Erythrocyte membrane proteins reactive with IgG (warm-reacting) anti- red blood cell autoantibodies: II. Antibodies coprecipitating band 3 and glycophorin A

In our initial immunochemical study of the red blood cell (RBC) membrane proteins targeted in 20 cases of warm-antibody autoimmune hemolytic anemia (AHA), RBC eluates of 6 patients mediated immunoprecipitation (IP) of both band 3 and glycophorin A (GPA). This dual IP pattern had previously been observed with murine monoclonal antibodies (MoAbs) against the high frequency blood group antigen, Wrb (Wright), suggesting that the Wrb epitope may depend on a band 3-GPA interaction. Earlier, anti-Wrb had been identified serologically as a prominent non-Rh specificity of AHA autoantibodies. In the present study, 6 autoantibody eluates immunoprecipitating band 3 and GPA from common Wr(b+) RBCs were retested, in parallel with murine anti-Wrb MoAbs, against very rare Wr(a+b-)En(a+)RBCs. One patient's autoantibodies were unreactive with the Wr(b-) RBCs by either IP or indirect antiglobulin test (IAT) and were judged to have "pure" anti- Wrb specificity. Two other patients' autoantibodies displayed both IP and serologic evidence for anti-Wrb as a major component in combination with one or more additional specificities. However, among 3 other patients whose autoantibodies coprecipitated band 3 and GPA, there was no reduction in IP or IAT reactivity with Wr(b-) RBCs in 2 and only slight reduction in the third. We conclude (1) that human anti-Wrb autoantibodies, like their murine monoclonal counterparts, coprecipitate band 3 and GPA from human RBCs; but (2) that not all antibodies with this IP behavior have anti-Wrb serologic specificity, as defined by this donor's Wr(b-) RBCs. The possibility of an additional (non-Wrb) RBC epitope dependent on a band 3-GPA interaction is raised.     

Magnitude, reproducibility, and association with baseline cardiac function of cardiac biomarker release in long-distance runners aged > or =55 years

Cardiac biomarker release after endurance exercise has been described in young athletes. Although older athletes are increasingly active in such sports, they have not previously been studied. Therefore, the aim of this study was to assess the magnitude and reproducibility of biomarker release in athletes aged > or =55 years. Forty-three healthy athletes (mean age 61 +/- 3.6 years) were assessed before and immediately after a 30-km cross-country race and studied with echocardiography at rest. The median N-terminal pro-brain natriuretic peptide (NT-proBNP; normal <194 ng/L) level was 42 ng/L (interquartile range 30 to 95) at baseline and 191 ng/L (interquartile range 114 to 308) after the race. Troponin T (normal <0.03 microg/L) was elevated in 19 subjects (44%) after the race. Twenty-two subjects had also been studied 3 years before at the same race, using an identical test protocol. Between the 2 races, strong correlations were seen for individual runners' postrace biomarker levels (NT-proBNP: r = 0.82, log transformed data; troponin T: Spearman's rho = 0.84; p <0.001 for both). The coefficient of variation for NT-proBNP release was 8.1%. Levels of NT-proBNP after the race were correlated with levels at baseline (r = 0.93, p <0.001) and with left ventricular mass index (r = 0.32, p = 0.03). Moreover, participants with elevated postrace NT-proBNP were significantly older (62.0 vs 59.8 years, p = 0.04). In conclusion, long-distance runners aged > or =55 years released NT-proBNP and troponin T in a reproducible fashion. The magnitude of NT-proBNP release during the race was correlated strongly with NT-proBNP baseline levels and was associated with left ventricular mass and age. These findings may suggest a potential adverse effect of long-distance running on cardiac function in certain participants in this age group.     

The rat extracellular superoxide dismutase dimer is converted to a tetramer by the exchange of a single amino acid.

Extracellular superoxide dismutase (EC-SOD) is a secreted Cu and Zn-containing glycoprotein. While EC-SOD from most mammals is tetrameric and has a high affinity for heparin and heparan sulfate, rat EC-SOD has a low affinity for heparin, does not bind to heparan sulfate in vivo, and is apparently dimeric. To examine the molecular basis of the deviant physical properties of rat EC-SOD, the cDNAs of the rat and mouse EC-SODs were isolated and the deduced amino acid sequences were compared with that of human EC-SOD. Comparison of the sequences offered no obvious explanation of the differences. Analysis of a series of chimeric and point mutated EC-SODs showed that the N-terminal region contributes to the oligomeric state of the EC-SODs, and that a single amino acid, a valine (human amino acid position 24), is essential for the tetramerization. This residue is replaced by an aspartate in the rat. Rat EC-SOD carrying an Asp --> Val mutation is tetrameric and has a high heparin affinity, while mouse EC-SOD with a Val --> Asp mutation is dimeric and has lost its high heparin affinity. Thus, the rat EC-SOD dimer is converted to a tetramer by the exchange of a single amino acid. Furthermore, the cooperative action of four heparin-binding domains is necessary for high heparin affinity. These results also suggest that tetrameric EC-SODs are not symmetrical tetrahedrons, but composed of two interacting dimers, further supporting an evolutionary relationship with the dimeric cytosolic Cu and Zn-containing SODs.     

Differences in EBV-specific antibody patterns at onset of infectious mononucleosis

The EBV-specific antibody patterns of infectious mononucleosis (IM) patients were analyzed in relation to the onset of symptoms and clinical parameters during the acute phase of the disease. The antibody patterns varied considerably on admission. Three groups of patients were identified: one had not yet attained peak antibody titers, the second was at the peak and the third had passed the peak pattern. Patients with a "peak" current pattern had significantly higher lymphocyte counts, ASAT, ALAT, serum IgG and serum IgA concentrations than patients of the third group. Unexpectedly, there was no difference between the groups with regard to duration of sore throat and general malaise before admission. It thus seems that the lymphocyte proliferation during IM closely parallels the course of the EBV-specific antibody responses, whereas the onset of IM does not closely correlate to a specific stage of the antibody pattern.     

Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis

To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3sMEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHIS in the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine subsitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors.     

Acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas and other malignancies in patients with hemophilia

Non-Hodgkin's lymphoma (NHL) is the most common human immunodeficiency virus (HIV)-associated malignancy in hemophiliacs. We studied the incidence and clinicopathologic features of NHL in 3,041 hemophiliacs followed at 18 US Hemophilia Centers between 1978 and 1989. Of the 1,295 (56.6%) who were HIV(+), 253 (19.5%) developed acquired immunodeficiency syndrome (AIDS), of whom 14 (5.5%) developed NHL. Three NHL occurred in HIV(-) hemophiliacs, for a 36.5-fold greater risk in HIV(+) than HIV(-) hemophiliacs (P < .001). The NHL incidence rate was 29-fold greater than in the US population by Surveillance, Epidemiology, and End Results (SEER) estimates (P < .001). Between 0 and 4 lymphomas have been observed per year between 1978 and 1989. At presentation 13 (92.9%) of the HIV(+) NHL were extranodal. Ten were stage IV, 1 stage II, and 3 stage IE. Ten (71.4%) were high-grade, 3 (21.4%) intermediate-grade, and 1 (7.1%) was a low-grade B-cell lymphoma. Epstein-Barr virus (EBV) DNA was detected in 36% by in situ hybridization, including one central nervous system (CNS) lymphoma. The mean CD4 cell count at NHL diagnosis was 64/mm3, the mean latency from initial HIV infection was estimated to be 59 months, and the median survival was 7 months. The incidence of basal cell carcinoma in HIV(+) hemophiliacs was 18.3-fold greater than in HIV(-) hemophiliacs (P < .001) and 11.4-fold greater than in the US population (P < .001). In conclusion, incidence rates of NHL and basal cell carcinoma in HIV(+) hemophiliacs are significantly increased over rates in HIV(-) hemophiliacs and over rates in the US population. Clinicopathologic presentation of NHL in HIV(+) hemophiliacs is similar to that in HIV(+) homosexual men.     

IgA antibodies to Epstein-Barr virus in infectious mononucleosis

The IgA anti-EBV (Epstein-Barr virus) response during the course of IM (infectious mononucleosis) was investigated. The IgA anti-VCA (viral capsid antigen) response was found not to be restricted to the early acute phase of the EBV infection as is the IgM anti-VCA response. Some patients with normal total serum IgA levels did not respond with measurable EBV specific IgA. These patients and those with low titers of IgA anti-VCA had shorter duration of sore throat than responders with high titers indicative of a strong correlation between the IgA anti-VCA titers and the duration of sore throat. In this way the EBV specific IgA response is unique since recent observations show that local oropharyngeal symptoms during IM appear poorly synchronized with the IgM and the IgG antibody responses. As EB virus is excreted into the oropharynx during IM, antigens are available for local EBV immunization. The results of the present study imply a possible local immunization process as a positive correlation was found between serum IgA anti-VCA and total salivary IgA.     

Production and characterization of monoclonal antibodies to the subunits of human phosphofructokinase: new tools for the immunochemical and genetic analyses of isozymes

Recently we have demonstrated that human phosphofructokinase (PFK; ATP: D-fructose-6-P, 1-phosphotransferase; EC.2.7.1.11) is under the control of three structural loci that code for M (muscle-type), L (liver-type), and P (platelet-type) subunits: random tetramerization of these subunits produces various isozymes. In this study, we have produced and characterized BALB/c hybridoma antibodies to the M- and L-type subunits of human PFK. The specific antibodies were detected by an enzyme- immunoprecipitation assay using Staphylococci-bearing protein A as an immunoadsorbent. Of the wells tested using red blood cell (RBC) PFK (M + L), 61% were positive. Only one M-specific hybridoma was identified. The one anti-M and 4 anti-L antibodies were characterized for their biochemical and immunochemical specificities. To define the combining specificities of these antibodies, we compared their reactivity and that of monospecific rabbit anti-M antiserum with muscle and liver PFKs from 15 different vertebrate species. The rabbit anti-M shows strong cross-reactivity with the muscle PFKs from all the species studied. In contrast, the monoclonal anti-M reacts exclusively with muscle PFKs from primates. Two of four anti-L antibodies react only with human L- PFK, whereas the other two react with that from a few other vertebrate species as well. Taken together, these data suggest that primate- specific antibodies recognize evolutionarily, recently acquired antigenic determinants, whereas the antibodies reactive with PFKs from distantly related species recognize conserved determinants. The differential immunoreactivities of muscle and liver PFKs strongly suggest the presence of distinct isozymes in all the vertebrate species studied. These studies demonstrate that it is feasible to produce and characterize monoclonal antibodies that distinguish among isozymes with structural and functional similarities. These antibodies provide sensitive tools in the analyses of isozyme structure, genetics, and related fields.