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PubMed2004

Use of in vitro cytoadherence assays in the comparative study of the virulence of isolates of Trichomonas vaginalis.

This study describes the use of bromo-deoxiuridine, a non-radioactive analogue of thymidine, to determine the adhesion of 40 T. vaginalis isolates, as compared with the clinical manifestations found in patients. Parasite-labeling was optimized and label detection by immunoassay was carried out. Bromo-deoxiuridine at 10 microM for 16 h produced the highest sensitivity. Once optimized, the assay was able to detect between 3.12 x 10(3) and 4 x 10(5) parasites, with a detection limit of 9.14 x 10(2), which is lower than with the use of [3H]-thymidine. The variation coefficients were 2.65% for repeatability and 3.8% for reproducibility. A correlation coefficient of 0.97 was found between the pattern curves obtained by both labeling procedures. The level of adhesion to HeLa cells was directly proportional to the severity of the clinical manifestations (P < 0.05).

Adolescent↗
PubMed2001

An experimental model of gastroschisis using fetoendoscopy: preliminary results and technical considerations.

BACKGROUND: The aim of this study was to show the feasibility of an experimental model of gastroschisis using fetoendoscopic surgery on sheep fetuses, and also to perform amnioinfusion until delivery using an in-dwelling intraamniotic catheter. METHODS: We analyzed the data from 18 pregnant ewes having 26 fetuses, which underwent surgery at 80 days of gestation (full term, 145 days). The fetendo technique was used to create a gastroschisis in 15 fetuses. The fetal abdominal wall was opened on the left side of the cord using scissors. The omentum and the intestinal loops were eviscerated using atraumatic forceps. Eleven fetuses were used as a control group. Twenty-one fetuses underwent amnioinfusion; a simple exteriorized catheter was used in seven cases and an intraamniotic catheter with an implantable port was used in the other 14. All ewes and fetuses were killed at the end of the experiment by an intravenous injection of pentotal; thereafter, the fetuses underwent necropsy. RESULTS: Twelve fetuses died and 14 survived (53.8%); seven of the 15 that underwent gastroschisis survived (46.7%). An amniotic infection occurred in nine fetuses (34.6%); of these, six died and three that were administered antibiotics survived. CONCLUSIONS: Our experience shows that this experimental model of gastroschisis is feasible and reproducible, and that a repeated amnioinfusion can be performed with an in-dwelling catheter in pregnant ewes. The use of an implantable port is safer than a simple exteriorized catheter.

Animals↗
PubMed1987

Protein kinase C: regulation of serotonin release from rat brain cortical slices.

The effects of phorbol esters on serotonin release were examined in an attempt to investigate the role of protein kinase C in the regulation of serotonin release. Rat brain parietal cortical slices were incubated with [3H]5-HT in the presence of pargyline in order to label the serotonin stores. Potassium stimulated (30 s) release and spontaneous [3H]5-HT efflux were examined in slices during superfusion with Krebs-Ringer solution containing chlorimipramine. Repeated K+ stimulations elicited reproducible responses with release ratios of approximately 1.0. Introduction of phorbol 12-myristate, 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu) 20 min prior to S2, or S3 resulted in dose-related increases in [3H]5-HT or [3H]NE release. PMA was slightly more potent (93% increase) than PDBu in potentiating K+-stimulated [3H]5-HT release. Phorbol and 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) which do not activate protein kinase C did not alter serotonin release. In contrast, basal [3H]5-HT and [3H]NE release were altered to a far lesser extent which was not always dose related. The response to the phorbol esters was reversible, Ca2+-dependent and reached maximal effect after 20 min of superfusion. The putative protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibited K+-induced [3H]5-HT release significantly (11%) but did not alter basal efflux. The PMA facilitation of serotonin release was, however, markedly prevented by the enzyme inhibitor. The effect of PMA on release was found not to be directly mediated through the prejunctional serotonin autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Intraerythrocytic administration of a synthetic Plasmodium antigen elicits antibody response in mice, without carrier molecules or adjuvants.

The synthetic peptide (NANP)40, reproducing the tandem-repeated epitope of the circumsporozoite protein of Plasmodium (Laverania) falciparum, was entrapped into murine, autologous erythrocytes by a hypotonic dialysis method. Mice immunized intravenously with minute amounts of encapsulated peptide produced considerable antibody titres. This result indicates that intraerythrocytic antigen administration may have a potential as an immunization system for humans, since it dispenses with adjuvants and carrier molecules.

Animals↗
PubMed1988

Characterization of surgical needles.

The acuity (sharpness) of surgical needle points was assessed by measuring the force required for repeat needle penetrations through a medium-gauge latex sheet glued to a perforated Plexiglas frame. The data on the variation in the applied force with repeat penetrations showed that needles obeyed the general relation: P = A + B.n; where P is the applied penetration force in grams, n is the number of penetrations and A and B are constants. Constant A characterized the needle-point acuity and B the maintenance of acuity. This relationship indicated both needle acuity and acuity maintenance with repeated passes through a reproducible target material. Determining the microhardness of needles provided data on their strength which helped to account for differences in the acuity of apparently similar needles. The tensile strength of the union between suture and needle was determined to evaluate the security of suture attachment.

Latex↗
PubMed1990

Hygienic hand disinfection tests in three laboratories.

A comparative study was made in three laboratories of a test for hygienic hand disinfection. Staphylococcus aureus was applied to the fingertips of a total of 74 volunteers (49 female and 25 male) and the effect of washing with three chlorhexidine preparations and one non-medicated soap was assessed after one and five applications. Fingertip inoculation is convenient and is a realistic representation of the in-use situation. Although significant differences were obtained between log10 reductions in test organisms using the same formulation in different centres, and different periods in the same centre, the maximum differences after a single application of a preparation were small, e.g. between centres 0.39 and between periods in the same centre 0.55, and after multiple applications the maximum difference between centres was 0.42 and between periods in the same centre it was 0.51. The differences between preparations were similar in all centres. This test compares well with other similar tests and products can be placed in rank order of effectiveness. It is concluded that this test, if carried out under the controlled conditions described, is sufficiently reproducible between laboratories and repeatable within laboratories to be used as a standard test.

Chlorhexidine↗
PubMed1993

Developmental toxicity of orally administered thiabendazole in Sprague-Dawley rats and New Zealand white rabbits.

The developmental toxicity of thiabendazole (TBZ) was assessed in studies in New Zealand white rabbits and Sprague-Dawley rats. The doses of TBZ for these studies were based on decreases in maternal weight gain in dose range-finding studies in pregnant females of each species. In rabbits, TBZ was administered orally at doses ranging from 24 to 600 mg/kg body weight/day in two separate studies. In rats, TBZ was administered at doses of 10, 40 or 80 mg/kg body weight/day. In all studies, TBZ was administered daily by gavage in aqueous suspension on gestation days 6 to 17. In the initial rabbit study, weight loss occurred in the 600 mg/kg/day group and weight gain decreased in the 120 mg/kg/day group. In addition, there were four complete litter resorptions and four abortions in the 120 and 600 mg/kg/day groups, respectively. To determine whether these changes (known to occur spontaneously in rabbits) were reproducible, the study was repeated. In this study, decreased maternal weight gain and decreased foetal weights were found at 600 mg TBZ/kg/day, but there was no evidence of selective developmental toxicity. Similarly, in rats TBZ produced decreased maternal weight gain (12 to 26%) associated with slight (5 to 7%) decreases in foetal weights at doses of 30 and 80 mg/kg/day. No changes were found at 10 mg TBZ/kg/day and no evidence of selective developmental toxicity or teratogenicity was found at any dose. On the basis of these results, it is concluded that TBZ is not teratogenic or selectivity foetotoxic in rats or rabbits.

Abnormalities, Drug-Induced↗
PubMed1995

Simultaneous determination of all-trans and 13-cis retinoic acids and their 4-oxo metabolites by adsorption liquid chromatography after solid-phase extraction.

All-trans retinoic acid (all-trans RA), the active metabolite of vitamin A, has been demonstrated to be an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (APL), the AML3 subtype of the FAB cytological classification. Complete remission is obtained by inducing terminal granulocytic differentiation of the leukemic cells. To study all-trans RA pharmacokinetics in patients with APL, a rapid, precise and selective high-performance liquid chromatographic (HPLC) assay was developed. This method is easy and shows good repeatability (C.V. = 8.41-12.44%), reproducibility (C.V. = 9.19-14.73%), accuracy (C.V. = 3.5-11%) and sensitivity with a detection limit of 5 pmol/ml. The analysis is performed using normal-phase HPLC in an isocratic mode with UV detection after solid-phase extraction on octadecyl (C18) columns. The mobile phase is hexane-dichloromethane-dioxane (78:18:4, v/v) containing 1% acetic acid.

Adsorption↗
PubMed1992

Uptake and utilization of [beta-11C]5-hydroxytryptophan in human brain studied by positron emission tomography.

The immediate precursor in the serotonin synthetic route, 5-hydroxytryptophan (5-HTP), labeled with 11C in the beta position, has become available for studies using positron emission tomography (PET) to examine serotonin formation in human brain. Normalized uptake and intracerebral utilization of tracer amounts of [beta-11C]5-HTP were studied twice in six healthy male volunteers, three of them before and after pharmacological pretreatments. The kinetic model defines regional utilization as the relative regional radioactivity accumulation rate. Repeat studies showed good reproducibility. Pretreatments with benserazide, p-chlorophenylalanine (PCPA), and unlabeled 5-HTP all significantly increased uptake of [beta-11C]5-HTP. The utilization rates in both striatal and frontal cortex were higher than those in the surrounding brain, indicating that PET studies using [beta-11C]5-HTP as a ligand quantitate selective processes in the utilization of 5-HTP. We tentatively interpret uptake and utilization as a measure of brain serotonin turnover, the selectivity of which was shown by pharmacological interventions in vivo.

5-Hydroxytryptophan↗
PubMed2004

A novel method to construct a third-generation biosensor: self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) nanospheres.

A novel method for fabrication of horseradish peroxidase (HRP) biosensor has been developed by self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) (St-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in thiol-functionalized poly(St-co-AA) nanosphere latex prepared by emulsifier-free emulsion polymerization of St with AA and function with dithioglycol to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups. Finally, horseradish peroxidase was immobilized on the surface of the gold nanoparticles. The sensor displayed an excellent electrocatalytical response to reduction of H2O2 without the aid of an electron mediator. The sensor was highly sensitive to hydrogen peroxide with a detection limit of 4.0 micromoll(-1), and the linear range was from 10.0 micromoll(-1) to 7.0 mmoll(-1). The biosensor retained more than 97.8% of its original activity after 60 days of use. Moreover, the studied biosensor exhibited good current repeatability and good fabrication reproducibility.

Acrylic Resins↗
PubMed2006

A disposable on-chip phosphate sensor with planar cobalt microelectrodes on polymer substrate.

Disposable microsensors on polymer substrates consisting of fully integrated on-chip planar cobalt (Co) microelectrodes, Ag/AgCl reference electrodes, and microfluidic channels have been designed, fabricated, and characterized for phosphate concentration measurement in aqueous solution. The planar Co microelectrode shows phosphate-selective potential response over the range from 10(-5) to 10(-2)M in acidic medium (pH 5.0) for both inorganic (KH(2)PO(4)) and organic (adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphates (ADP)) phosphate compounds. This microfabricated sensor also demonstrates significant reproducibility with a small repeated sensing deviation (i.e. relative standard deviation (R.S.D.)<1%) on a single chip and a small chip-to-chip deviation (i.e. R.S.D.<2.5%). Specifically, while keeping the high selectivity, sensitivity, and stability of a conventional bulk Co-wire electrode, the proposed phosphate sensor yields advantages such as ease of use, cost effectiveness, reduced analyte consumption, and ease of integrating into disposable polymer lab-on-a-chip devices. The capability to sense both inorganic and organic phosphate compounds makes this sensor applicable in diverse areas such as environmental monitoring, soil extract analysis, and clinical diagnostics.

Biosensing Techniques↗
PubMed2005

Long-lasting synapse formation in cultured rat hippocampal neurons after repeated PKA activation.

Recently, we reported that the repeated activation of cyclic-AMP-dependent protein kinase (PKA) in the rat hippocampus under tissue culture conditions induced the enhancement of excitatory postsynaptic potential (EPSP), which lasted more than 2 weeks and was accompanied by the formation of morphologically identifiable synapses. Here we examined whether an equivalent synapse formation is induced in dissociated cell cultures of rat hippocampal neurons. Brief (15-min) application of Sp-cAMPS (a membrane-permeable analog of cyclic AMP) induced an increase in the number of synaptic sites (identified by the apposition of immunocytochemically labeled pre- and postsynaptic structures). There were two types of increase: a short-lasting one that lasted less than 24 h after a single application of Sp-cAMPS, and a long-lasting one that lasted more than 2 weeks after repeated applications. The long-lasting increase in synaptic sites was dependent on the time and interval of application and was suppressed by Rp-cAMPS (a PKA inhibitor). The synapses were judged to be active based on the endocytosis of FM1-43, a fluorescent dye. Electron microscopy confirmed the increase in the number of synaptic ultrastructures. The present results show that the synaptogenesis induced by repeated PKA activation is reproducible in a neuronal network that is reconstituted under dissociated cell culture conditions. This experimental system, together with the synaptogenesis in the slice culture system described previously, serves as a good in vitro model for the analysis of the process of conversion from short-lasting plasticity (lasting for hours) into a long-lasting one (lasting for days-weeks).

Animals↗
PubMed2005

Raw material enzymatic activity determination: a specific case for validation and comparison of analytical methods--the example of superoxide dismutase (SOD).

Implementation of ICH guideline for validation of analytical methods was tested in the case of two enzymatic assays of determination of superoxide dismutase (SOD) activity. Analytical figures of merits of two tested methods (NBT reference method and the so called WST-1 method) demonstrate the feasibility of such approach. Nevertheless specification usually admitted for physicochemical method (e.g., HPLC), needs to be clearly enlarged, for instance, up to 10% for repeatability and 20% for reproducibility. On the other hand, the cross-validation performed between the two enzymatic activity determination techniques, though based on the same principle, clearly shows that the quality of results depends on small variation in the experimental conditions. This shows that an enzymatic activity determination should be strictly related to the technique used, especially in the pharmaceutical control quality field, and confirms that analytical figures of merits are strongly function of the technique used. Finally, it was demonstrated that both NBT reference method and WST-1 method give strictly similar results.

Animals↗
PubMed2006

Cortical changes to experimental sensitization of the human esophagus.

Topographical organization in the neocortex shows experience-dependent plasticity. We hypothesized that experimental sensitization of the esophagus results in changes of the topographical distribution of the evoked potentials and the corresponding dipole source activities to painful stimulation. An endoscopic method was used to deliver 35 electrical stimuli at the pain threshold to a fixed area of the mucosa in 10 healthy volunteer men and women. The stimulations were repeated after 30 min (reproducibility experiment), and after 60 min following perfusion of 200 ml 0.1 N hydrochloric acid (sensitization experiment). During stimulation the electroencephalogram was recorded from 64 surface electrodes. The sensitization resulted in a decrease in the pain threshold (F=6.2; P=0.004). The topographic distribution of the evoked potentials showed reproducible negative (N1, N2) and positive (P1, P2) components. After acid perfusion a reduced latency and a change in localization was seen for the P1 subdivided into frontal and occipital components (F=29.5, P<0.001; F=53.7, P<0.001). Furthermore the sensitization resulted in a reduction of the latency for P2 (F=6.2, P=0.009). The source analysis showed consistent dipolar activity in the bilateral opercular-insular cortex before and after acid perfusion. For the anterior cingulate dipole there was a reduction in latency (P=0.03) and a posterior shift (P=0.0002) following acid perfusion. The findings indicate that short-term sensitization of the esophagus results in central neuroplastic changes involving the cingulate gyrus, which also showed pathological activation in functional diseases of the gut, thus reflecting the importance of this region in visceral pain and hyperalgesia.

Adult↗
PubMed2006

Developmental toxicity evaluation of triclopyr butoxyethyl ester and triclopyr triethylamine salt in the CD rat.

Triclopyr (3,5,6-trichloro-2-pyridyloxyacetic acid) is an herbicide used extensively in the control of woody plants and broadleaf weeds, and is often formulated as a triethylamine salt (T-TEA) or butoxyethyl ester (T-BEE). This study evaluated the developmental toxicity of T-TEA or T-BEE in time-mated CD rats gavaged on gestation days 6-15 with 0, 30, 100 or 300 mg/kg body weight(bw)/day. The doses of each compound were equimolar and equivalent to 22, 76, 216 mg/kg bw/day of triclopyr, based on prior studies indicating rapid cleavage of the salt or ester and equivalent pharmacokinetics for the active ingredient. T-TEA caused maternal toxicity, evidenced by the death of one high-dose dam, reduced body weight gain, increased relative liver and kidney weights (300 mg/kg bw/day), reduced feed consumption, and increased water consumption (100 and 300 mg/kg bw/day). Developmental effects were limited to slightly decreased fetal body weight and reduced skeletal ossification at the high dose level. T-BEE caused similar, albeit slightly more severe, maternal toxicity, with three maternal deaths at 300 mg/kg bw/day, and slight maternal effects at 30 mg/kg bw/day. Due to an equivocal increase in malformations, which were mainly clustered in litters from three high dose dams with marked maternal toxicity, the T-BEE study was repeated using 30 dams/group, investigator-blind fetal evaluations, and an additional dose group (5 mg/kg bw/day). In the repeat study, the only reproducible fetal effect was an increased incidence of 14th thoracolumbar rib at 300 mg/kg bw/day. Overall analysis of the two T-BEE studies suggested that the fetal malformations unique to the initial study likely reflected a combination of spontaneous events, exacerbated by marked maternal toxicity. The combined weight of evidence from these developmental toxicity studies, coupled with their known pharmacokinetic equivalence, indicates that T-BEE and T-TEA are not selectively toxic to the fetus. The respective maternal toxicity no-observed effect levels (NOEL) for T-BEE and T-TEA were 5 and 30 mg/kg bw/day, while the NOEL for developmental toxicity was 100 mg/kg bw/day for both compounds.

Abnormalities, Drug-Induced↗
PubMed2006

A bladder-cooling reflex in the anaesthetised guinea-pig: a model of the positive clinical ice-water test.

INTRODUCTION: In patients with detrusor hyperreflexia, intravesical instillation of ice-cold water results in the development of involuntary bladder contractions at volumes less than normal cystometric capacity. This is referred to as a positive ice-water test (+IWT) and can be reversed by vanilloid receptor agonists and potentiated by menthol. The present study was designed to investigate the existence of an analogous cooling reflex in the guinea-pig bladder that could be used as a small animal model in order to test the effects of drugs on the reflex. METHODS: Bladder pressure and external urethral sphincter electromyogram (EUS EMG) were recorded in alpha-chloralose/urethane anaesthetised guinea-pigs during rapid infusion of cold or warm saline into the bladder with or without prior intravesical exposure to menthol or resiniferatoxin (RTX). RESULTS: The mean control micturition threshold volume (TV) of 2.58 ml at 38 degrees C was reduced to 1.52 ml in response to saline infusion at 3 degrees C (P=0.001). The cold-induced reduction in TV was reproducible during several subsequent repeat infusions at 38 degrees C and 3 degrees C and was accompanied by decreases in bladder voiding pressure. The duration of the micturition reflex was markedly increased following cold compared with warm saline infusion (mean 24.5 s at 3 degrees C, 10.2 s at 38 degrees C, P=0.001) and was associated with oscillations in bladder pressure and concomitant bursting activity in the EUS EMG. During step-wise decreases in infusate temperature from 38 degrees C to 23 degrees C, 15 degrees C, 7 degrees C and 3 degrees C, the threshold infusate temperature to elicit a significant reduction in TV was 15 degrees C. The reduction in TV at 3 degrees C was potentiated by intravesical infusion of 0.6 mM menthol whilst intravesical infusion of 500 nM RTX reversed the reduction in TV at 3 degrees C. DISCUSSION: These data suggest that a bladder-cooling reflex is present in the anaesthetised guinea-pig and represents a useful small animal model of the clinical +IWT.

Anesthesia↗
PubMed2000

Determination of inulin in meat products by high-performance liquid chromatography with refractive index detection.

Inulin is a naturally occurring carbohydrate with beneficial nutritional and technological properties. A high-performance liquid chromatographic (HPLC) method was developed for the quantitative determination of these beta-fructans in meat products, containing this type of additive. The method includes extraction of inulin with hot water, followed by hydrolysis with inulinase enzyme, and determination of the released fructose by HPLC with refractive index detection. An internal standard of rhamnose was used to quantify fructose. The method incorporates a sample blank (without inulinase hydrolysis) for each specimen to subtract contributions of free fructose and fructose from sucrose. The results showed good precision with average RSDs of 2.4% for repeatability and 5.2% for reproducibility. Analytical recovery ranged from 102 to 106%. Satisfactory linearity (r=0.999) was obtained.

Chromatography, High Pressure Liquid↗