P1PK: the blood group system that changed its name and expanded.

The antigens in the P1PK blood group system are carried on glycosphingolipids. The system currently includes three different antigens, P1, Pk, and NOR. The P1 antigen was disovered in 1927 by Landsteiner and Levine, and Pk and NOR were described in 1951 and 1982, respectively. As in the ABO system, naturally occurring antibodies of the immunoglobulin (Ig) M or IgG class, against the missing carbohydrate structures, can be present in the sera of people lacking the corresponding antigen. Anti-P1 is generally a weak and cold-reactive antibody not implicated in hemolytic transfusion reaction (HTR) or hemolytic disease of the fetus and newborn while Pk antibodies can cause HTR, and anti-NOR is regarded as a polyagglutinin. A higher frequency of miscarriage is seen in women with the rare phenotypes p, P1k, and P2k. Furthermore, the Pk and P1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. Why p individuals lack not only Pk and P expression but also P1 has been a longstanding enigma. Recently, it was shown that the same A4GALT-encoded galactosyltransferase synthesizes both the P1 and Pk antigens and that a polymorphism in a new exon in this gene predicts the P1 and P2 phenotypes.

A novel B(weak) hybrid allele lacks three enhancer repeats but generates normal ABO transcript levels.

BACKGROUND AND OBJECTIVES: Weak expression of A/B histo-blood group antigens is often explained by single nucleotide substitutions at the ABO locus. However, hybrid alleles containing segments from different ABO alleles can result in unexpected phenotypes and may complicate genotype analysis. We investigated the basis of weak B phenotype in a referred sample. MATERIALS AND METHODS: A healthy young woman was serologically phenotyped as AB(weak) and RBCs were characterized by flow cytometry. All seven ABO exons, five introns plus the 5'-region including the CCAAT-binding factor/Nuclear Factor Y (CBF/NF-Y) binding enhancer were sequenced. ABO transcript levels were measured in fresh peripheral blood samples. Expression of B antigen was semiquantified following transfection of HeLa cells. RESULTS: A new B(weak) allele with 53G>T resulted in a characteristic pattern of moderately weakened B antigen expression on RBCs. Its sequence revealed a novel hybrid between O(2) [O03] and B [B101] alleles with a crossingover region in intron 4 as defined by allele-specific polymorphisms. B transcript levels were similar to normal controls despite the O(2) -related single CBF/NF-Y-binding 43-bp motif in the enhancer region. Expression of the glycosyltransferase including the O(2) -specific Arg18Leu substitution resulted in a slight decrease in B-antigen-positive cells. CONCLUSION: We describe here the first hybrid between an O(2) and a B allele and characterized the associated decrease in B antigen expression. Although it lacks three enhancer repeat units compared to common B alleles, the resulting transcript level was unaltered. This study challenges previous suggestions that the number of 43-bp motifs in the ABO enhancer determines transcription rates in erythroid cells.

Wilms' tumor gene 1 protein represses the expression of the tumor suppressor interferon regulatory factor 8 in human hematopoietic progenitors and in leukemic cells.

Wilms' tumor gene 1 (WT1) is a transcription factor involved in developmental processes. In adult hematopoiesis, only a small portion of early progenitor cells express WT1, whereas most leukemias show persistently high levels, suggesting an oncogenic role. We have previously characterized oncogenic BCR/ABL1 tyrosine kinase signaling pathways for increased WT1 expression. In this study, we show that overexpression of BCR/ABL1 in CD34+ progenitor cells leads to reduced expression of interferon regulatory factor 8 (IRF8), in addition to increased WT1 expression. Interestingly, IRF8 is known as a tumor suppressor in some leukemias and we investigated whether WT1 might repress IRF8 expression. When analyzed in four leukemia mRNA expression data sets, WT1 and IRF8 were anticorrelated. Upon overexpression in CD34+ progenitors, as well as in U937 cells, WT1 strongly downregulated IRF8 expression. All four major WT1 splice variants induced repression, but not the zinc-finger-deleted WT1 mutant, indicating dependence on DNA binding. A reporter construct with the IRF8 promoter was repressed by WT1, dependent on a putative WT1-response element. Binding of WT1 to the IRF8 promoter was demonstrated by chromatin immunoprecipitation. Our results identify IRF8 as a direct target gene for WT1 and provide a possible mechanism for oncogenic effects of WT1 in leukemia.

Volume measurement of cerebral arteriovenous malformations from angiography.

We designed software for measuring the volume of cerebral arteriovenous malformations from angiography and validated it against prescription volumes in radiosurgery. We aimed to create a model for the risk for complications as a function of volume, based on established outcome prediction models for Gamma Knife radiosurgery, but without the need for dose planning. We created an application for computing the volume of cerebral arteriovenous malformations from the intersection of two X-ray cones in stereotactic space. Volume measurements were compared with prescription volumes from dose planning, in phantoms and in patients treated with Gamma Knife radiosurgery for cerebral arteriovenous malformations. Previous studies of 1128 treated patients were used to calculate the risk for complication as a function of the nidus volume. In 63 patients volumes measured with either method correlated, R2 = 0.85. Volume as measured with the intersecting cone model (ICM) correlated with predicted Gamma Knife radiosurgery complication rate, R2 = 0.84. The ICM can thus be used for measurement of AVM volumes less than 10 cm3 from angiography. Outcome models from Gamma Knife radiosurgery may be applied, but with reduced exactness. Standardised AVM volume measurement is valuable for comparing outcome and for quantification of volume reduction after therapy, notably embolisation. Thus the optimal management plan may be selected in conjunction with diagnostic or therapeutic angiography.

A repetitive sequence of Epstein-Barr virus nuclear antigen 6 comprises overlapping T cell epitopes which induce HLA-DR-restricted CD4(+) T lymphocytes.

Most human adults carry the Epstein-Barr virus (EBV) and develop immunological memory against the structural and the virus-encoded cellular proteins. The EBV nuclear antigen 6 (EBNA6) elicits cytotoxic T cell responses and it also maintains a persistent antibody response. The majority of sera from EBV-seropositive individuals reacts with a synthetic peptide, p63, comprising 21 amino acids of a repetitive region of EBNA6. CD4(+) T lymphocytes, with specificity for p63, could be recalled from the T cell repertoire of EBV carriers that expressed certain HLA-DR allotypes which were identified as good binders of p63 by an in vitro flow cytometric assay. Analysis of the HLA-DR/p63 interaction by molecular mechanics calculations indicated the presence of multiple overlapping epitopes which were predicted to bind in a HLA-DRB1 allo- and subtype-specific manner. Specific activation of p63-selected long-term CD4(+) T cell cultures resulted in a proliferative response, in the production of IL-2 and in the secretion of high levels of tumor necrosis factor as measured by bioassays. Proliferation and cytokine production of p63-specific T cells could be induced by p63-loaded HLA-DR-matched antigen-presenting cells and by B cells co-expressing relevant HLA-DR molecules and EBNA6. Our results show that peptides of an EBNA6 repeat region induce CD4(+) T cells which can react with EBNA6-carrying cells in many individuals. We suggest that these T(h) cells may be important in conditioning dendritic cells for initiation potent virus-specific immune responses, provide help for EBV-specific B cells, drive IgG isotype switch and support the sustained effector function of memory cytotoxic T lymphocytes.

Linear reduction of clonal cells in stem cell enriched grafts in transplanted multiple myeloma.

In 30 patients with multiple myeloma who were scheduled for peripheral blood stem-cell transplantation, a quantitative analysis of the stem cells following enrichment by anti-CD34 was carried out. To detect the cells of the specific myeloma clone, polymerase chain reaction (PCR) was performed using unique allele-specific oligo primers for the immunoglobulin heavy chain rearrangement. The clonogenic cells before and after stem-cell enrichment, were quantified by a limiting dilution assay and a highly sensitive semi-nested PCR combined with a real-time quantitative PCR. In order to accomplish a statistically adequate end-point analysis, a large number of PCR analyses (40 per sample) were performed. By this technique the lowest detection limit observed was one myeloma cell per 106 cells. Myeloma cells were detected in 29/30 samples from the CD34-enriched fraction. The CD34 selection procedure resulted in a median 28-fold enrichment of CD34+ haemopoietic precursor cells. The stem-cell selection reduced the median concentration of clonal cells per million total cells by half, with a highly significant linear relationship between the number of myeloma cells before and after stem cell enrichment. The median depletion of clonal cells by the overall procedure was 2.15 log units, corresponding to a reduction of the total quantity of clonal cells reinfused into the patients by at least 99.3%. We conclude that CD34+ cell enrichment led to a reliable tumour cell depletion of the order of 2 log, which may not be sufficient since the total number of tumour cells in the leukapheresis product was 7.2 log (median).

Genomic typing of the Kidd blood group locus by a single-tube allele-specific primer PCR technique.

The Kidd (JK) blood group system is clinically important in transfusion medicine. Alloantibodies to antigens in this system may be produced following blood transfusion or during pregnancy and can result in serious haemolytic transfusion reactions and haemolytic disease of the newborn (HDN). JK antigens on erythrocytes are carried by glycoproteins with the capacity to transport urea through cell membranes. cDNA complementary to mRNA transcribed at the JK locus was cloned in 1994. The molecular basis of the Jk(a)/Jk(b) blood group polymorphism was recently shown to be a single nucleotide substitution predicting an amino acid change (Asp280Asn) in an extracellular loop of the JK glycoprotein. After confirmation of the JK gene polymorphism we developed a rapid and robust technique for JK genotyping with allele-specific primers in a single-tube PCR. In addition, a 217 bp intron located at nucleotides 811-812 in the JK gene was found and sequenced. The genotyping test was validated with samples from 106 Caucasian Swedish and 13 Black South African random blood donors. Complete phenotype-genotype correlations were obtained. However, four Jk(a-b-) samples of Polynesian and Finnish origin typed as Jk(b)Jk(b). Potential use of the presented method can be predicted in clinical transfusion medicine including prenatal determination of the JK genotype in a fetus at risk for HDN caused by JK antibodies.

Clinical utility of immunoglobulin heavy chain gene rearrangement identification for tumour cell detection in multiple myeloma.

In an attempt to define the clinical utility of immunoglobulin heavy chain (IgH) gene rearrangement identification for tumour cell detection in multiple myeloma, we investigated 36 consecutive newly diagnosed patients intended for high-dose chemotherapy in a study protocol. After identification of the IgH rearrangement, an allele specific oligonucleotide (ASO) was constructed and used in a semiquantative PCR for minimal residual disease (MRD) evaluation. The myeloma-specific IgH gene rearrangement could be identified and an ASO primer constructed in 24 (67%) of the patients. All of these patients underwent transplantation; 22 were autologous, of whom three had PCR-negative stem cell harvests, and two were allogeneic. 10 patients achieved a clinical complete response (CR) and five were PCR negative in sequential bone marrow analyses. In patients not achieving CR, PCR negativity was occasionally found, but in general the PCR results reflected the clinical status of the patients. No consistent relationship between the bone marrow MRD status and the clinical course was found, and early relapses occurred also in PCR-negative patients.

Report from the HLA class II typing by PCR-SSP Multicentre Study.

Results from 360 HLA-DR and -DQ 'low-resolution' typings with polymerase chain reaction sequence-specific primers (PCR-SSP), performed by nine laboratories, were analysed for their overall utility in routinely defining the HLA-DR1-DR18, DR51-DR53 and DQ1-DQ9 specificities in less than 2.5 h. Thirty EDTA blood samples and 10 DNA samples were distributed and analysed by each laboratory. DNA was extracted using a rapid bromide salt extraction protocol. Complete HLA-DR and -DQ typings were performed, three by three, on pre-aliquoted 96-tube PCR trays. When compared with reference typing, 351/360 (98%) correct DR typings were obtained, whereas 320/360 (89%) of the DQ phenotypes were correctly assigned. The time for three complete HLA-DR and -DQ 'low-resolution' typings, including DNA extraction, ranged from 2.0 h to 2.3 h. Unfortunately, an unusually high level of PCR amplification failures was observed (3%), probably due to diffusion and a significant volume loss from some of the pre-aliquoted primer mixes. Consequently, only 52% of the typings were without any amplification failure, and 0-2 amplification failures where found in 88% of the PCR-SSP typings performed. The number of HLA-DR-DQ retypings needed was 7 and 8%, respectively, reflecting the low number of typings where allelic identification was directly affected by the relatively high level of amplification failures in this study. Thus, a 91-98% success rate of correctly identified HLA-DR and -DQ alleles could be maintained, even under suboptimal typing conditions.

An Ael allele-specific nucleotide insertion at the blood group ABO locus and its detection using a sequence-specific polymerase chain reaction.

Genomic DNA from each of four Acl individuals (genotypes AO1, AO1var, AO2) and one AclB individual was used as a template for amplifying exons 6 and 7 of the ABO genes, which were subsequently sequenced. In all the Ael alleles a single nucleotide insertion, compared to the A consensus sequence, was observed that would alter the amino acid sequence of the glycosyltransferase immediately after its postulated nucleotide sugar binding site and furthermore extend the translated protein by 37 amino acids (16 more than the A2 enzyme). A sequence-specific primer PCR assay was developed to detect the nucleotide insertion. It was possible to differentiate all 20 serologically defined Acl/AclB individuals available from 145 blood donors with normal ABO phenotypes and genotypes and 26 individuals with various A subgroups other than A1, A2 and Acl. This mutation explains the Acl phenotype and forms the basis of a method for detecting the Ael allele.

Sharing of MHC haplotypes among patients with systemic lupus erythematosus from unrelated Caucasian multicase families: disease association with the extended haplotype [HLA-B8, SC01, DR17].

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disease often clustered in families. We investigated the association between MHC haplotypes and SLE in multicase Caucasian families. METHODS: Ten consecutive families with 2 or more patients with SLE, in total 27 patients among 66 individuals, were studied. MHC haplotypes were determined by typing for HLA-A, B, C, DR, and DQ by serological and DNA methods. Complotypes were determined by protein typing and C4 gene polymorphism by DNA analysis. RESULTS: Fifty-four independent MHC haplotypes were found. Ten of the 31 haplotypes in the patients with SLE were examples of the extended haplotype [HLA-B8,SC01,DR17]. Six of these were found in 2 or more patients with SLE within the same family. All the 14 SLE sib-pairs in the families shared at least one haplotype and in 9 of the sib-pairs the shared haplotype was [HLA-B8,SCO1,DR17]. Three SLE associated haplotypes were [HLA-B7,SC31,DR15]. Four of the 27 patients with SLE were C4A deficient. Two C2 deficient siblings were homozygous for the haplotype [HLA-B18,S042,DR15]. CONCLUSION: We demonstrate that a very limited number of MHC haplotypes are associated with familial SLE. The haplotype [HLA-B8,SCO1,DR17] was closely related with the disease. There was no evidence suggesting familial SLE constitutes a disease subset. Determination of MHC haplotypes in multicase families is of value for assessment of disease susceptibility.

Granulocyte functions and Neisseria meningitidis: influence of properdin-deficient serum.

Granulocyte-mediated reactions such as opsonization, chemotaxis, and release of granulocyte myeloperoxidase and lactoferrin were studied in properdin-deficient and normal human serum incubated with serogroup A and W-135 meningococci. There were no differences between the sera when serogroup A meningococci were studied. Opsonic and chemotactic activity were impaired against serogroup W-135 meningococci in properdin-deficient serum. Restitution with properdin restored both activities. We found similar release of myeloperoxidase and lactoferrin from granulocytes challenged with serogroup A or W-135 meningococci in either sera. These findings are in accordance with the clinical observations of meningococcal infections caused by serogroup W-135 in properdin-deficient patients as well as the absence of infections caused by serogroup A meningococci.

Immune response to tetravalent meningococcal vaccine: opsonic and bactericidal functions of normal and properdin deficient sera.

Neisseria meningitidis serogroup W-135 appears to be a fairly common cause of infection associated with properdin deficiency or dysfunction, and anticapsular antibodies might be protective in these patients. For this reason, bactericidal and opsonophagocytic activities for serogroup W-135 were investigated before and four weeks after vaccination of two properdin-deficient adults with tetravalent meningococcal vaccine. In addition, the response of IgM, IgG and IgA class antibodies to the serogroups A, C, Y and W-135 was determined by ELISA. There was no evidence of poor antibody responses in the properdin-deficient persons. Vaccination promoted classical pathway-mediated killing in serum and opsonization of serogroup W-135 to the same extent as that seen in vaccinated controls. The increase of alternative pathway-mediated killing in the properdin-deficient sera was moderate, but vaccination clearly enhanced alternative pathway-mediated opsonophagocytosis in the sera. It was also shown that vaccination markedly reduced the requirement for properdin in alternative pathway-mediated killing of the meningococci.