Jan Borysowski

Bacteriophage endolysins as a novel class of antibacterial agents.

Endolysins are double-stranded DNA bacteriophage-encoded peptidoglycan hydrolases produced in phage-infected bacterial cells toward the end of the lytic cycle. They reach the peptidoglycan through membrane lesions formed by holins and cleave it, thus, inducing lysis of the bacterial cell and enabling progeny virions to be released. Endolysins are also capable of degrading peptidoglycan when applied externally (as purified recombinant proteins) to the bacterial cell wall, which also results in a rapid lysis of the bacterial cell. The unique ability of endolysins to rapidly cleave peptidoglycan in a generally species-specific manner renders them promising potential antibacterial agents. Originally developed with a view to killing bacteria colonizing mucous membranes (with the first report published in 2001), endolysins also hold promise for the treatment of systemic infections. As potential antibacterials, endolysins possess several important features, for instance, a novel mode of action, a narrow antibacterial spectrum, activity against bacteria regardless of their antibiotic sensitivity, and a low probability of developing resistance. However, there is only one report directly comparing the activity of an endolysin with that of an antibiotic, and no general conclusions can be drawn regarding whether lysins are more effective than traditional antibiotics. The results of the first preclinical studies indicate that the most apparent potential problems associated with endolysin therapy (e.g., their immunogenicity, the release of proinflammatory components during bacteriolysis, or the development of resistance), in fact, may not seriously hinder their use. However, all data regarding the safety and therapeutic effectiveness of endolysins obtained from preclinical studies must be ultimately verified by clinical trials. This review discusses the prophylactic and therapeutic applications of endolysins, especially with respect to their potential use in human medicine. Additionally, we outline current knowledge regarding the structure and natural function of the enzymes in phage biology, including the most recent findings.

Clinical aspects of phage therapy.

Phage therapy (PT) is a unique method of treatment of bacterial infections using bacteriophages (phages)-viruses that specifically kill bacteria, including their antibiotic-resistant strains. Over the last decade a marked increase in interest in the therapeutic use of phages has been observed, which has resulted from a substantial rise in the prevalence of antibiotic resistance of bacteria, coupled with an inadequate number of new antibiotics. The first, and so far the only, center of PT in the European Union is the Phage Therapy Unit (PTU) established at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland in 2005. This center continues the rich tradition of PT in Poland, which dates from the early 1920s. The main objective of this chapter is to present a detailed retrospective analysis of the results of PT of 153 patients with a wide range of infections resistant to antibiotic therapy admitted for treatment at the PTU between January 2008 and December 2010. Analysis includes the evaluation of both the efficacy and the safety of PT. In general, data suggest that PT can provide good clinical results in a significant cohort of patients with otherwise untreatable chronic bacterial infections and is essentially well tolerated. In addition, the whole complex procedure employed to obtain and characterize therapeutic phage preparations, as well as ethical aspects of PT, is discussed.

Phage as a modulator of immune responses: practical implications for phage therapy.

Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

Phage as a Modulator of Immune Responses

Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

Rapamycin, unlike cyclosporine A, enhances suppressive functions of in vitro-induced CD4+CD25+ Tregs

BACKGROUND A growing body of data shows that CD4(+)CD25(+) regulatory T cells (Tregs) can induce transplantation tolerance by suppressing immune responses to allograft antigens. However, both the generation and the suppressive capacity of CD4(+)CD25(+) Tregs can be substantially affected by different immunosuppressive drugs used in clinical transplantation. The goal of this study was to compare the effects of cyclosporine A and rapamycin on the induction and suppressive functions of human CD4(+)CD25(+) Tregs in vitro. METHODS CD4(+)CD25(+) Tregs were induced in two-way mixed lymphocyte reaction (MLR) in the presence of rapamycin (Treg-Rapa) or cyclosporine A (Treg-CsA). Tregs were identified in MLR cultures by flow cytometry using anti-CD4, anti-CD25, anti-CTLA-4, anti-CD122, anti-GITR mAbs and ant-PE-FOXP3 staining sets. Suppressive capacity of induced Tregs was evaluated by their capability to inhibit anti-CD3 Ab-triggered proliferation of peripheral blood mononuclear cells (PBMCs), as measured by flow cytometry. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS Although both rapamycin and cyclosporine A suppressed the induction of CD4(+)CD25(+) Tregs during MLRs, this effect was significantly more pronounced in cells cultured with cyclosporine. On the other hand, only rapamycin significantly decreased the percentage of CD4(+)CD25(+) Tregs which expressed GITR, a negative regulator of Tregs suppressive capacity. Importantly, Treg-Rapa, unlike Treg-CsA, displayed significant suppressive activity and were capable of inhibiting the proliferation of anti-CD3 Ab-activated PBMCs. This activity was likely mediated by TGF-beta1. CONCLUSIONS Rapamycin, unlike cyclosporine A, does not inhibit the function of CD4(+)CD25(+) Tregs. This implies that rapamycin could contribute to the development of transplantation tolerance by promoting the induction of functional CD4(+)CD25(+) Tregs. Moreover, our results suggest that rapamycin could be combined with functional Tregs.

Phage Therapy: Combating Infections with Potential for Evolving from Merely a Treatment for Complications to Targeting Diseases.

Antimicrobial resistance is considered to be one of the greatest challenges of medicine and our civilization. Lack of progress in developing new anti-bacterial agents has greatly revived interest in using phage therapy to combat antibiotic-resistant infections. Although a number of clinical trials are underway and more are planned, the realistic perspective of registration of phage preparations and their entering the health market and significantly contributing to the current antimicrobial crisis is rather remote. Therefore, in addition to planning further clinical trials, our present approach of phage treatment carried out as experimental therapy (compassionate use) should be expanded to address the growing and urgent needs of increasing cohorts of patients for whom no alternative treatment is currently available. During the past 11 years of our phage therapy center’s operation, we have obtained relevant clinical and laboratory data which not only confirm the safety of the therapy but also provide important information shedding more light on many aspects of the therapy, contributing to its optimization and allowing for construction of the most appropriate clinical trials. New data on phage biology and interactions with the immune system suggest that in the future phage therapy may evolve from dealing with complications to targeting diseases. However, further studies are necessary to confirm this promising trend.

Is phage therapy acceptable in the immunocompromised host

Over the last decade, bacteriophages (bacterial viruses) have emerged as the major alternative to antibiotics in the treatment of antibiotic-resistant infections. While a considerable body of evidence has accumulated for the efficacy and safety of phage therapy in immunocompetent patients, data remain relatively scarce regarding its use in the immunocompromised host. To our knowledge, the present article is the first to summarize all findings, of both experimental and clinical studies, that may be relevant to the employment of phage therapy in immunocompromised patients. The available data suggest that bacteriophages could also be an efficacious and safe therapeutic modality in such patients.

Effects of prophylactic administration of bacteriophages to immunosuppressed mice infected with Staphylococcus aureus

BackgroundBacteriophages can be successfully applied to treat infections caused by antibiotic-resistant bacteria. Until now no attempts have been undertaken to treat infections in immunosuppressed patients with phages. In this work we investigated the prophylactic efficacy of specific bacteriophages in CBA mice treated with cyclophosphamide (CP) and infected with Staphylococcus aureus.ResultsHigh numbers of bacterial colony-forming units in the organs as well as elevated tumor necrosis factor and interleukin-6 serum concentrations in CP-treated and S. aureus-infected mice were significantly lowered upon application of phages. The phages markedly increased the percentage of circulating neutrophils and immature cells from the myelocytic and lymphocytic lineages in CP-treated, S. aureus-infected mice as well as of myelocytes and immature neutrophils in the bone marrow. In addition, phages stimulated in such mice generation of specific agglutinins against S. aureus.ConclusionApplication of specific phages to immunosuppressed mice prior to infection with S. aureus proved very effective, suggesting a potential benefit of phage therapy in immunocompromised patients experiencing bacterial infections.

Phages and immunomodulation

In the past years, the microbiome and its role in the pathophysiology of diseases have gained great interest. The progress of our knowledge in this field opens completely novel prospects for treating disorders, including those which are most challenging to medicine today. Of special interest are studies on the interactions of the microbiome with the immune system. Only recently has the presence of bacteriophages in the microbiome been highlighted, and their potential role in maintaining normal immunity has gained increasing attention. We summarize the available data pointing to the potential impact of phages in maintaining immunological homeostasis.

Potential of Bacteriophages and Their Lysins in the Treatment of MRSA

Bacteriophages (phages) are viruses that specifically infect and kill bacteria. Lysins are enzymes of bacteriophage origin that cleave covalent bonds in peptidoglycan, thereby inducing rapid lysis of a bacterial cell. As potential antibacterial agents, phages and lysins have some important features in common, especially the capacity to kill antibiotic-resistant bacteria, a narrow antibacterial range, and lack of toxic effects on mammalian cells. In this article we present the staphylococcal phages and their lysins that can be used to combat methicillin-resistant Staphylococcus aureus (MRSA), one of today’s most dangerous pathogens. We also discuss the use of phages as vectors specifically delivering different antibacterial agents to bacterial cells. Experimental data show that both phages and lysins could be effective in the treatment of MRSA.