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PubMed1989

Genotype and the cryopreservation process affect the levels of aneuploidy and chromosome breakage in cultured human fibroblasts.

Spontaneous micronucleus frequencies were measured in 11 human fibroblast strains, with early-passage cells that had never been frozen and with cells of comparable population doublings that had been cryopreserved in liquid nitrogen. The mean micronucleus frequency of the 11 strains increased from 14.0 +/- 0.7 to 20.4 +/- 1.8/1250 mononucleated cells (P = 0.002) after the freeze-thaw process. The nature of this increase in micronucleus frequency was examined using an immunodetection assay for the in situ identification of kinetochores in micronuclei. The increase in micronucleus frequency occurred primarily in the kinetochore-positive fraction, which is indicative of aneuploidy, but also by an increase in chromosome breakage in several strains. The findings were reproducible in repeat biopsies from two donors. Plating efficiencies of the 11 strains were studied during 1-9 and 10-20 population doublings from primary outgrowth, before freezing and again after freeze-thaw. The mean plating efficiency of frozen-thawed cells before nine doublings was significantly lower than that of cells of similar ages that had never been frozen (P = 0.004). The four strains that had a greater than 25% decrease in plating efficiency post freeze-thaw also had the highest aneuploidy index post freeze-thaw, suggesting that chromosomal imbalance contributes to the observed reduction in growth. We conclude that the genotype and culture manipulations of a fibroblast strain influence the outcome of the micronucleus assay.

Aneuploidy↗
PubMed1994

Activity of Crohn disease assessed by measurement of superior mesenteric artery flow with Doppler US.

PURPOSE: To test the hypothesis that increased blood flow in the superior mesenteric artery (SMA) reflects disease activity in patients with Crohn disease. MATERIALS AND METHODS: Duplex Doppler sonographic measurements of SMA blood flow volume were obtained in 10 patients with active Crohn disease, 10 patients with chronic inactive Crohn disease, and 10 healthy volunteers. Disease activity was determined with clinical and laboratory indicators. RESULTS: Interstudy reproducibility of repeated SMA flow volume measurements was good (r = .98). A marked increase in SMA flow volume was noted in patients with active disease compared with patients with inactive disease and healthy volunteers: 1,588 mL/min +/- 576 versus 288 mL/min +/- 113 and 417 mL/min +/- 147, respectively (P < .05 for both comparisons). CONCLUSION: Activity of Crohn disease causes a substantial increase in SMA flow volume. Measurement of SMA blood flow may be an important noninvasive, readily available, inexpensive tool that can be used to monitor Crohn disease objectively.

Adolescent↗
PubMed1986

Measurement of function in isolated single smooth muscle cells.

Smooth muscle cells, isolated from the stomach of guinea pig, were anchored electrostatically to a poly-L-lysine-treated cover slip that formed the ceiling of a minichamber. The cells could be perfused at high flow rates for long periods without being dislodged. Contractile responses, measured by image-splitting micrometry, were obtained repeatedly and reproducibly at intervals of 5 s-5 min for up to 3 h. Peak response in single cells was attained in 1.5-2 min by comparison with peak response in suspensions of muscle cells (0.5 min). The delay was probably due to unstirred layers covering anchored single cells. Otherwise, the magnitude of response of single cells and their sensitivity to various agonists (C-terminal octapeptide of cholecystokinin, methionine-enkephalin, and acetylcholine), as expressed in the D50 values, were identical to those obtained from measurements on large populations (suspensions) of muscle cells. The identity of dose-response profiles of single cells and suspensions of cells, particularly the wide span of the dose-response curves and the low Hill coefficients (0.30-0.35), implied that receptor heterogeneity was an inherent property of each muscle cell rather than a reflection of differences between muscle cells. This simple, precise, and economical method offers notable advantages in studies of isolated smooth muscle cells.

Acetylcholine↗
PubMed1983

Method for cisterna magna perfusion of synthetic cerebrospinal fluid in the awake goat.

We developed a method to produce stable alterations in the ionic composition of the medullary chemoreceptor environment. A double-lumen catheter system (Hustead epidural needle and epidural catheter) was placed through a plastic cisternal guide tube into the cisterna magna of awake goats. A push-pull perfusion system using a modified infusion pump delivered matched cerebrospinal fluid (CSF) perfusate inflow and outflow of 3.1 ml/min. Ventilation changed within 15 min of the initiation of perfusion and reached steady state within 45-65 min. Steady-state ventilatory responses could be maintained for up to 240 min and were readily reversed in response to a change in [HCO-3]. Perfusions with normal mock CSF ( [HCO-3] = 23 meq/l) caused no change from nonperfused values. Over the range of CSF perfusate [HCO-3] used (13.5-34.4 meq/l), the gain of the steady-state ventilatory response averaged 0.6 Torr X meq-1 X l. [3H]inulin and [HCO-3] were equal in inflow and outflow by 20-30 min of perfusion indicating complete mixing of bulk CSF in the cistern. Anatomic study after methylene blue dye perfusion showed dye distribution to subarachnoid spaces of midbrain, cervical cord, cerebellum, medulla, and most of the cortex but not to any ventricles. This perfusion technique produces prolonged, stable, reproducible, and repeatable changes in the medullary chemoreceptor ionic environment of awake goats, is relatively atraumatic, and permits high flow through the cisternal subarachnoid space.

Animals↗
PubMed1989

In vivo regional diaphragm function in dogs.

A biplane videofluorographic system was used to track the position of metallic markers affixed to the abdominal surface of the left hemidiaphragm in supine anesthetized dogs. Regional shortening was determined from intermarker distances of rows of markers placed along muscle bundles in the ventral, middle, and dorsal regions of the costal diaphragm and of one row on the crural diaphragm. Considerable variability of regional shortening was seen in a given row, which was reproducible on repeat study in individual dogs but which differed between mechanical ventilation and spontaneous breathing. There were no consistent patterns among dogs. Regional shortening obtained from the change in length of rows extending from chest wall to central tendon showed no consistent differences among dogs during spontaneous breathing. At equal tidal volumes, all regions (except the ventral costal diaphragm) shortened more during spontaneous breathing than during mechanical ventilation.

Animals↗
PubMed2005

Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence: evidence against immortality as a constitutive trait of tumor cells.

Although in vitro establishment of new colorectal carcinoma (CRC) cell lines is an infrequent event, we have observed that primary cultures of CRC can be repeatedly and reproducibly initiated following in vitro plating of tumor-derived epithelial cells. These cultures, however, usually display a short life span as they undergo a limited number of cell passages before entering a state of irreversible growth arrest. In this study, we show that short-lived CRC primary cultures lack constitutive telomerase activity and undergo a senescence process characterized by progressive telomere shortening. Moreover, transduction of these cells with a retroviral vector encoding human telomerase reverse transcriptase (hTERT) is sufficient to reconstitute telomerase activity and allow immortalization. Detailed molecular characterization of hTERT-immortalized CRC cell lines confirms their individual tumor origin by showing expression of colonic epithelial differentiation markers, such as cytokeratin-20 (CK20), full match with class I and class II human leukocyte antigen genotyping of autologous B-lymphoblastoid cells, and presence of somatic mutations in key cancer genes (KRAS2, APC) identical to those of the corresponding autologous original tumor tissues. Moreover, functional characterization of hTERT-immortalized CRC cell lines shows that they have a transformed phenotype, being able to form colonies in soft agar and tumors in severe combined immunodeficient mice. Most interestingly, immunohistochemical analysis of original tumor tissues indicates that short-lived CRC primary cultures, although hTERT-negative in vitro, derive from hTERT-positive tumors. Taken together, our data show that, in a least subset of CRC, biochemical pathways involved in maintenance of telomere length, such as telomerase, are not activated in a constitutive way in all tumor cells.

Animals↗
PubMed1988

Eicosanoid release in polymorphous light eruption: selective UV-A-induced LTB4 generation by peripheral blood leukocytes.

The biochemical basis of ultraviolet-induced cutaneous inflammation in polymorphous light eruption (PLE) is not known. We investigated the potential role of eicosanoids--derivatives of arachidonic acid--as mediators of inflammation in this common disorder. 13 patients were investigated, in whom skin lesions could be experimentally reproduced by repeated UV-A but not UV-B irradiation. Peripheral blood leukocytes of these patients were irradiated with UV-A or UV-B, and the release of leukotriene B4 (LTB4), LTC4 and prostaglandin E2 (PGE2) was measured. Cells from PLE patients, but not healthy controls, released selectively high amounts of LTB4 in response to UV-A. The UV-A-induced LTB4 release was light-dose-dependent. This phenomenon was not observed with UV-B. The selective UV-A-induced release of LTB4 could play a major role in the pathophysiology of cutaneous inflammation in PLE.

Adult↗
PubMed1991

Technical procedures and software for magnification-corrected morphometry of optic disk photography.

In order to obtain a new diagnostic tool for the early diagnosis of glaucoma, we developed a method based on computerized analysis of the optic disk, cup and neuroretinal rim areas. A fundus camera, a personal computer and a graphic tablet are employed. Suitable software was developed for calculating the areas (disk, cup and rim) providing an index, the rim/disk ratio. The method revealed high repeatability and reproducibility. The simplicity and speed of the procedure make this test suitable for routine clinical use in the early diagnosis of glaucoma.

Glaucoma↗
PubMed1983

Measurement of xanthine oxidase activity in some human tissues. An optimized method.

Xanthine oxidase activity in human liver or jejunum homogenates was measured by using 8-14C-hypoxanthine (HX) as a substrate. Products of oxidation were separated by thin-layer chromatography on MN Polygram cellulose and located by autoradiography. The percentage of oxidation was measured by liquid scintillation. Main parameters of the enzyme reaction have been optimized. Repeatability and reproducibility of the overall procedure give a variation coefficient of 6.3 and 7.7%, respectively. Minimal detectable enzymatic activity corresponds to an oxidation percentage of 1% (HX) for liver and jejunum. The reliability and practicability of this method allow a rapid diagnosis of xanthine oxidase deficiency. Normal values obtained by this optimized method range between 25 and 42 mukat . kg-1 protein for liver and 18 and 40 mukat . kg-1 protein for jejunum.

Carbon Radioisotopes↗
PubMed1977

A new, noninvasive technique for inducing post-extrasystolic potentiation during echocardiography.

Left ventricular function was evaluated in 34 patients with the echocardiogram, and an external mechanical cardiac stimulator was used to induce a ventricular premature contraction (VPC) noninvasively. Extent of post-extrasystolic potentiation (PESP) was determined by comparing systolic dimensional shortening and ejection fraction of the sinus beat preceding the VPC to that of the potentiated beat which followed it. Using this technique, a VPC could be introduced into the cardiac cycle of 30 of the 34 patients, six of whom were free of obvious cardiac disease and 24 of whom had valvular, coronary or myopathic heart disease. The only complication observed was mild breast ecchymosis in a female patient. Systolic dimensional shortening and ejection fraction increased from control values by an average of 21% and 17% respectively, with a range of 0-100%. The degree of PESP was very reproducible in repeat studies and when the same patients were subsequently evaluated during a spontaneously occurring or catheter-induced VPC. The technique can safely and reliably induce post-extrasystolic potentiation during echocardiography and is a potentially important adjunct to the noninvasive evaluation of left ventricular function.

Adult↗
PubMed1996

Inhaled tryptase causes bronchoconstriction in sheep via histamine release.

Allergen-induced bronchoconstriction involves mast cell activation. Tryptase is a mast cell serine protease that is released during this process, but little is known about the action of tryptase in the airway. The purpose of this study was to determine: (1) if aerosolized tryptase causes bronchoconstriction, and (2) the mechanism by which this occurs. We measured mean pulmonary flow resistance (RL) in five allergic sheep before and after consecutive inhalations of 100 and 500 ng tryptase (in 2 ml total volume). Inhaled tryptase at 100 and 500 ng increased RL (mean +/- SE) by 33 +/- 12 and 122 +/- 8% (p < 0.05) over baseline. The response was reproducible upon repeat challenges. These studies were repeated in the same animals after pretreatment with aerosolized APC 366 (9 mg/3 ml), a specific tryptase inhibitor. In APC-366-treated sheep, tryptase increased RL by 10 +/- 3 and 6 +/- 2% (p < 0.05 versus control values) at 100 and 500 ng, respectively. The response to tryptase was also blocked by pretreating the sheep intravenously with the histamine H1-antagonist chlorpheniramine (2 mg/kg), in which RL increased only 5 +/- 4 and 7 +/- 6% after 100 and 500 ng tryptase. APC 366, however, did not block histamine-induced bronchoconstriction. Consistent with these findings was the observation that segmental bronchial challenge with tryptase (1 microgram) resulted in a significant increase in histamine levels in bronchoalveolar lavage. Inhaled tryptase (500 ng) also caused airway hyperresponsiveness to aerosolized carbachol 2 h after tryptase challenge. This tryptase-induced airway hyperresponsiveness could be blocked either by pretreating the sheep with APC 366 (30 min before challenge) or by treating the sheep 30 min after challenge. These results indicate that inhaled tryptase causes bronchoconstriction and airway hyperresponsiveness in allergic sheep by an event that may involve mast cell activation.

Administration, Inhalation↗
PubMed2006

Self-assembled monolayer of carboxyl-terminated poly(amido amine) dendrimer.

Adlayer formation and adsorption structure of 2.5th-generation poly(amido amine) dendrimer with carboxyl-terminated groups on solid substrates were investigated by atomic force microscopy, surface plasmon resonance spectroscopy, and surface enhanced infrared absorption spectroscopy. Dendrimer molecules are not uniformly adsorbed on solid surface but form aggregates with a width of approximately 100 nm and a height less than 1 nm. Adsorption reaches in equilibrium at 100-1000 sec, depending on the dendrimer concentration. The adsorption-desorption process is considerably reproducible and repeatable. Although the adsorption at equilibrium increases with dendrimer concentration and reaches maximum at neutral pH, monolayer is always maintained after the desorption with solvent. This indicates the formation of self-assembled monolayer. Such monolayer is preserved even at the variation of pH. Although most carboxylates are protonated at acidic pH, small amount of carboxylate remains even at acidic pH. The adsorption structure of dendrimer was illustrated.

Adsorption↗
PubMed2003

Flow cytoenzymology of intracellular tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRACP) is a cytochemical marker for hairy cell leukemia, macrophages, dendritic cells, and osteoclasts. Our purpose was to develop multicolor cytofluorometric methods to evaluate intracellular TRACP enzymic activity using a fluorogenic cytochemical reaction in combination with immunochemical stains for distinct surface membrane antigens. Monocyte-derived dendritic cells (DCs) were the model TRACP-expressing cells studied. Intracellular TRACP activity was disclosed using naphthol-ASBI phosphate as substrate with fast red-violet LB salt as coupler for the reaction product. Before the TRACP enzymic reaction, surface antigens, CD86 and CD11c of DCs, were bound with specific fluorescent antibodies to test compatibility of surface labeling and intracellular staining. TRACP activity varied in DCs from donor to donor but was reproducible on repeated examinations of each sample. Samples could be stained for simultaneous analysis of surface antigens and intracellular TRACP activity, provided certain technical details were observed. The TRACP reaction time should not exceed 9 min and the cell number should not exceed 2 x 10(5)/100 micro l test. Fluorescent surface labels did not affect the intensity of the TRACP stain, but the intensity of some surface labels may be diminished by elution of low-affinity antibodies during the TRACP reaction. Readjustment of the threshold settings in triple-labeled cells is needed to compensate for this phenomenon. Intracellular TRACP activity can be quantitated in subpopulations of cells within mixed cell populations by flow cytofluorometry using simple cytochemical methods in combination with fluorescent antibodies to cell-surface and other differentiation antigens. The cytochemical method should be useful for basic investigations of differentiation, maturation, and function of macrophages, DCs, and osteoclasts, and for diagnosis and management of hairy cell leukemia.

Acid Phosphatase↗
PubMedDate not supplied

Indirect calorimetry: comparison of hood and mask systems for measuring resting energy expenditure in healthy volunteers.

Controversy exists as to the validity and reliability of hood and mask systems in measuring indirect calorimetry. The purpose of this study was to evaluate the accuracy and reproducibility of repeat measurements of resting energy expenditure (REE) in volunteers. Paired REE measurements were performed in 23 subjects after an overnight fast using hood and mask systems. Lean body mass was calculated from four skinfold measurements and body weight determinations. Data were normalized to body weight and lean body mass and were calculated as percent predicted REE in paired tests taken within 5 minutes on the same subject. No significant difference in mean REE was noted between hood and mask systems. Linear regression analysis showed a strong positive correlation (r = 0.91, p less than 0.001) between hood and mask measurements of REE.

Adult↗
PubMed1978

Contrast enhancement of cranial lesions in computed tomography.

The time course of enhancement in apparently normal brain tissue, edema and focal lesions during 30 to 60 min after intravenous injection of vascular contrast medium was evaluated in a series of 41 patients with different intracranial lesions. The attenuation of apparently normal unenhanced brain tissue varied with the level of the scan, mainly an effect of beam hardening. Different types of enhancement response are discussed in terms of a 3-compartment model. The differential diagnostic potential of contrast enhancement in the early phase needs further evaluation using instruments with short scanning time, while the late phase of enhancement must be recorded with the use of a reliable head fixation to provide reproducibility of repeat measurements.

Adolescent↗
PubMed1998

Single-breath method for assessing the viscoelastic properties of the respiratory system.

In order to explain the time dependency of resistance and elastance of the respiratory system, a linear viscoelastic model (Maxwell body) has been proposed. In this model the maximal viscoelastic pressure (Pvisc.max) developed within the tissues of the lung and chest wall at the end of a constant-flow (V') inflation of a given time (tI) is given by: Pvisc,max = R2V'(1-e(-tI/tau2), where R2 and tau2 are, respectively, the resistance and time constant of the Maxwell body. After rapid airway occlusion at t1, tracheal pressure (Ptr) decays according to the following function: Ptr(t) = Pvisc(t) + Prs,st = Pvisc,max(etocc/tau2)+ Prs,st, where tocc/is time after occlusion and Prs,st is static re-coil pressure of the respiratory system. By fitting Ptr after occlusion to this equation, tau2 and Pvisc,max are obtained. Using these values, together with the V' and tI pertaining to the constant-flow inflation preceding the occlusion, R2 can be calculated from the former equation. Thus, from a single breath, the constants tau2, R2 and E2 (R2/tau2) can be obtained. This method was used in 10 normal anaesthetized, paralysed, mechanically ventilated subjects and six patients with acute lung injury. The results were reproducible in repeated tests and similar to those obtained from the same subjects and patients with the time-consuming isoflow, multiple-breath method described previously.

Adult↗
PubMed2006

The automatic recognition and counting of cough.

BACKGROUND: Cough recordings have been undertaken for many years but the analysis of cough frequency and the temporal relation to trigger factors have proven problematic. Because cough is episodic, data collection over many hours is required, along with real-time aural analysis which is equally time-consuming. A method has been developed for the automatic recognition and counting of coughs in sound recordings. METHODS: The Hull Automatic Cough Counter (HACC) is a program developed for the analysis of digital audio recordings. HACC uses digital signal processing (DSP) to calculate characteristic spectral coefficients of sound events, which are then classified into cough and non-cough events by the use of a probabilistic neural network (PNN). Parameters such as the total number of coughs and cough frequency as a function of time can be calculated from the results of the audio processing. Thirty three smoking subjects, 20 male and 13 female aged between 20 and 54 with a chronic troublesome cough were studied in the hour after rising using audio recordings. RESULTS: Using the graphical user interface (GUI), counting the number of coughs identified by HACC in an hour long recording, took an average of 1 minute 35 seconds, a 97.5% reduction in counting time. HACC achieved a sensitivity of 80% and a specificity of 96%. Reproducibility of repeated HACC analysis is 100%. CONCLUSION: An automated system for the analysis of sound files containing coughs and other non-cough events has been developed, with a high robustness and good degree of accuracy towards the number of actual coughs in the audio recording.

Journal Article↗