Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

Phage Therapy in the Resistance Era: Where Do We Stand and Where Are We Going?

PURPOSE: Widespread antibiotic-resistant bacteria are threatening the arsenal of existing antibiotics. Not only are antibiotics less likely to be effective today, but their extensive use continues to drive the emergence of multidrug-resistant pathogens. A new-old antibacterial strategy with bacteriophages (phages) is under development, namely, phage therapy. Phages are targeted bacterial viruses with multiple antibacterial effector functions, which can reduce multidrug-resistant infections within the human body. This review summarizes recent phage therapy clinical trials and patient cases and outlines the fundamentals behind phage treatment strategies under development, mainly through bench-to-bedside approaches. We discuss the challenges that remain in phage therapy and the role of phages when combined with antibiotic therapy. METHODS: This narrative review presents the current knowledge and latest findings regarding phage therapy. Relevant case reports and research articles available through the Scopus and PubMed databases are discussed. FINDINGS: Although recent clinical data suggest the tolerability and, in some cases, efficacy of phage therapy, the clinical functionality still requires careful definition. The lack of well-controlled clinical trial data and complex regulatory frameworks have driven the most recent human data generation on a single-patient compassionate use basis. These cases often include the concomitant use of antibiotics, which makes it difficult to draw conclusions regarding the effectiveness of phages alone. However, human data support using antibiotics as phage potentiators and resistance breakers; thus, phage adjuvants are a promising avenue for near-term clinical development. Current knowledge gaps exist on the appropriate routes of administration, phage selection, frequency of administration, dosage, phage resistance, and pharmacokinetic and pharmacodynamic properties of the phages. In addition, we highlight that some phage therapies have mild adverse effects in patients. IMPLICATIONS: Although more translational research is needed before the clinical implementation is feasible, phage therapy may well be pivotal in safeguarding humans against antibiotic-resistant infections.

Standardized bacteriophage purification for personalized phage therapy.

The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward-by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are being treated on an expanded-access emergency investigational new drug basis. To streamline the production of high-quality and clinically safe phage preparations, we developed a systematic procedure for medicinal phage isolation, liter-scale cultivation, concentration and purification. The 16- to 21-day procedure described in this protocol uses a combination of modified classic techniques, modern membrane filtration processes and no organic solvents to yield on average 23 mL of 1011 plaque-forming units (PFUs) per milliliter for Pseudomonas, Klebsiella, and Serratia phages tested. Thus, a single production run can produce up to 64,000 treatment doses at 109 PFUs, which would be sufficient for most expanded-access phage therapy cases and potentially for clinical phase I/II applications. The protocol focuses on removing endotoxins early by conducting multiple low-speed centrifugations, microfiltration, and cross-flow ultrafiltration, which reduced endotoxins by up to 106-fold in phage preparations. Implementation of a standardized phage cultivation and purification across research laboratories participating in phage production for expanded-access phage therapy might be pivotal to reintroduce phage therapy to Western medicine.

Topical VEGF-C/D Inhibition Prevents Lymphatic Vessel Ingrowth into Cornea but Does Not Improve Corneal Graft Survival.

Vascular endothelial growth factor-C/D (VEGF-C/D) regulates lymphangiogenesis. Ingrowth of lymphatic vessels is negatively associated with corneal transplantation success. In this study, we therefore analyzed the effect local blockade of VEGF-C/D has on inflamed corneas. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation. Mice were treated with a VEGF-C/D trap prior to transplantation. Topical inhibition of VEGF-C/D significantly reduced lymphatic vessel ingrowth, but increased Macrophage numbers in the cornea. Furthermore, corneal transplantation success was not improved by the topical application of the compound. This study demonstrates that local VEGF-C/D inhibition is insufficient to increases corneal transplantation success, likely due to interaction with immune cells.

Immune reactions after modern lamellar (DALK, DSAEK, DMEK) versus conventional penetrating corneal transplantation.

In the past decade, novel lamellar keratoplasty techniques such as Deep Anterior Lamellar Keratoplasty (DALK) for anterior keratoplasty and Descemet stripping automated endothelial keratoplasty (DSAEK)/Descemet membrane endothelial keratoplasty (DMEK) for posterior keratoplasty have been developed. DALK eliminates the possibility of endothelial allograft rejection, which is the main reason for graft failure after penetrating keratoplasty (PK). Compared to PK, the risk of endothelial graft rejection is significantly reduced after DSAEK/DMEK. Thus, with modern lamellar techniques, the clinical problem of endothelial graft rejection seems to be nearly solved in the low-risk situation. However, even with lamellar grafts there are epithelial, subepithelial and stromal immune reactions in DALK and endothelial immune reactions in DSAEK/DMEK, and not all keratoplasties can be performed in a lamellar fashion. Therefore, endothelial graft rejection in PK is still highly relevant, especially in the "high-risk" setting, where the cornea's (lymph)angiogenic and immune privilege is lost due to severe inflammation and pathological neovascularization. For these eyes, currently available treatment options are still unsatisfactory. In this review, we will describe currently used keratoplasty techniques, namely PK, DALK, DSAEK, and DMEK. We will summarize their indications, provide surgical descriptions, and comment on their complications and outcomes. Furthermore, we will give an overview on corneal transplant immunology. A specific focus will be placed on endothelial graft rejection and we will report on its incidence, clinical presentation, and current/future treatment and prevention options. Finally, we will speculate how the field of keratoplasty and prevention of corneal allograft rejection will develop in the future.

ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions.

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is a cell adhesion molecule of the immunoglobulin superfamily and has been implicated in diverse pathophysiological processes including T cell activation, leukocyte trafficking, and (lymph)angiogenesis. However, exploring the therapeutic potential of ALCAM blockade in immune-mediated inflammatory disorders has been difficult due to the lack of antibodies with blocking activity toward murine ALCAM. In this study, we identified and characterized a monoclonal antibody with high affinity and specificity for murine ALCAM. This antibody reduced in vitro T cell activation induced by antigen-presenting dendritic cells (DCs) as well as (trans)migration of murine DCs across lymphatic endothelial monolayers. Moreover, it reduced emigration of DCs from in vitro-cultured human skin biopsies. Similarly, antibody-based blockade of ALCAM reduced (lymph)angiogenic processes in vitro and decreased developmental lymphangiogenesis in vivo to levels observed in ALCAM-deficient mice. Since corneal allograft rejection is an important medical condition that also involves (lymph)angiogenesis, DC migration and T cell activation, we investigated the therapeutic potential of ALCAM blockade in murine corneal disease. Blocking ALCAM lead to DC retention in corneas and effectively prevented corneal allograft rejection. Considering that we also detected ALCAM expression in human corneal DCs and lymphatics, our findings identify ALCAM as a potential novel therapeutic target in human corneal allograft rejection.

Local VEGF-A blockade modulates the microenvironment of the corneal graft bed.

The microenvironment plays an important role in several immunological processes. Vascular endothelial growth factor-A (VEGF-A) not only regulates angiogenesis, but is known as a modulator of the immune microenvironment. Modulating the site of transplantation might be beneficial for subsequent transplant survival. In this study, we therefore analyzed the effect that a local blockade of VEGF-A in the inflamed cornea as the graft receiving tissue has on the immune system. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation, which is an optimal model for local drug application. Mice were treated with VEGFR1/R2 trap prior to transplantation. We analyzed corneal gene expression, as well as protein levels in the cornea and serum on the day of transplantation, 2 and 8 weeks later. Local VEGF depletion prior to transplantation increases the expression of pro-inflammatory as well as immune regulatory cytokines only in the corneal microenvironment, but not in the serum. Furthermore, local VEGFR1/R2 trap treatment significantly inhibits the infiltration of CD11c+ dendritic cells into the cornea. Subsequent increased corneal transplantation success was accompanied by a local upregulation of Foxp3 gene expression. This study demonstrates that locally restricted VEGF depletion increases transplantation success by modulating the receiving corneal microenvironment and inducing tolerogenic mechanisms.