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PubMed1982

Isolation and characterization of inflammatory cells from the human periapical granuloma.

Twelve histologically-confirmed periapical granulomas were evaluated by conventional immunologic rosette assays for the presence of T-lymphocytes and complement receptor-bearing lymphocytes. A technique for dispersing the granuloma cells into suspensions was adopted to facilitate performance of the assays which were not applicable to tissue sections. Differential cell counts by an acridine orange vital dye method disclosed that the cell suspensions contained 30% macrophages, 44% lymphocytes, 15% plasma cells, and 12% neutrophils. Complement receptor-bearing cells comprised 17.9%, and T cells comprised 34.5% of the unseparated inflammatory cells. This study provides the first direct evidence of a predominance of thymic-derived lymphocytes in the lymphocyte compartment of the periapical granuloma. Analysis of the data shows that cell-mediated immunity most likely plays a role in the pathogenesis of the periapical granuloma.

Cell Count↗
PubMed1999

Anatomical and functional connectivity of the transected ulnar nerve after intercostal neurotization in cats.

OBJECT: Acute transfer of three intercostal nerves to the ulnar nerve was performed in cats for histological and clinical evaluation of a distal muscle reinnervation. METHODS: Infraclavicular intercostal-ulnar communications were created after dividing the motor branches of the upper intercostal nerves in 14 adult cats. Reinnervation of distal forelimb muscles in the ulnar territory was assessed by electromyographic (EMG) studies and motor function rating each month until 18 months postsurgery. In five of these treated animals, and in tour controls, horseradish peroxidase (HRP) was applied to the ulnar or intercostal nerves to study the amount and distribution of retrograde motor neuron labeling in the spinal cord. Also, samples of reinnervated muscles and neurotized ulnar nerves were processed to assess regeneration. Simple ulnar transection without reconstruction led to permanent atrophy of ulnar muscles, lack of recovery according to EMG or clinical studies, and disappearance of the ulnar motor neuron pool. In contrast, ulnar neurotization with the intercostal nerves led to a high rate of functional recovery, which began 5 months postsurgery, and progressed from muscle activity synchronized with ventilatory movements to spontaneous movements that were independent of respiration. This recovery was accompanied by substantial retrograde labeling of intercostal motor neurons after HRP application in the ulnar nerve. Cell counts showed that practically the whole motor neuron pool of the involved intercostal nerves contributed to reinnervation of the transected ulnar nerve. CONCLUSIONS: These findings demonstrate that the use of intercostal nerves to neurotize long brachial plexus nerves can achieve long-lasting and successful reinnervation of distal forelimb muscles.

Animals↗
PubMed1992

Rapid measurement of protein kinase and phosphatase activities by slot-filtration.

A slot-filtration method has been developed for the detection and quantitation of protein kinase and phosphatase activities. In this technique, after kinase-dependent phosphorylation or phosphatase-dependent dephosphorylation of different substrates, samples are transferred under vacuum onto nitrocellulose using a slot-blotting apparatus. Non-incorporated or released radioactivity is then removed by filtration and washing under vacuum. Quantitation is performed by scintillation or Cerenkov counting of the excised membrane slots. Application of the method to the assay of four different protein kinases (protein kinase N, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinases type I and type III) and one phosphatase is presented. A number of protein substrates with varying molecular masses and isoelectric points were found suitable for the slot-filtration technique. The method is applicable to impure as well as purified kinase and phosphatase preparations, can be used over a wide range of concentrations of substrates, has a very low background of nonspecific ATP binding and provides highly reproducible data. The slot-filtration method can also be adapted for use with ion-exchange paper, particularly for assays using peptides as substrates. The technique, with either nitrocellulose or ion-exchange paper, can be used to rapidly process large numbers of samples and can be simultaneously applied to direct comparison of different kinases, phosphatases and/or substrates in the same experiment.

Animals↗
PubMed2005

Genetically engineered mouse melanoma and mouse Lewis lung carcinoma models.

Melanoma B16-F10 cells and Lewis lung carcinoma LL/2 cells were engineered with a bacterial gene -- chloramphenicol acetyl transferase (CAT) -- by establishing stable transductants. Expression of CAT in both cell types did not alter the ability of these cells to grow into tumors when injected subcutaneously into mice. In addition, the measurement of CAT levels in the lung using a simple ELISA assay revealed a close correlation with direct counting of metastatic nodules. Thus, the CAT-expressing cells will likely have wide ranging applications to quantify tumor metastasis especially in situations where visual counting is difficult. The availability of genetically labeled mouse B16-F10 melanoma and Lewis lung carcinoma cell lines will facilitate future studies of the mechanism and progression of cancer and the discovery of new therapies.

Animals↗
PubMed1985

First-third ejection fraction: is the first-pass radionuclide method accurate?

Previous reports have suggested that left ventricular first-third ejection fraction (EF) can be obtained from the left ventricular time-activity curve derived from first-pass radionuclide angiography based on Anger camera data. The validity of this technique was assessed by: a study of beat-to-beat variations in data from 15 patients in which electrocardiographic data were simultaneously recorded, and a computer simulation incorporating the application of Poisson statistics to appropriate count rate data. The results of patients studies showed no consistent trend in any first-third parameter obtained from consecutive beats in individual subjects, and unacceptably high statistical uncertainty in the calculation of the first-third ejection fraction. The weighted standard deviation of the first-third ejection fraction in each of 15 patients studied averaged 7.5 EF units, while first-third ejection fraction averaged 22.9 EF units. The relative error averaged 32%. The computer simulation indicated a high relative error of 47% associated with the first-third ejection fraction at typical end-diastolic count rates of 200 per frame from 1,000 computer Poisson randomizations of an appropriate analog volume curve. The results render the first-pass radiocardiographic method invalid for determining first-third ejection fraction.

Adult↗
PubMed1984

Two-dimensional filtering of SPECT images using the Metz and Wiener filters.

Two-dimensional filtering, both before and after reconstruction, has been applied to the processing of single photon emission computerized tomographic (SPECT) images. The filters investigated were the count-dependent Metz filter and Wiener filter, both of which automatically adapt to the image being processed. Using a SPECT phantom, with images reconstructed with these filters rather than the ramp, we observed a statistically significant increase (p less than 0.05) in the image contrast for solid Plexiglas spheres, and significant decrease (p less than 0.05) in the percent fractional standard deviation of counts in a region of uniform activity. The adaptability of these filters is demonstrated by a comparison of SPECT acquisitions of the phantom at two different count levels. An example of their application to clinical studies is presented. We conclude that two-dimensional digital image restoration with these techniques can produce a significant increase in SPECT image quality, with a small cost in processing time when these techniques are implemented on an array processor.

Filtration↗
PubMed1977

Organ distribution of 99mTc- and 51Cr-labeled autologous peripheral blood lymphocytes in rabbits.

The present series of experiments was designed to compare 99mTc with 51Cr as a lymphocyte label in rabbits and to correlate organ distribution patterns as determined by postmortem counting with those observed by 99mTc gamma imaging. The distribution of 51Cr and 99mTc-labeled, viable and nonviable, unreduced or SnCl2-reduced autologous lymphocytes was determined 2 hr following intravenous injection. Viable 99mTc-labeled lymphocytes localized primarily in the liver, lungs, and kidneys or remained circulating in the blood. These compartments accounted for 34-54% of the injected radioactivity. One to three percent of the injected dose was recovered from the spleen, thyroid, bladder, and stomach, and all remaining organs accounted for less than 1%. Reduced, nonviable cells showed increased localization in the lungs and liver (48%) compared to viable unreduced cells (15%). Chromium-51-labeled lymphocytes were distributed primarily to the liver, lungs, and spleen or remained circulating in the blood. Hepatic localization differed from that observed with viable, unreduced 99mTc-labeled lymphocytes (32 vs. 12%), but vascular compartmentalization was similar (18 vs. 15%). The distribution of pertechnetate and reduced 99mTc was different from that of 99mTc-labeled cells. The urine and blood accounted for 84% of the recovered radioactivity, and for 43-45% of the injected dose, indicating that the patterns we observed with 99mTc-labeled lymphocytes truly represented cell-associated radioactivity. Similarly, the distribution of Na251CrO4 differed from that of 51Cr-labeled cells. The kidneys and urine accounted for 26% of the injected dose, compared with 5% for 51Cr-labeled lymphocytes, and only trace amounts of radioactivity were found in the lungs and spleen. The distribution of 99mTc-labeled cells, as determined by scintigraphy 90 min after injection, correlated well with data obtained by postmortem counting, suggesting that 99mTc may be applicable as a lymphocyte label for diagnostic gamma-imaging procedures.

Animals↗
PubMed1995

Challenging consults: application of principles of physiology and biochemistry to the bedside. Osmotic diuresis: the importance of counting the number of osmoles excreted.

Polyuria is usually the result of a water diuresis or an osmotic diuresis. Traditionally, the assessment of the extracellular fluid (ECF) volume and the concentration of Na+ in plasma is sufficient to differentiate between the two. We present a case and our approach, which is based on calculations and quantitation of osmoles, to demonstrate the utility of this approach. A patient with diabetes mellitus, human T-cell lymphocyte virus, type 1 (HTLV-1) associated lymphoma, and hypercalcemia presented with marked ECF volume contraction and polyuria. A spot urine osmolality was 567 mOsm/kg H2O in the face of urine output of approximately 6 L/d. The initial diagnosis was an osmotic diuresis. However, a quantitative analysis revealed the enormous number of osmoles could not be accounted for physiologically. Hence, we postulated a water diuresis to be the cause of the polyuria. To confirm this hypothesis, we found that at different times during his hospitalization, the urine specific gravity ranged from 1.005 to 1.022, and urine output varied markedly over 8-h periods. Despite a plasma sodium of 147 mmol/L, the patient did not complain of thirst. Taken together, this suggested the presence of a hypothalamic lesion which caused central diabetes insipidus with variable output of antidiuretic hormone together with a blunted thirst response. Illustration of the utility of a quantitative approach to polyuria is the focus of the discussion.

Chlorides↗
PubMed2003

[The application of dual-color fluorescence in situ hybridization to the diagnosis of Klinefelter syndrome].

The objective of the work is to study the technique of dual-color fluorescence in situ hybridization(D-FISH) and its application value in the diagnosis of sex chromosomal count abnormality Klinefelter syndrome and establish an experimental approach to metaphase chromosome and interphase nucleus FISH technique. Biotin labeled alpha satellite X-chromosome DNA(pBamX7) probe and Digoxigenin labeled Y-chromosome long arm terminal repetitive sequence (pY3.4) probe were hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus in 19 cases of Klinefelter syndrome specimens. After being washed,the slides were treated with Avidin-FITC,Rhodamine-FITC and Anti-avidin,amplified with an additional layer and counter-stained with DAPI in an antifade solution. The hybridization signals,chromosomal or interphase nucleus settings were observed respectively with WIB, WIG and WU filters under fluorescence microscope Olympus AX-70,and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted. It was observed under the microscope that the Biotin labeled pBamX7 probe showed 2 green hybridization signals and that the Digoxigenin labeled pY3.4 probe showed 1 red hybridization signal. Chromosome or interphase nucleus counter-stained with DAPI showed blue. The average signal rate of chromosome and interphase nucleus hybridization was 95.89% and 95% respectively,significantly higher than the normal control (2.75%). Karyotype 47,XXY was confirmed,which agrees with the chromosomal findings. One case showed mosaic nuclei. XXY chromosome hybridization signal rate was 92% and XY hybridization signal rate was 6.7%, higher than the normal control rate of 4.17%. FISH is a valuable technique in diagnosing sex chromosomal count abnormality Klinefelter syndrome with the merits of fast speed, high sensitivity, strong signal,low background and multiple color. Therefore, FISH technique can find wide application and potential in prenatal diagnosis.

English Abstract↗
PubMedDate not supplied

Applications of low-light imaging to life sciences.

Photon imaging is a new technique for the quantitative analysis of bioluminescence and chemiluminescence and can be performed both at the macro and micro levels. The high sensitivity and spatial resolution of photon-counting cameras have resulted in the development of new applications in the life sciences. At the macro level, imaging is a valuable tool for the rapid identification of biological samples emitting long-lasting glows in assays using microtitre plates or filter formats (immunoassays, DNA probes, phagocytosis, gene expression, metabolite and drug analysis) and also for in vivo studies of promoter activity. At the micro level, low-light imaging can be used for analysing multiple analytes on micro sensors) and for advanced cell analysis (immunocytology, in situ hybridization, identification of cells or tissues expressing the luciferase gene, intracellular or intercellular protein traffic, metabolite analysis and imaging of Ca2+ flux and phosphorylation reactions). Two-dimensional photon-counting instrumentation is a versatile and powerful research tool for imaging and is complementary to conventional luminometers. The main applications to the life sciences involve many types of luminescence assays and can be performed on multiple samples in standard and non-standard formats. Photon-counting coupled to imaging is very helpful in selecting microorganisms or cells expressing bioluminescent genes. Measurements can be made in vitro and in vivo with a sensitivity comparable to that of phototube luminometers.

Animals↗
PubMedDate not supplied

Comparison of an antimicrobial adhesive drape and povidone-iodine preoperative skin preparation in dogs.

The antimicrobial efficacy of an adhesive drape applied after a 1-minute alcohol scrub was compared to a povidone-iodine (PI) skin preparation technique in dogs. Each technique was applied to both sides of 15 adult anesthetized dogs on premeasured, clipped areas of skin. Skin bacteria were quantified before, immediately after, and 1 hour after skin preparation. Predominant skin bacteria were isolated by swabbing the skin. The percentages of bacterial reduction immediately after and 1 hour after skin preparation, percentages of negative culture results, cultures with more than five colony-forming units, and the frequency of skin reactions were calculated and analyzed statistically. Drape adhesion was assessed subjectively. The percentage reduction in skin bacteria was significant for both techniques and comparable to that reported in humans. The adhesive drape was significantly less effective in both the immediate and 1-hour periods. Lift occurred in 66% of drape applications but was not associated with high bacterial counts. Acute contact dermatitis was more frequent after skin preparation with PI. There was no difference between the techniques in recovery of potential skin pathogens. The authors conclude that application of this antimicrobial adhesive drape after a 1-minute alcohol scrub is not as effective in the reduction of skin bacteria in dogs as is PI preparation of the skin.

Adhesives↗
PubMed1985

Quantitation of hepatitis B virus DNA (HBV DNA) in serum using the spot hybridization technique and scintillation counting.

The simple spot hybridization technique to detect HBV DNA in serum was modified to concentrate Dane particles by pelleting. This minimised interference by serum components and allowed larger volumes of sera (up to 500 microliters) to be used in serial dilution on borderline positive samples and increased the efficiency of filtering through a "Hybri.Dot' system. Quantification of HBV DNA by 32P-scintillation Cerenkov counting based on serial standards of cloned HBV DNA processed through the "Hybri.dot' was easy to perform and the assay had good sensitivity (detection limit 2 pg, mode 6.25 pg), precision and accuracy. The pellet-simple spot quantitative assay proved more sensitive than those involving lengthier extraction procedures or direct application of serum to hybridization filters. Scintillation counting curves were linear over a wider range (0-800 pg HBV DNA) than optical densitometry measurements (0-50 pg) of autoradiographs. There was an excellent correlation with DNA polymerase activity (r = 0.948; P less than 0.001) but the assay proved more sensitive since HBV DNA (greater than 45 pg cloned equivalents) was detected in 4 out of 14 DNA polymerase negative sera and in 7 of 7 borderline (DNA polymerase cpm range 116-303) samples. This sensitive, quantitative HBV DNA assay should be of considerable value in studies on infectivity and effectiveness of antiviral therapies.

DNA, Viral↗
PubMedDate not supplied

Survival of faecal coliforms and hygiene risks in soils treated with municipal sewage sludges.

An incubation experiment was conducted to monitor effect of sewage sludge application on changes in numbers of faecal coliforms in soils over time after sludge application and evaluate the hygiene risks. Soil faecal coliform counts were made after 1, 7, 14, 28, 56 and 84 days of incubation. The faecal coliform counts in the sludge-treated soils decreased substantially with time and were similar to those in the untreated controls after incubation for 56 days. Land application of air-dried sludges increased the hygiene risks due to the re-growth of faecal coliforms, and the counts of faecal coliforms in soil treated with air dried sludge from Suzhou (91% DM) were 50 times higher than in soils with fresh dewatered sludge from Suzhou (15% DM) after 7 days of incubation. The main factors affecting the changes in faecal coliform counts were sludge type and incubation time. Sludge type determined the faecal coliform counts and the ability of the faecal coliforms to re-establish, and indigenous microorganisms competed with the faecal coliforms for nutrients during the incubation process.

Animals↗
PubMed1993

[Subpopulation of leucocytes in the semen from fertile and infertile men--immunohistochemical and flow-cytometric analysis].

It is hard to differentiate leucocytes from immature germ cells among many round cells in ejaculated semen. Recently monoclonal antibodies (MoAbs) which recognize surface antigen of various subtypes of leucocytes are available. In this study we applied a panel of MoAbs for leucocyte subpopulations (granulocytes, T-lymphocytes, B-lymphocytes, Monocytes/Macrophages and pan-leucocytes). Semen smears from 64 mild oligozoospermic patients and 5 fertile volunteers were stained by immunohistological technique (avidin-biotin-immunoperoxidase method). We found highly varying total leucocyte numbers ranging from 1200 to 2.2 x 10(8) per ejaculate. When comparing semen from infertile patients with those from fertile donors, statistically significant higher medians of total leucocytes, total granulocytes and monocytes/macrophages were seen in infertile group. We compared a total number of leucocytes obtained from conventional Papanicolaou staining with that from immunohistochemical staining. Both were almost identical but the former gave a little bit higher value, because of the difficulty in differentiating leucocytes from immature germ cell by Papanicolaou staining. Moreover we performed an experiment to evaluate the applicability of flow-cytometry (FCM) to count leucocytes in semen. Cells in the semen smears were stained with MoAbs recognizing the surface antigen shared by every leucocyte, and percentage of the leucocytes which were thus stained with MoAbs was compared with that determined by FCM. Results from FCM constantly gave lower value than these from immunostaining. This is probably because FCM could not differentiate leucocytes from contaminated non cell substances (e.g. cell debris). From the present study it is not adequate to use FCM for the detection of leucocytes in semen as applied in haematology. Further techniques to remove non-cellular substances should be established for its application in semen analysis.

Antibodies, Monoclonal↗
PubMed2005

Melanocytic nevi in very young children: the role of phenotype, sun exposure, and sun protection.

BACKGROUND: Melanocytic nevi are strongly associated with cutaneous melanoma, yet little is known about factors influencing nevus development in the first years of life. OBJECTIVE: We sought to identify phenotypic and environmental factors associated with nevus counts in very young children. METHODS: In a cluster prevalence survey, full body nevus counts and phenotypic assessments were conducted on 193 children aged 1 to 3 years. Information on each child's sun exposure and sun protection practices was obtained through parental questionnaire. RESULTS: High total nevus counts were associated with heavy facial freckling, time spent outdoors on weekends in summer, and Caucasian ethnicity. Low nevus counts were associated with dark skin color, ability to tan, and frequent application of sunscreen. Frequent wearing of hats was specifically associated with low nevus counts on the face, but not at other sites. CONCLUSIONS: Nevi are common at a very young age among children in Queensland, Australia, and are associated with sun exposure and freckling. Diligent sun protection practices appear to reduce nevus burden, even after accounting for the effects of phenotype and sun exposure factors. Primary prevention strategies aimed at reducing sun exposure in very early life may be effective in reducing nevus prevalence and melanoma risk.

Australia↗
PubMedDate not supplied

On walks in molecular graphs.

Walks in molecular graphs and their counts for a long time have found applications in theoretical chemistry. These are based on the fact that the (i, j)-entry of the kth power of the adjacency matrix is equal to the number of walks starting at vertex i, ending at vertex j, and having length k. In recent papers (refs 13, 18, 19) the numbers of all walks of length k, called molecular walk counts, mwc(k), and their sum from k = 1 to k = n - 1, called total walk count, twc, were proposed as quantities suitable for QSPR studies and capable of measuring the complexity of organic molecules. We now establish a few general properties of mwc's and twc among which are the linear dependence between the mwc's and linear correlations between the mwc's and twc, the spectral decomposition of mwc's, and various connections between the walk counts and the eigenvalues and eigenvectors of the molecular graph. We also characterize the graphs possessing minimal and maximal walk counts.

Journal Article↗