Fc Interactions Research Articles

Cellular Uptake of rFVIIIFc in Liver Is Primarily Contributed by Sinusoidal Endothelial Cells.

AbstractAbstract 2219rFVIIIFc is a recombinant fusion protein consisting of human B-domain deleted factor VIII covalently linked to the Fc domain of IgG1. In hemophilia A patients, rFVIIIFc has been shown to display a ∼1.6-fold longer half-life than recombinant full length FVIII (Advate®) (Powell et al., 2012. Blood). This half-life extension can be attributed to a natural pathway mediated by the neonatal Fc receptor (FcRn) that re-circulates IgG molecules into the vascular system, as the long-lasting activity of rFVIIIFc is not observed in FcRn knockout mice.To identify the cell type that takes up and subsequently protects and recycles rFVIIIFc, we have recombinantly replaced the missing B-domain with a Halo tag in rFVIIIFc (rFVIIIFc-Halo) to allow visualization of the protein in the presence of fluorescently labeled Halo-ligand using confocal microscopy. Purified rFVIIIFc-Halo protein displayed similar specific activity and pharmacokinetic properties as rFVIIIFc in hemophilia A (HemA) mice, indicating that the addition of the Halo tag does not alter the functionality and the clearance mechanisms of rFVIIIFc. In quantitative whole body autoradiography studies (QWBA) in HemA mice with radiolabeled rFVIIIFc, we observed that 125I-rFVIIIFc is predominately distributed to the liver. Therefore, we selected primary liver cells isolated from HemA mice to study cellular uptake of rFVIIIFc.A co-culture of hepatocytes and non-parenchymal cells was isolated from HemA mice and prepared at a 1:1 ratio. Liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) in this culture were identified by fluorescently labeled antibodies to CD31 and F4/80 respectively. Both cell types effectively took up the fluorescently labeled AcLDL, confirming that the isolated LSECs and KCs retained the capacity for functional endocytosis in vitro. It was found that LSECs, as opposed to Kupffer cells or hepatocytes, are predominantly responsible for the cellular uptake of rFVIIIFc, as the localization of rFVIIIFc-Halo is apparent only in LSECs within 5 minutes after exposing 10 nM of rFVIIIFc-Halo to primary co-culture freshly isolated from HemA mice. In contrast, even with longer exposure time (up to 1 hour) and higher protein concentration (up to 40 nM), the localization of rFVIIIFc-Halo in Kupffer cells and hepatocytes still remains undetectable.Analysis of recombinant Halo-tagged factor VIII (rFVIII-Halo) yielded similar results, suggesting that the Fc-fusion does not alter the cellular uptake pathway of FVIII, which is consistent with the notion that the interaction of Fc with FcRn occurs at the intracellular level. Therefore, interestingly, both rFVIII-Halo and rFVIIIFc-Halo are internalized by LSEC that are the same cells reported to express FVIII by in situ hybridization studies (Hollestelle et al. 2001 Thromb Haemost). This study, together with recent findings that somatic cells in the liver are primarily responsible for rFVIIIFc recycling (Abstract by van der Flier et al), highlights the critical role of LSECs in the clearance of rFVIIIFc and suggests that rFVIIIFc is primarily recycled by FcRn in LSECs. The impact of VWF on the cellular uptake and recycling of the rFVIIIFc-VWF complex in liver cells may also be assessed utilizing this system. Disclosures:Tan:BiogenIdec: Employment. Chen:BiogenIdec: Employment. van der Flier:BiogenIdec: Employment. Liu:BiogenIdec: Employment. Light:biogenidec: Employment. Jiang:biogenidec: Employment.

Anti‐HIV‐1 antibody‐dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG

Antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV-1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV-specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV-1 envelope gp140 and elicited high titers of anti-gp140-binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ-receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose-dependent manner. Antibody-dependent killing was observed in the presence of anti-HIV-1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab')(2) fragments. ADCC activity was primarily mediated by CD14(+) monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte-mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti-HIV colostrum IgG have robust HIV-1-specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV-1 infection. This could assist the development of novel Ab-mediated approaches for prevention of HIV-1 transmission.

Fc–Fc interactions of human IgG4 require dissociation of heavy chains and are formed predominantly by the intra-chain hinge isomer

Human IgG4 antibodies are remarkable not only because they can dynamically exchange half-molecules (Fab-arm exchange) but also for their ability to interact with the Fc part of IgG4 and other IgG subclasses. This rheumatoid factor-like binding of IgG4 does not appear to take place spontaneously, because it is only observed to solid-phase or antigen-bound IgG. We hypothesized that Fc–Fc interactions might involve (partial) dissociation of heavy chains. We investigated the molecular basis of these Fc–Fc interactions, and found that the structural features important for the exchange reaction also control the Fc binding activity. In particular, if arginine-409 in the CH3–CH3 interface in IgG4 is mutated to lysine (the equivalent in IgG1), Fc–Fc interactions are formed 3 orders of magnitude less efficiently compared to the wild-type. This mutation was previously found to increase the CH3–CH3 interaction strength in IgG4. Furthermore, of the two hinge isomers of IgG4, the intra-chain (non-covalently linked) form was found to form Fc–Fc interactions, but not the inter-chain form. Together, these results demonstrate that Fc–Fc interactions of IgG4 involve (partial or complete) dissociation of heavy chains. The promiscuity to other IgG subclasses suggests that IgG4 might act as scavenger to IgG molecules with impaired structural integrity.

Coarse-Grained Modeling of the Self-Association of Therapeutic Monoclonal Antibodies

Coarse-grained computational models of two therapeutic monoclonal antibodies are constructed to understand the effect of domain-level charge-charge electrostatics on the self-association phenomena at high protein concentrations. The coarse-grained representations of the individual antibodies are constructed using an elastic network normal-mode analysis. Two different models are constructed for each antibody for a compact Y-shaped and an extended Y-shaped configuration. The resulting simulations of these coarse-grained antibodies that interact through screened electrostatics are done at six different concentrations. It is observed that a particular monoclonal antibody (hereafter referred to as MAb1) forms three-dimensional heterogeneous structures with dense regions or clusters compared to a different monoclonal antibody (hereafter referred to as MAb2) that forms more homogeneous structures (no clusters). These structures, together with the potential mean force (PMF) and radial distribution functions (RDF) between pairs of coarse-grained regions on the MAbs, are qualitatively consistent with the experimental observation that MAb1 has a significantly higher viscosity compared to MAb2, especially at concentrations >50 mg/mL, even though the only difference between the MAbs lies with a few amino acids at the antigen-binding loops (CDRs). It is also observed that the structures in MAb1 are formed due to stronger Fab-Fab interactions in corroboration with experimental observations. Evidence is also shown that Fab-Fc interactions can be equally important in addition to Fab-Fab interactions. The coarse-grained representations are effective in picking up differences based on local charge distributions of domains and make predictions on the self-association characteristics of these protein solutions. This is the first computational study of its kind to show that there are differences in structures formed by two different monoclonal antibodies at high concentrations.

Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs

AbstractDespite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation.

Atomic resolution model of the antibody Fc interaction with the complement C1q component

The globular C1q heterotrimer is a subunit of the C1 complement factor. Binding of the C1q subunit to the constant (Fc) part of antibody molecules is a first step and key event of complement activation. Although three-dimensional structures of C1q and antibody Fc subunits have been determined experimentally no atomic resolution structure of the C1q–Fc complex is known so far. Based on systematic protein–protein docking searches and Molecular Dynamics simulations a structural model of the C1q–IgG1–Fc-binding geometry has been obtained. The structural model is compatible with available experimental data on the interaction between the two partner proteins. It predicts a binding geometry that involves mainly the B-subunit of the C1q-trimer and both subunits of the IgG1–Fc-dimer with small conformational adjustments with respect to the unbound partners to achieve high surface complementarity. In addition to several charge–charge and polar contacts in the rim region of the interface it also involves nonpolar contacts between the two proteins and is compatible with the carbohydrate moiety of the Fc subunit. The model for the complex structure provides a working model for rationalizing available biochemical data on this important interaction and can form the basis for the design of Fc variants with a greater capacity to activate the complement system for example on binding to cancer cells or other target structures.

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Human immunoglobulin G4 (IgG4) is a poor trigger of effector functions and, therefore, is the preferred subclass for therapeutic monoclonal antibodies that merely aim to block their in vivo targets. An example is natalizumab, a recombinant IgG4 antibody directed against α4-integrin and used for treatment of multiple sclerosis. Efficient treatment requires that the pharmacokinetics of therapeutic monoclonal antibodies can be accurately monitored. For natalizumab, this requires special precautions due to recently reported structural peculiarities of human IgG4. Here we describe the development of an assay to determine serum levels of natalizumab. Compared with other IgG subclasses, human IgG4 possesses unique structural properties that influence its interactions in both in vivo and in vitro settings. Thus, IgG4 undergoes Fab arm exchange in vivo, resulting in effectively monovalent antibodies. Furthermore, IgG4 is able to bind to other human and nonhuman IgG via Fc interactions. We demonstrate how these features can interfere with measurement of specific IgG4 and describe how we addressed these issues, resulting in an assay that is not sensitive to Fab arm exchange by natalizumab or to IgG4 Fc interactions.

Irreversible Redox Reactions of Ferrocene/Ferrocenium on Self-Assembled Monolayer Modified Gold Electrode in Ionic Liquid BMIMBF4

Self-assembled monolayers of dodecanethiol on gold electrodes were characterized in an ionic liquid 1-butyl-4-methylimidazolium tetrafluoroborate containing redox probes ferrocene (Fc)/ferrocenium or others. An irreversible redox reaction of has been observed. The results may indicate a special double layer structure of an electrode/electrolyte interface and an interaction of imidazolium and Fc. Coverage of the monolayer has been determined by stripping voltammetry in .

Stromal Rescue of Drug Exposed CLL Cells Can Be Overcome by the Green Tea Extract Epigallocatechin 3 Gallate

AbstractAbstract 3610 PurposeChronic Lymphocytic Leukemia (CLL) is incurable with conventional chemotherapy treatments. Nurturing of leukemic CLL B cells by marrow microenvironment appears to be one important method of chemoresistance (NEJM 2005).Epigallocatechin 3 gallate (EGCG), the major catechin in green tea, induces caspase-dependent death CLL B-cells and down regulates anti-apoptotic proteins (Mcl-1; XIAP) critical for enhanced apoptosis resistance of CLL B-cells (Blood 2004). In Phase I and II clinical testing, we found EGCG induced a decline in absolute lymphocyte count and/or lymphadenopathy in the majority of Rai stage 0-II CLL patients (JCO 2009; ASCO 2010).In these phase I/II studies, trough plasma EGCG levels as high as 8 mM were achieved. Since it does not cause myelosuppression, EGCG has the potential to be combined with other chemotherapeutic agents used for CLL. We previously reported that pharmacologically achievable plasma levels of EGCG have an additive or synergistic effect when combined with fludarabine (F), chlorambucil (C), and FC in the majority of patients (ASH 2009). In this study, we evaluated i) the effect of pharmacologically achievable doses of EGCG when combined with bendamustine (B) and ii) the ability of pharmacologically achievable doses of EGCG to overcome in vitro stromal rescue of CLL B-cells exposed to B or FC. MethodsWe first evaluated the effects of EGCG combined with B to establish if this partnership would be additive or synergistic. Primary CLL cells were treated with B (12.5-100 mM) with or without 4 uM EGCG. After 48 hours cells were stained with annexin/PI, and viability analyzed by flow cytometry. Data were analyzed using the CalcuSyn program (Chou & Talay method) to determine the combination index (indicating antagonistic, additive or synergistic effects). Next, we evaluated the ability of 4 mM EGCG to overcome stromal rescue of CLL cells treated with B or FC. To eliminate the cytotoxic effects of FC and B on stromal cells, we treated CLL cells with FC or B for 24 hours, washed with PBS, and then cultured these drug exposed cells directly on the HS-5 human stromal cell line with or without 4 mM EGCG for 48 hours (FC) or 24 hours (B). Cells were harvested and viability of CD19+ cells was assessed by annexin/PI staining. To specifically evaluate the ability of 4 mM EGCG to overcome stromal protection, co-culture samples in which no stromal protection was observed were excluded. Similar experiments were conducted using primary bone marrow mesenchymal stromal cells (MSC) from CLL patients (Leuk Res, 2007). In these experiments the fludarabine/chlorambucil combination was used as an approximation of fludarabine/cyclophosphamide since the active form of cyclophosphamide (4-hydroxycyclophosphamide) rapidly breaks down in aqueous buffer and is not readily available for in vitro use and. ResultsWe previously observed additive or synergistic FC and EGCG interactions in a majority of patient samples (ASH 2009). 4 mM EGCG was less consistently beneficial when combined with increasing doses of B in the absence of stroma. Synergistic or mixed synergistic/additive effect was seen in 5 patients, mixed additive/antagonistic effects in 3, and antagonistic effects in 2. Given previously observed synergy with FC and additive/synergistic interactions with at least some B treated samples, we next explored the effects of EGCG on stromal cell rescue. Co-culture with HS-5 stromal cells significantly reduced both B and FC mediated cell death. However the addition of 4 μM EGCG significantly attenuated stromal cell rescue of CLL B-cells exposed to B and FC therapy (Fig. 1A and 1B respectively). Remarkably in the case of FC, 4 mM EGCG almost completely abrogated stromal rescue. Experiments using primary bone marrow MSC cultures from CLL patients yields similar results (n=3, data not shown). [Display omitted] [Display omitted] ConclusionsPharmacologically achievable doses of EGCG appear to reduce stromal rescue of CLL cells treated with B or combinations of purine nucleoside analog/alkylating agents. This occurs in the absence of detectable stromal cell kill by EGCG. These findings, coupled with the favorable toxicity profile and lack of myelosuppression in phase I/II trials of EGCG make it an attractive agent to test in combination with bendamustine or purine analogue/alkylating agent chemo-immunotherapy for patients with CLL. Disclosures:Shanafelt:Polyphenon Pharma: Research Funding.

Single and Binary Self-Assembled Monolayers of Phenyl- and Pentafluorophenyl-Based Silane Species, and Their Phase Separation with Octadecyltrichlorosilane

In this paper, we first present the study of the formation of phenyltrichlorosilane film and self-assembled monolayers of phenylalkyltrichlorosilane (PATCl), pentafluoro-phenylalkyltrichlorosilane (PFATCl), and a mixture of the two, on silicon covered by its native oxide. These monolayers are shown to grow in two steps with characteristic time constants. The first step is characterized by a similar time constant of growth for all the studied trichlorosilane molecules and attributed to chemisorption. The second step corresponds to the arrangement between molecules, accelerated by the presence of the short alkyl chain (3-4 carbon atoms), and by mixing phenyl and pentafluoro-phenyl terminal moieties, which is accounted for by hydrogen bonding CH···FC and/or attractive quadrupolar interactions within a face-to-face phenyl/pentafluoro-phenyl alternating stack arrangement. Such results should allow improvement of intermolecular stacking within conjugated molecular domains, which is particularly important for molecular electronic devices. In the second part, we studied how PATCl, PFATCl, and their mixture phase separate with octadecyltrichlorosilane (OTS) molecules in various ratios. The way to improve phase separation was studied modifying aromatic ring to ring as well as aromatic-aliphatic interactions. OTS island size and coverage are shown to be smaller with the aromatic phase that involves stronger ring to ring interactions, i.e., attractive interactions between the phenyl species by mixing phenyl and pentafluoro-phenyl rings. The best phase separation is obtained with PFATCl as the aromatic molecule. If nanoislands of aromatic molecules could not be observed in these experiments, we show that they are attainable by mixing OTS and aromatic small organotriethoxysilanes whose grafting kinetics is slower. These results pave the way to the control improvement of the composition and nanostructuration of SAMs, essential for their further use within molecular devices.

Traces of pFc’ in IVIG interact with human IgG Fc domains and counteract aggregation

To prevent multimer formation, intravenous immunoglobulin (IVIG) is often treated with traces of pepsin. So far, the mechanism behind this treatment has been unclear. Recently, we reported that human IgG4 binds other IgG molecules via Fc–Fc interactions. Here we show that IVIG treated with traces of pepsin (Nanogam) inhibits these interactions. We found that – besides IgG4 – peptides corresponding to IgG1 and IgG2 pFc’ (products of limited pepsin digestion) are responsible for the inhibitory action. Using radiolabeled pFc’, it was found that pFc’ binds directly to IgG1. Furthermore, recombinant CH3 fragments were found to also possess binding activity, and potencies of inhibition varied over 3 orders of magnitude amongst the subclasses, IgG4 being most potent. We propose that pFc’ formation explains how limited pepsin digestion diminishes adverse effects of IVIG. In particular, the presence of this fragment can enhance the stability of IgG products including IVIG and therapeutical monoclonal antibodies. Indeed, using a model system it was found that acid-induced aggregation of IgG is reduced in the presence of pFc’, suggesting a ‘chaperone-like’ activity of this fragment. Thus, pFc’ can modulate Fc interactions and may therefore reduce adverse effects of IVIG, in particular by preventing oligomerization.

Human IgG4 Binds to IgG4 and Conformationally Altered IgG1 via Fc-Fc Interactions

The Fc fragment of IgG4 can interact with the Fc fragment of another IgG molecule. This interaction is a confounding factor when measuring IgG4 rheumatoid factor levels. Recently, we demonstrated that half-molecules of IgG4 can exchange to form a bispecific Ab. We expected these two phenomena to be related and investigated the physicochemical aspects of IgG4 Fc-Fc interactions. We found that IgG4 is >99% monomeric by size-exclusion chromatography; therefore, IgG4 Fc-Fc interactions in the fluid phase (if any) would be short-lived. However, (125)I-labeled IgG4 does bind to IgG1 and IgG4 coupled to a solid phase. By contrast, IgG1 does not bind to coupled IgG4. Furthermore, conditions that induce partial unfolding/dissociation of the CH3 domains enhance IgG4 Fc binding, suggesting that Fc binding is primarily CH3 mediated. IgG4 slowly associates with both IgG4 and IgG1 coupled to a biosensor chip. Remarkably, subsequent dissociation was much faster for IgG4 than for IgG1. Moreover, after binding of an IgG4 mAb to Sepharose-coupled Ag, we observed additional binding of IgG4 with irrelevant specificity, whereas similar binding was not observed with Ag-bound IgG1. We propose that the IgG4-IgG4 Fc interaction resembles an intermediate of the Fab-arm (half-molecule) exchange reaction that is stabilized because one of the IgG4 molecules is coupled to a solid phase. By contrast, IgG4 Fc recognizes IgG1 only after a conformational change that renders CH3(IgG1) accessible to an interaction with the CH3(IgG4). Such Fc interactions may enhance Ag binding of IgG4 in vivo.

DC-SIGN and α2,6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells

Intravenous immunoglobulin (IVIg) is widely used to treat autoimmune diseases. Several mutually nonexclusive mechanisms are proposed to explain the beneficial effects of IVIg in patients (1, 2). Lately, Ravetch and colleagues (3) demonstrate that anti-inflammatory activity of IVIg is mediated mainly by antibodies that contain terminal α2,6-sialic acid linkages at the Asn297-linked glycan of Fc region.

New insights into intra- and intermolecular interactions of immunoglobulins: crystal structure of mouse IgG2b-Fc at 2.1-A resolution.

The structure of the Fc fragment of monoclonal antibody IgG2b from hybridom M75 of Mus musculus has been determined by single crystal X-ray diffraction. This is the first report of the structure of the murine immunoglobulin isotype IgG2b. The structure refined at 2.1 A resolution provides more detailed structural information about native oligosaccharides than was previously available. High-quality Fourier maps provide a clear identification of alpha-l-fucose with partial occupancy in the first branch of the antennary oligosaccharides. A unique Fc:Fc interaction was observed at the C(H)2-C(H)3 interface.

Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40

SGN-40 is a therapeutic antibody targeting CD40, which induces potent anti-lymphoma activities via direct apoptotic signalling cells and by cell-mediated cytotoxicity. Here we show antibody-dependent cellular phagocytosis (ADCP) by macrophages to contribute significantly to the therapeutic activities and that the antitumour effects of SGN-40 depend on Fc interactions.

The flavor-changing single-top quark production in the littlest Higgs model with T parity at the LHC

The littlest Higgs model with discrete symmetry named “T-parity” (LHT) is an interesting new physics model which does not suffer strong constraints from electroweak precision data. One of the important features of the LHT model is the existence of new source of FC interactions between the SM fermions and the mirror fermions. These FC interactions can make significant loop-level contributions to the couplings tcV, and furthermore enhance the cross sections of the FC single-top quark production processes. In this paper, we study some FC single-top quark production processes, p p → t c ¯ and p p → t V , at the LHC in the LHT model. We find that the cross sections of these processes strongly depend on the mirror quark masses. The processes p p → t c ¯ and p p → t g have large cross sections with heavy mirror quarks. The observation of these FC processes at the LHC is certainly the clue of new physics, and further precise measurements of the cross sections can provide useful information about the free parameters in the LHT model, specially about the mirror quark masses.

Fc-dependent expression of CD137 on human NK cells: insights into “agonistic” effects of anti-CD137 monoclonal antibodies

AbstractCD137 (4-1BB) is a costimulatory mol-ecule that can be manipulated for the treatment of cancer and autoimmune disease. Although it is known that agonistic antibodies (mAbs) against CD137 enhance the rejection of murine tumors in a natural killer (NK) cell– and T cell–dependent fashion, the mechanism for NK dependence is poorly understood. In this study, we evaluated the ability of 2 different glycoforms of a chimerized antihuman CD137 mAb, an aglycosylated (GA) and a low fucose form (GG), to react with human NK cells. Both mAbs bound similarly to CD137 and partially blocked the interaction between CD137 and CD137 ligand. However, unlike GA mAb, immobilized GG mAb activated NK cells and enhanced CD137 expression. These effects were seemingly dependent on Fc interaction with putative Fc receptors on the NK-cell surface, as only the immobilized Fc-fragment of GG was required for CD137 expression. Furthermore, CD137 expression could be enhanced with antibodies directed against non-CD137 epitopes, and the expression levels directly correlated with patterns of Fc-glycosylation recognized to improve Fc interaction with Fcγ receptors. Our data suggest that CD137 can be enhanced on NK cells in an Fc-dependent fashion and that expression correlates with phenotypic and functional parameters of activation.

Non FcR-binding murine antihuman CD3 monoclonal antibody is capable of productive TCR signalling and induces proliferation in the presence of costimulation.

CLB T3/4.A is a non FcR-binding CD3 mAb of the murine IgA isotype, which may be used as an alternative for the mitogenic OKT3 mAb in the treatment of acute cellular rejection after organ transplantation. We studied TCR signalling and T cell activation in response to T3/4.A in normal human PBMC in vitro. T3/4.A induced a rapid rise in free cytoplasmic Ca(2+), not different from the response to mitogenic CD3 mAb. However, protein tyrosine phosphorylation and, particularly, MAPK activation, were reduced as compared to mitogenic CD3 mAb. T3/4.A enhanced expression of both CD69 and CD25, but proliferation and detectable cytokine production did not occur. Addition of either CD28 mAb or IL-2 induced a strong proliferative response, which was accompanied by cytokine production. At higher mAb concentrations, T cell activation decreased, which correlated with TCR downmodulation. To exclude the possibility that activation by T3/4.A depends on interaction of murine IgA Fc with as yet unknown FcR, we showed that also with CD3 mAb F(ab')2 fragments upregulation of activation molecules occurred, as well as proliferation in the presence of costimulation. We conclude that the non FcR-binding murine IgA mAb T3/4.A acts as a partial agonist and leads to proliferation and cytokine production only in the presence of appropriate costimuli. These findings may explain the mitigated cytokine release syndrome observed in vivo with some nonmitogenic CD3 mAbs.

The role of anti‐dengue antibodies in the pathogenesis of Dengue disease

Dengue disease includes from mild dengue fever to severe dengue hemorrhagic fever (DHF), which is an important health problem in tropical or sub‐tropical areas. The immunopathogenesis of DHF is initiated by aberrant immune activation and autoantibody production caused by virus infection. There is a molecular mimicry between dengue antigens and self‐proteins. The anti‐dengue antibody cross‐react with platelet and endothelial cell, and would trigger the subsequent dysfunction of endothelial cells and hemorrhage during the acute infection. The antibody‐dependent enhancement theory plays a central role in the dengue disease. The enhancing antibody is dependent on the types of the target cells and the specificity of the enhancing antibody. The enhancing antibody can be either anti‐prM or anti‐E antibody. For anti‐E Ab‐mediated enhancement on monocytic cells, it can be concentration‐dependent: enhancing at sub‐neutralization level or enhance regardless of concentration by Fc and FcR interaction. For anti‐prM Ab‐mediated enhancement, it enhanced the dengue virion binding on both FcR or non‐FcR bearing cells with dual specificity. The anti‐dengue antibodies seem to be either enhancing antibody or the pathogenic antibody, and play a major role in the DHF pathogenesis.

Electrochemical Detection of a Dengue-related Oligonucleotide Sequence Using Ferrocenium as a Hybridization Indicator.

A simple method for electrochemical detection of a synthetic 20-bpoligonucleotide sequence related with dengue virus genome was developed. Acomplimentary DNA probe sequence was electrostatically immobilized onto a glassycarbon electrode modified with chitosan. Electrochemical detection of hybridizationbetween probe and target was performed by cyclic voltammetry, using ferrocene (Fc ) as ahybridization label. After hybridization, the peak current response of Fc oxidationincreased around 26%. A higher voltammetric decay rate constant (kd) and a lower half-lifeperiod (t1/2) for the interaction of Fc with dsDNA compared to those with ssDNAquantitatively characterize the different strengths of interaction with both types of DNA.By combining the simplicity of DNA immobilization onto a chitosan film and suitablevoltammetric detection of hybridization concomitant with ferrocene attachment, a gooddiscrimination between ssDNA and dsDNA was obtained.