Regression of line-10 hepatocellular carcinomas following treatment with water-soluble,microbial extracts combined with trehalose or arabinose mycolates

Summary Intratumor injections of the aqueous phase of phenol-water extracts of Re mutant Salmonella typhimurium (Re glycolipid) in combination with trehalose dimycolate at dose levels of 150 to 15 g were consistently and highly effective (65–93%) in producing regression of line-10 tumors in strain-2 guinea pigs. We observed that the rate of regression was more rapid than that seen after treatment with cell walls from Mycobacterium bovis strain Bacillus of Calmette and Guèrin (BCG). Arabinose mycolate could be substituted for trehalose dimycolate in the Re glycolipid-mycolate mixture without appreciably compromising antitumor activity, providing that the level of arabinose mycolate was not reduced below 15 g. In addition to the Re glycolipid preparation, similarly prepared aqueous extracts from Mycobacterium bovis strain BCG and strain AN5 in combination with trehalose dimycolate also possessed tumor-regressive activity. The activity of these last extracts was reduced when the arabinose mycolate was substituted for the trehalose dimycolate. The aqueous extract of a rickettsia, Coxiella burnetii, in combination with either trehalose dimycolate or arabinose mycolate was also active (50 and 80% tumor regression rates, respectively). Intracutaneous administration of Re glycolipid or aqueous extracts from BCG in combination with trehalose or arabinose mycolates did not produce life-threatening, clinical signs of toxicity in young mice. If additional toxicity studies demonstrate that adverse side effects can be satisfactorily controlled, these watersoluble extracts may prove beneficial in the treatment of spontaneous tumors of humans and other animals.     

Further studies on the structural requirements of agents for immunotherapy of the guinea pig line-10 tumor

Summary We made a comparative study of the in vivo binding of immunoglobulins (Ig) to a polyoma virus-induced ascitic tumor propagated in syngeneic or allogeneic mice. The Ig coat was found to appear more rapidly and to be denser in H 2-incompatible than in H 2-compatible mice. This suggests that antibodies were fixed specifically on strong normal transplantation antigens (H-2) recognized as non-self by allogeneic mice. Experiments with mice in which immunosuppression had been achieved by means of X-irradiation confirmed that the Ig fixed on SEWA cells are actively bound antibodies. The only mice that could fix Ig on tumor cells were those that had been specifically immunized against cell surface antigens shared by SEWA cells before irradiation, while mice hyperimmunized against nonrelated antigens could not.In partial fulfilment of doctorate thesis requirements     

Light and electron microscopic structure of golgi-stained neurons in the vertebrate brain (new rapid Golgi procedure)

Summary After application of a rapid, selective silver impregnation procedure for light (LM) and electron (EM) microscopy, individual neurons are distinguishable by a light silver precipitation. The silver content is sufficient that entire nerve cells can be observed light microscopically; on the other hand, electron microscopically the cytological details are still visible. Brains of mice were fixed by phosphate-buffered aldehyde perfusion, and pieces of tissue left in a 1 % K₂Cr₂O₇ solution for 13 h before impregnation in a 0.5 % AgNO₃ solution for 2h. Thick sections (30–50 m) of the impregnated tissue were cut; from these sections, suitably stained neurons were dissected out and re-embedded for ultrathin sectioning, thereby allowing observations on the same neurons at the EM level. A thin silver deposit was observed along the delimiting neuronal membrane, the microtubules and the smooth ER, including the spinal apparatus of the dendritic spines. The fine cytoplasmic details of the impregnated neurons and the surrounding tissue are well preserved and, therefore, suitable for subsequent determination of synaptic relationships of the impregnated neurons with the adjacent neuronal elements.     

Different structural requirements of endotoxic glycolipid for tumor regression and endotoxic activity

Endotoxic glycolipid extracted from the heptose-less mutant of $\text{Salmonella typhimurium}$ was treated with alkali and acid reagents. The glycolipid freed of all O-ester linked fatty acids by hydroxylamine had lost tumor regression activity and toxicity, whereas a partial removal of O-ester linked fatty acids by mild alkali did not impair with these activities. The glycolipid retained both activities after removal of 2-keto-3-deoxyotonate by sodium acetate (pH 4.5) but was rendered nontoxic while retaining antitumor activity when hydrolyzed by 0.1N HCl whereby 2-keto-3-deoxyoctonate and glycosidic phosphate was split off the glycolipid molecule. Nontoxic and tumor regressive fractions were separated by means of preparative thin layer chromatography of glycolipid hydrolyzed by mild acid. Thus, it was concluded that glycosidic bound phosphate and at least a portion of fatty acids of the lipid A moiety were essential for toxicity, but that this phosphate is not essential for tumor regression activity.     

Drone bees fixate the queen with the dorsal frontal part of their compound eyes

Summary Free flying drone bees (Apis mellifera carnia () were filmed from the side while chasing the honey bee queen or landing on a hive.Drones use the upper frontal part of the field of view of their eyes while chasing the queen. They fixate with the lower frontal part of the field of view of their eyes during landing. Drones keep a particular distance from the queen while chasing her.The diameter of ommatidial lenses in drones shows a maximum in the region where the queen is fixated.Niedersächsisches Landesinstitut für Bienenforschung und Bienenwirtschaftliche Betriebslehre, Wehrstrasse 4, D-3100 Celle, Federal Republic of GermanyLehrstuhl Entwicklungsphysiologie, Fakultät für Biologie, Universität Tübingen, Auf der Morgenstelle, D-7400 Tübingen, Federal Republic of GermanyWe thank Walter F. Gelinski for kind cooperation and Professor W. Engels for support.     

Delayed hypersensitivity and granulomatous response after immunization with protein antigens associated with a mycobacterial glycolipid and oil droplets.

A myocardial glycolipid (P3) mixed with protein antigens in oil-in-water emulsion induced lasting delayed hypersensitivity (DH) and granulomatous inflammation after intradermal injection into guinea pigs. This did not occur when P3 and bovine serum albumin (BSA) were given in Freund's incomplete adjuvant. The oil-in-water emulsions consisted of microscopic oil droplets suspended in aqueous medium. By separating oil and aqueous phases from BSA + P3 emulsion it was shown that antigen retained with oil droplets led to DH and granuloma formation. The association of antigen with oil droplets was P3 dependent and was quantitated with 125I-labeled BSA. The same phenomenon occurred with 125I-labeled rabbit gamma-globulin (RGG) + P3 emulsion. Fluorescein-conjugated RGG was observed in a particulate state within or on oil droplets in emulsion containing P3. These physical characteristics of antigen + P3 emulsion appeared to be important for immunogenicity.     

The first optic ganglion of the bee

Summary The arrangement of first and second order neurons in an optic cartridge and the topographical relationships of the second order neurons within a cartridge and to groups of surrounding cartridges have been analyzed in the visual system of the bee, Apis mellifera, from light and electron microscope studies on Golgi preparations. At the level of the monopolar cell body layer, the nine retinula cell fibres of each ommatidium, the six short visual fibres arranged in a circle surrounding the three long visual fibres, become cartridges as a consequence of the appearance of the second order neurons (L-fibres) which join the R-fibre bundles. Two of the four different L-fibre types, L-1 and L-2, remain together in the centre of the cartridge throughout the lamina. The axons of the L-3 and L-4 fibres, however, have their position integrated into the circle formed by the endings of the short visual fibres. On the basis of further examination of light and especially electron microscopical Golgi material, the different L-fibres can be classified into four types which appear in each cartridge. The clear stratification in the first synaptic region (A, B and C) seems to be the best criterion for a morphological classification since such a classification necessarily also includes a functional basis. According to a naming system based on the position of the lateral processes, L-fibres with side branches in strata A, B and C are called L-1 fibres. Fibres with lateral processes in strata A and B are L-2 fibres; monopolar cell fibres with branches only in the second stratum B are L-fibres of type 3; and all monopolar cells with branches only in stratum C are called L-4 fibres. In addition to the branching pattern covering only the parent cartridge, two of the four fibre types (L-2 and L-4) have long collaterals reaching neighbouring cartridges: L-2 in stratum A and L-4 in stratum C. These collaterals presumably form a substrate for lateral interactions.     

Tumor regression with Q fever rickettsiae and a mycobacterial glycolipid

Summary Regression of established tumors by Coxiella burneti, the rickettsial agent which causes Q fever, was studied using the transplantable line-10 tumor in strain 2 guinea pigs. Suspensions of formalin-killed, purified rickettsiae induced an average of 42% tumor regression after intratumor injection. The activity of C. burneti was enhanced by incorporation of the rickettsiae into oil droplet vaccines containing the mycobacterial glycolipid, P₃. Such vaccines induced 64% tumor regression. The activity of C. burneti that was enhanced by P₃ was found in phenol-water extracts of the rickettsiae. These extracts contained lipopolysaccharides which were less toxic than bacterial endotoxins, and they induced 63% tumor regression when combined with P₃. These lipopolysaccharides may provide agents of high therapeutic activity but relatively low toxicity for cancer immunotherapy.