Phase II study of vaccinia melanoma cell lysates (VMCL) as adjuvant to surgical treatment of stage II melanoma

Summary Patients with stage II melanoma were vaccinated with vaccinia virus-induced melanoma cell lysates (VMCL). The vaccine contained viable vaccinia virus, membranous fragments and no intact nuclei. A number of antigens defined by monoclonal antibodies were detected in the vaccine including the ganglioside GD3 and DR antigens. Administration of the vaccine was associated with depression of natural killer cell activity against melanoma and K562 target cells in the first 3–6 months of treatment. Leucocyte dependent antibody (LDA) activity against melanoma cells was induced or increased in titre in approximately half of the patients studied. Continued vaccination was associated in a number of patients with a decrease in LDA titres. Studies on a small sample of patients revealed that this was associated with the development of serum factors which inhibited LDA activity. LDA activity appeared directed to non-MHC antigens on melanoma cells which were of at least two specificities. One specificity which was shared with antigens on a number of non-melanoma carcinoma cells was removed by absorption on fetal brain and may be similar to oncofetal antigens described by other workers. Reactivity against melanocytes was induced in some patients and may underlie the development of vitiligo in several patients. These results suggest that vaccines prepared from VMCL may be a favourable method for increasing immune responses against melanoma.     

Enumeration of chlorine-stressed organisms with acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (AOINT)

Direct microscopic enumeration ofEnterobacter cloacae with the acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride technique (AOINT) was compared with spread plate counts on nonselective media to establish the usefulness of the former technique in the enumeration of chlorine-stressed cells. Results indicate that the techniques are comparable when the organisms are not stressed. However, AOINT is more sensitive than are plate counts in the detection of chlorine-stressed cells.     

UDPglucosyltransferase and its kinetic fluorimetric assay

A rapid, kinetic assay for UDPglucosyltransferase has been developed using 1-naphthol as substrate. It is based on the continuous fluorimetric monitoring of 1-naphthyl glucoside formation during the reaction at physiological pH. The conjugate is easily distinguished from aglycone, since their fluorimetric properties differ. Glucoside biosynthesis in vitro by microsomal preparations isolated from the gut and fat body of cockroaches Periplaneta americana and Leucophaea maderae, and from the green gland and hepatopancreas of the crayfish Astacus astacus, has been demonstrated. The effects of buffer, pH, MgCl2, UDP-glucuronic acid, UDP-N-acetylglucosamine, sodium cholate and sonication on the enzyme activity have been assessed. The kinetic parameters of 1-naphthol and UDP-glucose have also been determined.     

A rapid bioluminescence method for quantifying bacterial adhesion to polystyrene

Bioluminescence ATP analysis has been used to assess bacterial adhesion with hydrophobic polystyrene tubes as the attachment surface. The assay was performed at 37 degrees C and pH 6.8 with a 10 min incubation period. A variation of more than 200-fold was observed in the adherence capacity of 34 urinary isolates of Escherichia coli, and organisms could be classified as strongly or weakly adherent. All strains capable of strong adhesion possessed both type 1 fimbriae and flagella, and maximum adhesion was expressed during the exponential growth phase. Attachment was in all cases virtually eliminated by addition of 2.5% (w/v) D-mannose to the incubation buffer. Conversely, strains which were deficient in type 1 fimbriae or flagella, or both, were weakly adherent during all phases of growth. There was no correlation between adherence of E. coli to polystyrene and adherence to buccal or uroepithelial cells, but there was a significant association with adherence to uromucoid (P less than 0.002).     

Simultaneous determination of leu-enkephalin localization and [3H]γ-aminobutyric acid uptake in rat striatal cell cultures

The purpose of this study was to investigate the coexistence of gamma-aminobutyric acid (GABA) and leu-enkephalin in single neurons from the corpus striatum. Monolayer cell cultures, started from newborn rat corpus striatum, were grown in serum-free medium and examined using GABA autoradiography and leu-enkephalin immunocytochemistry in a double-label protocol. Examples of cells were found which were positive for one or the other neurotransmitter or for neither transmitter, but not for both. Furthermore, cells which appear similar by morphological criteria alone differed in transmitter specificity. We conclude that the two transmitters tested are not localized within single cells and that morphology alone is inadequate to identify functional cell classes in this area.     

Effect of Relative Humidity on Survival of Candida albicans and Other Yeasts

Individual blastospores of Candida albicans were deposited on the surface of 50-mm membranes (Millipore Corp.) and placed within sealed glass chambers at various relative humidities (RH). After 48 hr, virtually all cells maintained at 100 and 10% RH had survived, but 84% of the cells maintained at 60% RH failed to develop into colonies when transferred to Sabouraud medium. No morphological abnormalities could be observed in cells surviving low RH values, but their initial rate of multiplication after transfer to Sabouraud medium was greatly reduced, compared to that demonstrated by cells maintained at 100% RH. At 60% RH, the exposure time required to kill 50% of the blastospores was 2 to 3.5 days. The inimical effect of 60% RH was confirmed in a total of 21 isolates of C. albicans. No deleterious effect was noted when 12 other species of yeasts were subjected to 10, 60, and 100% RH. The single isolate of Candida brumptii and 1 out of the 20 isolates of Cryptococcus neoformans tested also failed to grow after blastospores had been exposed to 60% RH for 4 days.     

Allergenicity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations in Mice and Guinea Pigs

Guinea pigs were injected subcutaneously with mycobacterial ribosomal fraction incorporated in Freund's incomplete adjuvant and tested 6 and 12 weeks later by the intradermal injection of 0.5 μg (25 TU) of Purified Protein Derivative. No evidence of delayed-type hypersensitivity could be detected in these animals, although large necrotic reactions were obtained in guinea pigs sensitized with living, attenuated mycobacterial cells. Mice also were vaccinated by the intraperitoneal injection of mycobacterial ribosomal fraction or ribonucleic acid (RNA) and tested for sensitivity to tuberculin at various subsequent times. No evidence of true tuberculin hypersensitivity could be detected at any time, although what appeared to be small Arthus type reactions were seen in mice given the largest vaccinating doses. Attempts to recall tuberculin sensitivity in vaccinated mice by the intravenous injection, 4 weeks after vaccination of living cells, of either the virulent or attenuated mycobacterial strains were unsuccessful. Instead, when the virulent cells were injected, a suppression of footpad reactivity was noted in animals made sensitive to tuberculin by the previous intraperitoneal injection of viable attenuated mycobacterial cells. Both guinea pigs and mice, vaccinated as described above, were also skin tested or footpad tested, respectively, with 2 μg of the ribosomal fraction or RNA used for vaccination. No evidence of true tuberculin hypersensitivity could be obtained; instead, in guinea pig skin very small dermonecrotic areas were noted, and in mice swelling and redness of the footpad occurred to an equal extent in both vaccinated and nonvaccinated mice. The possible role of tuberculin hypersensitivity in acquired immunity to tuberculosis is discussed, and the conclusion is reached that its part, if any, is minor.     

Deoxyribonucleic Acid Homology and Relative Genome Size in Mycoplasma

The deoxyribonucleic acid homologies of Mycoplasma laidlawii type A and type B, M. pulmonis (#47 and #63), and M. hominis were determined by membrane methodology. The homology data revealed a difference in genome size between M. laidlawii type A and type B. This difference also held with stringent conditions of annealing (high temperature). Little or negligible homology was shown to exist between the M. laidlawii strains type A and type B and M. pulmonis strains 47 and 63 and M. hominis. M. hominis showed less than 10% homology to the M. pulmonis and M. laidlawii strains. Neither of the M. laidlawii strains showed more than 2% annealing to the M. pulmonis strains. Reaction rate studies are suggested as a means of demonstrating the phylogenetic relationship between the Mycoplasma and other microorganisms.     

Surfactants as Stimulants of Enzyme Production by Microorganisms

The addition of Tween 80 and sucrose monopalmitate, nonionic surfactants, to fungal cultures resulted in marked increases in yields of the enzymes cellulase, amylase, sucrase, beta-1 --> 3 glucanase, xylanase, purine nucleosidase, and benzoyl esterase. The action appears to be an effect of the surfactant on cell permeability.