The successful initiation of a primary antibody response in tissue culture has been reported by several workers (1--4). I n a previous report from this laboratory (3) it has been suggested that the sequential action of at least two different types of cells was responsible for the formation of antibody in tissue culture. One type was a mononucleated cell with phagocytic activity, referred to as a macrophage, and the other was a non-phagocytic cell belonging to the lymphocytic series. The macrophage, presumably a non-antibody--producing cell, was thought to interact with the antigen yielding a product which in some manner stimulated the production of specific antibody in the lymphocytic cells. The role of the macrophage, and the nature of the active substance(s) that result from the interaction of antigen and macrophages, were further investigated. The results of these experiments in which tissue culture and chick embryo techniques were used are presented in this paper. Materials and MahodsLymph Node Cdls.--Young Wistar strain rats (60 gin) obtained from Blue Spruce Farms, Altamount, New York, were used as the source of lymph node cells. The animals were sacrificed by a blow on the head and the two lumbar nodes, the renal node, and an intestinal node (5) were excised. The nodes from 10 rats were teased manually in 6 ml of a Special Buffer Solution (SBS) of the following composition: NaC10.8 per cent, KC1 0.04 per cent, Na~HPO4 0.0568 per cent, KHsPO4 0.0095 per cent, MgSO4 0.0060 per cent, phenol red 0.002 per cent, and CAC12.2 H20 0.014 per cent. The suspension of teased cells was then filtered through sterile gauze to remove tissue fragments as well as clumps of cells. Mter centrifuging the cells at 200 X g for 10 minutes in the cold, the cells were washed once with SBS and then counted. Approximately 20 X 106 cells were obtained per rat; not more than 5 per cent of these cells were macrophages.Rabbit lymph node cells were obtained by teasing the mesenteric and popllteal nodes in a similar manner. A much higher yield of lymph node cells (480 X 106) per rabbit was obtained; approximately 15 per cent of the cells were macrophages.lffacrophages.--Rat macrophages were obtained by injecting 5 ml of a beef heart infusion broth fortified with 10 per cent proteose-peptone No. 3 (6) intraperitoneally into 200 to 250 gin Wistar rats obtained from Blue Spruce Farms. The rats were sacrificed by a blow on

A new protein component has been demonstrated in myelin isolated from rat whole brain and from white matter dissected from bovine, dog and rabbit brain. It is also present in rabbit optic nerve. It does not appear to be present in other subcellular fractions of rat brain. It has a molecular weight of 20,540 ± 490(S.D.), as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. A rapid procedure for the isolation of myelin is also described.

Previous reports (1, 2) have described a system in which a culture of lymph node cells from non-immunized rats was stimulated, in vitro, to produce specific antibody. This stimulus was provided by a cell-free homogenate of peritoneal exudate ceils, consisting mainly of macrophages, which had been incubated with the antigen in question. The observations that both streptomycin and ribonuclease rendered the system ineffective directed attention to the possibility that nucleic acid was an essential factor. The experiments to be presented show that the specific stimulatory activity resides in a purified ribonucleic acid fraction of the cell-free homogenate.Tests for activity of the purified material were conducted with the aid of diffusion chambers. Such chambers, charged with non-immune rat lymph node ceils and the material to be tested, were inserted intraperitoneally into x-irradiated rats. In some experiments the material alone was placed into the chambers which were then inserted into non-irradiated recipients. Activity was measured by titrating the sera of the recipient rats for specific antibody. Material and MethodsAnimals.--200 to 250 gm Wistar rats were used as recipients for the diffusion chambers, and as the source of exudate cells, hereafter referred to as macrophages. 60 to 80 gm rats served as lymph node donors. The procedures for obtaining both macrophages and lymph node cells have been described in a previous paper (2).X-Irradiation Factors.--Rats were exposed to 500 r total body irradiation through the facilities of the Brookhaven National Laboratories. The x-ray factors were 250 KVP, 15 ma, 0.5 mm Cu -4-1.0 mm A1; distance from target to skin, 40 inches; roentgens in air per minute, 28.0. Macrophage-A ntigenInteraction.--The procedure for obtaining sterile cell-free homogenates of macrophage-bacteriophage T2 mixtures has been described (2). In brief, the macrophages

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Polymyxin B (PmB) blocks many of the toxic effects of lipopolysaccharide by mechanisms that are not yet understood. The production of tumor necrosis factor-alpha (TNF-alpha) by isolated rat alveolar macrophages in response to lipopolysaccharide and macrophage-activating factor was blocked by PmB at concentrations of 100, 10, and 1 micrograms/ml. Gentamicin enhanced rather than inhibited TNF production at the 100-micrograms/ml concentrations and had no effect at low concentration. Similar inhibitory effects were induced by PmB in an in vivo model in which rat macrophage TNF production was stimulated by intratracheally injected lipopolysaccharide. Because many of the effects of lipopolysaccharide are mediated by TNF, this inhibition provides a mechanism to explain the protection afforded by PmB against lipopolysaccharide-induced toxicity.

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