A three-year M.D. program for students with Ph.D. degrees.
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Curriculum↗
Biomedical subjects
H Chaplin
Publications and source records attributed to H Chaplin.
Comparisons of pooled polyclonal rabbit anti-human C3d with four monoclonal mouse anti-human C3ds. I. Preparation, purification and binding properties.
Performance characteristics of pooled rabbit IgG polyclonal anti-C3d are compared with one mouse IgM and three mouse IgG monoclonal anti-C3d antibodies (MAs). IgG MA,s employed singly or in combination, failed to precipitate C3d; by contrast, IgM MA and polyclonal anti-C3d precipitated C3d. Measurements of polyclonal anti-C3d concentration by chemical means and by 125I-C3d radioimmunoassay (RIA) agreed closely. RIA values were 50% of chemical measurement values for three of the four MAs. Use of sucrose density gradient ultracentrifugation to assess MA C3d/anti-C3d molar combining ratios for soluble anti-C3d/C3d was not possible because fast-sedimenting multimeric C3d/anti-C3d complexes did not form. Dissociation and competitive binding studies indicate that (1) two MAs had substantially lower affinities than the other anti-C3d antibodies, and (2) polyclonal anti-C3d recognizes more C3d epitopes than are recognized by individual MAs. The results demonstrate antigenic complexity of C3d fragment and illustrate the difficulties of predicting individual MA performance based on prior experience with polyclonal antibodies.
Animals↗
Comparisons of pooled polyclonal rabbit anti-human C3d with four monoclonal mouse anti-human C3ds. II. Quantitation of RBC-bound C3d, and characterization of antiglobulin agglutination reactions against RBC from 27 patients with autoimmune hemolytic anemia.
Labelled polyclonal IgG anti-C3d and monoclonal IgM and IgG anti-C3d antibodies (MAs) were employed at increasing antibody excess to measure the number of C3d molecules on human red blood cells (RBC) coated by complement in vitro and in vivo. Values for the number of C3d sites per cell determined with polyclonal anti-C3d were at least 4-fold higher than when MAs were used. The results suggest that the molar combining ratio for polyclonal anti-C3d with a single RBC-bound C3d fragment is more likely greater than 4.0 than 1.0 as previously assumed. Antiglobulin agglutination studies compared polyclonal and monoclonal anti-C3d antibodies against C3d-coated RBC from 27 patients with autoimmune hemolytic anemia. All four MAs showed striking prozones, requiring their use over a 25-fold higher range of dilutions than polyclonal anti-C3d. Polyclonal anti-C3d produced stronger agglutination reactions than any of the IgG MAs. Only the IgM MA produced agglutination as strong as, or stronger than, polyclonal anti-C3d. While IgM MA always gave the strongest MA agglutination reactions, no consistent ranking of the three IgG MAs was observed. Agglutination was not enhanced when all IgG MAs were combined; addition of IgG MAs to IGM MA reduced the strength of agglutination seen with IgM alone, suggesting blocking of IgM binding by competing IgG anti-C3d.
Anemia, Hemolytic, Autoimmune↗
Acute intravascular hemolytic anemia in the black rhinoceros: hematologic and immunohematologic observations.
To investigate the syndrome of acute intravascular hemolytic anemia in the black rhinoceros (Diceros bicornis), laboratory techniques used in the differential diagnosis of hemolytic anemias were performed on blood samples from 6 black rhinoceroses: 3 nonrelated healthy rhinoceroses, 1 rhinoceros with iron deficiency anemia, and 2 rhinoceroses with intravascular hemolysis. Osmotic fragility, erythrocyte membrane protein composition, hemoglobin electrophoresis, and hemoglobin stability did not distinguish between healthy and affected (anemia or hemolysis) rhinoceroses. Polyclonal antiglobulin reagents were prepared in rabbits, using whole rhinoceros serum and purified rhinoceros immunoglobulin G. These reagents were nonreactive against erythrocytes of the healthy and iron-deficient rhinoceroses. Reactions with RBC from the rhinoceros with fatal hemolytic anemia indicated increased membrane coating by the third component of complement; this was not evident in a second rhinoceros that survived a hemolytic event.
Anemia, Hemolytic↗
Further studies of the C3g component of the alpha 2D fragment of human C3.
Recent studies employing sheep red cells, rabbit anti-sheep cell IgM, guinea-pig R3 reagent and purified human C3, factor I, factor H and human serum have led to the discovery of a new C3b breakdown product antigen, C3g, which is present on the alpha 2D fragment of C3 but is removed by trypsinization. The present studies were undertaken to confirm the above findings in entirely human red cell and serum systems, including studies on cells from nine patients with a variety of complement-mediated autoimmune haemolytic anemias. The results confirm the recently reported findings in every respect. The presence of both C3d and C3g on all patient's red cells so far tested supports the concept that red cells coated by C3d alone are likely to be a laboratory artefact and that the alpha 2D fragment (C3d,g) is the natural end product of cell bound C3b breakdown in vivo.
Anemia, Hemolytic, Autoimmune↗
In vivo instability of red-blood-cell-bound C3d and C4d.
Until now, there have been no measurements of the in vivo stability of red-blood-cell-bound C3d and C4d subfragments of the third and fourth components of human complement. We have recently described a radiolabeled antiantiglobulin method for measuring RBC-bound C3d and have demonstrated that small amounts of C3d are present on RBC of all normal subjects tested. In the present study, the method was applied to follow the increments above baseline of RBC-bound C3d and C4d produced by autotransfusing 3 normal volunteers with 160-200 ml of RBC strongly coated in vitro by C3d and C4d. Posttransfusion measurements were carried out over 21-34 days. Immediate and long-term in vivo survival of the transfused RBC was unimpaired by C3d and C4d coating. Of the bound C3d antigen, 85%-95% disappeared from circulating RBC in 5-8 days; the remainder disappeared more slowly, with half-times in the range of 8-29 days. C4d antigen disappeared substantially more slowly, describable by a single exponential function in 2 of the 3 subjects, with half-times in the range of 12-31 days. Recognition of the in vivo instability of RBC-bound C3d helps in interpreting steady-state and changing levels of RBC C3d coating in a variety of alloimmune and autoimmune disorders.
Adult↗
Lymphoma in primary chronic cold hemagglutinin disease treated with chlorambucil.
Two patients with primary chronic cold hemagglutinin disease (CHAD) were treated with chlorambucil for ten and 7 1/2 years, with cumulative doses of 14 and 6.6 g, respectively. Both responded favorably, and malignant lymphoma eventually developed in both of them. An attempt to assess the incidence of lymphoma as part of the natural course of untreated CHAD failed for want of proof. Literature since 1970 reported no chlorambucil-associated lymphoma. Of 77 responses to a questionnaire mailed to 135 US hematology-oncology programs, 76 reported no examples of chlorambucil-associated lymphoma. One response, however, did relate that diffuse histiocytic lymphoma developed in three of 141 patients receiving prolonged chlorambucil therapy for polycythemia vera. Alkylating-agent therapy for CHAD might be associated with development of lymphoma.
Anemia, Hemolytic, Autoimmune↗
The proper use of previously frozen red blood cells for transfusion.
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Blood Preservation↗
Quantitation of red blood cell-bound C3d in normal subjects and random hospitalized patients.
A sensitive radiolabelled anti-antiglobulin method was devised and applied to quantitating red blood cell-bound C3d (RBC-C3d) in samples from 174 normal blood donors. C3d was demonstrable on all RBC examined; 98% of values fell over a broad range, with the highest values being approximately 3.5 X the lowest values (equivalent to 50-160 molecules of C3d per cell). RBC-C3d did not correlate with sex or age (over 18-65 years); indirect evidence suggests that values for the paediatric age group will fall in the same normal range. Studies on samples obtained weekly for 10-12 weeks from six adult males and six adult females indicated stable levels of RBC-C3 for individual subjects; i.e. high normals, mid normals and low normals remained in their characteristic range levels over the period of observation. For comparison, RBC-C3d was measured in samples from 313 randomly selected hospitalized adult patients. 33% of the values were above the normal range; 8% were elevated to a level likely to have been detectable by a direct anti-C3d antiglobulin test. The great majority of elevated values occurred in patients not ordinarily considered to have autoimmune conditions. The results provide background for studies of the aetiology and significance of RBC-C3d in health and disease.
Adult↗
Combined sickle cell disease and autoimmune hemolytic anemia.
Four patients experienced combined sickle cell disease and autoimmune hemolytic anemia within the past ten years. A fifth patient had positive direct antiglobulin test results without verified autoimmune hemolysis. Severely accelerated hemolysis was observed in four patients; anemia was severe, and the reticulocyte count rose into the 60% to 88% range. During the period of active autoimmune hemolysis, decline of the reticulocyte count rose into the 60% to 88% range. During the period of active autoimmune hemolysis, decline of the reticulocyte count into the 6% to 16% range was associated with rapid decrease in the hemoglobin level, requiring transfusion. All five patients were already alloimmunized by transfusions administered before onset of the autoimmune hemolytic anemia; two or more allospecificities were identified in four of five patients. The presence of autoantibody notably compromised compatibility testing; three patients experienced posttransfusion hemoglobinuria, and in vivo cross matching with 51Cr-labeled donor RBCs was employed on three occasions. All patients responded to corticosteroids; mercaptopurine was also administered to one patient. The direct antiglobulin test result reverted to negative in all patients after hospital discharge.
Adult↗
Aspirin-dipyridamole prophylaxis of sickle cell disease pain crises.
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Acute Disease↗
Assessment of complement binding by anti-D and anti-M antibodies employing labelled antiglobulin antibodies.
The presence of small amounts of C3d on freshly obtained normal red blood cells (RBC) was demonstrated by a radio-labelled antiglobulin technique; this increased 1.5-fold after incubation in fresh normal serum. No significant further increase in bound C3d was demonstrated for cDE/cDE, CDe/CDe, -D-/-D- RBC maximally sensitized with any of three potent anti-D antibodies. When anti-c, anti-D and anti-E antibodies were used in combination to sensitize cDE/cDE RBC, bound C3d increased by approximately 22%; however, a similar increase occurred with cde/cde RBC and was therefore considered non-Rh specific. Umbilical cord RBC from four infants severely affected by anti-D haemolytic disease of the newborn did not exhibit more bound C3d than cord RBC from normal controls. One serum containing IgM anti-M bound a small amount of complement in vitro; two IgG anti-M sera did not.
Antibodies, Anti-Idiotypic↗
Hematuria in hemoglobin S disorders.
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Anemia, Sickle Cell↗
Further observations on the preparation of antiglobulin reagents reacting with c3d and c4d on red cells.
A simplified method was developed for preparing rabbit red cells coated only with human C3d by the alternative pathway; these cells were injected into the donor rabbit to prepare an anti-C3d serum. Monkey red cells were sucessfully coated with human C4d, using a low-ionic-strength method, and these cells were injected into the donor monkey to prepare anti-human C4d. The reactions of these reagents were compared with those of antisera raised to complement-coated red cells by previously described methods and with those of antisera obtained by immunizing rabbits with purified soluble human C3d.
Animals↗
Rosette formation by sensitized human red cells- effects of source of peripheral leukocyte monolayers.
Employing standardized IgG-coated 51Cr-labelled human red blood cells (RBC), variation in rosette formation among monolayers obtained from 10 healthy donors was defined, as well as variation among 24 sequential monolayers obtained from one healthy donor and 8 sequential monolayers from a second healthy donor over a 4-month period. Rosette formation, expressed as eluate cpm per plate, average 220 (range 35-472) for the 10 donors; 24 determinations on one donor average 201 (range 59-420), 8 determinations on the second donor averaged 244 (range 69-395). Striking increases in rosette formation were observed employing monolayers from 4 of 4 normal subjects during the course of viral infections, with peak values exceeding 5, 8, 13 and 14 X the normal mean. Elevations were also observed with monolayers prepared from blood of selected patients with hematologic disorders; a remarkable value 168 X the normal mean was repeatedly obtained for a patient with myeloid metaplasia whose monolayer consisted almost exclusively of mature granulocytic rosette-forming cells. The results emphasize the influence of monolayer donor variables on rosette formation.
Acute Disease↗
Comparison of low-molecular-weight products following reaction of C3-C3b with C3b inactivator and with trypsin.
Substitution of trypsin for Konglutinogen-activating factor (KAF) in the procedure for cleaving C3d from C3-C3b substrate produced a relatively heterogeneous low-molecular-weight fraction (C3d-Tryp) which differed in a number of ways from the KAF-mediated cleavage product (C3d-KAF). The differences were demonstrable by agar and polyacrylamide gel electrophoresis, 125I-labelling, content of immunoreactive 125I-labelled C3d, inhibition of anti-complement antiglobulin reagents and rabbit immunization. By comparison with C3d-KAF, the C3d in C3d-Tryp was more heterogeneous and exhibited a faster electrophoretic mobility in agar at pH 8.6. By contrast to C3d-KAF, C3d-Tryp contained protein carrying C3c antigenic determinants.
Complement C3↗