Formation of IgE-Allergen-CD23 Complex Changes in Children Treated with Subcutaneous Immunotherapy for Japanese Cedar Pollinosis.

BACKGROUND: Subcutaneous immunotherapy (SCIT) is used to treat Japanese cedar (JC) pollinosis. The formation of IgE-allergen-CD23 complex after SCIT for JC pollinosis has not yet been fully elucidated. OBJECTIVE: The objective of this study was to investigate the formation of IgE-allergen-CD23 complex after SCIT for JC pollinosis. METHODS: Eleven patients were treated with 3-year SCIT for JC pollinosis at Sa-gamihara National Hospital from 2013 to 2014. Nasal and ocular symptoms (in terms of symptom scores) during the scattering of JC pollen and immunological changes were investigated. Levels of JC pollen-specific antibodies (IgE and IgG4) were measured by ImmunoCAP assays. To detect the changes in allergen-presenting ability of B cells, the levels of IgE-allergen-CD23 complexes in serum were measured by a cell-free, enzyme-linked immunosorbent-facilitated antigen-binding assay. RESULTS: The median (interquartile range) age of the subjects was 8 (6-10) years. Three patients (27%) had comorbid atopic dermatitis, and 5 patients (45%) had comorbid bronchial asthma. Before starting SCIT, the total IgE level was 373 (75-2,870) kU/L, and the level of JC pollen-specific IgE was 77.2 (15.4-528) kUA/L. Symptom scores improved significantly from the year after treatment. JC pollen-specific IgE levels did not change after 3 years of treatment. JC pollen-specific IgG4 levels increased significantly throughout the treatment period. The levels of IgE-allergen-CD23 complexes decreased significantly after 3 years of treatment. CONCLUSION: The ability of IgE-allergen complexes to bind to CD23 decreased after SCIT, suggesting that increasing levels of IgE-blocking antibodies, including IgG4, may play an important role in the mechanism of SCIT.

A Proliferation Inducing Ligand (APRIL) targeted antibody is a safe and effective treatment of murine IgA nephropathy.

IgA nephropathy (IgAN) is the most prevalent primary chronic glomerular disease for which no safe disease-specific therapies currently exist. IgAN is an autoimmune disease involving the production of autoantigenic, aberrantly O-glycosylated IgA1 and ensuing deposition of nephritogenic immune complexes in the kidney. A Proliferation Inducing Ligand (APRIL) has emerged as a key B-cell-modulating factor in this pathogenesis. Using a mouse anti-APRIL monoclonal antibody (4540), we confirm both the pathogenic role of APRIL in IgAN and the therapeutic efficacy of antibody-directed neutralization of APRIL in the grouped mouse ddY disease model. Treatment with 4540 directly translated to a reduction in relevant pathogenic mechanisms including suppressed serum IgA levels, reduced circulating immune complexes, significantly lower kidney deposits of IgA, IgG and C3, and suppression of proteinuria compared to mice receiving vehicle or isotype control antibodies. Furthermore, we translated these findings to the pharmacological characterization of VIS649, a highly potent, humanized IgG2κ antibody targeting and neutralizing human APRIL through unique epitope engagement, leading to inhibition of APRIL-mediated B-cell activities. VIS649 treatment of non-human primates showed dose-dependent reduction of serum IgA levels of up to 70%. A reduction of IgA+, IgM+, and IgG+ B cells was noted in the gut-associated mucosa of VIS649-treated animals. Population-based modeling predicted a favorable therapeutic dosing profile for subcutaneous administration of VIS649 in the clinical setting. Thus, our data highlight the potential therapeutic benefit of VIS649 for the treatment of IgAN.

Importance of Fc Receptor γ-Chain ITAM Tyrosines in Neutrophil Activation and in vivo Autoimmune Arthritis.

Activating Fcγ receptors associated with Fc receptor γ-chain (FcRγ) are critical for mediating neutrophil effector functions in immune complex-mediated autoimmune diseases. FcRγ contains ITAM tyrosines and the in vivo role of these tyrosines has not been defined in neutrophils and arthritis. In this study, the in vivo functions of FcRγ ITAM tyrosines were characterized using wild type and ITAM tyrosine mutant (Y65F/Y76F) transgenic mice crossed to an FcRγ-deficient genetic background. FcRγ-deficient neutrophils showed undetectable cell surface expression of the activating Fcγ receptor IV, defective immune complex-induced superoxide production, degranulation and spreading. Although the re-expression of both the wild type and the ITAM tyrosine mutant (Y65F/Y76F) FcRγ could restore activating Fcγ receptor expression of FcRγ-deficient neutrophils, only the wild type transgenic form could mediate Fcγ receptor-dependent effector functions. In contrast, neutrophils carrying ITAM tyrosine mutant FcRγ were unable to produce superoxide, mediate degranulation and perform active spreading. In addition, our results confirmed the protection of FcRγ-deficient mice from autoimmune arthritis. Importantly, the presence of the wild type FcRγ transgene, in contrast to the ITAM tyrosine mutant transgene, partially reversed autoimmune arthritis development. The reversing effect of the wild type transgene was even more robust when animals carried the wild type transgene in a homozygous form. Collectively, FcRγ ITAM tyrosines play a critical role in the induction of neutrophil effector responses, the initiation and progression of an autoantibody-induced experimental arthritis in vivo, indicating a signaling, rather than just a receptor stabilizing function of the molecule.

Targeting IgG in Arthritis: Disease Pathways and Therapeutic Avenues.

Rheumatoid arthritis (RA) is a polygenic and multifactorial syndrome. Many complex immunological and genetic interactions are involved in the final outcome of the clinical disease. Autoantibodies (rheumatoid factors, anti-citrullinated peptide/protein antibodies) are present in RA patients' sera for a long time before the onset of clinical disease. Prior to arthritis onset, in the autoantibody response, epitope spreading, avidity maturation, and changes towards a pro-inflammatory Fc glycosylation phenotype occurs. Genetic association of epitope specific autoantibody responses and the induction of inflammation dependent and independent changes in the cartilage by pathogenic autoantibodies emphasize the crucial contribution of antibody-initiated inflammation in RA development. Targeting IgG by glyco-engineering, bacterial enzymes to specifically cleave IgG/alter N-linked Fc-glycans at Asn 297 or blocking the downstream effector pathways offers new avenues to develop novel therapeutics for arthritis treatment.

Comparative assessment of immune complex-mediated hypersensitivity reactions with biotherapeutics in the non-human primate: Critical parameters, safety and lessons for future studies.

With the emergence of novel biotherapeutic formats and immunostimulatory biotherapeutics in cancer immunotherapy, an understanding of immune-complex (IC) mediated hypersensitivity reactions in toxicology studies - and their differentiation from pharmacology - remains key to the preclinical evaluation of these drugs. In this review we provide an in-depth evaluation and comparison of case examples where IC-mediated hypersensitivity reactions were observed in cynomolgus monkeys. We provide details of the parameters evaluated in each study to substantiate and guide the interpretation of these findings. Five study cases (1 therapeutic protein, 4 monoclonal antibodies) are discussed for which effects ranged from minor to fatal. Common characteristics are the high incidence of clinical signs, detectable antidrug antibodies, and accelerated drug clearance up to virtual loss of exposure. In our experience, measurement of cytokine levels in vivo and detection of complement (split products) were supportive markers in situations where coagulopathy was suspected to play a role in the observed effects. Recommendations are outlined to prepare for root-cause analysis of suspected hypersensitivity reactions. Overall, a thorough analysis of the findings has helped to start clinical trials despite major findings. The hypersensitivity reactions with our human(ized) immunoglobulins have not proven to be predictive for humans.

Altered glycan accessibility on native immunoglobulin G complexes in early rheumatoid arthritis and its changes during therapy.

The goal of this study was to investigate the glycosylation profile of native immunoglobulin (Ig)G present in serum immune complexes in patients with rheumatoid arthritis (RA). To accomplish this, lectin binding assays, detecting the accessibility of glycans present on IgG-containing immune complexes by biotinylated lectins, were employed. Lectins capturing fucosyl residues (AAL), fucosylated tri-mannose N-glycan core sites (LCA), terminal sialic acid residues (SNA) and O-glycosidically linked galactose/N-acetylgalactosamine (GalNac-L) were used. Patients with recent-onset RA at baseline and after 3-year follow-up were investigated. We found that native IgG was complexed significantly more often with IgM, C1q, C3c and C-reactive protein (CRP) in RA patients, suggesting alterations of the native structure of IgG. The total accessibility of fucose residues on captured immune complexes to the respective lectin was significantly higher in patients with RA. Moreover, fucose accessibility on IgG-containing immune complexes correlated positively with the levels of antibodies to cyclic citrullinated peptides (anti-CCP). We also observed a significantly higher accessibility to sialic acid residues and galactose/GalNAc glyco-epitopes in native complexed IgG of patients with RA at baseline. While sialic acid accessibility increased during treatment, the accessibility of galactose/GalNAc decreased. Hence, successful treatment of RA was associated with an increase in the SNA/GalNAc-L ratio. Interestingly, the SNA/GalNAc-L ratio in particular rises after glucocorticoid treatment. In summary, this study shows the exposure of glycans in native complexed IgG of patients with early RA, revealing particular glycosylation patterns and its changes following pharmaceutical treatment.

SEC Based Method for Size Determination of Immune Complexes of Therapeutic Antibodies in Animal Matrix.

Therapeutic monoclonal antibodies (mAbs) represent a milestone in pharmacological development. Their superiority is based on the combination of high specificity, low toxicity, and long half-life that characterizes biologics. If biologics have Achilles' heel, it is their potential immunogenicity. To better understand the impact of the size of immune complexes of mAbs on anti-drug antibody (ADA) dependent adverse reactions in Macaca fascicularis, we developed an efficient high-throughput size exclusion chromatography- (SEC-) based methodology that enables analysis of the size, size distribution, and ratio of free and ADA-complexed mAb in serum allowing for assessment of formation and clearance of circulating ADA-mAb immune complexes (CIC).

Novel vaccination approach for dengue infection based on recombinant immune complex universal platform.

Dengue infection is on the rise in many endemic areas of the tropics. Vaccination remains the most realistic strategy for prevention of this potentially fatal viral disease but there is currently no effective vaccine that could protect against all four known serotypes of the dengue virus. This study describes the generation and testing of a novel vaccination approach against dengue based on recombinant immune complexes (RIC). We modelled the dengue RIC on the existing Ebola RIC (Phoolcharoen, et al. Proc Natl Acad Sci USA 2011;108(Dec (51)):20695) but with a key modification that allowed formation of a universal RIC platform that can be easily adapted for use for other pathogens. This was achieved by retaining only the binding epitope of the 6D8 ant-Ebola mAb, which was then fused to the consensus dengue E3 domain (cEDIII), resulting in a hybrid dengue-Ebola RIC (DERIC). We expressed human and mouse versions of these molecules in tobacco plants using a geminivirus-based expression system. Following purification from the plant extracts by protein G affinity chromatography, DERIC bound to C1q component of complement, thus confirming functionality. Importantly, following immunization of mice, DERIC induced a potent, virus-neutralizing anti-cEDIII humoral immune response without exogenous adjuvants. We conclude that these self-adjuvanting immunogens have the potential to be developed as a novel vaccine candidate for dengue infection, and provide the basis for a universal RIC platform for use with other antigens.

Unconventional apoptosis of polymorphonuclear neutrophils (PMN): staurosporine delays exposure of phosphatidylserine and prevents phagocytosis by MΦ-2 macrophages of PMN.

Apoptosis of polymorphonuclear neutrophils (PMN) and subsequent 'silent' removal represents an important check-point for the resolution of inflammation. Failure in PMN clearance resulting in secondary necrosis-driven tissue damage has been implicated in conditions of chronic inflammation and autoimmunity. Apoptotic PMN undergo profound biophysical changes that warrant their efficient recognition and uptake by phagocytes before fading to secondary necrosis. In this study, we demonstrate that staurosporine (STS), a non-selective but potent inhibitor of cyclin-dependent kinase and protein kinase C, exerts a drastic impact on PMN apoptosis. PMN treated with STS underwent an unconventional form of cell death characterized by a delayed exposure of aminophospholipids, including phosphatidylserine (PS) and phosphatidylethanolamine and an increased exposure of neo-glycans. STS caused an impaired cellular fragmentation and accelerated DNA fragmentation. Phagocytosis of STS-treated PMN lacking PS on their surfaces was decreased significantly, which highlights the importance of PS for the clearance of apoptotic PMN. Specific opsonization with immune complexes completely restored phagocytosis of STS-treated PMN, demonstrating the efficiency of back-up clearance pathways in the absence of PS exposure.

Treating IgA nephropathy: quid novi?

IgA nephropathy is a common autoimmune renal disease resulting in kidney failure for patients with significant proteinuria. The therapeutic options are limited including non-specific treatment to reduce proteinuria accomplished by renin-angiotensin blockade. Strategies to control intrarenal inflammation include the administration of fish oil and for severe disease the use of immunosuppressive agents such as cyclophosphamide, glucocorticosteroids, and mycophenolate mofetil. In light of the limited option, there is an unmet need for novel therapeutic intervention in patients with progressive disease. Herein, we review the evidence for existing treatment choices and explore new immunopharmacologic agents being investigated for IgA nephropathy.

IgG antibodies in food allergy influence allergen-antibody complex formation and binding to B cells: a role for complement receptors.

Allergen-IgE complexes are more efficiently internalized and presented by B cells than allergens alone. It has been suggested that IgG Abs induced by immunotherapy inhibit these processes. Food-allergic patients have high allergen-specific IgG levels. However, the role of these Abs in complex formation and binding to B cells is unknown. To investigate this, we incubated sera of peanut- or cow's milk-allergic patients with their major allergens to form complexes and added them to EBV-transformed or peripheral blood B cells (PBBCs). Samples of birch pollen-allergic patients were used as control. Complex binding to B cells in presence or absence of blocking Abs to CD23, CD32, complement receptor 1 (CR1, CD35), and/or CR2 (CD21) was determined by flow cytometry. Furthermore, intact and IgG-depleted sera were compared. These experiments showed that allergen-Ab complexes formed in birch pollen, as well as food allergy, contained IgE, IgG1, and IgG4 Abs and bound to B cells. Binding of these complexes to EBV-transformed B cells was completely mediated by CD23, whereas binding to PBBCs was dependent on both CD23 and CR2. This reflected differential receptor expression. Upon IgG depletion, allergen-Ab complexes bound to PBBCs exclusively via CD23. These data indicated that IgG Abs are involved in complex formation. The presence of IgG in allergen-IgE complexes results in binding to B cells via CR2 in addition to CD23. The binding to both CR2 and CD23 may affect Ag processing and presentation, and (may) thereby influence the allergic response.

Immune markers in breast milk and fetal and maternal body fluids: a systematic review of perinatal concentrations.

Breastfeeding represents the continued exposure of the infant to the maternal immune environment.Uterine, perinatal, and postnatal exposure to immune factors may contribute to an infant's risk of developing immune-mediated disorders, including allergies. A PubMed search was conducted to review studies in humans and analyze concentrations of immune markers (TGF-beta, IFN-gamma, eotaxin, CCL5, CXCL10, TNF-alpha, MCP-1, IL-1beta, IL-4, IL-5, IL-6,IL-8, IL-10, IL-12, IL-13, sCD14, sIgA, IgG4, IgM) found in maternal serum, amniotic fluid, cord serum, colostrum, transition and mature milk. Concentrations of immune markers showed large variations across samples and studies. Reports documented conflicting results. Small sample sizes, differences in population characteristics, inconsistent sample collection times, and various sample collection and measurement methods may have led to wide variations in the concentrations of immune markers. Studies analyzing the associations between immune markers in maternal fluids and infant allergies remain inconclusive because of gaps in knowledge and a lack of standardized methods.

Therapeutic control of B cell activation via recruitment of Fcgamma receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold.

OBJECTIVE: To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications. METHODS: DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. RESULTS: DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice. CONCLUSION: This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.

A sensitive assay for the detection of IgE bound to the major birch pollen allergen, Bet v 1, in the form of immune complexes.

Allergen-IgE immune complexes, in which the IgE paratopes are occupied by allergen, remain undetected in standard IgE-detection assays which are based on capturing specific IgE by allergens.We describe an assay for the detection of immune complexes consisting of IgE bound to one of the most frequent environmental allergens, the major birch pollen allergen, Bet v 1. This assay is based on a Bet v 1-specific monoclonal antibody, Bip 1, which binds to an epitope on Bet v 1 that is distinct from the epitopes recognized by allergic patients' IgE. IgE-immune complexes formed with sera from birch pollen-allergic patients (n = 46) were undetectable with solid phase-bound allergen but could be captured by Bip 1 immobilized to nitrocellulose membranes or ELISA plates and traced with radioactively or enzymatically labelled anti-human IgE antibodies. The levels of IgE complexed with Bet v 1 measured in our assays were highly correlated with the amounts of non-complexed allergen-specific IgE as determined by the ImmunoCAP assay which is based on solid phase-bound IgE (r = 0.95; p < 0.01). Bet v 1-specific IgE could even be detected at serum dilutions below the cut off of the ImmunoCAP system (i.e., 0.35 kUA/L). We have thus developed a robust and sensitive assay for the detection and quantification of Bet v 1-IgE immune complexes which should be useful to measure allergen-bound IgE in human body fluids and in in vitro experiments.

Tyrosine kinases and inflammatory signalling.

The activity of tyrosine kinases is central to many cellular processes, and accumulating evidence suggests that their role in inflammation is no less profound. Three main tyrosine kinase families, the Src, Tec and Syk kinase families are intimately involved in TLR signalling, the critical first step in cellular recognition of invading pathogens and tissue damage. Their activity results in changes in gene expression in affected cells. Key amongst these genes are the cytokines, which orchestrate both the duration and extent of inflammation. Tyrosine kinases also play important roles in cytokine function, and are implicated in signalling through both pro- and anti-inflammatory cytokines such as TNF, IL-6 and IL-10. Thus, strategies to modulate tyrosine kinase activity have significant therapeutic potential in combating the chronic inflammatory state that is typical of many major health issues that face us today, including Rheumatoid Arthritis, Cardiovascular disease and cancer. Here we review current knowledge of the role of tyrosine kinases in inflammation with particular emphasis on their role in TLR signalling.

Substance abuse vaccines.

Conventional substance-abuse treatments have only had limited success for drugs such as cocaine, nicotine, methamphetamine, and phencyclidine. New approaches, including vaccination to block the effects of these drugs on the brain, are in advanced stages of development. Although several potential mechanisms for the effects of antidrug vaccines have been suggested, the most straightforward and intuitive mechanism involves binding of the drug by antibodies in the bloodstream, thereby blocking entry and/or reducing the rate of entry of the drug into the central nervous system. The benefits of such antibodies on drug pharmacodynamics will be influenced by both the quantitative and the qualitative properties of the antibodies. The sum of these effects will determine the success of the clinical applications of antidrug vaccines in addiction medicine. This review will discuss these issues and present the current status of vaccine development for nicotine, cocaine, methamphetamine, phencyclidine, and morphine.

Treatment of lupus-prone NZB/NZW F1 mice with recombinant soluble Fc gamma receptor II (CD32).

OBJECTIVES: Systemic lupus erythematosus (SLE) is a classical autoimmune disorder characterised by the production of IgG autoantibodies against double-stranded DNA (dsDNA). Activation of Fc gamma R-bearing effector cells by immune complexes (ICs) is a key event in SLE pathogenesis as lupus-prone NZB/NZW F(1) hybrids lacking activating Fc gamma receptors (Fc gamma R) are protected against inflammatory kidney damage despite glomerular deposition of ICs. Moreover, soluble Fc gamma Rs inhibit IC-caused Arthus reaction in vivo. Therefore, recombinant human soluble Fc gamma RII (CD32) was evaluated as a novel therapeutic strategy in lupus-like disease in NZB/NZW F(1) hybrids. METHODS: Binding of husCD32 to murine IgG was studied in vitro by binding to IgG-coated erythrocytes and inhibition of phagocytosis of IgG-opsonised murine erythrocytes. In order to examine therapeutic impact of husCD32 in vivo, female NZB/NZW F(1) mice were treated either from week 16 to 20 ("prophylactic", 150 microg/week husCD32) or continuously from week 24 ("therapeutic"; 100 microg/week husCD32) by subcutaneous injections. Controls received buffered saline. RESULTS: In vitro investigations of husCD32 revealed binding to murine erythrocytes coated with murine IgG. Moreover, husCD32 substantially diminished phagocytosis of murine IgG-opsonised murine red blood cells by peritoneal macrophages indicating disruption of IgG-Fc gamma R interaction. There was a therapeutic efficacy of husCD32 to attenuate lupus pathology indicated by significantly delayed onset of proteinuria and weight loss, reduced histopathological findings, delayed development of anaemia and improved survival by prophylactic application. Therapeutic treatment did not reverse nephritis but significantly prolonged survival despite apparent kidney damage. B cell count, concentration of IgG anti-dsDNA autoantibodies and deposition of glomerular ICs was not significantly affected by the application of husCD32. CONCLUSIONS: The results demonstrate binding properties of husCD32 to ICs in vitro and as a proof-of-principle therapeutic efficacy in inhibiting chronic murine lupus pathology in vivo.

Inhibitory effect of fulvic acid extracted from Canadian sphagnum peat on chemical mediator release by RBL-2H3 and KU812 cells.

Fulvic acid (FA) was extracted and purified from Canadian Sphagnum peat (CP-FA) and characterized by using an element analysis meter, Fourier transform infrared (FT-IR) spectroscopy, electron spin resonance (ESR) spectroscopy, and (13)C-nuclear magnetic resonance ((13)C-NMR) spectroscopy. To investigate the antiallergic effect of CP-FA, we incubated rat basophilic leukemia (RBL-2H3) cells with 0.001-10.0 microg/ml of CP-FA and determined the beta-hexosaminidase release inhibition at different response stages. The intracellular calcium [Ca(2+)](i) level was also determined by using Fluo 3-AM, a calcium-specific fluorescent probe, and the cytotoxicity of CP-FA was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The results revealed that RBL-2H3 cells incubated for 48 h with 0.001-10.0 microg/ml of CP-FA did not show any decreased viability. CP-FA inhibited the beta-hexosaminidase release by IgE-sensitized, antigen-stimulated RBL-2H3 cells at the antigen-antibody binding stage and the antibody-receptor binding stage. CP-FA also inhibited histamine release from A23187 plus PMA- or compound 48/80-stimulated KU812 cells. Furthermore, there was a decrease in the intracellular [Ca(2+)](i) level in IgE-sensitized cells incubated with CP-FA and stimulated with antigen. Our results show that CP-FA may be useful for the treatment or prevention of allergic diseases.

Sequential chemotactic and phagocytic activation of human polymorphonuclear neutrophils.

Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Delta[Ca(2+)](i) and DeltapH(i)) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca(2+)](i), mostly from intracellular stores, simultaneously with a drop in pH(i); these are followed by a drop in [Ca(2+)](i) and a rise in pH(i), with the latter being due to a Na(+)/H(+) antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca(2+)](i) is restored, 10(-7) M fMLP, previously shown to elicit maximal Delta[Ca(2+)](i) but no bactericidal functions, did not prevent the cells' responses with Delta[Ca(2+)](i) to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 mug/ml IC, previously shown to elicit maximal Delta[Ca(2+)](i) and bactericidal functions, exhibited no subsequent Delta[Ca(2+)](i) or DeltapH(i) to either stimulus. While exposure to 10(-7) M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10(-5) M fMLP did, presumably via the low-affinity receptor, using a different Ca(2+) source.

A structurally based approach to determine HLA compatibility at the humoral immune level.

HLAMatchmaker is a structurally based matching program. Each HLA antigen is viewed as a string of epitopes represented by short sequences (triplets) involving polymorphic amino acid residues in antibody-accessible positions. HLAMatchmaker determines which triplets are different between donor and recipient, and this algorithm is clinically useful in determining HLA mismatch acceptability. Triplets provide however an incomplete description of the HLA epitope repertoire and expanded criteria must be used including longer sequences and polymorphic residues in discontinuous positions. Such criteria should consider the structural basis of antibody-antigen interactions including contact areas and binding energy, the essence of antigenicity. This report describes the development of a structurally defined HLA epitope repertoire based on stereochemical modeling of crystallized complexes of antibodies and different protein antigens. This analysis considered also data in the literature about contributions of amino acid residues to antigen-antibody binding energy. The results have led to the concept that HLA antigens like other antigenic proteins have structural epitopes consisting of 15-22 residues that constitute the binding face with alloantibody. Each structural epitope has a functional epitope of about 2-5 residues that dominate the strength and specificity of binding with antibody. The remaining residues of a structural epitope provide supplementary interactions that increase the stability of the antigen-antibody complex. Each functional epitope has one or more non-self residues and the term "eplet" is used to describe polymorphic HLA residues within 3.0-3.5 A of a given sequence position on the molecular surface. Many eplets represent short linear sequences identical to those referred to as triplets but others have residues in discontinuous sequence positions that cluster together on the molecular surface. Serologically defined HLA determinants correspond well to eplets. The eplet version of HLAMatchmaker represents therefore a more complete repertoire of structurally defined HLA epitopes and provides a more detailed assessment of HLA compatibility.