Antibodies against TFPI and protein C are associated with a severe thrombotic phenotype in patients with and without antiphospholipid syndrome.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) antibodies, which have been reported in patients with antiphospholipid syndrome (APS), may impair TFPI activity and contribute to hypercoagulability, but their role in APS and in thrombosis remains undefined. OBJECTIVE/METHODS: We assessed the presence and avidity of TFPI IgG antibodies, associations with protein C IgG antibodies and associations with clinical disease severity, in 50 patients with thrombotic APS and 50 thrombotic control patients, on long term anticoagulation with warfarin. RESULTS: Thrombotic APS patients had a significantly higher prevalence of TFPI IgG antibodies (40%; 20/50) compared to thrombotic controls (18%; 9/50). TFPI antibodies were predominantly high avidity in APS (50%, 10/20 of positive patients) and strongly associated with a severe thrombotic phenotype (venous and arterial thromboembolism or recurrent thromboembolic episodes despite therapeutic anticoagulation) (odds ratio (OR): 12.0, 95%CI: 2.2-66.1, p = 0.004), while thrombotic control patients mainly showed low avidity antibodies (78%, 7/9 of positive patients). Coexistence of TFPI and protein C IgG antibodies, regardless of their avidity, was strongly associated with a more severe thrombotic phenotype in APS patients (OR: 20.2, 95%CI: 2.0-47.0, p < 0.0001) and also in thrombotic controls (OR: 75.0, 95%CI 1.2-195, p = 0.02). CONCLUSIONS: Coexistent TFPI and protein C IgG antibodies, irrespective of their avidity, may be a useful marker for a severe thrombotic phenotype in thrombotic patients. This suggests a possibly pathophysiological relationship between the two antibodies, predisposing to thrombosis with a possibly more general role in the development of thrombotic complications.

The automation of routine light transmission platelet aggregation.

INTRODUCTION: The investigation of platelet function by aggregometry requires specialist equipment and is labour intensive. We have developed an automated platelet aggregation method on a routine coagulation analyser. METHODS: We used a CS-2000i (Sysmex) with prototype software to perform aggregation in platelet-rich plasma (PRP), using the following agonists: ADP (0.5-10 µm), epinephrine (0.5-10 µm), collagen (0.5-10 mg/µL), ristocetin (0.75-1.25 mg/mL) and arachidonic acid (0.12-1.0 mm). Platelet agonists were from Hyphen Biomed, and an AggRAM aggregometer (Helena Biosciences) was used as the reference instrument. RESULTS: CS-2000i reaction cuvette stirrer speed was found to influence reaction sensitivity and was optimized to 800 rpm. There were no clinically significant changes in aggregation response when the PRP platelet count was 150-480 x 10(9) /L, but below this there were changes in the maximum amplitude (MA) and slope (rate). Dose response with each of the agonists was comparable between CS-2000i and an AggRAM aggregometer and normal subjects receiving antiplatelet drugs. Aggregation imprecision was similar on both the CS-2000i and AggRAM systems, with a cv for 2-5 µm ADP MA and slope varying between 3-12%. CONCLUSION: Our preliminary studies indicated that optimal sensitivity using the CS-2000i was obtained with a reaction cuvette stirrer speed of 800 rpm and a PRP platelet count of 200-300 x 10(9) /L; aggregation with a PRP count <100 x 10(9) /L showed poor sensitivity. Imprecision and detection of antiplatelet drug effects was similar between the CS-2000i and AggRAM. These data demonstrate that CS-2000i is comparable to a stand-alone aggregometer, although CS-2000i has the advantages of walk-away technology and also required a smaller sample volume than the AggRAM (44% less).

Lymphoid tissue inducer cells: bridges between the ancient innate and the modern adaptive immune systems.

Phylogeny indicates that adaptive immunity evolved first in diffusely distributed lymphoid tissues found in the lamina propria (LP) of the gut. B follicular structures appeared later, probably initially in isolated lymphoid follicles in the LP and then in organized lymphoid tissues such as lymph nodes and Peyer's patches. The development of these new lymphoid structures was enabled by gene duplication and evolution of new tumor necrosis family members. Here, we argue that lymphoid tissue inducer cells (LTis) had a pivotal role, not only in the development of organized lymphoid structures, but also in the subsequent genesis of the CD4-dependent class-switched memory antibody responses. In this review, we concentrate on the latter function: the sustenance by LTis of CD4 T-cell responses for protective immunity.

Dendritic cell subsets and costimulation for effector T-cell responses.

The past year has seen major advances in the understanding of dendritic cell biology and of the costimulatory molecules that dendritic cells use to prime effector T cells and memory T cells. Recent work has revealed the specialization between different dendritic cell subsets and how this relates to their different functions in optimizing T-cell help for antibody responses and inflammatory T-cell responses.

Co-stimulation and selection for T-cell help for germinal centres: the role of CD28 and OX40.

Given the importance of responding to infections with the right defensive strategy, much interest has focused on cytokine differentiation in CD4+ T cells. However, relatively little is known of the logistics of T-cell help for B cells. Here, Lucy Walker and colleagues propose key roles for CD28 and OX40 in coordinating the selection, expansion and migration of CD4+ T cells to B-cell follicles.

Epstein-Barr virus: Co-opting B-cell memory and migration.

Epstein-Barr virus, a B-lymphotropic human herpesvirus, persists in vivo by entering the long-lived memory B-cell compartment. Work with genetically modified mice suggests that the viral latent membrane protein LMP1 might allow infected B cells to access the memory compartment by an unusual route.

Developmental regulation of dendritic cell function.

1998 saw key advances in our understanding of the molecular mechanisms whereby immature dendritic cells recognise foreign pathogens in tissues and are induced to migrate to secondary lymphoid organs. In particular, there have been some key insights into how dendritic cells subsequently direct the evolution of immune responses by differential expression of co-stimulatory molecules.

Efficacy and safety of a seven-day, transdermal estradiol drug-delivery system: comparison with conjugated estrogens and placebo. The Transdermal Estradiol Patch Study Group.

OBJECTIVE: To evaluate the efficacy and safety of a new, seven-day, transdermal estradiol system in healthy postmenopausal women with hot flushes. METHODS: Two studies are described. In the first study, subjects were randomized to treatment with a 0.05 mg/day estradiol patch, a 0.1 mg/day estradiol patch, or a placebo patch; and in the second study, to treatment with either of the two estradiol patches or oral conjugated estrogens (as Premarin) 0.625 mg/day. Efficacy was evaluated on the basis of diary entries recording hot-flush frequency and severity. Subjects' and investigators' global assessments of treatment efficacy were recorded at follow-up visits. RESULTS: In Study 1, both the 0.05-mg and 0.1-mg estradiol patches were significantly more effective than placebo in reducing hot flushes and were associated with higher global assessments. In Study 2, all three active treatments produced a significant reduction in the number of hot flushes compared with base-line. There were no statistically significant between-group differences, although the response to the 0.1-mg estradiol patch was greater, and to the 0.05-mg estradiol patch less, than the response to conjugated estrogens. The patches were generally well tolerated. Skin irritation from the patch was the most common adverse experience in both studies. CONCLUSIONS: The new, seven-day, transdermal system effectively and safely treats post-menopausal vasomotor symptoms.

Relationships between the degree of cross-linking of surface immunoglobulin and the associated inositol 1,4,5-trisphosphate and Ca2+ signals in human B cells.

Cross-linking of surface immunoglobulin (Ig) receptors on human B cells leads to the activation of a tyrosine kinase. The activated tyrosine kinase subsequently phosphorylates a number of substrates, including phospholipase C-gamma. This enzyme breaks down phosphoinositol bisphosphate to form two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C and the release of intracellular Ca2+ respectively. We have used h.p.l.c. and flow cytometry to measure accurately the inositol phosphate turnover and Ca2+ release in anti-Ig-stimulated human B cells. In particular, we have examined the effect of dose of the cross-linking antibody on the two responses. The identity of putative messenger inositol phosphates has been verified by structural analysis, and the amounts of both inositol phosphates and Ca2+ present have been quantified. In the Ramos Burkitt lymphoma, which is very sensitive to stimulus through its Ig receptors, both inositol phosphate production and Ca2+ release were found to be related to the dose of anti-Ig antibody applied. This suggests that phospholipase C-mediated signal transduction in human B cells converts the degree of cross-linking of the immunoglobulin receptor quantitatively into intracellular signals.

Rapid signaling to B cells by antigen-specific T cells requires CD18/CD54 interaction.

This study reports early B and T cell signaling events during cognate interactions between a human B cell line pulsed with peptide and an Ag-specific T cell clone. As has been previously reported, peptide in the context of the appropriate class II molecule stimulated a rise in intracellular calcium [Ca2+]i in the Ag-specific T cell clone. The activation of the T cell clone was associated with a reciprocal rise in [Ca2+]i in the B cells. Engagement of receptors on the B cell surface by the T cell also was associated with inositol phospholipid turnover comparable to that elicited by stimulation through sIg. Early signaling events in B cells can therefore be stimulated in cognate interactions with Ag-specific T cells, without the direct engagement of Ig receptors. A class II deficient B lymphoblastoid mutant, 6.1.6, which was incapable of presenting peptide to the T cell clone, could be stimulated to produce a rise in [Ca2+]i if the T cell clone was activated by monoclonal antibodies to CD3. Therefore, the interaction of class II molecules on the B cell with the TCR and/or the CD4 accessory molecule was not essential for T-dependent B cell activation. However, T-dependent signalling of B cells was profoundly inhibited by mAb to CD18 (beta-chain of LFA-1) on the T cell or CD54 (ICAM-1) on the B cell, demonstrating the importance of this pair of adhesion molecules in early T-B cell interactions.

Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens.

Techniques which identify hapten-specific B cells in tissues have been used to determine the sites of B cell activation in rat spleens in response to T cell-dependent (TD) antigens and T cell-independent type-1 (TI-1) antigens. Surface-associated hapten binding by specific memory B cells and B blasts was distinguished from the strong cytoplasmic hapten binding by specific plasma cells and plasmablasts. Blast cells in S phase were identified in tissue sections by staining cells which had been pulse labeled in vivo with 5-bromo-2'-deoxyuridine. Hapten-specific B blast cells are found in three sites: (a) around interdigitating cells in the T cell-rich zones; (b) in the follicular dendritic cell network and (c) in association with macrophages in the red pulp. Hapten-binding memory B cells, which are not in cell cycle, accumulate in the marginal zones and to a lesser extent the follicular mantles in response to TD and TI-1 antigens. The hapten-specific blast response in T zones is confined to the first few days after antigen is given and is low for primary responses to TD antigens, but massive on secondary challenge, when marginal zone memory B cells migrate to the T zones. Both the primary and secondary T zone responses to TI-1 antigens are impressive and in these responses hapten-specific B blasts are also found in the splenic red pulp. The follicular response to TD antigens starts with a small number of B blasts (fewer than five) entering each follicle. These increase in number exponentially so that by the 4th day after immunization they fill the follicle. The oligoclonality of the response is shown in simultaneous responses to two haptens where 6%-31% of the follicles on day 3 after immunization contain blasts specific for only one of the two haptens. During the 4th day classical zonal pattern of germinal centers develops. The surface immunoglobulin-positive B blasts are lost from the follicle center, while one pole of the follicular dendritic cell network fills with surface immunoglobulin-negative centroblasts. Centroblasts do not increase in numbers but divide to give rise to centrocytes, which re-express sIg and migrate into the follicular dendritic cell network. Cell kinetic studies indicate that the centrocyte population is renewed from centroblasts every 7 h. Centrocytes either leave the germinal center within this time or die in situ.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of tyrosine phosphorylation in signal transduction through surface Ig in human B cells. Inhibition of tyrosine phosphorylation prevents intracellular calcium release.

Cross-linking surface Ig on human B cells, or the TCR complex on T cells leads to the rapid appearance of newly tyrosine phosphorylated proteins. This is associated with inositol phospholipid turnover and a rise in intracellular calcium. Incubation of human B or T lymphocytes with the tyrosine kinase inhibitors, herbimycin and genistein, inhibits new tyrosine phosphorylation after receptor-linked activation. This is associated with complete abrogation of the increase in intracellular calcium in these lymphocytes and inhibition of inositol phospholipid turnover. Herbimycin- and genistein-treated lymphocytes are nevertheless still capable of responding to aluminum fluoride with a rise in intracellular calcium. These data support the contention that a B cell-associated protein tyrosine kinase regulates signal transduction via phospholipase C. CD45, the membrane associated protein tyrosine phosphatase, and PMA that activates protein kinase C, both inhibit the calcium response in B lymphocytes induced by receptor cross-linking. PMA and cross-linking CD45 both induced the appearance of tyrosine phosphorylated proteins in human B cells, although the pattern is quite distinct from that seen when surface lg is cross-linked. However, the induction of new tyrosine phosphorylation by anti-mu does not appear to be affected by these reagents. Although this may reflect an insensitivity of the tyrosine phosphorylation assay, it could indicate that regulation of the calcium response and regulation of the tyrosine kinase can be independent processes.

Regulation of human B-cell activation and adhesion.

Human B lymphocyte differentiation is regulated by signals transmitted after binding of cytokines to their specific receptors and/or cross-linking of cell-cell adhesion receptors. In addition to surface immunoglobulin (sIg) receptors for antigen, a number of B cell-associated surface molecules have now been identified which may regulate activation and adhesion of B cells. These include members of the Ig supergene family such as CD19, CD22, B7/BB1, and BMC1, cell surface enzymes such as CD10, CD73, and CDw75, and proteins with multiple transmembrane domains such as CD20 and CD37. In this review we describe how several of these accessory molecules may affect signaling via antigen receptors and influence primary vs secondary immune responses. For instance, signaling via either CD21 or CD22 can augment responses to anti-Ig; the B cell activation marker B7/BB1 may function to trigger T cells via its ligand, CD28, to produce cytokines which in turn stimulate B cells; and the receptor, CD40, may transmit a signal to protect germinal center B cells from undergoing programmed cell death. Understanding how B cell accessory molecules regulate key interconnections during development may provide insights into the control and management of diseases with B-cell dysfunctions.

The role of class II molecules in human B cell activation. Association with phosphatidyl inositol turnover, protein tyrosine phosphorylation, and proliferation.

Cross-linking class II molecules on resting human B cells can initiate phosphatidyl inositol turnover and an increase in intracellular calcium concentration levels comparable with that seen with the cross-linking of surface Ig receptors. The calcium response is most evident on dense B cell fractions: buoyant cells are less responsive, even though the levels of class II expression are similar on dense and buoyant tonsillar B cells. Human B cell lines exhibit the same absence of correlation between intensity of the calcium signal and levels of surface class II expression, indicating that responsiveness is related to the state of differentiation of the cell rather than the amount of class II expressed. Cross-linking class II on normal B cells or B cell lines caused accumulation of inositol phosphates, suggesting class II induces calcium release from intracellular stores, rather than through direct regulation of calcium channels. The calcium response mediated through class II was completely abolished by bringing the protein tyrosine phosphatase, CD45, into close proximity with surface class II. This result indicated that protein tyrosine phosphorylation might regulate the signal transduced through this molecule. In support of this notion we found that tyrosine phosphorylation is induced when small dense tonsillar B cells are stimulated with either anti-Ig or with antibodies to class II. Finally, in B cell proliferation assays we show that cross-linking class II molecules on dense tonsillar B cells synergize strongly with suboptimal concentrations of PMA or IL-4. The significance of these results is discussed with regard to the cognate signal between B and T lymphocytes.

The evolution of B-cell clones.

This chapter identifies three forms of B-cell memory: (a) B blasts which characterize the established stage of the follicular response to TD antigens, (b) recirculating memory B cells, and (c) non-recirculating memory B cells of the marginal zones of the spleen and equivalent areas of other secondary lymphoid organs. The follicular B blasts show sustained proliferation driven by small amounts of antigen bound to FDCs. The probable relationships between these cells is summarized diagrammatically in Fig. 4. It is probable that follicular B blasts generate both the recirculating and marginal zone memory cells. The chapter by Gray and Leanderson in this volume cites data which indicate that the recirculating memory pool is not sustained for more than a few weeks in the absence of antigen. Data leading to the same conclusion for marginal zone memory B cells is set out in Sect. 5.1 of this chapter. Marginal zone memory B cells do not appear to move spontaneously to follicles for periodic renewal. They will only leave the marginal zone if a fresh supply of antigen reaches them in that site. Recirculating B cells are able to respond to antigen already held on FDCs. It is not known if they are able to displace B blasts of equivalent affinity for antigen which already occupy antigen-holding sites on FDCs. This could be a mechanism by which B blasts with high antigen affinity produced in one follicle could displace blasts of lower affinity in other follicles. Little is known of the factors which regulate the numbers of marginal zone and recirculating follicular memory B cells. In responses to hapten-protein conjugates, hapten-binding cells may approach 10% of marginal zone B cells but comprise well under 1% of recirculating follicular cells. The numbers of these memory cells do not increase if the recirculating pool of lymphocytes is depleted, indicating that the factors which regulate the number of memory B cells are independent of those which regulate the total size of the recirculating B-cell pool. A depleted peripheral B-cell pool can only be fully reconstituted by recruitment of newly produced virgin B cells. Data cited in Sect. 5.2 support the concept that this recruitment is at least partially independent of antigen-driven B-cell proliferation. Consequently, substantial proportions of the peripheral B-cell pools are likely to be either virgin cells or cells which have been recruited by antigen or anti-idiotype without entering cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Action of dexamethasone in an equine model of acute non-immune inflammation.

In a crossover study in seven New Forest ponies the actions of dexamethasone, at a dose rate of 0.06 mg kg-1 administered intravenously, were compared with those of a placebo treatment. Dexamethasone exerted expected effects on plasma and inflammatory exudate concentrations of cortisol and on blood glucose concentration and circulating leucocyte numbers, but it failed to affect exudate concentrations of the eicosanoids, prostaglandin E2, thromboxane B2, 6-keto-PGF1 alpha and leukotriene B4. These findings do not support the hypothesis that the anti-inflammatory actions of dexamethasone in the horse are mediated by inhibition of phospholipase A2.

CD23: a multi-functional receptor/lymphokine?

With the demonstration of identity between CD23 and the low affinity IgE Fc receptor (Fc epsilon RII), two previously separate avenues of immunological research have converged into one. Particularly in its guise as 'Blast-2' antigen, evidence has been mounting to implicate CD23 as an important molecule in B-cell growth regulation. It might seem pertinent, however, to question a role for an apparently isotype-specific immunoglobulin (Ig) receptor in general B-cell processes. In this article, John Gordon and colleagues attempt to reconcile the two, currently diverse, schools of thought regarding the primary function of CD23 and to provide a structural model that accounts for the biological pleiotropy observed.

B cell memory to thymus-independent antigens type 1 and type 2: the role of lipopolysaccharide in B memory induction.

Several studies have indicated that thymus-independent (TI) antigens, unlike their thymus-dependent (TD) counterparts, are poor at generating memory antibody responses (Immunol. Today 1981. 3:217). In contrast to this view, the present report shows that the TI type 1 (TI-1) antigen, 2,4,6-trinitrophenyl-lipopolysaccharide (TNP-LPS), elicits good secondary responses in rats. These secondary antibody responses are not only greater in magnitude than the primary responses, but display a different pattern of Ig classes with more IgG and IgA antibodies produced. In transfer experiments between congenic strains of rats which differ in their kappa light chain Ig allotype, it is shown that this memory is attributable to persistent B cell clones. The TI-2 antigen, 2,4-dinitrophenyl-hydroxyethyl starch (DNP-HES), given alone did not elicit B cell memory. However, when DNP-HES is presented to the immune system in association with LPS, the pattern of the anti-DNP response is similar to that elicited by TNP-LPS. The capacity to generate TI memory is associated with the appearance of hapten-specific B cells in the marginal zones of the spleen. Hapten-binding cells were induced in the marginal zones following immunization with TNP-LPS, but not by DNP-HES. However, concurrent immunization with DNP-HES and LPS which were not covalently linked was found to induce DNP-binding cells in the marginal zone. There is complete correlation between the appearance of hapten-binding memory B cells in the marginal zone and the capacity of these antigens to induce secondary responses.