bluuebunny/crossref_metadata_2025 huggingface dataset:
Usefulness of Four Different
Echinococcus granulosus
Recombinant Antigens for Serodiagnosis of Unilocular Hydatid Disease (UHD) and Postsurgical Follow-Up of Patients Treated for UHD
Authors: Hernández-González Ana , Muro Antonio , Barrera Inmaculada , Ramos Guillermo , Orduña Antonio , Siles-Lucas Mar
Published: November 2007

Four different recombinant antigens derived from
Echinococcus granulosus
, designated B1t, B2t, E14t, and C317, were tested with enzyme-linked immunosorbent assays (ELISAs) for the detection of specific immunoglobulin G (IgG) in patients with unilocular hydatid disease (UHD). The results were compared to those obtained with hydatid fluid and were subjected to receiver operator characteristic analysis. The diagnostic performance of the above-listed proteins was defined with respect to their specificity, sensitivity, and predictive values (PV); the influence of cyst location; and usefulness in the follow-up of surgical treatment for UHD and in the determination of whether or not patients have been surgically cured of UHD. The best diagnostic results were obtained with the anti-B2t IgG ELISA, with 91.2% sensitivity, 93% specificity, and high positive and negative PV (89.4 and 94.2, respectively). In addition, this diagnostic tool proved to be useful for the follow-up of surgically treated UHD patients. The anti-B2t IgG ELISA may find an application in the serodiagnosis of UHD in clinical laboratories.
Risk and Protective Factors for Leprosy Development Determined by Epidemiological Surveillance of Household Contacts
Authors: Goulart Isabela M. B. , Bernardes Souza Dulcinéa O. , Marques Carolina R. , Pimenta Vânia L. , Gonçalves Maria A. , Goulart Luiz R.
Published: November 2007

Household contacts of leprosy patients are the group with the highest risk of developing the disease, and although many risk or prevention factors have been identified, they have not been employed in leprosy-monitoring programs. This investigation aimed to establish the relative risks or the preventive effects of the presence of BCG vaccination, the Mitsuda test, and the ML-Flow assay. Household contacts (1,396) were monitored for a 5-year period. Twenty-eight contacts (2%) developed leprosy and had their clinical and operational classifications established. All immunological tests were performed, and intradermal BCG vaccination was given after the BCG scar count. Of the affected contacts, 75% developed the disease in the first year, and 71.4% were classified as having paucibacillary forms. Contacts of lepromatous leprosy patients presented a 3.8-fold-higher risk of developing leprosy. BCG vaccination and the Mitsuda test showed a protective effect against leprosy of 0.27 (at least one scar) and 0.16 (>7 mm), respectively, and the positive ML-Flow test indicated a relative risk approximately sixfold higher for occurrence of the disease. All unfavorable combinations of two and three assays generated significant risk values that ranged from 5.76 to 24.47, with the highest risk given by the combination of no BCG scar, negative Mitsuda test, and positive ML-Flow test. We suggest that the BCG vaccination may be given to stimulate Mitsuda test positivity, reducing the patient's risk of developing multibacillary forms. The high significance of these tests may have a great impact on programs to monitor contacts and should be used to improve early detection and treatment.
Modulation of Type 1 Diabetes Susceptibility by Tumor Necrosis Factor Alpha −308 G/A and Lymphotoxin Alpha +249 A/G Haplotypes and Lack of Linkage Disequilibrium with Predisposing
DQB1-DRB1
Haplotypes in Bahraini Patients
Authors: Stayoussef Mouna , Al-Jenaidi Fayza A. , Al-Abbasi Abduljabbar , Al-Ola Khadija , Khayyat Haya , Mahjoub Touhami , Almawi Wassim Y.
Published: November 2007

Tumor necrosis factor alpha (TNF-α) −308 G/A and lymphotoxin alpha (LTα) +249 A/G single-nucleotide polymorphisms were investigated in 228 type 1 diabetes mellitus (T1DM) patients and 240 controls. Only LTα +249G allele and +249G/+249G genotype frequencies were higher among patients, and no linkage disequilibrium was found between TNF-α/LTα alleles and susceptible/protective
DRB1-DQB1
haplotypes. TNF-α/LTα T1DM-susceptible (−308G/+249G) and protective (−308G/+249A) haplotypes were identified.
Discrimination of Antibody to Herpes B Virus from Antibody to Herpes Simplex Virus Types 1 and 2 in Human and Macaque Sera
Authors: Fujima Akikazu , Ochiai Yoshitsugu , Saito Aya , Omori Yuki , Noda Atsuya , Kazuyama Yukumasa , Shoji Hiroshi , Tanabayashi Kiyoshi , Ueda Fukiko , Yoshikawa Yasuhiro , Hondo Ryo
Published: November 2007

The antigenic cross-reactive characteristics of herpes B virus and herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are responsible for false-positive diagnoses by serological assays in humans and macaques. In the present study, we developed a fluorometric indirect enzyme-linked immunosorbent assay (ELISA) with recombinant herpes B virus glycoprotein D (gD) and HSV-1 and HSV-2 gG (gG-1 and gG-2, respectively) to discriminate between the three primate herpesvirus infections. The secreted form of gD, gDdTM, was used to detect antibody to herpes B virus gD. Sera positive for herpes B virus, HSV-1, and HSV-2 showed specific reactions to gD, gG-1, and gG-2, respectively. Sera collected from humans and rhesus macaques were investigated for the presence of antibodies to the recombinant proteins of the three herpesviruses. The results suggested that the approach is able to discriminate between herpes B virus and HSV infections. The ELISA was also found to be able to detect infections with multiple primate herpesviruses and may have the potential to identify a subsequent infection in individuals that have already been infected with another herpesvirus. In addition, we found evidence of a greater cross-reactivity of herpes B virus with HSV-1 than with HSV-2. It is suggested that the ELISA with the recombinant antigens is useful not only for the serodiagnosis of primate herpesvirus infections but also for elucidation of the seroprevalence of herpesviruses in humans and primates.
Nested PCR for Specific Diagnosis of
Taenia solium
Taeniasis
Authors: Mayta Holger , Gilman Robert H. , Prendergast Emily , Castillo Janeth P. , Tinoco Yeny O. , Garcia Hector H. , Gonzalez Armando E. , Sterling Charles R.
Published: November 2007

Taeniasis due to
Taenia solium
is a disease with important public health consequences, since the larval stage is not exclusive to the animal intermediate, the pig, but also infects humans, causing neurocysticercosis. Early diagnosis and treatment of
T. solium
tapeworm carriers is important to prevent human cysticercosis. Current diagnosis based on microscopic observation of eggs lacks both sensitivity and specificity. In the present study, a nested-PCR assay targeting the
Tso31
gene was developed for the specific diagnosis of taeniasis due to
T. solium
. Initial specificity and sensitivity testing was performed using stored known
T. solium
-positive and -negative samples. The assay was further analyzed under field conditions by conducting a case-control study of pretreatment stool samples collected from a population in an area of endemicity. Using the archived samples, the assay showed 97% (31/32) sensitivity and 100% (123/123) specificity. Under field conditions, the assay had 100% sensitivity and specificity using microscopy/enzyme-linked immunosorbent assay coproantigen testing as the gold standards. The Tso31 nested PCR described here might be a useful tool for the early diagnosis and prevention of taeniasis/cysticercosis.
Epidemiology of European Community-Associated Methicillin-Resistant
Staphylococcus aureus
Clonal Complex 80 Type IV Strains Isolated in Denmark from 1993 to 2004
Authors: Larsen A. R. , Böcher S. , Stegger M. , Goering R. , Pallesen L. V. , Skov R.
Published: November 2007

In Europe, community-associated methicillin-resistant
Staphylococcus aureus
(CA-MRSA) infections have been caused predominantly by isolates belonging to the “European CA-MRSA” clone (sequence type 80, staphylococcal cassette chromosome
mec
type IV). In this study, the epidemiology of European CA-MRSA was investigated on a nationwide scale, covering the period from 1993 to 2004. Denmark has been a low-prevalence country regarding MRSA since the mid-1970s but has experienced an increase in the number of new MRSA cases in recent years. Our results show that European CA-MRSA contributed to this increase. The isolates primarily caused skin and soft tissue infections (SSTIs) in patients outside hospitals, and transmission between household members was the predominant mode of spread. Although some of the isolates were found in hospitalized patients, nosocomial transmission seemed likely in only one instance, pointing to endogenous infections as an important factor. Compared to the CA-MRSA clone most common in the United States (USA300), the European CA-MRSA clone seems less well adapted to persist in hospital environments. Patients with a recent history of travel or family relation to the Mediterranean or Middle East were highly overrepresented. The epidemiological data indicated that the European CA-MRSA isolates were introduced into Denmark on multiple occasions, paralleled by an increasing level of genetic diversity of the isolates found during the study period. European CA-MRSA has previously been described as a rather uniform clone. However, we found pronounced, diverse pulsed-field gel electrophoresis subtypes, staphylococcal protein A gene (
spa
) types, and susceptibility patterns.
Identification of Adenoviruses in Specimens from High-Risk Pediatric Stem Cell Transplant Recipients and Controls
Authors: Zheng Xiaotian , Lu Xiaoyan , Erdman Dean D. , Anderson Evan J. , Guzman-Cottrill Judith A. , Kletzel Morris , Katz Ben Z.
Published: November 2007

Adenovirus infection is an important cause of morbidity and mortality in stem cell transplant recipients. We report species and type-specific analysis from a prospective study of high-risk adenovirus infections following hematopoietic progenitor cell transplantation prior to, during, and after treatment with cidofovir, as well as species analysis of contemporaneously collected samples from control patients. Nine different adenovirus types representing all six recognized species were identified, and mixed infections were commonly found in this group of patients.
Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions
Authors: Martró Elisa , González Victoria , Buckton Andrew J. , Saludes Verónica , Fernández Gema , Matas Lurdes , Planas Ramón , Ausina Vicenç
Published: November 2007

We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5′ noncoding region (5′NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5′NC reverse hybridization (method 2; InnoLiPA HCV II) and 5′NC sequencing (method 3; Trugene HCV 5′NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays.
Comparison of Linear Array and Line Blot Assay for Detection of Human Papillomavirus and Diagnosis of Cervical Precancer and Cancer in the Atypical Squamous Cell of Undetermined Significance and Low-Grade Squamous Intraepithelial Lesion Triage Study
Authors: Castle Philip E. , Gravitt Patti E. , Solomon Diane , Wheeler Cosette M. , Schiffman Mark
Published: November 2007

We evaluated Linear Array (LA), a newly commercialized PGMY09/11 L1 consensus primer PCR test that detects 37 human papillomavirus (HPV) genotypes by reverse line blot hybridization, for the detection of individual HPV genotypes and carcinogenic HPV and its clinical performance for detecting 2-year cumulative cervical precancer and cancer using archived specimens from the Atypical Squamous Cell of Undetermined Significance (ASCUS) and Low-Grade Squamous Intraepithelial Lesion Triage Study. LA testing was conducted on enrollment specimens from women referred because of an ASCUS Pap test. To gauge the performance of the new test, the results were compared to those of its prototype predecessor assay, Line Blot Assay (LBA), restricted to paired results (
n
= 3,335). LA testing was done masked to LBA results and clinical outcomes. The results of LA and LBA testing were compared for detection of carcinogenic HPV and clinical outcomes of cervical precancer and cancer. Overall, 50% and 55% of the women tested positive for carcinogenic HPV by LBA and LA, respectively (
P
< 0.0001). The percent agreement for carcinogenic HPV detection was 88%, percent positive agreement was 80%, and kappa was 0.76 for detection of carcinogenic HPV by the two assays. There was a significant increase in detection by LA for most of the 37 HPV genotypes targeted by both assays, including for 13 of 14 carcinogenic HPV genotypes. LA detected more multiple-genotype infections for all HPV genotypes among HPV-positive women (
P
< 0.0001) and for carcinogenic HPV genotypes among carcinogenic-HPV-positive women (
P
< 0.0001). LA was more sensitive (92.3% versus 87.1%;
P
= 0.003) and less specific (48.2% versus 54.0%;
P
< 0.0001) than LBA for 2-year cumulative cervical precancer and cancer as diagnosed by the Pathology Quality Control Group. In conclusion, we found LA to be a promising assay for the detection of HPV genotypes and carcinogenic HPV, and it may be clinically useful for the detection of cervical precancer and cancer in women with equivocal cytology.
Analysis of Typing Methods for Epidemiological Surveillance of both Methicillin-Resistant and Methicillin-Susceptible
Staphylococcus aureus
Strains
Authors: Faria Nuno A. , Carrico João A. , Oliveira Duarte C. , Ramirez Mário , de Lencastre Hermínia
Published: November 2007

Sequence-based methods for typing
Staphylococcus aureus
, such as multilocus sequence typing (MLST) and
spa
typing, have increased interlaboratory reproducibility, portability, and speed in obtaining results, but pulsed-field gel electrophoresis (PFGE), remains the method of choice in many laboratories due to the extensive experience with this methodology and the large body of data accumulated using the technique. Comparisons between typing methods have been overwhelmingly based on a qualitative assessment of the overall agreement of results and the relative discriminatory indexes. In this study, we quantitatively assess the congruence of the major typing methods for
S. aureus
, using a diverse collection of 198
S. aureus
strains previously characterized by PFGE,
spa
typing, MLST, and, in the case of methicillin-resistant
S. aureus
(MRSA), SCC
mec
typing in order to establish the quantitative congruence between the typing methods. The results of most typing methods agree in that MRSA and methicillin-susceptible
S. aureus
(MSSA) differ in terms of diversity of genetic backgrounds, with MSSA being more diverse. Our results show that
spa
typing has a very good predictive power over the clonal relationships defined by eBURST, while PFGE is less accurate for that purpose but nevertheless provides better typeability and discriminatory power. The combination of PFGE and
spa
typing provided even better results. Based on these observations, we suggest the use of the conjugation of
spa
typing and PFGE typing for epidemiological surveillance studies, since this combination provides the ability to infer long-term relationships while maintaining the discriminatory power and typeability needed in short-term studies.
Detection of Respiratory Viruses and Atypical Bacteria in Children's Tonsils and Adenoids
Authors: Drago Lorenzo , Esposito Susanna , De Vecchi Elena , Marchisio Paola , Blasi Francesco , Baggi Elena , Capaccio Pasquale , Pignataro Lorenzo
Published: November 2007

The tonsils and adenoids of 44 children were analyzed for the detection of respiratory syncytial virus, influenza virus, parainfluenza virus, adenovirus,
Chlamydophila pneumoniae
, and
Mycoplasma pneumoniae
. Viruses were detected in 47.7% of the children and 37.3% of the specimens, with adenovirus and parainfluenza viruses being the most frequently detected microorganisms.
Modulation of Human Immunodeficiency Virus Type 1 Infectivity through Incorporation of Tetraspanin Proteins
Authors: Sato Kei , Aoki Jun , Misawa Naoko , Daikoku Eriko , Sano Kouichi , Tanaka Yuetsu , Koyanagi Yoshio
Published: November 2007

Accumulating evidence indicates that human immunodeficiency virus type 1 (HIV-1) acquires various cellular membrane proteins in the lipid bilayer of the viral envelope membrane. Although some virion-incorporated cellular membrane proteins are known to potently affect HIV-1 infectivity, the virological functions of most virion-incorporated membrane proteins remain unclear. Among these host proteins, we found that CD63 was eliminated from the plasma membranes of HIV-1-producing T cells after activation, followed by a decrease in the amount of virion-incorporated CD63, and in contrast, an increase in the infectivity of the released virions. On the other hand, we found that CD63 at the cell surface was preferentially embedded on the membrane of released virions in an HIV-1 envelope protein (Env)-independent manner and that virion-incorporated CD63 had the potential to inhibit HIV-1 Env-mediated infection in a strain-specific manner at the postattachment entry step(s). In addition, these behaviors were commonly observed in other tetraspanin proteins, such as CD9, CD81, CD82, and CD231. However, L6 protein, whose topology is similar to that of tetraspanins but which does not belong to the tetraspanin superfamily, did not have the potential to prevent HIV-1 infection, despite its successful incorporation into the released particles. Taken together, these results suggest that tetraspanin proteins have the unique potential to modulate HIV-1 infectivity through incorporation into released HIV-1 particles, and our findings may provide a clue to undiscovered aspects of HIV-1 entry.
Nucleolar Targeting of Hepatitis Delta Antigen Abolishes Its Ability To Initiate Viral Antigenomic RNA Replication
Authors: Huang Wen-Hung , Chen Yen-Shun , Chen Pei-Jer
Published: November 2007

Hepatitis delta virus (HDV) is a small RNA virus that contains one 1.7-kb single-stranded circular RNA of negative polarity. The HDV particle also contains two isoforms of hepatitis delta antigen (HDAg), small (SHDAg) and large HDAg. SHDAg is required for the replication of HDV, which is presumably carried out by host RNA-dependent RNA polymerases. The localization and the HDAg and host RNA polymerase responsible for HDV replication remain important issues to be addressed. In this study, using recombinant SHDAg fused with a heterologous nucleolar localization sequence (NoLS) to confine its subcellular localization in nucleoli, we aimed to study the effect of SHDAg subcellular localization on HDV RNA replication. The initiation of genomic RNA synthesis from antigenomic template was hardly detectable when SHDAg was fused with the NoLS motif and localized mainly in nucleoli. In contrast, the initiation of antigenomic RNA synthesis was not affected. Drug treatment to release a SHDAg-NoLS mutant from nucleoli could partially restore the replication of HDV genomic RNA from antigenomic RNA. This also recovered the cointeraction between SHDAg and RNA polymerase II. These data strongly suggest that nuclear polymerase (RNA polymerase II) is involved in the synthesis of genomic RNA and that the synthesis of antigenomic RNA can occur in nucleoli. Our results support the idea that the replication of HDV genomic RNA or antigenomic RNA is likely to be carried out by different machineries in different subcellular localizations.
Extending the Cytoplasmic Tail of the Influenza A Virus M2 Protein Leads to Reduced Virus Replication In Vivo but Not In Vitro
Authors: Wu Wai-Hong , Pekosz Andrew
Published: November 2007

A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.
A
cis
Element between the TATA Box and the Transcription Start Site of the Major Immediate-Early Promoter of Human Cytomegalovirus Determines Efficiency of Viral Replication
Authors: Isomura Hiroki , Stinski Mark F. , Kudoh Ayumi , Nakayama Sanae , Murata Takayuki , Sato Yoshitaka , Iwahori Satoko , Tsurumi Tatsuya
Published: November 2007

The promoter of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV), also referred to as the CMV promoter, possesses a
cis
-acting element positioned downstream of the TATA box between positions −14 and −1 relative to the transcription start site (+1). We determined the role of the
cis
-acting element in viral replication by comparing recombinant viruses with the
cis
-acting element replaced with other sequences. Recombinant virus with the simian CMV counterpart replicated efficiently in human foreskin fibroblasts, as well as wild-type virus. In contrast, replacement with the murine CMV counterpart caused inefficient MIE gene transcription, RNA splicing, MIE and early viral gene expression, and viral DNA replication. To determine which nucleotides in the
cis
-acting element are required for efficient MIE gene transcription and splicing, we constructed mutations within the
cis
-acting element in the context of a recombinant virus. While mutations in the
cis
-acting element have only a minor effect on in vitro transcription, the effects on viral replication are major. The nucleotides at −10 and −9 in the
cis
-acting element relative to the transcription start site (+1) affect efficient MIE gene transcription and splicing at early times after infection. The
cis
-acting element also acts as a
cis
-repression sequence when the viral IE86 protein accumulates in the infected cell. We demonstrate that the
cis
-acting element has an essential role in viral replication.
Cytoplasmic Domain of Influenza B Virus BM2 Protein Plays Critical Roles in Production of Infectious Virus
Authors: Imai Masaki , Kawasaki Kazunori , Odagiri Takato
Published: November 2007

Influenza B virus BM2 is a type III integral membrane protein that displays H
+
ion channel activity. Analysis of BM2 knockout mutants has suggested that this protein is a necessary component for the capture of M1-viral ribonucleoprotein (vRNP) complex at the plasma membrane and for incorporation of vRNP complex into the virion during the assembly process. BM2 comprises 109 amino acid residues and possesses a longer cytoplasmic domain than the other 3 integral membrane proteins (hemagglutinin, neuraminidase, and NB). To explore whether the cytoplasmic domain of BM2 is important for infectious virus production, a series of BM2 deletion mutants lacking three to nine amino acid residues at the carboxyl terminus, BM2Δ107-109, BM2Δ104-109, and BM2Δ101-109, was generated by reverse genetics. Intracellular transport and incorporation into virions were indistinguishable between truncated BM2 proteins and wild-type BM2. The BM2Δ107-109 mutant produced levels of infectious virus similar to those of wild-type virus and displayed a spherical shape. However, the BM2Δ104-109 and BM2Δ101-109 mutants produced viruses containing dramatically reduced vRNP complex, as with BM2 knockout mutants, and formed enlarged, irregularly shaped virions. Moreover, gradient separation of membranes indicated that membrane association of M1 from mutants was greatly affected by carboxyl-terminal truncations of BM2. Studies of alanine substitution mutants further suggested that amino acid sequences in the 98-109 region are variable while those in the 86-97 region are a prerequisite for innate BM2 function. These results indicate that the cytoplasmic domain of the BM2 protein is required for firm association of the M1 protein with lipid membranes, vRNP complex incorporation into virions, and virion morphology.
Novel Plant Virus-Based Vaccine Induces Protective Cytotoxic T-Lymphocyte-Mediated Antiviral Immunity through Dendritic Cell Maturation
Authors: Lacasse Patrick , Denis Jérôme , Lapointe Réjean , Leclerc Denis , Lamarre Alain
Published: November 2007

Currently used vaccines protect mainly through the production of neutralizing antibodies. However, antibodies confer little or no protection for a majority of chronic viral infections that require active involvement of cytotoxic T lymphocytes (CTLs). Virus-like particles (VLPs) have been shown to be efficient inducers of cell-mediated immune responses, but administration of an adjuvant is generally required. We recently reported the generation of a novel VLP system exploiting the self-assembly property of the papaya mosaic virus (PapMV) coat protein. We show here that uptake of PapMV-like particles by murine splenic dendritic cells (DCs) in vivo leads to their maturation, suggesting that they possess intrinsic adjuvant-like properties. DCs pulsed with PapMV-like particles displaying the lymphocytic choriomeningitis virus (LCMV) p33 immunodominant CTL epitope (PapMV-p33) efficiently process and cross-present the viral epitope to p33-specific transgenic T cells. Importantly, the CTL epitope is also properly processed and presented in vivo, since immunization of p33-specific T-cell receptor transgenic mice with PapMV-p33 induces the activation of large numbers of specific CTLs. C57BL/6 mice immunized with PapMV-p33 VLPs in the absence of adjuvant develop p33-specific effector CTLs that rapidly expand following LCMV challenge and protect vaccinated mice against LCMV infection in a dose-dependent manner. These results demonstrate the efficiency of this novel plant virus-based vaccination platform in inducing DC maturation leading to protective CTL responses.
The Cigarette Smoke Carcinogen Benzo[
a
]pyrene Enhances Human Papillomavirus Synthesis
Authors: Alam Samina , Conway Michael J. , Chen Horng-Shen , Meyers Craig
Published: November 2007

Epidemiological studies suggest that cigarette smoke carcinogens are cofactors which synergize with human papillomavirus (HPV) to increase the risk of cervical cancer progression. Benzo[
a
]pyrene (B
a
P), a major carcinogen in cigarette smoke, is detected in the cervical mucus and may interact with HPV. Exposure of cervical cells to high concentrations of B
a
P resulted in a 10-fold increase in HPV type 31 (HPV31) viral titers, whereas treatment with low concentrations of B
a
P resulted in an increased number of HPV genome copies but not an increase in virion morphogenesis. B
a
P exposure also increased HPV16 and HPV18 viral titers. Overall, B
a
P modulation of the HPV life cycle could potentially enhance viral persistence, host tissue carcinogenesis, and permissiveness for cancer progression.
The Major Histocompatibility Complex Class II AllelesMamu-DRB1*1003and -DRB1*0306Are Enriched in a Cohort of Simian Immunodeficiency Virus-Infected Rhesus Macaque Elite Controllers
Authors: Giraldo-Vela Juan P. , Rudersdorf Richard , Chung Chungwon , Qi Ying , Wallace Lyle T. , Bimber Benjamin , Borchardt Gretta J. , Fisk Debra L. , Glidden Chrystal E. , Loffredo John T. , Piaskowski Shari M. , Furlott Jessica R. , Morales-Martinez Juan P. , Wilson Nancy A. , Rehrauer William M. , Lifson Jeffrey D. , Carrington Mary , Watkins David I.
Published: November 2007

The role of CD4+T cells in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication is not well understood. Even though strong HIV- and SIV-specific CD4+T-cell responses have been detected in individuals that control viral replication, major histocompatibility complex class II (MHC-II) molecules have not been definitively linked with slow disease progression. In a cohort of 196 SIVmac239-infected Indian rhesus macaques, a group of macaques controlled viral replication to less than 1,000 viral RNA copies/ml. These elite controllers (ECs) mounted a broad SIV-specific CD4+T-cell response. Here, we describe five macaque MHC-II alleles (Mamu-DRB*w606, -DRB*w2104, -DRB1*0306, -DRB1*1003, and -DPB1*06) that restricted six SIV-specific CD4+T-cell epitopes in ECs and report the first association between specific MHC-II alleles and elite control. Interestingly, the macaque MHC-II alleles,Mamu-DRB1*1003and -DRB1*0306, were enriched in this EC group (Pvalues of 0.02 and 0.05, respectively). Additionally,Mamu-B*17-positive SIV-infected rhesus macaques that also expressed these two MHC-II alleles had significantly lower viral loads thanMamu-B*17-positive animals that did not expressMamu-DRB1*1003and -DRB1*0306(Pvalue of <0.0001). The study of MHC-II alleles in macaques that control viral replication could improve our understanding of the role of CD4+T cells in suppressing HIV/SIV replication and further our understanding of HIV vaccine design.
Modification of Adenovirus Capsid with a Designed Protein Ligand Yields a Gene Vector Targeted to a Major Molecular Marker of Cancer
Authors: Belousova Natalya , Mikheeva Galina , Gelovani Juri , Krasnykh Victor
Published: November 2007

The future of genetic interventions in humans critically depends on the selectivity and efficiency of gene transfer to target tissues. The viral gene vectors explored to date cannot selectively transduce the desired targets. While substantial progress has been made in developing targeting strategies for adenovirus (Ad) vectors, future advances in this direction are severely limited by the shortage of naturally existing molecules available for use as targeting ligands. This shortage is due to fundamental and irresolvable differences at the level of both posttranslational modifications and intracellular trafficking between the Ad structural proteins and those natural proteins that are involved in interactions with the cell surface and could otherwise be considered as potential targeting ligands. We hypothesized that this problem could be resolved by altering the natural tropism of Ad vector through incorporation into its capsid of a rationally designed protein ligand, an affibody, whose structural, functional, and biosynthetic properties make it compatible with the Ad assembly process. We tested this hypothesis by redesigning the receptor-binding Ad protein, the fiber, using affibodies specific for human epidermal growth factor receptor type 2 (Her2), a major molecular marker of human tumors. The biosynthesis and folding of these fiber chimeras were fully compatible with Ad virion formation, and the resultant viral vectors were capable of selective delivery of a dual-function transgene to Her2-expressing cancer cells. By establishing the feasibility of this affibody-based approach to Ad vector targeting, the present study lays the foundation for further development of Ad vector technology toward its clinical use.
Flock House Virus Induces Apoptosis by Depletion of
Drosophila
Inhibitor-of-Apoptosis Protein DIAP1
Authors: Settles Erik W. , Friesen Paul D.
Published: November 2007

The molecular mechanisms by which RNA viruses induce apoptosis and apoptosis-associated pathology are not fully understood. Here we show that flock house virus (FHV), one of the simplest RNA viruses (family,
Nodaviridae
), induces robust apoptosis of permissive
Drosophila
Line-1 (DL-1) cells. To define the pathway by which FHV triggers apoptosis in this model invertebrate system, we investigated the potential role of
Drosophila
apoptotic effectors during infection. Suggesting the involvement of host caspases, the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluromethylketone (
z
-VAD-fmk) prevented FHV-induced cytopathology and prolonged cell survival. RNA interference-mediated ablation of the principal
Drosophila
effector caspase DrICE or its upstream initiator caspase DRONC prevented FHV-induced apoptosis and demonstrated direct participation of this intrinsic caspase pathway. Prior to the FHV-induced activation of DrICE, the intracellular level of inhibitor-of-apoptosis (IAP) protein DIAP1, the principal caspase regulator in
Drosophila melanogaster
, was dramatically reduced. DIAP1 was depleted despite
z
-VAD-fmk-mediated caspase inhibition during infection, suggesting that the loss of DIAP1 was caused by an upstream FHV-induced signal. The RNA interference-mediated knockdown of DIAP1 caused rapid and uniform apoptosis of DL-1 cells and thus indicated that DIAP1 depletion is sufficient to trigger apoptosis. Confirming this conclusion, the elevation of intracellular DIAP1 levels in stable
diap1
-transfected cells blocked caspase activation and prevented FHV-induced apoptosis. Collectively, our findings suggest that DIAP1 is a critical sensor of virus infection, which upon virus-signaled depletion relieves caspase inhibition, which subsequently executes apoptotic death. Thus, our study supports the hypothesis that altering the level or the activity of cellular IAP proteins is a general mechanism by which RNA viruses trigger apoptosis.
Identification of a Broadly Cross-Reacting and Neutralizing Human Monoclonal Antibody Directed against the Hepatitis C Virus E2 Protein
Authors: Perotti Mario , Mancini Nicasio , Diotti Roberta A. , Tarr Alexander W. , Ball Jonathan K. , Owsianka Ania , Adair R. , Patel Arvind H. , Clementi Massimo , Burioni Roberto
Published: November 2007

Identification of anti-hepatitis C virus (anti-HCV) human antibody clones with broad neutralizing activity is important for a better understanding of the interplay between the virus and host and for the design of an effective passive immunotherapy and an effective vaccine. We report the identification of a human monoclonal Fab (e137) able to bind the HCV E2 glycoprotein of all HCV genotypes but genotype 5. The results of antibody competition assays and testing the reactivity to alanine mutant E2 proteins confirmed that the e137 epitope includes residues (T416, W420, W529, G530, and D535) highly conserved across all HCV genotypes. Fab e137 neutralized HCV pseudoparticles bearing genotype 1a, 1b, and 4 E1-E2 proteins and to a lesser extent, genotype 2b. Fab e137 was also able to inhibit cell culture-grown HCV (genotype 2a). These data indicate that broadly cross-reacting and cross-neutralizing antibodies are generated during HCV infection.
Display of Heterologous Proteins on
gp64
null Baculovirus Virions and Enhanced Budding Mediated by a Vesicular Stomatitis Virus G-Stem Construct
Authors: Zhou Jian , Blissard Gary W.
Published: November 2007

The
Autographa californica
multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the
gp64
gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a
gp64
null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV
gp64
null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the
gp64
null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on
gp64
null AcMNPV budded virions. The combined use of
gp64
null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production.
Mutations in the U5 Region Adjacent to the Primer Binding Site Affect tRNA Cleavage by Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo
Authors: Oh Jangsuk , McWilliams Mary Jane , Julias John G. , Hughes Stephen H.
Published: November 2007

In retroviruses, the first nucleotide added to the tRNA primer defines the end of the U5 region in the right long terminal repeat, and the subsequent removal of this tRNA primer by RNase H exactly defines the U5 end of the linear double-stranded DNA. In most retroviruses, the entire tRNA is removed by RNase H cleavage at the RNA/DNA junction. However, the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase cleaves the tRNA 1 nucleotide from the RNA/DNA junction at the U5/primer binding site (PBS) junction, which leaves an rA residue at the U5 terminus. We made sequence changes at the end of the U5 region adjacent to the PBS in HIV-1 to determine whether such changes affect the specificity of tRNA primer cleavage by RNase H. In some of the mutants, RNase H usually removed the entire tRNA, showing that the cleavage specificity was shifted by 1 nucleotide. This result suggests that the tRNA cleavage specificity of the HIV-1 RNase domain H depends on sequences in U5.
Reduced fertility among shoe manufacturing workers
Authors: Sallmén M , Neto M , Mayan O N
Published: November 2007

Objectives:

To investigate whether fertility is reduced among female shoe manufacturing workers exposed to organic solvents.

Methods:

A retrospective study was conducted on time to pregnancy (TTP) among 250 Portuguese shoe manufacturing workers exposed to solvents and 250 unexposed women working in stores of food units and storehouses. Data on TTP and related factors were collected by face-to-face interviews. The participation rate was 92%, and 81% of the workers (197 exposed women and 209 unexposed women) provided data for the analyses. Exposure assessment was based on hygienic measurements in the workplaces. TTP data were analysed with discrete proportional hazards regression.

Results:

Female exposure to solvents was associated with reduced fertility (adjusted fecundability density ratio (FDR) 0.55, CI 0.40 to 0.74 for low exposure, and FDR 0.70, CI 0.52 to 0.94 for high exposure). The findings were robust in different sensitivity analyses. A slightly stronger association was found among women with regular menstrual cycles. Exposure for less than 6 years was more strongly associated with reduced fertility (FDR 0.50, CI 0.30 to 0.83 and FDR 0.50, CI 0.28 to 0.90 for low and high exposure, respectively) than at least 6 years of exposure (FDR 0.60, CI 0.39 to 0.92 and FDR 0.86, CI 0.57 to 1.29 for low and high exposure, respectively). There was an interaction between solvent exposure and female smoking or use of coffee, the exposed women who smoke or use coffee being highly fecund.

Conclusions:

The findings provide further evidence that exposure to organic solvents is hazardous for female reproduction. The observed association may be related to any of the following solvents commonly used in shoe manufacturing:n-hexane and hexane isomers, toluene, methyl ethyl ketone, acetone, ethyl acetate and dichloromethane.
Size distribution and total number concentration of ultrafine and accumulation mode particles and hospital admissions in children and the elderly in Copenhagen, Denmark
Authors: Andersen Z J , Wahlin P , Raaschou-Nielsen O , Ketzel M , Scheike T , Loft S
Published: November 2007

Objectives:

To study the association between short-term exposure to ultrafine particles and morbidity in Copenhagen, Denmark.

Methods:

We studied the association between urban background levels of the total number concentration of particles (NCtot, 6–700 nm in diameter) measured at a single site (15 May 2001 to 31 December 2004) and hospital admissions due to cardiovascular (CVD) and respiratory disease (RD) in the elderly (age ⩾65 years), and due to asthma in children (age 5–18 years). We examined these associations in the presence of PM10, PM2.5(particulate matter <10 and 2.5 µm in diameter, respectively) and ambient gasses. We utilised data on size distribution to calculate NCtotfor four modes with median diameters 12, 23, 57 and 212 nm, and NC100(number concentration of particles <100 nm in diameter) and examined their associations with health outcomes. We used a time series Poisson generalised additive model adjusted for overdispersion, season, day of the week, public holidays, school holidays, influenza, pollen and meteorology, with up to 5 days’ lagged exposure.

Results and conclusions:

The adverse health effects of particulate matter on CVD and RD hospital admissions in the elderly were mainly mediated by PM10and accumulation mode particles with lack of effects for NC100. For paediatric asthma, accumulation mode particles, NC100and nitrogen oxides (mainly from traffic related sources) were relevant, whereas PM10appeared to have little effect. Our results suggest that particle volume/mass from long-range transported air pollution is relevant for CVD and RD admissions in the elderly, and possibly particle numbers from traffic sources for paediatric asthma.
H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes
Authors: Pan Ting-Ting , Neo Kay Li , Hu Li-Fang , Yong Qian Chen , Bian Jin-Song
Published: November 2007

The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular Ca2+ handling in hydrogen sulfide (H2S)-preconditioned cardiomyocytes and its consequent effects on ischemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an H2S donor, 10−4 M) preconditioning (SP, 30 min). Intracellular Ca2+ was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKCα, PKCε, and PKCδ to membrane fraction but only translocation of PKCε and PKCδ was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca2+] transients, which were reversed by a selective PKC inhibitor chelerythrine. These data suggest that SP facilitated Ca2+ removal via both accelerating uptake of Ca2+ into sarcoplasmic reticulum and enhancing Ca2+ extrusion through Na+/Ca2+ exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca2+ overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKCα, PKCε, and PKCδ in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular Ca2+ handling.
Modulation of cardiac ischemia-sensitive afferent neuron signaling by preemptive C2 spinal cord stimulation: effect on substance P release from rat spinal cord
Authors: Ding XiaoHui , Ardell Jeffrey L. , Hua Fang , McAuley Ryan J. , Sutherly Kristopher , Daniel Jala J , Williams Carole A.
Published: November 2007

The upper cervical spinal region functions as an intraspinal controller of thoracic spinal reflexes and contributes to neuronal regulation of the ischemic myocardium. Our objective was to determine whether stimulation of the C2 cervical spinal cord (SCS) of rats modified the input signal at the thoracic spinal cord when cardiac ischemia-sensitive (sympathetic) afferents were activated by transient occlusion of the left anterior descending coronary artery (CoAO). Changes in c-Fos expression were used as an index of neuronal activation within the spinal cord and brain stem. The pattern of substance P (SP) release, a putative nociceptive transmitter, was measured using antibody-coated microprobes. Two SCS protocols were used: reactive SCS, applied concurrently with intermittent CoAO and preemptive, sustained SCS starting 15 min before and continuing during the repeated intermittent CoAO. CoAO increased SP release from laminae I and II in the T4 spinal cord above resting levels. Intermittent SCS with CoAO resulted in greater levels of SP release from deeper laminae IV–VII in T4 than CoAO alone. In contrast, SP release from laminae I and II was inhibited when CoAO was applied during preemptive, sustained SCS. Preemptive SCS likewise reduced c-Fos expression in the T4 spinal cord (laminae I–V) and nucleus tractus solitarius but increased expression in the intermediolateral cell column of T4 compared with CoAO alone. These results suggest that preemptive SCS from the high cervical region modulates sensory afferent signaling from the ischemic myocardium.