The leukocyte receptor CD84 inhibits Fc epsilon RI-mediated signaling through homophilic interaction in transfected RBL-2H3 cells.

PubMed

Oliver-Vila, Irene; Saborit-Villarroya, Ifigènia; Engel, Pablo; Martin, Margarita

2008-04-01

Signaling through the high-affinity receptor for immunoglobulin E (Fc epsilon RI) results in the coordinated activation of tyrosine kinases, thus leading to calcium mobilization, degranulation, and leukotriene and cytokine synthesis. Here, we show that CD84, a member of the CD150 family of leukocyte receptors, inhibits Fc epsilon RI-mediated mast cell degranulation in CD84-transfected rat basophilic leukaemia-2H3 mast cell line cells (RBL-2H3) through homophilic interaction. There was no reduction in overall protein phosphorylation following IgE triggering in CD84 RBL-2H3 cells. Indeed, phosphorylation of Dok-1 and c-Cbl increased in CD84 RBL-2H3, suggesting that inhibition is mediated by these molecules. MAP kinase phosphorylation (ERK1/2, JNK and p38) and cytokine synthesis were impaired in CD84 RBL-2H3. This inhibitory mechanism was independent of SAP and SHP-2 recruitment. Interestingly, CD84 mutants in tyrosines (Y279F and DeltaY324) reversed this inhibitory profile. These data suggest that CD84 may play a role in modulating Fc epsilon RI-mediated signaling in mast cells. Thus, CD84 could play a protective role against undesired allergic and inflammatory responses.

Attenuated atherosclerotic lesions in apoe-fc gamma-chain-deficient hyperlipidemic mouse model is associated with inhibition of Th17 cells and promotion of regulatory T cells

USDA-ARS?s Scientific Manuscript database

Though the presence of antioxidized low-density lipoprotein IgG is well documented in clinical and animal studies, the role for Fc gamma Rs to the progression of atherosclerosis has not been studied in detail. In the current study, we investigated the role for activating Fc gamma R in the progressio...

Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays▿

PubMed Central

Verma, Anita; Ngundi, Miriam M.; Meade, Bruce D.; De Pascalis, Roberto; Elkins, Karen L.; Burns, Drusilla L.

2009-01-01

Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fcγ) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcγ receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fcγ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcγ receptor blocking monoclonal antibodies, we found that at least three murine Fcγ receptor classes, IIB, III, and IV, can contribute to Fcγ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcγ receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output. PMID:19656993

IgA Fc receptors.

PubMed

Monteiro, Renato C; Van De Winkel, Jan G J

2003-01-01

The IgA receptor family comprises a number of surface receptors including the polymeric Ig receptor involved in epithelial transport of IgA/IgM, the myeloid specific IgA Fc receptor (FcalphaRI or CD89), the Fcalpha/muR, and at least two alternative IgA receptors. These are the asialoglycoprotein receptor and the transferrin receptor, which have been implicated in IgA catabolism, and tissue IgA deposition. In this review we focus on the biology of FcalphaRI (CD89). FcalphaRI is expressed on neutrophils, eosinophils, monocytes/macrophages, dendritic cells, and Kupffer cells. This receptor represents a heterogeneously glycosylated transmembrane protein that binds both IgA subclasses with low affinity. A single gene encoding FcalphaRI has been isolated, which is located within the leukocyte receptor cluster on chromosome 19. The FcalphaRI alpha chain lacks canonical signal transduction domains but can associate with the FcR gamma-chain that bears an activation motif (ITAM) in the cytoplasmic domain, allowing activatory functions. FcalphaRI expressed alone mediates endocytosis and recyling of IgA. No FcalphaRI homologue has been defined in the mouse, and progress in defining the in vivo role of FcalphaRI has been made using human FcalphaRI transgenic (Tg) mice. FcalphaRI-Tg mice demonstrated FcalphaRI expression on Kupffer cells and so defined a key role for the receptor in mucosal defense. The receptor functions as a second line of antibacterial defense involving serum IgA rather than secretory IgA. Studies in FcalphaRI-Tg mice, furthermore, defined an essential role for soluble FcalphaRI in the development of IgA nephropathy by formation of circulating IgA-FcalphaRI complexes. Finally, recent work points out a role for human IgA in treatment of infectious and neoplastic diseases.

Effect of leukocyte therapy on tumor necrosis factor-alpha and interferon-gamma production in patients with recurrent spontaneous abortion.

PubMed

Gharesi-Fard, Behrouz; Zolghadri, Jaleh; Kamali-Sarvestani, Eskandar

2008-03-01

Considering the deleterious role of T helper1 (Th1) cells in pregnancy outcome, a successful treatment for recurrent spontaneous abortion (RSA) should be able to make a significant shift away from Th1 responses. Although paternal leukocyte immunization has been used for treatment of RSA for years, because of methodological differences there is no consensus on the mechanism of action and effectiveness of this method. Twenty-five Iranian non-pregnant women with RSA and 16 non-pregnant control women with at least two successful pregnancies were included in this study. All cases were followed up after leukocyte therapy for pregnancy outcome. Mononuclear cells from women were co-cultured with the husband's mononuclear cells before and after immunotherapy. The levels of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were checked on culture supernatant by enzyme-linked immunosorbent assay method. The mean concentration of TNF-alpha was significantly higher in patients compared with that in normal controls (P=0.0001). After immunotherapy, the TNF-alpha level was only significantly decreased in women with successful outcome (P=0.0001). Immunotherapy also induced a significant reduction in the IFN-gamma level (P=0.009). The results of this investigation confirm the role of TNF-alpha in RSA and propose the assessment of TNF-alpha production as a valuable prognostic parameter for the prediction of abortion after leukocyte therapy.

Association of a coding polymorphism in Fc gamma receptor 2A and graft survival in re-transplant candidates.

PubMed

Arnold, Marie-Luise; Fuernrohr, Barbara G; Weiß, Katrin M; Harre, Ulrike; Wiesener, Michael S; Spriewald, Bernd M

2015-10-01

The family of Fc gamma receptors (FcγRs) is involved in mediating immunological effector functions. FcγRs are differentially expressed on immune cells and can act either activating or inhibitory, with FcγR2A belonging to the first group. The polymorphism H131R (rs1801274) in FCGR2A has been associated with acute rejection and can shift the overall balance between activating and inhibitory FcγRs. Anti-HLA allo-antibodies in transplant recipients have been identified as risk factor for organ survival after transplantation. In this study we genotyped FCGR2A H131R in 200 patients who had undergone kidney transplantation and experienced loss of graft function. FCGR2A polymorphism was related to graft survival and anti-HLA antibodies. Graft survival was calculated as the time interval between transplantation and return to chronic dialysis after transplantation. The gene frequency of FCGR2A R/R131 was found significantly more often in patients with earlier (⩽60months) compared to patients with later (>60months) graft failure. Overall patients homozygous for R/R131 had a significantly shorter graft survival, compared to H/H131 or H/R131 which is even more pronounced, when anti-HLA antibodies were present. These data suggest, that FCGR2A polymorphisms constitute a risk factor for graft loss following kidney transplantation and that this effect is related to anti-HLA antibodies. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake, immunoglobulin-mediated calcium increase, and neurotransmitter release in motor neurons

NASA Technical Reports Server (NTRS)

Mohamed, Habib A.; Mosier, Dennis R.; Zou, Ling L.; Siklos, Laszlo; Alexianu, Maria E.; Engelhardt, Jozsef I.; Beers, David R.; Le, Wei-dong; Appel, Stanley H.

2002-01-01

Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals. Copyright 2002 Wiley-Liss, Inc.

Registration of 'FC1028', 'FC1037, 'FC1038' and, 'FC1036' multigerm sugarbeet germplasm with multiple disease resistances

USDA-ARS?s Scientific Manuscript database

‘FC1028’, ‘FC1036’, ‘FC1037’, and ‘FC1038’ (PI 665053, PI 665054, PI 665055, PI 665056) sugarbeet germplasms (Beta vulgaris L.) were released from 20111027, 09-FC1036, 20111025, and 04-FC1038 seed lots, respectively, and tested under the designations 04-FC1028; 05-, 06-, 07-, 08-, 09-FC1036; 04-FC10...

Registration of FC1018, FC1019, FC1020, and FC1022, Sugarbeet Multigerm Pollinator Germplasms with Disease Resistance

USDA-ARS?s Scientific Manuscript database

‘FC1018’, ‘FC1019’, ‘FC1020’, and ‘FC1022’ (PI 658059, PI 658060, PI 658061, PI 658062, respectively) sugarbeet germplasm (Beta vulgaris L.) were released in 2009 from 05-FC1018, 05-FC1019, 07-/08-FC1020 and 05-FC101022 seed lots, respectively, and tested under those designations. They were develo...

Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

PubMed

Taupin, J L; Anderson, P

1994-12-01

The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

Combined Effects of Gamma Radiation and High Dietary Iron on Peripheral Leukocyte Distribution and Function

NASA Technical Reports Server (NTRS)

Crucian, Brian E.; Morgan, Jennifer L. L.; Quiriarte, Heather A.; Sams, Clarence F.; Smith, Scott M.; Zwart, Sara R.

2012-01-01

Both radiation and increased iron stores can independently increase oxidative damage, resulting in protein, lipid and DNA oxidation. Oxidative stress increases the risk of many health problems including cancer, cataracts, and heart disease. This study, a subset of a larger interdisciplinary investigation of the combined effect of iron overload on sensitivity to radiation injury, monitored immune parameters in the peripheral blood of rats subjected to gamma radiation, high dietary iron or both. Specific immune measures consisted of: (1) peripheral leukocyte distribution, (2) plasma cytokine levels and (3) cytokine production profiles following whole blood mitogenic stimulation

Nociceptive neuronal Fc-gamma receptor I is involved in IgG immune complex induced pain in the rat.

PubMed

Jiang, Haowu; Shen, Xinhua; Chen, Zhiyong; Liu, Fan; Wang, Tao; Xie, Yikuan; Ma, Chao

2017-05-01

Antigen-specific immune diseases such as rheumatoid arthritis are often accompanied by pain and hyperalgesia. Our previous studies have demonstrated that Fc-gamma-receptor type I (FcγRI) is expressed in a subpopulation of rat dorsal root ganglion (DRG) neurons and can be directly activated by IgG immune complex (IgG-IC). In this study we investigated whether neuronal FcγRI contributes to antigen-specific pain in the naïve and rheumatoid arthritis model rats. In vitro calcium imaging and whole-cell patch clamp recordings in dissociated DRG neurons revealed that only the small-, but not medium- or large-sized DRG neurons responded to IgG-IC. Accordingly, in vivo electrophysiological recordings showed that intradermal injection of IgG-IC into the peripheral receptive field could sensitize only the C- (but not A-) type sensory neurons and evoke action potential discharges. Pain-related behavioral tests showed that intradermal injection of IgG-IC dose-dependently produced mechanical and thermal hyperalgesia in the hindpaw of rats. These behavioral effects could be alleviated by localized administration of non-specific IgG or an FcγRI antibody, but not by mast cell stabilizer or histamine antagonist. In a rat model of antigen-induced arthritis (AIA) produced by methylated bovine serum albumin, FcγRI were found upregulated exclusively in the small-sized DRG neurons. In vitro calcium imaging revealed that significantly more small-sized DRG neurons responded to IgG-IC in the AIA rats, although there was no significant difference between the AIA and control rats in the magnitude of calcium changes in the DRG neurons. Moreover, in vivo electrophysiological recordings showed that C-nociceptive neurons in the AIA rats exhibited a greater incidence of action potential discharges and stronger responses to mechanical stimuli after IgG-IC was injected to the receptive fields. These results suggest that FcγRI expressed in the peripheral nociceptors might be directly activated

Complexes prepared from protein A and human serum, IgG, or Fc gamma fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion.

PubMed

Langone, J J; Das, C; Mainwaring, R; Shearer, W T

1985-01-01

Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of 125I-protein A and human 131I-IgG alone or in serum, and 131I-Fc gamma fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess 131I-IgG or 131I-Fc, all the 125I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess 125I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc gamma in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.

Deletion of the Fcγ Receptor IIb in Thymic Stromal Lymphopoietin Transgenic Mice Aggravates Membranoproliferative Glomerulonephritis

PubMed Central

Mühlfeld, Anja S.; Segerer, Stephan; Hudkins, Kelly; Carling, Matthew D.; Wen, Min; Farr, Andrew G.; Ravetch, Jeffrey V.; Alpers, Charles E.

2003-01-01

Engagement of immunoglobulin-binding receptors (FcγR) on leukocytes and other cell types is one means by which immunoglobulins and immune complexes activate effector cells. One of these FcγRs, FcγRIIb, is thought to contribute to protection from autoimmune disease by down-regulation of B-cell responsiveness and myeloid cell activation. We assessed the role of FcγRIIb in a mouse model of cryoglobulin-associated membranoproliferative glomerulonephritis induced by overexpression of thymic stromal lymphopoietin (TSLP). TSLP transgenic mice were crossbred with animals deficient for FcγRIIb on the same genetic background (C57BL/6). Renal pathology was assessed in female and male animals (wild-type, FcγRIIb−/−, TSLP transgenic, and combined TSLP transgenic/FcγRIIb−/− mice) after 50 and 120 days, respectively. FcγRIIb−/− mice had no significant renal pathology, whereas overexpression of TSLP induced a membranoproliferative glomerulonephritis, as previously established. TSLP transgenic FcγRIIb−/− mice appeared sick with increased mortality. Kidney function was significantly impaired in male mice corresponding to aggravated glomerular pathology with increases in glomerular matrix and cellularity. This resulted from both a large influx of infiltrating macrophages and increased cellular proliferation. These results emphasize the important role of FcγRIIb in regulating immune responses and suggest that modulation of Fcγ receptor activation or expression may be a useful therapeutic approach for treating glomerular diseases. PMID:12937154

In vivo gamma-rays induced initial DNA damage and the effect of famotidine in mouse leukocytes as assayed by the alkaline comet assay.

PubMed

Mozdarani, Hossein; Nasirian, Borzo; Haeri, S Abolghasem

2007-03-01

Ionizing radiation induces a variety of lesions in DNA, each of which can be used as a bio-indicator for biological dosimetry or the study of the radioprotective effects of substances. To assess gamma ray-induced DNA damage in vivo in mouse leukocytes at various doses and the effect of famotidine, blood was collected from Balb/c male mice after irradiation with 4 Gy gamma-rays at different time intervals post-irradiation. To assess the response, mice were irradiated with doses of gamma-rays at 1 to 4 Grays. Famotidine was injected intra-peritoneally (i.p) at a dose of 5 mg/kg at various time intervals before irradiation. Four slides were prepared from each sample and alkaline comet assay was performed using standard protocols. Results obtained show that radiation significantly increases DNA damage in leukocytes in a dose dependent manner (p < 0.01) when using appropriate sampling time after irradiation, because increasing sampling time after irradiation resulted in a time dependent disappearance of DNA damage. Treatment with only 5 mg/kg famotidine before 4 Gy irradiation led to almost 50% reduction in DNA damage when compared with those animals which received radiation alone. The radioprotective capability of famotidine might be attributed to radical scavenging properties and an anti-oxidation mechanism.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Jinghua; Marnell, Lorraine L.; Marjon, Kristopher D.

Pentraxins are a family of ancient innate immune mediators conserved throughout evolution. The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein, which are two of the acute-phase proteins synthesized in response to infection. Both recognize microbial pathogens and activate the classical complement pathway through C1q. More recently, members of the pentraxin family were found to interact with cell-surface Fc{gamma} receptors (Fc{gamma}R) and activate leukocyte-mediated phagocytosis. Here we describe the structural mechanism for pentraxin's binding to Fc{gamma}R and its functional activation of Fc{gamma}R-mediated phagocytosis and cytokine secretion. The complex structure between human SAP and Fc{gamma}RIIa reveals a diagonallymore » bound receptor on each SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. The 1:1 stoichiometry between SAP and Fc{gamma}RIIa infers the requirement for multivalent pathogen binding for receptor aggregation. Mutational and binding studies show that pentraxins are diverse in their binding specificity for Fc{gamma}R isoforms but conserved in their recognition structure. The shared binding site for SAP and IgG results in competition for Fc{gamma}R binding and the inhibition of immune-complex-mediated phagocytosis by soluble pentraxins. These results establish antibody-like functions for pentraxins in the Fc{gamma}R pathway, suggest an evolutionary overlap between the innate and adaptive immune systems, and have new therapeutic implications for autoimmune diseases.« less

Glucocorticoid-induced tumor necrosis factor receptor family-related ligand triggering upregulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and promotes leukocyte adhesion.

PubMed

Lacal, Pedro Miguel; Petrillo, Maria Grazia; Ruffini, Federica; Muzi, Alessia; Bianchini, Rodolfo; Ronchetti, Simona; Migliorati, Graziella; Riccardi, Carlo; Graziani, Grazia; Nocentini, Giuseppe

2013-10-01

The interaction of glucocorticoid-induced tumor necrosis factor receptor-family related (GITR) protein with its ligand (GITRL) modulates different functions, including immune/inflammatory response. These effects are consequent to intracellular signals activated by both GITR and GITRL. Previous results have suggested that lack of GITR expression in GITR(-/-) mice decreases the number of leukocytes within inflamed tissues. We performed experiments to analyze whether the GITRL/GITR system modulates leukocyte adhesion and extravasation. For that purpose, we first evaluated the capability of murine splenocytes to adhere to endothelial cells (EC). Our results indicated that adhesion of GITR(-/-) splenocytes to EC was reduced as compared with wild-type cells, suggesting that GITR plays a role in adhesion and that this effect may be due to GITRL-GITR interaction. Moreover, adhesion was increased when EC were pretreated with an agonist GITR-Fc fusion protein, thus indicating that triggering of GITRL plays a role in adhesion by EC regulation. In a human in vitro model, the adhesion to human EC of HL-60 cells differentiated toward the monocytic lineage was increased by EC pretreatment with agonist GITR-Fc. Conversely, antagonistic anti-GITR and anti-GITRL Ab decreased adhesion, thus further indicating that GITRL triggering increases the EC capability to support leukocyte adhesion. EC treatment with GITR-Fc favored extravasation, as demonstrated by a transmigration assay. Notably, GITRL triggering increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression and anti-ICAM-1 and anti-VCAM-1 Abs reversed GITR-Fc effects. Our study demonstrates that GITRL triggering in EC increases leukocyte adhesion and transmigration, suggesting new anti-inflammatory therapeutic approaches based on inhibition of GITRL-GITR interaction.

Immunoglobulin G1 Fc domain motions: implications for Fc engineering

PubMed Central

Frank, Martin; Walker, Ross C.; Lanzilotta, William N.; Prestegard, James H.; Barb, Adam W.

2014-01-01

The fragment crystallizable (Fc) region links the key pathogen identification and destruction properties of immunoglobulin G(IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fcγ receptors (FcγRs) on immune cells. The cellular response and committal to a damaging, though protective, immune response is tightly controlled at multiple levels. Control mechanisms are diverse and in many cases unclear, but one frequently suggested contribution originates in Fcγ receptor affinity being modulated through shifts in Fc conformational sampling. Here we report a previously unseen IgG1 Fc conformation. This observation motivated an extensive molecular dynamics (MD) investigation of polypeptide and glycan motions that revealed greater amplitude of motion for the N-terminal Cγ2 domains and N-glycan than previously observed. Residues in the Cγ2/Cγ3 interface and disulphide-bonded hinge were identified as influencing the Cγ2 motion. Our results are consistent with a model of Fc that is structurally dynamic. Conformational states that are competent to bind immune-stimulating FcγRs interconverted with Fc conformations distinct from those observed in FcγR complexes, which may represent a transient, nonbinding population. PMID:24522230

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry.

PubMed

Highland, Margaret A; Schneider, David A; White, Stephen N; Madsen-Bouterse, Sally A; Knowles, Donald P; Davis, William C

2016-06-01

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspecies comparative immune system analyses. This study describes cross-reactive mAbs that recognize leukocyte differentiation molecules (LDMs) and major histocompatibility complex antigens on DS and BHS leukocytes. Characterization of multichannel eosinophil autofluorescence in this study permitted cell-type specific gating of granulocytes for evaluating LDMs, specifically on neutrophils, by single-label FC. Evaluation of relative abundances of LDMs by flow cytometry revealed greater CD11a, CD11b, CD18 (β2 integrins) and CD 172a (SIRPα) on DS neutrophils and greater CD14 (lipopolysaccharide receptor) on BHS neutrophils. Greater CD25 (IL-2) was identified on BHS lymphocytes following Concavalin A stimulation. While DS and BHS have similar total peripheral blood leukocyte counts, BHS have proportionately more neutrophils. Published by Elsevier Ltd.

EphrinA1-Fc attenuates myocardial ischemia/reperfusion injury in mice

PubMed Central

DuSablon, Augustin; Parks, Justin; Whitehurst, K’Shylah; Estes, Heather; Chase, Robert; Vlahos, Eleftherios; Sharma, Uma; Wert, David

2017-01-01

EphrinA1, a membrane-bound receptor tyrosine kinase ligand expressed in healthy cardiomyocytes, is lost in injured cells following myocardial infarction. Previously, we have reported that a single intramyocardial injection of chimeric ephrinA1-Fc at the time of ischemia reduced injury in the nonreperfused myocardium by 50% at 4 days post-MI by reducing apoptosis and inflammatory cell infiltration. In a clinically relevant model of acute ischemia (30min)/reperfusion (24hr or 4 days) injury, we now demonstrate that ephrinA1-Fc reduces infarct size by 46% and completely preserves cardiac function (ejection fraction, fractional shortening, and chamber dimensions) in the short-term (24hrs post-MI) as well as long-term (4 days). At 24 hours post-MI, diminished serum inflammatory cell chemoattractants in ephrinA1-Fc-treated mice reduces recruitment of neutrophils and leukocytes into the myocardium. Differences in relative expression levels of EphA-Rs are described in the context of their putative role in mediating cardioprotection. Validation by Western blotting of selected targets from mass spectrometry analyses of pooled samples of left ventricular tissue homogenates from mice that underwent 30min ischemia and 24hr of reperfusion (I/R) indicates that ephrinA1-Fc administration alters several regulators of signaling pathways that attenuate apoptosis, promote autophagy, and shift from FA metabolism in favor of increased glycolysis to optimize anaerobic ATP production. Taken together, reduced injury is due a combination of adaptive metabolic reprogramming, improved cell survival, and decreased inflammatory cell recruitment, suggesting that ephrinA1-Fc enhances the capacity of the heart to withstand an ischemic insult. PMID:29236774

EphrinA1-Fc attenuates myocardial ischemia/reperfusion injury in mice.

PubMed

DuSablon, Augustin; Parks, Justin; Whitehurst, K'Shylah; Estes, Heather; Chase, Robert; Vlahos, Eleftherios; Sharma, Uma; Wert, David; Virag, Jitka

2017-01-01

EphrinA1, a membrane-bound receptor tyrosine kinase ligand expressed in healthy cardiomyocytes, is lost in injured cells following myocardial infarction. Previously, we have reported that a single intramyocardial injection of chimeric ephrinA1-Fc at the time of ischemia reduced injury in the nonreperfused myocardium by 50% at 4 days post-MI by reducing apoptosis and inflammatory cell infiltration. In a clinically relevant model of acute ischemia (30min)/reperfusion (24hr or 4 days) injury, we now demonstrate that ephrinA1-Fc reduces infarct size by 46% and completely preserves cardiac function (ejection fraction, fractional shortening, and chamber dimensions) in the short-term (24hrs post-MI) as well as long-term (4 days). At 24 hours post-MI, diminished serum inflammatory cell chemoattractants in ephrinA1-Fc-treated mice reduces recruitment of neutrophils and leukocytes into the myocardium. Differences in relative expression levels of EphA-Rs are described in the context of their putative role in mediating cardioprotection. Validation by Western blotting of selected targets from mass spectrometry analyses of pooled samples of left ventricular tissue homogenates from mice that underwent 30min ischemia and 24hr of reperfusion (I/R) indicates that ephrinA1-Fc administration alters several regulators of signaling pathways that attenuate apoptosis, promote autophagy, and shift from FA metabolism in favor of increased glycolysis to optimize anaerobic ATP production. Taken together, reduced injury is due a combination of adaptive metabolic reprogramming, improved cell survival, and decreased inflammatory cell recruitment, suggesting that ephrinA1-Fc enhances the capacity of the heart to withstand an ischemic insult.

Kinetics of leukocyte sequestration in the lungs of acutely septic primates: A study using sup 111 In-labeled autologous leukocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hangen, D.H.; Segall, G.M.; Harney, E.W.

1990-03-01

To further clarify the role of leukocytes in the pathogenesis of ARDS, we studied the localization and kinetics of leukocyte migration using 111In-labeled autologous white cell scans ({sup 111}In wbc scans) in four primates made acutely septic with infusions of Escherichia coli. Whole body images were obtained with a gamma camera and were acquired on computer every 15 min beginning immediately after the E. coli infusion. Simultaneous measurements of C5a and peripheral blood leukocyte count were also obtained. Within 5 min of initiating sepsis, three major events occurred: complement activation as measured by the production of C5a, a profound fallmore » in peripheral leukocyte count, and a significant increase in the sequestration of leukocytes in the lungs. The pulmonary sequestration reached a peak at 15 min with a mean of 152% of baseline activity. This sequestration consisted of a population that was predominantly neutrophils. Damage to the pulmonary capillary endothelium was demonstrated by an increase in extravascular lung water. The results support a role for neutrophils and complement as mediators in the pathogenesis of ARDS.« less

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

PubMed Central

Moore, Gregory L; Chen, Hsing; Karki, Sher

2010-01-01

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complementenhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens. PMID:20150767

Evaluation of the Combined Effects of Gamma Radiation and High Dietary Iron on Peripheral Leukocyte Distribution and Function

NASA Technical Reports Server (NTRS)

Crucian, Brian E.; Morgan, Jennifer L. L.; Quiriarte, Heather A.; Sams, Clarence F.; Smith, Scott M.; Zwart, Sara R.

2011-01-01

NASA is concerned with the health risks to astronauts, particularly those risks related to radiation exposure. Both radiation and increased iron stores can independently increase oxidative damage, resulting in protein, lipid and DNA oxidation. Oxidative stress increases the risk of many health problems including cancer, cataracts, and heart disease. This study, a subset of a larger interdisciplinary investigation of the combined effect of iron overload on sensitivity to radiation injury, monitored immune parameters in the peripheral blood of rats subjected to gamma radiation, high dietary iron or both. Specific immune measures consisted of (A) peripheral leukocyte distribution; (B) plasma cytokine levels; (C) cytokine production profiles following whole blood stimulation of either T cells or monocytes.

Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation.

PubMed

van Spriel, A B; Leusen, J H; van Egmond, M; Dijkman, H B; Assmann, K J; Mayadas, T N; van de Winkel, J G

2001-04-15

Receptors for human immunoglobulin (Ig)G and IgA initiate potent cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has previously been implicated in receptor cooperation with Fc receptors (FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was characterized by studying normal human PMNs, Mac-1-deficient mouse PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in Mac-1(-/-) PMNs and in human PMNs blocked with anti-Mac-1 monoclonal Ab (mAb). Mac-1(-/-) PMNs were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking difference in immunologic synapse formation between Mac-1(-/-) and wild-type PMNs. Also, respiratory burst activity could be measured outside membrane-enclosed compartments by using Mac-1(-/-) PMNs bound to Ab-coated tumor cells, in contrast to wild-type PMNs. In summary, these data document an absolute requirement of Mac-1 for FcR-mediated PMN cytotoxicity toward tumor targets. Mac-1(-/-) PMNs exhibit defective spreading on Ab-coated targets, impaired formation of immunologic synapses, and absent tumor cytolysis.

Notice of Release of FC1018, FC1019, FC1020 and FC1022 Multigerm Sugarbeet Germplasms with Multiple Disease Resistance

USDA-ARS?s Scientific Manuscript database

FC1018 (PI 658059) has excellent resistance to root-rotting strains (AG-2-2) of Rhizoctonia solani Kühn and carries the Rz1 gene, which confers resistance to some strains of Beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania. FC1018 has shown a moderate tolerance to cercospora ...

Rho is Required for the Initiation of Calcium Signaling and Phagocytosis by Fcγ Receptors in Macrophages

PubMed Central

Hackam, David J.; Rotstein, Ori D.; Schreiber, Alan; Zhang, Wei-jian; Grinstein, Sergio

1997-01-01

Phagocytosis of bacteria by macrophages and neutrophils is an essential component of host defense against infection. The mechanism whereby the interaction of opsonized particles with Fcγ receptors triggers the engulfment of opsonized particles remains incompletely understood, although activation of tyrosine kinases has been recognized as an early step. Recent studies in other systems have demonstrated that tyrosine kinases can in turn signal the activation of small GTPases of the ras superfamily. We therefore investigated the possible role of Rho in Fc receptor–mediated phagocytosis. To this end we microinjected J774 macrophages with C3 exotoxin from Clostridium botulinum, which ADP-ribosylates and inactivates Rho. C3 exotoxin induced the retraction of filopodia, the disappearance of focal complexes, and a global decrease in the F-actin content of J774 cells. In addition, these cells exhibited increased spreading and the formation of vacuolar structures. Importantly, inactivation of Rho resulted in the complete abrogation of phagocytosis. Inhibition of Fcγ receptor–mediated phagocytosis by C3 exotoxin was confirmed in COS cells, which become phagocytic upon transfection of the FcγRIIA receptor. Rho was found to be essential for the accumulation of phosphotyrosine and of F-actin around phagocytic cups and for Fcγ receptor–mediated Ca2+ signaling. The clustering of receptors in response to opsonin, an essential step in Fcγ-induced signaling, was the earliest event shown to be inhibited by C3 exotoxin. The effect of the toxin was specific, since clustering and internalization of transferrin receptors were unaffected by microinjection of C3. These data identify a role for small GTPases in Fcγ receptor–mediated phagocytosis by leukocytes. PMID:9294149

IgG Fc variant cross-reactivity between human and rhesus macaque FcγRs

PubMed Central

Boesch, Austin W.; Miles, Adam R.; Chan, Ying N.; Osei-Owusu, Nana Y.; Ackerman, Margaret E.

2017-01-01

ABSTRACT Non-human primate (NHP) studies are often an essential component of antibody development efforts before human trials. Because the efficacy or toxicity of candidate antibodies may depend on their interactions with Fcγ receptors (FcγR) and their resulting ability to induce FcγR-mediated effector functions such as antibody-dependent cell-meditated cytotoxicity and phagocytosis (ADCP), the evaluation of human IgG variants with modulated affinity toward human FcγR is becoming more prevalent in both infectious disease and oncology studies in NHP. Reliable translation of these results necessitates analysis of the cross-reactivity of these human Fc variants with NHP FcγR. We report evaluation of the binding affinities of a panel of human IgG subclasses, Fc amino acid point mutants and Fc glycosylation variants against the common allotypes of human and rhesus macaque FcγR by applying a high-throughput array-based surface plasmon resonance platform. The resulting data indicate that amino acid variation present in rhesus FcγRs can result in disrupted, matched, or even increased affinity of IgG Fc variants compared with human FcγR orthologs. These observations emphasize the importance of evaluating species cross-reactivity and developing an understanding of the potential limitations or suitability of representative in vitro and in vivo models before human clinical studies when either efficacy or toxicity may be associated with FcγR engagement. PMID:28055295

Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics

PubMed Central

Rath, Timo; Baker, Kristi; Dumont, Jennifer A.; Peters, Robert T.; Jiang, Haiyan; Qiao, Shuo-Wang; Lencer, Wayne I.; Pierce, Glenn F.; Blumberg, Richard S.

2016-01-01

Nearly 350 IgG-based therapeutics are approved for clinical use or are under development for many diseases lacking adequate treatment options. These include molecularly engineered biologicals comprising the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins) that confer the advantages of IgG, including binding to the neonatal Fc receptor (FcRn) to facilitate in vivo stability, and the therapeutic benefit of the specific effector functions. Advances in IgG structure-function relationships and an understanding of FcRn biology have provided therapeutic opportunities for previously unapproachable diseases. This article discusses approved Fc-fusion therapeutics, novel Fc-fusion proteins and FcRn-dependent delivery approaches in development, and how engineering of the FcRn–Fc interaction can generate longer-lasting and more effective therapeutics. PMID:24156398

Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

PubMed

Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

2016-01-01

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Distribution of FcγRIIa and FcγRIIIb Genotypes in Patients With Generalized Aggressive Periodontitis.

PubMed

de Souza, Rodrigo C; Colombo, Ana Paula V

2006-07-01

Polymorphisms in FcγR have been associated with different forms of periodontitis. This study determined the frequency of FcγRIIa and FcγRIIIb alleles/genotypes in patients with generalized aggressive periodontitis (GAgP). Thirty-one GAgP and 49 periodontally healthy Brazilian subjects participated in the study. Full-mouth periodontal examinations were carried out, and mouthwash samples were collected for human DNA isolation. FcγR genotyping was performed by polymerase chain reaction and hybridization with allele-specific oligonucleotide probes. Significant differences between groups were sought by Mann-Whitney, χ 2 , and Fisher exact tests and configural frequency analysis. FcγRIIa-H131 (53.8%) and FcγRIIIb-NA1 (75%) were the most prevalent alleles in this sample population. A significant overrepresentation of FcγRIIIb-NA2 was observed in the GAgP group, whereas FcγRIIIb-NA1 was detected more often in healthy individuals (odds ratio, 32.5; 95% confidence interval [CI], 10.6 to 99.8; P <0.001). No significant differences in the distribution of the FcγRIIa genotypes were observed between the groups. The prevalence of FcγRIIIb-NA2/NA2 was higher in GAgP patients, whereas FcγRIIIb-NA1/NA1 was predominant in the healthy group (χ 2 = 45.1; P <0.001). The combination of the genotypes FcγRIIIb-NA2/NA2 plus FcγRIIa-H/H131 was observed more frequently in GAgP subjects than expected from marginal frequencies (χ 2 = 12.5; P <0.001). The data suggest that the FcγRIIIb-NA2 allele and/or FcγRIIIb-NA2/NA2 genotype and the composite genotype FcγRIIIb-NA2/NA2 plus FcγRIIa-H/H131 may be associated with GAgP, whereas FcγRIIIb-NA1 and/or FcγRIIIb-NA1/NA1 may be related to periodontal health in this sample of the Brazilian population. © 2006 American Academy of Periodontology.

Identification of a unique IgG Fc binding site in human intestinal epithelium.

PubMed

Kobayashi, K; Blaser, M J; Brown, W R

1989-10-15

In experiments to determine whether serum antibodies in patients with Crohn's disease could be used as probes for detecting potentially etiologic Ag in the patients' tissues, we found that peroxidase (HRP)-labeled IgG from healthy persons, as well as from the patients, bound to normal colonic and small intestinal epithelium, mostly or entirely to goblet cells. The binding was due to a reaction involving the Fc region of IgG because HRP-labeled Fc fragments of IgG bound, but HRP-Fab, HRP-IgA, and HRP-bovine albumin did not, and because binding of HRP-IgG was inhibited competitively by unlabeled IgG or Fc fragments but not by IgG Fab fragments or IgA. These immunohistochemical results were confirmed by ELISA with microtiter wells coated with a sonicated homogenate from human colonocytes. The epithelial IgG Fc binding site was characterized by SDS-PAGE as consisting of a high Mr (greater than 200,000 Da) and a 78,000-Da component. It bound all four subclasses of human IgG and bound aggregated as well as monomeric IgG. It is distinct from known human Fc-gamma R by lack of recognition by mAb to those receptors and differences in affinity for various subclasses of human and murine IgG. This unique IgG Fc binding site might be involved in immunologic defense of the gut, perhaps by mediating reactions between foreign Ag and the contents of goblet cells.

Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules.

PubMed

Rees, Jennifer; Haig, David; Mack, Victoria; Davis, William C

2017-01-01

Flow cytometry was used to identify mAbs that recognize conserved epitopes on hamster leukocyte differentiation molecules (hLDM) and also to characterize mAbs developed against hLDM. Initial screening of mAbs developed against LDMs in other species yielded mAbs specific for the major histocompatibility (MHC) II molecule, CD4 and CD18. Screening of sets of mAbs developed against hLDM yielded 22 new mAbs, including additional mAbs to MHC II molecules and mAbs that recognize LDMs expressed on all leukocytes, granulocytes, all lymphocytes, all T cells, a subset of T cells, or on all B cells. Based on comparison of the pattern of expression of LDMs expressed on all hamster leukocytes with the patterns of expression of known LDMs in other species, as detected by flow cytometry (FC), four mAbs are predicted to recognize CD11a, CD44, and CD45. Cross comparison of mAbs specific for a subset of hamster T cells with a cross reactive mAb known to recognize CD4 in mice and one recognising CD8 revealed they recognize CD4. The characterization of these mAbs expands opportunities to use hamsters as an additional model species to investigate the mechanisms of immunopathogenesis of infectious diseases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor.

PubMed

Hinson, Shannon R; Clift, Ian C; Luo, Ningling; Kryzer, Thomas J; Lennon, Vanda A

2017-05-23

Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR's gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG-AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO.

Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor

PubMed Central

Hinson, Shannon R.; Clift, Ian C.; Luo, Ningling; Kryzer, Thomas J.; Lennon, Vanda A.

2017-01-01

Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR’s gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG–AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO. PMID:28461494

Engineering of Fc Fragments with Optimized Physicochemical Properties Implying Improvement of Clinical Potentials for Fc-Based Therapeutics.

PubMed

Yang, Chunpeng; Gao, Xinyu; Gong, Rui

2017-01-01

Therapeutic monoclonal antibodies and Fc-fusion proteins are successfully used in treatment of various diseases mainly including cancer, immune disease, and viral infection, which belong to the Fc-based therapeutics. In recent years, engineered Fc-derived antibody domains have also shown potential for Fc-based therapeutics. To increase the druggability of Fc-based therapeutic candidates, many efforts have been made in optimizing physicochemical properties and functions mediated by Fc fragment. The desired result is that we can simultaneously obtain Fc variants with increased physicochemical properties in vitro and capacity of mediating appropriate functions in vivo . However, changes of physicochemical properties of Fc may result in alternation of Fc-mediated functions and vice versa , which leads to undesired outcomes for further development of Fc-based therapeutics. Therefore, whether modified Fc fragments are suitable for achievement of expected clinical results or not needs to be seriously considered. Now, this question comes to be noticed and should be figured out to make better translation from the results of laboratory into clinical applications. In this review, we summarize different strategies on engineering physicochemical properties of Fc, and preliminarily elucidate the relationships between modified Fc in vitro and the subsequent therapeutic influence in vivo .

Advances in Therapeutic Fc Engineering – Modulation of IgG-Associated Effector Functions and Serum Half-life

PubMed Central

Saxena, Abhishek; Wu, Donghui

2016-01-01

Today, monoclonal immunoglobulin gamma (IgG) antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs) is achieved through both its antigen-binding fragment (Fab) and crystallizable fragment (Fc). Fab can specifically recognize tumor-associated antigen (TAA) and thus modulate TAA-linked downstream signaling pathways that may lead to the inhibition of tumor growth, induction of tumor apoptosis, and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cell-mediated phagocytosis. Moreover, Fc is the region interacting with the neonatal Fc receptor in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anticancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce the cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering-based mAbs under clinical trials. PMID:28018347

Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

PubMed

Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

2010-02-15

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

Engineering of Fc Fragments with Optimized Physicochemical Properties Implying Improvement of Clinical Potentials for Fc-Based Therapeutics

PubMed Central

Yang, Chunpeng; Gao, Xinyu; Gong, Rui

2018-01-01

Therapeutic monoclonal antibodies and Fc-fusion proteins are successfully used in treatment of various diseases mainly including cancer, immune disease, and viral infection, which belong to the Fc-based therapeutics. In recent years, engineered Fc-derived antibody domains have also shown potential for Fc-based therapeutics. To increase the druggability of Fc-based therapeutic candidates, many efforts have been made in optimizing physicochemical properties and functions mediated by Fc fragment. The desired result is that we can simultaneously obtain Fc variants with increased physicochemical properties in vitro and capacity of mediating appropriate functions in vivo. However, changes of physicochemical properties of Fc may result in alternation of Fc-mediated functions and vice versa, which leads to undesired outcomes for further development of Fc-based therapeutics. Therefore, whether modified Fc fragments are suitable for achievement of expected clinical results or not needs to be seriously considered. Now, this question comes to be noticed and should be figured out to make better translation from the results of laboratory into clinical applications. In this review, we summarize different strategies on engineering physicochemical properties of Fc, and preliminarily elucidate the relationships between modified Fc in vitro and the subsequent therapeutic influence in vivo. PMID:29375551

The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy.

PubMed

Sockolosky, Jonathan T; Szoka, Francis C

2015-08-30

Immunoglobulin G (IgG)-based drugs are arguably the most successful class of protein therapeutics due in part to their remarkably long blood circulation. This arises from IgG interaction with the neonatal Fc receptor, FcRn. FcRn is the central regulator of IgG and albumin homeostasis throughout life and is increasingly being recognized as an important player in autoimmune disease, mucosal immunity, and tumor immune surveillance. Various engineering approaches that hijack or disrupt the FcRn-mediated transport pathway have been devised to develop long-lasting and non-invasive protein therapeutics, protein subunit vaccines, and therapeutics for treatment of autoimmune and infectious disease. In this review, we highlight the diverse biological functions of FcRn, emerging therapeutic opportunities, as well as the associated challenges of targeting FcRn for drug delivery and disease therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Inhibition of th17 cells and promotion of tregs in fc gamma chain-deficient mice contributes to the attenuated atherosclerotic lesions

USDA-ARS?s Scientific Manuscript database

The presence of anti-oxLDL IgG is well documented in clinical and animal studies. However, the role for Fc Rs to the progression of atherosclerosis has not been studied in detail. In the present study, we investigated the role for activating Fc R in the progression of atherosclerosis using apoE-Fc -...

Influence of variants of Fc gamma receptors IIA and IIIA on the American College of Rheumatology and European League Against Rheumatism responses to anti-tumour necrosis factor alpha therapy in rheumatoid arthritis.

PubMed

Cañete, J D; Suárez, B; Hernández, M V; Sanmartí, R; Rego, I; Celis, R; Moll, C; Pinto, J A; Blanco, F J; Lozano, F

2009-10-01

Fc gamma receptor (Fc gammaR) polymorphism influences the affinity of the receptor for Ig, which may, in turn, affect the efficacy of Ig-based therapies. The relationship between functional single nucleotide polymorphisms (SNP) of the FCGR2A and FCGR3A genes and the response to anti-tumour necrosis factor (TNF)alpha therapy (infliximab) in patients with rheumatoid arthritis (RA) was assessed. A total of 91 patients with RA (89% female; 76.7% rheumatoid factor (RF) positive) starting therapy with infliximab were evaluated at 0, 6 and 30 weeks using the American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) response criteria and the 28-joint Disease Activity Score (DAS28) was evaluated using three parameters, including C-reactive protein (CRP) (DAS28 3v-CRP) changes during the follow-up. Genotyping of FCGR2A-R131H and FCGR3A-F158V polymorphisms was performed by allele-specific PCR and PCR sequence-based typing, respectively. The chi(2) and Fisher exact tests were used to show differences in the outcome variables, and analysis of variance (ANOVA) to analyse the evolution of DAS28 3v-CRP. A generalised linear models multivariable analysis was also performed. At week 6 of follow-up, the proportion of patients achieving 50% improvement as per ACR criteria (ACR50) and EULAR good responses were significantly higher among homozygotes of the low affinity FCGR3A allele (FF: 24.1% and VV-VF:2.2%; p = 0.003 and FF: 44.8% and VV-VF: 22.9%; p = 0.040, respectively). At week 30, homozygotes of the low affinity FCGR2A allele had a better ACR20 response (RR: 60% and HH-RH: 33.3%; p = 0.035). Changes in DAS28 3v-CRP during follow-up were consistent with those observed in ACR and EULAR responses. The response to anti-TNFalpha treatment with infliximab in patients with RA is influenced by the FCGR2A and FCGR3A genotypes. This effect is observed at different times in the follow-up (6 and 30 weeks, respectively) indicating the dynamic nature of the Fc gamma

Enhancement of antibody functions through Fc multiplications

PubMed Central

Wang, Qun; Cvitkovic, Romana; Bonnell, Jessica; Chang, Chien-Ying; Koksal, Adem C.; O'Connor, Ellen; Gao, Xizhe; Yu, Xiang-Qing; Wu, Herren; Stover, C. Kendall; Dall'Acqua, William F.; Xiao, Xiaodong

2017-01-01

ABSTRACT Antibodies carry out a plethora of functions through their crystallizable fragment (Fc) regions, which can be naturally tuned by the adoption of several isotypes and post-translational modifications. Protein engineering enables further Fc function modulations through modifications of the interactions between the Fc and its functional partners, including FcγR, FcRn, complement complex, and additions of auxiliary functional units. Due to the many functions embedded within the confinement of an Fc, a suitable balance must be maintained for a therapeutic antibody to be effective and safe. The outcome of any Fc engineering depends on the interplay among all the effector molecules involved. In this report, we assessed the effects of Fc multiplication (or tandem Fc) on antibody functions. Using IgG1 as a test case, we found that, depending on the specifically designed linker, Fc multiplication led to differentially folded, stable molecules with unique pharmacokinetic profiles. Interestingly, the variants with 3 copies of Fc improved in vitro opsonophagocytic killing activity and displayed significantly improved protective efficacies in a Klebsiella pneumoniae mouse therapeutic model despite faster clearance compared with its IgG1 counterpart. There was no adverse effect observed or pro-inflammatory cytokine release when the Fc variants were administered to animals. We further elucidated that enhanced binding to various effector molecules by IgG-3Fc created a “sink” leading to the rapid clearance of the 3Fc variants, and identified the increased FcRn binding as one strategy to facilitate “sink” escape. These findings reveal new opportunities for novel Fc engineering to further expand our abilities to manipulate and improve antibody therapeutics. PMID:28102754

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

PubMed

Rispens, Theo; Himly, Martin; Ooievaar-De Heer, Pleuni; den Bleker, Tamara H; Aalberse, Rob C

2010-04-16

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. 2010 Elsevier B.V. All rights reserved.

FcγRII-binding Centyrins mediate agonism and antibody-dependent cellular phagocytosis when fused to an anti-OX40 antibody

PubMed Central

Zhang, Di; Whitaker, Brian; Derebe, Mehabaw G.; Chiu, Mark L.

2018-01-01

ABSTRACT Immunostimulatory antibodies against the tumor necrosis factor receptors (TNFR) are emerging as promising cancer immunotherapies. The agonism activity of such antibodies depends on crosslinking to Fc gamma RIIB receptor (FcγRIIB) to enable the antibody multimerization that drives TNFR activation. Previously, Fc engineering was used to enhance the binding of such antibodies to Fcγ receptors. Here, we report the identification of Centyrins as alternative scaffold proteins with binding affinities to homologous FcγRIIB and FcγRIIA, but not to other types of Fcγ receptors. One Centyrin, S29, was engineered at distinct positions of an anti-OX40 SF2 antibody to generate bispecific and tetravalent molecules named as mAbtyrins. Regardless of the position of S29 on the SF2 antibody, SF2-S29 mAbtyrins could bind FcγRIIB and FcγRIIA specifically while maintaining binding to OX40 receptors. In a NFκB reporter assay, attachment of S29 Centyrin molecules at the C-termini, but not the N-termini, resulted in SF2 antibodies with increased agonism owing to FcγRIIB crosslinking. The mAbtyrins also showed agonism in T-cell activation assays with immobilized FcγRIIB and FcγRIIA, but this activity was confined to mAbtyrins with S29 specifically at the C-termini of antibody heavy chains. Furthermore, regardless of the position of the molecule, S29 Centyrin could equip an otherwise Fc-silent antibody with antibody-dependent cellular phagocytosis activity without affecting the antibody's intrinsic antibody-dependent cell-meditated cytotoxicity and complement-dependent cytotoxicity. In summary, the appropriate adoption FcγRII-binding Centyrins as functional modules represents a novel strategy to engineer therapeutic antibodies with improved functionalities. PMID:29359992

FcUni-RLuc: an engineered Renilla luciferase with Fc binding ability and light emission activity.

PubMed

Farzannia, A; Roghanian, R; Zarkesh-Esfahani, S H; Nazari, M; Emamzadeh, R

2015-03-07

A novel and advanced Fc-binding probe – FcUni-RLuc namely – has been produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence – HWRGWV – was fused to Renilla luciferase, and the purified probe was employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells.

Rapid desensitization of mice with anti-FcγRIIb/FcγRIII mAb safely prevents IgG-mediated anaphylaxis.

PubMed

Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Clay, Corey D; Morris, Suzanne C; Finkelman, Fred D

2013-12-01

Stimulatory IgG receptors (FcγRs) on bone marrow-derived cells contribute to the pathogenesis of several autoimmune and inflammatory disorders. Monoclonal antibodies that block FcγRs might suppress these diseases, but they can induce anaphylaxis. We wanted to determine whether a rapid desensitization approach can safely suppress IgG/FcγR-mediated anaphylaxis. Mice were injected with serially increasing doses of 2.4G2, a rat mAb that blocks the inhibitory FcγR, FcγRIIb, and the stimulatory receptor, FcγRIII. Rectal temperature was used to detect the development of anaphylaxis. Passive and active IgG-mediated anaphylaxis were evaluated in mice that had been rapidly desensitized with 2.4G2 or mock-desensitized in mice in which monocyte/macrophages, basophils, or neutrophils had been depleted or desensitized and in mice in which FcγRI, FcγRIII, and/or FcγRIV had been deleted or blocked. Rapid desensitization with 2.4G2 prevented 2.4G2-induced shock and completely suppressed IgG-mediated anaphylaxis. Rapid desensitization of ovalbumin-sensitized mice with 2.4G2 was safer and more effective than rapid desensitization with ovalbumin. 2.4G2 treatment completely blocked FcγRIII and removed most FcγRI and FcγRIV from nucleated peripheral blood cells. Because IgG(2a)-mediated anaphylaxis was partially FcγRI and FcγRIV dependent, the effects of 2.4G2 on FcγRI and FcγRIV were probably crucial for its complete inhibition of IgG(2a)-mediated anaphylaxis. IgG(2a)-mediated anaphylaxis was partially inhibited by depletion or desensitization of monocyte/macrophages, basophils, or neutrophils. IgG-mediated anaphylaxis can be induced by ligation of FcγRI, FcγRIII, or FcγRIV on monocycte/macrophages, basophils, or neutrophils and can be safely suppressed by rapid desensitization with anti-FcγRII/RIII mAb. A similar approach may safely suppress other FcγR-dependent immunopathology. Published by Mosby, Inc.

Fc-fusion Proteins in Therapy: An Updated View.

PubMed

Jafari, Reza; Zolbanin, Naime M; Rafatpanah, Houshang; Majidi, Jafar; Kazemi, Tohid

2017-01-01

Fc-fusion proteins are composed of Fc region of IgG antibody (Hinge-CH2-CH3) and a desired linked protein. Fc region of Fc-fusion proteins can bind to neonatal Fc receptor (FcRn) thereby rescuing it from degradation. The first therapeutic Fc-fusion protein was introduced for the treatment of AIDS. The molecular designing is the first stage in production of Fc-fusion proteins. The amino acid residues in the Fc region and linked protein are very important in the bioactivity and affinity of the fusion proteins. Although, therapeutic monoclonal antibodies are the top selling biologics but the application of therapeutic Fc-fusion proteins in clinic is in progress and among these medications Etanercept is the most effective in therapy. At present, eleven Fc-fusion proteins have been approved by FDA. There are novel Fc-fusion proteins which are in pre-clinical and clinical development. In this article, we review the molecular and biological characteristics of Fc-fusion proteins and then further discuss the features of novel therapeutic Fc-fusion proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

PubMed

Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

2015-01-01

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding*

PubMed Central

Andersen, Jan Terje; Dalhus, Bjørn; Viuff, Dorthe; Ravn, Birgitte Thue; Gunnarsen, Kristin Støen; Plumridge, Andrew; Bunting, Karen; Antunes, Filipa; Williamson, Rebecca; Athwal, Steven; Allan, Elizabeth; Evans, Leslie; Bjørås, Magnar; Kjærulff, Søren; Sleep, Darrell; Sandlie, Inger; Cameron, Jason

2014-01-01

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals. PMID:24652290

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

PubMed Central

King, Amy C.; Kavosi, Mania; Wang, Mengmeng; O'Hara, Denise M.; Tchistiakova, Lioudmila; Katragadda, Madan

2018-01-01

ABSTRACT A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics. PMID:28991504

Fc Gamma Receptor 3A Polymorphism and Risk for HIV-Associated Cryptococcal Disease

PubMed Central

Rohatgi, Soma; Gohil, Shruti; Kuniholm, Mark H.; Schultz, Hannah; Dufaud, Chad; Armour, Kathryn L.; Badri, Sheila; Mailliard, Robbie B.; Pirofski, Liise-anne

2013-01-01

ABSTRACT Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4+ T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4+ T cell decline, and nadir CD4+ T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation. PMID:23982074

HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

PubMed

Cox, Freek; Kwaks, Ted; Brandenburg, Boerries; Koldijk, Martin H; Klaren, Vincent; Smal, Bastiaan; Korse, Hans J W M; Geelen, Eric; Tettero, Lisanne; Zuijdgeest, David; Stoop, Esther J M; Saeland, Eirikur; Vogels, Ronald; Friesen, Robert H E; Koudstaal, Wouter; Goudsmit, Jaap

2016-01-01

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc-FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

Human FcγRIIA induces anaphylactic and allergic reactions

PubMed Central

Jönsson, Friederike; Mancardi, David A.; Zhao, Wei; Kita, Yoshihiro; Iannascoli, Bruno; Khun, Huot; van Rooijen, Nico; Shimizu, Takao; Schwartz, Lawrence B.; Daëron, Marc

2012-01-01

IgE and IgE receptors (FcϵRI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (FcγRIIIA and FcγRIV) expressed by neutrophils, rather than on IgE and FcϵRI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express FcγRIIIA or FcγRIV, but FcγRIIA. We therefore investigated the possible role of FcγRIIA in allergy by generating novel FcγRIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, FcγRIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking FcγRIIA in vivo abolished these reactions. We identified mast cells to be responsible for FcγRIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for FcγRIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after FcγRIIA engagement. IgG and FcγRIIA may therefore contribute to allergic and anaphylactic reactions in humans. PMID:22138510

Human FcγRIIA induces anaphylactic and allergic reactions.

PubMed

Jönsson, Friederike; Mancardi, David A; Zhao, Wei; Kita, Yoshihiro; Iannascoli, Bruno; Khun, Huot; van Rooijen, Nico; Shimizu, Takao; Schwartz, Lawrence B; Daëron, Marc; Bruhns, Pierre

2012-03-15

IgE and IgE receptors (FcεRI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (FcγRIIIA and FcγRIV) expressed by neutrophils, rather than on IgE and FcεRI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express FcγRIIIA or FcγRIV, but FcγRIIA. We therefore investigated the possible role of FcγRIIA in allergy by generating novel FcγRIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, FcγRIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking FcγRIIA in vivo abolished these reactions. We identified mast cells to be responsible for FcγRIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for FcγRIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after FcγRIIA engagement. IgG and FcγRIIA may therefore contribute to allergic and anaphylactic reactions in humans.

Associations between Fc gamma receptor IIA polymorphisms and the risk and prognosis of meningococcal disease.

PubMed

Domingo, Pere; Muñiz-Diaz, Eduardo; Baraldès, Maria A; Arilla, Marina; Barquet, Nicolau; Pericas, Roser; Juárez, Cándido; Madoz, Pedro; Vázquez, Guillermo

2002-01-01

In vitro studies have shown that the neutrophil Fc gamma receptor IIA (FcgammaRIIA) polymorphism influences the phagocytic capacity of neutrophils and the removal of encapsulated bacteria from the bloodstream. In particular, the R/R131 allotype is associated with less phagocytic activity. We performed a case-control study to determine the influence of the FcgammaRIIA polymorphism (R/R131, R/H131, H/H131) on the risk and outcome of meningococcal disease. The polymorphisms were measured in 130 patients with microbiologically proven meningococcal disease diagnosed from 1987 to 1998 (cases) and 260 asymptomatic sex-matched blood donors (controls). Clinical manifestations and complications of meningococcal disease were recorded, and a prognostic score (based on age, hemorrhagic diathesis, neurologic signs, and the absence of preadmission antibiotic) therapy was calculated. The distributions of FcgammaRIIA allotypes were similar in cases and controls. However, among patients with meningococcal infection, fulminant meningococcal disease (odds ratio [OR] = 3.9; 95% confidence interval [CI]: 1.0 to 16; P = 0.04) and meningococcemia without meningitis (OR = 3.0; 95% CI: 1.4 to 7.8; P = 0.004) were more common in those with the FcgammaRIIA-R/R131 allotype. Complications were also significantly more frequent in these patients. Of the 42 patients with the R/R131 allotype, 31 (74%) had an adverse prognostic score, compared with 7% (4 of 59) of those with the R/H131 allotype and 3% (1 of 29) of those with the H/H131 allotype (P <0.0001). The FcgammaRIIA-R/R131 allotype is associated with more severe forms of meningococcal disease.

Integrated profiling of Furanocoumarins (FC) in Grapefruit hybrids toward selection of low FC varieties

USDA-ARS?s Scientific Manuscript database

Furanocoumarins (FC) are a class of organic chemical components in grapefruits and other diet plants. Some of them in grapefruit juice can induce potentially adverse interactions with human drugs and in that patients may be advised to avoid the fruit and juice. To develop low FC grapefruit cultivars...

Reduced antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells of salivary polymorphonuclear leukocytes and inhibition of peripheral blood polymorphonuclear leukocyte cytotoxicity by saliva.

PubMed

Ashkenazi, M; Kohl, S

1990-06-15

Blood polymorphonuclear leukocytes (BPMN) have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against HSV-infected cells. Although HSV infections are frequently found in the oral cavity, the ADCC capacity of salivary PMN (SPMN) has not been studied, mainly because methods to isolate SPMN were not available. We have recently developed a method to isolate SPMN, and in this study have evaluated their ADCC activity against HSV-infected cells. SPMN were obtained by repeated washings of the oral cavity, and separated from epithelial cells by nylon mesh filtration. ADCC was quantitatively determined by 51Cr release from HSV-infected Chang liver cells. SPMN in the presence of antibody were able to destroy HSV-infected cells, but SPMN were much less effective in mediating ADCC than BPMN (3.4% vs 40.7%, p less than 0.0001). In the presence of antiviral antibody, SPMN were able to adhere to HSV-infected cells, but less so than BPMN (34% vs 67%), and specific antibody-induced adherence was significantly lower in SPMN (p less than 0.04). The spontaneous adherence to HSV-infected cells was higher for SPMN than BPMN. SPMN demonstrated up-regulation of the adhesion glycoprotein CD18, but down-regulation of the FcRIII receptor. Incubation with saliva decreased ADCC capacity of BPMN, up-regulated CD18 expression, and down-regulated FcRIII expression.

Mechanisms of lectin and antibody-dependent polymorphonuclear leukocyte-mediated cytolysis.

PubMed

Tsunawaki, S; Ikenami, M; Mizuno, D; Yamazaki, M

1983-04-01

The mechanisms of tumor lysis by polymorphonuclear leukocytes (PMNs) were investigated. In antibody-dependent PMN-mediated cytolysis (ADPC), sensitized tumor cells were specifically lysed via Fc receptors on PMNs. On the other hand, lectin-dependent PMN-mediated cytolysis (LDPC) caused nonspecific lysis of several murine tumors after recognition of carbohydrate moieties on the cell membrane of both PMNs and tumor cells. Both ADPC and LDPC depended on glycolysis, and cytotoxicity was mediated by reactive oxygen species; LDPC was dependent on superoxide and ADPC on the myeloperoxidase system. The participation of reactive oxygen species in PMN cytotoxicity was also demonstrated by pharmacological triggering with phorbol myristate acetate. These results indicate that reactive oxygen species have an important role In tumor killing by PMNs and that ADPC and LDPC have partly different cytolytic processes as well as different recognition steps.

Mechanisms and regulation of polymorphonuclear leukocyte and eosinophil adherence to human airway epithelial cells.

PubMed

Jagels, M A; Daffern, P J; Zuraw, B L; Hugli, T E

1999-09-01

Polymorphonuclear leukocytes (PMN) and eosinophils (Eos) are important cellular participants in a variety of acute and chronic inflammatory reactions in the airway. Histologic evidence has implicated direct interactions between these two subsets of leukocytes and airway epithelial cells during inflammation. A comprehensive characterization and comparison of physiologic stimuli and adhesion molecule involvement in granulocyte-epithelial-cell interactions done with nontransformed human airway epithelial cells has not been reported. We therefore examined the regulation and biochemical mechanisms governing granulocyte-epithelial-cell adhesion, using either purified PMN or Eos and primary cultures of human bronchial epithelial cells (HBECs). We investigated the involvement of a number of proinflammatory signals associated with allergic and nonallergic airway inflammation, as well as the contribution of several epithelial and leukocyte adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and members of the beta(1), beta(2), and beta(7) integrin families. ICAM-1 was expressed at low levels on cultured HBECs and was markedly upregulated after stimulation with interferon (IFN)-gamma or, to a lesser extent, with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1. VCAM-1 was not present on resting HBECs, and was not upregulated after stimulation with IFN-gamma, IL-1, IL-4, or TNF-alpha. PMN adhesion to HBECs could be induced either through activation of PMN with IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), or C5a, but not with IL-5 or by preactivation of HBECs with TNF-alpha or IFN-gamma. Blocking antibody studies indicated that PMN-HBEC adherence depended on beta(2) integrins, primarily alpha(M)beta(2) (Mac-1). Adherence of Eos to HBECs could be induced through activation of Eos with IL-5, GM-CSF, or C5a, but not with IL-8 or by prior activation of HBECs with TNF-alpha of IFN-gamma

Composition and functional capacity of blood mononuclear leukocyte populations from neonatal calves on standard and intensified milk replacer diets.

PubMed

Nonnecke, B J; Foote, M R; Smith, J M; Pesch, B A; Van Amburgh, M E

2003-11-01

Effects of increased dietary energy and protein on the composition and functional capacities of blood mononuclear leukocyte populations from milk replacer-fed calves were investigated. Holstein bull calves (average age: 4.2 d; n = 19) were assigned randomly to one of two treatment groups. Treatment 1 calves (n = 9) were fed a 20% crude protein, 20% fat milk replacer at a rate of 1.4% body weight of dry matter/d for 8 wk, whereas treatment 2 calves (n = 10) were fed a 30% crude protein, 20% fat milk replacer at a rate of 2.5% body weight of dry matter per day. Composition and functional capacities of mononuclear leukocyte populations from blood samples collected at 4, 18, 32, 46, and 60 d of age were characterized by flow cytometry and ex vivo cell function assays. From 11 to 60 d of age, the mean daily weight gain of treatment 2 calves (1.20 kg/d) was greater than daily weight gain of treatment 1 calves (0.55 kg/d). At 60 d of age, the mean body weight of treatment two calves was 53% (39 kg) greater than the mean body weight of treatment 1 calves. Total numbers of blood leukocytes and the composition of the mononuclear leukocyte population were unaffected by the plane of nutrition. Mitogen-induced DNA-synthesis and immunoglobulin M secretion also were unaffected by dietary treatment. Blood mononuclear leukocytes from calves on intensified diets, however, produced less interferon-gamma and more inducible nitric oxide, suggesting that increased dietary energy and protein affects specific aspects of leukocyte function associated with cell-mediated immunity. The impact of altered interferon-gamma and NO production on the calf s susceptibility to infectious disease are not known. Mononuclear leukocyte populations from all calves also demonstrated age-related changes in composition and functional capacity, likely reflecting natural exposure to infectious agents and maturation of the calfs immune system.

HAL/S-FC compiler system specifications

NASA Technical Reports Server (NTRS)

1976-01-01

This document specifies the informational interfaces within the HAL/S-FC compiler, and between the compiler and the external environment. This Compiler System Specification is for the HAL/S-FC compiler and its associated run time facilities which implement the full HAL/S language. The HAL/S-FC compiler is designed to operate stand-alone on any compatible IBM 360/370 computer and within the Software Development Laboratory (SDL) at NASA/JSC, Houston, Texas.

Correlation of leukocyte adhesiveness, adhesion molecule expression and leukocyte-induced contraction following balloon angioplasty

PubMed Central

Kennedy, Simon; McPhaden, Allan R; Wadsworth, Roger M; Wainwright, Cherry L

2000-01-01

The aim of this study was to examine the changes in leukocyte adhesion and leukocyte-induced contraction in balloon-injured rabbit subclavian artery and to correlate these changes with vessel morphology and expression of adhesion molecules on the injured arteries.Rabbits were anaesthetized and their left subclavian arteries were injured by balloon inflation and withdrawal followed by sacrifice at 2, 24, 48 h or 8 days after injury. The left and right subclavian arteries were removed and leukocytes were isolated from autologous rabbit blood. Leukocyte-induced contraction was measured in 5-HT precontracted artery rings and leukocyte adhesion was measured using 51Cr-labelled leukocytes. Immunocytochemistry using paraffin-embedded tissue was employed to detect changes in the expression of adhesion molecules on injured arteries.Autologous leukocytes caused a contraction of rabbit subclavian artery rings, which was prevented by L-NAME (10−3 M). Balloon-induced injury abolished the contractile response to leukocytes, which correlated with loss of carbachol-induced relaxationBalloon injury markedly enhanced the adhesiveness of the subclavian artery for leukocytes, most notably at 24 and 48 h after injury (1.7 and 1.8 fold respectively). Increased leukocyte adhesion at these two time points correlated with an upregulation of E-selectin, P-selectin and VCAM-1 expression on the remaining endothelium of the injured artery.Vessel morphology revealed that balloon inflation had induced an infiltration of inflammatory cells into the vessel wall, the greatest increase being seen at 24 h after injury.It is concluded that an increase in the expression of E-selectin, P-selectin and VCAM-1 following balloon-induced injury leads to enhanced leukocyte adhesion and migration into the injured vessel. PMID:10781003

CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

USDA-ARS?s Scientific Manuscript database

After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

The novel multispecies Fc-specific Pseudomonas exotoxin A fusion protein α-Fc-ETA' enables screening of antibodies for immunotoxin development.

PubMed

Klausz, Katja; Kellner, Christian; Derer, Stefanie; Valerius, Thomas; Staudinger, Matthias; Burger, Renate; Gramatzki, Martin; Peipp, Matthias

2015-03-01

Immunoconjugates that deliver cytotoxic payloads to cancer cells represent a promising class of therapeutic agents which are intensively investigated in various clinical applications. Prerequisites for the generation of effective immunoconjugates are antibodies which efficiently deliver the respective cytotoxic payload. To facilitate the selection of human or mouse antibodies that display favorable characteristics as immunotoxins, we developed a novel Pseudomonas exotoxin A (ETA)-based screening protein. The α-Fc-ETA' consists of a multispecies-specific Fc-binding domain antibody genetically fused to a truncated ETA version (ETA'). α-Fc-ETA' non-covalently bound to human and mouse antibodies but did not form immune complexes with bovine immunoglobulins. In combination with antibodies harboring human or mouse Fc domains α-Fc-ETA' inhibited proliferation of antigen-expressing tumor cells. The cytotoxic effects were strictly antibody dependent and were observed with low α-Fc-ETA' concentrations. Mouse antibodies directed against CD7 and CD317/HM1.24 that previously had been used for the generation of functional recombinant immunotoxins, also showed activity in combination with α-Fc-ETA' by inhibiting growth of antigen-positive myeloma and leukemia cell lines. In contrast, α-kappa-ETA', a similarly designed human kappa light chain-specific fusion protein, was only specifically active in combination with antibodies containing a human kappa light chain. Thus, the novel α-Fc-ETA' fusion protein is broadly applicable in screening antibodies and Fc-containing antibody derivatives from different species to select for candidates with favorable characteristics for immunotoxin development. Copyright © 2015 Elsevier B.V. All rights reserved.

The effect of FcγRIIA and FcγRIIB on coronary artery lesion formation and intravenous immunoglobulin treatment responses in children with Kawasaki disease

PubMed Central

Chang, Ling-Sai; Lo, Mao-Hung; Li, Sung-Chou; Yang, Ming-Yu; Hsieh, Kai-Sheng; Kuo, Ho-Chang

2017-01-01

Previous research has found patients with the FcγRIIIB NA1 variant having increased risk of intravenous immunoglobulin (IVIG) resistance in Kawasaki disease (KD). Our previous studies revealed that elevated FcγRIIA expression correlated with the susceptibility of KD patients. We conducted this research to determine whether and how Fcγ receptors affect the susceptibility, IVIG treatment response, and coronary artery lesions (CAL) of KD patients. The activating FcγRIIA and inhibitory FcγRIIB methylation levels of seven patients with KD and four control subjects were examined using HumanMethylation27 BeadChip. We enrolled a total of 44 KD patients and 10 control subjects with fevers. We performed real-time RT-PCR to determine the FcγRIIA and FcγRIIB expression levels, as well as a luciferase assay of FcγRIIA. We found a considerable increase in methylation of both FcγRIIA and FcγRIIB in KD patients undergoing IVIG treatment. Promoter methylation of FcγRIIA inhibited reporter activity in K562 cells using luciferase assay. The FcγRIIB mRNA expression levels were not found to increase susceptibility, CAL formation, or IVIG resistance. FcγRIIA mRNA expression levels were significantly higher in IVIG-resistant patients than in those that responded to IVIG during the pre-treatment period. Furthermore, the FcγRIIA/IIB mRNA expression ratio was considerably higher in KD patients with CAL than in those without CAL. FcγRIIA and FcγRIIB both demonstrated increased methylation levels in KD patients that underwent IVIG treatment. FcγRIIA expression influenced the IVIG treatment response of KD patients. The FcγRIIA/IIB mRNA expression ratio was greater in KD patients with CAL formation. PMID:27893416

The Major Histocompatibility Complex–related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan

PubMed Central

Chaudhury, Chaity; Mehnaz, Samina; Robinson, John M.; Hayton, William L.; Pearl, Dennis K.; Roopenian, Derry C.; Anderson, Clark L.

2003-01-01

The inverse relationship between serum albumin concentration and its half-life suggested to early workers that albumin would be protected from a catabolic fate by a receptor-mediated mechanism much like that proposed for IgG. We show here that albumin binds FcRn in a pH dependent fashion, that the lifespan of albumin is shortened in FcRn-deficient mice, and that the plasma albumin concentration of FcRn-deficient mice is less than half that of wild-type mice. These results affirm the hypothesis that the major histocompatibility complex–related Fc receptor protects albumin from degradation just as it does IgG, prolonging the half-lives of both. PMID:12566415

[Exceptional production of leukocyte-free erythrocyte concentrates using filtration with the BPF 4 BBS leukocyte filter].

PubMed

Zimmermann, B; Hillringhaus, I; Diekamp, U

1994-01-01

Leukocyte depletion prevents some undesirable effects of red cell transfusions. We evaluated the blood filter system BPF 4 BBS (PALL) by filtering 26 units of buffy coat-free red cell concentrates, prepared from 500 ml of whole blood and suspended in SAG-M-additive solution, over 8 min within 48 h after donation at room temperature. Filtration reduced the leukocyte content by more than 99.99% in 25 of the 26 units examined. In 20 instances, no leukocyte was found in the Nageotte counting chamber (2 x 50 microliters) corresponding to a total leukocyte count below 2.38 x 10(4). In 5 instances a single leukocyte was seen, corresponding to a total leukocyte count of (2.35 +/- 0.01) x 10(4). One unit contained 2.93 x 10(5) leukocytes after filtration, corresponding to a 99.98% leukocyte reduction. Red cell recovery was 92.5 +/- 3.8%. This system is well suited for routine use.

Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects

PubMed Central

Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Garrett, Logan; Forte, Carla; Woodward, Anne; Ng, Soo Bin; Born, Teresa; Retter, Marc; Manchulenko, Kathy; Sweet, Heather; Foltz, Ian N.; Wittekind, Michael; Yan, Wei

2010-01-01

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications. PMID:20400508

Transmembrane domain dependent inhibitory function of FcγRIIB.

PubMed

Wang, Junyi; Li, Zongyu; Xu, Liling; Yang, Hengwen; Liu, Wanli

2018-03-01

FcγRIIB, the only inhibitory IgG Fc receptor, functions to suppress the hyper-activation of immune cells. Numerous studies have illustrated its inhibitory function through the ITIM motif in the cytoplasmic tail of FcγRIIB. However, later studies revealed that in addition to the ITIM, the transmembrane (TM) domain of FcγRIIB is also indispensable for its inhibitory function. Indeed, recent epidemiological studies revealed that a non-synonymous single nucleotide polymorphism (rs1050501) within the TM domain of FcγRIIB, responsible for the I232T substitution, is associated with the susceptibility to systemic lupus erythematosus (SLE). In this review, we will summarize these epidemiological and functional studies of FcγRIIB-I232T in the past few years, and will further discuss the mechanisms accounting for the functional loss of FcγRIIB-I232T. Our review will help the reader gain a deeper understanding of the importance of the TM domain in mediating the inhibitory function of FcγRIIB and may provide insights to a new therapeutic target for the associated diseases.

The Role of CD38 in Fcγ Receptor (FcγR)-mediated Phagocytosis in Murine Macrophages*

PubMed Central

Kang, John; Park, Kwang-Hyun; Kim, Jwa-Jin; Jo, Eun-Kyeong; Han, Myung-Kwan; Kim, Uh-Hyun

2012-01-01

Phagocytosis is a crucial event in the immune system that allows cells to engulf and eliminate pathogens. This is mediated through the action of immunoglobulin (IgG)-opsonized microbes acting on Fcγ receptors (FcγR) on macrophages, which results in sustained levels of intracellular Ca2+ through the mobilization of Ca2+ second messengers. It is known that the ADP-ribosyl cyclase is responsible for the rise in Ca2+ levels after FcγR activation. However, it is unclear whether and how CD38 is involved in FcγR-mediated phagocytosis. Here we show that CD38 is recruited to the forming phagosomes during phagocytosis of IgG-opsonized particles and produces cyclic-ADP-ribose, which acts on ER Ca2+ stores, thus allowing an increase in FcγR activation-mediated phagocytosis. Ca2+ data show that pretreatment of J774A.1 macrophages with 8-bromo-cADPR, ryanodine, blebbistatin, and various store-operated Ca2+ inhibitors prevented the long-lasting Ca2+ signal, which significantly reduced the number of ingested opsonized particles. Ex vivo data with macrophages extracted from CD38−/− mice also shows a reduced Ca2+ signaling and phagocytic index. Furthermore, a significantly reduced phagocytic index of Mycobacterium bovis BCG was shown in macrophages from CD38−/− mice in vivo. This study suggests a crucial role of CD38 in FcγR-mediated phagocytosis through its recruitment to the phagosome and mobilization of cADPR-induced intracellular Ca2+ and store-operated extracellular Ca2+ influx. PMID:22396532

Comparison of technetium-99m-HM-PAO leukocytes with indium-111-oxine leukocytes for localizing intraabdominal sepsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mountford, P.J.; Kettle, A.G.; O'Doherty, M.J.

Technetium-99m-HM-PAO (({sup 99m}Tc)HM-PAO) leukocyte and indium-111-oxine (111In-oxine) leukocyte scanning were carried out simultaneously in 41 patients at 4 hr and 24 hr after reinjection to determine whether the 4-hr {sup 99m}Tc scan could replace the 24-hr {sup 111}In scan for detecting intraabdominal sepsis. Abdominal infection was confirmed in 12 cases. The 4-hr {sup 99}Tc-leukocyte scan, the 4-hr {sup 111}In-leukocyte scan, and the 24-hr {sup 111}In-leukocyte scan yielded a sensitivity of 100%, 67%, and 100%, respectively, and a specificity of 62%, 90%, and 86%, respectively. The 24-hr {sup 99m}Tc-leukocyte scan also produced a sensitivity of 100%, but it was falsely positivemore » in all 29 cases without infection due to physiologic bowel uptake. False-positive 4-hr {sup 99m}Tc-leukocyte scans were also produced by physiologic bowel uptake in seven cases all of whom had true-negative 4-hr and 24-hr {sup 111}In-leukocyte scans. Because of the high incidence of false-positive 4-hr ({sup 99m}Tc)HM-PAO leukocyte scans, it was concluded that they could not replace 24-hr {sup 111}In-leukocyte scans for detecting intraabdominal sepsis, and that serial {sup 99m}Tc leukocyte scans starting earlier than 4 hr after reinjection must be evaluated.« less

Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications

PubMed Central

Czajkowsky, Daniel M.; Andersen, Jan Terje; Fuchs, Anja; Wilson, Timothy J.; Mekhaiel, David; Colonna, Marco; He, Jianfeng; Shao, Zhifeng; Mitchell, Daniel A.; Wu, Gang; Dell, Anne; Haslam, Stuart; Lloyd, Katy A.; Moore, Shona C.; Sandlie, Inger; Blundell, Patricia A.; Pleass, Richard J.

2015-01-01

The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements. Using quality-by-design (QbD) approaches, we generated hexameric-Fc (hexa-Fc), a ~20 nm oligomeric Fc-based scaffold that we here show binds low-affinity inhibitory receptors (FcRL5, FcγRIIb, and DC-SIGN) with high avidity and specificity, whilst eliminating significant clinical limitations of monomeric Fc-fusions for vaccine and/or cancer therapies, in particular their poor ability to activate complement. Mass spectroscopy of hexa-Fc reveals high-mannose, low-sialic acid content, suggesting that interactions with these receptors are influenced by the mannose-containing Fc. Molecular dynamics (MD) simulations provides insight into the mechanisms of hexa-Fc interaction with these receptors and reveals an unexpected orientation of high-mannose glycans on the human Fc that provides greater accessibility to potential binding partners. Finally, we show that this biosynthetic nanoparticle can be engineered to enhance interactions with the human neonatal Fc receptor (FcRn) without loss of the oligomeric structure, a crucial modification for these molecules in therapy and/or vaccine strategies where a long plasma half-life is critical. PMID:25912958

Epistatic interactions between Fc (GM) and FcγR genes and the host control of human immunodeficiency virus replication.

PubMed

Deepe, Raymond N; Kistner-Griffin, Emily; Martin, Jeffrey N; Deeks, Steven G; Pandey, Janardan P

2012-03-01

Host genetic factors are thought to contribute to the interindividual differences in the control of human immunodeficiency virus (HIV) replication. The aim of the present investigation was to determine whether genes encoding GM and KM allotypes-genetic markers of immunoglobulin γ and κ chains, respectively-and those encoding Fcγ receptor (FcγR) IIa and IIIa are associated with the host control of HIV replication. A case-control design was employed among HIV-infected subjects, with a group that spontaneously controlled HIV replication ("controllers") as cases (n = 73) and those who did not control replication as controls (n = 100). Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, direct DNA sequencing, and TaqMan genotyping assays. In Caucasian Americans, certain combinations of FcγR and GM genotypes were differentially distributed between controllers and noncontrollers. Among the noncarriers of the FcγRIIa arginine allele, GM21 noncarriers had over 7-fold greater odds of being controllers than the carriers of this allele (odds ratio [OR] = 7.47). These GM determinants also interacted with FcγRIIIa alleles. Among the carriers of the FcγRIIIa valine allele, GM21 noncarriers had over 3-fold greater odds of being controllers than the carriers of this allele (OR = 3.26). These results demonstrate epistatic interactions of genes on chromosomes 14 (GM) and 1 (FcγR) in influencing the control of HIV replication. Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

FcγRIIa and FcγRIIIb polymorphisms and associations with clinical manifestations in systemic lupus erythematosus patients.

PubMed

Vigato-Ferreira, Isabel Cristina Costa; Toller-Kawahisa, Juliana Escher; Pancoto, João Alexandre Trés; Mendes-Junior, Celso Teixeira; Martinez, Edson Zangiacomi; Donadi, Eduardo Antônio; Louzada-Júnior, Paulo; Del Lama, José Eduardo Cavalcanti; Marzocchi-Machado, Cleni Mara

2014-11-01

In this study, we aimed to investigate whether the distribution of the FcγRIIa and FcγRIIIb polymorphisms determines susceptibility to systemic lupus erythematosus (SLE) and acts as predictors of SLE clinical manifestations in the Brazilian patients. A total of 157 patients that fulfilled the American College of Rheumatology classification criteria for SLE and 160 healthy volunteers were included in this study. FCGR2A and FCGR3B genotypes were determined by polymerase chain reaction-based allotyping methods with allele-specific primers; the clinical features were obtained from the patients' official medical records. In the case of FcγRIIa polymorphism, it was observed association of the allele FCGR2A-R-131 (p = 0.02, odds ratio (OR)=1.44) and genotype RR-131 (p = 0.03, OR = 2.09) with SLE. These associations were higher with allele (p < 0.01, OR = 1.67) as well genotype (p = 0.01, OR = 2.85) when lupus nephritis was considered. In contrast, the allele FCGR2A-H-131 was associated with susceptibility to arthritis and anti-DNA antibodies (p = 0.05 for both). As for FcγRIIIb polymorphism, skewing did not differ significantly between patients and controls, however the genotype FCGR3B*02*02 was associated with susceptibility to arthritis (p = 0.02) and malar rash (p = 0.03), but no association with nephritis was found. The results demonstrate that FcγRIIa polymorphism is associated with susceptibility to SLE in Brazilian patients, whereas for FcγRIIIb polymorphism no association was found. However, notably, both polymorphisms present allelic variants that influence the clinical manifestations and may contribute to the pathogenesis of the disease. In addition, to our knowledge, this is the first study considering the frequency of FcγRIIIb polymorphism in Brazilian SLE patients.

IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions.

PubMed

Lee, Chang-Han; Romain, Gabrielle; Yan, Wupeng; Watanabe, Makiko; Charab, Wissam; Todorova, Biliana; Lee, Jiwon; Triplett, Kendra; Donkor, Moses; Lungu, Oana I; Lux, Anja; Marshall, Nicholas; Lindorfer, Margaret A; Goff, Odile Richard-Le; Balbino, Bianca; Kang, Tae Hyun; Tanno, Hidetaka; Delidakis, George; Alford, Corrine; Taylor, Ronald P; Nimmerjahn, Falk; Varadarajan, Navin; Bruhns, Pierre; Zhang, Yan Jessie; Georgiou, George

2017-08-01

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Cryopreservation of Human Mucosal Leukocytes

PubMed Central

Shu, Zhiquan; Levy, Claire N.; Ferre, April L.; Hartig, Heather; Fang, Cifeng; Lentz, Gretchen; Fialkow, Michael; Kirby, Anna C.; Adams Waldorf, Kristina M.; Veazey, Ronald S.; Germann, Anja; von Briesen, Hagen; McElrath, M. Juliana; Dezzutti, Charlene S.; Sinclair, Elizabeth; Baker, Chris A. R.; Shacklett, Barbara L.; Gao, Dayong; Hladik, Florian

2016-01-01

Background Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. Methods and Findings To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10–15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. Conclusions Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes. PMID:27232996

Cryopreservation of Human Mucosal Leukocytes.

PubMed

Hughes, Sean M; Shu, Zhiquan; Levy, Claire N; Ferre, April L; Hartig, Heather; Fang, Cifeng; Lentz, Gretchen; Fialkow, Michael; Kirby, Anna C; Adams Waldorf, Kristina M; Veazey, Ronald S; Germann, Anja; von Briesen, Hagen; McElrath, M Juliana; Dezzutti, Charlene S; Sinclair, Elizabeth; Baker, Chris A R; Shacklett, Barbara L; Gao, Dayong; Hladik, Florian

2016-01-01

Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function*

PubMed Central

Ren, Li; Campbell, Amanda; Fang, Huiqing; Gautam, Shalini; Elavazhagan, Saranya; Fatehchand, Kavin; Mehta, Payal; Stiff, Andrew; Reader, Brenda F.; Mo, Xiaokui; Byrd, John C.; Carson, William E.; Butchar, Jonathan P.; Tridandapani, Susheela

2016-01-01

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function. PMID:26627823

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function.

PubMed

Ren, Li; Campbell, Amanda; Fang, Huiqing; Gautam, Shalini; Elavazhagan, Saranya; Fatehchand, Kavin; Mehta, Payal; Stiff, Andrew; Reader, Brenda F; Mo, Xiaokui; Byrd, John C; Carson, William E; Butchar, Jonathan P; Tridandapani, Susheela

2016-02-05

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function. © 2016 by The American Society for Biochemistry

The binding of an anti-PD-1 antibody to FcγRΙ has a profound impact on its biological functions.

PubMed

Zhang, Tong; Song, Xiaomin; Xu, Lanlan; Ma, Jie; Zhang, Yanjuan; Gong, Wenfeng; Zhang, Yilu; Zhou, Xiaosui; Wang, Zuobai; Wang, Yali; Shi, Yingdi; Bai, Huichen; Liu, Ning; Yang, Xiaolong; Cui, Xinxin; Cao, Yanping; Liu, Qi; Song, Jing; Li, Yucheng; Tang, Zhiyu; Guo, Mingming; Wang, Lai; Li, Kang

2018-04-23

Antibodies targeting PD-1 have been demonstrated durable anti-cancer activity in certain cancer types. However, the anti-PD-1 antibodies are less or not efficacious in many situations, which might be attributed to co-expression of multiple inhibitory receptors or presence of immunosuppressive cells in the tumor microenvironment. Most of the anti-PD-1 antibodies used in clinical studies are of IgG4 isotype with the S228P mutation (IgG4 S228P ). The functional impact by the interaction of anti-PD-1 IgG4 S228P antibody with Fc gamma receptors (FcγRs) is poorly understood. To assess the effects, we generated a pair of anti-PD-1 antibodies: BGB-A317/IgG4 S228P and BGB-A317/IgG4-variant (abbreviated as BGB-A317), with the same variable regions but two different IgG4 Fc-hinge sequences. There was no significant difference between these two antibodies in binding to PD-1. However, BGB-A317/IgG4 S228P binds to human FcγRI with high affinity and mediates crosslinking between PD-1 and FcγRI. In contrast, BGB-A317 does neither. Further cell-based assays showed that such crosslinking could reverse the function of an anti-PD-1 antibody from blocking to activating. More importantly, the crosslinking induces FcγRI + macrophages to phagocytose PD-1 + T cells. In a mouse model transplanted with allogeneic human cancer cells and PBMCs, BGB-A317 showed significant tumor growth inhibition, whereas BGB-A317/IgG4 S228P had no such inhibition. Immunohistochemistry study revealed an inverse correlation between FcγRI + murine macrophage infiltration and the density of CD8 + PD-1 + human T cells within tumors in the BGB-A317/IgG4 S228P -treated group. These evidences suggested that FcγRI + binding and crosslinking had negative impact on the anti-PD-1 antibody-mediated anti-cancer activity.

SPECIES SPECIFICITY OF LEUKOCYTIC PYROGENS

PubMed Central

Bornstein, Donald L.; Woods, James W.

1969-01-01

Polymorphonuclear neutrophilic leukocytes of the dog, cat, and goat release leukocytic pyrogen under the same conditions as the heterophile polymorphonuclear leukocytes of the rabbit. The characteristics of the febrile response to an intravenous injection of homologous leukocytic pyrogen in all four species are very similar: a brisk monophasic fever reaching a peak between 30 and 50 min with smooth defervescence to the baseline by 3 hr. Shivering, which is not obvious in the rabbit, is noted in the dog, cat, and goat during the first 30 min. Quantitative differences in response reveal the cat to be the most sensitive of of these species to homologous leukocytic pyrogen, followed by the rabbit, dog, and goat. The response to heterologous pyrogen is in most cases markedly diminished compared to that after equal doses of homologous protein, suggesting the operation of species specificity, although canine and feline pyrogen behaved very similarly in all tests. Species specificity of leukocytic pyrogen is probably related to amino acid substitutions in different species of a common mammalian protein effector molecule. PMID:5343431

Neonatal Fc receptor for IgG (FcRn) regulates cross-presentation of IgG immune complexes by CD8−CD11b+ dendritic cells

PubMed Central

Baker, Kristi; Qiao, Shuo-Wang; Kuo, Timothy T.; Aveson, Victoria G.; Platzer, Barbara; Andersen, Jan-Terje; Sandlie, Inger; Chen, Zhangguo; de Haar, Colin; Lencer, Wayne I.; Fiebiger, Edda; Blumberg, Richard S.

2011-01-01

Cross-presentation of IgG-containing immune complexes (ICs) is an important means by which dendritic cells (DCs) activate CD8+ T cells, yet it proceeds by an incompletely understood mechanism. We show that monocyte-derived CD8−CD11b+ DCs require the neonatal Fc receptor for IgG (FcRn) to conduct cross-presentation of IgG ICs. Consequently, in the absence of FcRn, Fcγ receptor (FcγR)-mediated antigen uptake fails to initiate cross-presentation. FcRn is shown to regulate the intracellular sorting of IgG ICs to the proper destination for such cross-presentation to occur. We demonstrate that FcRn traps antigen and protects it from degradation within an acidic loading compartment in association with the rapid recruitment of key components of the phagosome-to-cytosol cross-presentation machinery. This unique mechanism thus enables cross-presentation to evolve from an atypically acidic loading compartment. FcRn-driven cross-presentation is further shown to control cross-priming of CD8+ T-cell responses in vivo such that during chronic inflammation, FcRn deficiency results in inadequate induction of CD8+ T cells. These studies thus demonstrate that cross-presentation in CD8−CD11b+ DCs requires a two-step mechanism that involves FcγR-mediated internalization and FcRn-directed intracellular sorting of IgG ICs. Given the centrality of FcRn in controlling cross-presentation, these studies lay the foundation for a unique means to therapeutically manipulate CD8+ T-cell responses. PMID:21628593

Divergent Requirement of Fc-Fcγ Receptor Interactions for In Vivo Protection against Influenza Viruses by Two Pan-H5 Hemagglutinin Antibodies

PubMed Central

Wang, Shuangshuang; Ren, Huanhuan; Jiang, Wenbo; Chen, Honglin; Hu, Hongxing; Chen, Zhiwei

2017-01-01

ABSTRACT Recent studies have shown that Fc-Fcγ receptor (FcγR) interactions are required for in vivo protection against influenza viruses by broadly reactive anti-hemagglutinin (HA) stem, but not virus strain-specific, anti-receptor binding site (RBS), antibodies (Abs). Since only a few Abs recognizing epitopes in the head region but outside the RBS have been tested against single-challenge virus strains, it remains unknown whether Fc-FcγR interactions are required for in vivo protection by Abs recognizing epitopes outside the RBS and whether the requirement is virus strain specific or epitope specific. In the present study, we therefore investigated the requirements for in vivo protection using two pan-H5 Abs, 65C6 and 100F4. We generated chimeric Abs, 65C6/IgG2a and 100F4/IgG2a, which preferentially engage activating FcγRs, and isogenic forms, 65C6/D265A and 100F4/D265A, which do not bind FcγR. Virus neutralizing activity, binding, antibody-dependent cellular cytotoxicity (ADCC), and in vivo protection of these Abs were compared using three H5 strains, A/Shenzhen/406H/2006 (SZ06), A/chicken/Shanxi/2/2006 (SX06), and A/chicken/Netherlands/14015526/2014 (NE14). We found that all four chimeric Abs bound and neutralized the SZ06 and NE14 strains but poorly inhibited the SX06 strain. 65C6/IgG2a and 100F4/IgG2a, but not 65C6/D265A and 100F4/D265A, mediated ADCC against target cells expressing HA derived from all three virus strains. Interestingly, both 65C6/IgG2a and 65C6/D265A demonstrated comparable protection against all three virus strains in vivo; however, 100F4/IgG2a, but not 100F4/D265A, showed in vivo protection. Thus, we conclude that Fc-FcγR interactions are required for in vivo protection by 100F4, but not by 65C6, and therefore, protection is not virus strain specific but epitope specific. IMPORTANCE Abs play an important role in immune protection against influenza virus infection. Fc-FcγR interactions are required for in vivo protection by broadly

Divergent Requirement of Fc-Fcγ Receptor Interactions for In Vivo Protection against Influenza Viruses by Two Pan-H5 Hemagglutinin Antibodies.

PubMed

Wang, Shuangshuang; Ren, Huanhuan; Jiang, Wenbo; Chen, Honglin; Hu, Hongxing; Chen, Zhiwei; Zhou, Paul

2017-06-01

Recent studies have shown that Fc-Fcγ receptor (FcγR) interactions are required for in vivo protection against influenza viruses by broadly reactive anti-hemagglutinin (HA) stem, but not virus strain-specific, anti-receptor binding site (RBS), antibodies (Abs). Since only a few Abs recognizing epitopes in the head region but outside the RBS have been tested against single-challenge virus strains, it remains unknown whether Fc-FcγR interactions are required for in vivo protection by Abs recognizing epitopes outside the RBS and whether the requirement is virus strain specific or epitope specific. In the present study, we therefore investigated the requirements for in vivo protection using two pan-H5 Abs, 65C6 and 100F4. We generated chimeric Abs, 65C6/IgG2a and 100F4/IgG2a, which preferentially engage activating FcγRs, and isogenic forms, 65C6/D265A and 100F4/D265A, which do not bind FcγR. Virus neutralizing activity, binding, antibody-dependent cellular cytotoxicity (ADCC), and in vivo protection of these Abs were compared using three H5 strains, A/Shenzhen/406H/2006 (SZ06), A/chicken/Shanxi/2/2006 (SX06), and A/chicken/Netherlands/14015526/2014 (NE14). We found that all four chimeric Abs bound and neutralized the SZ06 and NE14 strains but poorly inhibited the SX06 strain. 65C6/IgG2a and 100F4/IgG2a, but not 65C6/D265A and 100F4/D265A, mediated ADCC against target cells expressing HA derived from all three virus strains. Interestingly, both 65C6/IgG2a and 65C6/D265A demonstrated comparable protection against all three virus strains in vivo ; however, 100F4/IgG2a, but not 100F4/D265A, showed in vivo protection. Thus, we conclude that Fc-FcγR interactions are required for in vivo protection by 100F4, but not by 65C6, and therefore, protection is not virus strain specific but epitope specific. IMPORTANCE Abs play an important role in immune protection against influenza virus infection. Fc-FcγR interactions are required for in vivo protection by broadly

Optimization on Fc for Improvement of Stability and Aggregation Resistance.

PubMed

Chen, Xiaobo; Zeng, Fang; Huang, Tao; Cheng, Liang; Liu, Huan; Gong, Rui

2016-01-01

Fc-based therapeutics including therapeutic full-size monoclonal antibodies (mAbs) and Fcfusion proteins represent fastest-growing market in biopharmaceutical industrial. However, one major challenge during development of Fc-based therapeutics is how to maintain their efficacy in clinic use. Many factors may lead to failure in final marketing. For example, the stability and aggregation resistance might not be high enough for bearing the disadvantages during fermentation, purification, formulation, storage, shipment and other steps in manufacture and sale. Low stability and high aggregation tendency lead to decreased bioactivity and increased risk of immunogenicity resulting in serious side effect. Because Fc is one of the major parts in monoclonal antibodies and Fc-fusion proteins, engineering of Fc to increase its stability and reduce or eliminate aggregation due to incorrect association are of great importance and could further extend the potential of Fc-based therapeutics. Lots of studies focus on Fc optimization for better physical and chemical characteristics and function by structured-based computer-aid rational design, high-throughput screening expression system selection and other methods. The identification of optimized Fc mutants increases the clinic potential of currently existed therapeutics mAbs and Fc-fusion proteins, and accelerates the development of new Fc-based therapeutics. Here we provide an overview of the related field, and discuss recent advances and future directions in optimization of Fc-based therapeutics with modified stability and aggregation resistance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Interferon-gamma promotes the survival and Fc epsilon RI-mediated histamine release in cultured human mast cells.

PubMed Central

Yanagida, M; Fukamachi, H; Takei, M; Hagiwara, T; Uzumaki, H; Tokiwa, T; Saito, H; Iikura, Y; Nakahata, T

1996-01-01

We examined the effects of interferon-gamma (IFN-gamma) on 100% pure human mast cells generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6). When mast cells were suspended in serum-free medium without any cytokine after the withdrawal of SCF and IL-6, they died over a period of 5 days because of apoptosis. IFN-gamma in the cultures suppressed apoptosis and prolonged their survival in a dose-dependent manner. This survival-promoting effect of IFN-gamma was blocked by neutralizing antibodies to IFN-gamma or to IFN-gamma receptor (IFN-gamma R). When mast cells were incubated with IFN-gamma in serum-free medium for more than 4 hr during sensitization, immunoglobulin E (IgE)/anti-IgE antibody-induced histamine release was effectively enhanced. Polymerase chain reaction (PCR) amplification of the alpha-chain of IFN-gamma R (IFN-gamma R alpha) yielded products of the correct size predicted from the sequence of the receptor. In addition, flow cytometry using anti-IFN-gamma R monoclonal antibodies (mAbs) indicated that these mast cells bear IFN-gamma R on their surface. These findings suggested that IFN-gamma activates human mast cells via specific receptors in certain aspects of inflammatory reactions. Images Figure 2 Figure 4 PMID:9014819

Optimization of protein-protein docking for predicting Fc-protein interactions.

PubMed

Agostino, Mark; Mancera, Ricardo L; Ramsland, Paul A; Fernández-Recio, Juan

2016-11-01

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc-protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc-binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc-protein complexes are available, numerous others have not yet been determined. Protein-protein docking could be used to investigate Fc-protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking-based structural bioinformatics approach is developed for predicting the structures of Fc-protein complexes. Based on the available set of X-ray structures of Fc-protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc-protein complexes, as well as in the design of peptides and proteins that target Fc. Copyright © 2016 John Wiley & Sons, Ltd.

The state diagram for cell adhesion under flow: leukocyte rolling and firm adhesion.

PubMed

Chang, K C; Tees, D F; Hammer, D A

2000-10-10

Leukocyte adhesion under flow in the microvasculature is mediated by binding between cell surface receptors and complementary ligands expressed on the surface of the endothelium. Leukocytes adhere to endothelium in a two-step mechanism: rolling (primarily mediated by selectins) followed by firm adhesion (primarily mediated by integrins). Using a computational method called "Adhesive Dynamics," we have simulated the adhesion of a cell to a surface in flow, and elucidated the relationship between receptor-ligand functional properties and the dynamics of adhesion. We express this relationship in a state diagram, a one-to-one map between the biophysical properties of adhesion molecules and various adhesive behaviors. Behaviors that are observed in simulations include firm adhesion, transient adhesion (rolling), and no adhesion. We varied the dissociative properties, association rate, bond elasticity, and shear rate and found that the unstressed dissociation rate, k(r)(o), and the bond interaction length, gamma, are the most important molecular properties controlling the dynamics of adhesion. Experimental k(r)(o) and gamma values from the literature for molecules that are known to mediate rolling adhesion fall within the rolling region of the state diagram. We explain why L-selectin-mediated rolling, which has faster k(r)(o) than other selectins, is accompanied by a smaller value for gamma. We also show how changes in association rate, shear rate, and bond elasticity alter the dynamics of adhesion. The state diagram (which must be mapped for each receptor-ligand system) presents a concise and comprehensive means of understanding the relationship between bond functional properties and the dynamics of adhesion mediated by receptor-ligand bonds.

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

PubMed Central

Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

2013-01-01

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

Decreased release of histamine and sulfidoleukotrienes by human peripheral blood leukocytes after wasp venom immunotherapy is partially due to induction of IL-10 and IFN-gamma production of T cells.

PubMed

Pierkes, M; Bellinghausen, I; Hultsch, T; Metz, G; Knop, J; Saloga, J

1999-02-01

Recent studies provide evidence that venom immunotherapy (VIT) alters the pattern of cytokine production by inducing an allergen-specific T-cell shift in cytokine expression from TH2 (IL-4, IL-5) to TH1 (IFN-gamma) cytokines and also inducing the production of IL-10. This study was carried out to analyze whether these changes in cytokine production of T cells already observed 1 week after the initiation of VIT in subjects with wasp venom allergy also influence the reactivity of effector cells, such as mast cells and basophils. All subjects included in this study had a history of severe systemic allergic reactions to wasp stings and positive skin test responses with venom and venom-specific IgE in the sera. Peripheral blood leukocytes were isolated before and after the initiation of VIT (rush therapy reaching a maintenance dose of 100 microg venom injected subcutaneously within 1 week) and preincubated with or without addition of IL-10, IFN-gamma, IL-10 + IFN-gamma, anti-IL-10, or anti-IFN-gamma. After stimulation with wasp venom, histamine and sulfidoleukotriene release were assessed by ELISA and compared with spontaneous release and total histamine content. After the induction of VIT, venom-induced absolute and relative histamine and sulfidoleukotriene release were reduced. This was at least partially due to the induction of IFN-gamma and IL-10 production, because (1) neutralization of IL-10 and IFN-gamma by mAbs partially restored the release after the initiation of VIT and (2) the addition of exogenous IFN-gamma and IL-10 caused a statistically significant diminution of the venom-induced histamine and sulfidoleukotriene release before VIT. Depletion of CD2(+) T cells also restored the releasability after VIT. These data indicate that T cells (producing IL-10 and IFN-gamma after VIT) play a key role for the inhibition of histamine and sulfidoleukotriene release of effector cells.

FcRn Rescues Recombinant Factor VIII Fc Fusion Protein from a VWF Independent FVIII Clearance Pathway in Mouse Hepatocytes

PubMed Central

van der Flier, Arjan; Liu, Zhan; Tan, Siyuan; Chen, Kai; Drager, Douglas; Liu, Tongyao; Patarroyo-White, Susannah; Jiang, Haiyan; Light, David R.

2015-01-01

We recently developed a longer lasting recombinant factor VIII-Fc fusion protein, rFVIIIFc, to extend the half-life of replacement FVIII for the treatment of people with hemophilia A. In order to elucidate the biological mechanism for the elongated half-life of rFVIIIFc at a cellular level we delineated the roles of VWF and the tissue-specific expression of the neonatal Fc receptor (FcRn) in the biodistribution, clearance and cycling of rFVIIIFc. We find the tissue biodistribution is similar for rFVIIIFc and rFVIII and that liver is the major clearance organ for both molecules. VWF reduces the clearance and the initial liver uptake of rFVIIIFc. Pharmacokinetic studies in FcRn chimeric mice show that FcRn expressed in somatic cells (hepatocytes or liver sinusoidal endothelial cells) mediates the decreased clearance of rFVIIIFc, but FcRn in hematopoietic cells (Kupffer cells) does not affect clearance. Immunohistochemical studies show that when rFVIII or rFVIIIFc is in dynamic equilibrium binding with VWF, they mostly co localize with VWF in Kupffer cells and macrophages, confirming a major role for liver macrophages in the internalization and clearance of the VWF-FVIII complex. In the absence of VWF a clear difference in cellular localization of VWF-free rFVIII and rFVIIIFc is observed and neither molecule is detected in Kupffer cells. Instead, rFVIII is observed in hepatocytes, indicating that free rFVIII is cleared by hepatocytes, while rFVIIIFc is observed as a diffuse liver sinusoidal staining, suggesting recycling of free-rFVIIIFc out of hepatocytes. These studies reveal two parallel linked clearance pathways, with a dominant pathway in which both rFVIIIFc and rFVIII complexed with VWF are cleared mainly by Kupffer cells without FcRn cycling. In contrast, the free fraction of rFVIII or rFVIIIFc unbound by VWF enters hepatocytes, where FcRn reduces the degradation and clearance of rFVIIIFc relative to rFVIII by cycling rFVIIIFc back to the liver sinusoid and

HAL/S-FC compiler system functional specification

NASA Technical Reports Server (NTRS)

1974-01-01

The functional requirements to be met by the HAL/S-FC compiler, and the hardware and software compatibilities between the compiler system and the environment in which it operates are defined. Associated runtime facilities and the interface with the Software Development Laboratory are specified. The construction of the HAL/S-FC system as functionally separate units and the interfaces between those units is described. An overview of the system's capabilities is presented and the hardware/operating system requirements are specified. The computer-dependent aspects of the HAL/S-FC are also specified. Compiler directives are included.

Effective therapy for a murine model of human anaplastic large-cell lymphoma with the anti-CD30 monoclonal antibody, HeFi-1, does not require activating Fc receptors

PubMed Central

Zhang, Meili; Yao, Zhengsheng; Zhang, Zhuo; Garmestani, Kayhan; Goldman, Carolyn K.; Ravetch, Jeffrey V.; Janik, John; Brechbiel, Martin W.; Waldmann, Thomas A.

2006-01-01

CD30 is a member of the tumor necrosis factor receptor family. Overexpression of CD30 on some neoplasms versus its limited expression on normal tissues makes this receptor a promising target for antibody-based therapy. Anaplastic large-cell lymphoma (ALCL) represents a heterogeneous group of aggressive non-Hodgkin lymphomas characterized by the strong expression of CD30. We investigated the therapeutic efficacy of HeFi-1, a mouse IgG1 monoclonal antibody, which recognizes the ligand-binding site on CD30, and humanized anti-Tac antibody (daclizumab), which recognizes CD25, in a murine model of human ALCL. The ALCL model was established by intravenous injection of karpas299 cells into nonobese diabetic/severe combined immuno-deficient (SCID/NOD) wild-type or SCID/NOD Fc receptor common γ chain–deficient (FcRγ–/–) mice. HeFi-1, given at a dose of 100 μg weekly for 4 weeks, significantly prolonged survival of the ALCL-bearing SCID/NOD wild-type and SCID/NOD FcRγ–/– mice (P < .01) as compared with the control groups. In vitro studies showed that HeFi-1 inhibited the proliferation of karpas299 cells, whereas daclizumab did not inhibit cell proliferation. We demonstrated that the expression of FcRγ on polymorphonuclear leukocytes and monocytes was not required for HeFi-1–mediated tumor growth inhibition in vivo, although it was required for daclizumab. PMID:16551968

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

PubMed

Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

2017-02-01

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differential Fc-receptor engagement drives an anti-tumor vaccinal effect

PubMed Central

DiLillo, David J.; Ravetch, Jeffrey V.

2015-01-01

Summary Passively-administered anti-tumor mAbs rapidly kill tumor targets via FcγR-mediated cytotoxicity (ADCC), a short-term process. However, anti-tumor mAb treatment can also induce a vaccinal effect, in which mAb-mediated tumor death induces a long-term anti-tumor cellular immune response. To determine how such responses are generated, we utilized a murine model of an anti-tumor vaccinal effect against a model neoantigen. We demonstrate that FcγR expression by CD11c+ antigen-presenting cells is required to generate anti-tumor T cell responses upon ADCC-mediated tumor clearance. Using FcγR-humanized mice, we demonstrate that anti-tumor huIgG1 must engage hFcγRIIIA on macrophages to mediate ADCC, but also engage hFcγRIIA, the sole hFcγR expressed by human DCs, to generate a potent vaccinal effect. Thus, while next-generation anti-tumor antibodies with enhanced binding to only hFcγRIIIA are now in clinical use, ideal anti-tumor antibodies must be optimized for both cytotoxic effects as well as hFcγRIIA engagement on DCs to stimulate long-term anti-tumor cellular immunity. PMID:25976835

FcγRIIb expression in early stage chronic lymphocytic leukemia.

PubMed

Bosch, Rosa; Mora, Alba; Vicente, Eva Puy; Ferrer, Gerardo; Jansà, Sonia; Damle, Rajendra; Gorlatov, Sergey; Rai, Kanti; Montserrat, Emili; Nomdedeu, Josep; Pratcorona, Marta; Blanco, Laura; Saavedra, Silvana; Garrido, Ana; Esquirol, Albert; Garcia, Irene; Granell, Miquel; Martino, Rodrigo; Delgado, Julio; Sierra, Jorge; Chiorazzi, Nicholas; Moreno, Carol

2017-11-01

In normal B-cells, B-cell antigen receptor (BCR) signaling can be negatively regulated by the low-affinity receptor FcγRIIb (CD32b). To better understand the role of FcγRIIb in chronic lymphocytic leukemia (CLL), we correlated its expression on 155 samples from newly-diagnosed Binet A patients with clinical characteristics and outcome. FcγRIIb expression was similar in normal B-cells and leukemic cells, this being heterogenous among patients and within CLL clones. FcγRIIb expression did not correlate with well known prognostic markers [disease stage, serum beta-2 microglobulin (B2M), IGHV mutational status, expression of ZAP-70 and CD38, and cytogenetics] except for a weak concordance with CD49d. Moreover, patients with low FcγRIIb expression (69/155, 44.5%) required therapy earlier than those with high FcγRIIb expression (86/155, 55.5%) (median 151.4 months vs. not reached; p=.071). These results encourage further investigation on the role of FcγRIIb in CLL biology and prognostic significance in larger series of patients.

Thermal decomposition of FC(O)OCH3 and FC(O)OCH2CH3.

PubMed

Berasategui, M; Argüello, G A; Burgos Paci, M A

2018-05-09

The thermal decomposition of methyl and ethyl formates has been extensively studied due to their importance in the oxidation of several fuels, pesticidal properties and their presence in interstellar space. We hitherto present the study of the thermal decomposition of methyl and ethyl fluoroformates, which could help in the elucidation of the reaction mechanisms. The reaction mechanisms were studied using FTIR spectroscopy in the temperature range of 453-733 K in the presence of different pressures of N2 as bath gas. For FC(O)OCH3 two different channels were observed; the unimolecular decomposition which is favored at higher temperatures and has a rate constant kFC(O)OCH3 = (5.3 ± 0.5) × 1015 exp[-(246 ± 10 kJ mol-1/RT)] (in units of s-1) and a bimolecular channel with a rate constant kFC(O)OCH3 = (1.6 ± 0.5) × 1011 exp[-(148 ± 10 kJ mol-1/RT)] (in units of s-1 (mol L)-1). However for ethyl formate, only direct elimination of CO2, HF and ethylene operates. The rate constants of the homogeneous first-order process fit the Arrhenius equation kFC(O)OCH2CH3 = (2.06 ± 0.09) × 1013 exp[-(169 ± 6 kJ mol-1/RT)] (in units of s-1). The difference between the mechanisms of the two fluoroformates relies on the stabilization of a six-centered transition state that only exists for ethyl formate. First principles calculations for the different channels were carried out to understand the dynamics of the decomposition.

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women: II. Identification of the molecule as a Fc-receptor-like molecule: a preliminary report.

PubMed

Aranha, C; Bordekar, A; Shahani, S

1998-11-01

Early pregnancy factor (EPF)-like activity from culture supernatants obtained from stimulated lymphocytes of pregnant women was characterized and identified. The enzyme-linked immunosorbent assay depending on the presence of "Fc" receptors on bovine spermatozoa was used to identify the EPF-like molecule purified by gel filtration and reverse-phase high-performance liquid chromatography. The results indicated that the crude lymphocyte culture supernatant, the EPF-positive G IV fraction obtained on gel filtration, and the EPF-positive reverse-phase high-performance liquid chromatography protein readily bound with the different concentrations of aggregated human gamma-globulin in a manner similar to that in which the standard control of aggregated human gamma-globulin binds to the bovine spermatozoa. EPF-like activity synthesized and secreted by lymphocytes during pregnancy may be a Fc-receptor-like molecule.

Past makes future: role of pFC in prediction.

PubMed

Fuster, Joaquín M; Bressler, Steven L

2015-04-01

The pFC enables the essential human capacities for predicting future events and preadapting to them. These capacities rest on both the structure and dynamics of the human pFC. Structurally, pFC, together with posterior association cortex, is at the highest hierarchical level of cortical organization, harboring neural networks that represent complex goal-directed actions. Dynamically, pFC is at the highest level of the perception-action cycle, the circular processing loop through the cortex that interfaces the organism with the environment in the pursuit of goals. In its predictive and preadaptive roles, pFC supports cognitive functions that are critical for the temporal organization of future behavior, including planning, attentional set, working memory, decision-making, and error monitoring. These functions have a common future perspective and are dynamically intertwined in goal-directed action. They all utilize the same neural infrastructure: a vast array of widely distributed, overlapping, and interactive cortical networks of personal memory and semantic knowledge, named cognits, which are formed by synaptic reinforcement in learning and memory acquisition. From this cortex-wide reservoir of memory and knowledge, pFC generates purposeful, goal-directed actions that are preadapted to predicted future events.

Measuring functional connectivity using MEG: Methodology and comparison with fcMRI

PubMed Central

Brookes, Matthew J.; Hale, Joanne R.; Zumer, Johanna M.; Stevenson, Claire M.; Francis, Susan T.; Barnes, Gareth R.; Owen, Julia P.; Morris, Peter G.; Nagarajan, Srikantan S.

2011-01-01

Functional connectivity (FC) between brain regions is thought to be central to the way in which the brain processes information. Abnormal connectivity is thought to be implicated in a number of diseases. The ability to study FC is therefore a key goal for neuroimaging. Functional connectivity (fc) MRI has become a popular tool to make connectivity measurements but the technique is limited by its indirect nature. A multimodal approach is therefore an attractive means to investigate the electrodynamic mechanisms underlying hemodynamic connectivity. In this paper, we investigate resting state FC using fcMRI and magnetoencephalography (MEG). In fcMRI, we exploit the advantages afforded by ultra high magnetic field. In MEG we apply envelope correlation and coherence techniques to source space projected MEG signals. We show that beamforming provides an excellent means to measure FC in source space using MEG data. However, care must be taken when interpreting these measurements since cross talk between voxels in source space can potentially lead to spurious connectivity and this must be taken into account in all studies of this type. We show good spatial agreement between FC measured independently using MEG and fcMRI; FC between sensorimotor cortices was observed using both modalities, with the best spatial agreement when MEG data are filtered into the β band. This finding helps to reduce the potential confounds associated with each modality alone: while it helps reduce the uncertainties in spatial patterns generated by MEG (brought about by the ill posed inverse problem), addition of electrodynamic metric confirms the neural basis of fcMRI measurements. Finally, we show that multiple MEG based FC metrics allow the potential to move beyond what is possible using fcMRI, and investigate the nature of electrodynamic connectivity. Our results extend those from previous studies and add weight to the argument that neural oscillations are intimately related to functional

The Neonatal Fc Receptor (FcRn) Enhances Human Immunodeficiency Virus Type 1 (HIV-1) Transcytosis across Epithelial Cells

PubMed Central

Gupta, Sandeep; Gach, Johannes S.; Becerra, Juan C.; Phan, Tran B.; Pudney, Jeffrey; Moldoveanu, Zina; Joseph, Sarah B.; Landucci, Gary; Supnet, Medalyn Jude; Ping, Li-Hua; Corti, Davide; Moldt, Brian; Hel, Zdenek; Lanzavecchia, Antonio; Ruprecht, Ruth M.; Burton, Dennis R.; Mestecky, Jiri; Anderson, Deborah J.; Forthal, Donald N.

2013-01-01

The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure. PMID:24278022

21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Leukocyte alkaline phosphatase test. 864.7660... Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used to identify the enzyme leukocyte alkaline phosphatase in neutrophilic granulocytes (granular...

21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Leukocyte alkaline phosphatase test. 864.7660... Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used to identify the enzyme leukocyte alkaline phosphatase in neutrophilic granulocytes (granular...

21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Leukocyte alkaline phosphatase test. 864.7660... Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used to identify the enzyme leukocyte alkaline phosphatase in neutrophilic granulocytes (granular...

In vitro agglutinin production by earthworm leukocytes.

PubMed

Stein, E A; Cooper, E L

1988-01-01

Leukocytes of the earthworm, Lumbricus terrestris, secrete agglutinins in vitro, as shown by measuring agglutinin titers of the culture medium and by observing secretory rosette formation by leukocytes with erythrocytes. Leukocytes form the highest percentages of secretory rosettes with rabbit erythrocytes (RBC) and with other RBC species in the order: rat, guinea pig, mouse, calf, sheep, horse, goat. Leukocytes displayed allotypic specificity by forming rosettes selectively with erythrocytes from different individual rabbits. Eight sugars inhibited rosette formation, along with the polysaccharide mannan and the glycoproteins thyroglobulin and bovine submaxillary mucin. Cyclohexamide did not affect rosette formation, suggesting that agglutinins may be preformed and stored in leukocytes prior to secretion. Leukocytes also formed E-type rosettes with erythrocytes, but apparently utilized different receptors from those of secretory rosettes since they were not inhibited by the same sugars.

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

PubMed

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

Computerized detection of leukocytes in microscopic leukorrhea images.

PubMed

Zhang, Jing; Zhong, Ya; Wang, Xiangzhou; Ni, Guangming; Du, Xiaohui; Liu, Juanxiu; Liu, Lin; Liu, Yong

2017-09-01

Detection of leukocytes is critical for the routine leukorrhea exam, which is widely used in gynecological examinations. An elevated vaginal leukocyte count in women with bacterial vaginosis is a strong predictor of vaginal or cervical infections. In the routine leukorrhea exam, the counting of leukocytes is primarily performed by manual techniques. However, the viewing and counting of leukocytes from multiple high-power viewing fields on a glass slide under a microscope leads to subjectivity, low efficiency, and low accuracy. To date, many biological cells in stool, blood, and breast cancer have been studied to realize computerized detection; however, the detection of leukocytes in microscopic leukorrhea images has not been studied. Thus, there is an increasing need for computerized detection of leukocytes. There are two key processes in the computerized detection of leukocytes in digital image processing. One is segmentation; the other is intelligent classification. In this paper, we propose a combined ensemble to detect leukocytes in the microscopic leukorrhea image. After image segmentation and selecting likely leukocyte subimages, we obtain the leukocyte candidates. Then, for intelligent classification, we adopt two methods: feature extraction and classification by a support vector machine (SVM); applying a modified convolutional neural network (CNN) to the larger subimages. If different methods classify a candidate in the same category, the process is finished. If not, the outputs of the methods are provided to a classifier to further classify the candidate. After acquiring leukocyte candidates, we attempted three methods to perform classification. The first approach using features and SVM achieved 88% sensitivity, 97% specificity, and 92.5% accuracy. The second method using CNN achieved 95% sensitivity, 84% specificity, and 89.5% accuracy. Then, in the combination approach, we achieved 92% sensitivity, 95% specificity, and 93.5% accuracy. Finally, the images

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

PubMed Central

Wang, Xiangdan; McKay, Patrick; Dutina, George; Hass, Philip E.; Nijem, Ihsan; Allison, David; Cowan, Kyra J.; Lin, Kevin; Quarmby, Valerie; Yang, Jihong

2017-01-01

ABSTRACT Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs. PMID:28001487

Fusion of the Fc part of human IgG1 to CD14 enhances its binding to gram-negative bacteria and mediates phagocytosis by Fc receptors of neutrophils.

PubMed

Vida, András; Bardoel, Bart; Milder, Fin; Majoros, László; Sümegi, Andrea; Bácsi, Attila; Vereb, György; van Kessel, Kok P M; van Strijp, Jos A G; Antal-Szalmás, Péter

2012-08-30

Microbial resistance to antimicrobial drugs is promoting a search for new antimicrobial agents that target highly conservative structures of pathogens. Human CD14 - a known pattern recognition receptor (PRR) which recognizes multiple ligands from different microbes might be a worthy candidate. The aim of our work was to create a CD14/Fc dimer protein and evaluate its whole bacteria binding and opsonizing capabilities. Fusion of CD14 with the fragment crystallisable (Fc) part of human IgG1 could not only lead to an artificial opsonin but the dimerization through the Fc part might also increase its affinity to different ligands. Human CD14 and the Fc part of human IgG1 was fused and expressed in HEK293 cells. A histidine tagged CD14 (CD14/His) was also expressed as control. Using flow cytometry we could prove that CD14/Fc bound to whole Gram-negative bacteria, especially to short lipopolysaccharide (Ra and Re) mutants, and weak interaction was observed between the fusion protein and Listeria monocytogenes. Other Gram-positive bacteria and fungi did not show any association with CD14/Fc. CD14/His showed about 50-times less potent binding to Gram-negative bacteria. CD14/Fc acted as an opsonin and enhanced phagocytosis of these bacteria by neutrophil granulocytes, monocyte-derived macrophages and dendritic cells. Internalization of bacteria was confirmed by trypan blue quenching and confocal microscopy. On neutrophils the Fc part of the fusion protein was recognized by Fc receptors (CD16, CD32), as determined by blocking experiments. CD14/Fc enhanced the killing of bacteria in an ex vivo whole blood assay. Our experiments confirm that PRR/Fc fusion proteins can give a boost to FcR dependent phagocytosis and killing provided the antimicrobial part binds efficiently to microbes. Copyright © 2012 Elsevier B.V. All rights reserved.

The Role of FcRn in Antigen Presentation

PubMed Central

Baker, Kristi; Rath, Timo; Pyzik, Michal; Blumberg, Richard S.

2014-01-01

Immunoglobulins are unique molecules capable of simultaneously recognizing a diverse array of antigens and themselves being recognized by a broad array of receptors. The abundance specifically of the IgG subclass and the variety of signaling receptors to which it binds render this an important immunomodulatory molecule. In addition to the classical Fcγ receptors that bind IgG at the cell surface, the neonatal Fc receptor (FcRn) is a lifelong resident of the endolysosomal system of most hematopoietic cells where it determines the intracellular fate of both IgG and IgG-containing immune complexes (IgG IC). Cross-linking of FcRn by multivalent IgG IC within antigen presenting cells such as dendritic cells initiates specific mechanisms that result in trafficking of the antigen-bearing IgG IC into compartments from which the antigen can successfully be processed into peptide epitopes compatible with loading onto both major histocompatibility complex class I and II molecules. In turn, this enables the synchronous activation of both CD4+ and CD8+ T cell responses against the cognate antigen, thereby bridging the gap between the humoral and cellular branches of the adaptive immune response. Critically, FcRn-driven T cell priming is efficient at very low doses of antigen due to the exquisite sensitivity of the IgG-mediated antigen delivery system through which it operates. FcRn-mediated antigen presentation has important consequences in tissue compartments replete with IgG and serves not only to determine homeostatic immune activation at a variety of sites but also to induce inflammatory responses upon exposure to antigens perceived as foreign. Therapeutically targeting the pathway by which FcRn enables T cell activation in response to IgG IC is thus a highly attractive prospect not only for the treatment of diseases that are driven by immune complexes but also for manipulating local immune responses against defined antigens such as those present during infections and

Lymphocytes with Immunoglobulin E Fc Receptors in Patients with Atopic Disorders

PubMed Central

Spiegelberg, H. L.; O'Connor, R. D.; Simon, R. A.; Mathison, D. A.

1979-01-01

Lymphocytes from normal nonallergic donors and patients with atopic disorders were analyzed for subpopulations bearing Fc receptors for immunoglobulin (Ig)E (Fcε) and IgG (Fcγ), surface IgM (sIgM) and IgD (sIgD), and for T cells forming spontaneous rosettes with sheep erythrocytes (E). The patients were divided into three groups according to serum IgE concentrations and systemic corticosteroid treatment. Group I consisted of 12 atopic patients with either normal or moderately increased IgE levels up to 4,000 U/ml. Four patients of group II and three of group III had 10,500-31,000 U/ml and severe atopic dermatitis. Patients of group III, but not I and II, were receiving corticosteroids systemically. The percentage (mean ±SD) and total number of Fcε+ lymphocytes were 1.2±0.5%, 41±24/mm3 in 12 normals; 1.6±0.9%, 59±43/mm3 in patients of group I: 7.0±2.0%, 187±67/mm3 in group II; and 0.3±0.1%, 13±5/mm3 in patients of group III. The increase in group II and decrease in group III of Fcε+ cells were statistically significantly different from the normal persons and patients of group I. In contrast, the patients did not differ significantly from the donors in sIgM+, sIgD+, Fcγ+, and E+ cell populations. As shown by depletion of sIg+ cells in four patients with atopic disorders, the great majority of the Fcε+ lymphocytes were B cells. However, two patients with elevated Fcε+ cell numbers had small numbers of mixed E- and Fcε-rosetting cells, presumably T cells. Two patients of group II were examined during an acute herpes simplex infection. Both showed an ≅80% decrease of Fcε+ cells at that time. No apparent correlation between numbers of Fcε+ cells and IgE level existed in patients of group I. Injection of an IgE myeloma protein into two monkeys did not significantly change their percentages of Fcε+ lymphocytes. The data indicate that Fcε+ lymphocytes are increased in patients with markedly elevated serum IgE and severe atopic disease, suggesting that

Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

PubMed Central

Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

2015-01-01

Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

Characterization and screening of IgG binding to the neonatal Fc receptor

PubMed Central

Neuber, Tobias; Frese, Katrin; Jaehrling, Jan; Jäger, Sebastian; Daubert, Daniela; Felderer, Karin; Linnemann, Mechthild; Höhne, Anne; Kaden, Stefan; Kölln, Johanna; Tiller, Thomas; Brocks, Bodo; Ostendorp, Ralf; Pabst, Stefan

2014-01-01

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding. PMID:24802048

The multiple faces of leukocyte interstitial migration

PubMed Central

Lämmermann, Tim; Germain, Ronald N.

2014-01-01

Spatiotemporal control of leukocyte dynamics within tissues is critical for successful innate and adaptive immune responses. Homeostatic trafficking and coordinated infiltration into and within sites of inflammation and infection rely on signaling in response to extracellular cues that in turn controls a variety of intracellular protein networks regulating leukocyte motility, migration, chemotaxis, positioning, and cell–cell interaction. In contrast to mesenchymal cells, leukocytes migrate in an amoeboid fashion by rapid cycles of actin polymerization and actomyosin contraction, and their migration in tissues is generally referred to as low adhesive and nonproteolytic. The interplay of actin network expansion, contraction, and adhesion shapes the exact mode of amoeboid migration, and in this review, we explore how leukocyte subsets potentially harness the same basic biomechanical mechanisms in a cell-type-specific manner. Most of our detailed understanding of these processes derives from in vitro migration studies in three-dimensional gels and confined spaces that mimic geometrical aspects of physiological tissues. We summarize these in vitro results and then critically compare them to data from intravital imaging of leukocyte interstitial migration in mouse tissues. We outline the technical challenges of obtaining conclusive mechanistic results from intravital studies, discuss leukocyte migration strategies in vivo, and present examples of mode switching during physiological interstitial migration. These findings are also placed in the context of leukocyte migration defects in primary immunodeficiencies. This overview of both in vitro and in vivo studies highlights recent progress in understanding the molecular and biophysical mechanisms that shape robust leukocyte migration responses in physiologically complex and heterogeneous environments. PMID:24573488

Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury

PubMed Central

Pyzik, Michal; Rath, Timo; Kuo, Timothy T.; Win, Sanda; Baker, Kristi; Hubbard, Jonathan J.; Grenha, Rosa; Gandhi, Amit; Krämer, Thomas D.; Mezo, Adam R.; McDonnell, Kevin; Nienaber, Vicki; Andersen, Jan Terje; Mizoguchi, Atsushi; Blumberg, Laurence; Purohit, Shalaka; Jones, Susan D.; Christianson, Greg; Lencer, Wayne I.; Sandlie, Inger; Kaplowitz, Neil; Roopenian, Derry C.; Blumberg, Richard S.

2017-01-01

The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn–albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity. PMID:28330995

[Mechanisms of leukocyte formation of endogenous pyrogen].

PubMed

Rybakina, E G; Sorokin, A V

1982-06-01

A study was made of the kinetics of endogenous pyrogen production by rabbit blood and exudate leukocytes and possible role played by the products of activated leukocytes in autoregulation of the process. It was established that accumulation of endogenous pyrogen in the cell precedes its release by stimulated cells. Then the processes of active pyrogen formation and release gel interdependent: pyrogen formed releases from the cell; the lowering of pyrogen concentration in the cell is accompanied by the decrease of its content in the medium. No stimulating effect of the products activated during leukocyte inflammation on pyrogen formation by blood leukocytes was discovered.

FcγRIII in ITP: it ain't over 'til it's over.

PubMed

McCrae, Keith R

2016-01-07

In this issue of Blood, Yu et al describe a novel anti–Fcγ receptor III (FcγRIII)-albumin fusion protein that inhibits the development of thrombocytopenia in a murine model of immune thrombocytopenia (ITP).1 The unique aspect of this protein is that it blocks FcγRIII-mediated uptake of antibody-coated platelets without activating FcγRIII and the associated inflammatory response.

Fusion protein of CDR mimetic peptide with Fc inhibit TNF-alpha induced cytotoxicity.

PubMed

Qin, Weisong; Feng, Jiannan; Li, Yan; Lin, Zhou; Shen, Beifen

2006-02-01

The variable regions of antibodies play central roles in the binding with antigens. Based on the model of a tumour necrosis factor-alpha (TNF-alpha) neutralizing monoclonal antibody (named as Z12) with TNF-alpha, heavy chain CDR2 (HCDR2) and light chain CDR3 (LCDR3) of Z12 were found to be the most responsible to bind with TNF-alpha. A mimetic peptide (PT) was designed based on the sequence derived from HCDR2 and LCDR3. Fusion protein PT-Fc was constructed by linking PT with Fc of human IgG1 through a flexible linker (GGGGGS). The primary structural characteristics of Fc and PT-Fc were analyzed, including the flexibility, hydrophilicity and epitopes. It was demonstrated that PT and Fc in the fusion protein possessed bio-function properly and non-interfering with each other. Furthermore, PT-Fc was expressed in Escherichia coli by fusion with thioredoxin (Trx). After trx-PT-Fc was cleaved with recombinant enterokinase, PT-Fc was obtained. The results of in vitro cytotoxic assays showed that both PT and PT-Fc could efficiently inhibit TNF-alpha induced apoptosis on L929 cells. At the same micromole concentration, the inhibition activity of PT-Fc was significantly higher than PT.

Durable Complete Responses in Some Recurrent High Grade Glioma Patients Treated with Toca 511 & Toca FC.

PubMed

Cloughesy, Timothy F; Landolfi, Joseph; Vogelbaum, Michael A; Ostertag, Derek; Elder, James B; Bloomfield, Stephen; Carter, Bob; Chen, Clark C; Kalkanis, Steven N; Kesari, Santosh; Lai, Albert; Lee, Ian Y; Liau, Linda M; Mikkelsen, Tom; Nghiemphu, Phioanh; Piccioni, David; Accomando, William; Diago, Oscar R; Hogan, Daniel J; Gammon, Dawn; Kasahara, Noriyuki; Kheoh, Thian; Jolly, Douglas J; Gruber, Harry E; Das, Asha; Walbert, Tobias

2018-05-12

Vocimagene amiretrorepvec (Toca 511) is an investigational gamma-retroviral replicating vector encoding cytosine deaminase that, when used in combination with extended-release 5-fluorocytosine (Toca FC), results preclinically in local production of 5-fluorouracil, depletion of immune-suppressive myeloid cells, and subsequent induction of anti-tumor immunity. Recurrent high grade glioma (rHGG) patients have a high unmet need for effective therapies that produce durable responses lasting more than 6 months. In this setting, relapse is nearly universal and most responses are transient. In this Toca 511 ascending-dose phase I trial (NCT01470794), HGG patients who recurred after standard of care underwent surgical resection, received Toca 511 injected into resection cavity wall followed by orally administered cycles of Toca FC. Among 56 patients, durable complete responses were observed. A subgroup was identified based on Toca 511 dose and entry requirements for the follow-up phase III study. In this subgroup, which included both IDH1-mutant and -wildtype tumors, the durable response rate is 21.7%. Median duration of follow-up for responders is 35.7+ months. As of August 25, 2017, all responders remain in response and are alive, 33.9+ to 52.2+ months after Toca 511 administration, suggesting a positive association of durable response with overall survival. Multi-year durable responses have been observed in rHGG patients treated with Toca 511 & Toca FC in a phase I trial and the treatment will be further evaluated in a randomized phase III trial. Among IDH1 mutant patients treated at first recurrence, there may be an enrichment of complete responders.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramsland, Paul A.; Farrugia, William; Bradford, Tessa M.

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. Fc{gamma}RIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of Fc{gamma}RIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of Fc{gamma}RIIa (Fc{gamma}RIIa-HR) and the Fc region of amore » humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence Fc{gamma}RIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for Fc{gamma}RIIa (IV.3), Fc{gamma}RIIb (X63-21), and a pan Fc{gamma}RII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of Fc{gamma}RIIa and Fc{gamma}RIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of Fc{gamma}RIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.« less

CD44 mediated hyaluronan adhesion of Toxoplasma gondii-infected leukocytes.

PubMed

Hayashi, Takeshi; Unno, Akihiro; Baba, Minami; Ohno, Tamio; Kitoh, Katsuya; Takashima, Yasuhiro

2014-04-01

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans and animals. Ingested parasites cross the intestinal epithelium, invade leukocytes and are then disseminated to peripheral organs. However, the mechanism of extravasation of the infected leukocytes remains poorly understood. In this study, we demonstrate that T. gondii-invaded human and mouse leukocytes express higher level of CD44, a ligand of hyaluronan (HA), and its expression on myeloid and non-myeloid leukocytes causes T. gondii-invaded human and mouse leukocyte to adhere to HA more effectively than non-invaded leukocytes. The specific adherence of parasite-invaded leukocytes was inhibited by anti CD44 antibody. Leukocytes of CD44 knockout mice did not show parasite-invaded leukocyte specific adhesion. Our results indicate that parasite-invaded leukocytes, regardless of whether myeloid or not, gain higher ability to adhere to HA than non-invaded leukocytes, via upregulation of CD44 expression and/or selective invasion to CD44 highly expressing cells. The difference in ability to adhere to HA between parasite-invaded cells and non-invaded neighboring cells might facilitate effective delivery of parasite-invaded leukocytes to the HA-producing endothelial cell surface and/or HA-rich extra cellular matrix. © 2013.

A novel factor H-Fc chimeric immunotherapeutic molecule against Neisseria gonorrhoeae

PubMed Central

Shaughnessy, Jutamas; Gulati, Sunita; Agarwal, Sarika; Unemo, Magnus; Ohnishi, Makoto; Su, Xia-Hong; Monks, Brian G.; Visintin, Alberto; Madico, Guillermo; Lewis, Lisa A.; Golenbock, Douglas T.; Reed, George W.; Rice, Peter A.; Ram, Sanjay

2015-01-01

Neisseria gonorrhoeae (Ng), the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including Ng, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18-20 (FH18-20). We explored the utility of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic. FH18-20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18-20 that eliminated complement activation on host cells, yet maintained binding to Ng, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18-20/Fc, but unlike FH18-20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical Ng isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10/15 (67%) strains and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant Ng. PMID:26773149

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow.

PubMed

Wang, Sheng; Ding, Miao; Xue, Boxin; Hou, Yingping; Sun, Yujie

2018-05-18

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

Structural characterization of the Man5 glycoform of human IgG3 Fc

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shah, Ishan S.; Lovell, Scott; Mehzabeen, Nurjahan

Immunoglobulin G (IgG) consists of four subclasses in humans: IgG1, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgG1, IgG3 exhibits a similar binding profile to Fcγ receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcγ receptor and C1q complement binding as well as maintaining optimal Fc conformation. We have determined themore » crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc2Man5 (Man5) high mannose glycoform at 1.8 Å resolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcγ receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function.« less

Rapid polyclonal desensitization with antibodies to IgE and FcεRIα

PubMed Central

Khodoun, Marat V.; Kucuk, Zeynep Yesim; Strait, Richard T.; Krishnamurthy, Durga; Janek, Kevin; Lewkowich, Ian; Morris, Suzanne C.; Finkelman, Fred D.

2013-01-01

Background Rapid desensitization,a procedure in which individuals allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky way to induce temporary tolerance. Objective To determine whether this approach can be adapted to suppress all IgE-mediated in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. Methods Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux and changes in cell number and FcεRI and IgE expression were evaluated. Results Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly slowlyinduced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer-lasting than rapid desensitization with antigen. Conclusion A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later, removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis. PMID:23632296

Effects of N-acetylcysteine and tirilazad mesylate on intestinal functional capillary density, leukocyte adherence, mesenteric plasma extravasation and cytokine levels in experimental endotoxemia in rats.

PubMed

Birnbaum, J; Lehmann, Ch; Klotz, E; Hein, O Vargas; Blume, A; Jubin, F; Polze, N; Luther, D; Spies, C D

2008-01-01

The study's objective was to determine the effects of the administration of N-acetylcysteine (NAC) and of tirilazad mesylate (TM) on intestinal functional capillary density, mesenteric plasma extravasation, leukocyte adherence and on cytokine release during experimental endotoxemia in rats. In a prospective, randomized, controlled animal study, 80 male Wistar rats were examined in 2 test series. Both series were divided into 4 groups. Group 1 served as control group (CON group). Group 2 (LPS group), group 3 (NAC group) and group 4 (TM group) received endotoxin infusions (10 mg/kg over 2 h). In NAC group 150 mg/kg body weight NAC was administered after the first 30 minutes of endotoxemia intravenously. In TM group, 10 mg/kg body weight TM was administered after the first 30 minutes of endotoxemia intravenously. Animals of the series 1 underwent studies of leukocyte adherence on submucosal venular endothelium of the small bowel wall and intestinal functional capillary density (FCD) in the intestinal mucosa and the circular as well as the longitudinal muscle layer by intravital fluorescence microscopy (IVM). Plasma levels of interleukin 1beta (IL-1beta), interferone gamma (IFN-gamma) and soluble intercellular adhesion molecule1 (s-ICAM 1) as well as white blood cell count (WBC) were estimated. In the animals of the series 2 mesenteric plasma extravasation was determined by IVM and plasma levels of tumor necrosis factor alpha (TNF-alpha), IL-4, IL-6, IL-10 and malondialdehyde (MDA) were estimated. After LPS administration, FCD in the villi intestinales was unchanged and in the longitudinal muscularis layer it was increased. There was no effect of NAC or TM administration on FCD.Although the plasma extravasation was not significantly influenced by LPS administration, TM administration resulted in a lower plasma extravasation in the TM group compared to the other groups. After endotoxin challenge, the firmly adherence of leukocytes to vascular endothelium as a parameter

Pathophysiologic roles of the fibrinogen gamma chain.

PubMed

Farrell, David H

2004-05-01

Fibrinogen binds through its gamma chains to cell surface receptors, growth factors, and coagulation factors to perform its key roles in fibrin clot formation, platelet aggregation, and wound healing. However, these binding interactions can also contribute to pathophysiologic processes, including inflammation and thrombosis. This review summarizes the latest findings on the role of the fibrinogen gamma chain in these processes, and illustrates the potential for therapeutic intervention. Novel gamma chain epitopes that bind platelet integrin alpha IIbbeta3 and leukocyte integrin alphaMbeta2 have been characterized, leading to the revision of former dogma regarding the processes of platelet aggregation, clot retraction, inflammation, and thrombosis. A series of studies has shown that the gamma chain serves as a depot for fibroblast growth factor-2 (FGF-2), which is likely to play an important role in wound healing. Inhibition of gamma chain function with the monoclonal antibody 7E9 has been shown to interfere with multiple fibrinogen activities, including factor XIIIa crosslinking, platelet adhesion, and platelet-mediated clot retraction. The role of the enigmatic variant fibrinogen gamma chain has also become clearer. Studies have shown that gamma chain binding to thrombin and factor XIII results in clots that are mechanically stiffer and resistant to fibrinolysis, which may explain the association between gammaA/gamma' fibrinogen levels and cardiovascular disease. The identification of new interactions with gamma chains has revealed novel targets for the treatment of inflammation and thrombosis. In addition, several exciting studies have shown new functions for the variant gamma chain that may contribute to cardiovascular disease.

Biomechanics of leukocyte rolling

PubMed Central

Sundd, Prithu; Pospieszalska, Maria K.; Cheung, Luthur Siu-Lun; Konstantopoulos, Konstantinos; Ley, Klaus

2011-01-01

Leukocyte rolling on endothelial cells and other P-selectin substrates is mediated by P-selectin binding to P-selectin glycoprotein ligand-1 expressed on the tips of leukocyte microvilli. Leukocyte rolling is a result of rapid, yet balanced formation and dissociation of selectin-ligand bonds in the presence of hydrodynamic shear forces. The hydrodynamic forces acting on the bonds may either increase (catch bonds) or decrease (slip-bonds) their lifetimes. The force-dependent ‘catch-slip’ bond kinetics are explained using the ‘two pathway model’ for bond dissociation. Both the ‘sliding-rebinding’ and the ‘allosteric’ mechanisms attribute ‘catch-slip’ bond behavior to the force-induced conformational changes in the lectin-EGF domain hinge of selectins. Below a threshold shear stress, selectins cannot mediate rolling. This ‘shear-threshold’ phenomenon is a consequence of shear-enhanced tethering and catch-bond enhanced rolling. Quantitative dynamic footprinting microscopy has revealed that leukocytes rolling at venular shear stresses (> 0.6 Pa) undergo cellular deformation (large footprint) and form long tethers. The hydrodynamic shear force and torque acting on the rolling cell are thought to be synergistically balanced by the forces acting on tethers and stressed microvilli, however, their relative contribution remains to be determined. Thus, improvement beyond the current understanding requires in silico models that can predict both cellular and microvillus deformation and experiments that allow measurement of forces acting on individual microvilli and tethers. PMID:21515934

Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins.

PubMed

Ha, Ji-Hee; Kim, Jung-Eun; Kim, Yong-Sung

2016-01-01

The monospecific and bivalent characteristics of naturally occurring immunoglobulin G (IgG) antibodies depend on homodimerization of the fragment crystallizable (Fc) regions of two identical heavy chains (HCs) and the subsequent assembly of two identical light chains (LCs) via disulfide linkages between each HC and LC. Immunoglobulin Fc heterodimers have been engineered through modifications to the CH3 domain interface, with different mutations on each domain such that the engineered Fc fragments, carrying the CH3 variant pair, preferentially form heterodimers rather than homodimers. Many research groups have adopted different strategies to generate Fc heterodimers, with the goal of high heterodimerization yield, while retaining biophysical and biological properties of the wild-type Fc. Based on their ability to enforce heterodimerization between the two different HCs, the established Fc heterodimers have been extensively exploited as a scaffold to generate bispecific antibodies (bsAbs) in full-length IgG and IgG-like formats. These have many of the favorable properties of natural IgG antibodies, such as high stability, long serum half-life, low immunogenicity, and immune effector functions. As of July 2016, more than seven heterodimeric Fc-based IgG-format bsAbs are being evaluated in clinical trials. In addition to bsAbs, heterodimeric Fc technology is very promising for the generation of Fc-fused proteins and peptides, as well as cytokines (immunocytokines), which can present the fusion partners in the natural monomeric or heterodimeric form rather than the artificial homodimeric form with wild-type Fc. Here, we present relevant concepts and strategies for the generation of heterodimeric Fc proteins, and their application in the development of bsAbs in diverse formats for optimal biological activity. In addition, we describe wild-type Fc-fused monomeric and heterodimeric proteins, along with the difficulties associated with their preparations, and discuss the

Humoral immunity provides resident intestinal eosinophils access to luminal antigen via eosinophil-expressed low affinity Fc gamma receptors

PubMed Central

Smith, Kalmia M.; Rahman, Raiann S.; Spencer, Lisa A.

2016-01-01

Eosinophils are native to the healthy gastrointestinal tract, and are associated with inflammatory diseases likely triggered by exposure to food allergens (e.g. food allergies and eosinophilic gastrointestinal disorders). In models of allergic respiratory diseases and in vitro studies, direct antigen engagement elicits eosinophil effector functions including degranulation and antigen presentation. However, it was not known whether intestinal tissue eosinophils that are separated from luminal food antigens by a columnar epithelium might similarly engage food antigens. Using an intestinal ligated loop model in mice, here we determined that resident intestinal eosinophils acquire antigen from the lumen of antigen-sensitized but not naïve mice in vivo. Antigen acquisition was immunoglobulin-dependent; intestinal eosinophils were unable to acquire antigen in sensitized immunoglobulin-deficient mice, and passive immunization with immune serum or antigen-specific IgG was sufficient to enable intestinal eosinophils in otherwise naïve mice to acquire antigen in vivo. Intestinal eosinophils expressed low affinity IgG receptors, and the activating receptor FcγRIII was necessary for immunoglobulin-mediated acquisition of antigens by isolated intestinal eosinophils in vitro. Our combined data suggest that intestinal eosinophils acquire lumen-derived food antigens in sensitized mice via FcγRIII antigen focusing, and may therefore participate in antigen-driven secondary immune responses to oral antigens. PMID:27683752

Endothelial cell regulation of leukocyte infiltration in inflammatory tissues

PubMed Central

Mantovani, A.; Introna, M.; Dejana, E.

1995-01-01

Endothelial cells play an important, active role in the onset and regulation of inflammatory and immune reactions. Through the production of chemokines they attract leukocytes and activate their adhesive receptors. This leads to the anchorage of leukocytes to the adhesive molecules expressed on the endothelial surface. Leukocyte adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. Adhesive molecules (such as PECAM) on the endothelial cell surface might also ‘direct’ leukocytes through the intercellular junction by haptotaxis. The information available on the molecular structure and functional properties of endothelial chemokines, adhesive molecules or junction organization is still fragmentary. Further work is needed to clarify how they interplay in regulating leukocyte infiltration into tissues. PMID:18475659

Cloning, characterization, and expression analysis of the DEAD-box family genes, Fc-vasa and Fc-PL10a, in Chinese shrimp ( Fenneropenaeus chinensis)

NASA Astrophysics Data System (ADS)

Zhou, Qianru; Shao, Mingyu; Qin, Zhenkui; Kyoung, Ho Kang; Zhang, Zhifeng

2010-01-01

RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication, DNA repair, and RNA processing. However, the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level. We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins, Fc-vasa and Fc-PL10a, from the testes of Chinese shrimp, Fenneropenaeus chinensis. The two predicted amino acid sequences contain all the conserved motifs characterized by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PL10 subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-vasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.

Genomic signatures characterize leukocyte infiltration in myositis muscles

PubMed Central

2012-01-01

Background Leukocyte infiltration plays an important role in the pathogenesis and progression of myositis, and is highly associated with disease severity. Currently, there is a lack of: efficacious therapies for myositis; understanding of the molecular features important for disease pathogenesis; and potential molecular biomarkers for characterizing inflammatory myopathies to aid in clinical development. Methods In this study, we developed a simple model and predicted that 1) leukocyte-specific transcripts (including both protein-coding transcripts and microRNAs) should be coherently overexpressed in myositis muscle and 2) the level of over-expression of these transcripts should be correlated with leukocyte infiltration. We applied this model to assess immune cell infiltration in myositis by examining mRNA and microRNA (miRNA) expression profiles in muscle biopsies from 31 myositis patients and 5 normal controls. Results Several gene signatures, including a leukocyte index, type 1 interferon (IFN), MHC class I, and immunoglobulin signature, were developed to characterize myositis patients at the molecular level. The leukocyte index, consisting of genes predominantly associated with immune function, displayed strong concordance with pathological assessment of immune cell infiltration. This leukocyte index was subsequently utilized to differentiate transcriptional changes due to leukocyte infiltration from other alterations in myositis muscle. Results from this differentiation revealed biologically relevant differences in the relationship between the type 1 IFN pathway, miR-146a, and leukocyte infiltration within various myositis subtypes. Conclusions Results indicate that a likely interaction between miR-146a expression and the type 1 IFN pathway is confounded by the level of leukocyte infiltration into muscle tissue. Although the role of miR-146a in myositis remains uncertain, our results highlight the potential benefit of deconvoluting the source of

Genomic signatures characterize leukocyte infiltration in myositis muscles.

PubMed

Zhu, Wei; Streicher, Katie; Shen, Nan; Higgs, Brandon W; Morehouse, Chris; Greenlees, Lydia; Amato, Anthony A; Ranade, Koustubh; Richman, Laura; Fiorentino, David; Jallal, Bahija; Greenberg, Steven A; Yao, Yihong

2012-11-21

Leukocyte infiltration plays an important role in the pathogenesis and progression of myositis, and is highly associated with disease severity. Currently, there is a lack of: efficacious therapies for myositis; understanding of the molecular features important for disease pathogenesis; and potential molecular biomarkers for characterizing inflammatory myopathies to aid in clinical development. In this study, we developed a simple model and predicted that 1) leukocyte-specific transcripts (including both protein-coding transcripts and microRNAs) should be coherently overexpressed in myositis muscle and 2) the level of over-expression of these transcripts should be correlated with leukocyte infiltration. We applied this model to assess immune cell infiltration in myositis by examining mRNA and microRNA (miRNA) expression profiles in muscle biopsies from 31 myositis patients and 5 normal controls. Several gene signatures, including a leukocyte index, type 1 interferon (IFN), MHC class I, and immunoglobulin signature, were developed to characterize myositis patients at the molecular level. The leukocyte index, consisting of genes predominantly associated with immune function, displayed strong concordance with pathological assessment of immune cell infiltration. This leukocyte index was subsequently utilized to differentiate transcriptional changes due to leukocyte infiltration from other alterations in myositis muscle. Results from this differentiation revealed biologically relevant differences in the relationship between the type 1 IFN pathway, miR-146a, and leukocyte infiltration within various myositis subtypes. Results indicate that a likely interaction between miR-146a expression and the type 1 IFN pathway is confounded by the level of leukocyte infiltration into muscle tissue. Although the role of miR-146a in myositis remains uncertain, our results highlight the potential benefit of deconvoluting the source of transcriptional changes in myositis muscle or other

Structural Heterogeneity and Functional Domains of Murine Immunoglobulin G Fc Receptors

NASA Astrophysics Data System (ADS)

Ravetch, Jeffrey V.; Luster, Andrew D.; Weinshank, Richard; Kochan, Jarema; Pavlovec, Amalia; Portnoy, Daniel A.; Hulmes, Jeffrey; Pan, Yu-Ching E.; Unkeless, Jay C.

1986-11-01

Binding of antibodies to effector cells by way of receptors to their constant regions (Fc receptors) is central to the pathway that leads to clearance of antigens by the immune system. The structure and function of this important class of receptors on immune cells is addressed through the molecular characterization of Fc receptors (FcR) specific for the murine immunoglobulin G isotype. Structural diversity is encoded by two genes that by alternative splicing result in expression of molecules with highly conserved extracellular domains and different transmembrane and intracytoplasmic domains. The proteins encoded by these genes are members of the immunoglobulin supergene family, most homologous to the major histocompatibility complex molecule Eβ. Functional reconstitution of ligand binding by transfection of individual FcR genes demonstrates that the requirements for ligand binding are encoded in a single gene. These studies demonstrate the molecular basis for the functional heterogeneity of FcR's, accounting for the possible transduction of different signals in response to a single ligand.

Matrix interference from Fc-Fc interactions in immunoassays for detecting human IgG4 therapeutics.

PubMed

Partridge, Michael A; Karayusuf, Elif Kabuloglu; Dhulipala, Gangadhar; Dreyer, Robert; Daly, Thomas; Sumner, Giane; Pyles, Erica; Torri, Albert

2015-01-01

An assay measuring an IgG4 biotherapeutic in human serum used a drug-specific monoclonal antibody (mAb) capture reagent and an antihuman IgG4 mAb as detection reagent. However, serum IgG4 binding to the capture mAb via Fc-interactions was detected by the anti-IgG4 mAb, causing high background. Two approaches were developed to minimize background; incorporating a mild acid sample preparation step or using the Fab of the capture antibody. Either strategy improved signal:noise dramatically, increasing assay sensitivity >20-fold. Biophysical analyses of antibody domains indicated that noncovalent Fc oligomers could inhibit the background. Matrix interference from human IgG4 binding to the capture mAb was reduced with a Fab fragment of the drug-specific capture antibody or by incorporating a mild acid sample treatment into the assay.

A dual positive and negative regulation of monocyte activation by leukocyte Ig-like receptor B4 depends on the position of the tyrosine residues in its ITIMs.

PubMed

Park, Mijeong; Liu, Robert W; An, Hongyan; Geczy, Carolyn L; Thomas, Paul S; Tedla, Nicodemus

2017-05-01

The leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory cell surface receptor, primarily expressed on mono-myeloid cells. It contains 2 C-type Ig-like extracellular domains and a long cytoplasmic domain that contains three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Data suggest that LILRB4 suppresses Fc receptor-dependent monocyte functions via its ITIMs, but relative contributions of the three ITIMs are not characterised. To address this, tyrosine (Tyr) residues at positions 337, 389 and 419 were single, double or triple mutated to phenylalanine and stably transfected into a human monocytic cell line, THP-1. Intact Tyr 389 was sufficient to maximally inhibit FcγRI-mediated TNF-α production in THP-1 cells, but, paradoxically, Tyr 337 significantly enhanced TNF-α production. In contrast, bactericidal activity was significantly enhanced in mutants containing Tyr 419 , while Tyr 337 markedly inhibited bacteria killing. Taken together, these results indicate that LILRB4 might have dual inhibitory and activating functions, depending on the position of the functional tyrosine residues in its ITIMs and/or the nature of the stimuli.

Rapid polyclonal desensitization with antibodies to IgE and FcεRIα.

PubMed

Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Lewkowich, Ian; Morris, Suzanne C; Finkelman, Fred D

2013-06-01

Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance. We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated. Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen. A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Fcgamma chain deficiency on hematopoietic cells ameliorates atherosclerosis in apoe-knockout mice by promoting Th2 responses

USDA-ARS?s Scientific Manuscript database

We have previously shown that oxLDL-immune complexes (oxLDL-IC) binding to Fcgamma receptors (Fc gamma R) expressed on human monocytes leads to induction of pro-inflammatory cytokines. Four classes of mouse Fc gamma Rs have been defined: Fc gamma RI, II, III, and IV. Functionally, Fc gamma Rs can be...

Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein.

PubMed

Oberst, Michael D; Augé, Catherine; Morris, Chad; Kentner, Stacy; Mulgrew, Kathy; McGlinchey, Kelly; Hair, James; Hanabuchi, Shino; Du, Qun; Damschroder, Melissa; Feng, Hui; Eck, Steven; Buss, Nicholas; de Haan, Lolke; Pierce, Andrew J; Park, Haesun; Sylwester, Andrew; Axthelm, Michael K; Picker, Louis; Morris, Nicholas P; Weinberg, Andrew; Hammond, Scott A

2018-05-01

Ligation of OX40 (CD134, TNFRSF4) on activated T cells by its natural ligand (OX40L, CD252, TNFSF4) enhances cellular survival, proliferation, and effector functions such as cytokine release and cellular cytotoxicity. We engineered a recombinant human OX40L IgG4P Fc fusion protein termed MEDI6383 that assembles into a hexameric structure and exerts potent agonist activity following engagement of OX40. MEDI6383 displayed solution-phase agonist activity that was enhanced when the fusion protein was clustered by Fc gamma receptors (FcγRs) on the surface of adjacent cells. The resulting costimulation of OX40 on T cells induced NFκB promoter activity in OX40-expressing T cells and induced Th1-type cytokine production, proliferation, and resistance to regulatory T cell (Treg)-mediated suppression. MEDI6383 enhanced the cytolytic activity of tumor-reactive T cells and reduced tumor growth in the context of an alloreactive human T cell:tumor cell admix model in immunocompromised mice. Consistent with the role of OX40 costimulation in the expansion of memory T cells, MEDI6383 administered to healthy nonhuman primates elicited peripheral blood CD4 and CD8 central and effector memory T-cell proliferation as well as B-cell proliferation. Together, these results suggest that OX40 agonism has the potential to enhance antitumor immunity in human malignancies. Mol Cancer Ther; 17(5); 1024-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Understanding the impact of Fc glycosylation on its conformational changes by molecular dynamics simulations and bioinformatics.

PubMed

Zhang, Yubo

2015-12-01

N-linked glycosylation of Fc at N297 plays an important role in its effector function, aberrance of which would cause disease pathogenesis. Here, we performed all-atom molecular dynamics simulations to explore the effects of Fc glycosylation on its dynamics behaviors. Firstly, equilibrium simulations suggested that Fc deglycosylation was able to induce residual flexibility in its CH2 domain. Besides, the free energy landscape revealed three minimum energy wells in deglycosylated Fc, representing its "open", "semi-closed" and "closed" states. However, we could only observe the "open" state of glycosylated Fc. Supportively, principal component analysis emphasized the prominent motion of delyclosylated Fc and dynamically depicted how it changed from the "open" state to its "closed" state. Secondly, we studied the recognition mechanism of the Fc binding to its partners. Energy decomposition analysis identified key residues of Fc to recognize its two partners P13 and P34. Evidently, electrostatic potential surfaces showed that electrostatic attraction helped to stabilize the interaction between Fc and its partners. Also, relative binding free energies explained different binding affinities in Fc-P13 and Fc-P34. Collectively, these results together provided the structural basis for understanding conformational changes of deglycosylated Fc and the recognition mechanism of the Fc binding to its partners.

Putative suppressing effect of IgG Fc-conjugated haemagglutinin (HA) stalk of influenza virus H7N9 on the neutralizing immunogenicity of Fc-conjugated HA head: implication for rational design of HA-based influenza vaccines.

PubMed

He, B; Xia, S; Yu, F; Fu, Y; Li, W; Wang, Q; Lu, L; Jiang, S

2016-02-01

The emergence of influenza A H7N9 in infection has posed a great threat to public health globally. Poor immunogenicity of H7N9 haemagglutinin (HA) is a major obstacle to the development of an effective H7N9 vaccine. Here, we found that the vaccine containing the H7HA head conjugated with IgG Fc (Hd-Fc) induced strong neutralizing antibody responses and protection against H7N9 infection, whilst the Fc-conjugated H7HA stalk (St-Fc)-based vaccine could not induce neutralizing antibodies, although the St-Fc-immunized mice were partially protected. The vaccines containing the full-length extracellular domain of HA conjugated with Fc and the mixture of Hd-Fc plus St-Fc induced significantly lower neutralizing antibody and haemagglutination inhibition titres than the Hd-Fc-based vaccine. These results suggest that the St-Fc may have inhibitory effects on the neutralizing immunogenicity of Hd-Fc. Therefore, the neutralizing domain(s), such as the receptor-binding domain, in the HA head should be kept and the non-neutralizing domain(s) in the HA stalk with the ability to potentially suppress the neutralizing immunogenicity of HA head should be removed from Fc-conjugated HA-based influenza vaccines to increase the neutralizing antibody response.

Chicken IgY Fc expressed by Eimeria mitis enhances the immunogenicity of E. mitis.

PubMed

Qin, Mei; Tang, Xinming; Yin, Guangwen; Liu, Xianyong; Suo, Jingxia; Tao, Geru; Ei-Ashram, Saeed; Li, Yuan; Suo, Xun

2016-03-21

Eimeria species are obligate intracellular apicomplexan parasites, causing great economic losses in the poultry industry. Currently wild-and attenuated- type anticoccidial vaccines are used to control coccidiosis. However, their use in fast growing broilers is limited by vaccination side effects caused by medium and/or low immunogenic Eimeria spp. There is, therefore, a need for a vaccine with high immunogenicity for broilers. The avian yolk sac IgY Fc is the avian counterpart of the mammalian IgG Fc, which enhances immunogenicity of Fc-fusion proteins. Here, we developed a stable transgenic Eimeria mitis expressing IgY Fc (Emi.chFc) and investigated whether the avian IgY Fc fragment enhances the immunogenicity of E. mitis. Two-week-old broilers were immunized with either Emi.chFc or wild type Eimeria and challenged with wild type E. mitis to analyze the protective properties of transgenic Emi.chFc. Chickens immunized with Emi.chFc had significantly lower oocyst output, in comparison with PBS, mock control (transgenic E. mitis expressing HA1 from H9N2 avian influenza virus) and wildtype E. mitis immunized groups after challenge, indicating that IgY Fc enhanced the immunogenicity of E. mitis. Our findings suggest that IgY Fc-expressing Eimeria may be a better coccidiosis vaccine, and transgenic Eimeria expressing Fc-fused exogenous antigens may be used as a novel vaccine-delivery vehicle against a wide variety of pathogens.

Increased circulating leukocyte-derived microparticles in ischemic cerebrovascular disease.

PubMed

He, Zhangping; Tang, Yanyan; Qin, Chao

2017-06-01

Circulating leukocyte-derived microparticles act as proinflammatory mediators that reflect vascular inflammation. In this study, we examined the hypothesis that the quantity of leukocyte-derived microparticles is increased in patients with ischemic cerebrovascular diseases, and investigated utility of various phenotypes of leukocyte-derived microparticles as specific biomarkers of vascular inflammation injury. Additionally we focused on identifying leukocyte-derived microparticles that may be correlated with stroke severity in acute ischemic stroke patients. The plasma concentration of leukocyte-derived microparticles obtained by a series of centrifugations of 76 consecutive patients with ischemic cerebrovascular diseases and 70 age-, sex-, and race-matched healthy controls were determined by flow cytometry. Significantly elevated numbers of leukocyte (CD45+), monocyte (CD14+), lymphocyte (CD4+), granulocyte (CD15+) derived microparticles were found in the plasma samples of patients ischemic cerebrovascular diseases, compared to healthy controls (p<0.05). Furthermore, the plasma levels of CD14+ microparticles were significantly correlated with stroke severity (r=0.355, p=0.019), cerebral vascular stenosis severity (r=0.255, p=0.025) and stroke subtype (r=0.242, p=0.036). No association with stroke was observed for other leukocyte-derived phenotypes. These results demonstrate that circulating leukocyte-derived microparticles amounts are increased in patients with ischemic cerebrovascular diseases, compared with healthy controls. As proinflammatory mediators, leukocyte-derived microparticles may contribute to vascular inflammatory and the inflammatory process in acute ischemic stroke. Levels of CD14+ microparticles may be a promising biomarker of ischemic severity and outcome of stroke in the clinic. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation by CD45 of the tyrosine phosphorylation of high affinity IgE receptor beta- and gamma-chains.

PubMed

Adamczewski, M; Numerof, R P; Koretzky, G A; Kinet, J P

1995-04-01

Previous studies using tyrosine phosphatase inhibitors have implicated tyrosine phosphatases in the signal transduction pathway initiated by aggregation of Fc epsilon RI, the high affinity receptor for IgE. To define more precisely a role for the tyrosine phosphatase CD45 in Fc epsilon RI-mediated signaling, we have transfected the three subunits of Fc epsilon RI into wild-type Jurkat and a CD45-deficient Jurkat derivative. Here we demonstrate that CD45 is necessary for the initiation of calcium flux through the transfected Fc epsilon RI. In contrast to the effect of phosphatase inhibitors, the tyrosine phosphorylation levels of beta and gamma after aggregation of Fc epsilon RI are surprisingly reduced, relative to wild-type Jurkat, in the CD45-deficient cells. After reconstitution of the CD45-deficient cells with a chimeric molecule containing the cytoplasmic phosphatase domains of CD45, both the base line and activation-induced tyrosine phosphorylation levels are increased. By examining Lck autophosphorylation, we find that Fc epsilon RI aggregation induces an increase in Lck enzymatic activity only in wild-type Jurkat and the CD45-deficient Jurkat reconstituted with chimeric CD45. This regulation of src-family tyrosine kinase activity may be the means by which CD45 controls aggregation-induced receptor phosphorylation.

Asymmetrical Fc Engineering Greatly Enhances Antibody-dependent Cellular Cytotoxicity (ADCC) Effector Function and Stability of the Modified Antibodies*

PubMed Central

Liu, Zhi; Gunasekaran, Kannan; Wang, Wei; Razinkov, Vladimir; Sekirov, Laura; Leng, Esther; Sweet, Heather; Foltz, Ian; Howard, Monique; Rousseau, Anne-Marie; Kozlosky, Carl; Fanslow, William; Yan, Wei

2014-01-01

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases. PMID:24311787

Synthesis of endogenous pyrogen by rabbit leukocytes.

PubMed

Moore, D M; Murphy, P A; Chesney, P J; Wood, W B

1973-05-01

Rabbit ieukocytes from peritoneal exudates and from blood were stimulated to form leukocyte pyrogen in the presence of radiolabeled amino acids. The stimuli used were endotoxin, phagocytosis, and tuberculin. The crude leukocyte pyrogen samples were purified; pyrogen from exudate cells was rendered homogeneous; pyrogen from blood cells was still contaminated with other proteins. All the purified pyrogens were radioactive; and for all it was shown that radioactivity and pyrogenic activity coincided on electrophoresis at pH 3.5 and pH 9 in acrylamide and on isoelectric focusing in acrylamide. Furthermore, pyrogens obtained from exudate cells stimulated in different ways, or from blood cells and exudate cells stimulated with endotoxin, appeared to be identical. These results suggest that leukocyte pyrogen was synthesized de novo from amino acid precursors and that leukocytes made the same pyrogen whatever the stimulus used to activate them.

Altered regulation of Fc gamma RII on aged follicular dendritic cells correlates with immunoreceptor tyrosine-based inhibition motif signaling in B cells and reduced germinal center formation.

PubMed

Aydar, Yüksel; Balogh, Péter; Tew, John G; Szakal, Andras K

2003-12-01

Aging is associated with reduced trapping of Ag in the form of in immune complexes (ICs) by follicular dendritic cells (FDCs). We postulated that this defect was due to altered regulation of IC trapping receptors. The level of FDC-M1, complement receptors 1 and 2, FcgammaRII, and FDC-M2 on FDCs was immunohistochemically quantitated in draining lymph nodes of actively immunized mice for 10 days after Ag challenge. Initially, FDC FcgammaRII levels were similar but by day 3 a drastic reduction in FDC-FcgammaRII expression was apparent in old mice. FDC-M2 labeling, reflecting IC trapping, was also reduced and correlated with a dramatic reduction in germinal center (GC) B cells as indicated by reduced GC size and number. Nevertheless, labeling of FDC reticula with FDC-M1 and anti-complement receptors 1 and 2 was preserved, indicating that FDCs were present. FDCs in active GCs normally express high levels of FcRs that are thought to bind Fc portions of Abs in ICs and minimize their binding to FcRs on B cells. Thus, cross-linking of B cell receptor and FcR via IC is minimized, thereby reducing signaling via the immunoreceptor tyrosine-based inhibition motif. Old FDCs taken at day 3, when they lack FcgammaRII, were incapable of preventing immunoreceptor tyrosine-based inhibition motif signaling in wild-type B cells but old FDCs stimulated B cells from FcgammaRIIB(-/-) mice to produce near normal levels of specific Ab. The present data support the concept that FcR are regulated abnormally on old FDCs. This abnormality correlates with a reduced IC retention and with a reduced capacity of FDCs to present ICs in a way that will activate GC B cells.

Indium-111 leukocyte imaging in patients with rheumatoid arthritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uno, K.; Matsui, N.; Nohira, K.

1986-03-01

This study evaluates the usefulness of labeled leukocyte imaging in patients with rheumatoid arthritis. In 33 patients, the incidence of pain and swelling in 66 wrist joints and 66 knee joints was compared with the accumulation of (/sup 111/In)leukocytes. No accumulation of (/sup 111/In)leukocytes was seen in any of the patients' wrists (0/12) or knee joints (0/14) when both pain and swelling were absent. In contrast, 93% (25/27) of wrist joints and 80% (24/30) of knee joints with both pain and swelling were positive by (/sup 111/In)leukocyte scintigraphy. There was little correlation between the stage of the disease, as determinedmore » by radiography, and (/sup 111/In)leukocyte accumulation. This study suggests that (/sup 111/In)leukocyte imaging may be a reliable procedure for monitoring the activity of rheumatoid arthritis, especially for confirming the lack of an ongoing inflammatory response.« less

Physiological levels of testosterone kill salmonid leukocytes in vitro

USGS Publications Warehouse

Slater, C.H.; Schreck, C.B.

1997-01-01

Adult spring chinook salmon (Oncorhynchus tshawytscha) elaborate high plasma concentrations of testosterone during sexual maturation, and these levels of testosterone have been shown to reduce the salmonid immune response in vitro. Our search for the mechanism of testosterone's immunosuppressive action has led to the characterization of an androgen receptor in salmonid leukocytes. In the present study we examined the specific effects that testosterone had on salmonid leukocytes. Direct counts of viable leukocytes after incubation with and without physiological levels of testosterone demonstrate a significant loss of leukocytes in cultures exposed to testosterone. At least 5 days of contact with testosterone was required to produce significant immunosuppression and addition of a 'conditioned media' (supernatant from proliferating lymphocytes not exposed to testosterone) did not reverse the immunosuppressive effects of testosterone. These data lead us to conclude that testosterone may exert its immunosuppressive effects by direct action on salmonid leukocytes, through the androgen receptor described, and that this action leads to the death of a significant number of these leukocytes.

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

PubMed Central

Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

PubMed

Konduru, Krishnamurthy; Shurtleff, Amy C; Bradfute, Steven B; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

DOE PAGES

Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; ...

2016-09-13

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulatedmore » with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 10 5–10 6 and neutralizing antibody titers of approximately 10 3 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deletedEBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-lengthGP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc,and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. In conclusion

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

DOE Office of Scientific and Technical Information (OSTI.GOV)

Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulatedmore » with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 10 5–10 6 and neutralizing antibody titers of approximately 10 3 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deletedEBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-lengthGP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc,and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. In conclusion

Sialylated IgG-Fc: a novel biomarker of chronic inflammatory demyelinating polyneuropathy.

PubMed

Wong, Anna Hiu Yi; Fukami, Yuki; Sudo, Makoto; Kokubun, Norito; Hamada, Shinsuke; Yuki, Nobuhiro

2016-03-01

Sialylation in Fc portion of IgG plays a crucial role in the pathogenesis of autoimmune diseases and the working mechanism of intravenous immunoglobulin (IVIG). We aim to test whether IgG-Fc sialylation is a biomarker of disease activity for chronic inflammatory demyelinating polyneuropathy (CIDP). By using specific lectins for sialylation, galactosylation and agalactosylation, lectin-enzyme assay and lectin blotting with pretreatment of IgG degradating enzyme of Streptococcus pyogenes were performed to compare the glycosylation levels of serum IgG-Fc (1) between patients of untreated CIDP (n=107) and normal control subjects (n=27), (2) among patients with untreated CIDP of different clinical severities and (3) before and after IVIG treatment of patients with CIDP (n=12). Sialylation and galactosylation of IgG-Fc were significantly reduced in patients with CIDP than normal control subjects (p=0.003 and 0.033, respectively), whereas agalactosylation was increased in CIDP (p=0.21). Ratios of sialylated/agalactosylated IgG-Fc levels were significantly reduced in CIDP (p<0.001) and inversely related to disease severity (p=0.044). After IVIG treatment, levels of sialylated IgG-Fc significantly increased (p=0.003). Sialylation of IgG-Fc is reduced in CIDP. Its level correlated with clinical severity and increased after IVIG treatment. Sialylated as well as ratio of sialylated/agalactosylated IgG-Fc could be new measures to monitor the disease severity and treatment status in CIDP. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Platelets Guide Leukocytes to Their Sites of Extravasation

PubMed Central

Puhr-Westerheide, Daniel; Pörnbacher, Michaela; Lauber, Kirsten; Krombach, Fritz; Reichel, Christoph Andreas

2016-01-01

Effective immune responses require the directed migration of leukocytes from the vasculature to the site of injury or infection. How immune cells “find” their site of extravasation remains largely obscure. Here, we identified a previously unrecognized role of platelets as pathfinders guiding leukocytes to their exit points in the microvasculature: upon onset of inflammation, circulating platelets were found to immediately adhere at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40-CD40L-dependent interactions. In this cellular crosstalk, ligation of PSGL-1 by P-selectin leads to ERK1/2 MAPK-dependent conformational changes of leukocyte integrins, which promote the successive extravasation of neutrophils and monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses. Our experimental data uncover a platelet-directed, spatiotemporally organized, multicellular crosstalk that is essential for effective trafficking of leukocytes to the site of inflammation. PMID:27152726

Inhibition of human polymorphonuclear leukocyte function by components of human colostrum and mature milk.

PubMed

Pickering, L K; Cleary, T G; Caprioli, R M

1983-04-01

To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 +/- 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-(14)C]glucose utilization, and Fc receptors were significantly (P < 0.05 to P < 0.001) less in PMNL exposed to whole human colostrum or colostral lipid than in non-lipid-exposed cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-(3)H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.

Mucosal CXCR4+ IgG plasma cells contribute to the pathogenesis of human ulcerative colitis through FcγR-mediated CD14 macrophage activation.

PubMed

Uo, Michihide; Hisamatsu, Tadakazu; Miyoshi, Jun; Kaito, Daiki; Yoneno, Kazuaki; Kitazume, Mina T; Mori, Maiko; Sugita, Akira; Koganei, Kazutaka; Matsuoka, Katsuyoshi; Kanai, Takanori; Hibi, Toshifumi

2013-12-01

Chronic inflammation characterised by IgG-producing plasma cell infiltration of colonic mucosa is a histological hallmark of ulcerative colitis (UC); however, whether its function is pathogenic or protective remains unclear. To explore the contribution of intestinal IgG plasma cells to UC pathogenesis. We isolated lamina propria mononuclear cells (LPMCs) from intestinal mucosa of UC patients and analysed the characteristics of intestinal plasma cells (expression profiles of differentiation molecules and chemokine receptors). We investigated the involvement of IgG-immune complex (IC)-Fc gamma receptor (FcγR) signalling in intestinal inflammation by examining the cytokine production by LPMCs in response to IgG-IC stimulation. IgG plasma cells that were markedly increased in number in the inflamed mucosa of UC patients showed a distinct expression profile (CD19(+)CD27(low), CCR10(low)CXCR4(high)) compared with IgA plasma cells (CD19(+/-)CD27(high), CCR10(high)CXCR4(-/low)). In vitro IgG-IC stimulation activated intestinal CD14 macrophages that were increased in number in the inflamed mucosa of UC patients via FcγRI and FcγRII, and induced the extensive production of pro-inflammatory cytokines such as tumour necrosis factor (TNF) and interleukin-1β (IL-1β), comparable to the effect of commensal bacteria stimulation. Co-stimulation with IgG-IC and commensal bacteria increased TNF and IL-1β production more than stimulation with the latter alone. Furthermore, IgG-IC notably up-regulated the expression of TL1A, whereas commensal bacteria specifically induced IL-23. Collectively, these results demonstrate a novel aspect of UC pathogenesis in which unique IgG plasma cells infiltrate the inflamed mucosa via CXCR4, and critically influence UC pathogenesis by exacerbating mucosal inflammation through the activation of 'pathogenic' intestinal CD14 macrophages via IgG-IC-FcγR signalling.

SYNTHESIS OF ENDOGENOUS PYROGEN BY RABBIT LEUKOCYTES

PubMed Central

Moore, Douglas M.; Murphy, Patrick A.; Chesney, P. Joan; Wood, W. B.

1973-01-01

Rabbit ieukocytes from peritoneal exudates and from blood were stimulated to form leukocyte pyrogen in the presence of radiolabeled amino acids. The stimuli used were endotoxin, phagocytosis, and tuberculin. The crude leukocyte pyrogen samples were purified; pyrogen from exudate cells was rendered homogeneous; pyrogen from blood cells was still contaminated with other proteins. All the purified pyrogens were radioactive; and for all it was shown that radioactivity and pyrogenic activity coincided on electrophoresis at pH 3.5 and pH 9 in acrylamide and on isoelectric focusing in acrylamide. Furthermore, pyrogens obtained from exudate cells stimulated in different ways, or from blood cells and exudate cells stimulated with endotoxin, appeared to be identical. These results suggest that leukocyte pyrogen was synthesized de novo from amino acid precursors and that leukocytes made the same pyrogen whatever the stimulus used to activate them. PMID:4573840

Advances in RNAi therapeutic delivery to leukocytes using lipid nanoparticles.

PubMed

Ramishetti, Srinivas; Landesman-Milo, Dalit; Peer, Dan

2016-11-01

Small interfering RNAs (siRNAs) therapeutics has advanced into clinical trials for liver diseases and solid tumors, but remain a challenge for manipulating leukocytes fate due to lack of specificity and safety issues. Leukocytes ingest pathogens and defend the body through a complex network. They are also involved in the pathogeneses of inflammation, viral infection, autoimmunity and cancers. Modulating gene expression in leukocytes using siRNAs holds great promise to treat leukocyte-mediated diseases. Leukocytes are notoriously hard to transduce with siRNAs and are spread throughout the body often located deep in tissues, therefore developing an efficient systemic delivery strategy is still a challenge. Here, we discuss recent advances in siRNA delivery to leukocyte subsets such as macrophages, monocytes, dendritic cells and lymphocytes. We focus mainly on lipid-based nanoparticles (LNPs) comprised of new generation of ionizable lipids and their ability to deliver siRNA to primary or malignant leukocytes in a targeted manner. Special emphasis is made on LNPs targeted to subsets of leukocytes and we detail a novel microfluidic mixing technology that could aid in changing the landscape of process development of LNPs from a lab tool to a potential novel therapeutic modality.

Live visualization of genomic loci with BiFC-TALE

PubMed Central

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-01

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP. PMID:28074901

Live visualization of genomic loci with BiFC-TALE.

PubMed

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-11

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP.

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody.

PubMed

Monnet, Céline; Jorieux, Sylvie; Souyris, Nathalie; Zaki, Ouafa; Jacquet, Alexandra; Fournier, Nathalie; Crozet, Fabien; de Romeuf, Christophe; Bouayadi, Khalil; Urbain, Rémi; Behrens, Christian K; Mondon, Philippe; Fontayne, Alexandre

2014-01-01

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen™) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling(®) platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.

Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor

PubMed Central

Burvenich, Ingrid J. G.; Farrugia, William; Lee, Fook T.; Catimel, Bruno; Liu, Zhanqi; Makris, Dahna; Cao, Diana; O'Keefe, Graeme J.; Brechbiel, Martin W.; King, Dylan; Spirkoska, Violeta; Allan, Laura C.; Ramsland, Paul A.; Scott, Andrew M.

2016-01-01

ABSTRACT IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic. PMID:27030023

Production of cytokines and stimulation of resistance to viral infection in human leukocytes by Scutellaria baicalensis flavones.

PubMed

Błach-Olszewska, Zofia; Jatczak, Bogna; Rak, Anna; Lorenc, Maria; Gulanowski, Bogdan; Drobna, Agnieszka; Lamer-Zarawska, Eliza

2008-09-01

Extracts of Scutellaria baicalensis display a wide spectrum of antiviral activity. It was of great interest to check the effect of baicalein and wogonin preparations on two important mechanisms of innate immunity: the secretion of cytokines and the natural resistance of human leukocytes to viral infection. To study the effect of S. baicalensis extracts on interferons (IFNs), tumor necrosis factor alpha (TNF-alpha), and interleukin (IL) production and virus replication, uninfected and vesicular stomatitis virus (VSV)-infected human peripheral blood leukocytes (PBLs) were used. Four pulverized preparations obtained from roots of Scutellaria and a Sigma-Aldrich preparation of purified baicalein were used in the study. RPMI extracts containing different amounts of baicalein and wogonin were used to study the effect on VSV replication in PBLs. PBLs express ex vivo individually differentiated cytokine-dependent resistance/innate immunity to viral infections. The degree of resistance was estimated on the basis of VSV replication in PBLs. The results obtained indicate that baicalein- and wogonin-containing extracts modulate cytokine production, that is inhibit IFN-alpha and IFN-gamma and stimulate TNF-alpha and IL (IL-12, IL-10) production. They also augment the resistance of PBLs to VSV. Extract from S. baicalensis containing baicalein and wogonin regulates the innate antiviral immunity by modulation of cytokine production and stimulation of human leukocyte resistance.

The expression of Fc and complement receptors in young, adult and aged mice.

PubMed Central

Vĕtvicka, V; Fornůsek, L; Zídková, J

1985-01-01

Age-dependent changes in the expression of Fc receptors (FcR) for different isotypes of immunoglobulins and receptors for C3b, C5b and C3bi fragments of complement on the membranes of peritoneal macrophages were studied with mice of different ages. An age-related increase in expression of Fc receptors for IgM, IgE, IgA, IgG2b and IgG3, and a decrease in the expression of Fc receptors for IgG1 was observed. The expression of FcR on macrophages of donors of different ages corresponded with Fc-receptor mediated phagocytosis. The highest number of C3b-binding macrophages was found in aged mice, in contrast to low numbers of C3bi-binding macrophages at this age. The percentage of C5b-binding macrophages was lowest in adult animals. We also observed effective inhibition of binding of the C3b component of complement by preincubation of macrophages with aggregated IgG and vice versa. These observations suggest that fluctuation in expression of Fc but not C receptors may be important to the generalized changes that occur in macrophage function during development and ageing. PMID:2931351

Leukocyte adhesion: High-speed cells with ABS.

PubMed

van der Merwe, P A

1999-06-03

In order to decide where to exit blood vessels and enter tissues, leukocytes roll along endothelial surfaces. Recent studies suggest that an 'automatic braking system' (ABS), involving selectin cell-adhesion molecules, enables leukocytes to roll at a fairly constant velocity despite large variations in blood flow rate.

Progress on research of chicken IgY antibody-FcRY receptor combination and transfer.

PubMed

Tian, Zehua; Zhang, Xiaoying

2012-10-01

The transfer of maternal immunoglobulins (Igs) plays a significant role in fetal initial humoral immunity, of which process has changed and diversified during the evolution of vertebrates. IgY is a key molecular in antibody evolution which links ancient Igs and mammalian Igs such as IgG and IgE. IgY's transfer to the embryo is a two-step receptor-mediated process, including the transfer from the maternal bloodstream to the yolk sac, and from the yolk sac to the embryo. IgY's neonatal Fc receptor (FcRY) mainly functions in the second process. This article reviews IgY's status in antibody evolution and IgY's structure and application. Furthermore, this review compares the binding and transferring mechanism between mammalian IgG, and IgG's neonatal Fc receptor and chicken IgY-FcRY. Details of IgY-FcRY combination, such as combining conditions required, IgY-FcRY binding stoichiometry and exact binding sites on both FcRY and IgY are discussed. Likewise, the endocytosis, the main mechanism of IgY-FcRY transfer and recycling mechanism are analyzed. Related knowledge might be important for better understanding antibody and receptor evolution, antibody-receptor interaction and antibody function. Furthermore, such kind of knowledge might be useful for antibody drug research and development.

Genetics Home Reference: leukocyte adhesion deficiency type 1

MedlinePlus

... adhesion deficiency type 1 Leukocyte adhesion deficiency type 1 Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Leukocyte adhesion deficiency type 1 is a ...

NRG1-Fc improves metabolic health via dual hepatic and central action.

PubMed

Zhang, Peng; Kuang, Henry; He, Yanlin; Idiga, Sharon O; Li, Siming; Chen, Zhimin; Yang, Zhao; Cai, Xing; Zhang, Kezhong; Potthoff, Matthew J; Xu, Yong; Lin, Jiandie D

2018-03-08

Neuregulins (NRGs) are emerging as an important family of signaling ligands that regulate glucose and lipid homeostasis. NRG1 lowers blood glucose levels in obese mice, whereas the brown fat-enriched secreted factor NRG4 protects mice from high-fat diet-induced insulin resistance and hepatic steatosis. However, the therapeutic potential of NRGs remains elusive, given the poor plasma half-life of the native ligands. Here, we engineered a fusion protein using human NRG1 and the Fc domain of human IgG1 (NRG1-Fc) that exhibited extended half-life in circulation and improved potency in receptor signaling. We evaluated its efficacy in improving metabolic parameters and dissected the mechanisms of action. NRG1-Fc treatment triggered potent AKT activation in the liver, lowered blood glucose, improved insulin sensitivity, and suppressed food intake in obese mice. NRG1-Fc acted as a potent secretagogue for the metabolic hormone FGF21; however, the latter was largely dispensable for its metabolic effects. NRG1-Fc directly targeted the hypothalamic POMC neurons to promote membrane depolarization and increase firing rate. Together, NRG1-Fc exhibits improved pharmacokinetic properties and exerts metabolic benefits through dual inhibition of hepatic gluconeogenesis and caloric intake.

NRG1-Fc improves metabolic health via dual hepatic and central action

PubMed Central

Kuang, Henry; Idiga, Sharon O.; Li, Siming; Chen, Zhimin; Cai, Xing; Zhang, Kezhong; Potthoff, Matthew J.; Xu, Yong; Lin, Jiandie D.

2018-01-01

Neuregulins (NRGs) are emerging as an important family of signaling ligands that regulate glucose and lipid homeostasis. NRG1 lowers blood glucose levels in obese mice, whereas the brown fat–enriched secreted factor NRG4 protects mice from high-fat diet–induced insulin resistance and hepatic steatosis. However, the therapeutic potential of NRGs remains elusive, given the poor plasma half-life of the native ligands. Here, we engineered a fusion protein using human NRG1 and the Fc domain of human IgG1 (NRG1-Fc) that exhibited extended half-life in circulation and improved potency in receptor signaling. We evaluated its efficacy in improving metabolic parameters and dissected the mechanisms of action. NRG1-Fc treatment triggered potent AKT activation in the liver, lowered blood glucose, improved insulin sensitivity, and suppressed food intake in obese mice. NRG1-Fc acted as a potent secretagogue for the metabolic hormone FGF21; however, the latter was largely dispensable for its metabolic effects. NRG1-Fc directly targeted the hypothalamic POMC neurons to promote membrane depolarization and increase firing rate. Together, NRG1-Fc exhibits improved pharmacokinetic properties and exerts metabolic benefits through dual inhibition of hepatic gluconeogenesis and caloric intake. PMID:29515030

Human IgG subclass cross-species reactivity to mouse and cynomolgus monkey Fcγ receptors.

PubMed

Derebe, Mehabaw G; Nanjunda, Rupesh K; Gilliland, Gary L; Lacy, Eilyn R; Chiu, Mark L

2018-05-01

In therapeutic antibody discovery and early development, mice and cynomolgus monkey are used as animal models to assess toxicity, efficacy and other properties of candidate molecules. As more candidate antibodies are based on human immunoglobulin (IgG) subclasses, many strategies are pursued to simulate the human system in the test animal. However, translation rate from a successful preclinical trial to an approved drug is extremely low. This may partly be due to differences in interaction of human IgG based candidate molecules to endogenous Fcγ receptors of model animals in comparison to those of human Fcγ receptors. In this study, we compare binding characteristics of human IgG subclasses commonly used in drug development (IgG1, IgG2, IgG4) and their respective Fc silent versions (IgG1σ, IgG2σ, IgG4 PAA) to human, mouse, and cynomolgus monkey Fcγ receptors. To control interactions between Fab and Fc domains, the test IgGs all have the same variable region sequences. We found distinct variations of interaction of human IgG subclasses to model animal Fcγ receptors in comparison to their human counterparts. Particularly, cynomolgus monkey Fcγ receptors showed consistently tighter binding to human IgGs than human Fcγ receptors. Moreover, the presumably Fc silent human IgG4 PAA framework bound to cynomolgus monkey FcγRI with nanomolar affinity while only very weak binding was observed for the human FcγRI. Our results highlighted the need for a thorough in vitro affinity characterization of candidate IgGs against model animal Fcγ receptors and careful design of preclinical studies. Copyright © 2018. Published by Elsevier B.V.

Induction of functional Fc receptors in P388 leukemia cells. Requirement for multiple differentiation signals.

PubMed

Cohen, D A; Stotelmyer, N L; Kaplan, A M

1985-04-01

The development of functional Fc receptors (FcR) during induced differentiation with the tumor promoter, phorbol myristate acetate (PMA), was studied in the murine tumor cell line, P388. PMA induced the appearance of FcR on the membranes of P388 cells as indicated by the binding of IgG-coated sheep red blood cells (IgG-SRBC). Concentrations of PMA as low as 1 ng/ml were sufficient to induce the expression of FcR as well as to inhibit cellular division and to induce adherence in the P388 tumor cell line; however, optimal FcR induction occurred at PMA concentrations of 10-100 ng/ml. Immunofluorescent analysis with heat-aggregated myeloma proteins indicated that PMA induced FcR which were capable of binding IgG2a and IgG2b immunoglobulins, but not IgG1. Adherence to a substratum was determined to be a second required signal for expression of FcR, since PMA induction of P388 tumor cells in teflon dishes failed to fully develop FcR and adherence of P388 cells to poly-L-lysine-coated culture dishes in the absence of PMA was insufficient for FcR expression. FcR which appeared after PMA induction were non-functional in the sense that membrane-bound IgG-SRBC were not ingested to any significant extent by the tumor cells. However, if FcR induction occurred in the presence conA-induced rat spleen cell culture supernatants, phagocytosis of membrane-bound erythrocytes occurred. These findings suggest that for the expression of FcR which are capable of particle internalization, at least three identifiable membrane-transmitted signals are required during differentiation.

Neonatal Fc receptor expression in dendritic cells mediates protective immunity against colorectal cancer.

PubMed

Baker, Kristi; Rath, Timo; Flak, Magdalena B; Arthur, Janelle C; Chen, Zhangguo; Glickman, Jonathan N; Zlobec, Inti; Karamitopoulou, Eva; Stachler, Matthew D; Odze, Robert D; Lencer, Wayne I; Jobin, Christian; Blumberg, Richard S

2013-12-12

Cancers arising in mucosal tissues account for a disproportionately large fraction of malignancies. Immunoglobulin G (IgG) and the neonatal Fc receptor for IgG (FcRn) have an important function in the mucosal immune system that we have now shown extends to the induction of CD8(+) T cell-mediated antitumor immunity. We demonstrate that FcRn within dendritic cells (DCs) was critical for homeostatic activation of mucosal CD8(+) T cells that drove protection against the development of colorectal cancers and lung metastases. FcRn-mediated tumor protection was driven by DCs activation of endogenous tumor-reactive CD8(+) T cells via the cross-presentation of IgG complexed antigens (IgG IC), as well as the induction of cytotoxicity-promoting cytokine secretion, particularly interleukin-12, both of which were independently triggered by the FcRn-IgG IC interaction in murine and human DCs. FcRn thus has a primary role within mucosal tissues in activating local immune responses that are critical for priming efficient anti-tumor immunosurveillance. Copyright © 2013 Elsevier Inc. All rights reserved.

Arithmetic of five-part of leukocytes based on image process

NASA Astrophysics Data System (ADS)

Li, Yian; Wang, Guoyou; Liu, Jianguo

2007-12-01

This paper apply computer image processing and pattern recognizition methods to solve the problem of auto classification and counting of leukocytes (white blood cell) in peripheral blood. In this paper a new leukocyte arithmetic of five-part based on image process and pattern recognizition is presented, which relized auto classify of leukocyte. The first aim is detect the leukocytes . A major requirement of the whole system is to classify these leukocytes to 5 classes. This arithmetic bases on notability mechanism of eyes, process image by sequence, divides up leukocytes and pick up characters. Using the prior kwonledge of cells and image shape information, this arithmetic divides up the probable shape of Leukocyte first by a new method based on Chamfer and then gets the detail characters. It can reduce the mistake judge rate and the calculation greatly. It also has the learning fuction. This paper also presented a new measurement of karyon's shape which can provide more accurate information. This algorithm has great application value in clinical blood test .

The FC-1D: The profitable alternative Flying Circus Commercial Aviation Group

NASA Technical Reports Server (NTRS)

Meza, Victor J.; Alvarez, Jaime; Harrington, Brook; Lujan, Michael A.; Mitlyng, David; Saroughian, Andy; Silva, Alex; Teale, Tim

1994-01-01

The FC-1D was designed as an advanced solution for a low cost commercial transport meeting or exceeding all of the 1993/1994 AIAA/Lockheed request for proposal requirements. The driving philosophy behind the design of the FC-1D was the reduction of airline direct operating costs. Every effort was made during the design process to have the customer in mind. The Flying Circus Commercial Aviation Group targeted reductions in drag, fuel consumption, manufacturing costs, and maintenance costs. Flying Circus emphasized cost reduction throughout the entire design program. Drag reduction was achieved by implementation of the aft nacelle wing configuration to reduce cruise drag and increase cruise speeds. To reduce induced drag, rather than increasing the wing span of the FC-1D, spiroids were included in the efficient wing design. Profile and friction drag are reduced by using riblets in place of paint around the fuselage and empennage of the FC-1D. Choosing a single aisle configuration enabled the Flying Circus to optimize the fuselage diameter. Thus, reducing fuselage drag while gaining high structural efficiency. To further reduce fuel consumption a weight reduction program was conducted through the use of composite materials. An additional quality of the FC-1D is its design for low cost manufacturing and assembly. As a result of this design attribute, the FC-1D will have fewer parts which reduces weight as well as maintenance and assembly costs. The FC-1D is affordable and effective, the apex of commercial transport design.

Gene expression profile of human Down syndrome leukocytes.

PubMed

Malagó, Wilson; Sommer, César A; Del Cistia Andrade, Camillo; Soares-Costa, Andrea; Abrao Possik, Patricia; Cassago, Alexandre; Santejo Silveira, Henrique C; Henrique-Silva, Flavio

2005-08-01

Identification of differences in the gene expression patterns of Down syndrome and normal leukocytes. We constructed the first Down syndrome leukocyte serial analysis of gene expression (SAGE) library from a 28 year-old patient. This library was analyzed and compared with a normal leukocyte SAGE library using the eSAGE software. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to validate the results. We found that a large number of unidentified transcripts were overexpressed in Down syndrome leukocytes and some transcripts coding for growth factors (e.g. interleukin 8, IL-8), ribosomaproteins (e.g. L13a, L29, and L37), and transcription factors (e.g., Jun B, Jun D, and C/EBP beta) were underexpressed. The SAGE data were successfully validated for the genes IL-8, CXCR4, BCL2A1, L13a, L29, L37, and GTF3A using RT-PCR. Our analysis identified significant changes in the expression pattern of Down syndrome leukocytes compared with normal ones, including key regulators of growth and proliferation, ribosomal proteins, and a large number of overexpressed transcripts that were not matched in UniGene clusters and that may represent novel genes related to Down syndrome. This study offers a new insight into transcriptional changes in Down syndrome leukocytes and indicates candidate genes for further investigations into the molecular mechanism of Down syndrome pathology.

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens.

PubMed Central

Schweitzer-Stenner, R; Tamir, I; Pecht, I

1997-01-01

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response. PMID:9168023

IgG receptor FcγRIIB plays a key role in obesity-induced hypertension.

PubMed

Sundgren, Nathan C; Vongpatanasin, Wanpen; Boggan, Brigid-Meghan D; Tanigaki, Keiji; Yuhanna, Ivan S; Chambliss, Ken L; Mineo, Chieko; Shaul, Philip W

2015-02-01

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB(+/+) mice developed obesity-induced hypertension, FcγRIIB(-/-) mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; FcγRIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet-fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an FcγRIIB-dependent process. Thus, we have identified a novel role for FcγRIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting FcγRIIB may potentially offer new means to treat hypertension in obese individuals. © 2014 American Heart Association, Inc.

IgG Receptor FcγRIIB Plays a Key Role in Obesity-Induced Hypertension

PubMed Central

Sundgren, Nathan C.; Vongpatanasin, Wanpen; Boggan, Brigid-Meghan D.; Tanigaki, Keiji; Yuhanna, Ivan S.; Chambliss, Ken L.; Mineo, Chieko; Shaul, Philip W.

2015-01-01

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB+/+ mice developed obesity-induced hypertension, FcγRIIB−/− mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; FcγRIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet–fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an FcγRIIB-dependent process. Thus, we have identified a novel role for FcγRIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting FcγRIIB may potentially offer new means to treat hypertension in obese individuals. PMID:25368023

Allelic Dependent Expression of an Activating Fc receptor on B cells Enhances Humoral Immune Responses

PubMed Central

Li, Xinrui; Wu, Jianming; Ptacek, Travis; Redden, David T; Brown, Elizabeth E; Alarcón, Graciela S; Ramsey-Goldman, Rosalind; Petri, Michelle A; Reveille, John D.; Kaslow, Richard A; Kimberly, Robert P; Edberg, Jeffrey C

2014-01-01

B cells are pivotal regulators of acquired immune responses and recent work in both experimental murine models and humans has demonstrated that subtle changes in the regulation of B cell function can significantly alter immunological responses. The balance of negative and positive signals in maintaining an appropriate B cell activation threshold is critical in B lymphocyte immune tolerance and autoreactivity. FcγRIIb (CD32B), the only recognized Fcγ receptor on B cells, provides IgG-mediated negative modulation through a tyrosine-based inhibition motif which down-regulates B cell receptor initiated signaling. These properties make FcγRIIb a promising target for antibody-based therapy. Here we report the discovery of allele-dependent expression of the activating FcγRIIc on B cells. Identical to FcγRIIb in the extracellular domain, FcγRIIc has a tyrosine-based activation motif in its cytoplasmic domain. In both human B cells and in B cells from mice transgenic for human FcγRIIc, FcγRIIc expression counterbalances the negative feedback of FcγRIIb and enhances humoral responses to immunization in mice and to BioThrax® vaccination in a human Anthrax vaccine trial. Moreover, the FCGR2C-ORF allele is associated with the risk of development of autoimmunity in humans. FcγRIIc expression on B cells challenges the prevailing paradigm of uni-directional negative feedback by IgG immune complexes via the inhibitory FcγRIIb, is a previously unrecognized determinant in human antibody/autoantibody responses, and opens the opportunity for more precise personalized use of B cell targeted antibody-based therapy. PMID:24353158

Structural Basis for FcγRIIa Recognition of Human IgG and Formation of Inflammatory Signaling Complexes

PubMed Central

Ramsland, Paul A.; Farrugia, William; Bradford, Tessa M.; Tan Sardjono, Caroline; Esparon, Sandra; Trist, Halina M.; Powell, Maree S.; Szee Tan, Peck; Cendron, Angela C.; Wines, Bruce D.; Scott, Andrew M.; Hogarth, P. Mark

2012-01-01

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag–Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly. PMID:21856937

Anti-influenza Hyperimmune Immunoglobulin Enhances Fc-functional Antibody Immunity during Human Influenza Infection.

PubMed

Vanderven, Hillary A; Wragg, Kathleen; Ana-Sosa-Batiz, Fernanda; Kristensen, Anne B; Jegaskanda, Sinthujan; Wheatley, Adam K; Wentworth, Deborah; Wines, Bruce D; Hogarth, P Mark; Rockman, Steve; Kent, Stephen J

2018-05-31

New treatments for severe influenza are needed. Passive transfer of influenza-specific hyperimmune pooled immunoglobulin (Flu-IVIG) boosts neutralising antibody responses to past strains in influenza-infected subjects. The effect of Flu-IVIG on antibodies with Fc-mediated functions, which may target diverse influenza strains, is unclear. We studied the capacity of Flu-IVIG, relative to standard IVIG, to bind to Fc receptors and mediate antibody-dependent cellular cytotoxicity in vitro. The effect of Flu-IVIG infusion, compared to placebo infusion, was examined in serial plasma samples from 24 subjects with confirmed influenza infection in the INSIGHT FLU005 pilot study. Flu-IVIG contains higher concentrations of Fc-functional antibodies than IVIG against a diverse range of influenza hemagglutinins. Following infusion of Flu-IVIG into influenza-infected subjects, a transient increase in Fc-functional antibodies was present for 1-3 days against infecting and non-infecting strains of influenza. Flu-IVIG contains antibodies with Fc-mediated functions against influenza virus and passive transfer of Flu-IVIG increases anti-influenza Fc-functional antibodies in the plasma of influenza-infected subjects. Enhancement of Fc-functional antibodies to a diverse range of influenza strains suggests that Flu-IVIG infusion could prove useful in the context of novel influenza virus infections, when there may be minimal or no neutralising antibodies in the Flu-IVIG preparation.

The role of Fc-receptors in the uptake and transport of therapeutic antibodies in the retinal pigment epithelium.

PubMed

Dithmer, Michaela; Hattermann, Kirsten; Pomarius, Prasti; Aboul Naga, Shereen Hassan; Meyer, Tim; Mentlein, Rolf; Roider, Johann; Klettner, Alexa

2016-04-01

In the ophthalmological clinic, intravitreally applied antibodies or Fc-containing fusion proteins are frequently used, but the biology and pharmacokinetics of these therapeutics in the retina are not well understood. We have previously shown intracellular uptake of Fc-containing molecules in RPE cells. In this study, we investigated the involvement of Fc-receptors, both Fcγ-receptors and the neonatal Fc-receptor (FcRn) in the uptake and intracellular trafficking of the VEGF-antagonists bevacizumab, aflibercept and the anti-CD20 antibody rituximab in three different model systems, primary porcine RPE cells, ARPE-19 cells and porcine RPE/choroid explants. The expression of Fcγ-receptors was tested in primary porcine RPE cells, and the expression of Fcγ-receptors I and II could be shown in RT-PCR and qRT-PCR, while the expression of FcRn was additionally confirmed in Western blot and immunocytochemistry. All three compounds, bevacizumab, rituximab and aflibercept, were taken up into the cells and displayed a characteristic time-dependent pattern, as shown in Western blot and immunohistochemistry. The uptake was not altered by the inhibition of Fcγ-receptors using different inhibitors (TruStain FcX, genistein, R406). However, the inhibition of FcRn with an antagonistic antibody reduced intracellular IgG in porcine RPE cells (rituximab) and ARPE-19 cells (bevacizumab, rituximab). Colocalisations between the tested compounds and myosin7a could be found. In addition, limited colocalization with FcRn and the tested compounds, as well as triple localization between compound, FcRn and myosin7a could be detected, indicating a role of myosin7a in FcRn mediated transport. However, the colocalizations are restricted to small fractions of the Fc-containing compounds. Furthermore, the FcRn is mainly found in the membrane section, where only minute amounts of the Fc-containing compounds are seen, suggesting a limited interaction. An apical to choroidal transport of Ig

Leukocyte diversity in resolving and nonresolving mechanisms of cardiac remodeling.

PubMed

Tourki, Bochra; Halade, Ganesh

2017-10-01

In response to myocardial infarction (MI), time-dependent leukocyte infiltration is critical to program the acute inflammatory response. Post-MI leukocyte density, residence time in the infarcted area, and exit from the infarcted injury predict resolving or nonresolving inflammation. Overactive or unresolved inflammation is the primary determinant in heart failure pathology post-MI. Here, our review describes supporting evidence that the acute inflammatory response also guides the generation of healing and regenerative mediators after cardiac damage. Time-dependent leukocyte density and diversity and the magnitude of myocardial injury is responsible for the resolving and nonresolving pathway in myocardial healing. Post MI, the diversity of leukocytes, such as neutrophils, macrophages, and lymphocytes, has been explored that regulate the clearance of deceased cardiomyocytes by using the classic and reparative pathways. Among the innovative factors and intermediates that have been recognized as essential in acute the self-healing and clearance mechanism, we highlight specialized proresolving mediators as the emerging factor for post-MI reparative mechanisms-translational leukocyte modifiers, such as aging, the source of leukocytes, and the milieu around the leukocytes. In the clinical setting, it is possible that leukocyte diversity is more prominent as a result of risk factors, such as obesity, diabetes, and hypertension. Pharmacologic agents are critical modifiers of leukocyte diversity in healing mechanisms that may impair or stimulate the clearance mechanism. Future research is needed, with a focused approach to understand the molecular targets, cellular effectors, and receptors. A clear understanding of resolving and nonresolving inflammation in myocardial healing will help to develop novel targets with major emphasis on the resolution of inflammation in heart failure pathology.-Tourki, B., Halade, G. Leukocyte diversity in resolving and nonresolving mechanisms

Modulation of leukocyte adhesion in rat mesenteric venules by aspirin and salicylate.

PubMed

Asako, H; Kubes, P; Wallace, J; Wolf, R E; Granger, D N

1992-07-01

Erythrocyte velocity, vessel diameter, leukocyte rolling velocity, and number of adherent and emigrated leukocytes were measured in postcapillary venules both before and during superfusion of rat mesentery with either aspirin or sodium salicylate. In some experiments, animals were treated with either a leukotriene (LT)-synthesis inhibitor (L-663,536), an LTD4 antagonist (MK-571), an LTB4 antagonist (SC-41930), misoprostol, or prostaglandin (PG) I2, then the aspirin protocol was repeated. Superfusion of aspirin but not sodium salicylate resulted in increased leukocyte adherence and a reduced leukocyte rolling velocity but did not affect leukocyte emigration. Aspirin-induced leukocyte adhesion was effectively prevented by the LT-synthesis inhibitor and LTB4 antagonist but not by the LTD4 antagonist. Misoprostol and PGI2 also prevented the aspirin-induced adhesion responses. Superfusion of the mesentery with either platelet-activating factor (PAF) or LTB4 enhanced leukocyte adherence and emigration while reducing leukocyte rolling velocity. Sodium salicylate prevented all of the adhesion responses elicited by LTB4. Although salicylate did not affect the PAF-induced leukocyte adherence and rolling responses, it completely prevented the increased leukocyte emigration. These results indicate that aspirin promotes, whereas sodium salicylate inhibits, leukocyte-endothelial cell adhesive interactions at therapeutically relevant concentrations.

Distribution and dosimetry of In-111 labeled leukocytes and platelets in humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodwin, D.A.; Finston, R.A.; Smith, S.I.

1981-06-01

The distribution of In-111 labeled leukocytes and platelets was studied by whole body gamma camera imaging in patients. Images were made approximately one hour and 24 hours after IV injection, and stored in digital form in computer memory. Estimates of the quantitative organ distribution were made from the geometric mean of the anterior and posterior region of interest counts after suitable background subtraction. Nearly quantitative retention of cell activity was observed with little or no excretion seen in either gut or kidneys. Mixed leukocytes distributed in spleen, liver and bone marrow in decreasing order of concentration, similar at both times,more » with a transient lung uptake noted at the one hour time only. The dose from 0.5 mCi In-111-WBC's was: liver, 1.4 rad; spleen, 8.5 rad; marrow, 2.3 rad. Lymphocytes had similar distribution with the addition of inguinal and cervical lymph nodes. The dose from 0.5 mCi In-111-lymphocytes was: liver, 0.8 rad; spleen, 6.7 rad; marrow and lymphatic tissue, 1.4 rad. Platelets distributed primarily in the blood pool with most of the remainder concentrating in the spleen, with a small amount in the penis. The dose from 0.5 mCi of In-111-platelets was: liver, 3.2 rad; spleen, 8.6 rad; and whole body, 0.3 rad.« less

Utilization of Fc Receptors as a Mucosal Vaccine Strategy against an Intracellular Bacterium, Francisella tularensis1

PubMed Central

Rawool, Deepak B.; Bitsaktsis, Constantine; Li, Ying; Gosselin, Diane R.; Lin, Yili; Kurkure, Nitin V.; Metzger, Dennis W.; Gosselin, Edmund J.

2013-01-01

Numerous studies have demonstrated that targeting Ag to Fc receptors (FcR) on APCs can enhance humoral and cellular immunity. However, studies are lacking that examine both the use of FcR-targeting in generating immune protection against infectious agents and the use of FcRs in the induction of mucosal immunity. Francisella tularensis is a category A intracellular mucosal pathogen. Thus, intense efforts are underway to develop a vaccine against this organism. We hypothesized that protection against mucosal infection with F. tularensis would be significantly enhanced by targeting inactivated F. tularensis live vaccine strain (iFt) to FcRs at mucosal sites, via intranasal immunization with mAb-iFt complexes. These studies demonstrate for the first time that: 1) FcR-targeted immunogen enhances immunogen-specific IgA production and protection against subsequent infection in an IgA-dependent manner, 2) FcγR and neonatal FcR are crucial to this protection, and 3) inactivated F. tularensis, when targeted to FcRs, enhances protection against the highly virulent SchuS4 strain of F. tularensis, a category A biothreat agent. In summary, these studies show for the first time the use of FcRs as a highly effective vaccination strategy against a highly virulent mucosal intracellular pathogen. PMID:18390739

The effects of stress on the enzymes of peripheral leukocytes

NASA Technical Reports Server (NTRS)

Leise, E. M.; Gray, I.

1973-01-01

Previous work showed an early response of rabbit and human leukocyte enzymes to the stress of bacterial infection. Since these represented a mixed population of leukocytes and since polymorphonuclear leukocytes (PMN) increased in these preparations, it was necessary to establish whether the observed increase in lactate dehydrenase (LDH) and protein was the result of an increase in any one particular cell type or in all cells. The need for the development of a simple reproducible method for the differential separation of peripheral leukocytes for the furtherance of our own studies was apparent. It was also becoming increasingly apparent that morphologically similar cells, such as small lymphocytes (L) and macrophages, were capable of different biological functions. A dextran gradient centrifugation method was developed which has provided an easily reproducible technique for separating L from PMN. During the course of this work, in which over 250 rabbits were examined, the pattern of daily leukocyte protein and enzyme variation became increasingly more apparent. This information could have some impact on future work with leukocyte enzymes, by our group and by other workers. The differences in normal protein and enzyme levels maintained by some individuals, and some inbred strains, were evaluated and reported separately. It has been shown that one type of leukocyte may react more to a given stress than other leukocytes.

Sex differences in leukocyte invasion in injured murine skeletal muscle.

PubMed

St Pierre Schneider, B; Correia, L A; Cannon, J G

1999-06-01

The three aims of this study were to describe the time course of leukocyte invasion in injured soleus muscles of male and female mice, to determine if differential subsets of leukocytes accumulate in intramyofiber and interstitial sites, and to determine if significant sex differences exist in invading leukocyte concentrations. Fifty sexually mature C57BL/6J mice (aged 11-12 weeks) underwent unilateral hindlimb muscle injury induced by lengthening contractions. This procedure models the muscle injury that can occur through strenuous exercise or overuse in humans. After 1, 3, 5, or 7 days of recovery, the injured and contralateral, uninjured solei were dissected and prepared for morphologic analysis. We found that leukocytes had invaded injured myofibers at 1-day postinjury for both sexes. Different subsets of leukocytes accumulated within damaged myofibers and the interstitium. Significantly fewer myofibers were invaded by acid phosphatase-positive leukocytes in females. Interstitial ER-BMDM1 leukocyte concentrations peaked in females at 7 days postinjury in comparison to 5 days postinjury in males. These findings expand nursing's knowledge base regarding the potential effect of gender on recovery from acute muscle injury.

Long-Term Leukocyte Filtration Should Be Avoided during Extracorporeal Circulation

PubMed Central

Tang, Jiali; Tao, Kaiyu; Zhou, Jing; Zhang, Chongwei; Gong, Lina; Luo, Nanfu

2013-01-01

Filtration during extracorporeal circulation (ECC) not only removes but also activates leukocytes; therefore, long-term leukocyte filtration may cause adverse effects. In the present study, we tested this hypothesis by priming ECC with 300 mL of canine blood and examining filtration effects in 3 groups (n = 6 each) during 60 min ECC. In the control group (Group C) blood was filtrated with an arterial filter for 60 min; in long-term (Group L) and short-term (Group S) groups, blood was filtrated with a leukocyte filter for 60 and 5 min. We found that about 90% of leukocytes were removed after 5 min of filtration in both Groups L and S. Although leukocyte count continued to reduce, mean fluorescent intensities of CD11/CD18, free hemoglobin, and neutrophil elastase increased in Group L and were higher than those in Groups C and S at 60 min. Leukocyte rupture, cytoplasmic leakage, and circulating naked nuclei were also found in Group L. The data support our hypothesis that long-term filtration can induce inflammation and lead to leukocyte destruction. PMID:24453424

IgA-mediated inhibition of human leucocyte function by interference with Fc gamma and C3b receptors.

PubMed

Saito, K; Kato, C; Katsuragi, H; Komatsuzaki, A

1991-09-01

The inhibitory effects of IgA from human colostrum, and IgA1 and IgA2 from human serum on the chemiluminescence (CL) response and phagocytosis of polymorphonuclear leucocytes (PML) to Staphylococcus epidermidis and the CL response to formylmethionyl-leucyl-phenylalanine (FMLP) were studied. The dose-dependent inhibition of the luminol-mediated CL response of human PML to the bacteria was observed in the presence of more than 0.1 mg/ml IgA from both colostrum and serum. The preincubation of PML with a solution of IgA enhanced the suppressive effect of IgA on the cells. Removal of IgA from the reaction mixture after preincubation resulted in recovery, with time, of the response of PML to the bacteria. The bacteria treated with IgA did not give rise to any inhibition of the response. The CL response of PML to FMLP was not affected by the presence of IgA in the reaction mixture. The decrease of phagocytic activity of PML in the presence of IgA resulted in a decrease of NADPH oxidase activity of PML after stimulation with the bacteria as compared with the absence of IgA. The effect of IgA on the receptors of Fc and C3b (CR1) on the surface of PML was measured by monitoring erythrocyte-antibody (EA) or erythrocyte-antibody-complement (EAC) rosette formation and by direct and indirect immunofluorescence techniques using anti-CR1 antibody and Fc-specific antibodies. The presence of IgA in the reaction mixture led to a quantitative decrease in CR1 and the ability to bind IgG to the surface of PML.

IgA-mediated inhibition of human leucocyte function by interference with Fc gamma and C3b receptors.

PubMed Central

Saito, K; Kato, C; Katsuragi, H; Komatsuzaki, A

1991-01-01

The inhibitory effects of IgA from human colostrum, and IgA1 and IgA2 from human serum on the chemiluminescence (CL) response and phagocytosis of polymorphonuclear leucocytes (PML) to Staphylococcus epidermidis and the CL response to formylmethionyl-leucyl-phenylalanine (FMLP) were studied. The dose-dependent inhibition of the luminol-mediated CL response of human PML to the bacteria was observed in the presence of more than 0.1 mg/ml IgA from both colostrum and serum. The preincubation of PML with a solution of IgA enhanced the suppressive effect of IgA on the cells. Removal of IgA from the reaction mixture after preincubation resulted in recovery, with time, of the response of PML to the bacteria. The bacteria treated with IgA did not give rise to any inhibition of the response. The CL response of PML to FMLP was not affected by the presence of IgA in the reaction mixture. The decrease of phagocytic activity of PML in the presence of IgA resulted in a decrease of NADPH oxidase activity of PML after stimulation with the bacteria as compared with the absence of IgA. The effect of IgA on the receptors of Fc and C3b (CR1) on the surface of PML was measured by monitoring erythrocyte-antibody (EA) or erythrocyte-antibody-complement (EAC) rosette formation and by direct and indirect immunofluorescence techniques using anti-CR1 antibody and Fc-specific antibodies. The presence of IgA in the reaction mixture led to a quantitative decrease in CR1 and the ability to bind IgG to the surface of PML. PMID:1834550

Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport.

PubMed

Stapleton, Nigel M; Armstrong-Fisher, Sylvia S; Andersen, Jan Terje; van der Schoot, C Ellen; Porter, Charlene; Page, Kenneth R; Falconer, Donald; de Haas, Masja; Williamson, Lorna M; Clark, Michael R; Vidarsson, Gestur; Armour, Kathryn L

2018-03-01

We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders. Here, we show that G1Δnab mutants retain pH-dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0-fold and 1.6-fold for the two antibodies investigated. The transport of the modified G1Δnab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild-type, except that no difference was observed with human umbilical vein endothelial cells. G1Δnab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1Δnab mutations are away from the FcRn-binding site, they have long-distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

A new C-type lectin (FcLec5) from the Chinese white shrimp Fenneropenaeus chinensis.

PubMed

Xu, Wen-Teng; Wang, Xian-Wei; Zhang, Xiao-Wen; Zhao, Xiao-Fan; Yu, Xiao-Qiang; Wang, Jin-Xing

2010-11-01

C-type lectins are one family of pattern recognition receptors (PRRs) that play important roles in innate immunity. In this work, cDNA and genomic sequences for a new C-type lectin (FcLec5) were obtained from the Chinese white shrimp Fenneropenaeus chinensis. FcLec5 cDNA contains an open reading frame of 1,008 bp and its genomic sequence is 1,137 bp with 4 exons and 3 introns. The predicted FcLec5 protein contains a signal peptide and two carbohydrate recognition domains (CRDs). The N-terminal CRD of FcLec5 has a predicted carbohydrate recognition motif of Gln-Pro-Asp (QPD), while the C-terminal CRD contains a motif of Glu-Pro-Gln (EPQ). Northern blot analysis showed that FcLec5 mRNA was specifically expressed in hepatopancreas. FcLec5 protein was expressed in hepatopancreas and secreted into hemolymph. Real-time PCR showed that FcLec5 transcript exhibited different expression profiles after immune-challenged with Vibrio anguillarum or White Spot Syndrome Virus (WSSV). Recombinant FcLec5 and its two individual CRDs could agglutinate most bacteria tested, and the agglutinating activity was Ca2+-dependent. Besides, the agglutinating activity to gram-negative bacteria is higher than that to gram-positive bacteria. Direct binding assay showed that recombinant FcLec5 could bind to all microorganisms tested (five gram-positive and four gram-negative bacteria, as well as yeast) in a Ca2+-independent manner. Recombinant FcLec5 also directly bound to bacterial peptidoglycan, lipopolysaccharide and lipoteichoic acids. These results suggest that FcLec5 may act as a PRR for bacteria via binding to bacterial cell wall polysaccharides in Chinese white shrimp.

The level of PPD-specific IFN-gamma-producing CD4+ T cells in the blood predicts the in vivo response to PPD.

PubMed

Martins, Marcia Valéria B S; Lima, Mônica Cristina B S; Duppre, Nadia C; Matos, Haroldo J; Spencer, John S; Brennan, Patrick J; Sarno, Euzenir N; Fonseca, Leila; Pereira, Geraldo M B; Pessolani, Maria Cristina V

2007-05-01

There are no reliable means for detecting subclinical mycobacterial infections. The recent sequencing of several mycobacterial genomes has now afforded new opportunities for the development of pathogen-specific diagnostic tests, critical in the context of leprosy and tuberculosis control. In the present study, we applied a multi-parametric flow cytometric analysis that allowed the investigation of T-cell functions in order to define immunological markers that measure previous exposure to mycobacteria. We compared the in vivo response to PPD, the gold standard skin test reagent for measuring previous exposure to Mycobacterium tuberculosis, with in vitro parameters of leukocyte activation in five PPD positive and five PPD negative healthy volunteers. PPD-stimulated peripheral leukocytes expressing CD4, CD69, cutaneous lymphocyte-associated antigen (CLA) and intracellular IFN-gamma were enumerated in whole blood and compared with the size of in vivo PPD-induced induration and IFN-gamma production levels as measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells. The reactivity to the tuberculin skin test (TST) was associated with markedly increased frequencies of PPD-responsive activated (CD69+) and IFN-gamma-producing CD4+T cells. Detection of PPD-specific IFN-gamma producing leukocytes was restricted to CD4+T cells and a subset of these cells was shown to express the skin homing molecule CLA. Multiple linear regression modeling of responses to PPD showed the highest association between skin test indurations and frequencies of PPD-responsive IFN-gamma-producing CD4+CD69+ T cells. Our data show that the in vitro enumeration of antigen-specific IFN-gamma-producing CD4+ T cells can provide an alternative to the in vivo tuberculin test for the detection of latent Mycobacterium tuberculosis infection. Moreover, the measurement of these immunological parameters can be useful for the screening of new specific antigens defined by the genome

Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

PubMed

Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

2017-07-12

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

Big insights from small volumes: deciphering complex leukocyte behaviors using microfluidics

PubMed Central

Irimia, Daniel; Ellett, Felix

2016-01-01

Inflammation is an indispensable component of the immune response, and leukocytes provide the first line of defense against infection. Although the major stereotypic leukocyte behaviors in response to infection are well known, the complexities and idiosyncrasies of these phenotypes in conditions of disease are still emerging. Novel tools are indispensable for gaining insights into leukocyte behavior, and in the past decade, microfluidic technologies have emerged as an exciting development in the field. Microfluidic devices are readily customizable, provide tight control of experimental conditions, enable high precision of ex vivo measurements of individual as well as integrated leukocyte functions, and have facilitated the discovery of novel leukocyte phenotypes. Here, we review some of the most interesting insights resulting from the application of microfluidic approaches to the study of the inflammatory response. The aim is to encourage leukocyte biologists to integrate these new tools into increasingly more sophisticated experimental designs for probing complex leukocyte functions. PMID:27194799

An agent-based model of leukocyte transendothelial migration during atherogenesis.

PubMed

Bhui, Rita; Hayenga, Heather N

2017-05-01

A vast amount of work has been dedicated to the effects of hemodynamics and cytokines on leukocyte adhesion and trans-endothelial migration (TEM) and subsequent accumulation of leukocyte-derived foam cells in the artery wall. However, a comprehensive mechanobiological model to capture these spatiotemporal events and predict the growth and remodeling of an atherosclerotic artery is still lacking. Here, we present a multiscale model of leukocyte TEM and plaque evolution in the left anterior descending (LAD) coronary artery. The approach integrates cellular behaviors via agent-based modeling (ABM) and hemodynamic effects via computational fluid dynamics (CFD). In this computational framework, the ABM implements the diffusion kinetics of key biological proteins, namely Low Density Lipoprotein (LDL), Tissue Necrosis Factor alpha (TNF-α), Interlukin-10 (IL-10) and Interlukin-1 beta (IL-1β), to predict chemotactic driven leukocyte migration into and within the artery wall. The ABM also considers wall shear stress (WSS) dependent leukocyte TEM and compensatory arterial remodeling obeying Glagov's phenomenon. Interestingly, using fully developed steady blood flow does not result in a representative number of leukocyte TEM as compared to pulsatile flow, whereas passing WSS at peak systole of the pulsatile flow waveform does. Moreover, using the model, we have found leukocyte TEM increases monotonically with decreases in luminal volume. At critical plaque shapes the WSS changes rapidly resulting in sudden increases in leukocyte TEM suggesting lumen volumes that will give rise to rapid plaque growth rates if left untreated. Overall this multi-scale and multi-physics approach appropriately captures and integrates the spatiotemporal events occurring at the cellular level in order to predict leukocyte transmigration and plaque evolution.

An agent-based model of leukocyte transendothelial migration during atherogenesis

PubMed Central

Bhui, Rita; Hayenga, Heather N.

2017-01-01

A vast amount of work has been dedicated to the effects of hemodynamics and cytokines on leukocyte adhesion and trans-endothelial migration (TEM) and subsequent accumulation of leukocyte-derived foam cells in the artery wall. However, a comprehensive mechanobiological model to capture these spatiotemporal events and predict the growth and remodeling of an atherosclerotic artery is still lacking. Here, we present a multiscale model of leukocyte TEM and plaque evolution in the left anterior descending (LAD) coronary artery. The approach integrates cellular behaviors via agent-based modeling (ABM) and hemodynamic effects via computational fluid dynamics (CFD). In this computational framework, the ABM implements the diffusion kinetics of key biological proteins, namely Low Density Lipoprotein (LDL), Tissue Necrosis Factor alpha (TNF-α), Interlukin-10 (IL-10) and Interlukin-1 beta (IL-1β), to predict chemotactic driven leukocyte migration into and within the artery wall. The ABM also considers wall shear stress (WSS) dependent leukocyte TEM and compensatory arterial remodeling obeying Glagov’s phenomenon. Interestingly, using fully developed steady blood flow does not result in a representative number of leukocyte TEM as compared to pulsatile flow, whereas passing WSS at peak systole of the pulsatile flow waveform does. Moreover, using the model, we have found leukocyte TEM increases monotonically with decreases in luminal volume. At critical plaque shapes the WSS changes rapidly resulting in sudden increases in leukocyte TEM suggesting lumen volumes that will give rise to rapid plaque growth rates if left untreated. Overall this multi-scale and multi-physics approach appropriately captures and integrates the spatiotemporal events occurring at the cellular level in order to predict leukocyte transmigration and plaque evolution. PMID:28542193

Penetration of equine leukocytes by merozoites of Sarcocystis neurona.

PubMed

Lindsay, David S; Mitchell, Sheila M; Yang, Jibing; Dubey, J P; Gogal, Robert M; Witonsky, Sharon G

2006-06-15

Horses are considered accidental hosts for Sarcocystis neurona and they often develop severe neurological disease when infected with this parasite. Schizont stages develop in the central nervous system (CNS) and cause the neurological lesions associated with equine protozoal myeloencephalitis. The present study was done to examine the ability of S. neurona merozoites to penetrate and develop in equine peripheral blood leukocytes. These infected host cells might serve as a possible transport mechanism into the CNS. S. neurona merozoites penetrated equine leukocytes within 5 min of co-culture. Infected leukocytes were usually monocytes. Infected leukocytes were present up to the final day of examination at 3 days. Up to three merozoites were present in an infected monocyte. No development to schizont stages was observed. All stages observed were in the host cell cytoplasm. We postulate that S. neurona merozoites may cross the blood brain barrier hidden inside leukocytes. Once inside the CNS these merozoites can egress and invade additional cells and cause encephalitis.

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry*

PubMed Central

Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D.

2015-01-01

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution. PMID:25378534

Leukocyte-reduced blood components: patient benefits and practical applications.

PubMed

Higgins, V L

1996-05-01

To review the various types of filters used for red blood cell and platelet transfusions and to explain the trend in the use of leukocyte removal filters, practical information about their use, considerations in the selection of a filtration method, and cost-effectiveness issues. Published articles, books, and the author's experience. Leukocyte removal filters are used to reduce complications associated with transfused white blood cells that are contained in units of red blood cells and platelets. These complications include nonhemolytic febrile transfusion reactions (NHFTRs), alloimmunization and refractoriness to platelet transfusion, transfusion-transmitted cytomegalovirus (CMV), and immunomodulation. Leukocyte removal filters may be used at the bedside, in a hospital blood bank, or in a blood collection center. Factors that affect the flow rate of these filters include the variations in the blood component, the equipment used, and filter priming. Studies on the cost-effectiveness of using leukocyte-reduced blood components demonstrate savings based on the reduction of NHFTRs, reduction in the number of blood components used, and the use of filtered blood components as the equivalent of CMV seronegative-screened products. The use of leukocyte-reduced blood components significantly diminishes or prevents many of the adverse transfusion reactions associated with donor white blood cells. Leukocyte removal filters are cost-effective, and filters should be selected based on their ability to consistently achieve low leukocyte residual levels as well as their ease of use. Physicians may order leukocyte-reduced blood components for specific patients, or the components may be used because of an established institutional transfusion policy. Nurses often participate in deciding on a filtration method, primarily based on ease of use. Understanding the considerations in selecting a filtration method will help nurses make appropriate decisions to ensure quality patient care.

Does leukocyte-poor or leukocyte-rich platelet-rich plasma applied with biopolymers have superiority to conventional platelet-rich plasma applications on chondrocyte proliferation?

PubMed

Yaşar Şirin, Duygu; Yılmaz, İbrahim; İsyar, Mehmet; Öznam, Kadir; Mahiroğulları, Mahir

2017-12-01

This study aims to investigate the possible effects of leukocyte concentration in the content of platelet-rich plasma (PRP) and the administration of PRP using a drug delivery system on chondrocyte proliferation in vitro conditions. Blood from nine male patients (mean age 65 years; range 49 to 81 years) with advanced stage osteoarthritis who had not responded to medical or conservative treatments and underwent total knee arthroplasty was used to prepare two formulations: PRP with low concentration leukocytes (2000-4000 leukocytes/µL) was designated as pure PRP (P-PRP), whereas PRP with high concentration leukocytes (9000-11000 leukocytes/µL) as leukocyte-rich PRP (L-PRP). Samples were divided into five groups as control group (group 1), chondrocyte cultures with P-PRP applied directly (group 2), chondrocyte cultures with L-PRP applied directly (group 3), chondrocytes co-cultured with P-PRP applied hydrogel (group 4), and chondrocytes co-cultured with L-PRP applied hydrogel (group 5). In all groups; cell morphology, viability and proliferation were compared with the expression of stage-specific embryonic antigen-1 (SSEA-1), a precondrocyte marker. Maximum cell proliferation and SSEA-1 expression occurred in group 4, with a statistically significant correlation between SSEA-1 expression and cell proliferation. Our study showed the importance of leukocyte concentration of PRP and efficiency of delivery systems such as hydrogel and that L-PRP administered with a delivery system is more efficient than conventional applications of PRP in the treatment of cartilage damage.

Columnar processing in primate pFC: evidence for executive control microcircuits.

PubMed

Opris, Ioan; Hampson, Robert E; Gerhardt, Greg A; Berger, Theodore W; Deadwyler, Sam A

2012-12-01

A common denominator for many cognitive disorders of human brain is the disruption of neural activity within pFC, whose structural basis is primarily interlaminar (columnar) microcircuits or "minicolumns." The importance of this brain region for executive decision-making has been well documented; however, because of technological constraints, the minicolumnar basis is not well understood. Here, via implementation of a unique conformal multielectrode recording array, the role of interlaminar pFC minicolumns in the executive control of task-related target selection is demonstrated in nonhuman primates performing a visuomotor DMS task. The results reveal target-specific, interlaminar correlated firing during the decision phase of the trial between multielectrode recording array-isolated minicolumnar pairs of neurons located in parallel in layers 2/3 and layer 5 of pFC. The functional significance of individual pFC minicolumns (separated by 40 μm) was shown by reduced correlated firing between cell pairs within single minicolumns on error trials with inappropriate target selection. To further demonstrate dependence on performance, a task-disrupting drug (cocaine) was administered in the middle of the session, which also reduced interlaminar firing in minicolumns that fired appropriately in the early (nondrug) portion of the session. The results provide a direct demonstration of task-specific, real-time columnar processing in pFC indicating the role of this type of microcircuit in executive control of decision-making in primate brain.

Leukocytes as carriers for targeted cancer drug delivery.

PubMed

Mitchell, Michael J; King, Michael R

2015-03-01

Metastasis contributes to over 90% of cancer-related deaths. Numerous nanoparticle platforms have been developed to target and treat cancer, yet efficient delivery of these systems to the appropriate site remains challenging. Leukocytes, which share similarities to tumor cells in terms of their transport and migration through the body, are well suited to serve as carriers of drug delivery systems to target cancer sites. This review focuses on the use and functionalization of leukocytes for therapeutic targeting of metastatic cancer. Tumor cell and leukocyte extravasation, margination in the bloodstream, and migration into soft tissue are discussed, along with the potential to exploit these functional similarities to effectively deliver drugs. Current nanoparticle-based drug formulations for the treatment of cancer are reviewed, along with methods to functionalize delivery vehicles to leukocytes, either on the surface and/or within the cell. Recent progress in this area, both in vitro and in vivo, is also discussed, with a particular emphasis on targeting cancer cells in the bloodstream as a means to interrupt the metastatic process. Leukocytes interact with cancer cells both in the bloodstream and at the site of solid tumors. These interactions can be utilized to effectively deliver drugs to targeted areas, which can reduce both the amount of drug required and various nonspecific cytotoxic effects within the body. If drug delivery vehicle functionalization does not interfere with leukocyte function, this approach may be utilized to neutralize tumor cells in the bloodstream to prevent the formation of new metastases, and also to deliver drugs to metastatic sites within tissues.

Neonatal Fc Receptor Regulation of Lung Immunoglobulin and CD103+ Dendritic Cells Confers Transient Susceptibility to Tuberculosis.

PubMed

Vogelzang, Alexis; Lozza, Laura; Reece, Stephen T; Perdomo, Carolina; Zedler, Ulrike; Hahnke, Karin; Oberbeck-Mueller, Dagmar; Dorhoi, Anca; Kaufmann, Stefan H E

2016-10-01

The neonatal Fc receptor (FcRn) extends the systemic half-life of IgG antibodies by chaperoning bound Fc away from lysosomal degradation inside stromal and hematopoietic cells. FcRn also transports IgG across mucosal barriers into the lumen, and yet little is known about how FcRn modulates immunity in the lung during homeostasis or infection. We infected wild-type (WT) and FcRn-deficient (fcgrt(-/-)) mice with Pseudomonas aeruginosa or Mycobacterium tuberculosis to investigate whether recycling and transport of IgG via FcRn influences innate and adaptive immunity in the lung in response to bacterial infection. We found that FcRn expression maintains homeostatic IgG levels in lung and leads to preferential secretion of low-affinity IgG ligands into the lumen. Fcgrt(-/-) animals exhibited no evidence of developmental impairment of innate immunity in the lung and were able to efficiently recruit neutrophils in a model of acute bacterial pneumonia. Although local humoral immunity in lung increased independently of the presence of FcRn during tuberculosis, there was nonetheless a strong impact of FcRn deficiency on local adaptive immunity. We show that the quantity and quality of IgG in airways, as well as the abundance of dendritic cells in the lung, are maintained by FcRn. FcRn ablation transiently enhanced local T cell immunity and neutrophil recruitment during tuberculosis, leading to a lower bacterial burden in lung. This novel understanding of tissue-specific modulation of mucosal IgG isotypes in the lung by FcRn sheds light on the role of mucosal IgG in immune responses in the lung during homeostasis and bacterial disease. Copyright © 2016 Vogelzang et al.

Hydrolysis and coordination behavior of ferrocenyl-phosphonodithiolate: Synthesis and structure of Cu 4[FcP(OCH 3)( μ-S)( μ3-S)] 4 [Fc = Fe( η5-C 5H 4)( η5-C 5H 5)

NASA Astrophysics Data System (ADS)

Liu, Shu-Lei; Wang, Xi-Ying; Duan, Taike; Leung, Wa-Hung; Zhang, Qian-Feng

2010-02-01

Treatment of the dimeric [FcP(S)( μ-S)] 2 [Fc = Fe( η5-C 5H 4)( η5-C 5H 5)] with the organic base Et 3N in methylene chloride solution resulted in the isolation of a multi-component compound [Et 3NH] 2[(FcPO 2S) 2CH 2][FcPS(OH) 2] 2·CH 2Cl 2 ( 1·CH 2Cl 2). The formation of the [(FcPO 2S) 2CH 2] 2- anion was due to the dechlorination of methylene chloride, it consists of two [FcPO 2S] 2- units bridging by a methylene group. Reaction of Na[FcP(OCH 3)S 2] with equal equivalent of [Cu(MeCN) 4][ClO 4] in methanol afforded a sole tetranuclear copper(I) complex Cu 4[FcP(OCH 3)( μ-S)( μ3-S)] 4 ( 2). The neutral complex 2 consists of a crystallographically centrosymmetric tetramer containing four CuS 3 arrays each of which has one μ-sulfur and two μ3-sulfur bridges.

Reproduction of the FC/DFC units in nucleoli.

PubMed

Smirnov, Evgeny; Hornáček, Matúš; Kováčik, Lubomír; Mazel, Tomáš; Schröfel, Adam; Svidenská, Silvie; Skalníková, Magdalena; Bartová, Eva; Cmarko, Dušan; Raška, Ivan

2016-04-25

The essential structural components of the nucleoli, Fibrillar Centers (FC) and Dense Fibrillar Components (DFC), together compose FC/DFC units, loci of rDNA transcription and early RNA processing. In the present study we followed cell cycle related changes of these units in 2 human sarcoma derived cell lines with stable expression of RFP-PCNA (the sliding clamp protein) and GFP-RPA43 (a subunit of RNA polymerase I, pol I) or GFP-fibrillarin. Correlative light and electron microscopy analysis showed that the pol I and fibrillarin positive nucleolar beads correspond to individual FC/DFC units. In vivo observations showed that at early S phase, when transcriptionally active ribosomal genes were replicated, the number of the units in each cell increased by 60-80%. During that period the units transiently lost pol I, but not fibrillarin. Then, until the end of interphase, number of the units did not change, and their duplication was completed only after the cell division, by mid G1 phase. This peculiar mode of reproduction suggests that a considerable subset of ribosomal genes remain transcriptionally silent from mid S phase to mitosis, but become again active in the postmitotic daughter cells.

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

PubMed

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

Protective effects of Fc-fused PD-L1 on two different animal models of colitis.

PubMed

Song, Mi-Young; Hong, Chun-Pyo; Park, Seong Jeong; Kim, Jung-Hwan; Yang, Bo-Gie; Park, Yunji; Kim, Sae Won; Kim, Kwang Soon; Lee, Ji Yeung; Lee, Seung-Woo; Jang, Myoung Ho; Sung, Young-Chul

2015-02-01

Programmed death-ligand 1 (PD-L1) has been shown to negatively regulate immune responses via its interaction with PD-1 receptor. In this study, we investigated the effects of PD-L1-Fc treatment on intestinal inflammation using two murine models of inflammatory colitis induced by dextran sulfate sodium (DSS) and T-cell transfer. The anti-colitis effect of adenovirus expressing Fc-conjugated PD-L1 (Ad/PD-L1-Fc) and recombinant PD-L1-Fc protein was evaluated in DSS-treated wild-type and Rag-1 knockout (KO) mice. We examined differentiation of T-helper cells, frequency of innate immune cells, and cytokine production by dendritic cells (DCs) in the colon from DSS-treated mice after PD-L1-Fc administration. In Rag-1 KO mice reconstituted with CD4 CD45RB(high) T cells, we assessed the treatment effect of PD-L1-Fc protein on the development of colitis. Administration of Ad/PD-L1-Fc significantly ameliorated DSS-induced colitis, which was accompanied by diminished frequency of interleukin (IL)-17A-producing CD4 T cells and increased interferon-γ-producing CD4 T cells in the colon of DSS-fed mice. The anti-colitic effect of PD-L1-Fc treatment was also observed in DSS-treated Rag-1 KO mice, indicating lymphoid cell independency. PD-L1-Fc modulated cytokine production by colonic DCs and the effect was dependent on PD-1 expression. Furthermore, PD-L1-Fc protein could significantly reduce the severity of colitis in CD4 CD45RB(high) T-cell-transferred Rag-1 KO mice. Based on the protective effect of PD-L1-Fc against DSS-induced and T-cell-induced colitis, our results suggest that PD-1-mediated inhibitory signals have a crucial role in limiting the development of colonic inflammation. This implicates that PD-L1-Fc may provide a novel therapeutic approach to treat inflammatory bowel disease. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Relationships between leukocytes and Hepatozoon spp. In green frogs, Rana clamitans.

PubMed

Shutler, Dave; Smith, Todd G; Robinson, Stephen R

2009-01-01

There are few published data on amphibian leukocyte profiles, and relationships between amphibian leukocytes and parasites are even less well known. Using counts from 35 pairs of blood smears taken 2 days apart, we tested for correlations between leukocyte proportions and infection intensities of Hepatozoon spp. (either Hepatozoon catesbianae or Hepatozoon clamatae) in green frogs (Rana clamitans). On average (SE), we counted 65.4 (1.7) lymphocytes, 14.0 (1.3) neutrophils, 19.3 (1.6) eosinophils, 0.9 (0.1) monocytes, and 0.4 (0.1) basophils per 100 leukocytes. All frogs harbored Hepatozoon spp. (median seven parasites per 100 leukocytes; range 1-250). Significant relationships were not observed between numbers of leukocytes and infection intensities of Hepatozoon spp. Among the possible explanations for these null results are that Hepatozoon spp. is benign, that Hepatozoon spp. is able to evade detection by the immune system, that Hepatozoon spp. is able to manipulate leukocyte investment, or that other unmeasured or undetected parasites were more important in affecting immune response.

Subcutaneous bioavailability of therapeutic antibodies as a function of FcRn binding affinity in mice

PubMed Central

Meng, Y Gloria; Hoyte, Kwame; Lutman, Jeff; Lu, Yanmei; Iyer, Suhasini; DeForge, Laura E; Theil, Frank-Peter; Fielder, Paul J; Prabhu, Saileta

2012-01-01

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies. PMID:22327433

Distinct Fcγ receptors mediate the effect of Serum Amyloid P on neutrophil adhesion and fibrocyte differentiation

PubMed Central

Cox, Nehemiah; Pilling, Darrell; Gomer, Richard H.

2014-01-01

The plasma protein Serum Amyloid P (SAP) reduces neutrophil adhesion, inhibits the differentiation of monocytes into fibroblast-like cells called fibrocytes, and promotes phagocytosis of cell debris by macrophages. Together, these effects of SAP reduce key aspects of inflammation and fibrosis, and SAP injections improve lung function in pulmonary fibrosis patients. SAP functions are mediated in part by Fcγ receptors, but the contribution of each Fcγ receptor is not fully understood. We found that amino acids Q55 and E126 in human SAP affect human fibrocyte differentiation and SAP binding to FcγRI. E126, K130 and Q128 affect neutrophil adhesion and SAP affinity for FcγRIIa. Q128 also affects phagocytosis by macrophages and SAP affinity for FcγRI. All the identified functionally significant amino acids in SAP form a binding site that is distinct from the previously described SAP-FcγRIIa binding site. Blocking FcγRI with an IgG blocking antibody reduces the SAP effect on fibrocyte differentiation, and ligating FcγRIIa with antibodies reduces neutrophil adhesion. Together, these results suggest that SAP binds to FcγRI on monocytes to inhibit fibrocyte differentiation, and binds to FcγRIIa on neutrophils to reduce neutrophil adhesion. PMID:25024390

Evaluate the contribution of the mixture components on the longevity and performance of FC-5.

DOT National Transportation Integrated Search

2014-05-01

The focus of the project was to evaluate how to improve the longevity of FDOTs FC-5 mixtures. In particular, what FC-5 mixture : components have the greatest impact on improving the cracking and durability of the FC-5 mixture. The data mining of F...

The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

PubMed

Chenoweth, Alicia M; Trist, Halina M; Tan, Peck-Szee; Wines, Bruce D; Hogarth, P Mark

2015-11-01

Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaluation of Human FcγRIIA (CD32) and FcγRIIIB (CD16) Polymorphisms in Caucasians and African-Americans Using Salivary DNA

PubMed Central

van Schie, Rob C. A. A.; Wilson, Mark E.

2000-01-01

Two classes of low-affinity receptors for the Fc region of immunoglobulin G (IgG) (FcγR) are constitutively expressed on resting human neutrophils. These receptors, termed FcγRIIa (CD32) and FcγRIIIb (CD16), display biallelic polymorphisms which have functional consequences with respect to binding and/or ingestion of targets opsonized by human IgG subclass antibodies. The H131-R131 polymorphism of CD32 influences binding of human IgG2 and, to a lesser extent, human IgG3 to neutrophils. The neutrophil antigen (NA1-NA2) polymorphism of CD16 influences the efficiency of phagocytosis of bacteria opsonized by human IgG1 and IgG3. These polymorphisms may influence host susceptibility to certain infectious and/or autoimmune diseases, prompting interest in the development of facile methods for determination of CD32 and CD16 genotype in various clinical settings. We previously reported that genomic DNA from saliva is a suitable alternative to DNA from blood in PCR-based analyses of CD32 and CD16 polymorphisms. In the present study, we utilized for the first time this salivary DNA-based methodology to define CD32 and CD16 genotypes in 271 Caucasian and 118 African-American subjects and to investigate possible linkage disequilibrium between certain CD32 and CD16 genotypes in these two ethnic groups. H131 and R131 gene frequencies were 0.45 and 0.55, respectively, among Caucasians and 0.59 among African-Americans. NA1 and NA2 gene frequencies were 0.38 and 0.62 among Caucasians and 0.39 and 0.61 among African-Americans. Since FcγRIIa and FcγRIIIb synergize in triggering neutrophils, we also assessed the frequency of different CD32 and CD16 genotype combinations in these two groups. In both groups, the R/R131-NA2/NA2 genotype combination was more common than the H/H131-NA1/NA1 combination (threefold for Caucasians versus sevenfold for African-Americans). Whether individuals with the combined R/R131-NA2/NA2 genotype are at greater risk for development of infectious and

Leukocytes as carriers for targeted cancer drug delivery

PubMed Central

Mitchell, Michael J

2017-01-01

Introduction Metastasis contributes to over 90% of cancer-related deaths. Numerous nanoparticle platforms have been developed to target and treat cancer, yet efficient delivery of these systems to the appropriate site remains challenging. Leukocytes, which share similarities to tumor cells in terms of their transport and migration through the body, are well suited to serve as carriers of drug delivery systems to target cancer sites. Areas covered This review focuses on the use and functionalization of leukocytes for therapeutic targeting of metastatic cancer. Tumor cell and leukocyte extravasation, margination in the bloodstream, and migration into soft tissue are discussed, along with the potential to exploit these functional similarities to effectively deliver drugs. Current nanoparticle-based drug formulations for the treatment of cancer are reviewed, along with methods to functionalize delivery vehicles to leukocytes, either on the surface and/or within the cell. Recent progress in this area, both in vitro and in vivo, is also discussed, with a particular emphasis on targeting cancer cells in the bloodstream as a means to interrupt the metastatic process. Expert opinion Leukocytes interact with cancer cells both in the bloodstream and at the site of solid tumors. These interactions can be utilized to effectively deliver drugs to targeted areas, which can reduce both the amount of drug required and various nonspecific cytotoxic effects within the body. If drug delivery vehicle functionalization does not interfere with leukocyte function, this approach may be utilized to neutralize tumor cells in the bloodstream to prevent the formation of new metastases, and also to deliver drugs to metastatic sites within tissues. PMID:25270379

Enhanced phagocytic activity of HIV-specific antibodies correlates with natural production of immunoglobulins with skewed affinity for FcγR2a and FcγR2b.

PubMed

Ackerman, Margaret E; Dugast, Anne-Sophie; McAndrew, Elizabeth G; Tsoukas, Stephen; Licht, Anna F; Irvine, Darrell J; Alter, Galit

2013-05-01

While development of an HIV vaccine that can induce neutralizing antibodies remains a priority, decades of research have proven that this is a daunting task. However, accumulating evidence suggests that antibodies with the capacity to harness innate immunity may provide some protection. While significant research has focused on the cytolytic properties of antibodies in acquisition and control, less is known about the role of additional effector functions. In this study, we investigated antibody-dependent phagocytosis of HIV immune complexes, and we observed significant differences in the ability of antibodies from infected subjects to mediate this critical effector function. We observed both quantitative differences in the capacity of antibodies to drive phagocytosis and qualitative differences in their FcγR usage profile. We demonstrate that antibodies from controllers and untreated progressors exhibit increased phagocytic activity, altered Fc domain glycosylation, and skewed interactions with FcγR2a and FcγR2b in both bulk plasma and HIV-specific IgG. While increased phagocytic activity may directly influence immune activation via clearance of inflammatory immune complexes, it is also plausible that Fc receptor usage patterns may regulate the immune response by modulating downstream signals following phagocytosis--driving passive degradation of internalized virus, release of immune modulating cytokines and chemokines, or priming of a more effective adaptive immune response.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

PubMed

Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

2018-04-01

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

Programming and memory dynamics of innate leukocytes during tissue homeostasis and inflammation.

PubMed

Lee, Christina; Geng, Shuo; Zhang, Yao; Rahtes, Allison; Li, Liwu

2017-09-01

The field of innate immunity is witnessing a paradigm shift regarding "memory" and "programming" dynamics. Past studies of innate leukocytes characterized them as first responders to danger signals with no memory. However, recent findings suggest that innate leukocytes, such as monocytes and neutrophils, are capable of "memorizing" not only the chemical nature but also the history and dosages of external stimulants. As a consequence, innate leukocytes can be dynamically programmed or reprogrammed into complex inflammatory memory states. Key examples of innate leukocyte memory dynamics include the development of primed and tolerant monocytes when "programmed" with a variety of inflammatory stimulants at varying signal strengths. The development of innate leukocyte memory may have far-reaching translational implications, as programmed innate leukocytes may affect the pathogenesis of both acute and chronic inflammatory diseases. This review intends to critically discuss some of the recent studies that address this emerging concept and its implication in the pathogenesis of inflammatory diseases. © Society for Leukocyte Biology.

Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

PubMed Central

Konduru, Krishnamurthy; Bradfute, Steven B.; Jacques, Jerome; Manangeeswaran, Mohanraj; Nakamura, Siham; Morshed, Sufi; Wood, Steven C.; Bavari, Sina

2011-01-01

Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use. PMID:21329775

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawamoto, Eiji; Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507; Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp

LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding ofmore » PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.« less

Structure of FcRY, an avian immunoglobulin receptor related to mammalian mannose receptors, and its complex with IgY

PubMed Central

He, Yongning; Bjorkman, Pamela J.

2011-01-01

Fc receptors transport maternal antibodies across epithelial cell barriers to passively immunize newborns. FcRY, the functional counterpart of mammalian FcRn (a major histocompatibility complex homolog), transfers IgY across the avian yolk sac, and represents a new class of Fc receptor related to the mammalian mannose receptor family. FcRY and FcRn bind immunoglobulins at pH ≤6.5, but not pH ≥7, allowing receptor–ligand association inside intracellular vesicles and release at the pH of blood. We obtained structures of monomeric and dimeric FcRY and an FcRY–IgY complex and explored FcRY's pH-dependent binding mechanism using electron cryomicroscopy (cryoEM) and small-angle X-ray scattering. The cryoEM structure of FcRY at pH 6 revealed a compact double-ring “head,” in which the N-terminal cysteine-rich and fibronectin II domains were folded back to contact C-type lectin-like domains 1–6, and a “tail” comprising C-type lectin-like domains 7–8. Conformational changes at pH 8 created a more elongated structure that cannot bind IgY. CryoEM reconstruction of FcRY dimers at pH 6 and small-angle X-ray scattering analysis at both pH values confirmed both structures. The cryoEM structure of the FcRY–IgY revealed symmetric binding of two FcRY heads to the dimeric FcY, each head contacting the CH4 domain of one FcY chain. FcRY shares structural properties with mannose receptor family members, including a head and tail domain organization, multimerization that may regulate ligand binding, and pH-dependent conformational changes. Our results facilitate understanding of immune recognition by the structurally related mannose receptor family and comparison of diverse methods of Ig transport across evolution. PMID:21746914

The mitochondria-targeted antioxidant MitoQ modulates oxidative stress, inflammation and leukocyte-endothelium interactions in leukocytes isolated from type 2 diabetic patients.

PubMed

Escribano-Lopez, Irene; Diaz-Morales, Noelia; Rovira-Llopis, Susana; de Marañon, Arantxa Martinez; Orden, Samuel; Alvarez, Angeles; Bañuls, Celia; Rocha, Milagros; Murphy, Michael P; Hernandez-Mijares, Antonio; Victor, Victor M

2016-12-01

It is not known if the mitochondria-targeted antioxidants such as mitoquinone (MitoQ) can modulate oxidative stress and leukocyte-endothelium interactions in T2D patients. We aimed to evaluate the beneficial effect of MitoQ on oxidative stress parameters and leukocyte-endothelium interactions in leukocytes of T2D patients. The study population consisted of 98 T2D patients and 71 control subjects. We assessed metabolic and anthropometric parameters, mitochondrial reactive oxygen species (ROS) production, glutathione peroxidase 1 (GPX-1), NFκB-p65, TNFα and leukocyte-endothelium interactions. Diabetic patients exhibited higher weight, BMI, waist circumference, SBP, DBP, glucose, insulin, HOMA-IR, HbA1c, triglycerides, hs-CRP and lower HDL-c with respect to controls. Mitochondrial ROS production was enhanced in T2D patients and decreased by MitoQ. The antioxidant also increased GPX-1 levels and PMN rolling velocity and decreased PMN rolling flux and PMN adhesion in T2D patients. NFκB-p65 and TNFα were augmented in T2D and were both reduced by MitoQ treatment. Our findings support that the antioxidant MitoQ has an anti-inflammatory and antioxidant action in the leukocytes of T2D patients by decreasing ROS production, leukocyte-endothelium interactions and TNFα through the action of NFκB. These data suggest that mitochondria-targeted antioxidants such as MitoQ should be investigated as a novel means of preventing cardiovascular events in T2D patients. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Human leukocytic pyrogen: purification and development of a radioimmunoassay.

PubMed

Dinarello, C A; Renfer, L; Wolff, S M

1977-10-01

Leukocytic pyrogen is a small endogenous protein that mediates fever. Because of the limitations of bioassays, circulating leukocytic pyrogen has not been demonstrated during fever in humans. The pyrogen was produced in vitro after phagocytosis of staphylococci by blood monocytes. Antibody against the pyrogen was obtained from rabbits immunized with leukocytic pyrogen and the antiserum was purified by solid-phase immunoadsorbants. Purified antibody to the pyrogen was attached to activated Sepharose 4B and used in conjunction with gel filtration to purify the pyrogen. The pyrogen was labeled with 125I and further purified by gel filtration and ion-exchange chromatography. The final preparation of 125I-labeled pyrogen demonstrated a homogeneous band during isoelectric focusing and other separation procedures. With antibody to pyrogen attached to Sepharose, less than 0.1 of a rabbit pyrogenic dose of human leukocytic pyrogen inhibited the binding of 125I-labeled pyrogen to this immunoadsorbant, and this inhibition was not affected by the presence of human serum. Thus, a radioimmunoassay for human leukocytic pyrogen has been developed that may be used to detect circulating pyrogen during fever in humans.

Access to site-specific Fc-cRGD peptide conjugates through streamlined expressed protein ligation.

PubMed

Frutos, S; Jordan, J B; Bio, M M; Muir, T W; Thiel, O R; Vila-Perelló, M

2016-10-12

An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners.

Fc-Mediated Anomalous Biodistribution of Therapeutic Antibodies in Immunodeficient Mouse Models.

PubMed

Sharma, Sai Kiran; Chow, Andrew; Monette, Sebastien; Vivier, Delphine; Pourat, Jacob; Edwards, Kimberly J; Dilling, Thomas R; Abdel-Atti, Dalya; Zeglis, Brian M; Poirier, John T; Lewis, Jason S

2018-04-01

A critical benchmark in the development of antibody-based therapeutics is demonstration of efficacy in preclinical mouse models of human disease, many of which rely on immunodeficient mice. However, relatively little is known about how the biology of various immunodeficient strains impacts the in vivo fate of these drugs. Here we used immunoPET radiotracers prepared from humanized, chimeric, and murine mAbs against four therapeutic oncologic targets to interrogate their biodistribution in four different strains of immunodeficient mice bearing lung, prostate, and ovarian cancer xenografts. The immunodeficiency status of the mouse host as well as both the biological origin and glycosylation of the antibody contributed significantly to the anomalous biodistribution of therapeutic monoclonal antibodies in an Fc receptor-dependent manner. These findings may have important implications for the preclinical evaluation of Fc-containing therapeutics and highlight a clear need for biodistribution studies in the early stages of antibody drug development. Significance: Fc/FcγR-mediated immunobiology of the experimental host is a key determinant to preclinical in vivo tumor targeting and efficacy of therapeutic antibodies. Cancer Res; 78(7); 1820-32. ©2018 AACR . ©2018 American Association for Cancer Research.

Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain*

PubMed Central

Vaughan, Andrew T.; Chan, Claude H. T.; Klein, Christian; Glennie, Martin J.; Beers, Stephen A.; Cragg, Mark S.

2015-01-01

Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes. PMID:25568316

Leukocyte integrin activation mediates transient neutropenia after G-CSF administration

PubMed Central

Tuschong, Laura; Bauer, Thomas R.; Yau, Yu Ying; Leitman, Susan F.; Hickstein, Dennis D.

2011-01-01

After administration of granulocyte colony-stimulating factor (G-CSF), there is a marked, albeit transient, drop in circulating neutrophils. To determine the role of leukocyte integrins in this disappearance, a dog having canine leukocyte adhesion deficiency (CLAD) or CLAD dogs who had undergone gene correction either by matched littermate allogeneic transplant or autologous gene therapy were evaluated. Shortly after G-CSF administration, a dramatic, yet transient, neutropenia was observed in the control littermates. This neutropenia was not as marked in the CLAD dogs. In all instances, it was CD18+ neutrophils that preferentially egressed from the circulation. The association of CD18 with this rapid loss suggested leukocyte integrin activation after G-CSF administration. To determine the activation status of the integrin, a monoclonal antibody recognizing the activated α-subunit cation binding domain (mAb24) was used to evaluate human leukocytes after G-CSF administration. Mirroring the dramatic decrease in circulating neutrophil numbers, there was a dramatic and specific increase in the activation of the α-subunit after G-CSF expression on polymorphonuclear leukocytes. This activation, like the drop in neutrophil count, was transient. These results demonstrate that the leukocyte integrin on circulating neutrophils is transiently activated after G-CSF administration and mediates the transient neutropenia observed after G-CSF administration. PMID:21844566

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies

PubMed Central

Richardson, Simone I.; Mabvakure, Batsirai; Mkhize, Nonhlanhla N.; Moore, Penny L.; Alter, Galit

2018-01-01

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies. PMID:29630668

Defining the chemokine basis for leukocyte recruitment during viral encephalitis.

PubMed

Michlmayr, Daniela; McKimmie, Clive S; Pingen, Marieke; Haxton, Ben; Mansfield, Karen; Johnson, Nicholas; Fooks, Anthony R; Graham, Gerard J

2014-09-01

The encephalitic response to viral infection requires local chemokine production and the ensuing recruitment of immune and inflammatory leukocytes. Accordingly, chemokine receptors present themselves as plausible therapeutic targets for drugs aimed at limiting encephalitic responses. However, it remains unclear which chemokines are central to this process and whether leukocyte recruitment is important for limiting viral proliferation and survival in the brain or whether it is predominantly a driver of coincident inflammatory pathogenesis. Here we examine chemokine expression and leukocyte recruitment in the context of avirulent and virulent Semliki Forest virus (SFV) as well as West Nile virus infection and demonstrate rapid and robust expression of a variety of inflammatory CC and CXC chemokines in all models. On this basis, we define a chemokine axis involved in leukocyte recruitment to the encephalitic brain during SFV infection. CXCR3 is the most active; CCR2 is also active but less so, and CCR5 plays only a modest role in leukocyte recruitment. Importantly, inhibition of each of these receptors individually and the resulting suppression of leukocyte recruitment to the infected brain have no effect on viral titer or survival following infection with a virulent SFV strain. In contrast, simultaneous blockade of CXCR3 and CCR2 results in significantly reduced mortality in response to virulent SFV infection. In summary, therefore, our data provide an unprecedented level of insight into chemokine orchestration of leukocyte recruitment in viral encephalitis. Our data also highlight CXCR3 and CCR2 as possible therapeutic targets for limiting inflammatory damage in response to viral infection of the brain. Brain inflammation (encephalitis) in response to viral infection can lead to severe illness and even death. This therefore represents an important clinical problem and one that requires the development of new therapeutic approaches. Central to the pathogenesis of

Immune-stimulatory effects of a bacteria-based probiotic on peripheral leukocyte subpopulations and cytokine mRNA expression levels in scouring holstein calves.

PubMed

Qadis, Abdul Qadir; Goya, Satoru; Yatsu, Minoru; Kimura, Atsushi; Ichijo, Toshihiro; Sato, Shigeru

2014-05-01

Subpopulations of peripheral leukocytes and cytokine mRNA expression levels were evaluated in scouring and healthy Holstein calves (age 10 ± 5 days; n=42) treated with a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum. The calves were assigned to the scouring or healthy group and then subdivided into pathogen-positive treated (n=8), pathogen-positive control (n=8), pathogen-negative treated (n=6), pathogen-negative control (n=6), healthy treated (n=6) and healthy control (n=8) groups. A single dose of the probiotic (3.0 g/100 kg body weight) was given to each calf in the treatment groups for 5 days. Blood samples were collected on the first day of scour occurrence (day 0) and on day 7. In the scouring calves, smaller peripheral leukocyte subpopulations and cytokine mRNA expression levels were noted on day 0. The numbers of CD3(+) T cells; CD4(+), CD8(+) and WC1(+) γδ T cell subsets; and CD14(+), CD21(+) and CD282(+) (TLR2) cells were significantly increased in the scouring and healthy treated calves on day 7. Furthermore, interleukin-6, tumor necrosis factor-alpha and interferon-gamma mRNA expression was elevated in the peripheral leukocytes of the scouring and healthy treated calves on day 7. The scouring calves given the probiotic recovered on day 7. A significantly smaller number of peripheral leukocytes and lower cytokine mRNA expression level might be induced by scouring in calves. Repeated probiotic administration might stimulate cellular immunity and encourage recovery from scouring in pre-weaning Holstein calves.

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

PubMed Central

Dumont, Jennifer A.; Liu, Tongyao; Low, Susan C.; Zhang, Xin; Kamphaus, George; Sakorafas, Paul; Fraley, Cara; Drager, Douglas; Reidy, Thomas; McCue, Justin; Franck, Helen W. G.; Merricks, Elizabeth P.; Nichols, Timothy C.; Bitonti, Alan J.; Pierce, Glenn F.

2012-01-01

Despite 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. PMID:22246033

Observing a fictitious stressful event: haematological changes, including circulating leukocyte activation.

PubMed

Mian, Rubina; Shelton-Rayner, Graham; Harkin, Brendan; Williams, Paul

2003-03-01

The aim of this study was to assess the effect of watching a psychological stressful event on the activation of leukocytes in healthy human volunteers. Blood samples were obtained from 32 healthy male and female subjects aged between 20 and 26 years before, during and after either watching an 83-minute horror film that none of the subjects had previously seen (The Texas Chainsaw Massacre, 1974) or by sitting quietly in a room (control group). Total differential cell counts, leukocyte activation as measured by the nitroblue tetrazolium (NBT) test, heart rate and blood pressure (BP) measurements were taken at defined time points. There were significant increases in peripheral circulating leukocytes, the number of activated circulating leukocytes, haemoglobin (Hb) concentration and haematocrit (Hct) in response to the stressor. These were accompanied by significant increases in heart rate, systolic and diastolic BP (P<0.05 from baseline). This is the first reported study on the effects of observing a psychologically stressful, albeit fictitious event on circulating leukocyte numbers and the state of leukocyte activation as determined by the nitrotetrazolium test.

Activatory and inhibitory Fcγ receptors augment rituximab-mediated internalization of CD20 independent of signaling via the cytoplasmic domain.

PubMed

Vaughan, Andrew T; Chan, Claude H T; Klein, Christian; Glennie, Martin J; Beers, Stephen A; Cragg, Mark S

2015-02-27

Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Leukocyte Trafficking in Cardiovascular Disease: Insights from Experimental Models.

PubMed

Jones, Daniel P; True, Harry D; Patel, Jyoti

2017-01-01

Chemokine-induced leukocyte migration into the vessel wall is an early pathological event in the progression of atherosclerosis, the underlying cause of myocardial infarction. The immune-inflammatory response, mediated by both the innate and adaptive immune cells, is involved in the initiation, recruitment, and resolution phases of cardiovascular disease progression. Activation of leukocytes via inflammatory mediators such as chemokines, cytokines, and adhesion molecules is instrumental in these processes. In this review, we highlight leukocyte activation with the main focus being on the mechanisms of chemokine-mediated recruitment in atherosclerosis and the response postmyocardial infarction with key examples from experimental models of cardiovascular inflammation.

Contour Detection of Leukocyte Cell Nucleus Using Morphological Image

NASA Astrophysics Data System (ADS)

Supriyanti, R.; Satrio, G. P.; Ramadhani, Y.; Siswandari, W.

2017-04-01

Leukocytes are blood cells that do not contain color pigments. Leukocyte function to the tool body’s defenses. Abnormal forms of leukocytes can be a sign of serious diseases such example is leukemia. Most laboratories still use cell morphology examination to assist the diagnosis of illness associated with white blood cells such example is leukemia because of limited resources, both infrastructure, and human resources as happens in developing nations, such as Indonesia. This examination is less expensive and quicker process. However, morphological review requires the expertise of a specialist clinical pathology were limited. This process is sometimes less valid cause in some cases trying to differentiate morphology blast cells into the type of myoblasts, lymphoblast, monoblast, or erythroblast thus potentially misdiagnosis. The goal of this research is to develop a detection device types of blood cells automatically as lower-priced, easy to use and accurate so that the tool can be distributed across all units in existing health services throughout Indonesia and in particular for remote areas. However, because the variables used in the identification of abnormal leukocytes are very complex, in this paper, we emphasize on the contour detection of leukocyte cell nucleus using the morphological image. The results show that this method is promising for further development.

Acute Fluoxetine Treatment Induces Slow Rolling of Leukocytes on Endothelium in Mice

PubMed Central

Herr, Nadine; Mauler, Maximilian; Witsch, Thilo; Stallmann, Daniela; Schmitt, Stefanie; Mezger, Julius; Bode, Christoph; Duerschmied, Daniel

2014-01-01

Objective Activated platelets release serotonin at sites of inflammation where it acts as inflammatory mediator and enhances recruitment of neutrophils. Chronic treatment with selective serotonin reuptake inhibitors (SSRI) depletes the serotonin storage pool in platelets, leading to reduced leukocyte recruitment in murine experiments. Here, we examined the direct and acute effects of SSRI on leukocyte recruitment in murine peritonitis. Methods C57Bl/6 and Tph1−/− (Tryptophan hydroxylase1) mice underwent acute treatment with the SSRI fluoxetine or vehicle. Serotonin concentrations were measured by ELISA. Leukocyte rolling and adhesion on endothelium was analyzed by intravital microscopy in mesentery venules with and without lipopolysaccharide challenge. Leukocyte extravasation in sterile peritonitis was measured by flow cytometry of abdominal lavage fluid. Results Plasma serotonin levels were elevated 2 hours after fluoxetine treatment (0.70±0.1 µg/ml versus 0.27±0.1, p = 0.03, n = 14), while serum serotonin did not change. Without further stimulation, acute fluoxetine treatment increased the number of rolling leukocytes (63±8 versus 165±17/0.04 mm2min−1) and decreased their velocity (61±6 versus 28±1 µm/s, both p<0.0001, n = 10). In Tph1−/− mice leukocyte rolling was not significantly influenced by acute fluoxetine treatment. Stimulation with lipopolysaccharide decreased rolling velocity and induced leukocyte adhesion, which was enhanced after fluoxetine pretreatment (27±3 versus 36±2/0.04 mm2, p = 0.008, n = 10). Leukocyte extravasation in sterile peritonitis, however, was not affected by acute fluoxetine treatment. Conclusions Acute fluoxetine treatment increased plasma serotonin concentrations and promoted leukocyte-endothelial interactions in-vivo, suggesting that serotonin is a promoter of acute inflammation. E-selectin was upregulated on endothelial cells in the presence of serotonin, possibly explaining the observed

Dose and dose rate effects of whole-body proton irradiation on leukocyte populations and lymphoid organs: part I

NASA Technical Reports Server (NTRS)

Gridley, Daila S.; Pecaut, Michael J.; Dutta-Roy, Radha; Nelson, Gregory A.

2002-01-01

The goal of part I of this study was to evaluate the effects of whole-body proton irradiation on lymphoid organs and specific leukocyte populations. C57BL/6 mice were exposed to the entry region of the proton Bragg curve to total doses of 0.5 gray (Gy), 1.5 Gy, and 3.0 Gy, each delivered at a low dose rate (LDR) of 1 cGy/min and high dose rate (HDR) of 80 cGy/min. Non-irradiated and 3 Gy HDR gamma-irradiated groups were included as controls. At 4 days post-irradiation, highly significant radiation dose-dependent reductions were observed in the mass of both lymphoid organs and the numbers of leukocytes and T (CD3(+)), T helper (CD3(+)/CD4(+)), T cytotoxic (CD3(+)/CD8(+)), and B (CD19(+)) cells in both blood and spleen. A less pronounced dose effect was noted for natural killer (NK1.1(+) NK) cells in spleen. Monocyte, but not granulocyte, counts in blood were highly dose-dependent. The numbers for each population generally tended to be lower with HDR than with LDR radiation; a significant dose rate effect was found in the percentages of T and B cells, monocytes, and granulocytes and in CD4(+):CD8(+) ratios. These data indicate that mononuclear cell response to the entry region of the proton Bragg curve is highly dependent upon the total dose and that dose rate effects are evident with some cell types. Results from gamma- and proton-irradiated groups (both at 3 Gy HDR) were similar, although proton-irradiation gave consistently lower values in some measurements.

Correlating the Impact of Well-Defined Oligosaccharide Structures on Physical Stability Profiles of IgG1-Fc Glycoforms.

PubMed

More, Apurva S; Toprani, Vishal M; Okbazghi, Solomon Z; Kim, Jae H; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

2016-02-01

As part of a series of articles in this special issue describing 4 well-defined IgG1-Fc glycoforms as a model system for biosimilarity analysis (high mannose-Fc, Man5-Fc, GlcNAc-Fc and N297Q-Fc aglycosylated), the focus of this work is comparisons of their physical properties. A trend of decreasing apparent solubility (thermodynamic activity) by polyethylene glycol precipitation (pH 4.5, 6.0) and lower conformational stability by differential scanning calorimetry (pH 4.5) was observed with reducing size of the N297-linked oligosaccharide structures. Using multiple high-throughput biophysical techniques, the physical stability of the Fc glycoproteins was then measured in 2 formulations (NaCl and sucrose) across a wide range of temperatures (10°C-90°C) and pH (4.0-7.5) conditions. The data sets were used to construct 3-index empirical phase diagrams and radar charts to visualize the regions of protein structural stability. Each glycoform showed improved stability in the sucrose (vs. salt) formulation. The HM-Fc and Man5-Fc displayed the highest relative stability, followed by GlcNAc-Fc, with N297Q-Fc being the least stable. Thus, the overall physical stability profiles of the 4 IgG1-Fc glycoforms also show a correlation with oligosaccharide structure. These data sets are used to develop a mathematical model for biosimilarity analysis (as described in a companion article by Kim et al. in this issue). Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Ischemia-reperfusion injury in the isolated rat lung. Role of flow and endogenous leukocytes.

PubMed

Seibert, A F; Haynes, J; Taylor, A

1993-02-01

Microvascular lung injury caused by ischemia-reperfusion (IR) may occur via leukocyte-dependent and leukocyte-independent pathways. Leukocyte-endothelial adhesion may be a rate-limiting step in IR lung injury. Leukocyte adhesion to microvascular endothelium occurs when the attractant forces between leukocyte and endothelium are greater than the kinetic energy of the leukocyte and the vascular wall shear rate. We hypothesized (1) that isolated, buffer-perfused rat lungs are not free of endogenous leukocytes, (2) that endogenous leukocytes contribute to IR-induced microvascular injury as measured by the capillary filtration coefficient (Kfc), and (3) that a reduction of perfusate flow rate would potentiate leukocyte-dependent IR injury. Sixty lungs were divided into four groups: (1) low-flow controls, (2) high-flow controls, (3) low-flow IR, and (4) high-flow IR. Microvascular injury was linearly related to baseline perfusate leukocyte concentrations at both low (r = 0.78) and high (r = 0.82) flow rates. Kfc in the high-flow IR group (0.58 +/- 0.03 ml/min/cm H2O/100 g) was less (p < 0.05) than Kfc in the low-flow IR group (0.82 +/- 0.07), and in both groups Kfc values were significantly greater than low-flow (0.34 +/- 0.03) and high-flow (0.31 +/- 0.01) control Kfc values after 75 min. Retention of leukocytes in the lung, evaluated by a tissue myeloperoxidase assay, was greatest in the low-flow IR group. We conclude (1) that isolated, buffer-perfused rat lungs contain significant quantities of leukocytes and that these leukocytes contribute to IR lung injury, and (2) that IR-induced microvascular injury is potentiated by low flow.

Pathophysiology of Relapsing Fever: Interaction of Borrelia Spirochetes with Blood Mononuclear Leukocytes Causes Production of Leukocytic Pyrogen and Tissue Thromboplastin.

DTIC Science & Technology

1980-12-10

AD-A093 BN6 CASE WESTERN RESERVE UNIV CLEVELAND OH DEPT OF MEDICINE FIG 6/5 PAT1OPHYSIOLOGY OF RELAPSING FEVERS INTERACTION OF BORRELtA SPI-ETC(U...NUMBER 4 Pathophysiology of Relapsing Fever : Interaction of Borrelia Spirochetes 0 with Blood Mononuclear Leukocytes Causes Production of Leukocytic...Relapsing fever caused by Borrelia spirochetes is characterized by episodes of spirochetemia, fever , and disseminated intravascular coagu- lation (DIC

FcγRIIa ligation induces platelet hypersensitivity to thrombotic stimuli.

PubMed

Berlacher, Mark D; Vieth, Joshua A; Heflin, Brittany C; Gay, Steven R; Antczak, Adam J; Tasma, Brian E; Boardman, Holly J; Singh, Navinderjit; Montel, Angela H; Kahaleh, M Bashar; Worth, Randall G

2013-01-01

Platelets are known for their important role in hemostasis, however their significance in other functions, including inflammation and infection, are becoming more apparent. Patients with systemic lupus erythematosus (SLE) are known to have circulating IgG complexes in their blood and are highly susceptible to thrombotic events. Because platelets express a single receptor for IgG, we tested the hypothesis that ligation of this receptor (FcγRIIa) induces platelet hypersensitivity to thrombotic stimuli. Platelets from SLE patients were considerably more sensitive to thrombin compared to healthy volunteers, and this correlated with elevated levels of surface IgG on SLE platelets. To test whether FcγRIIa ligation stimulated thrombin hypersensitivity, platelets from healthy volunteers were incubated with buffer or heat-aggregated IgG, then stimulated with increasing concentrations of thrombin. Interestingly, heat-aggregated IgG-stimulated platelets, but not buffer-treated platelets, were hypersensitive to thrombin, and hypersensitivity was blocked by an anti-FcγRIIa monoclonal antibody (mAb). Thrombin hypersensitivity was not due to changes in thrombin receptor expression (GPIbα or PAR1) but is dependent on activation of shared signaling molecules. These observations suggest that ligation of platelet FcγRIIa by IgG complexes induces a hypersensitive state whereby small changes in thrombotic stimuli may result in platelet activation and subsequent vascular complications such as transient ischemic attacks or stroke. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

BiFC Assay to Detect Calmodulin Binding to Plant Receptor Kinases.

PubMed

Fischer, Cornelia; Sauter, Margret; Dietrich, Petra

2017-01-01

Plant receptor-like kinases (RLKs) are regulated at various levels including posttranscriptional modification and interaction with regulatory proteins. Calmodulin (CaM) is a calcium-sensing protein that was shown to bind to some RLKs such as the PHYTOSULFOKINE RECEPTOR1 (PSKR1). The CaM-binding site is embedded in subdomain VIa of the kinase domain. It is possible that many more of RLKs interact with CaM than previously described. To unequivocally confirm CaM binding, several methods exist. Bimolecular fluorescence complementation (BiFC) and pull-down assays have been successfully used to study CaM binding to PSKR1 and are described in this chapter (BiFC) and in Chapter 15 (pull down). The two methods are complementary. BiFC is useful to show localization and interaction of soluble as well as of membrane-bound proteins in planta.

Fc functional antibodies in humans with severe H7N9 and seasonal influenza

PubMed Central

Vanderven, Hillary A.; Liu, Lu; Ana-Sosa-Batiz, Fernanda; Nguyen, Thi H.O.; Wan, Yanmin; Hogarth, P. Mark; Tilmanis, Danielle; Parsons, Matthew S.; Hurt, Aeron C.; Davenport, Miles P.; Kotsimbos, Tom; Cheng, Allen C.; Kedzierska, Katherine; Zhang, Xiaoyan; Xu, Jianqing; Kent, Stephen J.

2017-01-01

BACKGROUND. Both seasonal and novel avian influenza viruses can result in severe infections requiring hospitalization. Anti-influenza antibodies (Abs) with Fc-mediated effector functions, such as Ab-dependent cellular cytotoxicity (ADCC), are of growing interest in control of influenza but have not previously been studied during severe human infections. As such, the objective of this study was to examine Fc-mediated Ab functions in humans hospitalized with influenza infection. METHODS. Serum Ab response was studied in subjects hospitalized with either pandemic H7N9 avian influenza virus in China (n = 18) or circulating seasonal influenza viruses in Melbourne, Australia (n = 16). Recombinant soluble Fc receptor dimer ELISAs, natural killer (NK) cell activation assays, and Ab-dependent killing assays with influenza-infected target cells were used to assess the Fc functionality of anti-influenza hemagglutinin (HA) Abs during severe human influenza infection. RESULTS. We found that the peak generation of Fc functional HA Abs preceded that of neutralizing Abs for both severe H7N9 and seasonal influenza infections. Subjects who succumbed to complications of H7N9 infection demonstrated reduced HA-specific Fc receptor–binding Abs (in magnitude and breadth) immediately prior to death compared with those who survived. Subjects who recovered from H7N9 and severe seasonal influenza infections demonstrated increased Fc receptor–binding Abs not only against the homologous infecting strain but against HAs from different influenza A subtypes. CONCLUSION. Collectively, survivors of severe influenza infection rapidly generate a functional Ab response capable of mediating ADCC against divergent influenza viruses. Broadly binding HA Abs with Fc-mediated functions may be a useful component of protective immunity to severe influenza infection. FUNDING. The National Health and Medical Research Council ([NHMRC] grants 1023294, 1041832, and 1071916), the Australian Department of Health

HAL/S-FC compiler system functional specification

NASA Technical Reports Server (NTRS)

1974-01-01

Compiler organization is discussed, including overall compiler structure, internal data transfer, compiler development, and code optimization. The user, system, and SDL interfaces are described, along with compiler system requirements. Run-time software support package and restrictions and dependencies are also considered of the HAL/S-FC system.

In vivo imaging of leukocyte recruitment to glomeruli in mice using intravital microscopy.

PubMed

Kitching, A Richard; Kuligowski, Michael P; Hickey, Michael J

2009-01-01

Leukocytes mediate some forms of glomerulonephritis, particularly severe proliferative and crescentic forms. The renal glomerulus is one of the few sites within the microvasculature in which leukocyte recruitment occurs in capillaries. However, due to the difficulty of directly visualising the glomerulus, the mechanisms of leukocyte recruitment to glomerular capillaries are poorly understood. To overcome this, a murine kidney can be rendered hydronephrotic, by ligating one ureter, and allowing the mouse to rest for 12 weeks. This allows the visualisation of the glomerular microvasculature during inflammatory responses. In inflammation, in this example induced by anti-glomerular basement membrane (GBM) antibody, leukocytes can be observed undergoing adhesion in glomerular capillaries using intravital microscopy. Leukocyte adhesion can be quantitated using this approach. An observation protocol involving few, limited periods of epifluorescence avoids phototoxicity-induced leukocyte recruitment. The process of hydronephrosis does not alter the ability of anti-GBM-antibody to induce a glomerular inflammatory response. This approach allows detailed investigation of the mechanisms of leukocyte recruitment within glomeruli.

The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells

NASA Astrophysics Data System (ADS)

Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.

1989-06-01

Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

[Approaches to the immunological problems of leukocyte transfusions].

PubMed

Gualde, N; Malinvaud, G; Gaillard, S

1975-01-01

51 leukocyte transfusions from healthy donors and 3 from chronic myelogenous leukemia were given to 16 patients with acute leukemia and 4 with aplasia. During 14 transfusions we have observed clinicals reactions which are of immunological origin. The part of the transfusions and of the pregnancies in the presence of antibodies is argued. The necessity of utilisation of HL-A compatible donors during leukocyte transfusions is asserted.

Optimization and qualification of an Fc Array assay for assessments of antibodies against HIV-1/SIV.

PubMed

Brown, Eric P; Weiner, Joshua A; Lin, Shu; Natarajan, Harini; Normandin, Erica; Barouch, Dan H; Alter, Galit; Sarzotti-Kelsoe, Marcella; Ackerman, Margaret E

2018-04-01

The Fc Array is a multiplexed assay that assesses the Fc domain characteristics of antigen-specific antibodies with the potential to evaluate up to 500 antigen specificities simultaneously. Antigen-specific antibodies are captured on antigen-conjugated beads and their functional capacity is probed via an array of Fc-binding proteins including antibody subclassing reagents, Fcγ receptors, complement proteins, and lectins. Here we present the results of the optimization and formal qualification of the Fc Array, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. Assay conditions were optimized for performance and reproducibility, and the final version of the assay was then evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Leukocyte Trafficking in Cardiovascular Disease: Insights from Experimental Models

PubMed Central

2017-01-01

Chemokine-induced leukocyte migration into the vessel wall is an early pathological event in the progression of atherosclerosis, the underlying cause of myocardial infarction. The immune-inflammatory response, mediated by both the innate and adaptive immune cells, is involved in the initiation, recruitment, and resolution phases of cardiovascular disease progression. Activation of leukocytes via inflammatory mediators such as chemokines, cytokines, and adhesion molecules is instrumental in these processes. In this review, we highlight leukocyte activation with the main focus being on the mechanisms of chemokine-mediated recruitment in atherosclerosis and the response postmyocardial infarction with key examples from experimental models of cardiovascular inflammation. PMID:28465628

Benzenediamine analog FC-99 inhibits TLR2 and TLR4 signaling in peritoneal macrophage in vitro.

PubMed

Yang, Liu; Dou, Huan; Song, Yuxian; Hou, Yayi

2016-01-01

Inflammatory bowel disease (IBD) is an inflammatory disorder, characterized by abnormally increased expression of Toll-like receptors TLR2 and TLR4 in the colon and increased pro-inflammatory cytokine production by macrophages. In the present study, we explored the effect of FC-99, a novel benzenediamine analog, on dextran sulfate sodium (DSS)-induced mouse colitis and investigated its potential mechanism. The results revealed that FC-99 improved the colon morphology and the clinical parameters in DSS-induced mouse colitis. FC-99 inhibited the increase of DSS-induced T helper cells (Th) 1 and Th17 and enhanced the number of regulatory T cells (Treg) in mesenteric lymph nodes (MLN), but had no effect on Th2 cells. FC-99 also suppressed the DSS-induced secretion of interleukin (IL)-1β, IL-6, and the tumor necrosis factor (TNF)-α in the colon and hindered the infiltration of macrophages into colon lamina propria. Flow cytometric analysis also confirmed that FC-99 reduced CD11b(+)F4/80(+) colon macrophages, and down-regulated TNF-α level in situ. Moreover, FC-99 inhibited concentration-dependently the expression of TNF-α and IL-6 in vitro from mouse peritoneal macrophages, which were induced by TLR ligands: PamCSK4 and peptidoglycan (PGN, TLR2 ligand) as well as LPS (TLR4 ligand). Of note, FC-99 also suppressed the activation of TLR2 and TLR4 signaling pathways and the downstream nuclear factor-κB (NF-κB) in the DSS-induced mouse colitis. FC-99 improved the condition of DSS-induced mouse colitis by inhibiting the activation of TLR2 and TLR4 signaling pathways in macrophage. These results suggest that FC-99 may be developed as a new therapeutic drug for IBD. Copyright © 2015 Elsevier Inc. All rights reserved.

Relationship of Stress, Leukocyte Functions and Acute Ulcerative Gingivitis.

DTIC Science & Technology

1982-10-22

IONAL 1jjl (I Y AN[)AAU> I,)(, 4 •. . . . .i -AD (Report Number 3 , Lf) RELATIONSHIP OF STRESS, LEUKOCYTE FUNCTION AND ACUTE ULCERATIVE GINGIVITIS...AIk £It. KEY WORDS (C~mntm. a reers old. A *1acoa and Identit by block number) Acute Necrotic Ulcerative Gingivitis (ANUG)), Stress 4 Leukocyte

Track-weighted functional connectivity (TW-FC): a tool for characterizing the structural-functional connections in the brain.

PubMed

Calamante, Fernando; Masterton, Richard A J; Tournier, Jacques-Donald; Smith, Robert E; Willats, Lisa; Raffelt, David; Connelly, Alan

2013-04-15

MRI provides a powerful tool for studying the functional and structural connections in the brain non-invasively. The technique of functional connectivity (FC) exploits the intrinsic temporal correlations of slow spontaneous signal fluctuations to characterise brain functional networks. In addition, diffusion MRI fibre-tracking can be used to study the white matter structural connections. In recent years, there has been considerable interest in combining these two techniques to provide an overall structural-functional description of the brain. In this work we applied the recently proposed super-resolution track-weighted imaging (TWI) methodology to demonstrate how whole-brain fibre-tracking data can be combined with FC data to generate a track-weighted (TW) FC map of FC networks. The method was applied to data from 8 healthy volunteers, and illustrated with (i) FC networks obtained using a seeded connectivity-based analysis (seeding in the precuneus/posterior cingulate cortex, PCC, known to be part of the default mode network), and (ii) with FC networks generated using independent component analysis (in particular, the default mode, attention, visual, and sensory-motor networks). TW-FC maps showed high intensity in white matter structures connecting the nodes of the FC networks. For example, the cingulum bundles show the strongest TW-FC values in the PCC seeded-based analysis, due to their major role in the connection between medial frontal cortex and precuneus/posterior cingulate cortex; similarly the superior longitudinal fasciculus was well represented in the attention network, the optic radiations in the visual network, and the corticospinal tract and corpus callosum in the sensory-motor network. The TW-FC maps highlight the white matter connections associated with a given FC network, and their intensity in a given voxel reflects the functional connectivity of the part of the nodes of the network linked by the structural connections traversing that voxel. They

PET/CT with 18F-FDG- and 18F-FBEM-labeled leukocytes for metabolic activity and leukocyte recruitment monitoring in a mouse model of pulmonary fibrosis.

PubMed

Bondue, Benjamin; Sherer, Félicie; Van Simaeys, Gaetan; Doumont, Gilles; Egrise, Dominique; Yakoub, Yousof; Huaux, François; Parmentier, Marc; Rorive, Sandrine; Sauvage, Sébastien; Lacroix, Simon; Vosters, Olivier; De Vuyst, Paul; Goldman, Serge

2015-01-01

Idiopathic pulmonary fibrosis is characterized by a progressive and irreversible respiratory failure. Validated noninvasive methods able to assess disease activity are essential for prognostic purposes as well as for the evaluation of emerging antifibrotic treatments. C57BL/6 mice were used in a murine model of pulmonary fibrosis induced by an intratracheal instillation of bleomycin (control mice were instilled with a saline solution). At different times after instillation, PET/CT with (18)F-FDG- or (18)F-4-fluorobenzamido-N-ethylamino-maleimide ((18)F-FBEM)-labeled leukocytes was performed to assess metabolic activity and leukocyte recruitment, respectively. In bleomycin-treated mice, a higher metabolic activity was measured on (18)F-FDG PET/CT scans from day 7 to day 24 after instillation, with a peak of activity measured at day 14. Of note, lung mean standardized uptake values correlated with bleomycin doses, histologic score of fibrosis, lung hydroxyproline content, and weight loss. Moreover, during the inflammatory phase of the model (day 7), but not the fibrotic phase (day 23), bleomycin-treated mice presented with an enhanced leukocyte recruitment as assessed by (18)F-FBEM-labeled leukocyte PET/CT. Autoradiographic analysis of lung sections and CD45 immunostaining confirm the higher and early recruitment of leukocytes in bleomycin-treated mice, compared with control mice. (18)F-FDG- and (18)F-FBEM-labeled leukocyte PET/CT enable monitoring of metabolic activity and leukocyte recruitment in a mouse model of pulmonary fibrosis. Implications for preclinical evaluation of antifibrotic therapy are expected. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Comparison of Cytomegalovirus Loads in Plasma and Leukocytes of Patients with Cytomegalovirus Retinitis

PubMed Central

Jabs, Douglas A.; Forman, Michael; Enger, Cheryl; Jackson, J. Brooks

1999-01-01

Cytomegalovirus (CMV) DNA loads in paired leukocyte and plasma samples from 199 patient visits by 66 patients with CMV retinitis were determined. Leukocyte CMV load determinations had a greater range of values (mean, 24,587 copies/106 leukocytes; maximum, 539,000) than did plasma CMV load determinations (mean, 10,302 copies/ml; maximum, 386,000), and leukocyte viral loads were detectable in a greater proportion of patients at the time of diagnosis of CMV retinitis prior to initiation of anti-CMV therapy (82%) than were plasma viral loads (64%) (P = 0.0078). Agreement with CMV blood cultures was slightly better for plasma (κ = 0.68) than for leukocytes (κ = 0.53), due to a greater proportion of patients with detectable viral loads in leukocytes having negative blood cultures. PMID:10203500

Microglial Fc Receptors Mediate Physiological Changes Resulting From Antibody Cross-Linking of Myelin Oligodendrocyte Glycoprotein

PubMed Central

Marta, Cecilia B.; Bansal, Rashmi; Pfeiffer, Steven E.

2009-01-01

Antibodies to myelin oligodendrocyte glycoprotein (MOG) have been implicated in Multiple Sclerosis demyelination through activation of complement and/or macrophage-effector processes. We presented a novel mechanism, whereby MOG on oligodendrocytes, when cross-linked with anti-MOG and secondary antibody resulted in its repartitioning into lipid rafts, and changes in protein phosphorylation and morphology. Here, we show that similar events occur when anti-MOG is cross-linked with Fc receptors (FcRs) present on microglia but not with complement. These results indicate that FcRs are endogenous antigen/antibody cross-linkers in vitro, suggesting that FcRs could be physiologically relevant in vivo and possible targets for therapy in Multiple Sclerosis. PMID:18406472

Quantitative cumulative biodistribution of antibodies in mice: effect of modulating binding affinity to the neonatal Fc receptor.

PubMed

Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

2014-01-01

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.

Leukocyte Populations in Human Preterm and Term Breast Milk Identified by Multicolour Flow Cytometry

PubMed Central

Trend, Stephanie; de Jong, Emma; Lloyd, Megan L.; Kok, Chooi Heen; Richmond, Peter; Doherty, Dorota A.; Simmer, Karen; Kakulas, Foteini; Strunk, Tobias; Currie, Andrew

2015-01-01

Background Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood. Methods Sixty mothers of extremely preterm (<28 weeks gestational age), very preterm (28–31 wk), and moderately preterm (32–36 wk), as well as term (37–41 wk) infants were recruited. Colostrum (d2–5), transitional (d8–12) and mature milk (d26–30) samples were collected, cells isolated, and leukocyte subsets analysed using flow cytometry. Results The major CD45+ leukocyte populations circulating in blood were also detectable in breast milk but at different frequencies. Progression of lactation was associated with decreasing CD45+ leukocyte concentration, as well as increases in the relative frequencies of neutrophils and immature granulocytes, and decreases in the relative frequencies of eosinophils, myeloid and B cell precursors, and CD16- monocytes. No differences were observed between preterm and term breast milk in leukocyte concentration, though minor differences between preterm groups in some leukocyte frequencies were observed. Conclusions Flow cytometry is a useful tool to identify and quantify leukocyte subsets in breast milk. The stage of lactation is associated with major changes in milk leukocyte composition in this population. Fresh preterm breast milk is not deficient in leukocytes, but shorter gestation may be associated with minor differences in leukocyte subset frequencies in preterm compared to term breast milk. PMID:26288195

Fine-grained leukocyte classification with deep residual learning for microscopic images.

PubMed

Qin, Feiwei; Gao, Nannan; Peng, Yong; Wu, Zizhao; Shen, Shuying; Grudtsin, Artur

2018-08-01

Leukocyte classification and cytometry have wide applications in medical domain, previous researches usually exploit machine learning techniques to classify leukocytes automatically. However, constrained by the past development of machine learning techniques, for example, extracting distinctive features from raw microscopic images are difficult, the widely used SVM classifier only has relative few parameters to tune, these methods cannot efficiently handle fine-grained classification cases when the white blood cells have up to 40 categories. Based on deep learning theory, a systematic study is conducted on finer leukocyte classification in this paper. A deep residual neural network based leukocyte classifier is constructed at first, which can imitate the domain expert's cell recognition process, and extract salient features robustly and automatically. Then the deep neural network classifier's topology is adjusted according to the prior knowledge of white blood cell test. After that the microscopic image dataset with almost one hundred thousand labeled leukocytes belonging to 40 categories is built, and combined training strategies are adopted to make the designed classifier has good generalization ability. The proposed deep residual neural network based classifier was tested on microscopic image dataset with 40 leukocyte categories. It achieves top-1 accuracy of 77.80%, top-5 accuracy of 98.75% during the training procedure. The average accuracy on the test set is nearly 76.84%. This paper presents a fine-grained leukocyte classification method for microscopic images, based on deep residual learning theory and medical domain knowledge. Experimental results validate the feasibility and effectiveness of our approach. Extended experiments support that the fine-grained leukocyte classifier could be used in real medical applications, assist doctors in diagnosing diseases, reduce human power significantly. Copyright © 2018 Elsevier B.V. All rights reserved.

Endothelial Fcγ Receptor IIB Activation Blunts Insulin Delivery to Skeletal Muscle to Cause Insulin Resistance in Mice

PubMed Central

Tanigaki, Keiji; Chambliss, Ken L.; Yuhanna, Ivan S.; Sacharidou, Anastasia; Ahmed, Mohamed; Atochin, Dmitriy N.; Huang, Paul L.

2016-01-01

Modest elevations in C-reactive protein (CRP) are associated with type 2 diabetes. We previously revealed in mice that increased CRP causes insulin resistance and mice globally deficient in the CRP receptor Fcγ receptor IIB (FcγRIIB) were protected from the disorder. FcγRIIB is expressed in numerous cell types including endothelium and B lymphocytes. Here we investigated how endothelial FcγRIIB influences glucose homeostasis, using mice with elevated CRP expressing or lacking endothelial FcγRIIB. Whereas increased CRP caused insulin resistance in mice expressing endothelial FcγRIIB, mice deficient in the endothelial receptor were protected. The insulin resistance with endothelial FcγRIIB activation was due to impaired skeletal muscle glucose uptake caused by attenuated insulin delivery, and it was associated with blunted endothelial nitric oxide synthase (eNOS) activation in skeletal muscle. In culture, CRP suppressed endothelial cell insulin transcytosis via FcγRIIB activation and eNOS antagonism. Furthermore, in knock-in mice harboring constitutively active eNOS, elevated CRP did not invoke insulin resistance. Collectively these findings reveal that by inhibiting eNOS, endothelial FcγRIIB activation by CRP blunts insulin delivery to skeletal muscle to cause insulin resistance. Thus, a series of mechanisms in endothelium that impairs insulin movement has been identified that may contribute to type 2 diabetes pathogenesis. PMID:27207525

Structural basis for pH-insensitive inhibition of immunoglobulin G recycling by an anti-neonatal Fc receptor antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kenniston, Jon A.; Taylor, Brandy M.; Conley, Gregory P.

The neonatal Fc receptor FcRn plays a critical role in the trafficking of IgGs across tissue barriers and in retaining high circulating concentrations of both IgG and albumin. Although generally beneficial from an immunological perspective in maintaining IgG populations, FcRn can contribute to the pathogenesis of autoimmune disorders when an abnormal immune response targets normal biological components. We previously described a monoclonal antibody (DX-2507) that binds to FcRn with high affinity at both neutral and acidic pH, prevents the simultaneous binding of IgG, and reduces circulating IgG levels in preclinical animal models. Here, we report a 2.5 Å resolution X-raymore » crystal structure of an FcRn–DX-2507 Fab complex, revealing a nearly complete overlap of the IgG–Fc binding site in FcRn by complementarity-determining regions in DX-2507. This overlap explains how DX-2507 blocks IgG binding to FcRn and thereby shortens IgG half-life by preventing IgGs from recycling back into circulation. Moreover, the complex structure explains how the DX-2507 interaction is pH-insensitive unlike normal Fc interactions and how serum albumin levels are unaffected by DX-2507 binding. These structural studies could inform antibody-based therapeutic approaches for limiting the effects of IgG-mediated autoimmune disease.« less

Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis

PubMed Central

Savas, Jeffrey N.; De Wit, Joris; Comoletti, Davide; Zemla, Roland; Ghosh, Anirvan

2015-01-01

Ligand-receptor interactions represent essential biological triggers which regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol which couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared to previous approaches, our analysis increases sensitivity, shortens analysis duration, and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These “ecto-Fcs” are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion trap mass spectrometers. In four working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our “Ecto-Fc MS” approach outperforms antibody-based approaches and provides a reproducible and robust framework to identify extracellular ligand – receptor interactions. PMID:25101821

Regulation of Monoclonal Antibody Immunotherapy by FcγRIIB.

PubMed

Stopforth, Richard J; Cleary, Kirstie L S; Cragg, Mark S

2016-05-01

Monoclonal antibodies (mAb) are revolutionising the treatment of many different diseases. Given their differing mode of action compared to most conventional chemotherapeutics and small molecule inhibitors, they possess the potential to be independent of common modes of treatment resistance and can typically be combined readily with existing treatments without dose-limiting toxicity. However, treatments with mAb rarely result in cure and so a full understanding of how these reagents work and can be optimised is key for their subsequent improvement. Here we review how an understanding of the biology of the inhibitory Fc receptor, FcγRIIB (CD32B), is leading to the development of improved mAb treatments.

Polymeric capsule-cushioned leukocyte cell membrane vesicles as a biomimetic delivery platform

NASA Astrophysics Data System (ADS)

Gao, Changyong; Wu, Zhiguang; Lin, Zhihua; Lin, Xiankun; He, Qiang

2016-02-01

We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural cell membrane camouflaged polymeric multilayer capsules with the immunosuppressive and tumor-recognition functionalities of natural leukocytes provide a new biomimetic delivery platform for disease therapy.We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural

Crossing the Vascular Wall: Common and Unique Mechanisms Exploited by Different Leukocyte Subsets during Extravasation

PubMed Central

Schnoor, Michael; Alcaide, Pilar; Voisin, Mathieu-Benoit; van Buul, Jaap D.

2015-01-01

Leukocyte extravasation is one of the essential and first steps during the initiation of inflammation. Therefore, a better understanding of the key molecules that regulate this process may help to develop novel therapeutics for treatment of inflammation-based diseases such as atherosclerosis or rheumatoid arthritis. The endothelial adhesion molecules ICAM-1 and VCAM-1 are known as the central mediators of leukocyte adhesion to and transmigration across the endothelium. Engagement of these molecules by their leukocyte integrin receptors initiates the activation of several signaling pathways within both leukocytes and endothelium. Several of such events have been described to occur during transendothelial migration of all leukocyte subsets, whereas other mechanisms are known only for a single leukocyte subset. Here, we summarize current knowledge on regulatory mechanisms of leukocyte extravasation from a leukocyte and endothelial point of view, respectively. Specifically, we will focus on highlighting common and unique mechanisms that specific leukocyte subsets exploit to succeed in crossing endothelial monolayers. PMID:26568666

In vitro phagocytosis of several Candida berkhout species by murine leukocytes.

PubMed

Fontenla de Petrino, S E; Bibas Bonet de Jorrat, M E; Sirena, A

1985-03-01

In vitro phagocytosis of thirteen Candida berkhout species by rat leukocytes was studied to assess a possible correlation between pathogenicity and phagocytosis Yeast-leukocyte suspensions were mixed up for 3 h and phagocytic index, germ-tube formation and leukocyte candidacidal activity were evaluated. Highest values for phagocytosis were reached in all cases at the end of the first hour. Leukocyte candidacidal activity was absent. Only C. albicans produced germ-tubes. The various phagocytosis indices were determined depending on the Candida species assayed. Under these conditions, the more pathogenic species presented the lower indices of phagocytosis. It is determined that the in vitro phagocytic index may bear a close relationship with the pathogenicity of the Candida berkhout.

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

PubMed

Schneider, Sebastian; Zacharias, Martin

2012-05-01

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. Copyright © 2012 Elsevier Ltd. All rights reserved.

Peritoneal dialysis solution attenuates microvascular leukocyte adhesion induced by nitric oxide synthesis inhibition.

PubMed

White, R; Ram, S

1996-01-01

In the mesenteric microcirculation, inhibition of nitric oxide (NO) synthesis results in an inflammatory response through increased leukocyte adherence to the microvascular postcapillary venular endothelium. Recent studies have demonstrated that elevated concentrations of endogenous NO synthesis inhibitors are present in renal failure. How peritoneal dialysis solutions may affect leukocyte-endothelial interactions during inflammation induced by NO synthesis inhibition has been previously unknown. Using in vivo intravital microscopy of the rat mesenteric postcapillary venules, microvascular leukocyte adherence was quantitated during baseline conditions in which the mesentery was superfused with a buffer solution, followed by the superfusion of a NO synthesis inhibitor NG-nitro-L-ARGININE methyl ester (L-NAME) added to the buffer, followed by 4.25% Dianeal (4.25% D). When compared to baseline, L-NAME increased the mean number of adherent leukocytes by fivefold (2.2 +/- 0.9 vs 11.6 +/- 3.6 leukocytes/100 microns venule/10 min, p < 0.05), while 4.25% D quickly reversed the L-NAME-induced inflammatory response, returning the number of adherent leukocytes back to baseline values (11.6 +/- 3.6 vs 2.4 +/- 1.3 leukocytes/100 microns venule/ 10 min, p < 0.05). These results confirm that NO synthesis inhibition induces inflammation in mesenteric postcapillary venules. Superfusion of 4.25% D reverses leukocyte adhesion induced by NO synthesis inhibition. Thus, a standard peritoneal dialysis solution (4.25% D) reverses the leukocyte-adhesive effects of NO synthesis inhibition in the mesenteric microcirculation.

Identification and characterization of a Fc receptor activity on the Toxoplasma gondii tachyzoite.

PubMed

Vercammen, M; el Bouhdidi, A; Ben Messaoud, A; de Meuter, F; Bazin, H; Dubremetz, J F; Carlier, Y

1998-01-01

The Immunoglobulin (Ig) binding capacity of Toxoplasma gondii tachyzoites was investigated using fluorescence flow-cytometry analysis. Polyclonal mouse, human and rat immunoglobulins without specific anti-Toxoplasma activity bound to parasites in a concentration-dependent manner, saturating them at circulating serum concentrations. The immunoglobulin class and subclass specificity of binding was investigated using irrelevant monoclonal antibodies. IgM, IgA and IgG reacted with the parasite membrane. The attachment of mouse IgM to the parasite surface was hampered by mouse IgG1, IgG2a, IgG2b and IgG3. The binding of mouse IgG was proportionally reduced with increasing concentrations of mouse monoclonal IgM. The binding of murine immunoglobulin was diminished when in presence of human IgG. Purified Fc- but not Fab portions of immunoglobulins, fixed to parasites. Using labelled calibrated beads, the Ig binding capacity of parasites was estimated to be 6900 +/- 500 sites per tachyzoite. The Kd of the T. gondii Fc Receptor (FcR) activity was determined at 1.4 +/- 0.1 microM (mean +/- SEM). Such FcR activity was reduced by phospholipase C, trypsin and pronase treatment of the parasites. These data show a low affinity FcR activity on T. gondii tachyzoites which recognizes Ig of different species and isotypes and is likely supported by a glycosyl-phosphatidylinositol (GPI)-anchored surface protein of the parasite.

Person-specific theory of mind in medial pFC.

PubMed

Welborn, B Locke; Lieberman, Matthew D

2015-01-01

Although research on theory of mind has strongly implicated the dorsomedial pFC (incuding medial BA 8 and BA 9), the unique contributions of medial pFC (MPFC; corresponding to medial BA 10) to mentalizing remain uncertain. The extant literature has considered the possibility that these regions may be specialized for self-related cognition or for reasoning about close others, but evidence for both accounts has been inconclusive. We propose a novel theoretical framework: MPFC selectively implements "person-specific theories of mind" (ToMp) representing the unique, idiosyncratic traits or attributes of well-known individuals. To test this hypothesis, we used fMRI to assess MPFC responses in Democratic and Republican participants as they evaluated more or less subjectively well-known political figures. Consistent with the ToMp account, MPFC showed greater activity to subjectively well-known targets, irrespective of participants' reported feelings of closeness or similarity. MPFC also demonstrated greater activity on trials in which targets (whether politicians or oneself) were judged to be relatively idiosyncratic, making a generic theory of mind inapplicable. These results suggest that MPFC may supplement the generic theory of mind process, with which dorsomedial pFC has been associated, by contributing mentalizing capacities tuned to individuated representations of specific well-known others.

Tyrosine kinase Btk regulates E-selectin-mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) gamma2 and PI3Kgamma pathways.

PubMed

Mueller, Helena; Stadtmann, Anika; Van Aken, Hugo; Hirsch, Emilio; Wang, Demin; Ley, Klaus; Zarbock, Alexander

2010-04-15

Selectins mediate leukocyte rolling, trigger beta(2)-integrin activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to activation of an immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to integrin activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) gamma2- and phosphoinositide 3-kinase (PI3K) gamma-dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP(3)), and inducing E-selectin-mediated slow rolling. Inhibition of this signal-transduction pathway diminished Galpha(i)-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. Galpha(i)-independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk(-/-) and Plcg2(-/-) mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by E-selectin engagement.

Immune-Stimulatory Effects of a Bacteria-Based Probiotic on Peripheral Leukocyte Subpopulations and Cytokine mRNA Expression Levels in Scouring Holstein Calves

PubMed Central

QADIS, Abdul Qadir; GOYA, Satoru; YATSU, Minoru; KIMURA, Atsushi; ICHIJO, Toshihiro; SATO, Shigeru

2014-01-01

ABSTRACT Subpopulations of peripheral leukocytes and cytokine mRNA expression levels were evaluated in scouring and healthy Holstein calves (age 10 ± 5 days; n=42) treated with a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum. The calves were assigned to the scouring or healthy group and then subdivided into pathogen-positive treated (n=8), pathogen-positive control (n=8), pathogen-negative treated (n=6), pathogen-negative control (n=6), healthy treated (n=6) and healthy control (n=8) groups. A single dose of the probiotic (3.0 g/100 kg body weight) was given to each calf in the treatment groups for 5 days. Blood samples were collected on the first day of scour occurrence (day 0) and on day 7. In the scouring calves, smaller peripheral leukocyte subpopulations and cytokine mRNA expression levels were noted on day 0. The numbers of CD3+ T cells; CD4+, CD8+ and WC1+ γδ T cell subsets; and CD14+, CD21+ and CD282+ (TLR2) cells were significantly increased in the scouring and healthy treated calves on day 7. Furthermore, interleukin-6, tumor necrosis factor-alpha and interferon-gamma mRNA expression was elevated in the peripheral leukocytes of the scouring and healthy treated calves on day 7. The scouring calves given the probiotic recovered on day 7. A significantly smaller number of peripheral leukocytes and lower cytokine mRNA expression level might be induced by scouring in calves. Repeated probiotic administration might stimulate cellular immunity and encourage recovery from scouring in pre-weaning Holstein calves. PMID:24451928

Novel Confocal Microscopic and Flow Cytometric Based Assays to Visualize and Detect the (Beta)2-Adrenergic Receptor in Human Lymphocyte and Mononuclear Cell Populations

NASA Technical Reports Server (NTRS)

Salicru, A. N.; Crucian, B. E.; Nelman, M. A.; Sams, C. F.; Actor, J. K.; Marshall, G. D.

2006-01-01

The data show that immunophenotyping of leukocyte populations with (beta)2AR is possible with the commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the 2AR. The FC assay has applications for measuring alterations in total (beta)2AR in human leukocyte populations as changes in fluorescence. In addition, CM confirms that both surface and intracellular compartments stain positively for the (beta)2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.

Improved survival of newborns receiving leukocyte transfusions for sepsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cairo, M.S.; Rucker, R.; Bennetts, G.A.

To determine the role of polymorphonuclear (PMN) leukocyte transfusions in neonates with sepsis, 23 consecutive newborns were prospectively randomly selected during an 18-month period in a treatment plan to receive polymorphonuclear leukocyte transfusions with supportive care or supportive care alone. Thirteen neonates received transfusions every 12 hours for a total of five transfusions. Each transfusion consisting of 15 mL/kg of polymorphonuclear leukocytes was subjected to 1,500 rads of radiation. The polymorphonuclear leukocytes were obtained by continuous-flow centrifugation leukapheresis and contained 0.5 to 1.0 X 10(9) granulocytes per 15 mL with less than 10% lymphocytes. Positive findings on blood cultures weremore » obtained in 14/23 patients and seven were randomly selected for each treatment group. Absolute granulocyte counts were less than 1,500/microL in 13 patients but tibial bone marrow examinations revealed that the neutrophil supply pool was depleted in only three patients. The survival was significantly greater in the treatment group compared with the group that did not receive transfusions.« less

Occult purulent pericarditis detected by indium-111 leukocyte imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greenberg, M.L.; Niebulski, H.I.; Uretsky, B.F.

1984-05-01

Leukocyte imaging with indium-111 is a relatively new technique which, to this point in time, has been discussed almost exclusively in the radiologic literature. Although this procedure has been used mainly to detect intra-abdominal infection, the thorax is routinely imaged along with the abdomen, and therefore detection of cardiac disease may be feasible. This case report is of a young woman after liver transplantation who developed occult purulent pericarditis initially detected by a leukocyte scan with indium-111. This case demonstrates that striking pericardial uptake on a whole-body indium-111 leukocyte scan can occur with purulent pericarditis, and it reemphasizes how insidiouslymore » purulent pericarditis may present in an immunosuppressed patient.« less

[Construction and expression of a eukaryotic expression vector containing human CR2-Fc fusion protein].

PubMed

Li, Xinxin; Wu, Zhihao; Zhang, Chuanfu; Jia, Leili; Song, Hongbin; Xu, Yuanyong

2014-01-01

To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells. The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G418 selection. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting. Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass (Mr;) of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position. We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.

Effects of a Bacteria-Based Probiotic on Subpopulations of Peripheral Leukocytes and Their Cytokine mRNA Expression in Calves

PubMed Central

QADIS, Abdul Qadir; GOYA, Satoru; YATSU, Minoru; YOSHIDA, Yu-uki; ICHIJO, Toshihiro; SATO, Shigeru

2013-01-01

ABSTRACT Eight Holstein calves (10 ± 3 weeks) were used to examine the interaction between a bacteria-based probiotic agent (probiotic) and the function of peripheral blood mononuclear cells (PBMCs). The probiotic, consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum, was administered orally at 3.0 g/100 kg body weight to calves once daily for 5 consecutive days. Calves given the vehicle alone with no probiotic served as the control. In the treatment group, increases in numbers of CD282+ (TLR2) monocytes, CD3+ T cells and CD4+, CD8+ and WC1+ γδ T cell subsets were noted on day 7 post-placement compared to predose day and the control group. Expression of interleukin (IL)-6, interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) was elevated in peripheral leukocytes on days 7 and 14. These results suggest that peripheral blood leukocytes in healthy calves may be stimulated via the gastrointestinal microbiota, which was increased by the oral probiotic treatment, with overall stability of the rumen bacterial flora. The 5-day repeated administration of a bacteria-based probiotic may enhance cellular immune function in weaned calves. PMID:24131856

Effects of a bacteria-based probiotic on subpopulations of peripheral leukocytes and their cytokine mRNA expression in calves.

PubMed

Qadis, Abdul Qadir; Goya, Satoru; Yatsu, Minoru; Yoshida, Yu-Uki; Ichijo, Toshihiro; Sato, Shigeru

2014-03-01

Eight Holstein calves (10 ± 3 weeks) were used to examine the interaction between a bacteria-based probiotic agent (probiotic) and the function of peripheral blood mononuclear cells (PBMCs). The probiotic, consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum, was administered orally at 3.0 g/100 kg body weight to calves once daily for 5 consecutive days. Calves given the vehicle alone with no probiotic served as the control. In the treatment group, increases in numbers of CD282(+) (TLR2) monocytes, CD3(+) T cells and CD4(+), CD8(+) and WC1(+) γδ T cell subsets were noted on day 7 post-placement compared to predose day and the control group. Expression of interleukin (IL)-6, interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) was elevated in peripheral leukocytes on days 7 and 14. These results suggest that peripheral blood leukocytes in healthy calves may be stimulated via the gastrointestinal microbiota, which was increased by the oral probiotic treatment, with overall stability of the rumen bacterial flora. The 5-day repeated administration of a bacteria-based probiotic may enhance cellular immune function in weaned calves.

Infection in diabetic osteoarthropathy: use of indium-labeled leukocytes for diagnosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maurer, A.H.; Millmond, S.H.; Knight, L.C.

1986-10-01

Indium-111 labeled leukocyte imaging was compared with three-phase skeletal scintigraphy as a means of determining whether osteomyelitis was complicating diabetic osteoarthropathy. Three-phase scintigraphy demonstrated increased activity in both infected and noninfected osteopathic bone, with a sensitivity of 75% and a specificity of 56% for osteomyelitis. Leukocyte imaging had the same sensitivity but was most helpful for excluding infection (specificity, 89%) when three-phase imaging could not. Abnormal leukocyte localization was seen at the primary site of infection in all cases within 4 hours after injection. Disadvantages of leukocyte imaging included long preparation time, low count rates resulting in poor spatial resolution,more » and absence of bone landmarks, which made it difficult to differentiate soft tissue from bone infection.« less

Recombinant anthrax toxin receptor-Fc fusion proteins produced in plants protect rabbits against inhalational anthrax.

PubMed

Wycoff, Keith L; Belle, Archana; Deppe, Dorothée; Schaefer, Leah; Maclean, James M; Haase, Simone; Trilling, Anke K; Liu, Shihui; Leppla, Stephen H; Geren, Isin N; Pawlik, Jennifer; Peterson, Johnny W

2011-01-01

Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

PubMed

Jouvin, M H; Adamczewski, M; Numerof, R; Letourneur, O; Vallé, A; Kinet, J P

1994-02-25

Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

PubMed Central

Owen, Caroline A.

2008-01-01

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: 1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinase by cells; 2) the availability of surface binding sites for proteinases; and/or 3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: 1) concentrating the activity of proteinases to the immediate pericellular environment; 2) facilitating pro-enzyme activation; 3) increasing proteinase stability and retention in the extracellular space; 4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and 5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes. PMID:18329945

Hug tightly and say goodbye: role of endothelial ICAM-1 in leukocyte transmigration.

PubMed

Rahman, Arshad; Fazal, Fabeha

2009-04-01

Stable adhesion of leukocytes to endothelium is crucial for transendothelial migration (TEM) of leukocytes evoked during inflammatory responses, immune surveillance, and homing and mobilization of hematopoietic progenitor cells. The basis of stable adhesion involves expression of intercellular adhesion molecule-1 (ICAM-1), an inducible endothelial adhesive protein that serves as a counter-receptor for beta(2)-integrins on leukocytes. Interaction of ICAM-1 with beta(2)-integrins enables leukocytes to adhere firmly to the vascular endothelium and subsequently, to migrate across the endothelial barrier. The emerging paradigm is that ICAM-1, in addition to firmly capturing leukocytes, triggers intracellular signaling events that may contribute to active participation of the endothelium in facilitating the TEM of adherent leukocytes. The nature, duration, and intensity of ICAM-1-dependent signaling events may contribute to the determination of the route (paracellular vs. transcellular) of leukocyte passage; these aspects of ICAM-1 signaling may in turn be influenced by density and distribution of ICAM-1 on the endothelial cell surface, the source of endothelial cells it is present on, and the type of leukocytes with which it is engaged. This review summarizes our current understanding of the "ICAM-1 paradigm" of TEM with an emphasis on the signaling events mediating ICAM-1 expression and activated by ICAM-1 engagement in endothelial cells.

Loss of expression of the recycling receptor, FcRn, promotes tumor cell growth by increasing albumin consumption

PubMed Central

Khare, Priyanka; Schneider, Zita; Ober, Raimund J; Ward, Elizabeth Sally

2017-01-01

Tumor cells rely on high concentrations of amino acids to support their growth and proliferation. Although increased macropinocytic uptake and lysosomal degradation of the most abundant serum protein, albumin, in Ras-transformed cells can meet these demands, it is not understood how the majority of tumor cells that express wild type Ras achieve this. In the current study we reveal that the neonatal Fc receptor, FcRn, regulates tumor cell proliferation through the ability to recycle its ligand, albumin. By contrast with normal epithelial cells, we show that human FcRn is present at very low or undetectable levels in the majority of tumor cell lines analyzed. Remarkably, shRNA-mediated ablation of FcRn expression in an FcRn-positive tumor cell line results in a substantial growth increase of tumor xenografts, whereas enforced expression of this receptor by lentiviral transduction has the reverse effect. Moreover, intracellular albumin and glutamate levels are increased by the loss of FcRn-mediated recycling of albumin, combined with hypoalbuminemia in tumor-bearing mice. These studies identify a novel role for FcRn as a suppressor of tumor growth and have implications for the use of this receptor as a prognostic indicator and therapeutic target. PMID:27974681

Intranasal Introduction of Fc-Fused Interleukin-7 Provides Long-Lasting Prophylaxis against Lethal Influenza Virus Infection.

PubMed

Kang, Moon Cheol; Choi, Dong-Hoon; Choi, Young Woo; Park, Seong Jeong; Namkoong, Hong; Park, Ki Seok; Ahn, So-Shin; Surh, Charles D; Yoon, Sun-Woo; Kim, Doo-Jin; Choi, Jung-ah; Park, Yunji; Sung, Young Chul; Lee, Seung-Woo

2015-12-09

Influenza A virus (IAV) infection frequently causes hospitalization and mortality due to severe immunopathology. Annual vaccination and antiviral drugs are the current countermeasures against IAV infection, but they have a limited efficacy against new IAV variants. Here, we show that intranasal pretreatment with Fc-fused interleukin-7 (IL-7-mFc) protects mice from lethal IAV infections. The protective activity of IL-7-mFc relies on transcytosis via neonatal Fc receptor (FcRn) in the lung and lasts for several weeks. Introduction of IL-7-mFc alters pulmonary immune environments, leading to recruitment of T cells from circulation and their subsequent residency as tissue-resident memory-like T (TRM-like) cells. IL-7-mFc-primed pulmonary TRM-like cells contribute to protection upon IAV infection by dual modes. First, TRM-like cells, although not antigen specific but polyclonal, attenuate viral replication at the early phase of IAV infection. Second, TRM-like cells augment expansion of IAV-specific cytotoxic T lymphocytes (CTLs), in particular at the late phase of infection, which directly control viruses. Thus, accelerated viral clearance facilitated by pulmonary T cells, which are either antigen specific or not, alleviates immunopathology in the lung and mortality from IAV infection. Depleting a subset of pulmonary T cells indicates that both CD4 and CD8 T cells contribute to protection from IAV, although IL-7-primed CD4 T cells have a more prominent role. Collectively, we propose intranasal IL-7-mFc pretreatment as an effective means for generating protective immunity against IAV infections, which could be applied to a potential prophylaxis for influenza pandemics in the future. The major consequence of a highly pathogenic IAV infection is severe pulmonary inflammation, which can result in organ failure and death at worst. Although vaccines for seasonal IAVs are effective, frequent variation of surface viral proteins hampers development of protective immunity. In

A DFT-Elucidated Comparison of the Solution-Phase and SAM Electrochemical Properties of Short-Chain Mercaptoalkylferrocenes: Synthetic and Spectroscopic Aspects, and the Structure of Fc-CH2CH2-S-S-CH2CH2-Fc.

PubMed

Lewtak, Jan P; Landman, Marilé; Fernández, Israel; Swarts, Jannie C

2016-03-07

Facile synthetic procedures to synthesize a series of difficult-to-obtain mercaptoalkylferrocenes, namely, Fc(CH2)nSH, where n = 1 (1), 2 (2), 3 (3), or 4 (4) and Fc = Fe(η(5)-C5H5)(η(5)-C5H4), are reported. Dimerization of 1-4 to the corresponding disulfides 19-22 was observed in air. Dimer 20 (Z = 2) crystallized in the triclinic space group P1̅. Dimers 20-22 could be reduced back to the original Fc(CH2)nSH derivatives with LiAlH4 in refluxing tetrahydrofuran. Density functional theory (DFT) calculations showed that the highest occupied molecular orbital of 1-4 lies exclusively on the ferrocenyl group implying that the electrochemical oxidation observed at ca. -15 < Epa < 76 mV versus FcH/FcH(+) involves exclusively an Fe(II) to Fe(III) process. Further DFT calculations showed this one-electron oxidation is followed by proton loss on the thiol group to generate a radical, Fc(CH2)nS(•), with spin density mainly located on the sulfur. Rapid exothermic dimerization leads to the observed dimers, Fc(CH2)n-S-S-(CH 2)nFc. Reduction of the ferrocenium groups on the dimer occurs at potentials that still showed the ferrocenyl group ΔE = Epa,monomer - Epc,dimer ≤ 78 mV, indicating that the redox properties of the ferrocenyl group on the mercaptans are very similar to those of the dimer. (1)H NMR measurements showed that, like ferrocenyl oxidation, the resonance position of the sulfhydryl proton, SH, and others, are dependent on -(CH2)n- chain length. Self-assembled monolayers (SAMs) on gold were generated to investigate the electrochemical behavior of 1-4 in the absence of diffusion. Under these conditions, ΔE approached 0 mV for the longer chain derivatives at slow scan rates. The surface-bound ferrocenyl group of the metal-thioether, Fc(CH2)n -S-Au, is oxidized at approximately equal potentials as the equivalent CH2Cl2-dissolved ferrocenyl species 1-4. Surface coverage by the SAMs is dependent on alkyl chain length with the largest coverage obtained for 4, while

Role of Fc in Antibody-Mediated Protection from Ricin Toxin

PubMed Central

Pincus, Seth. H.; Das, Anushka; Song, Kejing; Maresh, Grace A.; Corti, Miriam; Berry, Jody

2014-01-01

We have studied the role of the antibody (Ab) Fc region in mediating protection from ricin toxicity. We compared the in vitro and in vivo effects of intact Ig and of Fab fragments derived from two different neutralizing Ab preparations, one monoclonal, the other polyclonal. Consistent results were obtained from each, showing little difference between Ig and Fab in terms of antigen binding and in vitro neutralization, but with relatively large differences in protection of animals. We also studied whether importing Ab into the cell by Fc receptors enhanced the intracellular neutralization of ricin toxin. We found that the imported Ab was found in the ER and Golgi, a compartment traversed by ricin, as it traffics through the cell, but intracellular Ab did not contribute to the neutralization of ricin. These results indicate that the Fc region of antibody is important for in vivo protection, although the mechanism of enhanced protection by intact Ig does not appear to operate at the single cell level. When using xenogeneic antibodies, the diminished immunogenicity of Fab/F(ab’)2 preparations should be balanced against possible loss of protective efficacy. PMID:24811206

Vitamin C Prevents Cigarette Smoke-Induced Leukocyte Aggregation and Adhesion to Endothelium in vivo

NASA Astrophysics Data System (ADS)

Lehr, Hans-Anton; Frei, Balz; Arfors, Karl-E.

1994-08-01

A common feature of cigarette-smoke (CS)-associated diseases such as atherosclerosis and pulmonary emphysema is the activation, aggregation, and adhesion of leukocytes to micro- and macrovascular endothelium. A previous study, using a skinfold chamber model for intravital fluorescence microscopy in awake hamsters, has shown that exposure of hamsters to the smoke generated by one research cigarette elicits the adhesion of fluorescently labeled leukocytes to the endothelium of arterioles and small venules. By the combined use of intravital microscopy and scanning electron microscopy, we now demonstrate in the same animal model that (i) CS-induced leukocyte adhesion is not confined to the microcirculation, but that leukocytes also adhere singly and in clusters to the aortic endothelium; (ii) CS induces the formation in the bloodstream of aggregates between leukocytes and platelets; and (iii) CS-induced leukocyte adhesion to micro- and macrovascular endothelium and leukocyte-platelet aggregate formation are almost entirely prevented by dietary or intravenous pretreatment with the water-soluble antioxidant vitamin C (venules, 21.4 ± 11.0 vs. 149.6 ± 38.7 leukocytes per mm^2, P < 0.01; arterioles, 8.5 ± 4.2 vs. 54.3 ± 21.6 leukocytes per mm^2, P < 0.01; aortas, 0.8 ± 0.4 vs. 12.4 ± 5.6 leukocytes per mm^2, P < 0.01; means ± SD of n = 7 animals, 15 min after CS exposure). No inhibitory effect was observed by pretreatment of the animals with the lipid-soluble antioxidants vitamin E or probucol. The protective effects of vitamin C on CS-induced leukocyte adhesion and aggregation were seen at vitamin C plasma levels (55.6 ± 22.2 μM, n = 7) that can easily be reached in humans by dietary means or supplementation, suggesting that vitamin C effectively contributes to protection from CS-associated cardiovascular and pulmonary diseases in humans.

Reward acts on the pFC to enhance distractor resistance of working memory representations.

PubMed

Fallon, Sean James; Cools, Roshan

2014-12-01

Working memory and reward processing are often thought to be separate, unrelated processes. However, most daily activities involve integrating these two types of information, and the two processes rarely, if ever, occur in isolation. Here, we show that working memory and reward interact in a task-dependent manner and that this task-dependent interaction involves modulation of the pFC by the ventral striatum. Specifically, BOLD signal during gains relative to losses in the ventral striatum and pFC was associated not only with enhanced distractor resistance but also with impairment in the ability to update working memory representations. Furthermore, the effect of reward on working memory was accompanied by differential coupling between the ventral striatum and ignore-related regions in the pFC. Together, these data demonstrate that reward-related signals modulate the balance between cognitive stability and cognitive flexibility by altering functional coupling between the ventral striatum and the pFC.

Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations.

PubMed

Martić, Sanela; Rains, Meghan K; Freeman, Daniel; Kraatz, Heinz-Bernhard

2011-08-17

The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.

Hydrodynamic delivery of plasmid DNA encoding human FcγR-Ig dimers blocks immune-complex mediated inflammation in mice.

PubMed

Shashidharamurthy, R; Machiah, D; Bozeman, E N; Srivatsan, S; Patel, J; Cho, A; Jacob, J; Selvaraj, P

2012-09-01

Therapeutic use and function of recombinant molecules can be studied by the expression of foreign genes in mice. In this study, we have expressed human Fcγ receptor-Ig fusion molecules (FcγR-Igs) in mice by administering FcγR-Ig plasmid DNAs hydrodynamically and compared their effectiveness with purified molecules in blocking immune-complex (IC)-mediated inflammation in mice. The concentration of hydrodynamically expressed FcγR-Igs (CD16A(F)-Ig, CD32A(R)-Ig and CD32A(H)-Ig) reached a maximum of 130 μg ml(-1) of blood within 24 h after plasmid DNA administration. The in vivo half-life of FcγR-Igs was found to be 9-16 days and western blot analysis showed that the FcγR-Igs were expressed as a homodimer. The hydrodynamically expressed FcγR-Igs blocked 50-80% of IC-mediated inflammation up to 3 days in a reverse passive Arthus reaction model. Comparative analysis with purified molecules showed that hydrodynamically expressed FcγR-Igs are more efficient than purified molecules in blocking IC-mediated inflammation and had a higher half-life. In summary, these results suggest that the administration of a plasmid vector with the FcγR-Ig gene can be used to study the consequences of blocking IC binding to FcγRs during the development of inflammatory diseases. This approach may have potential therapeutic value in treating IC-mediated inflammatory autoimmune diseases such as lupus, arthritis and autoimmune vasculitis.

Traffic of leukocytes in microfluidic channels with rectangular and rounded cross-sections.

PubMed

Yang, Xiaoxi; Forouzan, Omid; Burns, Jennie M; Shevkoplyas, Sergey S

2011-10-07

Traffic of leukocytes in microvascular networks (particularly through arteriolar bifurcations and venular convergences) affects the dynamics of capillary blood flow, initiation of leukocyte adhesion during inflammation, and localization and development of atherosclerotic plaques in vivo. Recently, a growing research effort has been focused on fabricating microvascular networks comprising artificial vessels with more realistic, rounded cross-sections. This paper investigated the impact of the cross-sectional geometry of microchannels on the traffic of leukocytes flowing with human whole blood through a non-symmetrical bifurcation that consisted of a 50 μm mother channel bifurcating into 30 μm and 50 μm daughter branches. Two versions of the same bifurcation comprising microchannels with rectangular and rounded cross-sections were fabricated using conventional multi-layer photolithography to produce rectangular microchannles that were then rounded in situ using a recently developed method of liquid PDMS/air bubble injection. For microchannels with rounded cross-sections, about two-thirds of marginated leukocytes traveling along a path in the top plane of the bifurcation entered the smallest 30 μm daughter branch. This distribution was reversed in microchannels with rectangular cross-sections--the majority of leukocytes traveling along a similar path continued to follow the 50 μm microchannels after the bifurcation. This dramatic difference in the distribution of leukocyte traffic among the branches of the bifurcation can be explained by preferential margination of leukocytes towards the corners of the 50 μm mother microchannels with rectangular cross-sections, and by the additional hindrance to leukocyte entry created by the sharp transition from the 50 μm mother microchannel to the 30 μm daughter branch at the intersection. The results of this study suggest that the trajectories of marginated leukocytes passing through non-symmetrical bifurcations are

Fc gamma receptor IIIb polymorphism and systemic lupus erythematosus: association with disease susceptibility and identification of a novel FCGR3B*01 variant.

PubMed

Santos, V C; Grecco, M; Pereira, K M C; Terzian, C C N; Andrade, L E C; Silva, N P

2016-10-01

The objective of this study was to evaluate the association between Fc gamma receptor IIIb polymorphism and susceptibility to systemic lupus erythematosus and clinical traits of the disease. Genomic DNA was obtained from 303 consecutive systemic lupus erythematosus patients and 300 healthy blood donors from the southeastern region of Brazil. The polymorphic region of the FCGR3B gene was sequenced and the alleles FCGR3B*01, FCGR3B*02 and FCGR3B*03 were analyzed. The FCGR3B*01 allele was more frequent in systemic lupus erythematosus patients (43.1%) while the FCGR3B*02 allele prevailed among controls (63.7%) (P = 0.001). The FCGR3B*03 allele was found equally in both groups. The FCGR3B*01/*01 (20.7%) and FCGR3B*01/*02 (41.1%) genotypes were more frequent among systemic lupus erythematosus patients (P = 0.028 and P = 0.012, respectively) while the FCGR3B*02/*02 genotype was more frequent in controls (45.5%) (P < 0.001). One variant of the FCGR3B*01 allele previously described in Germany was found in only one control. A new variant of the FCGR3B*01 allele with two substitutions (A227G/G277A) was found in one control. Three variants of the FCGR3B*02 allele previously described in African-Americans, Brazilians, Chinese and Japanese were found in ten 10 patients and two controls. In addition, several single nucleotide polymorphisms at non-polymorphic positions were identified in both patients and controls. Susceptibility to systemic lupus erythematosus was associated with the FCGR3B*01 allele, as well as with the FCGR3B*01/*01 and FCGR3B*01/*02 genotypes. No association was found between FCGR3B genotypes and clinical manifestations, disease severity or the presence of autoantibodies. © The Author(s) 2016.

Human Endogenous Retrovirus HERV-Fc1 Association with Multiple Sclerosis Susceptibility: A Meta-Analysis

PubMed Central

García-Montojo, Marta; Alcina, Antonio; Fedetz, María; Alloza, Iraide; Astobiza, Ianire; Leyva, Laura; Fernández, Oscar; Izquierdo, Guillermo; Antigüedad, Alfredo; Arroyo, Rafael; Álvarez-Lafuente, Roberto; Vandenbroeck, Koen; Matesanz, Fuencisla; Urcelay, Elena

2014-01-01

Background Human endogenous retroviruses (HERVs) are repetitive sequences derived from ancestral germ-line infections by exogenous retroviruses and different HERV families have been integrated in the genome. HERV-Fc1 in chromosome X has been previously associated with multiple sclerosis (MS) in Northern European populations. Additionally, HERV-Fc1 RNA levels of expression have been found increased in plasma of MS patients with active disease. Considering the North-South latitude gradient in MS prevalence, we aimed to evaluate the role of HERV-Fc1on MS risk in three independent Spanish cohorts. Methods A single nucleotide polymorphism near HERV-Fc1, rs391745, was genotyped by Taqman chemistry in a total of 2473 MS patients and 3031 ethnically matched controls, consecutively recruited from: Northern (569 patients and 980 controls), Central (883 patients and 692 controls) and Southern (1021 patients and 1359 controls) Spain. Our results were pooled in a meta-analysis with previously published data. Results Significant associations of the HERV-Fc1 polymorphism with MS were observed in two Spanish cohorts and the combined meta-analysis with previous data yielded a significant association [rs391745 C-allele carriers: pM-H = 0.0005; ORM-H (95% CI) = 1.27 (1.11–1.45)]. Concordantly to previous findings, when the analysis was restricted to relapsing remitting and secondary progressive MS samples, a slight enhancement in the strength of the association was observed [pM-H = 0.0003, ORM-H (95% CI) = 1.32 (1.14–1.53)]. Conclusion Association of the HERV-Fc1 polymorphism rs391745 with bout-onset MS susceptibility was confirmed in Southern European cohorts. PMID:24594754

Inhibition of glycosylation on a camelid antibody uniquely affects its FcγRI binding activity.

PubMed

Krahn, Natalie; Spearman, Maureen; Meier, Markus; Dorion-Thibaudeau, July; McDougall, Matthew; Patel, Trushar R; De Crescenzo, Gregory; Durocher, Yves; Stetefeld, Jörg; Butler, Michael

2017-01-01

Glycoengineering of mAbs has become common practice in attempts to generate the ideal mAb candidate for a wide range of therapeutic applications. The effects of these glycan modifications on the binding affinity of IgG mAbs for FcγRIIIa and their cytotoxicity are well known. However, little is understood about the effect that these modifications have on binding to the high affinity FcγRI receptor. This study analyzed the effect of variable N-glycosylation on a human-llama hybrid mAb (EG2-hFc, 80kDa) binding to FcγRI including a comparison to a full-sized IgG1 (DP-12, 150kDa). This was achieved by the addition of three glycosylation inhibitors (swainsonine, castanospermine, and kifunensine) independently to Chinese hamster ovary (CHO) cell cultures to generate hybrid and high mannose glycan structures. Biophysical analysis by circular dichroism, dynamic light scattering and analytical ultra-centrifugation confirmed that the solution-behaviour of the mAbs remained constant over multiple concentrations and glycan treatments. However, changes were observed when studying the interaction of FcγRI with variously glycosylated mAbs. Both mAbs were observed to have a decreased binding affinity upon treatment with swainsonine which produced hybrid glycans. Following de-glycosylation the binding affinity for EG2-hFc was only marginally reduced (6-fold) compared to a drastic (118-fold) decrease for DP-12. In summary, our data suggest that the relatively low molecular weight of chimeric EG2-hFc may contribute to its enhanced stability against glycan changes making it a highly suitable mAb candidate for therapeutic applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA.

PubMed

Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Kobayashi, Eiji; Fukuchi, Kaori; Tsukimoto, Mitsutoshi; Kojima, Shuji; Kohroki, Junya; Akimoto, Kazunori; Masuho, Yasuhiko

2013-07-05

The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA. Copyright © 2013 Elsevier Inc. All rights reserved.

Meisoindigo, but not its core chemical structure indirubin, inhibits zebrafish interstitial leukocyte chemotactic migration.

PubMed

Ye, Baixin; Xiong, Xiaoxing; Deng, Xu; Gu, Lijuan; Wang, Qiongyu; Zeng, Zhi; Gao, Xiang; Gao, Qingping; Wang, Yueying

2017-12-01

Inflammatory disease is a big threat to human health. Leukocyte chemotactic migration is required for efficient inflammatory response. Inhibition of leukocyte chemotactic migration to the inflammatory site has been shown to provide therapeutic targets for treating inflammatory diseases. Our study was designed to discover effective and safe compounds that can inhibit leukocyte chemotactic migration, thus providing possible novel therapeutic strategy for treating inflammatory diseases. In this study, we used transgenic zebrafish model (Tg:zlyz-EGFP line) to visualize the process of leukocyte chemotactic migration. Then, we used this model to screen the hit compound and evaluate its biological activity on leukocyte chemotactic migration. Furthermore, western blot analysis was performed to evaluate the effect of the hit compound on the AKT or ERK-mediated pathway, which plays an important role in leukocyte chemotactic migration. In this study, using zebrafish-based chemical screening, we identified that the hit compound meisoindigo (25 μM, 50 μM, 75 μM) can significantly inhibit zebrafish leukocyte chemotactic migration in a dose-dependent manner (p = 0.01, p = 0.0006, p < 0.0001). Also, we found that meisoindigo did not affect the process of leukocyte reverse migration (p = 0.43). Furthermore, our results unexpectedly showed that indirubin, the core structure of meisoindigo, had no significant effect on zebrafish leukocyte chemotactic migration (p = 0.6001). Additionally, our results revealed that meisoindigo exerts no effect on the Akt or Erk-mediated signalling pathway. Our results suggest that meisoindigo, but not indirubin, is effective for inhibiting leukocyte chemotactic migration, thus providing a potential therapeutic agent for treating inflammatory diseases.

A Two-pronged Binding Mechanism of IgG to the Neonatal Fc Receptor Controls Complex Stability and IgG Serum Half-life*

PubMed Central

Schoch, Angela; Larraillet, Vincent; Hilger, Maximiliane; Schlothauer, Tilman; Emrich, Thomas

2017-01-01

The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism of the neonatal Fc receptor (FcRn). For long serum half-life of therapeutic IgGs, the highly pH-dependent interaction with FcRn needs to be balanced to allow efficient FcRn binding and release at slightly acidic pH and physiological pH, respectively. Some IgGs, like the antibody briakinumab has an unusually short half-life of ∼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct FcRn interactions with both the Fc region and the Fab regions of briakinumab, and correlate the occurrence of excessive FcRn binding to an unusually strong Fab-FcRn interaction. PMID:28062799

Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

PubMed

Al-Alem, Linah; Puttabyatappa, Muraly; Rosewell, Kathy; Brännström, Mats; Akin, James; Boldt, Jeffrey; Muse, Ken; Curry, Thomas E

2015-09-01

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12-18 h) and late ovulatory (18-34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women.

Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

PubMed Central

Al-Alem, Linah; Puttabyatappa, Muraly; Rosewell, Kathy; Brännström, Mats; Akin, James; Boldt, Jeffrey; Muse, Ken

2015-01-01

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12–18 h) and late ovulatory (18–34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women. PMID:26125463

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

PubMed Central

Choe, Weonu; Durgannavar, Trishaladevi A.; Chung, Sang J.

2016-01-01

The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed. PMID:28774114

Biophysical stability of hyFc fusion protein with regards to buffers and various excipients.

PubMed

Lim, Jun Yeul; Kim, Nam Ah; Lim, Dae Gon; Eun, Chang-yong; Choi, Donghoon; Jeong, Seong Hoon

2016-05-01

A novel non-cytolytic hybrid Fc (hyFc) with an intact Ig structure without any mutation in the hyFc region, was developed to construct a long-acting agonistic protein. The stability of interleukin-7 (IL-7) fused with the hyFc (GXN-04) was evaluated to develop early formulations. Various biophysical methods were utilized and three different buffer systems with various pH ranges were investigated including histidine-acetate, sodium citrate, and tris buffers. Various excipients were incorporated into the systems to obtain optimum protein stability. Two evident thermal transitions were observed with the unfolding of IL-7 and hyFc. The Tm and ΔH increased with pH, suggesting increased conformational stability. Increased Z-average size with PDI and decreased zeta potential with pH increase, with the exception of tris buffer, showed aggregation issues. Moreover, tris buffer at higher pH showed aggregation peaks from DLS. Non-ionic surfactants were effective against agitation by outcompeting protein molecules for hydrophobic surfaces. Sucrose and sorbitol accelerated protein aggregation during agitation, but exhibited a protective effect against oxidation, with preferential exclusion favoring the compact states of GXN-04. The stability of GXN-04 was varied by basal buffers and excipients, hence the buffers and excipients need to be evaluated carefully to achieve the maximum stability of proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

House Dust Endotoxin and Peripheral Leukocyte Counts: Results from Two Large Epidemiologic Studies.

PubMed

Fessler, Michael B; Carnes, Megan U; Salo, Päivi M; Wilkerson, Jesse; Cohn, Richard D; King, Debra; Hoppin, Jane A; Sandler, Dale P; Travlos, Greg; London, Stephanie; Thorne, Peter; Zeldin, Darryl

2017-05-31

The peripheral leukocyte count is a biomarker of inflammation and is associated with human all-cause mortality. Although causes of acute leukocytosis are well-described, chronic environmental determinants of leukocyte number are less well understood. We investigated the relationship between house dust endotoxin concentration and peripheral leukocyte counts in human subjects. The endotoxin–leukocyte relationship was evaluated by linear regression in the National Health and Nutrition Examination Survey (NHANES) 2005–2006 (n=6,254) and the Agricultural Lung Health Study (ALHS; n=1,708). In the ALHS, we tested for a gene [Toll-like Receptor 4 ( TLR4 ), encoding the endotoxin receptor]-by-environment interaction in the endotoxin–leukocyte relationship using regression models with an interaction term. There is a statistically significant, positive association between endotoxin concentration and total leukocyte number [estimated change, 0.186×10 3 /μL (95% CI: 0.070, 0.301×10 3 /μL) per 10-fold change in endotoxin; p=0.004) in the NHANES. Similar positive associations were found for monocytes, lymphocytes, and neutrophils. Stratified analyses revealed possible effect modification by asthma and chronic obstructive pulmonary disease. We observed similar associations in the ALHS. For total leukocytes, there was suggestive evidence in the ALHS of a gene-by-environment interaction for minor allele carrier status at the TLR4 haplotype defined by rs4986790 and rs4986791 (interaction p=0.15). This is, to our knowledge, the first report of an association between house dust endotoxin and leukocyte count in a national survey. The finding was replicated in a farming population. Peripheral leukocyte count may be influenced by residential endotoxin exposure in diverse settings. https://doi.org/10.1289/EHP661.

House Dust Endotoxin and Peripheral Leukocyte Counts: Results from Two Large Epidemiologic Studies

PubMed Central

Carnes, Megan U.; Salo, Päivi M.; Wilkerson, Jesse; Cohn, Richard D.; King, Debra; Hoppin, Jane A.; Sandler, Dale P.; Travlos, Greg; London, Stephanie J.; Thorne, Peter S.; Zeldin, Darryl C.

2017-01-01

Background: The peripheral leukocyte count is a biomarker of inflammation and is associated with human all-cause mortality. Although causes of acute leukocytosis are well-described, chronic environmental determinants of leukocyte number are less well understood. Objectives: We investigated the relationship between house dust endotoxin concentration and peripheral leukocyte counts in human subjects. Methods: The endotoxin–leukocyte relationship was evaluated by linear regression in the National Health and Nutrition Examination Survey (NHANES) 2005–2006 (n=6,254) and the Agricultural Lung Health Study (ALHS; n=1,708). In the ALHS, we tested for a gene [Toll-like Receptor 4 (TLR4), encoding the endotoxin receptor]-by-environment interaction in the endotoxin–leukocyte relationship using regression models with an interaction term. Results: There is a statistically significant, positive association between endotoxin concentration and total leukocyte number [estimated change, 0.186×103/μL (95% CI: 0.070, 0.301×103/μL) per 10-fold change in endotoxin; p=0.004) in the NHANES. Similar positive associations were found for monocytes, lymphocytes, and neutrophils. Stratified analyses revealed possible effect modification by asthma and chronic obstructive pulmonary disease. We observed similar associations in the ALHS. For total leukocytes, there was suggestive evidence in the ALHS of a gene-by-environment interaction for minor allele carrier status at the TLR4 haplotype defined by rs4986790 and rs4986791 (interaction p=0.15). Conclusions: This is, to our knowledge, the first report of an association between house dust endotoxin and leukocyte count in a national survey. The finding was replicated in a farming population. Peripheral leukocyte count may be influenced by residential endotoxin exposure in diverse settings. https://doi.org/10.1289/EHP661 PMID:28599265

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding.

PubMed

Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia; Kuhlmann, Matthias; Cameron, Jason; Sørensen, Esben S; Wengel, Jesper; Howard, Kenneth A

2017-05-19

Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose hFcRn binding can be increased using a HB engineered albumin.

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding

NASA Astrophysics Data System (ADS)

Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia; Kuhlmann, Matthias; Cameron, Jason; Sørensen, Esben S.; Wengel, Jesper; Howard, Kenneth A.

2017-05-01

Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose hFcRn binding can be increased using a HB engineered albumin.

Study of terahertz-radiation-induced DNA damage in human blood leukocytes

NASA Astrophysics Data System (ADS)

Angeluts, A. A.; Gapeyev, A. B.; Esaulkov, M. N.; Kosareva, O. G.; Matyunin, S. N.; Nazarov, M. M.; Pashovkin, T. N.; Solyankin, P. M.; Cherkasova, O. P.; Shkurinov, A. P.

2014-03-01

We have carried out the studies aimed at assessing the effect of terahertz radiation on DNA molecules in human blood leukocytes. Genotoxic testing of terahertz radiation was performed in three different oscillation regimes, the blood leukocytes from healthy donors being irradiated for 20 minutes with the mean intensity of 8 - 200 μW cm-2 within the frequency range of 0.1 - 6.5 THz. Using the comet assay it is shown that in the selected regimes such radiation does not induce a direct DNA damage in viable human blood leukocytes.

The binding of human and guinea-pig IgG subclasses to homologous macrophage and monocyte Fc receptors.

PubMed Central

Alexander, M D; Andrews, J A; Leslie, R G; Wood, N J

1978-01-01

Guinea-pig IgG2 and IgT1 bind to contiguous Fc receptors on homologous peritoneal macrophages. Equilibrium association constants determined for the binding of human IgG subclasses to homologous peripheral blood monocytes show that the order of binding is IgG1 greater than IgG3 greater than IgG4 greater than IgG2. Direct binding and rosette assay techniques independently established that both guinea-pig IgG2 and human IgG bind to homologous macrophage-monocyte Fc receptors through a site present in whole Fc (CH2. CH3)2, but absent in pFc' subfragments (CH3)2. PMID:680795

Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia

PubMed Central

Li, June; van der Wal, Dianne E.; Zhu, Guangheng; Xu, Miao; Yougbare, Issaka; Ma, Li; Vadasz, Brian; Carrim, Naadiya; Grozovsky, Renata; Ruan, Min; Zhu, Lingyan; Zeng, Qingshu; Tao, Lili; Zhai, Zhi-min; Peng, Jun; Hou, Ming; Leytin, Valery; Freedman, John; Hoffmeister, Karin M.; Ni, Heyu

2015-01-01

Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa and/or the GPIb complex. Current theory suggests that antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc–FcγR interactions. However, we and others have demonstrated that anti-GPIbα (but not GPIIbIIIa)-mediated ITP is often refractory to therapies targeting FcγR pathways. Here, we generate mouse anti-mouse monoclonal antibodies (mAbs) that recognize GPIbα and GPIIbIIIa of different species. Utilizing these unique mAbs and human ITP plasma, we find that anti-GPIbα, but not anti-GPIIbIIIa antibodies, induces Fc-independent platelet activation, sialidase neuraminidase-1 translocation and desialylation. This leads to platelet clearance in the liver via hepatocyte Ashwell–Morell receptors, which is fundamentally different from the classical Fc–FcγR-dependent macrophage phagocytosis. Importantly, sialidase inhibitors ameliorate anti-GPIbα-mediated thrombocytopenia in mice. These findings shed light on Fc-independent cytopenias, designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of refractory ITP. PMID:26185093

Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.

PubMed

Kerppola, Tom K

2008-01-01

Protein interactions are a fundamental mechanism for the generation of biological regulatory specificity. The study of protein interactions in living cells is of particular significance because the interactions that occur in a particular cell depend on the full complement of proteins present in the cell and the external stimuli that influence the cell. Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragments. Numerous protein interactions have been visualized using the BiFC assay in many different cell types and organisms. The BiFC assay is technically straightforward and can be performed using standard molecular biology and cell culture reagents and a regular fluorescence microscope or flow cytometer.

Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

PubMed

Bhatti, L; Behle, K; Stevens, R H

1992-10-01

Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number

Antiviral Activity of Intranasally Applied Human Leukocyte Interferon

PubMed Central

Greenberg, Stephen B.; Harmon, Maurice W.; Johnson, Paul E.; Couch, Robert B.

1978-01-01

Previous studies in our laboratory have demonstrated that the development of antiviral activity of human leukocyte interferon (IF) in nasal epithelial cells is time and concentration dependent and that the loss of intranasally applied human leukocyte IF is rapid. The present studies compared the activity of IF applied intranasally either by nasal drops or by a saturated cotton pledget. Adult volunteers had IF applied to an area of nasal mucosa (2 by 2 cm2) either by repeated nose drops or by a saturated cotton pledget that was applied to the nasal mucosa and left in place for 1 h. Nasal epithelial cells scraped from the area of application, as well as the control, untreated side of the same volunteers, were challenged with vesicular stomatitis virus. No significant reduction in mean virus yield was found in volunteers who received 80,000 U by nose drops. Significant reduction (P < 0.025) in mean virus yield was found in cells obtained 4 h after 80,000, 50,000, or 20,000 U was applied by cotton pledget or in volunteers pretreated with oral antihistamines prior to receiving 80,000 U by nose drops. These experiments indicate that nasal epithelial cells can be made antiviral in vivo by application of human leukocyte IF. However, practical usefulness of human leukocyte IF for prophylaxis against respiratory viral infections may depend on the method of local application. PMID:214028

Enterococcus faecium FC-K Derived from Kimchi Is a Probiotic Strain That Shows Anti-Allergic Activity.

PubMed

Rho, Man-Kwang; Kim, Young-Eun; Rho, Hyun-In; Kim, Tae-Rahk; Kim, Yoon-Bum; Sung, Won-Kyung; Kim, Taw-Woo; Kim, Dae-Ok; Kang, Hee

2017-06-28

A rise in the occurrence of allergic diseases is attributed to the dysregulated balance of type 1/type 2 immunity, where type 2 T-helper (Th2) cells predominate over type 1 T-helper (Th1) cells, leading to an abnormally increased production of IgE in response to unharmful antigens. Kimchi, a traditional Korean fermented food, is a rich source of beneficial lactic acid bacteria. In this study, we investigated the ability of Enterococcus faecium FC-K derived from kimchi to induce type I immunity in the presence of Th2 polarizing conditions in vitro and in vivo. Stimulation of mouse peritoneal macrophages with E. faecium FC-K induced the production of tumor necrosis factor alpha, interleukin (IL)-6, and IL-12. Under the in vitro Th2 conditions in which splenic T cells were activated in the presence of IL-4, E. faecium FC-K enhanced the ability of T cells to produce interferon (IFN)-γ. Using the ovalbumin (OVA)-induced allergy model, male BALB/c mice receiving E. faecium FC-K reduced the serum level of total IgE, but not that of OVA-specific IgE. Furthermore, the population of activated splenic B cells during OVA immunization was decreased in E. faecium FC-K-treated mice, accounting for a reduction of total IgE in the serum. Restimulating splenocytes from OVA-immunized mice with OVA ex vivo resulted in an increased production of IFN-γ, with no effect on IL-4, in E. faecium FC-Ktreated mice. These observations provide the evidence that E. faecium FC-K can be a beneficial probiotic strain that can modulate the Th2-mediated pathologic response.

Role of Homologous Fc Fragment in the Potency and Efficacy of Anti-Botulinum Antibody Preparations.

PubMed

Torgeman, Amram; Ozeri, Eyal; Ben David, Alon; Diamant, Eran; Rosen, Osnat; Schwartz, Arieh; Barnea, Ada; Makovitzki, Arik; Mimran, Avishai; Zichel, Ran

2017-05-29

The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper-immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper-sensitivity reactions. New-generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs), which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti-botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400-mouse median lethal dose (MsLD50) of botulinum toxin, whereas the F(ab')2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper-immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti-botulinum toxin polyclonal horse F(ab')2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal-based to human-based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins.

Elevated leukocyte count is associated with periodontitis in Korean adults: the 2012-2014 KNHANES.

PubMed

Kwon, Y-J; Jeon, K-J; Chung, T-H; Lee, Y-J

2017-03-01

Both an elevated leukocyte count and periodontitis share well-recognized associations with cardiometabolic diseases. This cross-sectional study aimed to identify whether the leukocyte count is associated with periodontitis in a nationally representative Korean adult population. Data from 9391 participants (3659 males and 5732 females) enrolled in 2012-2014 Korean National Health and Nutrition Examination Survey were analyzed. Leukocyte quartiles were categorized as follows: 3000 ≤ Q1 ≤ 4870, 4880 ≤ Q2 ≤5790, 5800 ≤ Q3 ≤ 6840, and 6850 ≤ Q4 ≤ 10000 cells/μl. Periodontitis was defined as scoring greater than or equal to 'code 3' in at least one site according to the WHO's Community Periodontal Index. The odds ratios (ORs) and 95% confidence intervals (95% CIs) for periodontitis in each leukocyte count quartile were calculated using multiple logistic regression analyses. The prevalence of periodontitis was directly correlated with increasing leukocyte quartiles: 19%, 20.4%, 24.3%, and 30.3%. Compared with the lowest leukocyte quartile group, the OR (95% CI) for periodontitis of the highest leukocyte quartile was 1.558 (1.285-1.891) after controlling for confounding factors. An elevated leukocyte count was positively associated with the presence of periodontitis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

p,p'-DDE depresses the immune competence of chinook salmon (Oncorhynchus tshawytscha) leukocytes

USGS Publications Warehouse

Misumi, Ichiro; Vella, Anthony T.; Leong, Jo-Ann C.; Nakanishi, Teruyuki; Schreck, Carl B.

2005-01-01

p,p′-DDE, the main metabolite of DDT, is still detected in aquatic environments throughout the world. Here, the effects and mechanisms by which p,p′-DDE exposure might affect the immune system of chinook salmon (Oncorhynchus tshawytscha) was studied. Isolated salmon splenic and pronephric leukocytes were incubated with different concentrations of p,p′-DDE, and cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p′-DDE significantly reduced cell viability and proliferation and increased apoptosis. The effect of p,p′-DDE on pronephric leukocytes was more severe than on splenic leukocytes, likely because pronephric leukocytes had a higher proportion of granulocytes, cells that appear more sensitive to p,p′-DDE. The effect of p,p′-DDE on leukocytes appeared to vary between developmental stages or seasonal differences. The mitogenic response of leukocytes of chinook salmon exposed to p,p′-DDE in vivo exhibited a biphasic dose–response relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p′-DDE had a significantly lower percentage of Ig+ blasting cells than controls, although the response was biphasic. These results support the theory that exposure to chemical contaminants could lead to an increase in disease susceptibility and mortality of fish due to immune suppression.

A new gamma ray imaging diagnostic for runaway electron studies at DIII-D

NASA Astrophysics Data System (ADS)

Cooper, C. M.; Pace, D. C.; Eidietis, N. W.; Paz-Soldan, C.; Commaux, N.; Shiraki, D.; Hollmann, E. M.; Moyer, R. A.; Risov, V.

2015-11-01

A new Gamma Ray Imager (GRI) is developed to probe the electron distribution function with 2D spatial resolution during runaway electron (RE) experiments at DIII-D. The diagnostic is sensitive to 0.5 - 50 MeV gamma rays, allowing characterization of the RE distribution function evolution during RE dissipation from pellet injection. The GRI consists of a lead ``pinhole camera'' mounted on the midplane with 11x11 counter-current tangential chords 20 cm wide that span the vessel. Up to 30 bismuth germanate (BGO) scintillation detectors capture RE Bremsstrahlung radiation. Detectors operate in current saturation mode at 10 MHz, or the flux is attenuated for Pulse Height Analysis (PHA) capable of discriminating up to ~10k pulses per second. Digital signal processing routines combining shaping filters are performed during PHA to reject noise and record gamma ray energy. The GRI setup and PHA algorithms will be described and initial data from experiments will be presented. Work supported by the US DOE under DE-AC05-00OR22725, DE-FG02-07ER54917 & DE-FC02-04ER54698.

Targeting Mast Cells and Basophils with Anti-FcεRIα Fab-Conjugated Celastrol-Loaded Micelles Suppresses Allergic Inflammation.

PubMed

Peng, Xia; Wang, Juan; Li, Xianyang; Lin, Lihui; Xie, Guogang; Cui, Zelin; Li, Jia; Wang, Yuping; Li, Li

2015-12-01

Mast cells and basophils are effector cells in the pathophysiology of allergic diseases. Targeted elimination of these cells may be a promising strategy for the treatment of allergic disorders. Our present study aims at targeted delivery of anti-FcεRIα Fab-conjugated celastrol-loaded micelles toward FcεRIα receptors expressed on mast cells and basophils to have enhanced anti-allergic effect. To achieve this aim, we prepared celastrol-loaded (PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles using thin-film hydration method. The anti-FcεRIα Fab Fragment was then conjugated to carboxyl groups on drug-loaded micelles via EDC amidation reaction. The anti-FcεRIα Fab-conjugated celastrol-loaded micelles revealed uniform particle size (93.43 ± 12.93 nm) with high loading percentage (21.2 ± 1.5% w/w). The image of micelles showed oval and rod like. The anti-FcεRIα Fab-conjugated micelles demonstrated enhanced cellular uptake and cytotoxity toward target KU812 cells than non-conjugated micelles in vitro. Furthermore, diffusion of the drug into the cells allowed an efficient induction of cell apoptosis. In mouse model of allergic asthma, treatment with anti-FcεRIα Fab-conjugated micelles increased lung accumulation of micelles, and significantly reduced OVA-sIgE, histamine and Th2 cytokines (IL-4, IL-5, TNF-α) levels, eosinophils infiltration and mucus production. In addition, in mouse model of passive cutaneous anaphylaxis, anti-FcεRIα Fab-conjugated celastrol-loaded micelles treatment significantly decreased extravasated evan's in the ear. These results indicate that anti-FcεRIα Fab-conjugated celastrol-loaded micelles can target and selectively kill mast cells and basophils which express FcεRIα, and may be efficient reagents for the treatment of allergic disorders and mast cell related diseases.

Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line.

PubMed

Michelin, Severino; Gallegos, Cristina E; Dubner, Diana; Favier, Benoit; Carosella, Edgardo D

2009-12-01

Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of gamma-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of downregulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that gamma-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule.

Appearance of acute gouty arthritis on indium-111-labeled leukocyte scintigraphy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palestro, C.J.; Vega, A.; Kim, C.K.

1990-05-01

Indium-111-labeled leukocyte scintigraphy was performed on a 66-yr-old male with polyarticular acute gouty arthritis. Images revealed intense labeled leukocyte accumulation in a pattern indistinguishable from septic arthritis, in both knees and ankles, and the metatarsophalangeal joint of both great toes, all of which were involved in the acute gouty attack. Joint aspirate as well as blood cultures were reported as no growth; the patient was treated with intravenous colchicine and ACTH for 10 days with dramatic improvement noted. Labeled leukocyte imaging, repeated 12 days after the initial study, revealed near total resolution of joint abnormalities, concordant with the patient's clinicalmore » improvement. This case demonstrates that while acute gouty arthritis is a potential pitfall in labeled leukocyte imaging, in the presence of known gout, it may provide a simple, objective, noninvasive method of evaluating patient response to therapy.« less

Detection of murine cytomegalovirus DNA in circulating leukocytes harvested during acute infection of mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bale, J.F. Jr.; O'Neil, M.E.

1989-06-01

The authors used virus assay and in situ hybridization with a cloned fragment of the murine cytomegalovirus (MCMV) genome to study MCMV infection of circulating leukocytes harvested from 3-week-old BALB/c, C57BL/6, and C3H mice infected with MCMV intraperitoneally. Infectious virus or MCMV DNA was detected in leukocytes on days 1 through 21 of infection in BALB/c mice and on days 3 through 7 in C57BL/6 mice. On days 5 and 7, MCMV DNA or infectious virus was detected in the leukocytes of 17 (94%) of 18 BALB/c mice and 10 (59%) of 17 C57BL/6 mice. In both strains infection peakedmore » on days 5 and 7, when as many as 0.01 to 0.1% of the circulating leukocytes contained MCMV DNA. In C3H mice, however, infectious virus was rarely recovered from leukocyte fractions and MCMV DNA was detected in the circulating leukocytes of only one animal. Circulating leukocytes may have an important role in the dissemination of CMV infections in susceptible hosts.« less

A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma

NASA Astrophysics Data System (ADS)

Xia, Hui; Strachan, Briony C.; Gifford, Sean C.; Shevkoplyas, Sergey S.

2016-10-01

Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current ‘leukoreduction’ filters operate by trapping leukocytes within specialized filtration material, while allowing desired blood components to pass through. However, the continuous release of inflammatory cytokines from the retained leukocytes, as well as the potential for platelet activation and clogging, are significant drawbacks of conventional ‘dead end’ filtration. To address these limitations, here we demonstrate our newly-developed ‘controlled incremental filtration’ (CIF) approach to perform high-throughput microfluidic removal of leukocytes from platelet-rich plasma (PRP) in a continuous flow regime. Leukocytes are separated from platelets within the PRP by progressively syphoning clarified PRP away from the concentrated leukocyte flowstream. Filtrate PRP collected from an optimally-designed CIF device typically showed a ~1000-fold (i.e. 99.9%) reduction in leukocyte concentration, while recovering >80% of the original platelets, at volumetric throughputs of ~1 mL/min. These results suggest that the CIF approach will enable users in many fields to now apply the advantages of microfluidic devices to particle separation, even for applications requiring macroscale flowrates.

Recipient-Matching of Passenger Leukocytes Prolongs Survival of Donor Lung Allografts in Miniature Swine

PubMed Central

Madariaga, Maria Lucia L.; Michel, Sebastian G.; La Muraglia, Glenn M.; Sihag, Smita; Leonard, David A.; Farkash, Evan A.; Colvin, Robert B.; Cetrulo, Curtis L.; Huang, Christene A.; Sachs, David H.; Madsen, Joren C.; Allan, James S.

2014-01-01

Background Allograft rejection continues to be a vexing problem in clinical lung transplantation, and the role played by passenger leukocytes in the rejection or acceptance of an organ is unclear. Here we tested whether recipient-matching of donor graft passenger leukocytes would impact graft survival in a preclinical model of orthotopic left lung transplantation. Methods In the experimental group (Group 1), donor lungs were obtained from chimeric swine, in which the passenger leukocytes (but not the parenchyma) were MHC-matched to the recipients (n=3). In the control group (Group 2), both the donor parenchyma and the passenger leukocytes were MHC-mismatched to the recipients (n = 3). Results Lungs harvested from swine previously rendered chimeric by hematopoietic stem cell transplantation using recipient-type cells showed a high degree of passenger leukocyte chimerism by immunohistochemistry and flow cytometry. The chimeric lungs containing passenger leukocytes matched to the lung recipient (Group 1) survived on average 107 days (range 80–156). Control lung allografts (Group 2) survived on average 45 days (range 29–64; p<0.05). Conclusion Our data indicate that recipient-matching of passenger leukocytes significantly prolongs lung allograft survival. PMID:25757217

A multiscale SPH particle model of the near-wall dynamics of leukocytes in flow.

PubMed

Gholami, Babak; Comerford, Andrew; Ellero, Marco

2014-01-01

A novel multiscale Lagrangian particle solver based on SPH is developed with the intended application of leukocyte transport in large arteries. In such arteries, the transport of leukocytes and red blood cells can be divided into two distinct regions: the bulk flow and the near-wall region. In the bulk flow, the transport can be modeled on a continuum basis as the transport of passive scalar concentrations. Whereas in the near-wall region, specific particle tracking of the leukocytes is required and lubrication forces need to be separately taken into account. Because of large separation of spatio-temporal scales involved in the problem, simulations of red blood cells and leukocytes are handled separately. In order to take the exchange of leukocytes between the bulk fluid and the near-wall region into account, solutions are communicated through coupling of conserved quantities at the interface between these regions. Because the particle tracking is limited to those leukocytes lying in the near-wall region only, our approach brings considerable speedup to the simulation of leukocyte circulation in a test geometry of a backward-facing step, which encompasses many flow features observed in vivo. Copyright © 2013 John Wiley & Sons, Ltd.

Monitoring of benzene-induced hematotoxicity in mice by serial leukocyte counting using a microcavity array.

PubMed

Hosokawa, Masahito; Asami, Marie; Yoshino, Tomoko; Tsujimura, Noriyuki; Takahashi, Masayuki; Nakasono, Satoshi; Tanaka, Tsuyoshi; Matsunaga, Tadashi

2013-02-15

Monitoring of hematotoxicity, which requires serial blood collection, is difficult to carry out in small animals due to a lack of non-invasive, individual animal-appropriate techniques that enable enumeration of leukocyte subsets from limited amounts of whole blood. In this study, a microfluidic device equipped with a microcavity array that enables highly efficient separation of leukocytes from submicroliters of whole blood was applied for hematotoxicity monitoring in mice. The microcavity array can specifically separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Mouse leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered almost 100% of mouse leukocytes in 0.1 μL of whole blood without the effect of serial blood collection such as changes in body weight and total leukocyte count. We assessed benzene-associated hematotoxicity in mice using this system. Mice were administered with benzene once daily and the depression of leukocyte numbers induced in individual mice was successfully monitored from tail vein blood collected every other day for 2 weeks. Serial monitoring of the leukocyte number in individual mice will contribute to the understanding of hematotoxicity and reduction of the number of animal experiment trials. Copyright © 2012 Elsevier B.V. All rights reserved.

APC targeting enhances immunogenicity of a novel multistage Fc-fusion tuberculosis vaccine in mice.

PubMed

Soleimanpour, Saman; Farsiani, Hadi; Mosavat, Arman; Ghazvini, Kiarash; Eydgahi, Mohammad Reza Akbari; Sankian, Mojtaba; Sadeghian, Hamid; Meshkat, Zahra; Rezaee, Seyed Abdolrahim

2015-12-01

Numerous studies have demonstrated that targeting immunogens to FcγR on antigen-presenting cells (APCs) can selectively uptake and increase cellular immunity in vitro and in vivo. Therefore, the present study was conducted to evaluate immunogenicity of a novel multistage tuberculosis vaccine, a combination of an early and a dormant immunogenic protein, ESAT6 and HspX, fused to Fcγ2a fragment of mouse IgG2a to target all forms of tuberculosis. Codon-optimized genes consisting of ESAT6, a linker, and HspX fused either to mouse Fcγ2a (ESAT6:HspX:mFcγ2a) or 6× His-tag (ESAT6:HspX:His) were synthesized. The resulting proteins were then produced in Pichia pastoris. The fusion proteins were separately emulsified in dimethyldioctadecylammonium bromide(DDA)-trehalose-6,6-dibehenate(TDB) adjuvant, and their immunogenicity with and without bacille Calmette-Guérin (BCG) was assessed in C57BL/6 mice. Th1, Th2, Th17, and T-reg cytokine patterns were evaluated using the ELISA method. Both multistage vaccines induced very strong IL-12 and IFN-γ secretion from splenic cells; the Fc-tagged subunit vaccine induced a more effective Th1 immune response (IFN-γ, 910 pg/mL, and IL-12, 854 pg/mL) with a very low increase in IL-17 (∼0.1 pg/mL) and IL-4 (37 pg/mL) and a mild increase in TGF-β (543 pg/mL) compared to the BCG or ESAT6:HspX:His primed and boosted groups. The production of IFN-γ to ESAT6:HspX:Fcγ2a was very consistent and showed an increasing trend for IL-12 compared to the BCG or ESAT6:HspX:His primed and boosted groups. Fcγ2a used as a delivery vehicle supported the idea of selective uptake, inducing cross-presentation and forming a proper anti-tuberculosis response in context of Th1/Th2 and Th17/T-reg balances, which is important for protection and prevention of damage.

Uptake of apoptotic leukocytes by synovial lining macrophages inhibits immune complex-mediated arthritis.

PubMed

van Lent, P L; Licht, R; Dijkman, H; Holthuysen, A E; Berden, J H; van den Berg, W B

2001-11-01

Previously we have shown that synovial lining macrophages (SLMs) determine the onset of experimental immune complex-mediated arthritis (ICA). During joint inflammation, many leukocytes undergo apoptosis, and removal of leukocytes by SLMs may regulate resolution of inflammation. In this study we investigated binding and uptake of apoptotic leukocytes by SLMs and its impact on the onset of murine experimental arthritis. We used an in vitro model to evaluate phagocytosis of apoptotic cells on chemotaxis. Phagocytosis of apoptotic thymocytes resulted in a significant decrease (58%) of chemotactic activity for polymorphonuclear neutrophils (PMNs). If apoptotic cells were injected directly into a normal murine knee joint, SLMs resulted in a prominent uptake of cells. After ICA induction, electron micrographs showed that apoptotic leukocytes were evidently present in SLMs on days 1 and 2. Injection of apoptotic leukocytes into the knee joint 1 h before induction of ICA significantly inhibited PMN infiltration into the knee joint at 24 h (61% decrease). This study indicates that uptake of apoptotic leukocytes by SLM reduces chemotactic activity and inhibits the onset of experimental arthritis. These findings indicate an important mechanism in the resolution of joint inflammation.

Aliskiren Regulates Neonatal Fc Receptor and IgG Metabolism with Attenuation of Anti-GBM Glomerulonephritis in Mice.

PubMed

Kang, Ju Hyung; Baik, Haing Woon; Yoo, Seung-Min; Kim, Joo Heon; Cheong, Hae Il; Park, Chung-Gyu; Kang, Hee Gyung; Ha, Il-Soo

2016-01-01

Renin, in addition to its activation of the renin-angiotensin system, binds to the (pro)renin receptor (PRR) and triggers inflammatory and fibrogenic signaling in tissue. In addition, aliskiren, a direct renin inhibitor, has been shown to affect IgG metabolism by altering PRR and neonatal Fc receptors (FcRns). We investigated the effect of aliskiren on proteinuria, glomerular extracellular matrix, expressions of fibronectin, transforming growth factor β1 (TGF-β1), PRR, FcRn and renal metabolism of IgG in a mice model of anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). IgG deposition and expressions of FcRn and PRR were enhanced at glomeruli and urinary IgG levels increased in anti-GBM GN. Aliskiren attenuated anti-GBM GN with reduction of proteinuria and cortical expressions of fibronectin and TGF-β1. In addition, aliskiren suppressed the renal cortical expressions of FcRn and PRR. Aliskiren also reduced the glomerular IgG depositions and the urinary IgG levels albeit with increased circulating serum IgG levels. These results suggest that suppression of FcRn and PRR and regulation of IgG metabolism may be related to the attenuation of anti-GBM GN by aliskiren. © 2016 S. Karger AG, Basel.

Signal-activated phospholipase regulation of leukocyte chemotaxis.

PubMed

Cathcart, Martha K

2009-04-01

Signal-activated phospholipases are a recent focus of the rapidly growing field of lipid signaling. The extent of their impact on the pathways regulating diverse cell functions is beginning to be appreciated. A critical step in inflammation is the attraction of leukocytes to injured or diseased tissue. Chemotaxis of leukocytes, a requisite process for monocyte and neutrophil extravasation from the blood into tissues, is a critical step for initiating and maintaining inflammation in both acute and chronic settings. Recent studies have identified new important and required roles for two signal-activated phospholipases A2 (PLA2) in regulating chemotaxis. The two intracellular phospholipases, cPLA2alpha (Group IVA) and iPLA2beta (Group VIA), act in parallel to provide distinct lipid mediators at different intracellular sites that are both required for leukocytes to migrate toward the chemokine monocyte chemoattractant protein-1. This review will summarize the separate roles of these phospholipases as well as what is currently known about the influence of two other classes of intracellular signal-activated phospholipases, phospholipase C and phospholipase D, in regulating chemotaxis in eukaryotic cells, but particularly in human monocytes. The contributions of these phospholipases to chemotaxis both in vitro and in vivo will be highlighted.

Red pulp macrophages in the human spleen are a distinct cell population with a unique expression of Fc-γ receptors

PubMed Central

Bruggeman, Christine W.; den Haan, Joke M. M.; Mul, Erik P. J.; van den Berg, Timo K.; van Bruggen, Robin; Kuijpers, Taco W.

2018-01-01

Tissue-resident macrophages in the spleen play a major role in the clearance of immunoglobulin G (IgG)–opsonized blood cells, as occurs in immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA). Blood cells are phagocytosed via the Fc-γ receptors (FcγRs), but little is known about the FcγR expression on splenic red pulp macrophages in humans, with only a few previous studies that showed conflicting results. We developed a novel method to specifically isolate red pulp macrophages from 82 human spleens. Surface expression of various receptors and phagocytic capacity was analyzed by flow cytometry and immunofluorescence of tissue sections. Red pulp macrophages were distinct from splenic monocytes and blood monocyte–derived macrophages on various surface markers. Human red pulp macrophages predominantly expressed the low-affinity receptors FcγRIIa and FcγRIIIa. In contrast to blood monocyte–derived macrophages, red pulp macrophages did not express the inhibitory FcγRIIb. Red pulp macrophages expressed very low levels of the high-affinity receptor FcγRI. Messenger RNA transcript analysis confirmed this expression pattern. Unexpectedly and despite these differences in FcγR expression, phagocytosis of IgG-opsonized blood cells by red pulp macrophages was dependent on the same FcγRs as phagocytosis by blood monocyte–derived macrophages, especially in regarding the response to IV immunoglobulin. Concluding, we show the distinct nature of splenic red pulp macrophages in human subjects. Knowledge on the FcγR expression and usage of these cells is important for understanding and improving treatment strategies for autoimmune diseases such as ITP and AIHA. PMID:29692344

Fecal Leukocytes in Children Infected with Diarrheagenic Escherichia coli▿

PubMed Central

Mercado, Erik H.; Ochoa, Theresa J.; Ecker, Lucie; Cabello, Martin; Durand, David; Barletta, Francesca; Molina, Margarita; Gil, Ana I.; Huicho, Luis; Lanata, Claudio F.; Cleary, Thomas G.

2011-01-01

The purpose of this study was to determine the presence and quantity of fecal leukocytes in children infected with diarrheagenic Escherichia coli and to compare these levels between diarrhea and control cases. We analyzed 1,474 stool samples from 935 diarrhea episodes and 539 from healthy controls of a cohort study of children younger than 2 years of age in Lima, Peru. Stools were analyzed for common enteric pathogens, and diarrheagenic E. coli isolates were studied by a multiplex real-time PCR. Stool smears were stained with methylene blue and read by a blinded observer to determine the number of polymorphonuclear leukocytes per high-power field (L/hpf). Fecal leukocytes at >10 L/hpf were present in 11.8% (110/935) of all diarrheal episodes versus 1.1% (6/539) in controls (P < 0.001). Among stool samples with diarrheagenic E. coli as the only pathogen isolated (excluding coinfection), fecal leukocytes at >10 L/hpf were present in 8.5% (18/212) of diarrhea versus 1.3% (2/157) of control samples (P < 0.01). Ninety-five percent of 99 diarrheagenic E. coli diarrhea samples were positive for fecal lactoferrin. Adjusting for the presence of blood in stools, age, sex, undernutrition, and breastfeeding, enterotoxigenic E. coli (ETEC) isolation as a single pathogen, excluding coinfections, was highly associated with the presence of fecal leukocytes (>10 L/hpf) with an odds ratio (OR) of 4.1 (95% confidence interval [CI], 1.08 to 15.51; P < 0.05). Although diarrheagenic E. coli was isolated with similar frequencies in diarrhea and control samples, clearly it was associated with a more inflammatory response during symptomatic infection; however, in general, these pathogens elicited a mild inflammatory response. PMID:21325554

Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.

PubMed

Anumula, Kalyan Rao

2012-08-31

Typical clinical grade human IgG (intravenous immunoglobulin, IVIG), used for carbohydrate analysis, is derived from thousands of healthy donors. Quantitative high-resolution glycan profiles of IgG and its Fc-Fab fragments are presented here. Glycan profiles were established following digestions with Fc specific endoglycosidase S and generic PNGase F under denaturing and non-denaturing (native) conditions. The native PNGase F glycan profile of IgG was similar (but not identical) to that of Endo S. Endo S profiles did not contain the glycans with bisecting GlcNAc. PNGase F glycan profiles were the same for Fc fragments that were isolated from pepsin and Ide S protease digests. Both isolated Fab fragments and the previously deglycosylated IVIG (native conditions) yielded the same glycan profile. Glycan profiles were established using high resolution HPLC with 2-aminobenzoic acid (2AA) labeling. An accurate determination of sialylation levels can be made by this method. Carbohydrate content in Fc and Fab was determined using an internal standard and corrected for both protein and glycan recoveries. Fab portion contained about 14% of the total carbohydrate which translates to 2.3 sugar chains per mol in IVIG where 2 chains are located in the CH2 domain of the Fc. Fc glycans consisted of neutral (N) 84.5%; mono-sialylated (S1) 15% and di-sialylated (S2) 0.5%. In contrast, Fab contained N, 21%; S1, 43% and S2, 36%. The distribution of bisecting N-acetylglucosamine and fucose was found to be very different in various glycans (N, S1 and S2) found in Fab and Fc. Total IgG glycan profile (Fab plus Fc) contained N, 78.5%; S1, 17% and S2, 4.5%. Percent distribution of glycans G0, G1 and G2 (with 0, 1 and 2 two galactoses) was 26, 49 and 25 respectively within the 78% of the neutral glycans. Glycan profiles were nearly the same for various clinical grade IVIG preparations from various manufacturers. A fast HPLC profiling method was developed for the separation and quantitation

Paradoxical attenuation of leukocyte rolling in response to ischemia- reperfusion and extracorporeal blood circulation in inflamed tissue.

PubMed

Schäfer, Stephan C; Sehrt, Desiree N; Kamler, Markus; Jakob, Heinz; Lehr, Hans-Anton

2005-07-01

In contrast to acute preparations such as the exteriorized mesentery or the cremaster muscle, chronically instrumented chamber models allow one to study the microcirculation under "physiological" conditions, i.e., in the absence of trauma-induced leukocyte rolling along the venular endothelium. To underscore the importance of studying the naive microcirculation, we implanted titanium dorsal skinfold chambers in hamsters and used intravital fluorescence microscopy to study venular leukocyte rolling in response to ischemia-reperfusion injury or extracorporeal blood circulation. The experiments were performed in chambers that fulfilled all well-established criteria for a physiological microcirculation as well as in chambers that showed various extents of leukocyte rolling due to trauma, hemorrhage, or inflammation. In ideal chambers with a physiological microcirculation (<30 rolling leukocytes/mm vessel circumference in 30 s), ischemia-reperfusion injury and extracorporeal blood circulation significantly stimulated leukocyte rolling along the venular endothelium and, subsequently, firm leukocyte adhesion. In contrast, both stimuli failed to elicit leukocyte rolling in borderline chambers (30-100 leukocytes/mm), and in blatantly inflamed chambers with yet higher numbers of rolling leukocytes at baseline (>100 leukocytes/mm), we observed a paradoxical reduction of leukocyte rolling after ischemia-reperfusion injury or extracorporeal blood circulation. A similar effect was observed when we superfused leukotriene B4 (LTB4) onto the chamber tissue. The initial increase in leukocyte rolling in response to an LTB4 challenge was reversed by a second superfusion 90 min later. These observations underscore 1) the benefit of studying leukocyte-endothelial cell interaction in chronically instrumented chamber models and 2) the necessity to strictly adhere to well-established criteria of a physiological microcirculation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Szymanska, Ewelina; Korzeniowski, Marek; Raynal, Patrick

Receptor Fc{gamma}IIA (Fc{gamma}RIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P{sub 2} and PI(4,5)P{sub 2}-synthesizing PIP5-kinase I{alpha} to rafts contributes to Fc{gamma}RIIA signaling. A fraction of PIP5-kinase I{alpha} was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P{sub 2}. PIP5-kinase I{alpha} bound PI(4,5)P{sub 2}, and depletion of the lipid displaced PIP5-kinase I{alpha} from the DRM. Activation of Fc{gamma}RIIA in BHK transfectants led to recruitment of the kinase to the plasma membranemore » and enrichment of DRM in PI(4,5)P{sub 2}. Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After Fc{gamma}RIIA activation, PIP5-kinase I{alpha} and PI(4,5)P{sub 2} co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase I{alpha} and PI(4,5)P{sub 2} were present at the edges of electron-dense assemblies containing activated Fc{gamma}RIIA in their core. The data suggest that activation of Fc{gamma}RIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase I{alpha} and PI(4,5)P{sub 2}.« less

Chimeric antigen receptor T cells targeting Fc μ receptor selectively eliminate CLL cells while sparing healthy B cells.

PubMed

Faitschuk, Elena; Hombach, Andreas A; Frenzel, Lukas P; Wendtner, Clemens-Martin; Abken, Hinrich

2016-09-29

Adoptive cell therapy of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR)-modified T cells targeting CD19 induced lasting remission of this refractory disease in a number of patients. However, the treatment is associated with prolonged "on-target off-tumor" toxicities due to the targeted elimination of healthy B cells demanding more selectivity in targeting CLL cells. We identified the immunoglobulin M Fc receptor (FcμR), also known as the Fas apoptotic inhibitory molecule-3 or TOSO, as a target for a more selective treatment of CLL by CAR T cells. FcμR is highly and consistently expressed by CLL cells; only minor levels are detected on healthy B cells or other hematopoietic cells. T cells with a CAR specific for FcμR efficiently responded toward CLL cells, released a panel of proinflammatory cytokines and lytic factors, like soluble FasL and granzyme B, and eliminated the leukemic cells. In contrast to CD19 CAR T cells, anti-FcμR CAR T cells did not attack healthy B cells. T cells with anti-FcμR CAR delayed outgrowth of Mec-1-induced leukemia in a xenograft mouse model. T cells from CLL patients in various stages of the disease, modified by the anti-FcμR CAR, purged their autologous CLL cells in vitro without reducing the number of healthy B cells, which is the case with anti-CD19 CAR T cells. Compared with the currently used therapies, the data strongly imply a superior therapeutic index of anti-FcμR CAR T cells for the treatment of CLL. © 2016 by The American Society of Hematology.

Cutting Edge: The murine high-affinity IgG receptor FcγRIV is sufficient for autoantibody-induced arthritis.

PubMed

Mancardi, David A; Jönsson, Friederike; Iannascoli, Bruno; Khun, Huot; Van Rooijen, Nico; Huerre, Michel; Daëron, Marc; Bruhns, Pierre

2011-02-15

K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient W(sh) mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.

OPG-Fc but Not Zoledronic Acid Discontinuation Reverses Osteonecrosis of the Jaws (ONJ) in Mice

PubMed Central

de Molon, Rafael Scaf; Shimamoto, Hiroaki; Bezouglaia, Olga; Pirih, Flavia Q; Dry, Sarah M; Kostenuik, Paul; Boyce, Rogely W; Dwyer, Denise; Aghaloo, Tara L; Tetradis, Sotirios

2016-01-01

Osteonecrosis of the jaws (ONJ) is a significant complication of antiresorptive medications, such as bisphosphonates and denosumab. Antiresorptive discontinuation to promote healing of ONJ lesions remains highly controversial and understudied. Here, we investigated whether antiresorptive discontinuation alters ONJ features in mice, employing the potent bisphosphonate zoledronic acid (ZA) or the receptor activator of NF-κB ligand (RANKL) inhibitor OPG-Fc, utilizing previously published ONJ animal models. Mice were treated with vehicle (veh), ZA, or OPG-Fc for 11 weeks to induce ONJ, and antiresorptives were discontinued for 6 or 10 weeks. Maxillae and mandibles were examined by µCT imaging and histologically. ONJ features in ZA and OPG-Fc groups included periosteal bone deposition, empty osteocyte lacunae, osteonecrotic areas, and bone exposure, each of which substantially resolved 10 weeks after discontinuing OPG-Fc but not ZA. Full recovery of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclast numbers occurred after discontinuing OPG-Fc but not ZA. Our data provide the first experimental evidence demonstrating that discontinuation of a RANKL inhibitor, but not a bisphosphonate, reverses features of osteonecrosis in mice. It remains unclear whether antiresorptive discontinuation increases the risk of skeletal-related events in patients with bone metastases or fracture risk in osteoporosis patients, but these preclinical data may nonetheless help to inform discussions on the rationale for a “drug holiday” in managing the ONJ patient. PMID:25727550

Identification and quantification of human microcirculatory leukocytes using handheld video microscopes at the bedside.

PubMed

Uz, Zühre; van Gulik, Thomas M; Aydemirli, Mehtap Derya; Guerci, Philippe; Ince, Yasin; Cuppen, Diede V; Ergin, Bulent; Aksu, Ugur; de Mol, Bas A; Ince, Can

2018-03-08

Leukocyte recruitment and adhesion to the endothelium are hallmarks of systemic inflammation that manifest in a wide range of diseases. At present, no method is available to directly measure leukocyte kinetics at the bedside. In this study, we validate a new method to identify and quantify microcirculatory leukocytes observed by handheld vital microscopy (HVM) using space-time diagram (STD) analysis. Video clips (N=59) containing one capillary-post capillary venule (C-PCV) unit where leukocytes could be observed emanating from a capillary into a venule in cardiac surgery patients (N=20) were included. STD analysis and manual counting were used to quantify the number of leukocytes (total, rolling and non-rolling). Pearson's correlation and Bland-Altman analysis were used to determine agreement between the STDs and manual counting. For reproducibility, intra- and inter-observer coefficients of variation (CVs) were assessed. Leukocyte (rolling and non-rolling) and red blood cell velocities were assessed. The STDs and manual counting procedures for the quantification of rolling leukocytes showed good agreement (r=0.8197, P<0.0001), with a Bland-Altman analysis mean difference of -0.0 (-6.56; 6.56). The overall intra-observer CV for the STD method was 1.5%. The overall inter-observer CVs for the STD and the manual method were 5.6% and 9.4%, respectively. The non-rolling velocity was significantly higher than the rolling velocity (812{plus minus}519 µm/s vs 201{plus minus}149 µm/s, P=0.001). The STD results agreed with the manual counting procedure results, had a better reproducibility and could assess the leukocyte velocity. STD analysis using bedside HVM imaging presented a new methodology for quantifying leukocyte kinetics and functions in the microcirculation.

FC-99 ameliorates sepsis-induced liver dysfunction by modulating monocyte/macrophage differentiation via Let-7a related monocytes apoptosis.

PubMed

Zhao, Yarong; Zhu, Haiyan; Wang, Haining; Ding, Liang; Xu, Lizhi; Chen, Dai; Shen, Sunan; Hou, Yayi; Dou, Huan

2018-03-13

The liver is a vital target for sepsis-related injury, leading to inflammatory pathogenesis, multiple organ dysfunction and high mortality rates. Monocyte-derived macrophage transformations are key events in hepatic inflammation. N 1 -[(4-methoxy)methyl]-4-methyl-1,2-benzenediamine (FC-99) previously displayed therapeutic potential on experimental sepsis. However, the underlying mechanism of this protective effect is still not clear. FC-99 treatment attenuated the liver dysfunction in septic mice that was accompanied with reduced numbers of pro-inflammatory Ly6C hi monocytes in the peripheral blood and CD11b + F4/80 lo monocyte-derived macrophages in the liver. These effects were attributed to the FC-99-induced apoptosis of CD11b + cells. In PMA-differentiated THP-1 cells, FC-99 repressed the expression of CD11b, CD14 and caspase3 and resulted in a high proportion of Annexin V + cells. Moreover, let-7a-5p expression was abrogated upon CLP stimulation in vivo , whereas it was restored by FC-99 treatment. TargetScan analysis and luciferase assays indicated that the anti-apoptotic protein BCL-XL was targeted by let-7a-5p. BCL-XL was inhibited by FC-99 in order to induce monocyte apoptosis, leading to the impaired monocyte-to-macrophage differentiation. Murine acute liver failure was generated by caecal ligation puncture surgery after FC-99 administration; Blood samples and liver tissues were collected to determine the monocyte/macrophage subsets and the induction of apoptosis. Human acute monocytic leukemia cell line (THP-1) cells were pretreated with FC-99 followed by phorbol-12-myristate-13-acetate (PMA) stimulation, in order to induce monocyte-to-macrophage differentiation. The target of FC-99 and the mechanistic analyses were conducted by microarrays, qRT-PCR validation, TargetScan algorithms and a luciferase report assay. FC-99 exhibits potential therapeutic effects on CLP-induced liver dysfunction by restoring let-7a-5p levels.

FC-99 ameliorates sepsis-induced liver dysfunction by modulating monocyte/macrophage differentiation via Let-7a related monocytes apoptosis

PubMed Central

Zhao, Yarong; Zhu, Haiyan; Wang, Haining; Ding, Liang; Xu, Lizhi; Chen, Dai; Shen, Sunan; Hou, Yayi; Dou, Huan

2018-01-01

Background The liver is a vital target for sepsis-related injury, leading to inflammatory pathogenesis, multiple organ dysfunction and high mortality rates. Monocyte-derived macrophage transformations are key events in hepatic inflammation. N1-[(4-methoxy)methyl]-4-methyl-1,2-benzenediamine (FC-99) previously displayed therapeutic potential on experimental sepsis. However, the underlying mechanism of this protective effect is still not clear. Results FC-99 treatment attenuated the liver dysfunction in septic mice that was accompanied with reduced numbers of pro-inflammatory Ly6Chi monocytes in the peripheral blood and CD11b+F4/80lo monocyte-derived macrophages in the liver. These effects were attributed to the FC-99-induced apoptosis of CD11b+ cells. In PMA-differentiated THP-1 cells, FC-99 repressed the expression of CD11b, CD14 and caspase3 and resulted in a high proportion of Annexin V+ cells. Moreover, let-7a-5p expression was abrogated upon CLP stimulation in vivo, whereas it was restored by FC-99 treatment. TargetScan analysis and luciferase assays indicated that the anti-apoptotic protein BCL-XL was targeted by let-7a-5p. BCL-XL was inhibited by FC-99 in order to induce monocyte apoptosis, leading to the impaired monocyte-to-macrophage differentiation. Materials and Methods Murine acute liver failure was generated by caecal ligation puncture surgery after FC-99 administration; Blood samples and liver tissues were collected to determine the monocyte/macrophage subsets and the induction of apoptosis. Human acute monocytic leukemia cell line (THP-1) cells were pretreated with FC-99 followed by phorbol-12-myristate-13-acetate (PMA) stimulation, in order to induce monocyte-to-macrophage differentiation. The target of FC-99 and the mechanistic analyses were conducted by microarrays, qRT-PCR validation, TargetScan algorithms and a luciferase report assay. Conclusions FC-99 exhibits potential therapeutic effects on CLP-induced liver dysfunction by restoring let-7a-5

Cognitive-behavioral stress management reverses anxiety-related leukocyte transcriptional dynamics

PubMed Central

Antoni, Michael H.; Lutgendorf, Susan K.; Blomberg, Bonnie; Carver, Charles S.; Lechner, Suzanne; Diaz, Alain; Stagl, Jamie; Arevalo, Jesusa M.G.; Cole, Steven W.

2011-01-01

Background Chronic threat and anxiety are associated with pro-inflammatory transcriptional profiles in circulating leukocytes, but the causal direction of that relationship has not been established. This study tested whether a Cognitive-Behavioral Stress Management (CBSM) intervention targeting negative affect and cognition might counteract anxiety-related transcriptional alterations in people confronting a major medical threat. Methods 199 women undergoing primary treatment of Stage 0–III breast cancer were randomized to a 10-week CBSM protocol or an active control condition. 79 provided peripheral blood leukocyte samples for genome-wide transcriptional profiling and bioinformatic analyses at baseline, 6-, and 12-month follow-ups. Results Baseline negative affect was associated with > 50% differential expression of 201 leukocyte transcripts, including up-regulated expression of pro-inflammatory and metastasis-related genes. CBSM altered leukocyte expression of 91 genes by > 50% at follow-up (Group × Time interaction), including down-regulation of pro-inflammatory and metastasis-related genes and up-regulation of Type I interferon response genes. Promoter-based bioinformatic analyses implicated decreased activity of NF-κB/Rel and GATA family transcription factors and increased activity of Interferon Response Factors and the Glucocorticoid Receptor (GR) as potential mediators of CBSM-induced transcriptional alterations. Conclusions In early stage breast cancer patients, a 10-week CBSM intervention can reverse anxiety-related up-regulation of pro-inflammatory gene expression in circulating leukocytes. These findings clarify the molecular signaling pathways by which behavioral interventions can influence physical health and alter peripheral inflammatory processes that may reciprocally affect brain affective and cognitive processes. PMID:22088795

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA.

PubMed

Kobayashi, Eiji; Motoi, Sotaro; Sugiura, Masahito; Kajikawa, Masunori; Kojima, Shuji; Kohroki, Junya; Masuho, Yasuhiko

2014-09-01

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC. Copyright © 2014 Elsevier B.V. All rights reserved.

Comprehensive evaluation of leukocyte lineage derived from human hematopoietic cells in humanized mice.

PubMed

Takahashi, Masayuki; Tsujimura, Noriyuki; Otsuka, Kensuke; Yoshino, Tomoko; Mori, Tetsushi; Matsunaga, Tadashi; Nakasono, Satoshi

2012-04-01

Recently, humanized animals whereby a part of the animal is biologically engineered using human genes or cells have been utilized to overcome interspecific differences. Herein, we analyzed the detail of the differentiation states of various human leukocyte subpopulations in humanized mouse and evaluated comprehensively the similarity of the leukocyte lineage between humanized mice and humans. Humanized mice were established by transplanting human CD34(+) cord blood cells into irradiated severely immunodeficient NOD/Shi-scid/IL2Rγ(null) (NOG) mice, and the phenotypes of human cells contained in bone marrow, thymus, spleen and peripheral blood from the mice were analyzed at monthly intervals until 4 months after cell transplantation. The analysis revealed that transplanted human hematopoietic stem cells via the caudal vein homed and engrafted themselves successfully at the mouse bone marrow. Subsequently, the differentiated leukocytes migrated to the various tissues. Almost all of the leukocytes within the thymus were human cells. Furthermore, analysis of the differentiation states of human leukocytes in various tissues and organs indicated that it is highly likely that the human-like leukocyte lineage can be developed in mice. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oganesyan, Vaheh, E-mail: oganesyanv@medimmune.com; Mazor, Yariv; Yang, Chunning

In an effort to identify the critical structural features responsible for the high-affinity interaction of IgG1 Fc with FcγRI, the structure of the corresponding complex was solved at a resolution of 2.4 Å. The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Å using molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known majormore » energetic contribution of the Fc ‘LLGG’ motif at positions 234–237, and particularly of Leu235, via a ‘lock-and-key’ mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.« less

Optimization of ELISA Conditions to Quantify Colorectal Cancer Antigen-Antibody Complex Protein (GA733-FcK) Expressed in Transgenic Plant

PubMed Central

Ahn, Junsik; Lee, Kyung Jin

2014-01-01

The purpose of this study is to optimize ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment fused to KDEL, an ER retention motif (GA733-FcK) expressed in transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from transgenic plant expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG recognizing the human Fc existed in plant leaf extracts. For blocking buffer conditions, 3% BSA buffer clearly blocked the plate, compared to the 5% skim-milk buffer. For capture antibody, monoclonal antibody (MAb) CO17-1A was applied to coat the plate with different amounts (1, 0.5, and 0.25 μg/well). Among the amounts of the capture antibody, 1 and 0.5 μg/well (capture antibody) showed similar absorbance, whereas 0.25 μg/well of the capture antibody showed significantly less absorbance. Taken together, the optimized conditions to quantify plant-derived GA733-FcK were 0.5 μg/well of MAb CO17-1A per well for the capture antibody, 3% BSA for blocking buffer, and anti-human Fc conjugated HRP. To confirm the optimized ELISA conditions, correlation analysis was conducted between the quantified amount of GA733-FcK in ELISA and its protein density values of different leaf samples in Western blot. The co-efficient value R2 between the ELISA quantified value and protein density was 0.85 (p<0.01), which indicates that the optimized ELISA conditions feasibly provides quantitative information of GA733-FcK expression in transgenic plant. PMID:24555929

A benzenediamine derivative fc-99 attenuates lupus-like syndrome in MRL/lpr mice related to suppression of pDC activation.

PubMed

Ji, Jianjian; Fan, Hongye; Li, Fanlin; Li, Xiaojing; Dong, Guanjun; Gong, Wei; Song, Yuxian; Liu, Fei; Hua, Chunyan; Tan, Renxiang; Dou, Huan; Hou, Yayi

2015-12-01

Systemic lupus erythematosus (SLE) is an autoimmune disease with prominent chronic inflammatory aspects. Plasmacytoid dendritic cells (pDCs), which are the principal interferon-α (IFN-α)-producing cells, have known to be critically involved in SLE pathogenesis. Our previous research demonstrated that a benzenediamine derivative FC-99 possessed anti-inflammatory activities. However, the effects of FC-99 on SLE have not been investigated to date. In this study, we found that FC-99 attenuated lupus-like pathological symptoms and lupus nephritis as well as the expression of pro-inflammatory cytokines in kidneys of MRL/lpr mice. FC-99 also decreased both the total IgM, total IgG and anti-dsDNA IgG levels in sera and the activation of B cells in the PBMCs and spleens of MRL/lpr mice. Moreover, FC-99 inhibited the abnormal activation and number of pDCs from PBMCs and spleens and levels of IFN-α in MRL/lpr mice. Notably, FC-99 significantly suppressed the expression of IFN-inducible genes in peripheral blood mononuclear cells (PBMCs) and spleens from MRL/lpr mice. As expected, in vitro experiments demonstrated that FC-99 decreased both the activation and IFN-α production of pDCs and inhibited IRAK4 phosphorylation in pDCs upon TLR7 and TLR9 stimulation. We further confirm that the inhibition of FC-99 on B cell activation depended on level of pDCs-secreting IFN-α. These data indicate that FC-99 attenuated lupus-like syndrome in MRL/lpr mice related to suppression of pDC activation, especially pDCs-secreting IFN-α. This study suggests that FC-99 may be a potential therapeutic candidate for the treatment of SLE. Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

FcγRIIIa SNPs and haplotypes affect human IgG binding and association with lupus nephritis in African Americans

PubMed Central

Dong, Chaoling; Ptacek, Travis S; Redden, David T; Zhang, Kui; Brown, Elizabeth E.; Edberg, Jeffrey C.; McGwin, Gerald; Alarcón, Graciela S.; Ramsey-Goldman, Rosalind; Reveille, John D.; Vilá, Luis M.; Petri, Michelle; Qin, Aijian; Wu, Jianming; Kimberly, Robert P.

2014-01-01

Objective To investigate whether the FcγRIIIa-66R/H/L polymorphism influences net effective receptor function and to assess if the FCGR3A combined genotypes formed by FcγRIIIa-66R/H/L and FcγRIIIa-176F/V as well as copy number variation (CNV) confer risk for development of SLE and lupus nephritis. Methods FcγRIIIa variants, expressed on A20 IIA1.6 cells, were used in flow cytometry-based human IgG binding assays. FCGR3A SNP and CNV genotypes were determined by Pyrosequencing methodology in a cohort of 1728 SLE patients and 2404 healthy controls. Results The FcγRIIIa-66L/H/R (rs10127939) polymorphism influences ligand binding capacity in the context of the FcγRIIIa-176V (rs396991) allele. The low binding FcγRIIIa-176F allele was associated with SLE nephritis (p = 0.0609) in African Americans but not in European Americans (p > 0.10). Nephritis among African American SLE subjects was associated with FcγRIIIa low binding haplotypes containing the 66R/H/L and 176F variants (p = 0.03) and with low binding genotype combinations (p = 0.002). No association was observed in European American SLE patients. The distribution of FCGR3A CNV was not significantly different between controls and SLE patients with or without nephritis. Conclusion FcγRIIIa-66R/H/L influences ligand binding. The low binding haplotypes formed by 66R/H/L and 176F confer enhanced risk for lupus nephritis in African Americans. FCGR3A CNVs are not associated with SLE or SLE nephritis in either African Americans or European Americans. PMID:24782186

Flow cytometric analysis of leukocytes and reticulocytes stained with proflavine.

PubMed

Sagawa, H; Tatsumi, N

1997-12-01

Proflavine, an acridine analog for industrial use, was used to stain blood cells. A drop of blood treated with ethylenediaminetetraacetic acid-2K was mixed with a 0.00001% solution of the dye and observed immediately by fluorescence microscopy with a green filter. Leukocytes, platelets, and reticulocytes were stained but mature red blood cells were not. Chromatin in the nuclei of all leukocytes and nucleoli of lymphocytes and monocytes had greenish-yellow fluorescence, and the kind of cell could be identified by the tone and intensity of this color. Granules in granulocytes were in green. Reticular fine-granular or granulofibrous structures in the reticulocytes were brownish. The proflavine could be used routinely in clinical laboratories because this single stain makes possible simultaneous differentiation of leukocytes and counting of reticulocytes.

Key Spatial Relations-based Focused Crawling (KSRs-FC) for Borderlands Situation Analysis

NASA Astrophysics Data System (ADS)

Hou, D. Y.; Wu, H.; Chen, J.; Li, R.

2013-11-01

Place names play an important role in Borderlands Situation topics, while current focused crawling methods treat them in the same way as other common keywords, which may lead to the omission of many useful web pages. In the paper, place names in web pages and their spatial relations were firstly discussed. Then, a focused crawling method named KSRs-FC was proposed to deal with the collection of situation information about borderlands. In this method, place names and common keywords were represented separately, and some of the spatial relations related to web pages crawling were used in the relevance calculation between the given topic and web pages. Furthermore, an information collection system for borderlands situation analysis was developed based on KSRs-FC. Finally, F-Score method was adopted to quantitatively evaluate this method by comparing with traditional method. Experimental results showed that the F-Score value of the proposed method increased by 11% compared to traditional method with the same sample data. Obviously, KSRs-FC method can effectively reduce the misjudgement of relevant webpages.

Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

PubMed

Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

2015-09-01

A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

PubMed

Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

1985-05-01

During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

[Construction and eukaryotic expression of PVAX1-hPV58mE6E7fcGB composite gene vaccine].

PubMed

Wang, He; Yu, Jiyun; Li, Li

2013-10-01

To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.

75 FR 52528 - FC Landfill Energy, LLC; Supplemental Notice That Initial Market-Based Rate Filing Includes...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-26

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. ER10-2268-000] FC Landfill Energy, LLC; Supplemental Notice That Initial Market- Based Rate Filing Includes Request for Blanket... proceeding, of FC Landfill Energy, LLC's application for market-based rate authority, with an accompanying...

Subacute Low Dose Nerve Agent Exposure Causes DNA Fragmentation in Guinea Pig Leukocytes

DTIC Science & Technology

2005-10-01

1 SUBACUTE LOW DOSE NERVE AGENT EXPOSURE CAUSES DNA FRAGMENTATION IN GUINEA PIG LEUKOCYTES. Jitendra R. Dave1, John R. Moffett1, Sally M...DNA fragmentation in blood leukocytes from guinea pigs by ‘Comet’ assay after exposure to soman at doses ranging from 0.1LD50 to 0.4 LD50, once per...computer. Data obtained for exposure to soman demonstrated significant increases in DNA fragmentation in circulating leukocytes in CWNA treated guinea pigs as

A benzenediamine derivate FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting myeloid dendritic cell-secreted BAFF

PubMed Central

Ji, Jianjian; Xu, Jingjing; Li, Fanlin; Li, Xiaojing; Gong, Wei; Song, Yuxian; Dou, Huan; Hou, Yayi

2016-01-01

Myeloid dendritic cells (DCs) can produce B-cell-activating factor (BAFF) that modulates survival and differentiation of B cells and plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). Toll-like receptor 4 (TLR4) signaling has important functions in the process of BAFF production. Our previous study showed that a benzenediamine derivate FC-99 possesses anti-inflammation activity and directly interacts with interleukin-1 receptor-associated kinase 4 (IRAK4), which was a pivotal molecule in TLR4 signaling. In this study, we demonstrated that FC-99 attenuated lupus nephritis in the MRL/lpr mice. FC-99 also decreased the levels of total immunoglobulin G (IgG), total IgG2a and IgM in sera, as well as the activation of B cells in the spleens of MRL/lpr mice. Moreover, FC-99 inhibited abnormal activation of myeloid DCs in spleens and reduced the levels of BAFF in sera, spleens, and kidneys of MRL/lpr mice. Furthermore, upon TLR4 stimulation with lipopolysaccharide in vitro, FC-99 inhibited IRAK4 phosphorylation, as well as the activation and BAFF production in murine bone marrow-derived DCs. These data indicate that FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting DC-secreted BAFF, suggesting that FC-99 may be a potential therapeutic candidate for the treatment of SLE. PMID:27121231

Arachidonic acid, but not its metabolites, is essential for FcγR-stimulated intracellular killing of Staphylococcus aureus by human monocytes

PubMed Central

Zheng, L; Zomerdijk, T P L; Van Den Barselaar, M T; Geertsma, M F; Van Furth, R; Nibbering, P H

1999-01-01

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcγ receptor (FcγR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcγR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcγR-mediated killing process. The production of O−2 by monocytes upon FcγR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcγR cross-linking. Furthermore, FcγR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcγR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcγR-stimulated intracellular killing of S. aureus by monocytes. PMID:10233682

Evaluation of polyacrylonitrile electrospun nano-fibrous mats as leukocyte removal filter media.

PubMed

Pourbaghi, Raha; Zarrebini, Mohammad; Semnani, Dariush; Pourazar, Abbasali; Akbari, Nahid; Shamsfar, Reihaneh

2018-07-01

Removal of leukocytes from blood products is the most effective means for elimination of undesirable side effects and prevention of possible reactions in recipients. Micro-fibrous mats are currently used for removal of leukocytes from blood. In this study, samples of electrospun nano-fibrous mats were produced. The performance of the produced electrospun nano-fibrous mats as means of leukocytes removal from fresh whole blood was both evaluated and compared with that of commercially available micro-fibrous mats. In order to produce the samples, polyacrylonitrile (PAN) nano-fibrous mats were made under different electrospinning conditions. Mean fiber diameter, pore characterization and surface roughness of the PAN nano-fibrous mats were determined using image processing technique. In order to evaluate the surface tension of the fabricated mats, water contact angle was measured. The leukocyte removal performance, erythrocytes recovery percent and hemolysis rate of the nano-fibrous mats were compared. The effectiveness of nano-fibrous mats in removing leukocyte was established using both scanning electron microscope and optical microscope. Results showed that for given weight, the fabricated nano-fibrous mats were not only more efficient but also more cost-effective than their commercial counterparts. Results confirmed that changes in mean fiber diameter, the number of layer and weight of each layer in the absence of any chemical reaction or physical surface modification, the fabricated nano-fibrous mats were able to remove 5-log of leukocytes. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1759-1769, 2018. © 2017 Wiley Periodicals, Inc.

Impact of linker and conjugation chemistry on antigen binding, Fc receptor binding and thermal stability of model antibody-drug conjugates

PubMed Central

Acchione, Mauro; Kwon, Hyewon; Jochheim, Claudia M.; Atkins, William M.

2012-01-01

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHISNLC and aHISTPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate. PMID:22531451

Antibody Functional Assays as Measures of Fc Receptor-Mediated Immunity to HIV - New Technologies and their Impact on the HIV Vaccine Field

PubMed Central

Wines, Bruce D.; Billings, Hugh; Mclean, Milla R.; Kent, Stephen J.; Hogarth, P. Mark

2017-01-01

Background: There is now intense interest in the role of HIV-specific antibodies and the engagement of FcγR functions in the control and prevention of HIV infection. The analyses of the RV144 vaccine trial, natural progression cohorts, and macaque models all point to a role for Fc-dependent effector functions, such as cytotoxicity (ADCC) or phagocytosis (ADCP), in the control of HIV. However, reliable assays that can be reproducibly used across different laboratories to measure Fc-dependent functions, such as antibody dependent cellular cytotoxicity (ADCC) are limited. Method: This brief review highlights the importance of Fc properties for immunity to HIV, particular-ly via FcγR diversity and function. We discuss assays used to study FcR mediated functions of HIV-specific Ab, including our recently developed novel cell-free ELISA using homo-dimeric FcγR ecto-domains to detect functionally relevant viral antigen-specific antibodies. Results: The binding of these dimeric FcγR ectodomains, to closely spaced pairs of IgG Fc, mimics the engagement and cross-linking of Fc receptors by IgG opsonized virions or infected cells as the es-sential prerequisite to the induction of Ab-dependent effector functions. The dimeric FcγR ELISA reli-ably correlates with ADCC in patient responses to influenza. The assay is amenable to high throughput and could be standardized across laboratories. Conclusion: We propose the assay has broader implications for the evaluation of the quality of anti-body responses in viral infections and for the rapid evaluation of responses in vaccine development campaigns for HIV and other viral infections. PMID:28322167

Stress-induced enhancement of leukocyte trafficking into sites of surgery or immune activation

NASA Astrophysics Data System (ADS)

Viswanathan, Kavitha; Dhabhar, Firdaus S.

2005-04-01

Effective immunoprotection requires rapid recruitment of leukocytes into sites of surgery, wounding, infection, or vaccination. In contrast to immunosuppressive chronic stressors, short-term acute stressors have immunoenhancing effects. Here, we quantify leukocyte infiltration within a surgical sponge to elucidate the kinetics, magnitude, subpopulation, and chemoattractant specificity of an acute stress-induced increase in leukocyte trafficking to a site of immune activation. Mice acutely stressed before sponge implantation showed 200-300% higher neutrophil, macrophage, natural killer cell, and T cell infiltration than did nonstressed animals. We also quantified the effects of acute stress on lymphotactin- (LTN; a predominantly lymphocyte-specific chemokine), and TNF-- (a proinflammatory cytokine) stimulated leukocyte infiltration. An additional stress-induced increase in infiltration was observed for neutrophils, in response to TNF-, macrophages, in response to TNF- and LTN, and natural killer cells and T cells in response to LTN. These results show that acute stress initially increases trafficking of all major leukocyte subpopulations to a site of immune activation. Tissue damage-, antigen-, or pathogen-driven chemoattractants subsequently determine which subpopulations are recruited more vigorously. Such stress-induced increases in leukocyte trafficking may enhance immunoprotection during surgery, vaccination, or infection, but may also exacerbate immunopathology during inflammatory (cardiovascular disease or gingivitis) or autoimmune (psoriasis, arthritis, or multiple sclerosis) diseases. chemokine | psychophysiological stress | surgical sponge | wound healing | lymphotactin

Dark chocolate consumption improves leukocyte adhesion factors and vascular function in overweight men.

PubMed

Esser, Diederik; Mars, Monica; Oosterink, Els; Stalmach, Angelique; Müller, Michael; Afman, Lydia A

2014-03-01

Flavanol-enriched chocolate consumption increases endothelium-dependent vasodilation. Most research so far has focused on flow-mediated dilation (FMD) only; the effects on other factors relevant to endothelial health, such as inflammation and leukocyte adhesion, have hardly been addressed. We investigated whether consumption of regular dark chocolate also affects other markers of endothelial health, and whether chocolate enrichment with flavanols has additional benefits. In a randomized double-blind crossover study, the effects of acute and of 4 wk daily consumption of high flavanol chocolate (HFC) and normal flavanol chocolate (NFC) on FMD, augmentation index (AIX), leukocyte count, plasma cytokines, and leukocyte cell surface molecules in overweight men (age 45-70 yr) were investigated. Sensory profiles and motivation scores to eat chocolate were also collected. Findings showed that a 4 wk chocolate intake increased FMD by 1%, which was paralleled by a decreased AIX of 1%, decreased leukocyte cell count, decreased plasma sICAM1 and sICAM3, and decreased leukocyte adhesion marker expression (P<0.05 for time effect), with no difference between HFC and NFC consumption. Flavanol enrichment did affect taste and negatively affected motivation to consume chocolate. This study provides new insights on how chocolate affects endothelial health by demonstrating that chocolate consumption, besides improving vascular function, also lowers the adherence capacity of leukocytes in the circulation.

Accelerated dissociation of IgE:FcεRI complexes by disruptive inhibitors actively desensitizes allergic effector cells

PubMed Central

Eggel, Alexander; Baravalle, Günther; Hobi, Gabriel; Kim, Beomkyu; Buschor, Patrick; Forrer, Patrik; Shin, Jeoung-Sook; Vogel, Monique; Stadler, Beda M.; Dahinden, Clemens A.; Jardetzky, Theodore S.

2014-01-01

Background The remarkably stable interaction of immunoglobulin E (IgE) with its high-affinity receptor FcεRI on basophils and mast cells is critical for the induction of allergic hypersensitivity reactions. Due to the exceptionally slow dissociation rate of IgE:FcεRI complexes such allergic effector cells permanently display allergen-specific IgE on their surface and immediately respond to allergen challenge by releasing inflammatory mediators. We have recently described a novel macromolecular inhibitor that actively promotes the dissociation of IgE from FcεRI through a molecular mechanism termed facilitated dissociation. Objective Here, we assessed the therapeutic potential of this non-immunoglobulin based IgE inhibitor DARPin E2_79 as well as a novel engineered biparatopic DARPin bi53_79 and directly compared them to the established anti-IgE antibody omalizumab. Methods: IgE:FcεRI complex dissociation was analyzed in vitro using recombinant proteins in ELISA and surface plasmon resonance, ex vivo using human primary basophils with flow cytometry and in vivo using human FcεRI transgenic mice in a functional passive cutaneous anaphylaxis test. Results We show that E2_79 mediated removal of IgE from primary human basophils fully abrogates IgE-dependent cell activation and release of pro-inflammatory mediators ex vivo. Furthermore, we report that omalizumab also accelerates the dissociation of IgE from FcεRI albeit much less efficiently than E2_79. Using the biparatopic IgE targeting approach we further improved the disruptive potency of E2_79 by ~100 fold and show that disruptive IgE inhibitors efficiently prevent passive cutaneous anaphylaxis in mice expressing the human FcεRI alpha chain. Conclusion Our findings highlight the potential of such novel IgE inhibitors as important diagnostic and therapeutic tools to managing allergic diseases. PMID:24642143

Effects of conventional and new peritoneal dialysis fluids on leukocyte recruitment in the rat peritoneal membrane.

PubMed

Mortier, Siska; De Vriese, An S; McLoughlin, Rachel M; Topley, Nicholas; Schaub, Thomas P; Passlick-Deetjen, Jutta; Lameire, Norbert H

2003-05-01

Peritonitis remains an important cause of morbidity and technique failure in peritoneal dialysis (PD). Conventional peritoneal dialysate fluids (PDF) inhibit peritoneal leukocyte function in vitro and may thus adversely affect the immune response to peritonitis. New PDF have been designed with neutral pH, low glucose degradation product (GDP) contents, and bicarbonate as buffer. The present intravital microscopy study examined the effects of conventional and new PDF on leukocyte behavior in the peritoneal microcirculation of Wistar rats. The visceral peritoneum was superfused by a control solution (EBSS), a conventional (CAPD), or a new bicarbonate-buffered PDF with neutral pH and low GDP content (CAPD BicaVera). In addition, spent conventional and new PDF were tested. The number of rolling, adhering, and extravasated leukocytes and leukocyte rolling velocity were assessed at different time intervals after exposure to lipopolysaccharide (LPS) or cell-free supernatants of coagulase-negative staphylococci (CNS-CFS). Exposure to LPS or CNS-CFS dissolved in EBSS dramatically increased the number of rolling, adhering and extravasated leukocytes and decreased leukocyte rolling velocity. Superfusion by CAPD abolished the LPS- or CNS-CFS-induced leukocyte recruitment, whereas CAPD BicaVera had significantly fewer depressant effect. Spent PDF affected the leukocyte response in a similar way as fresh PDF. High lactate concentrations, GDP, and hypertonicity appeared to be mainly responsible for the inhibition of leukocyte recruitment. In conclusion, conventional PDF abolish in vivo leukocyte recruitment in response to potent inflammatory stimuli. Bicarbonate-buffered pH-neutral PDF with low GDP contents have fewer depressant effects and may therefore contribute to a better preservation of peritoneal host defense.

Exposure to Sodium Fluoride Produces Signs of Apoptosis in Rat Leukocytes

PubMed Central

Gutiérrez-Salinas, José; Morales-González, José A.; Madrigal-Santillán, Eduardo; Esquivel-Soto, Jaime; Esquivel-Chirino, César; González-Rubio, Manuel García-Luna y; Suástegui-Domínguez, Sigrit; Valadez-Vega, Carmen

2010-01-01

Fluoride is naturally present in the earth’s crust and can be found in rocks, coal, and clay; thus, it can be found in small quantities in water, air, plants, and animals. Therefore, humans are exposed to fluoride through food, drinking water, and in the air they breathe. Flouride is essential to maintain bone strength and to protect against dental decay, but if it is absorbed too frequently, it can cause tooth decay, osteoporosis, and damage to kidneys, bones, nerves, and muscles. Therefore, the present work was aimed at determining the effect of intake of sodium fluoride (NaF) as an apoptosis inducer in leukocytes of rats treated for eight weeks with 1 or 50 parts per million (ppm) NaF. Expression of p53, bcl-2, and caspade-3 were used as apoptotic and general metabolism indicators of leukocyte-like indicators of the (INT) oxidation system. Male rats were exposed to NaF (1 and 500 ppm) for eight weeks, and then sacrificed weekly to obtain blood samples. Expression of p53, bcl-2, and caspase-3 were determined in leukocytes by Western blot, and general metabolism of leukocytes was analyzed with a commercial kit. We found changes in the expression of the proteins described, especially when the animals received 50 ppm of NaF. These results indicate that NaF intoxication can be an apoptosis inducer in rat leukocytes treated with the compound for eight weeks. PMID:20957113

Measurement of leukocyte rheology in vascular disease: clinical rationale and methodology. International Society of Clinical Hemorheology.

PubMed

Wautier, J L; Schmid-Schönbein, G W; Nash, G B

1999-01-01

The measurement of leukocyte rheology in vascular disease is a recent development with a wide range of new opportunities. The International Society of Clinical Hemorheology has asked an expert panel to propose guidelines for the investigation of leukocyte rheology in clinical situations. This article first discusses the mechanical, adhesive and related functional properties of leukocytes (especially neutrophils) which influence their circulation, and establishes the rationale for clinically-related measurements of parameters which describe them. It is concluded that quantitation of leukocyte adhesion molecules, and of their endothelial receptors may assist understanding of leukocyte behaviour in vascular disease, along with measurements of flow resistance of leukocytes, free radical production, degranulation and gene expression. For instance, vascular cell adhesion molecule (VCAM-1) is abnormally present on endothelial cells in atherosclerosis, diabetes mellitus and inflammatory conditions. Soluble forms of intercellular adhesion molecule (ICAM-1) or VCAM can be found elevated in the blood of patients with rheumatoid arthritis or infections disease. In the second part of the article, possible technical approaches are presented and possible avenues for leukocyte rheological investigations are discussed.

Reaction of Methyl Fluoroformyl Peroxycarbonate (FC(O)OOC(O)OCH3) with Cl Atoms: Formation of Hydro-ChloroFluoro-Peroxides.

PubMed

Berasategui, Matias; Argüello, Gustavo A; Burgos Paci, Maxi A

2017-10-12

The products following Cl atom initiated reactions of FC(O)OOC(O)OCH 3 in 50-760 Torr of N 2 at 296 K were investigated using FTIR. Reaction of Cl atoms with methyl fluoroformyl peroxycarbonate proceeds mainly via attack at the methyl group, forming FC(O)OOC(O)OCH 2 • radicals. Further reaction of this kind of radical with Cl 2 forms three new compounds: FC(O)OOC(O)OCH 2 Cl, FC(O)OOC(O)OCHCl 2 , and FC(O)OOC(O)OCCl 3 , whose existence was characterized experimentally by FTIR spectroscopy assisted by ab initio calculations at the B3LYP/6-31++G(d,p) level. Relative rate techniques were used to measure k (Cl+FC(O)OOC(O)OCH3) = (4.0 ± 0.4) × 10 -14 cm 3 molecule -1 s -1 and k (Cl+FC(O)OOC(O)OCH2Cl) = (3.2 ± 0.3) × 10 -14 cm 3 molecule -1 s -1 . When the reaction is run in the presence of oxygen, the paths giving chlorinated peroxide formation are suppressed, and oxidation to (mainly) CO 2 and HCl takes place through highly oxidized intermediates with lifetimes long enough to be detected by FTIR spectroscopy.

Evaluation of ascitic soluble human leukocyte antigen-G for distinguishing malignant ascites from benign ascites.

PubMed

Sun, Juan; Chang, Yan-Xiang; Niu, Chun-Yan

2017-11-01

The overexpression of soluble human leukocyte antigen-G is associated with malignant tumours. The purpose of our study was to detect soluble human leukocyte antigen-G concentrations in ascites and to evaluate the value of ascitic soluble human leukocyte antigen-G for the diagnosis of malignant ascites. Enzyme-linked immunosorbent assay was used to detect soluble human leukocyte antigen-G levels in 64 patients with malignant ascites and 30 patients with benign ascites. Receiver operating characteristic curves were used to evaluate the diagnostic efficacy of ascitic soluble human leukocyte antigen-G for the detection of malignant ascites. Ascitic soluble human leukocyte antigen-G levels were significantly higher in the malignant ascites group than in the benign ascites group (20.718 ± 3.215 versus 12.467 ± 3.678 µg/L, t = 7.425, p < 0.001). The area under the receiver operating characteristic curve for ascitic soluble human leukocyte antigen-G was 0.957 (95% confidence interval, 0.872-0.992). At a cut-off value of 19.60 µg/L, the sensitivity and specificity of ascitic soluble human leukocyte antigen-G were 87.5% (95% confidence interval, 71.0%-96.5%) and 100% (95% confidence interval, 88.4%-100%), respectively. With respect to area under the receiver operating characteristic curve, sensitivity and specificity, ascitic carcinoembryonic antigen (0.810, 68.75% and 83.33%, respectively) and carbohydrate antigen 19-9 (0.710, 65.63% and 70%, respectively) significantly differed (all p < 0.05). In malignant ascites that were cytology-negative and biopsy-positive, the rate of positivity for ascitic soluble human leukocyte antigen-G was 75%, which was higher than the corresponding rates for ascitic carcinoembryonic antigen (31.25%) and carbohydrate antigen 19-9 (6.25%; both p < 0.05). In conclusion, The detection of ascitic soluble human leukocyte antigen-G exhibited good performance for diagnosing malignant ascites, and particularly those that

Inhibition of leukocyte-type 12-lipoxygenase by guava tea leaves prevents development of atherosclerosis.

PubMed

Takahashi, Yoshitaka; Otsuki, Akemi; Mori, Yoshiko; Kawakami, Yuki; Ito, Hideyuki

2015-11-01

Oxidation of low-density lipoprotein (LDL) is one of the crucial steps for atherosclerosis development, and an essential role of leukocyte-type 12-lipoxygenase expressed in macrophages in this process has been demonstrated. The biochemical mechanism of the oxidation of circulating LDL by leukocyte-type 12-lipoxygenase in macrophages has been proposed. The major ingredients in guava tea leaves which inhibited the catalytic activity of leukocyte-type 12-lipoxygenase were quercetin and ethyl gallate. Administration of extracts from guava tea leaves to apoE-deficient mice significantly attenuated atherogenic lesions in the aorta and aortic sinus. We recently showed that Qing Shan Lu Shui inhibited the catalytic activity of leukocyte-type 12-lipoxygenase. The major components inhibiting the enzyme contained in Qing Shan Lu Shui were identified to be novel monoterpene glycosides. The anti-atherogenic effect of the tea leaves might be attributed to the inhibition of leukocyte-type 12-lipoxygenase by these components. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative Study of 6-Mercaptopurine Metabolism in Human Leukemic Leukocytes and L1210 Cells

PubMed Central

Higuchi, Tomihiko; Nakamura, Toru; Uchino, Haruto; Wakisaka, Gyoichi

1977-01-01

Leukocytes from patients with leukemia and L1210 cells from mice were examined for the rate of formation and cellular concentration of phosphoribosylpyrophosphate, the rate of thioinosinic acid formation, and a number of selected enzymes involved in purine nucleotide synthesis. The amount of thioinosinic acid formed in L1210 cells was much higher than that in human leukemic leukocytes. In cell extracts, the synthesis of thioinosinic acid was similar in both cell types, and the amount of purine phosphoribosyltransferase was not rate limiting in either case. Much higher concentrations and rates of formation of phosphoribosylpyrophosphate were found in L1210 cells than in human leukemic leukocytes. The difference in response to 6-mercaptopurine between L1210 cells and human leukemic leukocytes might be attributed to their difference in supply of phosphoribosylpyrophosphate. Phosphoribosylpyrophosphate-amidotransferase was found to be high in L1210 cells, but was not detected in human leukemic leukocytes. PMID:921247

[Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

PubMed

Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

2016-01-01

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.