Neospora caninum NC-6 Argentina induces fetopathy in both serologically positive and negative experimentally inoculated pregnant dams

Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108?±?2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.     

TBX6-associated congenital scoliosis (TACS) as a clinically distinguishable subtype of congenital scoliosis: further evidence supporting the compound inheritance and TBX6 gene dosage model

PurposeTo characterize clinically measurable endophenotypes, implicating the TBX6 compound inheritance model.MethodsPatients with congenital scoliosis (CS) from China(N = 345, cohort 1), Japan (N = 142, cohort 2), and the United States (N = 10, cohort 3) were studied. Clinically measurable endophenotypes were compared according to the TBX6 genotypes. A mouse model for Tbx6 compound inheritance (N = 52) was investigated by micro computed tomography (micro-CT). A clinical diagnostic algorithm (TACScore) was developed to assist in clinical recognition of TBX6-associated CS (TACS).ResultsIn cohort 1, TACS patients (N = 33) were significantly younger at onset than the remaining CS patients (P = 0.02), presented with one or more hemivertebrae/butterfly vertebrae (P = 4.9 × 10^‒8), and exhibited vertebral malformations involving the lower part of the spine (T8–S5, P = 4.4 × 10^‒3); observations were confirmed in two replication cohorts. Simple rib anomalies were prevalent in TACS patients (P = 3.1 × 10^‒7), while intraspinal anomalies were uncommon (P = 7.0 × 10^‒7). A clinically usable TACScore was developed with an area under the curve (AUC) of 0.9 (P = 1.6 × 10^‒15). A Tbx6^{-/mh (mild-hypomorphic)} mouse model supported that a gene dosage effect underlies the TACS phenotype.ConclusionTACS is a clinically distinguishable entity with consistent clinically measurable endophenotypes. The type and distribution of vertebral column abnormalities in TBX6/Tbx6 compound inheritance implicate subtle perturbations in gene dosage as a cause of spine developmental birth defects responsible for about 10% of CS.     

Roles of two types of O6-methylguanine-DNA methyltransferases in DNA repair

Escherichia coli possesses 2 types of O⁶-methylguanine-DNA methyltransferases, one inducible and the other constitutive. These enzymes are coded by the ada and the ogt genes, respectively. Using a synthetic ogt-specific probe, we mapped ogt at 29.4 min, near the 5′-flanking region of the nirR gene, on the E. coli chromosome. To elucidate the roles of the 2 types of methyltransferases in DNA repair, we constructed mutant strains which lack either one or both of the genes. In either the ada⁺ or the ada⁻ background, the ogt mutation had no effect on cell survival after N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment. On the other hand, ada⁻ogt⁻ cells were more prone to mutation as compared to the ada⁻ogt⁺ cells exposed to MNNG. The frequency of spontaneous mutation of cells defective in either one or both of the genes was the same, however, the introduction of the ogt⁺ plasmid into the cells produced a 2–3-fold decrease in the frequency of spontaneous mutation. O⁶-methylguanine-DNA methyltransferases appear to eliminate premutagenic DNA lesions not only from cells exposed to alkylating agents but also from those grown in the absence of the agents.     

Detection of human herpesvirus-6 variants in pediatric brain tumors: Association of viral antigen in low grade gliomas

BackgroundHuman herpesvirus-6 (HHV-6) has been associated with a diverse spectrum of central nervous system (CNS) diseases and reported glial tropism.ObjectiveTo determine if HHV-6 is present in a series of pediatric brain tumors.Study designPediatric gliomas from 88 untreated patients represented in a tissue microarray (TMA) were screened for HHV-6 by nested polymerase chain reaction (PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) and compared to non-glial tumors (N = 22) and control brain (N = 32). Results were correlated with tumor grade and overall survival.ResultsHHV-6 U57 was detected by nested PCR in 68/120 (57%) tumors and 7/32 (22%) age-matched non-tumor brain (P = 0.001). HHV-6 U31 was positive in 73/120 (61%) tumors and 11/32 (34%) controls (P = 0.019). Seventy-two percent (43/60) of tumors were HHV-6 Variant A. HHV-6 U57 was confirmed by ISH in 83/150 (54%) tumors and 10/32 (31%) controls (P = 0.021), revealing a non-lymphocytic origin of HHV-6. HHV-6A/B gp116/64/54 late antigen was detected by IHC in 50/124 (40%) tumors and 6/32 (18%) controls (P = 0.013). Interestingly, 58% of low grade gliomas (N = 67) were IHC positive compared to 19% of high grade gliomas (N = 21, P = 0.002) and 25% of non-gliomas (N = 36, P = 0.001). HHV-6A/B gp116/64/54 antigen co-localized with glial fibrillary acidic protein, confirming the astrocytic origin of antigen. Overall, there was no primary association between HHV-6A/B gp116/64/54 antigen detection and survival (P = 0.861).ConclusionsWe provide the first reported series of HHV-6 detection in pediatric brain tumors. The predominance of HHV-6 in glial tumors warrants further investigation into potential neurooncologic disease mechanisms.     

Comparative Analysis of Bacillus subtilis Spores and Monophosphoryl Lipid A as Adjuvants of Protein-Based Mycobacterium tuberculosis-Based Vaccines: Partial Requirement for Interleukin-17A for Induction of Protective Immunity

The development of adjuvants for vaccines has become an important area of research as the number of protein-based vaccines against infectious pathogens increases. Currently, there are a number of adjuvant-based Mycobacterium tuberculosis vaccines in clinical trials that have shown efficacy in animal models. Despite these novel adjuvants, there is still a need to design new and more versatile adjuvants that have minimal adverse side effects but produce robust long-lasting adaptive immune responses. To this end, we hypothesized that Bacillus subtilis spores may provide the appropriate innate signals that are required to generate such vaccine-mediated responses, which would be sufficient to reduce the mycobacterial burden after infection with M. tuberculosis. In addition, we compared the response generated by B. subtilis spores to that generated by monophosphoryl lipid A (MPL), which has been used extensively to test tuberculosis vaccines. The well-characterized, 6-kDa early secretory antigenic target of M. tuberculosis (ESAT-6; Rv3875) was used as a test antigen to determine the T cell activation potential of each adjuvant. Inoculated into mice, B. subtilis spores induced a strong proinflammatory response and Th1 immunity, similar to MPL; however, unlike MPL formulated with dimethyldioctadecylammonium (DDA) bromide, it failed to induce significant levels of interleukin-17A (IL-17A) and was unable to significantly reduce the mycobacterial burden after pulmonary infection with M. tuberculosis. Further analysis of the activity of MPL-DDA suggested that IL-17A was required for protective immunity. Taken together, the data emphasize the requirement for a network of cytokines that are essential for protective immunity.     

MSH6 and PMS2 germ-line pathogenic variants implicated in Lynch syndrome are associated with breast cancer

PurposeAn association of Lynch syndrome (LS) with breast cancer has been long suspected; however, there have been insufficient data to address this question for each of the LS genes individually.MethodsWe conducted a retrospective review of personal and family history in 423 women with pathogenic or likely pathogenic germ-line variants in MLH1 (N = 65), MSH2 (N = 94), MSH6 (N = 140), or PMS2 (N = 124) identified via clinical multigene hereditary cancer testing. Standard incidence ratios (SIRs) of breast cancer were calculated by comparing breast cancer frequencies in our study population with those in the general population (Surveillance, Epidemiology, and End Results 18 data).ResultsWhen evaluating by gene, the age-standardized breast cancer risks for MSH6 (SIR = 2.11; 95% confidence interval (CI), 1.56–2.86) and PMS2 (SIR = 2.92; 95% CI, 2.17–3.92) were associated with a statistically significant risk for breast cancer whereas no association was observed for MLH1 (SIR = 0.87; 95% CI, 0.42–1.83) or MSH2 (SIR = 1.22; 95% CI, 0.72–2.06).ConclusionOur data demonstrate that two LS genes, MSH6 and PMS2, are associated with an increased risk for breast cancer and should be considered when ordering genetic testing for individuals who have a personal and/or family history of breast cancer.     

Regulation of cytokine gene expression by adjuvants in vivo

Antibody isotype affects biological activity of the antibodies and therefore should be considered in prevention of disease by vaccination. In previous reports, we demonstrated that adjuvants affect the antibody isotype switching process and favour the production of certain isotypes. The present study extends these findings and shows fundamental differences in the cytokine induction pattern according to the adjuvant used. Cytokine mRNA levels were determined by in situ RNA–RNA hybridization performed on splenocytes isolated from mice injected with different adjuvants. The results revealed that Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Al(OH)₃ and QuilA administration results in a type-2 (humoral) response, increasing IL-4, IL-5 and IL-13 gene expression, while poly I:C exhibits a type-1 (cell-mediated) response, increasing the production of interferon-gamma (IFN-γ), IL-2 and IL-6 mRNA. Finally, BeSO₄ and poly A:U augment IL-5 and IL-6 mRNA production, while lipopolysaccharide (LPS) and LiCl augment IL-6 and tumour necrosis factor-alpha (TNF-α) mRNA production. Also, the adjuvants appear capable of overcoming the inherent IL-2/IFN-γ and IL-4 dichotomy of C57Bl/6 and BALB/c mice, respectively, in response to cellular antigens such as Leishmania and herpessimplex virus (HSV). The overall data suggest that adjuvants direct the isotype switching process via induction of certain cytokines, a finding that can be useful in selection of the most efficient isotype of protective antibodies for disease prevention by vaccination.     

Nocardia farcinica Activates Human Dendritic Cells and Induces Secretion of Interleukin-23 (IL-23) Rather than IL-12p70

Studying the interaction of dendritic cells (DCs) with bacteria controlled by T-cell-mediated immune responses may reveal novel adjuvants for the induction of cellular immunity. Murine studies and the observation that nocardias infect predominantly immunosuppressed patients have suggested that these bacteria may possess an adjuvant potential. Moreover, adjuvants on the basis of the nocardial cell wall have been applied in clinical studies. Since the handling of adjuvants by DCs may determine the type of immune responses induced by a vaccine, the present study aimed at investigating the interaction of immature human monocyte-derived DCs with live or inactivated Nocardia farcinica in vitro and determining the cellular phenotypic changes as well as alterations in characteristic functions, such as phagocytosis, induction of T-cell proliferation, and cytokine secretion. Human DCs ingested N. farcinica and eradicated the bacterium intracellularly. DCs exposed to inactivated N. farcinica were activated, i.e., they developed a mature phenotype, downregulated their phagocytic capacity, and stimulated allogeneic T cells in mixed leukocyte reactions. Soluble factors were not involved in this process. To elucidate the potential adjuvant effect of N. farcinica on the induction of T-cell-mediated immune responses, we characterized the cytokines produced by nocardia-exposed DCs and detected substantial amounts of tumor necrosis factor alpha (TNF-α) and interleukin-12 p40 (IL-12p40). However, nocardia-treated DCs secreted only small amounts of IL-12p70, which were significantly smaller than the amounts of IL-23. Thus, N. farcinica activates DCs, but adjuvants based on this bacterium may have only a limited capacity to induce Th1 immune responses.     

Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N, 6-O-sulfated chitosan in vitro and in vivo

Bone morphogenetic protein-2 (BMP-2) has been widely used as an effective growth factor in bone tissue engineering. However, large amounts of BMP-2 are required to induce new bone and the resulting side effects limit its clinical application. Sulfated polysaccharides, such as native heparin, and heparan sulfate have been found to modulate BMP-2 bioactivity and play pivotal roles in bone metabolism. Whereas the direct role of chitosan modified with sulfate group in BMP-2 signaling has not been reported till now. In the present study, several sulfated chitosans with different positions were synthesized by regioselective reactions firstly. Using C2C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation from 6-O-sulfated chitosan (6SCS), while 2-N-sulfate was subsidiary group with less activation. Low dose of 2-N, 6-O-sulfated chitosan (26SCS) showed significant enhancement on the alkaline phosphatase (ALP) activity and the mineralization formation induced by BMP-2, as well as the expression of ALP and osteocalcin mRNA. Moreover, increased chain-length and further sulfation on 26SCS also resulted in a higher ALP activity. Dose-dependent effects on BMP-2 bioactivity were observed in both sulfated chitosan and heparin. Compared with native heparin, 26SCS showed much stronger simultaneous effects on the BMP-2 bioactivity at low dose. Stimulated secreted Noggin protein failed to block the function of BMP-2 in the presence of 26SCS. The BMP-2 ligand bound to its receptor was enhanced by low dose of 26SCS, whereas weakened by the increasing amounts of 26SCS. Furthermore, simultaneous administration of BMP-2 and 26SCS in vivo dose-dependently induced larger amounts of ectopic bone formation compared with BMP-2 alone. These findings clearly indicate that 26SCS is a more potent enhancer for BMP-2 bioactivity to induce osteoblastic differentiation in vitro and in vivo by promoting BMP-2 signaling pathway, suggesting that 26SCS could be used as the synergistic factor of BMP-2 for bone regeneration.     

Capsular polysaccharide gene diversity of pneumococcal serotypes 6A, 6B, 6C,and 6D

This study was performed to better understand the genetic diversity and evolutionary relatedness of pneumococcal serotypes 6A, 6B, 6C, and 6D. Multi-locus sequence typing (MLST) was performed for 160 serogroup 6 isolates from clinical specimens collected from children between 1991 and 2010. We identified 38 sequence types (STs) comprising five clonal complexes with 12 singletons. Although most STs were confined to a single serotype, some STs were shared by two serotypes, and one ST was shared by three serotypes. Many STs of serotype 6A showed genetic relatedness with those of serotype 6C or 6D in eBURST analysis. Five capsular polysaccharide (cps) genes – wchA, wciO, wciP, wzy, and wzx – were analysed in 74 isolates from our clinical samples and in 36 isolates from GenBank. There were several profiles and clades in each serotype on the analysis of the concatenated sequences of the five cps genes. Small genetic distances between serotypes 6A and 6B and between serotypes 6C and 6D were observed while serotype 6B with an indel sequence formed a distinct clade. When comparing the individual cps genes between the serotypes, there was also a high level of similarity in the wchA and wciO gene sequences between serotype 6C and serotype 6D. On the other hand, serotypes 6A and 6D had the most highly similar wzy and wzx gene sequences. The wzy sequences of serotype 6C were nearly identical (99.6%) to those of serotype 6A clade II strains. In conclusion, we revealed the diversity of the genetic background and cps sequences in each pneumococcal serotype of serogroup 6. Pneumococcal serotype diversity might be attributable to complex serial mutation and recombination events.     

Freund's adjuvants: relationship of arthritogenicity and adjuvanticity in rats to vehicle composition

Over a hundred compounds and natural materials were examined for their ability to induce arthritis in rats when mixed with heat-killed delipidated Mycobacteria tuberculosis. Many of these materials were also assessed for (CMI) adjuvant activity by their ability to induce allergic encephalomyelitis (EAE) in rats when mixed with guinea-pig spinal cord, both with and without added M. tuberculosis.

Cyclization and/or the presence of oxygen atoms, or double bonds reduced (or abolished) the arthritogenic potential and adjuvanticity of alkanes>C₁₀. Esters/triglycerides of fatty acids >C₁₂, retinol acetate (not palmitate) and vitamins E and K showed co-arthritogenic and adjuvant activity. Other active lipids included squalene and cholesterol oleate, which are both present in human sebum. Sebaceous lipids may therefore perhaps function as natural adjuvants if resorbed during abrasion and infection.

Squalane (perhydrosqualene), pristane and hexadecane were excellent substitutes for mineral oil in preparing arthritogenic adjuvants from various mycobacteria, C. rubrum and N. asteroides. These oily compounds were also very effective adjuvants per se, in the absence of bacterial material or emulsifier, for inducing EAE in Lewis rats.

     

Colony Morphology Variation of Burkholderia pseudomallei Is Associated with Antigenic Variation and O-Polysaccharide Modification

Burkholderia pseudomallei is a CDC tier 1 select agent that causes melioidosis, a severe disease in humans and animals. Persistent infections are common, and there is currently no vaccine available. Lipopolysaccharide (LPS) is a potential vaccine candidate. B. pseudomallei expresses three serologically distinct LPS types. The predominant O-polysaccharide (OPS) is an unbranched heteropolymer with repeating d-glucose and 6-deoxy-l-talose residues in which the 6-deoxy-l-talose residues are variably replaced with O-acetyl and O-methyl modifications. We observed that primary clinical B. pseudomallei isolates with mucoid and nonmucoid colony morphologies from the same sample expressed different antigenic types distinguishable using an LPS-specific monoclonal antibody (MAb). MAb-reactive (nonmucoid) and nonreactive (mucoid) strains from the same patient exhibited identical LPS banding patterns by silver staining and indistinguishable genotypes. We hypothesized that LPS antigenic variation reflected modification of the OPS moieties. Mutagenesis of three genes involved in LPS synthesis was performed in B. pseudomallei K96243. Loss of MAb reactivity was observed in both wbiA (encoding a 2-O-acetyltransferase) and wbiD (putative methyl transferase) mutants. The structural characteristics of the OPS moieties from isogenic nonmucoid strain 4095a and mucoid strain 4095c were further investigated. Utilizing nuclear magnetic resonance (NMR) spectroscopy, we found that B. pseudomallei 4095a and 4095c OPS antigens exhibited substitution patterns that differed from the prototypic OPS structure. Specifically, 4095a lacked 4-O-acetylation, while 4095c lacked both 4-O-acetylation and 2-O-methylation. Our studies indicate that B. pseudomallei OPS undergoes antigenic variation and suggest that the 9D5 MAb recognizes a conformational epitope that is influenced by both O-acetyl and O-methyl substitution patterns.     

The influence of adjuvants on the immunological response of the chicken: I. Effects on primary and secondary responses of various adjuvants in the primary stimulus

The effect of various adjuvant procedures on the antibody response (first 21 days) for human serum albumin (HSA) has been studied in the chicken. The lack of effect on the circulating antibody level contrasts with their action in some mammals.

The administration of depôt-type adjuvants failed to increase the peak circulating antibody levels (8–12 days after injection of antigen) by comparison with control birds. However, the circulating antibody level declined more slowly in birds given HSA in a water-in-oil emulsion than in birds given HSA in saline.

The administration of endotoxin and `surface active' adjuvants also failed to increase the peak circulating antibody levels over that of control birds. In three experiments there was significant depression of peak antibody levels in birds given endotoxin adjuvant in comparison to control birds.

The administration of HSA in Freund's complete adjuvants containing Mycobacterium tuberculosis or Mycobacterium avium did not result in elevation of peak antibody levels compared to those of control birds given HSA in saline or HSA in a water-in-oil emulsion.

Experiments to determine the effect of adjuvants from each of the main groups on the establishment of immunological memory were performed. Chickens were given adjuvant with the primary injection of HSA. A second injection of HSA without adjuvant was given 56 days later. None of the adjuvants used produced an increase in the peak antibody level attained during the secondary response compared to control birds.

     

A review of adjuvants for Leishmania vaccine candidates

Over the last decade, there has been a flurry of research on adjuvants for vaccines, and several novel adjuvants are now licensed products or in late stage clinical development. The success of adjuvants in enhancing the immune response to antigens has led many researchers to re-focus their vaccine development programs. Although several vaccine candidates have been tested against leishmaniasis, there is yet no effective vaccine against this parasitic disease. Recent research has documented that efforts to develop effective Leishmania vaccine have been limited due to lack of an appropriate adjuvant. In view of this, this review paper outlines some of the adjuvants that have been used in Leishmania vaccine candidates and cites a few of the responses obtained from these studies. The aim of the present review is to consolidate these findings to facilitate the application of these adjuvants in general and experimental vaccinology.     

Flagellin from Marinobacter algicola and Vibrio vulnificus activates the innate immune response of gilthead seabream

Adjuvants have emerged as the best tools to enhance the efficacy of vaccination. However, the traditional adjuvants used in aquaculture may cause adverse alterations in fish making necessary the development of new adjuvants able to stimulate the immune system and offer strong protection against infectious pathogens with minimal undesirable effects. In this respect, flagellin seems an attractive candidate due to its ability to strongly stimulate the immune response of fish. In the present study, we have evaluated the ability of recombinant flagellin from Marinobacter algicola (MA) and Vibrio vulnificus (Vvul), a non-pathogenic and a pathogenic bacteria, respectively, to stimulate the innate immune system of gilthead seabream (Sparus aurata L.) and compare the effect with that of the classical flagellin from Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, STF). Intraperitoneal injection of MA and Vvul resulted in a strong inflammatory response characterized by increased reactive oxygen species production and the infiltration of acidophilic granulocytes at the injection site. Interestingly, however, only flagellin from MA consistently induced the expression of the gene encoding pro-inflammatory interleukin-1β. These effects were further confirmed in vitro, where a dose-dependent activation of macrophages and acidophilic granulocytes by MA and Vvul flagellins was observed. In contrast, STF flagellin was found to be less potent in both in vivo and in vitro experiments. Our results suggest the potential use of MA and Vvul flagellins as immunostimulants and adjuvants for fish vaccination.     

Comparative biochemical studies on natural Trypanosoma evansi infection in she-camels

The biochemical changes associating Trypanosoma evansi infection in pregnant and non-pregnant camels were investigated. Based on pregnancy diagnosis and serological findings, camels were classified into four groups as non-pregnant healthy camels (N?=?6), non-pregnant camels infected with T. evansi (N?=?6), pregnant healthy camels (N?=?6), and pregnant camels infected with Trypanosoma evansi (N?=?8). The results revealed significant decreases (p?<?0.05) in serum total proteins, albumin and globulins levels, and significant increases (p?<?0.05) in serum total cholesterol and blood urea nitrogen (BUN) levels in pregnant camels infected with T. evansi compared with healthy pregnant camel. On the other hand, there were hyperproteinemia and hyperglobulinemia in healthy pregnant camel compared with non-pregnant camel. It could be concluded that the biochemical changes associating T. evansi infection in pregnant camels are hypoproteinemia, hypoalbuminemia, and hypoglobulinemia and increased serum total cholesterol and BUN levels.     

Anionic polymerization of 6-hexanelactam, 62. Fast catalytic systems in the anionic polymerization of 6-hexanelactam

With magnesium 6-hexanelactamate bromide (LMgBr) as a catalyst and the N-acyllactam as initiator an exceptionally high rate of polymerization of 6-hexanelactam is observed. Side reactions decreasing the concentrations of growth centres and activated monomer (lactam anion) are not affected to such an extent as to explain this acceleration. In the presence of poly(propylene oxide), the side reactions are largely suppressed with LMgBr. The results corroborate the assumption that the reactivity of the N-acyllactam growth centre is increased by coordination of a magnesium bromide ligand.     

Experimental induction of immune sialadenitis in guinea-pigs using different adjuvants

Groups of guinea-pigs were immunized with a saline extract of salivary gland combined with different adjuvants, either alone or in combination. Periductal inflammatory infiltrates were most commonly induced when two adjuvants were given in combination, whereas they were unusual when given alone. The severest degrees of sialadenitis were seen when carbonyl iron and Bordetella pertussis vaccine were used together.

The results suggest that this may be a useful model for the study of autoimmune sialadenitis.

     

Identification of N-linked carbohydrates from severe acute respiratory syndrome (SARS) spike glycoprotein

N-glycans were released from the SARS coronavirus (SARS-CoV) spike glycoprotein produced in Vero E6 cells and their structures were determined by a combination of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, negative ion electrospray collision-induced dissociation time-of-flight mass spectrometry and normal-phase high-performance liquid chromatography with exoglycosidase digestion. Major glycans were high-mannose (Man_5-9GlcNAc₂), hybrid and bi-, tri- and tetra-antennary complex with and without bisecting GlcNAc and core fucose. Complex glycans with fewer than the full complement of galactose residues were present and sialylation was negligible. Treatment with the glucosidase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) inhibited N-glycan processing as evidenced by the appearance of glycans of composition Glc₃Man_7-9GlcNAc₂. However, some complex glycans remained suggesting the presence of an α-endomannosidase. Our data in tissue culture indicate that inhibition of N-glycan processing may be considered as a therapeutic strategy against SARS CoV infections.     

Immunogenicity and In Vitro and In Vivo Protective Effects of Antibodies Targeting a Recombinant Form of the Streptococcus mutans P1 Surface Protein

Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a 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 multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1_39–512), 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 present study, we further investigated the immunological features of P1_39–512 in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1_39–512 preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1_39–512-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1_39–512 antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1_39–512 as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1.