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PubMed2004

Qualitative multiplex RT-PCR for simultaneous detection of hepatitis C virus and human immunodeficiency virus in plasma samples.

This report describes the development of a one-tube multiplex reverse transcriptase (RT)-PCR assay for the simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) in plasma samples. The assay was evaluated with two panels of HCV- and HIV-1-positive samples, as well as negative plasma specimens. Extraction and amplification of HCV and HIV-1 RNA from plasma samples were performed in a single reaction, and amplified genomes were detected with specific probes. Serial dilutions of the HCV and HIV-1 first World Health Organization International Standards were used to evaluate the sensitivity of the method. Two RNA controls were constructed to determine inter-assay variations and the sensitivity of the amplification step. The assay had good specificity and detected all the genotypes and subtypes tested. The analytical sensitivity of the entire assay was 100 IU/mL for HCV and 200 IU/mL for HIV-1, while the amplification step detected ten copies/reaction for HCV and 20 copies/reaction for HIV-1. The multiplex assay allowed the simultaneous extraction, amplification and detection of two virus genomes, thereby providing an important practical advantage and an efficient approach for analysing individual and pooled plasma donations.

Feasibility Studies↗
PubMed1995

Human pulp response after partial pulpotomy with two calcium hydroxide products.

Twenty human permanent teeth were used to study the pulpal response of two calcium hydroxide products, Dycal and Pulpdent Multi-Cal, after partial pulpotomy. Teeth were extracted at 4 months, fixed, and prepared for histologic examination. All 10 teeth treated with Dycal showed complete soft tissue healing and bridge formation. No stained bacteria were seen throughout the serial sections. One tooth treated with Dycal showed acceptable histologic results, dentin deposition in the root canal. Six cases dressed with Pulpdent Multi-Cal showed acceptable histologic results, whereas four teeth showed severe inflammation or necrosis associated with bacterial penetration into the pulp tissue. Clinically, one tooth treated with Pulpdent Multi-Cal showed pulpal pain and was extracted at 90 days. Our data support the thesis that human permanent pulps will promote tissue healing and dentin bridge formation as long as bacterial microleakage is excluded.

Bacteria↗
PubMed1954

The binding of penicillin in relation to its cytotoxic action. II. The reactivity with penicillin of resistant variants of streptococci, pneumococci, and staphylococci.

1. In a previous study, the differing sensitivity of bacterial strains as they occur in nature appeared to be correlated with their correspondingly differing reactivity with penicillin. Presumably, the over-all reactivity of the cell with penicillin paralleled that of the vulnerable cell component(s). However, when penicillin-resistant variants of these strains (Streptococcus pyogenes, Micrococcus pyogenes, Diplococcus pneumoniae, and Streptococcus faecalis) were produced by serial passage through increasing concentrations of antibiotic, this correlation between resistance and the ability of the cell to bind penicillin was no longer apparent. Some resistant variants bound more penicillin than the parent, sensitive cell (Streptococcus faecalis, Micrococcus pyogenes), some were unchanged in their reactivity (Diplococcus pneumoniae, Micrococcus pyogenes), and some bound less (Streptococcus pyogenes, Micrococcus pyogenes). One resistant variant of Micrococcus pyogenes at first showed enhanced reactivity with penicillin; on continued passage through antibiotic, there was a further increase in resistance, but now associated with a significantly decreased reactivity. In the case of Diplococcus pneumoniae, a resistant variant at first reacted normally with penicillin; on continued passage in antibiotic, its binding affinity for penicillin gradually decreased, but with no associated further increase in resistance. 2. The reactivity with penicillin of cell-free sonic extracts of the resistant variants paralleled that of the intact organisms. Permeability considerations therefore did not seem involved in the increased resistance produced by serial passage in antibiotic. 3. The penicillin-resistant variants did not have an enhanced capacity to degrade the free intracellular antibiotic. 4. Possible alternative explanations are discussed in the text.

Anti-Bacterial Agents↗
PubMed1987

Serum-free serial culture of adult human keratinocytes from suction-blister roof epidermis.

Coating cell culture flasks with natural extracellular matrix (ECM) enhanced the culture of adult human keratinocytes from suction-blister roof epidermis in an environment without fetal calf serum (FCS), bovine pituitary extracts or cellular feeder layers. A higher incidence of cell attachment on natural ECM was observed than on collagen and human fibronectins (HFN)-coated plastic dishes, and natural ECM was necessary for growth and proliferation of attached cells under the culture conditions used. Cells in primary culture grew to confluency on natural ECM-coated surfaces within about 14 days, and subsequent serial passage could be made up to fourth passage in collagen- and HFN-coated plastic flasks. Cultured keratinocytes in this serum-free environment formed colonies of small cuboidal, healthy cells with little keratinization or stratification and demonstrated antigenic characteristics of human basal cells.

Adolescent↗
PubMed1991

Kinetics of plasma fibronectin: increased lung tissue incorporation after postoperative bacteremia.

Fibronectin is an adhesive glycoprotein that may influence the permeability of the vascular barrier. We compared the plasma disappearance of purified human fibronectin (hFn) and its incorporation into lung tissue in control nonbacteremic and bacteremic sheep after surgery to determine the influence of postoperative bacteremia on the plasma clearance and lung deposition of hFn. Lymph fistulas were surgically prepared 48 h before either saline or bacterial challenge to allow for collection of timed samples of plasma, lung lymph, and peripheral lymph. On the day of the study, the sheep were anesthetized and given either 5 X 10(8) Pseudomonas aeruginosa in saline or saline alone. In parallel, they were infused intravenously with 100 mg of hFn, and the hFn concentration in plasma, lymph, and tissue extracts was subsequently determined by enzyme-linked immunosorbent assay over an 8-h experimental interval. Localization of endogenous sheep fibronectin (sFn) and the injected hFn in serial lung tissue samples was determined by dual-label immunofluorescence. In nonbacteremic control sheep, plasma hFn levels declined with an initial rapid slope (t1/2 = 0.53 +/- 0.19 min), followed by a second, gradual slope (t1/2 = 21.7 +/- 2.5 h), whereas the concentration of hFn in lung lymph and peripheral lymph rose exponentially with time. In bacteremic sheep, the early plasma disappearance of hFn was similar, but the second phase of plasma clearance was faster (t1/2 = 7.4 +/- 0.3 h). Lung tissue from control and bacteremic sheep contained the same level of extractable hFn. However, tissue extraction followed by immunofluorescence microscopy indicated that lung tissue from bacteremic sheep contained more nonextractable hFn than lung tissue from nonbacteremic sheep. Thus postoperative bacteremia that elicits acute lung vascular injury will increase the plasma disappearance of hFn and its incorporation into the lung tissue.

Animals↗
PubMed1985

Enzyme-antigen immunoassay for human placental alkaline phosphatase in serum and tissue extracts, and its application as a tumor marker.

In this enzyme-antigen immunoassay for human placental alkaline phosphatase (hPLAP; EC 3.1.3.1.) in serum and tissue extracts, polyclonal rabbit antiserum to mouse IgG2b is adsorbed to the wells of a microtiter plate, its excess binding sites are blocked, then it is incubated with murine monoclonal anti-hPLAP and mixed with serially diluted standard or sample antigen. The amount of antigen bound is determined by measuring its enzymic activity. The standard curve is linear for hPLAP concentrations of 0.2 to 1 U/L. The mean within-assay CV was 3.8% (SD 0.9%) for a serum sample and 6.1% (SD 3.0%) for a tissue extract. The respective mean between-assay CVs were: 6.7% (SD 2.0%), and 7.0% (SD 2.0%). Serum hPLAP concentrations, determined in four different dilutions, had a CV of 5.5%. We evaluated the method by standard additions and by comparing dilution curves for purified hPLAP, hPLAP in serum, and hPLAP in tissue extracts. The upper limit of activity in normal subjects was 0.1 U/L for serum samples, and 1.0 mU/g wet weight of tissue for tissue extracts. hPLAP activity was increased in 9.8% of all cancer patients, and in 40% of ovarian cancer patients. Almost half of the tumor biopsies were positive for hPLAP activity, and 94% of the biopsies from ovarian neoplasia had an increased activity of this isoenzyme. Of the nonmalignant tissues examined, normal lung tissue had the highest hPLAP activity.

Alkaline Phosphatase↗
PubMed1987

Occupational asthma from inhaled egg protein.

We evaluated workers and performed an industrial hygiene assessment at a plant where raw eggs are processed into powdered egg yolk and whole egg. Egg dust levels in the packaging room straddled the American Conference of Governmental Industrial Hygienists' (ACGIH) exposure guideline of 10 mg/m3 for nuisance dust. We obtained medical histories from 25 workers, and performed physical examinations, spirometry, and serial determinations of peak expiratory flow rate (PEFR) by portable meter every 3 hrs (while awake) for 7 days. We defined symptomatic bronchial lability to be a decrement in PEFR on any one day of 20% or more of the day's maximum, with concurrent symptoms. Skin-prick tests and serum assays for specific IgE by the radioallergosorbent (RAST) method were performed to assess sensitivity to commercial egg proteins, egg protein fractions, and freshly prepared extracts of whole egg powder and yolk. We classified participants as definite cases of asthma if both the examining physician diagnosed asthma and symptomatic bronchial lability was demonstrated by serial PEFR determinations. Definite noncases of asthma were those participants in whom the physician did not diagnose asthma and in whom symptomatic bronchial lability was not demonstrated by PEFR. All five definite cases, compared to three of 16 definite noncases of asthma, had one or more positive skin-prick tests to egg proteins. Four of five cases, compared to 0 of 14 noncases, who had serum determinations, had an elevated RAST to one or more of the egg proteins. This study demonstrates that occupational asthma associated with IgE-mediated allergy to egg proteins occurs among workers exposed to inhaled egg proteins.

Air Pollutants, Occupational↗
PubMed1980

The long-term period measurement, an instrumental method in phoniatry.

With the help of an Analog to Digital Converter we are able to perform serial and statistical analysis in the time domain. The difficulty in those long-term period measurements consists in the correct extraction of the periodicity in human voice. A simple method is the electroglottography, which, however, is not applicable in all cases. The analysis of the acoustic wave (translated via microphone) demands a lot of electronics if to be precise. Our investigation consists of a compromise between precision and amount of instrumentation, and we only took one parameter as a criterion. The possible error is negligible. We present the results of this measurement type using the patterns of one normal voice and eight different kinds of voice disorders.

Adolescent↗
PubMed1994

Antimicrobial activity of Ganoderma lucidum extract alone and in combination with some antibiotics.

Antimicrobial activity of GL (the aqueous extract from the carpophores of Ganoderma lucidum (FR)KARST) was tested in vitro against Gram positive and Gram negative bacteria by serial broth dilution method, and the antimicrobial activity was expressed by minimal inhibitory concentration (MIC). Among fifteen species of bacteria tested, the antimicrobial activity of GL was the most potent against Micrococcus luteus (MIC, 0.75 mg/ml). To investigate the effects of antimicrobial combinations of GL with four kinds of antibiotics (ampicillin, cefazolin, oxytetracycline and chloramphenicol), the fractional inhibitory concentration index (FICI) was determined by checkerboard assay for each strain. The antimicrobial combinations of GL with four antibiotics resulted in additive effect in most instances, synergism in two instances, and antagonism in two instances. Synergism was observed when GL was combined with cefazolin against Bacillus subtilis and Klebsiella oxytoca.

Anti-Bacterial Agents↗
PubMed1989

Infectivity of vesicles prepared from chilo iridescent virus inner membrane: evidence for recombination between associated DNA fragments.

Treatment of CIV particles with octylglucoside at high ionic strength leads to the solubilization of the inner viral membrane. Incubation of permissive cells (Cf124 cells) with vesicles obtained after dialysis of the detergent shows that this fraction is infectious. This infectivity, which is very low, could only be detected after two serial passages on permissive cells. This phenomenon is, however, reproducible. Isopycnic centrifugation analysis shows that some DNA cosediments with the vesicles. Extraction and purification of this DNA confirm the presence of a large DNA fragment of about 50.10(6) Da. Digestion with restriction endonucleases demonstrated that this DNA did not correspond to a particular fragment but to a population of DNA fragments of homogeneous size arising from various regions of the viral genome. Purified viral DNA was not infectious, the presence of DNA in the vesicles could not account therefore for their infectivity. Experiments of non-genetic reactivation of purified CIV DNA by UV-irradiated virus suggest that one (or several) structural component(s) of CIV particles must be involved in the first stages of the viral replication cycle. In addition, transfection of cells with large overlapping DNA fragments could generate infectious particles when the cells were superinfected with UV-irradiated virus. It can be supposed that the vesicle suspensions, which probably contain the reactivating factor, are composed of a population of vesicles which are all different in their DNA content. Infectivity of such suspensions would be the consequence of a recombination between large overlapping DNA fragments.

Aedes↗
PubMed2005

Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination.

We report a single, one-step PCR approach for detection and discrimination of Clonorchis sinensis and Opisthorchis viverrini in different life-stage forms (adults, metacercariae, and eggs) from fish intermediate hosts and from infected patients. Primers designed for species-specific PCR, amplifying portions of the mitochondrial (mt) genome, were also suitable for a multiplex PCR. The latter was a single, one-step reaction under high stringency conditions, using simultaneously 2 pairs of primers (1 pair for C. sinensis--product size 612 bp, and 1 pair for O. viverrini--product size 1357 bp). Assays using serially diluted templates demonstrated that as little as 0.78 ng of genomic DNA of either species could yield amplicons. Genomic DNA extracted from different life-stage forms including adult worms (of both species), eggs (of O. viverrini), eggs possibly of several trematode species (collected from patients infected with C. sinensis in Vietnam) and mixed metacercariae of common trematodes (collected from fishes in the C. sinensis endemic areas), yielded specific bands of the correct size and their identity was confirmed by sequence analysis. The multiplex PCR approach described here proved to be a species-specific, sensitive and fast tool for accurate diagnosis of clonorchiasis and/or opisthorchiasis, permitting the detection of their metacercariae in infected fishes or adult/eggs from patients in endemic areas.

Animals↗
PubMed1984

Grass pollen immunotherapy: a single year double-blind, placebo-controlled study in patients with grass pollen-induced asthma and rhinitis.

Fifteen grass pollen--sensitive asthmatic patients were selected from 200 patients with grass pollenosis on the basis of positive SPTs and RASTs that were restricted to grass pollens (except Bermuda grass), no previous IT, and residence and occupation in an area monitored by serial pollen counts. They underwent a double-blind trial of specific IT with a mixture of three grass pollen--aqueous extracts (velvet, sweet vernal, and timothy) or placebo. After 10 mo, the mean maintenance dose of pollen extract (assayed by RAST inhibition) in eight actively treated patients was 6000 RAST units (range 3000 to 8000) and the mean total dose was 18,700 RAST units (range 10,200 to 30,000). Results were assessment done by the following clinical and immunological data: (1) during the pollen season, daily symptom scores; (2) PD 20% FEV1, IgE antibody to timothy by RAST in serum and in nasal secretions, serum IgG antibody to purified timothy allergen D by solid-phase radioimmunoassay, and the four IgG subclass antibodies by enzyme immunoassay were all measured before treatment and before and after the pollen season. Symptom scores of both treated patients and controls correlated with pollen counts (R = 0.88, p less than 0.05 and R = 0.71, p less than 0.05, respectively). There was a significant difference between the mean symptom score values of treated patients versus controls (Kruskal-Wallis test, p less than 0.001). No significant differences or changes either in the PD 20% FEV1 or IgE antibody to timothy in serum and nasal secretions were found in the two groups before or after IT.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2002

Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay.

The availability of molecular protocols for the detection and quantitation of very low numbers of hepatitis C virus (HCV) particles in biological samples is an issue of interest in both clinical and analytical fields of HCV research. A sensitive and reproducible assay is described for HCV RNA quantitation using the TaqMan PCR fluorogenic real-time detection system to establish the levels of HCV RNA in chimpanzee plasma. Our TaqMan PCR protocol and synthetic full length HCV RNA template show that the threshold of sensitivity for our TaqMan PCR is two copies per reaction. As few as 10 genome copies per reaction could be quantitated maintaining a linear range. The accuracy of the TaqMan PCR test was comparable to commercial bDNA and Amplicor tests. The RNA standards of the laboratory were tested in parallel with a World Health Organization (WHO) International Standard for HCV RNA obtaining ratios of 2.7+/-0.7 RNA copies per HCV international unit (IU). Our method using RNA extracted from chimpanzee samples had an estimated sensitivity of 200 RNA copies/ml of plasma (approximately eight copies/reaction or 74 WHO IU/ml). Serial plasma samples from HCV-infected chimpanzees were analyzed using this methodology to evaluate its applicability, and RNA profiles were observed consistent with the evolution of the pathology in each animal. The present study therefore illustrates the high reproducibility, sensitivity and reliability of our TaqMan methodology, providing a useful method for HCV research to consistently detect and quantify viral RNA throughout a range of concentrations.

Animals↗
PubMed2000

Retinoic acid-induced apoptotic pathway in T-cell lymphoma: Identification of four groups of genes with differential biological functions.

Retinoic acid (RA) has been used to induce the regression of refractory T-cell lymphoma. In vitro and in vivo studies have shown that RA exerts this effect through the induction of apoptosis. This study was designed to investigate the molecular pathway of RA-induced apoptosis in T-lymphoma cell lines.RA-induced apoptosis was verified by morphology, flow cytometry, and DNA ladder analysis. Differential display method using a combination of 12 poly(A)-anchored primers and 20 arbitrary primers was adopted for gene cloning. Total RNAs were extracted from H9 cell line at 0, 6, 12, and 24 hours after All-trans RA (ATRA) treatment and the serial expression patterns of the candidate fragments were recognized. The cloned gene fragments were then analyzed and confirmed by Northern blot analysis on H9 and SR786 cell lines.ATRA-induced apoptosis of T-cell lymphoma was protein synthesis-dependent. The execution or irreversible phase of apoptosis appeared to occur at 6-12 hours of RA treatment. Among the 60,000 arbitrarily displayed bands, 25 of 250 candidate fragments were selected for further cloning and sequencing. A total of 14 clones could be matched to known genes and were categorized into four groups: A) transcription factors: prothymosin, CA150, p78 serine/threonine kinase, IL-1beta-stimulating gene, glucocorticoid receptor, MLN64/CAB1, gastrin-binding protein, and polypeptide from glioblastoma; B) chaperone: 90 kDa heat shock protein; C) ion channel: chloride channel protein 3; and D) cytoskeleton: cytovillin2/ezrin and vimentin. Another two clones of genes were of unrecognized functions. The remaining 11 clones belonged to unmatched or novel genes. The expression of these genes varied, either upregulated or downregulated, in response to ATRA treatment.RA-induced apoptosis may involve a cascade of genes that are related to transcription regulation, stress response, housekeeping, and the execution of apoptosis. The clarification of the RA-induced apoptotic pathway will help us to understand the molecular mechanism of cancer differentiation agents.

Apoptosis↗
PubMed2001

High-performance liquid chromatography assay for simultaneous determination of dextromethorphan and its main metabolites in urine and in microsomal preparations.

An HPLC method has been developed and validated for the determination of dextromethorphan, dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in urine samples. Deconjugated compounds were extracted on silica cartridges using dichloromethane/hexane (95:05, v/v) as an eluent. Chromatographic separation was accomplished on a Phenyl analytical column serially connected with a Nitrile analytical column. The mobile phase consisted of a mixture of an aqueous solution, containing 1.5% acetic acid and 0.1% triethylamine, and acetonitrile (75:25, v/v). Compounds were monitored using a fluorescence detector. Calibration curves were linear over the range investigated (0.2-8.0 microM) with correlation coefficients >0.999. The method was reproducible and precise. Coefficients of variation and deviations from nominal values were both below 10%. For all the analytes, recoveries exceeded 77% and the limits of detection were 0.01 microM. The validated assay proved to be suitable for the determination of DEM metabolic indexes reported to reflect the enzymatic activity of the cytochrome P450s, CYP2D6 and CYP3A, both in vivo, when applied to urine samples from patients, and in vitro, when applied to samples from the incubation of liver microsomes with dextromethorphan.

Aryl Hydrocarbon Hydroxylases↗
PubMed2003

Pharmacokinetics of all-trans retinoyl beta-glucuronide in rats following intraperitoneal and oral administration.

The purpose of this study was to examine the pharmacokinetics of a single dose (6.3 mumol, 3 mg) of all-trans retinoyl beta-glucuronide (RAG), when given either orally in corn oil or by intraperitoneal (i.p.) injection in dimethylsulfoxide (DMSO) to adult Sprague-Dawley rats. Following dosing, serial blood samples were collected at various times up to 48 hours from each rat via saphenous vein puncture. Retinoids were extracted from plasma samples and analyzed by high-performance liquid chromatography. In the plasma of i.p.-dosed rats (n = 6), a derivative of RAG, tentatively identified as the lactone of RAG (RAGL), was the major product found. RAGL persisted in the plasma for up to 48 hours. Much smaller concentrations of RAG and of retinoic acid (RA) were also present in the plasma at two to four hours, but generally not thereafter. In orally dosed rats (n = 6), neither RAG nor its products, except for occasional traces of the lactone, were detected. Plasma retinol levels decreased in both i.p.-injected and orally treated rats, the decrease being significant in orally dosed rats.

Administration, Oral↗
PubMed1980

Pregnancy-related changes in rat cervical glycosaminoglycans.

Non-pregnant and pregnant rats of known gestational age were killed at intervals and their uterine cervices were excised and digested with papain. Glycosaminoglycans thus extracted were separated by cellulose acetate electrophoresis and stained with Alcian Blue. Glycosaminoglycans were identified by comparison with standards and by serial degradation with chondroitin ABC lyase, butyl nitrite and leech hyaluronidase. Dermatan sulphate, hyaluronic acid and heparan sulphate were identified and quantitative determined by densitometry. The overall concentration of glycosaminoglycans changed little during pregnancy. A 3-fold total increase in uronic acid paralleled the increase in cervical weight. Hyaluronate content, however, increased 17-fold, and rose from 6% of total glycosaminoglycans in the non-pregnant state to 33% at term. Furthermore, the ratio of hyaluronate to hydroxyproline increased 10-fold. These changes are consistent with an accumulation of hyaluronate in the interstices between collagen fibres, resulting in the softening of this tissue that is seen in late pregnancy.

Animals↗
PubMed1987

Type VI collagen of the intervertebral disc. Biochemical and electron-microscopic characterization of the native protein.

The collagen framework of the intervertebral disc contains two major fibril-forming collagens, types I and II. Smaller amounts of other types of collagen are also present. On examination of the nature and distribution of these minor collagens within bovine disc tissue, type VI collagen was found to be unusually abundant. It accounted for about 20% of the total collagen in calf nucleus pulposus, and about 5% in the annulus fibrosus. It was discovered by serially digesting disc tissue with chondroitin ABC lyase and Streptomyces hyaluronidase that native covalent polymers of type VI collagen could be extracted. Electron micrographs of this material prepared by rotary shadowing revealed the characteristic dimensions of tetramers and double tetramers of type VI molecules, with their central rods and terminal globular domains. Molecular-sieve column chromatography on agarose under non-reducing non-denaturing conditions gave a series of protein peaks with molecular sizes equivalent to the tetramer, double tetramer and higher multimers. On SDS/polyacrylamide-gel electrophoresis after disulphide cleavage, these fractions of type VI collagen all showed a main band at Mr 140,000 and four lesser bands between Mr 180,000 and 240,000. On electrophoresis without disulphide cleavage in agarose/2.4% polyacrylamide only dimeric (six chains) and tetrameric (12 chains) forms of type VI molecules were present. The ability to extract all the type VI collagen of the tissue in 4 M-guanidinium chloride, and absence of aldehyde-mediated cross-linking residues on direct analysis, showed that, in contrast with most matrix collagens, type VI collagen does not function as a covalently cross-linked structural polymer.

Animals↗