This presentation Prof. Dr. Vladimir Trajkovski: IMUNOGENETIC ANALYSES IN PERSONS WITH AUTISM IN REPUBLIC OF MACEDONIA should presented in conference in Barcelona, but he wasn't there because it was cancelled.

1. UNIVERSITY „ST. CYRIL AND METHODIUS“ - SKOPJE
FACULTY OF PHYLOSOPHI - SKOPJE
INSTITUTE FOR SPECIAL EDUCATION AND REHABILITATION
IMUNOGENETIC ANALYSES IN
PERSONS WITH AUTISM IN
REPUBLIC OF MACEDONIA
Vladimir Trajkovski, M.D, Ph.D.
Barcelona
11.01.2003

2. 2
INTRODUCTION
• Autism is a developmental disorder of unknown etiology
characterized by severe communication, social, and
behavioral abnormalities.
• A number of factors have been implicated in the pathogenesis
of autism including, genetic, environmental, and
immunological factors.
• Immunologic evaluation of autistic children showed:
− depressed lymphocyte proliferation to mitogens,
− impaired cellular immunity of macrophages and NK cells,
− circulating autoantibodies against putative serotonin brain
receptors, myelin basic protein, glial fibrillary acid protein
and neuron-axon filament proteins,
− elevation of T-cell activation antigens such as soluble
interleukin-2, and soluble CD8,
− increased levels of DR+ activated T cells,
− the levels of IL-12 and IFN-γ are selectively elevated,
− alterations in serum immunoglobulin classes and subclasses,
− increased frequency of the C4B null allele and linkage to

3. 3
AIMS AND TASKS OF THE STUDY
1.Establishing the immunogenetic
structure - HLA-DNA typization of
persons with autism and their families;
2.Determination the level of
immunoglobulin classes and subclasses
in the serum;
3.Studying the specific allergic antibodies
in the serum.

4. 4
MATERIAL AND METHODS
• 50 persons with autism have been analyzed with
existing data in the Institute for Mental Health in
Skopje, Institute for Rehabilitation of Hear, Speech and
Voice in Skopje, Special Institute in Demir Kapija,
Special School „Dr. Zlatan Sremac“ in Skopje, Centers
for Social Work and Medical Centers in Macedonia.
• Blood has been taken for immunogenetic and
immunological analyzes from 35 persons with autism,
22 their biological brothers/sisters, 27 mothers and 23
fathers, as well from 98 healthy unrelated persons.
• Immunogenetic and immunological analyses have been
organized and made at the laboratories of the Institute of
Immunobiology and Human Genetics, Medical Faculty
in Skopje.
• DSM-IV and ICD-10 criteria was established by at least
two psychiatrists made diagnosis of infantile autism.

5. 5
MATERIAL AND METHODS
• Ten milliliters of venous blood was drawn from each donor
by the standard venipuncture in vacutaners with EDTA (K3).
At the time of blood drawing, none of autistic children were
receiving any medication or antipsychotic drug. Plasma
samples were separated by centrifugation and stored at –200
C
till the determination.
• Genomic DNA was extracted from whole blood using
standard proteinase K digestion method followed by salting-
out extraction and ethanol precipitation.
• HLA DNA typing was performed with high resolute
techniques established on 13th International
Histocompatibility Working Group.
• HLA DNA typing of class I genes (HLA-A, -B, and -C) was
performed using a Reverse Line Strip method (RLS), and the
Sequencing Based Typing method (SBT) was used for

6. 6
MATERIAL AND METHODS
• Serum specific allergens from the food (f76 alfa-
lactalbumin, f77 beta-lactglobulin, f78 casein, f79
gluten, f98 gliadin) have been determined with
automatic immunofluorescent devise with solid
phase called Pharmacia UniCAP 100.
• Serum immunoglobulin classes (IgA, IgG, IgM and
IgE) and subclasses (IgG1, IgG2, IgG3 and IgG4) are
determined immunonephelometric by automated
Dade-Behring Nephelometer Analyzer.
• The survey is realized in the period between the
April 2000 to the April 2002 year on the territory of
the Republic of Macedonia.

7. 7
MATERIAL AND METHODS
• The research data have been stored, classified and
processed with standard statistical programme SPSS for
Windows 7.5 version and Statgraphics Plus for
Windows 2.1 version.
• Descriptive statistic methods used in the study are:
central tendencies measures, variability and percentages.
• The differences between numeric variables have been
analyzed with Student’s t-test.
• Nonparametric tests of Kolmogorov-Smirnov, Mann-
Whitney and Kruskal-Wallis have been used in
asymmetric statistical distribution of data.
• Statistical analysis was performed with Arlequin v.2.000
software kindly provided by Excoffier and Slatkin. This
program calculated HLA-A, -C, -B, and –DRB1 allele
frequencies, haplotype frequencies.
• P values of 0.05 or less were considered significant.

8. 8
RESULTS
AND
DISCUSSION
about epidemiological
characteristics

9. 9
Country / Author Prevalence
Macedonia 0.25 / 10.000 (2002)
Trefferet et al. 0.7 / 10.000 (1970)
Honda et al.- Japan 21.1 / 10.000 (1996)
Japan - 21.610 1.3 / 1.000 children 3 years
England, Sweeden,
Danmark, France and USA
4 - 5 / 10.000
Bryson,Gilberg, Costello 10 / 10.000
New Scotland - 20.800 1/1.000 (children 6-14 years)
Rapin (Sweeden) 1-2/1.000 (children 3-17 years)
Croatia(Shvel) 7 / 10.000 (1986)
RESULTS

10. 10
RESULTS
PrevalencePrevalence
− The most comprehensible epidemiological study is Eric
Fombonne’s, where the author elaborates 23
epidemiological studies published on English from all
around the world in the period between 1966 and 19981966 and 1998.
In these studies have been included four million people,
from which 1533 persons1533 persons with autism. The estimation
of prevalence is in the range from 0,7 to 21 / 10.000,
with median value of 5,2/ 10.000 children.5,2/ 10.000 children.
− The rate of prevalence for preschool children is
estimated in 5 studies and it is 0,81, for school children
in 11 studies with value of 1,30 and rate of 0,99 for
adolescents estimated in 7 studies;

11. RESULTS
Gender
Male
72%
Female
28%
Coefficient of sex ratio is 2.5 male on 1 femaleCoefficient of sex ratio is 2.5 male on 1 female
person.person.
Gillberg C, Wing LGillberg C, Wing L (1999) male gender is more(1999) male gender is more
frequently affected compared to female (2.1:1 dofrequently affected compared to female (2.1:1 do
3.9:1);3.9:1);
FombonneFombonne (1999), male/female sex ratio varied from(1999), male/female sex ratio varied from
1.33 to 16.0 : 1 with median value 2.6.

12. Age
Median age in our study is 10 years.
Mean age of 10.8 years (SD = 5.77) is very similar
with French epidemiological study of Fombonne et
al., where mean age of the children with autism is
11.6 years (SD = 2.6).
4
13 13
5
2 2
0
2
4
6
8
10
12
14f
0-4 5-9 10-14 15-19 20-24 25-29

13. 13
RESULTS
AND
DISCUSSION
about immunogenetic structure
in persons with autism

14. 14
− The genes of HLA system are located on the short arm of 6th
chromosome (6p 21);
− The genes of HLA cover 3500 kb pairs of DNA;
− The human extended MHC cover a distance of 4
centimorgans of DNA (the frequencies of Crossing-over
within MHC is 4% in each meiosis).
− Several different HLA class I and class II genes encode
proteins with different ranges of abilities to bind pathogenic
peptides.
− The HLA is highly polymorphic (there are multiple forms-
alleles of an HLA gene.
− MHC genes and antigens are separated into 3 classes of
HLA:
class I: HLA-A, HLA-B, HLA-C
class II: HLA-DR, HLA-DP, HLA-DQ
class III: C2, BF, C4A and C4B
Structure of HLA molecules and genes

15. 15
Class II
B2 A2 B1 A1 A B B2 A2 B1 A1 B1 B2 B3 B4 B5 A
DP DN DQ DR
Class III
21B C4B 21A C4A Bf C2 A B B C E A G F
Class I
HLA system: The loci contents functional genes (black
rectangles) and pseudogenes (white rectangles).

16. 16
NS1.119 (12.9)23 (11.7)A*03
NS0.5718 (25.7)74 (37.7)A*02
70 (100.0)196 (100.0)Total
NS1.605 (7.1)9 (4.6)A*68
NS01 (0.5)A*66
NS5.742 (2.9)1 (0.5)A*33
NS0.452 (2.9)12 (6.1)A*32
NS2.831 (1.4)1 (0.5)A*31
NS1.411 (1.4)2 (1.0)A*30
NS0.551 (1.4)5 (2.5)A*29
NS0.552 (2.9)10 (5.1)A*26
NS1.892 (2.9)3 (1.5)A*25
NS1.8013 (18.6)22 (11.2)A*24
NS1.411 (1.4)2 (1.0)A*23
NS1.707 (10.0)12 (6.1)A*11
NS0.876 (8.6)19 (9.7)A*01
pOdds
ratio
OR
AF
N (%)
autism
AF
N (%)
control
HLA-A
alleles
HLA-A allele frequencies in persons with autism and control
subjects.

17. 17
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
allele frequencies
A*01 A*02 A*03 A*11 A*23
A*24 A*25 A*26 A*29 A*30
A*31 A*32 A*33 A*66 A*68
autism
control
HLA-A allele frequencies in persons with autism and
control subjects.

18. 1870 (100.0)196 (100.0)Total
NS03 (1.5)C*17
NS1.892 (2.9)3 (1.5)C*16
NS0.935 (7.1)15 (7.6)C*15
NS1.815 (7.1)8 (4.1)C*14
NS1.2814 (20.0)32 (16.3)C*12
NS8.733 (4.3)1 (0.5)C*08
NS0.5612 (17.1)53 (27.0)C*07
NS2.206 (8.6)8 (4.1)C*06
NS2.7108 (4.1)C*05
NS0.596 (8.6)27 (13.8)C*04
0.032.74*9 (12.9)10 (5.1)C*03
NS0.876 (8.6)19 (9.7)C*02
NS0.612 (2.9)9 (4.6)C*01
pOdds ratio
OR
AF
N (%)
autism
AF
N (%)
control
HLA-C
Alleles
HLA-C allele frequencies in persons with autism and control subjects.

19. 19
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
allele frequencies
C*01 C*02 C*03 C*04 C*05
C*06 C*07 C*08 C*12 C*14
C*15 C*16 C*17
autism
control
HLA-C allele frequencies in persons with autism and control
subjects.
∗

20. 20 NS01 (0.5)B*78
NS01 (0.5)B*58
NS5.742 (2.9)1 (0.5)B*57
NS01 (0.5)B*56
NS2.976 (8.6)6 (3.1)B*55
NS03 (1.5)B*53
NS2.445 (7.1)6 (3.1)B*52
NS1.1313 (18.6)33 (16.8)B*51
NS0.931 (1.4)3 (1.5)B*49
NS01 (0.5)B*45
NS0.684 (5.7)16 (8.2)B*44
NS03 (1.5)B*41
NS0.753 (4.3)11 (5.6)B*40
NS0.341 (1.4)8 (4.1)B*39
NS1.264 (5.7)9 (4.6)B*38
NS8.733 (4.3)1 (0.5)B*37
NS0.505 (7.1)26 (13.3)B*35
NS0.932 (2.9)6 (3.1)B*27
NS0.978 (11.4)23 (11.7)B*18
NS2.324 (5.7)5 (2.5)B*15
NS8.733 (4.3)1 (0.5)B*14
NS0.551 (1.4)5 (2.5)B*13
NS0.382 (2.9)14 (7.1)B*08
NS0.693 (4.3)12 (6.1)B*07
pOdds ratio
OR
AF
N (%)
autism
AF
N (%)
control
HLA-B
allele

21. 21
HLA-B allele frequencies in persons with autism and control.
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
allele frequencies
B*07 B*08 B*13 B*14 B*15 B*18 B*27 B*35
B*37 B*38 B*39 B*40 B*41 B*44 B*45 B*49
B*51 B*52 B*53 B*55 B*56 B*57 B*58 B*78
autism
control

22. 22
HLA-DRB1 allele frequencies in persons with autism and control
subjects.
70 (100.0)196 (100.0)Total
NS0.6110 (14.3)38 (19.4)DRB1*16
NS1.019 (12.9)25 (12.7)DRB1*15
NS2.443 (4.3)6 (3.1)DRB1*14
NS1.287 (10.0)18 (9.2)DRB1*13
NS03 (1.5)DRB1*12
NS0.8617 (24.3)47 (24.0)DRB1*11
NS2 (2.9)0DRB1*10
NS01 (0.5)DRB1*09
NS06 (3.1)DRB1*08
NS0.854 (5.7)13 (6.6)DRB1*07
NS0.543 (4.3)15 (7.6)DRB1*04
NS1.005 (7.1)14 (7.1)DRB1*03
0.0123.10*10 (14.3)10 (5.1)DRB1*01
pOdds ratio
OR
AF
N (%)
autism
AF
N (%)
control
HLA-
DRB1
allele

23. 23
HLA-DRB1 allele frequencies in persons with autism and
control subjects.
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
allele frequencies
DRB1*01 DRB1*03 DRB1*04 DRB1*07 DRB1*08
DRB1*09 DRB1*10 DRB1*11 DRB1*12 DRB1*13
DRB1*14 DRB1*15 DRB1*16
autism
control
∗

24. 24
Frequencies of most frequent haplotypes in persons with
autism and control subjects.
NS03 (1.5)A*11-C*12-B*52-DRB1*16
NS03 (1.5)A*02-C*06-B*13-DRB1*07
NS04 (2.0)A*02 C*07 B*18 DRB1*11
NS06 (3.1)A*01-C*07-B*08-DRB1*03
NS2 (2.9)0A*11-C*03-B*55-DRB1*14
NS2 (2.9)0A*24-C*03-B*55-DRB1*16
NS2 (2.9)0A*11-C*12-B*52-DRB1*15
p
HF
N (%)
autism
HF
N (%)
control
Haplotype

25. 25
DISCUSSION
− Stubbs and Magenis (1980) are the first who speak about the
association between autism and HLA. They have found
increased frequency of HLA-A10 antigen in 10 fathers of
children with autism.
− Reed Warren has found an amino acid sequence in the genes
of the MHC that is expressed more frequently in autistic
subjects than in controls. This sequence is responsible for
development of immunological response towards antigens
and it’s the major connection with other autoimmune
diseases.
− Reed Warren and Daniels report that extended haplotype
B44-SC30-DR4 is found in 40% of autistic subjects and their
mothers, opposite to 2% in control group;
− The third hyper variable region of DRB1 allele has strong
association with autism. HVR-3 DRB1*0401, DRB1*0404
and DRB1*0101 alleles are present in 23 from 50 (46%)
autistic subjects, compared to 6 from 79 (7.5%) normal
subjects.

26. 26
DISCUSSION
Stubbs et al. (1985) determined the HLA types of the parents
of 52 autistic probands. They found increased sharing of at
least one antigen among parents versus a control group from
the literature (75 versus 22 per cent).
Rogers et al. (1999) made linkage analysis, using genetic
marker loci in HLA region at multiplex families with autism.
They examined sharing of alleles identical by descent in 97
affected sib pairs from 90 families. Their results
demonstrated no deviation from the null expectation of 50%
sharing of alleles in this region.
Torres et al. (2002) have evaluated possible contributions of
HLA-DRB1 alleles to autism in 103 families of Caucasian
descent. The TDT indicated that autistic probands inherited
the DRB1*04 allele more frequently than expected (p=0.026)
from the fathers and they found protective association with
DRB1 *13 and *14 alleles.

27. 27
RESULTS
AND
DISCUSSION
about plasma concentration of
immunoglobulin classes and
subclasses

28. 28
<0.020.45±0.140.69±0.14*IgG4
NS0.45±0.090.46±0.07IgG3
NS2.34±0.522.44±0.38IgG2
NS8.09±0.608.45±0.82IgG1
NS12.39±0.9613.14±1.27IgG
NS1.20±0.151.36±0.31IgM
NS1.52±0.301.63±0.33IgA
pHealthy
brothers and
sisters
(n=21)
Autism
(n=35)
Antibodies
(g/L)
RESULTS
Plasma concentration of immunoglobulin classes and
subclasses.

29. 29
RESULTS
0
2
4
6
8
10
12
14
IgA IgM IgG IgG1 IgG2 IgG3 IgG4
Autism Control
Plasma concentration of immunoglobulin classes and
subclasses
∗

30. 30
Concentration of immunoglobulines in male and female
persons with autism
NS0.58±0.370.78±0.45IgG4
NS0.43±0.210.46±0.19IgG3
NS2.78±1.612.41±0.94IgG2
NS9.41±2.418.06±2.57IgG1
NS14.54±4.0213.04±3.99IgG
NS1.44±0.491.35±1.12IgM
NS2.00±1.151.44±0.72IgA
pFemale (n=8)Male (n=20)g/L
RESULTS

31. 31
Differences in concentration of Ig classes and subclasses
between Gupta et al. (1996) and our results
0
1
2
3
4
5
6
7
8
9
IgM IgA IgG1 IgG2 IgG3 IgG4
Trajkovski et al.
Gupta et al.
RESULTS
∗
∗
∗
∗
p<0.001 p<0.001p<0.002 p<0.009

32. 32
DISCUSSION
→Plyoplys AV, Greaves A, Yoshida W. (1989) didn’t
find abnormally increased concentrations of
immunoglobulins in the serum.
→Ferrari et al. (1988) found elevated IgG, IgM and IgA
antibody-titres in the serum of autistic patients, although
significance was only reached for IgG-titers.
→Gupta et al. Found in 20% of patients IgA deficiency
and low plasma levels of IgG subclass, but they didn’t
make inferential statistics.
→In our study children with autism have significantly
elevated values of IgG4 (p<0.02) compared to their
healthy brothers and sisters.
→Increased plasma level of IgG4 in the children with
autism could be connected with increased autoimmunity
and/or allergies in this children.

33. 33
RESULTS
AND
DISCUSSION
about plasma concentration of
specific allergic antibodies

34. 34
Plasma concentration of specific allergic IgE antibodies
(mgA/L)
0.35±0.020.35±0.000.35±0.000.52±0.74
f79 gluten
0.35±0.00*0.35±0.00*0.35±0.01*0.45±0.35*
f78 casein
0.35±0.00*0.35±0.00*0.35±0.00*0.43±0.26*
f77 beta-
lactglobulin
0.35±0.000.35±0.000.39±0.180.47±0.43
f76 alfa-
lactalbumin
Father
n=23
Mother
n=27
Brothers/
sisters
n=22
Autism
n=35
Allergens/ IgE
concentration
Values are expressed as mean± SD; p-significance of differences
between persons with autism, brothers/sisters, mother and father
(*p < 0 . 0 5) .

35. 35
0
0.1
0.2
0.3
0.4
0.5
0.6
f76 alfa-
lactalbumin
f77 beta-
lactglobulin
f78 casein f79 gluten
autism
brothers/sisters
mother
father
allergenes
Plasma concentration of specific allergic IgE
antibodies (mgA/L)
∗ ∗

36. 36
Plasma concentration of specific allergic IgG antibodies
(kU/L)
3.29±1.60†
4.18±1.67†
7.70±8.4012.83±21.06†
f98 gliadin
1.31±2.54*2.64±4.95*12.98±19.9722.84±26.79*
f78 casein
0.74±1.56*1.15±3.16*5.44±8.849.44±10.35*
f77 beta-
lactglobulin
0.86±2.01*1.07±2.20*9.27±20.9810.43±13.24*
f76 alfa-
lactalbumin
Father
n=23
Mother
n=27
Brothers-
sisters
n=21
Autism
n=35
Allergens/
IgG concentration
Values are expressed as mean± SD; p-significance of differences
between persons with autism, brothers/sisters, mother and father
(*p < 0.001); ( †
p < 0.04).

37. 37
0
5
10
15
20
25
f76 alfa-
lactalbumin
f77 beta-
lactglobulin
f78 casein f98 gliadin
autism
brothers/sisters
mother
father
Plasma concentration of specific allergic IgG antibodies
(kU/L)
allergenes
∗
∗
†

38. 38
Plasma concentration of specific allergic IgA
antibodies (kU/L)
1.67±0.891.76±0.691.37±0.801.55±0.81f98 gliadin
1.21±0.35†
1.27±0.51†
1.44±1.02*2.10±1.94*†
f78 casein
1.26±0.481.33±0.601.10±0.22*1.25±0.45*f77 beta-
lactglobulin
1.17±0.351.24±0.501.10±0.201.19±0.38f76 alfa-
lactalbumin
Father
n=23
Mother
n=27
Brothers-
sisters
n=22
Autism
n=35
Allergens/
IgA
concentration
Values are expressed as mean± SD; p-significance of differences
between persons with autism, brothers/sisters, mother and father
†

39. 39
0
0.5
1
1.5
2
2.5
f76 alfa-
lactalbumin
f77 beta-
lactglobulin
f78 casein f98 gliadin
autism
brothers/sisters
mother
father
Plasma concentration of specific allergic IgA antibodies
(kU/L)
allergenes
∗
∗
†

40. 40
Plasma concentration of total IgE antibodies (kU/L)
43.628.6†
27.85&
50.7&†
Median value
3523.0831.0125.03652Maximal value
4.73.852.03.37Minimal value
Father
n=23
Mother
n=27
Brothers
-sisters
n=22
Autism
n=35
Total IgE
antibodies
(kU/L)
Values are expressed as mean± SD; p-significance of
differences between persons with autism, brothers/sisters,
mother and father; (&
p < 0 . 0 2); (†
p < 0 . 0 4).

41. 41
DISCUSSION
• Reichelt et al. (1991) in 12 from 44 patients found high levels of
IgA antibodies against casein, gliadin and gluten. This results
confirm the hypothesis that excreted peptides in urine of patients
with autism are from the food. With elimination of gluten and
casein from the food (diet), they noticed improvement in
simptomatology, such as: decreased hyperactivity, increased
attention and concentration, better understanding, interest in new
activities and as well improvement in speech.
• Cade et al. report high titer of IgG antibodies against gliadin
found in 87% of patients with autism and 86% with
schizophrenia and high titer of IgG antibodies against casein
found in 90% of patients with autism and 93% of patients with
schizophrenia. High titer of IgA antibodies against gluten or
casein was found in 30% of children with autism, while 86% of
patients with schizophrenia had increased levels of IgA
antibodies against gluten and 67% against casein. Gluten/casein
free diet which was conducted by these authors shown
improvement in behavior in 81% of children with autism.

42. 42
• Plasma concentration of IgG antibodies against alfa-
laktalbumin, beta-lactoglobulin and casein is statistically
significant higher in autistic persons compared to their
parents (p < 0.001). This is according to high percentage
of patients who had specific IgG antibodies against
casein (Lucarelli et al.).
• Concentration of IgG antibodies against gliadin in
persons with autism was statistically significant higher
than the concentrations in fathers and mothers (p <0.04),
similar to findings of Cade et al.
• The finding of statistically significant higher
concentration of specific allergic IgG antibodies in
persons with autism compared to all control groups is
directly connected with finding of higher IgG4 subclass
concentration which shows involvement of allergies in
etiopathogenesis of the syndrome.

43. 43
• Mean value of plasma concentration of IgA antibodies
against beta-lactoglobulin in persons with autism shown
to be statistically significant higher compared to their
brothers and sisters (p < 0.05).
• The level of casein IgA antibodies in autistic group, also
shown statistically significant higher compared to
control group of fathers and mothers (p < 0.01) and
brothers/sisters (p < 0.05).
• This finding is very similar with results of Lucarelli et
al. who found higher levels of IgA against casein,
lactalbumin, beta-lactoglobulin and ovalbumin, with
results of Cade et al., who detected high titer of IgA
antibodies against gluten and casein and with findings of
Reichelt et al. who found in 27% of patients with autism
high levels of IgA antibodies against casein, gliadin and
gluten.

44. 44
• Specific allergic IgE antibodies against casein and beta-
lactoglobulin in persons with autism are with statistically
significant higher concentrations compared to their
brothers, sisters, fathers and mothers.
• Our results for autistic group compared to healthy
control subjects show higher concentrations of total IgE,
which is quite similar to the study of Peova S.
• For the first time in our study the level of IgE in persons
with autism has been determined (in 37% ⇑), and in
Lucarelli’s study 33% of autistic patients have high
values of total IgE.
• Patients with hyper IgE syndrome have: high levels of
IgE in serum, chronic dermatitis, recurrent
sinopulmonary tract infections, cold staphylococcal
abscesses. These symptomatology was present in our
autistic patients.

45. 45
• Serum IgE levels were highly correlative with serum IgG4
(r=0.75) in one study, but do not correlate significantly with
other IgG subclasses. In our study we found moderate
correlation between these two variables (r = 0.35; p = 0.04).
• The cytokine recombinant interleukin 4 (IL-4) enhanced not
only spontaneous IgE synthesis but also IgG4 synthesis in
cultures of lymphocytes from patients hyper IgE syndrome as
well as in healthy donors (p < 0.01).
• The disturbed regulation of IgE and IgG4 seen in patients
with hyper IgE syndrome maybe caused mainly by the
disturbed regulation of both cytokines. This effect of
recombinant IL-4 towards IgE and IgG4 was inhibited by low
concentrations of recombinant interferon-gamma.
• These our first results underline the existence of intolerance
or allergy to some food products especially casein and beta-
lactoglobulin and by that we confirm the hypothesis of
involvement of allergies and other immunological
disturbances in the etiopathogenesis of autism.

46. 46
CONCLUSIONS
• The prevalence rate of autism in Macedonia is 0.25
persons on 10.000 citizens;
• The male gender is 2.5 times more frequent then
female;
• Mean age of autistic persons is about 11 years;
• Imunogenetic analyses in autistic persons shows that
HLA-C*03 (p = 0.03) and HLA-DRB1*01 (p =
0.012) alleles are associated factors with autism;
• None of our patients carried allele or haplotype,
which were protective in our population;

47. 47
CONCLUSIONS
• Plasma concentration of IgG4 subclass immunoglobulin
is significantly higher in persons with autism (p < 0.02);
• The concentrations of IgA, IgM, total IgG, and subclasses
(IgG1, IgG2 and IgG3) are not significantly different
between autistic children and their brothers and sisters;
• Plasma concentration of immunoglobulins wasn’t
significantly different between males and females with
autism.
• Statistically significant higher plasma concentration of
IgG antibodies against alfa-lactalbumin, beta-
lactoglobulin and casein is found in autistic persons
compared to their parents (p < 0.001);
• The concentration of IgG gliadin antibodies in autistic
persons was found to be higher compared to their
mothers and fathers which is statistically significant
(p < 0.04);

48. 48
CONCLUSIONS
• Plasma concentration of specific allergic IgA antibodies
against beta-lactoglobulin in persons with autism is
significantly higher compared to brothers and sisters
(p < 0.05);
• The level of casein IgA antibodies in autistic group was
significantly higher compared with those of their
mothers and fathers (p < 0.01) and brothers/sisters
(p < 0.05);
• The level of casein and beta-lactoglobulin specific
allergic IgE antibodies in the serum of autistic patients
was significantly higher compared with those of their
brothers/sisters and their parents (p < 0.04);
• The level of total IgE antibodies was statistically
significant higher in autistic persons compared to their
mothers (p<0.04) and their brothers/sisters (p < 0.02).

49. 49
ACKNOWLEDGMENT
• Families of persons with autism;
• Institute of Special Education;
• Institute of Immunobiology and Human
Genetics;
• Ministry of Education and Science of
Macedonian Government;
• Institute of rehabilitation of hear, speech, and
voice;
• Institute of mental health;
• Special Institute Demir Kapija;