Antitumor effect of combined treatment of mice with cytostatic agents and bacteriophage T4.

The past few years have shown significant resurgent interest in the old concept of bacteriophage therapy. Some research groups continue to develop whole bacteriophage preparations as alternatives to antibiotic antibacterial treatment. However, improvements in the methods of purification of phage preparations open new opportunities in the successful treatment of antibiotic-resistant bacterial infections. An open question remains on whether bacteriophage preparations (BP) can be safely applied in antibacterial treatment of patients suffering from infections as a consequence of immunosuppression caused by anticancer chemotherapy. The aim of this study was to evaluate the potential modulating effect of bacteriophage T4 preparations administered to mice bearing s.c. or i.v. inoculated B16 melanoma and treated with conventional anticancer drugs, i.e. cyclophosphamide (CY), cisplatin (CPt) or 5-fluorouracil (5-FU). Treatment of mice with (BPT) T4 preparation slightly potentiated the antimetastatic effect of CY. Importantly, no combination of phage-cytostatic treatment resulted in a decrease in the antimetastatic or antitumour effects of an applied drug. This suggests the possibility of safe combination of bacteriophage preparations with popular antitumour drugs.

The effect of bacteriophages T4 and HAP1 on in vitro melanoma migration.

BACKGROUND: The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans). Bacteriophages are constantly present in mammalian bodies and the environment in great amounts. On the other hand, the perspective of the possible use of bacteriophage preparations for antibacterial therapies in cancer patients generates a substantial need to investigate the effects of phages on cancer processes. RESULTS: In these studies the migration of human and mouse melanoma on fibronectin was inhibited by purified T4 and HAP1 bacteriophage preparations. The migration of human melanoma was also inhibited by the HAP1 phage preparation on matrigel. No response of either melanoma cell line to lipopolysaccharide was observed. Therefore the effect of the phage preparations cannot be attributed to lipopolysaccharide. No differences in the effects of T4 and HAP1 on melanoma migration were observed. CONCLUSION: We believe that these observations are of importance for any further attempts to use bacteriophage preparations in antibacterial treatment. The risk of antibiotic-resistant hospital infections strongly affects cancer patients and these results suggest the possibility of beneficial phage treatment. We also believe that they will contribute to the general understanding of bacteriophage biology, as bacteriophages, extremely ubiquitous entities, are in permanent contact with human organisms.

Bacteriophage preparation inhibition of reactive oxygen species generation by endotoxin-stimulated polymorphonuclear leukocytes.

It has been known that administration of antibiotics may lead to excessive release of bacterial endotoxins and complicate clinical course of patients with Gram-negative infections. This concern may also apply to phages. Endotoxin may in turn activate neutrophils to produce reactive oxygen species (ROS) that are believed to play an important role in the pathogenesis of multiple organ dysfunction in the course of sepsis. We showed that a purified T4 phage preparation with low-endotoxin content could significantly diminish the luminol-dependent chemiluminescence (CL) of peripheral blood polymorphonuclear leukocytes (PMNs) both stimulated by lipopolysaccharides (LPSs) isolated from different Escherichia coli strains. This effect was also observed for live bacteria used for PMNs stimulation and was independent of bacterial susceptibility for T4-mediated lysis. Our data suggest, that phage-mediated inhibition of LPS- or bacteria-stimulated ROS production by PMNs may be attributed not only to phage-PMNs interactions, but also to phage-LPS interactions and bacterial lysis (in case of homologous phage). Interestingly, the T4 preparation did not influence ROS formation by PMNs stimulated with PMA. This suggests that the observed phenomena are also dependent upon the nature of activator. Bacteriophage-mediated inhibition of ROS formation by cells exposed to endotoxin provides new evidence for possible interactions between phages and mammalian cells. It helps in understanding the role of phages in our environment and may also be of important clinical significance.

Preparation of endotoxin-free bacteriophages.

Bacteriophages (phages) are bacterial viruses that interact with bacterial walls and invade bacterial cells. Moreover, they disturb bacterial metabolism and lead to bacteria lysis. In the case of Gram-negative bacteria crude phage cultures, apart from the phages themselves, the bacterial debris, bacterial proteins and nucleic acids contain endotoxins. These endotoxins (lipopolysaccharides) posses a high degree of toxicity in vitro and in vivo, and their removal is essential for safety in antibacterial bacteriophage therapy. An effective, scaleable purification of bacteriophages from endotoxins was accomplished by sequential ultrafiltration through polysulfone membrane (30 nm) followed by chromatography on sepharose 4B and Matrex Cellulofine Sulfate. The phage fraction after gel filtration chromatography routinely contained endotoxins in the 150-2500 EU/ml range. The procedure yielded bacteriophages contaminated with as little as 0.4-7 EU/ml (Limulus assay). This value lies within the permitted level for intravenous applications (5 EU/kg/h by European Pharmacopoeia, 1997).

Bacteriophages provide regulatory signals in mitogen-induced murine splenocyte proliferation.

The aim of this investigation was to reveal the regulatory properties of bacteriophage preparations in a model of mitogen-induced splenocyte proliferation in mice. We showed that sepharose 4B-purified preparations of the Staphylococcus aureus phage A20/R exhibited costimulatory activity in splenocyte proliferation induced by suboptimal (0.25 microg/ml) concentrations of ConA. On the other hand, the purified phage fraction was regulatory with regard to splenocyte proliferation induced by the optimal (2.5 microg/ml) ConA concentration. We also showed that the phage preparation can elicit IL-6 production in splenocyte cultures and enhance ConA-induced production of that cytokine. Furthermore, the phages preferentially induced IL-6 production in adherent splenocytes and increased levels of that cytokine in cultures of peritoneal cells from mice and rats. This phenomenon may explain the costimulatory activity of phages in the model described.