Mutational analysis of an autoantibody: differential binding and pathogenicity.

We have used site-directed mutagenesis to change amino acid residues in the heavy chain of the pathogenic R4A anti-double-stranded DNA (dsDNA) antibody and have looked for resultant alterations in DNA binding and in pathogenicity. The data demonstrate that single amino acid substitutions in both complementarity determining and framework regions alter antigen binding. Changes in only a few amino acids entirely ablate DNA specificity or cause a 10-fold increase in relative binding. In vivo studies in mice of the pathogenicity of the mutated antibodies show that a single amino acid substitution leading to a loss of dsDNA binding leads also to a loss of glomerular sequestration. Amino acid substitutions that increase relative affinity for dsDNA cause a change in localization of immunoglobulin deposition from glomeruli to renal tubules. These studies demonstrate that small numbers of amino acid substitutions can dramatically alter antigen binding and pathogenicity, and that the pathogenicity of anti-DNA antibodies does not strictly correlate with affinity for DNA.

The double edged sword of the immune response: mutational analysis of a murine anti-pneumococcal, anti-DNA antibody.

Anti-double-stranded (ds) DNA antibodies are not only an important diagnostic marker for SLE, but also play an important role in tissue injury. Microbial antigen may be a stimulus for the production of these antibodies. We isolated 99D.7E, an IgG2b monoclonal antibody from a nonautoimmune BALB/c mouse that is cross-reactive with both dsDNA and phosphorylcholine, the dominant hapten on the pneumococcal cell wall. While partially protective against a bacterial challenge, 99D.7E is also pathogenic to the kidney. To identify those molecular motifs that confer on anti-PC antibodies the potential for autoreactivity, we created a panel of 99D.7E mutants with single amino acid substitutions in the heavy chain, and examined the changes in antigen binding and renal deposition. Our results support the hypothesis that charge and affinity for dsDNA are not adequate predictors of the pathogenicity of anti-DNA antibodies. Differential renal damage from anti-dsDNA antibodies may be due to differences in fine specificity, rather than differential affinity for dsDNA. Importantly, high affinity IgG antibodies cross-reactive with bacterial and self antigen exist and can display pathogenic potential, suggesting that defects in peripheral regulation of B cells, activated by foreign antigen but cross-reactive with self antigen, might lead to autoimmune disorders.

Erythropoietin level and hematologic parameters in healthy adults.

Since the major physiological control of erythropoiesis is related to the eryhtropoietin (EPO) level, correlating the EPO level with hematologic parameters in healthy adults, which constitutes an inexpensive and simple routine laboratory report, would be very useful. Two hundred healthy adult blood donors, 100 males and 100 females, aged between 17 and 60 years old were randomly chosen. The EPO reference range was determined by enzyme linked immunosorbent assay (ELISA) using a reagent kit from Research & Development Systems Inc. The hematologic values for reticulocyte and red blood cell parameters were assessed using the Technicon H*3 RTC, an automated blood cell analyzer. The EPO reference range in the studied population was 2.21-20.95 mU/ml. The correlations between the EPO level and hematologic parameters were between -0.302 (p < 0.01) and 0.294 (p < 0.01). The results suggested that there were none or low correlations between the EPO level and hematologic parameters in healthy adults. According to our results, these parameters could not be used to indicate the level of EPO in healthy adults.

Reference ranges of reticulocytes in adults.

According to the International Committee for Standardization in Haematology (ICSH), we determined the reference values for reticulocytes using an automated blood cell analyzer Technicon H*3 RTC in 200 healthy adult blood donors, aged between 17 and 60 years, 100 of whom were male and 100 female. The parameters included reticulocyte count, and its corpuscular indices; mean reticulocyte corpuscular volume (MCVr), mean reticulocyte corpuscular hemoglobin concentration (CHCMr), mean reticulocyte hemoglobin content (CHr), reticulocyte distribution width (RDWr), reticulocyte hemoglobin distribution width (HDWr) and reticulocyte corpuscular hemoglobin concentration distribution width (CHDWr). The reference ranges were established by setting the reference limits at two standard deviations from the arithmetic reference mean.

Ratio of baseline erythropoietin (EPO) level and corrected reticulocyte count as an indicator for a favourable response to recombinant human erythropoietin (rhEPO) therapy in anaemic cancer patients.

PURPOSE: Recently, recombinant human erythropoietin (rhEPO) was introduced for the management of anemia in malignancy. To identify an indicator for a favourable response to rhEPO, 28 anaemic cancer patients undergoing chemotherapy and treated with rhEPO were evaluated. METHODS: Patients were classified into responder (16 of 28, 57%) and nonresponder (12 of 28, 43%) groups according to their responses to rhEPO therapy (response being defined as an increase in Hb level of > 2 g/dl from baseline without blood transfusion). RESULTS: Treatment with rhEPO showed significant improvements in the red blood cell (RBC) count, haemoglobin (Hb), packed cell volume (PCV), and reticulocyte count (ret. count) after 4 weeks. Upon analysing the baseline value of the EPO level and the corrected ret.count in these two groups, we found that the ratio of the EPO level and the corrected ret.count (EPO/ret.count) demonstrated a statistical significance (P = 0.03) in the prediction of response to rhEPO therapy. This ratio showed a sensitivity of 87.5%, specificity of 66.7%, and overall accuracy of 78.6%. CONCLUSION: Our study suggested that the baseline ratio of EPO/ret.count should be used as an indicator for a favourable response to rhEPO therapy.

Cross-reactive antibodies have both protective and pathogenic potential.

We report the characteristics of five IgM anti-phosphorylcholine (PC) Abs derived from nonautoimmune BALB/c mice. These Abs were chosen on the basis of their unique ability to cross-react with dsDNA. Three Abs protect mice from a lethal pneumococcal infection, and all five Abs deposit in glomeruli and cause renal dysfunction. Analysis of V region gene usage shows that none of the Abs is encoded by the V1 gene segment used to encode the dominant T15+ anti-PC response typically expressed in serum of PC-immunized BALB/c mice. The demonstration of the existence of noncanonical protective anti-PC Abs that also display pathogenic autoreactivity suggests that idiotypically restricted responses may serve to prevent the expression of Abs that react with both foreign and self Ags and so help maintain self-tolerance.

Erythropoietin level and hematologic parameters in healthy adults.

Since the major physiological control of erythropoiesis is related to the eryhtropoietin (EPO) level, correlating the EPO level with hematologic parameters in healthy adults, which constitutes an inexpensive and simple routine laboratory report, would be very useful. Two hundred healthy adult blood donors, 100 males and 100 females, aged between 17 and 60 years old were randomly chosen. The EPO reference range was determined by enzyme linked immunosorbent assay (ELISA) using a reagent kit from Research & Development Systems Inc. The hematologic values for reticulocyte and red blood cell parameters were assessed using the Technicon H*3 RTC, an automated blood cell analyzer. The EPO reference range in the studied population was 2.21-20.95 mU/ml. The correlations between the EPO level and hematologic parameters were between -0.302 (p < 0.01) and 0.294 (p < 0.01). The results suggested that there were none or low correlations between the EPO level and hematologic parameters in healthy adults. According to our results, these parameters could not be used to indicate the level of EPO in healthy adults.

Reference ranges of reticulocytes in adults.

According to the International Committee for Standardization in Haematology (ICSH), we determined the reference values for reticulocytes using an automated blood cell analyzer Technicon H*3 RTC in 200 healthy adult blood donors, aged between 17 and 60 years, 100 of whom were male and 100 female. The parameters included reticulocyte count, and its corpuscular indices; mean reticulocyte corpuscular volume (MCVr), mean reticulocyte corpuscular hemoglobin concentration (CHCMr), mean reticulocyte hemoglobin content (CHr), reticulocyte distribution width (RDWr), reticulocyte hemoglobin distribution width (HDWr) and reticulocyte corpuscular hemoglobin concentration distribution width (CHDWr). The reference ranges were established by setting the reference limits at two standard deviations from the arithmetic reference mean.