bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid detection of clonal expansion of T‐cell receptor‐beta gene in patients with HBV using the real‐time PCR with DNA melting curve analysis
Authors: Yang Jie‐Zuan , Li Ming‐Wei , Wang Jian‐Guo , Lu Hai‐Feng , Yao Xin‐Sheng , He Jian‐Qin , Li Lan‐Juan 
Published: January 2010

Aim:  The gene melting spectral pattern (GMSP) of PCR products from 24 T‐cell receptor beta chain variable (TCRBV) gene families was developed to determine sequence bias and feature of TCRBV CDR3 gene family.Methods:  The assay was based on reverse transcript quantitative polymerase chain reaction and their DNA melting curves.Results:  We discovered that the relatively conserved amino acid sequences X‐Q and X‐G are present in TCRBV CDR3 from patients with HBV. Further, the X of the X‐Q motif is preferentially E (glutamic acid), P (proline) or T (threonine) when accompanied by the BJ2.7, BJ1.5, or BJ2.3, respectively. The frequency of sequence bias in the TCRBV gene family showed a positive correlation with the T cell receptor excision circles (TRECs) content, and an inverse correlation with the HBV DNA loading.Conclusion:  These results suggest that the GMSP assay could be used to monitor the features of TCRBV gene distribution quickly, and facilitate the further study of HBV‐specific T cell in patients with HBV.
Changing the World by Changing Ourselves: Reflections from a Bunch of BINGOs
Authors: Shutt Cathy 
Published: January 2010

SummaryThis Practice Paper aims to contribute to ongoing reflections and debates taking place among aid practitioners about if, and how, big international NGOs (BINGOs) can be more effective agents of ‘progressive social change’. It summarises a series of conversations that took place among seven members of the Institute of Development Studies Participation Power and Social Change team and staff from eight BINGOs between July 2008 and March 2009.During the conversations, participants considered how internal and external factors influence the potential of BINGOs to contribute to shifts in power relations; greater realisation of rights; and enhanced economic, political and social justice for poor and vulnerable people. All of this was encapsulated in the term ‘progressive social change’. At the end of the process, participants agreed that there is considerable scope for many BINGOs to pursue a more progressive agenda. They recommended that similar conversations need to continue and branch out, both in topical range and in participants in order to stimulate the kind of reflection and organisational learning required to do so.This paper includes accounts of discussions, case studies shared by participants, inputs from academic critiques of BINGOs and practical tools to feed into such deliberations. It explores the types of changes that BINGOs are trying to achieve, the approaches they use – their models of change, and challenges and tensions commonly perceived to prevent BINGOs pursuing more radical social change agendas. Provocative questions are raised as a means to help practitioners identify changes that their organisations need to make in order to more actively pursue social, economic and political justice. In some instances inspiring examples from BINGO participants suggest means to do so. References to organisational theory, meeting discussions and BINGO case studies are used to interrogate assumptions about how large complex organisations behave and to identify lessons that may be used to inform efforts to transform BINGOs into more effective agents of progressive social change.
Single Lysophosphatidylcholine Components Exhibit Adjuvant Activities
In Vitro
and
In Vivo
Authors: Bach Guillaume , Perrin-Cocon Laure , Gerossier Estelle , Guironnet-Paquet Aurélie , Lotteau Vincent , Inchauspé Geneviève , Fournillier Anne 
Published: January 2010

ABSTRACT

Improving vaccine immunogenicity by developing new adjuvant formulations has long been a goal of vaccinologists. It has previously been shown that a natural mix of lysophosphatidylcholine (LPC) from chicken eggs promotes mature dendritic cell (DC) generation
in vitro
and primes antigen-specific immune responses in mice. In the present study, we dissected the adjuvant potentials of five individual LPC components found in the chicken egg mixture.
In vitro
analyses of the impact of the individual components on the maturation of human DCs were performed by means of phenotypic analysis, chemokine secretion analysis, and analysis of the ability of mature DC to stimulate T lymphocytes. Two components, C16:0-LPC and C18:0-LPC, were identified to be capable of the upregulation of expression of CD86, HLA-DR, and CD40 on
in vitro
-cultured monocyte-derived DCs from healthy donors. Both induced the release of chemokines to high concentrations (macrophage inflammatory protein 1, monocyte chemoattractant protein 1) or moderate concentrations (interleukin-8 [IL-8], gamma interferon-inducible protein 10). In addition, C16:0-LPC engaged naïve T cells to produce gamma interferon. This suggests that C16:0-LPC and C18:0-LPC have the capacity to promote, at least
in vitro
, a Th1-oriented response. The intravenous injection of C16:0-LPC or C18:0-LPC into mice resulted in the detectable secretion of IL-6 and IL-5 in sera. Both LPC components were tested for their capacities to act as adjuvants for two selected immunogens: the hepatitis B virus surface antigen and the hepatitis C virus NS3 helicase. The secretion of specific IgG1 was observed with either or both C16:0-LPC and C18:0-LPC, depending on the immunogen tested, and was observed at an efficiency comparable to that of alum. These data identify C16:0-LPC and C18:0-LPC as the active components of the LPC natural mixture. Although discrepancies between the results of the
in vitro
and
in vivo
analyses existed, studies with animals suggest that these components can trigger significant and specific humoral-mediated immunity.
Understanding the Molecular Epidemiology of the Footrot Pathogen
Dichelobacter nodosus
To Support Control and Eradication Programs
Authors: Buller N. B. , Ashley P. , Palmer M. , Pitman D. , Richards R. B. , Hampson D. J. 
Published: January 2010

ABSTRACT

The Gram-negative anaerobe
Dichelobacter nodosus
is the primary etiologic agent of ovine footrot. Few studies of the genetic diversity and epidemiology of
D. nodosus
have been done, despite the economic cost and welfare implications of the disease. This study examined a large collection of Australian isolates; 735 isolates from footrot-infected sheep from 247 farms in Western Australia (WA) were tested by pulsed-field gel electrophoresis (PFGE), and a subset of 616 isolates was tested by infrequent restriction site PCR (IRS-PCR). The genetic diversity of WA isolates was compared to that of 61 isolates from three other Australian states. WA isolates were genetically diverse, with 181 molecular types resolved by PFGE, resulting in a simple diversity ratio (SDR) of 1:4 and a Simpson's index of discrimination value (
D
) of 0.98. IRS-PCR resolved 77 molecular types (SDR = 1:8 and
D
= 0.95). The isolates were grouped into 67 clonal groups by PFGE (SDR = 1:11,
D
= 0.90) and 36 clonal groups by IRS-PCR (SDR = 1:17,
D
= 0.87). Despite the high genetic diversity, three common clonal groups predominated in WA and were found in other Australian states. On some farms, molecular type was stable over a number of years, whereas on other farms genetically diverse isolates occurred within a flock of sheep or within a hoof. This study provides a large database from which to appropriately interpret molecular types found in epidemiological investigations and to identify common and unknown types that may compromise footrot eradication or control programs.
Duplex Real-Time Reverse Transcriptase PCR Assays for Rapid Detection and Identification of Pandemic (H1N1) 2009 and Seasonal Influenza A/H1, A/H3, and B Viruses
Authors: Chidlow Glenys , Harnett Gerald , Williams Simon , Levy Avram , Speers David , Smith David W. 
Published: January 2010

ABSTRACT
Reports of a novel influenza virus type A (H1N1), now designated by the World Health Organization as pandemic (H1N1) 2009, emerged from the United States and Mexico in April 2009. The management of the pandemic in Australia required rapid and reliable testing of large numbers of specimens for the novel influenza strain and differentiation from seasonal influenza strains. A real-time reverse transcriptase PCR (RT-PCR) assay for the detection of pandemic (H1N1) 2009 was designed and used with existing real-time RT-PCR assays for seasonal influenza viruses A and B. MS2 coliphage was added to all samples and amplified as a quality control. Three duplex RT-PCR assays, each containing two primer pairs and corresponding 5′ nuclease probes, were initially evaluated on control material and stored samples and showed high sensitivity and specificity. More than 11,000 clinical samples were then tested for influenza A and B matrix gene targets and specific hemagglutinin gene targets for seasonal influenza A/H1, A/H3, and pandemic A (H1N1) 2009. Minimum sensitivities and specificities were 98.8% and 100%, respectively, for pandemic (H1N1) 2009, 81.5% and 98.9% for seasonal A/H1, and 96.3% and 99.6% for A/H3. Automated sample extraction facilitated the rapid processing of samples so that the assays allowed accurate, rapid, and cost-effective screening of large numbers of clinical samples.
Clostridium difficile
Testing in the Clinical Laboratory by Use of Multiple Testing Algorithms
Authors: Novak-Weekley Susan M. , Marlowe Elizabeth M. , Miller John M. , Cumpio Joven , Nomura Jim H. , Vance Paula H. , Weissfeld Alice 
Published: January 2010

ABSTRACT

The incidence of
Clostridium difficile
infection (CDI) has risen almost 3-fold in the United States over the past decade, emphasizing the need for rapid and accurate tests for CDI. The Cepheid Xpert
C. difficile
assay is an integrated, closed, nucleic acid amplification system that automates sample preparation and real-time PCR detection of the toxin B gene (
tcdB
). A total of 432 stool specimens from symptomatic patients were tested by a glutamate dehydrogenase (GDH) assay, a toxin A and B enzyme immunoassay (EIA), the Xpert
C. difficile
assay, and a cell culture cytotoxicity neutralization assay (CCCN). The results of these methods, used individually and in combination, were compared to those of toxigenic culture. Results for the Xpert
C. difficile
assay alone showed a sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 94.4, 96.3, 84.0, and 98.8%, while the EIA alone gave corresponding values of 58.3, 94.7, 68.9, and 91.9%, respectively. An algorithm using the GDH assay and the EIA (plus the CCCN if the EIA was negative) showed corresponding values of 83.1, 96.7, 83.1, and 96.1%. The Xpert
C. difficile
assay was statistically superior to the EIA (
P
, <0.001 by Fisher's exact test) and to the GDH-EIA-CCCN algorithm (
P
, 0.0363). Combining the GDH and Xpert
C. difficile
assays lowered both the sensitivity and the NPV of the Xpert assay. The GDH-EIA-CCCN procedure required, on average, 2 days to complete testing on GDH-positive results, while testing by the Xpert
C. difficile
assay was completed, on average, in less than 1 h. Xpert
C. difficile
testing yielded the highest sensitivity and NPV, in the least amount of time, of the individual- and multiple-test algorithms evaluated in this study.
Direct Ertapenem Disk Screening Method for Identification of KPC-Producing
Klebsiella pneumoniae
and
Escherichia coli
in Surveillance Swab Specimens
Authors: Lolans Karen , Calvert Karen , Won Sarah , Clark James , Hayden Mary K. 
Published: January 2010

ABSTRACT

Klebsiella pneumoniae
carbapenemase (KPC) production in Gram-negative bacilli is an increasing problem worldwide. Rectal swab surveillance is recommended as a component of infection prevention programs, yet few screening methods are published. We compared detection of KPC-producing
Klebsiella pneumoniae
and
Escherichia coli
in surveillance specimens by 2 methods: (i) inoculation of swabs in tryptic soy broth containing 2 μg/ml imipenem followed by plating to MacConkey agar (MAC) (method 1) and (ii) streaking swabs on MAC onto which a 10-μg ertapenem disk was then placed (method 2). Simulated rectal swab specimens of challenge isolates from a collection of well-characterized
K. pneumoniae
and
E. coli
strains and salvage rectal swab specimens collected from patients at 4 different health care facilities over a 7-month period were tested. The gold-standard comparator was
bla
KPC
PCR testing of isolates. Method 1 detected 4/9 (44%) KPC-positive challenge isolates. By method 2, 9/9 KPC-positive challenge isolates exhibited zones of inhibition of ≤27 mm; all KPC-negative isolates exhibited zones of inhibition greater than 27 mm. The sensitivity and specificity of method 1 for detection of KPC-positive
K. pneumoniae
and
E. coli
in 149 rectal swab specimens were 65.6% (95% confidence interval [CI], 46.8% to 80.8%) and 49.6% (95% CI, 40.3% to 58.9%), respectively. With method 2, a zone diameter of ≤27 mm had a sensitivity of 97.0% (95% CI, 82.5% to 99.8%) and specificity of 90.5% (95% CI, 83.3% to 94.9%) for detection of KPC in rectal swab specimens. Direct ertapenem disk testing is simpler, more sensitive, and more specific than selective broth enrichment with imipenem for detection of KPC-producing
K. pneumoniae
and
E. coli
in surveillance specimens.
Rapid Detection of Methicillin-Resistant
Staphylococcus aureus
(MRSA) in Diverse Clinical Specimens by the BD GeneOhm MRSA Assay and Comparison with Culture
Authors: Lucke Katja , Hombach Michael , Hug Melanie , Pfyffer Gaby E. 
Published: January 2010

ABSTRACT

The efficacy of the BD GeneOhm methicillin-resistant
Staphylococcus aureus
(MRSA) assay was assessed by analyzing nasal swabs and swabs from other body sites for the presence of MRSA in a low-prevalence area. From 681 patients with a high risk for MRSA carriage, 1,601 specimens were collected and transported in Amies agar. After discordant analysis, the sensitivity, specificity, positive predictive value, and negative predictive value of the BD GeneOhm MRSA assay were 84.3%, 99.2%, 88.4%, and 98.9%, respectively, compared to culture.
Real-Time PCR Testing for
mecA
Reduces Vancomycin Usage and Length of Hospitalization for Patients Infected with Methicillin-Sensitive Staphylococci
Authors: Nguyen David T. , Yeh Ellen , Perry Sharon , Luo Robert F. , Pinsky Benjamin A. , Lee Betty P. , Sisodiya Deepak , Baron Ellen Jo , Banaei Niaz 
Published: January 2010

ABSTRACT

Nucleic acid amplification tests (NAATs) have revolutionized infectious disease diagnosis, allowing for the rapid and sensitive identification of pathogens in clinical specimens. Real-time PCR testing for the
mecA
gene (
mecA
PCR), which confers methicillin resistance in staphylococci, has the added potential to reduce antibiotic usage, improve clinical outcomes, lower health care costs, and avoid emergence of drug resistance. A retrospective study was performed to identify patients infected with methicillin-sensitive staphylococcal isolates who were receiving vancomycin treatment when susceptibility results became available. Vancomycin treatment and length of hospitalization were compared in these patients for a 6-month period before and after implementation of
mecA
PCR. Among 65 and 94 patients identified before and after
mecA
PCR, respectively, vancomycin usage (measured in days on therapy) declined from a median of 3 days (range, 1 to 44 days) in the pre-PCR period to 1 day (range, 0 to 18 days) in the post-PCR period (
P
< 0.0001). In total, 38.5% (25/65) of patients were switched to β-lactam therapy in the pre-PCR period, compared to 61.7% (58/94) in the post-PCR period (
P
= 0.004). Patient hospitalization days also declined from a median of 8 days (range, 1 to 47 days) in the pre-PCR period to 5 days (range, 0 to 42 days) in the post-PCR period (
P
= 0.03). Real-time PCR testing for
mecA
is an effective tool for reducing vancomycin usage and length of stay of hospitalized patients infected with methicillin-sensitive staphylococci. In the face of ever-rising health care expenditures in the United States, these findings have important implications for improving outcomes and decreasing costs.
Gut Mucosal FOXP3+Regulatory CD4+T Cells and Nonregulatory CD4+T Cells Are Differentially Affected by Simian Immunodeficiency Virus Infection in Rhesus Macaques
Authors: Allers Kristina , Loddenkemper Christoph , Hofmann Jörg , Unbehaun Anett , Kunkel Désirée , Moos Verena , Kaup Franz-Josef , Stahl-Hennig Christiane , Sauermann Ulrike , Epple Hans-Jörg , Schneider Thomas 
Published: January 2010

ABSTRACTThe gastrointestinal tract represents a major site for human and simian immunodeficiency virus (HIV and SIV) replication and CD4+T-cell depletion. Despite severe depletion of mucosal CD4+T cells, FOXP3+regulatory CD4+T cells (Treg) are highly increased in the gut mucosa of chronically HIV-infected individuals and may contribute to HIV pathogenesis, either by their immunosuppressive function or as a significant target cell population for virus production. Little is known about the susceptibility of mucosal Tregto viral infection and the longitudinal effect of HIV/SIV infection on Tregdynamics. In this study, we determined the level of SIV infection in Tregand nonregulatory CD4+T cells (non-Treg) isolated from the colon of SIV-infected rhesus macaques. The dynamics of mucosal Tregand alterations in the mucosal CD4+T-cell pool were examined longitudinally. Our findings indicate that mucosal Tregwere less susceptible to productive SIV infection than non-Tregand thus were selectively spared from SIV-mediated cell death. In addition to improved survival, local expansion of Tregby SIV-induced proliferation of the mucosal CD4+T-cell pool facilitated the accumulation of mucosal Tregduring the course of infection. High frequency of mucosal Tregin chronic SIV infection was strongly related to a reduction of perforin-expressing cells. In conclusion, this study suggests that mucosal Tregare less affected by productive SIV infection than non-Tregand therefore spared from depletion. Although SIV production is limited in mucosal Treg, Tregaccumulation may indirectly contribute to viral persistence by suppressing antiviral immune responses.
Caspase 9 Is Essential for Herpes Simplex Virus Type 2-Induced Apoptosis in T Cells
Authors: Vanden Oever Michael J. , Han Jin-Young 
Published: January 2010

ABSTRACT
Herpes simplex virus type 2 (HSV-2) induces apoptosis in T cells by a caspase-dependent mechanism. Apoptosis can occur via extrinsic (death receptor) and/or intrinsic (mitochondrial) pathways. Here, we show that the initiator caspase for the intrinsic pathway is activated in T cells following HSV-2 exposure. To determine the respective contributions of intrinsic and extrinsic pathways, we assessed apoptosis in Jurkat cells that are deficient in caspase 8 or Fas-associating protein with death domain (FADD) for the extrinsic pathway and in cells deficient in caspase 9 for the intrinsic pathway. Our results indicate HSV-2-induced apoptosis in T cells occurs via the intrinsic pathway.
Autoexcision of Bacterial Artificial Chromosome Facilitated by Terminal Repeat-Mediated Homologous Recombination: a Novel Approach for Generating Traceless Genetic Mutants of Herpesviruses
Authors: Zhou Fuchun , Li Qiuhua , Wong Scott W. , Gao Shou-Jiang 
Published: January 2010

ABSTRACT

Infectious bacterial artificial chromosomes (BACs) of herpesviruses are powerful tools for genetic manipulation. However, the presence of BAC vector sequence in the viral genomes often causes genetic and phenotypic alterations. While the excision of the BAC vector cassette can be achieved by homologous recombination between extra duplicate viral sequences or loxP site-mediated recombination, these methods either are inefficient or leave a loxP site mark in the viral genome. Here we describe the use of viral intrinsic repeat sequences, which are commonly present in herpesviral genomes, to excise the BAC vector cassette. Using a newly developed
in vitro
transposon-based cloning approach, we obtained an infectious BAC of rhesus rhadinovirus (RRV) strain RRV26-95 with the BAC vector cassette inserted in the terminal repeat (TR) region. We showed that the BAC vector cassette was rapidly excised upon reconstitution in cells predominantly through TR-mediated homologous recombination. Genetic and phenotypic analysis showed that the BAC-excised virus was reversed to wild-type RRV. Using this autoexcisable BAC clone, we successfully generated an RRV mutant with a deletion of Orf50, which encodes a replication and transcription activator (RTA) protein. Together, these results illustrate the usefulness of TR for genetic manipulation of herpesviruses when combined with the novel transposon-based cloning approach.
Structure of Adenovirus Type 21 Knob in Complex with CD46 Reveals Key Differences in Receptor Contacts among Species B Adenoviruses
Authors: Cupelli Karolina , Müller Steffen , Persson B. David , Jost Marco , Arnberg Niklas , Stehle Thilo 
Published: January 2010

ABSTRACT
The complement regulation protein CD46 is the primary attachment receptor for most species B adenoviruses (Ads). However, significant variability exists in sequence and structure among species B Ads in the CD46-binding regions, correlating with differences in affinity. Here, we report a structure-function analysis of the interaction of the species B Ad21 knob with the two N-terminal repeats SCR1 and SCR2 of CD46, CD46-D2. We have determined the structures of the Ad21 knob in its unliganded form as well as in complex with CD46-D2, and we compare the interactions with those observed for the Ad11 knob-CD46-D2 complex. Surface plasmon resonance measurements demonstrate that the affinity of Ad21 knobs for CD46-D2 is 22-fold lower than that of the Ad11 knob. The superposition of the Ad21 and Ad11 knob structures in complex with CD46-D2 reveals a substantially different binding mode, providing an explanation for the weaker binding affinity of the Ad21 knob for its receptor. A critical difference in both complex structures is that a key interaction point, the DG loop, protrudes more in the Ad21 knob than in the Ad11 knob. Therefore, the protruding DG loop does not allow CD46-D2 to approach the core of the Ad21 knob as closely as in the Ad11 knob-CD46-D2 complex. In addition, the engagement of CD46-D2 induces a conformational change in the DG loop in the Ad21 knob but not in the Ad11 knob. Our results contribute to a more profound understanding of the CD46-binding mechanism of species B Ads and have relevance for the design of more efficient gene delivery vectors.