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Biomedical subjects

T N Nguyen

Publications and source records attributed to T N Nguyen.

At least 19 recordsLinked to original sources

White matter lesions, cognition, and recurrent hemorrhage in lobar intracerebral hemorrhage.

BACKGROUND: Accumulating evidence suggests that white matter lesions are associated with vascular cognitive impairment. The authors investigated the relationships between white matter lesions, cognitive impairment, and risk of recurrent hemorrhage in a prospectively identified cohort of patients with lobar intracerebral hemorrhage (ICH). METHODS: The authors collected clinical and genetic information on 182 consecutive patients age > or = 55 who had CT scan at admission for lobar ICH. White matter disease was graded on CT in all subjects and on MRI in a subset of 82 patients. All scans were interpreted blinded to clinical information. Survivors were followed for recurrent ICH by telephone interview. RESULTS: White matter damage was common (present on CT in 77%) and severe (advanced CT grade in 32%). White matter damage was correlated with the total number of hemorrhages on gradient-echo MRI and with risk of recurrent ICH. Subjects with cognitive impairment prior to their index ICH were more likely to have severe white matter damage on CT (OR 3.6, 95% CI 1.6 to 8.1, p = 0.003) and more likely to have advanced periventricular hyperintensities on MRI. The relationships between white matter damage and cognitive impairment were similar in the subset of 88 subjects meeting criteria for probable or definite cerebral amyloid angiopathy and remained independent after adjustment for age, cortical atrophy, and APOE genotype. CONCLUSIONS: White matter damage in lobar ICH is common and is associated with cognitive impairment. These data support the possibility that an underlying vasculopathy in lobar ICH patients, possibly cerebral amyloid angiopathy, can cause clinically important vascular dysfunction.

Cerebral Amyloid Angiopathy↗

Virulent Toxoplasma gondii strain RH promotes T-cell-independent overproduction of proinflammatory cytokines IL12 and gamma-interferon.

The aim of this study was the analysis of the cytokine response in BALB/c mice infected with the highly virulent RH or the weakly virulent Beverley strains of Toxoplasma gondii. Analysis of cytokine messages showed increased expression of IL12, IFN-gamma and TNF-alpha, but not IL4 mRNAs in spleen cells after infection with the T. gondii strains RH and Beverley. High levels of circulating IL12 and IFN-gamma were detected in the serum of mice infected with strain RH, although TNF-alpha levels remained low. In contrast, the same cytokines were detected at only low levels in the serum of mice infected with the Beverley strain. Administration of antibody against IL12 or IFN-gamma significantly delayed time to death of mice infected with strain RH compared to controls. T-Cell-deficient as well as normal mice were equally infected by strain RH, suggesting that T lymphocytes do not contribute to the response. Depletion of natural killer cells from the splenocyte population abolished the in vitro production of IFN-gamma. Together, our data suggest that the virulent strain RH induces in BALB/c mice a type 1 cytokine pattern with T-cell-independent overproduction of IL12 and IFN-gamma that may be involved in the pathogenesis of this micro-organism.

Animals↗

Safety and immunogenicity of a novel recombinant subunit respiratory syncytial virus vaccine (BBG2Na) in healthy young adults.

A novel recombinant respiratory syncytial virus (RSV) subunit vaccine, designated BBG2Na, was administered to 108 healthy adults randomly assigned to receive 10, 100, or 300 microg of BBG2Na in aluminum phosphate or saline placebo. Each subject received 1, 2, or 3 intramuscular injections of the assigned dose at monthly intervals. Local and systemic reactions were mild, and no evidence of harmful properties of BBG2Na was reported. The highest ELISA and virus-neutralizing (VN) antibody responses were evident in the 100- and 300-microg groups; second or third injections provided no significant boosts against RSV-derived antigens. BBG2Na induced > or 2-fold and > or =4-fold increases in G2Na-specific ELISA units in up to 100% and 57% of subjects, respectively; corresponding RSV-A-specific responses were 89% and 67%. Furthermore, up to 71% of subjects had > or =2-fold VN titer increases. Antibody responses to 2 murine lung protective epitopes were also highly boosted after vaccination. Therefore, BBG2Na is safe, well tolerated, and highly immunogenic in RSV-seropositive adults.

Adolescent↗

Lipophilic quaternary ammonium salt acts as a mucosal adjuvant when co-administered by the nasal route with vaccine antigens.

Nasal administration of vaccines is an attractive approach which offers several significant advantages over traditional intramuscular vaccine delivery. These advantages include easier administration and induction of immune responses in the mucosal secretions of the body. In this study we describe a new potent nasal adjuvant, dimethyldioctadecylammonium bromide (DDA), that induces both mucosal and systemic immune responses when co-administered with diphtheria toxoid (DT), tetanus toxoid (TT) and BBG2Na antigens. In particular, we show that the nasal delivery of recombinant fragment (BBG2Na) of the G protein of respiratory syncytial virus (RSV) mixed with DDA induces both local and systemic anti-RSV immune responses and protects against viral challenge. Furthermore, we provide evidence that the DDA+BBG2Na vaccine does not induce lung immunopathology upon subsequent RSV challenge.

Adjuvants, Immunologic↗

Development of potent non-carbohydrate imidazole-based small molecule selectin inhibitors with antiinflammatory activity.

A novel series of non-carbohydrate imidazole-based selectin inhibitors has been discovered via high-throughput screening using a P-selectin ELISA-based assay system. The initial lead 1 had an IC(50) of 17 microM in the P-selectin ELISA; this potency was significantly improved via an extensive SAR exploration. One of the current lead compounds (29) has an IC(50) of 300 nM in a P-selectin ELISA; it also has good activity in P- and E-selectin cell adhesion assays and shows efficacy in vivo. These compounds represent a novel series of sLe(X) mimetics with antiinflammatory activity. Their unique profile supports our interest in their further evaluation as drug candidates for the treatment of inflammation. Herein we describe the syntheses, optimization, and SAR of this series of novel potent selectin antagonists.

Animals↗

Development of a quantitative assay for residual host cell proteins in a recombinant subunit vaccine against human respiratory syncytial virus.

We have developed and validated a process-specific immunoligand assay based on the Threshold system for the quantification of residual host cell proteins (HCPs) in a recombinant subunit vaccine candidate against the human respiratory syncytial virus (hRSV). The industrial process of this vaccine produced in Escherichia coli, involved five chromatography steps for the production of clinical-grade batches. The clearance of non-product-related protein throughout the purification process was documented by the evaluation of the HCP content in the chromatographic fractions at each step of the downstream processing. The assay had a detection limit of 0.5 ng/ml of HCP equivalent to 10 parts per million (ppm). The quantification limit was 1.3 ng/ml of HCP, giving a sensitivity range of the assay of 10 to 30 ppm. To our knowledge, this is the first sensitive HCP assay reported for a vaccine.

Antibodies, Bacterial↗

Identification and characterisation of multiple linear B cell protectopes in the respiratory syncytial virus G protein.

Respiratory syncytial virus (RSV) is an important respiratory pathogen in man, against which no vaccine is available. However, recent evidence suggests that antibodies to the RSV F and G proteins may play an important role in disease prevention. We previously demonstrated that BBG2Na, a subunit vaccine candidate including residues 130-230 of the Long strain G protein, protects rodents against RSV challenge. Using a panel of monoclonal antibodies (MAb) and synthetic peptides, five linear B cell epitopes were identified that mapped to residues 152-163, 165-172, 171-187 (two over-lapping epitopes) and 196-204. Antibody passive transfer and peptide immunisation studies revealed that all were protective. Pepscan analyses of anti-RSV-A and BBG2Na murine polyclonal sera suggested stronger immunogenicity of some protective epitopes (protectopes) in the context of BBG2Na compared with live virus. However, all the identified murine B cell protectopes were conserved in RSV seropositive humans. Should these protectopes correspond with protection in humans, BBG2Na may constitute a very interesting vaccine candidate against RSV.

Amino Acid Sequence↗

Differential gene expression in Thermotoga neapolitana in response to growth substrate.

We have previously shown that beta-galactosidase activity expressed in Thermotoga neapolitana cells grown on lactose is subject to repression by glucose when they are grown on both substrates whereas beta-galactosidase and beta-glucosidase activities observed in cells grown on cellobiose are not repressed by growth on both glucose and cellobiose. To examine the differential expression of bgalA, bgalB, bglA and bglB in T. neapolitana, total RNA was isolated from cells growing on either glucose, lactose or cellobiose as the sole source of carbon and transcripts encoding these genes were quantitated by Northern blot analyses. BglA expression was induced by cellobiose while bglB was expressed under all three conditions at a lower level. Expression of the beta-galactosidase genes, bgalA and bgalB, was detected only in lactose-grown cells. beta-Glucosidase enzyme activity was only found in cell extracts of cellobiose-grown cells while beta-galactosidase activity was found in both lactose- and cellobiose-grown cell extracts. Our results show that in cellobiose-grown cells, the high beta-glucosidase activity is likely due to expression of bglA and that neither bgalA nor bgalB is responsible for the beta-galactosidase activity.

Blotting, Northern↗

Differential histopathology and chemokine gene expression in lung tissues following respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV- or BBG2Na-immunized mice.

A BALB/c mouse model of enhanced pulmonary pathology following vaccination with formalin-inactivated alum-adsorbed respiratory syncytial virus (FI-RSV) and live RSV challenge was used to determine the type and kinetics of histopathologic lesions induced and chemokine gene expression profiles in lung tissues. These data were compared and contrasted with data generated following primary and/or secondary RSV infection or RSV challenge following vaccination with a promising subunit vaccine, BBG2Na. Severe peribronchiolitis and perivascularitis coupled with alveolitis and interstitial inflammation were the hallmarks of lesions in the lungs of FI-RSV-primed mice, with peak histopathology evident on days 5 and 9. In contrast, primary RSV infection resulted in no discernible lesions, while challenge of RSV-primed mice resulted in rare but mild peribronchiolitis and perivascularitis, with no evidence of alveolitis or interstitial inflammation. Importantly, mice vaccinated with a broad dose range (20 to 0.02 microg) of a clinical formulation of BBG2Na in aluminium phosphate demonstrated histopathology similar to that observed in secondary RSV infection. At the molecular level, FI-RSV priming was characterized by a rapid and strong up-regulation of eotaxin and monocyte chemotactic protein 3 (MCP-3) relative gene expression (potent lymphocyte and eosinophil chemoattractants) that was sustained through late time points, early but intermittent up-regulation of GRO/melanoma growth stimulatory activity gene and inducible protein 10 gene expression, while macrophage inflammatory protein 2 (MIP-2) and especially MCP-1 were up-regulated only at late time points. By comparison, primary RSV infection or BBG2Na priming resulted in considerably lower eotaxin and MCP-3 gene expression increases postchallenge, while expression of lymphocyte or monocyte chemoattractant chemokine genes (MIP-1beta, MCP-1, and MIP-2) were of higher magnitude and kinetics at early, but not late, time points. Our combined histopathologic and chemokine gene expression data provide a basis for differentiating between aberrant FI-RSV-induced immune responses and normal responses associated with RSV infection in the mouse model. Consequently, our data suggest that BBG2Na may constitute a safe RSV subunit vaccine for use in seronegative infants.

Animals↗

Human Taf(II)130 is a coactivator for NFATp.

NFATp is one member of a family of transcriptional activators that regulate the expression of cytokine genes. To study mechanisms of NFATp transcriptional activation, we established a reconstituted transcription system consisting of human components that is responsive to activation by full-length NFATp. The TATA-associated factor (TAF(II)) subunits of the TFIID complex were required for NFATp-mediated activation in this transcription system, since TATA-binding protein (TBP) alone was insufficient in supporting activated transcription. In vitro interaction assays revealed that human TAF(II)130 (hTAF(II)130) and its Drosophila melanogaster homolog dTAF(II)110 bound specifically and reproducibly to immobilized NFATp. Sequences contained in the C-terminal domain of NFATp (amino acids 688 to 921) were necessary and sufficient for hTAF(II)130 binding. A partial TFIID complex assembled from recombinant hTBP, hTAF(II)250, and hTAF(II)130 supported NFATp-activated transcription, demonstrating the ability of hTAF(II)130 to serve as a coactivator for NFATp in vitro. Overexpression of hTAF(II)130 in Cos-1 cells inhibited NFATp activation of a luciferase reporter. These studies demonstrate that hTAF(II)130 is a coactivator for NFATp and represent the first biochemical characterization of the mechanism of transcriptional activation by the NFAT family of activators.

Animals↗

Deuterium-proton exchange on the native wild-type transthyretin tetramer identifies the stable core of the individual subunits and indicates mobility at the subunit interface.

Transthyretin is a human protein capable of amyloid formation that is believed to cause several types of amyloid disease, depending on the sequence deposited. Previous studies have demonstrated that wild-type transthyretin (TTR), although quite stable, forms amyloid upon dissociation from its native tetrameric form into monomers with an altered conformation. Many naturally occurring single-site variants of TTR display decreased stability in vitro, manifested by the early onset familial amyloid diseases in vivo. Only subtle structural changes were observed in X-ray crystallographic structures of these disease associated variants. In this study, the stability of the wild-type TTR tetramer was investigated at the residue-resolution level by monitoring (2)H-H exchange via NMR spectroscopy. The measured protection factors for slowly-exchanging amide hydrogen atoms reveal a stable core consisting of strands A, B, E, F, and interestingly, the loop between strands A and B. In addition, the faster exchange of amide groups from residues at the subunit interfaces suggests unexpected mobility in these regions. This information is crucial for future comparisons between disease-associated and wild-type tetramers. Such studies can directly address the regions of TTR that become destabilized as a consequence of single amino acid substitutions, providing clues to aspects of TTR amyloidogenesis.

Amides↗

Purification and characterization of beta-actin-rich tumor cell pseudopodia: role of glycolysis.

The MSV-MDCK-INV invasive variant of Moloney sarcoma virus (mos) transformed MDCK cells express multiple beta-actin-rich pseudopodia (P. U. Le et al., Cancer Res. 58, 1631-1635, 1998). We show here that the tips of these actively protruding cellular domains are morphologically distinct presenting numerous blebs and selectively pass through 1-microm-pore filters. The pseudopodia were purified from the underside of the filters and a major protein component was identified as the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). By confocal microscopy, GAPDH colocalized with actin in MSV-MDCK-INV pseudopodia localizing this glycolytic enzyme to this site of active actin polymerization. Inhibition of glycolysis with 2-deoxyglucose or oxamate induced a rapid transformation of beta-actin-rich pseudopodia into extended lamellipodia and prevented cell motility. A localized glycolytic supply of energy therefore regulates the formation of beta-actin-rich pseudopodial protrusions and thereby the motility of invasive tumor cells.

Actins↗

Influence of administration dose and route on the immunogenicity and protective efficacy of BBG2Na, a recombinant respiratory syncytial virus subunit vaccine candidate.

The immunogenicity and protective efficacy of BBG2Na, a novel recombinant respiratory syncytial virus subunit vaccine candidate, was assessed in BALB/c mice under various conditions of dose, administration route and number of immunisations. A single intra-peritoneal (i.p.) dose of 2 microg, or two doses of 0.2 microg, were sufficient to induce elevated RSV-A serum antibodies and sterilising lung protective immunity. Serum antibody titres were significantly boosted following second immunisations, but not a third. Of three routes of immunisation, i.p. induced the highest RSV-A antibody titres, followed in efficacy by the intra-muscular (i. m.) and subcutaneous (s.c.) routes. Nonetheless, all three routes induced comparable and sterilising lung protection. In contrast, upper respiratory tract protection was observed only after i.p. vaccination, although significant viral titre reductions were evident following i.m. or s.c. immunisations. Interestingly, Pepscan analyses indicated that antibody epitope usage was highest in i.p. and lowest in i.m. immunised mice, respectively. Nonetheless, all routes resulted in antibody responses to known lung protective epitopes (protectopes). Thus, the prevention of serious lower respiratory tract disease, the principle goal of a RSV vaccine, but not URT infection, is dose dependent but unlikely to be influenced by the route of BBG2Na administration.

Animals↗

Partial protection to respiratory syncytial virus (RSV) elicited in mice by intranasal immunization using live staphylococci with surface-displayed RSV-peptides.

A live bacterial vaccine-delivery system based on the food-grade bacterium Staphylococcus carnosus was used for delivery of peptides from the G glycoprotein of human respiratory syncytial virus, subtype A (RSV-A). Three peptides, corresponding to the G protein amino acids, 144-159 (denoted G5), 190-203 (G9) and 171-188 (G4 S), the latter with four cysteine residues substituted for serines, were expressed by recombinant means as surface-exposed on three different bacteria, and their surface accessibility on the bacteria was verified by fluorescence-activated cell sorting (FACS). Intranasal immunization of mice with the live recombinant staphylococci elicited significant anti-peptide as well as anti-virus serum IgG responses of balanced IgG1/IgG2a isotype profiles, and upon viral challenge with 10(5) tissue culture infectious doses(50) (TCID(50)), lung protection was demonstrated for approximately half of the mice in the G9 and G4 S immunization groups. To our knowledge, this is the first study in which protective immunity to a viral pathogen has been evoked using food-grade bacteria as vaccine-delivery vehicles.

Administration, Intranasal↗

Genetic variability determinants of Helicobacter pylori: influence of clinical background and geographic origin of isolates.

Helicobacter pylori has an unusual pattern of genetic variation, which complicates research on this organism. To gain a better understanding of the forces behind this phenomenon, the extent to which recombination and single point mutations affect genetic variability in H. pylori was quantified and the influence of both geographical distance and clinical background were assessed. Site-directed restriction-endonuclease digestion of 2 gene fragments was performed on 168 isolates from Montreal and Berlin. Allelic diversity was found to be much higher for H. pylori than for other bacterial species. This finding is consistent with those of previous studies on H. pylori that were conducted using other techniques. However, nucleotide diversity was within the range reported for other bacterial species. Phylogenetic analysis found no grouping of strains with clinical background or geographical origin. Recombination at a rate that resulted in linkage equilibrium within genes can explain these observations.

Alleles↗

Staphylococcal surface display and its applications.

Novel surface proteins can be introduced onto the bacterial cell surface by recombinant means. Here, we describe the development of such display systems for two food-grade bacteria, Staphylococcus carnosus and Staphylococcus xylosus, and present how such engineered bacteria can be used in different applications. A study will be described in which such staphylococci were employed as vaccine delivery vehicles to elicit protective antibody responses to respiratory syncytial virus (RSV). The use of surface-engineered staphylococci as novel microbial biocatalysts, as a new type of whole-cell diagnostic devices or for adsorption of metal ions with potential environmental or biosensor applications, will also be discussed.

Animals↗

Synthesis and characterization of Respiratory Syncytial Virus protein G related peptides containing two disulfide bridges.

Respiratory Syncytial Virus (RSV) is the most important cause of bronchiolitis and viral pneumonia in infants and young children. Approximately 100000 children are hospitalized in the USA each year as a result of RSV infections. During the research and development of subunit human Respiratory Syncytial Virus vaccines (hRSV), we have produced numerous synthetic peptides and recombinant proteins containing the four cysteines of the highly conserved central region of the G attachment protein. For several of these disulfide-containing peptides, all possible oxidized isomers were synthesized using various oxidation conditions and resulting in different ratios of isomers. Each isolated isomer was fully characterized by RP-HPLC, FZCE and ES-MS after purification by preparative RP-HPLC. The different cysteine pairings were unambiguously established after enzymatic digestion, LC-MS analysis and peptide microsequencing. These synthesis and analytical methods were developed for the characterization on one hand, of recombinant fusion protein BBG2Na which is currently being investigated in advanced clinical phases as a very promising vaccine candidate, and on the other hand, for peptides which were synthesized to be evaluated as conjugate vaccines or as immunochemical tools, after covalent coupling to carrier proteins. Furthermore, these studies allowed us to determine which of the different possible isomers was the most stable and probably the preferred form in native conditions. Finally, the different oxidation and analysis conditions, should be useful for disulfide pairing studies of other peptides and proteins having the same 'xCxxCxxxxxCxxxCx' framework, such as G proteins of non-human RSV strains, developed by other groups as veterinary vaccine candidates for example.

Amino Acid Sequence↗