Enhancement by IL-12 of the cytolytic T lymphocyte (CTL) response of mice immunized with tumor-specific peptides in an adjuvant containing QS21 and MPL.

Immunization of cancer patients with tumor-specific antigenic peptides is currently being tested in several clinical studies. We have examined the induction of CTL responses in mice after various modalities of peptide vaccination, to explore protocols that could be applied to humans. Our first model antigen was P198, which results from a point mutation in a normal gene. While two immunizations with peptide P198 in SBAS-1c adjuvant induced measurable CTL responses in less than 10% of DBA/2 mice, the addition of IL-12 to the peptide adjuvant mixture resulted in high CTL responses in nearly all mice. This strong enhancing effect of IL-12 was observed with 1,000 and 300 units and decreased gradually as the doses were reduced to 30 units. When IL-12 was replaced by other cytokines acting on T cells or antigen-presenting cells, such as IFN-gamma, IL-2, IL-6, IL-7, GM-CSF or MCP-3, no significant enhancing effect was observed. The same effect of IL-12 was obtained with peptide P1A, which is a major tumor-specific antigen of mastocytoma P815 and is encoded by a gene that is specifically activated in tumors.     

CD40 ligand-activated human monocytes amplify glomerular inflammatory responses through soluble and cell-to-cell contact-dependent mechanisms.

Monocytes/macrophages play a critical role in the initiation and progression of a variety of glomerulonephritides. We sought to define the interactions between physiologically activated human monocytes and glomerular mesangial cells (MC) by employing a cell culture system that permits the accurate assessment of the contribution of soluble factors and cell-to-cell contact. Human peripheral blood monocytes, primed with IFN-gamma and GM-CSF, were activated with CD40 ligand (CD40L) or TNF-alpha and cocultured with MC. CD40L-activated monocytes induced higher levels of IL-6, monocyte chemoattractant protein-1 (MCP-1) and ICAM-1 synthesis by MC. Separation of CD40L-activated monocytes from MC by a porous membrane decreased the mesangial synthesis of IL-6 by 80% and ICAM-1 by 45%, but had no effect on MCP-1. Neutralizing Abs against the beta 2 integrins, LFA-1 and Mac-1, decreased IL-6 production by 40 and 50%, respectively. Ligation of mesangial surface ICAM-1 directly enhanced IL-6, but not MCP-1, production. Simultaneous neutralization of soluble TNF-alpha and IL-1 beta decreased MCP-1 production by 55% in membrane-separated cocultures of MC/CD40L-activated monocytes. Paraformaldehyde-fixed CD40L-activated monocytes (to preserve membrane integrity but prevent secretory activity), cocultured with MC at various ratios, induced IL-6, MCP-1, and ICAM-1 synthesis by MC. Plasma membrane preparations from activated monocytes also induced mesangial IL-6 and MCP-1 synthesis. The addition of plasma membrane enhanced TNF-alpha-induced mesangial IL-6 production by approximately 4-fold. Together, these data suggest that the CD40/CD40L is essential for optimal effector function of monocytes, that CD40L-activated monocytes stimulate MC through both soluble factors and cell-to-cell contact mediated pathways, and that both pathways are essential for maximum stimulation of MC.     

Influence of concentration and structure of quaternary ammonium salts on their antifouling efficiency tested against a community of marine bacteria

With the view of incorporating quaternary ammonium salts (QAs) in marine paints, nineteen of these were tested against a community of marine bacteria, at a temperature and salinity close to those of seawater. The concentration of QAs and the length of the main substituting chain are the main parameters affecting the growth and adhesion of bacteria, but the nature of (i) the other chains, (ii) the counter‐ion and (iii) the rings when inserted in the QA molecule also influenced the bacteria. Increasing the concentration of the QAs decreased the growth rate of the bacteria, the maximum cell density at the plateau and the rate of adhesion. The effect of increasing the length of the main chain depended on the range of carbon numbers. Below 7 carbon atoms, the growth rate was not significantly modified, but the numbers of cells at the plateau increased in contrast with the adhesion rate which decreased rapidly. Increasing the length of the chain to between 7 and 16 carbon atoms resulted in a decrease in the growth rate, a decrease and then a stabilisation in the numbers of cells at the plateau and no further change in the adhesion rate. Possibly an increase in growth rate, adhesion rate and in the numbers of cells at the plateau may occur above 16 carbon atoms. In contrast, the length of the other chains influenced positively the cell concentration at the plateau, and more generally the efficiency of QAs decreased substantially when these chains had the same numbers of carbon atoms. QAs with iodide as counter‐ion were more effective than those with chloride or bromide and phenyl was more effective than benzyl as rings inserted in QAs. The minimum inhibitory concentrations (MIC) were often very high if compared to standard methods with laboratory strains, and this can be tentatively explained by the dominance of Gram— bacteria in the community assayed, the development of resistant strains in the cultures used with time and the presence of organic matter in the culture medium.     

Chemical, physicochemical and spectrophotometric properties of crystalline chlorophyll-protein complexes from Lepidium virginicum L

Two kinds of water-soluble chlorophyll-protein complexes were prepared from leaves of Lepidium virginicum L., one (CP661) from the plant cultivated in a green house from seeds collected near Mono Lake, CA, and the other (CP-663) from a plant collected at Narashino, Chiba, Japan, by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose and Sephacryl S-200. The chlorophyll . proteins were further purified by crystallization. CP661 has absorption peaks at 661, 468, 439, 419, 380, 339 and 272 nm. CP663 had absorption peaks at 663, 469, 438, 419, 379, 338 and 272 nm. Estimated molecular weights were 78 000 for CP661 and 80 000 for CP663 by gel filtration chromatography and 83 000 for CP661 and 107 000 for CP663 by an equilibrium sedimentation method. 1 mol chlorophyll . protein contained 4 mol chlorophyll a and b with ratios of 1.0 in CP661 and 1.6 to 1.9 in CP663, but no carotenoids. These characters are different from those of chlorophyll-protein complexes which are prepared from the thylakoid membranes of chloroplasts with detergents.     

An automated technique for monitoring carbon dioxide respired by insects*

ABSTRACT. A non-dispersive infrared gas analyser equipped with a Luft-type sonic detector and flow-through reference cell was automated to monitor the total volume of carbon dioxide (CO₂) respired by single insects or groups of insects. The infrared analyser was interfaced with an integrator for quantification, a microprocessor to control intermittent air flow through the insect respiration chambers, and a microcomputer for data storage and reduction. This technique has been used to monitor the CO₂ Output of diapausing and non-diapausing mature fifth instar larvae and of developing pupae of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). The resulting data were accurate, quantitative and reproducible.     

Effects of cholinergic drugs on longitudinal muscle contractions of Fasciola hepatica

Acetylcholine, cholinergic agonists and acetylcholinesterase inhibitors significantly decrease the amplitude and frequency of spontaneous longitudinal muscle contractions in Fasciola hepatica. In order of their effects on the inhibition of muscle contractions, the cholinergic agonists can be ranked as nicotine greater than carbachol greater than acetylcholine. High calcium ion concentration also causes a significant inhibition of contractions. Atropine, a cholinergic antagonist that acts on muscarinic receptors, significantly increases the amplitude and frequency of spontaneous contractions and completely reverses the effects of cholinomimetic drugs, including nicotine. In adult F. hepatica, the levels of acetylcholine and its precursor choline are 3.14 +/- 0.55 and 13.75 +/- 3.72 pmol/mg wet weight, respectively. The activities of choline acetyltransferase, specific acetylcholinesterase and the nonspecific cholinesterase are 1.25 +/- 0.19, 238.0 +/- 13.0, and 83.0 +/- 33.0 nmol/hr/mg protein, respectively.     

An adaptor strategy to subclone entire cDNA libraries as single insert recombinants

The current methods for subcloning entire cDNA libraries usually result in a significant portion of recombinants containing multiple inserts, since in most instances the inserts derived from the library to be subcloned are released as a bulk of self-ligatable DNA fragments. By use of an adaptor strategy, a method is presented to confer noncompatible ends to primarily self-ligatable inserts, resulting in efficient subcloning of entire libraries as single insert recombinants.