Errors in detection of subgroups in the ABO blood group system]

In the ABO blood group system, several subgroups have been described based on: 1) the difference of reactivities of the red cells with anti-A, anti-B, anti-A1, and anti-H, 2) the presence or absence of anti-A, anti-B, anti-A1, anti-H, and anti-HI in serum, and 3) the presence of A, B, H substances in the saliva of ABH secretors. Subgroups of A are more frequent in Caucasians than in Japanese, while those of B are more frequent in Japanese. Both the red cell typing (testing red cells for A and B antigens) and serum typing (testing the antibodies in the serum against red cells of known ABO groups) are important to identify and not to overlook these ABO subgroups. When transfusion is required in individuals with these subgroups, compatible blood products must be selected according to the presence or absence of antibodies active at 37 degrees C.     

Reactivity of monoclonal antibodies as blood grouping reagents

Many monoclonal antibodies against human red cell surface antigens have been widely utilized in Japan. Especially anti-A, anti-B, and anti-D are used in many clinical laboratories. However, demands of most medical technologists are as follows; the specificity of the monoclonal antibodies are similar or almost same to those of polyclonal ones. Therefore, commercial anti-A or anti-B is manufactured by mixing two or three monoclonal antibodies which react with various A or B antigens including ABO variants. We tested these antibodies as blood grouping reagent for evaluating ABO variants (table 3 and 4). We also evaluated several monoclonal anti-Ds by the use of partial Ds. One of 8 antibodies did not agglutinate with any partial D antigens (table 5). We think that red cells for transfusion to a patient with partial D must be D-negative red cells because we have found anti-D in some patients with partial D who were immunized by transfusion of D-positive red cells.     

Serologic characteristics of H-deficient phenotypes among Chinese in Hong Kong

BACKGROUND: The occasional presence of H-deficient red cells among both referred and donor blood samples prompted the mass screening of donated blood in Hong Kong for H-deficient phenotypes; 96 percent of the donors tested are Chinese from the southern province of Kwongtung. STUDY DESIGN AND METHODS: Donor blood was screened for H-deficient red cells with the use of Ulex europaeus. Lewis phenotyping was carried out on all H-deficient individuals, and saliva testing was performed on most such individuals. The thermal amplitude and potency of their anti-H and anti-HI in the serum were also estimated. RESULTS: Between 1984 and 1993, 28 H-deficient blood donors were identified; 16 H-deficient patient samples were also identified, and family studies revealed an additional 7 H-deficient subjects. The H-deficient red cells did not react with anti-H lectin, the levels of ABH substances in saliva were normal or near-normal, normal levels of A or B transferase were found in plasma, minute quantities of A or B (in persons who were genetically group A or B) were detected on the red cells, and anti-H or anti-HI was detected in the serum (about 66.7% of which reacted at 37 degrees C). Atypical anti-A or anti-B was demonstrated in 81.8 percent of the cases. CONCLUSION: The H-deficient phenotype among the Hong Kong Chinese seems to represent a homogeneous group. Despite the presence of normal quantities of ABH substance in the saliva, anti-H or anti-HI that was active at 37 degrees C was detected in most cases. The incidence of the H-deficient phenotype was 1 in 15,620.     

Conjugation of blocked ricin to an anti-CD19 monoclonal antibody increases antibody-induced cell calcium mobilization and CD19 internalization

CD19 (B4) is a signal transduction molecule restricted to the B-cell lineage and the target of the immunotoxin anti-B4-blocked ricin (anti-B4-bR), which is composed of the monoclonal antibody (MoAb) anti-B4 and the modified plant toxin blocked ricin. To explore the influence of conjugation of blocked ricin to anti-B4 on functional activation of CD19, we investigated the effects of anti-B4-bR, and that of unconjugated anti-B4, on intracellular calcium mobilization and ligand/receptor internalization. The data showed that anti-B4-bR was more potent than anti-B4 in triggering cell calcium mobilization. Two other immunotoxins that bind to the B-cell surface, anti-CD20-bR and anti-CD38-bR, were devoid of the calcium increasing effect of anti-B4-bR. Furthermore, anti-B4 conjugated to ricin A-chain was also without effect in Namalwa cells, indicating that the ricin B-chain component was required for anti-B4-bR effect. Anti-B4-bR-induced calcium mobilization was inhibited in the presence of lactose, yet the calcium response induced by cross-linking anti-B4-bR with a second step antibody was not affected. The extent of CD19 modulation induced by anti-B4-bR was higher than that induced by anti-B4, and lactose dampened the effect of the immunotoxin down to that of the MoAb. Moreover, the number of internalized immunotoxin molecules was higher than that of unconjugated MoAb. Although a mechanism involving dimerization of the immunotoxin cannot be excluded, our findings suggest that the residual binding activity of the blocked ricin B-chain to cell surface molecules plays an important role in the greater calcium fluxes and greater internalization rate of anti-B4-bR, and is of functional significance in the mechanism of intoxication of cells by the immunotoxin.     

Retroviral recombination is nonrandom and sequence dependent

The ABO blood group is the most clinically important human alloantigen system in transfusion medicine. The system involves three antigens A, B and H. H antigen is converted to either A or B by the activity of alpha1-->3-N-acetyl-galactosaminyl transferase (A transferase) or alpha1-->3 galactosyl transferase (B transferase). The O phenotype is the result of an inactive glycosyltransferase, which is unable to glycosylate the H antigen. The immunological properties of the ABO system were identified at the turn of the century; however, the genetic basis of the ABO system has only recently been characterized. This has enabled the development of a number of molecular ABO typing methods. Described here is a two-reaction multiplex allele-specific PCR (ASPCR) genotyping assay for the A1, A2, B, O1 and O2 subtypes. 11 different allele-specific oligonucleotide primers were selected to detect the presence or absence of the O1 associated G--> (-) deletion at base 261, the O2 associated G --> A substitution at base 802, the B associated G --> A substitution at base 803, and finally the A2 associated C --> (-) deletion at base 1059. A total of 122 peripheral blood samples were genotyped and serologically forward and reverse typed. A concordance rate of 98.4% (120/122 samples) was observed between the actual genotype and the serologically-based predicted genotype. These results indicate that this assay provides a rapid, accurate, and simple method for A(1,2)BO(1,2) genotyping that serves as a useful supplement to standard serological ABO typing.     

Race and intra-urban migration

Human blood group O cells were converted into B-active cells by incubation at either 4 degrees or 37 degrees with uridine diphosphate D-galactose and unfractionated serum from the Japanese tortoise (Clemmys japonica). The specificity of the converted cells was tested by their reactions with human anti-B, anti-A and rabbit anti-B sera. Under the conditions used, Bombay (Oh) type cells were not converted, and group-O foetal cells possessing a few H antigenic sites were only weakly converted into B-active cells. In addition, the agglutination titre of the converted cells with eel anti-H serum decreased. These results therefore indicate that the conversion depends upon the preference of H-substance on the red cells. The alpha-galactosyltransferase in tortoise serum thus resembles the transferase in human group-B serum and these results suggest that the mechanism of biosynthesis of blood group B substance in the tortoise is similar to that in humans.     

B7.2 has opposing roles during the activation versus effector stages of experimental autoimmune thyroiditis

APCs provide costimulatory and down-regulatory signals to Ag-activated T cells through interactions between B7.1 and B7.2 on APCs with either CD28 or CTL Ag-4 expressed on T cells. Recipients of mouse thyroglobulin (MTg)-primed spleen cells activated in the presence of anti-B7.2 had decreased experimental autoimmune thyroiditis (EAT) severity compared with recipients of cells cultured with control rat Ig or anti-B7.1. Blocking B7.2 during in vivo priming also suppressed the ability of MTg-primed spleen cells to transfer EAT, implicating a role for B7.2 for priming and in vitro activation of EAT effector cells. In contrast, administration of anti-B7.2 or anti-B7.2 Fab to recipients of MTg-activated spleen cells increased the severity of EAT compared with recipients receiving control Ig. Thyroids from anti-B7.2-treated recipients had increased expression of IL-4 mRNA compared with thyroids from rat Ig-treated controls. Both B7.1 and B7.2 molecules were expressed in the thyroids of mice with EAT, although B7.2 was more prevalent than B7.1. Administration of both anti-B7.1 and anti-B7.2 to recipient mice suppressed the development of EAT, while anti-B7.1 treatment alone had no effect on EAT severity. The suppression of EAT was not observed when anti-B7.1 and anti-B7.2 treatment was delayed until 7 days after cell transfer, suggesting a requirement for B7 in the initiation of EAT in recipient mice. These results suggest that costimulation is required during the effector phase of EAT and that B7.2 may have opposing roles in the activation versus effector stages of autoreactive T cells.     

Lack of effect of hypocalcemia on renal phosphate handling

A possible effect of decreased plasma ionized calcium concentration on renal phosphate handling was investigated in dogs with control of parathyroid hormone. Intrarenal artery infusion of either EDTA or sodium citrate decreased ionized calcium concentration 25 per cent in renal vein blood but had no significant effect on fractional phosphate excretion. Similarly, intravenous infusion of chelators had no significant effect on fractional phosphate excretion. It is concluded that acute decreases in ionized calcium have no significant effect on the renal handling of phosphate.     

Hydrops fetalis due to ABO incompatibility

Antenatal haemolysis in association with ABO incompatibility occurs very rarely. Two cases of hydrops fetalis in black infants caused by anti-B haemolysins are reported. The greater severity of ABO incompatibility in black African peoples may have important implications for antibody screening in this ethnic group.     

Sex-associated differences in cold-induced UCP1 synthesis in rodent brown adipose tissue

OBJECTIVE: To evaluate the usefulness of new ELISA for human parvovirus B19 (B19) antibodies and PCR for the diagnosis of acute onset of B19 polyarthritis. METHODS: We evaluated the reproducibility and sensitivity on the detection of anti-B19 antibody by ELISA using recombinant VP-1 and VP-2 (empty particle), and then studied for the prevalence of IgM and IgG B19 antibody in 125 samples for anti-B19 tests. The random study on anti-B19 antibody assay as well as PCR for B19-DNA was also performed in 130 cases with acute onset of arthritis excluding those with known origins, 224 with rheumatoid arthritis and 149 with other categories. RESULTS: The results by using B19-empty particle ELISA were reproducible and showed the assay was a sensitive way for clinical use. IgM anti-B19 antibodies were positive not only in all samples from erythema infectiosum, but also often in those from hemolytic anemia, pure red cell aplasia, fetal hydrops, hepatic injury, fever of unknown origin. Among 130 with acute onset of arthritis, 21 showed positive tests for IgM anti-B19 antibody and/or B19 DNA. On the other hand, 4 among 224 patients with rheumatoid arthritis were positive for IgM anti-B19 antibody, but all of 149 in control group were negative for IgM anti-B19 antibodies and for B19 DNA. CONCLUSION AND DISCUSSION: Anti-B19 ELISA using B19-empty particle which has been introduced as a routine test system, is a useful tool for the diagnosis of acute onset of B19 arthritis. An additional examination using PCR for B19 DNA may contribute for understanding persistent B19 polyarthritis or reactivation of B19 infection.     

Qualitative and quantitative effects of CD28/B7-mediated costimulation on naive T cells in vitro

The CD28/B7 system provides costimulatory signals necessary for optimal T cell activation. We have examined the effects of blocking B7.1 and/or B7.2 in an in vitro system using TCR transgenic T cells specific for myelin basic protein. Activation of naive T cells was found to be B7.2 dependent and not dependent on the presence of B7.1 molecules. However, increasing the strength of signal through the TCR using peptide analogues with higher affinity for MHC compensated for blockade of B7.2 molecules, suggesting that signal 1 alone can be sufficient for the activation of naive T cells. The role of B7 molecules in the differentiation of T cells was further investigated by restimulating T cells with fresh APC and peptide in B7-sufficient conditions. A down-regulation of IL-2 and IFN-gamma production by T cells primed in the presence of anti-B7.2 mAb was partially overcome when high affinity peptide analogues were used to restimulate T cells. In contrast, a significant down-regulation of the differentiation of cells producing Th-2 cytokines was observed in the presence of anti-B7 Abs. Differentiation of IL-4-secreting cells was influenced by both B7.1 and B7.2, while IL-5 secretion was totally dependent on B7.2. These results suggest that B7-mediated costimulation is essential for the development of Th-2-associated cytokines, the absence of which cannot be overcome by increasing the strength of the signal through the TCR.     

Leukocyte phenotypic changes in an in vitro model of ABO hemolytic transfusion reaction

BACKGROUND: ABO antigen-antibody interaction in the presence of peripheral blood leukocytes (white cells) results in the production of a variety of proinflammatory cytokines. However, although tumor necrosis factor alpha has been shown to be derived at least primarily from monocytes, the range of cells activated by this process has not previously been reported. Therefore, changes in mononuclear cell surface antigen expression were studied, to determine which subsets of white cells appeared to be activated in the setting of ABO incompatibility. STUDY DESIGN AND METHODS: Group O peripheral blood mononuclear cells (PBMCs) were incubated in autologous plasma with group A or O red cells (RBCs) for up to 24 hours. White cell expression of activation and adhesion markers was measured at 2 and 24 hours by flow cytometry, using direct or indirect fluorescein or phycoerythrin labeling. RESULTS: Expression of lymphocyte activation markers CD25, CDw108, and CD109 was equivalent when PBMCs incubated with group A and O RBCs were compared. However, after 2 hours, mean fluorescence of CD14 on PBMCs incubated with group A RBCs was 65 percent of that on PBMCs incubated with group O RBCs and remained similarly decreased at 24 hours. CD44 expression was upregulated on PBMCs exposed to both group A and O RBCs, but it was increased significantly more on monocytes exposed to group A RBCs. The ability to bind hyaluronic acid was induced in approximately 42 percent of CD14+ monocytes exposed to group A RBCs but in no cells exposed to group O RBCs. CONCLUSION: Downregulation of CD14 and increased binding of hyaluronic acid reflects monocyte activation in this model. No evidence of lymphocyte activation was found, supporting the hypothesis that ABO transfusion reactions primarily activate monocytes.     

Warm IgM anti-IT causing autoimmune haemolytic anaemia

A patient with autoimmune haemolytic anaemia due to an autoagglutinin of anti-IT specificity is reported. The antibody was of the IgM class and bound complement; unexpectedly, it reacted optimally at 37 degrees C. A prozone was noted when testing serial dilutions of the patient's serum against appropriate red cells in saline, but was not found when testing an eluate prepared from the patient's red cells. Investigations suggested that the prozone was due to a blocking antibody of high molecular weight, which appeared to be specific for I and IT.     

Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of beta-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon

The possibility that extracellular matrix-cell surface interaction might lead to signalling was examined in human mononuclear cells. In mononuclear cells loaded with Indo-1 acetoxymethylester, collagen binding elicited a 2-3 fold increase in cytosolic free calcium concentration within seconds. Plasma fibronectin, serum albumin and heparin did not cause any significant change in intracellular calcium levels. Collagen I, collagen IV and glucosylated collagen I evoked an increase in intracellular Ca++ both in the presence and absence of extracellular calcium. However, in the presence of 2mM EDTA, the increase in cytosolic free calcium concentration caused by collagen I and collagen IV was partly abolished, suggesting the requirement of a cation-dependent interaction of collagen with mononuclear cells. Glucosylated collagen induced intracellular calcium mobilization even in the presence of EDTA suggesting a cation-independent interaction. These results indicate that collagen binding induces rapid mobilization of calcium in human mononuclear cells, apparently from intracellular sources.     

Interactions in the complement-mediated lysis of blood group AB erythrocytes sensitized simultaneously with anti-A and anti-B monoclonal antibodies

The lysis of group AB erythrocytes by human complement was studied by different anti-A and anti-B IgM monoclonal antibodies (mabs) in a 51Cr-release assay. The concentration of membrane-bound immunoglobulin was detected by ELISA, and the amount of C1q and C3 bound to sensitized red cells was measured by using purified, 125I-labelled molecules. We have demonstrated that there is an exponential relationship between the concentration of the sensitizing IgM mabs and C1q binding to the sensitized AB cell. The efficiency of binding was related to the number of antibodies bound; thus, anti-A sensitized cells bound 3-6 times more C1q than anti-B sensitized cells did. AB cells, on the other hand, bound similar amounts of C3 whether anti-A or anti-B was present. The lytic efficiencies of the various IgM mabs during short incubation times were different, suggesting that the complement activation rates vary widely with different antibodies on the AB cell membrane. The binding of C1q to an antibody-sensitized target activates a cascade, whose components may migrate away from the sensitizing antibody; interactions between the activation processes generated by the anti-A and anti-B antibodies may thus occur. Choosing appropriate pairs of anti-A and anti-B mabs for the simultaneous sensitization of AB cells has indeed resulted in stimulation in some and inhibition in other combinations of mabs. It is suggested that stimulation is observed when the activated intermediates are produced in excess, whereas inhibition occurs when a shortage of activated intermediates prevents mutual utilization.     

The high-molecular-weight human mucin is the primary salivary carrier of ABH, Le(a), and Le(b) blood group antigens

Because many bacteria interact with the carbohydrate portions of receptor molecules, factors controlling glycosylation probably influence the ability of salivary components to mediate bacterial adherence/clearance. Important sources of diversity in glycosylation are the ABO, secretor, and Lewis genes, which code for glycosyltransferases that add specific sugar sequences to the termini of carbohydrate chains of glycolipids and glycoproteins. We identified, by Western blotting, salivary glycoproteins carrying the ABH and Le(a) or Le(b) antigens. Samples of whole, unstimulated saliva were obtained from 19 subjects whose blood group was determined by agglutination of red blood cells with specific antisera. After centrifugation, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Glycoproteins carrying blood group antigens were identified by staining the blot with monoclonal antisera specific for the A, B, H, Le(a), or Le(b) antigens. The most intensely staining component from all the samples migrated at the same position as the high-molecular-weight mucin. Saliva samples from the nonsecretors contained only the Le(a) antigen. Samples from the secretors contained one or more of the ABH antigens and, variably, the Le(b) antigen. In all cases, the salivary blood group antigens corresponded to those found on the red blood cells of the same subject. The functional consequences of the expression of blood group antigens on the high-molecular-weight mucin are not known, but their presence could modulate the adherence of certain oral microorganisms that interact preferentially with this molecule.     

Treatment of ABO blood type incompatibility caused early abortion with traditional Chinese medicine

This results of 30 cases with the history of abortion due to ABO blood type incompatibility treated with traditional Chinese medicine (TCM) were reported. The titer of serum IgG anti A/B antibody were determined in these patients. Antibody titer < or = 1:128 before pregnancy or < or = 1:64 after pregnancy were treated with TCM till delivery. 25 cases gave birth at full term, 5 cases were before term. Abortion, stillbirth or hemolytic newborn death were not found. The lower the maternal serum IgG anti A/B titer, the lower the incidence of hemolytic jaundice; while the earlier the treatment after pregnancy, the lower the occurrence of hemolytic jaundice also.     

Expression and function of B7-1 and B7-2 in hapten-induced contact sensitivity

We analyzed the expression and function of the co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86) during contact sensitivity reactions induced by the hapten 2,4-dinitrofluorobenzene (DNFB). In the normal skin, only a few epidermal Langerhans cells or dermal dendritic cells express B7-2. In contrast, following challenge with DNFB, expression of B7-2 is up-regulated in both epidermis and dermis. Importantly, B7-1 is induced later and at lower levels compared to B7-2. Intravenous injections of anti-B7-2 mAb, but not anti-B7-1 mAb partially inhibit the hapten-induced contact sensitivity reaction. Experiments in which mice are injected differentially with anti-B7-2 mAb, either before the afferent or before the efferent phase of the contact sensitivity response, suggest that B7-2 is important for successful antigen priming.