Individuals who are infected with human immunodeficiency virus (HIV) are known to have a high incidence of autoantibodies. In this study, serum samples from 100 individuals with HIV infection were ...tested for granulocyte antibodies (red cell antibodies, lymphocytotoxic antibodies, circulating immune complexes, and serum immunoglobulin G levels) by granulocyte agglutination (GA) and granulocyte immunofluorescence (GIF) assays. Granulocyte antibodies were detected in 66% of serum samples by GIF and in 21% of serum samples by GA. None of the positive sera reacted with granulocyte antigens of known specificity. Antibodies that reacted with red cell antigens other than ABO were detected in only three serum samples, but lymphocytotoxic antibodies were detected in 62% of patients. All serum samples were tested by immunoblotting with granulocyte plasma membranes. Only two samples were found to be positive; one sample reacted with a 58 kd protein and one reacted with a 55 kd protein, but neither serum sample immunoprecipitated any protein from granulocytes that were labeled at the cell surface with iodine 125. Since immune complexes that are bound to granulocyte membranes can be detected by GIF, circulating immune complex levels were measured in all 100 samples. Immune complexes were increased in GIF-reactive serum samples compared with GIF-nonreactive serum samples (23.3 +/- 19.5 micrograms Eq/ml mean +/- SD vs 9.6 +/- 8.1 micrograms Eq/ml, p less than 0.001) but not in GA-reactive serum samples compared with GA-nonreactive sera (24.4 +/- 21.3 micrograms Eq/ml versus 16.9 +/- 16.0 micrograms Eq/ml, p = 0.10).
A prospective study of 10 patients undergoing hemodialysis showed that less neutropenia and complement activation occurred with dialyzer reuse. Neutrophil counts fell 95% +/- 5% (SEM) with first use ...and 66% +/- 8% and 48% +/- 10% with second and third uses, respectively (p less than 0.05). The production of complement component C5a-desarg, as measured by the bioassay granulocyte aggregation, was decreased by 96% +/- 1% and 93% +/- 2% with second and third uses, respectively (p less than 0.05). We investigated the role of the dialyzer disinfectant formaldehyde in decreased neutropenia. In vitro, formaldehyde inhibited granulocyte aggregation and chemotaxis and the dialyzer membrane's ability to generate granulocyte aggregating activity; however, this occurred only at concentrations higher than those likely to obtain in patients. The ability of dialysis membranes to generate granulocyte aggregating activity in plasma was decreased 55% +/- 5% by their prior sequential preincubation in plasma and then formalin (p less than 0.05) and extensive rinsing, which is similar to the circumstances obtaining with dialyzer reuse. Preincubation of membranes in plasma or formalin alone resulted in no change in the membrane's ability to generate granulocyte aggregating activity. We conclude that the exposure of membranes to both plasma and formalin during dialysis and storage is responsible for the decreased C5a-desarg production with reuse, probably because plasma proteins are fixed to the membrane in such a way that interrupts free interaction between the membrane and plasma complement components.
Stimulated by a patient with dyspnea, thrombocytopenia, and leukopenia after sodium morrhuate sclerotherapy, we studied the effect of this agent on the plasma coagulation and complement systems, the ...formed elements of the blood, and cultured human endothelial cells. The addition of sodium morrhuate to citrated plasma did not cause clotting or shorten the prothrombin time or partial thromboplastin time. Incubation of a 1:100 dilution of the clinical sodium morrhuate preparation in heparinized plasma led to a modest rise in C3a. The addition of the drug (dilutions 1:50 to 1:300) to granulocytes caused prompt aggregation (and, at the higher concentrations, granulocyte cytotoxicity trypan blue exclusion; lactate dehydrogenase release), but the same dilutions failed to aggregate platelets. However, 0.05% morrhuate added to washed red blood cells caused a prompt 84.0% (+/- 0.8% SEM) hemolysis, rendering the supernatant buffer a potent platelet aggregant. Not only was this sclerosing agent toxic to granulocytes and red cells, but a 1:1000 dilution of the drug also caused the destruction of 35.5% (+/- 6.6%) of cultured endothelial cells as measured by chromium 51 release. Three other agents in current use (ethanolamine oleate, sodium tetradecyl sulfate, and polidocanol) were studied and found to cause effects qualitatively similar to those of sodium morrhuate. We conclude that these drugs cause phlebosclerosis not primarily through induction of plasma coagulation, but by directly damaging endothelium and red cells, triggering platelets, and aggregating granulocytes at the venous wall endothelium. These effects likely derive from the surfactant properties of sodium morrhuate as well as its high arachidonate content.
HLA-A02* has become an important target for cytotoxic T lymphocyte-based immunotherapy reflecting the high prevalence of this allele in patient populations. There are at least 26 different A*02 ...alleles, and their subtype specificity has significant functional implications for T-cell-mediated recognition of immunologic targets. We have developed a novel method for HLA-A*02 allelic screening using directed heteroduplex analysis (DHDA). DNA samples from Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (EBV-B) representing 10 different HLA-A*02 alleles (0201, 0202, 0204, 0205, 0206, 0208, 0210, 0211, 0216, 0217) were prepared. In addition, DNA was prepared from 81 individuals representing a wide variety of A*02 subtypes previously determined by sequence specific primer (SSP) polymerase chain reaction (PCR) including individuals heterozygous for two A*02 specificities. Probes and samples were generated by PCR amplification using HLA-A*02 specific primers encompassing exons 2 and 3, where most of the functionally significant allelic polymorphism is clustered. DHDA was performed by generating heteroduplex molecules composed of a fluorescein-labeled allelic probe sequence and an unlabeled allelic PCR product. Gel retardation was consistent for allele-probe combinations. We were able to identify several A*02 alleles prepared from EBV-B cell lines that, when used as probes, had very impressive specificity and sensitivity. Combinations of two probes were identified (0205 + 0211 and 0208 + 0211) that allowed differentiation of A*0201 alleles from all other A*02 alleles tested. All samples typed by probe combinations had DHDA typing and SSP typing confirmed by DNA sequencing. This study expands the molecular typing repertoire available to the modern HLA laboratory, and shows that DHDA has significant promise as a reliable screening method for HLA A*02 subtyping.
