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PubMed2006

Comparison of elicitation potential of chloroatranol and atranol--2 allergens in oak moss absolute.

Chloroatranol and atranol are degradation products of chloroatranorin and atranorin, respectively, and have recently been identified as important contact allergens in the natural fragrance extract, oak moss absolute. Oak moss absolute is widely used in perfumery and is the cause of many cases of fragrance allergic contact dermatitis. Chloroatranol elicits reactions at very low levels of exposure. In oak moss absolute, chloroatranol and atranol are present together and both may contribute to the allergenicity and eliciting capacity of the natural extract. In this study, 10 eczema patients with known sensitization to chloroatranol and oak moss absolute were tested simultaneously to a serial dilution of chloroatranol and atranol in ethanol, in equimolar concentrations (0.0034-1072 microM). Dose-response curves were estimated and analysed by logistic regression. The estimated difference in elicitation potency of chloroatranol relative to atranol based on testing with equimolar concentrations was 217% (95% confidence interval 116-409%). Both substances elicited reactions at very low levels of exposure. It is concluded that the differences in elicitation capacity between the 2 substances are counterbalanced by exposure being greater to atranol than to chloroatranol and that both substances contribute to the clinical problems seen in oak moss absolute-sensitized individuals.

Adult↗
PubMed1993

Relationship between development of diarrhea and the concentration of SN-38, an active metabolite of CPT-11, in the intestine and the blood plasma of athymic mice following intraperitoneal administration of CPT-11.

Severe diarrhea occurred during daily intraperitoneal administration of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) at a dose of 50 mg/kg in athymic mouse. Serial determination of CPT-11 and 7-ethyl-10-hydroxycamptothecin (SN-38), with the use of an on-line solid extraction HPLC system, demonstrated that much higher levels of the compounds are retained in the intestine and the blood plasma after five consecutive daily injections than after a single injection. Histologic examination of the gastrointestinal tract showed hemorrhagic colitis on day 7 and later after five consecutive daily injections of CPT-11. The direct cause of diarrhea associated with CPT-11 administration is considered to be enterocolitis caused by high levels of SN-38 and/or CPT-11 retained for a long period in the intestine.

Animals↗
PubMed1988

A comparison of the effect of timegadine, levamisole, and D-penicillamine on human neutrophil metabolism of endogenous arachidonic acid and chemotaxis.

The effect of timegadine, a novel experimental antirheumatic drug, on human neutrophil (PMN) 5-lipoxygenase activity and leukotriene B4 (LTB4) chemotaxis was compared with that of two second-line antiinflammatory drugs, D-penicillamine and levamisole. 1-14C-Arachidonic acid (AA) was incorporated into the purified cells until steady state conditions were obtained. After preincubation with serial dilutions of the three drugs, AA release and metabolism was stimulated with calcium ionophore A23187. The radioactive eicosanoids released were extracted and separated by thin-layer chromatography, followed by autoradiography and quantitative laser densitometry. Chemotaxis of PMNs towards LTB4 was measured in a modified Boyden chamber. Timegadine showed dose-dependent inhibition of both the 5-lipoxygenase pathway (IC50 3.4 x 10(-5) M), and of chemotaxis (IC50 3 x 10(-4) M). Inhibition of the release of AA from phospholipids, however, occurred only at therapeutically irrelevant doses (millimolar concentrations). Levamisole and D-penicillamine did not inhibit any of the cell functions investigated. Inhibition of both neutrophil motility and cellular synthesis of pro-inflammatory eicosanoids, may thus contribute to the clinical effects of timegadine in rheumatoid arthritis.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1998

Pharmacokinetic assessment of the sites of first-pass metabolism of BMS-181101, an antidepressant agent, in rats.

The relative contribution of the gut and the liver to the first-pass metabolism of BMS-181101 (3-[3-[4-(5-methoxy-4-pyrimidinyl)-1-piperazinyl]propyl]-5-fluoro-1H-ind ole dihydrochloride), a potential antidepressant agent, has been evaluated in rats. Nine male Sprague-Dawley rats were divided into three groups of three and each rat received a single 20 mg kg(-1) dose of [14C]BMS-181101 via a 30 min constant-rate intravenous infusion, a 30-min constant-rate intraportal infusion or oral gavage. Serial blood samples were collected for 8 h after dosing and plasma was analysed for unchanged BMS-181101 and total radioactivity. Extraction ratios for BMS-181101 by the gut and liver were calculated on the basis of ratios of the area under the plasma BMS-181101 concentration-time curve. The gut had a high intrinsic capacity for metabolizing BMS-181101-extraction ratios were 93% and 10% for the gut and liver, respectively. After oral administration BMS-181101 is sequentially exposed to the gut then the liver. As a result, the contribution of the gut to the overall first-pass effect (ca. 93%) was significantly greater than that of the liver (ca. 0.7%). The estimated total first-pass effect of 94% for BMS-181101 in rats is in excellent agreement with the observed absolute oral bioavailability of 6%. These results clearly illustrate the importance of metabolic activity in the gut for orally administered BMS-181101.

Administration, Oral↗
PubMed2002

Generation of kidney transcriptomes using serial analysis of gene expression.

Chronic renal disease initiation and progression remain incompletely understood. Genomewide expression monitoring should clarify the mechanisms which cause progressive renal disease by determining how clusters of genes coordinately change their activity. Serial analysis of gene expression (SAGE) is a technique of expression profiling which permits simultaneous and quantitative analysis of 9- to 13-bp sequence tags that correspond to unique mRNAs. Key principles of the technique are use of PCR in a manner to minimize distortion and serial concatenation of tags which facilitates sequencing and permits identification of many expressed genes in a single cDNA molecule. Tags are extracted from many concatenated sequences, counted using software, and identified by comparison with existing gene databases. In aggregate, gene expression profiles generated from a tag library comprise a transcriptome which represents a comprehensive and quantitative profile of genes expressed at the time of analysis. These global snapshots of gene expression patterns can better define basic cell biology and provide insights into disease pathogenesis by simultaneously determining the net consequences of gene-gene and gene-environment interactions on expression of thousands of genes. Rather than applying a priori assumptions (i.e., hypothesis testing), transcriptome analysis is hypothesis generating and requires no prior knowledge of gene expression. SAGE kidney transcriptomes, from normal animals and animals with progressive kidney disease, are being produced and can be analyzed for novel pathogenetic mechanisms. The use of SAGE and other genomic and proteomic tools should result in a better understanding of kidney disease pathogenesis and in identification of new therapeutic targets.

Animals↗
PubMed2000

Three-dimensional image and virtual dissection program of the brain made of Korean cadaver.

The three-dimensional (3D) structure of the brain needs to be understood for accurate diagnosis and treatment of brain diseases. The brain of a cadaver may not always be available for dissection when it is needed. To overcome this problem, we attempted to create a 3D image and virtual dissection program of the brain using a Korean cadaver. The brain extracted from a Korean male cadaver was embedded in gelatin solution. 130 MRI of the brain were taken and 130 serially-sectioned specimens were made. All of MRI and specimens were inputted into the computer, and 10 brain components were manually segmented. A 3D image and virtual dissection program of the brain was made. Various virtual dissection functions were established, such as 1) sectioning the 3D image of the brain at free angles to represent its plane as a real image, segmented image, and MRI, 2) identifying the brain components represented in the sectioned plane, and 3) rotating the 3D image of the whole brain or the selected brain components at free angles. The resulting virtual dissection program of the brain is helpful in better understanding the 3D location and shape of the brain components and it is expected to be used as a CD-title or through Internet as an educational tool for medical students and doctors.

Brain↗
PubMed1990

Comparative myocardial extraction of two technetium-labeled BATO derivatives (SQ30217, SQ32014) and thallium.

The transcapillary exchange of a new class of 99mTc-labeled compounds (BATO) were compared to 201Tl in isolated, blood perfused rabbit hearts. During variable blood flow (0.15-2.44 ml/min/g), peak and net extraction (Emax and Enet, respectively), and capillary permeability-surface area product (PScap) were determined with paired indicator-dilution techniques. Serial bolus injections of 201Tl, [111In]albumin, and [99mTc]BATO; chloro[tris(cyclohexanedionedioxime)methyl boronic acid]Tc (SQ30217, n = 8) and a hydroxy-substituted derivative (SQ32014, n = 5) were given to a total of 13 hearts. Mean (+/- s.d.) SQ30217 Emax and Enet were 0.72 +/- 0.09 and 0.55 +/- 0.18, respectively, which were higher than thallium values of 0.57 +/- 0.10 and 0.46 +/- 0.17 (p less than 0.03). Mean SQ30217 PScap was 1.1 +/- 0.4 ml/min/g and was also higher than corresponding thallium determinations (0.7 +/- 0.3; p less than 0.001). SQ32014 Emax, Enet, and PScap were all significantly less than thallium values (p less than 0.001). Thallium and SQ30217 values for Emax and PScap were closely correlated with blood flow (r greater than or equal to 0.73), whereas those for SQ32014 were weakly correlated (r = 0.09). A small clinical pilot study (n = 3) was performed, which showed that SQ32014 was a poor myocardial perfusion agent in man. In summary, transcapillary exchange of SQ30217 is greater than thallium, which in turn, is greater than SQ32014. Therefore, SQ30217 appears to have good clinical potential, but SQ32014 does not.

Adult↗
PubMed1995

Serial lecithin/sphingomyelin ratios and surfactant/albumin ratios in tracheal aspirates from term infants with respiratory failure receiving extracorporeal membrane oxygenation.

Serial tracheal aspirate samples were obtained for determination of lecithin/sphingomyelin (L/S) ratios from 47 term infants in respiratory failure. Phospholipids were extracted with Folch solution (chloroform:methanol, 2:1 by vol) and analyzed by HPLC with use of silica column and a mobile phase of acetonitrile:methanol:water (48:31:21, by vol). Surfactant/albumin (S/A) ratios were determined with the TDx Fetal Lung Maturity Assay (Abbott Labs). L/S ratios increased significantly over time in all patients (F = 19.42, P < 0.0001). The S/A ratio correlated with the L/S ratio (r = 0.554, P < 0.001). This study suggests that postnatal surfactant deficiency in term newborns with respiratory failure is a component of the newborn's lung injury.

Albumins↗
PubMed1995

Mycobacterium paratuberculosis and inflammatory bowel disease: frequency distribution in serial colonoscopic biopsies using the polymerase chain reaction.

An association between Mycobacterium paratuberculosis and Crohn's disease is suspected but the evidence remains controversial. Using a one-step DNA extraction procedure with the thermophilic protease PRETAQ and amplification by the polymerase chain reaction, M. paratuberculosis DNA was detected in 22% of patients with Crohn's disease, and in 13% of patients with ulcerative colitis. M. paratuberculosis DNA was not found in any biopsy tissue from control non-inflammatory bowel disease patients. The biopsy tissues in which M. paratuberculosis was detected all came from regions which were inflamed when viewed microscopically. Overall, 7.7% of biopsies from such inflamed areas were positive. This low frequency of detection could be explained on the basis of extremely low abundance of the organism in relation to the area of mucosa sampled, or be consistent with a non-aetiological role for M. paratuberculosis in inflammatory bowel disease.

Animals↗
PubMed1995

Histologic study of pulp capping using chlorhexidine in dogs.

This histological study was conducted to compare the pulp reactions to chlorhexidine and calcium hydroxide. Twenty permanent premolar teeth of beagle dogs were mechanically exposed under aseptic conditions. The pulp was then capped with one of the following: chlorhexidine 0.2% on the left side and calcium hydroxide (dycal) on the right side of the same dog. Maxillary second premolars were extracted from each dog three weeks after the pulp capping procedure. Maxillary third premolars and mandibular first premolars were extracted at 6 weeks and 9 weeks respectively. The mandibular second premolars were extracted three months after the pulp capping procedure. The specimens were fixed in 10% formalin and decalcified in 5% formic acid. Serial sections, 7 microns thick, were prepared and stained with hematoxylin and eosin. Teeth treated with chlorhexidine or with calcium hydroxide were well tolerated by the dental pulp at 3 and 6-week intervals. Pulps treated showed satisfactory pulp reaction and the odontoblastic layer appeared to be normal and in its configuration. Inflammatory response was not present at the 6-week time interval. At nine weeks, the inflammatory response was severe in those teeth treated with calcium hydroxide. There were no remarkable histological differences among the treatment groups at 12 and 15 weeks. These specimens showed complete loss of pulpal architecture. Regressive changes were seen and microscopic specimens revealed presence of spaces and intertwining bundles of films in the pulp.

Animals↗
PubMed2003

Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5'-->3' DNA polymerase and 3'-->5' exonuclease activity.

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (r Tth DNA Polymerase, XL) providing both 5'-->3' polymerase and 3'-->5' exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The r Tth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the r Tth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.

DNA Primers↗
PubMed2004

IgE antibody to Aspergillus fumigatus recombinant allergens in cystic fibrosis patients with allergic bronchopulmonary aspergillosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) is characterized by a heightened Th2 CD4+ T-cell response to Aspergillus fumigatus (Af) allergens and a hyper-immunoglobulin E (IgE) state compared with cystic fibrosis patients without ABPA. The IgE serologic differentiation of ABPA from atopic CF patients can be difficult. We propose as the reactivity with purified antigens varies qualitatively and quantitatively and that the antibody response is more specific than with crude Af antigen extract, the IgE responses to purified recombinant Af allergens may differentiate ABPA from atopic CF patients. METHODS: Serum IgE reactivity to seven recombinant purified allergens and to a crude extract of Af was measured in 15 ABPA, in 23 Af skin test positive (ST+), and in 19 Af skin test negative (ST-) CF patients. Four of the ABPA CF patients were studied before and after developing ABPA. Nine ABPA patients were studied during flares and remissions of ABPA. RESULTS: Allergic bronchopulmonary aspergillosis patients had significantly increased IgE reactivity to Asp f2, f3, f4, f6, and f16 compared with the Af ST+ and ST- non-ABPA CF patients. In the ABPA patients studied before and after developing ABPA, IgE reactivity also increased to Asp f2, f3, f4, and f6, and to the crude extract. In ABPA CF patients, IgE reactivity to Asp f1, f2, f3, and f6 significantly increased during periods of ABPA flares compared with periods of remission. Analysis of the receiver operating curve demonstrated that IgE reactivity to Asp f3 and f4 gave the best sensitivity and specificity and were better than IgE reactivity to a crude extract of Aspergillus. Furthermore, in ABPA patients studied during periods of remission the IgE reactivity to Asp f3 and f4 remained significantly elevated compared with Af ST+ non-ABPA patients. The IgE responses when considered either to be positive or negative to Asp f3 and f4 significantly differentiated ABPA from Af ST+ and ST- non-ABPA CF patients. In contrast, IgE reactivity was considered positive to the crude extract in 89% of ABPA, 61% of Af ST+, and 0% of Af ST- non-ABPA CF patients. CONCLUSIONS: Immunoglobulin E reactivity to a panel of purified Af allergens, especially to Asp f3 and f4, differentiates ABPA from atopic Af ST+ non-ABPA CF patients. Serial determinations of IgE reactivity to individual purified Aspergillus antigens, especially Asp f3, demonstrates that increases in IgE reactivity may provide improved distinction between stages of flares and remission compared with changes in IgE reactivity to a crude Aspergillus extract.

Allergens↗
PubMed2005

Development of a nested PCR method targeting a unique multicopy element, ISMap02, for detection of Mycobacterium avium subsp. paratuberculosis in fecal samples.

This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis that is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 10(2) CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 10(2) CFU/g for the IS900 element and 10(3) CFU/g for the ISMap02 element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.

Animals↗
PubMed1982

Studies of a patient with recurring thrombotic thrombocytopenic purpura.

A young woman with recurring bouts of thrombotic thrombocytopenic purpura presented an opportunity to study a single patient serially and in depth. This patient underwent 12 remissions in response to fresh frozen plasma, and as little as 250 ml of plasma or plasma from which cryoprecipitate had been extracted with efficacious. Neither the Lian factor nor alterations in either cold-insoluble globulin or protein C could be demonstrated. Evidence in support of the concept of "exhausted" platelets was generated, in that immediately before a thrombocytopenic relapse, platelet function was altered as manifested by bruising, prolongation of the bleeding time, and decreased platelet aggregation. The in vitro addition of normal plasma to such platelets did not improve aggregation. The antiplatelet agent sulfinpyrazone did not seem to add efficacy. Serial observations support the hypothesis that the progression of pathophysiologic events in this patient is: exhausted platelets, thrombocytopenia, hemolysis, renal perturbations, and central nervous system dysfunction.

Adolescent↗
PubMed1996

Diagnosis of Yersinia pseudotuberculosis infection by polymerase chain reaction.

BACKGROUND: The diagnosis of Yersinia pseudotuberculosis infection is usually based on serologic and/or bacteriologic tests. However, successfully culturing Y. pseudotuberculosis is difficult, and serologic tests in many cases require at least two serial sera obtained during 1-week intervals to confirm rising agglutination antibody titers. METHODS: We applied a nested polymerase chain reaction method for rapid diagnosis of Y. pseudotuberculosis infection. The DNAs extracted from the peripheral blood and urine of patients and from mountain water, a suspected source of infection, were used as templates for the polymerase chain reaction with consequent amplification of a fragment of the inv gene in the chromosomal DNA of Y. pseudotuberculosis. RESULTS: The overall rate of diagnosis with the polymerase chain reaction, which was based on a positive result with a single blood sample or one or more positive results with serial samples, was 93.3%. The polymerase chain reaction was also positive in two mountain water samples that were thought to be a source of infection. CONCLUSION: Based on our results the nested polymerase chain reaction method can be used clinically for rapid and precise diagnosis of Y. pseudotuberculosis infection.

Base Sequence↗
PubMed1989

Cholesterol and bile acid synthesis: utilization of D2O for metabolic studies.

Human fibroblasts and hepatoma (Hep G2) cells were grown in media containing 25% D2O. Cholesterol extracted from the cells and bile acids obtained from the media were analyzed by gas chromatography/mass spectrometry (GC/MS). Fibroblasts that were transferred serially in media containing D2O continued to grow and to synthesize cholesterol enriched in deuterium. The observed distribution of deuterium-enriched species of cholesterol corresponded to a distribution that was calculated based on C = 27, 13C = 1.107%, D2O/H2O = 0.25, hydrogen derived from water = 20, and is in agreement with the concept that deuterium incorporation occurs randomly and represents mostly the NADPD/NADPH ratio in the medium. The deuterium enrichment of cholesterol from hepatoma cells indicated a shift of the most abundant species from m/z 373 to m/z 375, which corresponds more closely to the derivation of 25 hydrogens from water and implies the formation of deuterated acetate in the medium. Analysis of chenodeoxycholic acid, the predominant bile acid synthesized by Hep G2 cells in vitro, indicates its derivation from both pre-formed and newly synthesized cholesterol and that A ring transformation from cholesterol utilizes deuterium derived from water. Analysis of the bile acids derived from hamster bile following the administration of D2O confirms that similar events occur in vivo.

Animals↗
PubMed1979

An iterative algorithm for the separation of action potential sequences.

Various iterative algorithms for separation of superimposed event sequences were designed, and their efficiency examined through simulation studies. It has been found that a double parallel-serial algorithm improved markedly the efficiency of the filtration process. For the same probability of detection of the event series to be extracted, the probability of 'impurity' events dropped by roughly 0.10, as compared to the non-iterative fixed window width algorithm. Conclusions are drawn regarding separation of motor unit action potential sequences from the total electromyogram.

Action Potentials↗
PubMed1983

Culture of retinal capillary cells using selective growth media.

Capillary fragments are isolated from the microvasculature of the bovine retina using limited collagenase digestion and sieving. The endothelial cells obtained from these capillary fragments are cultured in media containing platelet-poor plasma supplemented with retinal extract, which contains an endothelial cell mitogen. These cells form typical monolayers in vitro, contain Factor VIII antigen, and can be passaged serially. The pericytes obtained from these capillary fragments are cultured in media containing calf serum. These cells can also be passaged serially, are not contact-inhibited, and do not stain for Factor VIII antigen. Cultures of capillary cells such as these will allow the study of the individual cells from specific microvascular beds involved in various normal and pathological processes.

Animals↗