Automated cytofluorometry of solid malignant tumors after cell dispersal by means of intraperitoneal cultivation of cell complexes in young rats (author's transl)]

The use of isolated cells is a basic requirement besides data processing techniques to obtain optimum results with the automated flow cytofluorometry. Several techniques have been employed in order to get cell dispersal. But the claim for reproducible and representative cell samples cannot exactly be fulfilled: gross parts of cells are damaged by any dispersal method applied on solid tissues. Therefore we want to introduce a special method for cell isolation, which avoids mechanical and chemical damage of the treated cells. For this the intraperitoneal tolerance of growing tumor cells in young animals was used, in the species of which solid growth of such tumor cells is established. The investigations reported here were carried out with the Walker-tumor 256 on the rat as a model for solid tumor growth. Our interest was focused on the determination of cellular CNA distribution in tumor cells and contaminating somatic cells grown in the ascitic fluids. Material and Methods: Walker-tumors are permanently cultivated on Sprague-Dawley rats. The solid tumors (1 cm diameter) were removed under sterile conditions, cut with scissors, and resuspended in 0.9% saline. Ten rats (37 days old, body weight 90-120 g) had received an intraperitoneal injection of 1 ml Liquemin (5000 Roche). One million tumor cells could be obtained by such a procedure. After spontaneous sedimentation of cells in order to reduce the number of erythrocytes, the cells were processed and fixed according to Trujillo and Van Dilla (1972), digested with heated RNase (1 mg/ml tris-buffer; 0.18 M and pH 7.5), pepsin treated, and then stained with ethidium bromide (40 mug/ml tris-buffer) for 30 min in the cold. After rinsing with distilled water several times the cells were resuspended in buffer and measured within an hour immediately after passing a 10 mum filter to avoid cell aggregates...     

A computer-interfaced photometer and systematic spacing of duplicates to control within-plate enzyme-immunoassay variation

A Multiskan photometer for reading microtiter plate enzyme immunoassays was linked with a time sharing computer to facilitate control of assay variation and analysis of results. The interface that converted photometer output to RS-232-C format required changes to divide the output into segments short enough for input to the computer. To measure within-plate variation and investigate how the method of allocating sample duplicates to plate wells may affect the estimation of sample variance, uniformity tests were conducted with 47 plates. Coefficients of variation (CV) among wells within-plates ranged from 4.6 to 20.7% and in two-thirds of the plates exceeded 10%. Duplicates allocated to adjacent wells (method 1) gave consistently higher CV for sample means than duplicates allocated to opposite plate quadrants (method 2). In general, the CV by method 2 was about 30% smaller than that by method 1. Analysis of variance confirmed the effectiveness of the quadrant pattern of duplicate allocation as a method of controlling variation that arises from well position effects.     

Absence of fibula and ulna with oligodactyly, contractures, right-angle bowing of femora, abnormal facial morphology, cleft lip/palate and brain malformation in two sibs: a possibly new lethal syndrome

Absence of fibulae, and unilateral absence of ulna, associated with lateral oligodactyly, and rectangular bowing of the femora are the prominent features of a lethal syndrome studied in two children of related parents. Other malformations are cleft lip and palate and posterior midline abnormalities of the brain. One similar though not identical MCA-syndrome was reported. The literature is reviewed with respect to ulno-fibular malformations.     

Abeta-degrading endopeptidase,neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology

Metabolism of amyloid-beta peptide (Abeta) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Since neprilysin is the only rate-limiting catabolic peptidase proven by reverse genetics to participate in Abeta metabolism in vivo, we performed detailed immunohistochemical analysis of neprilysin in mouse brain using neprilysin-deficient mice as a negative control. The aim was to assess, at both the cellular and subcellular levels, where Abeta undergoes neprilysin-dependent degradation in the brain and how neprilysin localization relates to Abeta pathology in amyloid precursor protein (APP)-transgenic mice. In hippocampus, neprilysin was present in the stratum pyramidale and stratum lacunosum-moleculare of the CA1-3 fields and the molecular layer of the dentate gyrus. Confocal double immunofluorescence analyses revealed the subcellular localization of neprilysin along axons and at synapses. This observation suggests that after synthesis in the soma, neprilysin, a type II membrane-associated protein, is axonally transported to the terminals, where Abeta degradation is likely to take place. Among various cell types, GABAergic and metabotropic glutamate 2/3 receptor-positive neurons but not catecholaminergic or cholinergic neurons, expressed neprilysin in hippocampus and neocortex, implying the presence of a cell type-specific mechanism that regulates neprilysin gene expression. As expected, Abeta deposition correlated inversely with neprilysin expression in TgCRND8 APP-transgenic mice. These observations not only support the notion that neprilysin functions as a major Abeta-degrading enzyme in the brain but also suggest that down-regulation of neprilysin activity, which may be caused by aging, is likely to elevate local concentrations of Abeta at and around neuronal synapses.     

Exploring cancer register data to find risk factors for recurrence of breast cancer – application of Canonical Correlation Analysis

Background  

A common approach in exploring register data is to find relationships between outcomes and predictors by using multiple regression analysis (MRA). If there is more than one outcome variable, the analysis must then be repeated, and the results combined in some arbitrary fashion. In contrast, Canonical Correlation Analysis (CCA) has the ability to analyze multiple outcomes at the same time.     

Dynamic responses of oxygen uptake at the onset and end of moderate and heavy exercise in trained subjects.

Inconsistencies about dynamic asymmetry between the on- and off-transient responses in VO2 are found in the literature. Therefore the purpose of this study was to examine VO2 on- and off-transients during moderate- and heavy-intensity cycling exercise in trained subjects. Ten men underwent an initial incremental test for the estimation of ventilatory threshold (VT) and, on different days, two bouts of square-wave exercise at moderate (VT) intensities. VO2 kinetics in exercise and recovery were better described by a single exponential model (VT). For moderate exercise, we found a symmetry of VO2 kinetics between the on- and off-transients (i.e., fundamental component), consistent with a system manifesting linear control dynamics. For heavy exercise, a slow component superimposed on the fundamental phase was expressed in both the exercise and recovery, with similar parameter estimates. But the on-transient values of the time constant were appreciably faster than the associated off-transient, and independent of the work rate imposed (VT). Our results do not support a dynamically linear system model of VO2 during cycling exercise in the heavy-intensity domain.     

P-selectin inhibition suppresses muscle regeneration following injury

This investigation sought to determine if P-selectin-mediated mechanisms contributed to macrophage localization in damaged muscle, an essential process for muscle regeneration. Mice were injected intravenously (i.v.) with soluble P-selectin glycoprotein ligand-1 (sPSGL-1) at 5, 50, or 200 microg/mouse or with 100 microl vehicle alone, and then, lengthening contractions were induced in hindlimb plantar-flexor muscles. The contractions caused fiber damage in soleus muscles, with maximal invasion by CD11b+ mononuclear cells at 24 h post-injury and substantial accumulation of CD11b+ mononuclear cells in the extracellular matrix up to 7 days post-injury. sPSGL-1 treatment caused a dose-dependent decrease in the number of regenerating fibers (P=0.021), as determined by developmental myosin heavy chain (dMHC) expression. This expression was reduced 93% at 7 days post-injury by the highest dose of sPSGL-1, which had no significant influence on intrafiber or extracellular accumulation of cells expressing CD11b, a general marker for phagocytic cells. Additional mice were injected i.v. with 20 microg anti-P-selectin or isotype-control immunoglobulin G and were then subjected to lengthening contractions as before. At 7 days post-injury, soleus muscles from anti-P-selectin-treated mice contained 48% fewer mononuclear cells that bound ER-BMDM1 (P=0.019), a marker for mature macrophages and dendritic cells, and 84% fewer fibers expressing dMHC (P = 0.006), compared with muscles from isotype-injected, control mice. The number of CD11b+ cells was not significantly different between groups. The results are consistent with the concept that P-selectin is involved in the recruitment, maturation, and/or activation of cells that are critical for muscle fiber regeneration.     

Accuracy of reporting endocervical component adequacy--a continuous quality improvement project

Inaccurate reporting of the absence of an endocervical (EC) component on Pap smears often results in slide rescreens, amended reports, clinician dissatisfaction, and sometimes unnecessary repeat smears. Therefore, the accuracy of reporting EC component adequacy was selected as a quality indicator for the laboratory continuous quality improvement program (CQI). The process consisted of problem identification, analysis of the situation, collection of data, implementation of solutions, and evaluation of results. The objective of the study was to determine if the accuracy of reporting EC component adequacy on Pap smears improved after application of such a program. During the first phase, 150 Pap smears originally reported with the absence of an adequate EC component and 150 smears reported with the presence of an adequate EC component were rescreened to measure the baseline accuracy of EC component adequacy reporting. The improvement process was then implemented. A cause-and-effect diagram was developed and root cause was determined. A presentation was then made to the cytology staff. Criteria for EC component adequacy were reviewed, examples were shown, and standardized marking of EC component was implemented. Following improvement actions, a second audit of 150 Pap smears reported with the absence of an adequate EC component as well as 150 smears reported with the presence of an adequate EC component was undertaken to measure change in performance in assessing EC component adequacy. For the baseline rescreening, before initiation of the CQI program, 98% accuracy was achieved with smears that were reported as adequate for EC component present. However, the accuracy with smears reported as absence of an adequate EC component was only 71%, i.e., an adequate EC component was identified in almost 1/3 of these cases on rescreen. After the implementation of improvement actions, the accuracy with smears reported with the presence of EC component remained high (98%) and the accuracy of reporting the absence of EC component was 90%. The difference of the latter before and after the implementation was statistically significant (P = 0.015, z-test). The accuracy of reporting EC component adequacy increased following the CQI process. Using reporting EC component adequacy as an example, we demonstrate that by treating clinical problems as quality control issues and applying basic quality improvement tools, a positive outcome can be effected.