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PubMed2001

Diagnosing influenza: the value of clinical clues and laboratory tests.

OBJECTIVE: Our goal was to determine the utility of clinical clues, white blood cell (WBC) and differential counts, and a rapid antigen test for differentiating influenza from coexistent infectious diseases during influenza epidemics. STUDY DESIGN: Data were collected during 3 consecutive influenza outbreaks over a 2-year period. The information collected included date of onset, symptoms, vaccine status, WBC and differential counts, ZstatFlu test (ZymeTx, Oklahoma City, Ok), and influenza culture. Using culture positivity as the criterion for influenza diagnosis, we compared cases with noncases on each variable independently and by logistic regression. Receiver operating characteristic curves were plotted for WBC count, ZstatFlu, and their combination in an effort to determine the most useful diagnostic strategy. POPULATION: We included consecutive patients presenting to a family practice office with fever, cough, sore throat, myalgia, and/or headache during flu season. OUTCOMES MEASURED: The outcomes were sensitivity, specificity, and other measures of test accuracy. RESULTS: Culture-positive cases could not be reliably distinguished from those that were culture negative using symptoms or vaccination status. Both WBC count and ZstatFlu results discriminated fairly well, and their combination did somewhat better. Differential counts were not helpful. WBC counts above 8000 were associated with a low probability of influenza. The sensitivity and specificity of the ZstatFlu were 65% and 83%, respectively. CONCLUSIONS: Our data suggest that symptoms and vaccine status do not reliably identify patients with influenza. Use of WBC counts and the ZstatFlu test can be helpful. The sequence, combination, and criteria for use of these tests depend on tradeoffs between undertreatment of influenza cases and the overtreatment of noninfluenza cases, and the cost and benefit projections for individual patients.

Adult↗
PubMed2004

A targeted mutation of Nkd1 impairs mouse spermatogenesis.

Nkd1 is an antagonist of the canonical Wnt/beta-catenin signaling pathway. The EF-hand motif of Nkd1 is required for its inhibitory function. Early studies suggested that Nkd1 might play important roles in mouse embryonic development and tumorigenesis. We constructed Nkd1(-/-) mice whose Nkd1 protein lacked the EF-hand and was unable to inhibit Wnt/beta-catenin signaling. The homozygotes were viable and grew normally, but their fertility in males was reduced. In wild-type adult testes, Nkd1 mRNA was expressed more abundantly in the elongating spermatids than in the round spermatids. Lack of EF-hand caused reductions in the testis weight and sperm count by 30 and 60%, respectively. During testis development, Nkd1 mRNA expression started at the 25th day after birth, coincident with the onset of Wnt1 expression. Nuclear localization of beta-catenin increased in the elongating spermatids, suggesting that the mutant Nkd1 failed to inhibit the Wnt/beta-catenin pathway. These results suggest that deletion of the EF-hand from Nkd1 reduces the number of the elongating spermatids at haploid stage. In contrast, the mutant Nkd1 did not affect intestinal polyposis in Apc(Delta716) mice.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Nineteen additional unpredicted transcripts from human chromosome 21.

The identification of all human chromosome 21 (HC21) genes is a necessary step in understanding the molecular pathogenesis of trisomy 21 (Down syndrome). The first analysis of the sequence of 21q included 127 previously characterized genes and predicted an additional 98 novel anonymous genes. Recently we evaluated the quality of this annotation by characterizing a set of HC21 open reading frames (C21orfs) identified by mapping spliced expressed sequence tags (ESTs) and predicted genes (PREDs), identified only in silico. This study underscored the limitations of in silico-only gene prediction, as many PREDs were incorrectly predicted. To refine the HC21 annotation, we have developed a reliable algorithm to extract and stringently map sequences that contain bona fide 3' transcript ends to the genome. We then created a specific 21q graphical display allowing an integrated view of the data that incorporates new ESTs as well as features such as CpG islands, repeats, and gene predictions. Using these tools we identified 27 new putative genes. To validate these, we sequenced previously cloned cDNAs and carried out RT-PCR, 5'- and 3'-RACE procedures, and comparative mapping. These approaches substantiated 19 new transcripts, thus increasing the HC21 gene count by 9.5%. These transcripts were likely not previously identified because they are small and encode small proteins. We also identified four transcriptional units that are spliced but contain no obvious open reading frame. The HC21 data presented here further emphasize that current gene prediction algorithms miss a substantial number of transcripts that nevertheless can be identified using a combination of experimental approaches and multiple refined algorithms.

Chromosomes, Human, Pair 21↗
PubMed1998

Prevalence and genotypes of GB virus C/hepatitis G virus (GBV-C/HGV) and hepatitis C virus among patients infected with human immunodeficiency virus: evidence of GBV-C/HGV sexual transmission.

The development of new antiretroviral agents may improve survival of HIV-infected individuals, and therefore chronic viral hepatitis may become more relevant in these patients. The presence of GBV-C/HGV and hepatitis C virus (HCV) RNA were investigated by reverse transcriptase-nested polymerase chain reaction in plasma from 168 Spanish HIV-infected patients belonging to four different risk groups: intravenous drug users (IVDUs), hemophiliacs, homosexuals, and heterosexuals. GBV-C/HGV-RNA and HCV-RNA were detected in 18% and 43% of the patients, respectively. The prevalence of current infection with these viruses was notably high, 19% for GBV-C/HGV and 69% for HCV, among individuals with parenteral risk of infection (intravenous drug abusers and hemophiliacs), but sexual transmission with GBV-C/HGV was also suggested because 16.5% of patients with sexual risk, either homosexual or heterosexual, had GBV-C/HGV-RNA in plasma. Although investigation of GBV-C/HGV-RNA possibly underestimates the actual prevalence of infection with GBV-C/HGV, the above data suggest that sexual contact may play a relevant role in the spread of this virus. Phylogenetic analysis showed no evidence for clustering of NS3 sequences into different genotypes or subtypes of GBV-C/HGV.

Adolescent↗
PubMed1999

Drug-administration sequence does not change pharmacodynamics and kinetics of irinotecan and cisplatin.

In this study, 11 patients with solid tumors were randomized to receive irinotecan (CPT-11; 200 mg/m2) as a 90-min i.v. infusion, immediately followed by cisplatin (CDDP; 80 mg/m2) as a 3-h i.v. infusion in the first course and the reversed sequence in the second course or vice versa. No significant differences in any toxicity were observed between the treatment schedules (decrease in absolute neutrophil count, 74.7 +/- 18.3 versus 80.3 +/- 18.0%; P = 0.41). CPT-11 lactone clearance was similar to single agent data and not significantly different between study courses (60.4 +/- 17.1 versus 65.5 +/- 16.3 liter/h/m2; P = 0.66). The kinetic profiles of the major CPT-11 metabolites SN-38, SN-38 glucuronide, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidinolcarbonyloxycamptothecine (APC), and 7-ethyl-10-[4-N-(1-piperidino)-1-amino]carbonyloxycamptothecine (NPC) were also sequence independent (P > or = 0.20). In addition, CPT-11 had no influence on the clearance of nonprotein-bound CDDP (40.8 +/- 16.7 versus 50.3 +/- 18.6 liter/h/m2; P = 0.08) and the platinum DNA-adduct formation in peripheral leukocytes in either sequence (1.94 +/- 2.20 versus 2.42 +/- 1.62 pg Pt/microg DNA; P = 0.41). These data indicate that the toxicity of the combination CPT-11 and CDDP is schedule independent and that there is no mutual pharmacokinetic interaction.

Adult↗
PubMed1991

A pregnancy defect in the osteopetrotic (op/op) mouse demonstrates the requirement for CSF-1 in female fertility.

Correlative evidence suggests that maternal production of the mononuclear phagocyte growth factor colony stimulating factor-1 (CSF-1) regulates placental development. In order to study the role of CSF-1 in pregnancy the fertility of CSF-1-less osteopetrotic (op/op) mutant mice was investigated. Homozygous mutant crosses (op/op x op/op) were consistently infertile. As expected, op/op males were almost completely fertile when crossed with heterozygous females. Surprisingly, op/op females when mated to heterozygote males were fertile, although at a rate that was 46% of the rate for +/op females x op/op males. These data suggest that CSF-1 is required for pregnancy. However, a maternal CSF-1 source is not absolutely necessary in that pregnancies involving +/op fathers were partially rescued, suggesting that +/op fetuses and/or +/op seminal fluid provides CSF-1 or CSF-1-induced factors which compensate for the absence of maternally produced CSF-1. Despite the complete absence of CSF-1 in the uterus and placenta of op/op mice placental weights were normal, suggesting that proliferation of decidual cells and trophoblasts, both of which express the CSF-1 receptor, may not be solely regulated by CSF-1. Histochemical staining for F4/80 antigen was used to identify macrophages in the uterus and placenta. Uterine macrophages could not be detected in virgin op/op mice although they were abundant in +/op uteri. Interestingly, macrophages could be detected in op/op uteri as uncharacteristically rounded cells in early gestation, however, they were not maintained and no macrophages were apparent beyond Day 14 of pregnancy in op/op mice. Further studies in the osteopetrotic mouse will be useful in delineating those functions required for pregnancy that are regulated by CSF-1.

Animals↗
PubMed2006

Hyaluronic acid binding peptides prevent experimental staphylococcal wound infection.

Staphylococcus aureus is a major cause of surgical wound infections. The development of mechanisms of antimicrobial resistance by this and other bacterial pathogens has prompted the search for new approaches to treat infectious diseases. Hyaluronic acid binding peptides have been shown to modulate cellular trafficking during host responses and were assessed for their ability to treat and possibly prevent experimental surgical wound infections caused by S. aureus. Treatment with these peptides was highly efficacious in reducing the number of S. aureus cells at the wound site and ameliorated the inflammatory host response associated with these infections. These data suggest a novel approach for the treatment and prophylaxis of staphylococcal wound infections in the clinical setting.

Amino Acid Sequence↗
PubMed1995

Impaired survival of T cell receptor V gamma 3+ cells in interleukin-4 transgenic mice.

The mouse epidermis contains a network of Thy-1+ dendritic T cells. Most of these cells express a homogeneous T cell receptor (TCR) configuration (V gamma 3/V delta 1) with only negligible junctional diversity. Because fetal thymocytes are precursors of these dendritic epidermal T cells (DETC) and the addition of interleukin (IL)-4 to fetal thymic organ cultures causes an early arrest in thymopoiesis, we examined DETC development in transgenic (tg) mice expressing IL-4 under the control of major histocompatibility complex class I regulatory sequences. Immunohistologic examination of epidermal sheets and polymerase chain reaction analysis of total skin RNA from IL-4 tg mice failed to reveal TCR V gamma 3+ DETC and V gamma 3 mRNA, respectively. In contrast, the sizes of TCR gamma delta subpopulations in lymphoid organs were unchanged in these mice. Although the numbers and staining intensities of TCR V gamma 3+ thymocytes in early fetal (days 14-17) IL-4 tg mice were similar to those of littermate controls, we observed a preferential death of these cells in thymic organ cultures from IL-4 tg mice. We observed further that epidermal sheets prepared from 9-day-old mice whose mothers had been treated with an IL-4-neutralizing antibody from day 12 to day 18 of pregnancy contained DETC numbers similar to those of controls. However, upon termination of the anti-IL-4 treatment, DETC ceased to expand. We conclude that IL-4 impairs the survival of TCR V gamma 3+ cells.

Animals↗
PubMed1996

The expansion and selection of T cell receptor alpha beta intestinal intraepithelial T cell clones.

In conventional mice, the T cell receptor (TCR) alpha beta+ CD8 alpha alpha+ and CD8 alpha beta+ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Ep. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8 alpha alpha and CD8 alpha beta TCR alpha beta+ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCR alpha beta+ T cells than conventional mice. The TCR beta repertoire of the CD8 alpha alpha and the CD8 alpha beta IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show the intestinal microflora is not responsible for the repertoire oligoclonality of TCR alpha beta+ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8 alpha alpha and the CD8 alpha beta TCR alpha beta IEL clones have a heterogeneous size pattern, with clones containing from 3 x 10(3) cells up to 1.2 x 10(6) cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCR alpha beta IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.

Animals↗
PubMed1993

Potential for gene transfer from recombinant Escherichia coli K-12 used in bovine somatotropin production to indigenous bacteria in river water.

This study examined the transfer of the plasmid pBGH1, an expression vector for bovine somatotropin (BST), from Escherichia coli K-12 strain W3110G [pBGH1] to indigenous microorganisms present in flasks containing Missouri River water. Strain LBB269 is a nalidixic acid-resistant derivative of W3110G which was used as a plasmid-free control strain in these studies. Water samples were inoculated with strains W3110G [pBGH1] and LBB269; after 21 days of incubation the number of viable colony-forming units (CFU) of W3110G [pBGH1] and LBB269 were reduced from an initial level of about 1 x 10(7) CFU per ml to less than 1 CFU per 100 ml. At this time indigenous microbes resistant to both ampicillin and tetracycline (the antibiotic resistance markers on pBGH1) were isolated from 100 ml of water from each of the flasks inoculated with either strain W3110G [pBGH1] or LBB269. Plasmid DNA was isolated from these organisms and examined for sequences containing the gene for BST from pBGH1, using a polymerase chain reaction (PCR) assay. As expected, the day 0 sample from the flask inoculated with E. coli K-12 strain W3110G [pBGH1] gave a positive PCR response and the day 0 sample from the flask inoculated with E. coli K-12 strain LBB269 gave a negative PCR response. All of the day 21 samples containing indigenous microbes isolated from flasks that were inoculated with either W3110G [pBGH1] or LBB269 were negative in the PCR assay, indicating that the target sequence from pBGH1 was not present in any of these indigenous microorganisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Hypomethylation of DNA: a nongenotoxic mechanism involved in tumor promotion.

There is an abundant amount of information on the mechanisms of action of genotoxic chemicals that act as carcinogens and the role that mutations play in carcinogenesis. However, carcinogenesis is more than mutagenesis and many carcinogens are not mutagens. Thus, there is a need to consider nongenotoxic mechanisms that may be involved in carcinogenesis. In this paper, we review our working hypothesis that hypomethylation of DNA is an epigenetic, nongenotoxic mechanism that plays a role in tumor promotion by facilitating aberrant gene expression. The utility of employing experimental models that focus on relevant comparisons between sensitive and resistant strains of mice is emphasized. Additionally, aspects of DNA methylation in rodents and humans are compared and contrasted. We discuss hypomethylation of DNA as a secondary mechanism, that is expected to be threshold-exhibiting, and conclude by describing how this information may facilitate a rational approach towards risk assessment when dealing with nongenotoxic compounds that are carcinogenic in a bioassay.

Animals↗
PubMed1994

High sensitive DNA typing approaches for the analysis of forensic evidence: comparison of nested variable number of tandem repeats (VNTR) amplification and a short tandem repeats (STR) polymorphism.

The approach of using nested primers for the APO B variable number of tandem repeats (VNTR) increases the sensitivity of the polymerase chain reaction (PCR) to single cell level. Different experiments and a comparison to the short tandem repeats (STR) system VWA were carried out, to determine the applicability of this method to forensic samples. Nested amplification of the Apo B VNTR was affected by a strong tendency towards preferential amplification of the shorter alleles. This phenomenon was observed for DNA quantities as low as 100 pg and impaired, depending on the allele length, the results for mixed samples. As expected, VWA polymorphism showed less preferential amplification. The high sensitivity of both PCR systems is accompanied by an increased susceptibility to contamination. Using artificially contaminated bloodstains, the bloodstain genotype, the contamination or both genotypes could be found on one piece of evidence. Here a single analysis can lead to an incorrect result. Therefore a strategy for obtaining reliable results should consist of multiple stain extractions and the amplification of different stepped dilutions of the DNA solution.

Base Sequence↗
PubMed2005

Dynamics of indigenous yeast populations during spontaneous fermentation of wines from Mendoza, Argentina.

Fermentation of wine is a complex microbial reaction, which involves the sequential development of various species of yeasts and lactic acid bacteria. Of these, yeasts are the main group responsible for alcoholic fermentation. The aim of this work was to study, under industrial conditions, the evolution of yeast populations and to describe the individual evolution of the most important yeasts during three spontaneous fermentations of Malbec musts from Argentina. This work shows the significant participation of non-Saccharomyces yeasts during spontaneous fermentation of musts, with the ubiquitous presence of three main species: Kloeckera apiculata, Candida stellata and Metschnikowia pulcherrima.

Colony Count, Microbial↗
PubMed2005

Enumeration and characterization of culturable arsenate resistant bacteria in a large estuary.

Arsenic is a toxic element that exists in two major inorganic forms, arsenate and arsenite. A number of bacteria have been shown to resist arsenic exposure, and even more bacteria appear to possess the genes for arsenic resistance. In this study, the numbers of culturable arsenate-resistant bacteria present in water at three coastal sites in the Lake Pontchartrain estuary, Louisiana, was determined. Despite insignificant (less than 1.33 microM) levels of arsenic in this system, 20-50% of the viable count of bacteria showed appreciable arsenate resistance, suggesting that arsenic-resistant bacteria are common and widespread. A diverse array of arsenate-resistant isolates was obtained, with 16S rRNA sequence analysis indicating 37 different bacterial strains, representing six major bacterial groups. Many of these isolates were affiliated with groups of bacteria that have been poorly characterized in terms of arsenic resistance, such as the Betaproteobacteria or Flavobacteria. Some isolates were capable of tolerating very high (> 100 mM) levels of arsenate, although arsenite resistance was generally much lower. The results suggest that arsenic-resistant bacteria are common, even in environments with insignificant arsenic contamination, and that many different groups of aquatic bacteria show appreciable arsenic resistance.

Arsenates↗
PubMed2005

Macrolide and lincosamide resistance genes of environmental streptococci from bovine milk.

Environmental streptococcus isolates from bovine milk were identified to the species and strain level and screened for resistance to macrolide and lincosamide antibiotics by phenotypic and genotypic methods. Isolates were tested for resistance to erythromycin and pirlimycin by broth microdilution assays. Presence of ribosomal methylase genes (ermA, ermB, ermC) and efflux pump genes (mefA/E, msrA/C) was detected by polymerase chain reaction (PCR). Resistance to pirlimycin (minimum inhibitory concentration (MIC) = 8microg/ml) was detected in 6 of 13 Enterococcus isolates that were identified as E. faecium by API20Strep typing. msrC was detected in 10 enterococcal isolates but the detection of msrC was not associated with phenotypic resistance. msrC negative isolates were reclassified as Enterococcus mundtii based on sequencing of housekeeping genes. Resistance to erythromycin and pirlimycin (MIC > 16microg/ml) was detected in 4 of 4 Streptococcus dysgalactiae and 12 of 20 Streptococcus uberis isolates and was encoded by ermB. All Streptococcus isolates tested negative for ermA, ermC, mefA/E and msrA/C. Among ermB positive streptococci, three alleles were identified based on a 527 bp gene fragment. Each allele was detected in at least two herds. The same alleles have also been detected in other bacterial species from bovine and non-bovine hosts and farm soil, suggesting a theoretical potential for horizontal transfer of macrolide resistance genes on dairy farms.

Alleles↗
PubMedDate not supplied

Variation in virological parameters and antibody responses in macaques after atraumatic vaginal exposure to a pathogenic primary isolate of SIVmac251.

We developed an animal model for the male-to-female transmission of human immunodeficiency virus, consisting of an atraumatic vaginal application of simian immunodeficiency virus onto the intact vaginal mucosa of cynomolgus macaques. Different doses of a pathogenic isolate of SIVmac251, with or without seminal plasma, were infused into the vaginas of female macaques. Infection of macaques could be achieved after a single exposure to the virus. Two patterns of infection were underscored with no relation to the virus dose inoculated: in 50% of the monkeys, SIV was persistently recovered and a strong antibody response to SIV was evidenced in blood and vaginal secretions. In the other infected animals, SIV infection was only transiently evidenced and a weak systemic antibody response was detected. It appeared that the presence of seminal plasma may be implicated in this variability only when low doses of virus are inoculated. Sequence analysis of the env gene of SIV revealed that most of the persistently viraemic animals were infected with a viral variant different from that of transiently viraemic macaques.

Amino Acid Sequence↗
PubMedDate not supplied

[Synthesis and antibacterial activity of analogues of the N-terminal fragment of the sarcotoxin IA antimicrobial peptide].

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1-18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.

Amino Acid Sequence↗