The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin     

Methenamine-Silver Staining: a Simple and Sensitive Staining Method for Senile Plaques and Neurofibrillary Tangles

An improved methenamine-silver impregnation method is presented which exhibits sensitivity for amyloid substances comparable to that of anti-β protein immunostaining. In optimally treated sections, this technique stained both β-amyloid deposits and neurofibrillary tangles, which are known to have a β-pleated structure. This simple procedure allows a large number of sections to be stained for routine examination.     

Localization of the antigen for the monoclonal antibody ②9F11-B-E4 interspecific cross-reaction in the freshwater triclads

The localization of the antigen for monoclonal antibody 9F11-B-E₄ was clarified by immuno-electron microscopy. The antigens were localized on the mitochondria and Golgi bodies in the male germ cells and on the secretory granules of various glands cells in the penis bulb and subepidermal parenchymal tissue of Phagocata vivida. The results of the interspecific cross-reaction tests with seven other freshwater triclads showed that these secretory granules are species-specific. A positive interspecific reaction was showed with Dugesia (family Dugesiidae), but not with Polycelis within the same family Planariidae which suggests the position of Phagocata within the Planariidae needs to be reassesed.     

Maintaining genetic code through adaptations of tRNA synthetases to taxonomic domains

The universal genetic code is determined by the aminoacylation of tRNAs. In spite of the universality of the code, there are barriers to aminoacylation across taxonomic domains. These barriers are thought to correlate with the co-segregation of sequences of synthetases and tRNAs into distinct taxonomic domains. By contrast, we show here examples of eukaryote-like synthetases that are found in certain prokaryotes. The associated tRNAs have retained their prokaryote-like character in each instance. Thus, co-segregation of domain-specific synthetases and tRNAs does not always occur. Instead, synthetases make adaptations of tRNA-protein contacts to cross taxonomic domains.     

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.     

Production of S-lactoylglutathione by organic-solvent-extracted glyoxalase I from Hansenula mrakii

Summary Glyoxalase I was extracted from Hansenula mrakii IFO 0895 by incubating the cells with buffer solution containing 50% acetone (enzyme activity 35 units/g cells) or 50% ethyl acetate (enzyme activity 28 units/g cells) at 30°C for 10 h. Glyoxalase II was also extracted from the cells, although the activity of the enzyme was lost during incubation with organic solvents, especially at higher temperature (30°C). By using the organic-solvent-extracted fraction of H. mrakii, enzymatic production of S-lactoylglutathione was studied, and approximately 82 mmol/l (30 g/l) of S-lactoylglutathione was produced from 120 mmol/l glutathione. Offprint requests to: A. Kimura     

Simplified determination of lorazepam and oxazepam in biological fluids by gas chromatography—mass spectrometry

Lorazepam and oxazepam in plasma and urine were measued by gas chromatography—mass spectrometry. Oxazepam was used as an internal standard in the assay of lorazepam and vice versa. After removal of interfering substances with n-hexane, the drugs were extracted with benzene and converted to N₁,O₃-bistrimethylsilyl derivatives. Glucuronide forms of the drugs were extracted after hydrolysis with β-glucuronidase. A common fragment ion at m/e 429 was used to monitor the two drugs. The sensitivity was 2 ng/ml for both drugs, which was sufficient to determine plasma and urine concentrations after therapeutic doses to humans.