Ryszard Międzybrodzki

The influence of external factors on bacteriophages—review

The ability of bacteriophages to survive under unfavorable conditions is highly diversified. We summarize the influence of different external physical and chemical factors, such as temperature, acidity, and ions, on phage persistence. The relationships between a phage’s morphology and its survival abilities suggested by some authors are also discussed. A better understanding of the complex problem of phage sensitivity to external factors may be useful not only for those interested in pharmaceutical and agricultural applications of bacteriophages, but also for others working with phages.

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.

Transplantation of autologous olfactory ensheathing cells in complete human spinal cord injury.

Numerous studies in animals have shown the unique property of olfactory ensheathing cells to stimulate regeneration of lesioned axons in the spinal cord. In a Phase I clinical trial, we assessed the safety and feasibility of transplantation of autologous mucosal olfactory ensheathing cells and olfactory nerve fibroblasts in patients with complete spinal cord injury. Six patients with chronic thoracic paraplegia (American Spinal Injury Association class A-ASIA A) were enrolled for the study. Three patients were operated, and three served as a control group. The trial protocol consisted of pre- and postoperative neurorehabilitation, olfactory mucosal biopsy, culture of olfactory ensheathing cells, and intraspinal cell grafting. Patients clinical state was evaluated by clinical, neurophysiological, and radiological tests. There were no adverse findings related to olfactory mucosa biopsy or transplantation of olfactory ensheathing cells at 1 year after surgery. There was no evidence of neurological deterioration, neuropathic pain, neuroinfection, or tumorigenesis. In one cell-grafted patient, an asymptomatic syringomyelia was observed. Neurological improvement was observed only in transplant recipients. The first two operated patients improved from ASIA A to ASIA C and ASIA B. Diffusion tensor imaging showed restitution of continuity of some white matter tracts throughout the focus of spinal cord injury in these patients. The third operated patient, although remaining ASIA A, showed improved motor and sensory function of the first spinal cords segments below the level of injury. Neurophysiological examinations showed improvement in spinal cord transmission and activity of lower extremity muscles in surgically treated patients but not in patients receiving only neurorehabilitation. Observations at 1 year indicate that the obtaining, culture, and intraspinal transplantation of autologous olfactory ensheathing cells were safe and feasible. The significance of the neurological improvement in the transplant recipients and the extent to which the cell transplants contributed to it will require larger numbers of patients.

Functional regeneration of supraspinal connections in a patient with transected spinal cord following transplantation of bulbar olfactory ensheathing cells with peripheral nerve bridging.

Treatment of patients sustaining a complete spinal cord injury remains an unsolved clinical problem because of the lack of spontaneous regeneration of injured central axons. A 38-year-old man sustained traumatic transection of the thoracic spinal cord at upper vertebral level Th9. At 21 months after injury, the patient presented symptoms of a clinically complete spinal cord injury (American Spinal Injury Association class A-ASIA A). One of the patients olfactory bulbs was removed and used to derive a culture containing olfactory ensheathing cells and olfactory nerve fibroblasts. Following resection of the glial scar, the cultured cells were transplanted into the spinal cord stumps above and below the injury and the 8-mm gap bridged by four strips of autologous sural nerve. The patient underwent an intense pre- and postoperative neurorehabilitation program. No adverse effects were seen at 19 months postoperatively, and unexpectedly, the removal of the olfactory bulb did not lead to persistent unilateral anosmia. The patient improved from ASIA A to ASIA C. There was improved trunk stability, partial recovery of the voluntary movements of the lower extremities, and an increase of the muscle mass in the left thigh, as well as partial recovery of superficial and deep sensation. There was also some indication of improved visceral sensation and improved vascular autoregulation in the left lower limb. The pattern of recovery suggests functional regeneration of both efferent and afferent long-distance fibers. Imaging confirmed that the grafts had bridged the left side of the spinal cord, where the majority of the nerve grafts were implanted, and neurophysiological examinations confirmed the restitution of the integrity of the corticospinal tracts and the voluntary character of recorded muscle contractions. To our knowledge, this is the first clinical indication of the beneficial effects of transplanted autologous bulbar cells.

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.

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.

Effects of bacteriophages on free radical production and phagocytic functions

Reactive oxygen species (ROS) play a major role in mediating antibacterial functions of phagocytic cells. However, excessive ROS production may cause oxidative stress and tissue damage. Uncompensated ROS release has been implicated in a variety of disorders. Novel means of controlling elevated ROS production are urgently needed. We showed that homologous but not the heterologous phages inhibited, in a dose dependent manner, the degree of chemiluminescence in phagocytes induced by Escherichia coli. Treatment of the cells with the phages alone resulted in a small increase in ROS production. Homologous phages also facilitated phagocytosis when preincubated with bacteria. On the other hand, both homologous and heterologous phages inhibited phagocytosis following preincubation with phagocytic cells. The treatment of infected and uninfected mice with phages did not significantly alter the rate of phagocytosis by blood granulocytes and monocytes. In conclusion, we showed that bacteriophages can decrease ROS production by phagocytes. Although in some in vitro experimental models the phages tended to diminish phagocytosis, this phenomenon may be of little significance in clinical situations, since the process of eliminating bacteria in phage-treated patients is predominantly accomplished by both phages and phagocytes.

Eradication of Enterococcus faecalis by phage therapy in chronic bacterial prostatitis — case report

The treatment of three patients suffering from chronic bacterial prostatitis who were qualified for an experimental phage therapy protocol managed at the Phage Therapy Unit in Wrocław is described. They had previously been treated unsuccessfully with long-term targeted antibiotics, autovaccines, and laser biostimulation. Rectal application of phage lysates targeted against Enterococcus faecalis cultured from the prostatic fluid gave encouraging results regarding bacterial eradication, abatement of clinical symptoms of prostatitis, and lack of early disease recurrence.

The olfactory bulb and olfactory mucosa obtained from human cadaver donors as a source of olfactory ensheathing cells.

During the last decade, olfactory ensheathing cells (OECs) have been successfully applied in multiple experimental approaches aimed to repair damaged mammalian spinal cord. Some of these experiments have consequently been translated into clinical trials. Finding a reliable source of human OECs that is easily accessible and can ensure a sufficient number of cells is a major prerequisite for conducting studies on OEC‐mediated spinal cord regeneration. Here, we present a procedure for obtaining olfactory bulbs (OBs) and olfactory mucosa (OM) simultaneously from adult cadaver heart‐beating donors for OEC isolation and analyze some of the factors that may condition successful OEC culture. We show that the results of OEC culture from OBs (10 cases) correlated significantly with warm ischemia time (WIT) as well as the initial viability of the isolated cells. Efficient OEC culture was possible when the WIT for the OB was up to 20 min. Brain damage, assessed by determination of S100B serum level, was not related to the success of OEC culture from the OB. Cadaver OM (7 cases) was shown to be a more reliable source of human OECs than the OB. In most of the examined cases the efficacy of culturing OECs from cadaver OM obtained even 180 min after cardiac arrest was comparable to that of living patients. The method of obtaining OBs and OM from cadavers enables the use of an alternative source of primary adult human OECs for further preclinical and clinical studies on their neurotrophic properties.