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[Low level alpha activity measurements with pulse shape discrimination--application to the determination of alpha-nuclides in environmental samples].

Liquid scintillation counting of alpha rays with pulse shape discrimination was applied to the analysis of 226Ra and 239+240Pu in environmental samples and of alpha-emitters in/on a filter paper. The instrument used in this study was either a specially designed detector or a commercial liquid scintillation counter with an automatic sample changer, both of which were connected to the pulse shape discrimination circuit. The background counting rate in alpha energy region of 5-7 MeV was 0.01 or 0.04 cpm/MeV, respectively. The figure of merit indicating the resolving power for alpha- and beta-particles in time spectrum was found to be 5.7 for the commercial liquid scintillation counter.

Alpha Particles↗

Haemonchus spp. in sheep farmed under resource-poor conditions in South Africa--effect on haematocrit, conjunctival mucous membrane colour and body condition.

A longitudinal study was conducted on the differential faecal egg counts, haematocrits and body condition scores of sheep belonging to resource-poor farmers at Rust de Winter, Gauteng province, and Kraaipan, North West Province, South Africa. The animals were scored for level of anaemia using the FAMACHA method, an assay for the clinical evaluation of anaemia caused by Haemonchus spp. Periods of higher Haemonchus egg counts occurred from October to March for sheep at Rust de Winter and from September/October to February or April for sheep at Kraaipan. Lower haematocrit values were registered during these periods as was a higher incidence of anaemic conjunctival mucous membrane colour scores compared to the period April to September. No clear relationship between the faecal egg counts and the body condition scores was evident. Although wider application of the FAMACHA system in sheep raised by resource-poor farmers should be investigated, the present study indicates that this method may certainly prove to be a valuable worm control strategy for such livestock owners.

Anemia↗

Nuclear-labeling index analysis (NLIA), a software package used to perform accurate automation of cell nuclear-labeling index analysis on immunohistochemically stained rat liver samples.

The nuclear labeling index (labeled nuclei/100 nuclei) and the apoptotic index (apoptotic cells/100 cells) are important parameters of cell growth and death. Automatic counting of labeled nuclei is desirable since manual counting is tedious, time-consuming, and with a greater potential for inaccuracies. A nuclear-labeling index analysis (NLIA) software package was developed in this laboratory to perform the counting process automatically and accurately. This software package consists of an application program NLIA and a set of macros for obtaining nuclear data that is used in Scion Image. It is designed to work cooperatively with Scion Image, Adobe Photoshop, and Microsoft Office. NLIA has two basic functions: building nuclear data files and analyzing nuclear data. A color image captured from an immunohistochemically stained or autoradiographic sample is loaded into NLIA. Nuclear data can be entered into the program manually, automatically, or in combination. In the manual data entering mode, NLIA acts as an object-counting tool, while in the automatic mode it acts as a data picker: picking up the data generated by Scion Image into memory. A method to enter nuclear data (both labeled nuclei and unlabeled nuclei) in the automatic mode is described. The color image is processed in Adobe Photoshop, where the interested color ranges are selected and separated. These are then analyzed in Scion Image with the help of the macros for obtaining nuclear data. Since the advanced particle analysis function is used, the counting process is automatic and rapid. Data from thousands of nuclei can be obtained within seconds. To ensure the accuracy of the analysis, a nuclear data checking and edit feature is employed in NLIA: results of computer-generated counting can be compared with the original color image by overlaying the plot of counting results onto the original color image. In this way any computer counting mistakes can be easily discovered and corrected by the operator. Corrected nuclear data (including nuclear size, location, shape) are then stored in data files. These data files can be used in NLIA to obtain cell density and nuclear labeling indices. Because criteria for obtaining nuclear data (truncation diameter, shape factor) can be set by the operator in NLIA, nuclear size distribution and shape variation can be analyzed. This method provides a fast and accurate way to determine cell nuclear-labeling indices. Currently, Scion Image is a freeware on the internet, and NLIA software package is available from our lab home page. Methods presented here expand the Scion Image ability to analyze color images by using color separation techniques in a commercial graphic application. The instrumentation required can be relatively inexpensive, and the methods described may be useful in studies of cell kinetics, lesion growth, and tumor therapy.

Animals↗

Development of a method for measuring cell number: application to CNS primary neuronal cultures.

In the present work we further develop a method for counting cell number that is totally independent from the permeability or uptake conditions of the cells, from the state of activation of intracellular enzymes, and from cellular metabolism. We provide a visual characterization of the method and show that it is highly suitable for cells not growing as monolayers as well as for cultures containing numerous aggregates. We also extend the applicability of this method to CNS primary neuronal cultures and show its direct comparison with alternative means for cellular quantification. The technique is fast, does not require tedious procedures or long washes, and offers advantages such as a high sensitivity and no background.

Animals↗

Immunity, infection, malignancy and aging: possible immunity restoration and tumor prevention.

The incidence and gravity of infectious diseases in aged humans were known and feared before the age of antibiotics. Today the phenomenon, even if forgotten, still exists in old people's hospices where cross-infection flourishes. Although death in the elderly as a result of hospice-induced pneumonia may not attract public attention, a proportion of the public is still affected by death from cancer in the old. Their reaction is understandable, as it is at about 40 years of age that aging-related spontaneous tumors start to appear, after which their incidence tends to increase. The same phenomenon has been observed for spontaneous tumors in mice, in which they appear after 16 months of age. After this age, we have observed the decrease of T- and B-lymphocyte immune functions and their possible restoration parallel to tumor appearance prophylaxis, with the application of bestatin after the said 16 months. This cell regulator is also able to restore the CD4+ CD8- lymphocyte count in humans (possibly lowered after the age of 40 yr), but only if the CD4- CD8+ count is also reduced. A trial on the possible prevention or retardation of human tumor appearance by application of bestatin after the age of 40 yr is indicated.

Adult↗

Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy.

Here we discuss the application of scanning fluorescence correlation spectroscopy (SFCS) using continuous wave excitation to analyze membrane dynamics. The high count rate per molecule enables the study of very slow diffusion in model and cell membranes, as well as the application of two-foci fluorescence cross-correlation spectroscopy for parameter-free determination of diffusion constants. The combination with dual-color fluorescence cross-correlation spectroscopy with continuous or pulsed interleaved excitation allows binding studies on membranes. Reduction of photobleaching, higher reproducibility, and stability compared to traditional FCS on membranes, and the simple implementation in a commercial microscopy setup make SFCS a valuable addition to the pool of fluorescence fluctuation techniques.

Biological Transport, Active↗

Predictive inflammatory parameters in the diagnosis of acute appendicitis in children.

Acute appendicitis is the most common abdominal surgical emergency in children. In order to determine the related factors for early diagnosis of acute appendicitis in children, we analyzed retrospectively from January 2001 to December 2002 the hospital records of 260 consecutive patients under 18 years of age operated on because of suspected acute appendicitis. The patients group comprised 163 males (62.7%) and 97 females (37.3%) with a mean age of 11.1 +/- 4.3 years (range from 0 to 18 years old). The accurate diagnostic rate of acute appendicitis in children was 81.5 percent and negative appendicectomy rate was 18.5 percent. When combining leukocytosis, elevated neutrophil count and elevated C-reactive protein, the specificity and positive predictive value in diagnosing acute appendicitis were increased. According to the application of multivariate logistic regression analysis, the significantly related factors in acute appendicitis were elevated neutrophil count (Odds ratio = 3.79, 95% confidence interval = 1.732-8.293, p < 0.001) and elevated C-reactive protein concentration (Odds ratio = 2.81, 95% confidence interval = 1.231-6.390, p < 0.05). In conclusion, total neutrophil count and C-reactive protein may serve as predictive parameters for early diagnosis of acute appendicitis in children.

Acute Disease↗

The effectiveness of ultra-low-volume applications of malathion at a rate of 3 US fluid ounces per acre in controlling Aedes aegypti in Thailand.

Ultra-low-volume (ULV) aerial applications of malathion at a rate of 3 US fl oz per acre (219 ml/ha) for the control of Aedes aegypti and Culex pipiens fatigans were evaluated in 2 areas near Bangkok, Thailand. A housing project of the Royal Thai Air Force received 2 applications a week apart while the village of Bang Khen received 1 application. Bioassays, oil-sensitive red dye cards, landing-rate counts, ovitraps, and light-traps were used to evaluate the effects of the treatment. A comparison of pre- and post-treatment indices indicated good control of A. aegypti following all treatments. The results of these tests when compared with those of an earlier study using 6 US fl oz/acre (438 ml/ha) showed that the higher rate of application rate was more effective. Only fair control of C. p. fatigans was obtained. A comparison of light-trap collections before and after treatment demonstrated fairly good over-all control of other species of mosquitos for 3-4 days following each of the 2 treatments on the Air Force housing area.

Aedes↗

Utility of the three-part leukocyte differential count.

The utility of the automated three-part leukocyte differential count was evaluated in an acute care hospital setting. The manual method was specified in 92 of 223 (41%) requests for differentials on one day. For the others, 79 three-part differentials (60%) were completed; 19 (15%) were rejected for histogram abnormalities ("R-flags") or could not be computed; and 33 (25%) were rejected for out-of-range values that were later verified by slide review. The automated method missed 4 of 39 (10%) band elevations and 2 of 2 (100%) cases of eosinophilia reported on manual differentials, but those results had no apparent influence on patient management. In 98 cases of septicemia, automated and manual methods showed similar overall sensitivity (87% and 83%, respectively). Selectively combined with a qualitative slide review, the three-part differential was applicable to 84% of all specimens submitted for a differential count, with acceptable sensitivity and accuracy and substantial savings in personnel time.

Evaluation Studies as Topic↗

Exposure opportunity models for Agent Orange, dioxin, and other military herbicides used in Vietnam, 1961-1971.

Nearly 19.5 million gallons of herbicides were sprayed on the Republic of Vietnam between 1961 and 1971 for military purposes. Amounts of spray and patterns of applications are available in an electronic file called HERBS that contains records of 9141 defoliation missions, including detailed coordinates of US Air Force Ranch Hand aircraft flight paths, along with chemical agent and gallonage sprayed. Two classes of models for use in epidemiological and environmental studies that utilize the HERBS data for estimating relative exposure opportunity indices are presented: a discrete "hits" model that counts instances of proximity in time and space to known herbicide applications, and a continuous exposure opportunity index, E4, that takes into account type and amount of herbicide sprayed, distance from spray application, and time interval when exposure may have occurred. Both direct spraying and indirect exposure to herbicide (or dioxin) that may have remained in the local environment are considered, using a conservative first-order model for environmental disappearance. A correction factor for dermal versus respiratory routes of entry has been incorporated. E4 has a log-normal distribution that spans six orders of magnitude, thus providing a substantial amount of discrimination between sprayed and unsprayed areas. The models improve on earlier ones by making full use of the geometry of the HERBS spray flight paths of Ranch Hand aircraft. To the extent possible so many decades after the War, the models have been qualitatively validated by comparison with recent dioxin soil and biota samples from heavily contaminated areas of Vietnam, and quantitatively validated against adipose dioxin obtained in epidemiological studies of Vietnamese. These models are incorporated within a geographic information system (GIS) that may be used, as one would expect, to identify locations such as hamlets, villages, and military installations sprayed by herbicide. In a novel application, the GIS also facilitates quantitative risk assessment in epidemiological and ecological studies by applying the models within a framework of historical reconstruction of exposure history of individuals based upon their location histories.

2,4,5-Trichlorophenoxyacetic Acid↗

Pirenzepine prevents diethyl pyrocarbonate inhibition of central CO2 sensitivity.

Application by pledget of the M1-antimuscarinic receptor agent pirenzepine (40 mM) to the rostral chemosensitive areas of the ventrolateral medulla in anesthetized, paralyzed, vagotomized, glomectomized, and servoventilated cats inhibited the slope of the integrated phrenic response to CO2 by 32.5% (P less than 0.03) and the maximum value by 21.1% (P less than 0.01). Similar application of the imidazole-histidine blocking agent diethyl pyrocarbonate (DEPC) decreased the slope by 40.3% (P less than 0.01) and the maximum value by 29.3% (P less than 0.05). Both responses confirm previous results. DEPC treatment decreased the effectiveness of subsequent pirenzepine application such that although slope and maximum were further decreased, the values were not significantly different from those after DEPC. Pirenzepine treatment prevented any subsequent DEPC inhibitory effect. The results raise the possibility that the inhibitory effects of DEPC on CO2 chemosensitivity are via muscarinic receptors and that muscarinic receptor involvement in CO2 chemosensitivity requires the presence of imidazole-histidine. Analysis by scintillation counting of successive 100-micron sections of medulla after rostral area application of [3H]pirenzepine indicated that the pirenzepine and DEPC effects are most probably within 2.0 mm of the ventral surface as measured from the midline, well away from the dorsal and ventral respiratory group neurons.

Animals↗

Validation of the single-platform ISHAGE method for CD34(+) hematopoietic stem and progenitor cell enumeration in an international multicenter study.

BACKGROUND: Flow cytometric enumeration of CD34+ hematopoietic sterm and progenitor cells (HPC) is the reference point for undertaking apheresis and evaluation of adequacy for PBSC engraftment. An external quality assurance (EQA) scheme for CD34+ HPC enumeration has been operational in Belgium, Netherlands and Luxemburg (Benelux) since 1995. Within this group, a multicenter survey was held to validate the state-of-the-art methodology, i.e., multiparametric definition of HPC based on light scatter, expression of CD34 and CD45, and counting beads (i.e., 'single platform ISHAGE' method). METHODS: 'Real-time' EQA was used to monitor the application of the single-platform ISHAGE method by 36 participants. Three send-outs of stabilized blood with CD34+ cell counts 35-60 cells/microl were distributed to 36 participants, who were required to assay the samples on three occasions using the standard assay and their local techniques. These results were compared with thosed obtained by 111-116 UK NEQAS participants testing the same specimens. RESULTS: Using the single platform ISHAGE methods, between-laboratory coefficients of variations (CVs) as low as 10% were achieved. Intra-laboratory CVs were < 5% for approximately 50% of the participants. Local single-platform techniques yielded between-laboratory CVs as low as 9% in both Benelux and UK NEQAS cohorts. In contrast, the lowest between-laboratory CVs using dual-platform techniques were 17% (Benelux) and 21% (UK NEQAS), respectively. CONCLUSION: The single-platform ISHAGE method for CD34+ cell enumeration has been validated by an international group of 36 laboratories. The observed varation between laboratories allows a meaningful comparison of CD34+ cell enumeration.

Antigens, CD34↗

[Thyroid uptake error in four different gamma camera systems].

The thyroid uptake of (123)I and (131)I is generally measured by a gamma camera system. We evaluated the error in determining thyroid uptake caused by different methods of calculation among four gamma camera systems with various collimators. We first designed an original thyroid phantom that consisted of the thyroid and a body containing various levels of radioiodine activity. The applications for thyroid uptake equipped in two gamma camera systems performed calculations by the automatic method with background counts not subtracted from the capsule counts. When the size of the rectangular region of interest (ROI) for the capsule was set at 10x8 cm (a typical ROI size for the thyroid), percentages of thyroid uptake as calculated by the manual method with background counts subtracted from the capsule counts and thyroid counts were 52% to 57% when the value was set at 55% for (123)I; and 54.2% and 58.7%, respectively, when the value was set at 60% for (131)I. On the other hand, the percentages of thyroid uptake calculated by the automatic method with the application using two gamma camera systems with non-subtraction of background counts from the capsule counts were 46% and 50.5% when the value was set at 55%; and 49.6% when the value was set at 60%. The values calculated by the automatic method were underestimated as a result of background counts that were not subtracted from the capsule counts. When ROI size for the capsule was set at 4x4 cm, which is slightly larger than the capsule size, even thyroid uptake as determined by the automatic method using the application showed a difference of less 2% from the set values. There was no difference in thyroid uptake among the various kinds of collimators, high-resolution collimators, all-purpose collimators, and a suitable collimator for gamma-ray energy of (123)I.

Gamma Cameras↗

[Quantitative determination of fragment erythrocytes (schistocytes) in healthy subjects and patients after surgery].

Fragmented erythrocytes (schistocytes) were counted in blood subjects and from 50 patients who underwent various surgical procedures. The results showed to be as follows: 1. The average number of schistocytes in healthy controls of both sexes was 1-20/00 (medium in Thoma chamber 1.4%0/00, in blood smears 2.030/00, with a range from 0-50/00 and 0-60/00, respectively). 2. There was an increase in the number of schistocytes two hours after a surgical procedure (average 60/00), the degree of which correlated with the extent of the operation. Higher values of fragmented erythrocytes were counted after intraoperative blood transfusion (average 100/00). In most cases schistocyte values returned to normal at about 24 hours after surgery. 3. The highest schistocyte counts (18-350/00) were observed in two patients undergoing splenectomy with persistence of elevated counts for a longer period. 4. The average schistocyte number in stored blood of various age was 2.50/00. 5. Both counting of schistocytes in Thoma chambers and in blood smears are applicable, though the values obtained with the latter technique are slightly higher. Quantitative estimtion of schistocytes proves a useful method for evaluating disturbances in microcirculation.

Adult↗

Topical liposomal delivery of antibiotics in soft tissue infection.

A new drug delivery system (lipid microcarriers) was studied in an experimental model of infected soft tissue wounds. Superficial, nonlethal infection was produced in the adult rat by injecting 1 ml containing 10(8) colony-forming units (CFU) of Pseudomonas aeruginosa under the superficial fascia of the paraspinus muscle of a 2-cm2 excised wound. All wounds were dressed with N-Terface, a nonadherent wound material, and covered with Kontor sponge, an open-cell polyurethane sponge containing either normal saline (group I), free tobramycin (groups III and V), liposome-entrapped tobramycin (groups II and IV), silver sulfadiazene (group VI), or liposome-entrapped silver sulfadiazene (group VII). At 24, 48, and 72 hr postinjection, animals were sacrificed and colony-forming units of P. aeruginosa per gram of muscle tissue were determined. Group I had significantly higher colony-forming units of P. aeruginosa per gram than groups II and III at 48 and 72 hr and than groups IV and V at all times. One single dose of liposome-encapsulated silver sulfadiazine significantly decreased bacterial counts compared to untreated controls and, to a similar extent, compared to multiple applications of free drug. Colony-forming units in all treatment groups (II and III, IV and V, VI and VII) were similar at all time periods within equivalent dosages. The ability of one application of liposomal-entrapped antibiotics to result in a therapeutic effect that requires multiple applications of topically applied free antibiotics offers potential clinical advantages.

Administration, Topical↗

A modified murine local lymph node assay for the differentiation of contact photoallergy from phototoxicity by analysis of cytokine expression in skin-draining lymph node cells.

Since predictive differentiation of photoallergenic from phototoxic reactions, induced by low molecular weight compounds, represents a current problem, we tried to improve the differentiation between the two reactions by using a modified protocol of the local lymph node assay (LLNA). Briefly, groups of female BALB/c mice received compound solution or vehicle alone on the dorsum of both ears on 3 consecutive days. Immediately after compound application indicated groups of mice were exposed to a UVA light-dose of 10 J/cm2. Auricular lymph nodes draining the ear tissue were excised 24 h following the last exposure. Evaluation consisted of assessing lymph node weights and cell counts to monitor organ hyperplasia and in vivo-proliferative events following substance application. Furthermore, we analysed cytokine gene transcription in freshly prepared lymph node cells (LNC) and the cytokine release in vitro by restimulated CD4+ T-cells and antigen presenting cells (APC), both purified from the skin-draining lymph nodes. Both contact (photo) allergenic (oxazolone and tetrachlorosalicylanilide) and phototoxic substances (8-methoxypsoralen and acridine) caused a dose dependent increase in lymph node weights and cell counts pointing to an inflammatory process in the lymph nodes. Analysis of cytokine gene transcription ex vivo and cytokine release in vitro revealed that during the induction phase of contact (photo) allergy CD4+ T-cells produced IL-2 and IFN-gamma as well as IL-4 and IL-10, whereas IL-6 was derived from APC. In contrast, phototoxic reactions caused only an upregulation of IL-2 and IFN-gamma. Furthermore, we demonstrate that the release of IL-4 and IL-10 by CD4+ T-cells was clearly increased, whereas IL-6 and IFN-gamma expression was reduced or not changed following a challenge with contact (photo) allergens revealing an allergy-indicative shift in cytokine expression. In conclusion, our results show that contact photoallergenic reactions could be differentiated from phototoxic events by analysis of LNC cytokine expression patterns.

Animals↗

Therapeutic angiogenesis/arteriogenesis in the chronic ischemic rabbit hindlimb: effect of venous basic fibroblast growth factor retroinfusion.

Therapeutic induction of angiogenesis has been shown in experimental hindlimb ischemia. An alternative to targeting the ischemic hindlimb tissue via the severely stenosed or occluded artery consists in the intact venous system, e.g., by retroinfusion. We tested whether basic fibroblast growth factor (bFGF) enhances angiogenesis induction. Therefore, we applied bFGF retrogradely as compared to intramuscular application. Furthermore, we assessed whether bFGF-induced angiogenesis was enhanced by low-dose VEGF coapplication. Chronic hindlimb ischemia in rabbits was established by excision of the femoral artery at day 0 (d0). At d7, baseline collateral number in the ischemic limb and collateral flow velocity of contrast agent (frame count score) were assessed. Thereafter, saline solution (control group) or bFGF (20 microg/kg) with or without VEGF (10 microg/kg) was retroinfused through the femoral vein. Alternatively, bFGF (20 microg/kg) was injected into thigh and calf muscles. At d35, collateral growth and flow velocity were quantified, and tissue samples were analyzed for capillary density. In the untreated control group, capillary/muscle fiber (C/FM) ratio of the ischemic limb was 0.87 +/- 0.12, and collateral number as well as frame count score at -d35 did not change compared to d7 (107% +/- 7% and 109% +/- 10% of baseline, respectively). Retrograde application of bFGF induced capillary and collateral growth (C/FM ratio 1.56 +/- 0.19 and frame count 161% +/- 29% of baseline), resulting in enhanced flow velocity (143% +/- 13%), similar to the intramuscular application of bFGF. Additional low-dose VEGF retroinfusion did not further increase capillary/collateral growth (1.49 +/- 0.08 and 172% +/- 26%) nor perfusion velocity (149% +/- 7%). The authors conclude that bFGF retroinfusion is a feasible approach of inducing angiogenesis and arteriogenesis in an ischemic hindlimb, resulting in increased blood perfusion, which was not further extended by additional low-dose VEGF coapplication.

Animals↗

Gated TI-201 myocardial perfusion SPECT: application of energy window optimization.

PURPOSE: TI-201 scintigraphy is plagued with poor image quality because of the low-energy photons of TI-201 decay. Traditionally, a narrow 20% window centered on 71-72 keV has been used to improve sensitivity. Recent studies indicate that better imaging may be possible by optimizing the energy window to 34% centered on 77 keV. In this study, energy window optimization (EWO) was applied to gated TI-201 myocardial perfusion SPECT, and myocardial functional parameters were compared for gated TI-201 SPECT and gated Tc-99m sestamibi (Tc-99m MIBI) SPECT. METHODS: Count statistics for standard and optimal TI-201 myocardial scintigraphy were noted in 25 patients by assessing the total counts in a mid-ventricular slice of a rest-gated TI-201 myocardial SPECT study. The feasibility of performing functional studies with the application of EWO to TI-201 was assessed using the count statistics of a mid-ventricular slice of an optimized gated TI-201 SPECT study and a gated Tc-99m MIBI SPECT study. The functional parameters (ejection fraction, wall motion, and thickening) of TI-201 with EWO and Tc-99m MIBI were compared in 60 patients who underwent rest-gated TI-201 SPECT followed by poststress gated Tc-99m MIBI SPECT. The left ventricular ejection fraction was calculated using commercially available software, whereas wall thickness and motion were assessed by the consensus of two readers. RESULTS: The application of EWO increased available counts by more than 25%. It also resulted in sufficient counts being available to perform gated TI-201 SPECT without increasing acquisition times or the dose of TI-201. The average ejection fraction was 60.4% for gated TI-201 SPECT and 59.6% for gated Tc-99m MIBI SPECT (not significantly different). Overall, the image quality was rated excellent in 12% for TI-201 and Tc-99m MIBI and good in 50% and 62%, respectively, and poor in 38% and 26%, respectively. CONCLUSION: The application of EWO to TI-201 SPECT allows myocardial functional parameters to be assessed without having to increase the acquisition times or the administered dose of TI-201.

Adult↗