Birthweight of full-term infants is associated with cord blood CD34+ cell concentration.

AIM: CD34+ cell counts are used to define the haematopoietic stem cell potential of a given cord blood transplant. The aim was to test the hypothesis that high concentration of cord blood haematopoietic progenitor and stem cells could be a reflection of intrauterine growth, of which birthweight is an indicator. METHODS: Simple and multiple regression analyses were applied to test cord blood bank data on 1368 infants for associations of selected obstetric factors and cellular contents of cord blood. RESULTS: When groups were formed based on the extreme values (5th versus 95th percentiles) of a given variable, e.g. birthweight, the term infants having the highest birthweights were found to have statistically significantly higher median cord blood CD34+ cell concentrations. Also, infants in the top 50th percentile of relative birthweight had higher median CD34+ cell concentration than infants in the low 50th percentile. In multiple regression analysis, the correlation between birthweight and CD34+ cell concentration was statistically clearly significant. Notably, while an expected correlation between gestational age and nucleated cell concentration was found, there was no association between infant gestational age and CD34+ cell concentration. CONCLUSION: Haematopoietic progenitor and stem cells may reflect intrauterine growth and have a more central role in foetal development than has been reported earlier.

Associations among nucleated cell, CD34+ cell and colony-forming cell contents in cord blood units obtained through a standardized banking process.

BACKGROUND AND OBJECTIVES: Nucleated cell content is one of the main components used when evaluating cord blood (CB) units for clinical use. However, other indicators of the haematopoietic potential of a CB unit, such as CD34+ cell and colony-forming cell (CFU-TOT) content, have also been investigated. The aim of this study was to determine whether the CD34+ cell content could be used in selecting CB collections for banking. MATERIALS AND METHODS: The collection data, as well as cellular contents of 588 CB collections obtained using a standardized CB banking process, were analysed. RESULTS: Altogether, 526 CB units from the 588 collections accepted for processing were included in international search registries. The volume collected was, as expected, 69 ml (range 28-116 ml). The correlation between total CD34+ cell and CFU-TOT (n = 88) content in the CB collection was higher (r = 0.87) than the correlation between the total nucleated cell and CFU-TOT content (r = 0.69, both P < 0.0001). The correlations of pre- and postvolume reduction values of the total nucleated cell and CD34+ cell numbers were highly significant (r = 0.96, P < 0.0001, both). The total CFU-TOT content of the CB collection correlated significantly with the total CD34+ cell content of the CB unit before cryopreservation (but after volume reduction) (r = 0.89, P < 0.0001). CONCLUSIONS: CD34+ cell content predicts the haematopoietic potential of a CB unit better than nucleated cell content. Accordingly, the CD34+ cell content of CB could be used to select CB for banking purposes and for transplantation.

IL10.G microsatellites mark promoter haplotypes associated with protection against the development of reactive arthritis in Finnish patients.

OBJECTIVE: To investigate the association of microsatellites and single-nucleotide promoter polymorphisms (SNPs) in the gene for the cytokine interleukin-10 (IL-10) with susceptibility to and outcome of reactive arthritis (ReA). METHODS: From genomic DNA, IL-10 microsatellites G and R and IL-10 promoter polymorphisms at positions -1087 and -524 were typed by polymerase chain reaction, automated fragment length analysis, and restriction fragment digestion in 85 Finnish patients with ReA and 62 HLA-B27-positive Finnish controls. ReA patients had been followed up for 20 years. Genotypes and haplotypes of IL-10 were correlated with distinct features of the disease course, such as triggering agent, chronic arthritis, development of ankylosing spondylitis, and other chronic features. RESULTS: There was a significant decrease in the promoter alleles G12 (allele frequency 0.206 versus 0.033; corrected P < 0.001, odds ratio 0.14) and G10 (0.183 versus 0.092; P < 0.05, odds ratio 0.44) in the ReA group compared with the HLA-B27-positive controls. Chronic arthritis developed significantly more frequently in the B27-positive subjects than in the B27-negative subjects (P < 0.05) as well as in patients with [corrected] the IL10.G8 allele. No associations were observed for either SNP or for the IL10.R microsatellite polymorphism. CONCLUSION: IL10.G12 and G10 microsatellite alleles show a strong protective effect against the development of ReA in Finnish subjects. Since these polymorphic markers themselves do not have direct functional implications, they most likely mark promoter haplotypes with distinct functional properties, suggesting that differential production of IL-10 is an important susceptibility factor for the development of ReA.

Tumour necrosis factor microsatellites in reactive arthritis.

The purpose was to study tumour necrosis factor (TNF)-a, -b and -c microsatellites as potential new susceptibility markers for reactive arthritis (ReA). Fifty-nine patients typed for HLA-B27 were studied for frequencies of TNF microsatellite alleles and compared with allele frequencies determined from 285 random haplotypes and 46 healthy HLA-B27-positive controls. TNFa, -b and -c microsatellite sequences were amplified by the polymerase chain reaction, and the size of the product was defined by an automated sequencer. The frequencies of TNFa6 and -c1 alleles were found to be increased in patients with ReA, whereas TNFa11 and -c2 frequencies were decreased as compared to control haplotypes. The increase in the c1 allele in patients with ReA independently from HLA-B27 suggests that it might be a new susceptibility marker for the disease. The association of ReA with other alleles was due to a linkage disequilibrium with HLA-B27.

HLA-DQ2-negative celiac disease in Finland and Spain.

Genetic susceptibility to celiac disease (CD) is strongly associated with DQA1*0501 and DQB1*02 (= DQ2). To study whether CD patients without DQ2 share other MHC class II or TNF alleles, we screened DQ2-negative patients in Finland and Spain. Twelve of 84 (14%) Finnish patients and 13 of 189 (6%) Spanish patients were negative for DQ2. We observed that all but two of altogether 25 DQ2-negative patients had the DR4 DQ8 haplotype, or either DQA1*0501 or DQB1*02 alone. Also, all but three were positive for DRB4*01. The only patients without any of these alleles were both positive for DR 13. There was a clear difference between Finland and Spain: Ten (83%) of the 12 Finnish DQ2-negative patients but only five (38%) of the 13 Spanish patients had DRB1*03, DQA1*03, DQB1*0302 (= DQ8) alleles. Of the Spanish patients, eight (62%) had DQB1*02 without DQA1*0501 and three (23%) had DQA1*0501 without DQB1*02. None of the TNF, TAP, or DPB1 alleles was found to be significantly associated with CD. Our results indicate that in addition to the DQ2 heterodimer, the other major risk alleles for CD are DR4 DQ8, and either DQA1*0501 or DQB1*02 alone. Patients without these alleles appear to be very rare, only two (0.7%) were identified in altogether 253 patients tested.

Maternal DRB1*1501, DQA1*0102, DQB1*0602 haplotype in fetomaternal alloimmunization against human platelet alloantigen HPA-6b (GPIIIa-Gln489).

Fetomaternal incompatibility of platelet alloantigens may lead to alloimmunization and neonatal alloimmune thrombocytopenia (NAIT). Human platelet alloantigen (HPA) 6b, which associates with residue Gln 489 of platelet membrane glycoprotein IIIa, has been described as a cause of NAIT. We have studied the MHC genes of all available family members in the six thus far reported families with a thrombocytopenic newborn and fetomaternal HPA-6b incompatibility. Maternal HPA-6b antibodies could be detected in five mothers to the altogether seven thrombocytopenic male infants. The MHC genes HLA-DRB, -DQA1, -DQB1, -DPB1, TAP1,2 and HSP70-Hom were studied by using polymerase chain reaction (PCR)-based DNA analysis methods. All five mothers with detectable circulating HPA-6b antibodies at the time of delivery shared an identical DRB1*1501, DQA1*0102, DQB1*0602 haplotype. The sixth, HPA antibody negative mother and a HPA-6b-negative mother to a healthy HPA-6b+ child were negative for this haplotype. The frequency of DRB1*15-positive haplotype was increased in immunized mothers (100%) as compared with the general Finnish population (27%), but the association was not statistically significant after correction. We conclude that there is a potential association between the MHC haplotype DRB1*1501, DQA1*0102, DQB1*0602 and alloimmunization to the HPA-6b antigen and that this alloimmunization probably involves different HLA class II molecules from immunization to HPA-1a.

A comparative study of HLA genes in HLA-B27 positive ankylosing spondylitis and HLA-B27 positive peripheral reactive arthritis.

OBJECTIVE: To determine whether HLA-B27 positive patients with ankylosing spondylitis (AS) and reactive arthritis (ReA) share additional HLA factors that confer disease susceptibility. METHODS: HLA class I antigens were typed serologically, and class II antigens molecularly, in samples taken from 33 patients with AS, 30 patients with ReA, and 55 healthy HLA-B27 positive controls. RESULTS: There was no major difference between the HLA alleles in AS and ReA patients, but deviations were observed when compared with healthy controls, especially between the antigens that were probably encoded by genes in the non-B27 chromosome. CONCLUSION: These results suggest that both HLA class I and class II genes may influence the pathogenesis of HLA-B27 positive ReA, whereas class I antigens seem to be the major additional genetic factors in HLA-B27 positive AS.

An approach to mapping haplotype-specific recombination sites in human MHC class III.

Studies of the major histocompatibility complex (MHC) in mouse indicate that the recombination sites are not randomly distributed and their occurrence is haplotype-dependent. No data concerning haplotype-specific recombination sites in human are available due to the low number of informative families. To investigate haplotype-specific recombination sites in human MHC, we here describe an approach based on identification of recombinant haplotypes derived from one conserved haplotype at the population level. The recombination sites were mapped by comparing polymorphic markers between the recombinant and assumed original haplotypes. We tested this approach on the extended haplotype HLA A3; B47; Bf*F; C4A*1; C4B*Q0; DR7, which is most suitable for this analysis. First, it carries a number of rare markers, and second, the haplotype, albeit rare in the general population, is frequent in patients with 21-hydroxylase (21OH) defect. We observed recombinants derived from this haplotype in patients with 21OH defect. All these haplotypes had the centromeric part (from Bf to DR) identical to the original haplotype, but they differed in HLA A and B. We therefore assumed that they underwent recombinations in the segment that separates the Bf and HLA B genes. Polymorphic markers indicated that all break points mapped to two segments near the TNF locus. This approach makes possible the mapping of preferential recombination sites in different haplotypes.

TAP1 and TAP2 polymorphism in HLA-B27-positive subpopulations: no allelic differences in ankylosing spondylitis and reactive arthritis.

The polymorphic TAP1 and TAP2 genes encode subunits of the transporter that delivers peptides to the HLA class I molecules. Because the polymorphism of the TAP genes has been shown to affect peptide transport, it has been suggested that TAP genes are potential regulators of the immune response. We studied TAP1 and TAP2 polymorphism in two multifactorial HLA-B27-associated diseases, ankylosing spondylitis (N = 30) and reactive arthritis (N = 30), in order to establish whether TAP genes are involved in the different pathogenesis of these diseases. Healthy HLA-B27-positive individuals (N = 55) were chosen as the primary controls and 93 individuals represented the random Finnish population as secondary controls. We found differences between the random and HLA-B27-positive populations, thus suggesting that certain TAP alleles are prevalent in HLA-B27 haplotypes. No differences were found between the AS and ReA groups nor between either of them and the healthy HLA-B27-positive controls. Thus it seems unlikely that TAP polymorphism, ar the level studied, has a dominant role in the pathogenesis of these diseases. However, a family study is needed in order to determine whether the same TAP complexes are carried by the same haplotypes in these diseases.

Major histocompatibility complex class II and III in Addison's disease. MHC alleles do not predict autoantibody specificity and 21-hydroxylase gene polymorphism has no independent role in disease susceptibility.

The major autoantigens in Addison's disease have recently been shown to be members of the adrenal steroidogenic enzymes, such as 21OH. The genes encoding the 21OH enzyme are located in the class III segment of the MHC complex. Therefore, its identification as an autoantigen provides a novel link between MHC and susceptibility to this autoimmune disease. We have determined the MHC class II (DRB1, DQA1, DQB1, DPB1) and class III (TNF, HSP70, C4, 21OH) gene polymorphism in patients with Addison's disease. Also, we tested whether presence of autoantibodies against 21OH is associated with specific alleles in MHC. Our results show that patients with Addison's disease in association with APS2 or Addison's disease as an isolated form share highly similar MHC class II and class III alleles. A very strong association with HLA DRB1*0301, DQA1*0501, DQB1*0201, and DPB1*0101, as well as with the C4A + 21OHA gene deletion and TNFB*1 allele was observed. However, identical gene markers were observed also in controls matched for DRB1*0301, thus suggesting that the patient group did not carry MHC gene segments specific for Addison's disease. The presence of autoantibodies against 21OH was not found to be directly determined by the MHC alleles; rather it was associated with the clinical form of the disease.

HSP70-Hom NcoI polymorphism and HLA-associations in the Finnish population and in patients with ankylosing spondylitis or reactive arthritis.

The NcoI polymorphism of the HSP70-Hom gene was investigated in the Finnish population and in two HLA-B27-associated autoimmune diseases. The two HSP70-Hom alleles were shown to be strongly associated to some specific HLA-B/DR haplotypes in random Finnish population and the segregation of the alleles as a part of these haplotypes was confirmed in 18 families. In addition, the HSP70-Hom alleles of 31 patients with ankylosing spondylitis (AS) and 38 with reactive arthritis (ReA) were compared with each other and with 56 unrelated healthy HLA-B27 positive individuals. The results indicated that the HSP70-Hom polymorphic variation is not connected independently to the different pathogenesis of AS and ReA, as no statistically significant differences between the patient groups and/or controls could be found. The HSP70-Hom status was investigated also in 28 homozygous HLA typing cells and when compared with previously published results of HSP70-l and HSP70-2 polymorphisms, it appeared that these three MHC Class-III linked HSP70 genes segregated in fixed allelic combinations.

Foeto-maternal compatibility in HLA-DR, -DQ, and -DP loci in Finnish couples suffering from recurrent spontaneous abortions.

The polymorphism of Major Histocompatibility Complex (MHC) class II genes DRB, DQA, DQB, and DPA was studied by TaqI Restriction Fragment Length Polymorphism (RFLP) in recurrent spontaneous abortions (RSA). The study group consisted of 35 primary abortion (PA) couples (no children) and 15 secondary abortion (SA) couples (1-2 children before abortions). We found no increase in DR-DQ compatibility between the mother and the foetus in the Finnish RSA group. In contrast to findings in some other populations, foeto-maternal incompatibility was increased in the PA group. Thus, our results do not support the theory that increased MHC class II compatibility is a cause of abortions as such. The Finns are a small and relatively isolated population with a unique gene inheritance. Thus, one can speculate that, if the human MHC class II is in the linkage with disadvantageous 'fertility genes', and these genes might nonetheless still be clustered in only a few MHC haplotypes among the Finns. This would be the reason, that DR-DQ sharing is not seen. The presence of rare HLA alleles, such as DR2 and DR6, among the aborters also supports this. In addition, this study extends our previous findings on MHC class III in regards to PA and SA couples differing immunogenetically from each other. In MHC class II, this was most obvious in the DPA1 locus. The vast majority of SA women were heterozygous for the two most common DPA1 alleles (14.0 kb and 13.5 kb), resulting in significantly smaller chances for a DPA1 mismatched foetus to occur in the SA group than in the controls or in the PA women.

An HLA-DR typing protocol using group-specific PCR-amplification followed by restriction enzyme digests.

A simple PCR-based protocol for HLA-DR typing suitable for a routine practice is described. The method involves, first, a PCR amplification with seven different, group-specific (DR1, DR2, DR4, DR7, DR9, DR10, and DR3+5+6+8) primer-pairs, and second, typing of HLA-DR allele more exactly in DR1, DR2, DR4, and DR3+5+6+8 groups by digestion of PCR products with restriction enzymes distinguishing different HLA-DR types within each of the groups. Altogether 24 HLA-DR alleles, or any combination of these, can be typed. The whole procedure, starting from a blood sample, can be carried out during a single working-day. The method was tested by typing a set of homozygous cell lines, as well as a local panel previously typed by PCR/oligotyping. Also, 227 patients waiting for transplantation were typed to test the method in a routine setting. The results suggest that this kind of approach gives reliable HLA-DR types and works well in the routine use.

A new platelet alloantigen, Tua, on glycoprotein IIIa associated with neonatal alloimmune thrombocytopenia in two families.

We describe immunization of two mothers against a new platelet alloantigen, designated Tua, in association with thrombocytopenia in their first born children. The platelet-specific antibodies were identified by a glycoprotein-specific platelet protein assay with husband's platelets. Monoclonal antibodies against glycoprotein complex IIb/IIIa (AP2) and against glycoprotein IIb (SZ22) could be used to immobilize the antigen bearing protein. When monoclonal antibodies against glycoprotein Ib/IX (FMC25) or Ia/IIa (Gi9) were used, no platelet-specific antibodies were detectable. The previously described alloantigens on the glycoprotein IIb/IIIa complex (HPA 1,3,4, Sra and Vaa) were not responsible for the reaction. Immunochemical analysis by an immunoblot assay showed that the Tua antigen resides on GPIIIa but the antigen was destroyed by reduction of the protein. Altogether 10 individuals belonging to three unrelated families were shown to carry the antigen. The family studies within three generations indicated autosomal codominant inheritance. Thus the Tua antigen is apparently different from all previously published platelet alloantigens. One Tua positive blood donor was identified in a population study of approximately 150 individuals. This indicates a low frequency in the Finnish population. Extended population studies will be required to determine a more exact frequency of Tua antigen.