bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of
tcdB
Real-Time PCR in a Three-Step Diagnostic Algorithm for Detection of Toxigenic
Clostridium difficile
Authors: Larson Ann M. , Fung Angela M. , Fang Ferric C. 
Published: November 2009

ABSTRACT

Clostridium difficile
is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic
C. difficile
remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the
tcdB
gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for
C. difficile
glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified “gold standard” that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic
C. difficile
was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic
C. difficile
, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic
C. difficile
that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of specimens, although this approach is less sensitive than performing DPCR on all specimens.
Sputum
Mycobacterium tuberculosis
mRNA as a Marker of Bacteriologic Clearance in Response to Antituberculosis Therapy
Authors: Li L. , Mahan C. S. , Palaci M. , Horter L. , Loeffelholz L. , Johnson J. L. , Dietze R. , Debanne S. M. , Joloba M. L. , Okwera A. , Boom W. H. , Eisenach K. D. 
Published: November 2009

ABSTRACT

mRNA is a marker of cell viability. Quantifying
Mycobacterium tuberculosis
mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of
M. tuberculosis
viability.
Wild-Type MIC Distributions and Epidemiological Cutoff Values for the Echinocandins and
Candida
spp
Authors: Pfaller M. A. , Boyken L. , Hollis R. J. , Kroeger J. , Messer S. A. , Tendolkar S. , Jones R. N. , Turnidge J. , Diekema D. J. 
Published: November 2009

ABSTRACT

We tested a global collection of
Candida
sp. strains against anidulafungin, caspofungin, and micafungin, using CLSI M27-A3 broth microdilution (BMD) methods, in order to define wild-type (WT) populations and epidemiological cutoff values (ECVs). From 2003 to 2007, 8,271 isolates of
Candida
spp. (4,283
C. albicans
, 1,236
C. glabrata
, 1,238
C. parapsilosis
, 996
C. tropicalis
, 270
C. krusei
, 99
C. lusitaniae
, 88
C. guilliermondii
, and 61
C. kefyr
isolates) were obtained from over 100 centers worldwide. The modal MICs (in μg/ml) for anidulafungin, caspofungin, and micafungin, respectively, for each species were as follows:
C. albicans
, 0.03, 0.03, 0.015;
C. glabrata
, 0.06, 0.03, 0.015;
C. tropicalis
, 0.03, 0.03, 0.015;
C. kefyr
, 0.06, 0.015, 0.06;
C. krusei
, 0.03, 0.06, 0.06;
C. lusitaniae
, 0.05, 0.25, 0.12;
C. parapsilosis
, 2, 0.25, 1; and
C. guilliermondii
, 2, 0.5. 05. The ECVs, expressed in μg/ml (percentage of isolates that had MICs that were less than or equal to the ECV is shown in parentheses) for anidulafungin, caspofungin, and micafungin, respectively, were as follows: 0.12 (99.7%), 0.12 (99.8%), and 0.03 (97.7%) for
C. albicans
; 0.25 (99.4%), 0.12 (98.5%), and 0.03 (98.2%) for
C. glabrata
; 0.12 (98.9%), 0.12 (99.4%), and 0.12 (99.1%) for
C. tropicalis
; 0.25(100%), 0.03 (100%), and 0.12 (100%) for
C. kefyr
; 0.12 (99.3%), 0.25 (96.3%), and 0.12 (97.8%) for
C. krusei
; 2 (100%), 0.5 (98.0%), and 0.5 (99.0%) for
C. lusitaniae
; 4 (100%), 1 (98.6%), and 4 (100%) for
C. parapsilosis
; 16 (100%), 4 (95.5%), and 4 (98.9%) for
C. guilliermondii
. These WT MIC distributions and ECVs will be useful in surveillance for emerging reduced echinocandin susceptibility among
Candida
spp. and for determining the importance of various
FKS1
or other mutations.
Diagnosis of Congenital Toxoplasmosis by Using a Whole-Blood Gamma Interferon Release Assay
Authors: Chapey Emmanuelle , Wallon Martine , Debize Gisèle , Rabilloud Muriel , Peyron François 
Published: November 2009

ABSTRACT

Congenital toxoplasmosis in newborns is generally subclinical, but infected infants are at risk of developing ocular lesions. Diagnosis at birth relies mainly on serological tests. Cell-mediated immunity plays the major role in resistance to infection but is not routinely investigated for diagnostic purposes. Here, we describe a simple test based on the gamma interferon (IFN-γ) response after stimulation of whole blood by crude parasitic antigens. One milliliter of heparinized blood was centrifuged; plasma was kept for routine serological tests, and pellets were resuspended in culture medium. After 24 h of culture in the presence of crude
Toxoplasma gondii
antigen, the cells were centrifuged and the supernatant was assayed for IFN-γ. For 62 infants under 1 year of age born to mothers who were infected during pregnancy, the sensitivity and specificity of the test were 94% (with positive results for 16 of 17 infected infants) and 98% (with negative results for 44 of 45 uninfected infants), respectively. The false-negative result was for a treated baby who gave positive results after the withdrawal of treatment. The false positive was obtained for a 3-month-old baby. For a cohort of 124 congenitally infected patients between 1 and 30 years of age, the sensitivity of the assay was 100%. We present a simple test based on IFN-γ secretion to assess cell-mediated immunity in toxoplasmosis. As only 1 ml of blood is required to investigate humoral and cellular immunity, our assay is well adapted for the study of congenital toxoplasmosis in infants. Using purified antigens or recombinant peptides may improve the test performance.
Human Papillomavirus Type 8 E2 Protein Unravels JunB/Fra-1 as an Activator of the β4-Integrin Gene in Human Keratinocytes
Authors: Oldak Monika , Maksym Radoslaw B. , Sperling Tanya , Yaniv Moshe , Smola Hans , Pfister Herbert J. , Malejczyk Jacek , Smola Sigrun 
Published: November 2009

ABSTRACTThe papillomavirus life cycle parallels keratinocyte differentiation in stratifying epithelia. We have previously shown that the human papillomavirus type 8 (HPV8) E2 protein downregulates β4-integrin expression in normal human keratinocytes, which may trigger subsequent differentiation steps. Here, we demonstrate that the DNA binding domain of HPV8 E2 is sufficient to displace a cellular factor from the β4-integrin promoter. We identified the E2-displaceable factor as activator protein 1 (AP-1), a heteromeric transcription factor with differentiation-specific expression in the epithelium. β4-Integrin-positive epithelial cells displayed strong AP-1 binding activity. Both AP-1 binding activity and β4-integrin expression were coregulated during keratinocyte differentiation suggesting the involvement of AP-1 in β4-integrin expression. In normal human keratinocytes the AP-1 complex was composed of JunB and Fra-1 subunits. Chromatin immunoprecipitation assays confirmed that JunB/Fra-1 proteins interactin vivowith the β4-integrin promoter and that JunB/Fra-1 promoter occupancy is reduced during keratinocyte differentiation as well as in HPV8 E2 positive keratinocytes. Ectopic expression of the tethered JunB/Fra-1 heterodimer in normal human keratinocytes activated the β4-integrin promoter, while coexpression of HPV8 E2 reverted the JunB/Fra-1 effect. In summary, we identified a novel mechanism of human β4-integrin regulation that is specifically targeted by the HPV8 E2 protein mimicking transcriptional conditions of differentiation. This may explain the early steps of how HPV8 commits its host cells to the differentiation process required for the viral life cycle.
Early Events in Kaposi's Sarcoma-Associated Herpesvirus Infection of Target Cells
Authors: Chandran Bala 
Published: November 2009

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently identified member of the herpesvirus family, infects a variety of target cells in vitro and in vivo. This minireview surveys current information on the early events of KSHV infection, including virus-receptor interactions, involved envelope glycoproteins, mode of entry, intracellular trafficking, and initial viral and host gene expression programs. We describe data supporting the hypothesis that KSHV manipulates preexisting host cell signaling pathways to allow successful infection. The various signaling events triggered by infection, and their potential roles in the different stages of infection and disease pathogenesis, are summarized.
Evidence for an Activation Domain at the Amino Terminus of Simian Immunodeficiency Virus Vpx
Authors: Gramberg Thomas , Sunseri Nicole , Landau Nathaniel R. 
Published: November 2009

ABSTRACT
Vpx and Vpr are related lentiviral accessory proteins that enhance virus replication in macrophages and dendritic cells. Both proteins are packaged into virions and mediate their effects in the target cell through an interaction with an E3 ubiquitin ligase that contains DCAF1 and DDB1. When introduced into primary macrophages and dendritic cells in viruslike particles, Vpx can enhance the efficiency of a subsequent infection. Here, we confirm the ability of Vpx to enhance simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) infection of macrophages up to 100-fold by using single-cycle reporter viruses and by pretreatment of the cells with Vpx-containing viruslike particles. Vpx was also active in differentiated THP-1 cells but not in other cell lines. Induction of an antiviral state in macrophages with type I interferon significantly magnified the effect of Vpx on HIV-1 infection, suggesting that Vpx helps the virus to overcome an inducible intracellular restriction. Quantitative PCR quantitation of SIV and HIV-1 reverse transcripts in newly infected macrophages showed that the block was at an early step in reverse transcription. In spite of its structural similarity, Vpr was inactive. This difference allowed us to map the functional domains of Vpx with a panel of Vpr/Vpx chimeras. Analysis of the chimeras demonstrated that the amino-terminal domain of Vpx is important for the enhancement of infection. Fine mapping of the region indicated that amino acids at positions 9, 12, and 15 to 17 were required. Although the mutants failed to enhance infection, they retained their ability to interact with DCAF1. These findings suggest that the Vpx amino terminus contains an activation domain that serves as the binding site for a cellular restriction factor.
In Vitro
and
In Vivo
Studies of the RNA Conformational Switch in
Alfalfa Mosaic Virus
Authors: Chen Shih-Cheng , Olsthoorn René C. L. 
Published: November 2009

ABSTRACT

The 3′ termini of
Alfalfa mosaic virus
(AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme)
in vitro
is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3′ UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the
in vitro
and
in vivo
data clearly demonstrate the existence of the conformational switch in the 3′ UTR of AMV RNAs.
Characterization of T-Cell Responses in Macaques Immunized with a Single Dose of HIV DNA Vaccine
Authors: Arrode-Brusés Géraldine , Sheffer Darlene , Hegde Ramakrishna , Dhillon Sukbir , Liu Zhengian , Villinger François , Narayan Opendra , Chebloune Yahia 
Published: November 2009

ABSTRACTThe optimization of immune responses (IR) induced by HIV DNA vaccines in humans is one of the great challenges in the development of an effective vaccine against AIDS. Ideally, this vaccine should be delivered in a single dose to immunize humans. We recently demonstrated that the immunization of mice with a single dose of a DNA vaccine derived from pathogenic SHIVKU2(Δ4SHIVKU2) induced long-lasting, potent, and polyfunctional HIV-specific CD8+T-cell responses (G. Arrode, R. Hegde, A. Mani, Y. Jin, Y. Chebloune, and O. Narayan, J. Immunol.178:2318-2327, 2007). In the present work, we expanded the characterization of the IR induced by this DNA immunization protocol to rhesus macaques. Animals immunized with a single high dose of Δ4SHIVKU2DNA vaccine were monitored longitudinally for vaccine-induced IR using multiparametric flow cytometry-based assays. Interestingly, all five immunized macaques developed broad and polyfunctional HIV-specific T-cell IR that persisted for months, with an unusual reemergence in the blood following an initial decline but in the absence of antibody responses. The majority of vaccine-specific CD4+and CD8+T cells lacked gamma interferon production but showed high antigen-specific proliferation capacities. Proliferative CD8+T cells expressed the lytic molecule granzyme B. No integrated viral vector could be detected in mononuclear cells from immunized animals, and this high dose of DNA did not induce any detectable autoimmune responses against DNA. Taken together, our comprehensive analysis demonstrated for the first time the capacity of a single high dose of HIV DNA vaccine alone to induce long-lasting and polyfunctional T-cell responses in the nonhuman primate model, bringing new insights for the design of future HIV vaccines.
Antibody to Varicella-Zoster Virus Immediate-Early Protein 62 Augments Allodynia in Zoster via Brain-Derived Neurotrophic Factor
Authors: Hama Yuka , Shiraki Kimiyasu , Yoshida Yoshihiro , Maruyama Atsushi , Yasuda Makoto , Tsuda Masaaki , Honda Mariko , Takahashi Michiaki , Higuchi Hiroshi , Takasaki Ichiro , Daikoku Tohru , Tsumoto Tadaharu 
Published: November 2009

ABSTRACT
Varicella-zoster virus (VZV) expresses immediate-early protein 62 (IE62), and zoster is associated with neuropathic pain. Brain-derived neurotrophic factor (BDNF) is involved in the neuronal mechanism underlying pain hypersensitivity. Zoster is associated with prodrome and the robust production of booster antibody to VZV. We hypothesized that the intrathecal production of antibody to IE62 cross-reacting with BDNF and the nerve injury by skin lesions may augment allodynia in zoster by enhancing BDNF activity. One of three monoclonal antibodies against the 268-556 peptide of IE62 recognized BDNF. Immunological cross-reactivity between IE62 and BDNF and the effects of anti-IE62 monoclonal antibody (anti-IE62 MAb) cross-reactivity with BDNF on BDNF activity in cultured neurons were examined. Anti-IE62 MAb and anti-BDNF MAbs recognized the 414-429 peptide of IE62 and the BDNF dimer. Anti-IE62 MAb significantly augmented BDNF-related transcription in neurons and the morphological development of spinal dorsal horn neurons. Sera from patients recognized IE62 and BDNF and enhanced BDNF activity in neurons. The effect of anti-IE62 antibody on mechanical allodynia was characterized by the threshold of allodynia using von Frey filaments in a spinal nerve injury (SNI) in mice. The administration of anti-IE62 MAb to or immunization with cross-reacting IE62 protein to mice significantly enhanced mechanical allodynia on the side with SNI but not on the uninjured side. Anti-IE62 antibody augmented BDNF activity in neurons and allodynia in mice with SNI. The intrathecal production of anti-IE62 antibody augmenting BDNF activity and peripheral nerve injury by zoster may participate in the pathogenesis of allodynia in zoster.
Infection with Strains of
Citrus Tristeza Virus
Does Not Exclude Superinfection by Other Strains of the Virus
Authors: Folimonova Svetlana Y. , Robertson Cecile J. , Shilts Turksen , Folimonov Alexey S. , Hilf Mark E. , Garnsey Stephen M. , Dawson William O. 
Published: November 2009

ABSTRACT

Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of
Citrus tristeza virus
(CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases.
Phosphorylation of Serine 177 of the Small Hepatitis Delta Antigen Regulates Viral Antigenomic RNA Replication by Interacting with the Processive RNA Polymerase II
Authors: Hong Shiao-Ya , Chen Pei-Jer 
Published: November 2009

ABSTRACT

Recent studies revealed that posttranslational modifications (e.g., phosphorylation and methylation) of the small hepatitis delta antigen (SHDAg) are required for hepatitis delta virus (HDV) replication from antigenomic to genomic RNA. The phosphorylation of SHDAg at serine 177 (Ser
177
) is involved in this step, and this residue is crucial for interaction with RNA polymerase II (RNAP II), the enzyme assumed to be responsible for antigenomic RNA replication. This study demonstrated that SHDAg dephosphorylated at Ser
177
interacted preferentially with hypophosphorylated RNAP II (RNAP IIA), which generally binds at the transcription initiation sites. In contrast, the Ser
177
-phosphorylated counterpart (pSer
177
-SHDAg) exhibited preferential binding to hyperphosphorylated RNAP II (RNAP IIO). In addition, RNAP IIO associated with pSer
177
-SHDAg was hyperphosphorylated at both the Ser
2
and Ser
5
residues of its carboxyl-terminal domain (CTD), which is a hallmark of the transcription elongation isoform. Moreover, the RNAP II CTD kinase inhibitor 5,6-dichloro-1-β-
d
-ribofuranosyl-benzimidazole (DRB) not only blocked the interaction between pSer
177
-SHDAg and RNAP IIO but also inhibited HDV antigenomic replication. Our results suggest that the phosphorylation of SHDAg at Ser
177
shifted its affinity toward the RNA RNAP IIO isoform and thus is a switch for HDV antigenomic RNA replication from the initiation to the elongation stage.