Olli Penttilä

Human chronic lymphocytic leukemia: Karyotypes in different lymphocyte populations

Abstract Karyotypes of different lymphocyte populations from 10 patients with chronic lymphocytic leukemia (CLL) showed two types of chromosome abnormalities. In cultures containing mitogens for T cells (leukoagglutinin (LA)) as well as T and B cells (pokeweed mitogen (PWM); protein A (PA)) random structural aberrations or trisomies were seen. However, one patient had a clone with trisomy 12 in LA- as well as PA-cultured cells. This clone represented about 10–20% of all mitoses, and was observed over a time period of 4 years. Nonrandom rearrangements were found in lipopolysaccharide B (LPS)-stimulated B lymphocytes from one individual in 11 cells analyzed. All these cells had multiple chromosome rearrangements. The cells probably represented a clone, since the karyotype was almost identical in all 11 cells, and the following reciprocal translocations could be identified: t(6;7), t(7;13), and t(11;14).

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

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.

Studies of auricular catecholamines by fluorescence histochemistry in various heart diseases of man

A comparative histochemical and clinical study concerning the state of the intrinsic adrenergic innervation of the human atrial myocardium was carried out, using the glyoxylic acid-induced fluorescence histochemical method. Specimens from the right auricular appendage were obtained during open-heart surgery from patients suffering from 1. ischaemic heart disease (IHD), 2. atrial septal defect of the secundum type (ASD), and 3. left-sided univalvular or multivalvular heart disease (VHD) with or without congestive heart failure (CHF) experienced prior to surgery. In the IHD group the densities of both the perivascular and the “free” myocardial adrenergic nerve net were greater than in the ASD group and especially in the VHD/ CHF group. Secondly, the intensity of fluorescence of the adrenergic structures was generally higher in the IHD group than that in the VHD/CHF group. Further, the average size of the varicosities, the number of varicosities per given length of axon, and the proportional share of the large varicosities were greater in the IHD group than in the ASD and VHD/CHF groups. The difference between the IHD and ASD groups was not great but was obvious in any case. In some patients with VHD/CHF fluorescing axons were observed only occasionally, and the tiny varicosities exhibited a hardly discernible fluorescence. Thus the amount of noradrenaline (NA) in the adrenergic fibres in the IHD group seems to be higher than in the ASD and especially VHD/CHF groups. The high level of NA in the IHD group is assumed to constitute a contributory factor in both intracellular metabolic changes and the systemic changes typical of myocardial ischaemia and infarction. In one patient with IHD and in six patients with VHD/CHF with significantly higher heart volume (mean±SD) compared with the rest of the patients (P<0.001), huge local axonal accumulations of NA in the form of “droplet fibres” were found. These enlarged, bulging adrenergic axons are assumed to be a consequence of mechanical trauma with stretching or disruption of the axons due to myodegenerative processes. It is further assumed that these “droplet fibres” are relatively common in those patients with diseased myocardium. They may constitute an extra contributory factor to the tendency to arrhythmias so typical of patients of this kind, by increasing the excitability of non-automatic tissue.

Adrenergic innervation of the human gall bladder

SummaryAdrenergic innervation of the human gall bladder was studied using two specific fluorescence histochemical methods. Blue-green fluorescing varicose nerves were scarce and mostly followed the course of blood vessels as typical perivascular plexuses. However, some adrenergic nerves not associated with the vessels were occasionally seen, as well as structures suggestive of a pericellular arrangement of varicose adrenergic nerve terminals on non-fluorescing ganglion cells. A few enterochromaffin cells were seen in the epithelial lining, also in the deep invaginations obviously representing the Aschoff-Rokitansky sinuses. Occasionally, small rounded cells with a rounded, relatively large nucleus, and exhibiting a weak yellow-green to blue-green granular cytoplasmic fluorescence, were observed in the wall of the gall bladder. The possible functional and evolutionary significance of these neural and endocrine elements was discussed against the data on physiological and pharmacological studies obtained from the literature. It was concluded that their significance is, in all probability, secondary to the influence of the intestinal polypeptide hormones, vagal innervation and circulating catecholamines upon the normal function of the gall bladder. The glyoxylic acid-induced fluorescence histochemical method was found to be superior to the conventional formaldehyde technique in studies on human tissue.

Multi-score estimation of catecholamine fluorescence for clinical purposes.

Experience accumulated at multi-score semiquantitation of catecholamine fluorescence of glyoxylic acid-treated stretch preparations of human clinical specimens is presented. The methodology and criteria of quantitation are described in detail. For an example, comparison between 3 different methods for analyzing neural-bound noradrenaline in human myocardial tissue in various heart diseases (obtained during the course of cardiac surgery) is presented: Biochemical determination of tissue noradrenaline content multi-score estimation of catecholamine fluorescence of glyoxylic acid-treated stretch preparations microfluorimetric analysis of the same stretch preparations. The results show that the multi-score estimation method gives a reliable concept of the relative amounts of noradrenaline stored in the intrinsic adrenergic nerve net (corresponding closely to the individual and group differences observed at biochemical noradrenaline determination). In addition, possible regional differences, alterations in the structural integrity of the inbuilt intrinsic nerve net, and other structural changes (e.g. pathological catecholamine accumulations) are easily recognized, whereas biochemical estimation cannot give information on structural aspects, which may have important clinical repercussions. Microfluorimetry does not seem suitable for studies on human myocardial specimens for several reasons which are discussed. The method of multi-score estimation of catecholamine fluorescence described and discussed is recommended for other similar and related studies on human clinical materials.

Atrial Acetylcholinesterase Activity in Various Heart Diseases of Man

Distribution and activity of the acetylcholinesterase enzyme in the human atrial myocardium was studied histochemically in a clinical series of patients subjected to cardiac surgery for (1) uncomplicated atrial septal defect (ASD), (2) ischaemic heart disease (IHD), (3) mitral and/or aortic valvular disease (VHD) necessitating replacement with a prosthetic valve, without major symptoms or signs of myocardial incompensation, or (4) clinically overt congestive heart failure (CHF) due to VHD prior to cardiac surgery. In all specimens, a rich distribution of acetylcholinesterase-positive single axons and small fascicles, constituting a three-dimensional nerve net, was observed within the myocardial tissue. This nerve net was obviously mainly parenchymatous, i.e. unrelated to the blood vessels. Small groups of acetylcholinesterase-positive small nerve cells were observed in some specimens, with loosely woven fascicles of axons emerging from one pole of the ganglia. No differences in the distribution of the acetylcholinesterase activity or in the pattern of the inbuilt intrinsic nervous apparatus were observed in the various groups of patients. All specimens were completely devoid of non-specific cholinesterase activity. It was concluded that (I) the human atrial myocardium is richly supplied with cholinergic intrinsic (post-ganglionic vagal) axons and (II) the acetylcholinesterase activity is not a major determinant of the parasympathetic abnormalities associated with cardiac diseases, especially with myocardial pump failure.

Histochemically demonstrable monoamine oxidase activity in the adult human heart in various cardiac diseases

The present work was undertaken in order to study the role of monoamine oxidase (MAO) enzyme in the genesis of altered cardiac noradrenalin level in the human heart in various underlying pathologic conditions. The histochemical localization and the activity of MAO were studied in the right atrial appendage of man in ischemic heart disease, in valvular heart disease without or with congestive myocardial failure, and in hearts with an uncomplicated atrial septal defect. MAO was found to be localized mainly extraneuronally in the muscle cells, a little activity was detected in the connective tissue spaces, and nerves reacting positively were tentatively identified. There were no significant differences in MAO activity measured photometrically between the various heart disease groups. It seems that MAO enzyme plays only a small or no role in the genesis of the altered noradrenalin level in the human heart observed in ischemic heart disease or congestive cardiac failure.