Adrenergic mechanisms in the hyperglycaemia and hyperinsulinaemia of diabetic KK mice

Summary Simultaneous occurrence of hyperglycaemia (up to 300 mg/100 ml) and hyperinsulinaemia (up to 1200 U/ml) was evident in the fed state in genetically diabetic KK mice, indicating insulin resistance. The hyperinsulinaemia was enhanced by -adrenergic blockade and alleviated by B-blockade. Epinephrine-induced hyperglycaemia and isoproterenol-induced hyperinsulinaemia, both of which could be blocked by -adrenergic antagonists, were more marked in KK mice than in ICR mice. A possible significance of adrenergic mechanisms is discussed with respect to the aetiology of the metabolic disturbances characteristic of KK mice.     

Characterization of an acute T lymphoblastic leukemia expressing the γ/δ T-cell receptor

Summary Leukemic cells of a 20 year old patient, suffering from acute lymphoblastic leukemia, were characterized by surface marker and functional analysis. A significant cell population within this type of leukemia expresses concomitantly the CD4 and CD8 antigen on the same cell and might represent a new differentiation stage of T-cells with the / receptor. The leukemic cells show a distinct pattern of growth response to mitogens and lymphokines, which might correlate to their differentiation stage. Moreover, a natural killer-like activity can be induced in these cells by IL-2.Abbreviations FITC fluorescein isothiocyanate - PE phycoerythrin - IL-2 interleukin 2; - / TCR gamma/delta T cell receptor - NK natural killer - PBL peripheral blood lymphocytes - T-ALL acute T lymphoblastic leukemia - ConA concanavalin A - PMA phorbol myristate acetate - BM bone marrow - IL-2R IL-2 receptor - TdT terminal deoxynucleotidyl transferase Supported by the Deutsche Forschungsgemeinschaft (DFG Wi-728/3-1)     

Clinical evaluation of plasma des-γ-carboxy prothrombin as a marker protein of hepatocellular carcinoma in patients with tumors of various sizes

We measured des--carboxyprothrombin (DCP) (prothrombin induced by vitamin K absence or antagonist-II, abbreviated as PIVKA-II) by a newly developed enzyme immunoassay using an anti-DCP monoclonal antibody in 665 human subjects, of which 112 were patients with hepatocellular carcinoma (HCC). PIVKA-II was elevated to more than 0.1 AU/ml in 54 of the 112 patients (48.2%) with HCC, while it was positive only in 7.1% of those with liver cirrhosis and 3.1% of those with chronic hepatitis. Three patients with elevated PIVKA-II greater than 0.1 AU/ml who had been diagnosed as having liver cirrhosis by ultrasonography and computed tomography at the start of this study developed a diffuse type of HCC three or six months later, which was detected by angiography. No obvious correlation was observed between plasma PIVKA-II concentration and serum -fetoprotein (AFP) level in HCC patients. Of the 112 HCC patients, 40.2% showed an increase in AFP to above 200 ng/ml. In the remaining patients, 32.8% had a PIVKA-II concentration greater than 0.1 AU/ml. In these patients with a negative or low serum AFP concentration, PIVKA-II proved to be a valuable tumor marker for laboratory diagnosis of HCC. Among them, 59.8% tested positive for PIVKA-II and/or AFP. Thus, combination assay with PIVKA-II and AFP seems useful for increasing the accuracy of laboratory diagnosis of HCC. None of patients with a solitary tumor smaller than 2 cm had elevated PIVKA-II. In patients with larger-sized and multiple HCC, positive results of elevated PIVKA-II were more frequent than those of increased AFP. Thus, the determination of PIVKA-II may be more useful than AFP assay in patients with larger-sized and multiple tumors. The levels of plasma PIVKA-II concentration were higher in patients with larger-sized and multiple tumors than in those with smaller ones. In 20 patients, PIVKA-II decreased significantly after transcatheter arterial embolization (TAE) therapy, and in eight of these 20 patients it normalized after TAE. In conclusion, plasma PIVKA-II might be used as a valuable marker for the diagnosis and screening of HCC, especially in patients with negative or low AFP and in those with larger-sized and multiple tumors. However, its usefulness for mass screening of small HCC is limited.     

Influence of insulin on cyclic 3′,5′-AMP phosphodiesterase activity in liver,skeletal muscle,adipose tissue,and kidney

Summary Influence of insulin on liver glycogen metabolism and on lipolysis appears to be mediated by a decreased intracellular 3,5-AMP concentration. Reduced formation of 3,5-AMP had been shown in adipose tissue incubated with insulin. The influence of insulin on 3,5-AMP degradation has been investigated. — 3,5-AMP phosphodiesterase (PDE) activity was reduced in liver, adipose tissue and, insignificantly, in skeletal muscle of insulin deficient, i.e. alloxan diabetic or starved rats. I.V. injection of a low dose of insulin (0.5 U/kg) or stimulation of endogenous insulin secretion by injection of glucose led to a rapid increase of PDE activity in these tissues. 15 min after insulin injection liver PDE activity was increased. The maximal effect occurred after 30–45 min. Renal PDE activity was not decreased in alloxan diabetes, insulin injection has been found ineffective. —In vitro, there was an activating effect of crystalline insulin on PDE purified from beef heart. Insulin concentration required for duplication of enzyme activity was of the order of 2 · 10^–5 M. Treatment with actinomycin D nearly prevented stimulation of liver PDE by insulin. This may indicate that the action of insulin on PDE activity is essentially based on an increased enzyme synthesis. — Owing to the influence of insulin secretion on liver and adipose tissue 3,5-AMP concentration, glycogen metabolism and lipolysis can be quickly adapted to food intake.

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Der Einfluß von Insulin auf die 3,5-AMP-Phosphodiesterase-Aktivität in Leber, Skeletmuskulatur, Fettgewebe und Niere

Zusammenfassung An der Steigerung der Glykogensynthese der Leber und der Verminderung der Lipolyse durch Insulin ist eine Abnahme der 3,5-AMP-Konzentration wesentlich beteiligt. Die 3,5-AMP-Bildung ist in Fettgewebe, das mit Insulin inkubiert wird, vermindert. Insulin beeinflußt jedoch auch den 3,5-AMP-Abbau. -Die 3,5-AMP-Phosphodiesterase (PDE)-Aktivität des Fettgewebes, der Leber und, in geringerem Grade, der Skeletmuskulatur ist im Insulinmangel vermindert, d.h. bei alloxandiabetischen oder hungernden Ratten. I.v. Injektion von 0,5 E/kg Insulin oder eine erhöhte Abgabe von Insulin aus dem Pankreas nach Glucoseinjektion führen in diesen Geweben zu einem raschen Anstieg der PDE-Aktivität. Dieser ist in der Leber schon 15 min nach Insulingabe nachweisbar und erreicht nach 30–45 min sein Maximum. In der Niere ist kein Einfluß von Insulin auf die PDE-Aktivität nachweisbar. — Aus Rinderherz isolierte PDE wirdin vitro durch Insulin aktiviert, jedoch werden2 · 10^–5 M zur Verdopplung der Aktivität benötigt. Actinomycin D verhindert die Steigerung der Leber-PDE-Aktivität nach Insulininjektion. So kann die Wirkung des Hormons im wesentlichen auf eine gesteigerte PDE-Synthese zurückgeführt werden. — Durch diesen Einfluß der Insulininkretion auf die 3,5-AMP-Konzentration in Leber und Fettgewebe können Glykogenstoffwechsel und Lipolyse rasch an die Nahrungsaufnahme angepaßt werden.

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Influence de l'insuline sur l'activité de la 3,5-AMP-phosphodiestérase dans le foie, le muscle strié, le tissu adipeux et le rein

Résumé L'influence de l'insuline sur le métabolisme du glycogène hépatique et sur la lipolyse semble s'exercer par l'intermédiaire d'une diminution de la concentration de 3,5-AMP intracellulaire. Onamontré une diminution de la formation de 35-AMP dans le tissu adipeux incubé avec de l'insuline. L'influence de l'insuline sur la dégradation du 3,5-AMP est étudiée. — L'activité de la 3,5-AMP-phos-phodiestérase (PDE) est diminuée dans le foie, le tissu adipeux et, de façon non-significative, dans le muscle strié des rats qui manquent d'insuline, c-à-d les rats rendus diabétiques par l'alloxane ou les rats privés de nourriture. L'injection intraveineuse d'une faible dose d'insuline (0.5 U/kg) ou la stimulation de la sécrétion d'insuline endogène par une injection de glucose provoquent une augmentation rapide de l'activité de la phosphodiestérase dans ces tissus. 15 min après l'injection d'insuline, l'activité de la phosphodiesterase du foie est augmentée. L'effet maximum est atteint après 30–45 min. L'activité de la phosphodiestérase rénale n'est pas diminuée dans le diabète alloxanique, l'injection d'insuline s'est avérée inefficace.In vitro, l'insuline cristalline a un effet activant sur la phosphodiestérase purifiée du coeur de boeuf. La concentration d'insuline requise pour doubler l'activité de l'enzyme est de l'ordre de 2 · 10^–5 M. Le traitement avec actinomycin D empêche la stimulation par l'insuline de la PDE dans le foie. Ceci peut indiquer que l'action de l'insuline sur l'activité de la phosphodiestérase est essentiellement basée sur une synthèse accrue de l'enzyme. A cause de l'influence de la sécrétion d'insuline sur la concentration en 3,5-AMP du foie et du tissu adipeux, le métabolisme du glycogène et la lipolyse peuvent s'adapter rapidement à la prise de nourriture.

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Non-Standard Abbreviations G 6 P Glucose-6-phosphate - UDPG UDP-glucose - FFA non-esterifled, free fatty acids - 3,5-AMP cyclic adenosine-3,5-monophosphate - PDE 3,5-AMP phosphodiesterase This study was supported by the Deutsche Forschungsgemeinschaft.Deceased October 31, 1967.     

TCR1+ large granular lymphocyte proliferation in rheumatoid arthritis

The T-lymphoproliferative syndrome is characterized by a proliferation of large granular lymphocytes (LGL). It is often associated with neutropenia, and in 30% of cases with rheumatoid arthritis (RA). Phenotypic analysis has demonstrated that in most cases of RA with T-proliferative disease, the LGL represent T cells with a clonal rearrangement of the / T cell receptor (TCR2). Here, three patients with / TCR1⁺ LGL proliferation suffering from long-standing arthritis and neutropenia are described. The first patient with RA showed an expansion of a heterogeneous CD2⁺ CD16⁺ CD56^- LGL population, of which 30% coexpressed TCR1 with V1 rearrangement. The second patient with ankylosing spondylitis and RA was suffering from proliferation of TCR1⁺ (V9^-, V1^-), CD2⁺ CD16^- CD56^- LGL with low coexpression of CD8. The third patient with RA was suffering from a proliferation of TCR1⁺ (V1⁺, V9^-) CD4^- CE8^- CD16^- CD56^- lymphocytes. On the basis of these unusual findings, the pathogenetic role of TCR1⁺ T cells in RA is discussed.     

Ultrastructural changes in A-cells exposed to diabetic hyperglycaemia. Observations made on pancreas of chinese hamsters

Summary Pancreatic A-cells of chinese hamsters with diabetes of varying severity and duration were examined by electron microscopy. Two predominant changes were observed: 1. Lysosomal digestion of secretory granules (granulolysis, crinophagy) occurred in practically all A-cells of diabetic animals but was rarely observed in those of normoglycemic controls. This is considered a response of A-cells to the cessation of glucagon release secondary to hyperglycemia. 2. In relatively degranulated A-cells of ketotic diabetic animals, dilatation of the cisternae of the RER was seen together with accumulation of pale, flocculent material, possibly reflecting persisting or enhanced glucagon synthesis. In addition, numerous maturing secretory granules were seen in the cisternae of the Golgi complex. Since these apparently contradictory phenomena may be seen in the same cell, it is suggested that granulolysis may not only result from decreased hormone release secondary to hyperglycemia but that different and independent stimulatory signals may exist for glucagon synthesis, for glucagon release, and for the initiation of granulolysis.Supported in part by the Fonds national suisse de la Recherche scientifique (Grants. No. 4848.3 and 3.154.69).     

Overproduction of inhibitory hematopoietic cytokines by lipopolysaccharide-activated peripheral blood mononuclear cells in patients with aplastic anemia

The aim of this study was to measure the level of cytokines produced by peripheral blood mononuclear cells (PBMNC) in patients with aplastic anemia (AA) and to determine their effect on the clonal growth of normal bone marrow (BM) cells. Twenty-one patients with AA and 11 normal controls were enrolled in this study. Medium conditioned by PBMNC of AA patients in the presence of lipopolysaccharide (LPS) was found to be suppressive to the colony growth of normal BM cells. Thus, we further determined the presence in the PBMNC-conditioned medium (CM) of both inhibitory cytokines: macrophage inflammatory protein-1 (MIP-1), tumor necrosis factor- (TNF-), transforming growth factor-2 (TGF-2), and interferon- (IFN-), and stimulatory cytokines: interleukin-3 (IL-3) and stem cell factor (SCF). Spontaneous production of MlP-1 was higher in the AA patients than the normal controls (1887±174 pg/ml vs 1643±93 pg/ml), but the difference was not significant. After LPS stimulation, the production of MIP-1 was markedly increased in the AA patients, and its level was significantly higher than that of the normal controls (2360±149 pg/ml vs 1517±92 pg/ml, p=0.0022). The level of TNF was also higher in the AA patients. However, IFN-, TGF-2, SCF, and IL-3 were not detectable in the PBMNC-CM of either AA patients or normals. The myelopoietic suppressing effect of AA-PBMNC-CM from each AA patient was significantly blocked by pretreatment with anti-TNF-, resulting in a colony-forming enhancement of 174%±12%. A similar effect was noted in six of 11 AA patients by pretreatment with anti-MIP-1. We conclude that TNF and MIP-1 can be overproduced by the PBMNC of some AA patients, which may play a role in the progression of AA.     

Changes in the hydroxyproline,hexosamine and sialic acid of the diabetic human and β, β′-iminodipropionitrile-treated rat retinal vascular systems

Summary The retinal vasculature has been isolated from nondiabetic and diabetic post mortem human eyes by controlled trypsin digestion. Chemical analysis demonstrated increases in the hydroxyproline and hexosamine contents in diabetes. There was no general increase in the sialic acid content. These results have been related to histological preparations of sectors of the same retinas. Administration of, -iminodipropionitrile to rats caused a retinopathy characterised by endothelial cell proliferation, increased PAS-positivity and microaneurysms. Chemical analysis of retinal vascular systems from, -iminodipropionitrile-treated rats revealed increases in hydroxyproline, hexosamine and sialic acid contents.

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Änderungen des Gehaltes an Hydroxyprolin, Hexosamin und N- azetyl- Neuraminsäure in den Netzhautgefäen von menschlichen Diabetikern und mit , Iminodiproprionitril behandelten Ratten

Zusammenfassung Die Netzhautgefäße wurden post-mortal aus diabetischen und nichtdiabetischen Augen mit Hilfe einer kontrollierten Trypsin-Behandlung gewonnen. Bei der chemischen Analyse fand sich ein Anstieg des Hydroxyprolin- und Hexosamingehaltes in den diabetischen Augen. Der N-azetyl-Neuraminsäuregehalt war im allgemeinen nicht erhöht. Diese Resultate wurden in Beziehung gebracht zu den histologischen Befunden, die an den einzelnen Sektoren der gleichen Retina erhoben wurden. Verabreichung von, -Iminodiproprionitril an Ratten bewirkte eine Retinopathie unter den Zeichen einer Wucherung der Endothelzellen, verstärkter PAS-Anfärbbarkeit und Mikroaneurysmen. Die chemische Analyse der Netzhautgefäße von mit, -Iminodiproprionitril behandelten Ratten zeigte einen erhöhten Gehalt an Hydroxyprolin, Hexosamin und N-azetyl-Neuraminsäure.

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Variations de l'hydroxyproline, de l'hexosamine et de l'acide sialique dans les systèmes vasculaires rétiniens de l'homme diabétique et du rat traité par le , -iminodipropionitrile

Résumé Le système vasculaire rétinien a été isolé, par digestion trypsique contrôlée, après la mort, à partir d'yeux humains de non-diabétiques et de diabétiques. L'analyse chimique a révélé l'augmentation du contenu en hydroxyproline et en hexosamine, dans le diabète. Il n'y avait pas d'augmentation générale du contenu en acide sialique. Ces résultats ont été rapprochés des préparations histologiques de portions des mêmes rétines. L'administration de, -iminodipropionitrile à des rats provoquait une rétinopathie caractérisée par une prolifération des cellules endothéliales, une PAS-positivité augmentée et des microanévrismes. L'analyse chimique des systèmes vasculaires rétiniens des rats traités par le,-iminodipropionitrile, a révélé l'augmentation du contenu en hydroxyproline, en hexosamine et en acide sialique.

     

On the pathogenesis of preleukemic myelodysplastic syndromes

Summary After a single pulse dose of DMBA, rats develop bone-marrow hypoplasia, which is almost compensated for by regeneration after 16 weeks. Subsequently, dysplastic signs of hemopoiesis appear in all experimental animals as massive extrusion of normoblasts into the peripheral blood, red-cell anisoand poikilocytosis, nuclear deformities, atypical mitoses, and PAS-positivity, as well as megaloblastoid maturation dissociation of erythroblasts and nuclear and granulation anomalies of neutrophilic granulocytes and monocytes, comparable to human pseudo-Pelger cells and paraneutrophils. At the time of death (112-497 days after DMBA pulse) experimental animals showed hyperplastic bone marrow with increased granulopoietic/erythropoietic ratios and an augmented, mainly erythropoietic, hemopoiesis in the spleen, with splenomegaly in six rats. Splenic hemopoiesis is accompanied by white pulp atrophia. The cause of death was septicopyemia in three rats, anemia in three, and bleeding in one rat. None of the animals developed a leukemic blast phase. Myelodysplastic changes in this experiment are the same as have been shown to precede leukemia in rats treated with five DMBA pulses (Fohlmeister et al. 1981). Possible relations of myelodysplasia and leukemia are discussed.Supported by Deutsche Forschungsgemeinschaft (DFG)     

Erythromycin ameliorates renal injury via anti-inflammatory effects in experimental diabetic rats

Aims/hypothesis Recent studies have shown that the inflammatory process is involved in the pathogenesis of diabetic nephropathy. Fourteen-membered ring macrolides, including erythromycin, have anti-inflammatory, as well as antibacterial effects. The aim of this study was to investigate the renoprotective effects of erythromycin in streptozotocin (STZ)-induced diabetic rats.Methods STZ-induced diabetic rats were treated orally with erythromycin (5 mg/kg body weight) or vehicle every day for 8 weeks. To evaluate the effect of erythromycin treatment, we measured urinary albumin excretion, and examined the following in the kidney: histological changes, the expression of intercellular adhesion molecule-1 (ICAM-1), macrophage infiltration, and nuclear factor-kappa B (NF-B) activity.Results Erythromycin significantly reduced urinary albumin excretion without affecting blood glucose levels and blood pressure. Erythromycin also attenuated glomerular hypertrophy, mesangial expansion, macrophage infiltration and ICAM-1 expression in renal tissues. The expression of the gene encoding TGFB1 (also known as TGF-1), type IV collagen protein production and NF-B activity in renal tissues were increased in diabetic rats and reduced by erythromycin treatment.Conclusions/interpretation Erythromycin prevented renal injuries without changes of blood glucose levels and blood pressure in experimental diabetic rats. These results suggest that the renoprotective effects of erythromycin are based on its anti-inflammatory effect via suppression of NF-B activation. Modulation of microinflammation with erythromycin may provide a new approach for diabetic nephropathy.     

γ/δ Receptor-expressing T-cell clones from a cutaneous T-cell lymphoma suppress hematopoiesis

Summary Recently we described a cutaneous T-cell lymphoma expressing the / T-cell receptor [5]. The patient suffering from this lymphoma showed low numbers of myeloid and T cells in peripheral blood, while B and NK cells were relatively increased. In vitro culture of the patient's bone marrow (BM) cells revealed a significant suppression of myeloid/monocyte colony formation (GM-CFU) compared with normal controls. This was not due to infiltration of the BM with lymphoma cells. We speculated that a soluble factor either secreted or induced by the lymphoma cells might be responsible for the marked suppression of hematopoiesis in this patient. From a skin biopsy with infiltrating / T-lymphoma cells we established T-cell clones bearing the / T-cell receptor and resembling the phenotype of the lymphoma cells. The supernatant (SN) of these / T-cell clones reduced the number of colonies in a CFU-GM assay (using normal control BM) in comparison to SN of / T-cell clones established from the same biopsy. This suppression was seen mainly on day 7 of culture and was not neutralized by the addition of placenta-CM. The main mediator of this suppression seems to be IFN-,since it was detectable in high amounts in the SN of these / T-cell tumor clones as well as in the serum of the patient. In addition, anti-IFN- antibodies can reverse the T-cell SN-mediated suppression of CFU-GM. We conclude that high serum levels of interferon-, which is secreted in high amounts from / T-cells grown from a biopsy of a cutaneous lymphoma, can suppress hematopoiesis.Abbreviations TCR T-cell receptor - IFN- interferon- - SN supernatant - placenta CM placenta conditioned medium - BM bone marrow - CFU-GM myeloid/monocyte colony formation - NK cells natural killer cells - Ab antibody M. Wilhelm was supported by theDeutsche Forschungsgemeinschaft (DFG Wi 728-2)     

Decreased cyclic AMP and Insulin responses to glucose in pancreatic islets of diabetic chinese hamsters

Summary The dose as well as the time kinetics of insulin and adenosine-3,5-monophosphate (cyclic AMP) responses to glucose were compared in pancreatic islets isolated from normal and diabetic Chinese hamsters. The insulin content in diabetic islets was about one-half that in normal islets. Insulin release in diabetic islets incubated for 10 min with glucose 60–1000 mg/l00 ml was from one-third to one-half that in normal islets. Glucose 1000 mg/l00 ml stimulated three-fold increases in insulin release without increasing the accumulation of [³H] cyclic AMP in either normal or diabetic islets prelabelled with [³H] adenine. However, in the presence of 1.0 mM of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), glucose 150 mg/l00 ml elicited significant increases of insulin release (+ 134%) and [³H] cyclic AMP accumulation in islets (+ 44%) and incubation medium (+ 48%) of islets of normal but not diabetic hamsters. Also, in perifusion experiments with 0.1 mM IBMX, glucose 500 mg/l00 ml produced threefold greater increases in insulin release and two-fold greater increases in efflux of cyclic AMP in normal than diabetic islets. By contrast with the lesser effects of glucose in diabetic islets, 1.0 mM IBMX increased islet and medium cyclic AMP, as well as insulin release, similarly in normal and diabetic islets. It is suggested that the impairment of glucose induced insulin release in islets of the diabetic Chinese hamster may be due to a defective interaction of glucose with the adenylate cyclase-cyclic AMP system in the pancreatic B cell.This work was presented in part at a meeting of the European Association for the Study of Diabetes, Sept. 1975, Munich, Germanyon leave from the Department of Endocrinology, Karolinska Hospital, Stockholm     

Type I diabetes mellitus

Summary The major genetic susceptibility to insulin dependent (Type 1) diabetes is determined by genes in the HLA chromosomal region. An increased relative risk for developing the disease is observed in subjects who are HLA A1, A2, B8, B18, B15, B40, CW3, Bf^s, DW3, DW4, DRW3, DRW4 positive. There is an additive relative risk in subjects who possess two high risk HLA B alleles which has an important influence on the prevalence of the disease in sibships and possibly on the concordance rate in diabetic identical twins. There is also suggestive evidence that particular combinations of high risk HLA B alleles are associated with increased or persistent antibody production which may reflect enhanced or differential susceptibility. Certain factors (e. g. HLA B7, DW2 and DRW2) are associated with a significantly reduced risk and may exert a protective mechanism in Type I diabetes, by linkage disequilibrium with genes which reduce immune responsiveness. The significant increases and decreases in respect of the HLA B antigens are probably secondary to the corresponding HLA D and DRW associations which reflect a stronger linkage disequilibrium between the genes which determine these specificities and the putative genes which control susceptibility. Initial damage to the beta cells probably occurs a considerable time before the onset of symptoms and theoretically modification of the immune response early in the disease process may reduce the rate of beta cell destruction.     

Regulation of the release of tumour necrosis factor (TNF)α and soluble TNF Receptor by γ irradiation and interferon γ in Ewing's sarcoma/peripheral primitive neuroectodermal tumour cells

This study analyses the production of tumour necrosis factor (TNF) and soluble TNF receptor (sTNF-R) before and after exposure to irradiation and interferon (IFN) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNF-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-R_p55 and sTNF-R_p75 ELISA. The tumour cell lines released minimal amounts of TNF, prominent amounts of sTNF-R_p55 (7/12 cell lines) and no sTNF-R_p75. Exposure to irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNF release without changing sTNF-R_p55 and sTNF-R_p75 levels. Priming of cultures with recombinant human IFN (rhIFN) markedly enhanced TNF secretion in the radiation-responsive cell lines and had no influence on sTNF-R_p55 and sTNF-R_p75 levels. rhIFN affected the magnitude rather than the sensitivity of the radiation response. The TNF secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNF monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AACOCF3 (a specific inhibitor of phospholipase A₂) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated -irradiation-stimulated TNF release. The antioxidantsN-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited -irradiation-mediated TNF production. Collectively our findings indicate that IFN priming potentiates the secretion of bioactive TNF by ES/pPNET cells in response to irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the -irradiation-mediated intracellular signalling pathway leading to TNF production.     

Acute hematologic effects of interferon alpha,interferon gamma,tumor necrosis factor alpha and Interleukin 2

Summary This study was designed to investigate acute effects of various doses of the cytokines IFN-alpha, IFN-gamma Interleukin 2 and tumor necrosis factor alpha on white blood cell differential counts. Before initiation of phase II trials, a dose-determination phase was performed, where three different dose levels of each cytokine were applied as a single dose. White blood cell differential counts were assessed immediately before and 2, 12, 24, 48 and 168 h after injection. Patients enrolled suffered from metastatic cancer or chronic active hepatitis. In addition, IFN-alpha was administered to five healthy volunteers. Results indicate that cytokines cause rapid and transient changes in the numbers of leukocyte subsets. Hematologic changes were cell-type- and cytokine-specific: transient lymphopenia was observed after administration of all four cytokines, reaching a nadir 12 to 24 h after subcutaneous injection. Administration of TNF-alpha and IFN-gamma also caused transient monocytopenia. Neutrophilia developed after administration of Interleukin 2, IFN-alpha and TNF-alpha. We conclude that cytokines play a key role in the regulation of peripheral blood cell traffic by their capacity to influence homing patterns of peripheral blood leukocytes.     

Plasma immunoreactive glucagon fractions in four cases of glucagonoma: Increased “Large glucagon — immunoreactivity”

Summary Immunoreactive glucagon (IRG) fractions from plasma of 8 normal subjects and 4 patients with glucagon secreting tumors were studied by gel filtration techniques on Bio Gel P-30 and Sephadex G-50 columns. The pancreatic glucagon specific anti serum (30K) of Unger was utilized to measure IRG. Columns were calibrated with labelled albumin, proinsulin, insulin and glucagon. Four peaks were defined in normal and tumor bearing patients: peak I (>20000 mol. wt.), peak II (primarily 9000 mol. wt.), peak III pancreatic glucagon (3500 mol. wt.) and peak IV small glucagon (<3500 mol. wt.). Glucagonoma patients differed from our normal and reported normal subjects in that peak II contained most of the circulating IRG. The percent of IRG associated with peak II was 9.5–31.5% in normals and 39.1–61.2% in glucagonomas. Glucagon-like biological activity in an isolated hepatocyte system was demonstrated for all peaks. However, relative to immunoreactivity, peak II showed reduced activity (25–33%). Immunoassay of dilutions of all peaks revealed the probability of immuno determinants identical with porcine pancreatic glucagon. The presence of heterogeneous IRG peaks with biological glucagon-like activity suggest that the larger molecules may be prohormones. Further, it is possible that specific elevation of peak II may be a diagnostic feature of glucagonomas.Supported in part by NIH Grant #AM 17431-02.     

β-N-acetylglucosaminidase and β-glucuronidase activities in insulin-dependent diabetic subjects with retinopathy

Summary The serum activities of two lysosomal enzymes, -N-acetylglucosaminidase (EC 3.2.1.30, NAG) and -glucuronidase (EC 3.2.1.31, GLU), were determined in 41 insulin-dependent diabetics, 27 age-matched non-diabetic first-degree relatives of the diabetics and 103 age-matched non-diabetic blood-donors. The diabetics were divided into three groups on the basis of ophthalmoscopy: (1) no retinal abnormalities; (2) non-proliferative retinopathy; and (3) proliferative retinopathy. The activities of both serum enzymes were higher in diabetics (NAG 21.39 ±5.99; GLU 2.19±1.01) than in their relatives (NAG 17.22±3.99; GLU 1.62±0.61). The diabetics with non-proliferative retinopathy had higher serum enzyme levels (NAG 24.05±6.26; GLU 2.60 ±1.06) than diabetics without retinopathy (NAG 17.88±3.00; GLU 1.69±0.64), whereas no statistically significant difference was found in patients with the proliferative form of retinopathy (NAG 18.67±6.28; GLU 1.99±1.04). In diabetics a positive correlation was found between serum -N-acetylglucosaminidase activity and blood glucose (p < 0.01), but not between -glucuronidase and blood glucose. Furthermore, the activities of both enzymes in diabetics correlated with the plasma triglyceride level (p<0.05 for both correlations). No correlation was found between the enzyme levels and signs of other diabetic late complications.     

Inhibition by 16,16-Dimethyl Prostaglandin E2 of Tumor Necrosis Factor-α and Interleukin-1β Production and Messenger RNA Expression in Human Monocytes Stimulated by Helicobacter pylori

We investigated the effects of 16,16-dimethylprostaglandin E₂ on the production of tumornecrosis factor- and interleukin-1 in humanmonocytes stimulated with Helicobacter pylori. Monocytes isolated from human peripheral blood wereincubated for 24 hr with the extract of H. pyloridiluted 1:100 to 1:100,000 by volume, a combination ofthe extract and 16,16-dimethyl prostaglandinE₂, or a vehicle alone. The extract stimulated theproduction of tumor necrosis factor- andinterleukin-1 and the expression of theirmessenger RNA in a dose-dependent manner. 16,16-Dimethylprostaglandin E₂ inhibited the production of thesecytokines and their messenger RNA in the presence of H.pylori at doses higher than 10^-6 M,predominantly with tumor necrosis factor-. These data suggest that antiinflammatory effects ofprostaglandins on gastric mucosa are in part related totheir effects on inhibition of production ofproinflammatory cytokines by monocytes.     

Impairment of monocyte “lectin-like” receptor activity in Type 1 (insulin-dependent) diabetic patients

Summary In order to investigate whether the ability of peripheral blood monocytes to bind bacteria is impaired in diabetes, we studied carbohydrate-binding (lectin-like) receptors and the receptor for the Fc portion of immunoglobulin on monocytes from 25 male Type 1 (insulin-dependent) diabetic patients and 10 age-matched healthy control subjects. Peripheral blood monocytes from the diabetic patients expressed lower levels of lectin-like receptors compared to the control subjects, whereas the expression of the receptor for the Fc portion of immunoglobulin was similar in both populations. There was no correlation between the degree of lectin-like binding activity and plasma glucose concentration or glycaemic control. Recognition of unopsonized bacteria by the lectin-like receptor is impaired in Type 1 diabetes; this may affect the efficient elimination of potential pathogens.