In vitro propagated dendritic cells from patients with human-papilloma virus-associated preneoplastic lesions of the uterine cervix: use of Flt3 ligand

Dendritic cells (DC) are the most efficient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and accessibility in normal tissue. Substantial numbers of DC can be generated from peripheral blood cultured in the presence of interleukin-4 (IL-4) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). We showed in this study that substantial numbers of DC can be obtained from the peripheral blood of patients with (pre)neoplastic lesions of the uterine cervix. The procedure required relatively small blood samples (10 ml) and the presence of 100 U/ml IL-4 and 800 U/ml GM-CSF in the culture medium. There was no significant difference in the morphology, yield, phenotype and function of generated DC between patients with cervical (pre)neoplastic lesions and healthy individuals. When the hematopoietic factor Flt3 ligand (Flt3L, 40 ng/ml) was added, there was an average increase in the DC population of 26% compared to cultures with GM-CSF and IL-4 alone. Approximately 1.2 × 10⁶ cells with the characteristics of dendritic cells could be obtained when Flt3L was included in the medium. The addition of Flt3L did not modify the phenotypic profile of DC (HLA-DR⁺, CD1a⁺, CD4⁺, CD54⁺, CD80⁺, CD86⁺, CD40⁺, CD3⁻ and CD14⁻). In addition, Flt3L generated functional DC capable of stimulating the proliferation of alloreactive T cells. These results suggest that Flt3L, in association with GM-CSF and IL-4, provides an advantageous tool for the large-scale generation of DC and that an immunotherapy based on the use of DC generated in vitro is possible in patients with (pre)neoplastic lesions of the uterine cervix. Received: 8 January 1998 / Accepted: 30 April 1998     

On the Iron Requirement of Lactobacilli Grown in Chemically Defined Medium

The iron requirement of four strains of lactobacilli (L. acidophilus, L. delbrueckii subsp. bulgaricus, L. plantarum, and L. pentosus) was studied in a synthetic medium under aerobic or anaerobic conditions. Effects of iron salt and iron-chelated compounds were tested on bacterial growth in manganese-free or -supplemented media. No significant growth stimulation was observed in any condition. These results support the absolute manganese requirement for optimum growth of lactobacilli and the needless incorporation of iron in growth media. Received: 5 November 1997 / Accepted: 20 January 1998     

Gene Order of Encephalomyocarditis Virus as Determined by Studies with Pactamycin

Previous work has shown that translation of the encephalomyocarditis (EMC) viral ribonucleic acid (RNA) generates at least three primary products, polypeptides A, F, and C. The A and C polypeptides then undergo post-translational cleavages to complete the production of the stable viral polypeptides (delta, beta, gamma, alpha, G, I, F, H, and E). In this communication we show that A, F, and C are produced in equimolar amounts giving further support to the theory that the RNA of picornaviruses has only a single site for the initiation of protein synthesis. The biosynthesis of viral proteins in EMC virus-infected HeLa cells was studied in the presence of pactamycin at concentrations which preferentially inhibit the initiation of protein synthesis. The amount of each polypeptide formed during the residual period of protein synthesis observed after the addition of pactamycin was used as a criterion for ordering the genes on the viral RNA. The results obtained indicate that the primary gene products are ordered on the EMC viral RNA 5' --> 3' A-F-C and that the stable products are ordered delta-beta-gamma-alpha-G-I-F-H-E. Moreover, the intermediate chains B and epsilon map in the capsid region, whereas the intermediate chain D maps in the E region. This order is largely consistent with previously established relationships of the viral polypeptides and thus indicates that pactamycin is a valid tool for "genetic" mapping of polycistronic RNA molecules with single initiation sites.     

INFLUENCE OF CERTAIN PLANT GROWTH REGULATORS AND CROWN-GALL RELATED SUBSTANCES ON BUD FORMATION AND GAMETOPHORE DEVELOPMENT OF THE MOSS PYLAISIELLA SELWYNII

Bud formation and gametophore development were studied in the moss Pylaisiella selwynii (Kindb.) Steere and Anderson grown from spores in a liquid medium consisting of inorganic salts. Indoleacetic acid and ethrel increased bud formation within a narrow concentration range. Copious bud formation was obtained with the five cytokinins tested at concentrations varying from 5 X 10⁻⁶ to 5 X 10⁻¹⁴ M. Except for about 10 % of the buds obtained with 6-γ, γ-dimethylallylaminopurine at 5 times 10⁻¹⁴ m, the cytokinin-induced buds failed to develop into normal gametophores. Octopine, lysopine, and octopinic acid, substances obtained from crown-gall tumors, increased bud formation at 10⁻³ m. On lysopine-treated plants these buds developed into typical gametophores. Gemma-like structures were obtained with octopine but no gametophores. l -arginine and l -lysine, the amino acids which respectively occur in octopine and lysopine, failed to induce gametophore formation although buds were obtained with 10⁻³ m lysine. γ-Guanidinobutyric acid induced bud formation at 10⁻³ m, but these buds developed into highly abnormal gametophores. The failure of buds obtained with many of these treatments to develop into gametophores appeared to result from the formation of new cell walls in other than the normal geometrical relationship during initial divisions of the pro-bud. The relevance of the findings to the crown-gall problem is discussed.     

Structural Studies of Avian Myeloblastosis Virus: Comparison of Polypeptides in Virion and Core Component by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Two different systems of dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in separate laboratories detected analogous patterns of dye bands in virions of avian myeloblastosis virus (AMV). At least 11 of the dye bands co-migrated with the major polypeptides reported in Rous sarcoma virus. Particles with the morphology of the AMV core component, obtained after exposure of AMV to the nonionic surfactant Sterox SL, contained major polypeptides p12, p27, p60, p64, p91, and p98. The polypeptide p12 has been previously shown to be the major constituent of the inner ribonucleoprotein (RNP) of the AMV core, and has been designated p12(N). Two RNP polypeptides, p64 and p91, co-electrophoresed with purified AMV DNA polymerase and have now been designated p64(P) and p91(P). The polypeptide p27 has been identified as a probable constituent of the core shell, and has accordingly now been designated p27(C). In comparison to virions of AMV, the AMV core component contained a greatly reduced amount of polypeptide p15 and appeared to lack a major polypeptide, p19. Consequently, these polypeptides may be associated either with the exterior of the core shell or the interior of the viral envelope. Glycopeptides were not detected in AMV cores, in agreement with earlier reports that they reside in external projections from the viral envelope.     

N-Acetyl-β-d-hexosaminidase component A. Different forms in human tissues and fluids

1. Hexosaminidase A of human serum was resolved into two components, a minor form with properties identical with those of the single hexosaminidase A component of human liver, and a major form with significantly different properties. 2. The major serum hexosaminidase A form was eluted from a DEAE-cellulose column at a lower salt concentration than that required to elute the liver form. 3. A multiple-pass technique was used to elute the major serum enzyme A from a Sephadex G-150 column before that of liver enzyme A. 4. Clostridium perfringens neuraminidase converted the major component of serum hexosaminidase A into a form that was held less tightly by DEAE-cellulose, but the minor component of the A enzyme of serum, and the A enzyme of liver were not affected. 5. The hexosaminidase A from tears was similar to the A enzyme from serum, whereas those from several human tissues and from urine and lymph were similar to the liver form. 6. The A enzyme from serum may be derived from the A enzyme from liver by glycosylation before secretion.