The Distribution of HLA Alleles among Children with Atopic Asthma in Croatia

Allergic asthma is a multifactorial disease involving well known environmental factors and less identified genetic components. In several studies the HLA genes have been implicated in the development of asthma and atopy, but the importance of these associations remains unclear. The aim of the present study was to analyse the distribution of specificities at HLA class I loci (-A and -B) and HLA class II locus (-DRB1) in a group of 143 Croatian children with atopic asthma, regarding total serum IgE and specific IgE against common inhalant allergens, as well as their connection with different asthmatic phenotypes and to identify HLA genotype which increases the risk for atopy or asthma or which has a protective effect. As controls we used a group of 163 healthy unrelated individuals. HLA class I antigens were determined by serology, while DRB1 specificities were detected by polymerase-chain reaction amplification and hybridisation with sequence specific oligonucleotide probes method (PCR-SSOP). We found no significant correlation between any of the HLA-A antigens and asthma, atopy or associated atopic phenotypes. At HLA-B locus, HLA-B8 antigen was significantly increased among asthmatic patients (p=0.002), patients with high total serum IgE (p=0.002), as well as among patients sensitizated to Dermatophagoides pteronyssinus (Der p) (p=0.014) and among patients sensitizated to Der p + Dactylis glomerata (Dact g) or Ambrosia elatior (Amb a) (p=0.004). Among HLA-DRB1 specificities, HLA-DRB1*01 showed positive correlation with asthma and atopy (p=0.034), while HLA-DRB1*03 specificity was observed with significantly higher frequency among patients with total serum IgE 400 KU/L (p=0.048). HLA-DRB1*16 specificity was observed with significantly lower frequency among patients with asthma only in comparison to healthy controls (p=0.027) and to patients with asthma and allergic rhinitis (p=0.005). In conclusion, our data suggest that HLA specificities play a relevant role in predisposition to asthma, as well as in different clinical forms of atopic diseases. HLA-B8, HLA-DRB1*01 and HLA-DRB1*03 genotype increases the risk for atopic asthma and high serum IgE

The influence of tumor necrosis factor microsatellite polymorphisms on patient survival following hematopoietic stem cell transplantation

AIM: To investigate the influence of tumor necrosis factor (TNF) microsatellite polymorphisms on patient survival following hematopoietic stem cell transplantation. ----- METHODS: We analyzed TNFa, TNFb, and TNFd microsatellites among 100 patients who underwent allogeneic hematopoietic stem cell transplantation from a human leukocyte antigen (HLA)-identical sibling donor at the Internal Clinic of the University Hospital Center Zagreb in the period 2001-2009. The analysis was performed using polymerase chain reaction amplification and electrophoresis on a polyacrylamide gel in an automated sequencer. ----- RESULTS: There was no significant difference in patient survival with respect to the allele length at a given microsatellite. However, a significantly lower survival rate was noticed among patients who were positive for TNFa8 allele (P<0.001) and a significantly higher survival rate among those who were positive for TNFa10 allele (P=0.0220). ----- CONCLUSION: These results for the first time suggest an influence of TNFa microsatellite on patient survival following HSCT and indicate a need for further studies of this microsatellite

The diversity of extended gen haplotypes HLA-B*27 in Croatia

U ovom radu analizirani su produženi haplotipovi gena HLA-B*27 na uzorku od 42 obitelji iz Hrvatske. Svim ispitanicima uzeto je 2ml periferne krvi iz koje je izolirana DNA pomoću komercijalnog kita. Aleli gena HLA-B*27 određeni su lančanom reakcijom polimerazom s početnicama specifičnim za određeni alel (PCR-SSP), a aleli mikrosatelitskih lokusa D6S2927, STR_MICA i TNFa umnoženi su početnicama specifičnima za alele mikrosatelitskih lokusa (PCR-STR). Analizom produženih haplotipova HLA-A-B*27-D6S2927-STR_MICA-TNFa-DRB1 utvrđena je najjača neravnoteža udruživanja između alela gena HLA-B*27 i mikrosatelitskih alela D6S2927-1 i STR_MICA-A4. Analiza je pokazala da je rijetki alel B*2730 uvijek prisutan u produženom haplotipu: HLA-A3-B*2730-D6S2927-1-STR_MICA-A4-TNFa-9-DRB1*16. Rezultati ovog rada primijenit će se u daljnjim istraživanjima povezanosti mikrosatelitskih alela i spondiloartropatija te pomoći u boljem razumijevanju vezanja peptida na molekule HLA razreda I, kao i drugih procesa koji se javljaju tijekom imunološkog odgovora.In the present study the extended haplotypes of HLA-B*27 gene were analysed in the sample of 42 Croatian families. Peripheral blood (2ml) was collected from all the individuals and genomic DNA was extracted using a commercial kit. The HLA alleles were determined using the method PCR sequence specific primers (PCR-SSP) while the microsatellite alleles were amplified using the method PCR-STR. Analysis of HLA-A-B*27-D6S2927-STR_MICA-TNFa-DRB1 extended haplotypes demonstrated the strongest linkage disequilibrium between HLA-B*27 alleles and microsatellite alleles D6S2927-1 and STR_MICA-A4. Analysis indicated that the rare allele B*2730 is always present in the extended haplotype HLA-A3-B*2730-D6S2927-1-STR_MICA-A4-TNFa-9-DRB1*16. The results of the present study will be applied in future studies of association between microsatellite alleles and spondyloarthropathies and contribute to a better understanding of peptide binding to HLA class I molecules, as well as other aspects of immune response

The Distribution of HLA Alleles among Children with Atopic Asthma in Croatia

Allergic asthma is a multifactorial disease involving well known environmental factors and less identified genetic components. In several studies the HLA genes have been implicated in the development of asthma and atopy, but the importance of these associations remains unclear. The aim of the present study was to analyse the distribution of specificities at HLA class I loci (-A and -B) and HLA class II locus (-DRB1) in a group of 143 Croatian children with atopic asthma, regarding total serum IgE and specific IgE against common inhalant allergens, as well as their connection with different asthmatic phenotypes and to identify HLA genotype which increases the risk for atopy or asthma or which has a protective effect. As controls we used a group of 163 healthy unrelated individuals. HLA class I antigens were determined by serology, while DRB1 specificities were detected by polymerase-chain reaction amplification and hybridisation with sequence specific oligonucleotide probes method (PCR-SSOP). We found no significant correlation between any of the HLA-A antigens and asthma, atopy or associated atopic phenotypes. At HLA-B locus, HLA-B8 antigen was significantly increased among asthmatic patients (p=0.002), patients with high total serum IgE (p=0.002), as well as among patients sensitizated to Dermatophagoides pteronyssinus (Der p) (p=0.014) and among patients sensitizated to Der p + Dactylis glomerata (Dact g) or Ambrosia elatior (Amb a) (p=0.004). Among HLA-DRB1 specificities, HLA-DRB1*01 showed positive correlation with asthma and atopy (p=0.034), while HLA-DRB1*03 specificity was observed with significantly higher frequency among patients with total serum IgE 400 KU/L (p=0.048). HLA-DRB1*16 specificity was observed with significantly lower frequency among patients with asthma only in comparison to healthy controls (p=0.027) and to patients with asthma and allergic rhinitis (p=0.005). In conclusion, our data suggest that HLA specificities play a relevant role in predisposition to asthma, as well as in different clinical forms of atopic diseases. HLA-B8, HLA-DRB1*01 and HLA-DRB1*03 genotype increases the risk for atopic asthma and high serum IgE

Haplotipic associations of the two most common HLA-B*27 alleles in the Croatian population

Cilj ovog rada bio je analizirati produžene haplotipove dva najčešća podtipa gena HLA-B*27 (B*2702 i *2705) u hrvatskoj populaciji. Analizirana je skupina od 111 nesrodnih ispitanika pozitivnih za gen HLA-B*27, koji do početka ovog istraživanja nisu imali nikakve simptome ankilozantnog spondilitisa. Ukupan broj praćenih haplotipskih veza bio je 112 jer je jedna osoba bila homozigot za gen HLA-B*27. Podtip gena HLA-B*27 i geni na lokusu HLA-A i -DRB1 određeni su metodom PCR-SSP kod svih ispitanika. Od 7 različitih podtipova gena HLA-B*27 uočenih među našim ispitanicima, najčešći je bio alel B*2705 (61,6%) te alel B*2702 (30,4%), dok su preostali aleli (B*2701, B*2703, B*2704, B*2708 i B*2714) uočeni jedanput, odnosno, dvaput. Najčešći gen na lokusu HLA-A bio je HLA-A*02, kako unutar haplotipskih veza alela B*2702 tako i unutar veza alela B*2705. Nije uočena razlika u zastupljenosti gena na ovom lokusu između haplotipskih veza alela B*2702 i alela B*2705. Analiza haplotipskih veza HLA-B, -DRB1 ukazala je na jaku povezanost alela B*2702 i DRB1*16 koji su se zajedno pojavili s učestalošću od 44,1%, što je statistički značajno u usporedbi s učestalošću s kojom se DRB1*16 pojavio unutar haplotipskih veza alela B*2705 (4,0%; P<0,00001). Unutar skupine od 69 haplotipskih veza alela B*2705 uočena je statistički značajno povećana učestalost HLA-DRB1*01 u usporedbi sa skupinom (N=34) haplotipskih veza alela B*2702 (28,0% naspram 1,5%; P<0,00001).The aim of the present study was to analyze haplotypic associations of two the most common HLA-B*27 subtypes (B*2702 and *2705) in the Croatian population. One hundred and eleven unrelated HLA-B*27 positive individuals were included. None of them had any sign of ankylosing spondylitis. The total number of analyzed haplotypic associations was 112 because one individual was homozygous for HLA-B*27. HLA-B*27 alleles as well as HLA-A and DRB1 specificities were tested by PCR-SSP method. Among seven different HLA-B*27 alleles observed among our individuals, B*2705 was the most frequent (61.6%), followed by B*2702 (30.4%), while the frequency of all other observed alleles (B*2701, B*2703, B*2704, B*2708 i B*2714) was less than 2.0%. HLA-A*02 was the most frequent specificity at HLA-A locus in both groups of haplotypic associations (B*2702 and B*2705) and no difference in distribution of HLA-A genes was observed between two groups. Analysis of HLA-B*2702 haplotypic associations showed the high frequency of DRB1*16 (44.2%) in comparison to B*2705 haplotypic associations (4.0%) with significant P value (P<0.00001). HLA-DRB1*01 demonstrated significantly higher presence among 69 B*2705 haplotypic associations compared to 34 B*2702 haplotypic associations (28.0% vs. 1.5%; P<0.00001)

HEMATOPOIETIC STEM CELL TRANSPLANTATION IN TREATMENT OF AUTOIMMUNE DISEASES

Transplantacija krvotvornih matičnih stanica (TKMS) postala je učinkovita terapijska mogućnost za liječenje teških oblika autoimunosnih bolesti (AB). Krvotvorne matične stanice mogu se skupiti od samog bolesnika (autologno), jednojajčanog blizanca (singeno) te HLA-identičnog davatelja (alogeno). Alogenom transplantacijom mijenjaju se autoagresivne efektorne stanice imunosnog sustava bolesnika s neautoagresivnim stanicama davatelja, no ovaj oblik transplantacije manje je privlačan zbog moguće reakcije presatka protiv primatelja (GVHD, engl. graft-versus-host disease). S druge strane, autologna transplantacija dovodi do »resetiranja« imunosnog sata i ne uzrokuje GVHD, no zahtijeva prethodno kondicioniranje. Kao alternativa ovim oblicima liječenja razvila se i transplantacija mezenhimskih matičnih stanica (MMS) koje imaju znatan imunosupresivni učinak, ne zahtijevaju prethodno kondicioniranje i ne dovode do GVHD-a. Ipak, s obzirom na još i sad velik postotak smrtnih ishoda povezanih s liječenjem (TRM, engl. treatment related mortality), transplantaciju matičnih stanica treba sačuvati za bolesnike s najtežim oblicima AB-a, kojima se ovakvim načinom liječenja pruža čak i mogućnost potpunog izlječenja.Hematopoietic stem cell transplantation (HSCT) has become an effective therapeutic option for the treatment of severe cases of autoimmune diseases (AD). Hematopoietic stem cells (HSC) can be collected from the patient (autologous), identical twin (syngenic) or HLA identical donor (allogenous). In allogenous transplantation autoagressive immunological effector cells are substituted with non-autoagressive cells of the donor. Possible graft versus host reaction (GVHD) makes this type of transplantation less attractive. On the other hand, autologous transplantation can induce the »resetting« of immunological clock without any fear of GVHD, but it requires previous conditioning. As an alternative option, transplantation of mesenchymal stem cells (MSS) was developed. MSS has a strong immunosuppressive effect, while it doesn’t require previous conditioning, nor does it induce GVHD. Due to the treatment related mortality, these therapeutic option should remain reserved for the most severe cases of AD. Nevertheless, they present a great opportunity for these patients, and even a chance for full recovery

The influence of tumor necrosis factor microsatellite polymorphisms on patient survival following hematopoietic stem cell transplantation

Aim To investigate the influence of tumor necrosis factor (TNF) microsatellite polymorphisms on patient survival following hematopoietic stem cell transplantation. Methods We analyzed TNFa, TNFb, and TNFd microsatellites among 100 patients who underwent allogeneic hematopoietic stem cell transplantation from a human leukocyte antigen (HLA)-identical sibling donor at the Internal Clinic of the University Hospital Center Zagreb in the period 2001-2009. The analysis was performed using polymerase chain reaction amplification and electrophoresis on a polyacrylamide gel in an automated sequencer. Results There was no significant difference in patient survival with respect to the allele length at a given microsatellite. However, a significantly lower survival rate was noticed among patients who were positive for TNFa8 allele (P < 0.001) and a significantly higher survival rate among those who were positive for TNFa10 allele (P = 0.0220). Conclusion These results for the first time suggest an influence of TNFa microsatellite on patient survival following HSCT and indicate a need for further studies of this microsatellite