Different calculation methods for flow cytometric S-phase fraction: prognostic implications in breast cancer? The Swedish Society of Cancer Study Group.

S-phase fraction (SPF), estimated in the flow cytometric DNA histogram, is a prognostic factor in breast cancer. There are, however, some inherent difficulties in the estimation of SPF, such as the influence of debris, aggregates, and normal cells. Most of the available SPF calculation principles try to consider these difficulties, but so far no consensus has been reached with regard to which principle is to be recommended. The aim of the present study was to investigate the prognostic impact of SPF when estimated with different calculation methods in frozen breast cancer samples from 350 patients. Two nonparametric (Rman, Rmin/both rectangle) and three parametric (ACAS/DNA-base, ModFit, and MultiCycle) calculation methods, with and without correction for debris and aggregates, were used. The mean values for SPF varied from 4.3% (ACAS/DNA-base with correction for debris and aggregates) to 9.4% (MultiCycle without any correction for background). The pairwise correlation between methods varied considerably (R = 0.72-0.98). After categorization of SPF values into low SPF (lower two tertiles) and high SPF (upper tertile), all methods yielded statistically significant Pvalues for recurrence-free survival (median follow-up time 67 months), both univariately (0.0004-< 0.0001) and multivariately (0.048-0.0004), after adjusting for nodal status, tumor size, and estrogen receptor status. SPF with background correction did not yield lower P values than SPF without. Regardless of which method was used, SPF showed similar correlations with lymph node involvement, tumor size, and estrogen receptor content. In conclusion, as the mean value of SPF for different calculation methods varies, each laboratory must be restricted to use only one method. Background correction does not seem to improve the prognostic impact of SPF in DNA histograms. Based on the experiences obtained in the present study, S-phase calculation methods without background correction may therefore be the most suitable for routine evaluation of DNA histograms of fresh frozen breast cancer material (ModFit, MultiCycle, and Rman [the latter only for experienced operators]). The nonparametric Rmin, with an automatic setting of the region used for SPF calculation, may be an alternative, but suffers from the disadvantage of not being commercially available yet.

Ambulatory infusion of noradrenaline for long-term treatment of Shy-Drager syndrome.

A 70-year-old female patient with advanced Shy-Drager syndrome exhibited severe orthostatic hypotension, low serum catecholamine levels, and autonomic dysfunction. She was bedridden despite oral medication with fludrocortisone, etilefrin, dihydroergotamine, L-dopa, yohimbine, and amezinium methyl sulfate. Only intravenous application of noradrenaline (30 ng/kg/min) provided complete mobilization. After implantation of a port-a-cath system, intravenous noradrenaline treatment could be continued on an outpatient basis. Over the following 5 years, the patient was throughout sufficiently mobile and did not show any significant side effects of this treatment. However, during the 5th year she suffered from nonhemorrhagic brain stem infarction due to cerebral hypoperfusion after orthostatic stress in the absence of noradrenaline infusion. We conclude that ambulatory noradrenaline infusion is a new valuable tool for long-term treatment of advanced Shy-Drager syndrome.

Beta-adrenoceptors in the tree shrew brain. II. Time-dependent effects of chronic psychosocial stress on [125I]iodocyanopindolol bindings sites.

1. Stress in known to affect the functioning of the central noradrenergic system in a region-specific manner. The aim of the present investigation was to understand better the consequences of recurrent stressful experiences on central beta-adrenoceptors. 2. Alterations in the central nervous beta-adrenoceptor system resulting from different periods of psychosocial stress (PSS) were analyzed in male tree shrews (Tupaia belangeri) which were submitted to subordination stress for varying time periods. 3. In the first experiment, the whole number of beta-adrenoceptors was analyzed in the forebrains of subordinate animals and controls by in vitro autoradiography using 125I-iodocyanopindolol (125ICYP), while nonspecific binding of the radioligand to serotonin receptors was blocked with 100 microM 5HT. 4. PSS affects beta-adrenoceptors in a time-dependent manner. A decrease in receptor affinity occurred after just 21 days of PSS in cortical areas and in the hippocampus, indicating stress effects on the conformation of beta-adrenoceptors. After 30 days of PSS, the numbers of beta-adrenoceptors were significantly decreased in several cortical regions and in the olfactory area. 5. In the second experiment, we investigated the influence of PSS on both beta 1- and beta 2-adrenoceptors separately. 125ICYP binding was quantified in the presence of either ICI188.551 to block beta 2-adrenoceptors or in the presence of CGP20712A to block beta 1-adrenoceptors. 6. After 2, 10, 21, and 28 days of PSS, it become apparent that the two beta-adrenoceptor subtypes are regulated independently. Beta 1-adrenoceptors were transiently down-regulated after 2 days of PSS in the prefrontal cortex and in the olfactory area and were decreased after 28 days of PSS in the parietal cortex and the hippocampus. A transient up-regulation of beta 1-adrenoceptors occurred in the pulvinar nucleus after 10 days of PSS. Beta 2-adrenoceptors were transiently down-regulated after 2 days of PSS in the prefrontal cortex and up-regulated in the pulvinar nucleus after 28 days of PSS. 7. These data demonstrate that chronic psychosocial stress in subordinate tree shrews leads to time-dependent changes in the central nervous beta-adrenoceptors system. 8. The high regional variability in stress-induced beta-adrenoceptor regulation is supposed to be due to the complex mechanisms of intracellular beta-adrenoceptor sequestration, which includes down-regulation and/or reinsertion of receptors into the plasma membrane. These mechanisms may be important components of the regulatory apparatus which enables the individual to adapt to situations of recurrent stressful experiences by balancing the central nervous adrenoceptor number.

Beta-adrenoceptors in the tree shrew brain. I. Distribution and characterization of [125I]iodocyanopindolol binding sites.

1. The number and distribution pattern of beta-adrenergic receptors in the brain have been reported to be species specific. The aim of the present study was to describe binding of the beta-adrenoceptor ligand [125I]iodocyanopindolol in the brain of the tree shrew (Tupaia belangeri), a species which provides an appropriate model for studies of psychosocial stress and its consequences on central nervous processes. 2. 125I-Iodocyanopindolol (125ICYP) labeling revealed a high degree of nonspecific binding, which was due mainly to interactions of this ligand with serotonin binding sites. For a quantitative evaluation of beta 1- and beta 2-adrenoceptors, serotonin binding sites had to be blocked by 100 microM 5HT. 3. Binding of the radioligand to beta 1- and beta 2-adrenoceptors was characterized using the beta 1-specific antagonist CGP20712A and the beta 2-specific antagonist ICI118.551. beta 1-adrenoceptor binding is present in the whole brain, revealing low receptor numbers in most brain regions (up to 1.5 to 2.7 fmol/mg). A slight enrichment was observed in cortical areas (lateral orbital cortex: 4.0 +/- 0.7 fmol/mg) and in the cerebellar molecular layer (8.7 +/- 1.0 fmol/mg). 4. Competition experiments demonstrated high- and low-affinity binding sites with considerable variations in Ki values for CGP20712A, showing that various affinity states of beta 1-adrenoceptors are present in the brain (Ki: 0.61 nM to 67.1 microM). In the hippocampus, only low-affinity beta 1-adrenoceptors were detected (Ki: 1.3 +/- 0.2 microM). Since it is known that 125ICYP labels not only membrane bound but also internalized beta-adrenoceptors, it can be assumed that the large population of the low-affinity sites represents internalized receptors which may be abundant due to a high sequestration rate. 5. High numbers of beta 2-adrenoceptors are present in only a few brain structures of tree shrews (external layer of the olfactory bulb, 15.8 +/- 2.0 fmol/mg; claustrum, 19.3 +/- 1.5 fmol/mg; anteroventral thalamic nucleus, 19.4 +/- 1.5 fmol/mg; cerebellar molecular layer, 55.0 +/- 4.3 fmol/mg). Also for this class of beta-adrenoceptors, high- and low-affinity binding sites for the beta 2-selective antagonist ICI118.551 were observed, indicating that 125ICYP labels membrane bound and internalized beta 2-adrenoceptors. Only in the cerebellar molecular layer was a high percentage of high-affinity beta 2-adrenoceptors detected (Ki for ICI118.551 was 1.8 +/- 0.3 nM for 90% of the receptors). 6. In conclusion, beta 1- and beta 2-adrenoceptor binding can be localized and quantified by in vitro receptor autoradiography in the brains of tree shrews when serotonergic binding sites are blocked. Modulatory effects of long-term psychosocial conflict on the central nervous beta-adrenoceptor system in male tree shrews are described in the following paper.

Monoamine receptors in the prefrontal cortex of Tupaia belangeri during chronic psychosocial stress.

The prefrontal cortex has been reported to be involved in the regulation of emotional behaviour by integrating cognitive, emotional and autonomic information processes, and impairments of its functions are implicated in psychopathologies such as depression. Neuronal functioning in the prefrontal cortex is under the control of the noradrenergic and the serotonergic system which are both activated during stress. The present study aimed to quantify the effect of chronic psychosocial stress on alpha2-adrenoceptors, beta-adrenoceptors, and serotonin1A receptors in the prefrontal cortex. Male tree shrews (Tupaia belangeri) were subjected to subordination stress for 2, 10, 21 and 28 days, and binding sites for the alpha2-adrenergic antagonists 3H-rauwolscine and 3H-RX821002, for the beta-adrenergic antagonist 125I-iodocyanopindolol, and for the 5-hydroxytryptamine (5HT)1A receptor agonist 3H-8-hydroxy-2-(di-n-propylamino)tetralin were quantified by in vitro receptor autoradiography. Chronic psychosocial stress induced time-dependent receptor down- and upregulations. Beta-adrenoceptors were transiently reduced in numbers after just 2 days of psychosocial stress which is interpreted as agonist-mediated downregulation induced by high local concentrations of noradrenaline released from terminals originating from the locus coeruleus. Alpha2-adrenoceptors were transiently downregulated after 10 days, and upregulated after 28 days of psychosocial stress. These data indicate that the noradrenergic system adapts to the stress by counterbalancing its receptor numbers. 5HT1A receptors were only downregulated after 28 days of psychosocial stress, and thus react later than the noradrenergic receptors. In summary, our results show that monoaminergic receptors in the prefrontal cortex of tree shrews undergo dynamic changes during chronic psychosocial stress. These alterations probably have an impact on neuronal activity, and might contribute to the behavioural changes which have been previously described in subordinate male tree shrews.

A technique for automatic/interactive assessment of the proliferating fraction of neoplastic cells in solid tumors. A methodological study on the Ki-67 immunoreactive cells in human mammary carcinomas, including a comparison with the results of conventional S-phase fraction assessments by means of DNA cytometry.

Based on a computerized microscopy technique, a method has been devised which allows the practising pathologist to easily and rapidly assess quantitatively the relative number of actively proliferating neoplastic parenchymal cells in a tumor nodule. Our method has been tested on a series of 20 conventionally formalin-fixed and paraffin-embedded female mammary adenocarcinomas, using immunoreactivity with the MIB-1 monoclonal antibody against the cell proliferation antigen Ki-67. The values of the proportion of the MIB-1 immunoreactive cell nuclei were compared with those obtained DNA-cytometrically for the fraction of cells in the S-phase; a good correlation was found, although the MIB-1 values were consistently somewhat higher. A prerequisite for a success of the method was, of course, to achieve standardization of the MIB-1 immunostaining technique. By making simple adjustments of it, it could actually be improved to such an extent that almost the same color calibration and thresholding setup could be used. The measuring technique could be either interactive or automatic. The total number of immunoreactive and non-immunoreactive nuclei, as well as the total nuclear area of both cell types were registered in a computerized device. The data were accumulated sequentially for each measure field. To investigate the reproducibility of the immunostaining, two slides of each case were stained on different occasions. Each slide was measured three times; systemically randomly in the x- and y-axis-directions as well as in the subjectively defined histopathologically "most proliferative" area of the tumor. The values obtained were in good agreement with each other and obviously gave some valuable and objective supplementary pieces of information to that of the conventional clinical and histopathologic assessment of the degree of aggressiveness of a malignant neoplasm.

Monoamine receptors in the amygdaloid complex of the tree shrew (Tupaia belangeri).

Although it is well known that the mammalian amygdala comprises a heterogeneous complex of cytoarchitectonically and histochemically distinct nuclei, the association of these nuclei with different monoamine systems has not been described in detail. We therefore investigated the pattern of receptors for monoamines in the amygdala of the tree shrew (Tupaia belangeri). Binding sites for the alpha 2-adrenoceptor ligand (3H)rauwolscine, the alpha 1-adrenoceptor ligand (3H)prazosin, the beta-adrenoceptor ligand (125I)iodocyanopindolol, and the serotonin1A-receptor ligand (3H)8-hydroxy-2(di-n-propylamino)tetralin were visualized by in vitro autoradiography, and anatomically localized by comparing the autoradiograms to Nissl- and acetylcholinesterase-stained sections. To characterize binding of the radioligands pharmacologically, displacement experiments with different specific competitors were performed. Whereas the highest number of alpha 2-adrenergic binding sites was detected in the medial and the central nucleus as well as in the intercalated nuclei, the majority of serotonin1A binding sites was found in the magnocellular basal nucleus and the accessory basal nucleus, demonstrating a clear difference in the anatomy of the alpha 2-adrenergic and the serotonin1A receptor systems. In contrast, the pattern of alpha 1-adrenoceptor binding partially overlaps with that of both former receptor types. While the number of alpha-adrenergic and serotonin1A binding sites is relatively high in the tree shrew amygdala, there is a low number of beta-adrenergic binding sites in most nuclei. However, in the cortical nuclei, moderate to high numbers of binding sites for all radioligands are present. Therefore, according to our data on the tree shrew amygdala, which is anatomically similar to the amygdala of cats and primates, alpha 2-adrenoceptors cover primarily the medial part of the amygdaloid formation and serotonin1A-receptors predominantly occupy the basal nuclei, whereas alpha 1-adrenoceptors are present in both parts of the formation.

Pulmonary alveolar proteinosis in rats after administration of quartz: its possible role in morphogenesis of lung cancer.

In rats, primary peripheral lung tumors composed predominantly of alveolar type II cells have been induced by inhalation of alpha quartz. In our retrospective study on proliferation markers we evaluated lung specimens of 140 Wistar rats from larger experiments, which had been exposed to Dörentrup quartz (DQ12) by inhalation (10 mg/m3, 56 Weeks, 5 days/week, 7 h/day: n = 27) or intratracheal instillations (5 mg: n = 38; 20 mg: n = 10; 50 mg: n = 28; 15 x 3 mg: n = 12). In the last group 8/12 animals developed lung tumors. Animals were sacrificed 1-32 months after administration. For identification of an increased proliferation of alveolar type II cells the DNA content was monitored by microscopic (static) cytophotometry in histological slides. The argyrophil (AgNOR) method for the demonstration of nucleolar organizer regions (NOR) was used as second marker of type II cell proliferation. Measurements made 24 months after inhalation of DQ12 showed a slight increase of pneumocytic proliferation with 1.64 +/- 0.14 AgNOR/nucleus compared to the controls (1.23 +/- 0.04 mean AgNOR/nucleus). After intratracheal instillation of DQ12 a significant increase of AgNOR was found, e.g. 5 mg: 1.93 +/- 0.23 AgNOR/nucleus (6 months) and 1.96 +/- 0.19 (12 months); 50 mg: 1.77 +/- 15 (6 months) and 2.18 +/- 0.05 (12 months); 15 x 3 mg (+2 ml 2% polyvinylpyridine N-oxide s.c.): 1.81 +/- 0.13 AgNOR/nucleus (27-32 months). With the aid of the 2 c deviation index, i.e. the mean square deviation from the diploid DNA value, it was possible also to identify the pathologically increased proliferation of type II cells after intratracheal instillation of quartz: 0.02 +/- 0.01-0.06 +/- 0.04 c2 (controls); 0.07 +/- 0.04 c2 (5 mg/12 months); 0.12 +/- 0.08 c2 (15 x 3 mg/>27 months) and 0.68 +/- 0.48 c2 (50 mg/12 months). Only in the last group were nearly triploid values detected. Summarizing our results, intratracheal instillation and inhalation of quartz in rats regularly induces alveolar proteinosis and interstitial fibrosis in combination with a dose- and time-dependent increase of the type II cell proliferation rate. As mitogenesis increases carcinogenesis, alveolar proteinosis with increased pneumocytic proliferative activity might be a prerequisite for enhanced tumor development.

[Operated mechanical ileus of the small intestine]. / Der operierte mechanische Dünndarmileus.

Causes, pathophysiology, diagnosis, therapy and a retrospective analysis of small bowel obstruction were reviewed. 85 patients were operated during a 5-year period. The etiology of obstruction were adhesions (45 cases), hernia (20 cases) and 20 other cases. The mortality rate for the series was 8%. The most important factor of prognosis is the early operative treatment.

Cytophotometry.

Basic differences and similarities of flow and static cytophotometric instruments are viewed with regard to technical peculiarities and applicability in the fields of biology and tumor pathology. In flow systems, precision of the information concerning quantities of constituents is high, whereas precision of information concerning morphologic parameters is low. In static systems, morphologic identification by a trained operator can be used advantageously to classify any given cell interactively, thus replacing a large number of flow parameters. This is why the vast majority of automated static machines used in pathology are still more or less semiautomatic or interactive. Carefully performed specimen-adapted cell preparation procedures in which each step is strictly supervised, highly standardized staining methods, and internal controls are prerequisites in order to obtain reliable cytochemical results. In a number of human tumors with well-controlled cytopathologic and/or histopathologic and clinical data, quantitative cytochemical analysis has been demonstrated to provide diagnostic and prognostic information complementary to that obtained by conventional clinical and morphologic methods.

Monoclonal antibody-based immunoanalytical methods for detection of carcinogen-modified DNA components.

Hybridoma cell lines secreting monoclonal antibodies (Mab) directed against the products formed by reaction of alkylating N-nitroso carcinogens with DNA have been established by fusion of rat or mouse myeloma cells, respectively, with spleen cells of rats or mice immunized either with conjugates of various alkyl-ribonucleosides with suitable carrier proteins, or with alkylated DNA electrostatically complexed to carrier proteins. Due to their high affinity and specificity, some of these Mab detect very low amounts of the respective alkyl-deoxynucleosides (e.g., O6-methyl-2'-deoxyguanosine, O6-ethyl-2'-deoxyguanosine, O6-n-butyl-2'-deoxyguanosine, O6-isopropyl-2'-deoxyguanosine, O4-methyl-2'deoxythymidine, O4-ethyl-2'-deoxythymidine) and can be used in various types of immunoassays. With a competitive radioimmunoassay (RIA), specific DNA alkylation products can be quantitated in hydrolysates of cellular DNA, in body fluids, or in urine. The RIA is routinely applicable, reproducible, and sufficiently sensitive to permit the quantitation of femtomole amounts of modified nucleosides in small samples of DNA. When the alkyl-deoxynucleosides in question are separated from bulk DNA by high-performance liquid chromatography prior to analysis by RIA, very low levels of modification in DNA can be detected. The immuno-slot-blot (ISB), a noncompetitive solid-phase immunoassay, is more sensitive than the RIA. For analysis by ISB, alkylated DNA is heat-denatured and immobilized on nitrocellulose filters prior to exposure to the respective Mab and subsequent binding of a second (125I-labelled or biotinylated) antibody. In immunocytological analysis (ICA), the binding of Mab to alkyl-deoxynucleosides is visualized in individual cells by immunostaining of denatured nuclear DNA in situ (direct immunofluorescence; peroxidase-staining).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of monoclonal antibody-defined cell surface antigens during rat brain development.

Using single-cell suspensions of mechanically dissociated, prenatal BDIX-rat brain cells (13th, 15th, and 21st days after fertilization) for immunization, we have established a collection of 37 monoclonal antibodies (Mabs) directed against neural cell surface determinants. The developmental-stage-dependent expression of cell-surface antigens recognized by these Mabs was analyzed both on plasma membranes isolated from whole brains of BDIX rats (prenatal days 13-22 and adults) using an indirect 125I solid-phase radioimmunoassay, and on intact BDIX-rat brain cells (prenatal days 13-22) using a fluorescence-activated cell sorter. Different types of developmental stage-dependent profiles of Mab binding were found, these being indicative of the presence of neural cell surface determinants whose expression increases, decreases, or does not change with brain development. Some of the Mab-binding profiles showed transient changes as a function of developmental stage. These Mabs are currently being used for the characterization, reproducible identification, and isolation of neural cell subpopulations of the developing rat brain, with the aim of investigating the cell type dependence and developmental (differentiation) stage dependence of malignant transformation following pulse exposure to the carcinogen N-ethyl-N-nitrosourea at defined stages of brain development.

High-affinity monoclonal antibodies for the specific recognition and quantification of deoxynucleosides structurally modified by N-nitroso compounds.

The applicability of conventional radiochromatographic procedures to the detection and quantification of specific, carcinogen-induced structural modifications in the DNA of mammalian cells is limited by the necessity of using radioactively labelled agents and by the relatively large amounts of DNA required for analysis of low levels of DNA modification. Recently developed immunoanalytical methods have improved this situation considerably. High-affinity monoclonal antibodies (MAB), in combination with radio- and enzyme-immunoassays, now permit the sensitive detection of alkyldeoxynucleosides in small samples of hydrolysed DNA from tissues and cultured cells exposed previously to non-radioactive (e.g., environmental) alkylating N-nitroso carcinogens. Furthermore, MAB can be used to quantify by direct immunofluorescence (and with the aid of computer-based image analysis of electronically intensified fluorescence signals) specific alkylation products in the DNA of individual cells. With this method, the present detection limit for, e.g. O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) is of the order of 7 X 10(2) O6-EtdGuo molecules per diploid genome. Therefore, cells (e.g. from biopsy material) can now be monitored directly for the presence of specific carcinogen-DNA adducts, or with respect to their capacity to remove enzymatically such modified structures from DNA. In combination with transmission electron microscopy, MAB also permit the direct visualization of specific carcinogen-modified sites in DNA. Thus, O6-EtdGuo can be localized in double-stranded DNA molecules by the binding of a MAB specifically directed against this ethylation product.