C. Tschahargane

The Proliferative Index (PI) of Human Breast Cancer as Obtained by Flow Cytometry

As known from previous reports, the DNA synthesis fraction of mammary carcinoma cells is correlated with the course of the disease and response to adjuvant therapy. Quantitative parameters of the proliferative activity can be determined by the classic 3HTdR-labelling technique as well as by the more rapid flow cytometric (FCM) DNA analysis. The values of DNA-cytometric S-phase fractions in breast cancers reported up to now were consistently higher than those obtained by the 3HTdR labelling index (LI). This discrepancy was surmounted in the present study by corrections for systemic errors of the method. The fractions of cells in stages of the cell cycle as well as the DNA indices (DI) in 155 cases of primary resectable breast cancers were analyzed by DNA flow cytometry. The patients were aged between 24 and 88 years. As indicated by a bimodal age distribution with a decrease at age 60, the patient population was a representative of the generally known breast cancer incidence. 54% of the patients had aneuploid tumor cells with preferences of DNA indices 1.6 and 2.0. In the remaining 46%, no cell populations with deviating DNA content could be detected, in part possibly due to very small differences beyond the limits of detection. No correlations were found between age, menopausal status, histologic type of tumors, tumor size, fractions in stages of the cell cycle and proliferative index (PI = S + G2 + M in %), except a significant correlation between the S-phase fractions and the (G2 + M)-phase fractions in a ratio of approximately 1.(ABSTRACT TRUNCATED AT 250 WORDS)

The serum complement system--a mediator of acute pancreatitis.

The role of the complement system in the initial membrane lesion of acute pancreatitis was investigated. In the experimental sodium-taurocholate pancreatitis of the rat a sudden and steady decline of serum complement was observed. The deposition of 03 component of complement in acute pancreatitis could he demonstrated by immunofluorescence. To rule out mere deposition or activation of complement in the interstitial exsudative fluid, single acinar cells of rat and rabbit pancreatic tissue were prepared and transfered to culture medium. In contrast to heat inactivated serum and C6 deficient serum these cells were lysed by trypsin activated fresh serum. Consequently, an acute pancreatitis could be induced by activating exclusively the complement system by injection of cobra venom factor into the pancreatic duct of rats. The activated complement system is thought to be responsible for initial membrane lesion in exsudative inflammation, as could be shown in acute pancreatitis.

Morphological feedback effect on neurons of the nucl. arcuatus (sive infundibularis) and nucl. subventricularis hypothalami due to gonadal atrophy

We have correlated the light-microscopic features in the un-myelinated hypothalamus with gonadal atrophy in 15 women of 30-111 years of age and in 7 men between 29 and 82 years. In the postmenstrual cases there is a distinct concordance of gonadal atrophy and the manifestation of nucleolar changes (augmentation, multiplication and vacuolization) in many nerve ceils of the arcuate and subventricular nuclei. In younger, still fertile women this nucleolar finding was seen only rarely and sporadically, was limited to the arcuate nucleus and was absent in the subventricular nucleus. We interpret this nucleolar finding as a feedback effect. In man, too, this coincidence is obvious with age and in gonadal atrophy, with fewer nucleolar changes in old age than are seen in women. This difference is probably caused by a more rapid drop of the estradiol than of the testosterone level.-The hypertrophy of the subventricular nucleus (Sheehan and Kovacs) was also observed in our postmenstrual cases.

Maligner Granularzelltumor des Großhirnmarkes

SummaryThis is a report of the second known case of a primary malignant granular-cell tumor of the cerebral hemispherical white matter. Two cell types may be distinguished apparently representing different developmental stages of otherwise identical tumor cells. Quantitative histochemical and biochemical studies have shown that tumor cells were containing markedly elevated levels of DNA and RNA. Only few ribosomes and polysomes could be detected, however, by electron microscopy. While cytophotometry disclosed only slightly elevated cytoplasmic proteins, there was a distinct increase in total protein concentration of tumor tissue per unit weight, as measured by conventional techniques. It is suggested that this increase is mainly attributable to structural proteins of cellular and subcellular elements of multiplicating malignant cells. A ratio of RNA to DNA in excess of 1 was found for white matter derived from the central tumor, while the ratios of control tissue were lowered to values far below one due to postmortem changes greatly reducing tissue concentrations of RNA. No detailed characterization of RNA was attempted. The nuclear DNA content of tumor cells reached values equivalent to chromosomal hexadekaploidy. This was in sharp contrast to control data from an Abrikossoff tumor of the oral cavity and from a neurohypophyseal tumorette (“choristoma”), respectively, displaying diploidy only associated with a much lesser increase of cytoplasmic RNA and proteins. Qualitative lipid studies were consistent with a marked active demyelination of the tumor centre as indicated by a severe reduction of cerebrosides and sulfatides and the presence of cholesterol esters. In addition there was a striking loss of phosphatidylethanolamine and a lesser one of sphingomyelin of white matter of both the tumor-stricken and the contralateral unaffected hemispherical regions, possibly suggesting a generally disturbed metabolism of myelin. It is not clear whether these general changes were resulting from the presence of the unilateral tumor or from precocious cerebral involution.

Isolation of single cell nuclei from human epidermis for cytophotometric DNA--measurements.

SummaryDiluted acetic acid was applied to dissociate human epidermis into its constituents. A simple procedure to gain isolated single cells for cytophometric determination of their nuclear DNA content is described. The method is suited for single cell cytophotometric measurements and for cytofluorimetric flow systems likewise. The losses of Feulgen—stainable nuclear DNA during preparation were studied in dependence on the incubation time of the cells in acetic acid. The kinetics of extraction of nuclear DNA by diluted acetic acid follow a reaction of the first order. It was possible therefore to formulate a simple mathematical correction for the losses during the isolation of epidermal cells. Avoiding the disadvantage of cut nuclei in histological sections the method is used to study the DNA histograms of resting, regenerating and pathologically altered human epidermis.ZusammenfassungZur Auflösung menschlicher Epidermis in ihre zellulären Bestandteile erwies sich verdünnte Essigsäure als geeignet. Es wird eine einfache Prozedur zur Gewinnung von Einzelzellen für cytophotometrische Messungen des DNS-Gehaltes in den Zellkernen beschrieben. Diese Methode eignet sich gleichermaßen zur Herstellung von Zellsuspensionen für die Durchflußcytophotometrie wie auch von Ausstrichen auf Objektträger für die Einzelzell-Cytophotometrie. Die Verluste Feulgen-färbbarer Substanz während der Präparation wurden quantitativ erfaßt. Die Extraktion von DNS aus den Zellkernen durch verdünnte Essigsäure folgt näherungsweise einer Kinetik erster Ordnung. Demzufolge kann eine einfache Korrekturformel für die DNS-Verluste während der Präparation aufgestellt werden. Die Methode dient zur Untersuchung der DNS-Histogramme in der normal regenerierenden sowie der pathologisch veränderten menschlichen Epidermis, wobei die Nachteile angeschnittener Zellkerne in histologischen Schnitten weitgehend umgangen werden.

Simultaneous differential staining of nucleic acids and proteins in histological tissues by means of J-Band effect

SummaryA cationic carbocyanine dye, originally used in gel-electrophoresis, stains simultaneously DNA, RNA and proteins also in histological slides or smears. The resulting different colors are demonstrated by various spectral absorbance curves in cell nuclei and cytoplasm. The simple technique is suited also for quantitative cytophotometric determinations.

[Cerebral granular cell tumor in the rat after intraperitoneal application of aflatoxin b1 during pregnancy (author's transl)].

On a rat, which has spontaneous died 12 months after 4 times i.p. application of aflatoxin during pregnancy, there has been found a frontal tumor barely of the size of a pepper-corn which came out from the leptomeninx. A comparison to the Abrikossoff tumor concerning the light- and electron-microscope as well as the nucleic-acid concentration is justified. The protein content of the granula of the tumor cells is higher than the values known in the granular-cell myoblastoma of man.On a rat, which has spontaneous died 12 months after 4 times i.p. application of aflatoxin during pregnancy, there has been found a frontal tumor barely of the size of a pepper-corn which came out from the leptomeninx. A comparison to the Abrikossoff tumor concerning the light- and electron-microscope as well as the nucleic-acid concentration is justified. The protein content of the granula of the tumor cells is higher than the values known in the granular-cell myoblastoma of man.

Über die Eignung von Ethidiumbromid als Fluorochrom zur quantitativen Darstellung von Nukleinsäuren in histologischen Präparaten

SummaryIn order to test Ethidiumbromide1 as a marker for DNA in cells, slides with sections of human epidermis and smears of liver cells were stained in a buffered solution of this substance. The staining results demonstrated by photographs indicate, that Ethidiumbromide does not bind selectively to nucleic acids. As a consequence of the spectral properties of the stained tissue elements this fluorochrome does not seem to be suited for quantitative determinations of nucleic acids by measurements of the fluorescence intensity.ZusammenfassungMit dem in der Impulscytophotometrie zu DNS-Messungen benutzten Fluorochrom Ethidiumbromid1 wurden Schnittpräparate menschlicher Epidermis und Tupfpräparate menschlicher Leberzellen behandelt. Die an Fluoreszenz- und Durchlichtphotographien demonstrierten Färberesultate zeigen, daß keine nukleinsäurespezifische Bindung des Farbstoffes erfolgt. Aufgrund der spektralen Eigenschaften der mit dem Fluorochrom tingierten Zellelemente erscheint eine quantitative mikrofluorometrische Substanzmengen-bestimmung nur bedingt möglich.

Über den Altersformwandel der Zellkerne in der menschlichen Epidermis

SummaryThe abdominal epidermis of 23 male humans in different age groups was histologically, histochemically and karyometrically examined. This was done using Gallocyaninchromalum, Feulgen and UV methods in order to determine the average nucleic acid content and the volume of the epidermal cell nuclei. 6000 cell nuclei were examined photometrically and karyometrically and the results were statistically evaluated. The data obtained could contribute to the knowledge of age dependent changes. The results are as follows:1.In male individuals over 65 years of age, there is a reduction of nuclear volume and of the average nucleic acid content of approximately 35%. There also exists a reduction in the nuclear volume and the nucleic acid content from the cells of the stratum basale to the cells of the stratum spinosum of approximately 35% in each age group. The concentration of nucleic acid is for all practical purposes constant.2.There is a linear correlation between the amount of nucleic acid and the nuclear volume of epidermal cells. This correlation can be calculated in the form of a stochastic function. The latter is defined by calculating its coefficients.3.Knowing the normal values and their biological and age-dependent variations it is possible to differentiate between normal and pathological findings.ZusammenfassungDie Bauchhaut von 23 männlichen Versuchspersonen verschiedener Altersgruppen wurde histologisch, histochemisch und karyometrisch untersucht. Mit drei voneinander unabhängigen Methoden (Gallocyaninchromalaun-, Feulgen- und UV-Methode) wurden die mittlere Nukleinsäuremenge und das Volumen der epidermalen Zellkerne bestimmt. Auf diese Weise wurden etwa 6000 Zellkerne photometrisch und karyometrisch gemessen und die Ergebnisse statistisch ausgewertet. Die ermittelten Werte stellen einen Beitrag zur biorheutischen Metamorphose der menschlichen Epidermis dar. Folgende Befunde wurden erhoben.1.Bei männlichen Individuen über 64 Jahren kommt es zu einer Abnahme der Zellkernvolumina und des mittleren Nukleinsäuregehaltes um ca. 35%. Gleiche Verhältnisse ergeben sich für die Zellkerne von der letzten Reihe des Stratum spinosum im Vergleich zu Zellkernen des Stratum basale in jeder Altersgruppe. Auch hier nehmen die Nukleinsäuremenge und die Kernvolumina um 35% ab. Die Konzentration der Nukleinsäure bleibt im wesentlichen konstant.2.Zwischen Nukleinsäuremenge und den Kernvolumina der Epidermiszellen besteht eine lineare Korrelation. Diese läßt sich in Form stochastischer Punktionen unter Angaben der Korrelationskoeffizienten ausdrücken.3.Durch die Bestimmung von Normalwerten und deren biologischer wie auch altersabhängiger Variationen sind quantitative Abgrenzungen gegen pathologische Befunde möglich.

Microscopic and pulse cytophotometric investigation of a carcinoma of the jejunum in a seven year old child

In a seven year old boy with acute abdominal pain and intestinal bleeding, laparotomy revealed an invaginated carcinoma adenomatosum cylindrocellulare in the distal part of the jejunum. Histological and pulse cytophotometric investigations support the diagnosis of a highly differentiated but locally destructive and malignant tumor.