A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Nachweis der Plasmocytom-γ-Globuline und ihrer Individualspezifität mit der Stärkegel-Elektrophorese

Zusammenfassung Mit der Stärkegel-Elektrophorese nachSmithies wurden an 76 _ss- und 30 _1A-Plasmocytom-Seren folgende Befunde erhoben:1. Plasmocytom--Globuline sind im Gegensatz zu den Befunden der Papierelektrophorese nicht homogen, sondern aus mehreren Komponenten zusammengesetzt. Anordnung, relative Konzentration und Lokalisation dieser Komponenten sind für den einzelnen Patienten konstant und charakteristisch, also individualspezifisch.2. Die Heterogenität des _1A-Plasmocytom-Globulins ist durch höhermolekulare Anteile bedingt, die sich als distinkte Banden im Stärkegel nachweisen und durch Cysteamin dissoziieren lassen.3. Bei _ss-Plasmocytom-Globulinen werden zwei Typen beobachtet: a) In der Mehrzahl der Fälle sieht man multiple, maximal sieben bis neun Komponenten, die im Einzelserum durch gleiche Breite und regelmäßige Abstände gekennzeichnet sind; sie können sich annäherungsweise in ihrer relativen Konzentration einer Gauss-Verteilung unterordnen oder zeigen in anderen Fällen eine kathodenwärts zunehmende Konzentration. b) Bei ca. 1/4 der Seren wurde keine Unterteilung in mehrere Komponenten erreicht.4. Auch die nach Papainspaltung⁴⁴ isolierter _ss-Globuline entstehenden Fragmente sind in mehrere Komponenten unterteilt; charakteristische und ebenfalls individualspezifische Unterschiede zwischen normalen und abnormen _ss-Globulinen sind im S-Fragment nachweisbar⁴⁵.5. Die Stärkegel-Elektrophorese kann als zuverlässiges und reproduzierbares Verfahren die Immunoelektrophorese in der Erkennung abnormer -Globuline ergänzen bzw. ersetzen. Dies gilt besonders für den Nachweis und die Identifizierung von Bence-Jones-Proteinen im Harn und Serum und von sog. -Plasmocytomen.6. Die Untersuchungen zeigen, daß erworbene Anomalien der Proteinsynthese individualspezifisch sind. Die Befunde werden im Hinblick auf die Proteinsynthese in den Plasmazellen diskutiert.

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Summary Report on starch-gel electrophoresis studies on 76 sera of _ss-myeloma and 30 sera of _1A-myeloma. Most myeloma-_ss-globulins are not homogeneous but heterogeneous. Concentration and localization of these components are individually different. Heterogeneity of _1A-myelomaglobulins is dependent on higher molecular species, lacking after dissoziation by 2-mercaptoethanol. There exist two types of _ss-myeloma-globulins: a) in most cases starch-gel electrophoreses reveals multiple bands characterized by regularity of closely spaced periodic intervals; relative concentration of the bands may be distributed in some cases like a Gauss-curve or may increase in other cases to the cathodic end of the starch-gel strip. b) A single band was found in about 1/4–1/5 of the _ss-myeloma sera. — The papain-fragments of _ss-myeloma globulin also are composed of multiple components; individually specific differences were found in the S-fragments whereas F-fragments were identical with those of normal _ss-globulin. -myeloma often reveals an abnormal component in the post-albumin region (between albumin and transferrin), corresponding to the Bence-Jones protein of the urine.The findings favor the possibility that the single clone of plasma cells in myeloma forms multiple protein components. Consequently we may assume that the single plasma cell synthesizes a group of closely related proteins.

     

Interferon-γ for treatment of severe atopic eczema in two children

Two 4- and 5-year-old children suffering from refractory atopic dermatitis were treated with recombinant interferon- (rIFN-). rIFN- was injected at 50 g subcutaneously three times a week in the first child for 3 weeks, followed by three times 25 g in week 4. In the other child two treatment courses of 4 weeks were given after a break of 2 weeks. Therapy was well tolerated. In child one reductions in eczematous body surface and severity of lesions were observed, while no beneficial effect was seen in the other. Clinical chemistry data remained unchanged. Immunological studies performed in parallel showed a decrease in total serum IgE of 50% in child 1, a decrease in spontaneous in vitro IgE production, an increase in in vitro production of interleukin-6, and a normalization of previously decreased in vitro lymphocyte responses to several mitogens. While marked immunological changes were noted during IFN- treatment, clinical benefits were not encouraging. Diminished IFN- production has been claimed to be a major pathogenic factor in atopic eczema. Our results indicate that the pathogenesis is more complex. Clinically, we were unable to confirm previous observations in adults. Further studies are needed before IFN- can be recommended for therapy of pediatric atopic eczema.Abbreviations IFN- interferon- - IL interleukin     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

Sublocalization on chromosome 21 of human interferon-alpha receptor gene and the gene for an interferon-gamma response protein

The cellular responses to alpha and beta interferons (IFN- and -) are mediated through the IFN-/ (type I) receptor, while the response to IFN- is mediated through the IFN- (type II) receptor. The receptors for IFN-/ and IFN- are encoded by genes on human chromosomes 21 and 6q, respectively. The presence of chromosome 21q confers both ligand binding and responsiveness to human IFN-/, whereas chromosome 6q confers binding of Hu-IFN-, but not cellular responsiveness on somatic cell hybrids. Chromosome 6q (i.e., the Hu-IFN- receptor gene) and chromosome 21q are both necessary for the cellular response of somatic cell hybrids (from fibroblasts) to Hu-IFN-. It is conceivable that the factor mediating activity through the IFN- receptor is, in fact, the IFN- receptor, or that the two genes are distinct but part of an interferon response region. Here we more precisely localize on human chromosome 21 the genes for the IFN- receptor and for the factor(s) mediating the action of IFN- through the chromosome 6-encoded receptor. Hamster-human somatic cell hybrids containing various fragments of human chromosome 21 were used. The presence of the human IFN-/ receptor was determined by binding³²P-labeled human IFN- to cells, covalently cross-linking the [³²P]IFN--receptor complex, and analyzing it by SDS-polyacrylamide gel electrophoresis. The presence of the IFN- receptor-related factor mediating cellular responsiveness was determined by HLA induction in hybrid cells containing the IFN- receptor (chromosome 6q), a transfected copy of the human HLA-B7 gene, and various portions of chromosome 21. In all hybrids examined, the two genes cosegregate. Specifically, both genes are localized to the region of chromosome 21 containing the markers D21S58, D21S65, and GART and appear to be proximal to D21S58. The implications for IFN action are discussed.     

Anticonvulsant therapy and serumγ-glutamyl-transferase

Summary In 230 adult epileptic patients on long-term anticonvulsant therapy both serum drug levels and -glutamyl-transferase (GT) activities were determined and presented in groups according to the drug or drug combination in use. The GT activities measured never exceeded 250 U/l, there was a marked sex difference with lower values for females in all groups. The incidence of elevated GT values was between 90 and 100% for patients treated with diphenylhydantoin (DPH), phenobarbital (Pb), primidone (Prim) and combinations (Comb.), but lower for patients treated with carbamazepine (Cz). In this group with Cz-therapy mean value and standard deviation of GT values were also much lower than in the other groups. Only females with DPH alone and males with Pb alone showed a significant but slight correlation between drug levels and GT. These findings permit the assumption that serum GT is induced by treatment with DPH, Pb, Prim and Comb., to a lesser extent with Cz. Relating GT elevation to clinical data other than anticonvulsant drug therapy (e.g. age, type of seizures, duration of illness, history of intoxication) did not produce any subgroup with specific and consistent behaviour of serum GT. DPH-saturation did not appear to play an important part in GT elevation, rather the strongest stimulus for GT elevation seemed to derive from the initiation of anticonvulsant therapy and the highest levels of serum GT were reached between 1 and 4 weeks before the highest anticonvulsant levels. With long-term anticonvulsant therapy there appeared to be a direct relationship between serum GT and drug level for individual patients. Simultaneous estimations of serum drug levels and GT activities are helpful for monitoring patient compliance during long-term anticonvulsant therapy.     

Immunoelektrophoretische Studien an Myelomseren

Zusammenfassung 17 Seren von Kranken mit klinisch gesichertem Myelom wurden mit Hilfe der Papier- und der Immunoelektrophorese untersucht. Hierbei wurde jedes Serum 1. gegen ein Anti-Normalserum, 2. gegen ein Anti-Normalserum, aus welchem das -Globulin durch Absorption entfernt wurde, in 6 von den untersuchten Fällen auch 3. gegen das spezifische Antimyelomserum sowie 4. gegen das mit physiologischem -Globulin versetzte spezifische Antimyelomserum getestet.Es konnte festgestellt werden, daß sich Myelomfraktionen wie normale Globulinfraktionen verhalten, da sie I. bei Diffusion gegen Anti-Normalserum auspräcipitieren wie die entsprechenden physiologischen Fraktionen, und da II. der gegen die Myelomfraktionen gebildete Antikörper im entsprechenden Immunserum durch ein physiologisches Antigen — hier ein -Globulin — absorbiert wird, was darin zum Ausdruck kommt, daß Myelomfraktionen gegen ein Antimyelomserum nach Absorption durch -Globulinkeine Präcipitation mehr ergeben.Im einzelnen konnte die -Globulinfraktion von 9 papierelektrophoretisch reinen -Typen in 4 Fällen alseinheitliche, in weiteren 4 Fällen alsdoppelte und in einem Fall alsweitauslaufende Präcipitationslinie differenziert werden. Ferner waren zwei ₁-Myelome, ein ₂-, einM-Myelom, sowie drei ₂-Myelome immunoelektrophoretisch darstellbar. In einem papierelektrophoretisch normalen Serum konnte mit der hier angewandten immunologischen Methode eine im ₂-Bereich gelegene Präcipitationslinie nachgewiesen werden.Herrn Prof. Dr.H. Pette zum 70. Geburtstag.     

Isoenzymes of γ-glutamyl transpeptidase in liver diseases

Summary A new method for the separation of isoenzymes of-glutamyl-transpeptidase is described, using electrophoresis on acetate cellulose gel and a developing solution composed by-glutamyl-naphthylamide, and a colored diazonium compound.The method permits the separation of up to four different isoenzymes, which we called-GT₁,-GT₂,-GT₃,-GT₄, the first two showing an electrophoretic migration similar to that of ₁- and ₂-globulins and the other two to that of-globulins.The present technique has proved its usefulness in detecting isoenzymes in serum with values of total-glutamyl-transpeptidase higher than 80 U/L.The application of this method in 52 patients with different types of biliary obstruction and hepatocellular damage has shown that it provides new possibilities in differential diagnosis.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

Interferon-γ-like immunoreactivity in developing rat spinal ganglia neuronsin vivo andin vitro

Summary Interferon--like immunoreactivity was observed in a subpopulation of 16-day-old embryonic rat spinal ganglion neurons using two monoclonal antibodies directed against different epitopes of recombinant interferon-. During ontogenesis bothin vivo andin vitro, it was found that the strong immunoreactivity was confined to small neurons when neurons become morphologically distinct on the basis of size.In vivo, the interferon--immunoreactive neurons started to express major histocompatibility complex class I antigens after the first postnatal week, whilein vitro no such antigen could be detected. A quantitative Elisa method was developed to determine the levels of major histocompatibility complex class I and interferon-in vitro, whereby increased amounts of major histocompatibility complex class I antigen was detected after exposing the cultures to recombinant interferon- and Sendai virus. Sendai virus also caused a small increase in interferon- with a peak about 12 hours after infection. Thein vitro system will be used to study further the role of the putative neuronal interferon--like molecule in the regulation of cell growth, for induction of major histocompatibility complex antigens and in virus infection of sensory neurons.     

Isoproterenol and GTPγS inhibit L-type calcium channels of differentiating rat skeletal muscle cells

Summary In adult skeletal muscle, G-proteins have been shown to modulate the calcium channels both directly and through a cAMP-dependent phosphorylating mechanism. We have investigated the action of G-proteins on the L-type calcium current in cultured rat muscle cells (myoballs) under voltage clamp in whole cell or perforated patch modes. Intracellular photolytic release of 200 M GTPS inhibited the L-type calcium current. Inclusion of 500 M uncaged GTPS in the patch pipette in the whole cell configuration reduced the calcium current by a similar amount. Under perforated patch conditions external application of 10 M of the -adrenergic agonist isoproterenol also reduced the calcium current. Pretreatment of the cells with pertussis toxin reversed the effect of GTPS and removed that of isoproterenol. We conclude that rat myoballs contain -adrenergic receptors that inhibit the L-type calcium current, and that this inhibition is mediated by a pertussis toxinsensitive G-protein.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Elevated numbers of gamma-delta (γδ+) T lymphocytes in children with immune thrombocytopenic purpura

Immune thrombocytopenic purpura (ITP) in childhood is a heterogeneous clinical disorder characterized by immune-mediated platelet destruction. Although generally considered to involve autoreactive B lymphocytes which produce antiplatelet antibodies, there is increasing evidence that T lymphocytes also play an important role in this autoimmune process. We studied 11 children with acute ITP and 19 children with chronic ITP and observed elevated numbers of TCR+ T lymphocytes in several patients. In the three children with the highest elevations (TCR+/CD3+ percentage ranging from 37.8 to 48.1% at initial evaluation), the expanded cell population exclusively expressed the surface V2/V heterodimer and had enhanced,in vitro proliferation to mycobacterial extracts and IL-2. Analysis of the nucleotide sequences used by these TCR+ cells demonstrated a diverse set of VDDJC gene rearrangements, indicating polyclonal expansion of cells reminiscent of a superantigen response. There was a close correlation between the number of TCR+ T lymphocytes and the degree of thrombocytopenia in each patient. TCR78+ T lymphocytes may be important in the pathogenesis of immunemediated platelet destruction in some children with ITP.     

Autogenetic inhibition of γ-motoneurons in the spinal cat uncovered by Dopa injection

Summary The response of identified gastrocnemius -efferents to stretch of the triceps surae muscle was recorded in the acute spinal cat before and after the injection of Dopa. A strong tonic inhibition during static muscle extension was detectable only in those -efferents which had no resting and reflex activity in the spinal cat, but which responded to Dopa injection with an initially high discharge rate. In contrast -motoneurons with background and reflex activity before Dopa, and some previously quiescent units acquiring a low discharge rate after Dopa did not exhibit any signs of a stretch-dependent inhibition. It is concluded that the observed autogenetic inhibition is limited to the static -motoneurons. The responsiveness to stretch-evoked inhibition was found to be correlated with that to antidromic inhibition induced by repetitive stimulation of the ventral root. This could imply that the activation of -motoneurons by muscle stretch leads to a Renshaw inhibition of the -motoneurons. However, this pathway may be only partly responsible for the autogenetic inhibition of -motoneurons in the spinal cat after Dopa, since the inhibitory effects of muscle stretch were more pronounced than those obtained by antidromic activation of motor axons in the ventral root.     

Inhibitory effects ofγ-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones

The effects of mouse recombinant-interferon (-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg⁹-bradykinin and prostaglandin E₂ (PGE₂) have been studied using cultures of neonatal calvarial bones and analyzing the release of⁴⁵Ca from prelabelled bones as a paramenter of bone resorption. In addition, the effects of-IFN and indomethacin on formation of PGE₂ in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mol/l) totally abolished bradykinin (1 mol/l) induced⁴⁵Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE₂ (1 mol/l).-IFN (1000 U/ml) almost totally inhibited⁴⁵Ca release stimulated by bradykinin (1 mol/l), but the inhibitory effect could only be partially overcome by PGE₂.-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg⁹-bradykinin (1 mol/l) on⁴⁵Ca release. The stimulatory effects of PGE₂ (1 mol/l) on radioactive calcium mobilization was partially inhibited by-IFN (1000 U/ml), whereas indomethacin (1 mol/l) was without effect. The inhibitory effect of-IFN on⁴⁵Ca release stimulated by bradykinin and PGE₂ was dose-dependent with threshold for action at 3–30 U/ml. Comparative dose-response curves showed that-IFN was most potent as inhibitor of bradykinin induced⁴⁵Ca release. Bradykinin (1 mol/l) significantly stimulated PGE₂ formation by a mechanism that was completely inhibited by indomethacin (1 mol/l).-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE₂ formation. These data show that i)-IFN is a potent inhibitor of bone resorption induced by bradykinin and bradykinin analogues and ii) that the mechanism of action can be mainly explained by an inhibition of kinin induced prostaglandin biosynthesis. The results, however, also show that-IFN can inhibit bone resorption by mechanisms unrelated to prostaglandin formation.     

Recurrente Hemmung der Extensor-Fusimotoneurone?

Summary The antidromic effect of ventral root or muscle nerve stimulation has been studied on functionally isolated -efferents to the medial gastrocnemius muscle in the intercollicular decerebrate cat. Tetanic stimulation of the ventral root at levels below the threshold for the investigated -efferents decreases the activity of 10 out of 19 -motoneurones. This effect is greater in cats with intact contralateral dorsal roots. Tetanic stimulation of synergic muscular nerves inhibits some of the investigated -motoneurones, whereas tetanic stimulation of antagonistic muscular nerves fails to have any inhibitory effect. It is suggested that the recurrent inhibition of -motoneurones is mediated via at least one additional interneuron between the Renshaw cell and the -motoneuron.

     

γ-Glutamyltranspeptidase-Aktivität im Urin bei Gesunden und Nierenkranken

Zusammenfassung Die -Glutamyltranspeptidase-Aktivität (-GT) in der Niere ist bei chronischen Nephropathien und beim akuten Nierenversagen vermindert. Bei Gesunden wurde im Urin eine -GT-Aktivität von 12,7–37,7 mU/min ermittelt. Serum- und Urinaktivität verhielten sich unabhängig voneinander. Eine erhöhte -GT-Ausscheidung fand sich bei 75% der Nierenkranken mit normaler GFR, aber nur bei 11% mit eingeschränkter GFR.Zwischen -GT-Ausscheidung und Kreatininclearance bestand bei Gesunden (N=68,r=0,67) und Nierenkranken (N=114,r=0,71) eine statistisch hochsignifikante Korrelation. Bei Gesunden wurden deshalb die Toleranzgrenzen für beide Parameter gemeinsam berechnet. Bei Nierenkranken nahm die Enzymausscheidung proportional zur GFR und damit dem funktionsfähig verbleibenden Nierenparenchym ab. Die Relation -GT/C_Kreatinin erlaubt eine pathologisch gesteigerte Enzymabgabe aus den Tubuluszellen bei noch im Normalbereich liegender Gesamtausscheidung zu erkennen.     

Gamma-X-Globulin,eine immunoelektrophoretisch nachweisbare γ-Globulin-Komponente mit Beziehungen zum C-Reaktiven Protein

Zusammenfassung Bei der immunoelektrophoretischen Untersuchung pathologischer Seren mit Anti-Humanserum vom Pferd tritt im-Globulinbereich gelegentlich ein schwaches, in der Regel durch normale-Globuline überlagertes Immunpräcipitatsband auf, für das wir ein mit-X-Globulin bezeichnetes Paraprotein verantwortlich machen. Sein Nachweis gelingt wesentlich leichter, wenn das Anti-Humanserum zuvor mit normalem-Globulin oder normalem Serum in geeigneter Weise vorbehandelt wird.Das-X-Globulin findet sich vornehmlich in Seren von Patienten mit entzündlichen und neoplastischen Erkrankungen, und zwar unabhängig davon, ob ein europäisches oder zentralafrikanisches Krankengut untersucht wird.Es wird gezeigt, daß-X-Globulin ein Komponentensystem darstellt, das mit C-reaktivem Protein immunologisch verwandt ist.Die aus Pleurapunktat isolierte-X-Globulin-fraktion erweist sich im Geldiffusionstest auch immunologisch verwandt mit der Hauptfraktion normaler-Globuline.     

In vivo neutralization of tumor necrosis factor-alpha and interferon-gamma abrogates resistance to Yersinia enterocolitica infection in mice

Cytokines are important mediators of the inflammatory host response against infectious agents. In this study, the role of tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) in the elimination of a primary infection with highly virulent Yersinia enterocolitica serotype 0:8 strain WA-P has been investigated in C57BL/6 mice. The injection of anti-TNF- or anti-IFN- antibodies (serotherapy) prior to the intravenous challenge of a sublethal dose of Y. enterocolitica caused an increased bacterial net-growth in the spleens, although this effect was more pronounced for anti-TNF- treatment. The later treatment with anti-TNF- or anti-IFN- antibodies on day 3 post infection likewise abrogated resistance to Y. enterocolitica and, subsequently, led to death from progressive infection. Our data demonstrate for the first time that the endogenous production of both the cytokines TNF- and IFN- is required for the restriction of a primary Y. enterocolitica infection in mice.