Source Of Complement Research Articles

Variable sensitivity to complement-dependent cytotoxicity in murine models of neuromyelitis optica.

BackgroundStudies of neuromyelitis optica (NMO), an autoimmune disease of the central nervous system (CNS), have demonstrated that autoantibodies against the water channel aquaporin-4 (AQP4) induce astrocyte damage through complement-dependent cytotoxicity (CDC). In developing experimental models of NMO using cells, tissues or animals from mice, co-administration of AQP4-IgG and normal human serum, which serves as the source of human complement (HC), is required. The sensitivity of mouse CNS cells to HC and CDC in these models is not known.MethodsWe used HC and recombinant monoclonal antibodies (rAbs) against AQP4 to investigate CDC on mouse neurons, astrocytes, differentiated oligodendrocytes (OLs), and oligodendrocyte progenitors (OPCs) in the context of purified monocultures, neuroglial mixed cultures, and organotypic cerebellar slices.ResultsWe found that murine neurons, OLs, and OPCs were sensitive to HC in monocultures. In mixed murine neuroglial cultures, HC-mediated toxicity to neurons and OLs was reduced; however, astrocyte damage induced by an AQP-specific rAb #53 and HC increased neuronal and oligodendroglial loss. OPCs were resistant to HC toxicity in neuroglial mixed cultures. In mouse cerebellar slices, damage to neurons and OLs following rAb #53-mediated CDC was further reduced, but in contrast to neuroglial mixed cultures, astrocyte damage sensitized OPCs to complement damage. Finally, we established that some injury to neurons, OLs, and OPCs in cell and slice cultures resulted from the activation of HC by anti-tissue antibodies to mouse cells.ConclusionsMurine neurons and oligodendroglia demonstrate variable sensitivity to activated complement based on their differentiation and culture conditions. In organotypic cultures, the protection of neurons, OLs, and OPCs against CDC is eliminated by targeted astrocyte destruction. The activation of human complement proteins on mouse CNS cells necessitates caution when interpreting the results of mouse experimental models of NMO using HC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0767-4) contains supplementary material, which is available to authorized users.

Do Dose Numbers Matter?: Evaluation of Differing Infant and Toddler Meningococcal C Conjugate Vaccine Programs in Canadian Children.

The diversity of Canadian infant meningococcal C conjugate (MenC) vaccine programs is unique among countries providing MenC vaccines and offers a valuable opportunity to determine the optimal vaccine program. This longitudinal study assessed differences in seroprotection by 3 different vaccine schedules in children two years after receiving either 1 toddler MenC vaccine dose (1 dose), 1 infant and 1 toddler dose (2 doses), or 2 infant and 1 toddler MenC vaccine dose (3 doses). Three similar cohorts of healthy infants from 1, 2 and 3 dose program areas were enrolled before to their 12 month toddler dose and vaccinated with MenC-tetanus toxoid (MenC-TT) conjugate vaccine. Sera obtained 2 years later were assayed for serogroup C bactericidal activity using standardized procedures with rabbit as the exogenous complement source. Serum bactericidal activity titers ≥1:8 were considered protective. Results were available for 384 children. Rates of seroprotection at 36 months of age were significantly different between the 1 and 3 dose programs, but confidence intervals overlapped between the 1 and 2 dose programs and between the 2 and 3 dose programs: 1 dose 92% (95% confidence interval: 86%-96%) versus 99% (95%-100%) with 2 doses and 100% (97%-100%) with 3 doses. Geometric mean titers were significantly different at 12.1 (10.8-13.5), 32.4 (28.9-36.2) and 50.6 (45.7-55.9) in the 1, 2 and 3 dose programs, respectively. At 36 months of age, evidence of seroprotection remained for greater than 90% of participants. Our results indicate that 1 toddler dose or 1 infant plus 1 toddler dose with MenC-TT vaccine provides seroprotection against MenC disease in early childhood.

Low pH impairs complement-dependent cytotoxicity against IgG-coated target cells.

Local acidosis is a common feature of allergic, vascular, autoimmune, and cancer diseases. However, few studies have addressed the effect of extracellular pH on the immune response. Here, we analyzed whether low pH could modulate complement-dependent cytotoxicity (CDC) against IgG-coated cells. Using human serum as a complement source, we found that extracellular pH values of 5.5 and 6.0 strongly inhibit CDC against either B lymphoblast cell lines coated with the chimeric anti-CD20 mAb rituximab or PBMCs coated with the humanized anti-CD52 mAb alemtuzumab. Suppression of CDC by low pH was observed either in cells suspended in culture medium or in whole blood assays. Interestingly, not only CDC against IgG-coated cells, but also the activation of the complement system induced by the alternative and lectin pathways was prevented by low pH. Tumor-targeting mAbs represent one of the most successful tools for cancer therapy, however, the use of mAb monotherapy has only modest effects on solid tumors. Our present results suggest that severe acidosis, a hallmark of solid tumors, might impair complement-mediated tumor destruction directed by mAb.

Mechanisms responsible for differential bactericidal activities of human and rabbit complement for Neisseria meningitidis

Polysaccharide conjugate vaccines are available to prevent disease caused by Neisseria meningitidis serogroups A, C, W and Y. Meningococcal vaccine efficacy is assessed in clinical trials using serum bactericidal assays (SBAs). Baby rabbit serum is usually used as the source of complement for SBAs as it lacks endogenous bactericidal activity compared to human serum. Previous studies have shown that SBA activities determined with rabbit (rSBA) and human (hSBA) complement correlate poorly, possibly due to different interactions between antibody subclasses and the complement source, and species-specific interaction of Neisseria meningitidis with complement regulators. The aim of this project was to investigate the mechanisms responsible for differential bactericidal activities of human and rabbit complement against Neisseria meningitidis. The serum concentration of polysaccharide-specific antibody subclasses was measured following vaccination with quadrivalent meningococcal polysaccharide vaccines; data showed that the concentration of polysaccharide-specific IgG1 antibody correlated most significantly with hSBA titres whereas the concentration of polysaccharide-specific IgM antibody correlated most significantly with rSBA titres. The interaction of human IgM and IgG subclasses with human and rabbit complement was compared at the level of C1q and C3 using both binding and functional assays. These data define important differences in the ability of human antibody subclasses to fix human and rabbit complement. Specifically, polysaccharide-specific IgM contributes significantly more to bactericidal titres in rSBAs compared to hSBAs. As a consequence, rSBAs produce misleadingly high titres in individuals with large IgM responses to vaccination. Using a series of pathway-specific inhibitors, it was shown that the alternative pathway contributes significantly more to the bactericidal activity of rabbit complement towards Neisseria meningitidis than human complement. This project provides significant insight into the difficulties and challenges associated with the interpretation of rSBA data, enhances the understanding of antibody responses to meningococcal vaccines and will support improvements in the development and testing of meningococcal vaccines.

The Network Advantage of MNCs and Business Groups and Foreign Subsidiary Performance

Drawing on the literature on multinational corporations (MNCs) and business groups, we argue that the sources of multinational advantage and business group advantage complement each other in intern...

Antithymocyte Globulin at Clinically Relevant Concentration Kills Leukemic Blasts

Background/Rationale: Success of allogeneic hematopoietic cell transplantation (HCT) is limited due to graft-versus-host disease (GvHD) and leukemia relapse. Contrary to small molecule immunosuppressive drugs like cyclosporine or methotrexate, rabbit antithymocyte globulin (ATG) used for GvHD prophylaxis with myeloablative HCT conditioning does not increase relapse. The mechanism of the anti-GvHD effect of ATG has been studied extensively, however, the reason for ATG not increasing relapse is not known. We hypothesized that ATG has an anti-leukemic activity. Here we investigated the anti-leukemic activity of ATG in vitro at concentrations achieved in vivo with our standard ATG dose of 4.5 mg/kg (median 10 mg per liter of serum; range, 1-25). We focused on complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC).Methods: Blood or bone marrow from patients newly diagnosed with acute myeloid leukemia (AML, n=25) or acute lymphoid leukemia (ALL, n=4), obtained before induction chemotherapy, was used as the source of leukemic cells. We measured CDC and CIC of ATG, specifically against leukemic blasts at clinically achieved concentrations (1-25 mg/L) and an achievable concentration (50 mg/L). The leukemic blasts were identified as CD45dim/neg and side scatter low (SSclow) cells in case of AML-M0, M1 or M2 (according to British-American-French classification) or ALL. In case of AML-M4 or M5, we gated on CD45dim/neg and SSclow/intermediate cells. These cells could contain both leukemic and normal blasts and some post-blast (maturing/mature) cells. We minimized the fraction of normal blasts and post-blast cells included by introducing a "dump channel". The dump channel consisted of antibodies against antigens expressed by cells of lineages other than the leukemia lineage (except for antigens aberrantly expressed by the leukemic cells) and by post-blast cells. For most AML patients, the dump channel consisted of CD10, CD19, CD41a, CD235a, CD14 and CD16. For most ALL patients, the dump channel consisted of CD117, CD33, CD41a, CD235a, CD14 and CD16. Thus, leukemic blasts were defined as mononuclear cells that were CD45dim/neg and SSclow (or SSclow/intermediate for M4/M5) and did not express dump channel antigens. CDC was induced by incubation of the patient specimen with human serum (source of complement). CIC was induced by incubation of the patient specimen with heat-inactivated serum (no complement). For CDC dead cells were identified as 7-amino-actinomycin D positive (7AAD+). For CIC dead/dying cells were identified as Annexin V positive (Annexin V+).Results: ATG induced death of leukemic blasts both via CDC and CIC. Median 0.3%, 3.0%, 12.6% and 41.7% blasts were killed with 1, 10, 25 and 50 mg/L ATG, respectively, via CDC. Median 2.1%, 8.0%, 11.4% and 12.6% blasts were killed after 4 hour incubation with 1, 10, 25 and 50 mg/L ATG, respectively, via CIC. For both CDC and CIC, the percents were significantly higher than background (p<0.05). There was a high variability in the sensitivity of the leukemic blasts to ATG. The adjusted (background subtracted) percent of blasts killed ranged from 2.6% to 93.8% via CDC (Figure 1) and from 1.5% to 69.9% via CIC (Figure 2). When we arbitrarily defined a patient with resistant leukemic blasts as <10% (adjusted) leukemic blasts dead after exposure to 50 mg/L ATG, there were 3/28 patients with resistant leukemic blasts in case of CDC (Figure 1) and 9/23 patients in case of CIC (Figure 2).Conclusion: ATG at clinically relevant concentrations kills primary leukemic blasts in vitro. Some acute leukemias are highly sensitive and others relatively resistant to ATG. Whether this applies to in vivo needs to be determined. If yes, and given that the killing of leukemic cells is ATG concentration-dependent, conditioning regimens with high dose ATG could be developed for patients with leukemia sensitive to ATG to maximize the concurrent anti-GvHD and anti-relapse effects. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay.

Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing, we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Primary cultures of ECs were pre-treated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL-4-/IL-13-induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin.

Development of a serological assay to predict antibody bactericidal activity against non-typeable Haemophilus influenzae

BackgroundNon-typeable Haemophilus influenzae (NTHi) is a Gram negative microorganism residing in the human nasopharyngeal mucosa and occasionally causing infections of both middle ear and lower respiratory airways. A broadly protective vaccine against NTHi has been a long-unmet medical need, as the high genetic variability of this bacterium has posed great challenges.ResultsIn this study, we developed a robust serum bactericidal assay (SBA) to optimize the selection of protective antigens against NTHi. SBA takes advantage of the complement-mediated lysis of bacterial cells and is a key in vitro method for measuring the functional activity of antibodies. As a proof of concept, we assessed the bactericidal activity of antibodies directed against antigens known to elicit a protective response, including protein D used as carrier protein in the Synflorix pneumococcal polysaccharide conjugate vaccine. Prior to SBA screening, the accessibility of antigens to antibodies and the capacity of the latter to induce C3 complement deposition was verified by flow cytometry. Using baby rabbit serum as a source of complement, the proposed assay not only confirmed the bactericidal activity of the antibodies against the selected vaccine candidates, but also showed a significant reproducibility.ConclusionsConsidering the rapidity and cost-effectiveness of this novel SBA protocol, we conclude that it is likely to become an important tool to prove the capability of antibodies directed against recombinant antigens to induce NTHi in vitro killing and to both select new protective vaccine candidates, and predict vaccine efficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0420-x) contains supplementary material, which is available to authorized users.

Structural and functional studies of BapC protein of Bordetella pertussis

Bordetella pertussis, the causative agent of whooping cough, attaches to mucosal surface in upper respiratory tract, where it produces a variety of surface associated and secreted autotransporter molecules among others. In this study we have cloned newly identified member of autotransporter family BapC (B. pertussis autotransporter protein C); expressed it in Escherichia coli and characterized it for its different properties. We have also raised antisera to BapC protein; the antisera were used in immunofluorescence assay to determine the surface association of the protein. Results suggest that BapC in B. pertussis Taberman parent is surface exposed when compared with the respective BapC mutant. The neutralizing effect of anti-BapC serum was also evaluated in the presence of active complement proteins and results suggest that antiserum can potentiate the killing of B. pertussis cells in the presence of added source of complement. Structure of the protein was also studied, both α and β domains of the protein were modeled, β domain exhibits typical transmembrane β-barrel porin topology whereas α domain behaves as a characteristic bacterial autotransporter passenger domain.

Bactericidal monoclonal antibodies specific to the lipopolysaccharide O antigen from multidrug-resistant Escherichia coli clone ST131-O25b:H4 elicit protection in mice.

The Escherichia coli sequence type 131 (ST131)-O25b:H4 clone has spread worldwide and become responsible for a significant proportion of multidrug-resistant extraintestinal infections. We generated humanized monoclonal antibodies (MAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These MAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assay in vitro, MAbs induced >95% bacterial killing in the presence of human serum as the complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of action in vivo was investigated by using aglycosylated derivatives of the protective MAbs. The significant binding to live E. coli cells and the in vitro and in vivo efficacy were corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multidrug-resistant Gram-negative pathogens, passive immunization with bactericidal antibodies offers a therapeutic alternative to control infections caused by E. coli ST131-O25b:H4.

Seroprevalence of Antibody-Mediated, Complement-Dependent Opsonophagocytic Activity against Neisseria meningitidis Serogroup B in England.

The correlate of protection for the licensure of meningococcal vaccines is serum bactericidal activity. However, evidence indicates that a complex situation and other mechanisms, such as antibody-mediated, complement-dependent opsonophagocytosis (OP), may play a role in protection and should be investigated in order to understand immunity to this disease. In this study, a high-throughput flow cytometric opsonophagocytic assay (OPA) was optimized. The assay measures the presence of killed fluorescently labeled Neisseria meningitidis within human granulocytes (differentiated HL60 cells) by flow cytometry, using IgG-depleted pooled human plasma as an exogenous source of complement. This method was found to be reliable and correlated with the results of an opsonophagocytic killing assay. The OPA was used to measure OP activity in 1,878 serum samples from individuals ranging from 0 to 99 years of age against N. meningitidis strain NZ98/254 (B:4:P1.7-2,4). The levels of OP activity in individual serum samples varied greatly. OP activity showed an initial peak in the 6- to 12-month age group corresponding to a peak in disease incidence. The OP activity dropped in childhood until the late teenage years, although there was still a higher percentage of individuals with OP activity than with protective bactericidal antibody titers. OP activity reached a peak in the 30- to 39-year age group and then declined. This later peak in OP activity did not coincide with the young adults in whom peak serum bactericidal activity and disease incidence occurred. The demonstration of OP activity when disease incidence is low and when protective bactericidal antibody titers are not detected may indicate a role for OP in protection from meningococcal disease in these age groups.

Immune responses following meningococcal serogroups A, C, Y and W polysaccharide vaccination in C2-deficient persons: Evidence for increased levels of serum bactericidal antibodies

Complement C2 deficiency (C2D) is associated with immunological diseases and increased susceptibility to invasive infections caused by encapsulated bacteria such as Neisseria menigitidis. In this study we evaluate the immunogenicity of vaccination against N. menigitidis in C2D.C2D patients (n=22) and controls (n=52) were given a tetravalent meningococcal polysaccharide vaccine. Serum bactericidal antibody (SBA) titres (serogroups A, C, Y and W) were analysed using a rabbit complement source. Levels of IgG, IgM, and IgA, factor B, and factor H, polymorphisms of MBL and Fc-gamma receptors were determined.The C2D patients responded with an increased SBA titre to all four serogroups (p<0.001). The response rates define as SBA titres ≥8 were found to be between 85.7% and 92.5%. The post-vaccination titres for serogroups C, Y and W were equal to healthy controls. C2D patients with a history of invasive infection had a lower post-vaccination SBA titres both compared to healthy C2D persons (p=0.03) and compared to controls (p<0.0001). We found that the G2M*n/G2M*n genotype were associated with a higher SBA titres after immunization (p=0.03). None of the other investigated immunological factors appear to be important in influencing the vaccine responses. Autoimmune diseases in C2D did not affect the vaccine response.In general, vaccination against meningococci gave rise to antibody responses in the C2D patients that equal healthy controls. The response rate was lower to serogroup A and among C2D patients with history of invasive infections. The presence of G2M*n/G2M*n genotype was associated with higher SBA titres after immunization.

Endothelial progenitor cell-derived extracellular vesicles protect from complement-mediated mesangial injury in experimental anti-Thy1.1 glomerulonephritis.

Endothelial progenitor cells (EPCs) are known to induce tissue repair by paracrine mechanisms including the release of growth factors and extracellular vesicles (EVs), nanoparticles able to carry proteins and genetic information to target cells. The aim of this study was to evaluate whether EVs derived from EPCs may protect from complement-mediated mesangial injury in experimental anti-Thy1.1 glomerulonephritis. EVs were isolated by serial ultracentrifugation from supernatants of cultured human EPCs and characterized for their protein and RNA content. In vivo, EVs were injected i.v. in the experimental rat model of mesangiolytic anti-Thy1.1 glomerulonephritis evaluating renal function, proteinuria, complement activity and histological lesions. In vitro, the biological effects of EPC-derived EVs were studied in cultured rat mesangial cells incubated with anti-Thy1.1 antibody and rat or human serum as complement source. After i.v. injection in Thy1.1-treated rats, EVs localized within injured glomeruli and inhibited mesangial cell activation, leucocyte infiltration and apoptosis, decreased proteinuria, increased serum complement haemolytic activity (CH50) and ameliorated renal function. EV treatment decreased intraglomerular deposition of the membrane attack complex (MAC or C5b-9) and expression of smooth muscle cell actin and preserved the endothelial antigen RECA-1 and the podocyte marker synaptopodin. The protective effect of EVs was significantly reduced by pre-treatment with a high dose of RNase (1 U/mL), suggesting a key role for EV-carried RNAs in these mechanisms. Indeed, EPC-derived EVs contained different mRNAs coding for several anti-apoptotic molecules and for the complement inhibitors Factor H, CD55 and CD59 and the related proteins. The in vitro experiments aimed to investigate the mechanisms of EV protection indicated that EVs transferred to mesangial cell mRNAs coding for Factor H, CD55 and CD59 and inhibited anti-Thy1.1 antibody/complement-induced apoptosis and C5b-9/C3 mesangial cell deposition. EVs derived from EPCs exert a protective effect in Thy1.1 glomerulonephritis by inhibition of antibody- and complement-mediated injury of mesangial cells.

Trichinella spiralis: killing of newborn larvae by lung cells.

The migratory stage of Trichinella spiralis, the newborn larva (NBL), travels along the pulmonary microvascular system on its way to the skeletal muscle cells. The present work studies the capability of lung cells to kill NBL. For this purpose, in vitro cytotoxicity assays were performed using NBL, lung cell suspensions from Wistar rats, rat anti-NBL surface sera, and fresh serum as complement source. The cytotoxic activity of lung cells from rats infected on day 6 p.i. was compared with that from noninfected rats. Two and 20 h-old NBL (NBL2 and NBL20) were used as they had shown to exhibit different surface antigens altering their biological activity. Sera antibodies were analyzed by indirect immunofluorescence assay, and cell populations used in each assay were characterized by histological staining. The role of IgE in the cytotoxic attack against NBL was analyzed using heated serum. The FcεRI expression on cell suspensions was examined by flow cytometry. Results showed that lung cells were capable of killing NBL by antibody-dependent cell-mediated cytotoxicity (ADCC). Lung cells from infected animals yielded the highest mortality percentages of NBL, with NBL20 being the most susceptible to such attack. IgE yielded a critical role in the cytotoxic attack. Regarding the analysis of cell suspensions, cells from infected rats showed an increase in the percentage of eosinophils, neutrophils, and the number of cells expressing the FcεRI receptor. We conclude that lung cells are capable of killing NBL in the presence of specific antibodies, supporting the idea that the lung is one of the sites where the NBL death occurs due to ADCC.

Non-lytic autoantibody mediated effects on myelination are associated with increased pro-inflammatory chemokine expression

Multiple sclerosis is associated with intrathecal synthesis of immunoglobulins directed against sulphatide, a major CNS myelin glycolipid. To explore the clinical/pathological relevance of this response we investigated the effect of treating myelinating cultures with a sulphatide-reactive IgM mouse monoclonal antibody (O4). In the absence of an exogenous source of complement the mAb was unable to induce demyelination, but inhibited myelination, an effect associated with microglial proliferation and activation.

Shielding of a lipooligosaccharide IgM epitope allows evasion of neutrophil-mediated killing of an invasive strain of nontypeable Haemophilus influenzae.

ABSTRACTNontypeable Haemophilus influenzae is a frequent cause of noninvasive mucosal inflammatory diseases but may also cause invasive diseases, such as sepsis and meningitis, especially in children and the elderly. Infection by nontypeable Haemophilus influenzae is characterized by recruitment of neutrophilic granulocytes. Despite the presence of a large number of neutrophils, infections with nontypeable Haemophilus influenzae are often not cleared effectively by the antimicrobial activity of these immune cells. Herein, we examined how nontypeable Haemophilus influenzae evades neutrophil-mediated killing. Transposon sequencing (Tn-seq) was used on an isolate resistant to neutrophil-mediated killing to identify genes required for its survival in the presence of human neutrophils and serum, which provided a source of complement and antibodies. Results show that nontypeable Haemophilus influenzae prevents complement-dependent neutrophil-mediated killing by expression of surface galactose-containing oligosaccharide structures. These outer-core structures block recognition of an inner-core lipooligosaccharide epitope containing glucose attached to heptose HepIII-β1,2-Glc by replacement with galactose attached to HepIII or through shielding HepIII-β1,2-Glc by phase-variable attachment of oligosaccharide chain extensions. When the HepIII-β1,2-Glc-containing epitope is expressed and exposed, nontypeable Haemophilus influenzae is opsonized by naturally acquired IgM generally present in human serum and subsequently phagocytosed and killed by human neutrophils. Clinical nontypeable Haemophilus influenzae isolates containing galactose attached to HepIII that are not recognized by this IgM are more often found to cause invasive infections.

THU0535 Chronic TNF Alpha Primes Fibroblast-Like Synoviocytes in A Gene-Specific Manner by Removing the Chromatin Barrier

BackgroundFibroblast-like synoviocytes (FLS) have recently emerged as attractive therapeutic targets in rheumatoid arthritis (RA). FLS, during the chronic course of synovitis in RA, are a major source of inflammatory cytokines,...

Serum Bactericidal Assays To Evaluate Typhoidal and Nontyphoidal Salmonella Vaccines

Invasive Salmonella infections for which improved or new vaccines are being developed include enteric fever caused by Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B and sepsis and meningitis in young children in sub-Saharan Africa caused by nontyphoidal Salmonella (NTS) serovars, particularly S. enterica serovars Typhimurium and Enteritidis. Assays are needed to measure functional antibodies elicited by the new vaccines to assess their immunogenicities and potential protective capacities. We developed in vitro assays to quantify serum bactericidal antibody (SBA) activity induced by S. Typhi, S. Paratyphi A, S. Typhimurium, and S. Enteritidis vaccines in preclinical studies. Complement from various sources was tested in assays designed to measure antibody-dependent complement-mediated killing. Serum from rabbits 3 to 4 weeks of age provided the best complement source compared to serum from pigs, goats, horses, bovine calves, or rabbits 8 to 12 weeks of age. For S. Enteritidis, S. Typhimurium, and S. Typhi SBA assays to be effective, bacteria had to be harvested at log phase. In contrast, S. Paratyphi A was equally susceptible to killing whether it was grown to the stationary or log phase. The typhoidal serovars were more susceptible to complement-mediated killing than were the nontyphoidal serovars. Lastly, the SBA endpoint titers correlated with serum IgG anti-lipopolysaccharide (LPS) titers in mice immunized with mucosally administered S. Typhimurium, S. Enteritidis, and S. Paratyphi A but not S. Typhi live attenuated vaccines. The SBA assay described here is a useful tool for measuring functional antibodies elicited by Salmonella vaccine candidates.

Querying concepts in product data by means of query expansion

The Web has become the primary source of information containing both structured and unstructured information. A good example is e-commerce where products are usually described by technical specifications structured data and textual user reviews unstructured data. Both sources of information complement each other, covering quantifiable as well as perceived aspects of each product. In fact, for most searches users will have more or less abstract concepts in mind, as opposed to clear cut categorical information. In this paper, we develop a novel approach to reveal implicit product features for querying, by combining structured product data with natural-language product reviews. Using a self-supervised learning technique we progressively build a query-aware representation of the product domain under consideration. This representation can then effectively be used for intuitive querying. We performed extensive experiments confirming the effectiveness of our approach over real world product data. In particular, our evaluations show vastly improved precision and recall over the respective IR techniques.

HDAC6 Inhibition Increases CD20 Level and Improves The Efficacy Of Anti-CD20 Monoclonal Antibodies

CD20, an integrate membrane protein expressed on the surface of normal and malignant B-cells is widely used as a molecular target for monoclonal antibodies (mAbs) in the therapy of non-Hodgkin's lymphomas and chronic lymphocytic leukemia (CLL). Accumulating evidence indicates that CD20 can be modulated at several levels, both transcriptional and posttranscriptional and its up-regulation would result in increased efficacy of anti-CD20 mAbs. CD20 antigen has been reported to be regulated epigenetically e.g. by histone deacetylases (HDACs). The results of our preliminary experiments show that use of non-selective HDAC inhibitors as well as blocking the activity of a single HDAC isoform - HDAC6 leads to up-regulation of CD20 protein in B-cell lymphoma cell lines and increase of the efficacy of therapy with anti-CD20 mAbs. Since HDAC6 is engaged mainly in the acetylation of non-histone substrates and the observed up-regulation of CD20 molecule does not seem to rely on transcriptional mechanisms we postulate that HDAC6 is engaged in processes of CD20 trafficking or/and degradation. CD20 being a membrane bound protein is most probably undergoing endocytosis. However, this process and the role of HDAC6 in its regulation has not been explored so far.The aim of this study was to understand how the inhibition of HDAC6 activity influences CD20 level in normal and malignant B-cells. We wanted to determine the mechanisms underlying this phenomena.This study required use of B-cell lymphoma cell lines as well as lymphocytes infected with Epstein-Barr virus and normal B- lymphocytes. Several HDAC pan-inhibitors and HDAC6 inhibitors were tested. To assess the membrane level of CD20 antigen, FITC-anti-CD20 staining was performed followed by cytometric analysis. The influence of HDACi on total level of CD20 protein was assessed in Western blotting. The complement-dependent cytotoxicity (CDC) assay was performed using rituximab and ofatumumab as well as human serum as a source of complement. Cell cytotoxicity was assessed by propidium iodide staining followed by cytometric analysis. The influence of HDAC inhibition on the transcription of CD20 was examined by qRT-PCR using SyBR Green and hydrolysis probes. The activity of CD20 promoter after inhibition of HDAC was assessed in Dual Luciferase Assay. The colocalisation of CD20 with other proteins that may influence its trafficking/degradation was assessed using immunocytochemistry with specific antibodies and observed under confocal microscope.The results of our study strongly suggest that combining HDACi with anti-CD20 antibodies can be an effective therapeutic modality for patients suffering from B-cell malignancies. Extensive experiments aiming at determining what factors are engaged in the regulation of CD20 by HDAC6 are planned. [Display omitted] Disclosures:No relevant conflicts of interest to declare.