The effects of pyridine nucleotides on the activity of a calcium-activated nonselective cation channel in the rat insulinoma cell line,CRI-G1

The activity of a calcium-activated nonselective (Ca-NS⁺) channel in a rat insulinoma cell line (CRI-G1) is inhibited by pyridine nucleotides in excised patches. The effects of all four pyridine nucleotides tested, -NAD⁺, -NADH, -NADP⁺ and -NADPH were very similar when tested at 0.1 mm, and at 1 mm the phosphorylated forms, -NADP⁺ and -NADPH, appeared to be slightly more potent than -NAD⁺ and -NADH. All the pyridine nucleotides tested reduced both the open state probability of the channel and the number of functional channels observed in a single patch.The application of -NAD⁺, but not of the other nucleotides tested, to the cytoplasmic surface of isolated inside-out patches from CRI-G1 cells opened a novel nonselective cation channel (the -NAD⁺-NS⁺ channel). The activity of this new channel is calcium sensitive and may also be inhibited by AMP.     

“Differentiation Induction” culture of human leukemic myeloid cells stimulates high production of macrophage differentiation inducing factor

A suitable procedure for the production of human monokines was defined as differentiation-induction culture. Human monocytic leukemia THP-1 cells were well-differentiated from nonfunctional promonocytes into macrophage-like cells by the induction with a combination of mezerein, retinoic acid, and aMycoplasma fermentans extract. The differentiated THP-1 cells secreted a high amount of macrophage differentiation-inducing factor (DIF) activity and concomitantly produced other known monokines, such as tumor necrosis factor- (TNF-), interleukin-1 (IL-1) and interleukin-1 (IL-1), into the medium. These results suggest that other novel human monokines may also be found in the conditioned medium of THP-1 cells induced by the differentiation-induction culture conditions defined in this study. Macrophage DIF was purified to homogeneity and NH₂-terminal amino acid sequence analysis revealed that macrophage DIF is very similar or identical to human leukemia inhibitory factor (LIF). The cDNA encoding human LIF was isolated using the polymerase chain reaction, and a clone producing 3.7 g/10⁶ cells day recombinant LIF was selected from Chinese hamster ovary (CHO) cells which were transfected with the LIF cDNA. The recombinant LIF production in CHO cells was quantified using MTT reduction assay with M1 cells.     

Regulation of cellular Gsα levels and basal adenylyl cyclase activity by expression of the β2-adrenoceptor in neuroblastoma cell lines

Mouse neuroblastoma x rat glioma hybrid NG108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human 2 adrenoceptor cDNA under the control of the actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (N22) but not one expressing only low levels of the receptor (N17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg²⁺ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn²⁺. The elevated basal activity was partially reversed by addition of the -adrenoceptor antagonist propranolol. Agonist activation of N22 but not N17 cells led to a large selective down-regulation of cellular G_s levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [³H] forskolin was achieved with substantially greater potency (some 30 fold) in N22 cell membranes than in N17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [³H] forskolin binding which was equipotent in each of N22, N17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of G_s down-regulation in each cell type. Expression of an epitope tagged variant of G_s in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type G_s. Isolation of clones of NCB20 cells expressing high levels of the 2 adrenoceptor also resulted in a specific agonist-induced down-regulation of G_s.     

The effects of recombinant interleukin-1 and recombinant tumor necrosis factor on non-specific resistance to infection

Natural and synthetic immunomodulators that increase non-specific resistance to infection induce the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF). Therefore, we investigated the effect of IL-1 and of TNF on the survival of lethally-infected mice. Mice were injected with 1 × 10⁶ Klebsiella pneumoniae in the thigh muscle. When recombinant human IL-1 was given as a single i.p. injection 24 h before the infection, survival was increased. Using 80 ng IL-l per mouse, survival compared to control animals was 80% versus 20% 48 h after the infection (p < 0.001). No effect of IL-1 was observed when it was given &frac; h before or 6 h after the infection. IL-l proved to be at least as potent as IL-1.Numbers of bacteria cultured from the blood, thigh muscle, liver, spleen, and kidney were similar in IL-1-treated and control animals. Protection against death by IL-1 was also investigated in granulocytopenic mice with aPseudomonas aeruginosa infection. Administration of the cyclooxygenase-inhibitor, ibuprofen, did not affect the beneficial effect of IL-1. In this model human recombinant TNF was at least tenfold less active than IL-1.Pretreatment with IL-1 also had a significant effect on survival of mice that received a high dose of bacterial lipopolysaccharide.     

Curative effects of combination therapy with lentinan and interleukin-2 against established murine tumors,and the role of CD8-positive T cells

The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a 1–3 glucan with 1–6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8⁺ CTL but not by CD4⁺ T cells or NK1.1⁺ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.Abbreviations B6 C57BL/6 - BDF1 C57BL/6 × DBA/2 F1 - Lyt2 murine CD8, Lyt2.1. allele of murine CD8 - Lyt2.2 allele of murine CD8 - Lyt3 murine CD8 - L3T4 murine CD4     

Prenatal diagnosis of GM1-gangliosidosis: Biochemical manifestations in fetal tissues

Summary A prenatal diagnosis of G_M1-gangliosidosis was made in a pregnancy at risk, on the basis of a deficiency of -galactosidase activity demonstrated in cultured aminiotic fluid cells. Biochemical analyses were performed in the aborted fetus. G_M1-ganglioside -galactosidase activity was reduced to 1% of the control value in both the brain and liver of the affected fetus. Lamellar bodies suggestive of membranous cytoplasmic bodies were found in cells of basal ganglions, while the accumulation of G_M1-ganglioside in the brain was not remarkable.     

Purification, properties and possible gene assignment of an α1,3-fucosyltransferase expressed in human liver

1,3-Fucosyltransferase solubilized from human liver has been purified 40 000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with 1,3-fucosyltransferase activity and had a specific activity of 5–6 µmol min^–1 mg^–1 and anM _r 44 000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal1-4GlcNAc, NeuAc2-3Gal1-4GlcNAc and Fuc1-2Gal1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc1-2Gal1-4Glc and the Type 1 compound Gal1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc2-6Gal1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with theFuc-TVI cDNA, suggests a provisional identification ofFuc-TVI as the major 1,3-fucosyltransferase gene expressed in human liver.Died June, 1991     

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker

A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and -cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H^* were adapted to enable them to grow serum-free in the absence of cholesterol and -cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10⁶ cells ml^–1 producing 76.6 mg l^–1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.Abbreviations C cholesterol - CD cyclodextrin - dhfr dihydrofolate reductase - F68 Pluronic F68 - GS glutamine synthetase - MSX methionine sulphoximine - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - ADCC Antibody-dependant cellular cytotoxicity     

Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining

The expression of neutral glycosphingolipids and gangliosides has been studied in human skeletal and heart muscle using indirect immunofluorescence microscopy. Transversal and longitudinal cryosections were immunostained with specific monoclonal and polyclonal antibodies against the neutral glycosphingolipids lactosylceramide, globoside, Forssman glycosphingolipid, gangliotetraosylceramide, lacto-N-neotetraosylceramide and against the gangliosides G_M3(Neu5Ac) and G_M1(Neu5Ac). To confirm the lipid nature of positive staining, control sections were treated with methanol and chloroform:methanol (1:1) before immunostaining. These controls were found to be either negative or strongly reduced in fluorescence intensity, suggesting that lipid bound oligosaccharides were detected. In human skeletal muscle, lactosylceramide was found to be the main neutral glycosphinogolipid. Globoside was moderately expressed, lacto-N-neotetraosylceramide and gangliotetraosylceramide were minimally expressed and Forssman glycosphingolipid was not detected in human skeletal muscle. The intensities of the immunohistological stains of G_M3 and G_M1 correlated to the fact that G_M3 is the major ganglioside in skeletal muscle whereas G_M1 is expressed only weakly. In human heart muscle globoside was the major neutral glycosphingolipid. Lactosylceramide and lacto-N-neotetraosylceramide were moderately expressed, gangliotetraosylceramide was weakly expressed and the Forssman glycosphingolipid was not expressed at all in cardiac muscle. G_M3 and G_M1 were detected with almost identical intensity. All glycosphingolipids were present in plasma membranes as well as at the intracellular level. Abbreviations used: BSA, bovine serum albumin; DAPI, 4,6-diamidine-2-phenylindole-dihydrochloride; DTAF, fluorescein isothiocyanate derivative; GSL(s), glycosphingolipid(s); Neu5Ac,N-acetylneuraminic acid [50]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [51] and the nomenclature of Svennerholm [52]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse₃Cer, Gall-4Gall-4Glcl-1Cer; gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotetraosylceramide or GbOse₄Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; Forssman GSL or GbOse₃Cer, GalNAc1-3GalNAc1-3Gal1-4Gal1-4Gle1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M2, II³Neu5Ac-GgOse₃Cer; G_M1, II³Neu5Ac-GgOse₄Cer; G_D3 II³(Neu5Ac)₂-LacCer; G_D2, II³(Neu5Ac)₂-GgOse₃Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer.     

Human Immunodeficiency Virus Glycoprotein 160 Induces Cytokine mRNA Expression in the Rat Central Nervous System

1. Elevated proinflammatory cytokines within the central nervous system (CNS) of individuals infected with human immunodeficiency virus (HIV) may contribute to altered CNS processes prior to the onset of AIDS. Most studies of HIV-induced alterations in cytokine expression within the CNS have focused on interleukin (IL)-1 and tumor necrosis factor (TNF).2. We used a ribonuclease protection assay (RPA) to elucidate further the pattern of cytokine mRNA expression in the rat CNS in response to HIV envelope glycoprotein 160 (gp160). Male Sprague–Dawley rats were surgically implanted with a guide cannula directed into a lateral cerebral ventricle. HIV gp160 was injected intracerebroventricularly and rats were sacrificed immediately (time = 0) or at 1, 2, or 4 hr postinjection. Discrete brain regions were dissected, and peripheral glands removed. All tissues were frozen in liquid nitrogen until RNA extraction and assay.3. IL-1, IL-1, TNF-, and TNF mRNAs were constitutively expressed in brain tissues. Central administration of gp160 dramatically increased mRNA expression for IL-1 and TNF in the hypothalamus, hippocampus, brainstem, and cerebellum. Furthermore, although mRNA expression for IL-5, IL-6, and IL-10 was never detected under basal conditions, these mRNAs were increased in brain tissue after administration of gp160. Peak expression in each brain region was detected 2 hr after administration. Multiple cytokine mRNAs were detected in peripheral tissues, but their expression was not altered by central administration of gp160.4. Our results indicate that gp160 induces mRNA expression in brain for cytokines other than IL-1 and TNF. Screening for multiple cytokine mRNA in this manner provides extensive information concerning the particular cytokines that may be involved in HIV-induced pathologies and alterations in CNS processes.     

Phase 1B study to improve immune responses in head and neck cancer patients using escalating doses of 25-hydroxyvitamin D<Subscript>3</Subscript>

Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects. These defects are associated with a poor prognosis and are mediated, in part, by immune inhibitory CD34⁺ progenitor cells, whose numbers are increased in the peripheral blood of HNSCC patients. Immune inhibitory CD34⁺ cells are also present within HNSCC tumors. A phase IB clinical trial was conducted with HNSCC patients to determine if treatment with the differentiation-inducer 25-hydroxyvitamin D₃ could diminish CD34⁺ cell levels and improve a panel of immune parameters. Here we present the results of treatment with orally administered escalating doses (20, 40, 60 g) of 25-hydroxyvitamin D₃, with an emphasis on the six patients who received the maximum dosage of 60 g per day. Peripheral blood was collected at 0, 1, 2, 4, and 6 weeks, and assessed for markers of immune activity. Although no clinical responses were observed, results of this pilot study demonstrated that treatment of HNSCC patients with 25-hydroxyvitamin D₃ reduces the number of immune suppressive CD34⁺ cells, increases HLA-DR expression, increases plasma IL-12 and IFN- levels, and improves T-cell blastogenesis. In contrast, 25-hydroxyvitamin D₃ treatment did not modulate plasma IL-1, IL-2, IL-4, IL-6, IL-10, GM-CSF, or TGF- levels.Abbreviations GM-CSF granulocyte-macrophage colony-stimulating factor - high CD34⁺ patients patients with greater than 1% baseline CD34⁺ cell levels - HLA human leukocyte antigen - IFN interferon - IL interleukin - low CD34⁺ patients patients with less than 1% baseline CD34⁺ cell levels - OD optical density - TGF transforming growth factor     

Immunochemical analysis of anti-HLA-A2, HLA-A3, and HLA-B27 xenoantisera elicited with hybrids between human and murine cells

Rabbits were immunized with hybrids constructed with human and murine cells. Serological and immunochemical studies showed that xenoantiserum 1595 is operationally specific for HLA-A2, xenoantisera 0806 and 0746 for HLA-A3 and xenoantiserum 0745 for HLA-1327. The Fab2 blocking assay suggests a spatial relationship between allotypic determinants recognized by the xenoantisera and those reacting with the HLA-A, B-specific monoclonal antibodies (MoAb) Q1/28,6/3 1, Q6/64, and CR-1 and the anti- ₂-microglobulin ( 2-) MoAb NAMB-1.     

Association of Polymorphic Marker A1/A2 of Gene ITGB3 with Coronary Artery Disease and Myocardial Infarction

The distributions of the alleles and genotypes of polymorphic marker A1/A2 of gene ITGB3 encoding the 3 subunit of integrin IIb3 in groups of patients with coronary artery disease (CAD), including those who have had myocardial infarction (MI), and in the control group have been compared. Analysis using Fisher's test has not revealed significant differences between these groups with respect to the distributions of the alleles and genotypes of polymorphic marker A1/A2 of gene ITGB3. This indicates that this polymorphic marker is not associated with either CAD or MI in the Moscow population.     

Suppression of cytokine production by supernatants from CD8+ lymphocytes activated by mycobacterial fractions: The role of interleukins 4 and 6

Supernatants derived from CD8⁺ lymphocytes treated with mycobacterial components, or the partially purified carbohydrates from these supernatants, increased the production of IL-4 and IL-6 by mononuclear cells. The addition of anti-IL4 or anti-IL6 antibodies to LPS stimulated MN cells incubated with supernatants from CD8⁺ lymphocytes or carbohydrates resulted in the restoration of other cytokine production by these MN cells. Recombinant IL-4 and IL-6 on their own suppressed the production of IL-1, TNF-, IL-2 and IFN- by mononuclear cells. Such suppression could be reversed with antibodies to IL-4 and IL-6. The addition of rIL-4 and rIL-6 did not increase the suppression of cytokine production induced by suppressor supernatants or carbohydrates. Interleukin 4 decreased the production of IL-6 by MN cells; whilst IL-6 suppressed IL-4 production in a dose dependent manner. Both effects could be reversed with the appropriate antisera. Our results suggest that mycobacteria could evade host immunity by inducing the production of IL-4 and IL-6 by host mononuclear cells. These cytokines, in turn, would suppress the production of other cytokines necessary for effective cellular immunity.Abbreviations IL-1 interleukin 1 - IL-2 interleukin 2 - IL-4 interleukin 4 - IL-6 interleukin 6 - IFN- gamma interferon - TNF- tumour necrosis factor alpha - MN cells mononuclear cells - NAL's non-adherent lymphocytes - LPS lipopolysaccharide - PMA phorbol myristate acetate - RIA radioimmunoassay - ELISA enzyme-linked immunosorbent assay - rIL-4 recombinant interlukin-4 - rIL-6 recombinant interleukin-6 - U/ml units per millilitre - g/ml micrograms per millilitre - ng/ml nanograms per millilitre