Altered phosphorylation state of branched-chain 2-Oxo acid dehydrogenase in a branched-chain acyltransferase deficient human fibroblast cell line

Summary The abundance and phosphorylation state of the polypeptide constitutents of the human branched-chain 2-oxo acid dehydrogenase complex were examined in mitochondria from normal and maple syrup urine disease (MSUD) fibroblasts. In normal fibroblast mitochondria two forms of the E₁ subunit were observed: non-phosphorylated (E₁) and phosphorylated (E_1–P). About 40–50% of E₁ was present as E_1–P. The ability to quantitate the two forms of E₁ permitted examination of the association between decreased capacity to oxidize branched-chain 2-oxo acids and the phosphorylation state of E₁.Changes in phosphorylation state of E₁ were observed in MSUD fibroblasts as compared to control cells. Of particular interest was the absence of E_1–P in an MSUD fibroblast line which lacked the dihydrolipoyl acyltransferase (E₂) subunit of the dehydrogenase complex. In two MSUD cell lines deficient in E₁, the abundance of E_1–P appeared to be preferentially reduced. A fourth MSUD cell line contained normal quantities of E₃, E₂ and both forms of the E₁ polypeptide. Our results indicate that alterations in the abundance of dehydrogenase complex polypeptides in MSUD fibroblasts may influence the phosphorylation state of the E₁ polypeptide. They demonstrate the potential for examining simultaneously mutations which affect both the catalytic and regulatory components of the dehydrogenase complex.     

Maple syrup urine disease (MSUD): Screening for known mutations in Italian patients

Summary Maple syrup urine disease (MSUD) is an autosomal recessive disease due to deficiency of the branched-chain -ketoacid dehydrogenase (BCKDH) caused by a large number of mutations. In the present study, DNA from Italian patients and their relatives was examined for three point mutations (Y393N in the E₁ gene, T841G and G1031A in the E₂ gene) and two deletions (–G at the intron/exon border of exon 8 in the E₂ gene and an 11 bp deletion in exon 1 of the E₁ gene) using the polymerase chain reaction (PCR) followed by allele-specific oligonucleotide (ASO) hybridization, gene-scanning size analysis of fluorescent-tagged PCR products and/or automated DNA sequence analysis. Our results show that two different mutations account for 7 of the 20 mutant MSUD alleles. Two unrelated affected children, two of their parents and one sibling were carriers for the 11 bp deletion in the E₁ gene, one patient and her mother were heterozygous for Y393N in E₁, while T841G, G1031A and the –G deletion in E₂ were not detected. This study is the first attempt to characterize at a nucleic acid level MSUD mutations in Italy. Our results indicate that additional defects are present in the Italian population and that, unlike the Mennonites, a number of different MSUD mutations exist in Italians.     

A case of PDH-E1α mosaicism in a male patient with severe metabolic lactic acidosis

We have characterized a novel mutation in a male patient that affects the coding sequence of PDH-E₁ gene and changes arginine-141 to a leucine. This nucleotide substitution was found in about 75% of the studied DNA (fibroblasts, liver and muscle), a scenario that would indicate a case of E₁ mosaicism in a male patient. When the mutant E₁ protein was expressed in human skin fibroblasts with zero endogenous pyruvate dehydrogenase complex activity and E₁ protein expression, no significant restoration of activity was recorded, in contrast to the wild-type cDNA, even though both wild-type and mutant protein levels were comparable. We concluded that the R141L mutation is a severe one and that it must have occurred in one of the E₁ alleles during early embryogenesis.     

Mutation of the 97-197-197-1subunit of the pyruvate dehydrogenase complex,in relation to heterogeneity

Summary Pyruvate dehydrogenase complex was studied using bio- and immunochemical methods in cultured cells derived from two patients with the severe type and one patient with the mild type of pyruvate dehydrogenase complex deficiency. In patients 1 and 2, enzyme activity was all but undetectable and associated with the absence of E₁ subunit of the complex. Patient 3 had a slightly reduced level of enzyme activity, and this was associated with a larger form of E₁ subunit. The amount and size of E₁ mRNA in the three patients was similar to that of control samples. Thus, the severity of E₁ deficiency in these three patients is likely to depend on the type of mutation in the pyruvate dehydrogenase E₁ subunit and the synthesis and degradation rate of the subunit.     

Use of 4-trifluoromethylumbelliferyl-α-l-iduronide as a new substrate for detection of α-l-iduronidase deficiency in human tissues and for rapid prenatal diagnosis of Hurler disease

Summary Results are presented of -l-iduronidase assays in the leukocytes of normal individuals, patients with Hurler disease and heterozygous carriers. The assays were carried out using 4-methylumbelliferyl--l-iduronide and 4-trifluoromethylumbelliferyl--l-iduronide as substrates. It was shown that 4-trifluoromethylumbelliferyl--l-iduronide, along with the commonly used 4-methylumbelliferyl--l-iduronide, can serve as a specific substrate for -l-iduronidase and is therefore suitable for demonstrating the enzyme deficiency in patients with Hurler disease, as well as the decrease of enzyme activity in heterozygous disease carriers. Using the two substrates a prenatal diagnosis of Hurler disease in a fetus was made on the basis of the lack of enzyme activity in amniotic fluid cell cultures. The diagnosis was confirmed by the results of -l-iduronidase activity assay in fetal liver and kidney. It was found that 4-trifluoromethylumbelliferyl--l-iduronide is highly efficient for the rapid detection of -l-iduronidase deficiency directly in pieces of tissues and in placenta, which is important for the prenatal diagnosis of Hurler disease.     

Serum Levels of α1-Antitrypsin Predict Phenotypic Expression of the α1-Antitrypsin Gene

We conducted a retrospective analysis to determine if both ₁-antitrypsin serum level and phenotype need be studied when evaluating children for ₁-AT deficiency. We collected data from patients less than 19 years old who had both serum ₁-AT level and phenotype determined over a 9-year period (January 1992–December 2000). Eighty-eight patients were identified and 15 had the PiZZ phenotype. The serum ₁-AT level was below normal (normal 85–215 mg/dl) in all 15 PiZZ patients. Seventy-two of 73 non-PiZZ patients had normal or above normal serum levels. The sensitivity of the serum ₁-AT level was 100%, and the specificity was 99%. The serum ₁-AT level had a positive predictive value of 94% and a negative predictive value of 100%. We conclude that serum ₁-AT levels are highly predictive of the PiZZ phenotype. Determination of the serum ₁-AT level alone should be the initial test when evaluating for ₁-AT deficiency.     

Triplication (/ααα<Superscript>Anti3.7</Superscript>) or deletion (-α<Superscript>3.7</Superscript>/) association in Argentinian β-thalassemic carriers

Prevalence of alpha gene triplication or deletion in -thalassemia carriers was studied in 109 unrelated individuals in Rosario, Argentina. In different populations -^3.7 allele presents a higher prevalence than ^anti3.7; thus, -thalassemia associated with -thalassemia is more frequently observed. Nevertheless, this event was detected in only one case (0.9%), while the association with alpha triplication was present in two subjects (1.8%).     

Human Plasma Fibronectin Mediates Adhesion of U937 Cells by RGD and CS1

Fibronectin specifically binds to U937 cells (monocytic cell line) in a dose-dependent manner. The specific receptors for the RGD sequence have been identified as ₅₁ and _IIb3, and that for CS1 has been defined as ₄₁. RGDS, CS1 peptide, and two peptides together showed similar inhibitory activities on this adhesion, while the 29-kD dispase-digested fragment of the C-terminal heparin-binding domain did not. Thus, the adhesion of fibronectin to U937 cells is mainly mediated by RGDS in the cell-binding domain and CS1 in the alternatively spliced region. Flow cytometry using monoclonal antibodies revealed expressions of ₃₁, ₄₁, and ₅₁, and not expression of ₂₁. Adhesion of U937 cells to fibronectin-coated wells is specific and is inhibited by anti-₄₁ and anti- ₅₁ monoclonal antibodies. The IC-50 for anti-₅₁ antibody was almost a log lower than the value for anti-₄₁ antibody. These results demonstrated that interactions of RGDS and CS1 sequence of fibronectin with ₅₁ and ₄₁ on U937 cells were required for the adhesion of U937 cells to fibronectin. These results may provide further information to understand the mechanism(s) of tumor cell adhesion and atherogenesis.     

Capsular and ‘O’ serotype determinants of Bacteroides fragilis

Summary Forty-one strains ofBacteroides fragilis, 20 strains of otherBacteroides species and 14 strains of other genera were examined by the indirect immunofluorescent assay (IFA) using anticapsular serum. The sixty-oneBacteroides strains were O serotyped by direct agglutination tests using absorbed antisera raised against 23 strains, each with a different O antigenic determinant. All 41B. fragilis strains tested were positive by IFA with the anticapsular serum, but apart from one strain ofB. distasonis, none of the remaining 19 strains of other bacteroides, i. e.B. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, B. melaninogenicus group andB. ureolyticus, and none of the 14 other bacterial species examined were positive. The majority of strains of saccharolytic bacteroides tested reacted with one of the 23 O antisera and were designated as a specific O serotype; a fewBacteroides strains had multiple agglutination reactions indicating the presence of multiple antigenic determinants. All O serotypes gave positive IFA tests with their homologous O antisera. Common capsular determinants and O antigenic determinants appear to exist on the same strains ofB. fragilis. Serological typing ofB. fragilis and related species would be useful in epidemiological studies.

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Kapsel- und O-Determinanten von Bacteroides fragilis

Zusammenfassung 41 Stämme vonBacteroides fragilis, 20 Stämme andererBacteroides-Spezies und 14 Stämme anderer Genera wurden unter Verwendung von Kapsel-Antiserum mit dem indirekten Immunfluoreszenztest (IFA) untersucht. Die O-Serotypisierung der 61Bacteroides-Stämme erfolgte mit dem indirekten Agglutinationstest; dabei wurden absorbierte Antiseren gegen 23 Stämme verwendet, von denen jeder eine unterschiedliche O-Determinante aufwies. Alle untersuchten 41 Stämme vonB. fragilis waren im IFA mit Kapsel-Antiseren positiv; hingegen war mit Ausnahme eines Stammes vonB. distasonis keiner der übrigen Stämme andererBacteroides-Spezies positiv, das heißt der GruppeB. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, B. melaninogenicus undB. ureolyticus; von den anderen geprüften 14 Bakterienspezies war ebenfalls keine positiv. Die Mehrzahl der Stämme der untersuchten saccharolytischenBacteroides reagierte mit einem der 23 O-Antiseren und wurde einem spezifischen O-Serotyp zugeordnet; einigeBacteroides-Stämme wiesen mehrfache Agglutinations-reaktionen auf, was für das Vorliegen mehrerer Antigendeterminanten spricht. Bei denselben Stämmen vonB. fragilis scheinen gemeinsame Kapsel- und O-Antigendeterminanten vorzukommen. Für epidemiologische Untersuchungen dürfte die Serotypisierung vonB. fragilis und verwandten Spezies von Nutzen sein.

     

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.     

Identification ofN-acetyl-α-aminoadipic acid in the urine of a patient with α-aminoadipic and α-ketoadipic aciduria

Summary A 3.5-year-old Japanese boy with a mild speech disturbance excreted a large amount of -aminoadipic acid into the urine. The amino acid analysis using an amino acid analyser confirmed the presence of -aminoadipic acid in both urine and plasma.We detected -aminoadipic acid in the hydrolysate of the effluent and washwater fraction through cation exchange resin. This suggested the presence of acetylated derivatives and we identifiedN-acetyl--aminoadipic acid using liquid chromatography-atmospheric pressure ionization mass spectrometry (LC-API-MS). The concentrations of -aminoadipic acid andN-acetyl--aminoadipic acid in the urine of a patient with -aminoadipic aciduria were 376.9 nmol/mg creatinine and 18.1 nmol/mg creatinine, respectively. We also detected -ketoadipic acid in the urine of this patient using LC-API-MS.     

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.     

Pharmacologic Characteristics of Rabbit Esophageal Muscularis Mucosae In Vitro

The aim of this study was to investigate invitro the pharmacologic properties of rabbit esophagealmuscularis mucosae. Stimulation of its motor innervationelicited only atropinesensitive excitatory responses. This tissue was not refractory to histamine butappeared to be rapidly desensitized to its effects.Responses to adrenoreceptor agonists were produced byexcitatory ₁ and ₁adrenoreceptors, inhibitory ₃ receptors but not₂ receptors. The esophageal muscularismucosae was only weakly stimulated by neurokinins andbradykinin, and these responses were mediated via NK2,NK3, B1, and B2 receptors, respectively. Adenosine diphosphate andadenosine triphosphate produced 40% maximalcontractions through the activation of P2 receptors,whereas adenosine monophosphate and adenosine werewithout effect. Responses to prostaglandins E₂ andF₂ were 10% of the tissues'maximum response to acetylcholine. These datademonstrate that rabbit esophageal muscularis mucosaehas a simple excitatory innervation and is only weaklystimulated by a variety of pharmacologic agents. For thesereasons it is distinct from muscularis mucosae foundelsewhere in the rabbit gastrointestinaltract.     

Influence of interleukin-1β, tumour necrosis factor alpha and prostaglandin E2 on chondrogenesis and cartilage matrix breakdown in vitro

Inflammatory mediators such as the cytokines interleukin-1 (IL-1) or (TNF), and prostaglandins [predominantly prostaglandin E₂ (PGE₂)] are generally considered to be involved in the breakdown of cartilage matrix in chondrodestructive diseases, especially rheumatoid arthritis and osteoarthritis. Their mode of action is not yet completely understood. Blastemal cells or differentiated chondroblasts/chondrocytes of limb buds from mouse embryos (day 12) in organoid cultures provide an efficient system to investigate the mechanism of action of these substances. Using recombinant human IL-1, TNF and PGE₂ alone or together (in pairs) in this culture system, we found that none of these substances alone could affect chondrogenesis. TNF, however, when combined with IL-1, proved to be the more potent cytokine causing a transformation of embryonal chondrogenic cells into fibroblast-like cells and thus inhibiting the expression of the cartilage cell phenotype. This might be due to inhibition of both the morphgenetic and cytodifferentiation phases of chondrogenesis. The well-known synergistic interaction between both cytokines seems to be phase limited and may not occur in the postchondrogenesis phase. In addition, our results showed that TNF alone or combined with PGE₂ caused a marked breakdown of the cartilage matrix. These in vitro findings might be useful to elucidate the complexity of interactions between different cytokines and PGE₂ involved in cartilage destruction processes in vivo.Dedicated to Prof. Dr. H.-J. Merker on the occasion of his 65th birthday     

Bone marrow and peripheral blood globin chain biosynthesis in iron deficiency

Summary Globin chain synthesis was studied in 13 iron-deficient patients. The mean whole-cell globin / ratio in the peripheral blood of 11 patients was 1.05±0.06 which is similar to the value 0.99±0.08 obtained for 10 controls. The ratios odtained for stroma-free globin were not significantly different from those of whole cell preparations. In contrast, the / ratio of bone marrow was 0.73±0.14 in 10 iron deficient patients, which is significantly lower than that of controls. Two other patients had decreased / ratios in the peripheral blood, probably because of the presence of an -thalassemia gene. These results demonstrate a reduced rate of synthesis of chains relative to that of chains in the bone marrow of iron-deficient patients that is not demonstrable in the peripheral blood.This work was partly supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil     

The antigenicity of human hemoglobins

Summary The correlation of the antigenicities among native hemoglobins and their subunit chains were investigated by the absorption of antisera and the combination of urea added immunoelectrophoresis with double diffusion. Alphachain showed identity with Hb-F but partial identity with -chain and Hb-A. Beta-chain showed identity with Hb-A but -chain and Hb-F showed partial identity with this chain. Gamma-chain showed identity only with Hb-F and its antigenicity was considered as being different from those of - or -chains.The lines of -, -and -chains were reconfirmed from the facts that the appearance of them depended always on the existence of anti-Hb-A or anti-Hb-F antibodies in the absorbed antisera and the minor component lines of

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Zusammenfassung Die Zusammenhänge der Antigenität zwischen nativen Hämoglobinen und deren Unterketten wurden mit der Absorption der Antiseren und der Kombination der Harnstoff-Immunelektrophorese und Doppeldiffusion untersucht. Die -Kette zeigte Identität mit Hb-F, aber nur partielle Identität mit der -Kette und Hb-A. Die -Kette war in ihrer Antigenität mit Hb-A identisch, die -Kette und Hb-F waren teilweise identisch mit der -Kette. Die -Kette zeigte die Identität mit Hb-F; es wird angenommen, daß ihre Antigenität verschieden von der -oder -Ketten ist.Für das Auftreten der Linien der -, - und -Ketten müssen Anti-Hb-A-oder Anti-Hb-F-Antikörper in den absorbierten Antiseren vorhanden sein, außerdem fusionieren die schwächeren Linien der Doppeldiffusion nicht mit irgendwelchen Linien der Unterketten. Auch gereinigte - oder -Ketten wurden zur Feststellung ihrer Linien benutzt.

     

Protection by nitecapone against sodium taurocholate-induced damage to cultured gastric cells

The goal of this study was to observe if nitecapone protected against taurocholate-induced damage in primary cultured rat gastric mucosal cells, as well as in a well-differentiated human gastric epithelial cell line (MKN 28). Prostaglandins were measured to analyze the protection mechanism. In primary rat gastric mucosal cell culture, nitecapone 125–250 M protected the cells significantly against damage induced by sodidum taurocholate, increasing cell viability by 31–38%. In the human gastric epithelial cell line, in which mitochondrial activity was measured as an indication of cell viability, nitecapone (62.5–250 M) protected the cells against sodium taurocholate-induced damage by 12–20%. Prostaglandin E₂, thromboxane B₂, and 6-keto-prostaglandin F₁ measurements in the primary cultured rat gastric mucosal cells showed that nitecapone (125 M and 250 M) significantly stimulated prostaglandin E₂ production (84.7% and 61.0%, respectively), and inhibited thromboxane B₂ formation (50% at 250 M), while the 6-keto-prostaglandin F₁ formation was unaffected. Nitecapone had no effect on prostaglandin E₂ production in the MKN 28 epithelial cell line. Indomethacin or aspirin, at concentrations that did not affect cell viability, antagonized the stimulative effect of nitecapone on prostaglandin E₂ formation in the primary cultured rat gastric mucosal cells. Although the prostaglandin E₂ synthesis was blocked, nitecapone still protected against cell damage induced by taurocholate. These results demonstrated the direct and efficacious protection of nitecapone on gastric cell level and suggest that the cytoprotection by nitecapone against taurocholate may not be mediated through the mechanism of stimulated synthesis of prostaglandin E₂.     

The importance of granulocyte elastase in haematological diagnosis

Summary PMN elastase is a useful additional parameter in the differential diagnosis of the leukaemias. In all patients with myelocytic leukaemias there were elevated levels of elastase-₁-proteinase inhibitor (E-₁PI), while in the lymphatic leukaemias complexed elastase levels were decreased. The highest values were found in the peripheral blood plasma and bone marrow plasma of patients with CML. Despite high E-₁PI concentrations there were no signs of bleeding or consumption of plasmatic coagulation factors. In AML a wide range of E-₁PI levels was observed, extending from slightly elevated to four hundred-fold increased. In myeloblastic leukaemias without maturation (FAB M1) the concentrations of complexed elastase remained below 150 ng/ml. In myeloblastic leukaemias with maturation (FAB M2) the E-₁PI values ranged between 214 ng/ml and 850 ng/ml (x= 402 ± 69), and in myelo-monoblastic leukaemias (FAB M4) between 450 ng/ml and 720 ng/ml (x = 663 ± 72). The only case of promyelocytic leukaemia (FAB M3) exhibited an extremely high value of 4,550 ng/ml, while a monocytic leukaemia (FAB M5) showed an extremely low value of 5 ng/ml. During cytostatic therapy there was a rapid decrease in levels of complexed elastase, with E-₁PI values returning to normal in remission. In recidivating cases there was an increase of E-₁PI levels in AML and a decrease in ALL. There was a correlation between the E-₁PI concentrations in peripheral plasma and leukaemic bone marrow infiltration, so providing a good basis for monitoring remission from leukaemia and indicating relapse. It was also interesting to observe an extremely low E-₁PI level (5 ng/ml) in patients with myelodysplasia. Under Decortin/Plenastril therapy the concentration rose to 50 ng/ml. An E-₁PI level of 10 ng per ml was observed in one case of Ranitidine agranulocytosis. Under corticoid therapy the value returned to normal within eight days.     

Effects of tumour necrosis factor α on bone marrow aspirates of patients with acute myelogenous leukemia determined by flow-cytometric cell-cycle analysis

Summary Tumour necrosis factor (TNF) exerts cytotoxic and antiproliferative effects on neoplastic cells. It has been used as a therapeutic agent for solid tumours and haematological malignancies. We report on the ex vivo determination of the effect of recombinant human rhuTNF on bone marrow aspirates by a bromodeoxyuridine/propidium iodide method. Cell samples were drawn after 0.5, 2, 4, 6, 8, 10, 22, and 25 h from shortterm suspension bone marrow cultures from patients with acute myelogenous leukemia (AML). Flow-cytometric cell-cycle analysis was performed after double DNA staining with propidium iodide and anti-BrdU antibodies. By this method the effect of rhuTNF on cell proliferation can be evaluated after only 35 h. In about two-thirds of the bone marrow aspirates of AML an inhibiting effect on rhuTNF can be demonstrated, developing to its full extent after 10 h.Abbreviations TNF tumour necrosis factor - AML acute myelogenous leukemia     

Synergistic cytotoxicity of human recombinant tumour necrosis factor α combined with microtubule effectors

Summary Combinations of human recombinant tumour necrosis factor (rhTNF) with each of four different agents disturbing the microtubule system of the cellular cytoskeleton were tested for synergistic cytotoxic action against murine melanoma B16K and L-M(S) cells. In addition to the known microtubule effectors colchicine, vincristine, and taxol, the influence of the fluorenone-azomethine derivative-diphenylene-N-{p-[bis-(-hydroxyethyl)-amino]-phenyl}-nitrone (DHPN) on the rhTNF cytotoxicity was studied. Applying a novel computerbased isobole method [Suehnel J (1990) Antiviral Res 13:23–40] concentration ranges of synergistic, zero, and antagonistic interaction were found after in vitro combination of rhTNF with each of the drugs tested in a 72-h cytotoxicity assay. In contrast, a 24-h exposure of B16K cells to these combinations still did not inhibit in vitro colony formation to a greater extent than either drug alone. A preliminary in vivo experiment revealed an increased antitumour effect after treatment of established subcutaneous melanoma B16 tumours with a combination of rhTNF and DHPN.Abbreviations rhTNF human recombinant tumour necrosis factor - DHPN -diphenylene-N-{p-[bis-(-hydroxyethyl)-amino]-phenyl}-nitrone