CD14(hi)CD16+ monocytes phagocytose antibody-opsonised Plasmodium falciparum infected erythrocytes more efficiently than other monocyte subsets, and require CD16 and complement to do so.

PubMed

Zhou, Jingling; Feng, Gaoqian; Beeson, James; Hogarth, P Mark; Rogerson, Stephen J; Yan, Yan; Jaworowski, Anthony

2015-07-07

With more than 600,000 deaths from malaria, mainly of children under five years old and caused by infection with Plasmodium falciparum, comes an urgent need for an effective anti-malaria vaccine. Limited details on the mechanisms of protective immunity are a barrier to vaccine development. Antibodies play an important role in immunity to malaria and monocytes are key effectors in antibody-mediated protection by phagocytosing antibody-opsonised infected erythrocytes (IE). Eliciting antibodies that enhance phagocytosis of IE is therefore an important potential component of an effective vaccine, requiring robust assays to determine the ability of elicited antibodies to stimulate this in vivo. The mechanisms by which monocytes ingest IE and the nature of the monocytes which do so are unknown. Purified trophozoite-stage P. falciparum IE were stained with ethidium bromide, opsonised with anti-erythrocyte antibodies and incubated with fresh whole blood. Phagocytosis of IE and TNF production by individual monocyte subsets was measured by flow cytometry. Ingestion of IE was confirmed by imaging flow cytometry. CD14(hi)CD16+ monocytes phagocytosed antibody-opsonised IE and produced TNF more efficiently than CD14(hi)CD16- and CD14(lo)CD16+ monocytes. Blocking experiments showed that Fcγ receptor IIIa (CD16) but not Fcγ receptor IIa (CD32a) or Fcγ receptor I (CD64) was necessary for phagocytosis. CD14(hi)CD16+ monocytes ingested antibody-opsonised IE when peripheral blood mononuclear cells were reconstituted with autologous serum but not heat-inactivated autologous serum. Antibody-opsonised IE were rapidly opsonised with complement component C3 in serum (t1/2 = 2-3 minutes) and phagocytosis of antibody-opsonised IE was inhibited in a dose-dependent manner by an inhibitor of C3 activation, compstatin. Compared to other monocyte subsets, CD14(hi)CD16+ monocytes expressed the highest levels of complement receptor 4 (CD11c) and activated complement receptor 3 (CD11b) subunits. We show a special role for CD14(hi)CD16+ monocytes in phagocytosing opsonised P. falciparum IE and production of TNF. While ingestion was mediated by Fcγ receptor IIIa, this receptor was not sufficient to allow phagocytosis; despite opsonisation with antibody, phagocytosis of IE also required complement opsonisation. Assays which measure the ability of vaccines to elicit a protective antibody response to P. falciparum should consider their ability to promote phagocytosis and fix complement.

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

PubMed

McGonigal, Rhona; Cunningham, Madeleine E; Yao, Denggao; Barrie, Jennifer A; Sankaranarayanan, Sethu; Fewou, Simon N; Furukawa, Koichi; Yednock, Ted A; Willison, Hugh J

2016-03-02

Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

Coronative antibody tires in sera of healthy adults and experimentally infected volunteers.

PubMed

Bradburne, A F; Somerset, B A

1972-06-01

Six coronaviruses isolated in the U.S.A. have been inoculated into volunteers and all produced colds. Between 10 and 20% of infected volunteers developed heterologous antibody responses after these and other experimental infections with coronaviruses. The haemagglutination-inhibition test with the OC43 virus strain was found to detect antibody rises after infection with a variety of strains.Studies on normal adult sera taken between 1965 and 1970 revealed a high frequency of neutralizing antibody to one strain (229 E) and a frequency of HI antibody to strain OC43 which fluctuated from year to year. Complement-fixing antibodies to these two viruses were also found, revealing an apparent increase in the activity of coronaviruses in the general population of the U.K., during the winter of 1968-9.

Type specificity of complement-fixing antibody against herpes simplex virus type 2 AG-4 early antigen in patients with asymptomatic infection.

PubMed Central

Sherlock, C H; Ashley, R L; Shurtleff, M L; Mack, K D; Corey, L

1986-01-01

We evaluated the type specificity of complement-fixing (CF) antibody against the AG-4 early antigen of herpes simplex virus (HSV) type 2 (HSV-2) by comparing a commercial AG-4 CF kit (Simplex-2; Gene Link Australia, Inc., Princeton, N.J.) with quantal microneutralization (MN) and absorption-Western blotting in testing sera from patients with and without a history of genital herpes. Sera characterized as HSV type 1 (HSV-1) or HSV-2 positive or negative by MN were selected and tested by CF, and those with discordant results were further analyzed for specific antibodies by absorption with HSV-1 or HSV-2 antigen and Western blotting with heterologous HSV proteins. A total of 34 of 42 (81%) sera HSV-2 positive by MN, 19 of 43 (44%) sera HSV-1 positive by MN, and 0 of 19 sera negative by MN were positive by CF. Absorption-Western blotting showed that 12 of 18 (67%) sera HSV-1 positive by MN but positive by CF had no HSV-2-specific antibody and that all 7 sera HSV-2 positive by MN but negative by CF had HSV-2-specific antibody. When MN and absorption-Western blotting data were combined to analyze patients with no history of genital herpes, 7 of 19 (37%) with no HSV-2-specific antibody were positive by CF, and 7 of 27 (26%) with HSV-2-specific antibody were negative by CF. The positive and negative predictive values for the CF test were 78 and 75%, respectively, in this group. The presence of antibody to the HSV AG-4 antigen does not discriminate sufficiently between HSV-1- and HSV-2-infected patients to be of value in predicting HSV-2 infection in the absence of symptomatic disease. Images PMID:3023439

Non-HLA antibodies post-transplantation: clinical relevance and treatment in solid organ transplantation.

PubMed

Dragun, Duska; Hegner, Bjorn

2009-01-01

Antibodies and B cells are increasingly recognized as major modulators of allograft function and survival. Improved immunohistochemical and serologic diagnostic procedures have been developed to monitor antibody responses against HLA antigens during the last decade. Acute and chronic allograft rejection can occur in HLA-identical sibling transplants implicating the importance of immune response against non-HLA targets. Non-HLA anti-bodies may occur as alloantiboides, yet they seem to be predominantly autoantibodies. Antigenic targets of non-HLA antibodies described thus far include various minor histocompatibility antigens, vascular receptors, adhesion molecules, and intermediate filaments. Non-HLA antibodies may function as complement- and non-complement-fixing antibodies and they may induce a wide variety of allograft injuries, reflecting the complexity of their acute and chronic actions. Refined approaches considering the subtle mechanistic differences in the individual antibody responses directed against non-HLA antigens may help to define patients at particular risk for irreversible acute or chronic allograft injuries and improve over-all outcomes. We attempted to summarize the current state of research, development in diagnostic and therapeutic strategies, and to address some emerging problems in the area of humoral response against non-HLA antigens beyond ABO blood group and MHC class I chain-related gene A and B (MICA and MICB) antigens in solid organ transplantation. Copyright (c) 2009 S. Karger AG, Basel.

Serological reactions in Rhesus monkeys inoculated with the 17D strain of yellow fever virus.

PubMed

GROOT, H

1962-01-01

Haemagglutination-inhibition tests, which depend on the appearance of haemagglutination-inhibiting antibodies in the serum in virus infections, are in common use in the study of arthropod-borne diseases. This paper contains the results of an investigation into the appearance and pattern of haemagglutination-inhibiting antibodies in the serum of rhesus monkeys inoculated intracerebrally with the 17D strain of yellow fever virus during the testing of seed lots of yellow fever vaccine. These antibodies appeared on the tenth day after inoculation, and were still demonstrable four years later. In all of the eight monkeys tested complement-fixing and neutralizing antibodies against yellow fever antigens also developed, and in six out of the eight heterologous antigens developed.

Studies of the antibody nature of the rheumatoid factor

PubMed Central

Aho, K.; Harboe, M.; Leikola, J.

1964-01-01

The reaction of the rheumatoid factor (RF) with 7S γ-globulin antibodies of nine persons immunized with sheep erythrocytes was studied. All of a panel of rheumatoid sera with high Waaler-Rose titres agglutinated sheep cells sensitized with the human anti-sheep cell antibodies and O Rh positive cells sensitized with the `diagnostic' anti-CD serum Ripley. The RF measurable with these systems could be absorbed to diphtheria toxoid—human antitoxin precipitate, whereas the absorption with egg albumin—rabbit anti-egg albumin precipitate did not result in any appreciable titre reduction. However, the eluate from the rabbit precipitate was highly active in these systems, whereas Rh positive cells sensitized with anti-Rh sera suitable for Gm(a) typing were not regularly agglutinated. A markedly greater concentration of native than of heat-aggregated γ-globulin was needed for inhibition of the agglutination by the RF of cells heavily sensitized with the human anti-sheep cell antibodies. No appreciable difference in this respect was seen when using lightly sensitized cells. Only the heavily sensitized cells fixed complement. The `natural' 7S γ-globulin antibodies against sheep cells were neither suited for demonstration of RF nor did they fix complement. Sheep cells coated with 7S γ-globulin antibodies of a Gm(a+) person were agglutinated by a non-rheumatoid anti-Gm(a) serum, and this system was well suited for Gm(a) typing, whereas cells coated with antibodies of a Gm(a-) person were not agglutinated. Rheumatoid anti-Gm(a) sera agglutinated cells sensitized with antibodies of both Gm(a+) and Gm(a-) persons. Using cells coated with Gm(a+) antibodies, some distinction between Gm(a+) and Gm(a-) sera could be obtained under carefully controlled conditions. The use of a Gm(a-) coat resulted in a slight difference in the inhibiting capacity, which had no relation to the serum's Gm(a) type. The results suggest that the reaction of the RF with sheep cells heavily sensitized with human anti-sheep cell antibodies is essentially an interaction of the RF with immune aggregated γ-globulin, whereas when using lightly sensitized cells the individual γ-globulin molecules play a prominent role. PMID:14193154

Solid-Phase Radioimmunoassay of Total and Influenza-Specific Immunoglobulin G

PubMed Central

Daugharty, Harry; Warfield, Donna T.; Davis, Marianne L.

1972-01-01

An antigen-antibody system of polystyrene tubes coated with immunoglobulin antibody was used for quantitating immunoglobulins. A similar radioimmunoassay method was adapted for a viral antigen-antibody system. The viral system can be used for quantitating viruses and for measuring virus-specific antibodies by reacting with 125iodine-labeled anti-immunoglobulin G (IgG). Optimal conditions for coating the solid phase, specificity of the immune reaction, and other kinetics and sensitivities of the assay method were investigated. Comparison of direct and indirect methods of assaying for immunoglobulins or viral antibody indicates that the indirect method is more sensitive and can quantitate a minimum of 0.037 μg of IgG per ml. Results of solid-phase radioimmunoassay for influenza antibody correlate well with hemagglutinin antibody titers but not with complement-fixing antibody titers. Radioimmunoassay results for influenza antibody by solid phase are likewise in agreement with results by the carrier precipitate radioimmunoassay method. The simplicity, reproducibility, and versatility of the solid-phase procedure make it diagnostically useful. PMID:5062884

Resistance to Viral Challenge in the Days Immediately Following Vaccination.

DTIC Science & Technology

Powassan or yellow fever, by the intraperitoneal route into guinea pigs failed to induce visible signs of illness; however, with Japanese encephalitis... Powassan , and Banzi viruses, high titered complement-fixing antibodies developed. Since no disease and death could be used as a criterion of successful...infection of guinea pigs inoculated with Japanese encephalitis virus, or of hamsters inoculated with Powassan , an alternative method of determining

THE EFFECT OF ADRENOCORTICOTROPIC HORMONE ON INFLAMMATION DUE TO TUBERCULIN HYPERSENSITIVITY AND TURPENTINE AND ON CIRCULATING ANTIBODY LEVELS

PubMed Central

Osgood, Charles K.; Favour, Cutting B.

1951-01-01

The treatment with adrenocorticotropic hormone of guinea pigs sensitized with heat-killed tubercle bacilli caused suppression of their skin reactivity to tuberculin. Similar animals treated with saline did not show this change. Normal guinea pigs treated with adrenocorticotropic hormone showed suppression of inflammation, but not necrosis, produced by intracutaneous oil of turpentine. There was slight, but probably not significant, diminution of inflammation during saline administration. Tuberculin complement-fixing antibody titers were not altered by either adrenocorticotropic hormone or saline administration. Adrenocorticotropic hormone produced marked eosinopenia and lymphopenia in guinea pigs. PMID:14888823

Analysis of ovine colostrum to detect antibody against progressive pneumonia virus.

PubMed Central

Taylor, T B; Banowetz, G M; Schipper, I A; Gabrielson, D A

1982-01-01

Immunoglobulins were isolated and purified from the colostrum and serum of progressive pneumonia virus infected sheep and also from non-infected control sheep. Four classes of immunoglobulins were isolated from sheep colostrum (IgG1, IgG2, IgA and Ig10s). Three classes of immunoglobulins were isolated from sheep serum (IgG1, IgG2 and IgM). Low levels of virus neutralizing activity were demonstrated only in the whole serum and purified serum IgG1 preparations. No complement fixing activity was detected in any of the antibody preparations from colostrum. PMID:6284323

Immunization with Recombinantly Expressed LRP4 Induces Experimental Autoimmune Myasthenia Gravis in C57BL/6 Mice.

PubMed

Ulusoy, Canan; Çavuş, Filiz; Yılmaz, Vuslat; Tüzün, Erdem

2017-07-01

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ), characterized with muscle weakness. While MG develops due to acetylcholine receptor (AChR) antibodies in most patients, antibodies to muscle-specific receptor tyrosine kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4) may also be identified. Experimental autoimmune myasthenia gravis (EAMG) has been previously induced by both LRP4 immunization and passive transfer of LRP4 antibodies. Our aim was to confirm previous results and to test the pathogenic effects of LRP4 immunization in a commonly used mouse strain C57BL/6 (B6) using a recombinantly expressed human LRP4 protein. B6 mice were immunized with human LRP4 in CFA, Torpedo Californica AChR in CFA or only CFA. Clinical and pathogenic aspects of EAMG were compared among groups. LRP4- and AChR-immunized mice showed comparable EAMG clinical severity. LRP4-immunized mice displayed serum antibodies to LRP4 and NMJ IgG and complement factor C3 deposits. IgG2 was the dominant anti-LRP4 isotype. Cultured lymph node cells of LRP4- and AChR-immunized mice gave identical pro-inflammatory cytokine (IL-6, IFN-γ and IL-17) responses to LRP4 and AChR stimulation, respectively. Our results confirm the EAMG-inducing action of LRP4 immunization and identify B6 as a LRP4-EAMG-susceptible mouse strain. Demonstration of complement fixing anti-LRP4 antibodies in sera and complement/IgG deposits at the NMJ of LRP4-immunized mice indicates complement activation as a putative pathogenic mechanism. We have thus developed a practical LRP4-induced EAMG model using a non-conformational protein and a widely available mouse strain for future investigation of LRP4-related MG.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

PubMed Central

Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

2017-01-01

Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

Nephropathy associated with sickle cell anemia: an autologous immune complex nephritis. I. Studies on nature of glomerular-bound antibody and antigen identification in a patient with sickle cell disease and immune deposit glomerulonephritis.

PubMed

Strauss, J; Pardo, V; Koss, M N; Griswold, W; McIntosh, R M

1975-03-01

The nature of the glomerular-bound antibody and the putative antigen was investigated in one of the patients with sickle cell disease and immune deposit membranoproliferative glomerulonephritis by immunohistologic and glomerular antibody elution. Renal proximal tubular epithelial antigen was localized in association with immunoglobulins G (IgG), M (IgM), Clq fraction of the first component of complement (Clq) and the third component of complement (C3) in a granular pattern along the glomerular basement membrane of the patient's kidney. IgG and IgM were eluted from glomeruli. These immunoglobulins fixed to the proximal tubules of normal human kidney by direct immunofluorescence. This localization was abolished by absorption of the eluted immunoglobulins with renal tubular epithelial (RTE) antigen. The IgG eluted from the glomeruli blocked the fixation of rabbit anti-RTE antigen to normal proximal tubular brush border. These studies suggest that the nephritis in this patient was due to deposition of complexes or RTE antigen and specific antibody. An autologous immune complex nephritis may develop in some patients with sickle cell anemia secondary to RTE antigen released possibly after renal ischemia or some other phenomenon causing renal tubular damage.

The role of complement in myasthenia gravis: serological evidence of complement consumption in vivo.

PubMed

Romi, Fredrik; Kristoffersen, Einar K; Aarli, Johan A; Gilhus, Nils Erik

2005-01-01

Antibodies to the acetylcholine receptor (AChR) titin and the ryanodine receptor (RyR) occur in myasthenia gravis (MG). These antibodies are capable of complement activation in vitro. The involvement of the complement system should cause consumption of complement components such as C3 and C4 in vivo. Complement components C3 and C4 were assayed in sera from 78 AChR antibody-positive MG patients and 52 healthy controls. Forty-eight of the patient sera contained titin antibodies as well, and 20 were also RyR antibody-positive. MG patients with AChR antibody concentrations above the median (11.2 nmol/l) had significantly lower mean C3 and C4 concentrations in serum compared to those with AChR antibody concentrations below the median. Titin antibody-positive MG patients, titin antibody-negative early-onset MG patients, titin antibody-negative late-onset MG patients, and controls had similar C3 and C4 concentrations. Nor did mean C3 and C4 concentrations differ in MG patients with RyR antibodies. Patients with severe MG (grades 4 and 5) had similar C3 and similar C4 levels compared to those with mild MG (grades 1 and 2). An increased in vivo complement consumption was detected in MG patients with high AChR antibody concentrations, unrelated to MG severity and non-AChR muscle antibodies.

The prozone effect exerted by the complement-binding anti-Lea on anti-D.

PubMed

Joshi, Sanmukh R; Parekh, Kamlesh H

2017-01-01

Prozone phenomenon is seen with very high-titer antibodies in an immune serum. The prozone effect on anti-D by a low-titer anti-Le a was investigated associated with neonatal jaundice. Standard methods were used in investigations. The child was born at full-term developed mild jaundice. With weak direct antiglobulin test+, her indirect serum bilirubin was progressed to 27.5 mg/dL in 48 h. Anti-D and anti-Le a were detected in the mother. Both these antibodies were detected in the child's serum though the eluate from red blood cells (RBCs) contained only anti-D. Mother's anti-D was masked by anti-Le a if the RBCs possessed both the antigens together. Anti-D was revealed only with D-positive RBCs lacking Le a or if the serum was modified by mixing with Le a+ saliva or was heated at 56°C or fortified with citrate solution. An anti-D showed prozone effect exerted by the complement-fixing anti-Le a in the test.

Dengue-Immune Humans Have Higher Levels of Complement-Independent Enhancing Antibody than Complement-Dependent Neutralizing Antibody.

PubMed

Yamanaka, Atsushi; Konishi, Eiji

2017-09-25

Dengue is the most important arboviral disease worldwide. We previously reported that most inhabitants of dengue-endemic countries who are naturally immune to the disease have infection-enhancing antibodies whose in vitro activity does not decrease in the presence of complement (complement-independent enhancing antibodies, or CiEAb). Here, we compared levels of CiEAb and complement-dependent neutralizing antibodies (CdNAb) in dengue-immune humans. A typical antibody dose-response pattern obtained in our assay system to measure the balance between neutralizing and enhancing antibodies showed both neutralizing and enhancing activities depending on serum dilution factor. The addition of complement to the assay system increased the activity of neutralizing antibodies at lower dilutions, indicating the presence of CdNAb. In contrast, similar dose-response curves were obtained with and without complement at higher dilutions, indicating higher levels of CiEAb than CdNAb. For experimental support for the higher CiEAb levels, a cocktail of mouse monoclonal antibodies against dengue virus type 1 was prepared. The antibody dose-response curves obtained in this assay, with or without complement, were similar to those obtained with human serum samples when a high proportion of D1-V-3H12 (an antibody exhibiting only enhancing activity and thus a model for CiEAb) was used in the cocktail. This study revealed higher-level induction of CiEAb than CdNAb in humans naturally infected with dengue viruses.

MuSK induced experimental autoimmune myasthenia gravis does not require IgG1 antibody to MuSK.

PubMed

Küçükerden, Melike; Huda, Ruksana; Tüzün, Erdem; Yılmaz, Abdullah; Skriapa, Lamprini; Trakas, Nikos; Strait, Richard T; Finkelman, Fred D; Kabadayı, Sevil; Zisimopoulou, Paraskevi; Tzartos, Socrates; Christadoss, Premkumar

2016-06-15

Sera of myasthenia gravis (MG) patients with muscle-specific receptor kinase-antibody (MuSK-Ab) predominantly display the non-complement fixing IgG4 isotype. Similarly, mouse IgG1, which is the analog of human IgG4, is the predominant isotype in mice with experimental autoimmune myasthenia gravis (EAMG) induced by MuSK immunization. The present study was performed to determine whether IgG1 anti-MuSK antibody is required for immunized mice to develop EAMG. Results demonstrated a significant correlation between clinical severity of EAMG and levels of MuSK-binding IgG1+, IgG2+ and IgG3+ peripheral blood B cells in MuSK-immunized wild-type (WT) mice. Moreover, MuSK-immunized IgG1 knockout (KO) and WT mice showed similar EAMG severity, serum MuSK-Ab levels, muscle acetylcholine receptor concentrations, neuromuscular junction immunoglobulin and complement deposit ratios. IgG1 and IgG3 were the predominant anti-MuSK isotypes in WT and IgG1 KO mice, respectively. These observations demonstrate that non-IgG1 isotypes can mediate MuSK-EAMG pathogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site of Vulnerability in L1 Protein

PubMed Central

Kaever, Thomas; Meng, Xiangzhi; Matho, Michael H.; Schlossman, Andrew; Li, Sheng; Sela-Culang, Inbal; Ofran, Yanay; Buller, Mark; Crump, Ryan W.; Parker, Scott; Frazier, April; Crotty, Shane; Zajonc, Dirk M.; Peters, Bjoern

2014-01-01

ABSTRACT Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (MAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 MAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins, such as H3, D8, or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to nonneutralizing anti-L1 MAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and also could bind to virions. By using a variety of techniques, including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light-chain and heavy-chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies. IMPORTANCE Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history, but it presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic principle of successful vaccines and the development of next-generation, safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody-antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice. PMID:25031354

Role of humoral immune reactions as target for antirejection therapy in recipients of a spousal-donor kidney graft.

PubMed

Böhmig, G A; Regele, H; Säemann, M D; Exner, M; Druml, W; Kovarik, J; Hörl, W H; Zlabinger, G J; Watschinger, B

2000-04-01

Excellent graft outcome has been reported for spousal-donor kidney transplantation. In husband-to-wife transplantation, however, a tendency toward inferior graft survival has been described for recipients who were previously pregnant. In our series of spousal-kidney transplantations (nine transplantations; three female recipients), actual graft survival is 100% (median observation time, 339 days). Five patients experienced early allograft rejection. In four transplant recipients, rejection was easily reversible by conventional antirejection therapy. In a multiparous recipient, however, mild interstitial allograft rejection associated with early graft dysfunction was resistant to anticellular treatment (antilymphocyte antibody, tacrolimus rescue therapy). The particular finding of polymorphonuclear neutrophils in peritubular capillaries and the finding of diffuse capillary deposits of the complement split product, C4d, in a posttransplantation biopsy specimen suggested a role of antibody-mediated graft injury. Retrospective flow cytometry cross-matching showed the presence of preformed immunoglobulin G (IgG) antibodies to HLA class I antigens that were not detectable by pretransplantation lymphocytotoxic cross-match testing or screening for panel reactive antibodies. After transplantation, however, complement-fixing antibodies, also presumably triggered by reexposure to spousal-donor HLA antigens, could be detected in the patient's serum. These findings suggested antibody-mediated allograft rejection and led to the initiation of immunoadsorption therapy (14 sessions) with staphylococcal protein A. Selective removal of recipient IgG resulted in complete reversal of graft dysfunction. Our findings suggest that in husband-to-wife transplantation, donor-specific antibodies, presumably triggered by previous pregnancies, might occasionally induce sustained allograft dysfunction. Thus, in this particular setting, a detailed immunologic and histopathologic work-up regarding antibody-mediated allograft dysfunction is warranted because immunoadsorption may be a highly effective treatment modality.

[Comparative data on the formation of complement-binding and hemagglutinating antibodies to penicillin].

PubMed

Sluvko, A L

1976-10-01

Comparative data on production of complement-binding and hemagglutinating antibodies in the process of the antigenic effect of benzylpenicillin under experimental conditions are presented. 30 rabbit antisera and 3 sera of intact animals were studied. The hemagglutinating antibodies were determined in 19 antisera, high and reliable titers of the antipenicillin hemagglutinating antibodies being found only in 8 antisera. The antipenicillin complement-binding antibodies using complex antibiotic antibodies were also found in 19 antisera. The process of antibody production was more pronounced in the complement-binding reaction (CBR). Both types of the antibodies were detected simultaneously in 14 antisera. It is concluded that the CBR with the use of the penicillin complex antigenes on the stroma of the erythrocytes and in combination with the blood serum is a rather sensitive reaction for detection of antipenicillin antibodies.

Autoantibodies against Leydig cells in patients after spermatic cord torsion.

PubMed Central

Zanchetta, R; Mastrogiacomo, I; Graziotti, P; Foresta, C; Betterle, C

1984-01-01

This study is aimed at searching for the presence of circulating antibodies against frozen sections of human testis, ovary and trophoblast in patients that had spermatic cord torsion. Sixty-eight sera samples were studied. Nine patients (13.2%) were positive for organ specific anti-testis autoantibodies. Six patients were positive for antibodies against Leydig cells: five were positive only with the indirect immunofluorescence technique of complement fixing (ITT/CF), the sixth patient was positive only with the indirect immunofluorescence technique (ITT). The other three patients were positive for antibodies against germ line cells: two patients were positive with both techniques, the third was positive only with indirect immunofluorescence technique. Eight of these patients were negative for antibodies against adrenal cortex while only one case was positive with indirect immunofluorescence technique both on adrenal cortex and Leydig cells. Human lyophilized testis absorbed the reactive antibodies against Leydig cells and germ line cells, while adrenal cortex and lyophilized testosterone were ineffective. This study shows the identification of a specific antibody against Leydig cells and germ line cells in patients after spermatic cord torsion. PMID:6362937

Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

PubMed

Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

2016-01-01

Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

Complement deposition induced by binding of anti-contactin-1 auto-antibodies is modified by immunoglobulins.

PubMed

Appeltshauser, Luise; Weishaupt, Andreas; Sommer, Claudia; Doppler, Kathrin

2017-01-01

Inflammatory neuropathies associated with auto-antibodies against paranodal proteins like contactin-1 are reported to respond poorly to treatment with intravenous immunoglobulins (IVIG). A reason might be that IVIG interacts with the complement pathway and these auto-antibodies often belong to the IgG4 subclass that does not activate complement. However, some patients do show a response to IVIG, especially at the beginning of the disease. This corresponds with the finding of coexisting IgG subclasses IgG1, IgG2 and IgG3. We therefore aimed to investigate complement deposition and activation by samples of three patients with anti-contactin-1 IgG auto-antibodies of different subclasses as a potential predictor for response to IVIG. Complement deposition and activation was measured by cell binding and ELISA based assays, and the effect of IVIG on complement deposition was assessed by addition of different concentrations of IVIG. Binding of anti-contactin-1 auto-antibodies of all three patients induced complement deposition and activation with the strongest effect shown by the serum of a patient with predominance of IgG3 auto-antibodies. IVIG led to a reduction of complement deposition in a dose-dependent manner, but did not reduce binding of auto-antibodies to contactin-1. We conclude that complement deposition may contribute to the pathophysiology of anti-contactin-1 associated neuropathy, particularly in patients with predominance of the IgG3 subclass. The proportion of different auto-antibody subclasses may be a predictor for the response to IVIG in patients with auto-antibodies against paranodal proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

The in vivo mechanism of action of CD20 monoclonal antibodies depends on local tumor burden

PubMed Central

Boross, Peter; Jansen, J.H. Marco; de Haij, Simone; Beurskens, Frank J.; van der Poel, Cees E.; Bevaart, Lisette; Nederend, Maaike; Golay, Josée; van de Winkel, Jan G.J.; Parren, Paul W.H.I.; Leusen, Jeanette H.W.

2011-01-01

Background CD20 monoclonal antibodies are widely used in clinical practice. Antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct cell death have been suggested to be important effector functions for CD20 antibodies. However, their specific contributions to the in vivo mechanism of action of CD20 immunotherapy have not been well defined. Design and Methods Here we studied the in vivo mechanism of action of type I (rituximab and ofatumumab) and type II (HuMab-11B8) CD20 antibodies in a peritoneal, syngeneic, mouse model with EL4-CD20 cells using low and high tumor burden. Results Interestingly, we observed striking differences in the in vivo mechanism of action of CD20 antibodies dependent on tumor load. In conditions of low tumor burden, complement was sufficient for tumor killing both for type I and type II CD20 antibodies. In contrast, in conditions of high tumor burden, activating FcγR (specifically FcγRIII), active complement and complement receptor 3 were all essential for tumor killing. Our data suggest that complement-enhanced antibody-dependent cellular cytotoxicity may critically affect tumor killing by CD20 antibodies in vivo. The type II CD20 antibody 11B8, which is a poor inducer of complement activation, was ineffective against high tumor burden. Conclusions Tumor burden affects the in vivo mechanism of action of CD20 antibodies. Low tumor load can be eliminated by complement alone, whereas elimination of high tumor load requires multiple effector mechanisms. PMID:21880632

Pulmonary cytomegalovirus infection: detection by Gallium-67 imaging in the transplant patient

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

Hamed, I.A.; Wenzl, J.E.; Leonard, J.C.

1979-03-01

Cytomegalovirus (CMV) infection is a frequent complication during the first few months following renal transplantation. The diagnosis is sometimes difficult but may be made by viral culture, a fourfold rise in the CMV antibody titer, or by demonstration of the CMV inclusions in the affected tissue. An increased pulmonary uptake of gallium citrate Ga 67 has been demonstrated following renal transplantation in two patients, each of whom had a fourfold rise in CMV complement fixing antibody titer, one of whom additionally had CMV inclusion bodies in a lung biopsy specimen prior to clinical or radiological demonstration of the pulmonary involvement.more » Gallium imaging, therefore, appears to be a valuable noninvasive test for early diagnosis of CMV pulmonary infections.« less

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

PubMed

Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-08-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. © 2015 The Authors. American Journal of Transplantation Published by Wiley Periodicals, Inc.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA

PubMed Central

Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-01-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab′)2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. PMID:25904443

Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

PubMed

Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

2006-05-01

The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

PubMed

Winkler, Mark T; Bushey, Ryan T; Gottlin, Elizabeth B; Campa, Michael J; Guadalupe, Eross S; Volkheimer, Alicia D; Weinberg, J Brice; Patz, Edward F

2017-01-01

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

Determination of a Unique Epitope Binding Site for a Complement-Lysis- Enhancing Monoclonal Antibody, 3D12, on the Galactose Adherence Lectin of Entamoeba histolytica, Using BIAcore.

DTIC Science & Technology

1992-05-01

COMPLEMENT-LYSIS-ENHANCING MONOCLONAL ANTIBODY, 3D12, ON THE GALACTOSE ADHERENCE LECTIN OF ENTAMOEBA HISTOLYTICA, USING BIAcore Sheila J. Wood...Binding 5. FUNDING NUMBERS Site for a Complement-Lysis-Enhancing Monoclonal Antibody, 3D12, on the Galactose Adherence Lectin of Entamoeba Hiiutolitica...Mechani sms of pathogenicity used by Entamoeba histolytica to invade the bloodstream and cause liver abscess, include complement mediated lysis

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines.

PubMed

Kim, Kyoung Whun; Jeong, Soyoung; Ahn, Ki Bum; Yang, Jae Seung; Yun, Cheol-Heui; Han, Seung Hyun

2017-12-01

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.

Cobra venom factor immunoconjugates: effects of carbohydrate-directed versus amino group-directed conjugation.

PubMed

Zara, J; Pomato, N; McCabe, R P; Bredehorst, R; Vogel, C W

1995-01-01

Human IgM monoclonal antibody 16-88, derived from patients immunized with autologous colon carcinoma cells, was derivatized with two different cross-linkers, S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), which is carbohydrate-directed, and N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), which is amino group-directed. Two antibody functions, antigen binding and complement activation, were assayed upon derivatization with TPCH and SPDP. TPCH allowed for extensive modification (up to 17 TPCH molecules per antibody) without impairment of antigen binding activity, while this function was significantly compromised upon derivatization with SPDP. Antibody molecules derivatized with 16 SPDP residues showed almost complete loss of their antigen binding function. The complement activating ability of antibody 16-88 was significantly decreased after derivatization with TPCH or SPDP. In the case of SPDP derivatization, this decrease of the complement activating ability is predominantly a consequence of the impaired binding function. Upon conjugation of cobra venom factor (CVF), a nontoxic 137-kDa glycoprotein which is capable of activating the alternative pathway of complement, the antigen binding activity of SPDP-derivatized antibody was further compromised, whereas that of TPCH-derivatized antibody remained unaffected even after attachment of three or four CVF molecules per antibody. In both conjugates CVF retained good functional activity. CVF was slightly more active when attached to SPDP-derivatized antibody, suggesting a better accessibility of amino group-coupled CVF for its interaction with other complement proteins. These results indicate that carbohydrate-directed conjugation compromises the antibody function of complement activation, but allows for the generation of immunoconjugates with unimpaired antigen binding capability.(ABSTRACT TRUNCATED AT 250 WORDS)

NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways

PubMed Central

Yuen, Joshua; Pluthero, Fred G.; Douda, David N.; Riedl, Magdalena; Cherry, Ahmed; Ulanova, Marina; Kahr, Walter H. A.; Palaniyar, Nades; Licht, Christoph

2016-01-01

Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases. PMID:27148258

Immunity to human cytomegalovirus measured and compared by complement fixation, indirect fluorescent-antibody, indirect hemagglutination, and enzyme-linked immunosorbent assays.

PubMed Central

Brandt, J A; Kettering, J D; Lewis, J E

1984-01-01

The complement fixation test is currently the test employed most frequently to determine the presence of antibody to human cytomegalovirus. Several other techniques have been adapted for this purpose. A comparison of cytomegalovirus antibody titers was made between the complement fixation test, a commercially available enzyme-linked immunosorbent assay, an indirect immunofluorescent technique, and a modified indirect hemagglutination test. Forty-three serum samples were tested for antibodies by each of the above procedures. The enzyme-linked immunosorbent, immunofluorescent, and indirect hemagglutination assays were in close agreement on all samples tested; the titers obtained with these methods were all equal to or greater than the complement fixation titer for 38 of the 41 samples (92.6%). Two samples were anticomplementary in the complement fixation test but gave readable results in the other tests. The complement fixation test was the least sensitive of the procedures examined. The commercial enzyme-linked immunosorbent assay system was the most practical method and offered the highest degree of sensitivity in detecting antibodies to cytomegalovirus. PMID:6321544

Antibody mechanisms implicated in digestive disturbances following ingestion of soya protein in calves and piglets.

PubMed Central

Barratt, M E; Strachan, P J; Porter, P

1978-01-01

Serum antibody responses to ingested aqueous alcohol-extracted soya proteins were studied in thirty-six pre-ruminant calves. Characterization of this antibody showed it to be predominantly a complement-fixing IgG1 preciptin. No evidence of tolerance was seen; previously sensitized calves responded to reintroduction of a soya diet with marked increases in antibody levels. The soya antigen was shown to be resistant to proteolysis and, to a lesser degree, to the microbial action of rumen fluid. Biopsy studies showed that the feeding of soya protein resulted in morphological disturbances to the villi and lamina propria of the intestine. Physiological studies by Thirty-Vella loop perfusion in the pig showed that soya protein solutions resulted in significant inhibition of flow rates. The effect was only observed after previous sensitization with the soya antigen. This study shows the necessity of applying immunological criteria to the quality control of soya bean processing in order to ensure that the sensitizing agent is eliminated and the nutritional qualities of soya protein concentrates are optimized. Images FIG. 2a FIG. 2b PMID:565686

The role of the carbohydrate chains in complement (C3) fixation by solid-phase-bound human IgA.

PubMed Central

Nikolova, E B; Tomana, M; Russell, M W

1994-01-01

In contrast to antigen-antibody complexes containing native human IgA, solid-phase-deposited IgA activates the alternative complement pathway and binds C3b. To investigate the role of carbohydrate chains in this, various human IgA preparations were treated with neuraminidase alone or together with N-glycanase or O-glycanase, or with mixed glycosidases from the oral bacterium, Streptococcus mitis. Depletion of oligosaccharides was determined by carbohydrate analysis. Removal of sialic acid and N-linked glycan chains greatly increased the C3b-fixing properties of normal serum IgA1 and IgA2. Myeloma IgA1 and IgA2 proteins and secretory IgA had higher C3b-binding activity than normal serum IgA, and this was further increased by removal of sialic acid and N-linked glycans. Fc alpha and Fc alpha-SC fragments of myeloma and secretory IgA1, respectively, but not Fab alpha fragments, obtained by cleavage with bacterial IgA1 proteases and also free secretory component, fixed C3b by the alternative pathway. Images Figure 4 PMID:7927504

[Campylobacter jejuni infections in slaughterhouse workers].

PubMed

Mancinelli, S; Riccardi, F; Santi, A L; Palombi, L; Marazzi, M C

1988-01-01

Complement fixing (C.F.) antibodies to Campylobacter jejuni were detected in 83 slaughterhouse workers and 83 blood donors. Workers were aged 18-65 years (mean, 41.7 +/- 12.3) and had worked in the slaughterhouse for 2-40 years (mean, 17.5 +/- 5.1). C.F. antibodies were detected according to Mosimann's method and by including five antigens: Campylobacter jejuni, Yersinia enterocolitica types 03 and 09, Yersinia pseudotuberculosis and Brucella. Positive titers were found in 12.1% of workers and in 2.4% of control subjects (p less than 0.01); values ranged from 1:10 to 1:40. Frequent and close contact with animals or their products was significantly associated with seropositivity. No association was found with the time of employment. Sixty per cent of seropositive workers referred rheumatological symptoms. These findings confirm that slaughterhouse workers exposed to potential sources of C. jejuni have elevated titers of antibodies. Attention has, therefore, to be focused on breaking the chain of transmission as a means of control.

SUBCLINICAL INFECTION OF DOGS BY CANINE-ADAPTED MEASLES VIRUS EVIDENCED BY THEIR SUBSEQUENT IMMUNITY TO CANINE DISTEMPER VIRUS

PubMed Central

Moura, Roberto A.; Warren, Joel

1961-01-01

Moura, Roberto A. (Chas. Pfizer and Company, Inc., Terre Haute, Ind.) and Joel Warren. Subclinical infection of dogs by canine-adapted measles virus evidenced by their subsequent immunity to canine distemper virus. J. Bacteriol. 82:702–705. 1961.—Young dogs were inoculated with virulent measles virus which had been adapted to canine kidney or human amnion cell culture. None of the animals showed any clinical symptoms nor could virus be isolated from the blood, although measles-neutralizing and complement-fixing antibodies developed during convalescence. All dogs failed to develop antibody to canine distemper. However, when these and normal control animals were subsequently inoculated intracerebrally with virulent distemper virus, each of the controls succumbed to typical symptoms, whereas all of the measles-immune dogs survived. These results suggest that the cross-protection conferred by measles against canine distemper virus infection involves factors other than humoral antibody. The immunity persists for a considerable length of time. PMID:14476677

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

Delineating the Importance of Serum Opsonins and the Bacterial Capsule in Affecting the Uptake and Killing of Burkholderia pseudomallei by Murine Neutrophils and Macrophages

PubMed Central

Mulye, Minal; Bechill, Michael P.; Grose, William; Ferreira, Viviana P.; Lafontaine, Eric R.; Wooten, R. Mark

2014-01-01

Infection of susceptible hosts by the encapsulated Gram-negative bacterium Burkholderia pseudomallei (Bp) causes melioidosis, with septic patients attaining mortality rates ≥40%. Due to its high infectivity through inhalation and limited effective therapies, Bp is considered a potential bioweapon. Thus, there is great interest in identifying immune effectors that effectively kill Bp. Our goal is to compare the relative abilities of murine macrophages and neutrophils to clear Bp, as well as determine the importance of serum opsonins and bacterial capsule. Our findings indicate that murine macrophages and neutrophils are inherently unable to clear either unopsonized Bp or the relatively-avirulent acapsular bacterium B. thailandensis (Bt). Opsonization of Bp and Bt with complement or pathogen-specific antibodies increases macrophage-uptake, but does not promote clearance, although antibody-binding enhances complement deposition. In contrast, complement opsonization of Bp and Bt causes enhanced uptake and killing by neutrophils, which is linked with rapid ROS induction against bacteria exhibiting a threshold level of complement deposition. Addition of bacteria-specific antibodies enhances complement deposition, but antibody-binding alone cannot elicit neutrophil clearance. Bp capsule provides some resistance to complement deposition, but is not anti-phagocytic or protective against reactive oxygen species (ROS)-killing. Macrophages were observed to efficiently clear Bp only after pre-activation with IFNγ, which is independent of serum- and/or antibody-opsonization. These studies indicate that antibody-enhanced complement activation is sufficient for neutrophil-clearance of Bp, whereas macrophages are ineffective at clearing serum-opsonized Bp unless pre-activated with IFNγ. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis. PMID:25144195

Complement in Lupus Nephritis: New Perspectives.

PubMed

Bao, Lihua; Cunningham, Patrick N; Quigg, Richard J

2015-09-01

Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to self-antigens, the production of autoantibodies and deposition of complement-fixing immune complexes (ICs) in injured tissues. SLE is characterized by a wide range of clinical manifestations and targeted organs, with lupus nephritis being one of the most serious complications. The complement system consists of three pathways and is tightly controlled by a set of regulatory proteins to prevent injudicious complement activation on host tissue. The involvement of the complement system in the pathogenesis of SLE is well accepted; yet, its exact role is still not clear. Complement plays dual roles in the pathogenesis of SLE. On the one hand, the complement system appears to have protective features in that hereditary homozygous deficiencies of classical pathway components, such as C1q and C4, are associated with an increased risk for SLE. On the other hand, IC-mediated activation of complement in affected tissues is clearly evident in both experimental and human SLE along with pathological features that are logical consequences of complement activation. Studies in genetically altered mice have shown that lack of complement inhibitors, such as complement factor H (CFH) or decay-accelerating factor (DAF) accelerates the development of experimental lupus nephritis, while treatment with recombinant protein inhibitors, such as Crry-Ig, CR2-Crry, CR2-DAF and CR2-CFH, ameliorates the disease development. Complement-targeted drugs, including soluble complement receptor 1 (TP10), C1 esterase inhibitor and a monoclonal anti-C5 antibody (eculizumab), have been shown to inhibit complement safely, and are now being investigated in a variety of clinical conditions. SLE is an autoimmune disorder which targets multiple systems. Complement is centrally involved and plays dual roles in the pathogenesis of SLE. Studies from experimental lupus models and clinical trials support the use of complement-targeted therapy in the treatment of SLE.

Breaking down the complement system: a review and update on novel therapies.

PubMed

Reddy, Yuvaram N V; Siedlecki, Andrew M; Francis, Jean M

2017-03-01

The complement system represents one of the more primitive forms of innate immunity. It has increasingly been found to contribute to pathologies in the native and transplanted kidney. We provide a concise review of the physiology of the complement cascade, and discuss current and upcoming complement-based therapies. Current agents in clinical use either bind to complement components directly or prevent complement from binding to antibodies affixed to the endothelial surface. These include C1 esterase inhibitors, anti-C5 mAbs, anti-CD20 mAbs, and proteasome inhibitors. Treatment continues to show efficacy in the atypical hemolytic uremic syndrome and antibody-mediated rejection. Promising agents not currently available include CCX168, TP10, AMY-101, factor D inhibitors, coversin, and compstatin. Several new trials are targeting complement inhibition to treat antineutrophilic cystoplasmic antibody (ANCA)-associated vasculitis, C3 glomerulopathy, thrombotic microangiopathy, and IgA nephropathy. New agents for the treatment of the atypical hemolytic uremic syndrome are also in development. Complement-based therapies are being considered for targeted therapy in the atypical hemolytic uremic syndrome and antibody-mediated rejection, C3 glomerulopathy, and ANCA-associated vasculitis. A few agents are currently in use as orphan drugs. A number of other drugs are in clinical trials and, overall, are showing promising preliminary results.

Minimization of bacterial size allows for complement evasion and is overcome by the agglutinating effect of antibody

PubMed Central

Dalia, Ankur B.; Weiser, Jeffrey N.

2011-01-01

SUMMARY The complement system, which functions by lysing pathogens directly or by promoting their uptake by phagocytes, is critical for controlling many microbial infections. Here we show that in Streptococcus pneumoniae, increasing bacterial chain length sensitizes this pathogen to complement deposition and subsequent uptake by human neutrophils. Consistent with this, we show that minimizing chain length provides wild-type bacteria with a competitive advantage in vivo in a model of systemic infection. Investigating how the host overcomes this virulence strategy, we find that antibody promotes complement-dependent opsonophagocytic killing of Streptococcus pneumoniae and lysis of Haemophilus influenzae independent of Fc-mediated effector functions. Consistent with the agglutinating effect of antibody, F(ab′)2 but not Fab could promote this effect. Therefore, increasing pathogen size, whether by natural changes in cellular morphology or via antibody-mediated agglutination, promotes complement-dependent killing. These observations have broad implications for how cell size and morphology can affect virulence among pathogenic microbes. PMID:22100164

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

PubMed

Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

2016-06-15

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies. © 2016 UICC.

Complement in Non-Antibody-Mediated Kidney Diseases

PubMed Central

Angeletti, Andrea; Reyes-Bahamonde, Joselyn; Cravedi, Paolo; Campbell, Kirk N.

2017-01-01

The complement system is part of the innate immune response that plays important roles in protecting the host from foreign pathogens. The complement components and relative fragment deposition have long been recognized to be strongly involved also in the pathogenesis of autoantibody-related kidney glomerulopathies, leading to direct glomerular injury and recruitment of infiltrating inflammation pathways. More recently, unregulated complement activation has been shown to be associated with progression of non-antibody-mediated kidney diseases, including focal segmental glomerulosclerosis, C3 glomerular disease, thrombotic microangiopathies, or general fibrosis generation in progressive chronic kidney diseases. Some of the specific mechanisms associated with complement activation in these diseases were recently clarified, showing a dominant role of alternative activation pathway. Over the last decade, a growing number of anticomplement agents have been developed, and some of them are being approved for clinical use or already in use. Therefore, anticomplement therapies represent a realistic choice of therapeutic approaches for complement-related diseases. Herein, we review the complement system activation, regulatory mechanisms, their involvement in non-antibody-mediated glomerular diseases, and the recent advances in complement-targeting agents as potential therapeutic strategies. PMID:28748184

Guilty as charged: all available evidence implicates complement's role in fetal demise.

PubMed

Girardi, Guillermina

2008-03-01

Appropriate complement inhibition is an absolute requirement for normal pregancy. Uncontrolled complement activation in the maternal-fetal interface leads to fetal death. Here we show that complement activation is a crucial and early mediator of pregnancy loss in two different mouse models of pregnancy loss. Using a mouse model of fetal loss and growth restriction (IUGR) induced by antiphospholipid antibodies (aPL), we examined the role of complement activation in fetal loss and IUGR. We found that C5a-C5aR interaction and neutrophils are key mediators of fetal injury. Treatment with heparin, the standard therapy for pregnant patients with aPL, prevents complement activation and protects mice from pregnancy complications induced by aPL, and anticoagulants that do not inhibit complement do not protect pregnancies. In an antibody-independent mouse model of spontaneous miscarriage and IUGR (CBA/JxDBA/2) we also identified C5a as an essential mediator. Complement activation caused dysregulation of the angiogenic factors required for normal placental development. In CBA/JxDBA/2 mice, we observed inflammatory infiltrates in placentas, functional deficiency of free vascular endothelial growth factor (VEGF), elevated levels of soluble VEGF receptor-1 (sVEGFR-1, also known as sFlt-1; a potent anti-angiogenic molecule), and defective placental development. Inhibition of complement activation blocked the increase in sVEGFR-1 and rescued pregnancies. Our studies in antibody-dependent and antibody-independent models of pregnancy complications identified complement activation as the key mediator of damage and will allow development of new interventions to prevent pregnancy loss and IUGR.

Development of an Oral Barley Beta-Glucan Adjuvant That Augments the Tumoricidal Activity of Antibodies or Vaccines Used for the Immunotherapy of Breast Cancer

DTIC Science & Technology

2003-07-01

These Data provided strong evidence for the efficacy of an oral beta - glucan adjuvant for use in combination with anti-tumor antibodies such as...oral beta - glucan . The data showing that antibodies must activate complement and deposit iC3b on tumors means that antibodies that do not to activate complement would not benefit from oral beta - glucan .

Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

PubMed

Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2011-09-01

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

Activation of the classical pathway of complement by binding of bovine lactoferrin to unencapsulated Streptococcus agalactiae.

PubMed Central

Rainard, P

1993-01-01

The ability of lactoferrin (Lf) bound to Streptococcus agalactiae to interfere with the deposition of complement components on the bacterial surface was investigated by enzyme-linked immunosorbent assay (ELISA). By using a strain of S. agalactiae which activates the alternative pathway of complement in the absence of antibodies, it was found that pretreatment of bacteria with Lf shortened the lag phase preceding the deposition of C3 on bacteria. The kinetics of C3 deposition was comparable to that obtained by adding antibodies against S. agalactiae to agammaglobulinaemic precolostral calf serum (PCS) heated at 56 degrees for 3 min to inactivate the alternative pathway. Accelerated C3 deposition did not occur in the absence of Ca2+ ions. Deposition of C4 on bacteria occurred only when either antibodies or Lf were added to PCS. These results demonstrate that the interaction of lactoferrin with bacteria activated the classical pathway of complement in the absence of antibodies. The binding of purified C1q to bacteria was promoted in a dose-dependent manner by Lf, suggesting that recruitment of classical pathway of complement resulted from the interaction of C1q with Lf adsorbed to the bacterial surface. Phagocytosis of bacteria opsonized with heated PCS (at 56 degrees for 3 min) and Lf was comparable to that occurring in the presence of heated PCS and antibodies. In conclusion, Lf was able to substitute for antibodies in order to activate the classical pathway of complement and to opsonize unencapsulated S. agalactiae efficiently. PMID:8406591

Antibodies to PcpA and PhtD protect mice against Streptococcus pneumoniae by a macrophage- and complement-dependent mechanism.

PubMed

Visan, Lucian; Rouleau, Nicolas; Proust, Emilie; Peyrot, Loïc; Donadieu, Arnaud; Ochs, Martina

2018-02-01

Currently marketed Streptococcus pneumoniae (Spn) vaccines, which contain polysaccharide capsular antigens from the most common Spn serotypes, have substantially reduced pneumococcal disease rates but have limited coverage. A trivalent pneumococcal protein vaccine containing pneumococcal choline-binding protein A (PcpA), pneumococcal histidine triad protein D (PhtD), and detoxified pneumolysin is being developed to provide broader, cross-serotype protection. Antibodies against detoxified pneumolysin protect against bacterial pneumonia by neutralizing Spn-produced pneumolysin, but how anti-PhtD and anti-PcpA antibodies protect against Spn has not been established. Here, we used a murine passive protection sepsis model to investigate the mechanism of protection by anti-PhtD and anti-PcpA antibodies. Depleting complement using cobra venom factor eliminated protection by anti-PhtD and anti-PcpA monoclonal antibodies (mAbs). Consistent with a requirement for complement, complement C3 deposition on Spn in vitro was enhanced by anti-PhtD and anti-PcpA mAbs and by sera from PhtD- and PcpA-immunized rabbits and humans. Moreover, in the presence of complement, anti-PhtD and anti-PcpA mAbs increased uptake of Spn by human granulocytes. Depleting neutrophils using anti-Ly6G mAbs, splenectomy, or a combination of both did not affect passive protection against Spn, whereas depleting macrophages using clodronate liposomes eliminated protection. These results suggest anti-PhtD and anti-PcpA antibodies induced by pneumococcal protein vaccines protect against Spn by a complement- and macrophage-dependent opsonophagocytosis.

Radiobacteriolysis: a New Technique Using Chromium-51 for Assaying Anti- Vibrio cholerae Antibodies

PubMed Central

Blachman, Uzy; Clark, W. R.; Pickett, M. J.

1973-01-01

A new method for detecting and quantitating antibodies against Vibrio cholerae is described. The reaction involves the release of radiochromium from prelabeled vibrios in the presence of specific antibody and complement. The entire assay can be completed within 5 hr. The method is highly reproducible, immunologically specific, temperature- and complement-dependent, and significantly more sensitive than other methods currently used for titration of anti-Vibrio cholerae antibodies. The technique is also potentially applicable to titration of antibodies against other gram-negative bacteria. PMID:4570279

Complement fixation test to C burnetii

MedlinePlus

... complement fixation test; Coxiella burnetii - complement fixation test; C burnetii - complement fixation test ... a specific foreign substance ( antigen ), in this case, C burnetii . Antibodies defend the body against bacteria, viruses, ...

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.

Protection by phospholipids of Schistosoma mansoni schistosomula against the action of cytotoxic antibodies and complement.

PubMed

Billecocq, A

1987-09-01

Schistosoma mansoni schistosomula cultured in the presence of phospholipids showed a decreased sensitivity to the lethal complement-mediated action of anti-schistosome antibodies. Phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine had a protective action on the schistosomula transformed in vitro by passage through the skin or by a mechanical procedure. Phosphatidyl choline acted regardless of its fatty acid composition. Phosphatidyl serine and phosphatidic acid did not protect. Thus, it appears that phospholipids can play a role in parasite resistance to immune attack by cytotoxic antibodies and complement, and that this role is specific to certain phospholipid types.

Autoantibodies against complement components in systemic lupus erythematosus - role in the pathogenesis and clinical manifestations.

PubMed

Hristova, M H; Stoyanova, V S

2017-12-01

Many complement structures and a number of additional factors, i.e. autoantibodies, receptors, hormones and cytokines, are implicated in the complex pathogenesis of systemic lupus erythematosus. Genetic defects in the complement as well as functional deficiency due to antibodies against its components lead to different pathological conditions, usually clinically presented. Among them hypocomplementemic urticarial vasculitis, different types of glomerulonephritis as dense deposit disease, IgA nephropathy, atypical haemolytic uremic syndrome and lupus nephritis are very common. These antibodies cause conformational changes leading to pathological activation or inhibition of complement with organ damage and/or limited capacity of the immune system to clear immune complexes and apoptotic debris. Finally, we summarize the role of complement antibodies in the pathogenesis of systemic lupus erythematosus and discuss the mechanism of some related clinical conditions such as infections, thyroiditis, thrombosis, acquired von Willebrand disease, etc.

Effect of complement and its regulation on myasthenia gravis pathogenesis

PubMed Central

Kusner, Linda L; Kaminski, Henry J; Soltys, Jindrich

2015-01-01

Myasthenia gravis (MG) is primarily caused by antibodies directed towards the skeletal muscle acetylcholine receptor, leading to muscle weakness. Although these antibodies may induce compromise of neuromuscular transmission by blocking acetylcholine receptor function or antigenic modulation, the predominant mechanism of injury to the neuromuscular junction is complement-mediated lysis of the postsynaptic membrane. The vast majority of data to support the role of complement derives from experimentally acquired MG (EAMG). In this article, we review studies that demonstrate the central role of complement in EAMG and MG pathogenesis along with the emerging role of complement in T- and B-cell function, as well as the potential for complement inhibitor-based therapy to treat human MG. PMID:20477586

Complement and the control of HIV infection: an evolving story.

PubMed

Frank, Michael M; Hester, Christopher; Jiang, Haixiang

2014-05-01

Thirty years ago, investigators isolated and later determined the structure of HIV-1 and its envelope proteins. Using techniques that were effective with other viruses, they prepared vaccines designed to generate antibody or T-cell responses, but they were ineffective in clinical trials. In this article, we consider the role of complement in host defense against enveloped viruses, the role it might play in the antibody response and why complement has not controlled HIV-1 infection. Complement consists of a large group of cell-bound and plasma proteins that are an integral part of the innate immune system. They provide a first line of defense against microbes and also play a role in the immune response. Here we review the studies of complement-mediated HIV destruction and the role of complement in the HIV antibody response. HIV-1 has evolved a complex defense to prevent complement-mediated killing reviewed here. As part of these studies, we have discovered that HIV-1 envelope, on administration into animals, is rapidly broken down into small peptides that may prove to be very inefficient at provident the type of antigenic stimulation that leads to an effective immune response. Improving complement binding and stabilizing envelope may improve the vaccine response.

Demonstration of PDC-E1 subunits as major antigens in the complement-fixing fraction M4 and re-evaluation of PDC-E1-specific antibodies in PBC patients.

PubMed

Berg, Christoph P; Stein, Gerburg M; Klein, Reinhild; Pascu, Maria; Berg, Thomas; Kammer, Winfried; Priemer, Martin; Nordheim, Alfred; Schulze-Osthoff, Klaus; Gregor, Michael; Wesselborg, Sebastian; Berg, Peter A

2006-09-01

Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen. M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive (n=5) or negative (n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC). Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2(+)/E1(+) and the CFT-negative sera as PDC-E2(+)/E1(-). The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1alpha and E1beta antibodies at a frequency of 74% and 67%. CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target.

Subepidermal Blistering Induced by Human Autoantibodies to BP180 Requires Innate Immune Players in a Humanized Bullous Pemphigoid Mouse Model

PubMed Central

Liu, Zhi; Sui, Wen; Zhao, Minglang; Li, Zhuowei; Li, Ning; Thresher, Randy; Giudice, George J.; Fairley, Janet A.; Sitaru, Cassian; Zillikens, Detlef; Ning, Gang; Marinkovich, Peter; Diaz, Luis A.

2008-01-01

Bullous pemphigoid (BP) is a cutaneous autoimmune inflammatory disease associated with subepidermal blistering and autoantibodies against BP180, a transmembrane collagen and major component of the hemidesmosome. Numerous inflammatory cells infiltrate the upper dermis in BP. IgG autoantibodies in BP fix complement and target multiple BP180 epitopes that are highly clustered within a non-collagen linker domain, termed NC16A. Anti-BP180 antibodies induce BP in mice. In this study, we generated a humanized mouse strain, in which the murine BP180NC14A is replaced with the homologous human BP180NC16A epitope cluster region. We show that the humanized NC16A (NC16A+/+) mice injected with anti-BP180NC16A autoantibodies develop BP-like subepidermal blisters. The F(ab′)2 fragments of pathogenic IgG fail to activate complement cascade and are no longer pathogenic. The NC16A+/+ mice pretreated with mast cell activation blocker or depleting of complement or neutrophils become resistant to BP. These findings suggest that the humoral response in BP critically depends on innate immune system players. PMID:18922680

Relative susceptibility of Giardia muris trophozoites to killing by mouse antibodies of different isotypes.

PubMed

Heyworth, M F

1992-02-01

The aim of this work was to examine the ability of mouse IgA, IgG, and IgM anti-Giardia antibodies to kill Giardia muris trophozoites in the presence and absence of complement. Using a 2-color flow cytometry assay, binding of antibody to trophozoites was assessed with fluorescein-conjugated anti-mouse immunoglobulin, and percentages of killed trophozoites were quantified by staining with propidium iodide. Trophozoites were killed in the presence of complement by IgG3 and IgM anti-trophozoite monoclonal antibodies. Anti-trophozoite IgA, obtained from the intestinal lumen of G. muris-infected BALB/c mice, became bound to trophozoites in vitro but did not kill these organisms in the presence or absence of complement. The results suggest that clearance of G. muris infection by intestinal IgA directed against G. muris trophozoites does not involve antibody-dependent killing of trophozoites in the intestinal lumen.

Molluskan Hemocyanins Activate the Classical Pathway of the Human Complement System through Natural Antibodies

PubMed Central

Pizarro-Bauerle, Javier; Maldonado, Ismael; Sosoniuk-Roche, Eduardo; Vallejos, Gerardo; López, Mercedes N.; Salazar-Onfray, Flavio; Aguilar-Guzmán, Lorena; Valck, Carolina; Ferreira, Arturo; Becker, María Inés

2017-01-01

Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a Th1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropod Megathura crenulata [keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities: Fisurella latimarginata hemocyanin (FLH) and Concholepas concholepas hemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting, glycosylation status had a limited effect. Finally, our data open possibilities for further studies leading to the design of improved hemocyanin-based research tools for diagnosis and immunotherapy. PMID:28286504

Molluskan Hemocyanins Activate the Classical Pathway of the Human Complement System through Natural Antibodies.

PubMed

Pizarro-Bauerle, Javier; Maldonado, Ismael; Sosoniuk-Roche, Eduardo; Vallejos, Gerardo; López, Mercedes N; Salazar-Onfray, Flavio; Aguilar-Guzmán, Lorena; Valck, Carolina; Ferreira, Arturo; Becker, María Inés

2017-01-01

Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a T h 1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropod Megathura crenulata [keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities: Fisurella latimarginata hemocyanin (FLH) and Concholepas concholepas hemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting, glycosylation status had a limited effect. Finally, our data open possibilities for further studies leading to the design of improved hemocyanin-based research tools for diagnosis and immunotherapy.

Immunization with a Vaccine Combining Herpes Simplex Virus 2 (HSV-2) Glycoprotein C (gC) and gD Subunits Improves the Protection of Dorsal Root Ganglia in Mice and Reduces the Frequency of Recurrent Vaginal Shedding of HSV-2 DNA in Guinea Pigs Compared to Immunization with gD Alone ▿

PubMed Central

Awasthi, Sita; Lubinski, John M.; Shaw, Carolyn E.; Barrett, Shana M.; Cai, Michael; Wang, Fushan; Betts, Michael; Kingsley, Susan; DiStefano, Daniel J.; Balliet, John W.; Flynn, Jessica A.; Casimiro, Danilo R.; Bryan, Janine T.; Friedman, Harvey M.

2011-01-01

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation. PMID:21813597

Pneumococcal polysaccharides complexed with C3d bind to human B lymphocytes via complement receptor type 2.

PubMed Central

Griffioen, A W; Rijkers, G T; Janssens-Korpela, P; Zegers, B J

1991-01-01

The immunoregulatory function of the complement system has been the focus of many investigations. In particular, fragments of complement factor C3 have been shown to play a role in B-lymphocyte activation and proliferation, lymphokine production, and the generation of in vitro antibody production. Purified pneumococcal polysaccharides (PS) can induce direct activation of C3 via the alternative pathway. Using sera of C1q-deficient patients and healthy subjects, we demonstrated that C3d, a split product of C3 that is generated after degradation of iC3b, can be bound to PS antigens. The binding of C3d to PS can occur in the absence of specific antibodies. Subsequently, we showed that PS complexed with C3d can be recognized by complement receptor type 2 that is expressed on B cells. Treatment of B cells with a monoclonal antibody recognizing the C3d-binding site of complement receptor type 2 reduces the binding of PS-C3d to the cells. In addition, we showed that PS4 complexed with C3d exerted an increased immunogenicity compared with free PS4. Our results show that the complement system plays a role in the activation of PS-specific B cells, carrying membrane receptors for C3d. Consequently, the complement system plays a regulatory role in the antibody response to T-cell-independent type 2 antigens such as PS. PMID:1826897

Immunologic and hematologic responses in ponies with experimentally induced Strongylus vulgaris infection.

PubMed

Bailey, M; Martin, S C; Lloyd, S

1989-08-01

Immunologic and hematologic responses were examined in 4 ponies with experimentally induced Strongylus vulgaris infection and in 5 helminth-free ponies. Two ponies were inoculated with 200 larvae and 2 were inoculated with 700 larvae of S vulgaris and then were reinoculated with the same numbers of larvae 34 weeks later. Initial response of the ponies inoculated with S vulgaris was S vulgaris antigen-induced lymphocyte response that developed 1.5 to 3 weeks after inoculation and did not persist. Development of antigen-reactive lymphocytes was followed sequentially by a biphasic complement-fixing antibody response, then biphasic eosinophilia. Antibody titer to S vulgaris antigen was higher in ponies inoculated with 700 larvae, compared with that in ponies given 200 larvae of S vulgaris. Also, the second peak in antibody titer and in absolute number of eosinophils was observed earlier in ponies inoculated with 700 larvae, compared with ponies inoculated with 200 S vulgaris larvae, and subsided before or from about 24 weeks after inoculation. The prepatent period for S vulgaris infection was 24 to 25 weeks. After reinoculation with S vulgaris, a degree of increased lymphocyte responsiveness was apparent but, by 17 weeks after reinoculation, only the primary peak in the absolute number of eosinophils indicated an anamnestic response. Essentially, antibody was not detectable after reinoculation.

Ly6G-mediated depletion of neutrophils is dependent on macrophages.

PubMed

Bruhn, Kevin W; Dekitani, Ken; Nielsen, Travis B; Pantapalangkoor, Paul; Spellberg, Brad

2016-01-01

Antibody-mediated depletion of neutrophils is commonly used to study neutropenia. However, the mechanisms by which antibodies deplete neutrophils have not been well defined. We noticed that mice deficient in complement and macrophages had blunted neutrophil depletion in response to anti-Ly6G monoclonal antibody (MAb) treatment. In vitro, exposure of murine neutrophils to anti-Ly6G MAb in the presence of plasma did not result in significant depletion of cells, either in the presence or absence of complement. In vivo, anti-Ly6G-mediated neutrophil depletion was abrogated following macrophage depletion, but not complement depletion, indicating a requirement for macrophages to induce neutropenia by this method. These results inform the use and limitations of anti-Ly6G antibody as an experimental tool for depleting neutrophils in various immunological settings.

Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation.

PubMed

Yu, Feifan; Alesand, Veronica; Nygren, Per-Åke

2018-02-27

Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antibody-Mediated Complement C3b/iC3b Binding to Group B Streptococcus in Paired Mother and Baby Serum Samples in a Refugee Population on the Thailand-Myanmar Border

PubMed Central

Herbert, Jenny; Thomas, Stephen; Brookes, Charlotte; Turner, Claudia; Turner, Paul; Nosten, Francois; Le Doare, Kirsty; Hudson, Michael; Heath, Paul T.; Gorringe, Andrew

2015-01-01

Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal sepsis and meningitis. In this study, we determined antibody-mediated deposition of complement C3b/iC3b onto the bacterial cell surface of GBS serotypes Ia, Ib, II, III, and V. This was determined for 520 mother and umbilical cord serum sample pairs obtained at the time of birth from a population on the Thailand-Myanmar border. Antibody-mediated deposition of complement C3b/iC3b was detected to at least one serotype in 91% of mothers, despite a known carriage rate in this population of only 12%. Antibody-mediated C3b/iC3b deposition corresponded to known carriage rates, with the highest levels of complement deposition observed onto the most prevalent serotype (serotype II) followed by serotypes Ia, III, V, and Ib. Finally, neonates born to mothers carrying serotype II GBS at the time of birth showed higher antibody-mediated C3b/iC3b deposition against serotype II GBS than neonates born to mothers with no serotype II carriage. Assessment of antibody-mediated C3b/iC3b deposition against GBS may provide insights into the seroepidemiology of anti-GBS antibodies in mothers and infants in different populations. PMID:25589553

Anti-GK1 antibodies damage Taenia crassiceps cysticerci through complement activation.

PubMed

Núñez, Guadalupe; Villalobos, Nelly; Herrera, Cinthia P; Navarrete-Perea, José; Méndez, Adriana; Martinez-Maya, José J; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Aguilar, Laura; Del Arenal, Irene P

2018-06-06

Taeniasis-cysticercosis, a zoonosis caused by Taenia solium, is prevalent in underdeveloped countries, where marginalization promotes its continued transmission. Pig cysticercosis, an essential stage for transmission, is preventable by vaccination. An efficient multiepitope vaccine against pig cysticercosis, S3Pvac, was developed. Previous studies showed that antibodies against one of the S3Pvac components, GK-1, are capable of damaging T. solium cysticerci, inhibiting their ability to transform into the adult stage in golden hamster gut. This study is aimed to evaluate one of the mechanisms that could mediate anti-GK-1 antibody-dependent protection. To this end, pig anti-GK-1 antibodies were produced and purified by using protein A. Proteomic analysis showed that the induced antibodies recognized the respective native cysticercal protein KE7 (Bobes et al. Infect Immun 85:e00395-17, 2017) and two additional T. solium proteins (endophilin B1 and Gp50). A new procedure to evaluate cysticercus viability, based on quantifying the cytochrome c released after parasite damage, was developed. Taenia crassiceps cysticerci were cultured in the presence of differing amounts of anti-GK-1 antibody and complement in a saturating concentration, along with the respective controls. Cysticercus viability was assessed by recording parasite motility, trypan blue exclusion, and cytochrome c levels in cysticercal soluble extract. Anti-GK-1 antibody significantly increased cysticercus damage as measured by all three methods. Parasite evaluation by electron microscopy after treatment with anti-GK-1 antibody plus complement demonstrated cysticercus damage as shorter, capsule-severed microtrichia; a decrease in glycocalyx length with respect to untreated cysts; and disaggregated desmosomes. These results demonstrate that anti-GK-1 antibodies damage cysticerci through classic complement activation.

Switch from hapten-specific immunoglobulin M to immunoglobulin D secretion in a hybrid mouse cell line.

PubMed Central

Neuberger, M S; Rajewsky, K

1981-01-01

From a hybrid mouse cell line (B1-8) that secreted an IgM, lambda 1 anti-(4-hydroxy-3-nitrophenyl)acetyl antibody but that had no detectable surface IgM, selection for a variant with lambda 1 chains on the surface resulted in the isolation of a line that had switched from mu to delta expression. The surface and secreted Igs of this line were typed as IgD with two monoclonal antibodies, and the parental IgM and variant IgD molecules carried the same variable regions as judged by hapten-binding and idiotypic analysis. The surface and secreted delta chains of the IgD variant have apparent molecular weights of 64,000 and 61,000, respectively. However, the unglycosylated secreted delta polypeptide chain has a molecular weight of only 44,000. The secreted IgD exists predominantly in the delta 2 lambda A2 form, does not contain J protein, is relatively stable in serum, and does not fix complement. Images PMID:6940132

Congenital Chagas's disease in an urban population: investigation of infected twins.

PubMed

Hoff, R; Mott, K E; Milanesi, M L; Bittencourt, A L; Barbosa, H S

1978-01-01

In the Nordeste de Amaralina suburb of Salvador Bahia, Brazil, 47 of 285 pregnant women surveyed had complement fixing antibodies to Trypanosoma cruzi. At delivery T. cruzi was detected in one of 17 placentas from the sero-positive women. The offspring of this case were premature twins and T. cruzi was detected in the peripheral blood of each before death. At autopsy the gastro-intestinal tract and urinary bladder of both were severely affected. Immunofluorescence tests on cord sera, including the single case with T. cruzi in the placenta, were negative for IgM antibodies to T. cruzi. The mother of the infected twins and three of her living children, who were born and have resided in the city, were also infected with T. cruzi. Although the children had visited an area endemic for Chagas's disease for short periods, the mode of transmission in this family may have been transplacental. The value of the immunofluorescence test in the diagnosis of congenital Chagas's disease is discussed.

The effects of antibodies on cells

PubMed Central

Dumonde, D. C.; Bitensky, Lucille; Cunningham, G. J.; Chayen, J.

1965-01-01

Biochemical and histochemical methods were used to study the interaction of antibodies and complement with mouse Ehrlich ascites tumour cells. In the presence of complement, both iso- and hetero-antibodies caused cell lysis with penetration of antibodies into the damaged cells, as detected by immunofluorescence; the cells were then unable to support aerobic glycolysis though they retained their ability to consume oxygen in the presence of succinate. Under these conditions there was unmasking of phospholipid particularly at the cell surface, together with lysosomal changes resulting in diffuse staining for lysosomal acid—phosphatase. In the absence of complement, antibodies did not appear to penetrate the cells which respired normally and were not lysed. However, in these cells there was intense lysosomal activation accompanied by unmasking of cytoplasmic phospholipid; it appeared that an immune reaction confined to the cell surface was able to induce changes in the cytoplasm without acutely impairing the viability of the cell. ImagesFIGS. 1-8FIGS. 9-14 PMID:14245309

C1q complement component and -antibodies reflect SLE activity and kidney involvement.

PubMed

Horák, P; Hermanová, Z; Zadrazil, J; Ciferská, H; Ordeltová, M; Kusá, L; Zurek, M; Tichý, T

2006-07-01

The role of the complement system in the pathogenesis of systemic diseases is very ambivalent. In systemic lupus erythematosus (SLE), many abnormalities in the activation of the complement system have been reported. The most important antibodies formed against the complement system in SLE are the ones associated with the C1q component. The aim of this study was to assess separately the anti-C1q antibodies and C1q component in the serum from 65 patients with SLE, then in individuals with (n=33) and without (n=32) lupus nephritis and with active (n=36) and nonactive (n=29) form of the disease (European Consensus Lupus Activity Measurement, ECLAM>3, ECLAM

Binding of Free and Immune Complex-Associated Hepatitis C Virus to Erythrocytes Is Mediated by the Complement System.

PubMed

Salam, Kazi Abdus; Wang, Richard Y; Grandinetti, Teresa; De Giorgi, Valeria; Alter, Harvey J; Allison, Robert D

2018-05-09

Erythrocytes bind circulating immune complexes (IC) and facilitate IC clearance from the circulation. Chronic hepatitis C virus (HCV) infection is associated with IC-related disorders. In this study we investigated the kinetics and mechanism of HCV and HCV-IC binding to and dissociation from erythrocytes. Cell culture-produced HCV was mixed with erythrocytes from healthy blood donors and erythrocyte-associated virus particles were quantified. Purified complement proteins, complement-depleted serum, and complement receptor antibodies were used to investigate complement-mediated HCV-erythrocyte binding. Purified HCV-specific immunoglobulin G from a chronic HCV-infected patient was used to study complement-mediated HCV-IC-erythrocyte binding. Binding of HCV to erythrocytes increased 200 to 1,000 fold after adding complement active human serum in the absence of antibody. Opsonization of free HCV occurred within 10 minutes and peak binding to erythrocytes was observed at 20-30 minutes. Complement protein C1 was required for binding, while C2, C3 and C4 significantly enhanced binding. Complement receptor 1 (CR1, CD35) antibodies blocked the binding of HCV to erythrocytes isolated from chronically infected HCV patients and healthy blood donors. HCV-ICs significantly enhanced complement-mediated binding to erythrocytes compared to unbound HCV. Dissociation of complement-opsonized HCV from erythrocytes depended on the presence of Factor I. HCV released by Factor I bound preferentially to CD19+ B cells compared to other leukocytes. These results demonstrate that complement mediates the binding of free and IC-associated HCV to CR1 on erythrocytes, and provide a mechanistic rationale for investigating the differential phenotypic expression of HCV-IC-related disease. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

A randomized, first-in-human, healthy volunteer trial of BIVV009, a humanized antibody for the specific inhibition of the classical complement pathway.

PubMed

Bartko, Johann; Schoergenhofer, Christian; Schwameis, Michael; Firbas, Christa; Beliveau, Martin; Chang, Colin; Marier, Jean-Francois; Nix, Darrell; Gilbert, James C; Panicker, Sandip; Jilma, Bernd

2018-05-08

Aberrant activation of the classical complement pathway is the common underlying pathophysiology of orphan diseases such as bullous pemphigoid, antibody-mediated rejection of organ transplants, cold agglutinin disease and warm autoimmune haemolytic anaemia. Therapeutic options for these complement-mediated disorders are limited and BIVV009, a humanized monoclonal antibody directed against complement factor C1s, may be potentially useful for inhibition of the classical complement pathway. A phase-1, first-in-human, double-blind, randomized, placebo-controlled, dose-escalation trial of single and multiple doses of BIVV009 or placebo was conducted in 64 volunteers to evaluate safety, tolerability, pharmacokinetic, and pharmacodynamic profiles. Single and multiple infusions of BIVV009 were well tolerated without any safety concerns. BIVV009 exhibited a steep concentration-effect relationship with a Hill coefficient of 2.4, and an IC90 of 15.5 µg/mL. This study establishes the foundation for using BIVV009 as a highly selective inhibitor of the classical complement pathway in different diseases. This article is protected by copyright. All rights reserved. © 2018 American Society for Clinical Pharmacology and Therapeutics.

Acute energy deprivation in man: effect on serum immunoglobulins antibody response, complement factors 3 and 4, acute phase reactants and interferon-producing capacity of blood lymphocytes.

PubMed Central

Palmblad, J; Cantell, K; Holm, G; Norberg, R; Strander, H; Sunblad, L

1977-01-01

The effects of 10 days of total energy deprivation on serum levels of immunoglobulins, antibodies acute phase reactants and on interferon production were evaluated in fourteen healthy, normal-weight males. A significant depression was noted of the serum levels of complement factor 3, haptoglobin and orosomucoid. The titres of mercaptoethanol-sensitive specific antibodies to flagellin were higher in the subjects inoculated at the end of the starvation period than in controls and those inoculated at the start of the period. The serum levels of IgG, IgM, IgA, IgE, alpha-1-antitrypsin and complement factor 4, and the interferon-producing capacity of blood lymphocytes, were not changed. Thus, 10 days of total energy deprivation depresses the serum levels of several acute phase reactants and re-feeding may enhance antibody production. PMID:606438

[Possibilities of differentiation of antinuclear antibodies].

PubMed

Müller, W; Rosenthal, M; Stojan, B

1975-10-15

Antinuclear antibodies can give diagnostic informations according to their titre values, the belonging to different classes of immune globulins and on the basis of different patterns of immunofluorescence connection. The determination of granulocyte-specific antibodies which frequently appear in progressive chronic polyarthritis further contributes to the differential-diagnostic classification of diseases of the connective tissue. An antibody against extractable nuclear antigen is specific for the so-called mixed connective tissue disease, an antimitochondrial antibody for the pseudo-LE-syndrome. Moreover, the own examinations resulted in a particularly high and frequent ability of complement fixation of the antinuclear factors in systematic lupus erythematosus and sclerodermy. In contrast to this in the progressive chronic polyarthritis the complement fixation was clearly more insignificant.

STUDIES ON THE ANTIGENIC PROPERTIES OF COMPLEMENT

PubMed Central

Klein, Paul G.; Burkholder, Peter M.

1960-01-01

Sheep erythrocytes sensitized with amboceptor and persensitized thereafter with guinea pig complement are agglutinated by rabbit anti-guinea pig globulin and by immune sera obtained by injection of rabbits with fixed complement. In this agglutination neither C'1 nor C'2 takes part. Fixed C'4 acts as an agglutinogen. An additional agglutinogen, distinct from C'4, was found on persensitized cells. This additional agglutinogen appears to be distinct from hemolytically active C'3. PMID:14409703

Methanol extract of grain dust shows complement fixing activity and other characteristics similar to tannic acid.

PubMed Central

Skea, D; Broder, I

1986-01-01

We have found several similarities between tannic acid and grain dust extract prepared with methanol. Both formed a precipitate with IgG, and these interactions were inhibited by albumin. In addition, both preparations fixed complement; this activity was heat stable and was removed by prior adsorption of the preparations with hide powder. Adsorption with polyvinyl polypyrrolidone reduced the complement-fixing activity of tannic acid but not that of the methanol grain dust extract. The similarities between tannic acid and the methanol grain dust extract are consistent with the presence of a tannin or tanninlike material in grain dust. Images FIGURE 1. PMID:3709479

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

PubMed Central

Wright, J K; Tschopp, J; Jaton, J C; Engel, J

1980-01-01

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability. Images Fig. 2. PMID:6985362

Antibody mediated rejection associated with complement factor h-related protein 3/1 deficiency successfully treated with eculizumab.

PubMed

Noone, D; Al-Matrafi, J; Tinckam, K; Zipfel, P F; Herzenberg, A M; Thorner, P S; Pluthero, F G; Kahr, W H A; Filler, G; Hebert, D; Harvey, E; Licht, C

2012-09-01

Antibody mediated rejection (AMR) activates the classical complement pathway and can be detrimental to graft survival. AMR can be accompanied by thrombotic microangiopathy (TMA). Eculizumab, a monoclonal C5 antibody prevents induction of the terminal complement cascade (TCC) and has recently emerged as a therapeutic option for AMR. We present a highly sensitized 13-year-old female with end-stage kidney disease secondary to spina bifida-associated reflux nephropathy, who developed severe steroid-, ATG- and plasmapheresis-resistant AMR with TMA 1 week post second kidney transplant despite previous desensitization therapy with immunoglobulin infusions. Eculizumab rescue therapy resulted in a dramatic improvement in biochemical (C3; creatinine) and hematological (platelets) parameters within 6 days. The patient was proven to be deficient in complement Factor H-related protein 3/1 (CFHR3/1), a plasma protein that regulates the complement cascade at the level of C5 conversion and has been involved in the pathogenesis of atypical hemolytic uremic syndrome caused by CFH autoantibodies (DEAP-HUS). CFHR1 deficiency may have worsened the severe clinical progression of AMR and possibly contributed to the development of donor-specific antibodies. Thus, screening for CFHR3/1 deficiency should be considered in patients with severe AMR associated with TMA. © Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.

Update on hemolytic uremic syndrome: Diagnostic and therapeutic recommendations

PubMed Central

Salvadori, Maurizio; Bertoni, Elisabetta

2013-01-01

Hemolytic uremic syndrome (HUS) is a rare disease. In this work the authors review the recent findings on HUS, considering the different etiologic and pathogenetic classifications. New findings in genetics and, in particular, mutations of genes that encode the complement-regulatory proteins have improved our understanding of atypical HUS. Similarly, the complement proteins are clearly involved in all types of thrombotic microangiopathy: typical HUS, atypical HUS and thrombotic thrombocytopenic purpura (TTP). Furthermore, several secondary HUS appear to be related to abnormalities in complement genes in predisposed patients. The authors highlight the therapeutic aspects of this rare disease, examining both “traditional therapy” (including plasma therapy, kidney and kidney-liver transplantation) and “new therapies”. The latter include anti-Shiga-toxin antibodies and anti-C5 monoclonal antibody “eculizumab”. Eculizumab has been recently launched for the treatment of the atypical HUS, but it appears to be effective in the treatment of typical HUS and in TTP. Future therapies are in phases I and II. They include anti-C5 antibodies, which are more purified, less immunogenic and absorbed orally and, anti-C3 antibodies, which are more powerful, but potentially less safe. Additionally, infusions of recombinant complement-regulatory proteins are a potential future therapy. PMID:24255888

Experimental infection of horses with an attenuated Venezuelan equine encephalomyelitis vaccine (strain TC-83).

PubMed

Walton, T E; Alvarez, O; Buckwalter, R M; Johnson, K M

1972-05-01

Ten horses (Equus caballus) were vaccinated with strain TC-83 Venezuelan equine encephalomyelitis (VEE) virus vaccine. Febrile responses and leukopenia due to a reduction of lymphocytes and neutrophils were observed in all animals. Viremias were demonstrable in eight horses, with a maximum of 10(3.5) median tissue culture infectious dose units per ml of serum in two horses. Clinical illness with depression and anorexia were observed in five horses. Neutralizing (N), hemagglutination-inhibiting, and complement-fixing antibodies to the vaccine virus were demonstrable by 5, 6.5, and 7 days, respectively, after vaccination. Differential titrations of serum to six VEE strains revealed high titers of N antibody to vaccine virus, moderate titers to the epizootic Trinidad donkey no. 1 strain (VEE antigenic subtype I, variant A) from which TC-83 was derived, and low titers to two other epizootic strains (subtype I, variants B and C) in all horses at 1 month after vaccination; some animals responded with low levels of N antibody to the enzootic viruses (subtype I, variants D and E). Fourteen months after vaccination, six animals with detectable N antibody were challenged with MF-8 (subtype I, variant B), an epidemic-epizootic strain isolated in 1969 from a man in Honduras. All horses resisted challenge with the equine pathogenic strain of VEE. Marked increases of N antibody in most horses were demonstrable to some VEE strains when tested 1 month after challenge.

Experimental Infection of Horses with an Attenuated Venezuelan Equine Encephalomyelitis Vaccine (Strain TC-83)

PubMed Central

Walton, Thomas E.; Alvarez, Otto; Buckwalter, Ross M.; Johnson, Karl M.

1972-01-01

Ten horses (Equus caballus) were vaccinated with strain TC-83 Venezuelan equine encephalomyelitis (VEE) virus vaccine. Febrile responses and leukopenia due to a reduction of lymphocytes and neutrophils were observed in all animals. Viremias were demonstrable in eight horses, with a maximum of 103.5 median tissue culture infectious dose units per ml of serum in two horses. Clinical illness with depression and anorexia were observed in five horses. Neutralizing (N), hemagglutination-inhibiting, and complement-fixing antibodies to the vaccine virus were demonstrable by 5, 6.5, and 7 days, respectively, after vaccination. Differential titrations of serum to six VEE strains revealed high titers of N antibody to vaccine virus, moderate titers to the epizootic Trinidad donkey no. 1 strain (VEE antigenic subtype I, variant A) from which TC-83 was derived, and low titers to two other epizootic strains (subtype I, variants B and C) in all horses at 1 month after vaccination; some animals responded with low levels of N antibody to the enzootic viruses (subtype I, variants D and E). Fourteen months after vaccination, six animals with detectable N antibody were challenged with MF-8 (subtype I, variant B), an epidemic-epizootic strain isolated in 1969 from a man in Honduras. All horses resisted challenge with the equine pathogenic strain of VEE. Marked increases of N antibody in most horses were demonstrable to some VEE strains when tested 1 month after challenge. PMID:4637604

Monospecific high-affinity and complement activating anti-GM1 antibodies are determinants in experimental axonal neuropathy.

PubMed

Notturno, Francesca; Del Boccio, Piero; Luciani, Mirella; Caporale, Christina Michaela; Pieragostino, Damiana; Prencipe, Vincenza; Sacchetta, Paolo; Uncini, Antonino

2010-06-15

It has been difficult to replicate consistently the experimental model of axonal Guillain-Barré syndrome (GBS). We immunized rabbits with two lipo-oligosaccharides (LOS1 and LOS2) derived from the same C. jejuni strain and purified in a slightly different way. LOS1 did not contain proteins whereas several proteins were present in LOS2. In spite of a robust anti-GM1 antibody response in all animals the neuropathy developed only in rabbits immunized with LOS1. To explain this discrepancy we investigated fine specificity, affinity and ability to activate the complement of anti-GM1 antibodies. Only rabbits immunized with LOS1 showed monospecific high-affinity antibodies which activated more effectively the complement. Although it is not well understood how monospecific high-affinity antibodies are induced these are crucial for the induction of experimental axonal neuropathy. Only a strict adherence to the protocols demonstrated to be successful may guarantee the reproducibility and increase the confidence in the animal model as a reliable tool for the study of the human axonal GBS. Copyright 2010 Elsevier B.V. All rights reserved.

Leukemia diagnosis and testing of complement-fixing antibodies for bone marrow purging in acute lymphoid leukemia.

PubMed

Campana, D; Janossy, G

1986-12-01

In this paper a microplate method is described for diagnosing acute leukemia and for investigating the reactivity of monoclonal antibodies (MoAbs) against membrane antigens in combination with rabbit or murine antibodies to nuclear terminal transferase (TdT). The speed of this method facilitates the investigation of fresh leukemic cells from individual patients and assesses the cytolytic efficacy of the relevant MoAbs in the presence of complement (C'). Lymphoblasts (TdT+) are mixed in equal proportions with known numbers of "inert" cells, eg, RBC or nonleukemic bone marrow (BM). Following incubation with MoAbs and C' the ratio of residual TdT+ cells and inert cells is determined on cytospin preparations. Initially, percentages of TdT+ cells are counted in a unit volume of 5,000 inert cells, followed by the scanning of greater than 2 X 10(4) inert cells on entire slides. With this method more than 4 log cytoreduction of TdT + cells is detected. The method is also applicable for studying the cytolysis of malignant B cells by using mostly monoclonal lg expression rather than TdT for the identification of residual B cells. Ten representative patients selected from a group of greater than 100 are reported. In some cases cytoreduction of greater than 4 log with no identifiable residual TdT + cells is achieved by a single C'-fixing MoAb: anti-CD10 (RFAL3) in common acute lymphoid leukemia (ALL) and anti-CD7 (RFT2) in T cell ALL (T-ALL). Other cases require cocktails of anti-CD10, anti-CD19, and anti-CD24 in common ALL or anti-CD7 and anti-CD8 in T-ALL. In T-ALL a few TdT + cells remain that exhibit the features of normal TdT + BM cells (CD7-, HLA-DR+). This is particularly noticeable when patients are studied in partial remission or if nonleukemic BM is used as a source of inert cells. The methods described here contribute to establishing a range of MoAbs (ie, of IgM class) and techniques for efficient purging and to comparing the efficacy of "clean-up," in remission, of common ALL, T-ALL, and B cell malignancies.

Identification of a Naegleria fowleri Membrane Protein Reactive with Anti-Human CD59 Antibody

PubMed Central

Fritzinger, Angela E.; Toney, Denise M.; MacLean, Rebecca C.; Marciano-Cabral, Francine

2006-01-01

Naegleria fowleri, the causative agent of primary amebic meningoencephalitis, is resistant to complement lysis. The presence of a complement regulatory protein on the surface of N. fowleri was investigated. Southern blot and Northern blot analyses demonstrated hybridization of a radiolabeled cDNA probe for CD59 to genomic DNA and RNA, respectively, from pathogenic N. fowleri. An 18-kDa immunoreactive protein was detected on the membrane of N. fowleri by Western immunoblot and immunofluorescence analyses with monoclonal antibodies for human CD59. Complement component C9 immunoprecipitated with the N. fowleri “CD59-like” protein from amebae incubated with normal human serum. In contrast, a gene or protein similar to CD59 was not detected in nonpathogenic, complement-sensitive N. gruberi amebae. Collectively, our studies suggest that a protein reactive with antibodies to human CD59 is present on the surface of N. fowleri amebae and may play a role in resistance to lysis by cytolytic proteins. PMID:16428768

STUDIES ON SCRUB TYPHUS

PubMed Central

Smadel, Joseph E.; Rights, Fred L.; Jackson, Elizabeth B.

1946-01-01

A complement-fixing antigen specific for scrub typhus occurs in the body fluids and tissues of infected mice, white rats, and cotton rats. The specific serological substance is demonstrable only in those animals which develop a rapidly fatal disease after an incubation period of a few days. Such an experimental infection is induced in mice and rats by the intravenous injection of suspensions of yolk sac rich in R. orientalis. Ether extraction is an important step in the preparation of a complement-fixing antigen from tissues of mice dying with scrub typhus. The Imphal No. 8 and Calcutta strains of R. orientalis are indistinguishable on the basis of complement fixation and cross-immunity tests. The complement-fixing antigen in body fluids of infected mice and rats and in our preparations of tissues from such animals occurs as a soluble antigen. Under the proper conditions the soluble antigen can be stored or dehydrated without loss of serological activity. PMID:19871518

Classification of Blood-Group Antibodies as β2M or γ Globulin

PubMed Central

Adinolfi, M.; Polley, Margaret J.; Hunter, Denise A.; Mollison, P. L.

1962-01-01

Thirty selected blood-group antibodies (excluding anti-A and anti-B) have been classified as β2M (19S γ) globulin, γ (7S γ) globulin or mixtures, using the following three methods: fractionation on a DEAE-cellulose column; indirect anti-globulin tests, using specific anti-β2M-globulin and anti-γ-globulin sera; and treatment with 2-mercapto-ethanol. With only minor exceptions, results obtained with the three methods were in agreement. Most blood-group antibodies within the Le, MNSs and P systems appear to be `naturally occurring' and these were found to be β2M globulin. Blood-group antibodies within the Rh, K and Jk systems, which had arisen after an antigenic stimulus, were usually γ globulin but were occasionally β2M globulin. Antibodies composed of β2M globulin usually behave as agglutinins but may behave as incomplete antibodies (e.g. some examples of anti-Jka); conversely, antibodies composed of γ globulin usually behave as incomplete antibodies but may behave as agglutinins (e.g. an example of anti-M). The ability to bind complement seems to be related more to the blood-group specificity of the particular antibody than to its molecular size. For example, anti-Jka, when composed either of γ or β2M globulin, seems invariably to bind complement, whereas potent anti-M or anti-Rh, whether composed of γ or β2M globulin, do not bind complement. PMID:14011076

Evaluation of Streptococcus pneumoniae Type XIV Opsonins by Phagocytosis-Associated Chemiluminescence and a Bactericidal Assay

PubMed Central

Gardner, Susan E.; Anderson, Donald C.; Webb, Bette J.; Stitzel, Ann E.; Edwards, Morven S.; Spitzer, Roger E.; Baker, Carol J.

1982-01-01

The relative roles of serum factors required for opsonization of type XIV Streptococcus pneumoniae were investigated by means of luminol-enhanced chemiluminescence (CL), bactericidal, and immunofluorescence assays employing adult sera containing high (>1,000 ng of antibody nitrogen per ml) or low (<200 ng of antibody nitrogen per ml) antibody concentrations as determined by radioimmunoassay. Specific antibody concentration correlated directly with both total and heat-labile CL activity (P < 0.005) and with the bactericidal index (P < 0.05) at a serum concentration of 10%. The importance of specific antibody as an opsonin was confirmed by the abolition of CL activity and immunoglobulin immunofluorescence observed after absorption of heated sera with type XIV pneumococcal cells and by the dose response in CL and bactericidal activity observed with the addition of immunoglobulin G to hypogammaglobulinemic serum. A role for the classical complement pathway in opsonization was indicated by significantly greater CL integrals for high-antibody sera than for low-antibody sera depleted of factor D and by the bactericidal activity noted for untreated, but not magnesium ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid-chelated low-antibody sera. The alternative pathway contributed more than half of the CL activity of both high- and low-antibody sera. However, after magnesium ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid chelation, only sera with high antibody concentrations or agammaglobulinemic serum reconstituted with immunoglobulin G with high specific antibody levels supported significant bactericidal activity. Therefore, type-specific antibody and complement promote opsonization of type XIV S. pneumoniae, and this may occur via either complement pathway. These results suggest that CL is a suitable tool to delineate serum factors and their contribution to opsonization, but results must be related to other functional assays. PMID:6802760

The Perfect Storm: HLA Antibodies, Complement, FcγRs and Endothelium in Transplant Rejection

PubMed Central

Thomas, Kimberly A.; Valenzuela, Nicole M.; Reed, Elaine F.

2015-01-01

The pathophysiology of antibody-mediated rejection (AMR) in solid organ transplants is multi-faceted and predominantly caused by antibodies directed against polymorphic donor human leukocyte antigens (HLA). Despite the clearly detrimental impact of HLA antibodies (HLA-Ab) on graft function and survival, the prevention, diagnosis and treatment of AMR remain a challenge. Histological manifestations of AMR reflect signatures of HLA-Ab-triggered injury, specifically endothelial changes, recipient leukocytic infiltrate, and complement deposition. We review the interconnected mechanisms of HLA-Ab-mediated injury that might synergize in a “perfect storm” of inflammation. Characterization of antibody features that are critical for effector functions may help identify HLA-Ab more likely to cause rejection. We also highlight recent advancements that may pave the way for new, more effective therapeutics. PMID:25801125

Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

PubMed Central

Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

2016-01-01

Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

Development and Use of a Serum Bactericidal Assay Using Pooled Human Complement To Assess Responses to a Meningococcal Group A Conjugate Vaccine in African Toddlers

PubMed Central

Lynn, Freyja; Mocca, Brian; Borrow, Ray; Findlow, Helen; Hassan-King, Musa; Preziosi, Marie-Pierre; Idoko, Olubukola; Sow, Samba; Kulkarni, Prasad; LaForce, F. Marc

2014-01-01

A meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused by Neisseria meningitidis. Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC′) was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC′ and then used the SBA assay with hC′ (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12- to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC′ lots in two laboratories. hSBA titers of clinical trial sera were then determined using this complement sourcing method. Two different functional antibody tests were necessary for screening hC′. hSBA titers determined using three independent lots of pooled hC′ were within expected assay variation among lots and between laboratories. In African toddlers, PsA-TT elicited higher hSBA titers than meningococcal polysaccharide or Hib vaccines. PsA-TT immunization or PS challenge of PsA-TT-primed subjects resulted in vigorous hSBA memory responses, and titers persisted in boosted groups for over a year. Quantifying SBA using pooled hC′ is feasible and showed that PsA-TT was highly immunogenic in African toddlers. PMID:24671551

The duration of passive protection against Taenia ovis larvae in lambs.

PubMed

Heath, D D; Yong, W K; Osborn, P J; Parmeter, S N; Lawrence, S B; Twaalfhoven, H

1979-10-01

In an attempt to induce passive protection in lambs against Taenia ovis larvae that would last for the 15-20 weeks from birth to slaughter as fat lambs, one group of ewes was immunized by a series of injections of 2000, 4000, 8000, 16 000 and 32 000 activated oncospheres of Taenia ovis prior to parturition. Another group of ewes was not immunized. All ewes had previously grazed pasture lightly infected with T. ovis eggs. Most lambs from non-immunized ewes developed cysts after oral infection with T. ovis eggs. However, no lambs from immunized ewes developed cysts up to and including 6 weeks after birth. Between 8 and 16 weeks after birth a proportion of lambs were found to be susceptible to infection. By 18 weeks after birth all lambs were apparently susceptible. The 99% confidence band for the mean duration of demonstrable complement-fixing antibody titres was 6.2-7.8 weeks for lambs from immunized ewes. The persistence of maternal protective antibody in some lambs could possibly preclude successful active immunization of all lambs against T. ovis larvae before 18 weeks of age.

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

Non-Finite Complements in Russian, Serbian/Croatian, and Macedonian

ERIC Educational Resources Information Center

Kim, Bo Ra

2010-01-01

This study investigates the coherence properties of non-finite complements in Russian, Serbian/Croatian, and Macedonian. I demonstrate that Slavic non-finite complements do not project a uniform syntactic structure. The maximal projection of non-finite complements is not fixed but depends on the selectional properties of the matrix verb. I present…

Selective Blockade of Human Natural Killer Cells by a Monoclonal Antibody

NASA Astrophysics Data System (ADS)

Newman, Walter

1982-06-01

A murine monoclonal antibody, 13.1, which blocks human natural killer (NK) cell-mediated lysis, has been developed. Hybridoma 13.1 was derived by fusion of NS-1 cells with spleen cells from mice immunized with an enriched population of NK cells. Supernatants of growing hybridomas were screened for their ability to block NK cell-mediated lysis of K562 targets. Antibody 13.1 is an IgG1 with a single light chain type and it does not fix complement. The 13.1 antigen is expressed on all peripheral blood mononuclear cells, with an antigen density approximately 1/30th that of HLA antigen heavy chain. Pretreatment and washing experiments revealed that inhibition of cytotoxicity occurred at the effector cell level only. Significant blocking was achieved with nanogram quantities of antibody and was not due to toxic effects on NK cells. Likewise, controls with other antibodies of the same subclass demonstrated that blocking was not a consequence of mere binding to NK cells. When a panel of 17 NK cell-susceptible targets was tested, the lysis of only 5 of these was blocked, namely K562, HL-60, KG-1, Daudi, and HEL, a human erythroleukemic cell line. The lysis of 12 human B cell and T cell line targets was not inhibited. In addition to the demonstration that the 13.1 antigen is a crucial cell surface structure involved in NK lysis, a heterogeneity of target cell recognition has been revealed that argues for the proposition that individual NK cells have multiple recognitive capabilities.

T cells and cytokines in the pathogenesis of acquired myasthenia gravis.

PubMed

Milani, Monica; Ostlie, Norma; Wang, Wei; Conti-Fine, Bianca M

2003-09-01

Although the symptoms of myasthenia gravis (MG) and experimental MG (EAMG) are caused by autoantibodies, CD4(+) T cells specific for the target antigen, the nicotinic acetylcholine receptor, and the cytokines they secrete, have an important role in these diseases. CD4(+) T cells have a pathogenic role, by permitting and facilitating the synthesis of high-affinity anti-AChR antibodies. Th1 CD4(+) cells are especially important because they drive the synthesis of anti-AChR complement-fixing IgG subclasses. Binding of those antibodies to the muscle AChR at the neuromuscular junction will trigger the complement-mediated destruction of the postsynaptic membrane. Thus, IL-12, a crucial cytokine for differentiation of Th1 cells, is necessary for development of EAMG. Th2 cells secrete different cytokines, with different effects on the pathogenesis of EAMG. Among them, IL-10, which is a potent growth and differentiation factor for B cells, facilitates the development of EAMG. In contrast, IL-4 appears to be involved in the differentiation of AChR-specific regulatory CD4(+) T cells, which can prevent the development of EAMG and its progression to a self-maintaining, chronic autoimmune disease. Studies on the AChR-specific CD4(+) cells commonly present in the blood of MG patients support a crucial role of CD4(+) T cells in the development of MG. Circumstantial evidence supports a pathogenic role of IL-10 also in human MG. On the other hand, there is no direct or circumstantial evidence yet indicating a role of IL-4 in the modulatory or immunosuppressive circuits in MG.

Mapping of binding epitopes of a human decay-accelerating factor monoclonal antibody capable of enhancing rituximab-mediated complement-dependent cytotoxicity.

PubMed

Guo, Bo; Ma, Zheng-wei; Li, Hua; Xu, Gui-lian; Zheng, Ping; Zhu, Bo; Wu, Yu-Zhang; Zou, Qiang

2008-08-01

Complement-dependent cytotoxicity (CDC) is thought to be one of the most important mechanisms of action of therapeutic monoclonal antibodies (mAbs). The decay-accelerating factor (DAF) overexpressed in certain tumors limits the CDC effect of the therapeutic anticancer antibodies. The use of DAF blocking antibodies targeted specifically at cancer cells in combination with immunotherapeutic mAbs of cancer may improve the therapeutic effect in cancer patients. In this study, the lysis of Raji cells mediated by CDC was determined after blocking DAF function by anti-DAF polyclonal antibody and 3 mAbs (DG3, DG9, DA11) prepared in our laboratory, respectively, in the presence of the anti-CD20 chimeric mAb rituximab. The binding domains of the three anti-DAF mAbs were identified using yeast surface display technique, and the mimic epitopes of mAb DG3 were screened from a random phage-display nonapeptide library. The results showed that blocking DAF function by anti-DAF polyclonal antibody enhanced complement-mediated killing of Raji cells. Among the 3 mAbs against DAF, only DG3 was found to be able to remarkably enhance the CDC effect of the therapeutic mAb rituximab. DG3 bound to the third short consensus repeat (SCR) of DAF. Binding of DG3 to immobilized DAF was inhibited by mimic epitope peptides screened from the peptide library. Our results suggest that a higher level of DAF expressed by certain tumor cells is significant to abolish the CDC effect of therapeutic anticancer antibodies, and mAbs binding to SCR3 can enhance the complement-mediated killing of Raji cells. It is of significance to identify the DAF epitopes required in inhibiting CDC not only for better understanding of the relationship between the structure and function of DAF, but also for designing and developing anti-DAF mAbs capable of enhancing CDC.

The perfect storm: HLA antibodies, complement, FcγRs, and endothelium in transplant rejection.

PubMed

Thomas, Kimberly A; Valenzuela, Nicole M; Reed, Elaine F

2015-05-01

The pathophysiology of antibody-mediated rejection (AMR) in solid organ transplants is multifaceted and predominantly caused by antibodies directed against polymorphic donor human leukocyte antigens (HLAs). Despite the clearly detrimental impact of HLA antibodies (HLA-Abs) on graft function and survival, the prevention, diagnosis, and treatment of AMR remain a challenge. The histological manifestations of AMR reflect the signatures of HLA-Ab-triggered injury, specifically endothelial changes, recipient leukocytic infiltrate, and complement deposition. We review the interconnected mechanisms of HLA-Ab-mediated injury that might synergize in a 'perfect storm' of inflammation. Characterization of antibody features that are critical for effector functions may help to identify HLA-Abs that are more likely to cause rejection. We also highlight recent advances that may pave the way for new, more effective therapies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Complement System in Dermatological Diseases – Fire Under the Skin

PubMed Central

Panelius, Jaana; Meri, Seppo

2015-01-01

The complement system plays a key role in several dermatological diseases. Overactivation, deficiency, or abnormality of the control proteins are often related to a skin disease. Autoimmune mechanisms with autoantibodies and a cytotoxic effect of the complement membrane attack complex on epidermal or vascular cells can cause direct tissue damage and inflammation, e.g., in systemic lupus erythematosus (SLE), phospholipid antibody syndrome, and bullous skin diseases like pemphigoid. By evading complement attack, some microbes like Borrelia spirochetes and staphylococci can persist in the skin and cause prolonged symptoms. In this review, we present the most important skin diseases connected to abnormalities in the function of the complement system. Drugs having an effect on the complement system are also briefly described. On one hand, drugs with free hydroxyl on amino groups (e.g., hydralazine, procainamide) could interact with C4A, C4B, or C3 and cause an SLE-like disease. On the other hand, progress in studies on complement has led to novel anti-complement drugs (recombinant C1-inhibitor and anti-C5 antibody, eculizumab) that could alleviate symptoms in diseases associated with excessive complement activation. The main theme of the manuscript is to show how relevant the complement system is as an immune effector system in contributing to tissue injury and inflammation in a broad range of skin disorders. PMID:25688346

Effect of chlorphenesin on localized hemolysis in gel assay.

PubMed

Fukui, G M; Berger, F M; Chandlee, G C; Goldenbaum, E G

1968-10-01

Chlorphenesin, a simple glycerol ether, when added to Jerne plates greatly reduced the number of hemolytic plaques. This effect appeared to be related to dose, and was clearly demonstrable with antibody-forming spleen cells from mice that had been immunized either with sheep red blood cells or with penicillin G conjugated with Keyhole limpet hemocyanin. Chlorphenesin did not affect the antigen, destroy complement, or interfere with the interaction of complement and the antigen-antibody complexes. Incubation of spleen cell suspensions with chlorphenesin prior to plating was more effective in reducing the number of plaques than was addition of the substance to the plates. It may act by reducing the ability of antibodies to react with antigens or by affecting the release of antibodies from the spleen cells.

Effect of Chlorphenesin on Localized Hemolysis in Gel Assay

PubMed Central

Fukui, G. M.; Berger, F. M.; Chandlee, G. C.; Goldenbaum, E. G.

1968-01-01

Chlorphenesin, a simple glycerol ether, when added to Jerne plates greatly reduced the number of hemolytic plaques. This effect appeared to be related to dose, and was clearly demonstrable with antibody-forming spleen cells from mice that had been immunized either with sheep red blood cells or with penicillin G conjugated with Keyhole limpet hemocyanin. Chlorphenesin did not affect the antigen, destroy complement, or interfere with the interaction of complement and the antigen-antibody complexes. Incubation of spleen cell suspensions with chlorphenesin prior to plating was more effective in reducing the number of plaques than was addition of the substance to the plates. It may act by reducing the ability of antibodies to react with antigens or by affecting the release of antibodies from the spleen cells. PMID:5685993

Homologous species restriction of the complement-mediated killing of nucleated cells.

PubMed Central

Yamamoto, H; Blaas, P; Nicholson-Weller, A; Hänsch, G M

1990-01-01

The homologous restriction of complement (C) lysis is attributed to membrane proteins: decay-accelerating factor (DAF), C8 binding protein (C8bp) and P18/CD59. Since these proteins are also expressed on peripheral blood cells, species restriction was tested for in the complement-mediated killing of antibody-coated human leucocytes by human or rabbit complement. Killing was more efficient when rabbit complement was used. Preincubation of cells with an antibody to DAF abolished the difference. When C1-7 sites were first attached to the cells and either rabbit or human C8, C9 were added, the killing of monocytes and lymphocytes was equally efficient; only in polymorphonuclear neutrophils was a higher efficiency of rabbit C8, C9 seen. Thus, in contrast to haemolysis, restriction occurred predominantly at the C3 level and the action of the terminal complement components was not inhibited. Since C8bp isolated from peripheral blood cells showed essentially similar characteristics as the erythrocyte-derived C8bp, the failure of C8bp to inhibit the action of the terminal components on nucleated cells might reflect differences of the complement membrane interactions between erythrocytes or nucleated cells, respectively. Images Figure 5 PMID:1697561

HETEROGENETIC ANTIBODIES IN ACUTE HEPATITIS

PubMed Central

Eaton, Monroe D.; Murphy, William D.; Hanford, V. Lee

1944-01-01

A heterogenetic antibody showing fixation of complement with human liver and agglutination of sheep erythrocytes was found in certain cases of acute infective hepatitis. The antigen concerned in these reactions was apparently heat stable and alcohol soluble. Differences from other heterogenetic antigen-antibody systems have been noted. The possible relation of the heterogenetic antibody to liver damage was considered. PMID:19871386

Chlamydophila psittaci in free-living Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil.

PubMed

de Freitas Raso, Tânia; Seixas, Gláucia Helena Fernandes; Guedes, Neiva Maria Robaldo; Pinto, Aramis Augusto

2006-10-31

Chlamydophila psittaci (C. psittaci) infection was evaluated in 77 free-living nestlings of Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil. Tracheal and cloacal swab samples from 32 wild parrot and 45 macaw nestlings were submitted to semi-nested PCR, while serum samples were submitted to complement fixation test (CFT). Although all 32 Amazon parrot serum samples were negative by CFT, cloacal swabs from two birds were positive for Chlamydophila DNA by semi-nested PCR (6.3%); these positive birds were 32 and 45 days old. In macaws, tracheal and cloacal swabs were positive in 8.9% and 26.7% of the samples, respectively. Complement-fixing antibodies were detected in 4.8% of the macaw nestlings; macaw nestlings with positive findings were between 33 and 88 days old. These results indicate widespread dissemination of this pathogen in the two evaluated psittacine populations. No birds had clinical signs suggestive of chlamydiosis. To the best of our knowledge, this is the first report on C. psittaci in free-living Blue-fronted Amazon parrots and Hyacinth macaws in Brazil.

[The complement system in the pathogenesis of antineutrophil cytoplasm antibodies-associated vasculitis].

PubMed

Flores-Suárez, Luis F

2011-12-01

One of the main characteristics of the vasculitis associated with antineutrophil cytoplasm autoantibodies (AASV) is the absence of immune complex deposition in biopsies of affected tissues as well as a lack of complement depletion. However, in early stages of disease induced in animal models, it has been observed that the complement system may be involved in the generation of these diseases. There are various animal models which have been developed with the aim of knowing which are the pathogenic mechanisms in granulomatosis with polyangiitis (Wegener) (GPA) and microscopic polyangiitis (MPA), the latter being explained using these approaches in a more satisfactory manner, as there is lack of a model which reproduces the changes leading to a granulomatous vasculitis associated with antibodies against proteinase-3, as in GPA. This short review presents recent evidence of the presence of complement in biopsies of patients with AASV and the most recent animal models, which show the participation of complement in their etiology. Copyright © 2011 Elsevier España, S.L. All rights reserved.

Myasthenia gravis: the role of complement at the neuromuscular junction.

PubMed

Howard, James F

2018-01-01

Generalized myasthenia gravis (gMG) is a rare autoimmune disorder characterized by skeletal muscle weakness caused by disrupted neurotransmission at the neuromuscular junction (NMJ). Approximately 74-88% of patients with gMG have acetylcholine receptor (AChR) autoantibodies. Complement plays an important role in innate and antibody-mediated immunity, and activation and amplification of complement results in the formation of membrane attack complexes (MACs), lipophilic proteins that damage cell membranes. The role of complement in gMG has been demonstrated in animal models and patients. Studies in animals lacking specific complement proteins have confirmed that MAC formation is required to induce experimental autoimmune MG (EAMG) and NMJ damage. Complement inhibition in EAMG models can prevent disease induction and reverse its progression. Patients with anti-AChR + MG have autoantibodies and MACs present at NMJs. Damaged NMJs are associated with more severe disease, fewer AChRs, and MACs in synaptic debris. Current MG therapies do not target complement directly. Eculizumab is a humanized monoclonal antibody that inhibits cleavage of complement protein C5, preventing MAC formation. Eculizumab treatment improved symptoms compared with placebo in a phase II study in patients with refractory gMG. Direct complement inhibition could preserve NMJ physiology and muscle function in patients with anti-AChR + gMG. © 2017 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

Upregulation of CD59

PubMed Central

Griesemer, Adam D.; Okumi, Masayoshi; Shimizu, Akira; Moran, Shannon; Ishikawa, Yoshinori; Iorio, Justin; Arn, J. Scott; Yamada, Kazuhiko

2009-01-01

Background Survival of ABO-mismatched kidneys with stable renal function despite the persistence of anti-ABO antibodies is called accommodation. The mechanism of accommodation is unclear, but may involve complement regulatory proteins such as CD59. The development of alpha-1,3-Galactosyltransferase knock-out (GalT-KO) swine that produce anti-Gal antibodies provides a large animal model capable of determining the role of complement regulatory proteins in accommodation. Methods ELISA and antibody FACS were used to examine the rate of anti-Gal antibody expression as a function of age. MHC-matched kidneys were transplanted from Gal-positive MGH miniature swine to MGH GalT-KO swine with systemic immunosuppression. One recipient underwent adsorbtion of anti-Gal antibodies prior to transplantation. Graft survival, antibody and complement deposition patterns and CD59 expression were determined. Results Three animals rejected Gal-positive kidneys via humoral mechanisms. One animal with low titers of anti-Gal Ab displayed spontaneous accommodation and the animal that was treated with Ab adsorbtion also displayed accommodation. Rejected grafts had deposition of IgM, IgG, C3 and C5b-9 with low expression of CD59, while accommodated grafts had low deposition of C5b-9 and high expression of CD59. Re-transplantation of one accommodated graft to a naïve GalT-KO animal confirmed that changes in the graft were responsible for the lack of C5b-9 deposition. Conclusion GalT-KO miniature swine produce anti-Gal antibodies and titers increase with age. These anti-Gal antibodies can cause rejection of MHC matched kidneys unless accommodation occurs. CD59 upregulation appears to be involved in the mechanism of accommodation by preventing the formation of the MAC on the accommodated graft. PMID:19424030

Immune response and histology of humoral rejection in kidney transplantation.

PubMed

González-Molina, Miguel; Ruiz-Esteban, Pedro; Caballero, Abelardo; Burgos, Dolores; Cabello, Mercedes; Leon, Miriam; Fuentes, Laura; Hernandez, Domingo

2016-01-01

The adaptive immune response forms the basis of allograft rejection. Its weapons are direct cellular cytotoxicity, identified from the beginning of organ transplantation, and/or antibodies, limited to hyperacute rejection by preformed antibodies and not as an allogenic response. This resulted in allogenic response being thought for decades to have just a cellular origin. But the experimental studies by Gorer demonstrating tissue damage in allografts due to antibodies secreted by B lymphocytes activated against polymorphic molecules were disregarded. The special coexistence of binding and unbinding between antibodies and antigens of the endothelial cell membranes has been the cause of the delay in demonstrating the humoral allogenic response. The endothelium, the target tissue of antibodies, has a high turnover, and antigen-antibody binding is non-covalent. If endothelial cells are attacked by the humoral response, immunoglobulins are rapidly removed from their surface by shedding and/or internalization, as well as degrading the components of the complement system by the action of MCP, DAF and CD59. Thus, the presence of complement proteins in the membrane of endothelial cells is transient. In fact, the acute form of antibody-mediated rejection was not demonstrated until C4d complement fragment deposition was identified, which is the only component that binds covalently to endothelial cells. This review examines the relationship between humoral immune response and the types of acute and chronic histological lesion shown on biopsy of the transplanted organ. Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

Complement-fixing Activity of Fulvic Acid from Shilajit and Other Natural Sources

PubMed Central

Schepetkin, Igor A.; Xie, Gang; Jutila, Mark A.; Quinn, Mark T.

2008-01-01

Shilajit has been used traditionally in folk medicine for treatment of a variety of disorders, including syndromes involving excessive complement activation. Extracts of Shilajit contain significant amounts of fulvic acid (FA), and it has been suggested that FA is responsible for many therapeutic properties of Shilajit. However, little is known regarding physical and chemical properties of Shilajit extracts, and nothing is known about their effects on the complement system. To address this issue, we fractionated extracts of commercial Shilajit using anion exchange and size-exclusion chromatography. One neutral (S-I) and two acidic (S-II and S-III) fractions were isolated, characterized, and compared with standardized FA samples. The most abundant fraction (S-II) was further fractionated into three sub-fractions (S-II-1 to S-II-3). The van Krevelen diagram showed that the Shilajit fractions are products of polysaccharide degradation, and all fractions, except S-II-3, contained type II arabinogalactan. All Shilajit fractions exhibited dose-dependent complement-fixing activity in vitro with high potency. Furthermore, we found a strong correlation between complement-fixing activity and carboxylic group content in the Shilajit fractions and other FA sources. These data provide a molecular basis to explain at least part of the beneficial therapeutic properties of Shilajit and other humic extracts. PMID:19107845

Antibody ligation of murine Ly-6G induces neutropenia, blood flow cessation, and death via complement-dependent and independent mechanisms.

PubMed

Abbitt, Katherine B; Cotter, Matthew J; Ridger, Victoria C; Crossman, David C; Hellewell, Paul G; Norman, Keith E

2009-01-01

Ly-6G is a member of the Ly-6 family of GPI-linked proteins, which is expressed on murine neutrophils. Antibodies against Ly-6G cause neutropenia, and fatal reactions also develop if mice are primed with TNF-alpha prior to antibody treatment. We have investigated the mechanisms behind these responses to Ly-6G ligation in the belief that similar mechanisms may be involved in neutropenia and respiratory disorders associated with alloantibody ligation of the related Ly-6 family member, NB1, in humans. Neutrophil adhesion, microvascular obstruction, breathing difficulties, and death initiated by anti-Ly-6G antibodies in TNF-alpha-primed mice were shown to be highly complement-dependent, partly mediated by CD11b, CD18, and FcgammaR and associated with clustering of Ly-6G. Neutrophil depletion, on the other hand, was only partly complement-dependent and was not altered by blockade of CD11b, CD18, or FcgammaR. Unlike other neutrophil-activating agents, Ly-6G ligation did not induce neutropenia via sequestration in the lungs. Cross-linking Ly-6G mimicked the responses seen with whole antibody in vivo and also activated murine neutrophils in vitro. Although this suggests that the responses are, in part, mediated by nonspecific properties of antibody ligation, neutrophil depletion requires an additional mechanism possibly specific to the natural function of Ly-6G.

Association between anti-beta2 glycoprotein I antibodies and renal glomerular C4d deposition in lupus nephritis patients with glomerular microthrombosis: a prospective study of 155 cases.

PubMed

Shen, Y; Chen, X-W; Sun, C-Y; Dai, M; Yan, Y-C; Yang, C-D

2010-09-01

Glomerular microthrombosis (GMT) is a common vascular change in patients with lupus nephritis (LN). The mechanism underlying GMT is still unknown. In our previous study, we found that the level of IgG anti-beta2 glycoprotein I (beta2GPI) antibodies was higher in the LN-GMT group than in the LN-non-GMT group, which indicated that anti-beta2GPI antibodies may play a role in GMT formation. Many studies have demonstrated that the activation of the classical complement pathway may play a critical role in fetal loss and aPL-induced thrombosis formation. To investigate whether complement activation plays a role in GMT formation and to evaluate its relationship with aPL, we prospectively investigated deposition of C4d in 155 renal biopsy specimens of LN patients. The results revealed a strong relationship between the intensity of glomerular C4d staining and the presence of microthrombi (p < 0.001). The detection rate of IgG anti-beta2GPI antibodies was higher in the LN-GMT group than in the LN-non-GMT group (p < 0.05). Further, the intensity of glomerular C4d staining was significantly related with IgG anti-beta2GPI antibodies (p < 0.05). The results of our study suggest that anti-beta2GPI antibodies may play a role in GMT formation, and this process might involve complement activation.

Antigen-specific Immunotherapeutic Vaccine for Experimental Autoimmune Myasthenia Gravis

PubMed Central

Luo, Jie; Lindstrom, Jon

2014-01-01

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are caused by antibody-mediated autoimmune responses to muscle nicotinic acetylcholine receptors (AChRs) that impair neuromuscular transmission thereby causing muscle weakness. Previously, we discovered that i. p. injection of a therapeutic vaccine consisting of bacterially-expressed cytoplasmic domains of human AChR subunits reduced development of chronic EAMG in rats. Here we show that immunization with the therapeutic vaccine in adjuvant does not induce EAMG, thus is safe. Potency and efficacy of the therapeutic vaccine were greatly increased by administering repeated low doses subcutaneously in incomplete Freund’s adjuvant. Onset of chronic EAMG could be prevented. Established chronic EAMG could be rapidly reversed, modeling therapy of chronic MG. Therapy reduced pathological antibodies assayed by immune precipitation of a main immunogenic region chimera. Successfully treated rats exhibited long-term resistance to re-induction of EAMG, modeling a lasting cure of MG. A long-term effect of therapy was to change isotype of the pathogenic antibody response from IgG2b that fixes complement to IgG1 that does not. Prevention and reversal of chronic EAMG was not caused by the isotype switch, but the isotype switch may contribute to resistance to reinduction of EAMG. Immunization with AChR cytoplasmic domains in adjuvant is promising as a safe, antigen-specific, potent, effective, rapidly acting, and long lasting approach to therapy of MG. PMID:25288571

Isolation of circulating immune complexes using Raji cells. Separation of antigens from immune complexes and production of antiserum.

PubMed Central

Theofilopoulos, A N; Eisenberg, R A; Dixon, F J

1978-01-01

Raji cells were used for the isolation of complement-fixing antigen-antibody complexes from serum. Immune complexes bound to these cells were radiolabeled at the cell surface with lactoperoxidase. The complexes were then eluted from the cells with isotonic citrate buffer pH 3.2 or recovered by immunoprecipitation of cell lysates. The antigen and antibody moieties of the complexes were isolated by dissociating sucrose density gradient centrifugation or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of preformed immune complexes were successfully isolated from serum with this approach. In addition, these techniques were used to isolate and identify the antigens in immune complexes in the serum of rabbits with chronic serum sickness and rats with Moloney virus-induced sarcomas. Methods were also developed for the production of antisera against the antigenic moiety of immune complexes isolated from serum. Repeated challenge of rabbits with whole Raji cells with bound complexes or eluates from such cells resulted in antibody production against the antigens of the immune complexes, although reactivity against cellular and serum components was also elicited. Monospecific antisera against the antigens in immune complexes were produced by immunizing rabbits with the alum-precipitated antigen isolated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These techniques may be useful in isolating antigens in immune complex-associated diseases of unknown etiology. Images PMID:659616

Decreased complement mediated binding of antibody//sup 3/-dsDNA immune complexes to the red blood cells of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies

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

Taylor, R.P.; Horgan, C.; Buschbacher, R.

1983-06-01

The complement mediated binding of prepared antibody//sup 3/H-dsDNA immune complexes to the red blood cells obtained from a number of patient populations has been investigated. Patients with solid tumors have binding activity similar to that seen in a normal group of individuals. However, a significant fraction of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies have lowered binding activity compared with normal subjects. Quantitative studies indicate the lowered activity probably arises due to a decrease in complement receptors on the respective red blood cells. The potential importance and implications of these findings are briefly discussed.

IMMUNITY IN MUMPS

PubMed Central

Enders, John F.; Kane, Lewis W.; Cohen, Sidney; Levens, Jeanette H.

1945-01-01

Since the experimental data have already been discussed at some length, it is here necessary only to review those observations which appear to be of most significance. The manifestations of the disease we have produced in monkeys have in general been similar to those described by previous workers. It has been found that members of the species M. mulatta are regularly susceptible to infection with the virus of mumps, provided they have not been in contact with animals which have recently been infected. Such contact may be followed by the development of resistance to inoculation unattended by overt signs of infection. Following the intraparotid injection of virus into a susceptible animal, an antigen appears in the gland which reacts specifically in the complement fixation test with sera of monkeys which have recovered from an attack of mumps. This antigen has not been demonstrated in any other organ of the infected monkey which has been examined, nor does it persist or increase in amount when introduced into the brain or the testes. Attempts to propagate it in the tissues of other animal species including the developing hen's egg and tissue cultures have so far been uniformly unsuccessful. The antigen is relatively thermostabile, resisting a temperature of 65°C. for at least 20 minutes at the optimal hydrogen ion concentration. It is unimpaired by a concentration of formalin of 0.3 per cent. Together with the virus it may be preserved indefinitely in the frozen state. The maximal quantity of antigen in the gland, which may be considerable, is attained on or about the 5th day following inoculation of the virus. An antibody which reacts with the antigen appears in the serum from 8 to 14 days after infection. This antibody has not been found in the serum of normal monkeys which have had no opportunity for contact with the virus. It has been demonstrated during convalescence in the serum of all animals which have been examined. The antibody which is present in large amounts soon after recovery, although decreasing in concentration with the lapse of time, may continue to be present for many months. At 62°C. for 20 minutes its activity is not reduced and on storage at 4°C. its titer has remained unaltered for over 2 years. A similar factor which appears in human beings convalescent from mumps is less stabile, since its activity is reduced appreciably under these conditions. In the CO2 cabinet, however, both antibodies can be preserved for long periods of time. The definition of these reagents has afforded means whereby the simian infection with the virus of mumps, whether it is actively in progress or has occurred at some time in the past, can be recognized. Upon the establishment of these facts, the estimation of the quantity of antigen in the glands of vaccinated animals was employed as an index of their resistance in studies on the effect of the injection of formolized and alumprecipitated formolized suspensions of infected parotid gland. It has been shown that such vaccines, in which the virus has been rendered inactive, lead to the production of complement-fixing antibody. In about 60 per cent of the vaccinated animals, moreover, evidence of increased resistance was obtained as indicated by the partial or complete suppression of the formation of antigen in the inoculated gland. In certain instances no evidence of resistance was observed, yet antibody was found to be present. It is clear, therefore, that resistance is not determined solely by the presence of the complement-fixing antibody. The possibility, however, of its participation as a factor in immunity cannot be excluded. Employing the same criterion of resistance, the virus-neutralizing capacity of a pool of human mumps convalescent sera and of a globulin concentrate prepared from the pool has been studied. Neither of these materials was found to prevent entirely the formation of antigen, although each of them appeared to exert some inhibitory effect. PMID:19871448

Anti-C1q autoantibodies deposit in glomeruli but are only pathogenic in combination with glomerular C1q-containing immune complexes

PubMed Central

Trouw, Leendert A.; Groeneveld, Tom W.L.; Seelen, Marc A.; Duijs, Jacques M.G.J.; Bajema, Ingeborg M.; Prins, Frans A.; Kishore, Uday; Salant, David J.; Verbeek, J. Sjef; Kooten, Cees van; Daha, Mohamed R.

2004-01-01

Anti-C1q autoantibodies are present in sera of patients with several autoimmune diseases, including systemic lupus erythematosus (SLE). Strikingly, in SLE the presence of anti-C1q is associated with the occurrence of nephritis. We have generated mouse anti–mouse C1q mAb’s and used murine models to investigate whether anti-C1q autoantibodies actually contribute to renal pathology in glomerular immune complex disease. Administration of anti-C1q mAb JL-1, which recognizes the collagen-like region of C1q, resulted in glomerular deposition of C1q and anti-C1q autoantibodies and mild granulocyte influx, but no overt renal damage. However, combination of JL-1 with a subnephritogenic dose of C1q-fixing anti–glomerular basement membrane (anti-GBM) antibodies enhanced renal damage characterized by persistently increased levels of infiltrating granulocytes, major histological changes, and increased albuminuria. This was not observed when a non–C1q-fixing anti-GBM preparation was used. Experiments with different knockout mice showed that renal damage was dependent not only on glomerular C1q and complement activation but also on Fcγ receptors. In conclusion, anti-C1q autoantibodies deposit in glomeruli together with C1q but induce overt renal disease only in the context of glomerular immune complex disease. This provides an explanation why anti-C1q antibodies are especially pathogenic in patients with SLE. PMID:15343386

Effects of CTLA4-Fc on glomerular injury in humorally-mediated glomerulonephritis in BALB/c mice

PubMed Central

Kitching, A R; Huang, X R; Ruth, A-J; Tipping, P G; Holdsworth, S R

2002-01-01

The effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury. PMID:12067297

Effects of CTLA4-Fc on glomerular injury in humorally-mediated glomerulonephritis in BALB/c mice.

PubMed

Kitching, A R; Huang, X R; Ruth, A-J; Tipping, P G; Holdsworth, S R

2002-06-01

The effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury.

Reactivity of mouse antibodies against bromelain-treated mouse erythrocytes with thrombin-treated mouse platelets.

PubMed Central

Kawaguchi, S

1989-01-01

The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with mouse platelets before and after thrombin treatment was assessed by flow cytometry. Anti-BrMRBC antibodies could bind to thrombin-treated platelets, although normal platelets were also weakly reactive with the antibodies. The binding of anti-BrMRBC antibodies to platelets was confirmed by complement-dependent lysis. It is suggested that thrombin-activated platelets may be a real target for anti-BrMRBC antibodies. PMID:2467876

Characterization of Plasmodium vivax Transmission-Blocking Activity in Low to Moderate Malaria Transmission Settings of the Colombian Pacific Coast

PubMed Central

Arévalo-Herrera, Myriam; Solarte, Yezid; Rocha, Leonardo; Álvarez, Diego; Beier, John C.; Herrera, Sócrates

2011-01-01

Malaria infection induces antibodies capable of suppressing the infectivity of gametocytes and gametes, however, little is known about the duration of the antibody response, the parasite specificity, and the role of complement. We report the analyses of the transmission-blocking (TB) activity of sera collected from 105 Plasmodium vivax-infected and 44 non-infected individuals from a malaria endemic region of Colombia, using a membrane feeding assay in Anopheles albimanus mosquitoes. In infected donors we found that TB activity was antibody dose dependent (35%), lasted for 2–4 months after infection, and in 70% of the cases different P. vivax wild isolates displayed differential susceptibility to blocking antibodies. Additionally, in a number of assays TB was complement-dependent. Twenty-seven percent of non-infected individuals presented TB activity that correlated with antibody titers. Studies here provide preliminary data on factors of great importance for further work on the development of TB vaccines. PMID:21292881

Complement-dependent cytotoxicity (CDC) to detect Anti-HLA antibodies: old but gold.

PubMed

Saito, Patrícia Keiko; Yamakawa, Roger Haruki; Pereira, Lucieni Christina Marques da Silva; da Silva, Waldir Veríssimo; Borelli, Sueli Donizete

2014-07-01

The criterion (gold) standard to detect anti-human leukocyte antigen (HLA) antibodies is the complement-dependent cytotoxicity (CDC) assay. Recently, more sensitive methods have been used for the same purpose. This study analyzed 70 serum samples of patients with end-stage renal disease using CDC, CDC with the addition of anti-human globulin (CDC-AHG), CDC with the addition of dithiothreitol (CDC-DTT), and the recent solid-phase immunoassay (SPI; Labscreen PRA) to detect anti-HLA antibodies. Mean percent panel reactive antibodies (PRA) detected by SPI was 37.5% (±34.2) higher than the values detected by the other methods. Comparative analyses revealed significant difference between CDC and CDC-AHG, and between CDC and SPI (P < 0.0001), but not between CDC-AHG and SPI (P = 0.8026). Although the CDC-AHG method is "old," its performance to detect anti-HLA antibodies in the samples analyzed was comparable to the SPI in the evaluation of percent class I PRA. © 2014 Wiley Periodicals, Inc.

Complement research in the 18th-21st centuries: Progress comes with new technology.

PubMed

Sim, R B; Schwaeble, W; Fujita, T

2016-10-01

The complement system has been studied for about 120 years. Progress in defining this large and complex system has been dependent on the research technologies available, but since the introduction of protein chromatography, electrophoresis, and antibody-based assay methods in the 1950s and 60s, and sequencing of proteins and DNA in the 70s and 80s, there has been very rapid accumulation of data. With more recent improvements in 3D structure determination (nmr and X-ray crystallography), the structures of most of the complement proteins have now been solved. Complement research since 1990 has been greatly stimulated by the discoveries of the multiple proteins in the lectin pathway, the strong association of Factor H, C3, Factor B allelic variants with adult macular degeneration and atypical haemolytic uremic syndrome, and the introduction of the anti-C5 monoclonal antibody as a therapy for paroxysmal nocturnal hemoglobinuria and atypical haemolytic uremic syndrome. Potential new roles for complement in tissue development and the search for novel therapeutics suggest a very active future for complement research. Copyright © 2016 Elsevier GmbH. All rights reserved.

The important and diverse roles of antibodies in the host response to Borrelia infections.

PubMed

LaRocca, T J; Benach, J L

2008-01-01

Antibodies are of critical importance in the host response to tick-borne Borrelia species that cause relapsing fever and Lyme disease. Recent studies on the role of various B cell subsets in the host response to Borrelia, complement-independent, bactericidal antibodies, and diagnostics led to this review that focuses on the array of functions that antibodies to Borrelia can perform.

Milk complement and the opsonophagocytosis and killing of Staphylococcus aureus mastitis isolates by bovine neutrophils.

PubMed

Barrio, Maria Belén; Rainard, Pascal; Poutrel, Bernard

2003-01-01

Phagocytosis of bacteria by bovine polymorphonuclear neutrophils (PMN) has long been regarded as essential for host defense against mastitis infection. Complement-mediated opsonisation by complement component 3 (C3) binding is an important component of the innate immune system. We investigated the role of milk complement as an opsonin and its involvement in the phagocytosis and killing of Staphylococcus aureus isolates from cases of bovine mastitis by bovine blood PMN. We show that deposition of milk C3 component occurred on six different isolates of S. aureus and that the alternative pathway was the sole complement pathway operating in milk of uninflamed mammary gland. This deposition was shown to occur at the same location as the capsule, but not on capsular antigen. Milk complement enhanced the chemiluminescence response of PMN induced by S. aureus. Nevertheless, the association of S. aureus to cells and the overall killing of bacteria by bovine PMN were not affected by the presence of milk complement. Therefore, as all milk samples contained antibodies to capsular polysaccharide type 5 and to other surface antigens, it is likely that milk antibodies were responsible for these two phagocytic events. Results of this study suggest that the deposition of milk complement components on the surface of S. aureus does not contribute to the defence of the mammary gland against S. aureus.

A human anti-neuronal autoantibody against GABA B receptor induces experimental autoimmune agrypnia.

PubMed

Frisullo, Giovanni; Della Marca, Giacomo; Mirabella, Massimiliano; Caggiula, Marcella; Broccolini, Aldobrando; Rubino, Marco; Mennuni, Gioacchino; Tonali, Pietro Attilio; Batocchi, Anna Paola

2007-04-01

In the serum and cerebrospinal fluid of a patient with recurrent acute episodes of respiratory crises, autonomic symptoms and total insomnia (agrypnia), we identified a novel anti-neural complement fixing antibody directed against GABA(B) receptor (GABA(B)R). Patient purified IgG recognized a band of approximately 110 kDa on protein extracts of mouse cerebellum, cortex and brainstem and immunolabelled cultured Chinese hamster ovary (CHO) cells, transfected with human GABA(B)R1 and rat GABA(B)R2 receptors. Western blot analysis of transfected CHO homogenates showed the same band using both patient purified IgG and anti-GABA(B)R1 antibody. In order to verify the pathogenic role of these purified antibodies, we injected patient IgG intrathecally into cisterna magna of C57BL/6 mice pre-implanted with EEG electrodes and we observed severe ataxia followed by breathing depression and total suppression of slow wave sleep, as evidenced by EEG recording, in a dose-dependent manner. Immunohistochemistry on brain sections of mice injected with patient IgG showed the simultaneous presence of bound human IgG and C5b-9 deposits on Purkinje cells and cerebellar granular layer. After incubation with anti-GABA(B)R antibody, a marked reduction of receptor immunostaining was found with relative sparing of neuronal architecture. In conclusion we recognized an anti-neuronal autoantibody directed against GABA(B)R that is associated with autoimmune agrypnia and we showed that our patient purified IgG was able to induce in mice experimental autoimmune agrypnia characterized by a complex neurological syndrome affecting several CNS functions.

Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

PubMed Central

Tait, Brian D.

2016-01-01

This review outlines the development of human leukocyte antigen (HLA) antibody detection assays and their use in organ transplantation in both antibody screening and crossmatching. The development of sensitive solid phase assays such as the enzyme-linked immunosorbent assay technique, and in particular the bead-based technology has revolutionized this field over the last 10–15 years. This revolution however has created a new paradigm in clinical decision making with respect to the detection of low level pretransplant HLA sensitization and its clinical relevance. The relative sensitivities of the assays used are discussed and the relevance of conflicting inter-assay results. Each assay has its advantages and disadvantages and these are discussed. Over the last decade, the bead-based assay utilizing the Luminex® fluorocytometer instrument has become established as the “gold standard” for HLA antibody testing. However, there are still unresolved issues surrounding this technique, such as the presence of denatured HLA molecules on the beads which reveal cryptic epitopes and the issue of appropriate fluorescence cut off values for positivity. The assay has been modified to detect complement binding (CB) in addition to non-complement binding (NCB) HLA antibodies although the clinical relevance of the CB and NCB IgG isotypes is not fully resolved. The increase sensitivity of the Luminex® bead assay over the complement-dependent cytotoxicity crossmatch has permitted the concept of the “virtual crossmatch” whereby the crossmatch is predicted to a high degree of accuracy based on the HLA antibody specificities detected by the solid phase assay. Dialog between clinicians and laboratory staff on an individual patient basis is essential for correct clinical decision making based on HLA antibody results obtained by the various techniques. PMID:28018342

Altered Functionality of Anti-Bacterial Antibodies in Patients with Chronic Hepatitis C Virus Infection

PubMed Central

Lamontagne, Anne; Long, Ronald E.; Comunale, Mary Ann; Hafner, Julie; Rodemich-Betesh, Lucy; Wang, Mengjun; Marrero, Jorge; Di Bisceglie, Adrian M.; Block, Timothy; Mehta, Anand

2013-01-01

Background Using comparative glycoproteomics, we have previously identified a glycoprotein that is altered in both amount and glycosylation as a function of liver cirrhosis. The altered glycoprotein is an agalactosylated (G0) immunoglobulin G molecule (IgG) that recognizes the heterophilic alpha-gal epitope. Since the alpha gal epitope is found on gut enterobacteria, it has been hypothesized that anti-gal antibodies are generated as a result of increased bacterial exposure in patients with liver disease. Methods The N-linked glycosylation of anti-gal IgG molecules from patients with fibrosis and cirrhosis was determined and the effector function of anti-bacterial antibodies from over 100 patients examined. In addition, markers of microbial exposure were determined. Results Surprisingly, the subset of agalactosylated anti-gal antibodies described here, was impaired in their ability to mediate complement mediated lysis and inhibited the complement-mediated destruction of common gut bacteria. In an analysis of serum from more than 100 patients with liver disease, we have shown that those with increased levels of this modified anti-gal antibody had increased levels of markers of bacterial exposure. Conclusions Anti-gal antibodies in patients with liver cirrhosis were reduced in their ability to mediate complement mediated lysis of target cells. As bacterial infection is a major complication in patients with cirrhosis and bacterial products such as LPS are thought to play a major role in the development and progression of liver fibrosis, this finding has many clinical implications in the etiology, prognosis and treatment of liver disease. PMID:23750224

Expression of complement membrane regulators membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59) in human malignant gliomas.

PubMed Central

Mäenpää, A.; Junnikkala, S.; Hakulinen, J.; Timonen, T.; Meri, S.

1996-01-01

Gliomas are malignant brain tumors, which, despite recent progress in surgical and radiological treatment, still have a poor prognosis. Since gliomas apparently resist immunological clearance mechanisms, we became interested in examining bow gliomas resist killing by the human complement system. The resistance of human cells to complement-mediated damage is, in large part, mediated by specific inhibitors of complement:membrane cofactor protein (CD46), decay-accelerating factor (CD55), and protectin (CD59). In the present study we examined the expression of complement regulators in 14 human glioma tumors and in 7 glioma cell lines (U251, U87, HS683, U373, U138, U118, and H2). Protectin was found to be strongly expressed by all glioma tumors and cell lines. Northern blotting analysis demonstrated the typical pattern of four to five protectin mRNAs in the glioma cells. Except for blood vessels, the expression of decay-accelerating factor was weak or absent in the tumors in situ, whereas in the cell lines its expression varied, ranging from negative to intermediate. Membrane cofactor protein was moderately expressed by all the cell lines but only weakly in the tumors. Cell-killing experiments demonstrated that the glioma cell lines were exceptionally resistant to C-mediated lysis. Five of the seven cell lines (U373, HS683, U118, U138, and H2) resisted complement lysis under conditions where most other cell lines were sensitive to killing. Neutralization experiments using specific monoclonal antibodies indicated that protectin was functionally the most important complement regulator in the glioma cells. The killing of the U87 and U251 cells could be significantly increased by a blocking anti-protectin monoclonal antibody, whereas for the other cell lines only moderate or no response was observed. The H2 cell line resisted killing by all antibodies and by complement. These results show that protectin is the most important complement regulator on human glioma cells. The exceptional complement resistance of some glioma cell lines suggests that they may utilize other, hitherto less well characterized, mechanisms to resist complement killing. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 PMID:8644856

Complement-fixing activity of fulvic acid from Shilajit and other natural sources.

PubMed

Schepetkin, Igor A; Xie, Gang; Jutila, Mark A; Quinn, Mark T

2009-03-01

Shilajit has been used traditionally in folk medicine for the treatment of a variety of disorders, including syndromes involving excessive complement activation. Extracts of Shilajit contain significant amounts of fulvic acid (FA), and it has been suggested that FA is responsible for many therapeutic properties of Shilajit. However, little is known regarding the physical and chemical properties of Shilajit extracts, and nothing is known about their effects on the complement system. To address this issue, extracts of commercial Shilajit were fractionated using anion exchange and size-exclusion chromatography. One neutral (S-I) and two acidic (S-II and S-III) fractions were isolated, characterized and compared with standardized FA samples. The most abundant fraction (S-II) was further fractionated into three sub-fractions (S-II-1 to S-II-3). The van Krevelen diagram showed that the Shilajit fractions are the products of polysaccharide degradation, and all fractions, except S-II-3, contained type II arabinogalactan. All Shilajit fractions exhibited dose-dependent complement-fixing activity in vitro with high potency. Furthermore, a strong correlation was found between the complement-fixing activity and carboxylic group content in the Shilajit fractions and other FA sources. These data provide a molecular basis to explain at least part of the beneficial therapeutic properties of Shilajit and other humic extracts. (c) 2008 John Wiley & Sons, Ltd.

Coccidioidomycosis in the llama: case report and epidemiologic survey.

PubMed

Muir, S; Pappagianis, D

1982-12-01

An 8-year-old nongravid female llama with a 1-month history of progressive posterior paresis was referred because of suspected degenerative myelopathy secondary to copper deficiency or plant poisoning. Neurologic examination revealed loss of conscious proprioception and slightly depressed bilateral patellar reflexes. Electromyographic examination of hindlimb flexors and extensors did not elicit evidence of lower motor neuron disease. Possible fragmentation and mottling of the 10th thoracic vertebral body were noted radiographically. Results of a lumbar CSF tap, complete blood count, and fecal flotation were not diagnostic. In the face of poor prognosis, the llama was euthanatized. Postmortem and histologic evaluation revealed, in addition to disseminated visceral granulomas, an extradural pyogranulomatous mass compressing the cord laterally at the level of T-10. Large numbers of Coccidioides immitis were dispersed throughout the granulomas. Complement fixing antibody tests in 11 other herd members showed evidence of C immitis infection in three.

Autologous peripheral blood haematopoietic stem cell transplantation for systemic lupus erythematosus: the observation of long-term outcomes in a Chinese centre.

PubMed

Cao, Can; Wang, Menglei; Sun, Jing; Peng, Xuebiao; Liu, Qifa; Huang, Liang; Chai, Yanyan; Lai, Kuan; Chen, Pingjiao; Liu, Qingxiu; Li, Qian; Peng, Yusheng; Xiong, Hao; Zhang, Jing; Chen, Minghua; Zeng, Kang

2017-01-01

We aimed to evaluate the safety and long-term efficacy of autologous peripheral blood haematopoietic stem cell transplantation (APHSCT). We did not want to evaluate the efficacy of antibodies but rather the clinical response by investigating progression-free survival and serologic response by assessing autoantibody titres and complement levels. Overall, 22 patients with SLE (17 females; median age, 23 years) undergoing APHSCT were included. The 3-year progression-free survival (PFS) was 77.27% at our centre. We found that all the patients survived over three years. The 5-year PFS and overall survival (OS) rate was 67.90% and 95.20%. The titres of antinuclear antibody (ANA), anti-double-stranded deoxyribonucleic acid antibody (anti-dsDNA), anti-Sm antibody, and 24-h urinary protein significantly decreased, while complements 3 (C3) and C4 normalised at 100 days after transplantation (p<0.05). Kidney re-biopsy revealed a decrease in immune complex deposits in patients with remission. The incidence of CMV reactivation was 59.09% after transplantation in 3 years. Pregnancy and childbirth were reported in three female patients after transplantation. The risk of post-transplantation complications persisted for many years. Immunoablation followed by APHSCT has the potential to induce long-term clinical and serologic remissions despite withdrawal of immunosuppressive maintenance therapy. While relapses may occur, in our small cohort of patients we found no predictive markers for relapse development by analysing antibody and complement levels and urinary proteinuria.

DIRECT AND INDIRECT FLUORESCENT-ANTIBODY TECHNIQUES FOR THE PSITTACOSIS-LYMPHOGRANULOMA VENEREUM-TRACHOMA GROUP OF AGENTS1

PubMed Central

Ross, Martin R.; Borman, Earle K.

1963-01-01

Ross, Martin R. (Connecticut State Department of Health, Hartford) and Earle K. Borman. Direct and indirect fluorescent-antibody techniques for the psittacosis-lymphogranuloma venereum-trachoma group of agents. J. Bacteriol. 85:851–858. 1963.—Direct and indirect fluorescent-antibody (FA) techniques were developed for the detection of group antigen in infected tissue cultures and the titration of group antibody in human antiserum. The growth of the agent of meningopneumonitis (MP) in mouse embryo lung cell monolayers was followed by infectivity and complement-fixing (CF) antigen titrations, and cytological examination of FA stained cultures. Although infectivity and CF antigen reached a peak at 2 days and remained constant for an additional 3 days, only cells tested 2 to 3 days after infection were suitable for FA staining with labeled anti-MP serum because of excessive artifacts in the older cultures. Fluorescein isothiocyanate-labeled rooster and guinea pig anti-MP serums and human antipsittacosis serums were titrated in direct FA and hemagglutination-inhibition (HI) tests. The rooster conjugate showed brighter staining and higher antibody titers than the guinea pig or human conjugates and was more effective in detecting minimal amounts of virus antigen. FA staining reactions with 1 and 2 units of labeled rooster serum were inhibited by unlabeled rooster serum but clear-cut inhibition with human antipsittacosis serum could not be demonstrated. The indirect FA technique was successfully used for the titration of group antibody in human serum. A comparison of the indirect FA, HI, and CF tests showed the indirect FA technique to be intermediate in sensitivity between the HI and CF tests. None of the three tests showed significant cross reactions with human serums reactive for influenza A and B; parainfluenza 1, 2, and 3; respiratory syncytial virus; Q fever; or the primary atypical pneumonia agent. PMID:14044954

Effect of complement Factor H on anti-FHbp serum bactericidal antibody responses of infant rhesus macaques boosted with a licensed meningococcal serogroup B vaccine.

PubMed

Giuntini, Serena; Beernink, Peter T; Granoff, Dan M

2015-12-16

FHbp is a major serogroup B meningococcal vaccine antigen. Binding of complement Factor H (FH) to FHbp is specific for human and some non-human primate FH. In previous studies, FH binding to FHbp vaccines impaired protective anti-FHbp antibody responses. In this study we investigated anti-FHbp antibody responses to a third dose of a licensed serogroup B vaccine (MenB-4C) in infant macaques vaccinated in a previous study with MenB-4C. Six macaques with high binding of FH to FHbp (FH(high)), and six with FH(low) baseline phenotypes, were immunized three months after dose 2. After dose 2, macaques with the FH(low) baseline phenotype had serum anti-FHbp antibodies that enhanced FH binding to FHbp (functionally converting them to a FH(high) phenotype). In this group, activation of the classical complement pathway (C4b deposition) by serum anti-FHbp antibody, and anti-FHbp serum bactericidal titers were lower after dose 3 than after dose 2 (p<0.02). In macaques with the FH(high) baseline phenotype, the respective anti-FHbp C4b deposition and bactericidal titers were similar after doses 2 and 3. Two macaques developed serum anti-FH autoantibodies after dose 2, which were not detected after dose 3. In conclusion, in macaques with the FH(low) baseline phenotype whose post-dose 2 serum anti-FHbp antibodies had converted them to FH(high), the anti-FHbp antibody repertoire to dose 3 was skewed to less protective epitopes than after dose 2. Mutant FHbp vaccines that eliminate FH binding may avoid eliciting anti-FHbp antibodies that enhance FH binding, and confer greater protection with less risk of inducing anti-FH autoantibodies than FHbp vaccines that bind FH. Copyright © 2015 Elsevier Ltd. All rights reserved.

Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis.

PubMed

Bien, Christian G; Vincent, Angela; Barnett, Michael H; Becker, Albert J; Blümcke, Ingmar; Graus, Francesc; Jellinger, Kurt A; Reuss, David E; Ribalta, Teresa; Schlegel, Jürgen; Sutton, Ian; Lassmann, Hans; Bauer, Jan

2012-05-01

Classical paraneoplastic encephalitis syndromes with 'onconeural' antibodies directed to intracellular antigens, and the recently described paraneoplastic or non-paraneoplastic encephalitides and antibodies against both neural surface antigens (voltage-gated potassium channel-complexes, N-methyl-d-aspartate receptors) and intracellular antigens (glutamic acid decarboxylase-65), constitute an increasingly recognized group of immune-mediated brain diseases. Evidence for specific immune mechanisms, however, is scarce. Here, we report qualitative and quantitative immunopathology in brain tissue (biopsy or autopsy material) of 17 cases with encephalitis and antibodies to either intracellular (Hu, Ma2, glutamic acid decarboxylase) or surface antigenic targets (voltage-gated potassium channel-complex or N-methyl-d-aspartate receptors). We hypothesized that the encephalitides with antibodies against intracellular antigens (intracellular antigen-onconeural and intracellular antigen-glutamic acid decarboxylase groups) would show neurodegeneration mediated by T cell cytotoxicity and the encephalitides with antibodies against surface antigens would be antibody-mediated and would show less T cell involvement. We found a higher CD8/CD3 ratio and more frequent appositions of granzyme-B(+) cytotoxic T cells to neurons, with associated neuronal loss, in the intracellular antigen-onconeural group (anti-Hu and anti-Ma2 cases) compared to the patients with surface antigens (anti-N-methyl-d-aspartate receptors and anti-voltage-gated potassium channel complex cases). One of the glutamic acid decarboxylase antibody encephalitis cases (intracellular antigen-glutamic acid decarboxylase group) showed multiple appositions of GrB-positive T cells to neurons. Generally, however, the glutamic acid decarboxylase antibody cases showed less intense inflammation and also had relatively low CD8/CD3 ratios compared with the intracellular antigen-onconeural cases. Conversely, we found complement C9neo deposition on neurons associated with acute neuronal cell death in the surface antigen group only, specifically in the voltage-gated potassium channel-complex antibody patients. N-methyl-d-aspartate receptors-antibody cases showed no evidence of antibody and complement-mediated tissue injury and were distinguished from all other encephalitides by the absence of clear neuronal pathology and a low density of inflammatory cells. Although tissue samples varied in location and in the stage of disease, our findings strongly support a central role for T cell-mediated neuronal cytotoxicity in encephalitides with antibodies against intracellular antigens. In voltage-gated potassium channel-complex encephalitis, a subset of the surface antigen antibody encephalitides, an antibody- and complement-mediated immune response appears to be responsible for neuronal loss and cerebral atrophy; the apparent absence of these mechanisms in N-methyl-d-aspartate receptors antibody encephalitis is intriguing and requires further study.

Complement Interaction with Trypanosomatid Promastigotes in Normal Human Serum

PubMed Central

Domínguez, Mercedes; Moreno, Inmaculada; López-Trascasa, Margarita; Toraño, Alfredo

2002-01-01

In normal human serum (NHS), axenic promastigotes of Crithidia, Phytomonas, and Leishmania trigger complement activation, and from 1.2 to 1.8 × 105 C3 molecules are deposited per promastigote within 2.5 min. In Leishmania, promastigote C3 binding capacity remains constant during in vitro metacyclogenesis. C3 deposition on promastigotes activated through the classical complement pathway reaches a 50% maximum after ∼50 s, and represents >85% of total C3 bound. In C1q- and C2-deficient human sera, promastigotes cannot activate the classical pathway (CP) unless purified C1q or C2 factors, respectively, are supplemented, demonstrating a requirement for CP factor in promastigote C3 opsonization. NHS depleted of natural anti-Leishmania antibodies cannot trigger promastigote CP activation, but IgM addition restores C3 binding. Furthermore, Leishmania binds natural antibodies in ethylenediaminetetracetic acid (EDTA)-treated NHS; after EDTA removal, promastigote-bound IgM triggers C3 deposition in natural antibody-depleted NHS. Serum collectins and pentraxins thus do not participate significantly in NHS promastigote C3 opsonization. Real-time kinetic analysis of promastigote CP-mediated lysis indicates that between 85–95% of parasites are killed within 2.5 min of serum contact. These data indicate that successful Leishmania infection in man must immediately follow promastigote transmission, and that Leishmania evasion strategies are shaped by the selective pressure exerted by complement. PMID:11854358

Antibody-Mediated Rejection in Human Cardiac Allografts: Evaluation of Immunoglobulins and Complement Activation Products C4d and C3d as Markers

PubMed Central

Rodriguez, E. R.; Skojec, Diane V.; Tan, Carmela D.; Zachary, Andrea A.; Kasper, Edward K.; Conte, John V.; Baldwin, William M.

2005-01-01

Antibody-mediated rejection (AMR) in human heart transplantation is an immunopathologic process in which injury to the graft is in part the result of activation of complement and it is poorly responsive to conventional therapy. We evaluated by immunofluorescence (IF), 665 consecutive endomyocardial biopsies from 165 patients for deposits of immunoglobulins and complement. Diffuse IF deposits in a linear capillary pattern greater than 2+ were considered significant. Clinical evidence of graft dysfunction was correlated with complement deposits. IF 2+ or higher was positive for IgG, 66%; IgM, 12%; IgA, 0.6%; C1q, 1.8%; C4d, 9% and C3d, 10%. In 3% of patients, concomitant C4d and C3d correlated with graft dysfunction or heart failure. In these 5 patients AMR occurred 56–163 months after transplantation, and they responded well to therapy for AMR but not to treatment with steroids. Systematic evaluation of endomyocardial biopsies is not improved by the use of antibodies for immunoglobulins or C1q. Concomitant use of C4d and C3d is very useful to diagnose AMR, when correlated with clinical parameters of graft function. AMR in heart transplant patients can occur many months or years after transplant. PMID:16212640

Platelet-activating factor mediates monocyte chemoattractant protein-1 expression in glomerular immune injury.

PubMed

Jocks, T; Freudenberg, J; Zahner, G; Stahl, R A

1998-01-01

These studies were designed to determine the possible role of platelet-activating factor (PAF) in the production of monocyte chemoattractant protein-1 (MCP-1) in glomerular immune injury. The glomerular lesion was induced in isolated perfused rat kidneys by a rabbit anti-rat-thymocyte serum (ATS) and rat serum (RS) as a complement source. Perfusion of kidneys with ATS and RS results in the selective binding of the antiserum to the glomerular mesangium with consecutive intraglomerular activation of complement. Antibody binding and complement activation induced a significant increase in glomerular MCP-1 mRNA levels when assessed by Northern blotting or RT-PCR. Decomplemented RS or non antibody rabbit IgG had only moderate effects on glomerular MCP-1 mRNA levels. The PAF receptor antagonist WEB 2170 almost completely blocked the ATS and RS induced MCP-1 mRNA levels. Perfusion of control kidneys with PAF increased MCP-1 mRNA expression, an effect which was blocked by WEB 2170. Glomerular MCP-1 protein formation, assessed by Western blotting, was stimulated following ATS and RS and PAF, respectively, was blocked by WEB 2170. These data show that PAF, derived from glomerular resident cells following antibody and complement induced injury, stimulates MCP-1 expression. In addition to the direct effects on leukocyte adhesion and activation PAF may mediate inflammatory cell influx in glomerular injuries due to the release of MCP-1.

Pathologic highlights of dengue hemorrhagic fever in 13 autopsy cases from Myanmar.

PubMed

Aye, Khin Saw; Charngkaew, Komgrid; Win, Ne; Wai, Kyaw Zin; Moe, Kyaw; Punyadee, Nuntaya; Thiemmeca, Somchai; Suttitheptumrong, Aroonroong; Sukpanichnant, Sanya; Prida, Malasit; Halstead, Scott B

2014-06-01

Vascular permeability, thrombocytopenia, liver pathology, complement activation, and altered hemostasis accompanying a febrile disease are the hallmarks of the dengue hemorrhagic fever/dengue shock syndrome, a major arthropod-borne viral disease that causes significant morbidity and mortality throughout tropical countries. We studied tissues from 13 children who died of acute dengue hemorrhagic fever/dengue shock syndrome at the Childrens' Hospital, Yangon, Myanmar. Dengue viral RNA from each of the 4 dengue viruses (DENVs) was detected by reverse transcriptase polymerase chain reaction in 11 cases, and dengue viral proteins (envelope, NS1, or NS3) were detected in 1 or more tissues from all 13 cases. Formalin-fixed and frozen tissues were studied for evidence of virus infection using monoclonal antibodies against DENV structural and nonstructural antigens (E, NS1, and nonsecreting NS3). In the liver, DENV infection occurred in hepatocytes and Kupffer cells but not in endothelial cells. Liver damage was associated with deposition on hepatocytes of complement components of both classical and alternative pathways. Evidence of dengue viral replication was observed in macrophage-like cells in spleens and lymph nodes. No dengue antigens were detected in endothelial cells in any organ. Germinal centers of the spleen and lymph nodes showed a marked reduction in the number of lymphocytes that were replaced by eosinophilic deposits, which contained dengue antigens as well as immunoglobulins, and complement components (C3, C1q, and C9). The latter findings had previously been reported but overlooked as a diagnostic feature. Copyright © 2014 Elsevier Inc. All rights reserved.

A NEW SENSITIVE ASSAY FOR ANTIBODY AGAINST CELL SURFACE ANTIGENS BASED ON INHIBITION OF CELL-DEPENDENT ANTIBODY-MEDIATED CYTOTOXICITY

PubMed Central

Halloran, Phil; Schirrmacher, Volker; Festenstein, Hilliard

1974-01-01

Inhibition of cell-dependent antibody-mediated cytotoxicity has been investigated as a new assay for antibody against cell surface antigens. The cytotoxicity system consisted of effector cells (normal mouse spleen cells), target cells (61Cr-labeled chicken erythrocytes), and antitarget cell antibody. Addition of antibody against cell surface antigens in the effector cell population regularly inhibited the cytotoxicity measured in this system. This cytotoxicity inhibition assay (CIA) detected antibody with a variety of specificities: anti-H-2, anti-Thy 1.2, anti-immunoglobulin, and antimouse bone marrow-derived lymphocyte antigen. When the inhibition by anti-H-2 sera was analyzed using effector cells from congenic mice, the activity was found to be directed against specificities mapping in the H-2K, H-2D, and I regions of the H-2 complex, correlating well with the specificities characterized by complement-dependent assays. A comparison between the sensitivity of the CIA and complement-dependent lysis revealed that the CIA was 2–11 times more sensitive for anti-H-2 antisera and 20–780 times more sensitive for certain antisera against subpopulations of the spleen cells (i.e., T cells or B cells). The CIA proved to be precise, sensitive, and reliable. It may become a very useful antibody assay in various species including man. PMID:4547657

Use of hybridoma immunoglobulin switch variants in the analysis of the protective properties of anti-lipopolysaccharide antibodies in Escherichia coli K1 infection.

PubMed

Pelkonen, S; Pluschke, G

1989-10-01

Functional properties of rat immunoglobulins obtained from hybridoma isotype switch variants were studied in vivo in a rat model for neonatal bacterial sepsis. Escherichia coli 018:K1, a common cause of human neonatal sepsis and meningitis, was injected intravenously into 6-day-old rats after incubation with 018-specific antibodies IgM, IgG1, IgG2a, IgG2b, IgG2c, IgE and IgA. The clearance of bacteria treated with saline or IgE was low, whereas monoclonal antibodies of other isotypes triggered hepatic sequestration and killing of the K1 E. coli cells. All four IgG subclasses were more efficient than IgM and IgA. Comparable results were obtained upon injecting antibodies into rats with an established fulminating bacteraemia. IgM was inactive in animals depleted of complement with cobra-venom factor (CVF), whereas IgG2b was able to trigger hepatic clearance independently of complement.

A monoclonal antibody targeting amyloid β (Aβ) restores complement factor I bioactivity: Potential implications in age-related macular degeneration and Alzheimer's disease.

PubMed

Lashkari, Kameran; Teague, Gianna; Chen, Hong; Lin, Yong-Qing; Kumar, Sanjay; McLaughlin, Megan M; López, Francisco J

2018-01-01

Activation of the alternative complement cascade has been implicated in the pathogenesis of age related macular degeneration (AMD) and Alzheimer's disease (AD). Amyloid β (Aβ), a component of drusen, may promote complement activation by inhibiting CFI bioactivity. We determined whether Aβ reduced CFI bioactivity and whether antibodies against Aβ including a monoclonal antibody, GSK933776 could restore CFI bioactivity. We also measured CFI bioactivity in plasma of subjects with AMD and AD. In support of the GSK933776 development program in AMD (geographic atrophy), we developed a quantitative assay to measure CFI bioactivity based on its ability to cleave C3b to iC3b, and repeated it in presence or absence of Aβ and anti-Aβ antibodies. Using this assay, we measured CFI bioactivity in plasma of 194 subjects with AMD, and in samples from subjects with AD that had been treated with GSK933776 as part of the GSK933776 development program in AD. Aβ reduced the CFI bioactivity by 5-fold and pre-incubation with GSK933776 restored CFI bioactivity. In subjects with AMD, plasma CFI levels and bioactivity were not significantly different from non-AMD controls. However, we detected a positive linear trend, suggesting increasing activity with disease severity. In subjects with AD, we observed a 10% and 27% increase in overall CFI bioactivity after treatment with GSK933776 during the second and third dose. Our studies indicate that CFI enzymatic activity can be inhibited by Aβ and be altered in proinflammatory diseases such as AMD and AD, in which deposition of Aβ and activation of the alternative complement cascade are believed to play a key role in the disease process.

The structural requirements for immunoglobulin aggregates to localize in germinal centres.

PubMed Central

Embling, P H; Evans, H; Guttierez, C; Holborow, E J; Johns, P; Johnson, P M; Papamichail, M; Stanworth, D R

1978-01-01

The capacity of non-heat-aggregated monoclonal human immunoglobulins of different classes, to localize in murine splenic germinal centres within 24 h of intravenous injection has been investigated. It has been shown that at least trimerization of polyclonal IgG must occur before any germinal centre trapping is manifest. Studies of complement fixation by these IgG preparations in vivo, together with studies of the germinal centre trapping of various monoclonal immunoglobulins, have indicated that the sole structural requirement for germinal centre localization of immunoglobulin aggregates is the ability to fix complement. Results suggest that immunoglobulin aggregates are transported to germinal centres via membrane C3 receptors of mobile cells, and then are released with loss of complement to become fixed to dendritic macrophages by a separate mechanism. PMID:363602

Advances in Antibody Design

PubMed Central

Tiller, Kathryn E.; Tessier, Peter M.

2017-01-01

The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody–drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics. PMID:26274600

Influence of IgG Subclass on Human Antimannan Antibody-Mediated Resistance to Hematogenously Disseminated Candidiasis in Mice

PubMed Central

Nishiya, Casey T.; Boxx, Gayle M.; Robison, Kerry; Itatani, Carol; Kozel, Thomas R.

2015-01-01

Candida albicans is a yeast-like pathogen and can cause life-threatening systemic candidiasis. Its cell surface is enriched with mannan that is resistant to complement activation. Previously, we developed the recombinant human IgG1 antimannan antibody M1g1. M1g1 was found to promote complement activation and phagocytosis and protect mice from systemic candidiasis. Here, we evaluate the influence of IgG subclass on antimannan antibody-mediated protection. Three IgG subclass variants of M1g1 were constructed: M1g2, M1g3, and M1g4. The IgG subclass identity for each variant was confirmed with DNA sequence and subclass-specific antibodies. These variants contain identical M1 Fabs and exhibited similar binding affinities for C. albicans yeast and purified mannan. Yeast cells and hyphae recovered from the kidney of antibody-treated mice with systemic candidiasis showed uniform binding of each variant, indicating constitutive expression of the M1 epitope and antibody opsonization in the kidney. All variants promoted deposition of both murine and human C3 onto the yeast cell surface, with M1g4 showing delayed activation, as determined by flow cytometry and immunofluorescence microscopy. M1g4-mediated complement activation was found to be associated with its M1 Fab that activates the alternative pathway in an Fc-independent manner. Treatment with each subclass variant extended the survival of mice with systemic candidiasis (P < 0.001). However, treatment with M1g1, M1g3, or M1g4, but not with M1g2, also reduced the kidney fungal burden (P < 0.001). Thus, the role of human antimannan antibody in host resistance to systemic candidiasis is influenced by its IgG subclass. PMID:26573736

Antibodies against viruses: passive and active immunization

PubMed Central

Law, Mansun; Hangartner, Lars

2008-01-01

Summary of recent advances Antibodies, through passive or active immunization, play a central role in prophylaxis against many infectious agents. While neutralization is a primary function of antibodies in protection against most viruses, the relative contribution of Fc-dependent and complement-dependent antiviral activities of antibodies was found to vary between different viruses in recent studies. The multiple hit model explains how antibodies neutralize viruses and recent data on the stoichiometry of antibody neutralization suggest that the organization of viral surface proteins on viruses, in addition to virus size, influences the level of antibody occupancy required for neutralization. These new findings will improve our strategies in therapeutic antibody engineering and rational vaccine design. PMID:18577455

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21)

PubMed Central

Khandelwal, Sanjay; Lee, Grace M.; Hester, C. Garren; Poncz, Mortimer; McKenzie, Steven E.; Sachais, Bruce S.; Rauova, Lubica; Kelsoe, Garnett; Cines, Douglas B.; Frank, Michael

2016-01-01

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes. PMID:27412887

A monoclonal IgM smooth muscle antibody reactive with fibroblast stress fibres produced by immunization with Treponema pallidum.

PubMed Central

Strugnell, R A; Underwood, J R; Clarke, F M; Pedersen, J S; Chalmers, P J; Faine, S; Toh, B H

1983-01-01

A monoclonal IgM smooth muscle antibody secreted by a hybrid (MMI-1) of mouse plasmacytoma NS-1 with spleen cells from mouse immunized with Treponema pallidum was detected by indirect immunofluorescence tests on frozen tissue sections and on acetone fixed monolayers of rat and human fibroblasts. The antibody did not react with acetone fixed smears of T. pallidum but reacted with smooth muscle fibres and with striations of skeletal and cardiac muscle. In non-muscle cells, the antibody stained liver in a 'polygonal' pattern, thymus with accentuated staining of the thymic medulla, renal glomeruli and the brush border and peritubular fibrils of renal tubules. In fibroblast monolayers, the antibody stained stress fibres in an interrupted pattern. Immunoblotting with muscle proteins and the antibody showed labelling of a 100K molecule. The cellular distribution of the mouse monoclonal antibody is similar to that obtained with anti-actin antibody suggesting that the corresponding antigen may be an actin binding protein. Images Fig. 3 PMID:6347470

[Differentiation of nonspecific serological reactions in brucellosis].

PubMed

Khristoforov, L

1979-01-01

Differentiation of non-specific agglutination was performed by the complement binding reaction, Coombs' reaction, Hajdu reaction, the surface fixation and agglutination reaction and the reaction of complement binding with heterologic antigens. For that purpose the following were used: 1) Serums--antiglobulin against cattle globulin, 5720 serum of various animals which had manifested non-specific agglutination with brucella antigen and brucella serums of experimentally infected sheep, of naturally infected swine and of cattle--received from abroad. 2) Antigens--of Br. abortus 99, of bacteria heterologic to brucellae: Proteus vulgaris, Listeria monocytogenes, Staphylococcus albus, Escherichia coli, Streptococcus pyogenes, S. abortus ovis, for O and OH agglutination, water extraction antigens--for complement binding and concentrated suspensions of all bacteria used in brucellose and non-brucellose serum absorption. Highest number of non-specific reactions were observed in cattle serums and lowest--in goat serums. Titers with heterologic antigens were higher than these with brucella antigens. Often the serum having non-specific agglutiantion reacted not only with one, but with more heterologic antigens. Non-specific complement binding reactions were not produced in complete antibodies with the brucella antigen. Heterologic brucella antigens were exhausted more fully than heterologic complement binding antibodies. In their effectiveness (differentiation of non-specific agglutination with brucella antigen in cattle serum) the serological reactions studied rank as follows: complement binding reaction, slow agglutination with serums absorbed by heterologic antigens, surface fixation reaction, Coombs' reaction, and Hadju agglutination.

[Variations of peripheral blood autoantibody, immunoglobuliln, and complement levels in patients with non-lactational mastitis and their clinical significances].

PubMed

Xu, Rui; Guo, Qian-Qian; Yang, Le-Ping; Lai, Mi-Lin; Tong, Lin

2016-08-20

To detect the variations in peripheral blood levels of autoantibodies, immunoglobulilns and complements in patients with non-lactational mastitis and investigate whether non-lactational mastitis is an autoimmune disease with immune dysfunction. Seven-eight patients with non-lactational mastitis treated in our hospital between September 2013 and May 2015 and 88 healthy women (control) were examined for peripheral blood levels of antinuclear antibody (ANA), anti-histone antibody (AHA), immunoglobulins (IgA, IgM, and IgG) and complements (C3, C4, and total complements). s Of the 78 patients with non-lactational mastitis, 50 (64.10%) were positive of ANA showing mainly the granular and cytoplasmic granular fluorescence patterns, and the positivity rate was significantly higher than that in the control group (P<0.000). Twenty-eight (36.00%) of the patients were positive of AHA, a rate significantly higher than that in the control group (P<0.000). The levels of IgA, IgM, C4, and total complements levels were all significantly elevated in the patients compared with those in the control group (P<0.05). Patients with non-lactational mastitis have abnormal changes in peripheral blood levels of immunoglobulins and complements with high positivity rates for ANA and AHA, indicating that non-lactational mastitis is an autoimmune disease with immune dysfunction.

Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

PubMed Central

Medina, Eva; van Rooijen, Willemien J.; Spaan, András N.; van Kessel, Kok P. M.; Höök, Magnus; Rooijakkers, Suzan H. M.

2013-01-01

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein. PMID:24348255

PASSIVE HEMOLYSIS BY SERUM AND COBRA VENOM FACTOR: A NEW MECHANISM INDUCING MEMBRANE DAMAGE BY COMPLEMENT*

PubMed Central

Pickering, R. J.; Wolfson, M. R.; Good, R. A.; Gewurz, H.

1969-01-01

The studies presented here indicate that activation of the complement (C′) system by a foreign protein will cause membrane injury and passive lysis of unsensitized erythrocytes present at the time of the reaction. These observations suggest that in addition to the classical antibody-C′-induced cytolysis, there are alternative pathways or mechanisms for activation and participation of the terminal C′ components in the production of cell membrane injury. We have shown that a substance derived from cobra venom and eluted from a single protein band on polyacrylamide can promote lysis of unsensitized autologous or heterologous erythrocytes in the presence of fresh guinea pig serum and that this lysis-inducing activity and C′-inhibiting activity appear to reside in the same fractions. The lytic activity is prevented by several agents known to impair classical C′3 activity, but is unaffected by certain procedures which interfere with the function of C′ components C′1 and C′2, a suggestion that this reaction involves chiefly C′3-C′9. Further, the cobra venom (CV) factor depletes C′ activity in cobra serum, and the CV factor (with its 5S serum cofactor) converts purified C′3 to its inactive form,1 indicating that the reaction of this complex with the complement system occurs without participation of antibody. Therefore, since the lysis-inducing and C′-inhibiting activity of the CV factor appear to result from similar interactions with the complement system, these observations suggest that cell membrane damage and cell lysis can be accomplished through activation of the complement system by a mechanism involving little or no participation of classical antibody or C′ components C′1, 4, or 2. Images PMID:4978744

Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

PubMed

Schmitz, H

1981-01-01

An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

Prostaglandin E1 reduces the glomerular mRNA expression of monocyte-chemoattractant protein 1 in anti-thymocyte antibody-induced glomerular injury.

PubMed

Jocks, T; Zahner, G; Freudenberg, J; Wolf, G; Thaiss, F; Helmchen, U; Stahl, R A

1996-06-01

To study whether prostaglandins (PG) can regulate the mRNA expression of monocyte-chemoattractant protein 1 (MCP-1) in glomerular immune injury, MCP-1 mRNA levels were evaluated in anti-thymocyte antibody (ATS) -induced glomerular injury by Northern blotting and reverse transcription-polymerase chain reaction. Immune injury was induced in vivo by the intravenous application of ATS to male Wistar rats and in vitro by the perfusion of isolated rat kidneys with ATS and rat serum. In vivo 3 h and 5 days after antibody application, glomerular mRNA expression of MCP-1 was markedly enhanced compared with controls. In the isolated perfused kidney, antibody and complement also induced an increase in MCP-1 expression at 10 min and 60 min after antibody perfusion. When the rats were treated with PGE (250 micrograms, twice daily), the increase in MCP-1 expression was reduced. This was associated with a reduction of intraglomerular recruitment of monocytes/macrophages. In the isolated perfused kidneys, PGE1 (1 mg/L) prevented the antibody- and rat serum-stimulated increase in glomerular MCP-1 mRNA expression. These data demonstrate that PGE1 reduces glomerular MCP-1 mRNA expression in glomerulonephritis and in the isolated perfused rat kidney after induction of immune injury with antibody and complement. The data suggest that prostaglandins might mediate MCP-1 effects in glomerular immune injuries.

Antibody-mediated rejection in kidney transplantation: a review of pathophysiology, diagnosis, and treatment options.

PubMed

Kim, Miae; Martin, Spencer T; Townsend, Keri R; Gabardi, Steven

2014-07-01

Antibody-mediated rejection (AMR), also known as B-cell-mediated or humoral rejection, is a significant complication after kidney transplantation that carries a poor prognosis. Although fewer than 10% of kidney transplant patients experience AMR, as many as 30% of these patients experience graft loss as a consequence. Although AMR is mediated by antibodies against an allograft and results in histologic changes in allograft vasculature that differ from cellular rejection, it has not been recognized as a separate disease process until recently. With an improved understanding about the importance of the development of antibodies against allografts as well as complement activation, significant advances have occurred in the treatment of AMR. The standard of care for AMR includes plasmapheresis and intravenous immunoglobulin that remove and neutralize antibodies, respectively. Agents targeting B cells (rituximab and alemtuzumab), plasma cells (bortezomib), and the complement system (eculizumab) have also been used successfully to treat AMR in kidney transplant recipients. However, the high cost of these medications, their use for unlabeled indications, and a lack of prospective studies evaluating their efficacy and safety limit the routine use of these agents in the treatment of AMR in kidney transplant recipients. © 2014 Pharmacotherapy Publications, Inc.

The immunological response of the rat to infection with Taenia taeniaeformis. V. Sequence of appearance of protective immunoglobulins and the mechanism of action of 7Sgamma2a antibodies.

PubMed Central

Musoke, A J; Williams, J F

1975-01-01

Passive transfer of immunity to Taenia taeniaeformis was achieved with serum taken 14, 21, 49 and 63 days after infection. The protective capacity of serum collected at 14 and 21 days resided in the 7Sgamma2 immunoglobulins and appeared to be partics the infection progressed the range of chromatographic fractions showing protective capacity was extended to all those containing 7Sgamma2 and 7Sgamma1 immunoglobulins. Fractions enriched for gammaM did not confer protection. Immune serum containing 7Sgamma2a antibodies was able to kill developing parasites after they had left the intestine, and the hepatic postoncospheral forms retained their susceptibility to antibody over the first 5 days of growth. After that time they rapidly became insusceptible to antibody both in vivo and in vitro. Susceptibility to antibody-mediated attack was complement dependent. This appears to be the first time that complement has been demonstrated to play a role in immunity to a helminth infection in vivo. This finding is discussed in relation to the phenomenon of cestode parasite survival in immune animals. Images FIG. 1 PMID:1201860

Serum killing of Ureaplasma parvum shows serovar-determined susceptibility for normal individuals and common variable immuno-deficiency patients.

PubMed

Beeton, Michael L; Daha, Mohamed R; El-Shanawany, Tariq; Jolles, Stephen R; Kotecha, Sailesh; Spiller, O Brad

2012-02-01

Many Gram-negative bacteria, unlike Gram-positive, are directly lysed by complement. Ureaplasma can cause septic arthritis and meningitis in immunocompromised individuals and induce premature birth. Ureaplasma has no cell wall, cannot be Gram-stain classified and its serum susceptibility is unknown. Survival of Ureaplasma serovars (SV) 1, 3, 6 and 14 (collectively Ureaplasma parvum) were measured following incubation with normal or immunoglobulin-deficient patient serum (relative to heat-inactivated controls). Blocking monoclonal anti-C1q antibody and depletion of calcium, immunoglobulins, or lectins were used to determine the complement pathway responsible for killing. Eighty-three percent of normal sera killed SV1, 67% killed SV6 and 25% killed SV14; greater killing correlating to strong immunoblot identification of anti-Ureaplasma antibodies; killing was abrogated following ProteinA removal of IgG1. All normal sera killed SV3 in a C1q-dependent fashion, irrespective of immunoblot identification of anti-Ureaplasma antibodies; SV3 killing was unaffected by total IgG removal by ProteinG, where complement activity was retained. Only one of four common variable immunodeficient (CVID) patient sera failed to kill SV3, despite profound IgM and IgG deficiency for all; however, killing of SV3 and SV1 was restored with therapeutic intravenous immunoglobulin therapy. Only the classical complement pathway mediated Ureaplasma-cidal activity, sometimes in the absence of observable immunoblot reactive bands. Copyright © 2011 Elsevier GmbH. All rights reserved.

Anti-neutrophil cytoplasmic antibodies in new-onset systemic lupus erythematosus.

PubMed

Su, Fang; Xiao, Weiguo; Yang, Pingting; Chen, Qingyan; Sun, Xiaojie; Li, Tienan

2017-01-01

The clinical significance of anti-neutrophil cytoplasmic antibodies in patients with new-onset systemic lupus erythematosus, especially in systemic disease accompanied by interstitial lung disease remains to be elucidated. This study was designed to investigate the role of anti-neutrophil cytoplasmic antibodies in new-onset systemic lupus erythematosus patients. A hundred and seven patients with new-onset SLE were enrolled. Presence of anti-neutrophil cytoplasmic antibodies in the sera was assessed by indirect immunofluorescence as well as enzyme linked immunosorbent assay against proteinase-3 and myeloperoxidase. Clinical features and laboratory parameters of patients were also recorded. All patients were subjected to chest X-ray, chest high-resolution computed tomography and pulmonary function test. Forty-five systemic lupus erythematosus patients (45/107, 42%) were seropositive for anti-neutrophil cytoplasmic antibodies. Compared with anti-neutrophil cytoplasmic antibodies-negative patients, the anti-neutrophil cytoplasmic antibodies-positive patients had significantly higher incidence of renal involvement, anemia, and Raynaud's phenomenon as well as decreased serum level of complement 3/complement 4 and elevated erythrocyte sedimentation rate. In addition, there was a positive correlation between serum anti-neutrophil cytoplasmic antibodies level and disease activity of systemic lupus erythematosus. Furthermore, prevalence of interstitial lung disease in the anti-neutrophil cytoplasmic antibodies -positive patients (25/45, 55.6%) was obviously higher than that in the anti-neutrophil cytoplasmic antibodies-negative patients (15/62, 24.2%). The sample size was limited and the criteria for screening new-onset systemic lupus erythematosus patients might produce bias. The level of anti-neutrophil cytoplasmic antibodies in new-onset systemic lupus erythematosus patients correlates positively with the disease activity and the prevalence of interstitial lung disease.

Induction of suppressor cells in vitro by Candida albicans.

PubMed

Cuff, C F; Rogers, C M; Lamb, B J; Rogers, T J

1986-06-01

Normal splenocytes cultured with Formalin-killed Candida albicans were shown to acquire significant suppressor cell activity in a period of 3 days. These cells were found to suppress both the phytohemagglutinin-induced mitogen response as well as the anti-sheep erythrocyte antibody response. Experiments were carried out to determine the nature of the suppressor cell population. Results showed that these cells were not susceptible to treatment with anti-Thy 1 antibody and complement. Panning experiments showed that the suppressor cells were not plastic-adherent or Mac-1 antigen-positive. The suppressor cells were, however, adherent to anti-mouse immunoglobulin (F(ab')2-fragment)-coated dishes. Additional experiments showed that the suppressor cell activity was susceptible to treatment with monoclonal anti-Lyb 2.1 antibody and complement. These results suggest that the suppressor cell induced in vitro by Candida is a member of the B-lymphocyte lineage.

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

PubMed

Giuntini, Serena; Beernink, Peter T; Reason, Donald C; Granoff, Dan M

2012-01-01

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

Production and Characterization of High-Affinity Human Monoclonal Antibodies to Human Immunodeficiency Virus Type 1 Envelope Glycoproteins in a Mouse Model Expressing Human Immunoglobulins▿ †

PubMed Central

Sheppard, Neil C.; Davies, Sarah L.; Jeffs, Simon A.; Vieira, Sueli M.; Sattentau, Quentin J.

2007-01-01

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-197CN54), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-197CN54 Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies. PMID:17167037

Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins.

PubMed

Sheppard, Neil C; Davies, Sarah L; Jeffs, Simon A; Vieira, Sueli M; Sattentau, Quentin J

2007-02-01

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.

The Biology of IgG Subclasses and Their Clinical Relevance to Transplantation.

PubMed

Valenzuela, Nicole M; Schaub, Stefan

2018-01-01

Immunoglobulin G (IgG) is the dominant immunoglobulin and can be divided into 4 distinct subclasses. The evolution of IgG subclass switches is regulated by interaction with T cells and follows a 1-way direction (IgG3 → IgG1 → IgG2 → IgG4). Based on their structure, the 4 IgG subclasses can initiate different effector function such as complement activation, recruitment of various cells by Fc receptors, and agonistic signaling. Using current assays for HLA antibody detection as a template and replacing the generic reporter antibody with IgG subclass-specific reporter antibodies, it is possible to investigate the IgG subclasses of HLA antibodies. There are 15 different IgG subclass compositions possible. Based on the capability to activate the complement system and the class switch direction, 3 arbitrary patterns can be defined (ie, only complement-binding subclasses [IgG3 and/or IgG1], expansion to noncomplement-binding subclasses [IgG3 and/or IgG1 plus IgG2 and/or IgG4], and switch to noncomplement-binding subclasses [IgG2 and/or IgG4]). The latter group accounts for less than 5%, whereas the former 2 groups have a similar prevalence close to 50%. In the past 5 years, several studies correlated the IgG subclass pattern with occurrence of antibody-mediated rejection and allograft outcomes. Because of differences of the used IgG subclass assay, the time point of analyses, and the definition of outcomes, a clear picture has not emerged yet. Future needs are standardization of the assay, a more detailed knowledge of the initiated effector functions, and more well-designed clinical studies also looking at changes of the IgG subclass pattern over time.

Complement Activation in Inflammatory Skin Diseases

PubMed Central

Giang, Jenny; Seelen, Marc A. J.; van Doorn, Martijn B. A.; Rissmann, Robert; Prens, Errol P.; Damman, Jeffrey

2018-01-01

The complement system is a fundamental part of the innate immune system, playing a crucial role in host defense against various pathogens, such as bacteria, viruses, and fungi. Activation of complement results in production of several molecules mediating chemotaxis, opsonization, and mast cell degranulation, which can contribute to the elimination of pathogenic organisms and inflammation. Furthermore, the complement system also has regulating properties in inflammatory and immune responses. Complement activity in diseases is rather complex and may involve both aberrant expression of complement and genetic deficiencies of complement components or regulators. The skin represents an active immune organ with complex interactions between cellular components and various mediators. Complement involvement has been associated with several skin diseases, such as psoriasis, lupus erythematosus, cutaneous vasculitis, urticaria, and bullous dermatoses. Several triggers including auto-antibodies and micro-organisms can activate complement, while on the other hand complement deficiencies can contribute to impaired immune complex clearance, leading to disease. This review provides an overview of the role of complement in inflammatory skin diseases and discusses complement factors as potential new targets for therapeutic intervention. PMID:29713318

In vitro C3 mRNA expression in Pemphigus vulgaris: complement activation is increased by IL-1alpha and TNF-alpha.

PubMed

Feliciani, C; Toto, P; Amerio, P

1999-01-01

Pemphigus vulgaris (PV) is a potentially life-threatening disease, characterized immunohistologically by IgG deposits and complement activation on the surface of keratinocytes. Complement activation has been implicated in the pathogenesis with C3 deposits in about 90% of patients. In order to further elucidate the role of complement in PV and to define which cytokines play a role in C3 mRNA expression, we performed an in vitro study in human keratinocytes. Normal human epidermal keratinocytes (NHuK) were incubated with PV serum and C3 mRNA was measured. We previously had shown that IL-1alpha and TNF-alpha are expressed in PV in vivo and in vitro. Since cytokines are able to modulate complement activation, mRNA expression was evaluated in a similar experiment after pretreatment using antibodies against IL-1alpha and TNF-alpha. Incubation of NHuK with PV sera caused their detachment from the plates after 20-30 minutes with a complete acantholysis within 12 hours. An early C3 mRNA expression was seen after 30 minutes with a peak level after 1 hour. Blocking studies, using antibodies against human IL-1alpha and TNF-alpha in NHuK together with PV-IgG, showed reduction of in vitro induced acantholysis and inhibition of C3 mRNA expression. This study supports the hypothesis that complement C3 is important in PV acantholysis and that complement activation is increased by IL-1alpha and TNF-alpha.

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

PubMed Central

Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

2013-01-01

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella

PubMed Central

Boyd, Mary A.; Wang, Jin Y.; Tulapurkar, Mohan E.; Pasetti, Marcela F.; Levine, Myron M.; Simon, Raphael

2016-01-01

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity. PMID:26998925

Low complements and high titre of anti-Sm antibody as predictors of histopathologically proven silent lupus nephritis without abnormal urinalysis in patients with systemic lupus erythematosus.

PubMed

Ishizaki, Jun; Saito, Kazuyoshi; Nawata, Masao; Mizuno, Yasushi; Tokunaga, Mikiko; Sawamukai, Norifumi; Tamura, Masahito; Hirata, Shintaro; Yamaoka, Kunihiro; Hasegawa, Hitoshi; Tanaka, Yoshiya

2015-03-01

The aim of this study was to clarify the clinical characteristics and predictors of silent LN (SLN), a type of LN in SLE without abnormal urinalysis or renal impairment. Of 182 patients who underwent renal biopsy, 48 did not present with abnormal urinalysis or renal impairment at the time of biopsy. The patients with LN (SLN group, n = 36) and those without LN (non-LN group, n = 12) were compared with respect to their baseline characteristics. Bivariate analysis comprised Fisher's exact test and the Mann-Whitney test, whereas multivariate analysis employed binomial logistic regression analysis. LN was histopathologically identified in 36 of 48 patients. According to the International Society of Nephrology/Renal Pathology Society classification, 72% of the SLN patients were classified as having class I/II, with a further 17% having class III/IV. Bivariate analyses indicated that platelet count, serum albumin, complement components (C3 and C4), complement haemolytic activity (CH50), anti-Sm antibody titre and anti-ribonucleoprotein antibody titre were significantly different between groups. Multivariate analysis indicated that CH50 and C3 titres were significantly lower in the SLN group, whereas anti-Sm antibody titre was significantly higher. The cut-off titre, calculated based on the receiver operating characteristic curve for CH50, was 33 U/ml, with a sensitivity and specificity of 89% and 83%, respectively. The cut-off titre for anti-Sm antibodies was 9 U/ml, with a sensitivity and specificity of 74% and 83%, respectively. Low titres of CH50 and C3 and a high titre of anti-Sm antibody were identified as predictors of SLN. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Ganjam virus.

PubMed

Sudeep, A B; Jadi, R S; Mishra, A C

2009-11-01

Ganjam virus (GANV), a member of genus Nairovirus of family Bunyavirdae is of considerable veterinary importance in India. Though, predominantly tick borne, GANV was also isolated from mosquitoes, man and sheep. Neutralizing and complement fixing antibodies to GANV have been detected in animal and human sera collected from different parts of the country. Thirty three strains of GANV have been isolated from India, mainly from Haemaphysalis ticks. The virus replicated in certain vertebrate and mosquito cell lines and found pathogenic to laboratory animals. One natural infection and five laboratory-acquired infections in men were also reported. GANV is antigenically related to Nairobi sheep disease virus (NSDV) of Africa, which is highly pathogenic for sheep and goats causing 70-90 per cent mortality among the susceptible population. Recent molecular studies have demonstrated that GANV is an Asian variant of NSDV and both these viruses are related to the dreaded Crimean Congo haemorrhagic fever (CCHF) group viruses. The versatility of the virus to replicate in different arthropod species, its ability to infect sheep, goat and man makes it an important zoonotic agent.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

PubMed

Harris, Katherine E; Aldred, Shelley Force; Davison, Laura M; Ogana, Heather Anne N; Boudreau, Andrew; Brüggemann, Marianne; Osborn, Michael; Ma, Biao; Buelow, Benjamin; Clarke, Starlynn C; Dang, Kevin H; Iyer, Suhasini; Jorgensen, Brett; Pham, Duy T; Pratap, Payal P; Rangaswamy, Udaya S; Schellenberger, Ute; van Schooten, Wim C; Ugamraj, Harshad S; Vafa, Omid; Buelow, Roland; Trinklein, Nathan D

2018-01-01

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

PubMed Central

van den Bremer, Ewald TJ; Beurskens, Frank J; Voorhorst, Marleen; Engelberts, Patrick J; de Jong, Rob N; van der Boom, Burt G; Cook, Erika M; Lindorfer, Margaret A; Taylor, Ronald P; van Berkel, Patrick HC; Parren, Paul WHI

2015-01-01

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential. PMID:26037225

Banff study of pathologic changes in lung allograft biopsy specimens with donor-specific antibodies.

PubMed

Wallace, William Dean; Li, Ning; Andersen, Claus B; Arrossi, A Valeria; Askar, Medhat; Berry, Gerry J; DeNicola, Matthew M; Neil, Desley A; Pavlisko, Elizabeth N; Reed, Elaine F; Remmelink, Myriam; Weigt, S Sam; Weynand, Birgit; Zhang, Jennifer Q; Budev, Marie M; Farver, Carol F

2016-01-01

The diagnosis of antibody-mediated rejection (AMR) in the lung transplant is still an area under investigation. We performed a blinded multicenter study to determine if any statistically significant histologic findings in transbronchial biopsy specimens from lung transplant patients correlate with the presence of donor-specific antibodies (DSAs). We asked 9 pathologists with experience in lung transplantation to evaluate 161 lung transplant biopsy specimens for various histologic parameters. The findings were correlated with antibody status positive for DSAs, positive for non-DSAs, and no antibodies (NABs) present. The significance of each histologic variable was reviewed. We found no statistically significant association with acute cellular rejection, airway inflammation, or bronchiolitis obliterans and the presence or absence of antibodies. However, biopsy specimens with DSAs had a statistically significant difference vs NABs in the setting of acute lung injury, with or without diffuse alveolar damage (p = 0.0008), in the presence of capillary neutrophilic inflammation (p = 0.0014), and in samples with endotheliitis (p = 0.0155). In samples with complement 4d staining, there was a trend but no statistically significant difference between specimens associated with DSAs and specimens with NABs. Capillary inflammation, acute lung injury, and endotheliitis significantly correlated with DSAs. The infrequently observed diffuse staining for complement 4d limits the usefulness of this stain. Copyright © 2016 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

Antibodies against glucan, chitin, and Saccharomyces cerevisiae mannan as new biomarkers of Candida albicans infection that complement tests based on C. albicans mannan.

PubMed

Sendid, B; Dotan, N; Nseir, S; Savaux, C; Vandewalle, P; Standaert, A; Zerimech, F; Guery, B P; Dukler, A; Colombel, J F; Poulain, D

2008-12-01

Antibodies against Saccharomyces cerevisiae mannan (ASCA) and antibodies against synthetic disaccharide fragments of glucans (ALCA) and chitin (ACCA) are biomarkers of Crohn's disease (CD). We previously showed that Candida albicans infection generates ASCA. Here, we explored ALCA and ACCA as possible biomarkers of invasive C. albicans infection (ICI). ASCA, ALCA, ACCA, and Candida mannan antigen and antibody detection tests were performed on 69 sera obtained sequentially from 18 patients with ICIs proven by blood culture, 59 sera from CD patients, 47 sera from hospitalized subjects colonized by Candida species (CZ), and 131 sera from healthy controls (HC). ASCA, ALCA, and ACCA levels in CD and ICI patients were significantly different from those in CZ and HC subjects (P<0.0001). In ICI patients, these levels increased as infection developed. Using ASCA, ALCA, ACCA, and Platelia Candida tests, 100% of ICIs were detected, with the kinetics of the antibody response depending on the patient during the time course of infection. A large number of sera presented with more than three positive tests. This is the first evidence that the detection of antibodies against chitin and glucans has diagnostic value in fungal infections and that these tests can complement more specific tests. Future trials are necessary to assess the value of these tests in multiparametric analysis, as well as their pathophysiological relevance.

Systematic Search for Primary Immunodeficiency in Adults With Infections

ClinicalTrials.gov

2018-05-15

Complement Deficiency; Antibody Deficiency; Chronic Sinus Infection; Meningitis, Bacterial; Pneumonia, Bacterial; Otitis Media; Streptococcal Infection; Neisseria Infections; Haemophilus Influenza; Pneumococcal Infections

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.

Enhanced Shear-induced Platelet Aggregation Due to Low-temperature Storage

DTIC Science & Technology

2013-07-01

conjugated sheep polyclonal anti-VWF antibody , goat polyclonal anti-mouse FITC and PE secondary antibodies , and immunoglobulin G isotype control antibodies ...of fresh or stored PRP was incubated with 25 mg/mL FITC- conjugated AK2 or unconjugated SZ2 or VM16d antibodies for 20 minutes. In case of SZ2 and...VM16d, the primary antibody -bound PLTs were incubated with a PE- conjugated secondary antibody . The antibody - bound PLTs were then fixed in 400 mL of 1

Antibody titres at diagnosis and during follow-up of anti-NMDA receptor encephalitis: a retrospective study.

PubMed

Gresa-Arribas, Nuria; Titulaer, Maarten J; Torrents, Abiguei; Aguilar, Esther; McCracken, Lindsey; Leypoldt, Frank; Gleichman, Amy J; Balice-Gordon, Rita; Rosenfeld, Myrna R; Lynch, David; Graus, Francesc; Dalmau, Josep

2014-02-01

Anti-N-methyl-d-aspartate (NMDA) receptor encephalitis is a severe but treatable autoimmune disorder which diagnosis depends on sensitive and specific antibody testing. We aimed to assess the sensitivity and specificity of serum and CSF antibody testing in patients with anti-NMDA receptor encephalitis, and the relation between titres, relapses, outcome, and epitope repertoire. In this observational study, we used rat brain immunohistochemistry and cell-based assays (CBA) with fixed or live NMDA receptor-expressing cells to determine the sensitivity and specificity of antibody testing in paired serum and CSF samples. Samples were obtained at diagnosis from patients with anti-NMDA receptor encephalitis and from control participants worldwide. We deemed a patient to be antibody positive if their serum, their CSF, or both tested positive with both immunohistochemistry and CBA techniques; we determined titres with serial sample dilution using brain immunohistochemistry. We examined samples from 45 patients (25 with good outcome [modified Rankin Scale, mRS 0-2], ten with poor outcome [mRS 3-6], and ten with relapses) at three or more timepoints. We determined the epitope repertoire in the samples of 23 patients with CBA expressing GluN1-NMDA receptor mutants. We analysed samples from 250 patients with anti-NMDA receptor encephalitis and 100 control participants. All 250 patients had NMDA receptor antibodies in CSF but only 214 had antibodies in serum (sensitivity 100.0% [98.5-1000%] vs 85.6% [80.7-89.4%], p<0.0001). Serum immunohistochemistry testing was more often in agreement with CBA with fixed cells (77 [71%] of 108) than with CBA with live cells (63 [58%] of 108, p=0.0056). In multivariable analysis, CSF and serum titres were higher in patients with poor outcome than in those with good outcome (CSF dilution 340 vs 129, difference 211, [95% CI 1-421], p=0.049; serum dilution 7370 vs 1243, difference 6127 [2369-9885], p=0.0025), and in patients with teratoma than in those without teratoma (CSF 395 vs 110, difference 285 [134-437], p=0.0079; serum 5515 vs 1644, difference 3870 [548-7193], p=0.024). Over time there was a decrease of antibody titres in the 35 patients with good or poor outcome and samples followed at three timepoints regardless of outcome (from diagnosis to last follow-up: CSF 614 to 76, difference 538 [288-788]; serum 5460 to 1564, difference 3896 [2428-5362]; both p<0.0001). Relapses were associated with a change in titre more often in CSF than in serum (14 of 19 vs seven of 16, p=0.037). After recovery, 24 of 28 CSF samples and 17 of 23 serum samples from patients remained antibody positive. Patients' antibodies targeted a main epitope region at GluN1 aminoacid 369; the epitope repertoire did not differ between patients with different outcomes, and did not change during relapses. The sensitivity of NMDA receptor antibody testing is higher in CSF than in serum. Antibody titres in CSF and serum were higher in patients with poor outcome or teratoma than in patients with good outcome or no tumour. The titre change in CSF was more closely related with relapses than was that in serum. These findings emphasise the importance of including CSF in antibody studies, and that antibody titres can complement clinical assessments. Dutch Cancer Society, National Institutes of Health, McKnight Neuroscience of Brain Disorders award, the Fondo de Investigaciones Sanitarias, ErasmusMC fellowship, and Fundació la Marató de TV3. Copyright © 2014 Elsevier Ltd. All rights reserved.

Wall teichoic acids prevent antibody binding to epitopes within the cell wall of Staphylococcus aureus.

PubMed

Gautam, Samir; Kim, Taehan; Lester, Evan; Deep, Deeksha; Spiegel, David A

2016-01-15

Staphylococcus aureus is a Gram-positive bacterial pathogen that produces a range of infections including cellulitis, pneumonia, and septicemia. The principle mechanism in antistaphylococcal host defense is opsonization with antibodies and complement proteins, followed by phagocytic clearance. Here we use a previously developed technique for installing chemical epitopes in the peptidoglycan cell wall to show that surface glycopolymers known as wall teichoic acids conceal cell wall epitopes, preventing their recognition and opsonization by antibodies. Thus, our results reveal a previously unrecognized immunoevasive role for wall teichoic acids in S. aureus: repulsion of peptidoglycan-targeted antibodies.

Immunohistochemical detection of chlamydiae in formalin-fixed tissue sections: comparison of a monoclonal antibody with yolk derived antibodies (IgY).

PubMed

Kunz, U S; Pospischil, A; Paccaud, M F

1991-06-01

Immunohistological detection of chlamydiae in formalin-fixed and paraffin-embedded sections of various organs from several species is described. In a retrospective study, two antisera, a commercially available monoclonal murine antibody (IgMur) and vitelline immunoglobulins (IgY), extracted from the egg yolk of immunized hens, were compared and tested for their applicability under routine condition. Both antisera were applied to tissues from which chlamydiae had been isolated or in which the presence of chlamydiae had been suspected in specially stained sections. Antigen labelling was optimal with the monoclonal antibody. Vitelline immunoglobulins produced some unspecific reactions, especially in lung tissue sections. Because of the antigenic relationship between the vitelline antibodies and tissues of birds, IgY are not suitable for the detection of psittacosis on avian substrates, when using an indirect immunological method. Staining in other tissues e.g. intestine or placenta was of equal quality as that attained with monoclonal antibodies. Depending on the advantages and disadvantages in every individual case, one of the two antibodies may be chosen for further studies. Vitelline antibodies should be preferred with respect to animal welfare.

Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults

PubMed Central

MacLennan, Calman A.; Gilchrist, James J.; Gordon, Melita A.; Cunningham, Adam F.; Cobbold, Mark; Goodall, Margaret; Kingsley, Robert A.; van Oosterhout, Joep J. G.; Msefula, Chisomo L.; Mandala, Wilson L.; Leyton, Denisse L.; Marshall, Jennifer L.; Gondwe, Esther N.; Bobat, Saeeda; López-Macías, Constantino; Doffinger, Rainer; Henderson, Ian R.; Zijlstra, Eduard E.; Dougan, Gordon; Drayson, Mark T.; MacLennan, Ian C. M.; Molyneux, Malcolm E.

2013-01-01

Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibody protects against Salmonella bacteremia. We report that high titer antibodies specific for Salmonella lipopolysaccharide (LPS) associate with absent Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from target Salmonella, or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus our study indicates impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whilst serum killing of Salmonella is induced by antibodies against outer membrane proteins. PMID:20413503

Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein.

PubMed

Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2012-01-01

Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

Rituximab fails where eculizumab restores renal function in C3nef-related DDD.

PubMed

Rousset-Rouvière, Caroline; Cailliez, Mathilde; Garaix, Florentine; Bruno, Daniele; Laurent, Daniel; Tsimaratos, Michel

2014-06-01

Dense deposit disease (DDD), a C3 glomerulopathy (C3G), is a rare disease with unfavorable progression towards end-stage kidney disease. The pathogenesis of DDD is due to cytotoxic effects related to acquired or genetic dysregulation of the complement alternative pathway, which is at times accompanied by the production of C3 nephritic factor (C3NeF), an auto-antibody directed against the alternative C3 convertase. Available treatments include plasma exchange, CD20-targeted antibodies, and a terminal complement blockade via the anti-C5 monoclonal antibody eculizumab. We report here the case of an 8-year-old child with C3NeF and refractory DDD who presented with a nephritic syndrome. She tested positive for C3NeF activity; C3 was undetectable. Genetic analyses of the alternative complement pathway were normal. Methylprednisolone pulses and mycophenolate mofetil treatment resulted in complete recovery of renal function and a reduction in proteinuria. Corticosteroids were tapered and then withdrawn. Four months after corticosteroid discontinuation, hematuria and proteinuria recurred, and a renal biopsy confirmed an active DDD with a majority of extracapillary crescents. Despite an increase in immunosuppressive drugs, including methylprednisolone pulses and rituximab therapy, the patient suffered acute renal failure within 3 weeks, requiring dialysis. Eculizumab treatment resulted in a quick and impressive response. Hematuria very quickly resolved, kidney function improved, and no further dialysis was required. The patient received bimonthly eculizumab injections of 600 mg, allowing for normalization of renal function and reduction of proteinuria to <0.5 g per day. Since then, she continues to receive eculizumab. Complement regulation pathway-targeted therapy may be a specific and useful treatment for rapidly progressing DDD prior to the development of glomerulosclerosis. Our data provide evidence supporting the pivotal role of complement alternative pathway abnormalities in C3G with DDD.

T cell-independent and T cell-dependent immunoglobulin G responses to polyomavirus infection are impaired in complement receptor 2-deficient mice.

PubMed

Szomolanyi-Tsuda, Eva; Seedhom, Mina O; Carroll, Michael C; Garcea, Robert L

2006-08-15

Polyomavirus (PyV) infection induces protective T cell-independent (TI) IgM and IgG antibody responses in T cell-deficient mice, but these responses are not generated by immunization with viral proteins or virus like particles. We hypothesized that innate signals contribute to the generation of isotype-switched antiviral antibody responses. We studied the role of complement receptor (CR2) engagement in TI and T cell-dependent (TD) antibody responses to PyV using CR2-deficient mice. Antiviral IgG responses were reduced by 80-40% in CR2-/- mice compared to wild type. Adoptive transfer experiments demonstrated the need for CR2 not only in TD, but also in TI IgG responses to PyV. Transfer of CR2-/- B lymphocytes to SCID mice resulted in TI antiviral IgG responses that corresponded to 10% of that seen in wild-type B cell-reconstituted mice. Thus, our studies revealed a profound dependence of TI and TD antiviral antibody responses on CR2-mediated signals in PyV-infected mice, where the viral antigen is abundant and persistent.

Complement evasion by Bordetella pertussis: implications for improving current vaccines.

PubMed

Jongerius, Ilse; Schuijt, Tim J; Mooi, Frits R; Pinelli, Elena

2015-04-01

Bordetella pertussis causes whooping cough or pertussis, a highly contagious disease of the respiratory tract. Despite high vaccination coverage, reported cases of pertussis are rising worldwide and it has become clear that the current vaccines must be improved. In addition to the well-known protective role of antibodies and T cells during B. pertussis infection, innate immune responses such as the complement system play an essential role in B. pertussis killing. In order to evade this complement activation and colonize the human host, B. pertussis expresses several molecules that inhibit complement activation. Interestingly, one of the known complement evasion proteins, autotransporter Vag8, is highly expressed in the recently emerged B. pertussis isolates. Here, we describe the current knowledge on how B. pertussis evades complement-mediated killing. In addition, we compare this to complement evasion strategies used by other bacterial species. Finally, we discuss the consequences of complement evasion by B. pertussis on adaptive immunity and how identification of the bacterial molecules and the mechanisms involved in complement evasion might help improve pertussis vaccines.

Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

PubMed Central

Roit, Fabio Da; Engelberts, Patrick J.; Taylor, Ronald P.; Breij, Esther C.W.; Gritti, Giuseppe; Rambaldi, Alessandro; Introna, Martino; Parren, Paul W.H.I.; Beurskens, Frank J.; Golay, Josée

2015-01-01

The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3–3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs. PMID:25344523

Binding of Complement Factor H (FH) Decreases Protective Anti-FH Binding Protein Antibody Responses of Infant Rhesus Macaques Immunized With a Meningococcal Serogroup B Vaccine

PubMed Central

Granoff, Dan M.; Costa, Isabella; Konar, Monica; Giuntini, Serena; Van Rompay, Koen K. A.; Beernink, Peter T.

2015-01-01

Background. The meningococcal vaccine antigen, factor H (FH)–binding protein (FHbp), binds human complement FH. In human FH transgenic mice, binding decreased protective antibody responses. Methods. To investigate the effect of primate FH binding, we immunized rhesus macaques with a 4-component serogroup B vaccine (4CMenB). Serum FH in 6 animals bound strongly to FHbp (FHbp-FHhigh) and, in 6 animals, bound weakly to FHbp (FHbp-FHlow). Results. There were no significant differences between the respective serum bactericidal responses of the 2 groups against meningococcal strains susceptible to antibody to the NadA or PorA vaccine antigens. In contrast, anti-FHbp bactericidal titers were 2-fold lower in FHbp-FHhigh macaques against a strain with an exact FHbp match to the vaccine (P = .08) and were ≥4-fold lower against 4 mutants with other FHbp sequence variants (P ≤ .005, compared with FHbp-FHlow macaques). Unexpectedly, postimmunization sera from all 12 macaques enhanced FH binding to meningococci. In contrast, serum anti-FHbp antibodies elicited by 4CMenB in mice whose mouse FH did not bind to the vaccine antigen inhibited FH binding. Conclusions. Binding of FH to FHbp decreases protective anti-FHbp antibody responses of macaques to 4CMenB. Even low levels of FH binding skew the antibody repertoire to FHbp epitopes outside of the FH-binding site, which enhance FH binding. PMID:25676468

Complement Inhibitors from Scabies Mites Promote Streptococcal Growth – A Novel Mechanism in Infected Epidermis?

PubMed Central

Mika, Angela; Reynolds, Simone L.; Pickering, Darren; McMillan, David; Sriprakash, Kadaba S.; Kemp, David J.; Fischer, Katja

2012-01-01

Background Scabies is highly prevalent in socially disadvantaged communities such as indigenous populations and in developing countries. Generalized itching causes discomfort to the patient; however, serious complications can occur as a result of secondary bacterial pyoderma, commonly caused by Streptococcus pyogenes (GAS) or Staphylococcus aureus. In the tropics, skin damage due to scabies mite infestations has been postulated to be an important link in the pathogenesis of disease associated with acute rheumatic fever and heart disease, poststreptococcal glomerulonephritis and systemic sepsis. Treatment of scabies decreases the prevalence of infections by bacteria. This study aims to identify the molecular mechanisms underlying the link between scabies and GAS infections. Methodology/Principal Findings GAS bacteria were pre-incubated with blood containing active complement, phagocytes and antibodies against the bacteria, and subsequently tested for viability by plate counts. Initial experiments were done with serum from an individual previously exposed to GAS with naturally acquired anti-GAS antibodies. The protocol was optimized for large-scale testing of low-opsonic whole blood from non-exposed human donors by supplementing with a standard dose of heat inactivated human sera previously exposed to GAS. This allowed an extension of the dataset to two additional donors and four proteins tested at a range of concentrations. Shown first is the effect of scabies mite complement inhibitors on human complement using ELISA-based complement activation assays. Six purified recombinant mite proteins tested at a concentration of 50 µg/ml blocked all three complement activation pathways. Further we demonstrate in human whole blood assays that each of four scabies mite complement inhibitors tested increased GAS survival rates by 2–15 fold. Conclusions/Significance We propose that local complement inhibition plays an important role in the development of pyoderma in scabies infested skin. This molecular link between scabies and bacterial infections may provide new avenues to develop alternative treatment options against this neglected disease. PMID:22815998

Interaction between Pseudomonas aeruginosa and host defenses in cystic fibrosis.

PubMed

Marshall, B C; Carroll, K C

1991-03-01

The major causes of morbidity and mortality in cystic fibrosis are chronic pulmonary obstruction and infection. Mucoid Pseudomonas aeruginosa is the primary pathogen in up to 90% of these patients. Once Pseudomonas organisms colonize the airways, they are virtually never eradicated. No defect in systemic host defense has been elucidated, however, several mechanisms contribute to the breakdown in host defenses that allow persistence of this organism in the endobronchial space. These mechanisms involve both bacterial adaptation to an unfavorable host environment and impaired host response. P aeruginosa adapts to the host by expressing excessive mucoid exopolysaccharide and a less virulent form of lipopolysaccharide. These features make it less likely to cause systemic infection, yet still enable it to resist local host defenses. Mucociliary clearance becomes impaired due to abnormal viscoelastic properties of sputum, squamous metaplasia of the respiratory epithelium, and bronchiectasis. Despite a brisk antibody response to a variety of Pseudomonas antigens, several defects in antibody-mediated opsonophagocytosis have been identified. These include (1) development of antibody isotypes that are suboptimal at promoting phagocytosis, (2) formation of immune complexes that inhibit phagocytosis, and (3) proteolytic fragmentation of immunoglobulins in the endobronchial space. Complement-mediated opsonophagocytosis is also compromised by proteolytic cleavage of complement receptors from the cell surface of neutrophils and complement opsonins from the surface of Pseudomonas. The resultant chronic inflammation and infection lead to eventual obliteration of the airways.

Role of different pathways of the complement cascade in experimental bullous pemphigoid

PubMed Central

Nelson, Kelly C.; Zhao, Minglang; Schroeder, Pamela R.; Li, Ning; Wetsel, Rick A.; Diaz, Luis A.; Liu, Zhi

2006-01-01

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies directed against the hemidesmosomal proteins BP180 and BP230 and inflammation. Passive transfer of antibodies to the murine BP180 (mBP180) induces a skin disease that closely resembles human BP. In the present study, we defined the roles of the different complement activation pathways in this model system. Mice deficient in the alternative pathway component factor B (Fb) and injected with pathogenic anti-mBP180 IgG developed delayed and less intense subepidermal blisters. Mice deficient in the classical pathway component complement component 4 (C4) and WT mice pretreated with neutralizing antibody against the first component of the classical pathway, C1q, were resistant to experimental BP. These mice exhibited a significantly reduced level of mast cell degranulation and polymorphonuclear neutrophil (PMN) infiltration in the skin. Intradermal administration of compound 48/80, a mast cell degranulating agent, restored BP disease in C4–/– mice. Furthermore, C4–/– mice became susceptible to experimental BP after local injection of PMN chemoattractant IL-8 or local reconstitution with PMNs. These findings provide the first direct evidence to our knowledge that complement activation via the classical and alternative pathways is crucial in subepidermal blister formation in experimental BP. PMID:17024247

Complement component 5 contributes to poor disease outcome in humans and mice with pneumococcal meningitis

PubMed Central

Woehrl, Bianca; Brouwer, Matthijs C.; Murr, Carmen; Heckenberg, Sebastiaan G.B.; Baas, Frank; Pfister, Hans W.; Zwinderman, Aeilko H.; Morgan, B. Paul; Barnum, Scott R.; van der Ende, Arie; Koedel, Uwe; van de Beek, Diederik

2011-01-01

Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Disease outcome has been related to the severity of the proinflammatory response in the subarachnoid space. The complement system, which mediates key inflammatory processes, has been implicated as a modulator of pneumococcal meningitis disease severity in animal studies. Additionally, SNPs in genes encoding complement pathway proteins have been linked to susceptibility to pneumococcal infection, although no associations with disease severity or outcome have been established. Here, we have performed a robust prospective nationwide genetic association study in patients with bacterial meningitis and found that a common nonsynonymous complement component 5 (C5) SNP (rs17611) is associated with unfavorable disease outcome. C5 fragment levels in cerebrospinal fluid (CSF) of patients with bacterial meningitis correlated with several clinical indicators of poor prognosis. Consistent with these human data, C5a receptor–deficient mice with pneumococcal meningitis had lower CSF wbc counts and decreased brain damage compared with WT mice. Adjuvant treatment with C5-specific monoclonal antibodies prevented death in all mice with pneumococcal meningitis. Thus, our results suggest C5-specific monoclonal antibodies could be a promising new antiinflammatory adjuvant therapy for pneumococcal meningitis. PMID:21926466

Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients.

PubMed

Flechas, Ivonne D; Cuellar, Adriana; Cucunubá, Zulma M; Rosas, Fernando; Velasco, Víctor; Steindel, Mario; Thomas, María del Carmen; López, Manuel Carlos; González, John Mario; Puerta, Concepción Judith

2009-11-25

Antigen specificity and IgG subclass could be significant in the natural history of Chagas' disease. The relationship between the different stages of human Chagas' disease and the profiles of total IgG and its subclasses were thus analysed here; they were directed against a crude T. cruzi extract and three recombinant antigens: the T. cruzi kinetoplastid membrane protein-11 (rKMP-11), an internal fragment of the T. cruzi HSP-70 protein 192-433, and the entire Trypanosoma rangeli HSP-70 protein. Seventeen Brazilian acute chagasic patients, 50 Colombian chronic chagasic patients (21 indeterminate and 29 cardiopathic patients) and 30 healthy individuals were included. Total IgG and its subtypes directed against the above-mentioned recombinant antigens were determined by ELISA tests. The T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins were able to distinguish both acute from chronic chagasic patients and infected people from healthy individuals. Specific antibodies to T. cruzi crude antigen in acute patients came from IgG3 and IgG4 subclasses whereas IgG1 and IgG3 were the prevalent isotypes in indeterminate and chronic chagasic patients. By contrast, the specific prominent antibodies in all disease stages against T. cruzi KMP-11 and T. rangeli HSP-70 recombinant antigens were the IgG1 subclass. T. cruzi KMP-11 and the T. rangeli HSP-70 recombinant proteins may be explored together in the immunodiagnosis of Chagas' disease. Polarising the IgG1 subclass of the IgG response to T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins could have important biological effects, taking into account that this is a complement fixing antibody.

Effects of Pectic Polysaccharides Isolated from Leek on the Production of Reactive Oxygen and Nitrogen Species by Phagocytes

PubMed Central

Nikolova, Mariana; Ambrozova, Gabriela; Kratchanova, Maria; Denev, Petko; Kussovski, Veselin; Ciz, Milan

2013-01-01

Abstract The current survey investigates the effect of four polysaccharides isolated from fresh leek or alcohol insoluble substances (AIS) of leek on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) from phagocytes. The ability of the polysaccharides to activate serum complement was also investigated. Despite the lack of antioxidant activity, the pectic polysaccharides significantly decreased the production of ROS by human neutrophils. Polysaccharides isolated from AIS markedly activated RAW 264.7 macrophages for RNS production in a concentration-dependent manner. The Western blot analysis revealed that this effect was due to the stimulation of the inducible nitric oxide synthase protein expression of macrophages. The polysaccharides extracted from AIS with water showed the ability to fix serum complement, especially through the alternative pathway. It was found that the polysaccharide that has the highest complement-fixing effect is characterized by the highest content of uronic acids and the highest molecular weight. PMID:23905651

Eliminating Factor H-Binding Activity of Borrelia burgdorferi CspZ Combined with Virus-Like Particle Conjugation Enhances Its Efficacy as a Lyme Disease Vaccine.

PubMed

Marcinkiewicz, Ashley L; Lieknina, Ilva; Kotelovica, Svetlana; Yang, Xiuli; Kraiczy, Peter; Pal, Utpal; Lin, Yi-Pin; Tars, Kaspars

2018-01-01

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the most common tick-borne disease in the US and Europe. No potent human vaccine is currently available. The innate immune complement system is vital to host defense against pathogens, as complement activation on the surface of spirochetes results in bacterial killing. Complement system is inhibited by the complement regulator factor H (FH). To escape killing, B. burgdorferi produces an outer surface protein CspZ that binds FH to inhibit complement activation on the cell surface. Immunization with CspZ alone does not protect mice from infection, which we speculate is because FH-binding cloaks potentially protective epitopes. We modified CspZ by conjugating to virus-like particles (VLP-CspZ) and eliminating FH binding (modified VLP-CspZ) to increase immunogenicity. We observed greater bactericidal antibody titers in mice vaccinated with modified VLP-CspZ: A serum dilution of 1:395 (modified VLP-CspZ) vs 1:143 (VLP-CspZ) yielded 50% borreliacidal activity. Immunizing mice with modified VLP-CspZ cleared spirochete infection, as did passive transfer of elicited antibodies. This work developed a novel Lyme disease vaccine candidate by conjugating CspZ to VLP and eliminating FH-binding ability. Such a strategy of conjugating an antigen to a VLP and eliminating binding to the target ligand can serve as a general model for developing vaccines against other bacterial infectious agents.

Development of ELISA-detected anti-HLA antibodies precedes the development of bronchiolitis obliterans syndrome and correlates with progressive decline in pulmonary function after lung transplantation.

PubMed

Jaramillo, A; Smith, M A; Phelan, D; Sundaresan, S; Trulock, E P; Lynch, J P; Cooper, J D; Patterson, G A; Mohanakumar, T

1999-04-27

Development of anti-HLA antibodies after lung transplantation (LT) is thought to play an important role in the etiology of bronchiolitis obliterans syndrome (BOS). However, a cause-effect relationship between anti-HLA antibodies and BOS has not been established. This study was conducted to determine the temporal relationship between the development of anti-HLA antibodies and BOS after LT, and to determine the antigenic specificity of the antibodies developed in BOS patients. Sera from 15 BOS+ LT patients and 12 BOS- LT patients were obtained before LT and collected again at 6, 12, 24, 36, and 48 months after LT. Anti-HLA antibodies were detected by the PRA-STAT ELISA system and by complement-dependent cytotoxicity assays. Anti-HLA reactivity was further characterized by flow cytometry and absorption/elution with human platelets. When analyzed by ELISA, 10 of 15 BOS+ patients developed anti-HLA antibodies, whereas 0 of 12 BOS- patients developed anti-HLA antibodies (P<0.001). When analyzed by complement-dependent cytotoxicity, only 2 of 15 BOS+ patients developed anti-HLA antibodies and 1 of 12 BOS- patients developed anti-HLA antibodies (P = 0.99). There was a significant difference of 20.1 months between the time of anti-HLA antibody detection and the time of BOS diagnosis (P = 0.005). A progressive decrease in pulmonary function correlated with a progressive increase in the anti-HLA reactivity 36 months after LT. The anti-HLA reactivity was directed to one of the donor HLA class I antigens and to other unrelated HLA class I antigens. No anti-HLA reactivity was found against HLA class II molecules. Our study indicates that anti-HLA class I antibodies play an important role in the pathogenesis of BOS and that monitoring of anti-HLA class I antibody development by a highly sensitive assay such as the PRA-STAT ELISA after LT can provide an early identification of an important subset of LT patients with an increased risk of developing BOS.

Reduced bleeding events with subcutaneous administration of recombinant human factor IX in immune-tolerant hemophilia B dogs.

PubMed

Russell, Karen E; Olsen, Eva H N; Raymer, Robin A; Merricks, Elizabeth P; Bellinger, Dwight A; Read, Marjorie S; Rup, Bonita J; Keith, James C; McCarthy, Kyle P; Schaub, Robert G; Nichols, Timothy C

2003-12-15

Intravenous administration of recombinant human factor IX (rhFIX) acutely corrects the coagulopathy in hemophilia B dogs. To date, 20 of 20 dogs developed inhibitory antibodies to the xenoprotein, making it impossible to determine if new human FIX products, formulations, or methods of chronic administration can reduce bleeding frequency. Our goal was to determine whether hemophilia B dogs rendered tolerant to rhFIX would have reduced bleeding episodes while on sustained prophylactic rhFIX administered subcutaneously. Reproducible methods were developed for inducing tolerance to rhFIX in this strain of hemophilia B dogs, resulting in a significant reduction in the development of inhibitors relative to historical controls (5 of 12 versus 20 or 20, P <.001). The 7 of 12 tolerized hemophilia B dogs exhibited shortened whole blood clotting times (WBCTs), sustained detectable FIX antigen, undetectable Bethesda inhibitors, transient or no detectable antihuman FIX antibody titers by enzyme-linked immunosorbent assay (ELISA), and normal clearance of infused rhFIX. Tolerized hemophilia B dogs had 69% reduction in bleeding frequency in year 1 compared with nontolerized hemophilia B dogs (P =.0007). If proven safe in human clinical trials, subcutaneous rhFIX may provide an alternate approach to prophylactic therapy in selected patients with hemophilia B.

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice.

PubMed

Crudele, Julie M; Finn, Jonathan D; Siner, Joshua I; Martin, Nicholas B; Niemeyer, Glenn P; Zhou, Shangzhen; Mingozzi, Federico; Lothrop, Clinton D; Arruda, Valder R

2015-03-05

Emerging successful clinical data on gene therapy using adeno-associated viral (AAV) vector for hemophilia B (HB) showed that the risk of cellular immune response to vector capsid is clearly dose dependent. To decrease the vector dose, we explored AAV-8 (1-3 × 10(12) vg/kg) encoding a hyperfunctional factor IX (FIX-Padua, arginine 338 to leucine) in FIX inhibitor-prone HB dogs. Two naïve HB dogs showed sustained expression of FIX-Padua with an 8- to 12-fold increased specific activity reaching 25% to 40% activity without antibody formation to FIX. A third dog with preexisting FIX inhibitors exhibited a transient anamnestic response (5 Bethesda units) at 2 weeks after vector delivery following by spontaneous eradication of the antibody to FIX by day 70. In this dog, sustained FIX expression reached ∼200% and 30% of activity and antigen levels, respectively. Immune tolerance was confirmed in all dogs after challenges with plasma-derived FIX concentrate. Shortening of the clotting times and lack of bleeding episodes support the phenotypic correction of the severe phenotype, with no clinical or laboratory evidence of risk of thrombosis. Provocative studies in mice showed that FIX-Padua exhibits similar immunogenicity and thrombogenicity compared with FIX wild type. Collectively, these data support the potential translation of gene-based strategies using FIX-Padua for HB. © 2015 by The American Society of Hematology.

Targeted Lymphoma Cell Death by Novel Signal Transduction Modifications

DTIC Science & Technology

2007-07-01

monoclonal antibodies (mAbs) that bind the two NH2-terminal immunoglobulin domains of CD22 and specifically block the interaction of CD22 with its...ligand blocking mAbs that effectively crosslink CD22 have distinct functional properties and facilitate assembly of an effector protein complex. These...immune mechanisms such as antibody and complement dependent cellular cytotoxicity. We hypothesize that enhancing the intrinsic pro-apoptotic

Complement-binding anti-HLA antibodies are independent predictors of response to treatment in kidney recipients with antibody-mediated rejection.

PubMed

Viglietti, Denis; Bouatou, Yassine; Kheav, Vissal David; Aubert, Olivier; Suberbielle-Boissel, Caroline; Glotz, Denis; Legendre, Christophe; Taupin, Jean-Luc; Zeevi, Adriana; Loupy, Alexandre; Lefaucheur, Carmen

2018-05-22

A major hurdle to improving clinical care in the field of kidney transplantation is the lack of biomarkers of the response to antibody-mediated rejection (ABMR) treatment. To discover these we investigated the value of complement-binding donor-specific anti-HLA antibodies (DSAs) for evaluating the response to treatment. The study encompassed a prospective cohort of 139 kidney recipients with ABMR receiving the standard of care treatment, including plasma exchange, intravenous immunoglobulin and rituximab. Patients were systematically assessed at the time of diagnosis and three months after treatment initiation for clinical and allograft histological characteristics and anti-HLA DSAs, including their C1q-binding ability. After adjusting for clinical and histological parameters, post-treatment C1q-binding anti-HLA DSA was an independent and significant determinant of allograft loss (adjusted hazard ratio 2.57 (95% confidence interval 1.29-5.12). In 101 patients without post-treatment C1q-binding anti-HLA DSA there was a significantly improved glomerular filtration rate with significantly reduced glomerulitis, peritubular capillaritis, interstitial inflammation, tubulitis, C4d deposition, and endarteritis compared with 38 patients with posttreatment C1q-binding anti-HLA DSA. A conditional inference tree model identified five prognostic groups at the time of post-treatment evaluation based on glomerular filtration rate, presence of cg lesion and C1q-binding anti-HLA DSA (cross-validated accuracy: 0.77). Thus, circulating complement-binding anti-HLA DSAs are strong and independent predictors of allograft outcome after standard of care treatment in kidney recipients with ABMR. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Polyclonal and monoclonal antibodies in clinic.

PubMed

Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

2014-01-01

Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

In situ demonstration of tissue proliferative activity using anti-bromo-deoxyuridine monoclonal antibody.

PubMed Central

Veronese, S; Gambacorta, M; Falini, B

1989-01-01

Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody. Images Fig 1 Fig 1 PMID:2475528

Respiratory Syncytial Virus (RSV): Neutralizing Antibody, a Correlate of Immune Protection.

PubMed

Piedra, Pedro A; Hause, Anne M; Aideyan, Letisha

2016-01-01

Assays that measure RSV-specific neutralizing antibody activity are very useful for evaluating vaccine candidates, performing seroprevalence studies, and detecting infection. Neutralizing antibody activity is normally measured by a plaque reduction neutralization assay or by a microneutralization assay with or without complement. These assays measure the functional capacity of serum (or other fluids) to neutralize virus infectivity in cells as compared to ELISA assays that only measure the binding capacity against an antigen. This chapter discusses important elements in standardization of the RSV-specific microneutralization assay for use in the laboratory.

Species-Specific Immunodetection of an Entamoeba histolytica Cyst Wall Protein

PubMed Central

Kearney, Moira R.; Siddique, Abdullah; Ali, Ibne K.; Gilchrist, Carol A.; Arju, Tuhinur; Hoffstrom, Benjamin; Nguyen, Felicia K.; Petri, William A.; Haque, Rashidul; Cangelosi, Gerard A.

2016-01-01

Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection. PMID:27152855

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.

Immunization against parasitic diseases of fish.

PubMed

Woo, P T

1997-01-01

Parasitologists have not, in the past, exploited the immune system to protect fish against parasitic diseases. In the past few years, however, there has been an increased interest in adopting this strategy, and we have made steady and promising progress against a few parasites which are of economic importance. Amyloodinium ocellatum is an ectoparasitic dinoflagellate on brackish and marine fishes, which may also cause problems to aquarium fishes. Antiserum from fish inoculated intraperitoneally (i.p.) with living dinospores of the parasite immobilizes and agglutinates living dinospores; it also reduces parasite infectivity in cell culture. Cryptobia salmositica is a pathogenic haemoflagellate of salmonids on the Pacific coast of North America, causing mortality in semi-natural and intensive salmon culture facilities. A live attenuated vaccine inoculated i.p. protects susceptible juvenile and adult fish for at least 24 months. The protection involves production of complement fixing antibodies, phagocytosis, and antibody-dependent and antibody-independent T-cell cytotoxicity. A monoclonal antibody against a surface membrane glycoprotein (199-200 kDa is therapeutic in that it significantly reduces parasitaemias when inoculated into fish with acute disease. Ichthyophthirius multifiliis is an ectoparasitic ciliate of freshwater fishes with world wide distribution, usually causing disease when fish are stressed and/or when environmental conditions are favourable for parasite multiplication. Live theronts injected into the body cavity protect fish, and monoclonal antibodies with immobilizing activity upon parasites have been developed. There is some evidence of passive transfer of protective immunity from immune to naive fish, and to eggs. Diplostomum spathaceum is an intestinal parasite of gulls; the metacercaria stage of the parasite encyst and causes disease and mortality in numerous species of freshwater fish in Europe and in North America. Fish injected i.p. with sonicated/killed cercariae or metacercariae have fewer metacercariae in the eyes and survives longer. Lepeophtheirus salmonis and Caligus elongatus are parasitic copepods (sea lice), and they are important parasites of Atlantic salmon in cage cultures. A vaccine against fish lice is plausible, and the efficacy of about 20 candidate antigens in protecting fish is being tested.

Interaction between human blood platelets, viruses and antibodies. IV. Post-Rubella thrombocytopenic purpura and platelet aggregation by Rubella antigen–antibody interaction

PubMed Central

Myllylä, G.; Vaheri, A.; Vesikari, T.; Penttinen, K.

1969-01-01

A new method of measuring antibodies by observing sedimentation patterns of platelets has been compared with the complement fixation and haemagglutination inhibition techniques in ten cases of Rubella and seven cases of post-Rubella thrombocytopenic purpura. The method is based on the aggregation of platelets by the joint action of antibody and small size antigens. The platelet aggregation method gave exceptionally high titres in cases of post-Rubella thrombocytopenic purpura. Other serologic methods did not give these high titres. The hypothesis that small size virus antigen and antibody against it are both needed to induce thrombocytopenia during the recovery period is discussed. Large amounts of both may result in clinical symptoms. PMID:5814719

Complement inhibition by hydroxychloroquine prevents placental and fetal brain abnormalities in antiphospholipid syndrome.

PubMed

Bertolaccini, Maria Laura; Contento, Gregorio; Lennen, Ross; Sanna, Giovanni; Blower, Philip J; Ma, Michelle T; Sunassee, Kavitha; Girardi, Guillermina

2016-12-01

Placental ischemic disease and adverse pregnancy outcomes are frequently observed in patients with antiphospholipid syndrome (APS). Despite the administration of conventional antithrombotic treatment a significant number of women continue to experience adverse pregnancy outcomes, with uncertain prevention and management. Efforts to develop effective pharmacological strategies for refractory obstetric APS cases will be of significant clinical benefit for both mothers and fetuses. Although the antimalarial drug, hydroxychloroquine (HCQ) is increasingly used to treat pregnant women with APS, little is known about its efficacy and mechanism of action of HCQ. Because complement activation plays a crucial and causative role in placental ischemia and abnormal fetal brain development in APS we hypothesised that HCQ prevents these pregnancy complications through inhibition of complement activation. Using a mouse model of obstetric APS that closely resembles the clinical condition, we found that HCQ prevented fetal death and the placental metabolic changes -measured by proton magnetic resonance spectroscopy in APS-mice. Using 111 In labelled antiphospholipid antibodies (aPL) we identified the placenta and the fetal brain as the main organ targets in APS-mice. Using this same method, we found that HCQ does not inhibit aPL binding to tissues as was previously suggested from in vitro studies. While HCQ did not affect aPL binding to fetal brain it prevented fetal brain abnormal cortical development. HCQ prevented complement activation in vivo and in vitro. Complement C5a levels in serum samples from APS patients and APS-mice were lower after treatment with HCQ while the antibodies titres remained unchanged. HCQ prevented not only placental insufficiency but also abnormal fetal brain development in APS. By inhibiting complement activation, HCQ might also be an effective antithrombotic therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Relationship among antineutrophil cytoplasmic antibody, blood urea nitrogen and complement in patients with eosinophilic granulomatosis polyangiitis (Churg-Strauss syndrome).

PubMed

Kawakami, Tamihiro; Kimura, Satoko; Takeuchi, Sora; Soma, Yoshinao

2013-07-01

Eosinophilic granulomatosis with polyangiitis (EGPA), also known as Churg-Strauss syndrome, is an antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis characterized by a history of asthma, hypereosinophilia. The prevalence of ANCA in EGPA is less common than in other ANCA-associated vasculitis. Increasing evidence of complement activation in the pathogenesis of ANCA-associated vasculitis has been provided by studies in animal models. We examined EGPA patients with cutaneous manifestations as an initial sign and investigated the correlations among clinical, serological and histopathological findings. We focused on differences among ANCA, blood urea nitrogen and complement levels such as complement 3 (C3), C4 and total complement hemolytic activity (CH50). We retrospectively investigated the records of 22 patients (11 male and 11 female) with EGPA admitted to our hospital from 1997-2012. Ten of the 22 patients (46%) were positive for serum myeloperoxidase (MPO)-ANCA. In contrast, all the patients were negative for serum proteinase 3 ANCA. There was a significantly positive correlation between serum CH50 and C4 levels in patients with EGPA. Serum blood urea nitrogen (BUN) levels differed significantly between MPO-ANCA-positive and -negative patients. Serum CH50 levels were higher in MPO-ANCA-positive patients compared to negative patients. Serum BUN levels were higher in elevated CH50 patients compared to normal and low CH50-negative patients. We propose that positive findings for MPO-ANCA with CH50 high activity may be a risk factor for developing renal insufficiency. Assuming there are correlations between the presence of ANCA and complements, earlier diagnosis based on initial efficacious treatment for EGPA. © 2013 Japanese Dermatological Association.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

PubMed

Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin ( Ts -CRT), a Ca 2+ -binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts -CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts -CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts -CRT (r Ts -CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte-macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts -CRT on the surface of newborn larvae (NBL) of T. spiralis with anti- Ts -CRT antibody increased the C1q-mediated adherence of monocyte-macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis -expressed Ts -CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy

PubMed Central

Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin (Ts-CRT), a Ca2+-binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts-CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts-CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts-CRT (rTs-CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte–macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts-CRT on the surface of newborn larvae (NBL) of T. spiralis with anti-Ts-CRT antibody increased the C1q-mediated adherence of monocyte–macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis-expressed Ts-CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages. PMID:28620388

Coupling complement regulators to immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo

PubMed Central

HARRIS, C L; WILLIAMS, A S; LINTON, S M; MORGAN, B P

2002-01-01

Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59-Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function. PMID:12165074

Clinical roundtable monograph: Paroxysmal nocturnal hemoglobinuria: a case-based discussion.

PubMed

Szer, Jeff; Hill, Anita; Weitz, Ilene Ceil

2012-11-01

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disorder characterized by chronic intravascular hemolysis as the primary clinical manifestation and morbidities that include anemia, thrombosis, renal impairment, pulmonary hypertension, and bone marrow failure. The prevalence of the PNH clone (from <1-100% PNH granulocytes) is approximately 16 per million, and careful monitoring is required. The average age of onset of the clinical disease is the early 30s, although it can present at all ages. PNH is caused by the acquisition of a somatic mutation of the gene phosphatidylinositol glycan anchor (PIG-A) in a multipotent hematopoietic stem cell (HSC), with clonal expansion of the mutated HSC. The mutation causes a deficiency in the synthesis of glycosylphosphatidylinositol (GPI). In cells derived from normal HSCs, the complement regulatory proteins CD55 and CD59 are anchored to the hematopoietic cell membrane surface via GPI, protecting the cells from complement-mediated lysis. However, in patients with PNH, these 2 proteins, along with numerous other GPI-linked proteins, are absent from the cell surface of red cells, granulocytes, monocytes, and platelets, resulting in complement-mediated intravascular hemolysis and other complications. Lysis of red blood cells is the most obvious manifestation, but as other cell lineages are also affected, this complement-mediated attack contributes to additional complications, such as thrombosis. Eculizumab, a humanized monoclonal antibody against the C5 complement protein, is the only effective drug therapy for PNH patients. The antibody prevents cleavage of the C5 protein by C5 convertase, in turn preventing generation of C5b-9 and release of C5a, thereby protecting from hemolysis of cells lacking the CD59 surface protein and other complications associated with complement activation. Drs. Ilene C. Weitz, Anita Hill, and Jeff Szer discuss 3 recent cases of patients with PNH.

Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

PubMed

Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

Targeting complement-mediated immunoregulation for cancer immunotherapy.

PubMed

Kolev, Martin; Markiewski, Maciej M

2018-06-01

Complement was initially discovered as an assembly of plasma proteins "complementing" the cytolytic activity of antibodies. However, our current knowledge places this complex system of several plasma proteins, receptors, and regulators in the center of innate immunity as a bridge between the initial innate responses and adaptive immune reactions. Consequently, complement appears to be pivotal for elimination of pathogens, not only as an early response defense, but by directing the subsequent adaptive immune response. The discovery of functional intracellular complement and its roles in cellular metabolism opened novel avenues for research and potential therapeutic implications. The recent studies demonstrating immunoregulatory functions of complement in the tumor microenvironment and the premetastatic niche shifted the paradigm on our understanding of functions of the complement system in regulating immunity. Several complement proteins, through their interaction with cells in the tumor microenvironment and in metastasis-targeted organs, contribute to modulating tumor growth, antitumor immunity, angiogenesis, and therefore, the overall progression of malignancy and, perhaps, responsiveness of cancer to different therapies. Here, we focus on recent progress in our understanding of immunostimulatory vs. immunoregulatory functions of complement and potential applications of these findings to the design of novel therapies for cancer patients. Copyright © 2018 Elsevier Ltd. All rights reserved.

Using oligonucleotide aptamer probes for immunostaining of formalin-fixed and paraffin-embedded tissues

PubMed Central

Zeng, Zihua; Zhang, Peng; Zhao, Nianxi; Sheehan, Andrea M; Tung, Ching-Hsuan; Chang, Chung-Che; Zu, Youli

2011-01-01

For tissue immunostaining, antibodies are currently the only clinically validated and commercially available probes. Aptamers, which belong to a class of small molecule ligands composed of short single-stranded oligonucleotides, have emerged as probes over the last several decades; however, their potential clinical value has not yet been fully explored. Using cultured cells and an RNA-based CD30 aptamer, we recently demonstrated that the synthetic aptamer is useful as a specific probe for flow cytometric detection of CD30-expressing lymphoma cells. In this study, we further validated the use of this aptamer probe for immunostaining of formalin-fixed and paraffin-embedded lymphoma tissues. Using CD30 antibody as a standard control, we demonstrated that the synthetic CD30 aptamer specifically recognized and immunostained tumor cells of classical Hodgkin lymphoma and anaplastic large cell lymphoma, but did not react with background cells within tumor sites. Notably, the CD30 aptamer probe optimally immunostained lymphoma cells with lower temperature antigen retrieval (37 vs 96°C for antibody) and shorter probing reaction times (20 vs 90 min for antibody) than typical antibody immunostaining protocols. In addition, the CD30 aptamer probe showed no nonspecific background staining of cell debris in necrotic tissue and exhibited no cross-reaction to tissues that do not express CD30, as confirmed by a standard CD30 antibody staining. Therefore, our findings indicate that the synthetic oligonucleotide CD30 aptamer can be used as a probe for immunostaining of fixed tissue sections for disease diagnosis. PMID:20693984

Cloning of Helicobacter suis cholesterol α-glucosyltransferase and production of an antibody capable of detecting it in formalin-fixed, paraffin-embedded gastric tissue sections.

PubMed

Kawakubo, Masatomo; Horiuchi, Kazuki; Komura, Hitomi; Sato, Yoshiko; Kato, Masayoshi; Ikeyama, Meguru; Fukushima, Mana; Yamada, Shigenori; Ishizone, Satoshi; Matsumoto, Takehisa; Ota, Hiroyoshi; Sagara, Junji; Nakayama, Jun

2017-10-01

Helicobacter suis (H. suis), formerly called Helicobacter heilmannii type 1 (H. heilmannii), is a gram-negative bacterium of the Helicobacter species. This pathogen infects the stomach of humans and animals such as dogs, cats, pigs, and rodents, the latter giving rise to zoonotic infection. Here, we generated a H. suis-specific antibody useful for immunohistochemistry with formalin-fixed, paraffin-embedded tissue sections. To do so, we began by cloning the gene encoding H. suis cholesterol α-glucosyltransferase (αCgT). αCgT is the key enzyme responsible for biosynthesis of cholesteryl α-D-glucopyranoside (CGL), a major cell wall component of Helicobacter species including H. suis. The deduced amino acid sequence of H. suis αCgT had 56% identity with the corresponding Helicobacter pylori (H. pylori). We then developed a polyclonal antibody (anti-Hh-I205R) by immunizing rabbits with a 205 amino acid H. suis αCgT fragment. Immunohistochemistry with the anti-Hh-I205R antibody could differentiate H. suis from H. pylori in gastric mucosa sections derived from mice infected with either pathogen. We then probed formalin-fixed, paraffin-embedded sections of human gastric mucosa positive for H. suis infection with the anti-Hh-I205R antibody and detected positive staining. These results indicate that anti-Hh-I205R antibody is specific for H. suis αCgT and useful to detect H. suis in gastric specimens routinely analyzed in pathological examinations.

Reduction in erythrocyte-bound complement activation products and titres of anti-C1q antibodies associate with clinical improvement in systemic lupus erythematosus.

PubMed

Buyon, Jill; Furie, Richard; Putterman, Chaim; Ramsey-Goldman, Rosalind; Kalunian, Kenneth; Barken, Derren; Conklin, John; Dervieux, Thierry

2016-01-01

The relationship between cell-bound complement activation products (CB-CAPs: EC4d, EC3d), anti-C1q, soluble complement C3/C4 and disease activity in systemic lupus erythematosus (SLE) was evaluated. Per protocol, at baseline all SLE subjects enrolled in this longitudinal study presented with active disease and elevated CB-CAPs. At each monthly visit, the non-serological (ns) Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA-SLEDAI) and the British Isles Lupus Assessment Group (BILAG)-2004 index scores were determined as was a random urinary protein to creatinine ratio (uPCR). Short-form 36 (SF-36) questionnaires were also collected. All soluble markers were determined using immunoassays, while EC4d and EC3d were determined using flow cytometry. Statistical analysis consisted of linear mixed models with random intercept and fixed slopes. A total of 36 SLE subjects (mean age 34 years; 94% female) were enrolled and evaluated monthly for an average 11 visits per subject. Clinical improvements were observed during the study, with significant decreases in ns-SELENA-SLEDAI scores, BILAG-2004 index scores and uPCR, and increases in all domains of SF-36 (p<0.01). The longitudinal decrease in ns-SELENA-SLEDAI and BILAG-2004 index scores was significantly associated with reduced EC4d and EC3d levels, reduced anti-C1q titres and increased serum complement C3/C4 (p<0.05). The changes in uPCR significantly correlated with C3, C4, anti-C1q and EC4d, with EC4d outperforming C3/C4 by a multivariate analysis. The reduced EC4d or EC3d was associated with improvements in at least six out of the eight domains of SF-36 and outperformed C3/C4. Anti-dsDNA titres did not correlate with changes in disease activity. These data indicate that CB-CAPs and anti-C1q are helpful in monitoring patients with SLE.

IgGs are made for walking on bacterial and viral surfaces

NASA Astrophysics Data System (ADS)

Preiner, Johannes; Kodera, Noriyuki; Tang, Jilin; Ebner, Andreas; Brameshuber, Mario; Blaas, Dieter; Gelbmann, Nicola; Gruber, Hermann J.; Ando, Toshio; Hinterdorfer, Peter

2014-07-01

Binding of antibodies to their cognate antigens is fundamental for adaptive immunity. Molecular engineering of antibodies for therapeutic and diagnostic purposes emerges to be one of the major technologies in combating many human diseases. Despite its importance, a detailed description of the nanomechanical process of antibody-antigen binding and dissociation on the molecular level is lacking. Here we utilize high-speed atomic force microscopy to examine the dynamics of antibody recognition and uncover a principle; antibodies do not remain stationary on surfaces of regularly spaced epitopes; they rather exhibit ‘bipedal’ stochastic walking. As monovalent Fab fragments do not move, steric strain is identified as the origin of short-lived bivalent binding. Walking antibodies gather in transient clusters that might serve as docking sites for the complement system and/or phagocytes. Our findings could inspire the rational design of antibodies and multivalent receptors to exploit/inhibit steric strain-induced dynamic effects.

IMMUNOGLOBULINS IN DEFENSE, PATHOGENESIS AND THERAPY OF FUNGAL DISEASES

PubMed Central

Casadevall, Arturo; Pirofski, Liise-anne

2012-01-01

Summary Only two decades ago antibodies to fungi were thought to have little or no role in protection against fungal diseases. However, subsequent research has provided convincing evidence that certain antibodies can modify the course of fungal infection to the benefit or detriment of the host. Hybridoma technology was the breakthrough that enabled the characterization of antibodies to fungi, illuminating some of the requirements for antibody efficacy. As discussed in this review, fungal specific antibodies mediate protection through direct actions on fungal cells and through classical mechanisms such as phagocytosis and complement activation. Although mechanisms of antibody-mediated protection are often species-specific, numerous fungal antigens can be targeted to generate vaccines and therapeutic immunoglobulins. Furthermore, the study of antibody function against medically important fungi has provided fresh immunological insights into the complexity of humoral immunity that are likely to apply to other pathogens. PMID:22607798

Ripley-like serum and other anti-Rh sera in detection of Fc receptor-bearing human lymphocytes.

PubMed

Maślanka, K; Zupańska, B

1981-04-01

Three anti-Rh sera useful for the EAhum test were found (one comparable to Ri serum). It was shown that the usefulness of anti-Rh sera for the EAhum test depends on the amount of anti-Rh antibodies absorbed onto red cells demonstrated in the manual antiglobulin test. It does not depend on the quality of antibodies and the ability of complement binding.

Analysis of immune responses in genital tracts of mice immunised with purified ribosomal fractions of Neisseria gonorrhoeae.

PubMed Central

Kita, E; Kashiba, S

1984-01-01

Immunisation of ddY mice with the purified ribosomal fraction of Neisseria gonorrhoeae was found to protect against intravaginal challenge with homologous organisms. This protection correlated with the presence of bactericidal antibody to purified ribosomal fraction in serum as well as in vaginal secretions. Analysis of the vaginal fluids from control mice and those immunised with purified ribosomal fraction showed that the enhanced elimination of gonococci in immune mice might be because of an early response of leucocytes generated by the reaction mediated by antibody and complement. Absorption studies showed that there was at least one major protective antigen in purified ribosomal fraction, other than cell surface substances such as lipopolysaccharide, outer membrane proteins, and pili. Bactericidal assays mediated by antibody and complement showed that matched samples of serum and vaginal fluid from immune mice had comparable gonococcidal activity, which was augmented by the effect of progesterone. Although delayed hypersensitivity was produced in immune mice that were resistant to N gonorrhoeae, the exact role of cellular immunity could not be clarified in this study. These results suggest that antibody to purified ribosomal fraction plays a major part in protection against gonococcal infection in the genital tract, and that such protection may entail both cellular immunity and hormonal changes. PMID:6430462

Modulation of cytotoxic T lymphocyte, natural killer cell, antibody-dependent cellular cytotoxicity, and antibody-dependent complement-mediated cytotoxicity by Vernonia cinerea L. and vernolide-A in BALB/c mice via enhanced production of cytokines IL-2 and IFN-γ.

PubMed

Pratheeshkumar, Poyil; Kuttan, Girija

2012-02-01

Effect of Vernonia cinerea L. and vernolide-A on cell-mediated immune (CMI) response was studied in normal as well as tumor-bearing BALB/c mice. Administration of V. cinerea and vernolide-A significantly enhanced natural killer (NK) cell activity in both normal as well as tumor-bearing animals, and the activity was observed earlier than in tumor-bearing control animals. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were also enhanced significantly in both normal as well as tumor-bearing animals after V. cinerea and vernolide-A administration compared with untreated control tumor-bearing animals. Extract and vernolide-A showed a significant increase in cytotoxic T lymphocyte (CTL) production in both the in vivo and in vitro models. The level of cytokines such as interleukin (IL)-2 and interferon (IFN)-γ were also enhanced by the treatment of V. cinerea and vernolide-A in both normal as well as tumor-bearing animals. This study demonstrated that V. cinerea extract and vernolide-A stimulate the CTL, NK cell, ADCC, and ADCC through enhanced secretion of IL-2 and IFN-γ.

Biomarker-Based Metabolic Labeling for Redirected and Enhanced Immune Response.

PubMed

Li, Shanshan; Yu, Bingchen; Wang, Jiajia; Zheng, Yueqin; Zhang, Huajie; Walker, Margaret J; Yuan, Zhengnan; Zhu, He; Zhang, Jun; Wang, Peng George; Wang, Binghe

2018-06-01

Installation of an antibody-recruiting moiety on the surface of disease-relevant cells can lead to the selective destruction of targets by the immune system. Such an approach can be an alternative strategy to traditional chemotherapeutics in cancer therapy and possibly other diseases. Herein we describe the development of a new strategy to selectively label targets with an antibody-recruiting moiety through its covalent and stable installation, complementing existing methods of employing reversible binding. This is achieved through selective delivery of 1,3,4- O-acetyl- N-azidoacetylmannosamine (Ac 3 ManNAz) to folate receptor-overexpressing cells using an Ac 3 ManNAz-folate conjugate via a cleavable linker. As such, Ac 3 ManNAz is converted to cell surface glycan bearing an azido group, which serves as an anchor to introduce l-rhamnose (Rha), a hapten, via a click reaction with aza-dibenzocyclooctyne (DBCO)-Rha. We tested this method in several cell lines including KB, HEK-293, and MCF7 and were able to demonstrate the following: 1) Rha can be selectively installed to the folate receptor overexpressing cell surface and 2) the Rha installed on the target surface can recruit anti-rhamnose (anti-Rha) antibodies, leading to the destruction of target cells via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).

Determining antibody-binding site of streptococcal pyrogenic exotoxin B to protect mice from group a streptococcus infection.

PubMed

Tsao, Nina; Cheng, Miao-Hui; Yang, Hsiu-Chen; Wang, Yu-Chieh; Liu, Yi-Ling; Kuo, Chih-Feng

2013-01-01

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcal (GAS) infection. SPE B binds and cleaves antibody isotypes and further impairs the immune system by inhibiting complement activation. In this study, we examined the antibody-binding site of SPE B and used it to block SPE B actions during GAS infection. We constructed different segments of the spe B gene and induced them to express different recombinant fragments of SPE B. Using an enzyme-linked immunosorbent assay (ELISA), we found that residues 345-398 of the C-terminal domain of SPE B (rSPE B(345-398)), but not the N-terminal domain, was the major binding site for antibody isotypes. Using a competitive ELISA, we also found that rSPE B(345-398) bound to the Fc portion of IgG. The in vitro functional assays indicate that rSPE B(345-398) not only interfered with cleavage of antibody isotypes but also interfered with SPE B-induced inhibition of complement activation. Immunization of BALB/c mice using rSPE B(345-398) was able to induce production of a high titer of anti-rSPE B(345-398) antibodies and efficiently protected mice from GAS-induced death. These findings suggest that SPE B uses its C-terminal domain to bind the Fc portion of IgG and that immunization of mice with this binding domain (rSPE B(345-398)) could protect mice from GAS infection.

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell

PubMed Central

Wang, Qianli; Zhang, Junping; Guo, Zhongwu

2007-01-01

To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine. PMID:17892942

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell.

PubMed

Wang, Qianli; Zhang, Junping; Guo, Zhongwu

2007-12-15

To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine.

Controversies concerning the antiphospholipid syndrome in obstetrics.

PubMed

Camarena Cabrera, Dulce María Albertina; Rodriguez-Jaimes, Claudia; Acevedo-Gallegos, Sandra; Gallardo-Gaona, Juan Manuel; Velazquez-Torres, Berenice; Ramírez-Calvo, José Antonio

Antiphospholipid antibody syndrome is a non-inflammatory autoimmune disease characterized by recurrent thrombotic events and/or obstetric complications associated with the presence of circulating antiphospholipid antibodies (anticardiolipin antibodies, anti-β 2 glycoprotein-i antibodies, and/or lupus anticoagulant. Antiphospholipid antibodies are a heterogeneous group of autoantibodies associated with recurrent miscarriage, stillbirth, fetal growth restriction and premature birth. The diversity of the features of the proposed placental antiphospholipid antibodies fingerprint suggests that several disease processes may occur in the placentae of women with antiphospholipid antibody syndrome in the form of immune responses: inflammatory events, complement activation, angiogenic imbalance and, less commonly, thrombosis and infarction. Because of the disparity between clinical and laboratory criteria, and the impact on perinatal outcome in patients starting treatment, we reviewed the aspects of antiphospholipid antibody syndrome related to obstetric complications and seronegative antiphospholipid antibody syndrome, and their treatment in obstetrics. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

Exopolysaccharides Isolated from Hydrothermal Vent Bacteria Can Modulate the Complement System

PubMed Central

Courtois, Anthony; Berthou, Christian; Guézennec, Jean

2014-01-01

The complement system is involved in the defence against bacterial infection, or in the elimination of tumour cells. However, disturbances in this system contributes to the pathogenesis of various inflammatory diseases. The efficiency of therapeutic anti-tumour antibodies is enhanced when the complement system is stimulated. In contrast, cancer cells are able to inhibit the complement system and thus proliferate. Some marine molecules are currently being developed as new drugs for use in humans. Among them, known exopolyssacharides (EPSs) generally originate from fungi, but few studies have been performed on bacterial EPSs and even fewer on EPSs extracted from deep-sea hydrothermal vent microbes. For use in humans, these high molecular weight EPSs must be depolymerised. Furthermore, the over-sulphation of EPSs can modify their biological activity. The aim of this study was to investigate the immunodulation of the complement system by either native or over-sulphated low molecular weight EPSs isolated from vent bacteria in order to find pro or anti-activators of complement. PMID:24736648

Scabies Mite Peritrophins Are Potential Targets of Human Host Innate Immunity

PubMed Central

Holt, Deborah C.; Kemp, Dave J.; Fischer, Katja

2011-01-01

Background Pruritic scabies lesions caused by Sarcoptes scabiei burrowing in the stratum corneum of human skin facilitate opportunistic bacterial infections. Emerging resistance to current therapeutics emphasizes the need to identify novel targets for protective intervention. We have characterized several protein families located in the mite gut as crucial factors for host-parasite interactions. Among these multiple proteins inhibit human complement, presumably to avoid complement-mediated damage of gut epithelial cells. Peritrophins are major components of the peritrophic matrix often found in the gut of arthropods. We hypothesized that a peritrophin, if abundant in the scabies mite gut, could be an activator of complement. Methodology/Principal Findings A novel full length scabies mite peritrophin (SsPTP1) was identified in a cDNA library from scabies mites. The amino acid sequence revealed four putative chitin binding domains (CBD). Recombinant expression of one CBD of the highly repetitive SsPTP1 sequence as TSP-hexaHis-fusion protein resulted in soluble protein, which demonstrated chitin binding activity in affinity chromatography assays. Antibodies against a recombinant SsPTP1 fragment were used to immunohistochemically localize native SsPTP1 in the mite gut and in fecal pellets within the upper epidermis, co-localizing with serum components such as host IgG and complement. Enzymatic deglycosylation confirmed strong N- and O-glycosylation of the native peritrophin. Serum incubation followed by immunoblotting with a monoclonal antibody against mannan binding lectin (MBL), the recognition molecule of the lectin pathway of human complement activation, indicated that MBL may specifically bind to glycosylated SsPTP1. Conclusions/Significance This study adds a new aspect to the accumulating evidence that complement plays a major role in scabies mite biology. It identifies a novel peritrophin localized in the mite gut as a potential target of the lectin pathway of the complement cascade. These initial findings indicate a novel role of scabies mite peritrophins in triggering a host innate immune response within the mite gut. PMID:21980545

Downregulation of membrane complement inhibitors CD55 and CD59 by siRNA sensitises uterine serous carcinoma overexpressing Her2/neu to complement and antibody-dependent cell cytotoxicity in vitro: implications for trastuzumab-based immunotherapy.

PubMed

Bellone, S; Roque, D; Cocco, E; Gasparrini, S; Bortolomai, I; Buza, N; Abu-Khalaf, M; Silasi, D-A; Ratner, E; Azodi, M; Schwartz, P E; Rutherford, T J; Pecorelli, S; Santin, A D

2012-04-24

We evaluated the expression of CD46, CD55 and CD59 membrane-bound complement-regulatory proteins (mCRPs) in primary uterine serous carcinoma (USC) and the ability of small interfering RNA (siRNA) against these mCRPs to sensitise USC to complement-dependent cytotoxicity (CDC) and antibody (trastuzumab)-dependent cellular cytotoxicity (ADCC) in vitro. Membrane-bound complement-regulatory proteins expression was evaluated using real-time PCR (RT-PCR) and flow cytometry, whereas Her2/neu expression and c-erbB2 gene amplification were assessed using immunohistochemistry, flow cytometry and fluorescent in-situ hybridisation. The biological effect of siRNA-mediated knockdown of mCRPs on HER2/neu-overexpressing USC cell lines was evaluated in CDC and ADCC 4-h chromium-release assays. High expression of mCRPs was found in USC cell lines when compared with normal endometrial cells (P<0.05). RT-PCR and FACS analyses demonstrated that anti-mCRP siRNAs were effective in reducing CD46, CD55 and CD59 expression on USC (P<0.05). Baseline complement-dependent cytotoxicity (CDC) against USC cell lines was low (mean ± s.e.m.=6.8 ± 0.9%) but significantly increased upon CD55 and CD59 knockdown (11.6 ± 0.8% and 10.7 ± 0.9%, respectively, P<0.05). Importantly, in the absence of complement, both CD55 and CD59, but not CD46, knockdowns significantly augmented ADCC against USC overexpressing Her2/neu. Uterine serous carcinoma express high levels of the mCRPs CD46, CD55 and CD59. Small interfering RNA inhibition of CD55 and CD59, but not CD46, sensitises USC to both CDC and ADCC in vitro, and if specifically targeted to tumour cells, may significantly increase trastuzumab-mediated therapeutic effect in vivo.

Identification of anti-Lea by platelet complement fixation.

PubMed

Ando, B; Ibayashi, H

1986-01-01

Two anti-Lea sera which were able to detect Lea antigen on platelets were identified in a screening for anti-platelet antibodies by means of a platelet complement fixation test. These two antisera hemolyzed erythrocytes without enzyme treatment. The anti-Lea activity could be completely absorbed by red cells, platelets and lymphocytes of Le(a+b-) donors but not by cells from Le(a-b+) or Le(a-b-) donors. The antibody activity against red cells was eliminated by treatment of the antisera with dithiothreitol, thereby suggesting that the activity resided in the IgM class of immunoglobulins. As the anti-Lea was more reactive at 37 degrees C than at room temperature against both red cells and platelets, we suggest that transfusion of platelets of Lea-negative donors should be considered for patients with this type of anti-Lea.

Attenuation of S. aureus-induced bacteremia by human mini-antibodies targeting the complement inhibitory protein Efb

PubMed Central

Georgoutsou-Spyridonos, Maria; Ricklin, Daniel; Pratsinis, Haris; Perivolioti, Eustathia; Pirmettis, Ioannis; Garcia, Brandon L.; Geisbrecht, Brian V.; Foukas, Periklis G.; Lambris, John D.; Mastellos, Dimitrios C.; Sfyroera, Georgia

2015-01-01

Staphylococcus aureus (S. aureus) can cause a broad range of potentially fatal inflammatory complications (e.g. sepsis, endocarditis). Its emerging antibiotic resistance and formidable immune evasion arsenal have emphasized the need for more effective antimicrobial approaches. Complement is an innate immune sensor that rapidly responds to bacterial infection eliciting C3-mediated opsonophagocytic and immunomodulatory responses. Extracellular Fibrinogen-binding Protein (Efb) is a key immune evasion protein of S. aureus that intercepts complement at the level of C3. To date, Efb has not been explored as a target for monoclonal antibody (mAb)-based antimicrobial therapeutics. Herein we have isolated donor-derived anti-Efb IgGs that attenuate S. aureus survival through enhanced neutrophil killing. A phage library screen yielded mAbs (miniAbs) that selectively inhibit the interaction of Efb with C3 partly by disrupting contacts essential for complex formation. Surface Plasmon Resonance-based kinetic analysis enabled the selection of miniAbs with favorable Efb-binding profiles as therapeutic leads. MiniAb-mediated blockade of Efb attenuated S aureus survival in a whole blood model of bacteremia. This neutralizing effect was associated with enhanced neutrophil-mediated killing of S. aureus, increased C5a release and modulation of IL-6 secretion. Finally, these miniAbs afforded protection from S. aureus-induced bacteremia in a murine renal abscess model, attenuating bacterial inflammation in kidneys. Overall, these findings are anticipated to pave the way towards novel antibody-based therapeutics for S. aureus-related diseases. PMID:26342032

Identification of ’Rickettsia rickettsii’ in Formalin-Fixed, Paraffin-Processed Tissues by Immunofluorescence.

DTIC Science & Technology

1978-01-30

attachment of antibody to the rickettsiae. The technique is valuable in that a diagnosis of Rocky Mountain spotted fever can be confirmed from formalin-fixed tissues processed in a routine manner. (Author)

Immunoserological parameters in SLE: high-avidity anti-dsDNA detected by ELISA are the most closely associated with the disease activity.

PubMed

Andrejevic, Sladjana; Jeremic, Ivica; Sefik-Bukilica, Mirjana; Nikolic, Milos; Stojimirovic, Biljana; Bonaci-Nikolic, Branka

2013-11-01

We assessed the relationship between the serum levels of antibodies against double-stranded DNA (dsDNA), C1q, nucleosomes, histones, C3 and C4 complement components with one another, with organ involvement and overall disease activity in patients with systemic lupus erythematosus (SLE). One hundred seventy-five sera from 99 patients with SLE, 31 sera of patients with other connective tissue diseases, and 20 sera from healthy blood donors were tested. SLE disease activity was assessed by modified SLEDAI-2K (M-SLEDAI-2K), not including complement and anti-dsDNA descriptors. Anti-dsDNA antibodies were measured by indirect immunofluorescence on Crithidia luciliae (CLIFT), standard enzyme-linked immunosorbent assay (ELISA) and ELISA for high-avidity antibodies. The most significant risk factor for renal involvement were anti-C1q antibodies (OR = 3.88, p < 0.05), high-avidity anti-dsDNA antibodies for polyserositis (OR = 7.99, p < 0.01), anti-histone antibodies for joint involvement (OR = 2.75, p < 0.05), and low C3 for cytopenia (OR = 11.96, p < 0.001) and mucocutaneous lesions (OR = 3.32, p < 0.01). Multiple linear regression analysis showed that disease activity in SLE could be predicted by the levels of antibodies against dsDNA determined by standard (p < 0.05) and high-avidity (p < 0.001) ELISA, and inversely associated with concentration of C3 (p < 0.001). Using stepwise method, high-avidity anti-dsDNA antibodies were found to be in the closest association to M-SLEDAI-2K. Moreover, positive test for high-avidity anti-dsDNA antibodies appeared as an independent risk factor for moderately to severely active disease (M-SLEDAI-2K>5) (OR = 5.5, p < 0.01). The presence of high-avidity anti-dsDNA antibodies represented a risk for renal, joint, and most importantly for serosal involvement. Our results suggest that simple and reliable ELISA for high-avidity anti-dsDNA antibodies is the test of good clinical utility for the assessment of global SLE activity.

Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions.

PubMed

Alagesan, Swathi; Gaudana, Sandeep B; Sinha, Avinash; Wangikar, Pramod P

2013-11-01

Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several "proof of principle" studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by (13)C metabolic flux analysis ((13)C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report (13)C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of (12)C and (13)C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism's preferred mode under nitrogen-fixing conditions. The (13)C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism's distinct metabolic features under nitrogen-fixing and -non-fixing conditions.

Complement-Mediated Neutralization of Canine Distemper Virus In Vitro: Cross-Reaction between Vaccine Onderstepoort and Field KDK-1 Strains with Different Hemagglutinin Gene Characteristics

PubMed Central

Mochizuki, Masami; Motoyoshi, Megumi; Maeda, Ken; Kai, Kazunari

2002-01-01

The properties of neutralization of antigens of canine distemper virus Onderstepoort and a recent field isolate, KDK-1, were investigated with strain-specific dog sera. A conventional neutralization assay indicated antigenic dissimilarity between the strains; however, when guinea pig complement was included in the reaction mixture, the strains were neutralized with not only the homologous but also the heterologous antibodies. PMID:12093697

Interruption of the Sequential Release of Small and Large Molecules from Tumor Cells by Low Temperature During Cytolysis Mediated by Immune T-Cells or Complement

PubMed Central

Martz, Eric; Burakoff, Steven J.; Benacerraf, Baruj

1974-01-01

Specific lysis of tumor cells by thymus-derived lymphocytes from alloimmunized mice (T-effector specific lysis) was studied with target cells labeled with isotopes attached to both small (14C-labeled nicotinamide) and large (51Cr-labeled) molecules. The results confirm and extend previous reports that target cells release small molecules considerably earlier than large molecules during T-effector specific lysis. After interruption of T-effector specific lysis by specific antibody and complement directed against the killer cells, or by ethylenediaminetetraacetic acid, release of both isotopes continued, eventually reaching identical levels of specific release, the value of which represents the fraction of the target cell population which had been committed to die at the time these treatments were applied. On the other hand, release of both isotopes during T-effector specific lysis stops immediately when the cultures are cooled to 0°. Thus, while ethylenediaminetetraacetic acid or specific complement-mediated lysis of the killer cells merely prevents the initiation of any new damage to target cells, cooling to 0° also stops the lytic process in already-damaged target cells. The colloid osmotic phase of target cell lysis induced by specific antibody and complement was similarly stopped at 0° in tumor cells, but not in erythrocytes. Thus, in tumor target cells, both T-effector specific lysis and complement cause a sequential release of progressively larger molecules which can be immediately stopped at any point by cooling to 0°. PMID:4359327

BF*F allotype of the alternative pathway of complement: A marker of protection against the development of antiphospholipid antibodies in patients with systemic lupus erythematosus.

PubMed

Picceli, V F; Skare, T L; Nisihara, R M; Nass, F R; Messias-Reason, I T; Utiyama, S R R

2016-04-01

B factor (BF) from the alternative complement pathway seems to participate in the pathophysiology of systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). To study the allotypic variability of BF in SLE and their associations with clinical and autoantibodies profile. BF allotypes were determined by high-voltage agarose gel electrophoresis, under constant cooling, followed by immunofixation with anti-human BF antibody, in 188 SLE patients and 103 controls. Clinical and serological data were obtained from medical examination and records. No significant differences of BF variants between patients and controls were found, neither in relation to epidemiologic or clinical manifestations. Associations of phenotype BF SS07 and allotype BF*S07 were found with anticardiolipin IgM (aCl-IgM) antibodies (p = 0.014 and p = 0.009 respectively), but not with aCl-IgG, lupus anticoagulant (LA), anti β2GPI or clinical APS. A significant decrease in BF*F allotype (p = 0.043) and BF SF phenotype (p = 0.018) was detected in patients with anti-phospholipid antibodies as a whole (aCl-IgG, aCl-IgM, LA and anti β2GPI). There is a link between phenotype BF SS07 and allotype BF*S07 with aCl-IgM in SLE patients; BF*F allotype could be considered a marker of protection against the development of antiphospholipid antibodies in these patients. © The Author(s) 2015.

Distribution of anti-CD68 (EBM11) immunoreactivity in formalin-fixed, paraffin-embedded bovine tissues.

PubMed

Ackermann, M R; DeBey, B M; Stabel, T J; Gold, J H; Register, K B; Meehan, J T

1994-05-01

A commercially acquired anti-human macrophage antibody (anti-CD68; EBM11) was used in an immunocytochemical technique to detect macrophages in formalin-fixed, paraffin-embedded tissues from cattle, pigs, humans, rats, turkeys, dogs, and cats. In healthy cattle, the antibody labeled alveolar macrophages, pulmonary intravascular cells (presumably intravascular macrophages), and macrophage-like cells in other tissues. In bovine lungs infected with Pasteurella haemolytica, EBM11 antibody labeled 95% of alveolar macrophages and macrophages within alveolar septa but only 0-2% of streaming or "oat" leukocytes. Alveolar macrophages were also stained by EBM11 in pigs but not in rats, turkeys, dogs, and cats. The antibody also stained macrophage aggregates in the mesenteric lymph nodes and intestinal lamina propria of Mycobacterium paratuberculosis-infected cattle. This study shows that the anti-CD68 (EBM11) antibody is a useful marker of macrophages in normal bovine tissues or tissues from areas of acute or chronic inflammation that have been routinely processed. The study also adds strength to the growing evidence suggesting that streaming leukocytes seen in pneumonic pasteurellosis are neutrophils.

THE BIOLOGICAL ACTIVITY OF SOLUBLE ANTIGEN-ANTIBODY COMPLEXES

PubMed Central

Ishizaka, Kimishige; Ishizaka, Teruko; Campbell, Dan H.

1959-01-01

Soluble BSA-anti-BSA complexes, formed in antigen excess, give immediate skin reactions in normal guinea pigs. The mechanism of the reaction is not that of passive or reversed passive anaphylaxis. The complex itself is toxic. Skin activity of the complex depends on its composition. It has become obvious that the complex composed of two antigen molecules and one antibody molecule, (Ag2Ab), does not have the activity, whereas, Ag3Ab2 and more complicated complexes do. The role of complement as well as speculation on the structural changes of antibody-antigen complexes is presented. PMID:13620844

Conservation of nodulation genes between Rhizobium meliloti and a slow-growing Rhizobium strain that nodulates a nonlegume host

PubMed Central

Marvel, Deborah J.; Kuldau, Gretchen; Hirsch, Ann; Richards, Eric; Torrey, John G.; Ausubel, Frederick M.

1985-01-01

Parasponia, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with a Rhizobium species. The bacterial strain RP501 is a slow-growing strain of Rhizobium isolated from Parasponia nodules. Strain RP501 also nodulates the legumes siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata). Using a cosmid clone bank of RP501 DNA, we isolated a 13.4-kilobase (kb) EcoRI fragment that complemented insertion and point mutations in three contiguous nodulation genes (nodABC) of Rhizobium meliloti, the endosymbiont of alfalfa (Medicago sativa). The complemented R. meliloti nod mutants induced effective nitrogen-fixing nodules on alfalfa seedlings but not on siratro, cowpeas, or Parasponia. The cloned RP501 nodulation locus hybridized to DNA fragments carrying the R. meliloti nodABC genes. A 3-kb cluster of Tn5 insertion mutations on the RP501 13.4-kb EcoRI fragment prevented complementation of R. meliloti nodABC mutations. Images PMID:16593600

Coccidioides complement fixation

MedlinePlus

... antibodies are detected in the blood sample. Normal value ranges may vary slightly among different laboratories. Some labs use different measurements or test different samples. Talk to your health care provider about the meaning of your specific test results.

Studies of Altered Response to Infection Induced by Thermal Injury.

DTIC Science & Technology

1984-05-01

beginning to examine various prophylactic treatments for their efficacy in reversing a number of burn mediated immune defects. We expect even more interesting...with fluoresceinated antibody (OKM5) and FACS sorting or negative selection by Sephadex G-l0 passage and/or antibody and -3- complement treatment ... infectious episode. This mitogen hyperimmunity is typical of a normal immune system dealing with an infectious challenge. The Group II patients have a

Dose Rationalization of Pembrolizumab and Nivolumab Using Pharmacokinetic Modeling and Simulation and Cost Analysis.

PubMed

Ogungbenro, Kayode; Patel, Alkesh; Duncombe, Robert; Nuttall, Richard; Clark, James; Lorigan, Paul

2018-04-01

Pembrolizumab and nivolumab are highly selective anti-programmed cell death 1 (PD-1) antibodies approved for the treatment of advanced malignancies. Variable exposure and significant wastage have been associated with body size dosing of monoclonal antibodies (mAbs). The following dosing strategies were evaluated using simulations: body weight, dose banding, fixed dose, and pharmacokinetic (PK)-based methods. The relative cost to body weight dosing for band, fixed 150 mg and 200 mg, and PK-derived strategies were -15%, -25%, + 7%, and -16% for pembrolizumab and -8%, -6%, and -10% for band, fixed, and PK-derived strategies for nivolumab, respectively. Relative to mg/kg doses, the median exposures were -1.0%, -4.6%, + 27.1%, and +3.0% for band, fixed 150 mg, fixed 200 mg, and PK-derived strategies, respectively, for pembrolizumab and -3.1%, + 1.9%, and +1.4% for band, fixed 240 mg, and PK-derived strategies, respectively, for nivolumab. Significant wastage can be reduced by alternative dosing strategies without compromising exposure and efficacy. © 2017 American Society for Clinical Pharmacology and Therapeutics.

Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum.

PubMed

Muleme, Michael; Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M; Nguyen, Chelsea; Stevenson, Mark A; Wilks, Colin R; Firestone, Simon M

2016-06-01

Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum

PubMed Central

Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M.; Nguyen, Chelsea; Stevenson, Mark A.; Wilks, Colin R.; Firestone, Simon M.

2016-01-01

Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. PMID:27122484

Anti-CD40 antibody-mediated costimulation blockade promotes long-term survival of deep-lamellar porcine corneal grafts in non-human primates.

PubMed

Kim, Jaeyoung; Kim, Dong Hyun; Choi, Hyuk Jin; Lee, Hyun Ju; Kang, Hee Jung; Park, Chung-Gyu; Hwang, Eung-Soo; Kim, Mee Kum; Wee, Won Ryang

2017-05-01

Corneal xenotransplantation is an effective solution for the shortage of human donor corneas, and the porcine cornea may be a suitable candidate for the donor cornea because of its optical similarity with humans. However, it is necessary to administer additional immunosuppressants to overcome antigenic differences. We aimed to investigate the feasibility of porcine corneas with anti-CD40 antibody-mediated costimulation blockade in a clinically applicable pig-to-non-human primate corneal xenotransplantation model. Five Chinese rhesus macaques underwent deep-lamellar corneal transplantation using clinically acceptable sized (7.5 mm diameter) porcine corneal grafts. The anti-CD40 antibody was intravenously administered on a programmed schedule. Graft survival, central corneal thickness, and intraocular pressure were evaluated. Changes in effector and memory T and B cell subsets and anti-αGal and donor-specific antibodies were investigated in the blood, and the changes in complement levels in the aqueous humor and blood were evaluated. Memory cell profiles in the anti-CD40 antibody-treated group were compared with those from the anti-CD154 antibody-treated group or rejected controls presented in our previous report. The changes in anti-αGal, non-αGal, and donor-specific antibodies after 6 months were compared with baseline values. Anti-CD40 antibody-mediated costimulation blockade resulted in the successful survival of xenocorneal grafts (>389, >382, >236, >201, and >61 days), with 80% reaching 6 months of survival. Injection of anti-CD40 antibody considerably reduced the infiltration of inflammatory cells into the grafts and significantly blocked the complement response in the aqueous humor (P=.0159, Mann-Whitney U test). Systemic expansion of central or effector memory T cells was abrogated in the anti-CD40 antibody-treated primates compared with those in the rejected controls (P<.05, Mann-Whitney U test) or those in the anti-CD154 antibody-treated primates (P>.05, Mann-Whitney U test). The levels of anti-αGal, non-αGal, and donor-specific antibodies at 6 months were not significantly increased compared with baseline levels (P>.05, Wilcoxon signed rank test). An anti-CD40 antibody-mediated blockade appears to be effective immunosuppressive approach for porcine corneal deep-lamellar xenotransplantation in primates. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of alterations in glomerular charge on deposition of cationic and anionic antibodies to fixed glomerular antigens in the rat.

PubMed

Adler, S; Baker, P; Pritzl, P; Couser, W G

1985-07-01

Reduction of the negative charge of the glomerular capillary wall alters its charge- and size-selective properties. To investigate the effect of alteration in glomerular charge properties on antibody localization, we prepared cationic and anionic fractions of antibodies to subepithelial and glomerular basement membrane (GBM) antigens, and compared their deposition in normal rats and rats treated with protamine sulfate or aminonucleoside of puromycin to reduce capillary wall charge. IgG antibodies were eluted from kidneys of rats with active Heymann's nephritis (AICN), passive Heymann's nephritis (PHN), or anti-GBM nephritis (NTN), separated into cationic and anionic fractions, and radiolabeled with iodine 125 or iodine 131. Relative antibody content of each fraction was determined by incubation with an excess of glomerular antigen. Varying amounts of cationic and anionic IgG eluted from kidneys of rats with AICN or PHN were injected into 24 normal or protamine sulfate-treated rats. Glomerular binding of all antibodies was highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 4 hours was 1.08 +/- 0.07 for AICN eluate and 0.37 +/- 0.04 for PHN eluate. The ratios were not significantly different in animals pretreated with protamine sulfate (1.15 +/- 0.06 and 0.44 +/- 0.06, respectively; P greater than 0.05). Varying amounts of cationic and anionic IgG eluted from kidneys of rats with NTN were injected into 10 normal rats and four rats treated with aminonucleoside of puromycin. Glomerular binding of antibody was again highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 1 hour was 1.03 +/- 0.06, and was not significantly altered in rats treated with aminonucleoside of puromycin (1.05 +/- 0.03, P greater than 0.5). Proteinuria in PHN rats was also unaffected by treatment with protamine sulfate for 5 days (controls: 68 +/- 21 mg/day; protamine sulfate-treated: 65 +/- 14 mg/day; n = 25, P greater than 0.08). These results demonstrate that treatment to reduce glomerular polyanion does not significantly alter the ratio of cationic to anionic antibodies to fixed glomerular antigens that deposit in the glomerulus, or reduce proteinuria caused by deposition of antibody to a fixed subepithelial antigen.

The 100th anniversary of Wassermann-Neisser-Bruck reaction.

PubMed

Bialynicki-Birula, Rafal

2008-01-01

August Paul von Wassermann (1866-1925), German bacteriologist, together with Albert Neisser (1855-1916), German dermatologist and venereologist, and Carl Bruck (1879-1944), German dermatologist and venereologist, developed the first serologic test for the diagnosis of syphilis. They published their first article about it on May 10, 1906 (Dtsch Med Wochenschr 1906;32:745). They made use of the idea of the complement fixation test of Jules Bordet (1870-1961) and Octave Gengou (1875-1957), so the Wassermann reaction is sometimes called Bordet-Wassermann reaction. The study was done at the Berlin Institute of Infectious Diseases (Berliner Institut für Infektionskrankheiten) and at the Breslau (Wroclaw) Department of Dermatology. The Wassermann reaction was used in the diagnosis of syphilis. The antigen used in it was prepared empirically. Originally, the so-called antigens were extracts of human or monkey tissue rich in Treponema pallidum. The most active one was a liver extract of a syphilitic fetus. Later on, the active substance, referred to as cardiolipin, was found in normal nonsyphilitic tissues, including the heart (usually bovine heart). Karl Landsteiner (1868-1943) identified the antigen involved in the Wassermann reaction as a lipoid substance, which finally was identified as diphosphatidylglycerol. Wassermann antibodies, produced in the course of syphilis infection, are reactive with cardiolipin in the presence of lecithin and cholesterol. The antigen-antibody reaction produces immune complexes, which results in complement fixation via the classic pathway; this may be used to determine the serum level of antibodies (if <1 microg/mL). In the final step, indicator cells (erythrocytes) together with a subagglutinating amount of antibodies (antierythrocyte antibodies) are added to the mixture. If there remains any complement left, these cells will be lysed; if it has been consumed by immune complexes, the amount of the remaining complement will be insufficient to produce the lysis of the red cells. In the first experiment of Wassermann et al, the reaction was positive exclusively with the sera of patients with syphilis, but it was soon discovered that some other diseases gave positive results in nonsyphilitic individuals. First such cases were reported in 1909. With the discovery of new and more specific tests for syphilis, the complement fixation tests of Wassermann type gradually went into oblivion. A new era in venereology was started with the discovery of T pallidum by Fritz Schaudinn (1871-1906) and Erich Hoffmann (1868-1954) in 1905, and the development of serology of syphilis by Wassermann, Neisser, and Bruck in 1906. Although the Wassermann reaction is no longer in use now, it should be emphasized that it was one of the very first serodiagnostic tests ever used in medical practice. Carl Bruck said in the 1920s: "This fortunate and unique mistake constituted the basis of a very important discovery, whose significance was both theoretical and practical."

COMPLEMENT DEVIATION IN SCARLET FEVER WITH COMPARATIVE STUDIES OF THE WASSERMANN AND NOGUCHI SYSTEMS

PubMed Central

Kolmer, John A.

1911-01-01

The Wassermann reaction in scarlet fever per se is uniformly negative. The antigen of scarlet fever liver yields practically the same results as that of luetic liver, and both fail to deviate complement with scarlet fever antibodies. The Noguchi reaction in scarlet fever is practically negative. Sixteen cases, or 6.4 per cent. of 250 cases, were positive when active serum was used; with inactivated serum, but eleven, or 4.4 per cent., remained positive. Five of these eleven cases were also positive with the Wassermann system. In other words, sixteen cases, or 6.4 per cent., were positive according to the Noguchi system with active or inactivated serum or both, whereas with the Wassermann system only 2 per cent. were positive. The presence of anti-sheep hemolysin normally in human serum is one of the main disturbing factors in the Wassermann system; for this reason, complement and hemolysin (made by immunization of rabbits) require careful titration. A positive Wassermann reaction usually indicates the presence of syphilitic antibodies, and a negative Noguchi reaction, their absence, and both systems should be used in the examination of all cases. PMID:19867467

Monoclonal antibody specific for IDH1 R132H mutation.

PubMed

Capper, David; Zentgraf, Hanswalter; Balss, Jörg; Hartmann, Christian; von Deimling, Andreas

2009-11-01

IDH1 R132H mutations occur in approximately 70% of astrocytomas and oligodendroglial tumors. We developed a mouse monoclonal antibody targeting the IDH1 R132H mutation. Here, we show the high specificity and sensitivity of this antibody on Western blots and tissue sections from formalin fixed paraffin embedded tumor specimens. This antibody is highly useful for tumor classification, in detecting single infiltrating tumor cells and for the characterization of the cellular role of mutant IDH1 protein.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

PubMed Central

Ferreira, Viviana P.; Fazito Vale, Vladimir; Pangburn, Michael K.; Abdeladhim, Maha; Ferreira Mendes-Sousa, Antonio; Coutinho-Abreu, Iliano V.; Rasouli, Manoochehr; Brandt, Elizabeth A.; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Horácio Pereira, Marcos; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M. C.; Gontijo, Nelder F.; Collin, Nicolas; Valenzuela, Jesus G.

2016-01-01

Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

Meningococcal Polysaccharide Vaccine Failure in a Patient with C7 Deficiency and a Decreased Anticapsular Antibody Response

DTIC Science & Technology

2012-05-01

Because the patient had a history of meningococcal meningitis at age 10, archived serum was obtained for further analysis. Complement component C7...us.arrny.mil Submitted: 11/21/11; Revised: 01/17/ 12; Accepted: 01129/12 httpJ/dx.doi.orgl10.4161/hv.19517 did when he had meningitis ." The...found that 15 23% of adults with meningococcal meningitis had an underlying complement deficiency.3 The most common and most recencly described

Pharmacokinetics, safety and efficacy of a recombinant factor IX product, trenonacog alfa in previously treated haemophilia B patients.

PubMed

Collins, P W; Quon, D V K; Makris, M; Chowdary, P; Kempton, C L; Apte, S J; Ramanan, M V; Hay, C R M; Drobic, B; Hua, Y; Babinchak, T J; Gomperts, E D

2018-01-01

Trenonacog alfa (IB1001) is a recombinant factor IX (rFIX) manufactured in Chinese hamster ovary (CHO) cells. IB1001 was evaluated in a multicentre clinical trial with haemophilia B patients. The aim was to establish IB1001 pharmacokinetic non-inferiority to comparator rFIX, safety and efficacy in previously treated patients (PTPs) with haemophilia B. Subjects were severe or moderately severe haemophilia B adult and adolescent PTPs with no history of FIX inhibitors. IB1001 PK non-inferiority to comparator rFIX was demonstrated through ratio of AUC 0-∞ in 32 subjects. IB1001 was well tolerated in all 76 treated subjects; the most common adverse drug reaction was headache (2.6% of subjects) and there were no reports of FIX inhibitors. Transient non-inhibitory binding FIX antibodies and anti-CHO cell protein antibodies developed in 21% and 29% of subjects respectively; no safety concerns were associated with development of these antibodies. Prophylaxis (mean duration ± SD: 17.9 ± 9.6 months, mean dose: 55.5 ± 12.9 IU/kg, median 1.0 infusion per week) was effective in preventing bleeds (median annual bleed rate: 1.52, interquartile range: 0.0-3.46). One or two IB1001 infusions resolved 84% of the bleeds, while for 84% of treatments haemostatic efficacy of IB1001 was rated excellent or good. IB1001 haemostatic efficacy for all 19 major surgeries was rated adequate or better than adequate. IB1001 is safe and efficacious for treatment of bleeds, routine prophylaxis and perioperative management in haemophilia B patients. © 2017 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

Parvovirus B19 induced lupus-like syndrome with nephritis.

PubMed

Georges, Elodie; Rihova, Zuzana; Cmejla, Radek; Decleire, Pierre-Yves; Langen, Corinne

2016-12-01

We report a case of a 65-year-old man who developed an acute illness with fever, arthralgia and nephritic syndrome. Antinuclear antibodies were slightly positive and complement levels were low. Renal biopsy showed exudative diffuse proliferative endocapillary glomerulonephritis with diffuse immunoglobulin (IgG, IgA, IgM) and complement deposition (C3d, C4d, C1q) on immunofluorescence. The patient was first treated with corticosteroids and mycophenolate mofetil for suspected lupus with WHO class IV glomerulonephritis. The diagnosis was questioned and a diagnosis of parvovirus B19-associated nephritis was made based on elevation of serum IgM antibodies for parvovirus B19 and detection of parvovirus B19 DNA on renal biopsy. The immunosuppressive treatment was stopped and progressive spontaneous regression of clinical and laboratory abnormalities was observed. We conclude that human parvovirus B19 infection should be considered as a cause of lupus-like symptomatology and acute glomerulonephritis.

Serological tests for detecting Rift Valley fever viral antibodies in sheep from the Nile Delta.

PubMed Central

Scott, R M; Feinsod, F M; Allam, I H; Ksiazek, T G; Peters, C J; Botros, B A; Darwish, M A

1986-01-01

To determine the accuracy of serological methods in detecting Rift Valley fever (RVF) viral antibodies, we examined serum samples obtained from 418 sheep in the Nile Delta by using five tests. The plaque reduction neutralization test (PRNT) was considered the standard serological method against which the four other tests were compared. Twenty-four serum samples had RVF viral antibodies detected by PRNT. Hemagglutination inhibition and enzyme-linked immunosorbent assay antibodies to RVF virus were also present in the same 24 serum samples. Indirect immunofluorescence was less sensitive in comparison with PRNT, and complement fixation was the least sensitive. These results extend observations made with laboratory animals to a large field-collected group of Egyptian sheep. PMID:3533977

Optimization of Glial Fibrillary Acidic Protein Immunoreactivity in Formalin-fixed, Paraffin-Embedded Guinea Pig Brain Sections

DTIC Science & Technology

2003-09-01

fixed, paraffin-embedded guinea pig brain sections using a variety of commercially available GFAP antibody clones. Of the 7 clones tested for cross...determining neuropathological consequences in the guinea pig following exposure to chemical warfare nerve agent.

Complement and Antibody-Mediated Enhancement of Erythrocyte Invasion by Plasmodium Falciparum

DTIC Science & Technology

2016-04-01

Waki et al., 1995; Yoneto et al., 2001). However, the doses of antibody in those studies were much higher, usually given in milligram amounts and...erythrocyte receptor for Plasmodium falciparum PfRh4 invasion ligand. Proc. Natl. Acad. Sci. U. S. A. 107, 17327–17332. Waki , S., Uehara, S., Kanbe, K...infections. J. Immunol. 126, 1826–1828. Wold Health Organization, 2013. World Malaria Report 2013 (Geneva). Yoneto, T., Waki , S., Takai, T., Tagawa, Y

Interaction of non-human primate complement and antibodies with hypermucoviscous Klebsiella pneumoniae.

PubMed

Soto, Esteban; Marchi, Sylvia; Beierschmitt, Amy; Kearney, Michael; Francis, Stewart; VanNess, Kimberly; Vandenplas, Michel; Thrall, MaryAnna; Palmour, Roberta

2016-03-08

Emergent hypermucoviscosity (HMV) phenotypes of Klebsiella pneumoniae have been associated with increased invasiveness and pathogenicity in primates. In this study, we investigated the interaction of African green monkeys (AGM) (Chlorocebus aethiops sabaeus) complement and antibody with HMV and non-HMV isolates as in vitro models of primate infection. Significantly greater survival of HMV isolates was evident after incubation in normal serum or whole blood (p < 0.05) of AGM donors when compared to non-HMV strains. Greater survival of HMV strains (p < 0.05) was found after incubation in whole blood and serum from seropositive donors when compared to seronegative donor samples. Additionally, significantly greater amounts of K. pneumoniae were phagocytozed by AGM leukocytes when complement was active (p < 0.05), but no difference in uptake was observed when serum from seropositive or seronegative animals was used in challenged cells utilizing flow cytometry. Results demonstrate that interaction of cellular and humoral immune elements play a role in the in vitro killing of K. pneumoniae, particularly HMV isolates. Neither AGM serum, nor washed whole blood effectively killed HMV isolates; however, assays using heparinized whole blood of seronegative donors significantly reduced viability of HMV and non-HMV strains. The lack of bacterial killing observed in seropositive donors treatments could be at least partially associated with low IgG2 present in these animals. A better understanding of the pathogenesis of klebsiellosis in primates and host immune response is necessary to identify surface molecules that can induce both opsonizing and bactericidal antibody facilitating killing of Klebsiella, and the development of vaccines in human and animals.

Development of an opsonophagocytic killing assay for group a streptococcus.

PubMed

Jones, Scott; Moreland, Nicole J; Zancolli, Marta; Raynes, Jeremy; Loh, Jacelyn M S; Smeesters, Pierre R; Sriskandan, Shiranee; Carapetis, Jonathan R; Fraser, John D; Goldblatt, David

2018-05-15

Group A Streptococcus (GAS) or Streptococcus pyogenes is responsible for an estimated 500,000 deaths worldwide each year. Protection against GAS infection is thought to be mediated by phagocytosis, enhanced by bacteria-specific antibody. There are no licenced GAS vaccines, despite many promising candidates in preclinical and early stage clinical development, the most advanced of which are based on the GAS M-protein. Vaccine progress has been hindered, in part, by the lack of a standardised functional assay suitable for vaccine evaluation. Current assays, developed over 50 years ago, rely on non-immune human whole blood as a source of neutrophils and complement. Variations in complement and neutrophil activity between donors result in variable data that is difficult to interpret. We have developed an opsonophagocytic killing assay (OPKA) for GAS that utilises dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in current assays. We have standardised the OPKA for several clinically relevant GAS strain types (emm1, emm6 and emm12) and have shown antibody-specific killing for each emm-type using M-protein specific rabbit antisera. Specificity was demonstrated by pre-incubation of the antisera with homologous M-protein antigens that blocked antibody-specific killing. Additional qualifications of the GAS OPKA, including the assessment of the accuracy, precision, linearity and the lower limit of quantification, were also performed. This GAS OPKA assay has the potential to provide a robust and reproducible platform to accelerate GAS vaccine development. Copyright © 2018 Elsevier Ltd. All rights reserved.

Immunological properties of glycolipids from membranes of Acholeplasma laidlawii.

PubMed Central

Ryan, M D; Noker, P; Matz, L L

1975-01-01

Glycolipids, the predominant class of lipids in the membranes of Acholeplasma laidlawii, are the haptenic determinants that react with anti-A. Laidlawii serum to fix complement. The predominant complement-fixing activity of the membrane glycolipids was associated with the monoglucoysyl diglyceride, diglucosyl diglyceride, glycerlphosphoryl diglucosyl diglyceride (GPDD), and an unknown lipid B, which did not react with ninhydrin but release glucose and glycerol and traces of phosphorus upon hydrolysis. The glycolipids monoglucosyl diglyceride and diglucosyl diglyceride or GPDD and unknown lipid B were paired as a result of their cross-reactions with selective antisera prepared with the aid of reconstituted membrane complexes containing membrane lipids. Reconstituted membrane complexes assembled from [14C]monoglucosyl diglyceride and delipidated membrane proteins gave optimal complement fixation titers before saturation of the complexes with the ]14C]monoglucosyl diglyceride. The phosphoglycolipid of the membrane, GPDD, was anticomplementary as a pure lipid, a cholesterol liposome, and a reconstituted membrane complex. This anticomplementary activity, which was caused by 3 mug of pure GPDD, affected both human and guinea pig complement. Although human C1, C4, C3, and C5 were not inhibited by GPDD, C2 was inhibited 10-fold by reconstituted membrane complexes containing 150 mug of GPDD. A role for this phosphoglycolipid is discussed in the hypothetical mechanism of inhibition of C2 attachment to SAC1, 4 sites. PMID:1193716

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

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

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with onemore » GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.« less

[Killing effect of polymorphonuclear neutrophils on Trichomonas vaginalis].

PubMed

Zhao, Jian-Ling; Gao, Xing-Zheng; Qu, Ming

2008-10-30

To study the killing effect of polymorphonuclear neutrophils (PMNs) on Trichomonas vaginalis. The vaginal secretion from a patient with vaginitis was incubated in the liver infusion liquid medium to get T. vaginalis. One ml serum was collected from the patient and heated for 30 min at 56 degrees C to inactivate complement in serum, and was absorbed three times with the parasites at 0 degree C to make the serum free of antibodies. PMNs were separated from the patient's blood and purified with density gradient centrifugation and polymer accelerating sedimentation. NBT and safranin O were used to stain the sample. The interaction between PMNs and the parasites was observed under microscope. 300 trichomonads and 3x10(4) PMNs were incubated for 10, 20, 30, 40, 50, 60 minutes under the conditions of aerobic or anaerobic, with superoxide dismutase (SOD) and catalase (CAT) or without SOD and CAT, and with complement or without complement. They were then inoculated in solid medium for another five days under the anaerobic condition, and surviving organisms were enumerated. PMNs were observed to surround and kill a single trichomonad. In the petri-dish containing PMNs, the surviving rate of the parasites in anaerobic condition was 85%, only 3% in aerobic condition (P<0.01). SOD and CAT reduced the killing effect of PMNs, with a surviving rate of 98% and 94% respectively after 60 min incubation. Without SOD and CAT, the surviving rate is only 2% (P<0.05). PMNs in the serum without antibodies killed all the parasites, while the complement-inactivated serum fail to kill them. The trichomonacidal activity of PMNs relies on the presence of oxygen and complement in the serum of patient.

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay.

PubMed

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil; Kim, Dong-Myung; Yoo, Tae Hyeon; Kim, Yong-Sung

2015-11-27

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Natural IgM mediates complement-dependent uptake of Francisella tularensis by human neutrophils via CR1 and CR3 in nonimmune serum

PubMed Central

Schwartz, Justin T.; Barker, Jason H.; Long, Matthew E.; Kaufman, Justin; McCracken, Jenna; Allen, Lee-Ann H.

2012-01-01

A fundamental step in the life cycle of F. tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum, nor the receptors that mediate infection of neutrophils has been defined. Herein human neutrophil uptake of GFP-expressing F. tularensis strains LVS and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis whereas C5 was not. Second, we used purification and immuno-depletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-antigen and capsule as prominent targets of these antibodies on the bacterial surface. Finally, we demonstrate using receptor-blocking antibodies that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-antigen polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3-opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner. PMID:22888138

Protective immune responses against West Nile virus are primed by distinct complement activation pathways.

PubMed

Mehlhop, Erin; Diamond, Michael S

2006-05-15

West Nile virus (WNV) causes a severe infection of the central nervous system in several vertebrate animals including humans. Prior studies have shown that complement plays a critical role in controlling WNV infection in complement (C) 3(-/-) and complement receptor 1/2(-/-) mice. Here, we dissect the contributions of the individual complement activation pathways to the protection from WNV disease. Genetic deficiencies in C1q, C4, factor B, or factor D all resulted in increased mortality in mice, suggesting that all activation pathways function together to limit WNV spread. In the absence of alternative pathway complement activation, WNV disseminated into the central nervous system at earlier times and was associated with reduced CD8+ T cell responses yet near normal anti-WNV antibody profiles. Animals lacking the classical and lectin pathways had deficits in both B and T cell responses to WNV. Finally, and somewhat surprisingly, C1q was required for productive infection in the spleen but not for development of adaptive immune responses after WNV infection. Our results suggest that individual pathways of complement activation control WNV infection by priming adaptive immune responses through distinct mechanisms.

Cytology specimens offer an effective alternative to formalin-fixed tissue as demonstrated by novel automated detection for ALK break-apart FISH testing and immunohistochemistry in lung adenocarcinoma.

PubMed

Rosenblum, Frida; Hutchinson, Lloyd M; Garver, Joann; Woda, Bruce; Cosar, Ediz; Kurian, Elizabeth M

2014-11-01

Minimally invasive sampling by cytology or core needle biopsy often provides an initial diagnosis for treatment in patients with lung nodules. From these limited specimens, multiple molecular studies are frequently requested. Current guidelines from the US Food and Drug Administration recommend using formalin-fixed paraffin-embedded tissue sections for the detection of anaplastic lymphoma kinase (ALK) gene rearrangement by fluorescence in situ hybridization (FISH). The authors compared alcohol-fixed and formalin-fixed cytology specimens using a novel automated detection for ALK rearrangements by FISH and immunohistochemistry (IHC). ALK FISH testing was performed on 129 lung adenocarcinomas from 71 cytology cases and 58 biopsy/resection specimens using Papanicolaou staining with integrated cytomorphology. IHC with the ALK D5F3 antibody was performed on cases with residual material (88 of 129 cases). The mean age of the patients was 66 years; there were 62 women and 67 men. ALK gene rearrangement was present in 4% of cytology specimens (3 of 71 specimens) and 7% of surgical specimens (4 of 58 specimens). FISH in 13 cases was technically unsuccessful. Of the 7 FISH-positive cases, only 2 cytology cases (4%) and 2 surgical cases (6%) were found to be positive with the ALK antibody, demonstrating 80% concordance. The one case found to be negative for ALK by IHC demonstrated a variant rearrangement of the ALK 2p23 gene locus by FISH. The results of the current study validate the usefulness of alcohol-fixed and/or formalin-fixed cytology specimens for ALK rearrangement by a novel automated FISH method. IHC using the D5F3 antibody for ALK is specific in this limited cohort. The authors also demonstrated that alcohol-fixed cytology specimens can be used for ALK rearrangement by automated FISH, alone or in conjunction with IHC. © 2014 American Cancer Society.

[Topics of neuromyelitis optica].

PubMed

Miyamoto, Katsuichi

2014-01-01

Neuromyelitis optica (NMO) has been revealed the difference in the pathology of multiple sclerosis since the anti-aquaporin 4 (AQP4) antibody associated with NMO has been found. The clinical epidemiological study has been reviewed, NMO-related patient number in Japan is estimated to be about 4400 people. The antibody-positive patients against myelin-oligodendrocyte glycoprotein (MOG) are present in anti-AQP4 antibody-negative NMO patients. These patients have a characteristic with high frequency of optic neuritis and good response to therapy. In addition, by research in recent years, a new treatment such as anti-IL-6 therapy or anti-complement therapy has been attempted to NMO.

Donor Polymorphisms in Genes Related to B-Cell Biology Associated With Antibody-Mediated Rejection After Heart Transplantation.

PubMed

Marrón-Liñares, Grecia M; Núñez, Lucía; Crespo-Leiro, María G; Álvarez-López, Eloy; Barge-Caballero, Eduardo; Barge-Caballero, Gonzalo; Couto-Mallón, David; Pradas-Irun, Concepción; Muñiz, Javier; Tan, Carmela; Rodríguez, E Rene; Vázquez-Rodríguez, José Manuel; Hermida-Prieto, Manuel

2018-04-25

Heart transplantation (HT) is a well-established lifesaving treatment for endstage cardiac failure. Antibody-mediated rejection (AMR) represents one of the main problems after HT because of its diagnostic complexity and the poor evidence for supporting treatments. Complement cascade and B-cells play a key role in AMR and contribute to graft damage. This study explored the importance of variants in genes related to complement pathway and B-cell biology in HT and AMR in donors and in donor-recipient pairs.Methods and Results:Genetic variants in 112 genes (51 complement and 61 B-cell biology genes) were analyzed on next-generation sequencing in 28 donor-recipient pairs, 14 recipients with and 14 recipients without AMR. Statistical analysis was performed with SNPStats, R, and EPIDAT3.1. We identified one single nucleotide polymorphism (SNP) in donors in genes related to B-cell biology,interleukin-4 receptor subunitα (p.Ile75Val-IL4Rα), which correlated with the development of AMR. Moreover, in the analysis of recipient-donor genotype discrepancies, we identified another SNP, in this case inadenosine deaminase(ADA; p.Val178(p=)), which was related to B-cell biology, associated with the absence of AMR. Donor polymorphisms and recipient-donor discrepancies in genes related to the biology of B-cells, could have an important role in the development of AMR. In contrast, no variants in donor or in donor-recipient pairs in complement pathways seem to have an impact on AMR.

Blocking herpes simplex virus 2 glycoprotein E immune evasion as an approach to enhance efficacy of a trivalent subunit antigen vaccine for genital herpes.

PubMed

Awasthi, Sita; Huang, Jialing; Shaw, Carolyn; Friedman, Harvey M

2014-08-01

Herpes simplex virus 2 (HSV-2) subunit antigen vaccines targeting virus entry molecules have failed to prevent genital herpes in human trials. Our approach is to include a virus entry molecule and add antigens that block HSV-2 immune evasion. HSV-2 glycoprotein C (gC2) is an immune evasion molecule that inhibits complement. We previously reported that adding gC2 to gD2 improved vaccine efficacy compared to the efficacy of either antigen alone in mice and guinea pigs. Here we demonstrate that HSV-2 glycoprotein E (gE2) functions as an immune evasion molecule by binding the IgG Fc domain. HSV-2 gE2 is synergistic with gC2 in protecting the virus from antibody and complement neutralization. Antibodies produced by immunization with gE2 blocked gE2-mediated IgG Fc binding and cell-to-cell spread. Mice immunized with gE2 were only partially protected against HSV-2 vaginal challenge in mice; however, when gE2 was added to gC2/gD2 to form a trivalent vaccine, neutralizing antibody titers with and without complement were significantly higher than those produced by gD2 alone. Importantly, the trivalent vaccine protected the dorsal root ganglia (DRG) of 32/33 (97%) mice between days 2 and 7 postchallenge, compared with 27/33 (82%) in the gD2 group. The HSV-2 DNA copy number was significantly lower in mice immunized with the trivalent vaccine than in those immunized with gD2 alone. The extent of DRG protection using the trivalent vaccine was better than what we previously reported for gC2/gD2 immunization. Therefore, gE2 is a candidate antigen for inclusion in a multivalent subunit vaccine that attempts to block HSV-2 immune evasion. Herpes simplex virus is the most common cause of genital ulcer disease worldwide. Infection results in emotional distress for infected individuals and their partners, is life threatening for infants exposed to herpes during childbirth, and greatly increases the risk of individuals acquiring and transmitting HIV infection. A vaccine that prevents genital herpes infection will have major public health benefits. Our vaccine approach includes strategies to prevent the virus from evading immune attack. Mice were immunized with a trivalent vaccine containing an antigen that induces antibodies to block virus entry and two antigens that induce antibodies that block immune evasion from antibody and complement. Immunized mice demonstrated no genital disease, and 32/33 (97%) animals had no evidence of infection of dorsal root ganglia, suggesting that the vaccine may prevent the establishment of latency and recurrent infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Integration of multimodal transportation services : final report.

DOT National Transportation Integrated Search

2016-03-08

Flexible route paratransit services may complement as well as compete with conventional public transportation services (that : have fixed routes and schedules). Flexible routes are especially suitable for service areas or time periods with low demand...

49 CFR 37.127 - Complementary paratransit service for visitors.

Code of Federal Regulations, 2010 CFR

2010-10-01

... FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.127... that they are ADA paratransit eligible, under the criteria of § 37.125 of this part, in the...

49 CFR 37.141 - Requirements for a joint paratransit plan.

Code of Federal Regulations, 2010 CFR

2010-10-01

... INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.141 Requirements... effort: (1) A certification that the entity is committed to providing ADA paratransit service as part of...

C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies.

PubMed

Fuss, Alexander; Hope, Christopher M; Deayton, Susan; Bennett, Greg Donald; Holdsworth, Rhonda; Carroll, Robert P; Coates, P Toby H

2015-07-01

Acute antibody-mediated rejection can occur in absence of circulating donor-specific antibodies. Agonistic antibodies targeting the anti-angiotensin II type 1 receptor (anti-AT1 R) are emerging as important non-human leucocyte antigen (HLA) antibodies. Elevated levels of anti-angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure. We report 11 cases of biopsy-proven, Complement 4 fragment d (C4d)-negative, acute rejection occurring without circulating donor-specific anti-HLA antibodies. In eight cases, anti-angiotensin receptor antibodies were retrospectively examined. The remaining three subjects were identified from our centre's newly instituted routine anti-angiotensin receptor antibody screening. All subjects fulfilled Banff 2013 criteria for antibody-mediated rejection and all responded to anti-rejection therapy, which included plasma exchange and angiotensin receptor blocker therapy. These cases support the routine assessment of anti-AT1 R antibodies in kidney transplant recipients to identify subjects at risk. Further studies will need to determine optimal assessment protocol and the effectiveness of pre-emptive treatment with angiotensin receptor blockers. © 2015 Asian Pacific Society of Nephrology.

Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in manduca sexta.

PubMed

Willott, E; Trenczek, T; Thrower, L W; Kanost, M R

1994-12-01

We have made 140 monoclonal antibodies to hemocytes (insect blood cells) from Manduca sexta. Four of these antibodies, when used in immunofluorescent microscopy of fixed hemocytes, distinguish the four main morphologically distinct hemocyte types. Plasmatocytes, granular cells, and oenocytoids are each recognized by a unique antibody specific to that type; spherulocytes are recognized by an antibody that also binds to plasmatocytes. When used in flow cytometry with nonfixed hemocytes, three of the four antibodies bind their respective cells; the oenocytoid marker failed to bind to any hemocytes. This set of four monoclonal antibodies may be useful for labeling individual cell types and for separating the different hemocyte types for further study of hemocyte functions.

Development of a homogeneous immunoassay system using protein A fusion fragmented Renilla luciferase.

PubMed

Mie, Masayasu; Thuy, Ngo Phan Bich; Kobatake, Eiry

2012-03-07

A homogeneous immunoassay system was developed using fragmented Renilla luciferase (Rluc). The B domain of protein A was fused to two Rluc fragments. When complexes between an antibody and fragmented Rluc fusion proteins bind to target molecules, the Rluc fragments come into close proximity and the luminescence activity of fragmented Rluc is restored by complementation. As proof-of-principle, this fragmented Rluc system was used to detect E. coli homogeneously using an anti-E. coli antibody.

Detection of an enzootic plague focus by serological methods

PubMed Central

Cavanaugh, D. C.; Thorpe, B. D.; Bushman, J. B.; Nicholes, P. S.; Rust, J. H.

1965-01-01

Complement-fixation and haemagglutination tests, utilizing a highly purified, specific Fraction 1 antigen of Pasteurella pestis, have been employed to detect specific plague antibody in the sera of rodents resident in a sylvatic plague focus. The data show that while the isolation of P. pestis is seasonal and rather rare, antibodies can be detected for long periods of time and with great frequency in rodents surviving infection. The authors recommend that serological methods be incorporated into epidemiological surveys and control programmes involving rodent plague. PMID:14310906

DETECTION OF AN ENZOOTIC PLAGUE FOCUS BY SEROLOGICAL METHODS.

PubMed

CAVANAUGH, D C; THORPE, B D; BUSHMAN, J B; NICHOLES, P S; RUST, J H

1965-01-01

Complement-fixation and haemagglutination tests, utilizing a highly purified, specific Fraction 1 antigen of Pasteurella pestis, have been employed to detect specific plague antibody in the sera of rodents resident in a sylvatic plague focus. The data show that while the isolation of P. pestis is seasonal and rather rare, antibodies can be detected for long periods of time and with great frequency in rodents surviving infection. The authors recommend that serological methods be incorporated into epidemiological surveys and control programmes involving rodent plague.

Mesophilic Aeromonas sp. serogroup O:11 resistance to complement-mediated killing.

PubMed Central

Merino, S; Rubires, X; Aguilar, A; Albertí, S; Hernandez-Allés, S; Benedí, V J; Tomas, J M

1996-01-01

The complement activation by and resistance to complement-mediated killing of Aeromonas sp. strains from serogroup O:11 were investigated by using different wild-type strains (with an S-layer characteristic of this serogroup) and their isogenic mutants characterized for their surface components (S-layer and lipopolysaccharide [LPS]). All of the Aeromonas sp. serogroup O:11 wild-type strains are unable to activate complement, which suggested that the S-layer completely covered the LPS molecules. We found that the classical complement pathway is involved in serum killing of susceptible Aeromonas sp. mutant strains of serogroup O11, while the alternative complement pathway seems not to be involved, and that the complement activation seems to be independent of antibody. The smooth mutant strains devoid of the S-layer (S-layer isogenic mutants) or isogenic LPS mutant strains with a complete or rather complete LPS core (also without the S-layer) are able to activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b is rapidly degraded, and therefore the lytic membrane attack complex (C5b-9) is not formed. Isogenic LPS rough mutants with an incomplete LPS core are serum sensitive because they bind more C3b than the resistant strains, the C3b is not completely degraded, and therefore the lytic complex (C5b-9) is formed. PMID:8945581

Use of polyclonal/monoclonal antibody therapies in transplantation.

PubMed

Yeung, Melissa Y; Gabardi, Steven; Sayegh, Mohamed H

2017-03-01

For over thirty years, antibody (mAb)-based therapies have been a standard component of transplant immunosuppression, and yet much remains to be learned in order for us to truly harness their therapeutic capabilities. Current mAbs used in transplant directly target and destroy graft-destructive immune cells, interrupt cytokine and costimulation-dependent T and B cell activation, and prevent down-stream complement activation. Areas covered: This review summarizes our current approaches to using antibody-based therapies to prevent and treat allograft rejection. It also provides examples of promising novel mAb therapies, and discusses the potential for future mAb development in transplantation. Expert opinion: The broad capability of antibodies, in parallel with our growing ability to synthetically modulate them, offers exciting opportunities to develop better biologic therapeutics. In order to do so, we must further our understanding about the basic biology underlying allograft rejection, and gain better appreciation of how characteristics of therapeutic antibodies affect their efficacy.

Blood SC5b-9 complement levels increase at parturition during term and preterm labor.

PubMed

Segura-Cervantes, Enrique; Mancilla-Ramirez, Javier; Zurita, Luis; Paredes, Yuriria; Arredondo, José Luis; Galindo-Sevilla, Norma

2015-06-01

We explored the hypothesis that complement, an innate and adaptive immune effector, is active in the plasma of parturient women and is deposited on fetal membranes collected after delivery. A cross-sectional study was designed to evaluate complement activity at parturition. Pregnant women (n = 97) between 15 and 41 years of age were enrolled in a hospital protocol during the perinatal period to assess both SC5b-9 complement activity in blood and complement deposition on fetal membranes during parturition. Soluble SC5b-9 complement activity in plasma fractions was measured using a standard enzyme-linked immunosorbent assay (ELISA) that included specific anti-complement antibodies. Complement deposition on membranes was analyzed using immuno-dot blots and immunohistochemistry. Soluble SC5b-9 complement complex levels were increased in the plasma of women during term labor (TL; median 3361; range 1726-5670 ng/mL), preterm labor (PL; median 2958; range 1552-7092 ng/mL), and preterm premature rupture of membranes (PPROM; median 2272; range 167-6540 ng/mL) compared with pregnant women who were not in labor (P; median 1384; range 174-4570 ng/mL; P < 0.001, Kruskal-Wallis test). Active complement, as assessed by the C9 neo-antigen in C5b-9 complexes, was deposited on fetal membranes, with no difference between term and preterm delivery. The deposition of active complement on fetal membranes was confirmed by immunohistochemistry. Women who underwent non-labor-indicated Cesarean sections did not exhibit complement deposition. Soluble SC5b-9 complement complex levels increased in the plasma of women during parturition, and complement C5b-9 complexes were deposited on fetal membranes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

[Cytotoxicity of natural anti-HLA antibodies in Moroccan patients awaiting for kidney transplantation].

PubMed

Benseffaj, Nadia; Ouadghiri, Sanae; Bourhanbour, Asmaa Drissi; Zerrouki, Asmae Noor; Essakalli, Malika

2017-02-01

The presence of anti-HLA antibodies in the serum of a patient result from an immune response produced during an immunizing event as transfusion, pregnancy or graft. These antibodies can be cytotoxic by activating the complement pathway via C1q and may cause organ rejection during the transplant. Some male patients awaiting kidney transplantation are seropositive for anti-HLA antibodies when they have no immunizing antecedent event. These antibodies are qualified as natural antibodies. Our work is to assess the cytotoxicity of natural anti-HLA antibodies in patients followed at the immunology laboratory of the blood transfusion service and hemovigilance (STSH) as part of the kidney transplant. We evaluated the cytotoxicity of HLA antibodies detected in male Moroccan patients without immunization history using C1qScreen One Lambda reagent for Luminex™. Non-immunized men were positive for HLA antibodies screening in 25.4%. These antibodies are not cytotoxic. Our study showed a positivity rate of natural HLA antibody low than the literature (25.4% against 63%). It appears that these natural antibodies are not cytotoxic and their involvement in renal transplant remains to be determined. Copyright © 2016 Association Société de néphrologie. Published by Elsevier SAS. All rights reserved.

Discriminating the hemolytic risk of blood type A plasmas using the complement hemolysis using human erythrocytes (CHUHE) assay.

PubMed

Cunnion, Kenji M; Hair, Pamela S; Krishna, Neel K; Sass, Megan A; Enos, Clinton W; Whitley, Pamela H; Maes, Lanne Y; Goldberg, Corinne L

2017-03-01

The agglutination-based cross-matching method is sensitive for antibody binding to red blood cells but is only partially predictive of complement-mediated hemolysis, which is important in many acute hemolytic transfusion reactions. Here, we describe complement hemolysis using human erythrocytes (CHUHE) assays that directly evaluate complement-mediated hemolysis between individual serum-plasma and red blood cell combinations. The CHUHE assay is used to evaluate correlations between agglutination titers and complement-mediated hemolysis as well as the hemolytic potential of plasma from type A blood donors. Plasma or serum from each type A blood donor was incubated with AB or B red blood cells in the CHUHE assay and measured for free hemoglobin release. CHUHE assays for serum or plasma demonstrate a wide, dynamic range and high sensitivity for complement-mediated hemolysis for individual serum/plasma and red blood cell combinations. CHUHE results suggest that agglutination assays alone are only moderately predictive of complement-mediated hemolysis. CHUHE results also suggest that plasma from particular type A blood donors produce minimal complement-mediated hemolysis, whereas plasma from other type A blood donors produce moderate to high-level complement-mediated hemolysis, depending on the red blood cell donor. The current results indicate that the CHUHE assay can be used to assess complement-mediated hemolysis for plasma or serum from a type A blood donor, providing additional risk discrimination over agglutination titers alone. © 2016 AABB.

Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.

PubMed

Couch, Jessica A; Yu, Y Joy; Zhang, Yin; Tarrant, Jacqueline M; Fuji, Reina N; Meilandt, William J; Solanoy, Hilda; Tong, Raymond K; Hoyte, Kwame; Luk, Wilman; Lu, Yanmei; Gadkar, Kapil; Prabhu, Saileta; Ordonia, Benjamin A; Nguyen, Quyen; Lin, Yuwen; Lin, Zhonghua; Balazs, Mercedesz; Scearce-Levie, Kimberly; Ernst, James A; Dennis, Mark S; Watts, Ryan J

2013-05-01

Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.

Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells.

PubMed

Yamada, Tadaaki; Bando, Hideaki; Takeuchi, Shinji; Kita, Kenji; Li, Qi; Wang, Wei; Akinaga, Shiro; Nishioka, Yasuhiko; Sone, Saburo; Yano, Seiji

2011-12-01

Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC. © 2011 Japanese Cancer Association.

Antisperm antibodies in prepubertal boys and their reactivity with antigenic determinants on differentiated spermatozoa.

PubMed

Domagała, A; Kamieniczna, M; Kowalczyk, D; Kurpisz, M

1998-09-01

Antisperm antibodies induced in prepubertal boys with testicular failures were characterized by using four techniques of antibody detection. The reactivity of circulating antisperm antibodies in prepubertal boys and the reactivity of antibodies in sera samples of adult fertile and infertile males were compared against the same sperm antigenic pools (live or fixed spermatozoa, or sperm antigenic extracts). The incidence of antisperm antibodies in sera samples of 69 prepubertal boys with testicular failures and 21 samples obtained from adult, male individuals was assessed by indirect immunobead binding test (IDIBT), flow cytometry measurement, enzyme-linked immunosorbent assay, and Western blotting. Immunoblot analysis was performed by using sperm extracts of glycosylated and deglycosylated solubilized membrane antigens. Sera samples were studied in a group composed of healthy prepubertal boys (n = 7) and prepubertal boys with testicular failures (n = 69). Applied tests of antibody detection revealed striking differences in a group of boys with testicular pathology. With IDIBT, 7% of the sera samples were found positive, whereas with flow cytometry measurement, 48% of the sera samples were positive. Immunosorbent assay (fixed sperm) indicated 32% positive cases in the same group. The sera samples were found to be positive in 65% of immunoblotting reactions with glycosylated antigens and in 70% of immunoblotting reactions with deglycosylated antigens. All applied detection assays were clearly negative on sera samples from fertile, adult males. Western immunoblotting indicated an immunodominant antigenic determinant of 58 kDa. Tests of antibody detection with the use of live sperm (IDIBT and flow cytometry measurements) presented low sensitivity (8% and 48%, respectively) in a group of prepubertal boys. This observation underlines the difficulties in assigning the prospective prognosis of future fertility status in prepubertal boys with antisperm antibodies.

Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms

PubMed Central

Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada

2017-01-01

A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant–microbe interactions in the future. PMID:28654662

Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms.

PubMed

Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop

2017-01-01

A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

Immune system cells in healthy ferrets: an immunohistochemical study.

PubMed

Vidaña, B; Majó, N; Pérez, M; Montoya, M; Martorell, J; Martínez, J

2014-07-01

The ferret has emerged as an excellent animal model to characterize several physiologic and pathologic conditions. The distribution and characterization of different types of immune system cells were studied in healthy ferret tissues. Eight primary antibodies were tested for immunohistochemistry in formalin-fixed tissues: anti-CD3, anti-CD79α, anti-CD20, anti-HLA-DR, anti-lysozyme, anti-CD163, anti-SWC3, and anti-Mac387. The anti-CD3 antibody labeled T cells mainly in interfollicular and paracortical areas of lymph nodes, cortex and thymic medulla, and periarteriolar lymphoid sheaths in the spleen. The anti-CD79α and anti-CD20 antibodies immunolabeled B cells located in lymphoid follicles at lymph nodes, spleen, and Peyer patches. The CD79α and CD20 antibodies also labeled cells with nonlymphoid morphology in atypical B-cell locations. The anti-HLA-DR antibody labeled macrophages, some populations of B and T lymphocytes, and different populations of dendritic cells in lymph nodes, Peyer patches, spleen, and thymus. The anti-lysozyme antibody immunolabeled macrophages in the liver, lymph nodes, spleen, and thymus. The Mac-387, CD163, and SWC3 antibodies did not show any positive reaction in formalin-fixed or frozen tissues. To elucidate the origin of the uncommon CD79α/CD20 positive cells, a double immunohistochemistry was carried out using the anti-HLA-DR + the anti-CD79α, the anti-HLA-DR + the anti-CD20, and the anti-lysozyme + the anti-CD79α antibodies. Double labeling was mainly observed when the anti-HLA-DR + the anti-CD79α antibodies were combined. The immunohistologic characterization and distribution of these immune system cells in healthy ferret tissues should be of value in future comparative studies of diseases in ferrets. © The Author(s) 2013.

Young investigator challenge: Validation and optimization of immunohistochemistry protocols for use on cellient cell block specimens.

PubMed

Sauter, Jennifer L; Grogg, Karen L; Vrana, Julie A; Law, Mark E; Halvorson, Jennifer L; Henry, Michael R

2016-02-01

The objective of the current study was to establish a process for validating immunohistochemistry (IHC) protocols for use on the Cellient cell block (CCB) system. Thirty antibodies were initially tested on CCBs using IHC protocols previously validated on formalin-fixed, paraffin-embedded tissue (FFPE). Cytology samples were split to generate thrombin cell blocks (TCB) and CCBs. IHC was performed in parallel. Antibody immunoreactivity was scored, and concordance or discordance in immunoreactivity between the TCBs and CCBs for each sample was determined. Criteria for validation of an antibody were defined as concordant staining in expected positive and negative cells, in at least 5 samples each, and concordance in at least 90% of the samples total. Antibodies that failed initial validation were retested after alterations in IHC conditions. Thirteen of the 30 antibodies (43%) did not meet initial validation criteria. Of those, 8 antibodies (calretinin, clusters of differentiation [CD] 3, CD20, CDX2, cytokeratin 20, estrogen receptor, MOC-31, and p16) were optimized for CCBs and subsequently validated. Despite several alterations in conditions, 3 antibodies (Ber-EP4, D2-40, and paired box gene 8 [PAX8]) were not successfully validated. Nearly one-half of the antibodies tested in the current study failed initial validation using IHC conditions that were established in the study laboratory for FFPE material. Although some antibodies subsequently met validation criteria after optimization of conditions, a few continued to demonstrate inadequate immunoreactivity. These findings emphasize the importance of validating IHC protocols for methanol-fixed tissue before clinical use and suggest that optimization for alcohol fixation may be needed to obtain adequate immunoreactivity on CCBs. © 2016 American Cancer Society.

Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.

PubMed

Perruchini, Claire; Pecorari, Frederic; Bourgeois, Jean-Pierre; Duyckaerts, Charles; Rougeon, François; Lafaye, Pierre

2009-11-01

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

The identification of novel loci required for appropriate nodule development in Medicago truncatula.

PubMed

Domonkos, Agota; Horvath, Beatrix; Marsh, John F; Halasz, Gabor; Ayaydin, Ferhan; Oldroyd, Giles E D; Kalo, Peter

2013-10-11

The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules. Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed. The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later stages associated with bacterial release and nodule function.

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos.

PubMed

Rothschild, Sarah C; Francescatto, Ludmila; Tombes, Robert M

2016-02-19

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr. Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol. This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer's Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Quantitation of sperm bindable IgA and IgG in seminal fluid.

PubMed

Howe, S E; Lynch, D M

1986-05-01

Seminal fluid and serum from 95 infertile males were assayed for sperm bindable immunoglobulins using an indirect ELISA with whole target sperm. The ELISA method was compared to seminal fluid and serum immobilization and agglutination assays (functional assays). In this infertile group, the ELISA assay was positive in 22% of seminal fluids (greater than 1.2 fg IgA/sperm and greater than 0.3 fg IgG/sperm). The seminal fluid antibodies were IgA and had an accompanying elevated IgG component in 78% of patients. There was a 96% correlation between negative seminal fluid functional assays and negative ELISA, and a 95% correlation between positive seminal fluid functional assays and positive ELISA. Positive serum sperm antibody tests were found in 71% of the infertile males with positive seminal fluid sperm antibodies, but 29% of the infertile males with strongly positive IgA seminal fluid sperm antibodies showed normal levels of serum sperm antibodies by either ELISA or functional assays. The ELISA method gives reproducible quantitation of sperm antibodies in seminal fluid and correlates well with accepted functional assays. Comparisons with serum sperm antibody assays suggests that seminal fluid sperm antibody analysis complements the serum analysis of sperm antibodies.

Functional identification of pathogenic autoantibody responses in patients with multiple sclerosis

PubMed Central

Elliott, Christina; Lindner, Maren; Arthur, Ariel; Brennan, Kathryn; Jarius, Sven; Hussey, John; Chan, Andrew; Stroet, Anke; Olsson, Tomas; Willison, Hugh; Barnett, Susan C.; Meinl, Edgar

2012-01-01

Pathological and clinical studies implicate antibody-dependent mechanisms in the immunopathogenesis of multiple sclerosis. We tested this hypothesis directly by investigating the ability of patient-derived immunoglobulins to mediate demyelination and axonal injury in vitro. Using a myelinating culture system, we developed a sensitive and reproducible bioassay to detect and quantify these effects and applied this to investigate the pathogenic potential of immunoglobulin G preparations obtained from patients with multiple sclerosis (n = 37), other neurological diseases (n = 10) and healthy control donors (n = 13). This identified complement-dependent demyelinating immunoglobulin G responses in approximately 30% of patients with multiple sclerosis, which in two cases was accompanied by significant complement-dependent antibody mediated axonal loss. No pathogenic immunoglobulin G responses were detected in patients with other neurological disease or healthy controls, indicating that the presence of these demyelinating/axopathic autoantibodies is specific for a subset of patients with multiple sclerosis. Immunofluorescence microscopy revealed immunoglobulin G preparations with demyelinating activity contained antibodies that specifically decorated the surface of myelinating oligodendrocytes and their contiguous myelin sheaths. No other binding was observed indicating that the response is restricted to autoantigens expressed by terminally differentiated myelinating oligodendrocytes. In conclusion, our study identifies axopathic and/or demyelinating autoantibody responses in a subset of patients with multiple sclerosis. This observation underlines the mechanistic heterogeneity of multiple sclerosis and provides a rational explanation why some patients benefit from antibody depleting treatments. PMID:22561643

Antibody and complement reduce renal hemodynamic function in isolated perfused rat kidney.

PubMed

Jocks, T; Zahner, G; Helmchen, U; Kneissler, U; Stahl, R A

1996-01-01

To evaluate the effect of antibody and complement on renal hemodynamic changes, glomerular injury was induced in isolated perfused kidneys by an anti-thymocyte antibody (ATS) and rat serum (RS). Glomerular filtration rate (GFR), renal vascular resistance (RVR), and renal perfusate flow (RPF) were assessed over an 80-min period. The possible role of thromboxane (Tx) was tested by the application of the Tx synthesis inhibitor UK-38485 and the Tx receptor blocker daltroban. Perfusion of kidneys with ATS and RS significantly reduced GFR at 10 min (control, 501 +/- 111; ATS + RS, 138 +/- 86 ml.g kidney-1.min-1, significance of F = 0.000) after RS. Similarly, RPF (ml.g kidney-1.min-1) fell from 19.2 +/- 1.8 to 6.1 +/- 2.0 (significance of F = 0.000), whereas RVR (mmHg.ml-1.g.min) increased threefold from 5.2 +/- 0.4 to 17.9 +/- 5.0 at 10 min. These changes were ameliorated by the pretreatment of the rats with daltroban and UK-38485. Addition of erythrocytes to the perfusate increased RVR and GFR, whereas RPF decreased compared with cell-free perfused kidneys. ATS and RS in this preparation also decrease GFR and RPF. The hemodynamic alterations appeared without changes in filtration fraction. Compared with untreated, perfused control kidneys, glomerular Tx formation was significantly increased in ATS and RS perfused kidneys. These data demonstrate that antibody and RS induce impairment of renal hemodynamics, which are mediated by increased Tx formation.

Construction and characterization of an anti-CD20 mAb nanocomb with exceptionally excellent lymphoma-suppressing activity.

PubMed

Li, Hua-Fei; Wu, Cong; Chen, Ting; Zhang, Ge; Zhao, He; Ke, Chang-Hong; Xu, Zheng

2015-01-01

The CD20-directed monoclonal antibody rituximab (RTX) established a new era in the treatment of non-Hodgkin lymphoma (NHL); however, suboptimal response and/or resistance to RTX still limit its clinical merits. Although four effector mechanisms are validated to participate in CD20-based immunotherapy, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, caspase-dependent apoptosis, and lysosome-mediated programmed cell death (PCD), they could hardly be synchronously activated by any anti-CD20 mAb or mAb derivative until now. Herein, a novel mAb nanocomb (polyethylenimine polymer-RTX-tositumomab [PPRT nanocomb]) was firstly constructed through mass arming two different anti-CD20 mAbs (RTX and tositumomab) to one polymer by nanotechnology. Comparing with free mAbs, PPRT nanocomb possesses a comparable binding ability and reduced "off-rate" to surface CD20 of NHL cells. When treated by PPRT nanocomb, the caspase-dependent apoptosis was remarkably enhanced except for concurrently eliciting complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and lysosome-mediated PCD. Besides, "cross-cell link"-assisted homotypic adhesion by PPRT nanocomb further enhanced the susceptibility to PCD of lymphoma cells. Pharmacokinetic assays revealed that PPRT nanocomb experienced a relatively reduced clearance from peripheral blood compared with free antibodies. With the cooperation of all the abovementioned superiorities, PPRT nanocomb exhibits exceptionally excellent in vivo antitumor activities in both disseminated and localized human NHL xenotransplant models.

Construction and characterization of an anti-CD20 mAb nanocomb with exceptionally excellent lymphoma-suppressing activity

PubMed Central

Li, Hua-Fei; Wu, Cong; Chen, Ting; Zhang, Ge; Zhao, He; Ke, Chang-Hong; Xu, Zheng

2015-01-01

The CD20-directed monoclonal antibody rituximab (RTX) established a new era in the treatment of non-Hodgkin lymphoma (NHL); however, suboptimal response and/or resistance to RTX still limit its clinical merits. Although four effector mechanisms are validated to participate in CD20-based immunotherapy, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, caspase-dependent apoptosis, and lysosome-mediated programmed cell death (PCD), they could hardly be synchronously activated by any anti-CD20 mAb or mAb derivative until now. Herein, a novel mAb nanocomb (polyethylenimine polymer–RTX–tositumomab [PPRT nanocomb]) was firstly constructed through mass arming two different anti-CD20 mAbs (RTX and tositumomab) to one polymer by nanotechnology. Comparing with free mAbs, PPRT nanocomb possesses a comparable binding ability and reduced “off-rate” to surface CD20 of NHL cells. When treated by PPRT nanocomb, the caspase-dependent apoptosis was remarkably enhanced except for concurrently eliciting complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and lysosome-mediated PCD. Besides, “cross-cell link”-assisted homotypic adhesion by PPRT nanocomb further enhanced the susceptibility to PCD of lymphoma cells. Pharmacokinetic assays revealed that PPRT nanocomb experienced a relatively reduced clearance from peripheral blood compared with free antibodies. With the cooperation of all the abovementioned superiorities, PPRT nanocomb exhibits exceptionally excellent in vivo antitumor activities in both disseminated and localized human NHL xenotransplant models. PMID:26257518

Epstein-Barr virus/complement fragment C3d receptor (CR2) reacts with p53, a cellular antioncogene-encoded membrane phosphoprotein: detection by polyclonal anti-idiotypic anti-CR2 antibodies.

PubMed Central

Barel, M; Fiandino, A; Lyamani, F; Frade, R

1989-01-01

Epstein-Barr virus and the C3d fragment of the third component of complement are specific extracellular ligands for complement receptor type 2 (CR2). However, intracellular proteins that react specifically with CR2 and are involved in post-membrane signals remain unknown. We recently prepared polyclonal anti-idiotypic anti-CR2 antibodies (Ab2) by using the highly purified CR2 molecule as original immunogen. We showed that Ab2 contained anti-idiotypic specificities that mimicked extracellular domains of CR2 and detected two distinct binding sites on CR2 for its specific extracellular ligands, Epstein-Barr virus and C3d. We postulated that Ab2 might also contain specificities that could mimic intracellular domains of CR2. Here we report that Ab2, which did not react with Raji B-lymphoma cell surface components, detected specifically, among all components solubilized from Raji cell membranes, a single intracellular membrane protein of apparent molecular mass of 53 kDa. This protein was identified as the p53 cellular antioncogene-encoded membrane phosphoprotein by analyzing its antigenic properties with Pab1801, a monoclonal anti-p53 antibody, and by comparing its biochemical properties with those of p53. Additionally, solubilized and purified CR2 bound to solubilized p53 immobilized on Pab1801-Sepharose. p53, like CR2, was localized only in purified plasma membranes and nuclei of Raji cells. These data suggest strongly that p53, a cellular antioncogene-encoded phosphoprotein, reacted specifically with CR2 in Raji membranes. This interaction may represent one of the important steps through which CR2 could be involved in human B-lymphocyte proliferation and transformation. Images PMID:2557614

Pathogenesis and mechanisms of antibody-mediated hemolysis

PubMed Central

Flegel, Willy A

2015-01-01

Background The clinical consequences of antibodies to red blood cells (RBC) have been studied for a century. Most clinically relevant antibodies can be detected by sensitive in vitro assays. Several mechanisms of antibody-mediated hemolysis are well understood. Such hemolysis following transfusion is reliably avoided in a donor/recipient pair, if one individual is negative for the cognate antigen to which the other has the antibody. Study design and results Mechanisms of antibody-mediated hemolysis were reviewed based on a presentation at the Strategies to Address Hemolytic Complications of Immune Globulin Infusions Workshop addressing intravenous immunoglobulin (IVIG) and ABO antibodies. The presented topics included the rates of intravascular and extravascular hemolysis; IgM and IgG isoagglutinins; auto- and alloantibodies; antibody specificity; A, B, A,B and A1 antigens; A1 versus A2 phenotypes; monocytes/macrophages, other immune cells and complement; monocyte monolayer assay (MMA); antibody-dependent cell-mediated cytotoxicity (ADCC); and transfusion reactions due to ABO and other antibodies. Conclusion Several clinically relevant questions remained unresolved, and diagnostic tools were lacking to routinely and reliably predict the clinical consequences of RBC antibodies. Most hemolytic transfusion reactions associated with IVIG were due to ABO antibodies. Reducing the titers of such antibodies in IVIG may lower the frequency of this kind of adverse event. The only way to stop these events is to have no anti-A or anti-B antibodies in the IVIG products. PMID:26174897

[Humoral immune diseases: Cutaneous vasculitis and auto-immune bullous dermatoses].

PubMed

Wechsler, Janine

2018-02-01

Humoral immunity is the cause of multiple diseases related to antibodies (IgA, IgG, IgM) produced by the patient. Two groups of diseases are identified. The first group is related to circulating antigen-antibody complexes. The antigens are various. They are often unknown. These immune complexes cause a vascular inflammation due to the complement fixation. Consequently, this group is dominated by inflammatory vasculitis. In the second group, the pathology is due to the fixation in situ of antibodies to a target antigen of the skin that is no more recognized by the patient. This group is represented by the auto-immune bullous dermatoses. Copyright © 2017. Published by Elsevier Masson SAS.

Desensitization: Overcoming the Immunologic Barriers to Transplantation

PubMed Central

Choi, Jua; Vo, Ashley; Peng, Alice; Jordan, Stanley C.

2017-01-01

HLA (Human Leucocyte Antigen) sensitization is a significant barrier to successful kidney transplantation. It often translates into difficult crossmatch before transplant and increased risk of acute and chronic antibody mediated rejection after transplant. Over the last decade, several immunomodulatory therapies have emerged allowing for increased access to kidney transplantation for the immunologically disadvantaged group of HLA sensitized end stage kidney disease patients. These include IgG inactivating agents, anti-cytokine antibodies, costimulatory molecule blockers, complement inhibitors, and agents targeting plasma cells. In this review, we discuss currently available agents for desensitization and provide a brief analysis of data on novel biologics, which will likely improve desensitization outcomes, and have potential implications in treatment of antibody mediated rejection. PMID:28127571

[Streptomycin--an activator of persisting tick-borne encephalitis virus].

PubMed

Malenko, G V; Pogodina, V V; Karmysheva, V Ia

1984-01-01

The effect of streptomycin (C) on persistence of tick-borne encephalitis (TBE) virus in Syrian hamsters infected with 3 strains of the virus (41/65, Aina/1448, Vasilchenko ) intracerebrally or subcutaneously was studied. In the animals not given C the infectious virus could be detected in the brain for 8-14 days but not later although their organs (mostly brains and spleens) contained the hemagglutinating antigen and viral antigen detectable by immunofluorescence. Intramuscularly C was given twice daily for 13-35 days in a daily dose of 200 mg/kg. The C-treated hamsters yielded 7 virulent TBE virus strains: 3 from the brain, 3 from the spleen, and one from the blood. No virus could be isolated from the liver, kidneys, or lungs despite the use of various methods for isolation including tissue explantation. The activating effect of C was observed against the background of 4-fold decrease in the titre of complement-fixing and antihemagglutinating antibodies. C exerted its activating effect both at early (70 days) and late (9 months) stages of TBE virus persistence. The activating effect of C appears to be due to its immunosuppressive properties and neurotoxic action on the CNS.

Antibody Therapeutics in Oncology.

PubMed

Wold, Erik D; Smider, Vaughn V; Felding, Brunhilde H

2016-03-01

One of the newer classes of targeted cancer therapeutics is monoclonal antibodies. Monoclonal antibody therapeutics are a successful and rapidly expanding drug class due to their high specificity, activity, favourable pharmacokinetics, and standardized manufacturing processes. Antibodies are capable of recruiting the immune system to attack cancer cells through complement-dependent cytotoxicity or antibody dependent cellular cytotoxicity. In an ideal scenario the initial tumor cell destruction induced by administration of a therapeutic antibody can result in uptake of tumor associated antigens by antigen-presenting cells, establishing a prolonged memory effect. Mechanisms of direct tumor cell killing by antibodies include antibody recognition of cell surface bound enzymes to neutralize enzyme activity and signaling, or induction of receptor agonist or antagonist activity. Both approaches result in cellular apoptosis. In another and very direct approach, antibodies are used to deliver drugs to target cells and cause cell death. Such antibody drug conjugates (ADCs) direct cytotoxic compounds to tumor cells, after selective binding to cell surface antigens, internalization, and intracellular drug release. Efficacy and safety of ADCs for cancer therapy has recently been greatly advanced based on innovative approaches for site-specific drug conjugation to the antibody structure. This technology enabled rational optimization of function and pharmacokinetics of the resulting conjugates, and is now beginning to yield therapeutics with defined, uniform molecular characteristics, and unprecedented promise to advance cancer treatment.

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

PubMed

Moulton, Elizabeth A; Bertram, Paula; Chen, Nanhai; Buller, R Mark L; Atkinson, John P

2010-09-01

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

Epitope-Specific Suppression of IgG Responses by Passively Administered Specific IgG: Evidence of Epitope Masking.

PubMed

Bergström, Joakim J E; Xu, Hui; Heyman, Birgitta

2017-01-01

Specific IgG, passively administered together with particulate antigen, can completely prevent induction of antibody responses to this antigen. The ability of IgG to suppress antibody responses to sheep red blood cells (SRBCs) is intact in mice lacking FcγRs, complement factor 1q, C3, or complement receptors 1 and 2, suggesting that Fc-dependent effector functions are not involved. Two of the most widely discussed explanations for the suppressive effect are increased clearance of IgG-antigen complexes and/or that IgG "hides" the antigen from recognition by specific B cells, so-called epitope masking. The majority of data on how IgG induces suppression was obtained through studies of the effects on IgM-secreting single spleen cells during the first week after immunization. Here, we show that IgG also suppresses antigen-specific extrafollicular antibody-secreting cells, germinal center B-cells, long-lived plasma cells, long-term IgG responses, and induction of memory antibody responses. IgG anti-SRBC reduced the amount of SRBC in the spleens of wild-type, but not of FcγR-deficient mice. However, no correlation between suppression and the amount of SRBC in the spleen was observed, suggesting that increased clearance does not explain IgG-mediated suppression. Instead, we found compelling evidence for epitope masking because IgG anti-NP administered with NP-SRBC suppressed the IgG anti-NP, but not the IgG anti-SRBC response. Vice versa, IgG anti-SRBC administered with NP-SRBC, suppressed only the IgG anti-SRBC response. In conclusion, passively transferred IgG suppressed all measured parameters of an antigen-specific antibody/B cell response and an important mechanism of action is likely to be epitope masking.

Serum bactericidal assay for the evaluation of typhoid vaccine using a semi-automated colony-counting method.

PubMed

Jang, Mi Seon; Sahastrabuddhe, Sushant; Yun, Cheol-Heui; Han, Seung Hyun; Yang, Jae Seung

2016-08-01

Typhoid fever, mainly caused by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening disease, mostly in developing countries. Enzyme-linked immunosorbent assay (ELISA) is widely used to quantify antibodies against S. Typhi in serum but does not provide information about functional antibody titers. Although the serum bactericidal assay (SBA) using an agar plate is often used to measure functional antibody titers against various bacterial pathogens in clinical specimens, it has rarely been used for typhoid vaccines because it is time-consuming and labor-intensive. In the present study, we established an improved SBA against S. Typhi using a semi-automated colony-counting system with a square agar plate harboring 24 samples. The semi-automated SBA efficiently measured bactericidal titers of sera from individuals immunized with S. Typhi Vi polysaccharide vaccines. The assay specifically responded to S. Typhi Ty2 but not to other irrelevant enteric bacteria including Vibrio cholerae and Shigella flexneri. Baby rabbit complement was more appropriate source for the SBA against S. Typhi than complements from adult rabbit, guinea pig, and human. We also examined the correlation between SBA and ELISA for measuring antibody responses against S. Typhi using pre- and post-vaccination sera from 18 human volunteers. The SBA titer showed a good correlation with anti-Vi IgG quantity in the serum as determined by Spearman correlation coefficient of 0.737 (P < 0.001). Taken together, the semi-automated SBA might be efficient, accurate, sensitive, and specific enough to measure functional antibody titers against S. Typhi in sera from human subjects immunized with typhoid vaccines. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

Harada, M.; Odaka, K.; Kondo, K.

The effects of activated lymphocytes were studied in the regulation of in vitro hematopoiesis. Peripheral blood lymphocytes stimulated by concanavalin A (Con A) were cocultured with normal bone marrow cells in the assay system of hematopoietic stem cells. Con-A-stimulated lymphocytes and their supernatants showed significant suppression of in vitro growth of myeloid and erythroid progenitor cells (CFU-C, CFU-E, and BFU-E). Suppressive activity detected in the T-cell fraction was completely abolished by treatment with OKT3 or OKT8 monoclonal antibody and complement and 20 Gy radiation but not OKT4 or OKIa1 antibody and complement. These observations indicate that peripheral blood lymphocytes canmore » be induced by Con-A stimulation to become suppressor T cells for myeloid and erythroid progenitor cells that are OKT8 positive, Ia negative, and radiosensitive. Together with our previous observation that CFU-C suppressor cells induced by alloantigen stimulation are radioresistant and OKT8- and Ia-positive T cells, it is suggested that in vitro hematopoiesis may be regulated by heterogeneous subpopulations of activated T-lymphocytes.« less

The alternative complement pathway control protein H binds to immune complexes and serves their detection

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

Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.

1983-01-01

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound /sup 125/I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of /sup 125/I-H; when fresh serum was chelated with 10 mM EDTA, /sup 125/I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samplesmore » from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), /sup 125/I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while /sup 125/I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.« less

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.

New Milestones Ahead in Complement-Targeted Therapy

PubMed Central

Ricklin, Daniel; Lambris, John D.

2017-01-01

The complement system is a powerful effector arm of innate immunity that typically confers protection from microbial intruders and accumulating debris. In many clinical situations, however, the defensive functions of complement can turn against host cells and induce or exacerbate immune, inflammatory, and degenerative conditions. Although the value of inhibiting complement in a therapeutic context has long been recognized, bringing complement-targeted drugs into clinical use has proved challenging. This important milestone was finally reached a decade ago, yet the clinical availability of complement inhibitors has remained limited. Still, the positive long-term experience with complement drugs and their proven effectiveness in various diseases has reinvigorated interest and confidence in this approach. Indeed, a broad variety of clinical candidates that act at almost any level of the complement activation cascade are currently in clinical development, with several of them being evaluated in phase 2 and phase 3 trials. With antibody-related drugs dominating the panel of clinical candidates, the emergence of novel small-molecule, peptide, protein, and oligonucleotide-based inhibitors offers new options for drug targeting and administration. Whereas all the currently approved and many of the proposed indications for complement-targeted inhibitors belong to the rare disease spectrum, these drugs are increasingly being evaluated for more prevalent conditions. Fortunately, the growing experience from preclinical and clinical use of therapeutic complement inhibitors has enabled a more evidence-based assessment of suitable targets and rewarding indications as well as related technical and safety considerations. This review highlights recent concepts and developments in complement-targeted drug discovery, provides an overview of current and emerging treatment options, and discusses the new milestones ahead on the way to the next generation of clinically available complement therapeutics. PMID:27321574

Complement in Action: An Analysis of Patent Trends from 1976 Through 2011.

PubMed

Yang, Kun; Deangelis, Robert A; Reed, Janet E; Ricklin, Daniel; Lambris, John D

2013-01-01

Complement is an essential part of the innate immune response. It interacts with diverse endogenous pathways and contributes to the maintenance of homeostasis, the modulation of adaptive immune responses, and the development of various pathologies. The potential usefulness, in both research and clinical settings, of compounds that detect or modulate complement activity has resulted in thousands of publications on complement-related innovations in fields such as drug discovery, disease diagnosis and treatment, and immunoassays, among others. This study highlights the distribution and publication trends of patents related to the complement system that were granted by the United States Patent and Trademark Office from 1976 to the present day. A comparison to complement-related documents published by the World Intellectual Property Organization is also included. Statistical analyses revealed increasing diversity in complement-related research interests over time. More than half of the patents were found to focus on the discovery of inhibitors; interest in various inhibitor classes exhibited a remarkable transformation from chemical compounds early on to proteins and antibodies in more recent years. Among clinical applications, complement proteins and their modulators have been extensively patented for the diagnosis and treatment of eye diseases (especially age-related macular degeneration), graft rejection, cancer, sepsis, and a variety of other inflammatory and immune diseases. All of the patents discussed in this chapter, as well as those from other databases, are available from our newly constructed complement patent database: www.innateimmunity.us/patent .

Complement in action: an analysis of patent trends from 1976 through 2011.

PubMed

Yang, Kun; DeAngelis, Robert A; Reed, Janet E; Ricklin, Daniel; Lambris, John D

2013-01-01

Complement is an essential part of the innate immune response. It interacts with diverse endogenous pathways and contributes to the maintenance of homeostasis, the modulation of adaptive immune responses, and the development of various pathologies. The potential usefulness, in both research and clinical settings, of compounds that detect or modulate complement activity has resulted in thousands of publications on complement-related innovations in fields such as drug discovery, disease diagnosis and treatment, and immunoassays, among others. This study highlights the distribution and publication trends of patents related to the complement system that were granted by the United States Patent and Trademark Office from 1976 to the present day. A comparison to complement-related documents published by the World Intellectual Property Organization is also included. Statistical analyses revealed increasing diversity in complement-related research interests over time. More than half of the patents were found to focus on the discovery of inhibitors; interest in various inhibitor classes exhibited a remarkable transformation from chemical compounds early on to proteins and antibodies in more recent years. Among clinical applications, complement proteins and their modulators have been extensively patented for the diagnosis and treatment of eye diseases (especially age-related macular degeneration), graft rejection, cancer, sepsis, and a variety of other inflammatory and immune diseases. All of the patents discussed in this chapter, as well as those from other databases, are available from our newly constructed complement patent database: www.innateimmunity.us/patent.

Chemical studies on damages of Escherichea coli by the immune bactericidal reaction. II. Release of phosphatidylethanolamine from a phospholipase A-deficient mutant of E. coli during the immune bactericidal reaction.

PubMed

Inoue, K; Yano, K; Amano, T

1974-12-01

When an antibody-sensitized, phospholipase A-deficient mutant of Escherichia coli B/SM was treated with complement in the absence of lysozyme, bacterial phosphatidylethanolamine (PE) was liberated into the lipid fraction of the surrounding medium, but only traces of its degradation products were found in this fraction. Therefore, most of the degradation of bacterial PE to FFA and LPE observed in the usual immune bactericidal reaction (Inoue et al., 1974) must be the result of the action of bacterial phospholipase A which is activated or becomes accessible to its substrate on formation of lesions by complement. The mechanism of complement-mediated formation of membrane lesions is discussed on the basis of these results.

Unusual Features of Vaccinia Virus Extracellular Virion Form Neutralization Resistance Revealed in Human Antibody Responses to the Smallpox Vaccine

PubMed Central

Benhnia, Mohammed Rafii-El-Idrissi; Maybeno, Matthew; Blum, David; Aguilar-Sino, Rowena; Matho, Michael; Meng, Xiangzhi; Head, Steven; Felgner, Philip L.; Zajonc, Dirk M.; Koriazova, Lilia; Kato, Shinichiro; Burton, Dennis R.; Xiang, Yan; Crowe, James E.; Peters, Bjoern

2013-01-01

The extracellular virion form (EV) of vaccinia virus (VACV) is essential for viral pathogenesis and is difficult to neutralize with antibodies. Why this is the case and how the smallpox vaccine overcomes this challenge remain incompletely understood. We previously showed that high concentrations of anti-B5 antibodies are insufficient to directly neutralize EV (M. R. Benhnia, et al., J. Virol. 83:1201–1215, 2009). This allowed for at least two possible interpretations: covering the EV surface is insufficient for neutralization, or there are insufficient copies of B5 to allow anti-B5 IgG to cover the whole surface of EV and another viral receptor protein remains active. We endeavored to test these possibilities, focusing on the antibody responses elicited by immunization against smallpox. We tested whether human monoclonal antibodies (MAbs) against the three major EV antigens, B5, A33, and A56, could individually or together neutralize EV. While anti-B5 or anti-A33 (but not anti-A56) MAbs of appropriate isotypes were capable of neutralizing EV in the presence of complement, a mixture of anti-B5, anti-A33, and anti-A56 MAbs was incapable of directly neutralizing EV, even at high concentrations. This remained true when neutralizing the IHD-J strain, which lacks a functional version of the fourth and final known EV surface protein, A34. These immunological data are consistent with the possibility that viral proteins may not be the active component of the EV surface for target cell binding and infectivity. We conclude that the protection afforded by the smallpox vaccine anti-EV response is predominantly mediated not by direct neutralization but by isotype-dependent effector functions, such as complement recruitment for antibodies targeting B5 and A33. PMID:23152530

Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

PubMed

Mujib, Shariq; Liu, Jun; Rahman, A K M Nur-Ur; Schwartz, Jordan A; Bonner, Phil; Yue, Feng Yun; Ostrowski, Mario A

2017-08-15

Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS. IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and eradication of HIV-1 in infected humans remains uncertain. In this study, we tested the ability of bnAbs to directly recognize and eliminate primary human CD4 T cells infected with diverse HIV-1 strains representative of the global epidemic by antibody-dependent pathways. We also tested several combinations of bnAbs in our assays in order to maximize the clearance of infected cells. We show that the ability of bnAbs to identify and kill infected cells is highly variable and that only a few of them are able to exert this function. Our data will help guide the formulation of bnAbs to test in future human trials aimed at the development of a cure. Copyright © 2017 American Society for Microbiology.

scFv-based “grababody” as a general strategy to improve recruitment of immune effector cells to antibody-targeted tumors

PubMed Central

Cai, Zheng; Fu, Ting; Nagai, Yasuhiro; Lam, Lian; Yee, Marla; Zhu, Zhiqiang; Zhang, Hongtao

2013-01-01

Recruitment of immune cells to tumor cells targeted by a therapeutic antibody can heighten the antitumor efficacy of the antibody. For example, p185her2/neu-targeting antibodies not only downregulate the p185her2/neu kinase (ERBB2) but also trigger complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) through the antibody Fc region. Here we describe a generalized strategy to improve immune cell recruitment to targeted cancer cells, using a modified scFv antibody we call a “grababody” that binds the target protein and endogenous immunoglobulins. The model system we used to illustrate the utility of this platform recognizes p185her2/neu and includes an IgG binding domain. The recombinant scFv grababody that was created recruited circulating human IgGs and attracted immune cells carrying Fc receptors to tumor cells that expressed p185her2/neu. The presence of the IgG binding domain significantly enhanced CDC and ADCC activity and improved anti-tumor activity in vivo. Our results illustrate a novel general approach to improve antibody-like proteins for therapeutic applications. PMID:23396586

Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

PubMed

Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

2015-01-01

Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future.

Immunofluorescence Staining — EDRN Public Portal

Cancer.gov

Direct immunofluorescence method is used to detect the deposit of immunoglobulins, complement components, fibrinogen, etc. in tissues. This technique is usually performed on frozen sections. The primary antibody is conjugated to fluorescein binds directly with the antigen and can be detected by the fluorescent tag using a fluorescent microscope.

Monoclonal antibody (AFH1) immunoreactive on morphologically abnormal basal melanocytes within dysplastic nevi, nevocellular nevus nests, and melanoma.

PubMed

Aronson, P J; Ito, K; Fukaya, T; Hashimoto, K; Mehregan, A H

1988-04-01

The mouse monoclonal antibody AFH1 was produced using formalin-fixed, sham paraffin-embedded human melanoma cell culture line A375 as immunogen. Reactivity of this antibody was assessed by immunohistochemical techniques against formalin- or acid alcohol-fixed paraffin-embedded tissue as well as formalin- or acid alcohol-fixed unembedded lesions. Ninety-seven nevomelanocytic lesions, neurofibromas, epithelial lesions, and a plasmacellular infiltrate were evaluated. AFH1 was immunoreactive on 54 of 55 nevocytic lesions (98.2%), 15 of 16 primary melanomas (93.7%), a lentigo maligna, and nests in 21 of 21 dysplastic nevi (100%). Of 100 consecutive basal melanocytes of intraepidermal melanoma cells counted in each lesion, mean AFH1 immunoreactivity for nonnested basal melanocytes in nevocellular nevi was 3.8%; for dysplastic nevi, 13.8%; and for intraepidermal melanoma cells, 78.0%. When nonnested basal melanocytes were subdivided into cytologically normal and abnormal cell groups, AFH1 immunoreactivity was 9.4% and 72.6%, respectively. AFH1 recognition of the lentiginous portion of dysplastic nevi corresponds statistically to the appearance of abnormal melanocyte cytology, nest formation, or both. Using 50% immunoreactive nonnested melanocytes as the criterion, AFH1 seems to distinguish primary melanoma from dysplastic nevi with a sensitivity of 93.8% and a specificity of 95.8%.

Synergy between the classical and alternative pathways of complement is essential for conferring effective protection against the pandemic influenza A(H1N1) 2009 virus infection

PubMed Central

Rattan, Ajitanuj; Pawar, Shailesh D.; Nawadkar, Renuka; Kulkarni, Neeraja

2017-01-01

The pandemic influenza A(H1N1) 2009 virus caused significant morbidity and mortality worldwide thus necessitating the need to understand the host factors that influence its control. Previously, the complement system has been shown to provide protection during the seasonal influenza virus infection, however, the role of individual complement pathways is not yet clear. Here, we have dissected the role of intact complement as well as of its individual activation pathways during the pandemic influenza virus infection using mouse strains deficient in various complement components. We show that the virus infection in C3-/- mice results in increased viral load and 100% mortality, which can be reversed by adoptive transfer of naïve wild-type (WT) splenocytes, purified splenic B cells, or passive transfer of immune sera from WT, but not C3-/- mice. Blocking of C3a and/or C5a receptor signaling in WT mice using receptor antagonists and use of C3aR-/- and C5aR-/- mice showed significant mortality after blocking/ablation of C3aR, with little or no effect after blocking/ablation of C5aR. Intriguingly, deficiency of C4 and FB in mice resulted in only partial mortality (24%-32%) suggesting a necessary cross-talk between the classical/lectin and alternative pathways for providing effective protection. In vitro virus neutralization experiments performed to probe the cross-talk between the various pathways indicated that activation of the classical and alternative pathways in concert, owing to coating of viral surface by antibodies, is needed for its efficient neutralization. Examination of the virus-specific complement-binding antibodies in virus positive subjects showed that their levels vary among individuals. Together these results indicate that cooperation between the classical and alternative pathways not only result in efficient direct neutralization of the pandemic influenza virus, but also lead to the optimum generation of C3a, which when sensed by the immune cells along with the antigen culminates in generation of effective protective immune responses. PMID:28301559

Antibody persistence for up to 5 years after a fourth dose of Haemophilus influenzae type b and Neisseria meningitidis serogroups C and Y-tetanus toxoid conjugate vaccine (HibMenCY-TT) given at 12-15 months of age.

PubMed

Marshall, Gary S; Blatter, Mark; Marchant, Colin; Aris, Emmanuel; Mesaros, Narcisa; Miller, Jacqueline M

2013-06-01

A 4-dose series of recently licensed Haemophilus influenzae type b-meningococcal serogroups C and Y-tetanus toxoid conjugate vaccine (HibMenCY-TT) was immunogenic with a clinically acceptable safety profile in infants, with antibodies persisting in most participants for 1 year following dose 4. This study assessed antibody persistence up to 5 years after vaccination. Participants had received HibMenCY-TT or Hib-TT at 2, 4 and 6 months of age. At age 12-15 months, HibMenCY-TT vaccinees received a fourth HibMenCY-TT dose (HibMenCY x 4 group), whereas those who received Hib-TT received a fourth dose of either Hib-TT (Hib) or HibMenCY-TT (HibMenCY x 1). Blood samples were collected 1 month and 1, 3 and 5 years after the last dose for measurement of antipolyribosylribitol phosphate (the Hib capsular polysaccharide) antibodies and serum bactericidal activity (human complement source) against meningococcal serogroups C and Y. Five years after the fourth dose, the percentages of children with antipolyribosylribitol phosphate ≥0.15 μg/mL in HibMenCY x 4, HibMenCY x 1 and Hib groups were 98.8% (95% confidence interval: 93.5%-100%), 97.3% (85.8%-99.9%) and 92.3% (79.1%-98.4%), respectively. The percentages with human complement serum bactericidal activity ≥1:8 for meningococcal serogroup C were 82.9% (72.5%-90.6%), 73.5% (55.6%-87.1%) and 21.1% (9.6%-37.3%), respectively. The percentages with human complement serum bactericidal activity ≥1:8 for serogroup Y were 69.5% (58.4%-79.2%), 54.3% (36.6%-71.2%) and 18.4% (7.7%-34.3%), respectively. HibMenCY-TT given as a 4-dose series or as a single dose at 12-15 months of age induced immune responses for all 3 antigens that lasted for up to 5 years after vaccination in more than half of recipients.

Spectrum of primary immunodeficiency disorders in Sri Lanka

PubMed Central

2013-01-01

Background While primary immunodeficiencies (PID has been recognized in the west for decades, recognition has been delayed in the third world. This study attempts to detail the spectrum of PID, the therapy provided, and constraints in the diagnosis and treatment in a middle income country such as Sri Lanka. Methods Nine hundred and forty two patients with recurrent infections and features suggestive of immune deficiency, referred from the entire country in a 4 year period, to the sole immunology unit in Sri Lanka were included. The following tests were performed. Full blood counts, serum Immunoglobulin and complement C3 and C4 levels, functional antibody levels, enumeration of lymphocyte subsets, in vitro and in vivo T cell functional assays,, nitroblue tetrazolium assay to diagnose chronic granulomatous disease, hair shaft assay to diagnose Griscelli syndrome. Sequencing of the common gamma chain to identify x linked severe combined immune deficiency, and X linked agammaglobulinemia was confirmed by assaying for Btk mutations by single sequence conformation polymorphism. HIV/AIDS was excluded in all patients. Results Seventy three patients were diagnosed with a primary immune deficiency. The majority (60.27%) had antibody deficiency. Common variable immune deficiency was the commonest (28.76%), followed by X linked agammaglobulinemia (XLA) (20.54%). Five patients had possible hyper IgM syndrome. Ten patients had severe combined immune deficiency (SCID), including 2 with x linked SCID, in addition to DiGeorge syndrome (2), ataxia telangiectasia (6), autosomal dominant hyper IgE syndrome (2), chronic granulomatous disease (4), leucocyte adhesion deficiency type 1 (2) and Griscelli syndrome (3). Patients with autoinflammatory, innate immune and complement defects could not be identified due to lack of facilities. Conclusions Antibody deficiency is the commonest PID, as in the west.IgA deficiency is rare. Autoinflammatory diseases, innate immune and complement deficiencies could not be identified due to lack of diagnostic facilities. Lack of awareness of PID among adult physicians result in delay in treatment of adult patients. While treatment of antibody deficiencies provided in state hospitals has extended life expectancy, there is no treatment available for severe T cell defects. PMID:24373416

Management of children undergoing cardiac transplantation with high Panel Reactive Antibodies.

PubMed

Asante-Korang, Alfred; Jacobs, Jeffrey P; Ringewald, Jeremy; Carapellucci, Jennifer; Rosenberg, Kristin; McKenna, Daniel; McCormack, Jorge; Wilmot, Ivan; Gjeldum, Abigail; Lopez-Cepero, Mayra; Sleasman, John

2011-12-01

Highly sensitised children in need of cardiac transplantation have overall poor outcomes because of increased risk for dysfunction of the cardiac allograft, acute cellular and antibody-mediated rejection, and vasculopathy of the cardiac allograft. Cardiopulmonary bypass and the frequent use of blood products in the operating room and cardiac intensive care unit, as well as the frequent use of homografts, have predisposed potential recipients of transplants to allosensitisation. The expansion in the use of ventricular assist devices and extracorporeal membrane oxygenation has also contributed to increasing rates of allosensitisation in candidates for cardiac transplantation. Antibodies to Human Leukocyte Antigen can be detected before transplantation using several different techniques, the most common being the "complement-dependent lymphocytotoxicity assays". "Solid-phase assays", particularly the "Luminex® single antigen bead method", offer improved specificity and more detailed information regarding specificities of antibodies, leading to improved matching of donors with recipients. Allosensitisation prolongs the time on the waiting list for potential recipients of transplantation and increases the risk of complications and death after transplantation. Aggressive reduction of antibodies to Human Leukocyte Antigen in these high-risk patients is therefore of vital importance for long-term survival of the patient and cardiac allograft. Strategies to decrease Panel Reactive Antibody or percent reactive antibody before transplantation include plasmapheresis, intravenous administration of immunoglobulin, and specific treatment to reduce B-cells, particularly Rituximab. These strategies have resulted in varying degrees of success. Antibody-mediated rejection and cardiac allograft vasculopathy are two of the most important complications of transplantation in patients with high Panel Reactive Antibody. The treatment of antibody-mediated rejection in recipients of cardiac transplants is largely empirical and includes the use of high-dose corticosteroids, plasmapheresis, intravenous administration of immunoglobulins, anti-thymocyte globulin, and Rituximab. Cardiac allograft vasculopathy is believed to be secondary to chronic complement-mediated endothelial injury and chronic vascular rejection. The use of proliferation signal inhibitors, such as sirolimus and everolimus, has been shown to delay the progression of cardiac allograft vasculopathy. In some non-sensitised recipients of cardiac transplants, the de novo formation of antibodies to Human Leukocyte Antigen after transplantation may increase the likelihood of adverse clinical outcomes. The use of serial testing for donor-specific antibodies after cardiac transplantation may be advisable in patients with frequent episodes of rejection and patients with history of sensitisation. Allosensitisation before transplantation can negatively influence outcomes after transplantation. A high incidence of antibody-mediated rejection and graft vasculopathy can result in graft failure and decreased survival. Current strategies to decrease allosensitisation have helped to expand the pool of donors, improve times on the waiting list, and decrease mortality. Centres of transplantation offering desensitisation are currently using plasmapheresis to remove circulating antibodies; intravenous immunoglobulin to inactivate antibodies; cyclophosphamide to suppress B-cell proliferation; and Rituximab to deplete B-lymphocytes. Similar approaches are also used to treat antibody-mediated rejection after transplantation with promising results.

Differential mechanisms of complement-mediated neutralization of the closely related paramyxoviruses simian virus 5 and mumps virus

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

Johnson, John B.; Capraro, Gerald A.; Parks, Griffith D.

2008-06-20

The complement system is an important component of the innate immune response to virus infection. The role of human complement pathways in the in vitro neutralization of three closely related paramyxoviruses, Simian Virus 5 (SV5), Mumps virus (MuV) and Human Parainfluenza virus type 2 (HPIV2) was investigated. Sera from ten donors showed high levels of neutralization against HPIV2 that was largely complement-independent, whereas nine of ten donor sera were found to neutralize SV5 and MuV only in the presence of active complement pathways. SV5 and MuV neutralization proceeded through the alternative pathway of the complement cascade. Electron microscopy studies andmore » biochemical analyses showed that treatment of purified SV5 with human serum resulted in C3 deposition on virions and the formation of massive aggregates, but there was relatively little evidence of virion lysis. Treatment of MuV with human serum also resulted in C3 deposition on virions, however in contrast to SV5, MuV particles were lysed by serum complement and there was relatively little aggregation. Assays using serum depleted of complement factors showed that SV5 and MuV neutralization in vitro was absolutely dependent on complement factor C3, but was not dependent on downstream complement factors C5 or C8. Our results indicate that even though antibodies exist that recognize both SV5 and MuV, they are mostly non-neutralizing and viral inactivation in vitro occurs through the alternative pathway of complement. The implications of our work for development of paramyxovirus vectors and vaccines are discussed.« less

Effect of increased CRM₁₉₇ carrier protein dose on meningococcal C bactericidal antibody response.

PubMed

Lee, Lucia H; Blake, Milan S

2012-04-01

New multivalent CRM(197)-based conjugate vaccines are available for childhood immunization. Clinical studies were reviewed to assess meningococcal group C (MenC) antibody responses following MenC-CRM(197) coadministration with CRM(197)-based pneumococcal or Haemophilus influenzae type b conjugate vaccines. Infants receiving a total CRM(197) carrier protein dose of ∼50 μg and concomitant diphtheria-tetanus-acellular pertussis (DTaP)-containing vaccine tended to have lower MenC geometric mean antibody titers and continued to have low titers after the toddler dose. Nevertheless, at least 95% of children in the reported studies achieved a MenC serum bactericidal antibody (SBA) titer of ≥ 1:8 after the last infant or toddler dose. SBA was measured using an assay with a baby rabbit or human complement source. Additional studies are needed to assess long-term antibody persistence and MenC CRM(197) conjugate vaccine immunogenicity using alternative dosing schedules.

Obstetric antiphospholipid syndrome.

PubMed

Esteve-Valverde, E; Ferrer-Oliveras, R; Alijotas-Reig, J

2016-04-01

Obstetric antiphospholipid syndrome is an acquired autoimmune disorder that is associated with various obstetric complications and, in the absence of prior history of thrombosis, with the presence of antiphospholipid antibodies directed against other phospholipids, proteins called cofactors or PL-cofactor complexes. Although the obstetric complications have been related to the procoagulant properties of antiphospholipid antibodies, pathological studies of human placenta have shown the proinflammatory capacity of antiphospholipid antibodies via the complement system and proinflammatory cytokines. There is no general agreement on which antiphospholipid antibodies profile (laboratory) confers the greatest obstetric risk, but the best candidates are categories I and IIa. Combined treatment with low doses of aspirin and heparin achieves good obstetric and maternal outcomes. In this study, we also review the therapeutic possibilities in refractory cases, although the likelihood of progressing to other autoimmune diseases is low. We briefly comment on incomplete obstetric antiphospholipid syndrome, also known as antiphospholipid antibody-mediated pregnancy morbidity syndrome. Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Medicina Interna (SEMI). All rights reserved.

Allogeneic major histocompatibility complex-mismatched equine bone marrow-derived mesenchymal stem cells are targeted for death by cytotoxic anti-major histocompatibility complex antibodies.

PubMed

Berglund, A K; Schnabel, L V

2017-07-01

Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Controversy exists, however, over whether major histocompatibility complex (MHC)-mismatched MSCs are recognised by the recipient immune system and targeted for death by a cytotoxic antibody response. To determine if cytotoxic anti-MHC antibodies generated in vivo following MHC-mismatched MSC injections are capable of initiating complement-dependent cytotoxicity of MSCs. Experimental controlled study. Antisera previously collected at Days 0, 7, 14 and 21 post-injection from 4 horses injected with donor MHC-mismatched equine leucocyte antigen (ELA)-A2 haplotype MSCs and one control horse injected with donor MHC-matched ELA-A2 MSCs were utilised in this study. Antisera were incubated with ELA-A2 MSCs before adding complement in microcytotoxicity assays and cell death was analysed via eosin dye exclusion. ELA-A2 peripheral blood leucocytes (PBLs) were used in the assays as a positive control. Antisera from all 4 horses injected with MHC-mismatched MSCs contained antibodies that caused the death of ELA-A2 haplotype MSCs in the microcytotoxicity assays. In 2 of the 4 horses, antibodies were present as early as Day 7 post-injection. MSC death was consistently equivalent to that of ELA-A2 haplotype PBL death at all time points and antisera dilutions. Antisera from the control horse that was injected with MHC-matched MSCs did not contain cytotoxic ELA-A2 antibodies at any of the time points examined. This study examined MSC death in vitro only and utilized antisera from a small number of horses. The cytotoxic antibody response induced in recipient horses following injection with donor MHC-mismatched MSCs is capable of killing donor MSCs in vitro. These results suggest that the use of allogeneic MHC-mismatched MSCs must be cautioned against, not only for potential adverse events, but also for reduced therapeutic efficacy due to targeted MSC death. © 2016 The Authors Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

PubMed

Monahan, Paul E; Sun, Junjiang; Gui, Tong; Hu, Genlin; Hannah, William B; Wichlan, David G; Wu, Zhijian; Grieger, Joshua C; Li, Chengwen; Suwanmanee, Thipparat; Stafford, Darrel W; Booth, Carmen J; Samulski, Jade J; Kafri, Tal; McPhee, Scott W J; Samulski, R Jude

2015-02-01

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100-500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

PubMed

Prechl, József; Papp, Krisztián; Hérincs, Zoltán; Péterfy, Hajna; Lóránd, Veronika; Szittner, Zoltán; Estonba, Andone; Rovero, Paolo; Paolini, Ilaria; Del Amo, Jokin; Uribarri, Maria; Alcaro, Maria Claudia; Ruiz-Larrañaga, Otsanda; Migliorini, Paola; Czirják, László

2016-01-01

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

The protective role of immunoglobulins in fungal infections and inflammation.

PubMed

Elluru, Sri Ramulu; Kaveri, Srini V; Bayry, Jagadeesh

2015-03-01

Increased incidence of fungal infections in the immunocompromised individuals and fungi-mediated allergy and inflammatory conditions in immunocompetent individuals is a cause of concern. Consequently, there is a need for efficient therapeutic alternatives to treat fungal infections and inflammation. Several studies have demonstrated that antibodies or immunoglobulins have a role in restricting the fungal burden and their clearance. However, based on the data from monoclonal antibodies, it is now evident that the efficacy of antibodies in fungal infections is dependent on epitope specificity, abundance of protective antibodies, and their isotype. Antibodies confer protection against fungal infections by multiple mechanisms that include direct neutralization of fungi and their antigens, inhibition of growth of fungi, modification of gene expression, signaling and lipid metabolism, causing iron starvation, inhibition of polysaccharide release, and biofilm formation. Antibodies promote opsonization of fungi and their phagocytosis, complement activation, and antibody-dependent cell toxicity. Passive administration of specific protective monoclonal antibodies could also prove to be beneficial in drug resistance cases, to reduce the dosage and associated toxic symptoms of anti-fungal drugs. The longer half-life of the antibodies and flexibilities to modify their structure/forms are additional advantages. The clinical data obtained with two monoclonal antibodies should incite interests in translating pre-clinical success into the clinics. The anti-inflammatory and immunoregulatory role of antibodies in fungal inflammation could be exploited by intravenous immunoglobulin or IVIg.

THE SEROLOGICAL SPECIFICITY OF PARTICULATE COMPONENTS DERIVED FROM VARIOUS NORMAL MAMMALIAN ORGANS

PubMed Central

Henle, Werner; Chambers, Leslie A.; Groupé, Vincent

1941-01-01

1. Particles derived from filtrates of organ suspensions by high speed centrifugation were serologically active as shown by agglutination and complement fixation techniques. Particles from brain, liver, lung, kidney, heart muscle, spleen, testicle, and pancreas of various species have been studied. 2. All particles showed a certain degree of organ specificity with the exception of pancreas. Cross-reactions occurred between the particles from various organs from one species, which were more marked when complement fixation technique was employed than by the agglutination test. However, agglutination always appeared earlier and was stronger, and complement fixation was positive in higher dilutions of antigen in the presence of homologous antiserum than with heterologous antisera. 3. The cross-reactions did not depend on the occasional precipitins for serum and the agglutinins for the red cells of the species from which the particles were derived, nor did they bear a relation to Wassermann and Forssman antibodies present in some of the sera. 4. The organ specific differentiation of the particles from various organs could more clearly be demonstrated by two means: The antiserum could be diluted in such a way that only the homologous reaction still showed a positive result while the cross-reactions had become negative; or the cross-reacting antibodies could be absorbed by heterologous particles and the homologous reaction was still more or less intact. 5. In addition to the organ specific differentiation, most particles were found to exhibit species specificity. While the particles derived from kidney, lung, testicle, and heart muscle aggregated only in the presence of the antiserum against the corresponding organ particles from the homologous species, brain particles reacted with brain antisera against both homologous and heterologous species alike. Absorption of brain particle antisera with brain preparations from a heterologous species removed all antibodies. Liver particle preparations showed an intermediate position in that all liver preparations with the exception of rabbit liver particles were aggregated by any liver particle antiserum. However, absorption with liver particles from a heterologous species left a distinct species specific reaction in the serum. 6. The antigens involved are all destroyed by heating to 100° C. for a few minutes with the exception of brain particles, which after 20 minutes at 100° C. still gave complement fixation almost to the same strength as the untreated controls. 7. Alcoholic and ether extracts of brain reacted with the brain particle antisera only. All alcoholic or ether extracts of other organs gave no complement fixation. None of the various other organ particle antisera tested contained antibodies for these extracts. 8. The relationship between the heat-stable and the alcohol-soluble brain particle antigen studied by absorption technique revealed that there were two antigens present, both organ specific and independent of the species, the one alcohol- and ether-soluble, the other not soluble in these solvents but heat stable. Some of the sera showed besides a few species specific antibodies. 9. Preliminary evidence has been gathered to show that no iso-immunization could be obtained with any one of the organ particles. As far as cytotoxic activity of the sera is concerned only the kidney particle antisera have been studied for nephrotoxins; these failed to reveal any such activity in the mouse. PMID:19871150

EVALUATING THE IMPACTS OF COINFECTION ON IMMUNE SYSTEM FUNCTION OF THE DEER MOUSE ( PEROMYSCUS MANICULATUS) USING SIN NOMBRE VIRUS AND BARTONELLA AS MODEL PATHOGEN SYSTEMS.

PubMed

Lehmer, Erin M; Lavengood, Kathryn; Miller, Mason; Rodgers, Jacob; Fenster, Steven D

2018-01-01

:  Simultaneous infections with multiple pathogens can alter the function of the host's immune system, often resulting in additive or synergistic morbidity. We examined how coinfection with the common pathogens Sin Nombre virus (SNV) and Bartonella sp. affected aspects of the adaptive and innate immune responses of wild deer mice ( Peromyscus maniculatus). Adaptive immunity was assessed by measuring SNV antibody production; innate immunity was determined by measuring levels of C-reactive protein (CRP) in blood and the complement activity of plasma. Coinfected mice had reduced plasma complement activity and higher levels of CRP compared to mice infected with either SNV or Bartonella. However, antibody titers of deer mice infected with SNV were more than double those of coinfected mice. Plasma complement activity and CRP levels did not differ between uninfected deer mice and those infected with only Bartonella, suggesting that comorbid SNV and Bartonella infections act synergistically, altering the innate immune response. Collectively, our results indicated that the immune response of deer mice coinfected with both SNV and Bartonella differed substantially from individuals infected with only one of these pathogens. Results of our study provided unique, albeit preliminary, insight into the impacts of coinfection on immune system function in wild animal hosts and underscore the complexity of the immune pathways that exist in coinfected hosts.

Epstein-Barr virus glycoprotein gH/gL antibodies complement IgA-viral capsid antigen for diagnosis of nasopharyngeal carcinoma

PubMed Central

Tang, Lin-Quan; Zhang, Hua; Li, Yan; Liu, Wan-Li; Zhong, Qian; Zeng, Mu-Sheng; Huang, Xiao-Ming

2016-01-01

To determine whether measuring antibodies against Epstein-Barr virus (EBV) glycoprotein gH/gL in serum could improve diagnostic accuracy in nasopharyngeal carcinoma (NPC) cases, gH/gL expressed in a recombinant baculovirus system was used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in two independent cohorts. Binary logistic regression analyses were performed using results from a training cohort (n = 406) to establish diagnostic mathematical models, which were validated in a second independent cohort (n = 279). Levels of serum gH/gL antibodies were higher in NPC patients than in healthy controls (p < 0.001). In the training cohort, the IgA-gH/gL ELISA had a sensitivity of 83.7%, specificity of 82.3% and area under the curve (AUC) of 0.893 (95% CI, 0.862-0.924) for NPC diagnosis. Furthermore, gH/gL maintained diagnostic capacity in IgA-VCA negative NPC patients (sensitivity = 78.1%, specificity = 82.3%, AUC = 0.879 [95% CI, 0.820 - 0.937]). Combining gH/gL and viral capsid antigen (VCA) detection improved diagnostic capacity as compared to individual tests alone in both the training cohort (sensitivity = 88.5%, specificity = 97%, AUC = 0.98 [95% CI, 0.97 - 0.991]), and validation cohort (sensitivity = 91.2%, specificity = 96.5%, AUC = 0.97 [95% CI, 0.951-0.988]). These findings suggest that EBV gH/gL detection complements VCA detection in the diagnosis of NPC and aids in the identification of patients with VCA-negative NPC. PMID:27093005

Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens

PubMed Central

Cywes-Bentley, Colette; Skurnik, David; Zaidi, Tanweer; Roux, Damien; DeOliveira, Rosane B.; Garrett, Wendy S.; Lu, Xi; O’Malley, Jennifer; Kinzel, Kathryn; Zaidi, Tauqeer; Rey, Astrid; Perrin, Christophe; Fichorova, Raina N.; Kayatani, Alexander K. K.; Maira-Litràn, Tomas; Gening, Marina L.; Tsvetkov, Yury E.; Nifantiev, Nikolay E.; Bakaletz, Lauren O.; Pelton, Stephen I.; Golenbock, Douglas T.; Pier, Gerald B.

2013-01-01

Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A β-(1→6)–linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology. PMID:23716675

Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential

PubMed Central

Lohmueller, Jason J.; Sato, Shuji; Popova, Lana; Chu, Isabel M.; Tucker, Meghan A.; Barberena, Roberto; Innocenti, Gregory M.; Cudic, Mare; Ham, James D.; Cheung, Wan Cheung; Polakiewicz, Roberto D.; Finn, Olivera J.

2016-01-01

MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a wide range of binding affinities. These antibodies bind hypoglycosylated MUC1 on human cancer cell lines and tumor tissues but show no reactivity against fully-glycosylated MUC1 on normal cells and tissues. We found that several antibodies activate complement-mediated cytotoxicity and that T cells carrying chimeric antigen receptors with the antibody variable regions kill MUC1+ target cells, express activation markers, and produce interferon gamma. Fully-human and tumor-specific, these antibodies are candidates for further testing and development as immunotherapeutic drugs. PMID:27545199

Short report: Changes in West Nile virus seroprevalence and antibody titers among Wisconsin mesopredators 2003-2006

USGS Publications Warehouse

Docherty, D.E.; Samuel, M.D.; Egstad, K.F.; Griffin, K.M.; Nolden, C.A.; Karwal, L.; Ip, Hon S.

2009-01-01

After the 2001 occurrence of West Nile virus (WNV) in Wisconsin (WI), we collected sera, during 2003-2006, from south-central WI mesopredators. We tested these sera to determine WNV antibody prevalence and geometric mean antibody titer (GMAT). Four-fold higher antibody prevalence and 2-fold higher GMAT in 2003-2004 indicated greater exposure of mesopredators to WNV during the apparent epizootic phase. The period 2005-2006 was likely the enzootic phase because WNV antibody prevalence fell to a level similar to other flaviviruses. Our results suggest that, in mesopredators, vector-borne transmission is the primary route of infection and WNV antibodies persist for < 1 year. Mesopredators may be sensitive indicators of West Nile virus spill-over into humans and horses. Mesopredator sero-surveys may complement dead crow surveillance by providing additional data for the timing of public health interventions. Research is needed to clarify the dynamics of WNV infection in these mammals and their role as potential WNV amplifiers. Copyright ?? 2009 by The American Society of Tropical Medicine and Hygiene.

A commercial ELISA detects high levels of human H5 antibody but cross-reacts with influenza A antibodies.

PubMed

Stelzer-Braid, Sacha; Wong, Bruce; Robertson, Peter; Lynch, Garry W; Laurie, Karen; Shaw, Robert; Barr, Ian; Selleck, Paul W; Baleriola, Cristina; Escott, Ros; Katsoulotos, Gregory; Rawlinson, William D

2008-10-01

Commercial serological assays to determine influenza A H5N1 infection are available, although the accuracy and reproducibility of these are not reported in detail. This study aimed to assess the validity of a commercial ELISA H5 hemagglutinin (HA) antibody kit. A commercial ELISA for detection of antibodies towards influenza A H5 HA was evaluated using human sera from vaccinated individuals. The ELISA was used to screen 304 sera with elevated influenza A complement fixation titres collected between the period 1995-2007. The ELISA was found to be accurate for sera with high levels of anti-H5 antibodies, and would be useful in clinical settings where a rapid result is required. Thirteen of the stored sera were positive using the ELISA, but were confirmed as negative for H5N1 exposure using further serological tests. Absorption studies suggested that antibodies towards seasonal H3N2 and H1N1 influenza may cross-react with H5 antigen, giving false positive results with the ELISA.

Chemical Synthesis of GM2 Glycans, Bioconjugation with Bacteriophage Qβ, and the Induction of Anticancer Antibodies

PubMed Central

Yin, Zhaojun; Dulaney, Steven; McKay, Craig S.; Baniel, Claire; Kaczanowska, Katarzyna; Ramadan, Sherif; Finn, M. G.

2016-01-01

The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qβ. Although the copper-catalyzed azide–alkyne cyclo-addition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside. PMID:26538065

Anti-Ig autoantibody and complement-mediated destruction of neoplastic cells

NASA Technical Reports Server (NTRS)

Towmey, J. J.

1976-01-01

Some immune response are effected through immunoglobulins (Ig), of which five classes have been recognized, namely, IgA, IgD, IgE, IgG, and IgM. Auto-antibodies associated with rheumatoid arthritis, termed rheumatoid factors (RF) react with antigenic determinants on IgG heavy chains. RF has predominant but not complete IgM specificity. This auto-antibody response was not detected in treated patients with primary brain tumors (where tissue is sequestered from the immune system by an intact bloodbrain barrier) or with multiple myeloma where humoral immunity is usually impaired. In addition, the prevalence of RF is not increased with solid tumors prior to initiation of chemotherapy or radiotherapy. It is proposed that RF is related to prior chemotherapy or radiotherapy of tumors anatomically accessible to immunologic tissues capable of antibody responses. A primary IgG response occurs, antigen-antibody complexes form, complexed IgG becomes immunologic, and an RF response results.

Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.

PubMed

van Vuuren, B Jansen; Bergseth, G; Mollnes, T E; Shaw, A M

2014-01-15

Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env

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

Do Kwon, Young; Pancera, Marie; Acharya, Priyamvada

As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. In this paper, we present the structure of the ligand-free HIV-1–Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer boundmore » by a single CD4 without the typical antigenic hallmarks of CD4 induction. Finally, antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.« less

Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env

DOE PAGES

Do Kwon, Young; Pancera, Marie; Acharya, Priyamvada; ...

2015-06-22

As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. In this paper, we present the structure of the ligand-free HIV-1–Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer boundmore » by a single CD4 without the typical antigenic hallmarks of CD4 induction. Finally, antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.« less

Diagnosis of primary antibody and complement deficiencies in young adults after a first invasive bacterial infection.

PubMed

Sanges, S; Wallet, F; Blondiaux, N; Theis, D; Vérin, I; Vachée, A; Dessein, R; Faure, K; Viget, N; Senneville, E; Leroy, O; Maury, F; Just, N; Poissy, J; Mathieu, D; Prévotat, A; Chenivesse, C; Scherpereel, A; Smith, G; Lopez, B; Rosain, J; Frémeaux-Bacchi, V; Hachulla, E; Hatron, P-Y; Bahuaud, M; Batteux, F; Launay, D; Labalette, M; Lefèvre, G

2017-08-01

Screening for primary immunodeficiencies (PIDs) in adults is recommended after two severe bacterial infections. We aimed to evaluate if screening should be performed after the first invasive infection in young adults. Eligible patients were retrospectively identified using hospital discharge and bacteriology databases in three centres during a 3-year period. Eighteen to 40-year-old patients were included if they had experienced an invasive infection with encapsulated bacteria commonly encountered in PIDs (Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), Neisseria gonorrhoeae (NG), Haemophilus influenzae (HI), or group A Streptococcus (GAS)). They were excluded in case of general or local predisposing factors. Immunological explorations and PIDs diagnoses were retrieved from medical records. Serum complement and IgG/A/M testings were systematically proposed at the time of study to patients with previously incomplete PID screening. The study population comprised 38 patients. Thirty-six had experienced a first invasive episode and a PID was diagnosed in seven (19%): two cases of common variable immunodeficiency revealed by SP bacteraemia, one case of idiopathic primary hypogammaglobulinaemia, and two cases of complement (C6 and C7) deficiency revealed by NM meningitis, one case of IgG2/IgG4 subclasses deficiency revealed by GAS bacteraemia, and one case of specific polysaccharide antibody deficiency revealed by HI meningitis. Two patients had previously experienced an invasive infection before the study period: in both cases, a complement deficiency was diagnosed after a second NM meningitis and a second NG bacteraemia, respectively. PID screening should be considered after a first unexplained invasive encapsulated-bacterial infection in young adults. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

PubMed Central

Khattab, Ayman; Barroso, Marta; Miettinen, Tiera; Meri, Seppo

2015-01-01

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood. PMID:25679788

49 CFR 37.147 - Considerations during FTA review.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Considerations during FTA review. 37.147 Section 37.147 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.147 Considerations...

49 CFR 37.133 - Subscription service.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Subscription service. 37.133 Section 37.133 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.133 Subscription service. (a) This...

49 CFR 37.143 - Paratransit plan implementation.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Paratransit plan implementation. 37.143 Section 37.143 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.143 Paratransit plan...

49 CFR 37.121 - Requirement for comparable complementary paratransit service.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Requirement for comparable complementary paratransit service. 37.121 Section 37.121 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route...

49 CFR 37.129 - Types of service.

Code of Federal Regulations, 2010 CFR

2010-10-01

... DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.129 Types of service. (a) Except as provided in this section, complementary paratransit service for ADA paratransit eligible persons shall be origin-to-destination service. (b) Complementary paratransit service for ADA paratransit eligible persons...

49 CFR 37.133 - Subscription service.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 1 2013-10-01 2013-10-01 false Subscription service. 37.133 Section 37.133 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.133 Subscription service. (a) This...

49 CFR 37.133 - Subscription service.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 1 2011-10-01 2011-10-01 false Subscription service. 37.133 Section 37.133 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.133 Subscription service. (a) This...

Experimental autologous immune deposit nephritis in rats associated with mercuric chloride administration.

PubMed

Kelchner, J; McIntosh, J R; Boedecker, E; Guggenheim, S; McIntosh, R M

1976-09-15

Serial administration of mercuric chloride to rats was followed by development of antibodies to tubular basement membrane and renal tubular epithelial antigen (RTE) and glomerulonephritis characterized by granular deposits of hosts IgG, C3 and RTE along the glomerular capillary walls. The glomerular fixed antibody was directed against RTE. These studies suggest that tubular injury by mercury may lead to release of RTE and autosensitization and subsequent antibody production to this antigen result in formation of and glomerular deposition of circulating immunopathogenic complexes (RTE-anti-RTE) and glomerular morphologic alterations.

Hepatic Subcellular Compartmentation of Cytoplasmic Phosphoenolpyruvate Carboxykinase Determined by Immunogold Electron Microscopy

NASA Astrophysics Data System (ADS)

Gao, Kuixiong; Cardell, Emma Lou; Morris, Randal E.; Giffin, Bruce F.; Cardell, Robert R.

1995-08-01

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 [mu]m) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 [mu]m) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles; hepatocytes in pericentral regions have a diffuse subcellular distribution of PEPCK and thus more scattered gold particles. When normal serum replaced the first antibody in the immunogold staining procedures, the background was very low.

Pathogenetic mechanisms in dengue haemorrhagic fever: Report of an international collaborative study*

PubMed Central

1973-01-01

In a study of 55 persons with dengue haemorrhagic fever—36 of whom showed the dengue shock syndrome—clinical, haematological, virological, and serological changes were correlated with serial measurements of complement components and immunopathological studies. Viruses dengue-1 or dengue-2 were isolated from the sera of 9 patients. Serological responses indicative of secondary dengue virus infections were observed in 53 patients; 2 (infants) had primary infections. During the acute phase of the disease, dengue antibody titres rose logarithmically. Marked depression of complement components, especially C3, was observed. Activation of both the classical and alternative complement pathways was demonstrated, with depression of both C4 and C3 proactivator levels in most instances, although in some cases it appeared that one mechanism was involved to a greater extent than the other. The level of depression of C3 was correlated with the severity of the disease. Relatively stable transferrin levels indicated that depletion of complement proteins was not primarily due to extravasation. Fibrinogen levels were depressed and fibrinogen split products were found in the plasma. The accumulated data provide further evidence of the central role that activated complement components play in the pathogenesis of dengue haemorrhagic fever. PMID:4575523

Interlaboratory ring trial to evaluate CFT proficiency of European laboratories for diagnosis of glanders in equids.

PubMed

Laroucau, K; Colaneri, C; Jaÿ, M; Corde, Y; Drapeau, A; Durand, B; Zientara, S; Beck, C

2016-06-18

To evaluate the routine complement fixation test (CFT) used to detect Burkholderia mallei antibodies in equine sera, an interlaboratory proficiency test was held with 24 European laboratories, including 22 National Reference Laboratories for glanders. The panels sent to participants were composed of sera with or without B mallei antibodies. This study confirmed the reliability of CFT and highlighted its intralaboratory reproducibility. However, the sensitivity of glanders serodiagnosis and laboratory proficiency may be improved by standardising critical reagents, including antigens, and by developing a standard B mallei serum. British Veterinary Association.

Effects of FVIII immunity on hepatocyte and hematopoietic stem cell–directed gene therapy of murine hemophilia A

PubMed Central

Lytle, Allison M; Brown, Harrison C; Paik, Na Yoon; Knight, Kristopher A; Wright, J Fraser; Spencer, H Trent; Doering, Christopher B

2016-01-01

Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV. PMID:26909355

Production, characterization and applications for Toxoplasma gondii-specific polyclonal chicken egg yolk immunoglobulins.

PubMed

Ferreira Júnior, Álvaro; Santiago, Fernanda M; Silva, Murilo V; Ferreira, Flávia B; Macêdo Júnior, Arlindo G; Mota, Caroline M; Faria, Matheus S; Silva Filho, Hercílio H; Silva, Deise A O; Cunha-Júnior, Jair P; Mineo, José R; Mineo, Tiago W P

2012-01-01

Toxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model. In this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg) and purified egg yolk antibodies (IgY) by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers. Due to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii.

Phosphorylcholine Allows for Evasion of Bactericidal Antibody by Haemophilus influenzae

PubMed Central

Clark, Sarah E.; Snow, Julian; Li, Jianjun; Zola, Tracey A.; Weiser, Jeffrey N.

2012-01-01

The human pathogen Haemophilus influenzae has the ability to quickly adapt to different host environments through phase variation of multiple structures on its lipooligosaccharide (LPS), including phosphorylcholine (ChoP). During colonization with H. influenzae, there is a selection for ChoP+ phase variants. In a murine model of nasopharyngeal colonization, this selection is lost in the absence of adaptive immunity. Based on previous data highlighting the importance of natural antibody in limiting H. influenzae colonization, the effect of ChoP expression on antibody binding and its bactericidal activity was investigated. Flow cytometric analysis revealed that ChoP+ phase variants had decreased binding of antibody to LPS epitopes compared to ChoP− phase variants. This difference in antibody binding correlated with increased survival of ChoP+ phase variants in the presence of antibody-dependent, complement-mediated killing. ChoP+ phase variants were also more resistant to trypsin digestion, suggesting a general effect on the physical properties of the outer membrane. Moreover, ChoP-mediated protection against antibody binding correlated with increased resilience of outer membrane integrity. Collectively, these data suggest that ChoP expression provides a selective advantage during colonization through ChoP-mediated effects on the accessibility of bactericidal antibody to the cell surface. PMID:22396641

Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

PubMed

Mahan, Alison E; Jennewein, Madeleine F; Suscovich, Todd; Dionne, Kendall; Tedesco, Jacquelynne; Chung, Amy W; Streeck, Hendrik; Pau, Maria; Schuitemaker, Hanneke; Francis, Don; Fast, Patricia; Laufer, Dagna; Walker, Bruce D; Baden, Lindsey; Barouch, Dan H; Alter, Galit

2016-03-01

Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

49 CFR 37.153 - FTA waiver determination.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false FTA waiver determination. 37.153 Section 37.153 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.153 FTA waiver determination. (a...

49 CFR 37.135 - Submission of paratransit plan.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Submission of paratransit plan. 37.135 Section 37.135 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.135 Submission of...

Transfer of T-cell mediated immunity to Hymenolepis nana from mother mice to their neonates.

PubMed

Asano, K; Okamoto, K

1992-01-15

Administration of lymph node cells from Hymenolepis nana-infected mice into lactating mothers, or directly suckling neonates successfully transferred immunity to the neonates. The capacity of lymph node cells to transfer immunity was completely abrogated by pretreatment with anti-Thy-1.2 monoclonal antibody and complement.

Complement and Antibody-Mediated Enhancement of Erythrocyte Invasion by Plasmodium Falciparum

DTIC Science & Technology

2015-09-01

Long, C. A. (1998) J. Immunol. 161, 1908-1912. 40. Waki , S., Uehara, S., Kanbe, K., Nariuch, H. & Suzuki, M. (1995) Parasite Immunol. 17, 503-508...41. Yoneto, T., Waki , S., Takai, T., Tagawa, Y., Iwakura, Y., Mizuguchi, J., Nariuchi, H. & Yoshimoto, T. (2001) J. Immunol. 166, 6236-6241. 42

Probing the Energetics of Antigen-Antibody Recognition by Titration Microcalorimetry

PubMed

Jelesarov; Leder; Bosshard

1996-06-01

Our understanding of the energetics that govern antigen-antibody recognition lags behind the increasingly rapid accumulation of structural information on antigen-antibody complexes. Thanks to the development of highly sensitive microcalorimeters, the thermodynamic parameters of antigen-antibody interactions can now be measured with precision and using only nanomole quantities of protein. The method of choice is isothermal titration calorimetry, in which a solution of the antibody (or antigen) is titrated with small aliquots of the antigen (or antibody) and the heat change accompanying the formation of the antigen-antibody complex is measured with a sensitivity as high as 0.1 μcal s-1. The free energy of binding (DeltaG), the binding enthalpy (DeltaH), and the binding entropy (DeltaS) are usually obtained from a single experiment, and no spectroscopic or radioactive label must be introduced into the antigen or antibody. The often large and negative change in heat capacity (DeltaCp) accompanying the formation of an antigen-antibody complex is obtained from DeltaH measured at different temperatures. The basic theory and the principle of the measurements are reviewed and illustrated by examples. The thermodynamic parameters relate to the dynamic physical forces that govern the association of the freely moving antigen and antibody into a well-structured and unique complex. This information complements the static picture of the antigen-antibody complex that results from X-ray diffraction analysis. Attempts to correlate dynamic and static aspects are discussed briefly.

Neisseria meningitidis: pathogenesis and immunity.

PubMed

Pizza, Mariagrazia; Rappuoli, Rino

2015-02-01

The recent advances in cellular microbiology, genomics, and immunology has opened new horizons in the understanding of meningococcal pathogenesis and in the definition of new prophylactic intervention. It is now clear that Neissera meningitidis has evolved a number of surface structures to mediate interaction with host cells and a number of mechanisms to subvert the immune system and escape complement-mediated killing. In this review we report the more recent findings on meningococcal adhesion and on the bacteria-complement interaction highlighting the redundancy of these mechanisms. An effective vaccine against meningococcus B, based on multiple antigens with different function, has been recently licensed. The antibodies induced by the 4CMenB vaccine could mediate bacterial killing by activating directly the classical complement pathway or, indirectly, by preventing binding of fH on the bacterial surface and interfering with colonization. Copyright © 2014 The Author. Published by Elsevier Ltd.. All rights reserved.

Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

PubMed

Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y; Cheung, Nai-Kong V

2015-05-22

Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A New Approach for the Treatment of Arthritis in Mice with a Novel Conjugate of an anti-C5aR1 antibody and C5 siRNA

PubMed Central

Mehta, Gaurav; Scheinman, Robert I.; Holers, V. Michael; Banda, Nirmal K.

2015-01-01

Rheumatoid Arthritis (RA) is an inflammatory autoimmune joint disease in which the complement system plays an important role. Of the several components of complement, current evidence points to C5 as the most important inducer of inflammation. Several groups generated antibodies or siRNAs or small molecule inhibitors against C5 and C5aR1 (CD88) which have showed some efficacy in RA in animal models. However, none of these candidate therapeutics has moved from bench to bedside. Here we test in CAIA a new therapeutic strategy using a novel anti-C5ab-C5 siRNA conjugate. We first demonstrate that while C5aR2 or C5L2 (GPR77) plays no role in collagen antibody induced arthritis (CAIA), C5aR1 contributes to pathogenesis. We demonstrate that injection of siRNAs blocking either C5, C5aR1 or the combination decreased clinical disease activity (CDA) in mice with CAIA by 45%, 51% and 58%, respectively. Anti-C5 antibody (BB5.1) has only limited efficacy nonetheless significantly reduced arthritis up to 66%. We then generated a novel anti-C5aR1ab-protamine-C5siRNA conjugate. Here we show for the first time that while unconjugated antibody plus siRNAs reduce arthritis by 19%, our an anti-C5aR1ab - protamine - C5 siRNA conjugate was effective in reducing arthritis by 83% along with a parallel decrease in histopathology, C3 deposition, neutrophils and macrophages in the joints of mice with CAIA. These data suggest that by targeting anti-C5 siRNAs to the receptor for its C5a activation fragment (C5aR1), a striking clinical effect can be realized. PMID:25917104

Molecular characterization of the rhesus rhadinovirus (RRV) ORF4 gene and the RRV complement control protein it encodes.

PubMed

Mark, Linda; Spiller, O Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A; Wong, Scott W; Damania, Blossom; Blom, Anna M; Blackbourn, David J

2007-04-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes▿

PubMed Central

Mark, Linda; Spiller, O. Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A.; Wong, Scott W.; Damania, Blossom; Blom, Anna M.; Blackbourn, David J.

2007-01-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model. PMID:17287274

Complement is activated in progressive multiple sclerosis cortical grey matter lesions.

PubMed

Watkins, Lewis M; Neal, James W; Loveless, Sam; Michailidou, Iliana; Ramaglia, Valeria; Rees, Mark I; Reynolds, Richard; Robertson, Neil P; Morgan, B Paul; Howell, Owain W

2016-06-22

The symptoms of multiple sclerosis (MS) are caused by damage to myelin and nerve cells in the brain and spinal cord. Inflammation is tightly linked with neurodegeneration, and it is the accumulation of neurodegeneration that underlies increasing neurological disability in progressive MS. Determining pathological mechanisms at play in MS grey matter is therefore a key to our understanding of disease progression. We analysed complement expression and activation by immunocytochemistry and in situ hybridisation in frozen or formalin-fixed paraffin-embedded post-mortem tissue blocks from 22 progressive MS cases and made comparisons to inflammatory central nervous system disease and non-neurological disease controls. Expression of the transcript for C1qA was noted in neurons and the activation fragment and opsonin C3b-labelled neurons and glia in the MS cortical and deep grey matter. The density of immunostained cells positive for the classical complement pathway protein C1q and the alternative complement pathway activation fragment Bb was significantly increased in cortical grey matter lesions in comparison to control grey matter. The number of cells immunostained for the membrane attack complex was elevated in cortical lesions, indicating complement activation to completion. The numbers of classical (C1-inhibitor) and alternative (factor H) pathway regulator-positive cells were unchanged between MS and controls, whilst complement anaphylatoxin receptor-bearing microglia in the MS cortex were found closely apposed to cortical neurons. Complement immunopositive neurons displayed an altered nuclear morphology, indicative of cell stress/damage, supporting our finding of significant neurodegeneration in cortical grey matter lesions. Complement is activated in the MS cortical grey matter lesions in areas of elevated numbers of complement receptor-positive microglia and suggests that complement over-activation may contribute to the worsening pathology that underlies the irreversible progression of MS.

49 CFR 37.137 - Paratransit plan development.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Paratransit plan development. 37.137 Section 37... WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.137 Paratransit plan... the plan to identify any person or entity (public or private) which provides a paratransit or other...

49 CFR 37.139 - Plan contents.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 1 2013-10-01 2013-10-01 false Plan contents. 37.139 Section 37.139 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.139 Plan contents. Each plan shall contain the following information: (a)...

49 CFR 37.139 - Plan contents.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 1 2014-10-01 2014-10-01 false Plan contents. 37.139 Section 37.139 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.139 Plan contents. Each plan shall contain the following information: (a)...

49 CFR 37.139 - Plan contents.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 1 2012-10-01 2012-10-01 false Plan contents. 37.139 Section 37.139 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.139 Plan contents. Each plan shall contain the following information: (a)...

49 CFR 37.139 - Plan contents.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Plan contents. 37.139 Section 37.139 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.139 Plan contents. Each plan shall contain the following information: (a)...

49 CFR 37.139 - Plan contents.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 1 2011-10-01 2011-10-01 false Plan contents. 37.139 Section 37.139 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.139 Plan contents. Each plan shall contain the following information: (a)...

49 CFR 37.145 - State comment on plans.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false State comment on plans. 37.145 Section 37.145 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.145 State comment on plans. Each...

49 CFR 37.125 - ADA paratransit eligibility: Process.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false ADA paratransit eligibility: Process. 37.125... INDIVIDUALS WITH DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.125 ADA... § 37.121 of this part shall establish a process for determining ADA paratransit eligibility. (a) The...

49 CFR 37.149 - Disapproved plans.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Disapproved plans. 37.149 Section 37.149... DISABILITIES (ADA) Paratransit as a Complement to Fixed Route Service § 37.149 Disapproved plans. (a) If a plan... shall amend its plan consistent with this information and resubmit the plan to the appropriate FTA...

Blockade of the antigen-antibody reaction using benzil condensation with the guanidyl residue of arginine.

PubMed

Montero, C; Segura, D I; Gutierrez, M

1991-03-01

Benzil blockade of the guanidyl group of arginine was tried on sections of paraffin-embedded tissue fixed in two different fixatives, in an attempt to evaluate the relevance of this amino acid to the reaction of several proteins with their corresponding antibodies. The two fixatives were 10% formaldehyde, and Bouin's fluid without acetic acid. Both polyclonal and monoclonal antibodies against proteins or peptides (lysozyme, adrenocorticotropic hormone, growth hormone, placental lactogen, and prolactin) were used on human biopsies or material from autopsies. The blockade was effective when monoclonal antibodies were used, whereas no effect or only a small decrease of the intensity of the reaction was observed with polyclonal antibodies. The least definitive result was obtained with prolactin, where a complete blockade was never achieved with monoclonal antibodies. Calcitonin, a peptide that does not contain arginine, was used as a control not susceptible to benzil blockade; no blockade of immunostaining was observed.

A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer

PubMed Central

Elvington, Michelle; Huang, Yuxiang; Morgan, B. Paul; Qiao, Fei; van Rooijen, Nico; Atkinson, Carl

2012-01-01

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer. PMID:22442351

Comparison and Correlation of Neisseria meningitidis Serogroup B Immunologic Assay Results and Human Antibody Responses following Three Doses of the Norwegian Meningococcal Outer Membrane Vesicle Vaccine MenBvac

PubMed Central

Findlow, Jamie; Taylor, Stephen; Aase, Audun; Horton, Rachel; Heyderman, Robert; Southern, Jo; Andrews, Nick; Barchha, Rita; Harrison, Ewan; Lowe, Ann; Boxer, Emma; Heaton, Charlotte; Balmer, Paul; Kaczmarski, Ed; Oster, Philipp; Gorringe, Andrew; Borrow, Ray; Miller, Elizabeth

2006-01-01

The prediction of efficacy of Neisseria meningitidis serogroup B (MenB) vaccines is currently hindered due to the lack of an appropriate correlate of protection. For outer membrane vesicle (OMV) vaccines, immunogenicity has primarily been determined by the serum bactericidal antibody (SBA) assay and OMV enzyme-linked immunosorbent assay (ELISA). However, the opsonophagocytic assay (OPA), surface labeling assay, whole blood assay (WBA), and salivary antibody ELISA have been developed although correlation with protection is presently undetermined. Therefore, the aim of the study was to investigate further the usefulness of, and relationships between, MenB immunologic assays. A phase II trial of the OMV vaccine, MenBvac, with proven efficacy was initiated to compare immunologic assays incorporating the vaccine and six heterologous strains. Correlations were achieved between the SBA assay, OMV ELISA, and OPA using human polymorphonuclear leukocytes and human complement but not between an OPA using HL60 phagocytic cells and baby rabbit complement. Correlations between the surface labeling assay, the SBA assay, and the OMV ELISA were promising, although target strain dependent. Correlations between the salivary antibody ELISA and other assays were poor. Correlations to the WBA were prevented since many samples had results greater than the range of the assay. The study confirmed the immunogenicity and benefit of a third dose of MenBvac against the homologous vaccine strain using a variety of immunologic assays. These results emphasize the need for standardized methodologies that would allow a more robust comparison of assays between laboratories and promote their further evaluation as correlates of protection against MenB disease. PMID:16861642

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

Use of eculizumab in crescentic IgA nephropathy: proof of principle and conundrum?

PubMed Central

Ring, Troels; Pedersen, Birgitte Bang; Salkus, Giedrius; Goodship, Timothy H.J.

2015-01-01

IgA nephropathy (IgAN) is characterized by a variable clinical course and multifaceted pathophysiology. There is substantial evidence to suggest that complement activation plays a pivotal role in the pathogenesis of the disease. Therefore, complement inhibition using the humanized anti-C5 monoclonal antibody eculizumab could be a rational treatment. We report here a 16-year-old male with the vasculitic form of IgAN who failed to respond to aggressive conventional therapy including high-dose steroids, cyclophosphamide and plasma exchange and who was treated with four weekly doses of 900 mg eculizumab followed by a single dose of 1200 mg. He responded rapidly to this treatment and has had a stable creatinine around 150 µmol/L (1.67 mg/dL) for >6 months. However, proteinuria was unabated on maximal conventional anti-proteinuric treatment, and a repeat renal biopsy 11 months after presentation revealed severe chronic changes. We believe this case provides proof of principle that complement inhibition may be beneficial in IgAN but also that development of chronicity may be independent of complement. PMID:26413271

Complement, lymphocytotoxins and immune complexes in infectious mononucleosis: serial studies in uncomplicated cases

PubMed Central

Charlesworth, J. A.; Quin, J. W.; Macdonald, G. J.; Lennane, R. J.; Boughton, C. R.

1978-01-01

Serial studies of complement, immunoglobulins, lymphocytotoxins and immune complexes were performed in thirteen patients with uncomplicated infectious mononucleosis (IM). Two methods were used to detect immune complexes: a C1q-binding assay (C1q-BA) and the Raji-cell radioimmunoassay (RIA). Patients were followed until there was complete serological recovery. Individual complement components were normal or elevated but three patients showed initial reduction in total haemolytic activity. IgG, IgM, and IgA rose moderately during the acute phase. All sera showed thymocyte-specific cytotoxic activity at some time during the acute phase but were negative by 6 months. The C1q-BA was positive initially in twelve patients but had returned to normal by 6 months. The standard Raji RIA was negative in fifty out of fifty-five samples tested and it is proposed that this reflects the predominant IgM antibody response in these patients. In contrast, incorporation of a multispecific anti-immunoglobulin into this assay yielded data that was frequently positive; these correlated highly with that of the C1q-BA (P<0·001). Lymphocytotoxic activity correlated with the C1q-BA (P<0·001) and the modified Raji RIA (P<0·05). Patterns of lymphocytotoxicity and immune complex reactivity suggested an inverse relationship between these two parameters. It is proposed that this lymphocytotoxicity leads to production of antibody of restricted class permitting enhanced clearance of immune complexes. PMID:737909

Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies.

PubMed

Sierralta, W D; Thole, H H

1996-05-01

The unmasking of estradiol receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the receptor's domain E, showed a preference for the cytoplasmic receptor. In stroma, all antibodies detected more receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the receptor differ according to the functional uterine cell type.

High-resolution definition of humoral immune response correlates of effective immunity against HIV.

PubMed

Alter, Galit; Dowell, Karen G; Brown, Eric P; Suscovich, Todd J; Mikhailova, Anastassia; Mahan, Alison E; Walker, Bruce D; Nimmerjahn, Falk; Bailey-Kellogg, Chris; Ackerman, Margaret E

2018-03-26

Defining correlates of immunity by comprehensively interrogating the extensive biological diversity in naturally or experimentally protected subjects may provide insights critical for guiding the development of effective vaccines and antibody-based therapies. We report advances in a humoral immunoprofiling approach and its application to elucidate hallmarks of effective HIV-1 viral control. Systematic serological analysis for a cohort of HIV-infected subjects with varying viral control was conducted using both a high-resolution, high-throughput biophysical antibody profiling approach, providing unbiased dissection of the humoral response, along with functional antibody assays, characterizing antibody-directed effector functions such as complement fixation and phagocytosis that are central to protective immunity. Profiles of subjects with varying viral control were computationally analyzed and modeled in order to deconvolute relationships among IgG Fab properties, Fc characteristics, and effector functions and to identify humoral correlates of potent antiviral antibody-directed effector activity and effective viral suppression. The resulting models reveal multifaceted and coordinated contributions of polyclonal antibodies to diverse antiviral responses, and suggest key biophysical features predictive of viral control. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from a dual-host phage display vector.

PubMed

Shang, Yonglei; Tesar, Devin; Hötzel, Isidro

2015-10-01

A recently described dual-host phage display vector that allows expression of immunoglobulin G (IgG) in mammalian cells bypasses the need for subcloning of phage display clone inserts to mammalian vectors for IgG expression in large antibody discovery and optimization campaigns. However, antibody discovery and optimization campaigns usually need different antibody formats for screening, requiring reformatting of the clones in the dual-host phage display vector to an alternative vector. We developed a modular protein expression system mediated by RNA trans-splicing to enable the expression of different antibody formats from the same phage display vector. The heavy-chain region encoded by the phage display vector is directly and precisely fused to different downstream heavy-chain sequences encoded by complementing plasmids simply by joining exons in different pre-mRNAs by trans-splicing. The modular expression system can be used to efficiently express structurally correct IgG and Fab fragments or other antibody formats from the same phage display clone in mammalian cells without clone reformatting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A teleost CD46 is involved in the regulation of complement activation and pathogen infection.

PubMed

Li, Mo-Fei; Sui, Zhi-Hai; Sun, Li

2017-11-03

In mammals, CD46 is involved in the inactivation of complement by factor I (FI). In teleost, study on the function of CD46 is very limited. In this study, we examined the immunological property of a CD46 molecule (CsCD46) from tongue sole, a teleost species with important economic value. We found that recombinant CsCD46 (rCsCD46) interacted with FI and inhibited complement activation in an FI-dependent manner. rCsCD46 also interacted with bacterial pathogens via a different mechanism to that responsible for the FI interaction, involving different rCsCD46 sites. Cellular study showed that CsCD46 was expressed on peripheral blood leukocytes (PBL) and protected the cells against the killing effect of complement. When the CsCD46 on PBL was blocked by antibody before incubation of the cells with bacterial pathogens, cellular infection was significantly reduced. Consistently, when tongue sole were infected with bacterial pathogens in the presence of rCsCD46, tissue dissemination and survival of the pathogens were significantly inhibited. These results provide the first evidence to indicate that CD46 in teleosts negatively regulates complement activation via FI and protects host cells from complement-induced damage, and that CD46 is required for optimal bacterial infection probably by serving as a receptor for the bacteria.

Preservation of renal function in atypical hemolytic uremic syndrome by eculizumab: a case report.

PubMed

Giordano, Mario; Castellano, Giuseppe; Messina, Giovanni; Divella, Claretta; Bellantuono, Rosa; Puteo, Flora; Colella, Vincenzo; Depalo, Tommaso; Gesualdo, Loreto

2012-11-01

Genetic mutations in complement components are associated with the development of atypical hemolytic uremic syndrome (aHUS), a rare disease with high morbidity rate triggered by infections or unidentified factors. The uncontrolled activation of the alternative pathway of complement results in systemic endothelial damage leading to progressive development of renal failure. A previously healthy 8-month-old boy was referred to our hospital because of onset of fever, vomiting, and a single episode of nonbloody diarrhea. Acute kidney injury with preserved diuresis, hemolytic anemia, and thrombocytopenia were detected, and common protocols for management of HUS were followed without considerable improvement. The persistent low levels of complement component C3 led us to hypothesize the occurrence of aHUS. In fact, the child carried a specific mutation in complement factor H (Cfh; nonsense mutation in 3514G>T, serum levels of Cfh 138 mg/L, normal range 350-750). Given the lack of response to therapy and the occurrence of kidney failure requiring dialysis, we used eculizumab as rescue therapy, a monoclonal humanized antibody against the complement component C5. One week from the first administration, we observed a significant improvement of all clinical and laboratory parameters with complete recovery from hemodialysis, even in the presence of systemic infections. Our case report shows that complement inhibiting treatment allows the preservation of renal function and avoids disease relapses during systemic infections.

Autoantibodies and their antigens in autoimmune hepatitis.

PubMed

Bogdanos, Dimitrios P; Mieli-Vergani, Giorgina; Vergani, Diego

2009-08-01

Autoantibody detection assists in the diagnosis and allows differentiation of autoimmune hepatitis (AIH) type 1 (AIH-1), characterized by antinuclear antibody (ANA) and/or smooth muscle antibody (SMA), and type 2 (AIH-2), distinguished by the presence of antibodies to liver-kidney microsome type 1 (anti-LKM1) and/or antibodies to liver cytosol type 1 (anti-LC1). Detection of atypical perinuclear antineutrophil cytoplasmic antibodies (pANCA) and anti-soluble liver antigen (SLA) antibodies can act as an additional pointer toward the diagnosis of AIH, particularly in the absence of the conventional autoantibodies. Routine autoantibody testing by indirect immunofluorescence has been recently complemented by molecular assays based on purified or recombinant antigens. Although the AIH-1-specific ANA and SMA targets need better definition, those of anti-LKM1 and anti-LC1 in AIH-2 have been clearly identified; the fine specificity of antibody reactivity and its clinical relevance to disease pathogenesis are the focus of ongoing investigation. This article critically discusses the current knowledge of the diagnostic and clinical significance of AIH-related autoantibody reactivities, focusing on key issues that the physician needs to be aware of to be able to request the appropriate testing and to interpret correctly the laboratory results within the clinical context of the patient. Copyright Thieme Medical Publishers.

Surrogate target cells expressing surface anti-idiotype antibody for the clinical evaluation of an internalizing CD22-specific antibody.

PubMed

Leung, Shui-On; Gao, Kai; Wang, Guang Yu; Cheung, Benny Ka-Wa; Lee, Kwan-Yeung; Zhao, Qi; Cheung, Wing-Tai; Wang, Jun Zhi

2015-01-01

SM03, a chimeric antibody that targets the B-cell restricted antigen CD22, is currently being clinically evaluated for the treatment of lymphomas and other autoimmune diseases in China. SM03 binding to surface CD22 leads to rapid internalization, making the development of an appropriate cell-based bioassay for monitoring changes in SM03 bioactivities during production, purification, storage, and clinical trials difficult. We report herein the development of an anti-idiotype antibody against SM03. Apart from its being used as a surrogate antigen for monitoring SM03 binding affinities, the anti-idiotype antibody was engineered to express as fusion proteins on cell surfaces in a non-internalizing manner, and the engineered cells were used as novel "surrogate target cells" for SM03. SM03-induced complement-mediated cytotoxicity (CMC) against these "surrogate target cells" proved to be an effective bioassay for monitoring changes in Fc functions, including those resulting from minor structural modifications borne within the Fc-appended carbohydrates. The approach can be generally applied for antibodies that target rapidly internalizing or non-surface bound antigens. The combined use of the anti-idiotype antibody and the surrogate target cells could help evaluate clinical parameters associated with safety and efficacies, and possibly the mechanisms of action of SM03.

Propionibacterium acnes-derived insoluble immune complexes in sinus macrophages of lymph nodes affected by sarcoidosis.

PubMed

Suzuki, Yoshimi; Uchida, Keisuke; Takemura, Tamiko; Sekine, Masaki; Tamura, Tomoki; Furukawa, Asuka; Hebisawa, Akira; Sakakibara, Yumi; Awano, Nobuyasu; Amano, Tomonari; Kobayashi, Daisuke; Negi, Mariko; Kakegawa, Tomoya; Wada, Yuriko; Ito, Takashi; Suzuki, Takashige; Akashi, Takumi; Eishi, Yoshinobu

2018-01-01

Propionibacterium acnes is thought to be a causative agent of sarcoidosis. Patients with sarcoidosis have circulating immune complexes. We attempted to detect P. acnes-derived immune complexes in sarcoid lesions. We evaluated formalin-fixed and paraffin-embedded lymph node samples from 38 sarcoidosis patients and 90 non-sarcoidosis patients (27 patients with necrotizing lymphadenitis, 28 patients with reactive lymphadenitis, 16 patients with colon cancer, 19 patients with gastric cancer) by immunohistochemistry using anti-human immunoglobulins (IgG, IgA, and IgM) and complement (C1q and C3c) antibodies, and a P. acnes-specific monoclonal antibody (PAB antibody) that reacts with the membrane-bound lipoteichoic acid of P. acnes. Small round bodies (SRBs) bound to IgA, IgM, or IgG were detected in sinus macrophages, in 32 (84%), 32 (84%), or 11 (29%) sarcoid samples, respectively, and in 19 (21%), 26 (29%), or no (0%) control samples, respectively. Some of these insoluble immune complexes (IICs) also bound to C1q and C3c. We developed a microwave treatment followed by brief trypsin digestion (MT treatment) to detect PAB-reactive SRBs bound to immunoglobulins (IIC-forming P. acnes). MT treatment revealed abundant IIC-forming P. acnes in most (89%) of the sarcoid samples and sparse distribution in some (20%) of the control samples with lymphadenitis, but no IIC-forming P. acnes was detected in control samples without inflammation. IIC-forming P. acnes were mostly bound to both IgA and IgM. The PAB-reactive antigen and immunoglobulins were both located at the peripheral rim of the IIC-forming P. acnes. Conventional electron microscopy identified many SRBs (0.5-2.0 μm diameter) in sinus macrophages of sarcoid lymph nodes with many IIC-forming P. acnes, some of which were in phagolysosomes with a degraded and lamellar appearance. P. acnes-derived IICs in sinus macrophages were frequent and abundant in sarcoid lymph nodes, suggesting a potential etiologic link between sarcoidosis and this commensal bacterium.

Extended Antiphospholipid Antibodies Screening in Systemic Lupus Erythematosus Patients.

PubMed

Dima, Alina; Caraiola, Simona; Jurcut, C; Balanescu, Eugenia; Balanescu, P; Ramba, Doina; Badea, Camelia; Pompilian, V; Ionescu, R; Baicus, Anda; Baicus, C; Dan, G A

2015-01-01

The antiphospholipid syndrome (APS) is one of the most encountered autoimmunity in systemic lupus erythematosus (SLE) patients and pathogenesis of these two seems to be intricate. To investigate the association of antiphospholipid antibodies (APLAs) titer with the presence of secondary APS diagnosis in SLE patients. 65 patients fulfilling the 2012 Systemic Lupus Collaborating International Clinics (SLICC) SLE's criteria were included. The APS diagnosis was sustained according to the 2006 Sydney APS's criteria. Three groups of patients were defined: SLE patients with secondary APS, SLE with history of positive "criteria" APLAs but without APS clinical features, respectively SLE patients without positive APLAs or clinical APS criteria. An extended APLAs panel was searched in all cases: both IgM and IgG of anticardiolipin antibodies (aCL), anti-P2 glycoprotein I antibodies (aβ2GPI), antiphosphatidylethanolamine antibodies (aPE), antiphosphatidylserine antibodies (aPS), respectively antiprothrombin antibodies (aPT). Results. Only the aβ2GPI, both IgM and IgG serotypes, had significantly higher titers in patients with SLE and secondary APS compared to no APS (with/ without positive APLAs): median (min; max) 7.0 (0.0-300.0) vs. 1.0 (0.0-28.0) vs. 1.0 (0.0-12.0), respectively 3.0 (0.0-79.0) vs. 1.0 (0.0-3.0) vs. 1.0 (0.0-12.0) (p<0.001, Kruskal-Wallis test)]. Also, in regression logistic models, only the aβ2 GPI (IgG and IgM ) were identified as risk factors for secondary APS diagnosis in the SLE patients: OR(95%CI) 5.9 (2.2-15.7), respectively 1.3 (1.1-1.5). In regard with the SLE markers, the IgG serotypes of the "non-criteria" APLAs analyzed (aPS, aPT, aPE) were correlated with the antiDNA titers while the IgM serotypes inversely associated with the complement C3 levels. IgG aβ2 GPI are accompanied by almost 6-fold increase risk of secondary APS when screening SLE patients. On the contrary, the "non-criteria" APLAs do not seem associated with the APS diagnosis in SLE patients. Some correlates of the "non-criteria" APLAs with the antiDNA and complement C3 levels were also observed.

[Immunologic and transplantation biology studies in patients with terminal renal failure].

PubMed

Lanzer, G

1985-01-01

Best possible tissue compatibility is a fundamental precondition for a successful organ transplantation. The desirable tissue compatibility is mainly defined by transplantation-antigens of the donor and the recipient and therefore--beside ABO antigens--the characteristics of HLA-system are the focal point of the preoperative immunological diagnosis. Based on the results of comperative examinations it is demonstrated, that HLA-antisera have such a widespread biological variability, that they lead sometimes to a faulty diagnosis which in turn causes the biological importance of the HLA-system to be doubted. The preoperative immunologic diagnosis should include an estimation of the risk factors in the patient. The consideration of the crossmatch between donor and recipient is a decisive factor in organ transplantation. The starting point is the preoperative antibody monitoring which checks the patients serum reactions against a panel of blood donors to see, whether the patient is a "high" or "low"-- responder to allogenetic stimuli. A positive reaction in the crossmatch is brought about by different kinds of antibodies whereby only in the presence of auto-antibody or cold-reactive B-cell antibody a transplantation may take place. The antibody characterisation in preoperative diagnosis is supported by the results of the immunologic antibody monitoring, whereby--because of the results here presented-- it can be confirmed, that through the knowledge of the antibody specifities which have been checked in a positive crossmatch transplantation on highly sensitized patients can take place with a prospect of success. Supplementing the "Eurotransplant" results the HLA-DRw6 antigen is shown not only to be an indication of risk in transplantation, but may also be an aetiopathogenetic factor. In the presentated statistics it can be shown, that in patients suffering from glomerulonephritis or pyelonephritis and requiring dialysis treatment the HLA - DRw6 antigen occur more frequently than in the control group of healthy blood donors. In glomerulonephritis patients there is additionally a significant change in distribution favourable to HLA - DRw10 shown. The determination of genetically caused risk factors is appreciably supported by characterisation of lymphocyte subpopulations and diagnosis of changes in the Complement-system. Changes in T-lymphocyte subpopulations pointing to proceedings of immunostimulation and conditions of the activated Complement-system represent warning signals in organ transplantation.

Characterization of a monoclonal antibody against P57, the C3/C3b-cleaving proteinase expressed in human erythrocyte membranes.

PubMed

Fiandino-Tirel, A; Barel, M; Lyamani, F; Gauffre, A; Hermann, J; Frade, R

1991-08-01

A monoclonal antibody was raised against p57, a serine proteinase, characterized by an apparent molecular weight of 57 kDa, and purified from human erythrocyte membranes. P57 proteinase cleaves the human third component of complement, C3. The antibody selected, MP1, of IgG2a isotype, precipitated specifically the p57 antigen which carried the C3/C3b-cleaving activity present in membrane crude extract of human erythrocytes. P57 proteinase eluted from MP1-sepharose was inhibited by 5 x 10(-4) M PMSF, enhanced by 0.5% SDS and generated C3 fragments identical to those generated by membrane crude extract of human erythrocytes. All these properties were identical to those of the p57 previously purified by biochemical procedures. In addition, 5000 binding sites were detected on cell surface. This MP1 monoclonal antibody will be helpful to analyse the role of p57 in human erythrocytes.

Chemical Synthesis of GM2 Glycans, Bioconjugation with Bacteriophage Qβ, and the Induction of Anticancer Antibodies.

PubMed

Yin, Zhaojun; Dulaney, Steven; McKay, Craig S; Baniel, Claire; Kaczanowska, Katarzyna; Ramadan, Sherif; Finn, M G; Huang, Xuefei

2016-01-01

The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qβ. Although the copper-catalyzed azide-alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of an enzyme-linked-immunosorbent-assay technique for accurate identification of poorly preserved silks unearthed in ancient tombs.

PubMed

Zheng, Qin; Wu, Xiaofeng; Zheng, Hailing; Zhou, Yang

2015-05-01

We report the preparation of a specific fibroin antibody and its use for the identification of unearthed ancient silk relics. Based on the 12-amino-acid repeat sequence "GAGAGSGAGAGS", which is found in fibroin of the silkworm Bombyx mori, a specific antibody against fibroin was prepared in rabbits through peptide synthesis and carrier-protein coupling. This antibody was highly specific for fibroin found in silk. Using this antibody we have successfully identified four silk samples from different time periods. Our results reveal, for the first time, a method capable of detecting silk from a few milligrams of archaeological fabric that has been buried for thousands of years, confirming that the ancient practice of wearing silk products while praying for rebirth dated back to at least 400 BCE. This method also complements current approaches in silk detection, especially for the characterization of poorly preserved silks, promoting the investigation of silk origins and of ancient clothing cultures.

Application of histone modification-specific interaction domains as an alternative to antibodies.

PubMed

Kungulovski, Goran; Kycia, Ina; Tamas, Raluca; Jurkowska, Renata Z; Kudithipudi, Srikanth; Henry, Chisato; Reinhardt, Richard; Labhart, Paul; Jeltsch, Albert

2014-11-01

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies. © 2014 Kungulovski et al.; Published by Cold Spring Harbor Laboratory Press.

[Monoclonal antibodies in diagnosis of acute leukemias].

PubMed

Krawczyńska, A; Robak, T

1996-01-01

Immunophenotyping has become an essential component for the study of acute myeloblastic (AML) and lymphoblastic (ALL) leukaemias. The recent development of highly specific monoclonal antibodies (Mc Ab) to differentiation antigens (CD) of haematopoetic cells have made it readily available to clinical laboratories in most major hospitals. Immunophenotyping complements standard morphology by providing information on lineage, stage of differentiation and clonality. In addition some of the flow cytometry findings have independent prognostic significance. Monoclonal antibodies useful in defining lineage (B-cell versus T-cell) and stages of differentiation of ALL. It can be also used in identifying characteristic feature of AML and aiding in lineage determination in acute leukaemias that are morphologically undifferentiated. Surface immunophenotyping is especially helpful for recognizing mixed lineage acute leukaemia and diagnosing certain rare entities such as erythroleukaemia (M6), acute megakaryocytic leukaemia (M7) and minimally differentiation acute myeloid leukaemia.

Constitutive production of catalytic antibodies to a Staphylococcus aureus virulence factor and effect of infection.

PubMed

Brown, Eric L; Nishiyama, Yasuhiro; Dunkle, Jesse W; Aggarwal, Shreya; Planque, Stephanie; Watanabe, Kenji; Csencsits-Smith, Keri; Bowden, M Gabriela; Kaplan, Sheldon L; Paul, Sudhir

2012-03-23

Antibodies that recognize microbial B lymphocyte superantigenic epitopes are produced constitutively with no requirement for adaptive immune maturation. We report cleavage of the Staphylococcus aureus virulence factor extracellular fibrinogen-binding protein (Efb) by catalytic antibodies produced with no exposure to the bacterium and reduction of the catalytic antibody activity following infection. IgG catalytic antibodies that specifically hydrolyzed Efb via a nucleophilic catalytic mechanism were found in the blood of healthy humans and aseptic mice free of S. aureus infection. IgG hydrolyzed peptide bonds on the C-terminal side of basic amino acids, including a bond located within the C3b-binding domain of Efb. Efb digested with the IgG lost its ability to bind C3b and inhibit complement-dependent antibody-mediated red blood cell lysis. In addition to catalysis, the IgG expressed saturable Efb binding activity. IgG from S. aureus-infected mice displayed reduced Efb cleaving activity and increased Efb binding activity compared with uninfected controls, suggesting differing effects of the infection on the antibody subsets responsible for the two activities. IgG from children hospitalized for S. aureus infection also displayed reduced Efb cleavage compared with healthy children. These data suggest a potential defense function for constitutively produced catalytic antibodies to a putative superantigenic site of Efb, but an adaptive catalytic response appears to be proscribed.

[Pathomechanism of Autoantibody Production in the Nervous System Diseases].

PubMed

Shimizu, Fumitaka; Kanda, Takashi

2018-04-01

Antibodies to different brain and peripheral nerve proteins have recently been found to be associated with several different autoimmune diseases. They can bind to either neuronal or non-neuronal antigens and may have a pathogenic role by themselves or in synergy with other inflammatory mediators after penetrating the blood-brain barrier or the blood-nerve barrier. In this review, we will describe the association with the impairment of immune tolerance, innate immunity, and autoantibody production of myasthenia gravis (MG), systemic lupus erythematosus (SLE), and Guillain-Barré syndrome (GBS). Impairment of central tolerance, which is characterized by the repertoire selection of immature T-lymphocytes in the thymus, is seen in patients with MG who are positive for anti-Ach R antibodies. Impairment of peripheral tolerance due to activation of autoreactive T-cells and suppression of regulatory T-cells is seen in SLE. In addition, molecular mimicry between the lipooligosaccharides of Campylobacter jejuni and gangliosides of the peripheral nerves results in the production of anti-gangliosides antibodies in GBS. Next, we will describe the antibody-mediated pathology in neuromyelitis optica and anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. The binding of anti-aquaporin-4 antibodies or anti-NMDAR antibodies to their respective targets initiates target internalization and complement- or antibody-dependent cellular cytotoxicity of the target cells. Further understanding of antibody-mediated pathology may suggest novel therapeutic strategies.

Production, Characterization and Applications for Toxoplasma gondii-Specific Polyclonal Chicken Egg Yolk Immunoglobulins

PubMed Central

Ferreira Júnior, Álvaro; Santiago, Fernanda M.; Silva, Murilo V.; Ferreira, Flávia B.; Macêdo Júnior, Arlindo G.; Mota, Caroline M.; Faria, Matheus S.; Filho, Hercílio H. Silva; Silva, Deise A. O.; Cunha-Júnior, Jair P.; Mineo, José R.; Mineo, Tiago W. P.

2012-01-01

Background Toxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model. Methodology/Principal Findings In this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg) and purified egg yolk antibodies (IgY) by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers. Conclusions/Significance Due to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii. PMID:22808150

Comparative Assessment of Clinically Utilized CD20-directed Antibodies in Chronic Lymphocytic Leukemia Cells Reveals Divergent NK cell, Monocyte and Macrophage Properties

PubMed Central

Rafiq, Sarwish; Butchar, Jonathon P.; Cheney, Carolyn; Mo, Xiaokui; Trotta, Rossana; Caligiuri, Michael; Jarjoura, David; Tridandapani, Susheela; Muthusamy, Natarajan; Byrd, John C.

2013-01-01

CD20 is a widely validated, B cell specific target for therapy in B cell malignancies. Rituximab is an anti-CD20 antibody that when combined with chemotherapy prolongs survival of CLL patients. Ofatumumab and GA101 (obinutuzumab) are CD20-directed antibodies now being developed as alternative agents to rituximab in CLL based upon different properties of enhanced direct cell death (DCD), NK cell-mediated antibody dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). Despite wide spread study, ofatumumab and GA101 have not been directly compared to one another, nor studied for interaction with monocytes and macrophages that are critical to CD20-mediated antibody efficacy in murine models. In CLL cells, we show that DCD is greatest with GA101 and CDC with ofatumumab. GA101 promotes enhanced NK cell activation and ADCC at high antibody concentrations. Ofatumumab has superior antibody dependent cellular phagocytosis (ADCP) with monocyte derived macrophages (MDM). GA101 demonstrated reduced activation of monocytes with diminished pERK, TNF-α release, and FcγRIIa recruitment to lipid rafts. These data demonstrate GA101 and ofatumumab are superior to rituximab against CLL cells via different mechanisms of potential tumor elimination. These findings bear relevance to potential combination strategies with each of these anti-CD20 antibodies in the treatment of CLL. PMID:23418626

Validation of 31 of the most commonly used immunohistochemical antibodies in cytology prepared using the Cellient(®) automated cell block system.

PubMed

Montgomery, Eric; Gao, Chen; de Luca, Julie; Bower, Jessie; Attwood, Kristropher; Ylagan, Lourdes

2014-12-01

The Cellient(®) cell block system has become available as an alternative, partially automated method to create cell blocks in cytology. We sought to show a validation method for immunohistochemical (IHC) staining on the Cellient cell block system (CCB) in comparison with the formalin fixed paraffin embedded traditional cell block (TCB). Immunohistochemical staining was performed using 31 antibodies on 38 patient samples for a total of 326 slides. Split samples were processed using both methods by following the Cellient(®) manufacturer's recommendations for the Cellient cell block (CCB) and the Histogel method for preparing the traditional cell block (TCB). Interpretation was performed by three pathologists and two cytotechnologists. Immunohistochemical stains were scored as: 0/1+ (negative) and 2/3+ (positive). Inter-rater agreement for each antibody was evaluated for CCB and TCB, as well as the intra-rater agreement between TCB and CCB between observers. Interobserver staining concordance for the TCB was obtained with statistical significance (P < 0.05) in 24 of 31 antibodies. Interobserver staining concordance for the CCB was obtained with statistical significance in 27 of 31 antibodies. Intra-observer staining concordance between TCB and CCB was obtained with statistical significance in 24 of 31 antibodies tested. In conclusions, immunohistochemical stains on cytologic specimens processed by the Cellient system are reliable and concordant with stains performed on the same split samples processed via a formalin fixed-paraffin embedded (FFPE) block. The Cellient system is a welcome adjunct to cytology work-flow by producing cell block material of sufficient quality to allow the use of routine IHC. © 2014 Wiley Periodicals, Inc.

Innate Immune Mechanisms in Transplant Allograft Vasculopathy

PubMed Central

Jane-wit, D; Fang, C; Goldstein, DR

2016-01-01

Purpose of Review Allograft vasculopathy (AV) is the leading cause of late allograft loss following solid organ transplantation. Ischemia reperfusion injury (IRI) and donor specific antibody (DSA)-induced complement activation confer heightened risk for AV via numerous innate immune mechanisms including MyD88, HMGB1, and complement induced non-canonical NF-kB signaling. Recent Findings The role of MyD88, a signal adaptor downstream of the toll-like receptors (TLR), has been defined in an experimental heart transplant model, which demonstrated that recipient MyD88 enhanced AV. Importantly, triggering receptor on myeloid receptor 1(Trem1), a MyD88 amplifying signal, was present in rejecting human cardiac transplant biopsies and enhanced the development of AV in mice. HMGB1, a nuclear protein that activates TLRs, also enhanced the development of AV. Complement activation elicits assembly of membrane attack complexes (MAC) on endothelial cells which activate non-canonical NF-kB signaling, a novel complement effector pathway that induces pro-inflammatory genes and potentiates endothelial cell mediated alloimmune T cell activation, processes which enhance AV. Summary Innate immune mediators including HMGB1, MyD88, and non-canonical NFκB signaling via complement activation contribute to AV. These pathways represent potential therapeutic targets to reduce AV after solid organ transplantation. PMID:27077602

Mannose-binding lectin-associated serine protease 2 (MASP-2) contributes to poor disease outcome in humans and mice with pneumococcal meningitis.

PubMed

Kasanmoentalib, E Soemirien; Valls Seron, Mercedes; Ferwerda, Bart; Tanck, Michael W; Zwinderman, Aeilko H; Baas, Frank; van der Ende, Arie; Brouwer, Matthijs C; van de Beek, Diederik

2017-01-03

Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Disease outcome has been related to the severity of the pro-inflammatory response in the subarachnoid space. The complement system, which mediates key inflammatory processes, has been implicated as a modulator of pneumococcal meningitis disease severity in animal studies. We investigated mannose-binding lectin-associated serine protease (MASP-2) levels in cerebrospinal fluid (CSF) samples derived from the diagnostic lumbar puncture, which was available for 307 of 792 pneumococcal meningitis episodes included in our prospective nationwide cohort study (39%), and the association between these levels and clinical outcome. Subsequently, we studied the role of MASP-2 in our experimental pneumococcal meningitis mouse model using Masp2 -/- mice and evaluated the potential of adjuvant treatment with MASP-2-specific monoclonal antibodies in wild-type (WT) mice. MASP-2 levels in cerebrospinal fluid of patients with bacterial meningitis were correlated with poor functional outcome. Consistent with these human data, Masp2-deficient mice with pneumococcal meningitis had lower cytokine levels and increased survival compared to WT mice. Adjuvant treatment with MASP-2-specific monoclonal antibodies led to reduced complement activation and decreased disease severity. MASP-2 contributes to poor disease outcome in human and mice with pneumococcal meningitis. MASP-2-specific monoclonal antibodies can be used to attenuate the inflammatory response in pneumococcal meningitis.

Diagnosis of Helicobacter pylori infection by using pyloriset EIA-G and EIA-A for detection of serum immunoglobulin G (IgG) and IgA antibodies.

PubMed Central

Granberg, C; Mansikka, A; Lehtonen, O P; Kujari, H; Grönfors, R; Nurmi, H; Räihä, I; Ståhlberg, M R; Leino, R

1993-01-01

We evaluated the performance of new enzyme immunoassay (EIA) kits (Pyloriset; Orion Corporation, Orion Diagnostica, Espoo, Finland) for the detection of immunoglobulin G (IgG) and IgA antibodies to Helicobacter pylori in serum. Serum samples from 195 patients with upper abdominal complaints were collected. Biopsy specimens of the gastric mucosae were taken for histological analysis and bacterial culture. The sensitivity, specificity, and positive and negative predictive values, and efficacy of the Pyloriset EIA-G in detecting IgG antibodies to H. pylori were 92, 84, 88, 90, and 89%, respectively, when compared with those of the reference methods used. The corresponding data for detection of IgA antibodies were 80, 89, 89, 79, and 84%, respectively. The overall prevalence of defined H. pylori positivity was 54%. Moreover, the antibody tests showed a very good correlation with the biopsy findings. IgG antibodies were found in 93% of sera from patients with documented gastritis and H. pylori positivity, whereas only 4% of the sera from patients with documented gastritis and H. pylori-negative patients was positive. The results obtained for IgA antibodies were 81 and 6%, respectively. We conclude that the Pyloriset EIA-G, the test for IgG antibodies, is a good and reliable test for the detection of antibodies to H. pylori and as an indication of H. pylori infection. The determination of IgA antibodies may be used as a test that complements the IgG antibody assay. PMID:8314985

Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen.

PubMed

Byrne, Guerard W; Du, Zeji; Stalboerger, Paul; Kogelberg, Heide; McGregor, Christopher G A

2014-01-01

Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. The porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes, respectively. The B4GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4GALNT2 in human HEK cells (HEK-B4T) results in increased binding of antibody to the B4GALNT2 enzyme, and increased reactivity with anti-Sd(a) and DBA. HEK-B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO:CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non-Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK-B4T cells. The functional isolation of the porcine B4GALNT2 gene from a PAEC expression library, the pattern of B4GALNT2 gene expression and its sensitization of HEK-B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4GALNT2 produces a new immunogenic non-Gal glycan which contributes in part to the non-Gal immune response detected after pig-to-baboon cardiac xenotransplantation. © 2014 The Authors. Xenotransplantation Published by John Wiley & Sons Ltd.

Chemical characterization and complement fixation of pectins from Cola cordifolia leaves.

PubMed

Austarheim, Ingvild; Christensen, Bjørn E; Aas, Hoai Thi Nguyen; Thöle, Christian; Diallo, Drissa; Paulsen, Berit S

2014-02-15

Defatted leaves from the medicinal tree Cola cordifolia were extracted with 50% EtOH, 50 °C and 100 °C water. The polysaccharide rich extracts were fractionated and the structure of the polysaccharides elucidated. Linkage analysis of the polysaccharides indicates a rhamnogalacturonan type I backbone where both Rha and parts of GalA are substituted in position 3, indicating a highly branched polymer with short side chains. The purified fractions were tested for complement fixation, macrophage stimulating activity and anti-adhesion activity towards Helicobacter pylori. Here we report on complex and polydisperse types of pectins (Mw: 3-1300 kDa) as well as the presence of low Mw (<3 kDa) acidic oligosaccharides. The fractions showed a moderate complement fixing activity and no macrophage activating effects after LPS removal. Anti-adhesion activity towards H. pylori was not found. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

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

Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes

PubMed Central

Fernie-King, Barbara A; Seilly, David J; Willers, Christine; Würzner, Reinhard; Davies, Alexandra; Lachmann, Peter J

2001-01-01

Streptococcal inhibitor of complement (SIC) was first described in 1996 as a putative inhibitor of the membrane attack complex of complement (MAC). SIC is a 31 000 MW protein secreted in large quantities by the virulent Streptococcus pyogenes strains M1 and M57, and is encoded by a gene which is extremely variable. In order to study further the interactions of SIC with the MAC, we have made a recombinant form of SIC (rSIC) in Escherichia coli and purified native M1 SIC which was used to raise a polyclonal antibody. SIC prevented reactive lysis of guinea pig erythrocytes by the MAC at a stage prior to C5b67 complexes binding to cell membranes, presumably by blocking the transiently expressed membrane insertion site on C7. The ability of SIC and clusterin (another putative fluid phase complement inhibitor) to inhibit complement lysis was compared, and found to be equally efficient. In parallel, by enzyme-linked immunosorbent assay both SIC and rSIC bound strongly to C5b67 and C5b678 complexes and to a lesser extent C5b-9, but only weakly to individual complement components. The implications of these data for virulence of SIC-positive streptococci are discussed, in light of the fact that Gram-positive organisms are already protected against complement lysis by the presence of their peptidoglycan cell walls. We speculate that MAC inhibition may not be the sole function of SIC. PMID:11454069

Complement Inhibition Alleviates Paraquat-Induced Acute Lung Injury

PubMed Central

Sun, Shihui; Wang, Hanbin; Zhao, Guangyu; An, Yingbo; Guo, Yan; Du, Lanying; Song, Hongbin; Qiao, Fei; Yu, Hong; Wu, Xiaohong; Atkinson, Carl; Jiang, Shibo; Tomlinson, Stephen

2011-01-01

The widely used herbicide, paraquat (PQ), is highly toxic and claims thousands of lives from both accidental and voluntary ingestion. The pathological mechanisms of PQ poisoning–induced acute lung injury (ALI) are not well understood, and the role of complement in PQ-induced ALI has not been elucidated. We developed and characterized a mouse model of PQ-induced ALI and studied the role of complement in the pathogenesis of PQ poisoning. Intraperitoneal administration of PQ caused dose- and time-dependent lung damage and mortality, with associated inflammatory response. Within 24 hours of PQ-induced ALI, there was significantly increased expression of the complement proteins, C1q and C3, in the lung. Expression of the anaphylatoxin receptors, C3aR and C5aR, was also increased. Compared with wild-type mice, C3-deficient mice survived significantly longer and displayed significantly reduced lung inflammation and pathology after PQ treatment. Similar reductions in PQ-induced inflammation, pathology, and mortality were recorded in mice treated with the C3 inhibitors, CR2-Crry, and alternative pathway specific CR2-fH. A similar therapeutic effect was also observed by treatment with either C3a receptor antagonist or a blocking C5a receptor monoclonal antibody. Together, these studies indicate that PQ-induced ALI is mediated through receptor signaling by the C3a and C5a complement activation products that are generated via the alternative complement pathway, and that complement inhibition may be an effective clinical intervention for postexposure treatment of PQ-induced ALI. PMID:21421909

Sequestration of host-CD59 as potential immune evasion strategy of Trichomonas vaginalis.

PubMed

Ibáñez-Escribano, Alexandra; Nogal-Ruiz, Juan José; Pérez-Serrano, Jorge; Gómez-Barrio, Alicia; Escario, J Antonio; Alderete, J F

2015-09-01

Trichomonas vaginalis is known to evade complement-mediated lysis. Because the genome of T. vaginalis does not possess DNA sequence with homology to human protectin (CD59), a complement lysis restricting factor, we tested the hypothesis that host CD59 acquisition by T. vaginalis organisms mediates resistance to complement killing. This hypothesis was based on the fact that trichomonads are known to associate with host proteins. No CD59 was detected on the surface of T. vaginalis grown in serum-based medium using as probe anti-CD59 monoclonal antibody (MAb). We, therefore, infected mice intraperitoneally with live T. vaginalis, and trichomonads harvested from ascites were tested for binding of CD59. Immunofluorescence showed that parasites had surface CD59. Furthermore, as mouse erythrocytes (RBCs) possess membrane-associated CD59, and trichomonads use RBCs as a nutrient source, organisms were co-cultured with murine RBCs for one week. Parasites were shown to have detectable surface CD59. Importantly, live T. vaginalis with bound CD59 were compared with batch-grown parasites without surface-associated CD59 for sensitivity to complement in human serum. Trichomonads without surface-bound CD59 had a higher level of killing by complement than did parasites with surface CD59. These data show that host CD59 acquired onto the surface by live T. vaginalis may be an alternative mechanism for complement evasion. We describe a novel strategy by T. vaginalis consistent with host protein procurement by this parasite to evade the lytic action of complement. Copyright © 2015 Elsevier B.V. All rights reserved.

Increased activity of the complement system in the liver of patients with alcoholic hepatitis.

PubMed

Shen, Hong; French, Barbara A; Liu, Hui; Tillman, Brittany C; French, Samuel W

2014-12-01

Inflammation has been suggested as a mechanism underlying the development of alcoholic hepatitis (AH). The activation of the complement system plays an important role in inflammation. Although it has been shown that ethanol-induced activation of the complement system contributes to the pathophysiology of ethanol-induced liver injury in mice, whether ethanol consumption activates the complement system in the human liver has not been investigated. Using antibodies against C1q, C3, and C5, the immunoreactivity of the complement system in patients with AH was examined by immunohistochemistry and quantified by morphometric image analysis. The immunoreactivity intensity of C1q, C3, and C5 in patients with AH was significantly higher than that seen in normal controls. Further, the gene expression of C1q, C3, and C5 was examined using real-time PCR. There were increases in the levels of C1q and C5, but not C3 mRNA in AH. Moreover, the immunoreactivity of C5a receptor (C5aR) also increased in AH. To explore the functional implication of the activation of the complement system in AH, we examined the colocalization of C5aR in Mallory-Denk bodies (MDBs) forming balloon hepatocytes. C5aR was focally overexpressed in the MDB forming cells. Collectively, our study suggests that alcohol consumption increases the activity of the complement system in the liver cells, which contributes to the inflammation-associated pathogenesis of AH. Copyright © 2014 Elsevier Inc. All rights reserved.

Fixed-Cell Imaging of Schizosaccharomyces pombe.

PubMed

Hagan, Iain M; Bagley, Steven

2016-07-01

The acknowledged genetic malleability of fission yeast has been matched by impressive cytology to drive major advances in our understanding of basic molecular cell biological processes. In many of the more recent studies, traditional approaches of fixation followed by processing to accommodate classical staining procedures have been superseded by live-cell imaging approaches that monitor the distribution of fusion proteins between a molecule of interest and a fluorescent protein. Although such live-cell imaging is uniquely informative for many questions, fixed-cell imaging remains the better option for others and is an important-sometimes critical-complement to the analysis of fluorescent fusion proteins by live-cell imaging. Here, we discuss the merits of fixed- and live-cell imaging as well as specific issues for fluorescence microscopy imaging of fission yeast. © 2016 Cold Spring Harbor Laboratory Press.

Immunohistochemical investigation of the cross-reactivity of selected cell markers in formalin-fixed, paraffin-embedded lymphoid tissues of Franciscana (Pontoporia blainvillei).

PubMed

Díaz-Delgado, J; Ressio, R; Groch, K R; Catão-Dias, J L

2018-06-01

A considerable amount of knowledge on natural and anthropogenic pathologic conditions affecting different cetacean species has been gained over the last decades. Nonetheless, the immunopathological bases for most of these processes have been poorly documented or remain unknown. Comparative immunopathological investigations in these species are precluded by the limited number of specific antibodies, most of which are not commercially available, and the reduced spectrum of validated and/or cross-reactive ones. To partially fill in this gap of knowledge, a set of commercially available primary antibodies were tested for cross-reactivity against leukocytes and cytokines in formalin-fixed, paraffin-embedded (FFPE) lymphoid tissues (lymph nodes, spleen and thymus) of three bycaught, apparently healthy and fresh Franciscanas (Pontoporia blainvillei) using immunohistochemistry. On the basis of similar region specificity within the lymphoid organs, cellular morphology and staining pattern with human control tissues, 13/19 primary antibodies (caspase 3, CD3, CD57, CD68, FoxP3, HLA-DRα, IFNγ, IgG, IL4, IL10, Lysozyme, TGFβ and PAX-5) exhibited satisfactory cross-reactivity. Our results expand the spectrum of suitable cross-reactive primary antibodies in FFPE cetacean tissues. Further comparative immunopathological studies focused on infectious diseases and ecotoxicology may benefit from establishment of baseline expression of immunologically relevant molecules in various cetaceans species. Copyright © 2018 Elsevier B.V. All rights reserved.

[Histopathological diagnosis of amyloidosis].

PubMed

Hoshii, Yoshinobu

2006-05-01

For the diagnosis of amyloidosis, histological evidence of amyloid deposition is essential. Histologically, an amyloid deposit is stained orange red with Congo red and shows green birefringence under polarized light. When amyloidosis is clinically suspected, endoscopic biopsy of the stomach, duodenum or colon, or aspiration biopsy of abdominal fat is usually performed. If clinicians suspect amyloidosis, they should advise pathologists. Identification of the chemical type of amyloid is necessary with respect to treatment and prognosis. Immunohistochemical examination of amyloid in formalin-fixed, paraffin-embedded sections is simple to perform in most pathological laboratories. In Japan, almost all cases of systemic amyloidosis are classified as AL, AA, ATTR or Abeta2M amyloidosis, so the use of anti-immunoglobulin light chain, anti-amyloid A, anti-transthyretin and anti-beta2 microglobulin antibody is recommended for the classification of systemic amyloidosis. Formic acid pretreatment, which is often used for immunohistochemical detection of amyloidosis, is useful and easy for antigen retrieval. Amyloid deposits of AL amyloidosis are sometimes not immunostained well with commercial anti-immunoglobulin light chain antibody. Previously, we generated polyclonal antibodies against synthetic peptides corresponding to positions 118-134 of immunoglobulin lambda light chain and positions 116-133 of immunoglobulin kappa light chain. These antibodies are very useful for detecting AL amyloidosis because they react with amyloid deposits on formalin-fixed, paraffin-embedded specimens in almost all AL amyloidosis cases. Exact diagnosis and typing of amyloidosis are necessary for therapy.

Healthcare Worker Seroconversion in SARS Outbreak

PubMed Central

Ooi, Eng-Eong; Tan, Hiang-Khoon; Ong, Kong-Wee; Sil, Bijon Kumar; Teo, Melissa; Ng, Timothy; Soo, Khee-Chee

2004-01-01

Serum samples were obtained from healthcare workers 5 weeks after exposure to an outbreak of severe acute respiratory syndrome (SARS). A sensitive dot blot enzyme-linked immunosorbent assay, complemented by a specific neutralization test, shows that only persons in whom probable SARS was diagnosed had specific antibodies and suggests that subclinical SARS is not an important feature of the disease. PMID:15030691

Structural features and complement-fixing activity of pectin from three Brassica oleracea varieties: white cabbage, kale, and red kale.

PubMed

Samuelsen, Anne Berit; Westereng, Bjørge; Yousif, Osman; Holtekjølen, Ann Katrin; Michaelsen, Terje E; Knutsen, Svein H

2007-02-01

Leaves of different cabbage species are used both as food and as wound healing remedies in traditional medicine. This supposed wound healing activity might be connected to presence of immunomodulating water soluble polysaccharides. To study this, three different cabbage varieties, white cabbage (W), kale (K), and red kale (RK), were pretreated with 80% ethanol and then extracted with water at 50 degrees C and 100 degrees C for isolation of polysaccharide-containing fractions. The fractions were analyzed for monosaccharide composition, glycosidic linkages, Mw distribution, protein content, and phenolic compounds and then tested for complement-fixing activity. All fractions contained pectin type polysaccharides with linkages corresponding to homogalacturonan and hairy regions. Those extracted at 50 degrees C contained higher amounts of neutral side chains and were more active in the complement-fixation test than those extracted at 100 degrees C. The fractions can be ranged by decreasing activity: K-50 > RK-50 > W-50 approximately = K-100 > RK100 approximately = W-100. Studies on structure-activity relationships (SAR) employing multivariate statistical analysis strongly suggest that the magnitude of the measured activity is influenced by the content of certain side chains in the polymers. High activity correlates to large neutral side chains with high amounts of (1-->6)- and (1-->3,6)-linked Gal and low amounts of (1-->4)-linked GalA but not on molecular weight distribution of the polymers.

Antigen S1, encoded by the MIC1 gene, is characterized as an epitope of human CD59, enabling measurement of mutagen-induced intragenic deletions in the AL cell system

NASA Technical Reports Server (NTRS)

Wilson, A. B.; Seilly, D.; Willers, C.; Vannais, D. B.; McGraw, M.; Waldren, C. A.; Hei, T. K.; Davies, A.; Chatterjee, A. (Principal Investigator)

1999-01-01

S1 cell membrane antigen is encoded by the MIC1 gene on human chromosome 11. This antigen has been widely used as a marker for studies in gene mapping or in analysis of mutagen-induced gene deletions/mutations, which utilized the human-hamster hybrid cell-line, AL-J1, carrying human chromosome 11. Evidence is presented here which identifies S1 as an epitope of CD59, a cell membrane complement inhibiting protein. E7.1 monoclonal antibody, specific for the S1 determinant, was found to react strongly with membrane CD59 in Western blotting, and to bind to purified, urinary form of CD59 in ELISAs. Cell membrane expression of S1 on various cell lines always correlated with that of CD59 when examined by immunofluorescent staining. In addition, E7.1 antibody inhibited the complement regulatory function of CD59. Identification of S1 protein as CD59 has increased the scope of the AL cell system by enabling analysis of intragenic mutations, and multiplex PCR analysis of mutated cells is described, showing variable loss of CD59 exons.

Gal knockout and beyond.

PubMed

Zhong, R

2007-01-01

Recently, Galalpha1-3Galbeta1-4GlcNAc (Gal) knockout (k/o) pigs have been developed using genetic cloning technologies. This remarkable achievement has generated great enthusiasm in xenotransplantation studies. This review summarizes the current status of nonhuman primate experiments using Gal k/o pig organs. Briefly, when Gal k/o pig organs are transplanted into primates, hyperacute rejection does not occur. Although graft survival has been prolonged up to a few months in some cases, the overall results were not better than those using Gal-positive pig organs with human complement regulatory protein transgenes. Gal k/o pig kidneys rapidly developed rejection which was associated with increased anti-non-Gal antibodies. Although the precise mechanisms of Gal k/o pig organ rejection are not clear, it could result from incomplete deletion of Gal, up-regulation of new antigen (non-Gal antigen) and/or production of non-Gal antibodies. Future work in xenotransplantation should place emphasis on further modification of donors, such as combining human complement regulatory genes with Gal k/o, deleting non-Gal antigens and adding protective/surviving genes or a gene that inhibits coagulation. Induction of donor-specific T- and B-cell tolerance and promotion of accommodation are also warranted.

Antibody localization in the glomerular basement membrane may precede in situ immune deposit formation in rat glomeruli.

PubMed

Agodoa, L Y; Gauthier, V J; Mannik, M

1985-02-01

The administration of cationized antibodies, specific to human serum albumin, into the renal artery of rats caused transient presence of IgG in glomeruli by immunofluorescence microscopy. Intravenous infusion of appropriate doses of antigen after the injection of cationized antibodies resulted in immune deposit formation in glomeruli that persisted through 96 hr. By electron microscopy, these deposits were located in the subepithelial area. The injection of large doses of antigen produced immune deposits which were present in glomeruli for only a few hours, presumably due to formation of only small-latticed immune complexes. The presented data indicate that cationic antibodies bound to the fixed negative charges of the glomerular basement membrane can interact with circulating antigen to form immune deposits in glomeruli. This mechanism may be important because anionic antigens have been shown to induce the synthesis of cationic antibodies.

A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining.

PubMed

Staudt, Nicole; Müller-Sienerth, Nicole; Fane-Dremucheva, Alla; Yusaf, Shahnaz P; Millrine, David; Wright, Gavin J

2015-01-02

Cell surface receptors and secreted proteins play important roles in neural recognition processes, but because their site of action can be a long distance from neuron cell bodies, antibodies that label these proteins are valuable to understand their function. The zebrafish embryo is a popular vertebrate model for neurobiology, but suffers from a paucity of validated antibody reagents. Here, we use the entire ectodomain of neural zebrafish cell surface or secreted proteins expressed in mammalian cells to select monoclonal antibodies to ten different antigens. The antibodies were characterised by Western blotting and the sensitivity of their epitopes to formalin fixation was determined. The rearranged antigen binding regions of the antibodies were amplified and cloned which enabled expression in a recombinant form from a single plasmid. All ten antibodies gave specific staining patterns within formalin-treated embryonic zebrafish brains, demonstrating that this generalised approach is particularly efficient to elicit antibodies that stain native antigen in fixed wholemount tissue. Finally, we show that additional tags can be easily added to the recombinant antibodies for convenient multiplex staining. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides

PubMed Central

Wiertsema, Selma P.; Corscadden, Karli J.; Mateus, Tulia; Mullaney, Gemma L.; Zhang, Guicheng; Richmond, Peter C.; Thornton, Ruth B.

2016-01-01

ABSTRACT The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group (P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children. PMID:28031178

Epitope and isotype specificities of antibodies to β-amyloid peptide for protection against Alzheimer's disease-like neuropathology

PubMed Central

Bard, Frédérique; Barbour, Robin; Cannon, Catherine; Carretto, Robert; Fox, Michael; Games, Dora; Guido, Teresa; Hoenow, Kathleen; Hu, Kang; Johnson-Wood, Kelly; Khan, Karen; Kholodenko, Dora; Lee, Celeste; Lee, Mike; Motter, Ruth; Nguyen, Minh; Reed, Amanda; Schenk, Dale; Tang, Pearl; Vasquez, Nicki; Seubert, Peter; Yednock, Ted

2003-01-01

Transgenic PDAPP mice, which express a disease-linked isoform of the human amyloid precursor protein, exhibit CNS pathology that is similar to Alzheimer's disease. In an age-dependent fashion, the mice develop plaques containing β-amyloid peptide (Aβ) and exhibit neuronal dystrophy and synaptic loss. It has been shown in previous studies that pathology can be prevented and even reversed by immunization of the mice with the Aβ peptide. Similar protection could be achieved by passive administration of some but not all monoclonal antibodies against Aβ. In the current studies we sought to define the optimal antibody response for reducing neuropathology. Immune sera with reactivity against different Aβ epitopes and monoclonal antibodies with different isotypes were examined for efficacy both ex vivo and in vivo. The studies showed that: (i) of the purified or elicited antibodies tested, only antibodies against the N-terminal regions of Aβ were able to invoke plaque clearance; (ii) plaque binding correlated with a clearance response and neuronal protection, whereas the ability of antibodies to capture soluble Aβ was not necessarily correlated with efficacy; (iii) the isotype of the antibody dramatically influenced the degree of plaque clearance and neuronal protection; (iv) high affinity of the antibody for Fc receptors on microglial cells seemed more important than high affinity for Aβ itself; and (v) complement activation was not required for plaque clearance. These results indicate that antibody Fc-mediated plaque clearance is a highly efficient and effective process for protection against neuropathology in an animal model of Alzheimer's disease. PMID:12566568

Clinical cytometry and progress in HLA antibody detection.

PubMed

Bray, Robert A; Tarsitani, Christine; Gebel, Howard M; Lee, Jar-How

2011-01-01

For most solid organ and selected stem cell transplants, antibodies against mismatched HLA antigens can lead to early and late graft failure. In recognition of the clinical significance of these antibodies, HLA antibody identification is one of the most critical functions of histocompatibility laboratories. Early methods employed cumbersome and insensitive complement-dependent cytotoxicity assays with a visual read-out. A little over 20 years ago flow cytometry entered the realm of antibody detection with the introduction of the flow cytometric crossmatch. Cytometry's increased sensitivity and objectivity quickly earned it popularity as a preferred crossmatch method especially for sensitized recipients. Although a sensitive method, the flow crossmatch was criticized as being "too sensitive" as false positive reactions were a know drawback. In part, the shortcomings of the flow crossmatch were due to the lack of corresponding sensitive and specific HLA antibody screening assays. However, in the mid 1990s, solid phase assays, capable of utilizing standard flow cytometers, were developed. These assays used microparticles coated with purified HLA molecules. Hence, the era of solid-phase, microparticle technology for HLA antibody detection was born permitting the sensitive and specific detection of HLA antibody. It was now possible to provide better correlation between HLA antibody detection and the flow cytometric crossmatch. This flow-based technology was soon followed by adaptation to the Luminex platform permitting a mutltiplexed approach for the identification and characterization of HLA antibodies. It is hoped that these technologies will ultimately lead to the identification of parameters that best correlate with and/or predict transplant outcomes. Copyright © 2011 Elsevier Inc. All rights reserved.

Antiphospholipid Syndrome Nephropathy: From Pathogenesis to Treatment.

PubMed

Tektonidou, Maria G

2018-01-01

Kidney damage is a well-recognized complication of the antiphospholipid syndrome (APS), either primary or systemic lupus erythematosus (SLE)-associated APS. Kidney involvement in APS involves a variety of manifestations, such as renal artery thrombosis or stenosis, renal vein thrombosis, allograft loss due to thrombosis after kidney transplantation, and injury to the renal microvasculature, also known as APS nephropathy. Biopsy in patients with APS nephropathy includes acute thrombotic microangiopathy lesions and chronic intrarenal vascular lesions such as interlobular fibrous intimal hyperplasia, arterial and arteriolar recanalizing thrombosis, fibrous arterial occlusion, and focal cortical atrophy. The most frequent clinical features are hypertension, microscopic hematuria, proteinuria (ranging from mild to nephritic levels), and renal insufficiency. It is uncertain whether antiphospholipid antibodies or other factors are implicated in the development of APS nephropathy, and whether it is driven mainly by thrombotic or by inflammatory processes. Experimental models and clinical studies of thrombotic microangiopathy lesions implicate activation of the complement cascade, tissue factor, and the mTORC pathway. Currently, the management of APS nephropathy relies on expert opinion, and consensus is lacking. There is limited evidence about the effect of anticoagulants, and their use remains controversial. Treatment approaches in patients with APS nephropathy lesions may include the use of heparin based on its role on complement activation pathway inhibition or the use of intravenous immunoglobulin and/or plasma exchange. Targeted therapies may also be considered based on potential APS nephropathy pathogenetic mechanisms such as B-cell directed therapies, complement inhibition, tissue factor inhibition, mTOR pathway inhibition, or anti-interferon antibodies, but prospective multicenter studies are needed to address their role.

Polyclonal antibody against a complement-activating pectin from the roots of Angelica acutiloba.

PubMed

Wang, N L; Kiyohara, H; Matsumoto, T; Otsuka, H; Hirano, M; Yamada, H

1994-10-01

Anti-sera against a complement-activating pectin (AR-2IIb), which was purified from the roots of Angelica acutiloba Kitagawa, were obtained by immunization of rabbits, and a polyclonal anti-AR-2IIb antibody of the IgG class was purified by affinity chromatography on AR-2IIb-immobilized Sepharose and Protein G-Sepharose. Periodate oxidation of AR-2IIb significantly reduced its inhibitory activity on the reactivity of AR-2IIb to anti-AR-2IIb-IgG, but pronase digestion of AR-2IIb did not affect its inhibitory activity. Other pharmacologically active pectins from A. autiloba, Bupleurum falcatum, and Glycyrrhiza uralensis and the complement-activating pectic arabinogalactan from A. autiloba also showed significant inhibitory activities on the reactivity of AR-2IIb to anti-AR-2IIb-IgG, but these inhibitory activities were lower than that of AR-2IIb. Other pectins, polygalacturonic acid, arabinogalactan, galactan, and araban tested had negligible inhibitory activity. Endo-a-(1-->4)-polygalacturonase digestion of AR-2IIb indicated that its "ramified" region (rhamnogalacturonan core possessing neutral oligosaccharide side-chains) contained epitopes for anti-AR-2IIb-IgG, but that 2-keto-3-deoxyoctulosonic acid (KDO)-containing regions and oligogalacturonides obtained from AR-2IIb were not recognized by anti-AR-2IIb-IgG. Although carboxyl-reduction of galacturonic acid in the "ramified" region decreased the inhibitory activity of the "ramified" on its reactivity to anti-AR-2IIb, an acidic tetrasaccharide unit in the rhamnogalacturonan core had negligible inhibitory activity.

Circulating heavy IgM in IgM nephropathy.

PubMed

Disciullo, S O; Abuelo, J G; Moalli, K; Pezzullo, J C

1988-09-01

IgM nephropathy (IgMN) causes nephrotic syndrome and is characterized by IgM mesangial deposits. It is speculated that these deposits are derived from circulating IgM aggregates or immune complexes, either of which would have a molecular weight heavier than that of normal IgM. To test this hypothesis the sera of 11 patients with IgMN, five patients with nephrotic syndrome of other etiologies, and 13 normal controls were analysed for such heavy IgM. The serum samples were passed over a Biogel A5M molecular sieve column and the fractions were tested for IgM concentration by enzyme linked immunosorbent assay (ELISA). The column effluent from the void volume to the IgM peak was divided into four equal regions, and the average IgM concentrations in each region were compared. The IgMN group had significantly higher IgM concentrations than normal controls in the heaviest region (0.81 +/- 0.84 vs. 0.32 +/- 0.17 micrograms/ml; P = 0.01) and in the lightest region (95.8 +/- 59.5 vs. 46.3 +/- 41.2 micrograms/ml; P = 0.02). Although the IgMN group appeared to have about double the IgM levels of the nephrotic control group in all four regions, this was only significant in the lightest (19S) region. In serum samples from two IgMN patient methods known to break antigen antibody bonds eliminated the heavy IgM; in one case we used gel filtration in potassium thiocyanate and in another ultracentrifugation at pH 2.8. In addition, the heavy IgM in this second patient exhibited complement fixation activity in a sandwich ELISA for IgM-C3 complexes. We conclude that IgMN patients have circulating heavy IgM, which by preliminary studies probably consists of complement fixing IgM immune complexes.

PULMONARY MYCOTIC INFECTIONS—Allergic and Immunologic Factors

PubMed Central

Keeney, Edmund L.

1954-01-01

The mechanisms of immunity and allergy, at play in every infectious disease, must be comprehended before the pathogenesis of an infection can be appreciated. Immunity, allergy and serology are concerned with specific antigen-antibody reactions. In immunity the principal concern is with the final disposition of antigen (agglutination, lysis, and phagocytosis). In allergy attention is focused upon tissue damage resulting from antigen-antibody union. In serology interest is devoted to the presence of antibody as evaluated by certain visible in vitro reactions—precipitin, agglutination, opsonization and complement fixation tests. There are two types of allergic reaction—the immediate or anaphylactic type and the delayed type or the allergic disease of infection. Neither kind takes part in the mechanism of immunity. At this time the allergic antibody and the immune antibody must be considered as two different and distinct antibodies. Skin and serologic tests are important diagnostic aids in certain pulmonary mycotic infections—for example, coccidioidomycosis, blastomycosis, histoplasmosis and moniliasis. Clinical expressions of allergy may appear in coccidioidomycosis, histoplasmosis and moniliasis. Pulmonary mycoses are divided into three groups, that is, the endogenous mycoses (actinomycosis, moniliasis, geotrichosis), the endogenous-exogenous mycoses (cryptococcosis, aspergillosis, mucormycosis) and the exogenous mycoses (nocardiosis, coccidioidomycosis, histoplasmosis, North American blastomycosis). The diagnosis and treatment of the important mycotic infections that invade lung tissue are discussed. PMID:13209369

Seroprevalence for Coxiella burnetii, Francisella tularensis, Brucella abortus and Brucella melitensis in Austrian adults: a cross-sectional survey among military personnel and civilians.

PubMed

Tobudic, Selma; Nedomansky, Klara; Poeppl, Wolfgang; Müller, Maria; Faas, Angelus; Mooseder, Gerhard; Allerberger, Franz; Stanek, Gerold; Burgmann, Heinz

2014-04-01

The prevalence of Coxiella burnetii, Francisella tularensis, Brucella abortus, and Brucella melitensis infections in Austria and the exposure risk of military personnel were assessed in an exploratory nationwide cross-sectional seroprevalence survey in 526 healthy adult individuals, 222 of which were soldiers and 304 were civilians. Screening for IgA/IgG antibodies to C. burnetii (Phase I) and IgG/IgM antibodies to C. burnetii (Phase II), and to F. tularensis was done with commercial enzyme-linked immunosorbent assays. To detect antibodies against B. abortus and B. melitensis, an in-house complement fixation test was used. Overall, 11 individuals (2.0%) showed antibodies to C. burnetii, 3 individuals (0.5%) were seropositive for F. tularensis, and one (0.3%) individual was borderline positive. All individuals positive or borderline for F. tularensis tested negative for antibodies against C. burnetii. All individuals tested negative for antibodies against B. melitensis/B. abortus. There were no significant differences between the seroprevalence of C. burnetii and F. tularensis among military personnel and civilians. Our data demonstrate serological evidence of a low rate of exposure to C. burnetii and F. tularensis among the Austrian adult population and military personnel. Copyright © 2014 Elsevier GmbH. All rights reserved.

Guidelines for pre-clinical assessment of the acetylcholine receptor-specific passive transfer myasthenia gravis model - recommendations for methods and experimental designs

PubMed Central

Kusner, Linda L.; Losen, Mario; Vincent, Angela; Lindstrom, Jon; Tzartos, Socrates; Lazaridis, Konstantinos; Martinez-Martinez, Pilar

2015-01-01

Antibodies against the muscle acetylcholine receptor (AChR) are the most common cause of myasthenia gravis (MG). Passive transfer of AChR antibodies from MG patients into animals reproduces key features of human disease, including antigenic modulation of the AChR, complement-mediated damage of the neuromuscular junction, and muscle weakness. Similarly, AChR antibodies generated by active immunization in experimental autoimmune MG models can subsequently be passively transferred to other animals and induce weakness. The passive transfer model is useful to test therapeutic strategies aimed at the effector mechanism of the autoantibodies. Here we summarize published and unpublished experience using the AChR passive transfer MG model in mice, rats and rhesus monkeys, and give recommendations for the design of preclinical studies in order to facilitate translation of positive and negative results to improve MG therapies. PMID:25743217

[The alterations of proteins glycosylation in rheumatic diseases].

PubMed

Chludzińska, Anna; Chrostek, Lech; Cylwik, Bogdan

2012-08-01

The alterations in glycosylation of serum glycoproteins were reported in several pathological conditions including rheumatic diseases. The many studies demonstrated the occurrence of some differentially glycosylated plasma immunoglobulins, especially IgG in rheumatoid arthritis. The most characteristic features are the decrease in galactose content, the presence of N-acetylglucosamine and the increase in fucose content. The structure of oligosaccharides attached to the antibody Fc region affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. The changes in immunoglobulin glycosylation was suggested to be important in the etiology of rheumatoid athritis and correlated with the disease severity. In addition to impaired glycosylation of imunoglubulins, in rheumatic diseases exist the disturbances in glycosylation of both acute-phase and non acute-phase response, such as alpha-1 acid glycoprotein, haptoglobin and alpha-2 macroglobulin. The alterations in glycosylation of these glycoproteins were also correlated with the disease activity.

Extended low-resolution structure of a Leptospira antigen offers high bactericidal antibody accessibility amenable to vaccine design

PubMed Central

Tseng, Andrew; Suguiura, Igor Massahiro de Souza; McDonough, Sean P; Sritrakul, Tepyuda; Li, Ting; Lin, Yi-Pin; Gillilan, Richard E

2017-01-01

Pathogens rely on proteins embedded on their surface to perform tasks essential for host infection. These obligatory structures exposed to the host immune system provide important targets for rational vaccine design. Here, we use a systematically designed series of multi-domain constructs in combination with small angle X-ray scattering (SAXS) to determine the structure of the main immunoreactive region from a major antigen from Leptospira interrogans, LigB. An anti-LigB monoclonal antibody library exhibits cell binding and bactericidal activity with extensive domain coverage complementing the elongated architecture observed in the SAXS structure. Combining antigenic motifs in a single-domain chimeric immunoglobulin-like fold generated a vaccine that greatly enhances leptospiral protection over vaccination with single parent domains. Our study demonstrates how understanding an antigen’s structure and antibody accessible surfaces can guide the design and engineering of improved recombinant antigen-based vaccines. PMID:29210669

[Parasitic and viral marker detection in pregnant adolescents and their newborn infants at risk].

PubMed

Contreras, M C; Escaff, V; Salinas, P; Saavedra, T; Suárez, M

1995-01-01

We have investigated the prevalence of antibodies against Toxoplasma gondii, Trypanosoma cruzzi, Hepatitis B virus, cytomegalovirus, rubella virus, and human immunodeficiency virus in 139 adolescent pregnant women and in their high risk newborn children. The methods employed were the Sabin and Feldman reaction, complement fixation reaction, ELISA, and xenodiagnostic 30.9% of the pregnant group were seropositive for T. gondii, both mothers and newborns were IgM-negative. Two mothers (1.4%) presented anti T. cruzii antibodies, and one newborn child had circulating parasites. Related to the virological studies, 93.5% of the population were anti CMV antibodies positive and all their newborns were IgM (-) 90.6% of the adolescents were rubella positive and one was seropositive to VIH. We conclude that the prevalence found in this group of adolescent pregnant women are not significantly different to the one reported for the general pregnant women population.

Absence of cytotoxic antibody to human immunodeficiency virus-infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gp120

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

Nara, P.L.; Robey, W.G.; Gonda, M.A.

1987-06-01

The presence of antibody-dependent complement-mediated cytotoxicity (ACC) was assessed in humans and chimpanzees, which are capable of infection with human immunodeficiency virus isolate HTLV-IIIb, and examined in the goat after immunization with the major viral glycoprotein (gp120) of HTLV-IIIb. In infected humans no antibody mediating ACC was observed regardless of the status of disease. Even healthy individuals with high-titer, broadly reactive, neutralizing antibodies has no ACC. In contrast, chimpanzees infected with HTLV-IIIb, from whom virus could be isolated, not only had neutralizing antibody but also antibodies broadly reactive in ACC, even against distantly related human immunodeficiency virus isolates, as wellmore » as against their own reisolated virus. In the goat, the gp120 of HTLV-IIIb induced a highly type-specific response as measured by both ACC and flow cytofluorometry of live infected H9 cells. Normal human cells were not subject to ACC by animal anti-HTLV-III gp120-specific sera. Induction of ACC and neutralizing antibody were closely correlated in the animal experimental models but not in humans. The presence of ACC in gp120-inoculated goats and HTLV-III-infected chimpanzees represent a qualitative difference that may be important in the quest for the elicitation of a protective immunity in humans.« less

Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

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

Sjoewall, Christopher; Wetteroe, Jonas; Bengtsson, Torbjoern

2007-01-05

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fc{gamma} receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficientlymore » down-regulated at CRP levels >150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.« less

Effect of complement depletion by cobra venom factor on fowlpox virus infection in chickens and chicken embryos.

PubMed Central

Ohta, H; Yoshikawa, Y; Kai, C; Yamanouchi, K; Taniguchi, H; Komine, K; Ishijima, Y; Okada, H

1986-01-01

The course of infection with an attenuated strain of fowlpox virus (FPV), which is known to induce antibody-independent activation of complement via the alternative pathway, was investigated in 1- to 3-day-old chickens and 14-day-old chicken embryos by treatment with cobra venom factor (CVF). CVF was found to inhibit complement activity transiently via the alternative pathway but not via the classical pathway. In chickens treated with CVF, virus growth in the skin was enhanced, and pock lesions tended to disseminate, leading to fatal infection in some birds. Histologically, an acute inflammation at an early stage of infection (within 3 days) was inhibited, and virus content in the pock lesion was increased. In chicken embryos with immature immune capacities, CVF treatment caused changes in pock morphology from clear pocks to diffuse ones, an increase in virus content in the pock, and inhibition of cell infiltration. Thus, FPV infection was aggravated in both CVF-treated chickens and chicken embryos. These results are discussed in relation to roles of complement in the elimination of virus at an early stage of FPV infection. Images PMID:3003397

MURI: Adaptive Waveform Design for Full Spectral Dominance

DTIC Science & Technology

2011-03-11

a three- dimensional urban tracking model, based on the nonlinear measurement model (that uses the urban multipath geometry with different types of ... the time evolution of the scattering function with a high dimensional dynamic system; a multiple particle filter technique is used to sequentially...integration of space -time coding with a fixed set of beams. It complements the

Antibody-dependent enhancement of HIV-1 infection in human term syncytiotrophoblast cells cultured in vitro.

PubMed Central

Tóth, F D; Mosborg-Petersen, P; Kiss, J; Aboagye-Mathiesen, G; Zdravkovic, M; Hager, H; Aranyosi, J; Lampé, L; Ebbesen, P

1994-01-01

We examined if Fc receptor-mediated antibody-dependent enhancement (FcR-ADE) or complement-mediated antibody-dependent enhancement (C'-ADE) of virus infection can contribute to increasing replication of HIV-1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR-ADE and C'-ADE may result in enhanced virus release from HIV-1-infected ST cells. We show that FcR-ADE of HIV-1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'-ADE uses both CD4 and CR2-like receptors. FcR-ADE seems to be more efficient in enhancing HIV-1 replication than C'-ADE. While FcR-ADE leads to increased internalization of HIV-1, C'-ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR-ADE are reactive with the gp120 viral envelope antigen, whereas antibodies involved in C'-ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR-ADE and C'-ADE may contribute to the spread of HIV-1 from mother to the fetus. PMID:8004808

Maternal antibody reactivity to lymphocytes of offspring with autism.

PubMed

Bressler, Joseph P; Gillin, Pam K; O'Driscoll, Cliona; Kiihl, Samara; Solomon, Megan; Zimmerman, Andrew W

2012-11-01

The study examined whether maternal serum antibodies from mothers of autistic children preferentially bind to lymphocytes of their autistic children compared with unaffected siblings. In a previous study, maternal serum antibodies from mothers mediated cytotoxicity with complement to lymphocytes of their autistic children. Here, maternal serum antibody binding was examined by flow cytometry. We compared levels of mothers' serum binding against peripheral blood monocytes of their autistic children vs unaffected siblings. Because the level of binding to peripheral blood monocytes could be low, binding was examined in specific lymphocyte subpopulations. In 19 samples, the mean level of maternal serum immunoglobulin G binding to CD4 and CD8 T cells, B cells, natural killer cells, and macrophages was not significantly different from the mean level of binding to unaffected siblings. The percentages of different subpopulations were not significantly different between autistic children and unaffected siblings, although a trend (P < 0.1) emerged, i.e., autistic children displayed a higher percentage of natural killer cells and a lower percentage of B cells. These findings cast doubt on a direct effect of maternal antibodies, but do not preclude potential intrauterine pathogenic immune mechanisms in autism. Copyright © 2012 Elsevier Inc. All rights reserved.

PD-1 suppresses development of humoral responses that protect against Tn-bearing tumors

PubMed Central

Haro, Marcela A.; Littrell, Chad A.; Yin, Zhaojun; Huang, Xuefei; Haas, Karen M.

2017-01-01

Tn is a carbohydrate antigen uniquely exposed on tumor mucins and thus, an ideal target for immunotherapy. However, it has been difficult to elicit protective antibody responses against Tn antigen and other tumor associated carbohydrate antigens. Our study demonstrates this can be attributed to PD-1 immuno-inhibition. Our data show a major role for PD-1 in suppressing mucin- and Tn-specific B-cell activation, expansion, and antibody production important for protection against Tn-bearing tumor cells. These Tn/mucin-specific B cells belong to the innate-like B-1b cell subset typically responsible for T cell–independent antibody responses. Interestingly, PD-1–mediated regulation is B cell–intrinsic and CD4+ cells play a key role in supporting Tn/mucin-specific B cell antibody production in the context of PD-1 deficiency. Mucin-reactive antibodies produced in the absence of PD-1 inhibition largely belong to the IgM subclass and elicit potent antitumor effects via a complement-dependent mechanism. The identification of this role for PD-1 in regulating B cell–dependent antitumor immunity to Tn antigen highlights an opportunity to develop new therapeutic strategies targeting tumor associated carbohydrate antigens. PMID:27856425

Antibody-dependent enhancement of HIV-1 infection in human term syncytiotrophoblast cells cultured in vitro.

PubMed

Tóth, F D; Mosborg-Petersen, P; Kiss, J; Aboagye-Mathiesen, G; Zdravkovic, M; Hager, H; Aranyosi, J; Lampé, L; Ebbesen, P

1994-06-01

We examined if Fc receptor-mediated antibody-dependent enhancement (FcR-ADE) or complement-mediated antibody-dependent enhancement (C'-ADE) of virus infection can contribute to increasing replication of HIV-1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR-ADE and C'-ADE may result in enhanced virus release from HIV-1-infected ST cells. We show that FcR-ADE of HIV-1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'-ADE uses both CD4 and CR2-like receptors. FcR-ADE seems to be more efficient in enhancing HIV-1 replication than C'-ADE. While FcR-ADE leads to increased internalization of HIV-1, C'-ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR-ADE are reactive with the gp120 viral envelope antigen, whereas antibodies involved in C'-ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR-ADE and C'-ADE may contribute to the spread of HIV-1 from mother to the fetus.

Anti-CD22 and anti-CD79b antibody-drug conjugates preferentially target proliferating B cells.

PubMed

Fuh, Franklin K; Looney, Caroline; Li, Dongwei; Poon, Kirsten A; Dere, Randall C; Danilenko, Dimitry M; McBride, Jacqueline; Reed, Chae; Chung, Shan; Zheng, Bing; Mathews, William Rodney; Polson, Andrew; Prabhu, Saileta; Williams, Marna

2017-04-01

CD22 and CD79b are cell-surface receptors expressed on B-cell-derived malignancies such as non-Hodgkin's lymphoma (NHL). An anti-mitotic agent, monomethyl auristatin E, was conjugated to anti-CD22 and anti-CD79b antibodies to develop target-specific therapies for NHL. The mechanism of action (MOA) and pharmacological and pharmacokinetic (PK) profiles of these antibody-drug conjugates (ADCs) were investigated in cynomolgus monkeys. Animals were administered anti-CD22 or anti-CD79b ADCs, respective unconjugated antibodies or vehicle. Pharmacodynamic effects on total and proliferating B cells and serum PK were then assessed. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the ADCs were evaluated in vitro. Depletion of B cells was observed after administration of either ADC or the respective unconjugated antibodies. An extended duration of depletion was observed in animals administered ADCs. Similarly, preferential depletion of proliferating B cells in blood and germinal centre B cells in spleen were only observed in animals administered ADCs. Serum PK profiles of ADCs and respective unconjugated antibodies were comparable. In vitro, anti-human CD22 and anti-human CD79b antibodies showed no or only moderate ADCC activity, respectively; neither antibody had CDC activity. The findings support the proposed MOA: initial depletion of total B cells by antibody-mediated opsonization, followed by preferential, sustained depletion of proliferating B cells by the auristatin conjugate due to its anti-mitotic action. Delivering potent anti-mitotic agents to B cells via the specificity of monoclonal antibodies provides a means to eliminate pathogenic B cells in NHL with improved risk-benefit profiles over traditional chemotherapeutics. © 2016 Genentech. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)*

PubMed Central

Bader, Sabine; Zajac, Magdalena; Friess, Thomas; Ruge, Elisabeth; Rieder, Natascha; Gierke, Berthold; Heubach, Yvonne; Thomas, Marlene; Pawlak, Michael

2015-01-01

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues. PMID:26106084

Anti-Jo-1 antibody-positive patients show a characteristic necrotizing perifascicular myositis.

PubMed

Mescam-Mancini, Lénaig; Allenbach, Yves; Hervier, Baptiste; Devilliers, Hervé; Mariampillay, Kuberaka; Dubourg, Odile; Maisonobe, Thierry; Gherardi, Romain; Mezin, Paulette; Preusse, Corinna; Stenzel, Werner; Benveniste, Olivier

2015-09-01

Idiopathic inflammatory myopathies can be classified as polymyositis, dermatomyositis, immune-mediated necrotizing myopathy, sporadic inclusion body myositis or non-specific myositis. Anti-Jo-1 antibody-positive patients are assigned to either polymyositis or dermatomyositis suggesting overlapping pathological features. We aimed to determine if anti-Jo-1 antibody-positive myopathy has a specific morphological phenotype. In a series of 53 muscle biopsies of anti-Jo-1 antibody-positive patients, relevant descriptive criteria defining a characteristic morphological pattern were identified. They were tested in a second series of anti-Jo-1 antibody-positive patients and compared to 63 biopsies from patients suffering from other idiopathic inflammatory myopathies. In anti-Jo-1 antibody-positive patients, necrotic fibres, which strongly clustered in perifascicular regions, were frequently observed. Sarcolemmal complement deposition was detected specifically in perifascicular areas. Inflammation was mainly located in the perimysium and around vessels in 90.6%. Perimysial fragmentation was observed in 90% of cases. Major histocompatibility complex class I staining was diffusely positive, with a perifascicular reinforcement. Multivariate analysis showed that criteria defining perifascicular pathology: perifascicular necrosis, atrophy, and perimysial fragmentation allow the distinction of anti-Jo-1 antibody-positive patients, among patients suffering from other idiopathic inflammatory myopathies. Anti-Jo-1 antibody-positive patients displayed perifascicular necrosis, whereas dermatomyositis patients exhibited perifascicular atrophy. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Successful Renal Transplantation Across HLA Barrier: Report from India.

PubMed

Aggarwal, G; Tiwari, A K; Dorwal, P; Chauhan, R; Arora, D; Dara, R C; Kher, V

2017-01-01

Organ donors are sometimes found "unsuitable" due to the presence of donor-specific anti-HLA antibodies in the recipient. In recent years, improved desensitization protocols have successfully helped to overcome HLA incompatibility hurdle. We present three cases where optimum desensitization was achieved in patients with the donor-specific anti-HLA antibody (DSA) leading to successful renal transplantation. All patient-donor pair underwent HLA typing, complement dependent cytotoxicity crossmatch (CDC-XM), flow cytometry XM (FC-XM), and panel reactive antibody. If any of the three tests was positive, single antigen bead assay was performed to determine the specificity of the anti-HLA antibody (s). Patients with DSA were offered organ-swap or anti-HLA antibody desensitization followed by transplantation. Desensitization protocol consisted of single dose rituximab and cascade plasmapheresis (CP) along with standard triple immunosuppression. The target DSA mean fluorescence index (MFI) was <500, along with negative CDC-XM and FC-XM for both T- and B-cells. Three patients with anti-HLA DSA, who did not find a suitable match in organ swap program, consented to anti-HLA antibody desensitization, followed by transplantation. Mean pre-desensitization antibody MFI was 1740 (1422-2280). Mean number of CP required to achieve the target MFI was 2.3 (2-3). All the three patients are on regular follow-up and have normal renal function test at a mean follow-up of 8 months. This report underlines successful application of desensitization protocol leading to successful HLA-antibody incompatible renal transplants and their continued normal renal functions.

[Production of the monoclonal antibodies to the rabies virus nucleoprotein].

PubMed

Gribencha, S V; Kozlov, A Iu; Kostina, L V; Elakov, A L; Losich, M A; Tsibezov, V V; Zaberezhnyĭ, A D; Aliper, T I

2013-01-01

Five hybridomas secreting monoclonal antibodies (MAbs) for the nucleocapsid protein of the rabies virus were obtained through the fusion of the SP2/0 murine myeloma cells with splenocytes of BALB/c mice immunized with fixed rabies virus (CVS strain). All hybridomas secret MAbs of the IgG class that display different specificity to the nucleocapsids of rabies and rabies-related viruses. MAbs 2ell showed the specificity for the prevalent in Russia rabies viruses that are similar to commercially available anti-rabies conjugate.

Acquisition of Heat Stable Enzymes from Thermophilic Microorganisms: Peroxidases, Ureases, and Glucose Oxidases.

DTIC Science & Technology

1992-04-01

foetus antibody (approx. 43.8 mg/ mL) (Nakane). After conjugation, unconjugated antibody and urease were separated from the IgG-Urease conjugate by...using fixed T. foetus organisms were incubated in 10 mM citrate buffer, pH 7, as a control or IgG-Urease in the same citrate buffer for 15 min. Organisms...Table 15. T. foetus - Anti T. foetus Model Immunoassay Relative Urease Activity Reaction (Absofbances @590 nm) T. foetus + Buffer Control 0.501 IgG-Urease

Structural and functional studies on Ribonuclease S, retro S and retro-inverso S peptides

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

Pal-Bhowmick, Ipsita; Pati Pandey, Ramendra; Jarori, Gotam K.

2007-12-21

Ribonuclease S peptide and S protein offer a unique complementation system to understand the finer features of molecular recognition. In the present study the S peptide (1-16), and its retro and retro-inverso analogs have been analyzed for their structural and biological attributes. RPHPLC, CD, and NMR analyses have revealed that the physicochemical and conformational properties of the S peptide are distinct from those of its retro and retro-inverso analogs. On the functional side, while the S peptide complemented the S protein to give RNase activity, was recognized by anti-S peptide antibodies and induced T cell proliferation, neither the retro normore » the retro-inverso S peptides could do so.« less

Detection of AA76, a Common Form of Amyloid A Protein, as a Way of Diagnosing AA Amyloidosis.

PubMed

Sato, Junji; Okuda, Yasuaki; Kuroda, Takeshi; Yamada, Toshiyuki

2016-01-01

Reactive amyloid deposits consist of amyloid A (AA) proteins, the degradation products of serum amyloid A (SAA). Since the most common species of AA is the amino terminal portion produced by cleavage between residues 76 and 77 of SAA (AA76), the presence of AA76 in tissues could be a consequence of AA amyloid deposition. This study assessed the diagnostic significance of the detection of AA76 for AA amyloidosis using two different approaches. Biopsy specimens (n=130 from 54 subjects) from gastroduodenal mucosa or abdominal fat (n=9 from 9 subjects) of patients who had already been diagnosed with or were suspected of having AA amyloidosis were used. Fixed mucosal sections were subjected to immunohistochemistry using a newly developed antibody recognizing the carboxyl terminal end of AA76 (anti-AA76). The non-fixed materials from gastroduodenal mucosa or abdominal fat were subjected to immunoblotting for detection of the size of AA76. Among the gastroduodenal specimens (n=115) from already diagnosed patients, the positive rates of Congo red staining, immunohistochemistry using anti-AA76, and immunoblotting were 68.4%, 73.0%, and 92.2%, respectively. The anti-AA76 did not stain the supposed SAA in the blood or leakage, which was stained by anti-SAA antibody. AA76 was not detected either by immunohistochemistry or by immunoblot in the materials from patients in whom AA amyloidosis had been ruled out. In the abdominal fat, the immunoblot detected AA76 in 8 materials from 8 already diagnosed patients and did not in 1 patient whose gastroduodenal mucosa was negative. In conclusion, the detection of AA76 may alter the ability to diagnose AA amyloidosis. In immunohistochemistry for fixed specimens, the new anti-AA76 antibody can improve the specificity. Immunoblot for non-fixed materials, which can considerably improve the sensitivity, should be beneficial for small materials like abdominal fat. © 2016 by the Association of Clinical Scientists, Inc.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation.

PubMed

Kim, Sook Young; Son, Myoungsun; Lee, Sang Eun; Park, In Ho; Kwak, Man Sup; Han, Myeonggil; Lee, Hyun Sook; Kim, Eun Sook; Kim, Jae-Young; Lee, Jong Eun; Choi, Ji Eun; Diamond, Betty; Shin, Jeon-Soo

2018-01-01

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia-reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

PubMed Central

Kim, Sook Young; Son, Myoungsun; Lee, Sang Eun; Park, In Ho; Kwak, Man Sup; Han, Myeonggil; Lee, Hyun Sook; Kim, Eun Sook; Kim, Jae-Young; Lee, Jong Eun; Choi, Ji Eun; Diamond, Betty; Shin, Jeon-Soo

2018-01-01

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia–reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation. PMID:29696019

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections.

PubMed

Bolognesi, Maddalena Maria; Manzoni, Marco; Scalia, Carla Rossana; Zannella, Stefano; Bosisio, Francesca Maria; Faretta, Mario; Cattoretti, Giorgio

2017-08-01

Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.

The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase.

PubMed

Edgren, Tomas; Nordlund, Stefan

2004-04-01

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.

Combination of two anti-CD5 monoclonal antibodies synergistically induces complement-dependent cytotoxicity of chronic lymphocytic leukaemia cells.

PubMed

Klitgaard, Josephine L; Koefoed, Klaus; Geisler, Christian; Gadeberg, Ole V; Frank, David A; Petersen, Jørgen; Jurlander, Jesper; Pedersen, Mikkel W

2013-10-01

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non-overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti-CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab-induced CD5 internalization and degradation. Importantly, an anti-CD5 mAb combination enhanced CDC of CLL cells when combined with the anti-CD20 mAbs rituximab and ofatumumab as well as with the anti-CD52 mAb alemtuzumab. These results suggest that an anti-CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5-expressing haematological or lymphoid malignancies. © 2013 John Wiley & Sons Ltd.

3β-Hydroxysterol Δ24-Reductase on the Surface of Hepatitis C Virus-Related Hepatocellular Carcinoma Cells Can Be a Target for Molecular Targeting Therapy

PubMed Central

Saito, Makoto; Takano, Takashi; Nishimura, Tomohiro; Kohara, Michinori; Tsukiyama-Kohara, Kyoko

2015-01-01

In our previous study, we demonstrated that 3β-hydroxysterol Δ24-reductase (DHCR24) was overexpressed in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), and that its expression was induced by HCV. Using a monoclonal antibody against DHCR24 (2-152a MAb), we found that DHCR24 was specifically expressed on the surface of HCC cell lines. Based on these findings, we aimed to establish a novel targeting strategy using 2-152a MAb to treat HCV-related HCC. In the present study, we examined the antitumor activity of 2-152a MAb. In the presence of complement, HCC-derived HuH-7 cells were killed by treatment with 2-152a MAb, which was mediated by complement-dependent cytotoxicity (CDC). In addition, the antigen recognition domain of 2-152a MAb was responsible for the unique anti-HCV activity. These findings demonstrate the feasibility of using 2-152a MAb for antibody therapy against HCV-related HCC. In addition, surface DHCR24 on HCC cells exhibited a functional property, agonist-induced internalization. We showed that 2-152a MAb-mediated binding of a cytotoxic agent (a saponin-conjugated secondary antibody) to surface DHCR24 led to significant cytotoxicity. This suggests that surface DHCR24 on HCC cells can function as a carrier for internalization. Therefore, surface DHCR24 could be a valuable target for HCV-related HCC therapy, and 2-152a MAb appears to be useful for this targeted therapy. PMID:25875901

Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120.

PubMed

Omairi-Nasser, Amin; Mariscal, Vicente; Austin, Jotham R; Haselkorn, Robert

2015-08-11

The filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Several fra gene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N- media. Cryopreservation combined with electron tomography were used to investigate the role of three fra gene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.

Expression of recombinant CD59 with an N-terminal peptide epitope facilitates analysis of residues contributing to its complement-inhibitory function.

PubMed

Zhou, Q; Zhao, J; Hüsler, T; Sims, P J

1996-10-01

CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immunodominant epitopes of CD59 are known to be sensitive to disruption of native tertiary structure, complicating immunological measurement of expressed mutant constructs for structure function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of this complement inhibitor, independent of CD59 antigen, an 11-residue peptide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted before the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cell lines expressing 0 to > 10(5) CD59/cell. The TAG-CD59 fusion protein was confirmed to be GPI-anchored, N-glycosylated and showed identical complement-inhibitory function to wild-type CD59, lacking the TAG peptide sequence. Using this construct, the contribution of each of four surface-localized aromatic residues (4Y, 47F, 61Y, and 62Y) to CD59's complement-inhibitory function was examined. These assays revealed normal surface expression with complete loss of complement-inhibitory function in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 62Y --> S mutants retained approximately 40% of function of wild-type CD59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surface aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function.

Androgen Deprivation Enhances PLZF-Repressed Cistrome that Promotes the Castration-Resistant Phenotype

DTIC Science & Technology

2014-10-01

Briefly, 10 million cells were used per IP. Cells were fixed with 1% formaldehyde solution. DNA was sonicated and subjected to immunoprecipitation with... formaldehyde solution. Total chromatin and RNAs were sonicated and subjected to immunoprecipitation with the same AR and Med1 antibodies used in ChIP...were fixed with 1% formaldehyde . Cell pellets were lysed and resuspended in restriction buffer for BstY1 and 0.1% SDS for 10 min at 65 °C. Triton X

Use of AN Eosinophil Specific Monoclonal Antibody in Assessing Eosinophil Function.

NASA Astrophysics Data System (ADS)

Minkoff, Marjorie Sue

A monoclonal antibody to an eosinophil specific determinant is very important in assessing eosinophil function during helminthic infection. Eosinophils induced by Schistosoma mansoni infection in BALB/c mice were used to induce C57B1/6 immunocytes for production of hybridomas secreting eosinophil monoclonal antibodies. These antibodies were shown to react with an eosinophil surface epitope but not with neutrophils or macrophages as determined by ELISA, immunodiffusion, immunofluorescence, and immunoblot assay. Affinity chromatography with eosinophil chemotactic factor-sepharose consistently selected out a { rm M_ R} 67,000 protein from solubilized eosinophil membrane antigens but not from neutrophil and macrophage antigens. In vitro studies showed that the eosinophil-specific monoclonal antibodies abrogated antibody-dependent eosinophil -mediated killing of S. mansoni schistosomula using mouse, rat or human eosinophils. Neutrophil and macrophage killing activities were unaffected. The monoclonal antibodies effected complement-dependent lysis of mouse and rat eosinophils but not of human eosinophils. ECF-treated eosinophils showed enhanced killing of schistosomula which was blocked by the monoclonal antibody. Murine and human eosinophils preincubated with monoclonal antibody exhibited decreased chemotaxis to ECF at optimal chemotactic concentrations. The monoclonal antibody also blocked eosinophil binding to ECF- sepharose beads. In vivo induction of peripheral blood eosinophilia by injection of S. mansoni eggs was suppressed by injections of monoclonal antibodies 2CD13 and 2QD45 in mouse and rat experimental models. Eosinophilia induced by keyhole limpet hemocyanin- cyclophosphamide treatment was also suppressed by monoclonal antibody in both murine and rat systems. Pulmonary granulomas in mice given egg injection and monoclonal antibody were smaller and contained fewer eosinophils than those granulomas from mice given eggs only. In immuno-biochemical studies, the monoclonal antibody 2QD45 specifically immunoprecipitated the {rm M_ R} 67,000 ECF-binding protein from ^{125}{rm I}-labeled mouse, rat, and human eosinophils as assessed by SDS-PAGE and autoradiography. Two-dimensional gel electrophoresis showed that this ECF-binding protein has a lower PI point than either mouse or bovine albumin.

Model of a ternary complex between activated factor VII, tissue factor and factor IX.

PubMed

Chen, Shu-wen W; Pellequer, Jean-Luc; Schved, Jean-François; Giansily-Blaizot, Muriel

2002-07-01

Upon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

Anti-aquaporin-4 autoantibodies in systemic lupus erythematosus persist for years and induce astrocytic cytotoxicity but not CNS disease.

PubMed

Alexopoulos, Harry; Kampylafka, Eleni I; Fouka, Penelope; Tatouli, Ioanna; Akrivou, Sofia; Politis, Panagiotis K; Moutsopoulos, Haralampos M; Tzioufas, Athanasios G; Dalakas, Marinos C

2015-12-15

Anti-aquaporin-4 autoantibodies are specific for the neuromyelitis optica spectrum disorders (NMOSD) and they have also been described in patients with systemic lupus erythematosus (SLE) with neurological signs consistent with NMOSD. Our objective was to test for the presence and pathogenicity of anti-AQP4 antibodies in SLE patients without neurological disease. Sera from 89 non-CNS-SLE patients were screened for anti-AQP4 autoantibodies. Two of the 89 patients were positive. Archived samples dating back 11 years were also positive. A brain and spinal cord MRI did not reveal any NMOSD-compatible lesions. An in vitro cytotoxicity assay showed that either sera or purified IgG from these patients induced a complement-mediated damage in cultured astrocytes comparable to antibodies obtained from typical NMO patients. We conclude that AQP4-antibodies can be present in SLE patients and persist for many years, without concurrent clinical or radiological NMOSD signs. It is unclear why the anti-AQP4 antibodies did not induce CNS disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Thermostability of heterophile antibodies from human sera infected with Schistosoma mansoni and geo-helminths. An immuno-metric statistical analysis.

PubMed

Chamone, Munir; Atuncar, Gregorio S; Coelho, Paulo Marcos Zech

2006-01-01

Antibody in human sera that induces lysis of sheep erythrocytes in hemolytic assay was investigated. The present study showed that the presence in serum of the thermostable cytolytic anti-sheep red blood cells antibodies is dependent on the Schistosoma mansoni infection, and this is more frequent in adults than in children. The thermostable characteristic of hemolysins in normal sera was not dependent on the presence of Ascaris lumbricoides, Trichuris trichiura or hookworm geo-helminths. Further, thermostable complement-activating heterophile antibodies were noticed in children in association with massive number of S. mansoni eggs. The results were obtained by using the z- and the chi-square tests. The z-test allows us to formulate a one-sided alternative, i.e., a tendency of one of the attributes. On the other hand, the chi-square test analyzes the independence between attributes by using a contingency table. Besides the obtained results being interesting in the field of schistosomiasis mansoni, they can provide a new insight into the use of statistics in medical science.

THE ANTIBODY-FORMATION BY POLYSACCHARIDS.

PubMed

Nishimura, S

1929-09-30

1. By complement fixation tests, it has been clearly demonstrated that the sera of rabbits immunized with inulin, soluble starch and dextrine contain specific antibodies. 2. All these immune sera gave a negative precipitation reaction. 3. The kind of dextrine which has a construction very near to starch has an antigenic property, but those in a state of further decomposition do not give rise to antibodies. 4. All the three kinds of polysaccharids have power to produce antibodies without any vehicle. Dextrine is the only one of the three that gives rise to immune bodies more readily when pig serum is added to it. 5. Regarded as antigens, inulin stood first and soluble starch and dextrine next in order. 6. All three kinds of polysaccharids that were employed gave a negative protein color reaction. All of them, however, contained nitrogen. It has been proved that the large portion of the nitrogen contained in the soluble starch is derived from its protein contents. 7. It is suggested that in the production of immune bodies by these three kinds of polysaccharids, proteins might play the part of the vehicle. This is, however, still to be determined.

Proprietary arabinogalactan extract increases antibody response to the pneumonia vaccine: a randomized, double-blind, placebo-controlled, pilot study in healthy volunteers.

PubMed

Udani, Jay K; Singh, Betsy B; Barrett, Marilyn L; Singh, Vijay J

2010-08-26

Arabinogalactan from Larch tree (Larix spp.) bark has previously demonstrated immunostimulatory activity. The purpose of this study was to test the hypothesis that ingestion of a proprietary arabinogalactan extract, ResistAid™, would selectively enhance the antibody response to the pneumococcal (pneumonia) vaccine in healthy adults. This randomized, double-blind, placebo-controlled, parallel group pilot study included 45 healthy adults who had not previously been vaccinated against Streptococcus pneumoniae. The volunteers began taking the study product or placebo (daily dosage 4.5 g) at the screening visit (V1-Day 0) and continued over the entire 72 day study period. After 30 days the subjects received the 23-valent pneumococcal vaccine (V2). They were monitored the following day (V3-Day 31), as well as 21 days (V4-Day 51) and 42 days (V5-Day 72) after vaccination. Responses by the adaptive immune system (antigen specific) were measured via pneumococcal IgG antibodies (subtypes 4, 6B, 9V, 14, 18C, 19F, and 23F) and salivary IgA levels. Responses by the innate immune system (non-specific) were measured via white blood cell counts, inflammatory cytokines and the complement system. Vaccination significantly increased pneumococcal IgG levels as expected. The arabinogalactan group demonstrated a statistically significant greater IgG antibody response than the placebo group in two antibodies subtypes (18C and 23F) at both Day 51 (p = 0.006 and p = 0.002) and at Day 72 (p = 0.008 and p = 0.041). These same subtypes (18C and 23F) also demonstrated change scores from baseline which were significant, in favor of the arabinogalactan group, at Day 51 (p = 0.033 and 0.001) and at Day 72 (p = 0.012 and p = 0.003). Change scores from baseline and mean values were greater in the arabinogalactan group than placebo for most time points in antibody subtypes 4, 6B, 9V, and 19F, but these differences did not reach statistical significance. There was no effect from the vaccine or arabinogalactan on salivary IgA, white blood cell count, inflammatory cytokines or complement. The proprietary arabinogalactan extract (ResistAid), tested in this randomized, double-blind, placebo-controlled, parallel-group pilot study, increased the antibody response of healthy volunteers to the 23-valent pneumococcal vaccine compared to placebo. ISRCTN98817459.

Proprietary arabinogalactan extract increases antibody response to the pneumonia vaccine: a randomized, double-blind, placebo-controlled, pilot study in healthy volunteers

PubMed Central

2010-01-01

Background Arabinogalactan from Larch tree (Larix spp.) bark has previously demonstrated immunostimulatory activity. The purpose of this study was to test the hypothesis that ingestion of a proprietary arabinogalactan extract, ResistAid™, would selectively enhance the antibody response to the pneumococcal (pneumonia) vaccine in healthy adults. Methods This randomized, double-blind, placebo-controlled, parallel group pilot study included 45 healthy adults who had not previously been vaccinated against Streptococcus pneumoniae. The volunteers began taking the study product or placebo (daily dosage 4.5 g) at the screening visit (V1-Day 0) and continued over the entire 72 day study period. After 30 days the subjects received the 23-valent pneumococcal vaccine (V2). They were monitored the following day (V3-Day 31), as well as 21 days (V4-Day 51) and 42 days (V5-Day 72) after vaccination. Responses by the adaptive immune system (antigen specific) were measured via pneumococcal IgG antibodies (subtypes 4, 6B, 9V, 14, 18C, 19F, and 23F) and salivary IgA levels. Responses by the innate immune system (non-specific) were measured via white blood cell counts, inflammatory cytokines and the complement system. Results Vaccination significantly increased pneumococcal IgG levels as expected. The arabinogalactan group demonstrated a statistically significant greater IgG antibody response than the placebo group in two antibodies subtypes (18C and 23F) at both Day 51 (p = 0.006 and p = 0.002) and at Day 72 (p = 0.008 and p = 0.041). These same subtypes (18C and 23F) also demonstrated change scores from baseline which were significant, in favor of the arabinogalactan group, at Day 51 (p = 0.033 and 0.001) and at Day 72 (p = 0.012 and p = 0.003). Change scores from baseline and mean values were greater in the arabinogalactan group than placebo for most time points in antibody subtypes 4, 6B, 9V, and 19F, but these differences did not reach statistical significance. There was no effect from the vaccine or arabinogalactan on salivary IgA, white blood cell count, inflammatory cytokines or complement. Conclusions The proprietary arabinogalactan extract (ResistAid™), tested in this randomized, double-blind, placebo-controlled, parallel-group pilot study, increased the antibody response of healthy volunteers to the 23-valent pneumococcal vaccine compared to placebo. Trial Registration ISRCTN98817459 PMID:20796315

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

PubMed Central

2010-01-01

Trastuzumab (Herceptin®), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab. PMID:18941743

Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides.

PubMed

Kirkham, Lea-Ann S; Wiertsema, Selma P; Corscadden, Karli J; Mateus, Tulia; Mullaney, Gemma L; Zhang, Guicheng; Richmond, Peter C; Thornton, Ruth B

2017-03-01

The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group ( P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children. Copyright © 2017 Kirkham et al.

IgM anti-myeloperoxidase antibody-secreting lymphocytes are present in the peripheral repertoire of lupus mice but rarely differentiate into IgG-producing cells

PubMed Central

Vittecoq, O; Brard, F; Jovelin, F; Le Loet, X; Tron, F; Gilbert, D

1999-01-01

Two IgM, κ anti-myeloperoxidase (MPO) monoclonal antibodies, 6D6 and 9B5, bound to MPO in a solid-phase enzyme-linked immunosorbent assay were derived from the splenocytes of (NZB × NZW) F1 and MRL/lpr-lpr mice, respectively. 6D6 gave a characteristic perinuclear immunofluorescence staining pattern on ethanol-fixed human neutrophils, bound to the native form of MPO by immunoblotting and had a high constant affinity for MPO as demonstrated by real-time specific interaction. 9B5 produced a cytoplasmic immunofluorescence staining pattern, reacted with the heavy chain of MPO and had a low constant affinity for MPO. The heavy-and light-chain variable region genes of monoclonal antibodies (mAb) 6D6 and 9B5 were sequenced and found to be highly homologous to germline genes and to contain negatively charged amino acids in the complementarity determining regions. IgM MPO-binding activity was observed in most BW and MRL/lpr-lpr mouse sera, which may correspond to polyclonal activation of B cells, whereas IgG anti-MPO antibodies could be rarely detected. Thus, this study indicates that (i) BW and MRL/lpr-lpr mice do not delete IgM anti-MPO secreting B cells, do not maintain these B cells in a state of anergy, but most individuals are not able to spontaneously induce the class-switching of this autoantibody population; (ii) IgM anti-MPO antibodies can recognize different epitopes on MPO and produce different immunofluorescence staining pattern on ethanol-fixed human neutrophils, as demonstrated by the immunochemical properties of the two lupus-mouse derived mAb. PMID:10540169

Detection of hepatitis "C" virus in formalin-fixed liver tissue by nested polymerase chain reaction.

PubMed

Sallie, R; Rayner, A; Portmann, B; Eddleston, A L; Williams, R

1992-08-01

Interpretation of antibody to hepatitis C virus (HCV) in patients with liver disease is difficult due to false-positive reactivity in some conditions. To evaluate the feasibility of HCV in archival material, HCV was sought in formalin-fixed, paraffin-embedded liver biopsy specimens. Nested polymerase chain reaction was used to detect hepatitis C virus in formalin-fixed, paraffin-embedded liver biopsy specimens after total RNA was extracted from tissue by proteinase K digestion and phenol/chloroform purification. The relative efficiency of amplification of HCV RNA from formalin-fixed material was estimated semiquantitatively by serial dilution of cDNA synthesised from RNA extracted from fresh and formalin-fixed sections from the same liver. Although HCV RNA could be detected in formalin-fixed liver tissue by nested PCR in 5/5 cases in which HCV was detected in serum, amplification was approximately 5-fold less efficient than when HCV was amplified from fresh tissue. Nevertheless, nested PCR of HCV from formalin-fixed liver tissue represents a useful technique in addressing some important questions related to the pathogenesis of liver disease.

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics.

PubMed

Devanaboyina, Siva Charan; Lynch, Sandra M; Ober, Raimund J; Ram, Sripad; Kim, Dongyoung; Puig-Canto, Alberto; Breen, Shannon; Kasturirangan, Srinath; Fowler, Susan; Peng, Li; Zhong, Haihong; Jermutus, Lutz; Wu, Herren; Webster, Carl; Ward, E Sally; Gao, Changshou

2013-01-01

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a "buffering" effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0-7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.

Histologic evaluation of post-implantation immediate C4d deposition in 13 intestinal grafts: correlation with cell-based crossmatching, cold ischemia time, and preservation injury.

PubMed

López-García, P; Calvo Pulido, J; Colina, F; Ballestin Carcavilla, C; Jiménez-Romero, C; Martinez González, M A; Ibarrola de Andrés, C; López-Alonso, G; Cambra Molero, F; Justo Alonso, I; Moreno-González, E

2014-01-01

C4d deposits are predictive of humoral rejection in kidney and heart transplantation. The aim of this study was to identify C4d deposit patterns in intestinal mucosa of the grafts on biopsy specimens obtained immediately after implantation and to detect if it could be a valuable tool to predict humoral or acute rejection. A second objective was to search for a statistically significant relationship between positive C4d deposition and other collected variables. Thirteen immediately post-transplantation mucosal graft biopsy specimens, formalin fixed, underwent immunohistochemical stain for C4d deposits. Diffuse intense staining of capillary endothelium was considered positive and absent, focal or weak stains as negative. Preservation injury grade and cold ischemia times were registered for each case. Donor-specific preformed antibodies were detected by complement dependent cytotoxicity serologic technique (crossmatching). Another 19 endoscopic follow-up biopsy specimens from days 2 to 6 were also evaluated. Statistical studies were made using the index of correlation ρ (Spearman's test). Diffuse intense C4d deposits were observed in 2 grafts, focal and weak in 5, and completely negative in 6. The mean cold ischemia time was 327 ± 101 minutes. Two cases showed diffuse positive deposits, 1 had a positive crossmatch and the cold ischemia time was 360 minutes whereas the other had not preformed antibodies and its cold ischemia time was 475 minutes. Humoral or acute rejection was not observed in follow-up mucosal biopsy specimens. There was no statistically significant relationship between the C4d deposition, cold ischemia time, crossmatching results, and preservation injury degree. In conclusion, C4d deposition was not a helpful tool for diagnosis of humoral rejection and prediction of acute rejection during the early post-transplantation period. Copyright © 2014 Elsevier Inc. All rights reserved.

Neutrophil extracellular traps can activate alternative complement pathways.

PubMed

Wang, H; Wang, C; Zhao, M-H; Chen, M

2015-09-01

The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). ANCAs activate primed neutrophils to release neutrophil extracellular traps (NETs), which have recently gathered increasing attention in the development of AAV. The relationship between NETs and alternative complement pathway has not been elucidated. The current study aimed to investigate the relationship between NETs and alternative complement pathway. Detection of components of alternative complement pathway on NETs in vitro was assessed by immunostain and confocal microscopy. Complement deposition on NETs were detected after incubation with magnesium salt ethyleneglycol tetraacetic acid (Mg-EGTA)-treated human serum. After incubation of serum with supernatants enriched in ANCA-induced NETs, levels of complement components in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Complement factor B (Bb) and properdin deposited on NETs in vitro. The deposition of C3b and C5b-9 on NETs incubated with heat-inactivated normal human serum (Hi-NHS) or EGTA-treated Hi-NHS (Mg-EGTA-Hi-NHS) were significantly less than that on NETs incubated with NHS or EGTA-treated NHS (Mg-EGTA-NHS). NETs induced by ANCA could activate the alternative complement cascade in the serum. In the presence of EGTA, C3a, C5a and SC5b-9 concentration decreased from 800·42 ± 244·81 ng/ml, 7·68 ± 1·50 ng/ml, 382·15 ± 159·75 ng/ml in the supernatants enriched in ANCA induced NETs to 479·07 ± 156·2 ng/ml, 4·86 ± 1·26 ng/ml, 212·65 ± 44·40 ng/ml in the supernatants of DNase I-degraded NETs (P < 0·001, P = 0·008, P < 0·001, respectively). NETs could activate the alternative complement pathway, and might thus participate in the pathogenesis of AAV. © 2015 British Society for Immunology.

[Immunodetection of bacteriophages by a piezoelectric resonator with lateral electric field].

PubMed

Gulii, O I; Zaitsev, B D; Shikhabudinov, A M; Teplykh, A A; Borodina, I A; Pavlii, S A; Larionova, O S; Fomin, A S; Staroverov, S A; Dykman, L A; Ignatov, O V

2016-01-01

It has been demonstrated that electroacoustic analysis with polyclonal antibodies can be used for bacteriophage detection. The frequency dependences of the real and imaginary parts of electrical impedance of a resonator with a viral suspension with antibodies were shown to be essentially different from the dependences of a resonator with control viral suspension without antibodies. It was shown that ΦAl-Sp59b bacteriophages were detected with the use of antibodies in the presence of foreign virus particles. The ΦAl-Sp59b bacteriophage content in the analyzed suspension was ~1010–106 phages/mL; the time of analysis was no more than 5 min. The optimally informative parameter for obtaining reliable information was the change in the real or imaginary part of electrical impedance at a fixed frequency near the resonance upon the addition of specific antibodies to the analyzed suspension. It was demonstrated that the interaction between bacteriophages and antibodies can be recorded, offering good prospects for the development of a biological sensor for liquid-phase identification and virus detection.

Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes

PubMed Central

Posch, Wilfried; Cardinaud, Sylvain; Hamimi, Chiraz; Fletcher, Adam; Mühlbacher, Annelies; Loacker, Klaus; Eichberger, Paul; Dierich, Manfred P.; Pancino, Gianfranco; Lass-Flörl, Cornelia; Moris, Arnaud; Saez-Cirion, Asier; Wilflingseder, Doris

2014-01-01

Background Control of HIV is suggested to depend on potent effector functions of the virus-specific CD8+ T-cell response. Antigen opsonization can modulate the capture of antigen, its presentation, and the priming of specific CD8+ T-cell responses. Objective We have previously shown that opsonization of retroviruses acts as an endogenous adjuvant for dendritic cell (DC)–mediated induction of specific cytotoxic T lymphocytes (CTLs). However, in some HIV-positive subjects, high levels of antibodies and low levels of complement fragments coat the HIV surface. Methods Therefore we analyzed the effect of IgG opsonization on the antigen-presenting capacity of DCs by using CD8+ T-cell proliferation assays after repeated prime boosting, by measuring the antiviral activity against HIV-infected autologous CD4+ T cells, and by determining IFN-γ secretion from HIV-specific CTL clones. Results We find that DCs exposed to IgG-opsonized HIV significantly decreased the HIV-specific CD8+ T-cell response compared with the earlier described efficient CD8+ T-cell activation induced by DCs loaded with complement-opsonized HIV. DCs exposed to HIV bearing high surface IgG levels after incubation in plasma from HIV-infected subjects acted as weak stimulators for HIV-specific CTL clones. In contrast, HIV opsonized with plasma from patients exhibiting high complement and low IgG deposition on the viral surface favored significantly higher activation of HIV-specific CD8+ T-cell clones. Conclusion Our ex vivo and in vitro observations provide the first evidence that IgG opsonization of HIV is associated with a decreased CTL-stimulatory capacity of DCs. PMID:23063584