Spectroscopic studies on IgG aggregate formation.

Spectroscopic techniques have been used to examine the effects of IgG of heating, irradiation by ultraviolet light and exposure to glutaraldehyde. Relatively few changes were observed in treated IgG which remained unaggregated but several significant size-dependent changes were observed in aggregated IgG. These results suggest that IgG aggregate formation by cross-linking with glutaraldehyde involves the least perturbation of the basis IgG structure, whereas aggregate formation by heating involves the most, with ultraviolet irradiation occupying an intermediate position.

The ex vivo function and expression of function-associated antigens of peripheral blood neutrophils and monocytes.

The availability of recombinant human granulocyte and granulocyte-macrophage colony-stimulating factors (rhG-CSF and rhGM-CSF) has prompted many studies of the analysis of antigen expression and function of monocytes and neutrophils from patients receiving these factors as therapeutic agents. Preparatory procedures for leukocytes are known to alter antigen expression and so function. We therefore investigated the use of a novel procedure in which live leukocytes are analyzed by flow cytometry without isolation from blood. The expression levels of CD11b, CD13, CD14, CD16, and CD18 antigens and L-selectin (TQ1 and Leu-8 epitopes) on neutrophils and monocytes from 15 normal individuals were determined and compared with a previously used method in which the leukocytes were fixed and the erythrocytes lysed before analysis. Significant differences for the apparent expression of CD11b, CD18, and L-selectin were observed between the two methods. The reasons for this were investigated. Since the new method allowed analysis of live cells, we also investigated whether modulation of antigen expression could be determined following receptor agonist interaction. This was found to be easily achievable, and we advocate using the new procedure where possible for the ex vivo analysis of function and function-associated antigens on monocytes and neutrophils.

A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood.

A novel procedure has been developed for the quantitation by flow cytometry of function-associated antigens on neutrophils and monocytes in unlysed, unfixed, peripheral blood samples. Freshly drawn blood anticoagulated with the serine esterase inhibitor, phenylmethylsulphonyl fluoride, is mixed with the vital nucleic acid stain, LDS-751, labelled with monoclonal antibodies for 5 min at 4 degrees C, diluted and analysed in a five-parameter flow cytometer. The three major leucocyte subpopulations (neutrophils, lymphocytes and monocytes) can be resolved in real time on the basis of their side light scattering and staining intensity with LDS-751 in the FL3 channel (erythrocytes and platelets stain very weakly), whilst the fluorescence intensity due to bound fluorescein isothiocyanate- or phycoerythrin-labelled antibody is monitored simultaneously in the FL1 or FL2 channels respectively. This procedure avoids potential artefacts that can occur due to the use of fixatives, erythrocyte lysing agents, or anticoagulants which are also divalent metal ion chelators. It should be widely applicable for the quantitation of those function-associated antigens, such as adhesion molecules and immune complex receptors, whose surface expression can be rapidly upregulated following activation, as well as for the quantitation of those leucocyte surface antigens whose expression is not subject to rapid modulation.

Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes.

This study investigated the effects of commonly used procedures for the isolation of leucocytes from human blood in comparison with cells in whole blood on the surface expression of CD11b and L-selectin (adhesion molecules which are known to be increased and decreased respectively by cell activation). Washing of granulocytes or monocytes with Hanks' buffered salt solution after separation by either dextran sedimentation or density gradient centrifugation, increased surface expression of CD11b (p < 0.05). The number of monocytes bearing CD11b was enhanced (p < 0.05) by dextran sedimentation and two layer density gradient centrifugation (Histopaque). The increase in CD11b expression on granulocytes was associated with enhanced binding of the cells to endothelial monolayers that were either untreated (r = 0.902; p < 0.001) or treated with tumour necrosis factor alpha (TNF-alpha) (r = 0.68; p = 0.004). The expression of L-selectin was reduced on granulocytes that had been isolated by dextran sedimentation followed by hypotonic lysis of contaminating erythrocytes. All isolates of granulocytes demonstrated a loss of L-selectin following activation with fMLP though this effect was less marked with cells subjected to erythrocyte lysis. The various separation methods had little effect on expression or distribution of CD11b or L-selectin on lymphocytes. We conclude that isolation of lymphocytes by density gradient centrifugation and of granulocytes and monocytes by dextran-sedimentation and centrifugation using Histopaque gradients, but avoiding washing and the use of hypotonic erythrocyte lysis, are appropriate techniques for studying the expression and function of adhesion molecules.

How should CD34+ cells be analysed? A study of three classes of antibody and five leucocyte preparation procedures.

For patients undergoing stem cell transplantation after intensive marrow ablative therapy it is important to enumerate the CD34+ stem cells in peripheral blood so that the harvest can be timed in order to maximize the number of cells collected by leucophoresis for subsequent haematopoietic reconstitution. The use of rapid flow cytometric techniques for the determination CD34+ leucocyte numbers has been advocated, although there is no consensus as to the best method. In this study, we have examined the effects of preparation procedures for flow cytometry on the binding of four CD34 antibodies (Immu-133, QBEND-10, HPCA2 and BIRMA-K3) to the three classes of epitopes on leucocytes. Whole blood, bone marrow and leucophoresis samples were analysed either directly after labelling with a vital nuclear dye (LDS-751) and fluorochrome-conjugated antibodies or after additional erythrocyte lysis and leucocyte fixation using four commercially available reagents (Q-Prep, OptiLyse B, OptiLyse C and FACS Lysing Solution). By comparison with the results obtained from viable leucocytes in unmanipulated samples, it was found that the binding of all four antibodies could be affected by lysis and fixation procedures and that the binding of the class I antibody Immu-133 was most markedly decreased. We conclude that CD34+ cells are best analysed using a whole blood procedure in which nucleated cells are identified by their side light scatter and the fluorescence associated with a vital nuclear dye (in this instance LDS-751) and the CD34+ cells are detected with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies.

Soluble IgG aggregates produced by heating remain stable on freeze-drying.

IgG aggregates produced by heating gamma globulin solutions were freeze-dried, kept at 4 degrees C and reconstituted up to 4 months later. By comparison with frozen (-20 degrees C) preparations, only minimal changes in biological reactivity and in physical integrity occurred during this period. These results demonstrate that freeze-dried preparations of heat-aggregated IgG are potentially useful as a reference reagent for the comparative evaluation and standardisation of immune complex assays.

The production of small IgG aggregates by glutaraldehyde cross-linking.

Reaction conditions have been determined for the production of soluble IgG polymers in the size range 10 S to 30 S by covalent cross-linking with glutaraldehyde. This size range is comparable with that of the immune complexes which are frequently found in the circulation of patients with certain autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. The yield of IgG aggregates in this size range is far greater than has been reported for cross-linking by other bifunctional reagents or for aggregation by heating. Glutaraldehyde cross-linked IgG polymers are stable and biologically reactive. They can also be labelled with fluorescein and freeze-dried with minimal loss of integrity or reactivity.

Centrifugation of normal and rheumatoid arthritis blood on Ficoll-Hypaque and Ficoll-Nycodenz solutions.

Attempts to use the rapid single-step Ficoll-Hypaque centrifugation procedure for the purification of mononuclear and polymorphonuclear leucocytes from the blood of normal individuals and rheumatoid arthritis patients have sometimes been unsuccessful, largely because the erythrocytes would not sediment through the centrifugation medium. Re-evaluation of the factors (e.g. Ficoll concentration, temperature, and ratio of the diatrizoate salts) which affect these separations showed that under our conditions it was advantageous to use a medium with a lower viscosity (Ficoll concentration) and/or a higher osmotic strength (increased sodium diatrizoate: meglumine diatrizoate) than had been recommended previously (Ferrante and Thong, 1978; 1980; Ferrante et al., 1982). Higher osmotic strength media must be used for separating the components of blood from rheumatoid arthritis patients than from normal individuals because rheumatoid arthritis erythrocytes have a lower buoyant density than normal erythrocytes.

Flow cytometric analysis of different adhesion molecules expression on circulating CD14- and CD64- human dendritic cell precursors.

Blood dendritic cell precursors (DCps) are identified as mononuclear leukocytes expressing HLA-DR but lacking the characteristic antigens associated with T cells (CD3), NK cells (CD16 and CD56) and B cells (CD 19). Dendritic cell precursors are distinguished from monocytes by their lack of expression of CD64 rather than of CD14. This study investigated whether CD14- DCps differed from CD64-DCps, which were predominantly CD14+, in their expression of five well-characterised adhesion molecules. There were significantly fewer cells expressing CD11b, CD18 and CD29 in the CD64-DCp population compared with CD14- DCps, and this CD64- DCp subpopulation also had a lower expression of CD11b and CD18. Our results suggest that the two DC precursor subpopulations may differ from one another in their binding characteristics to blood vessel walls and to other leukocytes.

The leucocytosis of exercise. A review and model.

Exercise is known to induce an immediate leucocytosis, the magnitude of which is related, in most instances, to the intensity and duration of the work. On finishing exercise, however, the leucocyte count may change in any one of several different ways. The pattern of postexercise changes in the leucocyte count is determined mainly by the time which has elapsed since beginning exercise, rather than the work intensity or the total work done, if, for example, exercise has been intermittent. Consideration of, firstly, the circumstances under which the plasma concentrations of catecholamines and cortisol have been found separately to correlate with the leucocyte count at the finish of exercise, and, secondly, the effects on the leucocyte count of exogenous administration of these substances has led us to develop a model which can satisfactorily account for all of the principal changes in the leucocyte count that have been noted during and after exercise. It is proposed that catecholamines produced during exercise act to increase the ratio of circulating to non-circulating leucocytes, while cortisol acts, by a mechanism which involves a time lag, to increase the total number of leucocytes in the vascular compartment. Examination of previously published reports shows that many contain results which support this model. Using the model as a basis, some predictions are made that can be tested experimentally, and some experiments are suggested which should help elucidate the mode of action of catecholamines and cortisol.

Plasma naltrexone kinetics after intravenous bolus administration in dogs and monkeys.

This investigation generated data characterize a specific electron-capture GLC assay reported previously for naltrexone and applied the method to a determination of naltrexone pharmacokinetics. Extraction efficiencies are reported for the assay, and mass spectral evidence indicates that naltrexone forms a triester when derivatized for electron-capture GLC with pentafluoropropionic anhydride and a base catalyst. Plasma level-time data for intravenous naltrexone at two dose levels in monkeys yielded no evidence of dose-dependent kinetics. A two-compartment open pharmacokinetic model was fitted to plasma level-time data for naltrexone in two dogs and yielded a total body clearance of 51-55 ml/min/kg. Urine collected for 0-24 hr contained 36% of the dose as naltrexone conjugates with less than 1% as unchanged naltrexone. Plasma level-time data for intravenous naltrexone in six monkeys yielded an average terminal half-life of 7.8 hr and a total body clearance of 64 ml/min/kg. The total body clearance for naltrexone was greater than the hepatic plasma or blood flow in both dogs and monkeys. This finding, together with the extremely low renal excretion of naltrexone, suggests the existence of elimination mechanisms besides liver metabolism and renal excretion.

An eclectic inpatient treatment model for Vietnam and Desert Storm veterans suffering from posttraumatic stress disorder.

This paper describes the therapeutic components of an eclectic, intensive inpatient treatment strategy for Vietnam and Desert Storm veterans with posttraumatic stress disorder. A specific treatment model was devised by this author. The procedure was a collaborative effort: the staff and the patients participated in this preparatory phase treatment program at the Tripler Posttraumatic Stress Unit, Tripler Army Medical Center, Honolulu, Hawaii. The basic treatment is based on group therapy, utilizing educational, cognitive-behavioral therapy, gestalt therapy, and individualized psychotherapy, and eye movement desensitization strategies. The timing and sequencing of these treatments are a critical part of the model, and we have come to refer to it as the layered model because the treatments are layered, much like the delicious parfait dessert.

Novel anticoagulants for flow cytometric analysis of live leucocytes in whole blood.

Enzyme inhibitors have been compared with conventional anticoagulants for the flow cytometric analysis of live leucocytes in whole blood. At 18 to 20 degrees C in vitro, PPACK (60 microM), hirudin (10 units ml-1), leupeptin (20 microg ml-1) and aprotinin (50 microg ml-1) inhibited blood clotting for 4 h or more, whereas PMSF (8 mg ml-1) or AEBSF (5 mg ml-1) inhibited clotting for only 20 or 40 min, respectively. When labelled with CD11b antibodies and analysed immediately ex vivo at 4 degrees C, the percentages of lymphocytes, monocytes, and polymorphs which stained positively and their mean fluorescence intensities were similar, irrespective into which anticoagulant blood was collected. Less than 1.5-fold increases in expression occurred on monocytes and polymorphs when blood anticoagulated with enzyme inhibitors or conventional anticoagulants was kept at 4 degrees C, or when blood anticoagulated with citrate, heparin, or hirudin was kept at 18 to 20 degrees C for 1 h before labelling and analysis, whereas approximately 2-fold increases in expression occurred in blood kept with K3EDTA, leupeptin, or aprotinin and more than 3-fold increases in blood kept with AEBSF or PPACK at 18 to 20 degrees C for 1 h. Further studies showed that leupeptin could be used effectively as the anticoagulant when investigating functional responses of live leucocytes in whole blood samples by flow cytometry and for the isolation of leucocytes with minimal modulation of adhesion molecules.

Coxsackievirus B4 infection of the mouse pancreas: acute and persistent infection.

The course of infection of a pancreas-adapted isolate of coxsackievirus B4 was followed over a 10 month period in a murine model. Following intraperitoneal inoculation a typical acute infection was seen in nine of 10 inbred mouse strains. Virus rapidly infected the exocrine pancreas, titres peaking 3 to 4 days post-infection (p.i.). Lesions were almost exclusively confined to pancreatic acinar cells and varied in severity among the inbred strains. Virus shed into the blood-stream was not cell-associated. Evidence of persistent infection was found in nine mouse strains and infective virus was recovered from the pancreas of seven strains for up to 10 months p.i. Approximately 28% of pancreases examined beyond the acute phase showed focal inflammation and 22% showed focal necrosis (cell death). Virus was occasionally recovered from other organs (heart, liver and spleen), but lesions were rarely seen. Virus-specific antigen was localized to small groups of pancreatic acinar cells using an indirect immunogold silver staining technique. These observations suggested that the virus persists in pancreatic tissues because it seems unlikely that virus disseminated from distant sites would cause such localized infection. In three of these strains, the course of infection may have been influenced by superinfection with mouse hepatitis virus.

Peripheral blood neutrophils in inflammatory bowel disease: morphological evidence of in vivo activation in active disease.

Morphological evidence of activation in vivo of circulating neutrophils in patients with inflammatory bowel disease (IBD) was sought by quantitative light microscope examination of toluidine blue-stained preparations made from peripheral venous bloods that had been fixed immediately ex vivo. The proportion of spherical (unactivated) circulating neutrophils was reduced in active Crohn's disease (73%; 46-96 (median; range), n = 11) compared with inactive Crohn's (90%; 45-99; n = 18, P less than 0.01) and normal subjects (94%; 44-98; n = 13, P less than 0.05). There tended to be fewer spherical neutrophils in active ulcerative colitis (77%; 13-96; n = 17) than in quiescent colitis (88%; 57-99, n = 13, P less than 0.1) or normal subjects (P less than 0.05). Activated neutrophils occur in the circulating pool of patients with active IBD and can be detected by light microscopy of peripheral venous blood leucocyte preparations.

Leucocyte and erythrocyte counts during a multi-stage cycling race ('the Milk Race').

Venous blood samples were taken from eight competitors in mid-evening after a racing day, and in the early morning before the next day's race, three times during the course of the Milk Race, 1992. These were used to gather information about the changes in circulating leucocyte levels in response to the exceptionally high sustained daily workload required during a major multi-stage race. The primary objective was to provide knowledge of 'normal' values against which future clinical judgements of abnormality might be made in these unusual circumstances. During the race, estimated energy output was about 25 MJ (6000 kCal)/day. The mean total circulating leucocyte numbers (per litre of blood), and those of individual leucocyte classes (neutrophil, lymphocyte, monocyte, eosinophil and basophil) were all inside the normal range both in the morning and in the evening. Evening counts were, however, 30-50% higher than morning counts, for all classes except eosinophils. We conclude that individual clinical decisions about leucocyte levels can best be made using normal (sedentary man) values if a morning sample is taken.

An evaluation of the Cell-Dyn 1000.

The Cell-Dyn 1000 is a new eight parameter fully automatic cell counter. This was evaluated following the ICSH guidelines. Scientific evaluation was generally satisfactory, largely conforming to the manufacturer's specifications. A number of potential microbiological safety hazards were noted; many could be overcome by minor modifications. Throughput and start-up/close-down times were acceptable. However, the reliability of the tested machine was felt to be less than satisfactory although this may be corrected by further minor machine modifications.

Effect of fixation on quantification of the expression of leucocyte function-associated surface antigens.

The surface expression of adhesion molecules and other function-associated antigens on peripheral blood leucocytes may be measured by flow cytometry. However, quantification of these antigens is difficult, because their expression can be rapidly and artefactually modulated if the cells are activated in vitro. Consequently, it is common, when analyzing these antigens either: (1) to label leucocytes in whole blood at 4 degrees C, to lyse erythrocytes and then fix the leucocytes, or (2) to fix the leucocytes in whole blood, to lyse the erythrocytes, and then label the leucocytes. We have compared the mean fluorescence intensity (MFI) values for CD11b, CD18, and L-selectin (Leu-8 and TQ1 epitopes) on human peripheral blood leucocytes, using these two approaches. In addition, we have simultaneously evaluated how anticoagulants (acid citrate, K3EDTA, and heparin) and the presence or absence of divalent metal ions (Ca2+ and Mg2+) affect the expression levels of these antigens. The results for all four epitopes varied markedly depending on the preparation procedure used but were less affected by the choice of anticoagulant and whether divalent cations were or were not present in the media used for cell preparation and labelling. Comparison of the results obtained using these procedures, which involve fixation with formaldehyde, with those obtained by a recently developed procedure in which unfixed leucocytes were labelled with the vital nuclear dye LDS-751 and antibodies together, then analysed in unlysed whole blood at 4 degrees C, showed that formaldehyde-based preparation techniques underestimated the expression (MFI) of CD18, Leu-8, and TQ1. It is recommended that, whenever practicable, measurements are made on unfixed cells stained using the newer procedure.