Hong Lioe Ko

Effect of Three Strains of Propionibacteria (P. granulosum, P. avidum, P. acnes) and Cell-Wall Preparations on Lymphocytes and Macrophages

The effect of the strains of Propionibacteria (P. granulosum, P. avidum, P. acnes) on functional and metabolic activities of mouse lymphocytes and rabbit macrophages was examined in vivo and in vitro. In some of the experiments bacterial cell walls isolated from the above bacteria were tested and compared with the activity of whole bacteria. In isolated lymph-node lymphocytes influx of 86Rb and its inhibition by ouabaine, release of 86Rb and 51Cr from prelabelled cells, as well as spontaneous and PHA-stimulated incorporation of 3H-uridine were tested after treatment with 10 or 50 microgram/ml of Propionibacteria (whole cells or cell walls). Phagocytosis of 32P-labelled Staphylococci and reduction of Nitro-BT were examined in isolated rabbit peritoneal macrophages treated with the above concentrations of Propionibacteria. Clearance of 32P-labelled Staphylococci from peripheral blood in rabbits treated with 5 mg per kg body weight of Propionibacteria was also studied. All three strains of Propionibacteriae stimulated 86Rb influx into lymphocytes, the effect being observed only in concentrations leading to parallel increase of 86Rb and 51Cr release from prelabelled cells. No significant differences in activity between the three tested strains of bacteriae were found. Propionibacteria of a dosage of 50 microgram/ml significantly increased spontaneous and PHA-stimulated incorporation of 3H-uridine into lymphocytes. All three strains of Propionibacteria markedly stimulated both phagocytosis and intracellular digestion (as measured by the ability to reduce Nitro-BT) in isolated peritoneal macrophages. This was accompanied by faster clearance of Staphylococci from peripheral blood of rabbits treated with 5 mg per kg body weight of Propionibacteriae. The obtained results showed direct stimulation of lymphocytic and macrophagic functional activity by whole cells and isolated cell walls of all three tested strains of Propionibacteria.

The correlation of susceptibility of differentPropionibacterium strains to macrophage killing and antitumor activity

The stimulation of the reticuloendothelial system and antitumor activity againstsarcoma-L-1 of three different strains ofPropionibacterium was investigated. The bacteria injected intraperitoneally with a single dose of 1 mg per mouse appeared to possess different effectiveness in both assays used. The same strains were examined for their susceptibility to phagocytosis and intracellular killing by murine peritoneal macrophages. The results obtained showed correlations betweenPropionibacterium resistance to degradation by phagocytic cells and their antitumor and stimulatory activities. It is concluded that the potency of antitumor and stimulatory activities of different strains ofPropionibacterium are closely related to their cell wall structure.

The in vivo cytostatic effect induced by Propionibacterium granulosum on murine tumor cells.

SummaryThe cytostatic action of Propionibacterium granulosum was studied in a mouse sarcoma in vivo. Kinetic analysis of tumor cells 28 days after tumor implantation and systemic immunotherapy showed that the cell cycle time was identical in both treated and untreated tumors. P. granulosum treatment resulted in a marked prolongation of the S phase and shortening of the G1 phase of the cell cycle. A pronounced drop in the number of labeled interphases and the reduction of the growth fraction were observed in tumors obtained from mice given injection of P. granulosum. Cloning efficiency of tumor cells from P. granulosum treated animals was quantitatively similar to that of control animals and only differences in the size of lung colonies were observed.

Blood group phenotype determines lectin-mediated adhesion of Pseudomonas aeruginosa to human outer ear canal epithelium

Pseudomonas aeruginosa is the most frequent bacterial pathogen causing acute diffuse otitis externa. In a recent prospective phase II study we demonstrated that lectin-mediated bacterial adhesion can be blocked by receptor-analogue carbohydrates in patients suffering from Pseudomonas aeruginosa-induced acute otitis externa. In this investigation, human ABO blood group antigens were analysed on outer ear canal epithelial cells with standard routine histological procedures by monoclonal antibodies for the blood groups A and B, and with Ulex europaeus I lectin for the blood group O, respectively. In all cases (n = 20) the blood groups could be shown immunohistologically. P. aeruginosa-specific adhesion and inhibition assays were performed in the presence of N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), D-mannose and A-like substance. Outer ear canal tissue sections were incubated with P. aeruginosa (strain PA 60), presenting lectin-specificity for GalNAc. Sections from patients presenting with blood group A were closely settled with bacteria in the presence of non-specific GlcNAc, D-mannose and PBS however, GalNAc and A-like substance inhibited the microbial adhesion. Amongst others, P. aeruginosa present adhesion molecules (lectins) with specificity for GalNAc. Thus, the correlation between blood group A phenotype and P. aeruginosa-induced acute diffuse otitis externa was investigated. Statistical evaluation proved a highly significant association. These data support the hypothesis that P. aeruginosa lectins with GalNAc specificity apparently adhere to GalNAc moieties, representing the terminal blood group A-determinant and further indicate that patients presenting with blood group A may have a genetic disposition for this form of otitis externa.

Action of three species of Propionibacteria (P. granulosum, P. acnes, P. avidum) on experimental tumor systems in mice.

The antitumor activity of three different species of Propionibacteria (P. granulosum, P. avidum and P. acnes) against murine Sarcoma 180 and Moloney-Sarcoma-Virus-induced tumor (MSV tumor) was investigated. All three Propionibacteria injected intraperitoneally or intratumorally in multiple doses of 1 mg per mouse appeared to be effective regarding the retardation of growth and the stimulation of regression of Sarcoma 180 in CFW mice. Moreover, the application of Propionibacteria significantly prolonged the survival of Sarcoma-180-bearing mice. No significant differences were found in antitumor effect against Sarcoma 180 between P. granulosum, P. Avidum and P. acnes, but in general, intratumoral injections of all three strains were more effective than intraperitoneal applications. All three strains of Propionibacteria were equally ineffective regarding the retardation of growth and the stimulation of regression of MSV tumors in adult NMRI mice, as assessed after intraperitoneal injections of P. granulosum, P. avidum or P. acnes (1 mg per mouse) 3 days prior to inoculation of virus particles.

Influence of Pseudomonas aeruginosa Proteases on Prothrombin, Plasminogen and Fibrinogen

Out of 136 strains of Pseudomonas aeruginosa tested, 101 produced a procoagulant protease and 70 a protease with fibrinolytic activity. Strain No. 1800 producing both these proteases served as a source of the crude enzyme mixture. The crude enzyme was isolated from the supernatant and subjected to fractionation by isoelectric focusing. Five active components were isolated. Activities of individual proteases were compared. It was found that for prothrombin activation a protease characterized by isoelectric point of 7.0 is responsible; this protease is different from both collagenase and elastase. Protease available at isoelectric point of 8.8 is plasminlike and is also not identical with collagenase and elastase.

Macrophage and T Lymphocyte Content of Tumors in Mice Treated with Propioni bacterium

Macrophage and T lymphocyte content of sarcoma tumors in mice treated with Propionibacterium granulosum (PG) by different routes was investigated and compared with the effectiveness of the therapy. Two different methods for tumor macrophage detection, the Fc receptor analysis and phagocytic properties, were employed. Indirect fluorescent staining of theta-antigen on lymphocytes was used to evaluate the proportion of tumor T cells. Systemic treatment with PG appeared to be effective if given soon after tumor implantation; however, this route of administration failed to affect the tumor growth if given when the tumor was already advanced. The therapeutic efficiency of intralesional PG injection was almost as effective as early systemic treatment. We obtained evidence indicating that effectiveness of PG therapy was associated with an increase of the macrophage and T cell content of tumors.

Hemolysin of Propionibacterium avidum.

Thiol-activated extracellular hemolysin produced by a strain of Propionibacterium avidum was partially purified and several properties were examined. The hemolysin was heat labile and inactivated by proteases and deoxyribonuclease. Hemolysin adsorbed to erythrocytes at 0 degrees C. Cu++ at 1 mM inhibited hemolysis partially. Lecithin prevented the activity completely. The hemolytic spectrum was relatively wide and the activity was high with rabbit, dog, horse, and pig erythrocytes.

Changed murine lymphocyte trapping after treatment with Propionibacterium granulosum

SummaryThe trapping of lymphocytes occurring at different times after a single injection of Propionibacterium granulosum was studied. Labeled syngeneic lymphocytes injected into Propionibacterium-treated recipients showed a different pattern of localization from that observed in untreated animals. A pronounced decrease in homing to the lymph nodes and spleen and an increase in localization in the liver were found. The extent of trapping in the liver corresponded to the increase in weight of this organ. Whole-body irradiation with 400 R, used to achieve autologic lymphocyte depletion, did not change the localization of labeled cells. However, macrophage damage caused by silica resulted in diminished trapping in the liver in Propionibacterium-treated animals and increased accumulation of labeled lymphocytes in the spleen and lymph nodes.

Importance of lectins for the prevention of bacterial infections and cancer metastases.

Adhesion of bacteria and of metastasizing tumour cells have much in common, especially the participation of lectins in this process. In the future it might be possible to inhibit the metastatic process and bacterial adhesion by blocking with lectins specific for appropriate (oligo) saccharides or glycoconjugates. Initial clinical trials are very promising.