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- Immunogenicity and in vitro and in vivo protective effects of antibodies targeting a recombinant form of the Streptocccus mutans P1 surface protein. [JOURNAL ARTICLE]
- Infect Immun 2014 Sep 15.
Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185 kDa) and complex multi-domain protein considered a promising target antigen for anti-caries vaccines. Previous observations showed that a recombinant P1 fragment (P139-512) produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the current study, we further investigated the immunological features of P139-512 after parenteral administration of mice in combination with different adjuvants: alum, a derivative of the heat labile toxin (LT), and Salmonella flagellin (FliCi). Our results demonstrated that recombinant P139-512 preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Co-administration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclasses switching. Importantly P139-512-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P139-512 antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P139-512 as a potential candidate for the development of anti-caries vaccines, and as a tool for functional studies of S. mutans P1.
- Tenascin-R promotes assembly of the extracellular matrix of perineuronal nets via clustering of aggrecan. [Journal Article]
- Philos Trans R Soc Lond B Biol Sci 2014 Oct 19; 369(1654)
Perineuronal nets (PNs) in the brains of tenascin-R-deficient (tn-r(-/-)) mice develop in temporal concordance with those of wild-type (tn-r(+/+)) mice. However, the histological appearance of PNs is abnormal in adult tn-r(-/-) mice. Here, we investigated whether similar defects are also seen in dissociated and organotypic cultures from hippocampus and forebrain of tn-r(-/-) mice and whether the structure of PNs could be normalized. In tn-r(-/-) cultures, accumulations of several extracellular matrix molecules were mostly associated with somata, whereas dendrites were sparsely covered, compared with tn-r(+/+) mice. Experiments to normalize the structure of PNs in tn-r(-/-) organotypic slice cultures by depolarization of neurons, or by co-culturing tn-r(+/+) and tn-r(-/-) brain slices failed to restore a normal PN phenotype. However, formation of dendritic PNs in cultures was improved by the application of tenascin-R protein and rescued by polyclonal antibodies to aggrecan and a bivalent, but not monovalent form of the lectin Wisteria floribunda agglutinin. These results show that tenascin-R and aggrecan are decisive contributors to formation and stabilization of PNs and that tenascin-R may implement these functions by clustering of aggrecan. Proposed approaches for restoration of normal PN structure are noteworthy in the context of PN abnormalities in neurological disorders, such as epilepsy, schizophrenia and addiction.
- Peptide inhibitor of complement c1, a novel suppressor of classical pathway activation: mechanistic studies and clinical potential. [Journal Article, Review]
- Front Immunol 2014.:406.
The classical pathway of complement plays multiple physiological roles including modulating immunological effectors initiated by adaptive immune responses and an essential homeostatic role in the clearance of damaged self-antigens. However, dysregulated classical pathway activation is associated with antibody-initiated, inflammatory diseases processes like cold agglutinin disease, acute intravascular hemolytic transfusion reaction (AIHTR), and acute/hyperacute transplantation rejection. To date, only one putative classical pathway inhibitor, C1 esterase inhibitor (C1-INH), is currently commercially available and its only approved indication is for replacement treatment in hereditary angioedema, which is predominantly a kinin pathway disease. Given the variety of disease conditions in which the classical pathway is implicated, development of therapeutics that specifically inhibits complement initiation represents a major unmet medical need. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement. In vitro studies have demonstrated that these peptide inhibitors of complement C1 (PIC1) bind to the collagen-like region of the initiator molecule of the classical pathway, C1q. PIC1 binding to C1q blocks activation of the associated serine proteases (C1s-C1r-C1r-C1s) and subsequent downstream complement activation. Rational design optimization of PIC1 has resulted in the generation of a highly potent derivative of 15 amino acids. PIC1 inhibits classical pathway mediated complement activation in ABO incompatibility in vitro and inhibiting classical pathway activation in vivo in rats. This review will focus on the pre-clinical development of PIC1 and discuss its potential as a therapeutic in antibody-mediated classical pathway disease, specifically AIHTR.
- Synthesis, micellization and lectin binding of new glycosurfactants. [JOURNAL ARTICLE]
- Carbohydr Res 2014 Aug 5.:31-36.
Here we report the preparation and physico-chemical characterization of carbohydrate-decorated micelles and their interaction with lectins. A library of biosourced amphiphiles was prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC) between alkynyl sugars (lactose, N-acetyl-d-glucosamine) and azido-functionalized poly(ethylene glycol) esters (N3-PEG900-decanoate (C10) and -dodecanoate (C12)). In water, these glycoconjugates self-assemble into micelles of homogeneous nanometric size (11nm) as evidenced by scattering techniques (DLS for light, and SAXS for X-ray). A comparative study with previously synthesized octadecanoate counterparts pointed out that that nature of the fatty acid has no significant influence on the particle size but only affects their compactness. These findings are in favor of a possible bulk preparation from lipid mixtures such as those encountered in renewable vegetable oils. The presence of the carbohydrate epitopes on the surface of the micelles and their bioavailability for lectin targeting were also evidenced by light scattering measurements using wheat germ agglutinin (WGA) and peanut (Arachis hypogaea) (PNA) lectins, supporting possible application as targeted drug nanocarriers.
- The interaction of N-trifluoroacetylgalactosamine and its derivatives with winged bean (Psophocarpus tetragonolobus) basic agglutinin reveals differential mechanism of their recognition: a fluorine-19 nuclear magnetic resonance study. [JOURNAL ARTICLE]
- Glycoconj J 2014 Sep 4.
Here, we show the binding results of a leguminosae lectin, winged bean basic agglutinin (WBA I) to N-trifluoroacetylgalactosamine (NTFAGalN), methyl-α-N-trifluoroacetylgalactosamine (MeαNTFAGalN) and methyl-β-tifluoroacetylgalactosamine (MeβNTFAGalN) using (19) F NMR spectroscopy. No chemical shift difference between the free and bound states for NTFAGalN and MeβNTFAGalN, and 0.01-ppm chemical shift change for MeαNTFAGalN, demonstrate that the MeαNTFAGalN has a sufficiently long residence time on the protein binding site as compared to MeβNTFAGalN and the free anomers of NTFAGalN. The sugar anomers were found in slow exchange with the binding site of agglutinin. Consequently, we obtained their binding parameters to the protein using line shape analyses. Aforementioned analyses of the activation parameters for the interactions of these saccharides indicate that the binding of α and β anomers of NTFAGalN and MeαNTFAGalN is controlled enthalpically, while that of MeβNTFAGalN is controlled entropically. This asserts the sterically constrained nature of the interaction of the MeβNTFAGalN with WBA I. These studies thus highlight a significant role of the conformation of the monosaccharide ligands for their recognition by WBA I.
- Oxidative damage in the gastrocnemius of patients with peripheral artery disease is myofiber type selective. [Journal Article]
- Redox Biol 2014.:921-8.
Peripheral artery disease (PAD), a manifestation of systemic atherosclerosis that produces blockages in the arteries supplying the legs, affects approximately 5% of Americans. We have previously, demonstrated that a myopathy characterized by myofiber oxidative damage and degeneration is central to PAD pathophysiology.In this study, we hypothesized that increased oxidative damage in the myofibers of the gastrocnemius of PAD patients is myofiber-type selective and correlates with reduced myofiber size.Needle biopsies were taken from the gastrocnemius of 53 PAD patients (28 with early PAD and 25 with advanced PAD) and 25 controls. Carbonyl groups (marker of oxidative damage), were quantified in myofibers of slide-mounted tissue, by quantitative fluorescence microscopy. Myofiber cross-sectional area was determined from sarcolemma labeled with wheat germ agglutinin. The tissues were also labeled for myosin I and II, permitting quantification of oxidative damage to and relative frequency of the different myofiber Types (Type I, Type II and mixed Type I/II myofibers). We compared PAD patients in early (N=28) vs. advanced (N=25) disease stage for selective, myofiber oxidative damage and altered morphometrics.The carbonyl content of gastrocnemius myofibers was higher in PAD patients compared to control subjects, for all three myofiber types (p<0.05). In PAD patients carbonyl content was higher (p<0.05) in Type II and I/II fibers compared to Type I fibers. Furthermore, the relative frequency and cross-sectional area of Type II fibers were lower, while the relative frequencies and cross-sectional area of Type I and Type I/II fibers were higher, in PAD compared to control gastrocnemius (p<0.05). Lastly, the type II-selective oxidative damage increased and myofiber size decreased as the disease progressed from the early to advanced stage.Our data confirm increased myofiber oxidative damage and reduced myofiber size in PAD gastrocnemius and demonstrate that the damage is selective for type II myofibers and is worse in the most advanced stage of PAD.
- Pig Spermatozoa Defect in Acrosome Formation Caused Poor Motion Parameters and Fertilization Failure through Artificial Insemination and In vitro Fertilization. [Journal Article]
- Asian-Australas J Anim Sci 2014 Oct; 27(10):1417-25.
The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not sufficient to select normal and fertilizable spermatozoa. In conclusion, the present study suggests that the acrosome staining and detailed motion characteristics such as progressiveness, VCL, and VSL should be included in determining semen quality together with primary parameters for successful AI and high breeding performance in the swine industry.
- Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen. [JOURNAL ARTICLE]
- Xenotransplantation 2014 Sep 1.
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.
- Prospective study on Norovirus infection among allogeneic stem cell transplant recipients: Prolonged viral excretion and viral RNA in the blood. [JOURNAL ARTICLE]
- J Clin Virol 2014 Aug 13.
Human caliciviruses (Norovirus and Sapovirus) are important acute gastroenteritis agents. The Norovirus (NoV) disease is usually self-limited; however, prolonged viral excretion and complications have been reported, mainly in immunosuppressed individuals.In this prospective study, we have monitored allogeneic stem cell transplant (ASCT) patients for human calicivirus infection.Ten ASCT patients were monitored for NoV and sapoviruses (SaV) infection, for a period of five months to a maximum of one year. Prolonged NoV excretion and long term viral RNA in the blood were assessed by multiplex RT-PCR targeting region C of the viral capsid. Secretor status of the patients was determined by enzyme immunoassay using Ulex Europaeus agglutinin. Partial genomic sequencing and phylogenetic analysis were performed to characterize the viral genotypes.NoV was detected in six out of ten patients (60%). Prolonged viral excretion in feces (mean of 61.6 days) and long term presence of NoV RNA in the sera (mean of 33.6 days) of the patients were observed. SaV was not detected in any of the samples. All patients had diarrhea, vomiting and fever during NoV positivity. All NoV-positive samples were characterized as GI.3 NoV. Three Nov-infected patients presented with acute intestinal graft versus host disease.This study brings important information on NoV course of infection in ASCT patients. It also provides evidence for long term viral RNA in the blood highlighting the importance of the inclusion of NoV screening in the routine testing performed before transplantation and during follow-up of these patients.
- Serum gp73 is also a biomarker for diagnosing cirrhosis in population with chronic HBV infection. [JOURNAL ARTICLE]
- Clin Biochem 2014 Aug 25.
To clarify the role of Golgi membrane glycoprotein 73 (gp73) in evaluating the progression of chronic hepatitis B virus (HBV) infection.Participants included 958 controls, 421 chronic hepatitis B, 944 hepatic cirrhosis, and 127 hepatocellular carcinoma (HCC) patients. All the patients, with the exception of the controls, were diagnosed HBsAg positive. Serum biomarkers, including gp73, alpha-fetoprotein (AFP), alpha-l-fucosidase, and Lens culinaris agglutinin-reactive fraction of AFP, were determined.The patients with Hepatic cirrhosis gp73 levels over 150ng/mL had an odds ratio of 3.21 (95% CI: 2.07-5.00). In hepatic cirrhosis patients, serum gp73 correlated with the Child-Pugh score. gp73 is a marker for diagnosing cirrhosis in the hepatitis populations. When the cut-off was set at 75.5ng/mL, the sensitivity, specificity, and AUC were 75.6% (95% CI: 71.30%-79.62%), 60.3% (95% CI: 56.95%-63.63%) and 0.72 (95% CI: 0.69-0.75), respectively.The variation trend of gp73 in chronic liver disease may indicate that monitoring of serum gp73 is helpful to diagnose cirrhosis in population with chronic HBV infection.