about 10 in both the indigenous
and immigrant populations. However there was some
evidence that the two groups might have different
risk factors for conversion on the skin test.
Most notably at the IL4-590 locus where there was
a significant risk of conversion on the skin test
associated with the C allele in the indigenous
group but not the Ladino group. It is not
uncommon for associations found in one ethnic
group to be missing in another group. However ,
the sample sizes in this study were small and it
will be important to determine if the results
observed here are reproduced in other samples
form the same population as well as in other
ethnic groups to determine whether these
observations were a real risk factor or whether
they are artefacts of small sample
sizes. Genetic basis for conversion on the skin
test? Since the micoarray study was undertaken on
cells stimulated with ConA rather than a
Leishmania antigen the results provide evidence
that there is a genetic component to the risk of
conversion on the skin test. This may have
important implications for the interpretation of
the results from this widely used diagnostic and
epidemiological test.
References and Acknowledgements We gratefully
acknowledge the participants in this study and
Milo Henstermann and all the others who conducted
the field work. This work was funded by the
Wellcome Trust. Davies, C.R. and Gavgani, A.S.M.
(1999) Age, acquired immunity and the risk of
visceral leishmaniasis a prospective study in
Iran. Parasitology 119247-257. Noyes, H.A.,
Reyburn, H., Bailey, J.W., and Smith, D. (1998) A
nested-PCR-based schizodeme method for
identifying Leishmania kinetoplast minicircle
classes directly from clinical samples and its
application to the study of the epidemiology of
Leishmania tropica in Pakistan. Journal of
Clinical Microbiology 362877-2881. Perrey, C.,
Pravica, V., Sinnott, P.J., and Hutchinson, I.V.
(1998) Genotyping for polymorphisms in
interferon-gamma, interleukin-10, transforming
growth factor-beta 1 and tumour necrosis
factor- alpha genes a technical report.
Transplant Immunology 6193-197 Turner, D.M.,
Williams, D.M., Sankaran, D., Lazarus, M.,
Sinnott, P.J., and Hutchinson, I.V. (1997) An
investigation of polymorphism in the
interleukin-10 gene promoter. European Journal of
Immunogenetics 241-8.