Three-dimensional reconstruction of transmitter-identified central neurons by “en bloc” immunofluorescence histochemistry and confocal scanning microscopy

Summary A new method for three-dimensional reconstruction of transmitter-identified neurons is presented which involves en bloc immunofluorescence histochemistry and confocal scanning microscopy. The technique was applied to different types of neurons in the rat brain and lamprey spinal cord. Thick sections or tissue blocs (50–200 m thick) were incubated with antisera against neuropeptides or monoaminergic markers, followed by fluorescent secondary antibodies. Three-dimensional reconstructions were obtained by scanning the preparations in sequential focal planes with a thin laser beam, while sampling the emitted light in each focal plane. The method is convenient and can be applied to a wide variety of neuron types. The reconstructions obtained are accurate since the optical serial sections of the specimen are perfectly aligned, and optic disturbances such as halo phenomena do not occur.     

Cyclic AMP-resistant mutants of S49 mouse lymphoma cells hemizygous for expression of regulatory subunit of type I cyclic AMP-dependent protein kinase

For use in studies of the functional organization of regulatory (R) subunit of type I cAMP-dependent protein kinase, 84 independent cyclic AMP-resistant mutants were isolated from sublines of S49 mouse lymphoma cells that are hemizygous for expression of the R subunit. Mutants were characterized by two-dimensional gel analysis of the R subunits, assays of kinase activation, and assays of cAMP-binding. All but eight of the mutants had kinases with increased apparentK _as for cAMP-dependent activation, and studies with site-selective cAMP analogs revealed considerable phenotypic diversity among these mutants. Forty-nine of the mutants had charge-shift lesions that mapped to regions of the R subunit polypeptide implicated in cAMP-binding. Twenty-five of the charge-shift mutants expressed only mutant R subunits, and the lesions in most of these isolates inhibited binding of cAMP to mutated cAMP-binding sites. The remainder of the charge-shift mutants expressed both mutant R subunit and R subunit with wild-type gel mobilities. The origin of these heterozygous mutants from parental hemizygous cells remains a puzzle.     

Fluorescence histochemical and electron-microscopical observations on the innervation of the atrial myocardium of the adult human heart

Summary The existence of both adrenergic and cholinergic innervation of the atrial myocardium of the adult human heart was demonstrated by means of fluorescence induced by formaldehyde or glyoxylic acid and by electron microscopy.The adrenergic fluorescing axons (1) followed the course of blood vessels as typical perivascular nerve plexuses, and (2) formed a three-dimensional fairly dense nerve net obviously not related to the blood vessels. The varicosities frequently came into close apposition on myocardial cells.Several types of nerve terminals were differentiated at electron microscopy: (1) an adrenergic type containing small (diameter 450–700 Å) dense-cored vesicles and usually (in various proportions) small empty and/or large (900–1500 Å) dense-cored vesicles, (2) a cholinergic type containing small (ca. 500 Å) empty vesicles and occasionally also some large (mean diameter ca. 1200 Å) dense-cored vesicles, (3) a pale type containing only a few or no vesicles, (4) a disintegrated type containing degenerated mitochondria, autophagic vacuoles, and occasional normal-looking mitochondria, (5) nerve terminals containing a large number of mitochondria in addition to varying vesicle populations, and (6) a (possibly baroreceptive type of) nerve terminal containing myelinlike lamellated structures. The disintegrated and the pale types of nerve terminals possibly represent different stages of axonal degeneration, or may correspond to diminution in the transmitter substance concentration under certain pathophysiologic conditions, respectively. Nerve terminals crowded with mitochondria may be sensory and involved in mechano-or chemoreceptive functions.In preliminary experiments convincing evidence was obtained that the glyoxylic acid-induced fluorescence histochemical method will be suitable for comparative studies on (human) clinical specimens, e.g., for analyzing the degree of the functional activity of the intrinsic adrenergic innervation of the myocardium under various pathophysiologic conditions. The modification which appeared most appropriate for such studies is described in detail, and is proposed for use as a standard method in other similar or related studies on human clinical series. The essential criteria for analyzing the specimens at fluorescence microscopy are suggested as well.     

Further observations on the morphogenesis of human rotavirus in MA 104 cells

Summary Human rotavirus KUN strain was cultivated in a fetal rhesus monkey kidney cell line, MA 104 cells. Four types of virus particles in cells infected with KUN strain were clearly identified: nucleoid cores, single-shelled particles, double-shelled particles, and membrane band, enveloped particles. Enveloped particles were found only in the thin sections of infected cells. When first visible, the virus precursors appeared at the ribosome free membrane of rough endoplasmic reticulum (RER), increasing in size while simultaneously being coated with nucleocapsid, inner shell. Single-shelled particles were also synthesized within bundles of filaments of viroplasm in the cytoplasma. During subsequent virus maturation two types of budding processes were observed. Double-shelled particles arising at the RER membrane entered the cisternae of the RER through an exocytosis-like process. In contrast, the enveloped particles developed in the cisternae by being completely enclosed with RER membrane, and later during cytolysis released the single-shelled particles. These enveloped virus particles appeared to be the result of inefficient virus maturation at the last stage of outer capsid formation.With 11 Figures     

Summation and adaptation in stimulation of the motor cortex in adult and newborn animals

Summary In rabbits and their progeny an investigation was made of summation and adaptation of the neurones of the motor cortex; unipolar stimulation was used, and the muscular response recorded. Two types of summation curves were found: exponential and Y-shaped,, and were related to the direct and indirect summation of stimulation. In adults, Y-shaped summation curves were more frequent, whereas in rabbits aged 24 h exponential curves preponderted. We found very little capacity for adaptation at any age.(Presented by Active Member AMN SSSR A. F. Turom) Translated from Byulleten Éksperimental'noi Biologii i Medisiny, Vol. 55, No. 6, pp. 3–7, June, 1963     

Disturbance of delayed match-to-sample in macaques by tetanization of anterior commissure versus limbic system or basal ganglia

Summary Three pig-tailed macaques were trained to select (match) from a pair of colored images that which they had seen (sample) and responded to 5–15 s previously. The anterior commissure (AC) and/or its radiation, various loci in basal ganglia, hippocampal formation and control areas, (splenium of corpus callosum, precentral gyrus, insular cortex), totalling 40 loci, were each tetanized for 4 s during presentation of the sample image, during the delay period, or when the monkey was required to select the matching image. For several loci in the hippocampal formation tetanization at any phase of the task reduced matching to chance levels and gave evidence of electrical after-discharge; but other comparable hippocampal loci had little or no effect. Response to sample or match stimuli were absent during tetanization of basal ganglia or anterior commissure. When finally made, upon cessation of tetanization, responses were equally correct for basal ganglia and control sites, but for AC were at chance levels.     

Foraging strategies ofDrosophila melanogaster: A chromosomal analysis

Two larval foraging strategies inDrosophila melanogaster were identified, rover and sitter. Rovers traverse a large area while feeding whereas sitters cover a small area. The difference between rovers and sitters was analyzed genetically by chromosomal substitutions between isogenic stocks. Differences in larval locomotor behavior (crawling behavior) can be attributed to the second chromosome, the rover strategy being dominant over the sitter strategy. Differences in feeding rate (shoveling behavior) are affected additively by both the second and third chromosomes. Natural populations ofDrosophila larvae were sampled three times over a 2-month period; rovers and sitters were at constant frequencies in these populations. The two foraging strategies are discussed in the light of resource utilization in environments where food is distributed continuously or discontinuously.     

Evidence for a direct relationship between mitochondrial genome organization and regeneration ability in hexaploid wheat somatic tissue cultures

Summary Embryogenic and non-embryogenic long-term callus cultures of hexaploid wheat exhibit differences in the organization of their mitochondrial genome. Embryogenic and non-embryogenic fractions of callus cultures initiated from immature embryos of the wheat cultivar Chinese Spring have been isolated and subsequently subcultured. DNA-DNA hybridization experiments using labelled cloned wheat mitochondrial DNA fragments have shown that the mitochondrial DNA organization of embryogenic subcultures derived from embryogenic parts of Chinese Spring calli is closely related to that of the initial Chinese Spring calli, while non-embryogenic subcultures derived from non-embryogenic fragments of Chinese Spring calli exhibit a mitochondrial DNA organization similar to that found in non-embryogenic calli derived from cultivar Aquila. In addition, somatic tissue cultures initiated from three other non-embryogenic wheat cultivars (Talent, Thésée and Capitole) display mitochondrial DNA arrangements similar to those found in cultivar Aquila. These results strongly suggest that, in wheat callus cultures, a particular mitochondrial genome organization is correlated with the ability of cultured cells to regenerate whole plants.Abbreviations mtDNA mitochondrial DNA - ctDNA chloroplast DNA - rRNA ribosomal RNA - kb kilobase pair - cv cultivar - 2,4-D 2,4-dichlorophenoxyacetic acid     

Beitrag zur Frage einer gestörten Nierenfunktion bei exogen-alimentärer Fettsucht

Zusammenfassung Bei 15 nierengesunden Fettsüchtigen und 17 normalgewichtigen Personen bestimmten wir simultan Inulin- und Paraaminohippursäure-Clearance. Außerdem untersuchten wir die Beziehung zwischen den absoluten Clearance-Werten einerseits sowie tatsächlicher und idealer Körperoberfläche andererseits.Nach unseren Untersuchungen werden die Werte der Nieren-Clearance (ml/min/1,73 m²) Adipöser erheblich von der gewählten Bezugsgröße — der tatsächlichen oder idealen Körperoberfläche — beeinflußt. Durch Regressionsanalysen ließ sich zeigen, daß bei überernährten Probanden zwischen Glomerulumfiltrat und ihrer idealen Körperoberfläche ein signifikanter Zusammenhang bestand. Keinerlei Beziehung ergab sich für Inulin-Clearance und tatsächliche Körperoberfläche sowie für den effektiven Nierenplasmastrom einerseits und tatsächliche bzw. ideale Körperoberfläche andererseits. Die Frage, welche der Bezugsgrößen bei klinischen Clearance-Bestimmungen Fettsüchtiger zu verwenden ist, beantworteten wir sowohl für Inulin- als auch PAH-Clearance zugunsten der idealen Körperoberfläche.Unter Berücksichtigung der idealen Körperoberfläche lag das Glomerulumfiltrat der Fettsüchtigen im Mittel signifikant über dem des Vergleichskollektivs, während ihre PAH-Clearance durchschnittlich normal, bei 2 von 15 Adipösen erniedrigt ausfiel. Die Filtrationsfraktion war gegenüber der normalgewichtiger Personen im Mittel signifikant erhöht. Als Grund der veränderten Nieren-Clearance Fettsüchtiger werden funktionelle Einflüsse diskutiert.

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Summary Renal clearance rates (ml/min) of inulin (C_In) and paraamino-hippuric acid (C_PAH) were determined simultaneously in 15 obese patients with healthy kidneys and 17 persons of normal weight. C_In and C_PAH were computed to the standard unit 1,73 m² body surface area. Individual surface areas are calculated on the basis of real and ideal body weight. Our investigations show that the rates of renal clearance (ml/min/1,73 m²) depend to a considerable extent upon the selected surface area-actual or ideal body surface. Analysis indicates that in obese patients a significant connection exists between glomerulum filtration (C_In) and the ideal surface area. No connection was established for inulin clearance (C_In) and actual surface area, nor for C_PAH and real and ideal body surface area. In the determination of clinical clearance rates C_In and C_PAH both should in cases of obesity be computed to the ideal surface area. The mean glomerular filtration of obese persons was found to lie significantly above the mean value of persons of normal weight. C_PAH was normal except in two patients which had lowered paraamino-hippuric acid clearances. The filtration fraction was on average significantly above that of the persons of normal weight. Functional influences are discussed as the reason for the different renal clearance values of obese persons.

     

Amygdalar inputs to ADH-secreting supraoptic neurones in rats

Summary Effects of amygdala stimulation on the discharge activity of antidromically identified supraoptic neurosecretory neurones were studied in male rats anaesthetized with urethane. Stimulation of the medial and the basal amygdala produced excitation or inhibition of discharge activity both in phasically firing (phasic) and in continuously firing (continuous) neurones. More phasic neurones were excited than were inhibited after medial amygdala stimulation. On the other hand, fewer continuous neurones were excited by stimulation of the either amygdala area than were inhibited. This difference of responsiveness between phasic and continuous neurones is statistically significant. Synaptic inputs to supraoptic neurosecretory neurones after amygdala stimulation were also observed in rats with a lesion of the stria terminalis. Supraoptic nucleus stimulation activated antidromically 14 of the 336 amygdala neurones tested. Since phasic neurones have been identified as ADH-secreting neurones, it is concluded that ADH-secreting neurones in the rat supraoptic nucleus receive predominantly excitatory synaptic inputs from the medial amygdala and these amygdalar synaptic inputs are mediated by pathways which are at least in part monosynaptic and are not included in the stria terminalis.Supported by the grants nos. 56440079, 56121007 and 56770057 from the Ministry of Education, Science and Culture, Japan     

The distribution of lanthanum in tight junctions of the kidney tubule

Summary The penetration of either ionic or colloidal lanthanum into the tight junctions of different tubular segments of the rat kidney was studied with the electron microscope. After perfusing the tracer through the aorta it was found that ionic and colloidal lanthanum penetrated the tight junctions of both the proximal and distal convoluted tubules, while the junctions of the collecting tubules were free of tracer. An attempt to correlate junctional tightness and electrical resistance in these epithelia is presented.     

Electron microscopic observations on the cilio-zonular border area of the human eye with particular reference to the aging changes

Summary The basement membrane at the inner surface of the nonpigmented ciliary epithelium is the direct continuation of the basement membrane at the inner surface of the neural retina. It shows a remarkable transformation during life. In the early childhood it is one-layered, whereas in the adult eye it forms a multi-layered network. This may be caused by aging processes or by biomechanical forces. Within the mashes of the aged basement membrane network, granular and lamellar deposits, probably lipids, are interposed.The nonpigmented ciliary epithelium cells are not absolutely free of pigment. Electronmicroscopically we found melanin granules and melanosomes also in the nonpigmented cells of the pars plana corporis ciliaris of a 9 month old child. With increasing age, the nonpigmented ciliary epithelium cells contain an increasing number of lipid droplets, lipofuscins and vacuoles.Presented in part at the 43th meeting of the Vereinigung Rhein-Mainischer Augenärzte, Marburg, May 30–31, 1970.     

Modeling the vestibulo-ocular reflex of the squirrel monkey during eccentric rotation and roll tilt

Model simulations of the squirrel monkey vestibulo-ocular reflex (VOR) are presented for two motion paradigms: constant velocity eccentric rotation and roll tilt about a naso-occipital axis. The model represents the implementation of three hypotheses: the internal model hypothesis, the gravito-inertial force (GIF) resolution hypothesis, and the compensatory VOR hypothesis. The internal model hypothesis is based on the idea that the nervous system knows the dynamics of the sensory systems and implements this knowledge as an internal dynamic model. The GIF resolution hypothesis is based on the idea that the nervous system knows that gravity minus linear acceleration equals GIF and implements this knowledge by resolving the otolith measurement of GIF into central estimates of gravity and linear acceleration, such that the central estimate of gravity minus the central estimate of acceleration equals the otolith measurement of GIF. The compensatory VOR hypothesis is based on the idea that the VOR compensates for the central estimates of angular velocity and linear velocity, which sum in a near-linear manner. During constant velocity eccentric rotation, the model correctly predicts that: (1) the peak horizontal response is greater while facing-motion than with back-to-motion; (2) the axis of eye rotation shifts toward alignment with GIF; and (3) a continuous vertical response, slow phase downward, exists prior to deceleration. The model also correctly predicts that a torsional response during the roll rotation is the only velocity response observed during roll rotations about a nasooccipital axis. The success of this model in predicting the observed experimental responses suggests that the model captures the essence of the complex sensory interactions engendered by eccentric rotation and roll tilt.This research was supported by NASA contracts NASW-3651, NAG2-445, NAS9-16523, the NASA Graduate Student Researchers Program, and the GE Forgivable Loan Fund. Preliminary findings were presented in a PhD thesis (Merfeld 1990) and at the Sixteenth Barany Society Meeting in Tokyo in 1990 (Merfeld et al. 1991).     

On the existence of a lyotropic and osmotic type of collagen swelling produced by representative electrolytes and anelectrolytes

Summary The influence of NaI, NaCl, urea, glucose and sucrose on venous tissue volume were studied with regard to osmotic (electrostatic and dehydrating) and lyotropic effects. NaI leads to the highest volume increase and to the extreme irreversibility of this change. The tested anelectrolytes cause a decrease of tissue volume over the whole range of concentration with the exception that, beyond 1.0 osM, urea causes an increase. The large reversibility of this increase is pointed out. A strict discrimination between osmotic and lyotropic volume change is questioned.     

Coronary blood flow in rats native to simulated high altitude and in rats exposed to it later in life

Summary In rats exposed to a simulated high altitude of 3500 m for their whole prenatal and postnatal life a severe cardiac hypertrophy develops. In rats born and first staying 5 weeks at sea level and then being exposed to simulated high altitude, only a unilateral right cardiac hypertrophy occurs. In both groups nutritional coronary blood flow was estimated in left ventricle, right ventricle, and septum and was compared with control animals of similar age. Coronary blood flow was measured at hypoxia in all groups. At first cardiac output was determined by the Fick principle, then⁸⁶Rb was applied and the animals were killed after 55 sec. Activity of⁸⁶Rb was measured in both cardiac ventricles and septum and the fractional uptake was calculated. According to Sapirstein (1956, 1958) the distribution of⁸⁶Rb follows the distribution of cardiac output and from both these data the nutritional blood flow to the parts of the heart may be estimated.Cardiac output was similar in rats exposed to simulated high altitude later in life (newcomers) and in control animals, but it was significantly lower in rats born in the low pressure chamber (natives).Fractions of cardiac output supplying cardiac ventricles and septum in rats from both hypoxic groups were significantly higher than in control animals. In the natives they were significantly higher than in the newcomers. The fractions of cardiac output in both newcomers and natives remained significantly higher than those of the control animals, also when calculated per gram of heart tissue.Nutritional coronary blood flow (in ml/min) was higher in both ventricles and septum of the newcomers and in the right ventricle of the natives, and lower in the septum of the natives, when compared with control animals. Coronary blood flow per gram of heart tissue (in ml/min·g) was significantly higher in all cardiac parts of the newcomers, but it was about the same in all cardiac parts of the natives when compared with controls.The importance of observed changes concerning myocardial tissue oxygenation is analyzed by using Krogh's cylindrical tissue model.Presented in part at the XXVIth International Congress of Physiological Sciences, New Delhi, India, October 20–26, 1974.     

The antigenetic properties of “human leukemic factor” cultivated on the chorioallantoic membrane of the chick embryo

Summary It was demonstrated with the aid of anaphylactic reaction with desensitization that the specific leukemic factor is present in the allantoic fluid of chik's embryos into which the blood of leukemic patients was injected. This factor is absent in the normal allantoic fluid. Human leukemic factor may be cultivated for a long time (up to 19 passages) on the chrion-allantoic membrane of developing chick's embryos.Presented by Active Member Acad. Med. Sci. USSR L.A. Zil'ber     

Eine neue Bestimmung der Neutralfette im Blutserum und Gewebe

Zusammenfassung Die Neutralfette im Blutserum und Gewebe lassen sich über Glycerin exakt ermitteln. Freies und nach alkoholischer Verseifung nachweisbares Gesamt-Glycerin werden mit Glycerokinase in einer dreistufigen enzymatischen Nachweisreaktion im Mikroverfahren bestimmt. Die Differenz wird als Glycerid-Glycerin angesprochen. Reaktionsprinzip, Durchführung der Methode und Berechungsweise werden ausführlich besprochen.Für freies Glycerin werden 0,2 ml Serum, für Gesamtglycerin etwa 0,007–0,015 ml Serum äquivalente Hydrolysatmengen in den Test eingesetzt. Bei einer Empfindlichkeit von 0,0008 µMol (wenn bei 340 nm, bzw. 0,0015 µMol Glycerin, wenn bei 366 nm gemessen wird), können noch 1/20 bis 1/10 des im Normalserum vorhandenen freien bzw. Gesamtglycerins mit den Standardansätzen erfaßt werden. Die obere Nachweisgrenze reicht dann bis 0,15 bzw. 0,3 µMol Glycerin, das entspricht der 8- bzw. 15fachen Normalmenge für freies und Gesamtglycerin. In den angegebenen Grenzen liegen die über Glyceridglycerin nachweisbaren Triglyceridkonzentrationen. Empfindlichkeit und obere Nachweisgrenze können durch andere Serum- bzw. Hydrolysatmengen in weiten Grenzen variiert und der jeweiligen Fragestellung angepaßt werden.

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Summary Triglycerides in blood serum and tissue can be identified exactly by means of glyceride-glycerol. Free and total glycerol, measurable after alcoholic saponification, are determined by glycerolkinase in a three-step enzymatic proof-reaction by means of micro-methods. The difference between total and free glycerol is called glycerideglycerol. Reaction principles, working instructions and the calculation will be discussed in detail.The sensitivity of the standard test runs to 0,0008 respeccively 0,0015 µMol glycerol or 1/20 resp. 1/10 of the normal contentrations. The upper proof limit is at 0,15 respectively 0,3 µMol glycerol per test. This is equal to 8–15 times more than the normal of free and total glycerol. Sensitivity and upper proof limit can easily by varied and adapted to the formulation of the question.

     

A Proposed Heuristic for Communicating Heritability Estimates to the General Public, with Obesity as an Example

It is well established that many continuously distributed traits have a heritable component. However, it is often difficult to communicate to the general public the meaning of quantitative estimates of heritability. To address this problem, the present paper introduces a heuristic for communicating heritability to nonscientific audiences. This heuristic involves adopting an extremely simplified model of inheritance and artificially (and somewhat arbitrarily) defining a cutoffs of low environmental risk and affectation status. Using body weight and obesity as an example, we present a table which gives estimates of the proportion of obese persons who are genetically obese assuming varying levels of environmental risk for obesity and relative body weight scores for defining obesity. The resulting statistic may prove useful for lay audiences in understanding a heritability estimate.     

Prehension in the pigeon

Summary Eating in the pigeon involves a series of jaw movements some of which serve a prehensile function; i.e., they are utilized in the grasping and manipulation of objects. These prehensile behaviors are extremely brief (30–80 ms), produce an adjustment of jaw opening amplitude to the size of the food object, are mediated by an effector system involving a relatively small number of muscles and are amenable to both reflexive and voluntary control. This combination of structural simplicity and functional complexity suggests that the pigeon's jaw movements may provide a useful model system for the study of motor control mechanisms in targeted movements. The present report provides a classification of jaw opening movements occurring during eating and a preliminary determination of the extent to which each movement class is scaled to the size of the food object. Jaw movements were monitored during responses to spherical food pellets of six different sizes (3.2–11.1 mm in diameter) using a transducing system which produces a continuous record of gape (i.e., interbeak distance). Assignment to movement classes was then carried out using a computer-assisted scoring program. Functions relating jaw opening amplitude to target size were determined for each movement class. Four jaw movement classes were identified: Prepecks (just prior to pecking), Grasps (opening movements made during pecking but prior to contact with the target), Mandibulations (movements serving to position and transport the object within the buccal cavity) and Swallows. For two of these movement classes (Grasps, Mandibulations) jaw opening amplitude is scaled to pellet size but the scaling functions differ in ways that reflect the functional requirements of the two behaviors. However, for both movements, the data suggest that information about initial gape is used to control opening amplitude. It is concluded that during Grasping, the adjustment of opening amplitude to stimulus size involves visual inputs and open-loop control mechanisms, while for Mandibulation, that adjustment involves tactile input and closed-loop mechanisms.     

On the terms “reticulosis” and “reticulum cell sarcoma” with regard to the modern concept of the monocyte macrophage system

Summary The terms reticulosis and reticulum cell sarcoma (= malignant lymphoma, histiocytic type) are discussed regarding the modern concept of the monocyte macrophage system which today has replaced the ancient theory of the reticuloendothelial system. The monocyte macrophage system is not independent, but closely related to the myeloid system. Thus, a third blood forming system as was believed in the case of RES does not exist. Phagocytic reticulum cells of the various hematopoietic organs are highly activated monocyte-derived macrophages. All those conditions formerly termed reticuloses have been found to belong either to the myeloid or to the lymphatic system. Considering the reticulum cell sarcomas or malignant histiocytic lymphomas, most of them seem to be of lymphatic rather than of macrophage origin, representing highgrade malignant lymphomas, possibly immunoblastic sarcomas. No relationship between these tumours and the monocyte macrophage system has been established, so far. Therefore, the terms reticulosis and reticulum cell sarcoma should be no longer used in order to avoid confusion, in order to stimulate sufficient diagnostic efforts which will really clarify such cases, and in order to give full credit to modern results of hematopathology.