Comparisons of Pooled Polyclonal Rabbit Anti-Human C3d with Four Monoclonal Mouse Anti-Human C3ds

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.     

Experience of a combined apheresis, blood collection, and blood transfusion unit in a large tertiary care medical center

The Barnes Hospital Apheresis Blood Collection and Blood Transfusion Unit is part of Barns Hospital Blood Bank. Because of its size and complexity, we report our experience which may be useful to administrators and physicians involved in the planning or management of similar services. From 1985 through 1988 we collected platelets from 1,976 different donors, the majority of which (87%) were community donors. Sixty-nine percent of 1,976 donors donated in 1988 an average of 4.9 times. Of 6,568 apheresis products collected. 1.1% were discarded because of positive screening tests and 0.7% were discarded because of outdating or presence of fibrin clot. In 1988 a total of nine cell separators were used. All donor apheresis were done with seven blood separators, and on average a separator produced an apheresis product every 4.5 worked hours. All therapeutic apheresis (338) were done on two separators. Most of them (88%) were performed during work hours. In 1988 donor and therapeutic apheresis were done by 17 1/2 full-time employees (FTEs) during work hours. Considering the Workload Unit Value per procedure given by the College of American Pathologists (CAP) and that each FTE worked 1,864 hours per year, the worked hour productivity for donor and therapeutic apheresis was 78.2%. Blood collections, therapeutic bleeds, and outpatient transfusions (1,127, 114 and 1,745 respectively) were accomplished by two FTEs, for a worked hour productivity of 35.5%. Because 95.1% of total worked units was produced by efficient donor and therapeutic apheresis activities, overall efficiency remained high at 73.8%.     

Course of depressive symptoms and medication adherence after acute coronary syndromes: an electronic medication monitoring study.

OBJECTIVES: We tested whether improvements in depressive symptoms precede improved adherence to aspirin in patients with acute coronary syndromes (ACS). BACKGROUND: Depression is associated with medication nonadherence in patients with ACS, but it is unclear whether changes in depression impact on adherence. METHODS: Electronic medication monitoring was used to measure adherence to aspirin during a 3-month period in a consecutive cohort of 172 patients (25 to 85 years) recruited within 1 week of hospitalization for ACS. Depressive symptom severity was assessed using the Beck Depression Inventory (BDI) during hospitalization and at 1 and 3 months after hospitalization. Adherence was defined as the percentage of days aspirin was taken as prescribed. RESULTS: Depression severity in hospital was associated with nonadherence in a gradient fashion: 15% of non-depressed patients (BDI score 0 to 4), 29% of mildly depressed patients (BDI score 10 to 16), and 37% of patients with moderately-to-severely depressive symptoms (BDI score >16) took aspirin less than 80% of the time (p = 0.03). A cross-lagged path analytic model revealed that improvements in depressive symptoms in the first month after the ACS were associated with improvements in adherence rates in the subsequent 2 months (standardized direct effect -0.32, p = 0.016). CONCLUSIONS: Diagnosis and treatment of depressive symptoms may improve medication adherence in patients after ACS.     

The effect of pH on the aerobic and hypoxic cytotoxicity of SR4233 in HT-29 cells.

We have observed that low pH can substantially potentiate the cytotoxic effect of the bioreductive drug SR4233 in aerobic HT-29 human tumour cells. No such potentiation was observed under hypoxic conditions. This pH effect might be relevant both to the therapeutic effectiveness and to the normal tissue toxicity of this new agent.     

Diagnostic role of fine-needle aspiration of bone lesions in patients with a previous history of malignancy

At the present time fine-needle aspiration (FNA) is considered a routine diagnostic procedure in evaluating neoplastic vs. nonneoplastic lesions in many organs, with high sensitivity and specificity. The purpose of this study was to assess the utility of FNA in areas of diagnostic difficulty and its limitations in evaluating bone lesions in patients with a previous history of malignancy. From 1989 to 2000, 249 CT-guided FNAs of bone lesion were performed at our institutions; 187/249 (75.1%) patients had a previous history of malignancy. Aspirated material was air-dried for Diff-Quik stain or fixed in ethanol for Papanicolaou staining. Subsequent surgical tissue was available in 69/187 (36.9%) of the cases. There were 114 males and 73 females, ages 14-86 yr (mean, 64 yr). The primary tumor site was lung 49, genitourinary 46, breast 31, gastrointestinal 28, hematopoietic 26, soft tissue/skin 5, and thyroid 2. There were 125 FNAs of the vertebral spine, 19 from the pelvis, 11 from the ribs, 9 from the sternum, 5 from the femur, and 18 from miscellaneous bone sites. Out of 187, 166 (88.7%) were malignant aspirates confirming the patients' primary malignancies. The most common malignancy encountered was adenocarcinoma, 126/187 (67.4%). Surgical tissue was available for review in 69 patients and the results were in agreement with the FNAs diagnosis in all cases. Nine out of 187 (4.8%) cases were diagnosed as marrow elements on cytological material. These patients have been followed for 1-9 yr and have failed to reveal signs or symptoms of clinical recurrence. Three out of 187 (1.6%) cases showed osteomyelitis. Nine out of 187 (4.8%) were unsatisfactory specimens, with biopsy follow-up available in four cases, showing three metastatic tumors and one case of osteomyelitis. FNA of metastatic bone lesions is a major step in pretreatment diagnosis. On satisfactory specimens, the cytological diagnosis viewed in the clinical-radiological context proves to be similar to surgical diagnosis. FNA is an excellent technique with a high accuracy rate in assessing metastatic bone lesions.     

Interleukin-1 (IL-1) production in a mouse tissue chamber model of inflammation. II. Identification of (tissue) macrophages as the IL-1 producing cells and the effect of anti-inflammatory drugs

We have used our newly described mouse tissue chamber model [1], to investigate the process of IL-1 production in more detail. The inflammatory reaction in the tissue surrounding the implanted chambers was investigated histologically and by using the polymerase chain reaction (PCR). The inflammatory response included influx of leucocytes into the granuloma surrounding the tissue chamber, expression of IL-1 on macrophages present in the inflamed tissue and an increase in the mRNA coding for IL-1 and IL-6 proteins in the granuloma. The effects of three anti-inflammatory or immunosuppressive drugs, prednisolone, indomethacin and cyclosporin A, on IL-1 and PGE₂ production in zymosan andBordetella-pertussis-vaccine (BPV)-challenged tissue chambers were also examined. Oral treatment with prednisolone and cyclosporin A of zymosan-challenged animals showed a dose-dependent reduction of IL-1 concentrations, but no effect of indomethacin. Both prednisolone and indomethacin dose-dependently reduced PGE₂ concentrations to control levels, while cyclosporin A was effective only at the highest dose tested (100 mg/kg/day p.o.). In drug-treated BPV-challenged animals, prednisolone and cyclosporin A also showed a dose-dependent reduction of IL-1, while indomethacin was again ineffective. Prednisolone and indomethacin also dose-dependently reduced the PGE₂ concentrations to control levels, whereas cyclosporin A was effective only at the highest dose tested (100 mg/kg/day p.o.).This model will be useful for investigating the mechanisms controlling the production of IL-1 from the mRNA level to the secretion of mature biologically active protein [1], and in the search for new drugs which could selectively interfere with this process.     

The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding.

We have found that amino acid residues necessary for C1q and Fc gamma R binding of human IgG1 are located in the N-terminal region of the CH2 domain, residues 231-238, using a matched set of engineered antibodies based on the anti-HLA-DR antibody L243. Changing the leucine 235 in the CH2 region of IgG3 and IgG4 to glutamic acid was already known to abolish Fc gamma RI binding. We have confirmed this for IgG1 and also found a concomitant abolition of human complement lysis with retention of Fc gamma RIII-mediated function. Changing the glycine at 237 to alanine of IgG1 also abolished Fc gamma RI binding and reduced human complement lysis and Fc gamma RIII-mediated function. Exchanging the whole region 233-236 with the sequence found in human IgG2, abolished Fc gamma RI binding and human complement lysis and reduced Fc gamma RIII-mediated function of IgG1. In contrast, a change in the previously described C1q-binding motif, from lysine at 320 to alanine, had no effect on IgG1-mediated complement lysis.     

Quantification of antibodies to the C3d subcomponent of human C3.

Purified soluble C3d has been employed to measure the concentration of anti-C3d antibodies in immune rabbit sera. Multiple batches of C3d, prepared from C3-C3b substrate by treatment with C3b-inactivator (KAF), after labelling with 125I, retained 80% immunoreactivity, and were stable on storage at -50 degrees and +4 degrees. Concentrations of anti-C3d were determined by Scatchard analysis of equilibrium concentrations of bound and free C3d in a mixture of 125I-labelled C3d and anti-C3d. Separation of bound from free C3d was by G-75 Sephadex filtration. Assuming a 1:1 molar ratio in the antibody-C3d complex, anti-C3d antibody concentrations for four rabbit whole antisera and four IgG preparations fell in the range 288-2433 microgram/ml, with Ko values of 6-2 X 10(8)-2-9 X 10(9) litres/mol. One commercial antiglobulin-serum contained 3-6 microgram anti C3d/ml and had a Ko value of 1-7 X 10(8) litres/mol. Values for anti-C3d concentrations measured independently by an indirect method employing 125I-labelled sheep anti-rabbit IgG averaged 20% lower than those obtained with 125I-labelled C3d. Antibody concentrations were correlated with antiglobulin agglutination titres against C3d-coated red cells; a titre of 1 was given by an anti-C3d concentration of 0-5 microgram/ml.