Download A Variant of the Endothelial Nitric Oxide Synthase Gene is a Risk

18s
Medical Research Society
Young Investigator Communications YI-Y46
for other risk factors. eNOS Asp298 was not associated with EH and there was
no correlationbetween this variant and BP in either EH or normotensive
subjects.
Y I CLINICAL XENOTRANSPLANTATION OF PORCINE
ORGANS: IMMUNE BARRIERS BEYOND HYPERACUTE
REJECTION
Conclusions: Three common variants of the NOS 3 gene (two in the promoter
and a third, which predicts an eNOS variant, eNOS Asp298) have the potential
to influence expression or function of the encoded e w e . Homozygosity for
eNOS Asp298 was a risk factor for atherosclerotic CAD in this UK population.
Studies investigating the biochemistry of this novel variant are in progress.
A DORLING AND RI LECHLER
Department of Immunology, Royal Postgraduate Medical
School, Hmersmith Hospital, London W12 ONN, England
The
use
of
organs
from
animal
donors
(xenotransplantation) is a potential solution to the
chronic shortage of allogeneic organs and currently the
pig is thought to be the most suitable donor for man.
However, porcine organs are rejected rapidly by a
vascular process called hyperacute rejection (HAR) which
has so far prevented clinical xenotransplantation. It
is likely that this barrier will be overcome in the near
future by the application of novel strategies, probably
involving the use of organs from transgenic pigs. Data
from animal models indicates that multiple other immune
mechanisms will contribute to the rejection of
xenografts once HAR has been bypassed.
Little was known of these 'post-HAR' immune-mediated
events in the specific human anti-pig combination at the
start of this work. We have described two aspects of
these mechanisms.
First, the phenomenon of
'accommodation', whereby xenografts acquire in vivo
resistance to HAR and delayed xenograft rejection, has
been explored in an in vitro model utilising
immortalised porcine endothelial cells. The results
indicate that human anti-pig antibodies induce a
concentration-dependent and time-dependent change in
porcine endothelial cells compatible with the
development of accommodation.
Secondly, the in vitro human anti-porcine T cell
response has been documented in detail, with particular
emphasis on quantitative and qualitative comparisons
with the in vitro T cell alloresponse. The results of
this work, which indicate that the response to porcine
xenografts is likely to be significantly stronger than
that against allografts, have important implications for
the level of conventional immunosuppression that may be
necessary to prevent xenograft rejection, and provide an
important basis for the development of strategies to
promote xenograft-specific immunosuppression and
tolerance.
Y2
A VARIANT OF THE ENDOTHELIAL NITRIC OXIDE SYNTHASE
GENE IS A RISK FACTOR FOR CORONARY ATHEROSCLEROSIS.
AD HINGORANI, C LIANG, J FATIBENE, A PARSONS, R HOPPER,D
TRUTWEIN, NG STEPHENS, KM O'SHAUGHNESSY, MJ BROWN.
Clinical PharmacologyUnit, University of Cambridge, Addenbrooke's Hospital,
Cambridge CB2 2QQ, UK
Background: Endotheliumderived nitric oxide (eN0) is a potent vasodilator
with anti-aggregatory and anti-proliferative effects. eN0 is synthesised from
arginine by endothelial nitric oxide synthase (eNOS), encoded by the NOS 3
gene on chromosome 7. A reduction in bioactive eN0 may contribute to the
development of ischemic heart disease and essential hypertension (EH),both of
which.have a heritable component. We hypothesised that polymorphic variants
of the NOS 3 gene might be responsible for reduced NO production and thereby
predsiposeto these diseases.
Methods: DNA variants of the NOS 3 gene were sought using single strand
conformation polymorphism analysis and DNA sequencing. Genotype and
allele frequencies of the four bi-allelic polymorphisms identified were
determined in unrelated population controls. Case-control studies were used to
assess the relationship between a G+T 894 polymorphism and coronary artery
disease (CAD) and EH.
Results: Two variants were identified in the gene promoter at -1468 (T+A)
and -922 (A+G) bp from the transcription start site. and another (A-S) was
located 30 bp from the start of exon 12. The fourth variant (894 G+T), in exon
7 of the gene, predicts a Glu+Asp amino acid substitution at codon 298. There
was an excess of eNOS Asp298 homozygotes among CAD cases compared to
healthy controls ( 3 6 % ~10%. p<O.OoOl). In comparison to Glu298
homozygotes, the odds ratio for coronary artery disease in Asp298 homozygotes
was 4.2 (95% CI: 2.3-7.7) increasing to 7.6 (95% CI: 2.3-17.9) after adjustment
Y3 IDENTIFICATION OF CD8+ CYTOTOXIC T LYMPHOCYTES
(CTL) SPECIFIC FOR Plasmodium foldpanan AND Mycohacteriiim
riibercirlosisIN NATURALLY INFECTED HUMANS
A LALVANI. M AIWO, R BROOKES, and AVS HILL
Nuffield Department of Clinical Medicine. University of Oxford. John
Radcliffe Hospital, Oxford OX3 9DU
CD8+ CTL are a crucial element of immunity to viruses. Evidence from
animal models points to a protective role for CTL against M .
tuberculosis and the liver stage of P.folcipamn, yet CTL specific for
mycobacterial or protozoal pathogens have not been identified in man.
Using a reverse immunogenetic approach, selected preerythrocytic
antigens of P.,fulcipanun and secreted antigens of M. tubercwlosis were
scarined with HLA class 1 allelespecific peptide motifs. Approximately
200 peptides congruent with these motifs were synthesized. Pools of
peptides, sorted by HLA class I motifs, were used to stimulate
peripheral blood lymphocytes in titro from donors with the
corresponding HLA class I type. CTL cognate for a candidate epitope
preferentially proliferate and are detected by assays of effector function.
The "Cr release CTL assay was used and, for tuberculosis, a highly
sensitive ELISPOT assay for single cell peptide-specific interferongamma release was developed.
P. folcipamn-derived peptides were screened in naturally exposed
partially immune Africans in the Gambia. Extending the molecular
analysis of the protective association of HLA-B53 with severe malaria
which yielded 3 Cn epitopes, this work identified 8, largely
conserved, CI'L epitopes from 4 pre-erythrocytic antigens restricted by
several different HLA class I alleles. These epitopes were also
recognised in Tanzanians in an area of more intense P. foldpunan
transmission and the C l l recognised endogenously processed antigen.
2 CD8+ (JIZ epitopes were identified in ESAT-6, a secreted antigen
specific for M. tuberculosis complex but absent in BCG. CTL exhibited
MHC class I-restricted peptidespecific interfaon-gamma release and
lytic activity and recognised endogenously processed antigen. CD8+
CTL were detected only in healthy contacts and patients with
lymphadenitis (where the bacillus is contained by a vigorous host
response) but in none of the patients with pulmonary or extrapulmonary
disease, suggesting that CD8+ CTL are associated with protection.
These studies establish that CD8+ Cn specific for non-viral
intracellular pathogens are induced during natural infection in man. This
work endorses current efforts to develop mL-inducing vaccines against
P. fakiponon and M. tuberculosis and identifies candidate antigens for
inclusion in subunit vaccines.
Y4
TWO-STAGE GENOME-WIDE SEARCH I N
INFLAMMATORY B O W E L DISEASE: S T R O N G
E V I D E N C E F O R S U S C E P T I B I L I T Y LOCI ON
CHROMOSOMES 3,7 AND 12
Jack Satsa i1.3, Miles Parkefii.3, Eduoard Louis'*3, Ken WelsQ,
John I Bel?', Derek P Jewel1 .
Nuffield Departments of Medicine' and Sur ery', Oxford
Radcliffe Hospiyls, Oxford and the Wellcome g u s t Centre for
Human Genetics , Oxford
Concordance rates in siblings and twin pairs have provided
strong evidence that genetic predisposition is important in the
gthogenesis of the inflammatory bowel diseases (IBD).
owever, neither Crohn's disease (CD) nor ulcerative colitis (UC)
has a simple Mendelian pattern of inheritance; the model which
seems most pertinent to inflammatory bowel disease is that of a
heterogeneous group of polygenic disorders. In the present study,
a systematic two-stage search of the human enome for
suscepiibility genes in inflammatory bowel &ease was
performed, involving 186 affected siblin pairs.
In the first stage, 89 sibling pairs wi%i IBD were genotyped at
260 microsatellite markers spanning the 22 autosomes, using
fluorescence labelled primers for polymerase chain reaction, and
semi-automated DNA fragment sizing technology. Allele sharing