Distribution pattern of α and β myosin in normal and diseased human ventricular myocardium

Summary All fibers in three normal, four dilated, and two ischemic human ventricles were classified according to their myosin content using three sets of monoclonal antibodies each specific for one myosin heavy chain isoform (, and ). Numerous fibers contained only myosin heavy chain (denoted as fibers), others contained either and , or and myosin heavy chain (denoted as and fibers, respectively). The percentages of fibers were systematically determined along the walls of seven homologous regions of the ventricular myocardium.In all ventricles, there was an -fiber transmural gradient, with less fiber in the subendocardium than in the subepicardium. More fibers were found in the right than in the left ventricular wall but there was no difference between the mid-portion and the apex of the free wall of each ventricle. The diseased ventricles contained a lower fiber percentage than the normal hearts. fibers were very rare in the normal ventricles (less than 5%) and almost inexistent in pathological hearts. The correlation between the mean fiber percentages of the diseased hearts and their cardiac indices (r=0.88, P<0.05) suggests that the small amount of myosin distributed in a large number of ventricular fibers could play a role in the contractile performance of the heart. In conclusion, this study provides evidence for 1) an fiber transmural gradient, and 2) a lower myosin ratio in discased than in normal human ventricle.This work was supported in part by L'Institut National de la Santé et de la Recherche Médicale 101 rue de Tolbiac, 75013 Paris     

Desensitization pattern of cardiac β-adrenoceptor subtypes by prolonged in vivo infusion of pindolol and celiprolol in rats

Summary The regulatory effects of pindolol and celiprolol on cardiac -adrenoceptor density were studied in vivo in order to assess the subtype selectivity of their partial agonistic activity (PAA). The substances were continuously administered to rats for 1 week by means of implanted osmotic minipumps. The density of -adrenoceptor subtypes were estimated from ICYP saturation binding experiments performed on cardiac ventricular plasma membranes in the presence of a highly selective antagonist (CGP 20172 A or ICI 118,551). Both antagonists were employed at concentrations as high as to block one subtype only without affecting the complementary subtype. For control purposes, rats were also treated with isoprenaline (0.4 mg/kg/h) and propranolol (0.15 mg/kg/h), or vehicle. Pindolol (0.036 mg/kg/h) and celiprolol (0.36 mg/kg/h) reduced the density of ventricular ₂-adrenoceptors by 46% and 23%, respectively, which — in the case of pindolol — was significant when compared to the non-treated controls. Both compounds, however, produced a small, but distinct increase in the number of ₁-adrenoceptors by approximately 26%. This finding is in contrast to the propranolol-induced upregulation of both ₁- and ₂-adrenoceptors by approximately 80%. Since supramaximal doses of each drug were administered, a significant smaller increase of ₁-adrenoceptors by pindolol and celiprolol —as compared to the increase produced by propranolol — can be interpreted as evidence for a PAA of pindolol and celiprolol on ₁-adrenoceptors as well. Isoprenaline as a full agonist caused a marked loss of of both -adrenoceptor subtypes. Although it exhibits equal affinity at both subtypes the decrease amounted to 80% of the ₂- but only to 54% of the ₁-adrenoceptors density. This indicates that the down-regulation of cardiac -adrenoceptors in general seems to be more pronounced at the ₂-than at the ₁-adrenoceptors population. We conclude that the subtype desensitization pattern of agents with intrinsic activity precludes the determination of subtype-selectivity, since ₁- and ₂-adrenoceptors appear to differ in their sensitivity presumably as a result of subtype specific baseline desensitization produced by endogenous catecholamines.     

Effect of metoprolol on activity of β-adrenoceptor coupled to guanine nucleotide binding regulatory proteins in adriamycin-induced cardiotoxicity

Summary Prevention of cardiotoxicity without interfering with the therapeutic efficacy of adriamycin is a very crucial question. We have investigated the activity of -adrenoceptor coupled to guanine nucleotide binding regulatory proteins (G-proteins) and Ca²⁺-ATPase activity in experimental adriamycin-induced cardiotoxicity and the influence of metoprolol treatment on these variables. Adriamycin was administered to rats intravenously as a single dose of 6 mg/kg, and metoprolol was continuously given by means of implanted osmotic pumps. -adrenoceptor characteristics were measured by radioligand-binding experiments and by basal and stimulated adenylyl cyclase activity. Northern blot and dot blot analysis was used to quantify G-protein mRNA. It was shown that adriamycin did not induce any change in the total -adrenoceptor density, nor did the high affinity agonist binding to -adrenoceptor change. Adriamycin did not induce any alteration in the amount of mRNA encoding for stimulatory (Gs) or inhibitory (Gi) G-proteins. Also, basal and stimulated adenylyl cyclase activities were identical in the different experimental groups. In contrast, the Ca²⁺-ATPase was shown to increase in adriamycin-treated rats compared to control rats (45 ± 3.8 versus 23 ± 1.2 mol Pi/mg/h, P < .01). Metoprolol was shown to normalize this increase (29 ± 2.1 mol Pi/mg/h). Thus, it may be concluded that in experimental adriamycin-induced cardiotoxicity, despite Ca²⁺-overloading, the -adrenoceptor-G protein-adenylyl cyclase system remains intact. Metoprolol seems to prevent Ca²⁺-overloading independently of the -adrenoceptors studied here.     

Immunolocalization of Transforming Growth Factor-α and Epidermal Growth Factor Receptor in the Rat Gastroduodenal Area

The maintenance of gastrointestinal epitheliumintegrity requires a fine balance between proliferationand differentiation as well as protection againstgastric acid secretion. Transforming growthfactor- (TGF-) regulates these functions bybinding to epidermal growth factor receptor (EGF-R).This study was designed to identify the localization ofTGF- and EGF-R in the rat gastroduodenal region. In the stomach, the surface and gastric pitcells showed staining for TGF- antibodies in thecytoplasm and basolateral and apical membranes.TGF- and EGF-R were observed in the supranuclearregion of the cells lining the gland. In the duodenum,the enterocytes coexpressed both TGF- and EGF-Rin the supranuclear area. The EGF-R was also observed inthe apical membrane. Brunner's glands were positive for both TGF- and EGF-R antibodies. Ourresults demonstrate the coexpression of TGF- andEGF-R in the rat gastroduodenal area, which suggests afunctional role for them in the establishment and maintenance of the epithelialrenewal.     

Contractile proteins in globally “stunned” rabbit myocardium

Summary The isolated working rabbit heart preparation was used to study whether the contractile machinery remains unchanged in globally stunned myocardium. The function of the heart has been measured in nonischemic and postischemic conditions. The effect of isoprenaline or calcium chloride administration in both conditions was also studied. Myocardial contractile function was significantly depressed after 20-min global ischemia and returned to normal after CaCl₂ and supranormal values after isoprenaline administration. From hearts used in experiments myofibrils were prepared and their ATPase activity was determined. It was observed that myofibrils prepared from stunned myocardium showed about 50 % increase in ATPase activity in the presence of CaCl₂. Subjection of the heart to ischemia caused a decrease in calcium sensitivity of the myofibrillar ATPase. Myofibrils obtained from ischemic hearts but subjected to isoprenaline or CaCl₂ administration exhibited increased calcium sensitivity over that of control heart. These effects were accompanied by changes in the extent of phosphorylation of troponin I (TNI) and myosin light chains. The modification of contractile apparatus in the postischemic period described in this paper may contribute to the overall mechanism of myocardial stunning.     

Influence of β-adrenergic stimulation on the fast sodium current in the intact rat papillary muscle

Summary The loose-patch-clamp technique was used on intact cardiac papillary muscle of the rat to examine whether the fast sodium inward current is influenced by the -adrenergic stimulant isoproterenol (ISO) or by 8-bromo-3,5-cyclic adenosine monophosphate (8-Br-cAMP), respectively.The amplitude of evoked by test pulses of 5 ms to a transmembrane potential of 0 mV and its time to peak were analyzed. The availability of was tested with conditioning pulses of 2.5 s to potentials between –130 mV and –50 mV.The potential of half-maximal availability was slightly shifted to more negative values by 1 M ISO (2.0 mV, n.s.), as well as by 50 M 8-Br-cAMP (4.0 mV; p < 0.05).The peak amplitude of elicited from strongly negative potentials was increased by ISO (18 %, n.s.), while 8-Br-cAMP exerted no directional effect. Depolarizing conditioning pulses (–60 mV) decreased to 13.3 % of the maximal attainable current under control conditions, while ISO decreased to 9.1 % of control (p < 0.1). Corresponding values under the influence of 8-Br-cAMP were 11.4 % and 8.3 % (p < 0.05). Moreover, in the presence of ISO there was a significant shortening of the time to peak of (0.56 ms to 0.50 ms at –80 mV conditioning potential, p < 0.05) which could not be detected in the presence of 8-Br-cAMP.These observations confirm the assumption of a depressant influence of -adrenergic stimulation on cardiac fast sodium inward current also in the depolarized intact myocardium, and suggest that this influence includes cAMP-dependent as well as cAMP-independent pathways.Preliminary results have been published at the spring meeting of the Deutsche Physiologische Gesellschaft, 5–8 March 1991, Freiburg i Br     

Characteristics of lysosomal phosphomannosyl-enzyme receptors in the rat heart

Summary The receptor system recognizing mannose 6-phosphate groups of lysosomal enzymes has been characterized, e. g. in fibroblasts and liver cells. The purpose of this study was to demonstrate the presence of a phosphomannosyl receptor system in rat heart muscle. The characterization of receptors was accomplished with -N-acetylglucosaminidase (-GA) secreted by rat embryo fibroblasts after ammonium chloride stimulation. The receptor binding of ligand enzymes was saturated by adding increasing concentrations of -GA and the binding increased linearly when the content of membrane protein was increased. The binding of -GA was inhibited by mannose and glucose phosphates, especially mannose 6-phosphate. Mannose itself did not inhibit binding of the enzyme, showing that the binding was not mediated by mannose receptors. Alkaline phosphatase treatment of -GA decreased the binding of ligand enzymes to receptors. Alkaline conditions increased the dissociation of receptorligand complexes, whereas the dissociation was minimal between pH 5.5 and 6.5. The proportion of endogenous -GA activity in membranes probably representing receptor-bound location, varied between 40 and 55% of the total activity in various parts of rat cardiac muscle. The differences in the content of phosphomannosyl receptors, however, were insignificant between various cardiac muscle samples. At the organelle level the highest specific binding capacity, as well as the highest endogenous ß-GA activity, was in the sarcolemmal fraction. These results suggest that phosphomannosyl receptors also function in the endocytosis and transport of lysosomal enzymes in cardiomyocytes, as well as in several other cell types studied.     

Transforming Growth Factor-α Precursors in Human Colon Carcinoma Cells

Among the proteins of the epidermal growth factor family, transforming growth factor- (TGF-) may be an especially reliable indicator of metastasis or prognosis in human colorectal carcinomas. Moreover, anomalous forms of TGF- have been detected in several tissues of cancer origin, suggesting a role of these forms in the development of the disease. This study was designed to identify the presence of TGF- precursors in different colon cancer cell lines by mean of immunocytochemistry and western blotting techniques. Pro-TGF- was detected in all cell lines tested. Staining for pro-TGF- was observed in cytoplasm. Monoclonal antibody to TGF- detected two bands of 20 and 21 kDa. Polyclonal antibody to pro-TGF- revealed five bands ranging from 15 to 24 kDa. All these proteins were also detected in nonmalignant cells expressing a transfected rat pro-TGF- gene. In conclusions, transformation in these human colon carcinoma cells is not due to the presence of anomalous forms of TGF- precursors.     

Mechanisms and Roles of Neutrophil Infiltration in Stress-Induced Gastric Injury in Rats

Water-immersion and restraint stress is associated with an increase in neutrophil infiltration into the gastric mucosa, but the mechanism responsible for this infiltration is unclear. We investigated the involvement of intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor- (TNF-) in neutrophil infiltration in stress-induced gastric injury in rats. Rats were administered neutralizing antibody against ICAM-1 or TNF- and were subjected to induction of gastric injury by 6-hr water-immersion and restraint stress. To evaluate the relationship between gastric acid and neutrophil infiltration, some rats were given cimetidine before administration of stress. Neutralizing antibodies inhibited both the lesion formation and the increase in myeloperoxidase activity induced by stress. Expression of ICAM-1 on endothelial cells was increased by stress, accompanied by an increase of TNF--positive cells. Antibody against TNF- inhibited this increase in ICAM-1 expression. Cimetidine almost completely inhibited gastric lesions, but did not affect myeloperoxidase activity. In conclusion, neutrophil infiltration in stress-induced gastric injury may be mediated by ICAM-1 and TNF-, but not gastric acid, and may play crucial roles in the progression of gastric injury.     

Role of Central Interleukin-1β in Gastrointestinal Motor Disturbances Induced by Lipopolysaccharide in Sheep

Cytokines are involved in the symptoms of theacute phase response induced by infectious diseases inhumans as well as in animals, and interleukin-1(IL-1 ) has a pivotal role in these changes. The role of central IL-1 in the gastrointestinalhypomotility and fever evoked by intravenousadministration of lipopolysaccharide (LPS) and themechanisms involved, were investigated in sheep as anexperimental model. LPS (0.1 g/kg, intravenously)induced gastrointestinal hypomotility and fever thatwere significantly reduced by priorintracerebroventricular administration of IL-1receptor antagonist protein (IL-1ra, 2 g/kg). The effects of LPS were mimickedby intracerebroventricular IL-1 (50 ng/kg),whereas IL-1 injected intravenously at the samedose only caused a slight and transient fever withoutmodifying the gastrointestinal motility. Priorintracerebroventricular administration of thecyclooxygenase inhibitor indomethacin (100 g/kg) butnot the corticotropin-releasing factor (CRF) receptorantagonist -helical CRF_9-41 (5 g/kg) blocked alleffects evoked by both LPS and IL-1. These resultssuggest that in sheep, LPS induces digestive motordisturbances through a central release of IL-1 andprostaglandins.     

β-Adrenoceptor and adenylate cyclase regulation in cardiac myocyte growth

Summary We studied the effect of growth on -adrenergic receptor properties of neonatal rat heart myocytes cultured in serum-free medium with transferrin and insulin. Growth was induced by addition of 1 M (–)-norepinephrine for two days, 200 nM of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for two days, or 30 nM T₃ for six days. The K_d values for -receptor binding (¹²⁵I-ICYP) were unaffected by growth. The maximum number of -receptor binding sites calculated as sites/cell was increased 1.47-fold by T₃ (p<.005), but was decreased to 54% of control values by (–)-norepinephrine (p<.005); TPA had no effect on either Kd or B_max values. (–)-Isoproterenol-stimulated adenylate cyclase activity was augmented only in membranes from T₃-treated cells and was reduced by 69% in membranes from (–)-norepinephrine treated cells. TPA had no effect on(–)-isoproterenol-stimulated adenylate cyclase activity. We conclude that the mechanisms controlling -adrenergic receptor number may be distinct from those controlling growth, since receptor number does not correlate with cell enlargement. Furthermore, in (–)-norepinephrine-stimulated growth, which we have shown previously is an ₁-adrenoceptor mediated response, -adrenergic signal transduction is modulated in a directionally opposite fashion.Supported by grants from the Veterans Administration Research Service, American Heart Association California Affiliate, and National Heart, Lung, and Blood Institute Grant HL31113. Dr. Simpson is a Clinical Investigator of the Veterans Administration Research Service.     

Serum β2-Microglobulin in Chronic Hepatitis C

₂-Microglobulin(₂-MG) plays a key role in influencingthe immune response to viral infections as it is anintegrating part of the main histocompatibility system(HLA). We attempted to evaluate the changes in class I HLA antigens bycomparing the serum ₂-MG behavior in agroup of patients affected by chronic hepatitis C withthat observed in a group of healthy controls. Our studyrevealed that the patients presented higher serumbeta-MG levels than healthy controls (P = 0.0003).beta-MG levels were correlated with duration and degreeof the disease. Furthermore, there was a statisticallysignificant correlation between ₂-MG andHCV RNA levels, while no correlations were observedbetween serum ₂-MG levels and HCVgenotypes. The increment in serum ₂-MGvalues accompanying the progression of liver disease may be an expression ofaugmented production rather than alteredexcretion.     

Cellular and molecular pharmacology of 4′-epidoxorubicin in HeLa Cells

Summary The effects on cellular DNA and cytotoxicity produced by doxorubicin (Dx) and its epimer 4-epidoxorubicin (4E-Dx) were investigated in cultured HeLa cells. 4E-Dx was 2.3 times more cytotoxic than Dx after 1 h of treatment, but the two anthracyclines were equally cytotoxic on longer-term (24h) drug exposure. The different kinetics of cell lethality were related to pharmacodynamic differences between the two drugs. In fact, cellular uptake and efflux rates of 4E-Dx were faster than those of Dx on 1 h of drug exposure but similar after 24 h of treatment. 4E-Dx caused more protein-concealed strand breaks in DNA (single and double) than did Dx, despite a lower potency for free-radical formation. The degree of strand breakage by 4E-Dx was not a linear function of exposure time and, in fact, the rate of strand-break induction declined continuously with time. In contrast, Dx caused an almost linear increase in DNA single-strand breaks with time during 1 h of drug exposure; this was apparently due to its slower uptake. There was little repair of the DNA single-strand breaks produced by Dx upon postincubation for 5 h in a drug-free medium, whereas DNA lesions caused by 4E-Dx were removed with a t _1/2 of about 1.7h. These findings underline the importance of the cellular pharmacokinetics of anthracyclines in relation to their cytotoxic and DNA-damaging effects.Abbreviations used DX doxorubicin - 4E-Dx 4-epidoxorubicin     

Deleterious effects of digitalis on reperfusion-induced arrhythmias and myocardial injury in ischemic rat hearts: possible involvements of myocardial Na+ and Ca2+ imbalance

Summary Isolated rat hearts were made ischemic for 25 min after an initial recirculating perfusion, followed by 30 min of reperfusion. In some hearts, interventions including administration of ouabain and/or high [K⁺] in the buffer were performed during the first 10 min of reperfusion.During ischemia, intracellular Na⁺ (Na_i) increased from 15 to 64 [mol/g dry weight (dwt). During reperfusion, Na_i declined rapidly (at 10 min of reperfusion: 48 nol/g dwt, at 30 min: 25 mol/g dwt) and regular rhythm was recovered within 10 min in hearts without any intervention during reperfusion.⁴⁵Ca²⁺ uptake increased from 0.8 to 7.5 mol/g dwt after 30 min of reperfusion. Ventricular function recovered by 45 %.A 10-min perfusion with 10 or 50 M of ouabain increased Na_i (17 to 21 or 27 mol/g dwt) with increased left-ventricular (LV) contractile function, but these effects were reversed by combination of high perfusate [K⁺] (20 mM) in non-ischemic hearts.A 10-min reperfusion with ouabain retarded or stopped the decline in Na_i (at 10 min of reperfusion: 54 or 63 mol/g dwt, at 30 min: 32 or 40 mol/g dwt). These amounts of ouabain also increased the incidence of ventricular tachyarrhythmias during reperfusion to 30 % or 50 %, and increased the duration of ventricular fibrillation from 6.5 to 11.5 or 18.0 min.⁴⁵Ca²⁺ uptake reached to 8.8 or 10.0 mol/g dwt, and function recovered only 35 % or 28 %. When high perfusate [K⁺] was combined with ouabain during reperfusion, the retarded decline in Nai, augmented⁴⁵Ca²⁺ uptake, and reduced recovery of function caused by ouabain alone were attenuated. These results suggest that digitalis has toxic effects on reperfused ischemic hearts by inhibition of rapid active outward transport of previously elevated Na_i and potentiation of Ca²⁺ overload.The work was supported in part by grant HL 37936 from the National Heart, Lung and Blood Institute. J. R. Neely was deceased on November 29, 1988     

Gene expression of cytokines suppressing hematopoietic progenitor cells in lymphoid malignancies

Summary The expression of cytokine genes for tumor necrosis factor (TNF), lymphotoxin and transforming growth factor (TGF), all of which are known to suppress normal hematopoiesis, was investigated in 32 patients with lymphoid malignancies using Northern blot analysis. Messenger RNA (mRNA) for TNF, lymphotoxin and TGF was detected in 9 cases, 2 cases and 7 cases, respectively. When the relationship between cytokine gene expression and surface phenotype was analyzed, the expression of CD19 correlated significantly with expression of the TNF gene (P<0.05). This suggests that B cell malignancies are likely to produce TNF. When the hematological parameters of patients expressing and not expressing the gene were compared, the expression of TNF mRNA was found to correlate with more profound anemia in acute lymphoblastic leukemia (P<0.05). Both granulocyte and platelet counts were lower in patients expressing TNF mRNA; however, the decreases were not significant. Neither lymphotoxin nor TGF gene expression correlated significantly with any hematological parameter.Abbreviations TNF tumor necrosis factor - TGF transforming growth factor - IL interleukin - ALL acute lymphoblastic leukemia - CLL chronic lymphocytic leukemia - ATLL adult T-cell leukemia/lymphoma - NHL non-Hodgkin's lymphoma - MM multiple myeloma Partly supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan (60770949, 63015063, 02256102, 03670325) and from the Fukuoka Anti-Cancer-Society.     

Expression and secretion of alpha1-proteinase inhibitor are regulated by proinflammatory cytokines in human pancreatic islet cells

Aims/hypothesis Alpha1-proteinase inhibitor (1-PI) has been considered a key player in inflammatory processes. In humans, the main production site of 1-PI is the liver, but other tissues, including pancreatic islets, also synthesise this molecule. The aims of this study were to assess the islet cell types that produce 1-PI, to determine whether 1-PI is actually secreted by islet cells, and to assess how its production and/or secretion are regulated.Methods Expression of 1-PI in human islet cells was assessed by immunofluorescence, electron microscopy and western blotting. Release of 1-PI was analysed by reverse haemolytic plaque assay and ELISA. The effects of cytokines on 1-PI synthesis and secretion were tested.Results Immunofluorescence showed that alpha and delta cells do express 1-PI, whereas beta cells do not. By electron microscopy, we demonstrated a colocalisation of 1-PI with glucagon and somatostatin within secretory granules. Immunolabelling also revealed localisation of 1-PI within the Golgi apparatus, related vesicles and lysosomal structures. The expression of 1-PI in islet cells was also demonstrated by western blotting and ELISA of protein extracts. ELISA and reverse haemolytic plaque assay showed that 1-PI is secreted into the culture medium. Treatment of islet cells with IL-1 and oncostatin M for 4 days increased the production and release of 1-PI.Conclusions/interpretation Our results demonstrate that 1-PI is expressed by the alpha and delta cells of human islets, and that proinflammatory cytokines enhance the production and release of this inhibitor.     

Lack of α2-Antiplasmin Enhances ADP Induced Platelet Micro-Aggregation through the Presence of Excess Active Plasmin in Mice

Background: The role of 2-antiplasmin (2-AP) on platelet aggregation was investigated using mice deficient in 2-AP (2-AP^–/–) or using wild type mice (2-AP^+/+). Methods: Blood samples were taken from each mouse under anesthesia with ether and platelet rich plasma (PRP) was prepared. Platelet aggregation induced by various doses of ADP (0.3–30 M) was detected using a laser-light scattering (LS) system. Aggregated forms were observed using a scanning electron microscopy (SEM). Results: Dose-dependent platelet aggregation was not different in both types of mice. However, platelet micro-aggregate formation in 2-AP^–/– mice induced by low dose of ADP (1.0 M) markedly increased compared to the situation in wild type mice. Aggregated form detected by SEM showed supported data from LS analysis. When washed platelets of 2-AP^+/+ mice were resuspended in plasma of 2-AP^–/– mice, platelet micro-aggregation was also increased. On the contrary, when washed platelets of 2-AP^–/– mice were suspended in plasma of 2-AP^+/+ mice, platelet micro-aggregation did not change. In separate experiments, tPA (1.0 g/ml) was added to PRP before the stimulation of ADP. tPA had no effect on platelet aggregation in 2-AP^+/+ mice, however platelet micro-aggregation in 2-AP^–/– mice was markedly increased by the treatment with tPA. Moreover, the amount of released ATP from stimulated platelets was increased in 2-AP^–/– mice treated with tPA. Conclusion: Lack of 2-AP increased platelet micro-aggregation, and plasmin plays an important role in the formation of platelet aggregation when 2-AP knockout mice are used. Consequently, the reduction of 2-AP could be a risk factor for the activation of platelets resulting in thrombus formation.     

Effects of human α-interferon on granulocyte-macrophage progenitor cells (CFU-GM) in vitro

Summary Two preparations of human interferon (IFN)- were assessed for their influence on granulocyte-macrophage progenitor cells (CFU-GM) in vitro. Both highly purified human IFN- Ly and recombinant IFN- 2a suppressed CFU-GM colony formation in a dose-dependent manner using low-density bone-marrow target cells. Suppression of CFU-GM colony formation was accompanied by an increase in clusters. However, depletion of monocytes, T lymphocytes and B lymphocytes from low-density bone-marrow cells resulted in insensitivity of progenitor cells to IFN-. These results demonstrate that the effects of human IFN- on myeloid progenitor cells (CFU-GM) are mediated by accessory cells within the bone marrow.     

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.     

Effects on infarct size of reperfusion and pretreatment with β-blockade and calcium antagonists

Summary The mass of tissue at risk and the myocardial infarct developed was studied in dogs subjected to either 24-h occlusion of the left anterior descending coronary artery or 2-h occlusion followed by 22-h reperfusion. The mass of tissue at risk was defined under anaesthesia at the time of occlusion using the microsphere technique. Twenty-four hours later the hearts were removed, sliced transversely and stained with 2,3,5-triphenyltetrazolium chloride to define the infarcted tissue. All myocardial tissue was mapped and cut into small pieces for weighing and radioactive counting. Radioactivity was present in all tissue, including the infarct. In the centre of the the infarct, counts remained low and then increased very rapidly with distance just beyond the edge. Tissue at risk from infarction was taken as that with less than 15% of the peak left ventricular (non-ischaemic) counts.A linear relationship was found between the mass of the left ventricular infarct and the left ventricular mass of tissue at risk. The effect of 22 hours reperfusion was examined by this method and expressed by a regression equation. There was a significant decrease in slope for the regression line of the reperfusion data, (p<0.05, analysis of covariance), indicating less infarcted tissue for each gram of underperfused tissue.None of the drug pretreatments explored had any effect on infarct size in the 24-h occlusion model. With reperfusion, propranolol and flunarizine diminished infarct size compared with reperfusion only (p <0.05 for reduced slope, the new slope being not significantly different from zero). The effect of diltiazem was not so marked. Thus infarct size can be reduced with pretreatment, as long as the myocardium is reperfused.S. Torr and M. Main were supported by grants from the Science and Engineering Research Council, UK; A.J. Drake-Holland was supported by a fellowship from the Janssen Research Foundation.M.I.M. Noble is supported by the Garfield Weston Trust as the Weston Professor of Cardiovascular Medicine.