Adsorption of coagulation proteins from whole blood on to polymer materials: relation to platelet activation.

A combination of methods, immunoassays of plasma proteins and platelet release of beta-thromboglobulin and chromogenic substrates for enzymatically active coagulation factors, was used to measure the reactions of coagulation proteins upon contact between whole blood and artificial surfaces as a function of time and surface material. Four types of well-known polymer surfaces, polyvinylchloride, polytetrafluoroethylene, polyurethane and silicone rubber, were investigated to elucidate if a simple and fast in vitro experimental set-up can be of guidance in the selection of materials for use in vivo. Platelets were activated at the polymer surfaces whereas the coagulation enzymes showed little activity on the polymer surfaces tested. There was a correlation between the adsorption of adhesins (fibrinogen, fibronectin and factor VIII-related antigen) at the surfaces and the release of beta-thromboglobulin from platelets, suggesting that adsorption of adhesins is a major determinant of blood compatibility of polymer materials. Significant differences between the surfaces were seen--polyurethane being the surface with the least protein adsorbed and least platelet activation initiated. This study shows that it is possible to make a first in vitro choice of possible blood compatible artificial surfaces before expensive and cumbersome in vivo experiments.     

Spontaneous and in vitro induced apoptosis of lymphocytes and neutrophils in patients with alcohol dependence

Spontaneous and induced apoptosis of neutrophils and lymphocytes was studied in alcoholics during the abstinent syndrome and in healthy individuals. In alcoholics, the levels of lymphocyte and neutrophil apoptosis at the receptor and cellular levels were higher than in healthy subjects. Blood cells from alcohol addicts and normal individuals similarly react to stimuli (hyperthermia and synthetic glucocorticoid prednisolone) in vitro.     

Spontaneous and experimental myofibrillar hypoplasia and its relation to splayleg in newborn pigs

The relation of myofibrillar hypoplasia to clinical splayleg was studied. A strain of Belgian Landrace sows was selected for this study because they produced pigs which had no myofibrillar hypoplasia. Myofibrillar hypoplasia could nevertheless by induced experimentally in these animals by dexamethasone treatment of the sows during late pregnancy. The lesion was observed without clinical signs and was compared to the myofibrillar hypoplasia in clinical cases of splayleg. The differences between these 2 groups may account for the appearance of clinical signs. These differences included the maturity of the myofibrils and the degree of autophagolysosomal glycogen breakdown.     

Immunomodulatory effect of glutoxim on some activities of isolated human neutrophils and in whole blood

Activity of Glutoxim, a sulfur-containing hexapeptide with immunomodulating effect on lymphocytes, was studied on human neutrophils. Commercial available agent containing the substance, Glutoxim, was used. At the doses of 1, 3, 6 or 100 µg/mL the drug stimulated the superoxide anion and hypochlorous acid generation in resting neutrophils but inhibited them in cells stimulated with zymosan or PMA. The only inhibiting effect was observed on nitric oxide production in LPS-stimulated RAW 264.7 cells. The drug also influenced neutrophil chemotaxis showing chemoattractant activity on cells and inhibiting fMLP-induced chemotaxis. These effects on neutrophils confirm and extend range of Glutoxim immunomodulating abilities.     

A quantitative assay of oxidative metabolism by neutrophils in whole blood using flow cytometry

A large number of purified and washed PMNLs are required to monitor generation of active oxygen derivatives in most in vitro studies and this can preclude investigations in small children. The present method has enabled us to measure the oxidative burst (generation of hydrogen peroxide) of PMNLs in a small amount of whole blood using 2′,7′-dichlorofluorescin diacetate, phorbol myristate acetate and flow cytometry. Optimal conditions for this determination were evaluated and the reaction was found to be independent of the absolute numbers of PMNLs and other types of cell in whole blood. The present method will be of value in investigations of the leukocyte metabolism of patients not only with chronic granulomatous disease (CGD) but also with various infectious diseases.     

Spontaneous histamine release from washed leukocytes and whole blood in atopic and non-atopic individuals

The ability of basophils from patients with allergic rhinitis, extrinsic asthma, chronic idiopathic urticaria and atopic dermatitis to release histamine spontaneously in vitro was studied. Spontaneous release in the presence and absence of 30% D2O was investigated from both washed leukocytes and whole blood. Compared with controls histamine release (HR) from washed leukocytes was significantly enhanced in allergic rhinitis patients only, whereas in the other groups only a certain percentage of patients was found to have high spontaneous HR. In whole blood experiments HR in all groups was within the normal range. Our data indicate that enhanced spontaneous mediator release from washed basophils in vitro does not necessarily prove this mechanism to be of pathophysiological relevance in vivo.     

Recombinant fusion proteins for haemagglutination-based rapid detection of antibodies to HIV in whole blood

Recombinant fusion proteins, consisting of a monovalent anti-human RBC monoclonal antibody B6, and conserved immunodominant peptide of HIV-1 envelope glycoprotein gp41 or HIV-2 envelope glycoprotein gp36, have been designed and purified after over-expression in E. coli. These fusion proteins are Fab-based and were obtained by assembling the light chain with Fd (variable domain and the first constant domain of the heavy chain) or Fd fusions containing HIV-derived peptide, and following a protocol of in vitro denaturation of inclusion bodies and subsequent renaturation to assemble functional Fab. Using a multistep column chromatographic procedure, monomeric Fab and Fab fusion proteins containing HIV-derived peptide were purified to high degree, free of aggregates. The yield of various proteins on the laboratory scale (1-2 l of shake flask culture) was in the range of tens of milligram. Purified anti-human RBC Fab fusion proteins containing sequences derived from HIV-1 gp41 and HIV-2 gp36 were highly specific for detection of antibodies to HIV-1 and HIV-2, respectively. The described design, expression and purification protocols will make it possible to produce specific recombinant reagents in large quantities for agglutination-based rapid detection of antibodies to HIV in whole blood.     

Identification of caspase-10 in human neutrophils and its role in spontaneous apoptosis

In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis of human polymorphonuclear neutrophils (PMN). Whereas TNF-alpha-mediated apoptosis was almost absent in the presence of the caspase-8 inhibitor Z-Ac-Ala-Glu-Val-Asp-7-fluoromethyl ketone (Z-AEVD-FMK), the inhibitor had no effect on spontaneous apoptosis, suggesting that spontaneous apoptosis was independent of caspase-8. Subsequently, we identified different isoforms of caspase-10 in human PMN and found high expression of caspase-10/b and/or -10/d and low expression of caspase-10/a and -10/c at the mRNA level. At the protein level, freshly isolated PMN showed high expression of caspase-10/b and -10/d as well as moderate expression of caspase-10/a and -10/c. Upon spontaneous apoptosis, caspase-10/b was down-regulated, which was accompanied by the appearance of a specific 47-kDa caspase-10/b cleavage product and an increased caspase-10 activity. In contrast, no down-regulation of caspase-10/a, -10/c, or -10/d was observed, suggesting that spontaneous apoptosis was associated with a differential activation of caspase-10/b. This was confirmed by the finding that spontaneous apoptosis was inhibited in the presence of Z-Ile-Glu-Thr-Asp (Z-IETD)-FMK, which blocks caspase-10. However, no down-regulation of caspase-10 isoforms was observed in the presence of TNF-alpha, suggesting that caspase-10 was not involved in TNF-alpha-induced apoptosis. Taken together, our study demonstrates that spontaneous and TNF-alpha-mediated apoptosis of PMN have different molecular requirements. Whereas TNF-alpha-mediated apoptosis depends on the activation of caspase-8, spontaneous apoptosis requires the activation of caspase-10/b. This finding may reveal that PMN apoptosis in different (patho-) physiological settings results from distinct molecular mechanisms.     

Simultaneous measurement by flow cytometry of phagocytosis and hydrogen peroxide production of neutrophils in whole blood

Bacterial ingestion and hydrogen peroxide production by polymorphonuclear leukocytes (PMN) were simultaneously measured using flow cytometry and 2',7'-dichlorofluorescein diacetate and fluorescent Staphylococcus aureus. Only 100 microliters of a whole blood specimen were required for these determinations, and the results were found to be independent of the absolute numbers of PMN, making the purification and adjustment of PMN numbers unnecessary. A positive correlation between phagocytosis and hydrogen peroxide production by individual PMN was demonstrated in healthy adults.     

Adherence of Staphylococcus epidermidis to extracellular matrix proteins and effects of fibrinogen-bound bacteria on oxidase activity and apoptosis in neutrophils

Staphylococcus epidermidis often causes foreign-body infections such as those associated with hip prostheses, but the underlying pathogenic mechanisms are not fully understood. We performed spectrophotometry to study the ability of S. epidermidis to bind to immobilised fibrinogen, fibronectin, vitronectin, and collagen. The strains were isolated from infected hip prostheses or from normal flora and the well-known protein-binding strain Staphylococcus aureus Cowan was used as positive control. We also analysed the interaction between neutrophils and a fibrinogen-bound prosthesis-derived strain of S. epidermidisby measuring chemiluminescence to determine the neutrophil oxidative response and binding of annexin V to indicate neutrophil apoptosis. We found that binding of S. epidermidis to extracellular matrix proteins varied under different growth conditions, and that prosthesis isolates adhered better to vitronectin than did strains from normal flora. The oxidative response caused by fibrinogen-bound S. epidermidis was not above the background level, which was in marked contrast to the distinct response induced by fibrinogen-associated S. aureus Cowan. Furthermore, fibrinogen-adhering S. epidermidis retarded neutrophil apoptosis. We conclude that surface-bound S. epidermidis induces only a weak inflammatory response, which in combination with the ability of the adherent bacteria to retard neutrophil apoptosis may contribute to low-grade inflammation and loosening of prostheses.     

Tumor necrosis factor-alpha gene expression in human whole blood

Tumor necrosis factor-alpha (TNF) is recognized as a principal mediator of a variety of pathophysiologic and immunologic events. Lipopolysaccharide (LPS) challenge, either in vitro or in vivo, results in significant TNF production. In this study we present data demonstrating LPS-induced TNF mRNA expression and bioactivity using an in vitro tissue system of whole blood (WB). The kinetics of LPS-induced TNF production by WB was significantly accelerated as compared to isolated cultured peripheral blood monocytes (PBM). At post-LPS challenge, plasma from WB demonstrated a rapid rise in TNF bioactivity, peaking by 4 hr (1,021 units/ml/10(6) cells), plateauing between 4 and 8 hr, and then decreasing over the next 16 hr. In contrast, the highest measured TNF bioactivity from PBM did not occur until the 24-hr time-point (175 units/ml/10(6) cells). Whole blood buffy-coat TNF mRNA was assessed by Northern blot analysis, and demonstrated significant TNF mRNA accumulation at 1 hr and a peak 2 hr post-LPS challenge. By 8 hr TNF mRNA was undetectable. Concomitant administration of LPS with either prostaglandin E2 (10(-6)M) or Dexamethasone (10(-6)M) resulted in significant suppression of LPS-induced TNF production. This data supports WB as a useful in vitro medium for the molecular and cellular analysis of TNF. As specialized connective tissue, WB may provide an important environment to study the pharmacologic manipulation of TNF mRNA and bioactivity.     

Spontaneous histamine release in whole blood in patients before and after 4 months of specific immunotherapy

Spontaneous histamine release (SHR) in whole blood was assessed before and after 4 months of specific immunotherapy (SIT) for allergic rhinoconjunctivitis in 32 patients. Spontaneous histamine release was significantly enhanced (P < 0.05) in patients prior to immunotherapy compared with 20 controls. Spontaneous histamine release decreased significantly in patients after 4 months of specific immunotherapy (P < 0.04) and almost reached the same values as spontaneous histamine release in controls. Clinical success of treatment after 4 months was seen in 15 patients (improvement > or =50%), 10 of whom showed a significant decrease in spontaneous histamine release. Decrease of spontaneous histamine release after 4 months indicates the efficacy of specific immunotherapy already at an early stage of treatment. Assessment of spontaneous histamine release appears to be a useful and easily performable method for monitoring success of treatment of patients during specific immunotherapy.     

Preclinical evaluation of innate immunity to baculovirus gene therapy vectors in whole human blood

Interactions of gene therapy vectors with human blood components upon intravenous administration have a significant effect on vector efficacy and patient safety. Here we describe methods to evaluate these interactions and their effects in whole human blood, using baculovirus vectors as a model. Opsonisation of baculovirus particles by binding of IgM and C3b was demonstrated, which is likely to be the cause of the significant blood cell-associated virus that was detected. Preventing formation of the complement C5b-9 (membrane attack) complex maintained infectivity of baculovirus particles as shown by studying the effects of two specific complement inhibitors, Compstatin and a C5a receptor antagonist. Formation of macroscopic blood clots after 4 h was prevented by both complement inhibitors. Pro- and anti-inflammatory cytokines Il-1β, IL-6, IL-8 and TNF-α were produced at variable levels between volunteers and complement inhibitors showed patient-specific effects on cytokine levels. Whilst both complement inhibitors could play a role in protecting patients from aggressive inflammatory reactions, only Compstatin maintained virus infectivity. We conclude that this ex vivo model, used here for the first time with infectious agents, is a valuable tool in evaluating human innate immune responses to gene therapy vectors or to predict the response of individual patients as part of a clinical trial or treatment. The use of complement inhibitors for therapeutic viruses should be considered on a patient-specific basis.     

Ligation of intercellular adhesion molecule 3 induces apoptosis of human blood eosinophils and neutrophils

BACKGROUND: Intercellular adhesion molecule 3 (ICAM-3) is highly expressed on human granulocytes, including eosinophils and neutrophils, but the functions of ICAM-3 in these cells are not well understood. OBJECTIVE: Our studies test the hypothesis that ICAM-3 regulates granulocyte apoptosis. METHODS: Intercellular adhesion molecule 3 was activated by a mAb that recognizes an ICAM-3 epitope that binds its ligand, CD11a/CD18. In some experiments with eosinophils, recombinant human IL-5 or GM-CSF was added to mimic in vivo antiapoptotic conditions. Staining with annexin V-fluorescein isothiocyanate and propidium iodide identified apoptotic cells. RESULTS: Binding of ICAM-3 increased apoptosis of both eosinophils (18 and 48 hours) and neutrophils (18 hours). At 18 hours, eosinophil apoptosis increased from 31.4% +/- 3.5 SE (IgG control) to 45.2% +/- 3.8 SE (anti-ICAM-3), and neutrophil apoptosis increased from 48% +/- 4.1 SE (IgG control) to 55.3% +/- 4.5 SE (anti-ICAM-3). At 48 hours, eosinophil apoptosis increased 2-fold under baseline conditions and also in the presence of recombinant human IL-5 or GM-CSF. In both eosinophils and neutrophils, incubation with a blocking antibody against CD18 integrins blunted ICAM-3-induced apoptosis. In eosinophils, blocking peptides for caspases 8 and 9, proteases critical to apoptosis, also decreased ICAM-3-induced apoptosis to control levels. CONCLUSION: Through its effect on eosinophil and neutrophil apoptosis, ICAM-3 may be an important anti-inflammatory molecule that can oppose the proinflammatory effects of IL-5 and GM-CSF. CLINICAL IMPLICATIONS: These findings provide a mechanism for apoptotic clearance of eosinophils and neutrophils involved in allergic inflammation that, unlike necrosis, does not cause nonspecific tissue injury.