bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Aggregation-Specific Enzyme-Linked Immunosorbent Assay: Detection of Conformational Differences between Recombinant PrP Protein Dimers and PrP
Sc
Aggregates
Authors: Pan Tao , Chang Binggong , Wong Poki , Li Chaoyang , Li Ruliang , Kang Shin-Chung , Robinson John D. , Thompsett Andrew R. , Tein Po , Yin Shaoman , Barnard Geoff , McConnell Ian , Brown David R. , Wisniewski Thomas , Sy Man-Sun 
Published: September 2005

ABSTRACT

The conversion of the normal cellular prion protein, PrP
C
, into the protease-resistant, scrapie PrP
Sc
aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrP
Sc
aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrP
Sc
aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease.
Shared Alterations in NK Cell Frequency, Phenotype, and Function in Chronic Human Immunodeficiency Virus and Hepatitis C Virus Infections
Authors: Meier Ute-Christiane , Owen Rachel E. , Taylor Elizabeth , Worth Andrew , Naoumov Nikolai , Willberg Christian , Tang Kwok , Newton Phillipa , Pellegrino Pierre , Williams Ian , Klenerman Paul , Borrow Persephone 
Published: September 2005

ABSTRACTHuman immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3−CD56+NK subsets in HIV+and HCV+patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56dimcell fraction compared to CD56brightcells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56dimNK subset were observed in HIV+patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV+and HCV+patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.
Inactivation of Retroviruses with Preservation of Structural Integrity by Targeting the Hydrophobic Domain of the Viral Envelope
Authors: Raviv Yossef , Viard Mathias , Bess Julian W , Chertova Elena , Blumenthal Robert 
Published: September 2005

ABSTRACT
We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.
Protection of Mice against Lethal Infection with Highly Pathogenic H7N7 Influenza A Virus by Using a Recombinant Low-Pathogenicity Vaccine Strain
Authors: de Wit Emmie , Munster Vincent J. , Spronken Monique I. J. , Bestebroer Theo M. , Baas Chantal , Beyer Walter E. P. , Rimmelzwaan Guus F. , Osterhaus Albert D. M. E. , Fouchier Ron A. M. 
Published: September 2005

ABSTRACT
In 2003, an outbreak of highly pathogenic avian influenza occurred in the Netherlands. The avian H7N7 virus causing the outbreak was also detected in 88 humans suffering from conjunctivitis or mild respiratory symptoms and one person who died of pneumonia and acute respiratory distress syndrome. Here we describe a mouse model for lethal infection with A/Netherlands/219/03 isolated from the fatal case. Because of the zoonotic and pathogenic potential of the H7N7 virus, a candidate vaccine carrying the avian hemagglutinin and neuraminidase proteins produced in the context of the high-throughput vaccine strain A/PR/8/34 was generated by reverse genetics and tested in the mouse model. The hemagglutinin gene of the recombinant vaccine strain was derived from a low-pathogenicity virus obtained prior to the outbreak from a wild mallard. The efficacy of a classical nonadjuvanted subunit vaccine and an immune stimulatory complex-adjuvanted vaccine was compared. Mice receiving the nonadjuvanted vaccine revealed low antibody titers, lack of clinical protection, high virus titers in the lungs, and presence of virus in the spleen, liver, kidneys, and brain. In contrast, mice receiving two doses of the immune stimulatory complex-adjuvanted vaccine revealed high antibody titers, clinical protection, ∼1,000-fold reduction of virus titers in the lungs, and rare detection of the virus in other organs. This is the first report of an H7 vaccine candidate tested in a mammalian model. The data presented suggest that vaccine candidates based on low-pathogenicity avian influenza A viruses, which can be prepared ahead of pandemic threats, can be efficacious if an effective adjuvant is used.
Effect of Hemagglutinin Glycosylation on Influenza Virus Susceptibility to Neuraminidase Inhibitors
Authors: Mishin Vasiliy P. , Novikov Dmitri , Hayden Frederick G. , Gubareva Larisa V. 
Published: September 2005

ABSTRACT
Inhibition of neuraminidase (NA) activity prevents release of progeny virions from influenza-infected cells and removal of neuraminic (sialic) acid moieties from glycans attached to hemagglutinin (HA). Neuraminic acid moieties situated near the HA receptor-binding site can reduce the efficiency of virus binding and decrease viral dependence on NA activity for replication. With the use of reverse genetics technique, we investigated the effect of glycans attached at Asn 94a, 129, and 163 on the virus susceptibility to NA inhibitors in MDCK cells and demonstrated that the glycan attached at Asn 163 plays a dominant role in compensation for the loss of NA activity.
Minimal T-Cell-Stimulatory Sequences and Spectrum of HLA Restriction of Immunodominant CD4
+
T-Cell Epitopes within Hepatitis C Virus NS3 and NS4 Proteins
Authors: Gerlach J. T. , Ulsenheimer A. , Grüner N. H. , Jung M.-C. , Schraut W. , Schirren C.-A. , Heeg M. , Scholz S. , Witter K. , Zahn R. , Vogler A. , Zachoval R. , Pape G. R. , Diepolder H. M. 
Published: September 2005

ABSTRACT

The hepatitis C virus (HCV)-specific CD4
+
T-cell response against nonstructural proteins is strongly associated with successful viral clearance during acute hepatitis C. To further develop these observations into peptide-based vaccines and clinical immunomonitoring tools like HLA class II tetramers, a detailed characterization of immunodominant CD4
+
T-cell epitopes is required. We studied peripheral blood mononuclear cells from 20 patients with acute hepatitis C using 83 overlapping 20-mer peptides covering the NS3 helicase and NS4. Eight peptides were recognized by ≥40% of patients, and specific CD4
+
T-cell clones were obtained for seven of these and three additional, subdominant epitopes. Mapping of minimal stimulatory sequences defined epitopes of 8 to 13 amino acids in length, but optimal T-cell stimulation was observed with 10- to 15-mers. While some epitopes were presented by different HLA molecules, others were presented by only a single HLA class II molecule, which has implications for patient selection in clinical trials of peptide-based immunotherapies. In conclusion, using two different approaches we identified and characterized a set of CD4
+
T-cell epitopes in the HCV NS3-NS4 region which are immunodominant in patients achieving transient or persistent viral control. This information allows the construction of a valuable panel of HCV-specific HLA class II tetramers for further study of CD4
+
T-cell responses in chronic hepatitis C. The finding of immunodominant epitopes with very constrained HLA restriction has implications for patient selection in clinical trials of peptide-based immunotherapies.
Circularization of the Herpes Simplex Virus Type 1 Genome upon Lytic Infection
Authors: Strang Blair L. , Stow Nigel D. 
Published: September 2005

ABSTRACT

For many years, the generally accepted model for the replication of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1) incorporated initial circularization of linear molecules in the cell nucleus. Ensuing DNA synthesis resulted in the generation of head-to-tail concatemers which were subsequently cleaved into monomeric units and packaged into the nascent viral capsid. Recently, however, it has been proposed that circularization of HSV-1 genomes does not occur at the onset of lytic infection and moreover that this event is specifically inhibited by the HSV-1 transcriptional transactivator, ICP0 (S.A. Jackson and N.A. DeLuca, Proc. Natl. Acad. Sci. USA 100:7871-7876, 2003). To further investigate genome circularization, we have generated HSV-1 derivatives in which the viral
a
sequences, which contain the cleavage-packaging signals, have been replaced by a minimal packaging element located in the thymidine kinase gene. In contrast to wild-type HSV-1, fusion of the genomic termini of these viruses produces a novel fragment in circular or concatemeric DNA which can be detected by Southern blot hybridization. Utilizing these viruses, we demonstrate that fusion of the genomic termini occurred rapidly upon infection and in the presence of inhibitors of viral DNA or protein synthesis. We provide evidence indicating that the end joining represented circularization rather than concatemerization of input molecules and that circularized molecules functioned as templates for replication. Since the termini of these viruses lack direct repeats, our findings indicate that circularization can be mediated by direct end-to-end ligation of linear input genomes.
Genomewide Screening Reveals High Levels of Insertional Polymorphism in the Human Endogenous Retrovirus Family HERV-K(HML2): Implications for Present-Day Activity
Authors: Belshaw Robert , Dawson Anna L. A. , Woolven-Allen John , Redding Joanna , Burt Austin , Tristem Michael 
Published: September 2005

ABSTRACT

The published human genome sequence contains many thousands of endogenous retroviruses (HERVs) but all are defective, containing nonsense mutations or major deletions. Only the HERV-K(HML2) family has been active since the divergence of humans and chimpanzees; it contains many members that are human specific, as well as several that are insertionally polymorphic (an inserted element present only in some human individuals). Here we perform a genomewide survey of insertional polymorphism levels in this family by using the published human genome sequence and a diverse sample of 19 humans. We find that there are 113 human-specific HERV-K(HML2) elements in the human genome sequence, 8 of which are insertionally polymorphic (11 if we extrapolate to those within regions of the genome that were not suitable for amplification). The average rate of accumulation since the divergence with chimpanzees is thus approximately 3.8 × 10
−4
per haploid genome per generation. Furthermore, we find that the number of polymorphic elements is not significantly different from that predicted by a standard population genetic model that assumes constant activity of the family until the present. This suggests to us that the HERV-K(HML2) family may be active in present-day humans. Active (replication-competent) elements are likely to have inserted very recently and to be present at low allele frequencies, and they may be causing disease in the individuals carrying them. This view of the family from a population perspective rather than a genome perspective will inform the current debate about a possible role of HERV-K(HML2) in human disease.
The Transmembrane Domain of the Respiratory Syncytial Virus F Protein Is an Orientation-Independent Apical Plasma Membrane Sorting Sequence
Authors: Brock Sean C. , Heck Josh M. , McGraw Patricia A. , Crowe James E. 
Published: September 2005

ABSTRACT
The processes that facilitate transport of integral membrane proteins though the secretory pathway and subsequently target them to particular cellular membranes are relevant to almost every field of biology. These transport processes involve integration of proteins into the membrane of the endoplasmic reticulum (ER), passage from the ER to the Golgi, and post-Golgi trafficking. The respiratory syncytial virus (RSV) fusion (F) protein is a type I integral membrane protein that is uniformly distributed on the surface of infected nonpolarized cells and localizes to the apical plasma membrane of polarized epithelial cells. We expressed wild-type or altered RSV F proteins to gain a better understanding of secretory transport and plasma membrane targeting of type I membrane proteins in polarized and nonpolarized epithelial cells. Our findings reveal a novel, orientation-independent apical plasma membrane targeting function for the transmembrane domain of the RSV F protein in polarized epithelial cells. This work provides a basis for a more complete understanding of the role of the transmembrane domain and cytoplasmic tail of viral type I integral membrane proteins in secretory transport and plasma membrane targeting in polarized and nonpolarized cells.
Attenuated Rabies Virus Activates, while Pathogenic Rabies Virus Evades, the Host Innate Immune Responses in the Central Nervous System
Authors: Wang Zhi W. , Sarmento Luciana , Wang Yuhuan , Li Xia-qing , Dhingra Vikas , Tseggai Tesfai , Jiang Baoming , Fu Zhen F. 
Published: September 2005

ABSTRACT
Rabies virus (RV) induces encephalomyelitis in humans and animals. However, the pathogenic mechanism of rabies is not fully understood. To investigate the host responses to RV infection, we examined and compared the pathology, particularly the inflammatory responses, and the gene expression profiles in the brains of mice infected with wild-type (wt) virus silver-haired bat RV (SHBRV) or laboratory-adapted virus B2C, using a mouse genomic array (Affymetrix). Extensive inflammatory responses were observed in animals infected with the attenuated RV, but little or no inflammatory responses were found in mice infected with wt RV. Furthermore, attenuated RV induced the expression of the genes involved in the innate immune and antiviral responses, especially those related to the alpha/beta interferon (IFN-α/β) signaling pathways and inflammatory chemokines. For the IFN-α/β signaling pathways, many of the interferon regulatory genes, such as the signal transduction activation transducers and interferon regulatory factors, as well as the effector genes, for example, 2′-5′-oligoadenylate synthetase and myxovirus proteins, are highly induced in mice infected with attenuated RV. However, many of these genes were not up-regulated in mice infected with wt SHBRV. The data obtained by microarray analysis were confirmed by real-time PCR. Together, these data suggest that attenuated RV activates, while pathogenic RV evades, the host innate immune and antiviral responses.
Identification of Two N-Linked Glycosylation Sites within the Core of the Simian Immunodeficiency Virus Glycoprotein Whose Removal Enhances Sensitivity to Soluble CD4
Authors: Pikora Cheryl , Wittish Christine , Desrosiers Ronald C. 
Published: September 2005

ABSTRACT
Using PCR mutagenesis to disrupt the NXT/S N-linked glycosylation motif of the Env protein, we created 27 mutants lacking 1 to 5 of 14 N-linked glycosylation sites within regions of gp120 lying outside of variable loops 1 to 4 within simian immunodeficiency virus strain 239 (SIV239). Of 18 mutants missing N-linked glycosylation sites predicted to lie within 10 Å of CD4 contact sites, the infectivity of 12 was sufficient to measure sensitivity to neutralization by soluble CD4 (sCD4), pooled immune sera from SIV239-infected rhesus macaques, and monoclonal antibodies known to neutralize certain derivatives of SIV239. Three of these 12 mutants (g3, lacking the 3rd glycan at position 79; g11, lacking the 11th glycan at position 212; and g3,11, lacking both the 3rd and 11th glycans) were approximately five times more sensitive to neutralization by sCD4 than wild-type (WT) SIV239. However, these same mutants were no more sensitive to neutralization than WT by pooled immune sera. The other 9 of 12 replication-competent mutants in this group were no more sensitive to neutralization than the WT by any of the neutralizing reagents. Six of the nine mutants that did not replicate appreciably had three or more glycosylation sites eliminated; the other three replication-deficient strains involved mutation of site 15. Our results suggest that elimination of glycan attachment sites 3 and 11 enhanced the exposure of contact residues for CD4. Thus, glycans at positions 3 and 11 of SIV239 gp120 may be particularly important for shielding the CD4-binding site from antibody recognition.
Lys-34, Dispensable for Integrase Catalysis, Is Required for Preintegration Complex Function and Human Immunodeficiency Virus Type 1 Replication
Authors: Lu Richard , Vandegraaff Nick , Cherepanov Peter , Engelman Alan 
Published: September 2005

ABSTRACT

Retroviral integrases (INs) function in the context of preintegration complexes (PICs). Two conserved Lys residues in the N-terminal domain of human immunodeficiency virus type 1 (HIV-1) IN were analyzed here for their roles in integration and virus replication. Whereas HIV-1
K46A
grew like the wild type, HIV-1
K34A
was dead. Yet recombinant IN
K34A
protein functioned in in vitro integration assays, and Vpr-IN
K34A
efficiently transcomplemented the infectivity defect of an IN active site mutant virus in cells. HIV-1
K34A
was therefore similar to a number of previously characterized mutant viruses that failed to replicate despite encoding catalytically competent IN. To directly analyze mutant PIC function, a sensitive PCR-based integration assay was developed. HIV-1
K34A
and related mutants failed to support detectable levels (<1% of wild type) of integration. We therefore concluded that mutations like K34A disrupted higher-order interactions important for PIC function/maturation compared to the innate catalytic activity of IN enzyme.
Infection of Nonhuman Primates with Recombinant Human Metapneumovirus Lacking the SH, G, or M2-2 Protein Categorizes Each as a Nonessential Accessory Protein and Identifies Vaccine Candidates
Authors: Biacchesi Stéphane , Pham Quynh N. , Skiadopoulos Mario H. , Murphy Brian R. , Collins Peter L. , Buchholz Ursula J. 
Published: September 2005

ABSTRACT
Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the ΔSH virus was only marginally less efficient than that of wild-type HMPV, whereas the ΔG and ΔM2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The ΔG and ΔM2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.
Endothelial function and inflammation in coronary artery disease
Authors: Tousoulis D , Charakida M , Stefanadis C 
Published: September 2005

Abstract
Evidence supports the central role of endothelium and inflammation in all phases of the atherosclerotic process. Clinical studies have shown their prognostic potential for the development of ischaemic events and for adverse outcome after acute coronary syndromes. Reduction in inflammatory levels and improving endothelial function by traditional and novel treatment strategies were associated with a proportional reduction in cardiovascular events. However, randomised controlled trials are required to explore further whether drugs targeting the inflammatory process and endothelial function will constitute a reasonable adjunctive treatment for patients with coronary artery disease.
Antithrombotic treatment in atrial fibrillation
Authors: Lip G Y H , Boos C J 
Published: September 2005

Abstract
Atrial fibrillation (AF) can significantly increase morbidity and mortality. It is gaining in clinical and economic importance, being the most commonly encountered tachyarrhythmia in clinical practice. Stroke is the most serious complication. Evidence from AF antithrombotic treatment trials is reviewed, risk stratification of patients with AF is discussed, and recommendations for anticoagulation are presented.