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Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Intervertebral disc (IVD) degeneration is a complex process characterized by elevated concentrations of proinflammatory cytokines and proteolytic enzymes. Because of pro-healing constituents, we hypothesized that fibrin sealant (FS) can reduce inflammation and augment soft tissue healing within the damaged or degenerative IVD. To test this, human and porcine nucleus pulposus (NP) and annulus fibrosus (AF) cells were extracted from tissues and encapsulated into alginate beads (NP cells) and type I collagen sponges (AF cells). Half of the alginate and collagen scaffolds were embedded in FS. To provoke inflammatory behaviours, the constructs were cultured with and without continuous IL-1α (10 ng/ml) for 4, 7 and 14 days. ELISA was used to quantify the cellular synthesis (ng/µg DNA) of clinically relevant cytokines, proteolytic enzymes and growth factors. In NP cell constructs with IL-1α, the syntheses of TNFα, IL-1β, IL-6, IL-8 was elevated at all culture durations. In the presence of FS, secretion of several pro-inflammatory cytokines were significantly reduced [IL-6 and IL-8 (porcine); and TNFα, IL-1β, IL-6, IL-8 (human)]. Consistent with these reductions, human NP cultures exposed to FS and FS + IL-1α synthesized significantly reduced amounts of MMP-1 and -3 compared to constructs with IL-1α. For porcine and human AF cells, there were no significant differences in the synthesis of the inflammatory or proteolytic cytokines relative to controls (without IL-1α) at any culture duration. However, the porcine AF cells exposed to FS synthesized elevated amounts of the anti-inflammatory cytokine IL-4. The results suggest that FS may have beneficial effects for patients with degenerated intervertebral discs., (Copyright © 2012 John Wiley & Sons, Ltd.)

Objective: Assess the safety and efficacy of intradiscal fibrin sealant in adults with chronic discogenic low back pain., Design: Prospective, nonrandomized Food and Drug Administration approved pilot study., Setting: Three centers in the United States., Subjects: Fifteen adults with chronic, single, or contiguous two-level lumbar discogenic pain confirmed through meticulous provocation discography., Interventions: Volume- and pressure-controlled intradiscal delivery of BIOSTAT BIOLOGX(®) Fibrin Sealant with the Biostat(®) Delivery Device into symptomatic lumbar disc(s)., Outcome Measures: Assessments were performed at baseline, 72 hours, and 1, 4, 13, 26, 52, and 104 weeks following intervention. Potential adverse events were evaluated with serial assessment of neurological status, radiographic, and magnetic resonance imaging (MRI). Efficacy measures included serial assessments of low back pain visual analog scale (VAS) measurements and the Roland-Morris Disability Questionnaire (RMDQ)., Results: Safety neurological assessments, X-ray, and MRI showed no significant changes. Adverse events were reported in nine subjects. Two instances of low back muscle spasm and one case of discitis were the only events considered related to the procedure or product., Efficacy: Mean low back pain VAS scores (mm) decreased from 72.4 (95% confidence interval 64.6-80.3) at baseline to 31.7 (17.4-46.1), 35.4 (17.7-53.1), and 33.0 (16.3-49.6); mean RMDQ score improved from 15.2 (12.7-17.7) at baseline to 8.9 (5.3-12.5), 6.2 (3.4-9.1), and 5.6 (2.9-8.4) at 26, 52, and 104 weeks, respectively., Conclusion: Intradiscal injection of BIOSTAT BIOLOGX Fibrin Sealant with the Biostat Delivery Device appears safe and may improve pain and function in selected patients with discogenic pain., (Wiley Periodicals, Inc.)

The current study tests a model that depicts the relationships among coping strategies (active, distraction, avoidance, and support seeking) and anxiety symptoms. SEM is used to test if the relationship between these variables is mediated by coping efficacy. A large sample of Canadian children (N = 506) aged 8 to 11 years (boys = 249, girls = 245, unknown gender = 12) participated in the study. Results showed that coping efficacy is a partial mediator of the relations between active coping strategies and anxiety symptoms, however support was not found for it to be an effective mediator for other coping strategies. This study contributes to the understanding of childhood anxiety by highlighting the importance of the relationship between anxiety and the methods children use to cope with stress and how perceptions of their coping abilities influence this relationship.

We report the use of a matched set of mice/human chimaeric antibodies, directed against the 5-iodo-4-hydroxyl-3-nitrophenacetyl (NIP) hapten, to investigate the roles of different human isotypes in antibody-mediated eosinophil-dependent killing of schistosomula. The chimaeric antibodies consist of mouse VH, VL and CL regions with human gamma 1, gamma 2, gamma 3 (2 allotypes), gamma 4, alpha 2, mu or epsilon CH regions and were used in in vitro assays with human eosinophils and NIP-coated S. mansoni schistosomula. Some anti-NIP isotypes mediated high levels of killing, which was specific for NIP-coated larvae, and we suggest that these antibodies will be a valuable tool for studies on the role of antibody isotypes in anti-schistosome immune effector mechanisms. In particular, this method directly demonstrated, for the first time, that IgA is highly effective in mediating the killing of metazoan parasites by human eosinophils.

This review summarizes the personal experiences of the authors and their colleagues during ten years of field and laboratory studies on human immunity to Schistosoma mansoni infections. There is evidence for the very slow development with age of an acquired resistance to reinfection (demonstrable after chemotherapy of the primary infection) distinguishable from a lack of reinfection due to reduced exposure. The implications of this immunity for the design of chemotherapy programs targeted at infected schoolchildren are discussed. Observational studies on the mechanisms of immunity have demonstrated a marked helminthocidal capacity of eosinophils. Subsequent correlative studies have indicated a role of IgM and IgG2 "blocking" antibodies in maintaining the continued susceptibility of young children, and of IgE antibodies in mediating protection in older individuals. Some problems in studying human immunity, and the implications for vaccine development, are also discussed.

IgG antibodies and eosinophils kill schistosomula of Schistosoma mansoni in vitro, and there is now evidence to suggest that the main factor that contributes to the expression of purified IgG effector function is the degree of activation of the donor's eosinophils. This study was designed to identify serum-derived activating factors in sera from individuals infected with S. mansoni. Such activating factors may be responsible for enhancing eosinophil cytotoxicity against schistosomula. Serum-borne mediators were prepared by fractionation of sera from infected individuals by gel filtration high-performance liquid chromatography. The eosinophil-stimulating activity of these mediators was assayed by a new method which depends on the increased expression of the CR3 alpha chain (CD11b) on the surface of activated eosinophils. Sera from infected individuals exhibited different levels of eosinophil activation, and activation appeared to be due to several serum factors, including interleukin 5. In conclusion, our results suggest that eosinophil-activating factors present in infection sera may not only be responsible for enhancing eosinophil cytotoxicity but also be necessary for its expression.

The observation that activation of eosinophils in vitro with PAF increases the surface expression of the alpha chain of the complement receptor CR3 (CD11b) has been extended to other eosinophil activating factors. CD11b may be detected on activated eosinophils by reaction with mouse monoclonal anti-human CD11b IgG, following the addition of urease-conjugated sheep anti-mouse IgG. CD11b levels were increased on eosinophils after incubation with (a) recombinant colony stimulating factors, IL-3, GM-CSF and IL-5, at concentrations of 100 U/ml, or (b) with eosinophil activating factors, recombinant TNF alpha (1000 U/ml), EAF purified from mononuclear cell supernatants and PAF (10(-6) M). CD11b levels were not affected by IL-1 alpha, IL-2 or IFN-gamma. Unstimulated neutrophils had higher levels of CD11b than unstimulated eosinophils, but neutrophil CD11b was unaffected by IL-3, GM-CSF and IL-5 and was only slightly affected by TNF, EAF and PAF. Polyclonal rabbit antibodies to IL-3 and TNF neutralised their CD11b enhancing activities. The PAF antagonists WEB 2086 and WEB 2170 neutralised the CD11b enhancing activity of PAF. We conclude that measurement of CD11b expression on eosinophils is a convenient method for the assay of eosinophil activating activity.

A protein of apparent molecular weight 55000, designated protein 3, becomes newly detectable on the eosinophil surface as a specific consequence of interaction with antigen-antibody complexes immobilized in agar layers. The effect of various agents upon this interaction has been determined by monitoring the appearance of this protein by lactoperoxidase-catalysed iodination. Other parameters that have been measured include: the attachment of eosinophils to the agar layers and their subsequent degranulation, as measured by the release of granule peroxidase, and the degree of spreading of the eosinophils, as assessed by electron microscopy. Attachment of eosinophils to antibody-coated layers is inhibited by heat-aggregated immunoglobulin G (IgG), suggesting that this attachment is mediated via eosinophil Fc receptors. In addition, agents, such as the eosinophil chemotactic factor Ala-Gly-Ser-Glu, that enhance the expression of Fc receptors also enhance the appearance of protein 3, while agents, such as hydrocortisone, that inhibit the expression of Fc receptors reduce its appearance. It is concluded that the appearance of protein 3 parallels the expression of Fc receptors. Attempts to block the Fc region of the bound antibody with staphylococcal protein A were not successful. These experiments indicated that the Fc region of bound IgG has different binding sites for protein A and for the Fc receptor. The correlation between the appearance of protein 3 and subsequent degranulation of the eosinophils was confirmed by the use of agents, such as cytochalasin D and levamisole, that enhance both the appearance of protein 3 and degranulation. Conversely, hydrocortisone reduces the appearance of protein 3 and inhibits degranulation. Protein 3 does not appear when eosinophils adhere to agar layers coated with concanavalin A instead of antibody and the eosinophils do not degranulate. Addition of the calcium ionophore A23187, while causing the release of granule peroxidase, does not elicit the appearance of protein 3. These observations provided additional evidence that the appearance of protein 3 is a specific consequence of the interaction of eosinophils with antibody-coated surfaces. The fact that protein 3 appears at the eosinophil surface as a direct consequence of the interaction with antibody suggests that this protein is closely associated with the eosinophil Fc receptor. The enhancement of the appearance of protein 3 in the presence of cytochalasin D indicates that the movement and reorientation of both this protein and the Fc receptor are constrained by association with cytoplasmic microfilaments.

The regularly arranged surface protein, the a-protein, of Acinetobacter 199A has been shown to have phospholipase A2 activity. Since half of the a-protein synthesised by Acinetobacter 199A is secreted into the growth medium, the bacteria are producing extracellular phospholipase A2.

Incubation of eosinophils with schistosoma of Schistosoma mansoni in vitro induces changes in the eosinophil membrane. Cell surface proteins were blocked with unlabelled iodide and then, after incubation with schistosomula, newly accessible proteins were detected by lactoperoxidase catalysed iodination with radioactive iodide. Normal turnover restores several proteins to the eosinophil surface, but the schistosomula specifically induced the appearance of a protein of mol. wt 18K. This protein has previously been detected when eosinophils interact with antibody coated agar layers. Soluble factors released by schistosomula induced the appearance of enhanced levels of 16 and 18K proteins. It has previously been shown that proteins involved in binding of eosinophils to IgG coated antigen coupled cellulose have mol. wts of 16K and 18K.

Eosinophil activating factor (EAF) is a 40 kD protein released from cultured, unstimulated human monocytes which enhances the IgG-dependent eosinophil-mediated cytotoxicity of helminthic larvae. We have recently shown that eosinophils elaborate substantial quantities of leukotriene C4 (LTC4) during incubation with IgG-coated particles and now report that EAF, partially purified by sequential chromatography on Sephacryl S-200 and DEAE-cellulose, enhanced this IgG-dependent LTC4 production by human eosinophils in a dose- and time-dependent fashion. LTC4 production by normal density eosinophils, separated on discontinuous metrizamide gradients, was significantly increased after incubation with several dilutions of EAF (P less than 0.05), although an increase was not seen with low density cells. The enhancement was similar in degree to that seen when normal density eosinophils were activated with the bacterial analogue, f-met-leu-phe (fMLP). EAF produced a time-dependent increase in LTC4 which was significantly greater (P less than 0.01) than the control. Sulphidopeptide leukotriene (LT) generation was validated by reverse phase high pressure liquid chromatography (RP-HPLC). These results indicate that there is a firm association between monocytes, eosinophils and LTC4; an observation which may be of relevance to mechanisms in chronic asthma and related disorders, and in immune reactions against migrating helminthic larvae.

A new cell line has been produced by fusing human cervical keratinocytes with HeLa cells. This cell line secretes eosinophil-activating activity upon stimulation with tumour necrosis factor (TNF). About one-third of the eosinophil-activating activity co-purifies with eosinophil-activating factor (EAF) from mononuclear cell supernatants. The purification procedure indicates that it resembles EAF in molecular weight and acidity. It also resembles EAF in its effect on eosinophils. Not only does it enhance the cytotoxic activity of eosinophils to antibody-coated schistosomula of Schistosoma mansoni, but it also increases the oxidative activity of eosinophils, as measured by reduction of nitroblue tetrazolium, and changes the morphology of eosinophils, affecting the distribution of F-actin in the cell.

The bacteriolytic and bactericidal effects of the human proteinases cathepsin B, cathepsin D, cathepsin G, and elastase were investigated. Cathepsin G and elastase were 5 to 10% as active as egg white lysozyme in the lysis of Micrococcus lysodeikticus. All four enzymes slowly lysed the lysozyme-resistant Staphylococcus aureus. The gram-negative Acinetobacter 199A was rendered sensitive to lysozyme by all of the proteinases. Only elastase caused marked proteolysis of the outer membrane, which would permit access by lysozyme to the underlying peptidoglycan. When the surface layer of regularly arranged a protein was removed, however, the outer membrane proteins became susceptible to the other proteinases. Cathepsin G, elastase, and cathepsin D were bactericidal to Acinetobacter 199A. The bactericidal activity of cathepsin D was shown to be dependent on enzymatic activity, unlike that of cathepsin G, which was related to its cationic nature.

Trypanosome phagocytosis by normal polymorphonuclear leucocytes was studied in the presence of the pregnancy-associated alpha 2-glycoprotein (PAG). A serum fraction containing PAG at a concentration of 13.5 micrograms/ml significantly inhibited the uptake of Trypanosoma dionisii when contrasted with an identically obtained male serum fraction devoid of PAG (P less than .01). This finding of diminished trypanosome uptake in physiologic concentrations of PAG could account for the suggested increase in the frequency and severity of protozoal infections during gestation.

Human peripheral blood eosinophils attach to and flatten down onto antibody-coated surfaces and subsequently degranulate. An antibody-coated surface was prepared by treating a layer of agar, containing tetanus toxoid antigen and eosinophil chemotactic factor (ECF), with human anti-tetanus immunoglobin. Changes in eosinophil surface proteins during attachment to the antibody-coated agar layer were detected by lactoperoxidase catalysed iodination. Purified eosinophils were pre-treated with unlabelled iodide, lactoperoxidase and hydrogen peroxide to block pre-existing accessible tyrosine residues on the cell surface. They were then allowed to interact with the agar layer, and subsequently treated with lactoperoxidase and 125I-labelled iodide to label newly accessible surface proteins. Separation of the radioactive proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed that, while incubation of the cells in suspension restored the major proteins to the cell surface, interaction with the antibody-coated agar layer caused the appearance of additional proteins of apparent molecular weight 55K, 30K, 28K and 18K. The 55K, 28K and 18K proteins were greatly reduced when antibody was absent, but the 55K protein was distinguishable from immunoglobulin G (IgG) heavy chain, since it could be detected in low amounts even in the absence of antibody. It was found in purified plasma membranes and it could be separated from IgG heavy chain by iso-electric focusing. The possibility is discussed that this protein is either linked to the receptor for the Fc portion of IgG, or that it is itself the receptor. The 18K protein required both antibody and ECF for maximum expression, but was seen in limited amounts with ECF alone. Possibly it is concerned with an ECF-mediated recognition of IgG. Unlike the 55K protein, it binds concanavalin A. Plasma membranes were prepared from eosinophils by lysis in borate, followed by purification on a glass-bead column. Both the 55K and the 18K proteins were found to be major components of the eosinophil membrane.

Acinetobacter sp. strain MJT/F5/199A carries an array of tetragonally arranged subunits on its outer surface. The subunits can be detached from isolated cell walls by incubation with 1 M urea or by washing with water after treatment with 10 mM ethylenediaminetetraacetic acid or ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid. After removal of the urea, they reaggregate into the same ordered array at air-water interfaces in the presence of MgCl(2). The detached subunits were characterized as an acidic protein of molecular weight 65,000. They represent one-fifth of the total cell wall protein.

Monocytes from moderately eosinophilic individuals secrete material that enhances the cytotoxic activity of eosinophils against antibody-coated schistosomula of Schistosoma mansoni. This material is not a single substance, but can be fractionated into several active components of different size and different charge. Gel filtration of mononuclear cell supernatants separated the eosinophil-activating activity into a major component of molecular mass of 40 kDa and a minor component of molecular mass of less than 10 kDa. The major component exhibited further heterogeneity on fractionation by high performance liquid chromatography. The bulk of the eosinophil-activating activity could be separated from both colony-stimulating factor (CSF) alpha activity and from tumor necrosis factor (TNF) activity. However, human recombinant CSF alpha (GM-CSF), human recombinant TNF and rabbit tumor necrosis serum all had eosinophil-activating activity when tested against schistosomula. Eosinophils were not activated by interleukin 1, interleukin 2, interferon-alpha, lipopolysaccharide or phorbol myristate acetate.

The formation of the components of the cell envelope of Acinetobacter sp. 199A was investigated by measuring the incorporation of [3H]leucine into protein, [14C]galactose into lipopolysaccharide, 32P into phospholipid, and [3H]diaminopimelic acid into peptidoglycan. Whereas the lipopolysaccharide and intrinsic protein of the outer membrane were stable, some of the regularly arranged surface protein, the alpha-protein, was lost into the growth medium. Only newly synthesized alpha-protein was lost. The peptidoglycan of the murein layer was also labile. Selective inhibition of the formation of individual components of the cell envelope with penicillin, chloramphenicol, and bacitracin showed that incorporation of protein into the outer membrane required the simultaneous formation of complete lipopolysaccharide. The converse was not true: protein synthesis was not required for lipopolysaccharide incorporation. Formation of the outer membrane and the murein layer proceeded independently.

Trypomastigotes of Trypanosoma dionisii, a stercorarian trypanosome from bats, are effectively killed by neutrophils from human peripheral blood but are less sensitive to the cytotoxic action of human monocytes. The mechanism of killing appears to involve peroxidase and hydrogen peroxide. Trypomastigotes are as effective as epimastigotes in inducing the formation of hydrogen peroxide by effector cells. They are, however, less sensitive than epimastigotes to the cytotoxic effect of peroxidase and hydrogen peroxide. They are therefore susceptible to the high concentrations of peroxidase found in the phagosome of the neutrophil, but resist the lower levels encountered in monocytes.

Although IgG antibodies and eosinophils have been shown to kill schistosomula of Schistosoma mansoni in vitro, very little data exist that describe the role of each IgG antibody isotype in this event. This study was designed to test the role of each IgG subclass in the eosinophil-dependent killing reaction. IgG antibodies purified by protein G or protein A affinity chromatography demonstrated a killing effect only in the presence of eosinophils activated in vivo or normal eosinophils activated in vitro by eosinophil activating factor. Purification of each IgG isotype allowed confirmation of these results and demonstrated that the killing effect was associated with IgG1 and IgG3 antibodies. IgG2 antibodies expressed a dual function: 1) an effector function with activated eosinophils and 2) a blocking function with normal eosinophils. IgG4 antibodies, whatever the source of eosinophils, blocked the killing mediated by IgG effector antibodies. These findings are discussed in relation to immunity and susceptibility to reinfection in human schistosomiasis.

Responses of blood platelets to bacterial endotoxin lipopolysaccharide (LPS) have been correlated with changes in the molecular organization and composition of the platelet plasma membrane proteins. Binding of LPS, which occurred in the absence of Ca2+, was distinguished from platelet aggregation and degranulation, which required Ca2+ and plasma proteins. Changes in membrane organization were detected by double-labelling with [125I] and [131I] iodide, mediated by lactoperoxidase and hydrogen peroxide. Changes in total membrane composition were detected by gel electrophoresis of isolated membranes. Binding of LPS was associated with increased accessibility of a protein of mol. wt. 80000 to iodination. After aggregation and degranulation there was, in addition, increased accessibility of proteins of mol. wt. 68000 and 48000. Isolated membranes from LPS-stimulated platelets contained more of a protein of mol. wt. 200000 and less of a protein of mol. wt. 220000 than control membranes prepared from unstimulated platelets in the presence of cAMP and aminophylline. The relationship of the modified plasma membrane proteins to the contractile proteins of the platelet and their possible redistribution in the cell during aggregation and secretion is discussed.

Rabbit peritoneal polymorphonuclear leucocytes were induced to aggregate by a variety of bacterial species. In the absence of serum, Gram-negative bacteria were more effective at inducing aggregation than Gram-positive. The most effective micro-organism tested, Acinetobacter sp. 199A, was readily phagocytosed and also induced extracellular secretion of the granule enzymes peroxidase and lysozyme. Isolated endotoxin from this bacterial species was highly effective in inducing aggregation and granule enzyme release. Endotoxin-induced aggregation was associated with a large increase in the amount of lactoperoxidase-catalysed iodination of surface protein. Only one iodinatable protein was detected, of molecular weight 150 000. It is postulated that phagocytosis of Gram-negative bacteria, followed by granule enzyme release, accelerates the rate of membrane recycling and that this brings new adhesive protein to the surface more rapidly.

The cell-mediated resistance of human leucocytes to Trypanosoma dionisii, a bat parasite related to T. cruzi, was investigated. Human peripheral blood neutrophils and monocytes were cytotoxic to T. dionisii as assessed by electron microscopy and by induction of 99mTc release from trypanosomes pre-labelled with [99mTc] pertechnetate. The enhancement of cytotoxicity by specific antiserum varied considerably from one individual to another. Neither blood lymphocytes nor blood eosinophils induced 99mTc release from T. dionisii. The trypanosomes were readily phagocytosed by neutrophils and monocytes even in the absence of added antiserum but the rate was enchanced when antiserum was present. Eosinophils also phagocytosed T. dionisii but only in the presence of antiserum. Investigation by electron microscopy revealed that T. dionisii is rapidly destroyed in the phagocytic vacuole of enutrophils and monocytes and by eosinophils. Phagocytosis, ultrastructural damage and induction of 99mTc release occurred more rapidly in neutrophils than in monocytes.

Proteins involved in the attachment of eosinophils to immobilized antigen-antibody complexes were isolated. Eosinophils, purified from normal human peripheral blood, were surface labelled with 125I-iodide in the presence of lactoperoxidase and hydrogen peroxide. Immobilized immune complexes were prepared by covalent coupling of tetanus toxoid antigen to cellulose and treatment of the fixed antigen with human anti-tetanus IgG. Intact cells were allowed to interact with the antigen-antibody complex and the cells were then lysed in situ with a salt solution containing the non-ionic detergent NP40. After exhaustive washing to remove unattached proteins, the bound proteins were eluted with a buffer containing mercaptoethanol with or without SDS. A major protein of mol. wt 16K and a minor protein of mol. wt 18K were isolated. These proteins were unaffected by the temperature of attachment of eosinophils to the fixed IgG antigen complexes or by the presence of protease inhibitors and did not therefore appear to be proteolytic cleavage products.

The cytotoxic activity of eosinophils, as measured by their ability to kill antibody-coated schistosomula of Schistosoma mansoni, is enhanced by a factor (eosinophil-activating factor, EAF) which is secreted by peripheral blood monocytes from certain moderately eosinophilic individuals. The secretion of this factor by monocytes is inhibited by methylprednisolone (1 microgram/ml), beclomethasone (10 micrograms/ml) and theophylline (100 micrograms/ml), but not by sodium cromoglycate or salbutamol. Methylprednisolone, beclomethasone and theophylline do not inhibit either eosinophil cytotoxic activity or enhancement of eosinophil cytotoxic activity by EAF at these concentrations.

Clostridum thermosaccharolyticum and Clostridium thermohydrosulfuricum possess as outermost cell wall layer a tetragonal or hexagonal ordered array of macromolecules. The subunits of the surface layer can be detached from isolated cell walls with urea (8M) or guanidine-HCl (4 to 5 M). Triton X-100, dithiothreitol, ethylenediaminetetracetate, and KCl (3 M) had no visible effect on the regular arrays. Sodium dodecyl sulfate-polyacrylamide electrophroesis showed that, in both organisms, the surface layer is composed of glycoprotein of molecular weight 140,000. The glycoprotein from both microorganisms has a predominantly acidic amino acid composition and an acidic isoelectric point after isoelectric focusing on polyacrylamide gels. The glycocomponent is composed of glucose, galactose, mannose, and rhamnose.

A model has been developed to simulate the surface of an antibody-coated schistosomulum. It consists of a layer of agar, containing antigen (tetanus toxoid) and a chemotactic factor (ECF). Some layers were coated with human anti-tetanus immunoglobulin. The mode of adherence of human eosinophils and neutrophils to these agar layers and the subsequent degranulation of the cells exactly paralleled the interaction of these cell types with antibody-coated schistosomula of Schistosoma mansoni. In particular, eosinophils made much more intimate contact than did neutrophils, and lysosomal enzymes were secreted extracellularly by direct fusion of granules with the plasma membrane of the cell. Biochemical evidence was also obtained for the secretion of enzymes during degranulation and the rate of enzyme release was found to be enhanced in the presence of specific antibody. This model, non-phagocytosable surface has the potential to provide basic information of the mode of action of effector cells in cell-mediated cytotoxic reactions against a wide range of parasites by incorporation of different factors into the agar layers.

Acinetobacter 199A carries on the outer surface of its outer membrane a layer of regularly arranged protein subunits. The isolated surface protein assembles into the same regular array even in the absence of the underlying outer membrane. Cl- minus is required for this self-assembly. Evidence is presented that the interaction of the surface protein with the outer membrane involves the linking of a carboxyl group in the surface protein to a negatively charged group in the outer membrane protein, via a divalent cation. The surface protein could be detached from the outer membrane by the protein perturbant urea, by the chelating agent EDTA and by replacing Mg-2+ with Na+. It could not be detached by treatment with phospholipases A anc D or the detergents Tween 80 and sodium deoxycholate. The conditions favourable for reattachment of surface protein to the cell wall were the presence of divalent cations and a pH of 3-5. Conversion of carboxyl groups in the surface protein to amine with carbodiimide and ethylene diamine interfered with reattachment. The surface protein did not attach to isolated cell wall lipid or lipopolysaccharide.

Evidence is presented that T lymphocyte cytotoxicity is mediated by hydrogen peroxide (H2O2). At a concentration of 5 x 10(-4) M H2O2 induced 51Cr release from pre-labelled P815 mastocytoma cells. H2O2 was generated when T lymphocytes from mouse spleen were exposed to P815 cells. The concentration of H2O produced was apparently one thousand times lower than the concentration required to lyse the P815 cells. This suggests that the H2O2 is produced and acts at a highly localized site on the target cell. Sulphydryl groups on the target cell were particularly sensitive both to H2O2 and to spleen cell attack. The activity of the spleen cells was inhibited by cyanide and azide and by reducing agents which protected the target cells. Cytotoxicity was enhanced by agents which prevented H2O2 breakdown.

Cell-mediated immunity represents an important host defence mechanism against protozoal infections. The effector cells directly involved are neutrophils, macrophages and, ultimately, activated macrophages. Within this simple scheme there are, however, considerable variations in activity. Effector cells from different animal species, and even from different strains of the same species, may be more or less effective in controlling a certain protozoal infection. Different protozoa differ in their susceptibility to cell-mediated killing according to genus, species, strain and morphological form. The most susceptible morphological form is that which occurs in the insect vector, and which has not yet adapted to protect itself from the vertebrate host. Epimastigotes of Trypanosoma and promastigotes of Leishmania are readily killed by phagocytic cells, while the corresponding trypomastigote and amastigote forms are considerably more resistant. Protozoa which live in macrophages, such as amastigotes of Leishmania, endozoites (tachyzoites) of Toxoplasma and amastigotes of reticulotropic strains of T. cruzi, have developed a remarkable resistance to the microbicidal activity of the host cell. Conversely, amastigotes of myotropic strains of T. cruzi, which live in muscle cells, have not developed this resistance to cell-mediated killing by macrophages. Readily accessible protozoa, such as T. brucei trypomastigotes and Plasmodium merozoites in the bloodstream, while they lack the marked resistance developed by reticulotropic protozoa, have a partial protection since they are attacked by phagocytic cells only when specific antibody is present. Granulocyte-mediated killing can be largely attributed to neutrophils. Eosinophils appear to play only a minor role and compete ineffectually when neutrophils are also present. The only group of protozoal species which may be significantly controlled by eosinophils are the stercorarian species of Trypanosoma. In vitro experiments show that antibody-coated trypomastigotes of T. cruzi can be killed by eosinophils, although there is little evidence that this occurs in vivo. Interestingly, this is the only species that has been reported to be susceptible to the major basic protein of eosinophils, a toxic component of the lysosomal granules which is very active against helminths. Neutrophils are not very active against endozoites of Toxoplasma gondii, Trypanosoma, trypomastigotes of salivarian Trypanosoma, free merozoites of Plasmodium, and promastigotes and amastigotes of Leishmania.(ABSTRACT TRUNCATED AT 400 WORDS)

Endotoxin lipopolysaccharide (LPS) from Acinetobacter 199A induced aggregation of blood platelets from immune adherence-positive species (rat, rabbit) but not from immune adherence-negative species such as pig and man. Aggregation occurred in 2 phases: the first was not accompanied by secretion of platelet constituents, was apparently a consequence of C3 activation, and was selectively inhibited by EGTA. The second phase of aggregation was associated with secretion of platelet granule contents, and with a lesser amount of cytoplasmic leakage. Secondary aggregation was abolished by the sulphydryl alkylating agent N-ethylmaleimide, and by agents which increased the level of cyclic AMP in platelets, such as prostaglandin E1 (a stimulator of adenylate cyclase) and methyl xanthines (inhibitors of phosphodiesterase). Secondary aggregation was partly inhibited by agents which block platelet prostaglandin biosynthesis (e.g. aspirin, indomethacin). Primary aggregation was unaffected by these inhibitors at concentrations which blocked secondary aggregation.

Antibody-dependent cell-mediated cytotoxicity (ADCC) of infectious bovine rhinotracheitis (IBR)-infected bovine kidney cells (MDBK) by neutrophils was demonstrated. Neutrophils from bovine and sheep mammary exudate and peripheral blood, and also from human peripheral blood, were all active in the presence of anti-IBR antibody. The component of the ruminant neutrophil granules which was responsible for cytotoxicity appeared to be cationic protein since purified cationic protein lysed the virus-infected cells and heparin inhibited cytotoxicity. Human neutrophil cytotoxicity to herpes simplex virus (HSV)-infected human Chang liver cells was also inhibited by heparin. Human neutrophil cytotoxicity to IBR-infected bovine kidney cells did not appear to be mediated by cationic protein since it was inhibited by the chelators of oxidative intermediates DMSO, thiourea, tryptophane, benzoate and mannitol, and not by heparin.

Mononuclear cells from blood samples from asthmatic patients were tested for their ability to produce two eosinophil activating factors, EAF and TNF. High levels of EAF were produced by some but not by all patients. The influence of external factors on EAF production was evaluated. Patients receiving prednisolone (10-30 mg/day) tended to produce only low levels of EAF. Prednisolone has been shown to inhibit production of EAF in vitro by isolated mononuclear cells, at concentrations of 0.1-1 micrograms steroid/ml. Incubation of mononuclear cells with certain allergens enhances EAF production in sensitive individuals. Little or no production of TNF was observed in the patients examined.

The mechanism of the cytotoxic reaction of leukocytes to Trypanosoma dionisii was investigated. Cytotoxicity was measured by release of [99mTc]pertechnetate from labeled protozoa. Both granulocytes and lymphocytes were found to be cytotoxic to antibody-coated T. dionisii. The reaction was inhibited by diethyldithiocarbamate and by potassium cyanide, both of which inhibit myeloperoxidase. Myeloperoxidase from azurophil granules was toxic to T. dionisii, provided that hydrogen peroxide was also present. Hydrogen peroxide formation was induced in granulocytes and, to a lesser extent, in lymphocytes by antibody-coated T. dionisii. Inhibition of this hydrogen peroxide formation by treatment of the effector cell surface with p-diazobenzenesulfonic acid inhibited cytotoxicity. It is therefore concluded that granulocytes, and probably also lymphocytes, kill T. dionisii with hydrogen peroxide by a peroxidase-mediated reaction. Although hydrogen peroxide and myeloperoxidase alone were also cytotoxic to the lymphoblastoid cell line CLA4, it seems unlikely that this is the cytotoxic mechanism for this process because these cells were unable to induce hydrogen peroxide formation.

Plasma membrane changes during the interaction of human eosinophils with large, antibody-coated, non-phagocytosable surfaces have been investigated in a model system. Human peripheral blood eosinophils were incubated with layers of agar into which teranus toxoid (ECF), were incorporated. Changes in organization of the eosinophil plasma membrane proteins during interaction with the agar layer were detected by lactoperoxidase-catalysed iodination with [125]iodide. A protein of apparent mol. wt 55 000 became newly accessible on the eosinophil surface as a specific consequence of interaction with antigen-antibody complexes in the agar layer. This protein appeared in the early attachnent phase of the interaction which preceded extracellular degranulation. Cytochalasin D enhanced its appearance, while Mg2+-deficiency prevented it. A second newly accessible protein of apparent mol. wt 58 000 was blocked when ECF was present and may therefore be a receptor for ECF. Other proteins of apparent mol. wt 68 000 and 46 000 newly appeared at the surface of eosinophils even after incubation in suspension, apparently as a consequence of the rapid cycling of membrane components which occurs in eosinophils.

The role of thiols in T cell-mediated cytotoxicity was investigated by studying the thiols of the target cell and cytotoxic cell separately. Agents which protect the reduced thiols of the target cell inhibit their lysis by the cytotoxic T cells; thiol reactive reagents may be directly toxic to the target cell. The thiol groups of the effector cell are also important, since pre-treatment with thiol reactive reagents inhibits killing.

A protein (eosinophil-activating factor, EAF), which enhances the capacity of human peripheral blood eosinophils to kill antibody-coated schistosomula of Schistosoma mansoni, has been partially purified from supernatants of cultured peripheral blood mononuclear cells by sequential chromatography on Sephacryl S200 and DEAE-cellulose. This protein is acidic with a molecular mass on gel filtration of 40 +/- 7 kDa. It not only enhances the activity of eosinophils against schistosomula but also increases their ability to lyse antibody-coated, herpes simplex virus-infected Chang liver cells. It enhances the production of superoxide and hydrogen peroxide by eosinophils that occurs both spontaneously and in response to opsonized zymosan. However, increased respiratory burst activity does not appear to be responsible for the enhancement of eosinophil-mediated killing of schistosomula, since a comparable or greater increase in hydrogen peroxide production is induced by column fractions that have little or no effect on schistosomulum killing. EAF enhances eosinophil degranulation, both spontaneously and after incubation with opsonized zymosan. Enhanced degranulation is associated with release of eosinophil peroxidase and eosinophil cationic protein. These findings suggest that EAF enhances the capacity of eosinophils to kill parasites by increasing the extent of eosinophil degranulation and the amount of toxic granule proteins that are secreted.

To distinguish between direct and indirect involvement of oxygen metabolites in CTL cytotoxicity a comparison was made of cloned CTLs and mixed cells from mouse spleen. Tumour cells could be protected from cloned CTLs and from spleen CTLs by the thiol protecting reducing agents DTT and DETC. Cytotoxic activity was inhibited by diversion of reducing power from NAD(P)H to artificial electron acceptors and by the inhibitor of NAD(P) linked enzymes cibacron blue. Although H2O2 formation could be detected during the lysis of P815 by spleen CTLs it did not prove to be a necessary requirement for cytolysis since it was not formed when glutaraldehyde-treated P815 cells were lysed. Of the scavengers of toxic oxygen metabolites tested only the hydroxyl radical scavenger sodium benzoate inhibited cytotoxicity.

Growing cells of Streptococcus mutans Ingbritt incorporate radioactive mevalonate into unsaponifiable lipid. Of the radioactive lipid 40% was shown by chromatography and mass spectrometry to be C(50) and C(55) prenol.

The incorporation of (14)C-mevalonic acid by Lactobacillus plantarum predominantly into C(55) prenol made it possible to determine the distribution of (14)C-prenol between all its derivatives. In logarithmic-phase cells, 25% of the prenol was free, 31% was as monophosphate, 4% as pyrophosphate, 12% as peptidoglycan precursor, and 28% as glyco-phospho-prenol. The glyco-phospho-prenol contained rhamnose, and probably glucose, galactose, and ribitol phosphate, and it may, therefore, be involved in polysaccharide and teichoic acid biosynthesis. The proportion of free prenol increased, up to 73%, as the cell culture aged. Free prenol was also formed when cells were incubated in buffer. The free prenol was readily reutilized when cells were returned to growth medium.

Endogenous dolichol was shown to function as a natural acceptor of mannose residues by using regenerating rat liver containing [(3)H]dolichol. When subcellular fractions from this liver were incubated with GDP-[(14)C]mannose a double-labelled lipid, which represented 30% of the total [(14)C]mannolipid, could be isolated. This lipid was shown to be identical with the dolichol phosphate mannose formed from exogenous dolichol phosphate, by chromatography, stability to alkali and by chemical cleavage to mannose and dolichol derivatives. It was formed by the rough endoplasmic reticulum and mitochondria. If it is concerned in glycoprotein synthesis this would suggest that it functions in the formation of both secreted and mitochondrial glycoproteins. When both the dolichol and retinol of rat tissue were radioactive they made similar contributions to the synthesis of the lipid by liver microsomal fractions and intestinal epithelial cells.