What is your diagnosis? Compression fracture of the 12th thoracic vertebra.
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Animals↗
Biomedical subjects
J H Cox
Publications and source records attributed to J H Cox.
Highly stable expression of a foreign gene from rabies virus vectors.
A reverse genetics approach was applied to generate a chimeric nonsegmented negative strand RNA virus, rabies virus (RV) of the Rhabdoviridae family, that expresses a foreign protein. DNA constructs containing the entire open reading frame of the bacterial chloramphenicol acetyltransferase (CAT) gene and an upstream RV cistron border sequence were inserted either into the nontranslated pseudogene region of a full-length cDNA copy of the RV genome or exchanged with the pseudogene region. After intracellular T7 RNA polymerase-driven expression of full-length antigenome RNA transcripts and RV nucleoprotein, phosphoprotein and polymerase from transfected plasmids, RVs transcribing novel monocistronic mRNAs and expressing CAT at high levels, were recovered. The chimeric viruses possessed the growth characteristics of standard RV and were genetically stable upon serial cell culture passages. CAT activity was still observed in cell cultures infected with viruses passaged for more than 25 times. Based on the unprecedented stability of the chimeric RNA genomes, which is most likely due to the structure of the rhabdoviral ribonucleoprotein complex, we predict the successful future use of recombinant rhabdovirus vectors for displaying foreign antigens or delivering therapeutic genes.
Animals↗
CD4+ T-lymphocyte lines developed from HIV-1-seropositive patients recognize different epitopes within the V3 loop.
To define the epitopes present within the V3 loop sequence recognized by five HIV-1 envelope-specific T-cell lines, a panel of V3 LAI peptides bearing sequential truncations from both the N- and C-terminus was synthesized and tested for their ability to induce proliferation. Each individual T-cell line had a different pattern of response against the truncated V3 peptides, demonstrating the presence of a cluster of CD4+ T-cell epitopes within the V3 loop. To assess the ability of these envelope-specific T-cell lines to recognize and proliferate in response to V3 loops of different viral strains, they were tested against a panel of heterologous V3 loop peptides derived from different viral genotypes within and outside of HIV-1 clade B. There was no proliferative response against heterologous V3 loops by any of the lines, demonstrating that recognition of the V3 epitopes is highly strain specific. One of the defined epitopes was shown to elicit a cytotoxic response as well, suggesting the multifaceted role that the CD4+ T cell might play in HIV-1 disease.
Amino Acid Sequence↗
Presentation of endogenous and exogenous antigens is not affected by inactivation of E1 ubiquitin-activating enzyme in temperature-sensitive cell lines.
Little is known regarding the mechanism by which MHC class I-associated peptides are generated. Proteins can be targeted for degradation by the covalent attachment of ubiquitin. The first step in ubiquitin conjugation to proteins is its binding to E1 ubiquitin-activating enzyme. To study the role of ubiquitin-targeted protein degradation in Ag processing, we used two mutant cell lines with temperature-sensitive E1 proteins, and a recombinant vaccinia virus expressing wild-type human E1. One of the cell lines examined (hamster ts20 cells) was previously reported to have a minimal capacity after a 1-h incubation at 41 degrees C to present osmotically loaded OVA to a T cell hybridoma, as assessed by IL-2 release. Even after incubating the same cells for 1 h at 43 degrees C, we failed to detect an E1-related decrease in the presentation of biosynthesized or osmotically loaded OVA to splenic T cells, as measured by target cell lysis. We introduce the use of mouse tsA1S9 cells to Ag-processing studies and provide the initial biochemical characterization of their defect in protein ubiquitination. Relative to parental L929 cells, after thermal inactivation of E1, these cells actually demonstrate enhanced presentation of endogenous or exogenous viral Ags to T cells. Our findings do not support a role for protein ubiquitination in Ag processing, and indicate that either the temperature-sensitive cell lines examined do not exhibit a sufficient reduction in ubiquitin-conjugating activity to affect the generation of antigenic peptides, or that ubiquitin-targeted proteolysis is not essential for processing the two exogenous and six endogenous Ags examined.
Animals↗
Single- and multiple-dose-response relationships of beta-carotene in cystic fibrosis.
Concentrations of carotenoids are low in patients with cystic fibrosis (CF) and are associated with essential fatty acid deficiency and increased markers of inflammation. We conducted single- and multiple-dose studies of beta-carotene supplementation in patients with CF. Dose-proportional increases in beta-carotene concentrations were found, although clearance was independent of dose. Large doses of beta-carotene were necessary to achieve normal plasma levels.
Adjuvants, Immunologic↗
Establishment and characterization of human immunodeficiency virus type 1 (HIV-1) envelope-specific CD4+ T lymphocyte lines from HIV-1-seropositive patients.
Human immunodeficiency virus type 1 (HIV-1) gp160-, gp120-, and tetanus toxoid-specific CD4+ T lymphocyte lines were developed from 11 HIV-1-seropositive volunteers enrolled in a vaccine therapy trial. Of the 20 HIV-1 envelope-specific T cell lines, 9 were challenged with a panel of overlapping peptides spanning the gp120LAI sequence. The most frequently recognized regions were amino acids 74-105 in the C1 region and 306-328 in the V3 region. When tested against a panel of divergent HIV-1 envelopes, 55% of the envelope-specific lines were able to recognize gp120MN, while only 22% recognized gp120SF2. Cytotoxicity testing with HIV-1 envelope antigen or peptides demonstrated killing by all 3 envelope-specific lines tested. Supernatants from 2 of 9 lines had high titers of p24 gag antigen, which did not seem to interfere with functional properties.
Amino Acid Sequence↗
Expression of adenovirus E3/19K protein does not alter mouse MHC class I-restricted responses to vaccinia virus.
The adenovirus E3/19K glycoprotein forms a tight complex with most human and certain mouse MHC class I allomorphs, retaining them in the endoplasmic reticulum by virtue of its cytosolic carboxyl terminal amino acids. This prevents the presentation of viral antigens at the cell surface to class I-restricted cytotoxic T lymphocytes (CTL). In adenovirus infection of cotton rats, E3/19K appears to act as an anti-inflammatory and/or immunosuppressive factor. Further studies of the role of E3/19K in adenovirus pathogenesis have been hampered by the lack of sufficient knowledge concerning the immune system of the cotton rat and by the poor correlation between adenovirus infection in mice and humans. We therefore addressed the function of this adenovirus glycoprotein in virus pathogenesis by infecting B10.HTG (H-2KdDb) mice with a vaccinia virus (VV) recombinant encoding E3/19K. The Kd and Db allomorphs normally present VV antigens to CTL and have high affinity for E3/19K. Infected mice were examined for the kinetics of virus replication in various tissues and the generation of natural killer (NK) cell and CTL responses. It was found that expression of E3/19K by vaccinia virus had no detectable effect on CTL responses, NK responses, or viral replication. These findings suggest that immune modulating proteins evolve to exploit unique circumstances of the host immune response to a given virus.
Adenovirus E3 Proteins↗
Association of human class I MHC alleles with the adenovirus E3/19K protein.
A panel of HLA-A and -B locus products was analyzed for their ability to associate with the adenovirus E3/19K (E19) protein in a co-immunoprecipitation assay. Three general categories of binding were identified. HLA-A2.1 and -B7 bind very well to E19. Compared with A2.1, 6- to 30-fold less E19 was associated with HLA-A3, -A1, and -Aw69; 50- to 150-fold less E19 was associated with HLA-Aw68, -B27, and -Bw58. Digestion with endoglycosidase H indicated that all levels of association resulted in inhibition of intracellular transport and processing, however, a fraction of Aw68, B27, and Bw58 escaped from intracellular retention. In contrast to the human class I molecules analyzed, transport of the murine H-2Dd molecule was not inhibited in the presence of E19. Hybrid class I molecules, in which exons encoding domains of A2.1 and H-2Dd had been exchanged, were used to define the regions of A2.1 required for E19 association. The alpha 1 and alpha 2 domains of A2.1 contain the minimum residues necessary for both stable association with E19 and subsequent inhibition of transport. A hybrid construct containing only the alpha 2 domain of A2.1 associated weakly with E19, but its post-translational processing was completely inhibited. In contrast, although a construct containing only the alpha 1 domain of A2.1 also associated weakly with E19, its intracellular transport was slowed rather than completely inhibited. Taken together, these results indicate that residues in both the alpha 1 and alpha 2 domains of A2.1 and Dd can influence stable binding of E19, with the phenotypic changes dominated by the origin of the alpha 2 domain.
Adenovirus Early Proteins↗
TAP (transporter associated with antigen processing)-independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxyl-terminus of the peptide.
Under most circumstances, cell surface MHC class I molecules display peptides derived from a cytosolic pool of proteins. The efficient presentation of such peptides requires the functioning of two MHC gene products [TAP1 and TAP2 (transporter-associated with Ag processing 1 and 2)] that form a complex that facilitates transmembrane movement of peptides from the cytosol to the endoplasmic reticulum, the site of peptide association with class I molecules. It has been previously shown that peptides can be presented in a TAP-independent manner in association with HLA A2.1 or H-2 Kd if they are expressed COOH-terminal to an endoplasmic reticulum insertion/signal sequence derived from the adenovirus E3/19K glycoprotein (Anderson et al., 1991. J. Exp. Med. 174: 489; Eisenlohr et al., 1992. Cell 71: 963). We show that: 1) the E3/19K signal sequence greatly enhances the presentation of each of four additional peptides tested in association with H-2 Kb or Kk, 2) the E3/19K signal sequence can be substituted by a signal sequence derived from beta-IFN, and 3) the E3/19K signal sequence does not function when located at the COOH terminus of antigenic peptides. These findings indicate that first, many peptides require TAP for efficient presentation to T cells, second, expression of peptides COOH-terminal to signal sequences is a generally applicable method of bypassing the TAP-dependence of peptide presentation and third, the leader sequence does not act to bypass TAP simply by increasing the hydrophobic nature of peptides.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Nuclear localization of a double-stranded RNA-binding protein encoded by the vaccinia virus E3L gene.
We produced a B cell hybridoma (TW2.3) from vaccinia virus-infected mice that secreted a monoclonal antibody (MAb) reactive with a 25-kDA early viral protein that was localized by laser scanning confocal microscopy to the nucleus and cytoplasmic viral factory regions of infected cells. By cell-free translation of mRNA selected by hybridization to a complete library of vaccinia virus DNA fragments, the immunoreactive polypeptide was mapped to open reading frame E3L. The RNA start site of an early promoter was located 26 nucleotides upstream of the first methionine codon of E3L. Evidence was obtained that translation initiation occurs in vivo and in vitro at both the first and second methionine codons to produce major and minor polypeptides of 25 and 19 kDa, respectively. Both polypeptides bound double-stranded RNA, confirming the recent report of H.-W. Chang, J. C. Watson, and B. L. Jacobs (Proc. Natl. Acad. Sci. USA 89, 4825-4829, 1992). Other vaccinia virus proteins were not required for the nuclear localization of the E3L protein, since MAb TW2.3 bound to the nuclei of uninfected cells that were transfected with the E3L gene under the control of the SV40 early promoter. We also demonstrated that the E3L protein can bind to nuclei of aldehyde fixed and detergent permeabilized uninfected cells. This binding was abrogated by treatment of the cells with RNase but not DNase. The nuclear and cytoplasmic locations of the double-stranded RNA binding protein are consistent with multiple functions in the vaccinia virus infectious cycle.
Amino Acid Sequence↗
Carotenoid levels in normal children and in children with cystic fibrosis.
The carotenoids are potent antioxidants with the ability to quench singlet oxygen and other toxic oxygen species. We studied 17 patients with cystic fibrosis (CF) and 10 normal children to assess plasma levels of four carotenoids, beta-carotene, alpha-carotene, lutein, and lycopene, by high-performance liquid chromatography. We found significantly lower plasma levels of specific carotenoids in children with CF than in normal control subjects. The standardization of carotenoid levels for total cholesterol did not significantly attenuate these differences. No differences in total carotene intake were apparent between the groups. Carotenoid levels did not correlate with fat absorption or measures of adiposity in children with CF. Additionally, levels of selected carotenoids correlated negatively with serum IgG levels, an indirect measure of inflammation. The differences in plasma carotenoid levels between children with CF and normal children may be due to rapid turnover of carotenoids, perhaps through quenching of toxic oxygen species in inflammatory states of CF. Studies assessing supplementation of these antioxidants should be considered.
Adolescent↗
Hyperkalaemic periodic paralysis: diagnosing the disease in the headlines.
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Animals↗
Molecular analysis of rabies-related viruses from Ethiopia.
From brain samples collected from domestic animals in Ethiopia, two rabies-related viruses were isolated. According to their reactivity pattern with anti-nucleocapsid monoclonal antibodies, they were characterized as Lagos bat virus (isolate Eth-58) and Mokola virus (isolate Eth-16). This classification was confirmed by neutralization experiments with Mokola and Lagos bat specific antisera. Two potent anti-rabies vaccines were unable to protect mice against the two rabies-related viruses. In order to investigate molecular relationships to classical rabies virus, cDNA cloning and sequencing was performed. The RNA genome of both viruses comprises 12 kilobases (kb) and has an organization similar to that of rabies virus with the gene order 3'-N-P-M-G-L-5'. Using virus-specific cDNA as probes in heterologous hybridization experiments, the RNAs of other members of lyssavirus serotypes 2 and 3 were detected. From hybridization experiments and sequence analysis of the 3' terminal 5,5 kb of the genomes, Eth-16 and Eth-58 viruses were shown to be equally genetically distant from rabies virus with 60% nucleotide identity; Eth-16 and Eth-58 had 68% homology.
Animals↗
Isolation and characterization of 115 street rabies virus isolates from Ethiopia by using monoclonal antibodies: identification of 2 isolates as Mokola and Lagos bat viruses.
There were 115 isolates of rabies viruses recovered by tissue culture technique from 119 animal brains collected in Ethiopia. By using 17 selected antinucleocapsid monoclonal antibodies (MAbs), 113 isolates were classic street rabies viruses (serotype 1). An isolate of feline origin (Eth-16) was a Mokola virus (serotype 3) and another isolate (Eth-58, obtained from a rabid dog) was serotype 2 (Lagos bat virus). None of the 16 antiglycoprotein MAbs used neutralized the Eth-16 isolate, whereas Eth-58 was neutralized by 1 (TERA543). Antirabies vaccines prepared from Pitman-Moore and Pasteur virus strains protected mice against homologous challenge, but neither was protective against the 2 rabies-related virus isolates. The isolation of Mokola and Lagos bat viruses from domestic animals in eastern Africa is of public and veterinary concern mainly due to lack of effective vaccines against these agents and the difficulty of proper diagnosis.
Animals↗
Detection of rabies antibody by ELISA and RFFIT in unvaccinated dogs and in the endangered Simien jackal (Canis simensis) of Ethiopia.
Varying levels of rabies antibody have been detected both by Enzyme Linked Immunosorbent Assay (ELISA) and Rapid Fluorescent Focus Inhibition Test (RFFIT) in the sera collected from wild and domestic canids in the Bale Mountains National Park (BMNP) of Southern Ethiopia. Rabies antibody was detected in 80% (8 out of 10) of domestic dog samples, 13.3% (2 out of 15) of Simien jackal samples and in one common jackal. Rabies virus was isolated from one dog in an area where contact with the Simien jackal could possibly occur. All samples examined from wild rodents as possible reservoir hosts for rabies were found negative. The presence of large proportion of susceptible Simien jackals in the population should be a cause of great concern in saving this endangered species from the ravages of rabies.
Animals↗
Rabies virus neuritic paralysis: immunopathogenesis of nonfatal paralytic rabies.
Two pathogenetically distinct disease manifestations are distinguished in a murine model of primary rabies virus infection with the Evelyn-Rokitnicky-Abelseth strain, rabies virus neuritic paralysis (RVNP) and fatal encephalopathogenic rabies. RVNP develops with high incidence in immunocompetent mice after intraplantar infection as a flaccid paralysis restricted to the infected limb. The histopathologic correlate of this monoplegia is a degeneration of the myelinated motor neurons of the peripheral nerve involved. While, in this model, fatal encephalopathogenic rabies develops only after depletion of the CD4 subset of T lymphocytes and without contribution of the CD8 subset, RVNP is identified as an immunopathological process in which both the CD4 and CD8 subsets of T lymphocytes are critically implicated.
Animals↗
Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation.
The E3/19K glycoprotein of adenovirus functions to diminish recognition of adenovirus-infected cells by major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTLs) by binding intracellular class I molecules and preventing them from reaching the plasma membrane. In the present study we have characterized the nature of the interaction between E3/19K and the H-2Kd (Kd) molecule. An E3/19K molecule genetically engineered to terminate six residues from its normal COOH terminus (delta E19), was found to associate with Kd in a manner indistinguishable from wild-type E3/19K. Unlike E3/19K, however, delta E19 was transported through the Golgi complex to the plasma membrane, where it could be detected biochemically and immunocytochemically using a monoclonal antibody specific for the lumenal domain of E3/19K. Importantly, delta E19 also differed from E3/19K in being unable to prevent the presentation of Kd-restricted viral proteins to CTLs. This is unlikely to be due to delta E19 having a lower avidity for Kd than E3/19K, since delta E19 was able to compete with E3/19K for Kd binding, both physically, and functionally in nullifying the E3/19K blockade of antigen presentation. These findings indicate that the ability of E3/19K to block antigen presentation is due solely to its ability to retain newly synthesized class I molecules in the endoplasmic reticulum.
Adenovirus Early Proteins↗