Vesna Kerhin-Brkljačić

An association between serum testosterone level and HLA phenotype

The concentration of testosterone was determined in sera of 122 HLA-typed women. Subsequently the women were classified in the category below or above the mean serum testosterone concentration. When the frequencies of HLA antigens were compared in these two categories of women, it was found that HLA-B5 and HLA-B12 antigens were significantly increased in the category of women with serum testosterone level above the mean concentration (P less than 0.01 and P less than 0.05, respectively). The frequency of HLA-B8 antigen was significantly decreased in the same category of women (P less than 0.05). The comparisons of the mean testosterone values of each HLA antigen and the variance analysis have also shown significant differences between the mean of HLA-B8 antigen and the means of other HLA antigens--HLA-A2, A3, A9, B5, B12 and Bw35. These results gave further conclusive evidence that gene(s) inside HLA region influence either the androgen hormone metabolism itself or cellular sensitivity to hormonal action as it has been presented for congenital adrenal hyperplasia.

Enumeration of Haemagglutinin‐specific CD8+ T Cells after Influenza Vaccination Using MHC Class I Peptide Tetramers

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‐γ+ 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.

Outcome of influenza vaccination in combat-related post-traumatic stress disorder (PTSD) patients

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.

HLA-B27 subtypes in Croatian patients with ankylosing spondylitis

Sir, The association between ankylosing spondylitis (AS) and HLA-B27 is one of the strongest known and it has been con® rmed in various populations. HLAB27 is found in about 96% of AS patients, while only 4 ± 12% of healthy Caucasians are HLA-B27 positive (1). The study of the association between B27 subtypes and AS gave different results in various populations. The results available indicate that alleles B*2701, *2702, *2704, *2705, and *2707 are occurring in AS patients (1,2). Negative association of B*2706 and *2709 subtypes has been described in patient groups of the Thai and Sardinian population, respectively (2). The frequency of the B27 antigen in healthy Croatians is about 12%, while in AS patients it is approximately 90%. The aims of the study was to analyse the distribution of HLA class I antigens and B27 haplotypes in a group of 119 B27+ AS patients and a group of 165 B27+ healthy individuals, and to examine the frequency of B27 subtypes in B27+ AS patients and B27+ healthy controls. A total of 119 unrelated B27+ AS patients and 165 B27+ healthy individuals were included in this study. For all individuals HLA genotyping was performed using segregation data of parental haplotypes. The AS patients were clinically diagnosed according to the New York criteria of 1986. All the included families had been living in the same area for three generations at least. HLA-A and -B typing was performed using the standard microlymphocytotoxity test (MLCT) on local trays (3). PCR-SSP typing for the B27 subtypes was performed using Dynal HLA-B27 high resolution kit (DYNAL, Oslo, Norway) (4). Comparison of HLA class I antigen frequencies between both B27+ groups (AS patients and B27+ healthy individuals) and random controls, revealed an increased frequency of HLA-B44 antigen in random healthy controls (p= 0.65 and p= 0.59, respectively). Results of HLA-A and -B antigen frequencies showed a quite similar distribution in both B27+ groups. An increased frequency of HLAA9 antigen, observed among Basque, Indian, and USA AS patients, was not found in Croatian AS patients (9.1% vs 8.8%, p40.05) (5). In this study we did not observe HLA-B60 antigen more often than expected in B27+ AS patients as was suggested by Robinson et al (2.8% vs 3.9%, p40.05). Namely, they reported that B60 increases susceptibility to AS in B27+ patients while it does not seem to be a predisposing factor in the absence of B27 antigen (6). Our results indicate that there are no additional HLA-B alleles that are involved in a synergistic effect with B27 in a population of Croatian AS patients. This lack of agreement about association between AS and other HLA class I alleles could be explained by genetic characteristic of each population reported, but also support the hypothesis that B27 itself is a major genetic susceptibility factor for AS. The ® nding of lower frequency of the HLA-A26, -B27 haplotype in the group of AS patients in comparison to B27+ healthy controls (2.0% vs 5.9%,p50.05) suggest the potentially protective role of this haplotype in the Croatian AS population. This hypothesis needs to be con® rmed on a larger group of patients bearing B27 haplotypes. Molecular typing of HLA-B27 subtypes was performed in a group of 45 unrelated AS patients and in a group of 38 B27+ healthy controls. The Croatian population showed quite similar distribution of B27 subtypes as in other Caucasians reported so far (1). AS patients possessed similar B27 subtypes as B27+ control individuals. The B*2705 allele was the most common allele in AS patients and B27+ controls (83.3% and 73.7%, respectively), while B*2702 was the second most common allele in both groups (12.5% and 23.7%, respectively). The data from different populations suggest that both B*2702 and B*2705 are positively associated with AS (1,2). The other two subtypes observed in Croatian AS patients, B*2701 and B*2704, are previously reported as alleles associated with AS. B*2704 subtype is positively associated with AS in Oriental populations but its presence is also reported in the Mary population, a Finno-Ugric population from Russia. Thus, it seems that this allele is not only present in Orientals, as was suggested by Lopez-Larrea and further supported by this study (2). At this moment it is not possible to make conclusion for positive or non-association between B*2701 and AS because the publishing data are based on a small number of AS patients. Present study did not demonstrate any of B27 alleles as susceptible or protective genetic marker for AS in the Croatian population and it ® ts into the frame of the oldness of the association between AS Zorana Grubic , National Referral Organ Transplantation and Tissue Typing Center, University Hospital Center Zagreb, KisÏ patic eva 12, HR-10000, Zagreb, CroatiaA total of 119 unrealted B27+ AS patients and 165 B27+ healthy controls were included in this study. For all individuals HLA genotyping was performed using segregation data of parental haplotypes. The AS patients were clinically diagnosed according to the New York criteria of 1986. HLA-A and -B typing was performed using the standard MLCT test on local trays. PCR-SSP typing for the B-27 subtypes was performed using Dynal HLA-B27 high resolution kit. Present study did not demonstarte any of B27 alleles as susceptible or protective genetic marker for AS in the Croatian population and it fits into the frame of the oldness of the association between AS and B27. AS appears to be older than the origin of the B27 subtypes.