Showing papers by "Karolinska Institutet published in 1972"

Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells.

Abstract: By using the two criteria (a) high density of immunoglobulin determinants on the cell surface and (b) presence of receptors for C'3 on the cell surface for defining bone marrow-derived lymphocytes, it is indirectly shown that all or at least a major population of human thymus-derived lymphocytes under certain conditions will form nonimmune rosettes with sheep red blood cells (SRBC). Almost all thymocytes tested from two different donors formed rosettes. The SRBC rosettes are not formed by virtue of immunoglobulin receptors and form only around living cells. Positive bivalent ions are required for rosette formation since EDTA will block rosette formation. Sodium iodoacetate will also block rosette formation demonstrating the dependence on an intact glycolytic pathway. Rosette formation is temperature dependent and will not appear at 37°C. Trypsin treatment of lymphocytes will abolish their SRBC-binding ability which cannot be restored by treating them with fresh donor serum or fetal calf serum, but which will reappear after culturing the lymphocytes. It is suggested that these rosettes are formed by a rapidly released or metabolized receptor substance on the living cell surface which behaves as a trypsin-sensitive structure produced by the cells themselves.

Late prenatal ontogeny of central monoamine neurons in the rat: Fluorescence histochemical observations

Abstract: The early ontogeny of the monoamine neuron systems in the rat brain has been analysed using Falck-Hillarp fluorescence histochemistry. Serial sagittal sections of embryos with a crown rump length between 7 and 13 mm, approximately corresponding to gestational days 12 to 15 were obtained from mothers treated with a monoamine oxidase inhibitor given in order to increase the monoamine levels of the embryos. 5-hydroxytryptamine (5-HT)-neurons made their first appearance in the 8 mm embryo, dopamine (DA)-neurons in the 9 mm embryo, and noradrenaline (NA)-neurons in the 11 mm embryo. Small, rounded, weakly fluorescent cell bodies forming sparse aggregations appeared first. Fluorescent processes of two types soon appeared. Short processes from the cell bodies were running perpendicular to the long axis of the brain stem within the cell groups, while long slender axon bundles could be traced ascending through the met- and mesencephalon and into the prosencephalon as well as descending in the myelencephalon andspinal cord. In the 12 mm embryo the primordial DA cell formation of the substantia nigra with its striatal projections, the 5-HT neuron formations of the caudal mesencephalon, met- and myelencephalon as well as the NA neurons of the met- and myelencephalon are relatively well developed. It is concluded that the monoamine-neurons develop mechanisms for synthesis and storage of amines at a very early stage during ontogeny, thus recapitulating the phylogeny of these old systems. Likewise, monoamine oxidase is present early. The presence of neurotransmitters specifying the different developing neurons long before development of their nerve terminal areas and therefore before the establishment of normal synaptic function may indicate a role of these substances during ontogeny other than transmission of nerve impulses.

Isolation from Porcine‐Intestinal Wall of a Vasoactive Octacosapeptide Related to Secretin and to Glucagon

Abstract: A polypeptide composed of 28 amino acid residues has been isolated from porcine upper intestinal wall. In addition to exhibiting several biological activities which may be attributed to a relaxant effect on vascular and systemic smooth muscle it stimulates exocrine pancreatic secretion in a secretin-like fashion, with an apparent efficiency, on a molar basis, of 5–10% of that of secretin and also gives rise to hyperglycaemia with about 30% of the efficiency of glucagon. Structurally it resembles secretin and glucagon in having a histidyl-seryl sequence N-terminally. Of the amino acid residues commonly occurring in mammalian proteins the residues of cysteine/cystine, glycine, proline and tryptophan are absent from it.

Temperatures Measured in Human Cortical Bone when Drilling

Abstract: Temperature measurements have been made in cortical bone while drilling under controlled laboratory conditions. Cortical temperatures greater than 100 degrees centigrade were frequently recorded when drilling if no specific provisions for cooling were made. The force applied to the drill was found to be much more important than drilling speed as a factor in both the magnitude and duration of cortical temperature elevations. Increases in the force applied to the drill were associated with decreases in the maximum temperatures and the durations of temperature elevation. Worn drills caused much greater temperature changes than new drills. All forms of irrigation that allowed the stream of irrigating fluid to be directed to the point of penetration of the cortex were effective in limiting the increases in cortical temperature. Tapping did not appear to cause significant temperature elevations.

Induction of immunoglobulin and antibody synthesis in vitro by lipopolysaccharides.

Abstract: Lipopolysaccharides (LPS) from E. coli bacteria were found to be mitogenic for bone marrowderived (B) lymphocytes, but had no effect on thymusderived (T) lymphocytes. When added to normal spleen cells in culture, LPS selectively stimulated the secretion of 19 S proteins, whereas there was no demonstrable increase of 7 S protein synthesis. Spleen cell cultures treated with various doses of LPS exhibited atypical dose response curve with regard to induction of DNA synthesis, 10 μg/ml being optimal, higher and lower concentrations giving lower responses. When direct antibody producing cells to horse and sheep red cells were studied in normal spleen cell cultures exposed to different concentrations of LPS in vitro, it was found that their number increased in parallel with stimulation of DNA synthesis. In contrast, the number of antibody producing cells to LPS itself did not parallel activation of DNA synthesis. Spleen cells from LPS tolerant animals responded with increased numbers of antibody producing cells to heterologous red cells after treatment with LPS in vitro to the same extent as normal spleen cells. Thus, a B cell mitogen, such as LPS, could activate division in B cells, resulting in an increased number of cells producing antibodies to non-cross-reacting antigens, mimicing the effect of specific antigen.

Sensitivity of Epstein-Barr Virus (EBV) Producer and Non-Producer Human Lymphoblastoid Cell Lines to Superinfection With EB-virus

Abstract: Twenty-nine lymphoblastoid lines and one IgE-producing myeloma line of human origin were exposed to Epstein-Barr virus (EBV) concentrates in vitro. The adsorption of the virus to the outer cell membrane was assessed by counting the number of direct membrane fluorescence-positive cells immediately after infection and by the direct radioimmune membrane labelling method. Reference reagents were derived for both tests from the same serum (“Agnes”), containing antibodies against EB-viral envelope and capsid antigens. The intracellular course of infection was followed by counting the number of cells that responded with the development of early antigen (EA) 48 h after infection. The 11 lymphoblastoid lines that produced no EBV-determined membrane and early antigens adsorbed the virus, although there were quantitative differences between them. EA-positive cells appeared in significant numbers in only seven of them, however. Four lines remained EA negative in spite of a relatively good adsorbing capacity. The IgE-producing myeloma line showed neither virus adsorption nor EA development. Eighteen lymphoblastoid lines were “producers”, or “abortive producers”, i.e.a small proportion of the cells continuously generated two or three of the immunofluorescence-detectable viral antigens, MA, EA and VCA. Nine lines failed to adsorb significant virus quantities and showed no certain increase of EA-positive cells. The resistance of these lines to superinfection is probably determined at the level of viral receptors. Five lines showed a relatively good virus adsorption, but this was not followed by any significant increase in the number of EA-positive cells. Four lines showed good adsorption and also responded with a significant increase in the number of EA-positive cells. The same responses can thus be found to EBV-superinfection in producer and non-producer lines, but the producer lines show a strong preponderance of superinfection-resistant lines with an adsorption block at the receptor level. Sensibilite des lignees cellulaires lymphoblastoides humaines productrices ou non productrices de virus D'epstein-Barr (VEB) a la surinfection par le veb Vingt-neuf lignees lymphoblastoides et une lignee de myelome produisant des IgE, toutes d'origine humaine, ont ete exposees a des concentres de virus d'Epstein-Barr (VEB) in vitro. L'absorption du virus sur la membrane cellulaire exterieure a ete evaluee d'apres le nombre de cellules reagissant positivement a la fluorescence directe de la membrane immediatement apres l'infection et par la methode directe de marquage radioimmunitaire de la membrane. Pour les deux tests, les reactifs de reference provenaient du měme serum (“Agnes”) qui contenait des anticorps contre les antigenes des capsides et de l'enveloppe virale EB. Le cours intracellulaire de l'infection a ete observe d'apres le nombre de cellules qui reagissaient au developpement de l'antigene precoce (EA) 48 heures apres l'infection. Les onze lignees lymphoblastoides qui produisaient des EA ou des antigenes de la membrane non determines par le VEB absorbaient le virus, mais il y avait entre elles des differences quantitatives. Toutefois, les cellules EA-positives n'etaient tres nombreuses que dans 7 lignees. Quatre lignees sont restees EA-negatives en depit d'une capacite d'absorption relativement bonne. Dans la lignee de myelome produisant des IgE, on n'a observe ni adsorption du virus, ni developpement de l'EA. Dix-huit lignees lymphoblastoides etaient “productrices” ou “productrices abortives”, c'est-a-dire qu'une faible proportion des cellules produisaient continuellement deux ou trois antigenes viraux decelables par immunofluorescence, MA, RA et VCA. Neuf lignees n'adsorbaient pas de grosses quantites de virus et ne presentaient pas d'augmentation certaine des cellules EA-positives. La resistance de ces lignees a la surinfection est probablement determinee au niveau des recepteurs viraux. Cinq lignees se caracterisaient par une adsorption virale relativement bonne, mais il ne s'ensuivait aucune augmentation notable du nombre de cellules EA-positives. Dans quatre lignees, l'adsorption etait bonne et le nombre de cellules EA-positives augmentait considerablement. On peut donc observer les měmes reponses a la surinfection par le VEB dans les lignees productrices et non productrices, mais on constate parmi les premieres une forte predominance de lignees resistant a la surinfection avec un blocage de l'adsorption au niveau du recepteur.

Evidence for a receptor recognizing antigen complexed immunoglobulin on the surface of activated mouse thymus lymphocytes.

Abstract: The distribution in the mouse of lymphoid cells carrying receptors for IgG or IgG-Ag was investigated. B lymphocytes were found to have receptors reacting with both IgG and IgG-Ag. Resting T lymphocytes did not react with IgG-Ag. T lymphocytes activated by passage through irradiated, allogeneic mice had a receptor reacting with IgG-Ag, but not with IgG.

Lymphocyte mediated cytotoxicity in vitro. Induction and inhibition by humoral antibody and nature of effector cells.

Abstract: During the past decade, experimental evidence has been brought forward in many laboratories that lymphocytes are capable of destroying appropriate target cells in vitro. These cytotoxic effects of lymphocytes have been assumed to reflect their effector role in tissue damaging immune responses such as occur in allograft rejection, tumor surveillance and certain autoimmune diseases (Perlmann & Holm 1969). Lymphocyte mediated cytotoxicity in vitro is complex and may involve a number of different pathways. The contention that a cytotoxic reaction is lymphocyte induced does not necessarily imply that the lymphocytes which start the reaction also are the cytotoxic effector cells, nor that the latter are lymphocytes at all. In addition, even in those instances in which there is evidence that both inducerand effector cells are lymphocytic, lymphocytes of different origin and function may participate in the cytotoxic reaction. Work with mice in alloimmune situations, involving the H2 system, has provided evidence that thymus derived lymphocytes (T-cells) are required for cytotoxicity (Blomgren et at. 1970, Cerottini et al. 1970a,b, Golstein et al. 1972a). In a few instances available data suggest that T-cells may be both inducerand effector cells (Golstein et al. I972a,b). Recent evidence also indicates that humoral antibodies may induce cytotoxicity of thymus-independent lymphoid cells (Harding et al. 1971, Van Boxel ct al.

PPD tuberculin--a B-cell mitogen.

Abstract: Spleen cells from non-immunized mice and guinea-pigs are triggered to DNA synthesis in vitro by PPD tuberculin. The responding cells are shown to be B lymphocytes.

Autoradiographic identification of cerebral and cerebellar cortical neurons accumulating labeled gamma-aminobutyric acid ( 3 H-GABA).

Abstract: Small amounts of labeled gamma-aminobutyric acid (3H-GABA), a putative neurotransmitter, were injected stereotaxically into the cerebral and cerebellar cortices of rats pretreated with aminooxyacetic acid (AOAA), a drug which prevents GABA breakdown. After fixation by aldehyde perfusion the tissue was processed for light and electron microscopic autoradiography.

Separation of two forms of rabbit metallothionein by isoelectric focusing.

Abstract: Rabbits were given repeated injections of cadmium chloride. Cadmium- and zinc-containing protein fractions were obtained from the livers of these animals by precipitation procedures and Sephadex G-75 chromatography. The protein thus obtained showed several characteristics similar to those of the earlier described protein metallothionein. Further separation by isoelectric focusing showed two main protein peaks with isoelectric points at 3.9 and 4.5 respectively. Amino acid analysis of these two forms showed similar content of most amino acids [residues per cent.: cysteine (28%), aspartate (8%), threonine (5-6%), serine (12%), glycine (7%), alanine (13%), methionine (2%), isoleucine (2%)] but with a small difference in content of lysine (12 and 13% respectively), proline (9 and 5% respectively) and glutamate (2 and 4% respectively). The two forms of the protein both contained cadmium, but only the one with pI4.5 contained also significant amounts of zinc.

Separation of cells according to surface antigens by the use of antibody-coated columns. Fractionation of cells carrying immunoglobulins and blood group antigen.

Abstract: It has been found possible to separate cells according to their surface anti gens by the use of antibody-coated columns. High efficiency columns were made by double-layer principles, first coating beads with antigen followed by antibody in excess. Such columns could be shown to contain a high amount of free antigen-binding sites for the relevant antigens. Lymphoid cells were thus fractionated according to their surface concentration of immunoglobu lin and a highly selective retention of mouse B lymphocytes was observed when filtering spleen cells through an anti-immunoglobulin column prepared according to the above procedure. No evidence of retention of mouse T lymphoid cells was observed in the same system. By the use of anti-gamma-2a immunoglobulin columns, it was found possible to deplete a population from memory cells potentially capable of synthesizing gamma-2a antibodies. No evidence was found that columns prepared in the described manner would function through combining with receptors on lymphoid cells for antibody-antigen complexes. By using anti-A blood group columns, it was possible to selectively retain cells (erythrocytes or kidney cells) with A blood group anti gen on their surface. High-avidity immune antibodies were found to be more efficient than ‘natural’ anti-A antibodies in this test. No evidence was found of anti-A antibodies being adsorbed on to the passing cells as tested by in vitro serological tests and tissue culture experiments. The applications of a technique for separating cells according to their surface antigens are con sidered obvious.

Mitogens as probes for immunocyte activation and cellular cooperation.

Abstract: 2. Proliferative responses to mitogens 2.1 Selective effects of mitogens on T and B lymphocytes 2.2 Activation of B cells by Con A 2.2.1 Evidence for a soluble T cell factor 2.2.2 Support for the local concentration hypothesis 2.3 Cap formation does not correlate with lymphocyte activation 2.4 High dose unresponsiveness to Con A is reversible 2.5 Quantitation of the number of activating mitogen molecules 2.6 A model for mitogen-induced lymphocyte stimulation

Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology

Abstract: Three enzymes in the catecholamine synthesis—dopa decarboxylase (DDC), dopamine-β-oxidase (DBH) and phenylethanolamine-N-methyltransferase (PNMT)—have been isolated. Subsequently antibodies to these enzymes were prepared and used in immunohistochemical studies mainly with the aim to elucidate methodological problems. The indirect immunofluorescent technique was used throughout the study. It was found that cryostat sectioning followed by fixation in acetone or alcohol, a standard procedure in immunohistochemistry, was successful only with antibodies to the granule bound enzyme DBH. With antibodies to DDC and PNMT, two cytoplasmic enzymes, on the other hand, the results were hampered by diffusion artefacts. These drawbacks could be prevented by a brief aldehyde fixation, preferably by perfusion before cryostat sectioning. The best results were obtained with formalin followed by hydroxyadipaldehyde and acrolein. However, after glutaraldehyde fixation no specific fluorescence at all was observed. Freezing of fresh adrenals, followed by freeze-drying, treatment with formaldehyde vapours and paraffin embedding was tested but consistent results were only obtained with antibodies to PNMT. A new instrument, the Vibratome®, which allows sectioning of unembedded fixed or unfixed tissue, was used and successful results were obtained with all three antibodies. Furthermore, the possibility with this instrument to combine the immunohistochemical technique e.g. with the formaldehyde fluorescence method for visualization of monoamines is demonstrated. It is emphasized that the Vibratome ® technique may be a valuable tool for immunohistochemical studies on the central nervous system.

Coding of Amplitude and Frequency Modulated Sounds in the Cochlear Nucleus of the Rat

Abstract: The responses of single units in the cochlear nucleus of the rat to sinusoidally amplitude- and frequency-modulated tones and amplitude-modulated broadband noise were studied. The distribution of discharges within a cycle of modulation was determined from cycle histograms locked to the modulation wave. In response to amplitude-modulated tones and broadband noise, all units investigated showed a peak in the degree of modulation of the spike frequency within the modulation frequency range from 50 to 200 Hz. In many units the relationship between the degree of modulation of the stimulus sound and of the modulation of the resulting spike train was almost unchanged over a wide range of sound intensities. In other units, enhancement of modulation within a certain range of modulation frequency became more pronounced when the sound intensity was increased. This was mainly due to a suppression of modulation at lower modulation frequencies. The shape of the histograms was nearly sinusoidal even at modulation depths which resulted in nearly 100% modulation of the neural discharge frequency. The amount of modulation of the discharge frequency in response to frequency-modulated tones was dependent on the frequency of the tone in relation to the CF of the unit.

Activation of b lymphocytes by locally concentrated concanavalin a.

Abstract: B lymphocytes are not stimulated by soluble concanavalin A (Con A). However, Con A cross-linked at the bottom of tissue culture petri dishes could activate DNA synthesis in B cells. Addition of free Con A to such cultures inhibited the proliferative response to insoluble Con A. T lymphocytes were not activated by locally concentrated Con A, although they responded to soluble Con A. In the presence of both soluble and cross-linked Con A there was a shift in the dose response curve to soluble Con A, the magnitude of which varied with the concentration of cross-linked Con A.

Cells mediating specific in vitro cytotoxicity. II. Probable autonomy of thymus-processed lymphocytes (T cells) for the killing of allogeneic target cells.

Abstract: In order to investigate whether only T cells are involved in a cell-mediated cytotoxic system in vitro, we tested the cytotoxicity of immune killing cell populations as deprived as possible of B cells. Educated thymus cells, immune spleen cells purified by filtration through a column of beads coated with antimouse Ig antiserum, and finally educated thymus cells further purified by filtration through such a column fully retained their specific cytotoxic activity. This very strongly suggests that only T cells are involved in the killing of target cells by allogeneic immune cells in vitro, in this system. Receptor-bearing cells involved in killing in the present system are thus very probably T cells. This point was further strengthened by the demonstration of specific adsorption, on the relevant monolayers, of each of the three above mentioned killing cell populations.

“Ayahuasca,” the South American hallucinogenic drink: An ethnobotanical and chemical investigation

Abstract: The Sharanahua and Culina, small Indian tribes located in the southwestern Amazon basin, use a hallucinogenic drink for medicinal and social purposes. This decoction, called “Ayahuasca” in Peru, is prepared from Banisteriopsis Caapi stems and Psychotria sp. leaves. These plants have been botanically identified on the basis of voucher herbarium specimens and investigated for alkaloid content by means of a gas chromatography-mass spectrometry technique. A list of other occasional plant admixtures is given. Harmine, Harmaline, Tetrahydroharmine, Harmol and 6-Methoxytryptamine have been found in Banisteriopsis Caapi. Dimethyltryptamine, Monomethyltryptamine and 2-methyl-1,2,3,4-tetrahydro-β-carboline have been found in Psychotria viridis and Psychotria carthaginensis. Harmine, Harmaline, Tetrahydroharmine and Dimethyltryptamine have been found in the drink. Quantitative calculations show the amount of each alkaloid administered in the Ayahuasca drink.