bluuebunny/crossref_metadata_2025 huggingface dataset:
N-Linked Glycosylation of CXCR4 Masks Coreceptor Function for CCR5-Dependent Human Immunodeficiency Virus Type 1 Isolates
Authors: Chabot Donald J. , Chen Hong , Dimitrov Dimiter S. , Broder Christopher C.
Published: July 2002

The chemokine receptors CXCR4 and CCR5 are the principal coreceptors for infection of X4 and R5 human immunodeficiency virus type 1 (HIV-1) isolates, respectively. Here we report on the unexpected observation that the removal of the N-linked glycosylation sites in CXCR4 potentially allows the protein to serve as a universal coreceptor for both X4 and R5 laboratory-adapted and primary HIV-1 strains. We hypothesize that this alteration unmasks existing common extracellular structures reflecting a conserved three-dimensional similarity of important elements of CXCR4 and CCR5 that are involved in HIV envelope glycoprotein (Env) interaction. These results may have far-reaching implications for the differential recognition of cell type-dependent glycosylated CXCR4 by HIV-1 isolates and their evolution in vivo. They also suggest a possible explanation for the various observations of restricted virus entry in some cell types and further our understanding of the framework of elements that represent the Env-coreceptor contact sites.
Adeno-Associated Virus Serotype 4 (AAV4) and AAV5 Both Require Sialic Acid Binding for Hemagglutination and Efficient Transduction but Differ in Sialic Acid Linkage Specificity
Authors: Kaludov Nikola , Brown Kevin E. , Walters Robert W. , Zabner Joseph , Chiorini John A.
Published: July 2002

Adeno-associated virus serotype 4 (AAV4) and AAV5 have different tropisms compared to AAV2 and to each other. We recently reported that α2-3 sialic acid is required for AAV5 binding and transduction. In this study, we characterized AAV4 binding and transduction and found it also binds sialic acid, but the specificity is significantly different from AAV5. AAV4 can hemagglutinate red blood cells from several species, whereas AAV5 hemagglutinates only rhesus monkey red blood cells. Treatment of red blood cells with trypsin inhibited hemagglutination for both AAV4 and AAV5, suggesting that the agglutinin is a protein. Treatment of Cos and red blood cells with neuraminidases also indicated that AAV4 bound α2-3 sialic acid. However, resialylation experiments with neuraminidase-treated red blood cells demonstrated that AAV4 binding required α2–3 O-linked sialic acid, whereas AAV5 required N-linked sialic acid. Similarly, resialylation of sialic acid-deficient CHO cells supported this same conclusion. The difference in linkage specificity for AAV4 and AAV5 was confirmed by binding and transduction experiments with cells incubated with either N-linked or O-linked inhibitors of glycosylation. Furthermore, AAV4 transduction was only blocked with soluble α2-3 sialic acid, whereas AAV5 could be blocked with either α2–3 or α2-6 sialic acid. These results suggest that AAV4 and AAV5 require different sialic acid-containing glycoproteins for binding and transduction of target cells and they further explain the different tropism of AAV4 and AAV5.
Human Hepatitis B Virus Polymerase Interacts with the Molecular Chaperonin Hsp60
Authors: Park Sung Gyoo , Jung Guhung
Published: July 2002

Previous studies showed that hepatitis B virus polymerase (HBV Pol) interacts with host factors such as the Hsp90 complex, which is a critical step in viral genome replication. In this report, we propose that another chaperone, Hsp60, interacts with human HBV Pol and that this is a very important step for maturation of human HBV Pol into the active state. In the immunoprecipitation of recombinant human HBV Pol expressed in insect cells with the recombinant baculovirus expression system, the 60-kDa protein was coimmunoprecipitated with Pol and the protein was identified as Hsp60 through peptide sequencing and immunogenic analysis with an anti-Hsp60 antibody. In vitro experiments showed that Hsp60 strongly affected human HBV Pol activity in that (i) blocking of Hsp60 by the protein-specific antibody reduced human HBV Pol activity, (ii) the activity was increased by addition of Hsp60 in the presence of ATP, and (iii) ATP synergistically activated human HBV Pol with Hsp60. In vivo experiments showed that inhibition of Hsp60 in cells by a mutant Hsp60, CΔ540, resulted in the reduction of human HBV Pol activity. In summary, our results indicate that the interaction is significant for conversion of human HBV Pol into the active state.
Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented intrans
Authors: Grassmann Claus W. , Isken Olaf , Tautz Norbert , Behrens Sven-Erik
Published: July 2002

The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5′ and 3′ untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act incisor intrans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued intrans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to becisdominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.
Interaction of Classical Swine Fever Virus with Membrane-Associated Heparan Sulfate: Role for Virus Replication In Vivo and Virulence
Authors: Hulst M. M. , van Gennip H. G. P. , Vlot A. C. , Schooten E. , de Smit A. J. , Moormann R. J. M.
Published: July 2002

Passage of native classical swine fever virus (CSFV) in cultured swine kidney cells (SK6 cells) selects virus variants that attach to the surface of cells by interaction with membrane-associated heparan sulfate (HS). A Ser-to-Arg change in the C terminus of envelope glycoprotein E
rns
(amino acid 476 in the open reading frame of CSFV) is responsible for selection of these HS-binding virus variants (M. M. Hulst, H. G. P. van Gennip, and R. J. M. Moormann, J. Virol. 74:9553–9561, 2000). In this investigation we studied the role of binding of CSFV to HS in vivo. Using reverse genetics, an HS-independent recombinant virus (S-ST virus) with Ser
476
and an HS-dependent recombinant virus (S-RT virus) with Arg
476
were constructed. Animal experiments indicated that this adaptive Ser-to-Arg mutation had no effect on the virulence of CSFV. Analysis of viruses reisolated from pigs infected with these recombinant viruses indicated that replication in vivo introduced no mutations in the genes of the envelope proteins E
rns
, E1, and E2. However, the blood of one of the three pigs infected with the S-RT virus contained also a low level of virus particles that, when grown under a methylcellulose overlay, produced relative large plaques, characteristic of an HS-independent virus. Sequence analysis of such a large-plaque phenotype showed that Arg
476
was mutated back to Ser
476
. Removal of HS from the cell surface and addition of heparin to the medium inhibited infection of cultured (SK6) and primary swine kidney cells with S-ST virus reisolated from pigs by about 70% whereas infection with the administered S-ST recombinant virus produced in SK6 cells was not affected. Furthermore, E
rns
S-ST protein, produced in insect cells, could bind to immobilized heparin and to HS chains on the surface of SK6 cells. These results indicated that S-ST virus generated in pigs is able to infect cells by an HS-dependent mechanism. Binding of concanavalin A (ConA) to virus particles stimulated the infection of SK6 cells with S-ST virus produced in these cells by 12-fold; in contrast, ConA stimulated infection with S-ST virus generated in pigs no more than 3-fold. This suggests that the surface properties of S-ST virus reisolated from pigs are distinct from those of S-ST virus produced in cell culture. We postulate that due to these surface properties, in vivo-generated CSFV is able to infect cells by an HS-dependent mechanism. Infection studies with the HS-dependent S-RT virus, however, indicated that interaction with HS did not mediate infection of lung macrophages, indicating that alternative receptors are also involved in the attachment of CSFV to cells.
Fine Definition of the Epitope on the gp41 Glycoprotein of Human Immunodeficiency Virus Type 1 for the Neutralizing Monoclonal Antibody 2F5
Authors: Parker Carol E. , Deterding Leesa J. , Hager-Braun Christine , Binley James M. , Schülke Norbert , Katinger Hermann , Moore John P. , Tomer Kenneth B.
Published: July 2002

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.
Gamma Interferon Can Prevent Herpes Simplex Virus Type 1 Reactivation from Latency in Sensory Neurons
Authors: Liu Ting , Khanna Kamal M. , Carriere Brian N. , Hendricks Robert L.
Published: July 2002

We recently demonstrated that CD8+T cells could block herpes simplex virus type 1 (HSV-1) reactivation from latency in ex vivo trigeminal ganglion (TG) cultures without destroying the infected neurons. Here we establish that CD8+T-cell prevention of HSV-1 reactivation from latency is mediated at least in part by gamma interferon (IFN-γ). We demonstrate that IFN-γ was produced in ex vivo cultures of dissociated latently infected TG by CD8+T cells that were present in the TG at the time of excision. Depletion of CD8+T cells or neutralization of IFN-γ significantly enhanced the rate of HSV-1 reactivation from latency in TG cultures. When TG cultures were treated with acyclovir for 4 days to insure uniform latency, supplementation with recombinant IFN-γ blocked HSV-1 reactivation in 80% of cultures when endogenous CD8+T cells were present and significantly reduced and delayed HSV-1 reactivation when CD8+T cells or CD45+cells were depleted from the TG cultures. The effectiveness of recombinant IFN-γ in blocking HSV-1 reactivation was lost when its addition to TG cultures was delayed by more than 24 h after acyclovir removal. We propose that when the intrinsic ability of neurons to inhibit HSV-1 gene expression is compromised, HSV-specific CD8+T cells are rapidly mobilized to produce IFN-γ and perhaps other antiviral cytokines that block the viral replication cycle and maintain the viral genome in a latent state.
Porcine Encephalomyocarditis Virus Persists in Pig Myocardium and Infects Human Myocardial Cells
Authors: Brewer Laurie A. , Lwamba Humphrey C. M. , Murtaugh Michael P. , Palmenberg Ann C. , Brown Corrie , Njenga M. Kariuki
Published: July 2002

Recent advances toward using pig tissues in human transplantation have made it necessary to determine the risk of transmitting zoonotic viruses from pigs to humans or vice versa. We investigated the suitability of the porcine encephalomyocarditis virus (EMCV) model for such studies by determining its ability to persist in pigs, escape detection by routine serological methods, and infect human cells. Intraperitoneal inoculation of 5-week-old pigs with EMCV-30, a strain isolated from commercial pigs, resulted in acute cellular degeneration, infiltration of lymphocytes, and apoptosis in myocardium in 13 of 15 (86.7%) pigs during the acute phase of disease (3 to 21 days postinfection), followed by less-severe lymphocytic infiltration and apoptosis in 5 of 10 (50%) pigs during the chronic phase of the disease (day 45 to 90 postinfection). In the brain, lymphocytic infiltration, neuronal degeneration, and gliosis were observed in 26 to 33% of pigs in the acute phase of disease whereas perivascular cuffing was the predominant feature during chronic disease. EMCV antigens and RNA were demonstrated in the myocardium and brain during the chronic phase of disease. Analysis of 100 commercial pigs that were negative for EMCV antibodies identified two pig hearts positive for EMCV RNA. Porcine EMCV productively infected primary human cardiomyocytes as demonstrated by immunostaining using a monoclonal antibody specific for EMCV RNA polymerase, which is expressed only in productively infected cells, and by a one-step growth curve that showed production of 100 to 1,000 PFU of virus per cell within 6 h. The findings that porcine EMCV can persist in pig myocardium and can infect human myocardial cells make it an important infectious agent to screen for in pig-to-human cardiac transplants and a good model for xenozoonosis.
CD154 Costimulated Ovine Primary B Cells, a Cell Culture System That Supports Productive Infection by Bovine Leukemia Virus
Authors: Van den Broeke A. , Cleuter Y. , Beskorwayne T. , Kerkhofs P. , Szynal M. , Bagnis C. , Burny A. , Griebel P.
Published: July 2002

Bovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (CD40 ligand) and γ-chain-common cytokines to support the growth of B cells isolated from ovine lymphoid tissues. Integrated provirus, extrachromosomal forms, and viral transcripts were detected in BLV-exposed populations of immature, rapidly dividing surface immunoglobulin M-positive B cells from sheep ileal Peyer's patches and also in activated mature B cells isolated from blood. Conclusive evidence of direct B-cell infection by BLV was obtained through the use of cloned B cells derived from sheep jejunal Peyer's patches. Finally, inoculation of sheep with BLV-infected cultures proved that infectious virus was shed from in vitro-infected B cells. Collectively, these data confirm that a variety of ovine B-cell populations can support productive infection by BLV. The development of ovine B-cell cultures permissive for BLV infection provides a controlled system for investigating B-cell leukemogenic processes and the pathogenesis of BLV infection.
Characterization of Herpes Simplex Viruses Selected in Culture for Resistance to Penciclovir or Acyclovir
Authors: Sarisky Robert T. , Quail Matthew R. , Clark Philip E. , Nguyen Tammy T. , Halsey Wendy S. , Wittrock Robert J. , Bartus Joan O'Leary , Van Horn Marion M. , Sathe Ganesh M. , Van Horn Stephanie , Kelly Michael D. , Bacon Teresa H. , Leary Jeffry J.
Published: July 2002

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv
r
clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv
r
mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.
DNase Protection Analysis of Retrovirus Integrase at the Viral DNA Ends for Full-Site Integration In Vitro
Authors: Vora Ajaykumar , Grandgenett Duane P.
Published: July 2002

Retrovirus intasomes purified from virus-infected cells contain the linear viral DNA genome and integrase (IN). Intasomes are capable of integrating the DNA termini in a concerted fashion into exogenous target DNA (full site), mimicking integration in vivo. Molecular insights into the organization of avian myeloblastosis virus IN at the viral DNA ends were gained by reconstituting nucleoprotein complexes possessing intasome characteristics. Assembly of IN–4.5-kbp donor complexes capable of efficient full-site integration appears cooperative and is dependent on time, temperature, and protein concentration. DNase I footprint analysis of assembled IN-donor complexes capable of full-site integration shows that wild-type U3 and other donors containing gain-of-function attachment site sequences are specifically protected by IN at low concentrations (<20 nM) with a defined outer boundary mapping ∼20 nucleotides from the ends. A donor containing mutations in the attachment site simultaneously eliminated full-site integration and DNase I protection by IN. Coupling of wild-type U5 ends with wild-type U3 ends for full-site integration shows binding by IN at low concentrations probably occurs only at the very terminal nucleotides (<10 bp) on U5. The results suggest that assembly requires a defined number of avian IN subunits at each viral DNA end. Among several possibilities, IN may bind asymmetrically to the U3 and U5 ends for full-site integration in vitro.
Herpes Simplex Virus Type 1 Entry Is Inhibited by the Cobalt Chelate Complex CTC-96
Authors: Schwartz Jennifer A. , Lium Erik K. , Silverstein Saul J.
Published: July 2002

The CTC series of cobalt chelates display in vitro and in vivo activity against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). The experiments described here identify the stage in the virus life cycle where CTC-96 acts and demonstrate that the drug inhibits infection of susceptible cells. CTC-96 at 50 μg/ml has no effect on adsorption of virions to Vero cell monolayers. Penetration assays reveal that CTC-96 inhibits entry of the virus independent of gC and cellular entry receptors. This observation was supported by the failure to detect the accumulation of virus-specified proteins and α mRNA transcripts when CTC-96 is present at the onset of infection. Moreover, virion-associated αTIF does not accumulate in the nucleus of cells infected in the presence of CTC-96. CTC-96 targets the initial fusion event between the virus and the cell and also inhibits cell-to-cell spread and syncytium formation. Furthermore, CTC-96 inhibits plaque formation by varicella-zoster virus and vesicular stomatitis virus as efficiently as by HSV-1. Collectively, these experiments suggest that CTC-96 is a broad-spectrum inhibitor of infection by enveloped viruses and that it inhibits HSV-1 infection at the point of membrane fusion independent of the type of virus and cellular receptors present.
Reverse Genetics Demonstrates that Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Essential for Replication in Cell Culture
Authors: Neumann Gabriele , Feldmann Heinz , Watanabe Shinji , Lukashevich Igor , Kawaoka Yoshihiro
Published: July 2002

Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.
Early Pathogenesis of Transmucosal Feline Immunodeficiency Virus Infection
Authors: Obert Leslie A. , Hoover Edward A.
Published: July 2002

To identify the early target cells and tissues in transmucosal feline immunodeficiency virus (FIV) infection, cats were exposed to a clade C FIV isolate via the oral-nasal or vaginal mucosa and multiple tissues were examined by virus isolation coculture (VI), DNA PCR, catalyzed tyramide signal-amplified in situ hybridization (TSA-ISH), and immunohistochemistry between days 1 and 12 postinoculation (p.i.). FIV RNA was detected in tonsil and oral or vaginal mucosa as early as 1 day p.i. by TSA-ISH and in retropharyngeal, tracheobronchial, or external iliac lymph nodes and sometimes in spleen or blood mononuclear cells by day 2, indicating that regional and distant spread of virus-infected cells occurred rapidly after mucosal exposure. By day 8, viral RNA, DNA, and culturable virus were uniformly detected in regional and distant tissues, connoting systemic infection. TSA-ISH proved more sensitive than DNA PCR in detecting early FIV-infected cells. In mucosal tissues, the earliest demonstrable FIV-bearing cells were either within or subjacent to the mucosal epithelium or were in germinal centers of regional lymph nodes. The FIV+cells were of either of two morphological types, large stellate or small round. Those FIV RNA+cells which could be colabeled for a phenotype marker, were labeled for either dendritic-cell-associated protein p55 or T-lymphocyte receptor antigen CD3. These studies indicate that FIV crosses mucous membranes within hours after exposure and rapidly traffics via dendritic and T cells to systemic lymphoid tissues, a pathway similar to that thought to occur in the initial phase of infection by the human and simian immunodeficiency viruses.
Identification and Characterization of the UL56 Gene Product of Herpes Simplex Virus Type 2
Authors: Koshizuka Tetsuo , Goshima Fumi , Takakuwa Hiroki , Nozawa Naoki , Daikoku Tohru , Koiwai Osamu , Nishiyama Yukihiro
Published: July 2002

The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.
Deficiencies in the Acute-Phase Cell-Mediated Immune Response to Viral Antigens Are Associated with Development of Chronic Woodchuck Hepatitis Virus Infection following Neonatal Inoculation
Authors: Menne Stephan , Roneker Carol A. , Roggendorf Michael , Gerin John L. , Cote Paul J. , Tennant Bud C.
Published: July 2002

In vitro proliferation of peripheral blood mononuclear cells was used to measure virus-specific cell-mediated immunity (vCMI) following neonatal woodchuck hepatitis virus (WHV) infection. Fifteen neonates were inoculated with the W8 strain of WHV. In 11, infection was resolved, and 4 became chronic carriers. Nineteen neonates were inoculated with the W7 strain and all became chronic carriers. Seven age-matched uninfected woodchucks served as controls. Virologic and vCMI profiles among the W8 and W7 infections were compared and related to the outcome of infection. Resolving woodchucks had robust, acute-phase vCMI to WHV antigens (core, surface, and x) and to several nonoverlapping core peptides. The acute-phase vCMI was associated temporally with the clearance of viral DNA and of surface antigen from serum at 14 to 22 weeks postinfection. In contrast, in approximately half of the W8 and W7 infections that progressed to chronicity, no significant acute-phase vCMI was detected. In the remaining carriers, acute-phase vCMI was observed, but it was less frequent and incomplete compared to that of resolved woodchucks. Serum viral load developed less rapidly in those carriers that had evidence of acute-phase vCMI, but it was still increased compared to that of resolving woodchucks. Thus, vigorous and multispecific acute-phase vCMI was associated with resolution of neonatal WHV infection. Absent or incomplete acute-phase vCMI was associated with the progression to chronic infection. By analogy, these results suggest that the onset of chronic hepatitis B virus (HBV) infection in humans may be associated with deficiencies in the primary T-cell response to acute HBV infection.
Solution Structure of the Viral Receptor Domain of Tva and Its Implications in Viral Entry
Authors: Wang Qing-Yin , Huang Wen , Dolmer Klavs , Gettins Peter G. W. , Rong Lijun
Published: July 2002

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue, cysteine-rich motif called the LDL-A module. Here we report the solution structure of the LDL-A module of Tva, determined by nuclear magnetic resonance (NMR) spectroscopy. Although the carboxyl terminus of the Tva LDL-A module has a structure similar to those of other reported LDL-A modules, the amino terminus adopts a different conformation. The LDL-A module of Tva does not contain the signature antiparallel β-sheet observed in other LDL-A modules, and it is more flexible than other reported LDL-A modules. The LDL-A structure of Tva provides mechanistic insights into how a simple viral receptor functions in retrovirus entry. The side chains of H38 and W48 of Tva, which have been identified as viral contact residues by mutational analysis, are solvent exposed, suggesting that they are directly involved in EnvA binding. However, the side chain of L34, another potential viral contact residue identified previously, is buried inside of the module and forms the hydrophobic core with other residues. Thus L34 likely stabilizes the Tva structure but is not a viral interaction determinant. In addition, we propose that the flexible amino-terminal region of Tva plays an important role in determining specificity in the Tva-EnvA interaction.
Recent advances in cystic fibrosis
Authors: Doull Iolo J M
Published: July 2002

The median life expectancy for cystic fibrosis is now over 30 years, and it is projected that in newborn infants it will become more than 40 years. The identification of the cystic fibrosis gene and its product, cystic fibrosis transmembrane conductance regulator (CFTR), has widened the spectrum of the disease from the classical case of the infant with cystic fibrosis to the elderly childless man with unexplained bronchiectasis. There is increasing evidence of the advantages of newborn screening for cystic fibrosis and subsequent specialist care. Management concentrates on optimising nutritional status and preventing lung infection and inflammation.
Factors associated with hip joint rotation in former elite athletes
Authors: Kettunen Jyrki A , Kujala Urho M , Räty Heli , Videman Tapio , Sarna Seppo , Impivaara Olli , Koskinen Seppo
Published: July 2002

Objectives—To study factors associated with passive hip rotation range of motion (ROM) in former elite male athletes.

Methods—Athletes were interviewed about hip pain, disability, lifetime occupational loading, and athletic training. The passive hip rotation was measured with a Myrin inclinometer in 117 former elite male long distance runners, soccer players, weight lifters, and shooters aged 45–68 years. Magnetic resonance imaging was used to detect hip osteoarthritis.

Results—There were no differences in passive hip rotation ROM between the four athlete groups nor between diverging lifetime loading patterns associated with occupational or athletic activities. Among the subjects without hip osteoarthritis, hip pain, and hip disability according to a stepwise linear regression analysis, the only factor that was associated with the passive hip rotation ROM was body mass index (BMI), explaining about 21% of its variation. Subjects with high BMI had lower passive hip rotation ROM than those with low BMI. There was no right-left difference in the mean passive hip rotation ROM in subjects either with or without hip osteoarthritis as determined by magnetic resonance imaging. Nevertheless, hip rotation ROM was clearly reduced in a few hips with severe caput deformity.

Conclusions—Long term loading appears to have no association with passive hip rotation ROM. On the other hand, the hip rotation value was lower in subjects with high BMI than in those with low BMI. A clear right-left difference in hip rotation was found only in those subjects who, according to our magnetic resonance imaging criteria, had severe hip osteoarthritis. These findings should be taken into account when hip rotation ROM is used in the clinical assessment of hip joints.
Correlation of bone scintigraphy and histological findings in medial tibial syndrome
Authors: Bhatt R , Lauder I , Finlay D B , Allen M J , Belton I P
Published: July 2002

Objective—To correlate bone scintigraphy and histopathological findings in patients with medial tibial syndrome.

Methods—Twenty patients (32 limbs) with a clinical diagnosis of medial tibial syndrome had surgery. Bone scintigraphy before the operation was compared with the histological appearance of bone and periosteal specimens obtained at surgery.

Results—Delayed bone scintigraphy showed normal appearance in 11 limbs, characteristic diffuse tubular pattern uptake in 16 limbs, and focal uptake in five. Periosteal histology disclosed fibrous thickening as the most common finding associated with increased vascularity, occasionally with chronic inflammatory cell infiltration, haemosiderin, and acid mucopolysaccharide deposition. Loss of osteocytes was the main finding of bone histology associated with some enlargement of lacunae and lamellar structure disruption. A grading system was used to score normal and abnormal histological appearance. For analysis the findings were regrouped to provide tables using Fisher's exact test. There was no correlation between bone scintigraphy and the histology of bone and periosteum, but two interesting observations were noted. Those cases with periosteal thickening had mostly normal bone scan appearance (p = 0.0028). Those cases with low levels of osteocyte loss had mostly abnormal bone scintigraphy.

Conclusion—Abnormal histological appearance of bone and periosteum is a feature of medial tibial syndrome. These histological findings show poor correlation with bone scintigraphy. The exact pathogenesis of this syndrome remains unclear.
Suprascapular neuropathy in volleyball players
Authors: Witvrouw E , Cools A , Lysens R , Cambier D , Vanderstraeten G , Victor J , Sneyers C , Walravens M
Published: July 2002

Background—Suprascapular nerve entrapment with isolated paralysis of the infraspinatus muscle is uncommon. However, this pathology has been reported in volleyball players. Despite a lack of scientific evidence, excessive strain on the nerve is often cited as a possible cause of this syndrome. Previous research has shown a close association between shoulder range of motion and strain on the suprascapular nerve. No clinical studies have so far been designed to examine the association between excessive shoulder mobility and the presence of this pathology.

Aim—To study the possible association between the range of motion of the shoulder joint and the presence of suprascapular neuropathy by clinically examining the Belgian male volleyball team with respect to several parameters.

Methods—An electromyographic investigation, a clinical shoulder examination, shoulder range of motion measurements, and an isokinetic concentric peak torque shoulder internal/external rotation strength test were performed in 16 professional players.

Results—The electrodiagnostic study showed a severe suprascapular neuropathy in four players which affected only the infraspinatus muscle. In each of these four players, suprascapular nerve entrapment was present on the dominant side. Except for the hypotrophy of the infraspinatus muscle, no significant differences between the affected and non-affected players were observed on clinical examination. Significant differences between the affected and non-affected players were found for range of motion measurements of external rotation, horizontal flexion and forward flexion, and for flexion of the shoulder girdle (protraction); all were found to be higher in the affected players than the non-affected players.

Conclusions—This study suggests an association between increased range of motion of the shoulder joint and the presence of isolated paralysis of the infraspinatus muscle in volleyball players. However, the small number of patients in this study prevents definite conclusions from being drawn.
International gastroenterology research: subject areas, impact, and funding
Authors: Lewison G , Grant J , Jansen P
Published: July 2002

AIMS

To examine the volume and potential impact of gastroenterology research outputs from 1985 to 1998 from 14 developed countries; the overlap with research in cancer, infectious diseases, and genetics; and the funding sources for this research. To determine if countries' research outputs correlated with their burden of corresponding diseases and inputs to their research.

METHODS

Selective retrieval of papers from the Science Citation Index and manual look up of a sample to determine funding sources. Classification of journals by four categories of research level (clinical/basic) and potential impact (low/high).

RESULTS

Gastroenterology represents about 8% of world biomedical research but over 11% in Italy, Japan, and Spain. Its potential impact is highest (but declining) for the USA. It has increased noticeably in most European countries, particularly in Finland. Gastroenterology research has become more clinical in Japan, Spain, Australia, and the Netherlands but more basic in Canada, Germany, Finland, Israel, and South Africa. Funding comes primarily from national governments, followed by national private non-profit sources and industry but little industrial funding occurs in some countries. There is a strong and positive correlation between reported deaths from gastrointestinal neoplasms and countries' outputs of research in gastrointestinal oncology.

CONCLUSIONS

Bibliometric analysis can reveal differences between countries in their research in a subject when a common methodology is applied to an international database. Variations in research methods in different countries can plausibly explain some of the variation in the potential impact of the work.
Histological identification ofHelicobacter pylori: comparison of staining methods
Authors: Rotimi O , Cairns A , Gray S , Moayyedi P , Dixon M F
Published: July 2002

Aim—To determine whether two recently described staining methods (the modified McMullen's and theHelicobacter pylorisilver stain HpSS methods) used for the histological identification of H pylori organisms are superior to two established techniques (the modified Giemsa and anti-H pyloriantibody immunostain) in terms of availability, reproducibility, rapidity, sensitivity, and cost.

Methods—Histological sections from 63 paired gastric biopsies from adult patients previously investigated for dyspepsia were stained with the four methods and these were assessed blindly and independently by two observers. Of the 63 patients, 30 were originally negative in all tests forH pyloriinfection, 30 were positive, and the remaining three cases had discordant results using a combination of five tests (rapid biopsy urease test, urea breath test, culture, serology, and histology).

Results—Interobserver agreement was best with the antibody method (98%), followed by the McMullen's (90%), Giemsa (87%), and HpSS (85%). Of the 60 “gold standard” positive and negative cases, 30 were positive by the modified Giemsa stain, 29 by the McMullen's method, 29 by HpSS, and 30 by the antibody stain. However, there were two false positives with the HpSS method. The modified Giemsa is the cheapest and easiest to perform technically.

Conclusions—WhenH pyloriare present, careful examination will almost always reveal them, whichever of these stains is used. However, the modified Giemsa stain is the method of choice because it is sensitive, cheap, easy to perform, and reproducible.
No detection of enteroviral genome in the myocardium of patients with arrhythmogenic right ventricular cardiomyopathy
Authors: Calabrese Fiorella , Angelini Annalisa , Thiene Gaetano , Basso Cristina , Nava Andrea , Valente Marialuisa
Published: July 2002

Aims—Despite the evidence of familial occurrence, chromosomal gene mapping, and apoptosis as a mechanism of myocyte death, the aetiopathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC) remains speculative. Because of the frequent histological finding of focal inflammatory infiltrates, the hypothesis of an infective myocarditis aetiology has been put forward. The aim of this investigation was to test this hypothesis. The presence of enteroviruses was investigated by a highly sensitive and specific molecular technique.

Methods—Endomyocardial tissue samples from 20 patients with ARVC (11 male, nine female; mean age, 40 years; SD, 16) and 20 control subjects with other cardiac diseases were analysed using reverse transcription and nested polymerase chain reaction (PCR). Myocardial samples obtained from four patients with enteroviral myocarditis and coxsackie B3 virus infected cells were used as positive controls.

Results—Endomyocardial biopsy was diagnostic for ARVC in all patients: myocardial atrophy was seen, with less than 45% residual myocytes. Foci of inflammatory infiltrates were seen in four biopsies, and the cells were identified by immunohistochemistry as mainly T cells. All samples, from both patients with ARVC and subjects with other cardiac diseases, were negative for enteroviral genome by means of nested PCR.

Conclusion—These findings indicate that enteroviruses are not involved in the aetiopathogenesis of ARVC. Future molecular studies should investigate the presence of other infective agents, as well as their possible role in triggering apoptosis.
Ethical approval for research involving geographically dispersed subjects: unsuitability of the UK MREC/LREC system and relevance to uncommon genetic disorders
Authors: Lewis Julia C , Tomkins Susan , Sampson Julian R
Published: July 2002

Objectives—To assess the process involved in obtaining ethical approval for a single-centre study involving geographically dispersed subjects with an uncommon genetic disorder.

Design—Observational data of the application process to 53 local research ethics committees (LRECs) throughout Wales, England and Scotland. The Multicentre Research Ethics Committee (MREC) for Wales had already granted approval.

Results—Application to the 53 LRECs required 24,552 sheets of paper and took two months of the researcher's time. The median time taken for approval was 39 days with only seven (13%) of committees responding within the recommended 21 days. In at least nineteen cases (36%) a subcommittee considered the application. Thirty-three committees (62%) accepted the proposal without amendments but, of the remainder, four (8%) requested changes outside of the remit of LRECs.

Discussion—Difficulties still exist with the system for obtaining ethical approval for studies involving a single centre but with patients at multiple sites, as is often required for genetic observational research. As such studies differ from true multicentre studies, it may be advantageous to develop a separate and specific process of application to ensure that resources are not unnecessarily expended in the quest for ethical approval.
Death, dying and donation: organ transplantation and the diagnosis of death
Authors: Kerridge I H , Saul P , Lowe M , McPhee J , Williams D
Published: July 2002

Refusal of organ donation is common, and becoming more frequent. In Australia refusal by families occurred in 56% of cases in 1995 in New South Wales, and had risen to 82% in 1999, becoming the most important determinant of the country's very low organ donation rate (8.9/million in 1999).

Leading causes of refusal, identified in many studies, include the lack of understanding by families of brain death and its implications, and subsequent reluctance to relegate the body to purely instrumental status. It is an interesting paradox that surveys of the public continue to show considerable support for organ donation programmes—in theory we will, in practice we won't (and don't).

In this paper we propose that the Australian community may, for good reason, distrust the concept of and criteria for “whole brain death”, and the equation of this new concept with death of the human being. We suggest that irreversible loss of circulation should be reinstated as the major defining characteristic of death, but that brain-dead, heart-beating entities remain suitable organ donors despite being alive by this criterion. This presents a major challenge to the “dead donor rule”, and would require review of current transplantation legislation. Brain dead entities are suitable donors because of irreversible loss of personhood, accurately and robustly defined by the current brain stem criteria.

Even the dead are not terminally ill any more.1
The butler(s) DID it - dissociative identity disorder in cinema
Authors: Byrne Peter
Published: July 2002

Beginning with classic Hollywood melodramas of the 1940s, cinema has maintained a prolific output of films with their own take on mental illnesses–none more so than the rare syndrome of dissociative identity disorder (DID). DID films are a popular and enduring genre, whose influence can be seen throughout mainstream cinema. Exploration of DID in cinema is a useful exercise in that it touches on issues in film studies, psychiatry and the mythology around mental illness. Despite “detective story” narratives and conformity to the codes of the psychological thriller, DID representations correspond closely to contemporary thinking about its phenomenology and aetiology. That said, some films confuse DID with schizophrenia, and many reinforce two other misconceptions within film psychiatry: mental illness as violence and the belief that every mentally ill person harbours one “great dark secret”. Those sceptical about DID have suggested that popular written accounts increase its profile: so too, powerful cinematic images may suggest the possibility of DID to susceptible people, including clinicians. Recent DID films reflect the real debate within psychiatry about the diagnostic validity and scientific basis of DID.
Epidemiological survey of workers exposed to inorganic germanium compounds
Authors: Swennen B , Mallants A , Roels H A , Buchet J P , Bernard A , Lauwerys R R , Lison D
Published: July 2002

OBJECTIVES

To assess occupational exposure to inorganic germanium (Ge) in workers from a producing plant, and to assess the health of these workers, with a special focus on respiratory, kidney, and liver functions.

METHODS

Cross sectional study of 75 workers exposed to Ge and 79 matched referents. Exposure was characterised by measuring air and urine concentrations of the element during a typical working week, and health was assessed by a questionnaire, clinical examination, lung function testing, chest radiography, and clinical chemistry in serum and urine, including high and low molecular weight urinary proteins.

RESULTS

Airborne concentrations of Ge (inhalable fraction) ranged from 0.03 to 300 μg/m, which was reflected by increased urinary excretion of Ge (0.12–200 μg/g creatinine, after the shift at the end of the working week). Lung, liver, and haematological variables were not significantly different between referents and workers exposed to Ge. A slightly higher urinary concentration of high molecular weight proteins (albumin and transferrin) was found in workers exposed to Ge, possibly reflecting subclinical glomerular changes. No relation was found between the intensity or duration of exposure and the urinary concentration of albumin. No difference between referents and workers exposed to Ge was found for other renal variables.

CONCLUSIONS

Measurement of urinary Ge can detect occupational exposure to inorganic Ge and its compounds. It is prudent to recommend the monitoring of renal variables in workers exposed to Ge.