Download THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) AND ITS

THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) AND ITS
ROLE IN AUTOIMMUNE DISEASES
Dr. Vatay Ágnes
Program leader: Prof. Dr. Füst György
Semmelweis University, Budapest
Molecular medical sciences
Experimental and clinical immunology Program
2004
INTRODUCTION
The products of the major histocompatibility complex (MHC) gene region play
an important role in the regulation of the intact immune response. There is a
relationship between the genes encoded in this region and the development of
some autoimmune diseases, like type 1 diabetes mellitus, inflammatory bowel
disease (IBD), psoriasis, etc…
There is a strong linkage disequilibrium between the genes encoded within this
region; some alleles of the MHC I and II region (HLA -genes) are inherited
together with some alleles of the genes encoded in the MHC III region.
Our scientific work was complex: first of all our aim was to examine this
linkage disequilibrium in two healthy Caucasian population, and second, to
determine the relationship between some autoimmune diseases, like type 1
diabetes mellitus or inflammatory bowel disease and some genes encoded in the
MHC region.
Because of the linkage disequilibrium seen inside the MHC region, extended
well-conserved gene linkages maintained, called ancestral haplotypes. In the
limelight stays the 8.1 ancestral haplotype (HLA-A1, B8, TNFa2b3, C2C, BfS,
C4AQ0, C4B1, HERV(C4)0, TNFA-308*2, DRB1*0301, DQA1*0501,
DQB1*0201), its relationship between different autoimmune diseases is well
known. Our study group have reported earlier, that the short C4B1 gene is part
of the 8.1 ancestral haplotype, which is part of the monomodular RCCX
structure.
The relationship between some MHC II (HLA -DR and DQ) alleles and the type
1 diabetes is well known. No earlie r data is available about the relationship
between these alleles and the Latent Autoimmune Diabetes in Adult (LADA),
which is the latent form of type 1 autoimmune diabetes, or whether some other
genes encoded in this region, like tumour necrosis factor alpha (TNF-? ) could
confluence the course of these diseases. As it was shown earlier, there is a
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strong relationship between the development of type 1 diabetes mellitus and the
HLA -DR4/DQ8, and HLA -DR3/DQ2 haplotype, while the presence of the
HLA -DR13/DQ6 and the HLADR11/DQ7 haplotype is protective against the
disease.
The relationship between the IBD and the MHC genes is
contradictive; some authors have reported a relationship between certain HLA
alleles and the IBD subgroups (Crohn disease and colitis ulcerosa), while others
have not confirmed this finding. The role of TNF-? is complex in the
pathogenesis of IBD: the anti-TNF-? monoclonal antibody treatment is
effective in some patients; and some authors have shown relationship between
the development of the disease and the TNF-? gene promoter polymorphism,
while others have not confirmed it.
AIMS
Our scientific work was complex: first of all our aim was to examine this
linkage disequilibrium in two healthy Caucasian population, and second, to
determine the relationship between some autoimmune diseases, like type 1
diabetes mellitus or inflammatory bowel disease and some genes encoded in the
MHC region.
1. The TNF-a promoter polymorphism, the RCCX modul variants; the
relationship between the C4A and C4B variants and the RCCX modul
variants
The RP-C4-CYP21-TNX (RCCX) modules and the TNF gene cluster are
probably the most polymorphic genomic regions in the human central MHC.
Using definitive methods for genotypic and phenotypic analyses of complement
components C4A and C4B, determination of the RCCX length variants, and
SSP-PCR/RFLP analyses of TNFA promoter polymorphisms at positions ?308
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and ?238, we studied the complex relationships between the C4 and TNFA
polymorphisms in two normal Caucasian populations.
2. The MHC II (DR, DQ) and MHC II (TNF-a) genes in type 1
autoimmune diabetes
The aim of the study was to determine whether there is any genetic difference
in the MHC II and MHC III region between the type 1 diabetes mellitus and its
latent form, the LADA. In the development of the two disease entities the
autoimmune courses play an important role; while the clinical appearance is
different: the type 1 diabetes has rapid progression which leads rapidly to the
exhaustion of the pancreatic ? cells, while in LADA the progression is slow.
We determined the HLA -DR and DQ haplotypes and the promoter
polymorphism of the tumour necrosis factor alpha (TNF-? ) gene at position –
238 and –308 in type 1 diabetes and in LADA patients, and in the healthy
Hungarian population.
3. The TNF-a promoter polymorphism in inflammatory bowel disease. The
effect of the TNF-? gene promoter polymorphism on the plasma CRP
levels.
The aim of this study was to determine the alleles encoded in the TNF-?
promoter region at position –238 and –308 in patients with Crohn’s disease and
ulcerative colitis. The measurement of plasma CRP level is an important
diagnostic tool of disease activity. Since TNF-? is one major regulator of the of
hepatic CRP production we found it interesting to test whether a relationship
between TNF-? promoter polymorphism and plasma CRP levels exists in IBD
patients in different clinical status.
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METHODS
Patients and controls
a)
We examined 69 type 1 diabetes patients and 42 LADA patients (WHO
criteria: no younger than 35 years of age at the onset of the diabetes, had
autoantibody positivity for either ICA or GAD, and did not require
insulin treatment for at least 6 months after the onset)
b)
We examined 125 patients with inflammatory bowel disease; 74 patients
with Crohn disease, 51 ulcerative colitis.
c)
As control data of 336 kidney donors and samples of 138 healthy
Hungarian volunteers were used for the MHC- and for the TNF alpha
promoter polymorphism determination, respectively
d)
Examining RCCX modul and the TNF-? promoter polymorphism one
normal Caucasian group comprised 80 females recruited form central
Ohio. The second group consisted of 125 healthy people recruited from
Budapest, Hungary.
Analytical techniques
Genomic DNA was isolated accoriding to Miller (Miller et al; N A Res 1988;
16:1215). The HLA -DR and DQ alleles were detected by PCR-SSP method.
The TNF alpha promoter polymorphism at position –238 and –308 was
determined by PCR-RFLP using the primers suggested by Day (Diabetologia
1998; 41:430), the PCR products were digested at 37 ?C with NcoI and MspI.
For Studying the RCCX modules we used Southern blot analysis, the DNA
probes used for these determinations as well as the method for detection of C4
mutants were described previously (Blanchong et al; Int Immunopharm. 2001;
1:365) The complement C4 alloptipization was made according to Sim
(Biochem J 1986; 239:763) The plasma C4 concentration and the C4A and
C4B proteins were analysed by radial immundiffusion method. C-reactive
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protein
serum
concentrations
were
measured
by
particle
enhanced
immunoturbidimetric assay (Roche).
Statistical analysis was performed using the GraphPad Prism V 3.00 for
Windows software package (GraphPad Software). Frequencies were compared
by the chi-square test, Fisher exact test or chi-square test for trend. Logistic
regression was applied to evaluate potential confounding by co-variables
(Statistica software). The median CRP levels of the groups were compared by
Mann-Whitney t-test. P-values less than 0.05 were considered significant.
RESULTS
1. Relationship between the complement component C4A and C4B
diversities and the TNF-? promoter polymorphism in two Caucasian
population
The allelic frequencies of TNFA ? 308A in haploid genomes were almost the
same in the two examined population: 16.4% and 17.5% in the Hungarians and
Ohioans, respectively. By contrast, there was significant difference in the
frequencies of the TNFA ? 238A alleles in the Budapest and Ohio populations,
which were 1.6% and 10%, respectively (p=0.0003).
We analysed the distribution of the mono-, bi- and trimodular RCCX
haplotypes in the Ohio and the Budapest cohort with and without the presence
of the TNF alpha –308 A allele. The RCCX modul consists of four, tandemly
repeated genes: RP, complement component 4 (C4A and C4B), steroid-21hydroxilase (CYP21), and tenascin X (TNX) gene. The duplication or deletion
of the C4 gene is always escorted by the duplication or deletion of the other
genes within the RCCX modul. One to three RCCX modules may be present in
an MHC haplotype, although quadrimodular RCCX haplotypes with four C4
genes have been demonstrated both in Caucasian and Asian individuals. The
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number of C4 genes present in a diploid genome, i.e., the gene dosage,
predominantly varies from 2 to 6 among different individuals. About threequarters of the C4 genes in the Caucasian populations have the endogenous
retrovirus HERV-K(C4) incorporated into the intron 9, creating a dichotomy of
C4 gene sizes which are either 20.6 kb or 14.2 kb in length. Each C4 gene may
code for a C4A protein or a C4B protein. In both populations there were
marked differences in the RCCX modular structure between the TNF alpha –
308 A allele carriers (– 308 AA and GA) and those who not carried the
mentioned allele (GG homozygous) (p<0,0001). 95.2% of the Hungarian
subjects with mono-S were carriers of the ?308A allele, and 94.1% of the Ohio
subjects with mono-S are carriers of the ? 308A allele.
The striking association of the mono-S RCCX structures with ?308A prompted
us to further look into the complement C4 genotypes and phenotypes and
compare the frequency of the probable C4AQ0 B1 haplotype among TNFA
?308A carriers and non-carriers. The results were very similar in the two
populations. There were 6 homozygous TNFA ?308A carriers in the two
populations. At least one probable C4AQ0 B1 haplotype occurred in all of these
subjects. Among the GA heterozygotes, 60% (35/58) had the probable C4AQ0
B1 haplotype while it occurred rarely, 7% (10/141) among the GG
homozygotes (p<0.0001).
We previously found that TNFA ?238A strongly associated with C4A6 in
patients with coronary artery disease. The frequency of C4A6 B1 haplotype
among TNFA ?238A carriers and non-carriers in these two populations of
healthy subjects were compared; the probable C4A6 B1 haplotypes were found
to be clustered among carriers of the ?238A allele.
The mean levels of the C4A proteins were lower in the –308 A carriers than in
the non-carriers and the difference was highly significant (p<0.001). A
significant difference was also found with the total C4 serum concentrations
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between the carriers and non-carriers (p=0.023) . By contrast the levels of the
C4B proteins did not differ (p=0.875) between the carriers and non-carriers of
the TNFA –308A allele.
2. Associations of type 1 diabetes and LADA with some DQ and DR alleles
Type 1 diabetes mellitus was found to be positively associated with the
presence of DQB1*0201 (DQ2), DQB1*0302 (DQ8), DRB1*03 (DR3) and
DRB1*04 (DR4) alleles, and negatively associated with DQB1*0301 (DQ7)
and DRB1*11 (DR11) alleles. The DQ2 allele (p=0.001, OR=2.4 ?1.4-4.4?) the
DQ8 allele (p=0.00001, OR=4 ?2.1-7.4?), the DRB1*03 allele (p=0.00009,
OR=3.2 ?1.8-5.6?), and the DR4 allele was significantly more frequent
(p<0.00001, OR=4.4 ?2.4-8?) in type 1 diabetic patients compared to control
group. In contrast, significant negative association was observed in the
occurrence of DQ7 and DR11 alleles (p=0.00009 and p=0.001, OR=0.15 ?0.070.3? and OR=0.05 ?0.01-0.2?, respectively) among patients with type 1 diabetes
when compared to control group.
LADA was positively associated with DQ8 (p=0.0006, OR=3.6 ?1.7-7.5?) and
DR4 (p=0.0002, OR=3. 7 ?1.9-7.4?) and negatively associated with DQ7
(p=0.012, OR=0.38 ?0.18-0.8?) and DR11 (p=0.006, OR=0.28 ?0.1-0.7?) alleles
compared to the control group, while no significant association was detected
between the presence of DR3 and DQ2 alleles and LADA compared to the
controls
3. Associations of type 1 diabetes and LADA with some HLA-DR/DQ
haplotypes
In type 1 diabetes the haplotype frequencies of HLA -DR3/DQ2 and HLA DR4/DQ8 were significantly higher (p=0.00008 and p=0.00001, OR=3.2 ?1.85.7? and OR=4.1 ?2.2-7.6?, respectively), while the frequency of HLA -
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DR11/DQ7 haplotype was significantly lower (p=0.00006, OR=0.05 ?0.010.2?) than in control group. There was a significant positive association
between LADA and haplotype frequency of HLA -DR4/DQ8 (p=0.0005,
OR=3.66 ?1.7-7.6?), too, while there was no significant difference at the
frequency of HLA -DR3/DQ2 haplotype between LADA and the control group.
The frequency of HLA -DR11/DQ7 haplotype was significantly lower
(p=0.007, OR=0.28 ?0.1-0.7?) in LADA than in control group similarly to type
1 diabetes.
4. Decreased frequency of the TNF alpha promoter polymorphism in
patients with LADA
The frequency of G? A substitution at position –308 was (which is associated
with elevated TNF-? production) significantly lower in patients with LADA
when compared to the control group (p=0.0179). Similar difference was
observed when patients with LADA and type I diabetes (p=0.0222) were
compared. No significant difference was observed, however, between type 1
diabetes and control group.
5. The presence of the 8.1 ancestral haplotype in type 1 diabetes and in
LADA
We analysed the results with Arlequin software in order to calculate the
maximum likelihood allele frequencies, and we have found strong linkage
disequilibrium between the HLA -DQ, DR and the A allele of a part of the TNF
? promoter region at position –308 (p=0.0004). The presence of the HLA DR3/DQ2/ TNF-? – 308 A allele, previously described as the 8.1 ancestral
haplotype was significantly less frequent among LADA patients than in the
type 1 diabetic group (allele frequency: 0.0427 vs. 0.138, p=0.018), while the
DR11/DQ7/ TNF-? –308 G allele was mo re frequent among LADA patients
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compared to the type 1 diabetic group (allele frequency: 0.075 vs. 0.022,
p=0.039).
6. The TNF alpha promoter polymorphism in inflammatory bowel disease.
Relationship between CRP level, clinical parameters and the TNF alpha
carrier status.
The number of the –308 A allele carriers was significantly lower in patients
with IBD (all of the carriers were GA heterozygotes) (16%) when compared to
the control group (both GA heterozygous and AA homozygous) (33%,
p=0.0009). Similar difference was observed when compared the patients with
Crohn’s disease (15%) to the healthy population (p=0.0035). The same
tendency was observed when ulcerative colitis patients (18%) and the healthy
controls were compared (p=0.036). No significant difference was observed
between Crohn’s disease and ulcerative colitis in the carrier frequency of the
TNF2 allele.
We have found that the median CRP level was significantly higher in the active
phase of the disease (median CRP=18.46 mg/l) compared to the inactive phase
(median CRP=2.4 mg/l, p=0.002) among the -308 A (TNF2) allele carriers,
while this difference was not significant when compared the CRP levels in the
active (median CRP=9.95 mg/l) and inactive phase (median CRP=6.1 mg/l,
p=0.084) among those who do not carry the mentioned allele (wild type, -308
GG homozygous patients).
DISCUSSION
The aim of our study was to examine the MHC gene region located on the short
arm of the chromosome 6 in two healthy Caucasian population, in type 1
diabetic patients, LADA patients and inflammatory bowel disease.
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We have compared the characteristics of the RCCX modules in subjects with
and without the TNFA ?308A allele. Marked differences were found in several
properties of the RCCX modules between the TNFA ?308A carriers and noncarriers: monomodular RCCX modules with one short C4B gene is a
characteristic for carriers of the TNFA ?308A allele. When the complement
C4A and total C4 protein concentrations in serum were compared between the
TNFA ? 308A carriers and non-carriers, there were significantly lower
concentrations of C4A and total C4 among the carriers. Complete or partial
deficiencies of C4A had been noted as important risk factors of systemic lupus
erythematosus (SLE). A significant increase was found in the frequency of
TNFA ? 308A in the SLE patients. The paradox is that TNFA ? 308A is likely to
increase the expression levels of TNF-? , which was shown to be protective
against the pathogenesis of the lupus disease in animal experiments. To
understand the roles of TNF-? and C4 in autoimmune diseases, it is relevant to
segregate the two categories of the TNFA ?308A, i.e., with and without mono-S
RCCX structures (or in other words, with and without C4A deficiency).
The association between the TNFA ?308A allele, the C4AQ0 phenotype and
monomodular RCCX structure with a short C4B1 gene in the Caucasians with
HLA -A1/B8/DR3 is described as the 8.1 ancestral haplotype (AH-8.1). The
AH-8.1 is associated with an increased risk to develop a variety of diseases
including autoimmune diseases. The mechanism of this association is, however,
a highly controversial issue. Several data indicate that carriers of the AH-8.1
are high producers of TNF-? , sELAM, cortisol and IL-10 and consequently to
predominance of the type 2 cytokine response. Monomodular RCCX module
with a short C4B gene that means the absence of C4A is a characteristic feature
of the AH-8.1. Since the ability of C4A for handling immune complexes is
better than that of C4B, a carriage of the AH-8.1 may be associated with
impaired handling of circulating immune complexes, decreased ability of
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immune complexes to bind to complement receptors, and a reduced ability to
inhibit immune precipitation. All of these procedures may contribute to the
tissue inflammation in SLE and other autoimmune diseases.
We also observed strong linkage disequilibrium was also observed between the
C4A6 B1 haplotype and the TNFA ?238A allele in both populations.
According to our study there are differences in the genetic background between
the two diabetes groups. This conclusion is based on the following
observations: The frequency of the HLA -DR4/DQ8 haplotype was higher
among both type 1 diabetes and LADA compared to the control population, and
the HLA -DR11/DQ7 haplotype was found to be protective against both forms
of autoimmune diabetes. In contrast, the frequency of the HLA -DR3/DQ2
haplotype differed in type 1 diabetes and LADA this type was more frequent in
patients with type 1 diabetes.
We have shown that there was a significant difference in TNF alpha promoter
polymorphism at position –308. The number of TNF-? –308 A carriers was
significantly lower in LADA patients compared to both type 1 diabetic patients
and controls, while the presence of TNF-? –308 A allele was about the same in
type 1 diabetes and in controls. The DR3/DQ2/ TNF ? –308 A allele, known to
be the part of the 8.1 ancestral haplotype, was significantly less frequent, while
the DR11/DQ7/ TNF-? –308 G was more frequent among LADA patients
compared to the type 1 diabetic group
The destruction of pancreatic beta cells is more rapid in type 1 diabetes than in
LADA. It is tempting to speculate that this difference is related to our present
findings on the differences of the genetic background of the two groups. The
final destruction of pancreatic beta cells is caused by inflammation, in the
patients carrying TNF ? –308 A allele the inflammation is more progressive,
than in patients not carrying the mentioned allele. The low presence of TNF ? –
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308 A allele (known to be associated with high amount of TNF-? ) in LADA
could be one of the factors responsible for the relatively slow progression.
We found association between IBD and the TNF alpha –308 polymorphism; in
Hungarian Caucasian subjects the frequency of the –308 A allele was decreased
in IBD patients and this association was more pronounced in CD patients. Most
importantly we report in this study on significantly higher median CRP levels
in the active phase of the disease compared to the inactive phase among the 308 A allele carriers, while this difference was not significant when compared
the CRP levels in the active and inactive phase among the -308 GG
homozygous patients. This observation indicates that TNF carrier status may
significantly influence disease course as reflected by plasma CRP levels.
Expression of CRP is regulated mainly at transcriptional level, interleukin-6
(IL-6) is the principal inducer of the CRP gene, while IL-1, glucocorticoids and
certain other factors with IL-6 enhance its effect. IL-6 is tightly regulated at the
level of expression, IL-1 and TNF-? activate the IL-6 gene transcription.
Hence, increased IL-1/TNF-? production and decreased estrogen synthesis
enhance IL-6 expression and consequently CRP production. This could be one
of the factors responsible for the observed differences. The G ? A substitution
at the promoter region at position -308 is associated with elevated TNF alpha
secretion, which could lead to the persisting of the microbial agent in the
luminar flora, which can be the triggering factor of the disease.
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The most important findings of the Ph.D. study, conclusions.
1.
We have found strong association between the TNF alpha –308 A allél and
the mono-S RCCX structure C4AQ0 C4B1 haplotype in both Caucasian
populations. The association between the TNFA ?308A allele, the C4AQ0
phenotype and monomodular RCCX structure with a short C4B1 gene in the
Caucasians with HLA A1 B8 DR3 is described as the 8.1 ancestral
haplotype (AH-8.1). The AH-8.1 is associated with an increased risk to
develop a variety of diseases including autoimmune diseases .
2.
We also observed strong linkage disequilibrium was also observed between
the C4A6 B1 haplotype and the TNFA ? 238A allele in both populations.
3.
We have shown that there are marked differences in respect of the TNF-?
promoter polymorphism at position –308 between type 1 diabetes mellitus
and LADA. The number of the –308 A allele carriers was decreased in
LADA patients compared to type 1 diabetes and the healthy control group.
The frequency of the HLA -DR3/DQ2 haplotype was significantly higher
among type 1 diabetic patients than in control group, while there was no
significant difference between LADA and the control group. The presence
of the HLA -DRB1*03/DQB1*02/ A allele at position –308 at the TNF ?
promoter region, previously described as the 8.1 ancestral haplotype was
significantly less frequent among LADA patients than in the type 1 diabetic
group
(allele
frequency:
0.0427
vs.
0.138,
p=0.018),
while
the
DRB1*11/DQB1*0301/ G allele at position –308 at the TNF ? promoter
region was more frequent among LADA patients compared to the type 1
diabetic group.
4.
We found association between IBD patients and the TNF alpha –308
polymorphism in Hungarian Caucasian subjects the frequency of the –308 A
allele was decreased in IBD patients and this association was more
pronounced in CD patients.
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5.
Most importantly we report in this study on significantly higher median
CRP levels in the active phase of the disease compared to the inactive phase
among the -308 A allele carriers, while this difference was not significant
when compared the CRP levels in the active and inactive phase among the 308 GG homozygous patients This observation indicates that TNF carrier
status may significantly influence disease course as reflected by plasma
CRP levels. The G ? A substitution at the promoter region at position -308
is associated with elevated TNF alpha secretion, which could lead to the
persisting of the microbial agent in the luminar flora. As an important novel
observation we report here a possible modifying effect of TNF-? carrier
status on disease course through the tight interaction with plasma CRP
levels.
Published own articles summarised in the Ph.D. work:
1.
Vatay A, Rajczy K, Pozsonyi E, Hosszufalusi N, Prohaszka Z, Fust G,
Karadi I, Szalai C, Grosz A, Bartfai Z, Panczel P: Differences in the
genetic background of latent autoimmune diabetes in adults (LADA)
and type 1 diabetes mellitus. Immunol Lett. 2002 Nov 1;84(2):109115.
2.
Vatay A, Yang Y, Chung EK, Zhou B, Blanchong CA, Kovacs M,
Karadi I, Fust G, Romics L L, Varga L, Yu CY, Szalai C: Relationship
between complement components C4A and C4B diversities and two
TNFA
promoter
polymorphisms
in
two
healthy
Caucasian
populations. Hum Immunol. 2003 May;64(5):543-52.
3.
Vatay A, Bene L, Kovacs A, Prohaszka Z, Szalai C, Romics L, Fekete
B, Karadi I, Fust G: Relationship between the tumor necrosis factor
15
alpha polymorphism and the serum C-reactive protein levels in
inflammatory bowel disease. Immunogenetics. 2003 Jul;55(4):247-52.
Other published literature:
Yang Y, Chung EK, Zhou B, Blanchong CA, Yu CY, Fust G, Kovacs M, Vatay
A, Szalai C, Karadi I, Varga L: Diversity in intrinsic strengths of the human
complement system: serum C4 protein concentrations correlate with C4 gene
size and polygenic variations, hemolytic activities, and body mass index. J
Imunol. 2003 Sep 1;171(5):2734-45.
Hosszufalusi N, Vatay A, Rajczy K, Prohaszka Z, Pozsonyi E, Horvath L,
Grosz A, Gero L, Madacsy L, Romics L, Karadi I, Fust G, Panczel P: Similar
genetic features and different islet cell autoantibody pattern of latent
autoimmune diabetes in adults (LADA) compared with adult-onset type 1
diabetes with rapid progression. Diabetes Care. 2003 Feb;26(2):452-7.
Kocsis J, Veres A, Vatay A, Duba J, Karadi I, Fust G, Prohaszka Z: Antibodies
against the human heat shock protein hsp70 in patients with severe coronary
artery disease. Immunol Invest. 2002 Aug-Nov;31(3-4):219-31.
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