bluuebunny/crossref_metadata_2025_split huggingface dataset:
Widespread Occurrence of
Pneumocystis carinii
in Commercial Rat Colonies Detected Using Targeted PCR and Oral Swabs
Authors: Icenhour Crystal R. , Rebholz Sandra L. , Collins Margaret S. , Cushion Melanie T. 
Published: July 2002

ABSTRACT

The genus
Pneumocystis
contains a family of fungal organisms that infect a wide variety of mammalian species. Although it is a cause of pneumonia in immunocompromised hosts, recent evidence suggests that these organisms colonize nonimmunosuppressed hosts. Detection of cryptic colonization with
Pneumocystis
becomes important in animal studies when infection-free animals are necessary. Provocation by chronic immunosuppression, histology, and serology has been widely used to detect the presence of
Pneumocystis
in rat colonies, requiring lengthy time periods and/or postmortem tissue. We conducted a study to evaluate the use of PCR amplification of oral swabs for the antemortem detection of
Pneumocystis
in 12 rat groups from three commercial vendors. Sera were collected upon arrival, and the oral cavity was swabbed for PCR analysis. Ten of these groups of rats were then housed in pairs under barrier and immunosuppressed to provoke
Pneumocystis
growth. Once moribund, the rats were sacrificed, and the lungs were collected to evaluate the presence of
Pneumocystis
by PCR and microscopic enumeration. DNA was extracted from oral swabs and lung homogenates, and PCR was performed using primers targeting a region within the mitochondrial large-subunit rRNA of
Pneumocystis carinii
f. sp.
carinii
. Upon receipt, 64% of rats were positive for
P. carinii
f. sp.
carinii
-specific antibodies, while
P. carinii
f. sp.
carinii
DNA was amplified from 98% of oral swabs. Postmortem PCR analysis of individual lungs revealed
P. carinii
f. sp.
carinii
DNA in all rat lungs, illustrating widespread occurrence of
Pneumocystis
in commercial rat colonies. Thus, oral swab/PCR is a rapid, nonlethal, and sensitive method for the assessment of
Pneumocystis
exposure.
Pretreatment with Urea-Hydrochloric Acid Enhances the Isolation of
Helicobacter pylori
from Contaminated Specimens
Authors: Song Qunsheng , Zirnstein Gerald W. , Swaminathan Bala , Gold Benjamin D. 
Published: July 2002

ABSTRACT

Human saliva seeded with
H. pylori
was incubated in urea-HCl and then cultured on nonselective media. Pretreatment with 0.06 N HCl–0.08 M urea for 5 min at 37°C resulted in reproducible isolation of
H. pylori
, even at low inocula (≤10
2
CFU/ml of saliva), despite the presence of large numbers of contaminating organisms.
Development of Conventional and Real-Time PCR Assays for Detection of
Legionella
DNA in Respiratory Specimens
Authors: Rantakokko-Jalava Kaisu , Jalava Jari 
Published: July 2002

ABSTRACT

The development and validation of a PCR assay based on the use of new 16S ribosomal DNA (rDNA)-targeted primers to detect
Legionella
DNA in respiratory specimens are described. The assay was originally developed as conventional PCR followed by electrophoretic detection and was then adapted to Lightcycler format with SYBR Green I detection and melting curve analysis. The 73
Legionella pneumophila
strains tested were amplified with both applications. In addition, 21 and 23 out of 27 other
Legionella
strains were found positive by conventional and real-time PCR assays, respectively, including the clinically important species
L. micdadei,
L. bozemaniae
,
and
L. dumoffii
. Two DNA purification methods were compared using artificially seeded clinical specimens: a standard organic extraction method and a commercial kit based on adsorption of DNA to silica particles. The detection limit of the assay varied from 2 CFU to >200,000 CFU per ml of clinical specimen, depending on the background sample (i.e., pooled sputa or BAL fluids) and the DNA purification method, the silica method achieving lower detection limits. Analysis of 77 clinical samples (66 bronchoalveolar lavage fluid and 11 sputum samples) by conventional PCR yielded results that were consistent with
Legionella
culture results. The melting curve analysis in the Lightcycler system readily detected the specific amplification products. However, run-to-run variations in the measured melting temperatures required normalization against the standard sample in each run. The results obtained with the clinical specimens were similar to those obtained with conventional PCR, but more samples are required to determine whether the system can be applied to routine screening of samples for the presence of
Legionella
DNA.
Existence of Distinct GB Virus C/Hepatitis G Virus Variants with Different Tropism
Authors: Fogeda Marta , López-Alcorocho Juan Manuel , Bartolomé Javier , Arocena Carlos , Martín Maria Angeles , Carreño Vicente 
Published: July 2002

ABSTRACT
To study the existence of GB virus C/hepatitis G virus (GBV-C/HGV) variants with different tropism, we have analyzed the heterogeneity and quasispecies composition of GBV-C/HGV isolated from in vitro-infected peripheral blood mononuclear cells (PBMC) and from sera, livers, and PBMC from two chronically infected patients. For this purpose, the GBV-C/HGV 5′ noncoding region (5′NCR) was amplified by reverse transcription-PCR and the amplified products were cloned and sequenced. These analyses showed that the master 5′NCR sequences isolated from the in vitro-infected PBMC and from the PBMC isolated from the patient whose serum was used as the inoculum were identical but different from that of the inoculum. Furthermore, phylogenetic analysis revealed that all PBMC sequences grouped together into a branch which was separate from those of the inoculum. For one of the two chronically infected patients, all the sequences from the PBMC and one from the liver clustered into a single branch while the sequences from the serum and all the other liver sequences grouped together in the other branch. For the other patient, the sequences from the serum and PBMC and three sequences from the liver grouped together into one branch, while the remaining five sequences from the liver were separated in a different cluster. In conclusion, our results support the existence of different GBV-C/HGV variants with different tissue tropism.
Sequences in the 5′ and 3′ R Elements of Human Immunodeficiency Virus Type 1 Critical for Efficient Reverse Transcription
Authors: Ohi Yuki , Clever Jared L. 
Published: July 2002

ABSTRACT
The genome of human immunodeficiency virus type 1 (HIV-1) contains two direct repeats (R) of 97 nucleotides at each end. These elements are of critical importance during the first-strand transfer of reverse transcription, during which the minus-strand strong-stop DNA (−sssDNA) is transferred from the 5′ end to the 3′ end of the genomic RNA. This transfer is critical for the synthesis of the full-length minus-strand cDNA. These repeats also contain a variety of other functional domains involved in many aspects of the viral life cycle. In this study, we have introduced a series of mutations into the 5′, the 3′, or both R sequences designed to avoid these other functional domains. Using a single-round infectivity assay, we determined the ability of these mutants to undergo the various steps of reverse transcription utilizing a semiquantitative PCR analysis. We find that mutations within the first 10 nucleotides of either the 5′ or the 3′ R sequence resulted in virions that were markedly defective for reverse transcription in infected cells. These mutations potentially introduce mismatches between the full-length −sssDNA and 3′ acceptor R. Even mutations that would create relatively small mismatches, as little as 3 bp, resulted in inefficient reverse transcription. In contrast, virions containing identically mutated R elements were not defective for reverse transcription or infectivity. Using an endogenous reverse transcription assay with disrupted virus, we show that virions harboring the 5′ or the 3′ R mutations were not intrinsically defective for DNA synthesis. Similarly sized mismatches slightly further downstream in either the 5′, the 3′, or both R sequences were not detrimental to continued reverse transcription in infected cells. These data are consistent with the idea that certain mismatches within 10 nucleotides downstream of the U3-R junction in HIV-1 cause defects in the stability of the cDNA before or during the first-strand transfer of reverse transcription leading to the rapid disappearance of the −sssDNA in infected cells. These data also suggest that the great majority of first-strand transfers in HIV-1 occur after the copying of virtually the entire 5′ R.
LMP1 of Epstein-Barr Virus Induces Proliferation of Primary Mouse Embryonic Fibroblasts and Cooperatively Transforms the Cells with a p16-Insensitive CDK4 Oncogene
Authors: Yang Xinhai , Sham Jonathan S. T. , Ng M. H. , Tsao Sai-Wah , Zhang Dekai , Lowe Scott W. , Cao Liang 
Published: July 2002

ABSTRACT

The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-associated malignancies including nasopharyngeal carcinoma and Hodgkin's lymphoma. Previous work demonstrates that the LMP1 gene of EBV is sufficient to transform certain established rodent fibroblast cell lines and to induce the tumorigenicity of some human epithelial cell lines. In addition, LMP1 plays pleiotropic roles in cell growth arrest, differentiation, and apoptosis, depending on the background of the target cells. To examine the roles of LMP1 in cell proliferation and growth regulation in primary culture cells, we constructed a recombinant retrovirus containing an LMP1 gene. With this retrovirus, LMP1 was shown to stimulate the proliferation of primary mouse embryonic fibroblasts (MEF cells). It has a mitogenic activity for MEF cells, as demonstrated by an immediate induction of cell doubling time. In addition, it significantly extends the passage number of MEF cells to more than 30 after retroviral infection, compared with less than 5 for uninfected MEF cells. Furthermore, LMP1 cooperates with a p16-insensitive CDK4

R24C

oncogene in transforming MEF cells. Our results provide the first evidence of the abilities of the LMP1 gene, acting alone, to effectively induce the proliferation of primary MEF cells and of its cooperativity with another cellular oncogene in transforming primary cells.
Mutations in the PPPY Motif of Vesicular Stomatitis Virus Matrix Protein Reduce Virus Budding by Inhibiting a Late Step in Virion Release
Authors: Jayakar Himangi R. , Murti K. Gopal , Whitt Michael A. 
Published: July 2002

ABSTRACT
The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.
Host Sequences Flanking the Human T-Cell Leukemia Virus Type 1 Provirus In Vivo
Authors: Leclercq India , Mortreux Franck , Cavrois Marielle , Leroy Arnaud , Gessain Antoine , Wain-Hobson Simon , Wattel Eric 
Published: July 2002

ABSTRACT
Human pathogenic retroviruses do not have common loci of integration. However, many factors, such as chromatin structure, transcriptional activity, DNA-protein interaction, CpG methylation, and nucleotide composition of the target sequence, may influence integration site selection. These features have been investigated by in vitro integration reactions or by infection of cell lines with recombinant retroviruses. Less is known about target choice for integration in vivo. The present study was conducted in order to assess the characteristics of cellular sequences targeted for human T-cell leukemia virus type 1 (HTLV-1) integration in vivo. Sequencing integration sites from ≥200 proviruses (19 kb of sequence) isolated from 29 infected individuals revealed that HTLV-1 integration is not random at the level of the nucleotide sequence. The virus was found to integrate in A/T-rich regions with a weak consensus sequence at positions within and without of the hexameric repeat generated during integration. These features were not associated with a preference for integration near active regions or repeat elements of the host chromosomes. Most or all of the regions of the genome appear to be accessible to HTLV-1 integration. As with integration in vitro, integration specificity in vivo seems to be determined by local features rather than by the accessibility of specific regions.
Efficient Lymphoreticular Prion Propagation Requires PrPcin Stromal and Hematopoietic Cells
Authors: Kaeser Pascal S. , Klein Michael A. , Schwarz Petra , Aguzzi Adriano 
Published: July 2002

ABSTRACTIn most prion diseases, infectivity accumulates in lymphoreticular organs early after infection. Defects in hematopoietic compartments, such as impaired B-cell maturation, or in stromal compartments, such as abrogation of follicular dendritic cells, can delay or prevent lymphoreticular prion colonization. However, the nature of the compartment in which prion replication takes place is controversial, and it is unclear whether this compartment coincides with that expressing the normal prion protein (PrPc). Here we studied the distribution of infectivity in splenic fractions of wild-type and fetal liver chimeric mice carrying the gene that encodes PrPc(Prnp) solely on hematopoietic or on stromal cells. We fractionated spleens at various times after intraperitoneal challenge with prions and assayed infectivity by bioassay. Upon high-dose challenge, chimeras carrying PrPcon hematopoietic cells accumulated prions in stroma and in purified splenocytes. In contrast, after low-dose challenge ablation ofPrnpin either compartment prevented splenic accumulation of infectivity, indicating that optimal prion replication requires PrPcexpression by both stromal and hematopoietic compartments.
Analysis of Virus-Specific CD4+T Cells during Long-Term Gammaherpesvirus Infection
Authors: Flaño Emilio , Woodland David L. , Blackman Marcia A. , Doherty Peter C. 
Published: July 2002

ABSTRACTMajor histocompatibility complex class II-mediated antigen presentation after intranasal infection with murine gammaherpesvirus 68 differs in mediastinal lymph nodes and spleen. Evidence that virus-specific CD4+T cells were being stimulated was found as late as 6 to 8 months after infection, and cells specific for the viral gp15067–83and ORF11168–180peptides were maintained as a fairly stable proportion of the total response.
Detection of Diverse Hepatitis C Virus (HCV)-Specific Cytotoxic T Lymphocytes in Peripheral Blood of Infected Persons by Screening for Responses to All Translated Proteins of HCV
Authors: Wong David K. H. , Dudley Darryll D. , Dohrenwend Paul B. , Lauer Georg M. , Chung Raymond T. , Thomas David L. , Walker Bruce D. 
Published: July 2002

ABSTRACTBroadly directed hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been identified from liver-infiltrating lymphocytes but have been more difficult to assess in peripheral blood of infected persons. To enhance the detection of CTL from peripheral blood mononuclear cells (PBMC), we cocultured PBMC with autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines that had been infected with recombinant vaccinia virus constructs so that they expressed the entire translated polyprotein of HCV-H, a type 1a strain. These stimulated cells from HCV-infected as well as exposed seronegative persons were then cloned at limiting dilution and tested for HCV-specific CTL activity using a standard51Cr release assay. HCV-specific CTL were detected in PBMC from seven of nine persons with chronic hepatitis, including five of seven in whom CTL had previously been detected from liver biopsy specimens but not PBMC. In a single person with chronic HCV infection, CTL directed against as many as five different epitopes were detected in peripheral blood and were similar in specificity to those detected in liver tissue. This technique was used to evaluate eight subjects identified to be at high risk for HCV exposure due to continued injection drug abuse; no evidence of CTL in PBMC was found. We conclude that CTL can be detected in PBMC from the majority of persons with chronic HCV infection but are present at lower levels or absent in exposed but persistently seronegative persons. The high degree of concordance of HCV epitopes identified from liver and PBMC suggests that this strategy is a reasonable alternative to liver biopsy for characterizing the CTL response to HCV in chronically infected persons.
Identification and Antigenicity of Broadly Cross-Reactive and Conserved Human Immunodeficiency Virus Type 1-Derived Helper T-Lymphocyte Epitopes
Authors: Wilson Cara C. , Palmer Brent , Southwood Scott , Sidney John , Higashimoto Yuichiro , Appella Ettore , Chesnut Robert , Sette Alessandro , Livingston Brian D. 
Published: July 2002

ABSTRACTHuman immunodeficiency virus (HIV)-specific helper T lymphocytes (HTL) play a key role in the immune control of HIV type 1 (HIV-1) infection, and as such are an important target of potential HIV-1 vaccines. In order to identify HTL epitopes in HIV-1 that might serve as vaccine targets, conserved HIV-1-derived peptides bearing an HLA-DR binding supermotif were tested for binding to a panel of the most representative HLA-DR molecules. Eleven highly cross-reactive binding peptides were identified: three in Gag and eight in Pol. Lymphoproliferative responses to this panel of peptides, as well as to the HIV-1 p24 and p66 proteins, were evaluated with a cohort of 31 HIV-1-infected patients. All 11 peptides were recognized by peripheral blood mononuclear cells from multiple HIV-infected donors. Many of the responsive HIV-infected subjects showed recognition of multiple peptides, indicating that HIV-1-specific T-helper responses may be broadly directed in certain individuals. A strong association existed between recognition of the parental recombinant HIV-1 protein and the corresponding HTL peptides, suggesting that these peptides represent epitopes that are processed and presented during the course of HIV-1 infection. Lastly, responses to the supermotif peptides were mediated by CD4+T cells and were restricted by major histocompatibility complex class II molecules. The epitopes described herein are potentially important components of HIV-1 therapeutic and prophylactic vaccines.
Regulation of Human Immunodeficiency Virus Type 1 Infection, β-Chemokine Production, and CCR5 Expression in CD40L-Stimulated Macrophages: Immune Control of Viral Entry
Authors: Cotter Robin L. , Zheng Jialin , Che Myhanh , Niemann Douglas , Liu Ying , He Johnny , Thomas Elaine , Gendelman Howard E. 
Published: July 2002

ABSTRACTMononuclear phagocytes (MP) and T lymphocytes play a pivotal role in the host immune response to human immunodeficiency virus type 1 (HIV-1) infection. Regulation of such immune responses can be mediated, in part, through the interaction of the T-lymphocyte-expressed molecule CD40 ligand (CD40L) with its receptor on MP, CD40. Upregulation of CD40L on CD4+peripheral blood mononuclear cells during advanced HIV-1 disease has previously been reported. Based on this observation, we studied the influence of CD40L-CD40 interactions on MP effector function and viral regulation in vitro. We monitored productive viral infection, cytokine and β-chemokine production, and β-chemokine receptor expression in monocyte-derived macrophages (MDM) after treatment with soluble CD40L. Beginning 1 day after infection and continuing at 3-day intervals, treatment with CD40L inhibited productive HIV-1 infection in MDM in a dose-dependent manner. A concomitant and marked upregulation of β-chemokines (macrophage inhibitory proteins 1α and 1β and RANTES [regulated upon activation normal T-cell expressed and secreted]) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) was observed in HIV-1-infected and CD40L-treated MDM relative to either infected or activated MDM alone. The addition of antibodies to RANTES or TNF-α led to a partial reversal of the CD40L-mediated inhibition of HIV-1 infection. Surface expression of CD4 and the β-chemokine receptor CCR5 was reduced on MDM in response to treatment with CD40L. In addition, treatment of CCR5- and CD4-transfected 293T cells with secretory products from CD40L-stimulated MDM prior to infection with a CCR5-tropic HIV-1 reporter virus led to inhibition of viral entry. In conclusion, we demonstrate that CD40L-mediated inhibition of viral entry coincides with a broad range of MDM immune effector responses and the down-modulation of CCR5 and CD4 expression.
Alkaloids from Papaver pinnatifidum MORIS
Authors: Slavík Jiří , Slavíková Leonora 
Published: July 2002

Oripavine (Ia) was isolated from Papaver pinnatifidum MORIS (tetraploid, 2n = 28) of the section Rhoeadium SPACH as the major alkaloid. In a low yield, protopine and (+)-isocorydine were obtained together with a novel levorotatory alkaloid PP 1 of the composition C17H19NO3 and m.p. 179 °C, probably of a normorphinane type. Small amounts of thebaine, allocryptopine, scoulerine, isoboldine, and papaverrubines A, C and D, berberine, and corytuberine were also detected by thin layer chromatography.
Evaluation of Heterodimerization Constant from Solid-Liquid and Vapor-Liquid Equilibrium Data
Authors: Malijevská Ivona 
Published: July 2002

Activity coefficients obtained from the solid-liquid equilibrium data were used to fit the isobaric vapor-liquid equilibrium data to evaluate the vapor-phase equilibrium constant of heterodimerization of the system propanoic acid-trifluoroethanoic acid. The found hetero-dimerization constant is several times higher than that estimated on the basis of the "double-geometric-mean" rule and its temperature dependence has the form ln KAB = 7 196.7/T - 26.80.