Factor H (fH), a key alternative complement pathway regulator, is a cofactor for factor I-mediated cleavage of C3b. fH consists of 20 short consensus repeat (SCR) domains. Sialic acid binding domains ...have previously been localized to fH SCRs 6-10 and 13. To examine fH binding on a sialylated microbial surface, we grew Neisseria gonorrhoeae in the presence of 5'-cytidinemonophospho-N-acetylneuraminic acid, which sialylates lipooligosaccharide and converts to serum resistance gonococci previously sensitive to nonimmune serum killing. fH domains necessary for binding sialylated gonococci were determined by incubating organisms with recombinant human fH (rH) and nine mutant rH molecules (deletions spanning the entire fH molecule). rH and all mutant rH molecules that contained SCRs 16-20 bound to the sialylated strain; no mutant molecule bound to serum-sensitive nonsialylated organisms. Sialic acid was demonstrated to be the fH target by flow cytometry that showed a fourfold increase in fH binding that was reversed by neuraminidase-mediated cleavage of sialic acid off gonococci. Functional specificity of fH was confirmed by decreased total C3 binding and almost complete conversion to iC3b on sialylated gonococci. Sialic acid can therefore bind fH uniquely through SCRs 16-20. This blocks complement pathway activation for N. gonorrhoeae at the level of C3.
Despite considerable advances in the understanding of the pathogenesis of meningococcal disease, this infection remains a major cause of morbidity and mortality globally. The role of the complement ...system in innate immune defenses against invasive meningococcal disease is well established. Individuals deficient in components of the alternative and terminal complement pathways are highly predisposed to invasive, often recurrent meningococcal infections. Genome-wide analysis studies also point to a central role for complement in disease pathogenesis. Here we review the pathophysiologic events pertinent to the complement system that accompany meningococcal sepsis in humans. Meningococci use several often redundant mechanisms to evade killing by human complement. Capsular polysaccharide and lipooligosaccharide glycan composition play critical roles in complement evasion. Some of the newly described protein vaccine antigens interact with complement components and have sparked considerable research interest.
Purpose - The purpose of this empirical paper is to determine the effects of line extension introductions on parent brand equity.Design methodology approach - The paper uses a cross-sectional sample ...of 318 supermarket brands. A system of equations is proposed and estimated using seemingly unrelated regression.Findings - Brands benefit from line extension introductions, but only high equity brands benefit from innovation. Low equity brands benefit from the solo advertising of their new line extensions.Practical implications - The results suggest that there are two routes for improving brand equity; high equity brands can introduce innovative products, while low equity parent brands may improve brand equity by supporting new line extensions with solo advertising.Originality value - The paper is important in identifying the effects of new product introduction and innovation on brand equity.
The pathogenesis of malaria, an insect-borne disease that takes millions of lives every year, is still not fully understood. Complement receptor 1 (CR1) has been described as a receptor for ...Plasmodium falciparum, which causes cerebral malaria in humans. We investigated the role of CR1 in an experimental model of cerebral malaria. Transgenic mice expressing human CR1 (hCR1(+)) on erythrocytes were infected with Plasmodium berghei ANKA and developed cerebral malaria. No difference in survival was observed in hCR1(+) mice compared to wild-type mice following infection with P. berghei ANKA; however, hCR1 detection was significantly diminished on erythrocytes between days 7 and 10 postinfection. hCR1 levels returned to baseline by day 17 postinfection in surviving animals. Immunoblot assays revealed that total erythrocyte hCR1 levels were diminished, confirming that immune complexes in association with erythrocyte hCR1 were likely removed from erythrocytes in vivo by clearance following immune adherence. Decreases in hCR1 were completely dependent on C3 expression, as mice treated with cobra venom factor (which consumes and depletes C3) retained hCR1 on erythrocytes during C3 depletion through day 7; erythrocyte hCR1 decreases were observed only when C3 levels recovered on day 9. B-cell-deficient mice exhibit a marked increase in survival following infection with P. berghei ANKA, which suggests that immune complexes play a central role in the pathogenesis of experimental cerebral malaria. Together, our findings highlight the importance of complement and immune complexes in experimental cerebral malaria. IMPORTANCE Cerebral malaria is a deadly complication of infection with Plasmodium falciparum. Despite its high prevalence, relatively little is understood about its pathogenesis. We have determined that immune complexes are generated and deposited on erythrocytes specifically expressing human complement receptor 1 in a mouse model of cerebral malaria. We also provide evidence demonstrating the importance of immunoglobulins in the pathogenesis of cerebral malaria in mice. These findings may have important implications in human cerebral malaria.
Complement is a key arm of the innate immune defenses against the pathogenic neisseriae. We previously identified lipooligosaccharide on Neisseria meningitidis as an acceptor for complement C4b. ...Little is known about other neisserial targets for complement proteins C3 and C4, which covalently attach to bacterial surfaces and initiate opsonization and killing. In this study we demonstrate that Neisseria gonorrhoeae porin (Por) 1B selectively binds C4b via amide linkages and C3b via ester linkages. Using strains expressing hybrid Por1A/1B molecules, a region spanned by loops 4 and 5 of Por1B was identified as the preferred binding site for C4b. We also identified the opacity protein (Opa), a major adhesin of pathogenic neisseriae, as a target for C4b and C3b on both N. meningitidis and N. gonorrhoeae. Using N. gonorrhoeae variants that predominantly expressed individual Opa proteins, we found that all Opa proteins tested (A, B, C, D, E, F, and I) bound C4b and C3b via amide and ester linkages, respectively. Amide linkages with Por1B and Opa were confirmed using serum containing only the C4A isoform, which exclusively forms amide linkages with targets. While monomers and heterodimers of C4Ab were detected on bacterial targets, C4Bb appeared to preferentially participate in heterodimer (C5 convertase) formation. Our data provide another explanation for the enhanced serum sensitivity of Por1B-bearing gonococci. The binding of C3b and C4b to Opa provides a rationale for the recovery of predominantly "transparent" (Opa-negative) neisserial isolates from persons with invasive disease, where the bacteria encounter high levels of complement.
Both Toll-like receptor 4 (TLR4)– and MD-2–deficient mice succumb to otherwise nonfatal Gram-negative bacteria inocula, demonstrating the pivotal role played by these proteins in antibacterial ...defense in mammals. MD-2 is a soluble endogenous ligand for TLR4 and a receptor for lipopolysaccharide (LPS). LPS-bound MD-2 transmits an activating signal onto TLR4. In this report, we show that both recombinant and endogenous soluble MD-2 bind tightly to the surface of live Gram-negative bacteria. As a consequence, MD-2 enhances cellular activation, bacterial internalization, and intracellular killing, all in a TLR4-dependent manner. The enhanced internalization of MD-2–coated bacteria was not observed in macrophages expressing Lpsd, a signaling-incompetent mutant form of TLR4, suggesting that the enhanced phagocytosis observed is dependent on signal transduction. The data confirm the notion that soluble MD-2 is a genuine opsonin that enhances proinflammatory opsonophagocytosis by bridging live Gram-negative bacteria to the LPS transducing complex. The presented results extend our understanding of the role of the TLR4/MD-2 signaling axis in bacterial recognition by phagocytes.
Neisseria gonorrhoeae isolated from patients with disseminated infection are often of the porin (Por1A) serotype and resist killing by nonimmune normal human serum. The molecular basis of this ...resistance (termed stable serum resistance) in these strains has not been fully defined but is not related to sialylation of lipooligosaccharide. Here we demonstrate that Por1A bearing gonococcal strains bind more factor H, a critical downregulator of the alternative complement pathway, than their Por1B counterparts. This results in a sevenfold reduction in C3b, which is >75% converted to iC3b. Factor H binding to isogenic gonococcal strains that differed only in their porin serotype, confirmed that Por1A was the acceptor molecule for factor H. We identified a surface exposed region on the Por1A molecule that served as the binding site for factor H. We used gonococcal strains with hybrid Por1A/B molecules that differed in their surface exposed domains to localize the factor H binding site to loop 5 of Por1A. This was confirmed by inhibition of factor H binding using synthetic peptides corresponding to the putative exposed regions of the porin loops. The addition of Por1A loop 5 peptide in a serum bactericidal assay, which inhibited binding of factor H to the bacterial surface, permitted 50% killing of an otherwise completely serum resistant gonococcal strain. Collectively, these data provide a molecular basis to explain serum resistance of Por1A strains of N. gonorrhoeae.
The field of gene therapy has made considerable progress over the past several years. Adeno-associated virus (AAV) vectors have emerged as promising and attractive tools for in vivo gene therapy. ...Despite the recent clinical successes achieved with recombinant AAVs (rAAVs) for therapeutics, host immune responses against the vector and transgene product have been observed in numerous preclinical and clinical studies. These outcomes have hampered the advancement of AAV gene therapies, preventing them from becoming fully viable and safe medicines. The human immune system is multidimensional and complex. Both the innate and adaptive arms of the immune system seem to play a concerted role in the response against rAAVs. While most efforts have been focused on the role of adaptive immunity and developing ways to overcome it, the innate immune system has also been found to have a critical function. Innate immunity not only mediates the initial response to the vector, but also primes the adaptive immune system to launch a more deleterious attack against the foreign vector. This Review highlights what is known about innate immune responses against rAAVs and discusses potential strategies to circumvent these pathways.
Single phase nano and micro crystalline silicon films deposited using SiF
4/H
2 plasma at different H
2 dilution levels were studied at initial and terminal stages of film growth with spectroscopic ...ellipsometry (SE), Raman scattering (RS) and atomic force microscopy (AFM). The analysis of data obtained from SE elucidates the microstructural evolution with film growth in terms of the changes in crystallite sizes and their volume fractions, crystallite conglomeration and film morphology. The effect of H
2 dilution on film microstructure and morphology, and the corroborative findings from AFM studies are discussed. Our SE results evince two distinct mean sizes of crystallites in the material after a certain stage of film growth. The analysis of Raman scattering data for such films has been done using a bimodal size distribution of crystallite grains, which yields more accurate and physically rational microstructural picture of the material.
We isolated serologically identical (by serovar determination and porin variable region VR typing) strains of Neisseria gonorrhoeae from an infected male and two of his monogamous female sex ...partners. One strain (termed 398078) expressed the L1 (Galalpha1 rightwards arrow 3Gal{szligbeta}1 rightwards arrow 4Glc{szligbeta}1 rightwards arrow 4HepI) lipooligosaccharide (LOS) structure exclusively; the other (termed 398079) expressed the lacto-N-neotetraose (LNT; Gal{szligbeta}1 rightwards arrow 4GlcNAc{szligbeta}1 rightwards arrow 3Gal{szligbeta}1 rightwards arrow 4Glc{szligbeta}1 rightwards arrow 4HepI) LOS structure. The strain from the male index case expressed both glycoforms and exhibited both immunotypes. Nuclear magnetic resonance analysis revealed that sialic acid linked to the terminal Gal of L1 LOS via an alpha2 rightwards arrow 6 linkage and, as expected, to the terminal Gal of LNT LOS via an alpha2rightwards arrow 3 linkage. Insertional inactivation of the sialyltransferase gene (known to sialylate LNT LOS) abrogated both L1 LOS sialylation and LNT LOS sialylation, suggesting a bifunctional nature of this enzyme in gonococci. Akin to our previous observations, sialylation of the LNT LOS of strain 398079 enhanced the binding of the complement regulatory molecule, factor H. Rather surprisingly, factor H did not bind to sialylated strain 398078. LOS sialylation conferred the LNT LOS-bearing strain complete (100%) resistance to killing by even 50% nonimmune normal human serum (NHS), whereas sialylation of L1 LOS conferred resistance only to 10% NHS. The ability of gonococcal sialylated LNT to bind factor H confers high-level serum resistance, which is not seen with sialylated L1 LOS. Thus, serum resistance mediated by sialylation of gonococcal L1 and LNT LOS occurs by different mechanisms, and specificity of factor H binding to sialylated gonococci is restricted to the LNT LOS species.
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