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Automated contrast fluorescence cytotoxicity testing does not require background correction
Abstracts
125 input and the generation of various clinical reports (percent reactive antibody (PRA) reports, graphs of PRA vs. time, etc.). Benchmark statistics will be presented for a variety of microcomputer hardware configurations. Although the program will run on a standard IBM PC, we reco m m e n d a computer with an 80286 processor chip, 80287 math coprocessor chip, a hard disk, and sufficient RAM to support a large RAM-disk to run the program (e.g., IBM PC AT enhanced or equivalent). Implementation of highspeed coprocessor boards in a standard IBM will be discussed. Time and RAM requirements which are dependent on panel size, number of alleles tested, and sample size, will be discussed. Sample input and output will be presented.
THE IDENTIFICATION OF AN AUTOANTIBODY IN PATIENTS WITH PRIMARY VASCULITIS. James Cerilli, Lauren Brasile, Jolene Clarke, and Joel Kremer; Albany Medical College, Albany, N Y Although little is known of the etiology and pathogenesis of primary vasculitis, recent evidence has implicated a possible immunological mechanism. If true, it is important to identify the antigen responsible for initiating the immunologically mediated events. While immune complexes have been associated with this disease, most research has centered on exogenous agents such as drugs or infectious agents as the causal antigen. Previously, the attempts to demonstrate vascularwall antigens and antibodies in vasculitis have proven to be inconclusive. Our studies have previously demonstrated that the VEC contains a specific antigen system and this system is ubiquitous throughout the vascular tree. We have now demonstrated an autoanti-vascular endothelial cell antibody in 6/7 patients with primary vasculitis. This autoantibody is highly cytotoxic, complement fixing, and specific to antigens expressed exclusively on vascular endothelial cells and peripheral blood monocytes. The antibody is not absorbed with T or B lymphocytes nor with platelets. The seventh patient with no detectable autoantibody had been receiving serial plasmapheresis treatments and had experienced clinical remission. The presence of autoantibody in these six patients was not associated with an increased frequency of any HLA antigen phenotype. This observation supports our previously reported finding in patients with clinically significant peripheral vascular disease. In such patients autoanti-VEC antibody was found in 4 6 % of the peripheral vascular disease patients as compared to 9 % in the age-matched controls and has never being found in young normal controls. The high frequency of autoanti-vascular endothelial cell antibody in these patients with primary vasculitis suggests an autoimmune mechanism specifically directed against an antigen system represented on the VEC and monocyte but not on lymphocytes may be involved in the pathogenesis of vasculitis. The sera from these six patients are currently being screened against a 20-member vascular endothelial cell panel to further define the specificity of the autoantibody.
DIABETES PREVENTION WITH CYCLOSPORIN A IN EXPERIMENTAL ANIMAL MODEL SHOWS SEX DIFFERENCES IN DRUG REACTIVITY. C. Gardell and D. Gardell; Department of Health and Welfare, Ottawa, Ontario, Canada The anitlymphocytic properties of Cyclosporin A (CyA) have opened up a wide variety o f clinical and experimental applications including the possibility of preventing distinct immunological disease. The disease suppression properties of CyA were investigated using the BB rat model. This strain has a 6 0 - 8 0 % incidence of spontaneous insulin-dependent diabetes peaking between 80 and 120 days o f age. Clinical symptoms are similar to those of human Type I juvenile
126
Annual AACHT Meeting, 198 ~, onset diabetes. Following initial dose titration trials, the animals received a loading dose of 10 mg/kg/day for 4 weeks, which was later reduced to immunosuppressive maintenance levels o f 8 mg/kg/day. This lower close achieved a satisfactory blood level, comparably to human therapeutic levels, and avoided all clinical signs ~)i toxicity. 40 male and 40 female BB rats, all 34-days-old, were randomly divided into two groups of 20 males and 20 females each and atloted to the treated ~r control group. 75% of the controls developed spontaneous diabetes by day 105, whereas none of the CyA-treated animals showed any diabetic symptoms at 128 days. Following the protocol, 50% of the treated rats were randomly taken ~>f! CyA treatment. One male and four females in this group developed the disease The continuously treated animals showed no signs of diabetes at the end of theexperiment at 189 days of age. The analysis of the clinical data and detailed anatomical and histological studies of all animals demonstrated: (1) C yA treatment at immunosuppressive levels prevented the development of clinical and histological signs o f the spontaneous diabetes in BB rats. (2) Lymphocytic thyroiditis. common in the BB rat, occurred less often in the CyA-treated group, and ir~ some treated animals, the thymus showed signs of lymphocytic depIetion. (:~'. T h e r e was no clinical evidence of drug toxicity. (4) Histological lesions, as re. vealed by mild tubular necrosis and hydronephrosis due to toxicity, were limited to the kidneys o f CyA-treated female rats. (5) A trend toward different reactivitof males and females was found: females appeared more prone to develop the disease, more sensitive to possible drug toxicity of CyA, and less able to absorb the drug from the gastrointestinal tract. Furthermore, CyA may be less efficiem in the female than in the male BB rat. The reactivity pattern is suggestive of ~ higher susceptibility to diabetes in female BB rats and of a close associatio,~ between female sex and autoimmune disease.
AUTOMATED CONTRAST FLUORESCENCE CYTOTOXICITY TESTING DOES NOT RE QUIRE BACKGROUND CORRECTION. D. Gardell and G. Rock; Canadian Red Cross BTS. Ottawa. Ontario, Canada
Automated cytotoxicity testing using two sequential measurements of fluoresce~,.: colors requires mathematical correction for the background fuorescence and fi,r the difference in number and distribution of cells. We tested the hypothesis tha~ data correction is not necessary in a fully automated system for measurement t)~cytotoxicity. Negative and positive cytotoxic reactions were tested by incubating 2 x 10 -~ cells/well with the live celt indicator, fluorescein diacetate ~FDA) tot green fluorescence and, simultaneously, with complement and antibod y, followed by ethidium bromide (EB) for red fluorescence of damaged cells In -= 120). AH epifluorescence microscope, featuring an automated filter changing mechanism (Zeiss) and a scanning procedure which provided the same distribution and nux~ber of cells for green and red (LI) measurements, was used. LI were either stabilized for background fluorescence using ethylene diaminetetra acetate ( E D T A / N a C I / o r measured without correcting the background. Evaluation of the tests using raw data for green and red measurements of negative and positivt~ controls showed a significant decline in fluorescem Ll for stabilized (20.67e7¢,) and uncorrected medium fluorescence (25.82%). Serial measurements of the fluorescein LI between 1 and 28 hr after terminating a test resulted in an even stronger decrease of 35.76%. The increase in EB fluorescence between the negative and positive reacuon was 41.76% for tests with background correction. and 30.47% for the uncorrected LI data. The difference between both groups was not significant. Serial data acquisition for EB for the same test showed an increase in LI o f 30.15%. When comparing the cytotoxic reactivity of serial HLA antibody dilutions in the standard N I H tests to automated FDA/EB readings without the background correction, an analysis of variance showed good corrc-
125 input and the generation of various clinical reports (percent reactive antibody (PRA) reports, graphs of PRA vs. time, etc.). Benchmark statistics will be presented for a variety of microcomputer hardware configurations. Although the program will run on a standard IBM PC, we reco m m e n d a computer with an 80286 processor chip, 80287 math coprocessor chip, a hard disk, and sufficient RAM to support a large RAM-disk to run the program (e.g., IBM PC AT enhanced or equivalent). Implementation of highspeed coprocessor boards in a standard IBM will be discussed. Time and RAM requirements which are dependent on panel size, number of alleles tested, and sample size, will be discussed. Sample input and output will be presented.
THE IDENTIFICATION OF AN AUTOANTIBODY IN PATIENTS WITH PRIMARY VASCULITIS. James Cerilli, Lauren Brasile, Jolene Clarke, and Joel Kremer; Albany Medical College, Albany, N Y Although little is known of the etiology and pathogenesis of primary vasculitis, recent evidence has implicated a possible immunological mechanism. If true, it is important to identify the antigen responsible for initiating the immunologically mediated events. While immune complexes have been associated with this disease, most research has centered on exogenous agents such as drugs or infectious agents as the causal antigen. Previously, the attempts to demonstrate vascularwall antigens and antibodies in vasculitis have proven to be inconclusive. Our studies have previously demonstrated that the VEC contains a specific antigen system and this system is ubiquitous throughout the vascular tree. We have now demonstrated an autoanti-vascular endothelial cell antibody in 6/7 patients with primary vasculitis. This autoantibody is highly cytotoxic, complement fixing, and specific to antigens expressed exclusively on vascular endothelial cells and peripheral blood monocytes. The antibody is not absorbed with T or B lymphocytes nor with platelets. The seventh patient with no detectable autoantibody had been receiving serial plasmapheresis treatments and had experienced clinical remission. The presence of autoantibody in these six patients was not associated with an increased frequency of any HLA antigen phenotype. This observation supports our previously reported finding in patients with clinically significant peripheral vascular disease. In such patients autoanti-VEC antibody was found in 4 6 % of the peripheral vascular disease patients as compared to 9 % in the age-matched controls and has never being found in young normal controls. The high frequency of autoanti-vascular endothelial cell antibody in these patients with primary vasculitis suggests an autoimmune mechanism specifically directed against an antigen system represented on the VEC and monocyte but not on lymphocytes may be involved in the pathogenesis of vasculitis. The sera from these six patients are currently being screened against a 20-member vascular endothelial cell panel to further define the specificity of the autoantibody.
DIABETES PREVENTION WITH CYCLOSPORIN A IN EXPERIMENTAL ANIMAL MODEL SHOWS SEX DIFFERENCES IN DRUG REACTIVITY. C. Gardell and D. Gardell; Department of Health and Welfare, Ottawa, Ontario, Canada The anitlymphocytic properties of Cyclosporin A (CyA) have opened up a wide variety o f clinical and experimental applications including the possibility of preventing distinct immunological disease. The disease suppression properties of CyA were investigated using the BB rat model. This strain has a 6 0 - 8 0 % incidence of spontaneous insulin-dependent diabetes peaking between 80 and 120 days o f age. Clinical symptoms are similar to those of human Type I juvenile
126
Annual AACHT Meeting, 198 ~, onset diabetes. Following initial dose titration trials, the animals received a loading dose of 10 mg/kg/day for 4 weeks, which was later reduced to immunosuppressive maintenance levels o f 8 mg/kg/day. This lower close achieved a satisfactory blood level, comparably to human therapeutic levels, and avoided all clinical signs ~)i toxicity. 40 male and 40 female BB rats, all 34-days-old, were randomly divided into two groups of 20 males and 20 females each and atloted to the treated ~r control group. 75% of the controls developed spontaneous diabetes by day 105, whereas none of the CyA-treated animals showed any diabetic symptoms at 128 days. Following the protocol, 50% of the treated rats were randomly taken ~>f! CyA treatment. One male and four females in this group developed the disease The continuously treated animals showed no signs of diabetes at the end of theexperiment at 189 days of age. The analysis of the clinical data and detailed anatomical and histological studies of all animals demonstrated: (1) C yA treatment at immunosuppressive levels prevented the development of clinical and histological signs o f the spontaneous diabetes in BB rats. (2) Lymphocytic thyroiditis. common in the BB rat, occurred less often in the CyA-treated group, and ir~ some treated animals, the thymus showed signs of lymphocytic depIetion. (:~'. T h e r e was no clinical evidence of drug toxicity. (4) Histological lesions, as re. vealed by mild tubular necrosis and hydronephrosis due to toxicity, were limited to the kidneys o f CyA-treated female rats. (5) A trend toward different reactivitof males and females was found: females appeared more prone to develop the disease, more sensitive to possible drug toxicity of CyA, and less able to absorb the drug from the gastrointestinal tract. Furthermore, CyA may be less efficiem in the female than in the male BB rat. The reactivity pattern is suggestive of ~ higher susceptibility to diabetes in female BB rats and of a close associatio,~ between female sex and autoimmune disease.
AUTOMATED CONTRAST FLUORESCENCE CYTOTOXICITY TESTING DOES NOT RE QUIRE BACKGROUND CORRECTION. D. Gardell and G. Rock; Canadian Red Cross BTS. Ottawa. Ontario, Canada
Automated cytotoxicity testing using two sequential measurements of fluoresce~,.: colors requires mathematical correction for the background fuorescence and fi,r the difference in number and distribution of cells. We tested the hypothesis tha~ data correction is not necessary in a fully automated system for measurement t)~cytotoxicity. Negative and positive cytotoxic reactions were tested by incubating 2 x 10 -~ cells/well with the live celt indicator, fluorescein diacetate ~FDA) tot green fluorescence and, simultaneously, with complement and antibod y, followed by ethidium bromide (EB) for red fluorescence of damaged cells In -= 120). AH epifluorescence microscope, featuring an automated filter changing mechanism (Zeiss) and a scanning procedure which provided the same distribution and nux~ber of cells for green and red (LI) measurements, was used. LI were either stabilized for background fluorescence using ethylene diaminetetra acetate ( E D T A / N a C I / o r measured without correcting the background. Evaluation of the tests using raw data for green and red measurements of negative and positivt~ controls showed a significant decline in fluorescem Ll for stabilized (20.67e7¢,) and uncorrected medium fluorescence (25.82%). Serial measurements of the fluorescein LI between 1 and 28 hr after terminating a test resulted in an even stronger decrease of 35.76%. The increase in EB fluorescence between the negative and positive reacuon was 41.76% for tests with background correction. and 30.47% for the uncorrected LI data. The difference between both groups was not significant. Serial data acquisition for EB for the same test showed an increase in LI o f 30.15%. When comparing the cytotoxic reactivity of serial HLA antibody dilutions in the standard N I H tests to automated FDA/EB readings without the background correction, an analysis of variance showed good corrc-