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With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-gamma+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.

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Post-traumatic stress disorder (PTSD) is an anxiety disorder that can occur after exposure to extreme traumatic experience such as war trauma, and is accompanied by fear, helplessness or horror. Exposure to trauma can result in immune dysregulation and influence susceptibility to infectious disease as well as vaccine efficacy. The aim of the study was to determine the relation of psychological stress and the immune response to influenza vaccination in combat-related PTSD patients (n = 28). Detection of anti-viral antibody titre was performed by inhibition of haemagglutination assay. Ex vivo tetramer staining of CD8(+) T lymphocytes was used to monitor T cells specific for human leucocyte antigen (HLA)-A*0201-restricted influenza A haemagglutinin antigens before and after vaccination. Twenty patients showed a fourfold antibody titre increase to one or both influenza A viral strains, and 18 of them showed the same response for both influenza B viral strains. Ten of 15 healthy controls showed a fourfold rise in antibody titre to both influenza A viral strains and eight of them showed the same response for both influenza B viral strains. HLA-A*0201(+) PTSD patients (n = 10) showed a significant increase of influenza-specific CD8 T cells after vaccination. Although those PTSD patients had a lower number of influenza-specific CD8(+) T cells before vaccination compared to HLA-A*0201(+) healthy controls (n = 6), there was no difference in influenza A antibody titre between PTSD patients and control subjects before vaccination. The generated humoral and cellular immune response in PTSD patients argues against the hypothesis that combat-related PTSD in war veterans might affect protection following influenza vaccination.

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The aim of the present study was to investigate polymorphism of D6S2927, STR_MICA, D6S2793, TNFa (D6S2792), TNFb and TNFd (D6S2789) microsatellites and linkage disequilibria between these loci and human leucocyte antigen (HLA)-B (previously tested) for better characterisation of extended HLA haplotypes. A total of 176 healthy unrelated Croatians were studied using polymerase chain reaction amplification and electrophoresis on 6% polyacrylamide gel in ALFexpress sequencer. Eight HLA-B/D6S2927 haplotypic associations (B*07/D6S2927-4, B*08/D6S2927-3, B*18/D6S2927-3, B*27/D6S2927-1, B*35/D6S2927-5, B*38/D6S2927-4, B*51/D6S2927-2 and B*61/D6S2927-1) showed strong association (P < 0.001, D > 0.5). Among 88 different HLA-B/STR_MICA haplotypic associations, seven combinations (B*07/STR_MICA-A5.1, B*08/STR_MICA-A5.1, B*15/STR_MICA-A5, B*18/STR_MICA-A4, B*27/STR_MICA-A4, B*38/STR_MICA-A9 and B*51/STR_MICA-A6) demonstrated high linkage (D> or = 0.3) with significant P value (P < 0.001). Strong associations were also observed for five HLA-B/D6S2793 haplotypes (B*07/D6S2793-CA17, B*08/D6S2793-CA24, B*13/D6S2793-CA18, B*14/D6S2793-CA14 and B*27/D6S2793-CA14). HLA-B*08/TNFb3 and HLA-B*50/TNFb7 were the strongest associations for HLA-B/TNFb. Nine HLA-B/TNFa combinations were observed with significant P value (B*07/TNFa11, B*08/TNFa2, B*13/TNFa7, B*18/TNFa10, B*27/TNFa6, B*37/TNFa9, B*38/TNFa10, B*39/TNFa13 and B*44/TNFa4). Out of six HLA-B/TNFd haplotypic associations with strong D value, HLA-B*08/TNFd2 and B*37/TNFd3 showed the highest statistical significance (P < 0.0001). These results provide data on the region around the HLA-B that is very attractive because of its contribution to genetic susceptibility for many HLA-associated diseases and therefore this information will help in all further HLA-B locus-associated disease studies.

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In this study we monitored mixed chimerism in 36 patients with various hematologic disorders. All of them underwent a classic conditioning regimen, 31 patients for related bone marrow transplantation (BMT) and 5 patients for unrelated BMT. DNA was isolated from peripheral blood, and samples were polymerase chain reaction (PCR) amplified for 5 short tandem repeat (STR) loci (TH01, VWA31, FES/FPS, F13A01, and SE33) and for one variable number of tandem repeats locus (D1S80). Samples were run on a 6% polyacrylamide gel in an automated ALFexpress sequencer. In all 36 donor-recipient pairs we found differences for at least two STR loci. In most cases the difference was observed for SE33 and D1S80 loci. Mixed chimerism (MC) was detected in 18 patients: 4 with unrelated BMT and 14 with related sibling donors. In 11 patients MC was detected in the early period after BMT, but was soon followed by full donor chimerism (FDC) in peripheral blood. In 5 cases patients MC appearing after FDC was established, and was predictive for the relapse. One patient showed alternating MC and FDC, but at the end showed only recipient cells and graft rejection. In conclusion, the PCR-STR analysis is a highly informative, fast, and simple screening method for monitoring chimerism in a BMT program.

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In this study, we have analysed the distribution of HLA class II alleles and the extended haplotype HLA-Cw-B-DRB1-DQA1-DQB1 in Croatian patients with type I and type II psoriasis by hybridization with specific oligonucleotide probes. Type I psoriasis showed a significant association with the DRB1*0701 [P < 0.00001; relative risk (RR) = 5.83], DQA1*0201 (P < 0.00001; RR = 6.12), DQB1*0201 (P = 0.0006; RR = 3.29) and DQB1*0303 alleles (P = 0.0008; RR = 7.51). A negative correlation with type I disease was observed for the DQA1*0102 allele (P = 0.002; RR = 0.26). Type II psoriasis did not show any association with any class II alleles. The extended haplotype HLA-Cw*0602-B57-DRB1*0701-DQA1*0201-DQB1*0201 was present at a significantly higher frequency in type I patients (P < 0.00001; RR = 7.72). However, this haplotype was not detected at all in patients with type II psoriasis. In conclusion, the extended haplotype HLA-Cw*0602-B57-DRB1*0701-DQA1*0201-DQB1*0201 is a risk haplotype for type I disease in the Croatian population. This particular haplotype has not been reported previously in association with psoriasis in any other ethnic groups.

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The purpose of the present study was to investigate polymorphism of HLA class II haplotypic associations (HLA-DRB1, -DQA1, -DQB1) and DQCAR alleles in 78 Croatian patients with psoriasis. Patients were divided into two groups according to a family history of disease and age of onset: type I (positive family history and early onset) and type II (negative family history and late onset). The difference in frequency of HLA class II haplotypic associations between type I patients and controls was observed for the following combinations: HLA-DRB1*0701, -DQA1*0201, -DQB1*02 (23.6% vs. 7.2%; p < 0.001), HLA-DRB1*0701, -DQA1*0201, -DQB1*0303 (8.5% vs. 1.3%; p = 0.0018) and HLA-DRB1*1601, -DQA1*0102, -DQB1*0502 (2.8% vs. 9.3%; p = 0.06). The difference between type II psoriasis and controls for association: HLA-DRB1*1501, -DQA1*0102, -DQB1*0602 is not significant (20.0% vs. 8.9%; p = 0.06). The significantly higher frequency of DQCAR 113bp and 119bp alleles in patients with type Ipsoriasis is a result of linkage disequlibrium of these alleles with both HLA-DRB1*0701 haplotypic associations. Analysis ofDQCAR alleles in the HLA-DRB1*0701 haplotypic associations in patients with psoriasis vulgaris and matched controls did not reveal any difference in polymorphism of DQCAR alleles. These data suggest that HLA-DRB*0701 haplotypic combinations are associated with type I but not for type II psoriasis in the Croatian population. DQCAR polymorphism is not useful genetic marker to distinguish susceptible HLA class II haplotypic association.

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AIM: To investigate the polymorphism of DQCAR alleles and their association with HLA-DRB1, -DQA1, -DQB1 haplotypic associations in the Croatian population. METHODS: Blood samples were collected from 135 healthy unrelated donors from Zagreb area previously typed for HLA class II alleles (DRB1, DQA1, DQB1). The DQCAR samples were run on a standard denaturing sequencing gel in a DNA sequencer and the sequences were analyzed and compared. RESULTS: Among 10 different DQCAR alleles found in the population of Croatia, the most frequent were DQCAR 103 bp (41.5%), 121 bp (13.7%), 111 bp (11.9%), and 99 bp (10.7%). DQCAR alleles 101 bp, 115 bp, 123 bp, and 125 bp were not observed. Comparison of DQCAR allele frequencies between Croatians and other populations did not reveal any significant difference. The study proved a little diversity in DQ1 haplotypic associations. Among 141 examined DQ1 associations, 120 were DQCAR 103 bp, whereas the remaining 21 were DQCAR 107 bp. The DRB1*07 haplotypic association showed the highest diversity of DQCAR alleles (111 bp, 113 bp, 117 bp, 119 bp, and 121 bp). Three unusual haplotypic combinations were found: HLA-DRB1*0401, -DQA1*0301, -DQB1*0302, -DQCAR119bp; HLA-DRB1*0408, -DQA1*0301, -DQB1*0304, -DQCAR117bp; and HLA-DRB1*0701, -DQA1*0201, -DQB1*02, -DQCAR 105bp. CONCLUSION: Specific DQCAR alleles observed in association with common Caucasoid haplotypes are also found in the Croatian population, but in new and unusual associations. These associations have not been reported in other populations, which suggests that they might be a characteristic of Croatians.

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We describe for the first time extended haplotypes in a Croatian population. The present study gives the HLA-A, -B, -DRB1, -DQA1 and -DQB1 allele and haplotype frequencies in 105 families with at least two offspring. All individuals were studied by conventional serology for HLA class I antigens (A and B), while class II alleles (DRB1, DQA1, DQB1) were typed using the PCR-SSOP method. HLA genotyping was performed by segregation in all 105 families. For extended haplotype analysis, 420 independent parental haplotypes were included. Fourteen HLA-A, 18 HLA-B, 28 DRB1, 9 DQA1 and 11 DQB1 alleles were found in the studied population. Most of the DRB1 alleles in our population had an exclusive association with one specific DQA1-DQB1 combination. This strong linkage disequilibrium within the HLA class II region is often extended to the HLA-B locus. A total of 10 HLA-A, -B, -DRB1, -DQA1, -DQB1 haplotypes were observed with a frequency

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The HLA class I polymorphism was studied in a sample of the Albanian population. Ninety-three unrelated healthy Albanians were typed for HLA-A, -B and -Cw antigens by standard microlyphocytotoxicity test. The antigens with the highest frequencies were: HLA-A2 (34.4%), A3 (14.5%) and A1 (12.4%); B51 (19.3%), B35 (12.4%) and B18 (10.2%); Cw4 (16.2%), Cw7 (16.2%) and Cw6 (10.8%). The HLA haplotypes with high frequency in Albanians included A2-B51 (4.3%), A2-B18 (2.4%), A2-B35 (2.4%), Cw4-B35 (7.6%), and Cw7-B18 (6.5%), which are not significantly different from the other neighboring populations. Low frequency of HLA-A1-B8 haplotype (1.1%) is noted in the Albanian population. The frequency of HLA-B27 antigen (1.1%) is one of the lowest frequencies observed in Caucasians. Such results are important in studies of HLA-A1-B8, HLA-B27 and disease associations. These findings should be also useful in understanding the origin of Albanians, representing a base for future studies about HLA polymorphism in the Albanian population.

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In this study the immunogenetic relationships among 141 unrelated HLA-B27+ patients with ankylosing spondylitis (AS) and 792 members of their families were studied. Two control groups, with at least one B27+ parent were used (families undergoing transplantation program and triplet families undergoing paternity testing). All subjects were typed for HLA-A and -B antigens by microlyphocytotoxity test (MLCT) on local typing trays. The frequency of HLA-A and -B alleles was equal in the all tested groups. The segregation of all tested genes was regular regarding to the total number of positive and negative siblings, while regarding to the sex of sibs was irregular for HLA-B27 and -B5 gene. The statistical significance (p < 0.05) was found when ratio between B27+ and B27- sons in AS group was compared with the same ration in control families. In AS group was detected statistical significant (p < 0.01) high number of B5+ than B5- daughters and statistical significant (p < 0.05) less number of B5+ sons. HLA-B21 was shown to be decreased among B27+ AS patients. A synergistic effect between additional HLA-B alleles and B27 was not observed. The distribution of B27 haplotypes in AS and control families was similar except for haplotype HLA-A10, B27 which was significant (p < 0.001) less present in AS families.

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Polymorphism at the level of two microsatellite loci (D6S273 and TNFa) was studied in Croatian population. The most frequent alleles at D6S273 locus are D6S273 134 bp and 136 bp, while at TNFa locus two most frequent alleles are TNFa 117 bp and 99 bp. This study confirms the irregularity in distribution of microsatellite alleles in different populations with the predominance of two or three alleles on these two investigated microsatellite loci.

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The HLA class II alleles (DRB1, DRB3, DRB5, DQA1, and DQB1) and haplotypic associations were studied in the population of the island of Krk using the PCR-SSOP method and the 12th International Histocompatibility Workshop primers and probes. Allele and haplotypic frequencies were compared with the general Croatian population. Significant differences were observed between the population of the island of Krk and Croatians for: a) three broad specificities at DRB1 locus (DRB1*01, *15, and *07), b) one allele at DRB3 locus (DRB3*0301), c) one allele at DQA1 locus (DQA1*0201), d) one allele at DQB1 locus (DQB1*0303). Four unusual haplotypic associations, which have not yet been described in the Croatian population, DRB1*1301-DQA1*0103-DQB1*0607, DRB1*1302-DQA1*0102-DQB1*0605, DRB1*1305-DQA1*0102-DQB1*0605 and DRB1*1305-DQA1*0103-DQB1*0603 were observed in the population from the island of Krk.

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The HLA-A*02 allele is the most heterogeneous allele at HLA-A locus with 22 different subtypes so far identified. All of these subtype polymorphisms are located in alpha 1 and alpha 2 domains which are responsible for peptide biding and HLA restricted recognition by T-cell receptor. The aim of the present study was to determine the frequency of different HLA-A*02 alleles in 33 healthy unrelated Croatians. HLA-A*02 subtyping has also been retrospectively performed in 2 recipient-unrelated donor pairs and in 4 recipient-HLA phenotypically identical parent pairs. All subjects, previously typed as HLA-A2 by serology were tested using HLA-A*02 ARMS-PCR kit which discriminates 17 different A*02 alleles. Among 17 A*02 alleles we have found 4 different A*02 subtypes in healthy unrelated Croatians. The most frequent A*02 allele was A*0201 (84%). The frequency of the remaining A*02 alleles were as follows: A*0205 (3%), A*0207 (6%) and A*0213 (6%). Among 6 tested bone-marrow transplantation (BMT) pairs, only one has been found to be A*02 subtype incompatible (A*0201/A*0205). Four different A*02 alleles are found in Croatian population with the predominance of A*0201. However these results suggest that A*02 subtyping is also necessary for optimal matching of HLA-A*02 positive donor-recipient pairs in HLA incompatible BMT.

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The DRB1, DRB3, DRB5, DQA1 and DQB1 allele polymorphisms were analysed in 3 western and 3 eastern villages of the island of Hvar using PCR-SSOP method and 12th International Workshop primers and probes. Three DQB1 alleles (*0304, *0305, *0607) detected in the population of the island of Hvar (HP) have not yet been observed in general Croatian population (GCP). Significant differences were observed between two regions of Hvar for: a) DRB1*0701 allele (p < 0.001), b) DQA1*0201 allele (p < 0.01), and c) DRB1*0101-DQA1*0101-DQB1*0501 haplotypic association (p < 0.05). Two unusual haplotypic associations, which have not yet been described in general Croatian population (GCP), DRB1*0101-DQA1*0102-DQB1*0501 and DRB1*1501-DQA1 *0102-DQB1*0604 were observed in the population from the island of Hvar (HP). Measures of genetic kinship and genetic distances revealed isolation and clusterization which coincides with the known ethnohistorical, as well as biological and biocultural data obtained from a series of previous investigations. The five studied village subpopulations formed two clusters (East-West) to which the far eastern village (with the highest rii of 0.0407) joined later, thus indicating possible impact of historical immigrations from the mainland.

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