Salmonella enterica Serovar Typhimurium Increases Functional PD-L1 Synergistically with Gamma Interferon in Intestinal Epithelial Cells via Salmonella Pathogenicity Island 2.

Nontyphoidal serovars of Salmonella enterica are pathogenic bacteria that are common causes of food poisoning. Whereas Salmonella mechanisms of host cell invasion, inflammation, and pathogenesis are mostly well established, a new possible mechanism of immune evasion is being uncovered. Programmed death ligand 1 (PD-L1) is an immunosuppressive membrane protein that binds to activated T cells via their PD-1 receptor and thereby halts their activation. PD-L1 expression plays an essential role in the immunological tolerance of self-antigens but is also exploited for immune evasion by pathogen-infected cells and cancer cells. Here, we show for the first time that Salmonella infection of intestinal epithelial cells causes the induction of PD-L1. The increased expression of PD-L1 through Salmonella infection was seen in both human and rat intestinal epithelial cell lines. We determined that cellular invasion by the bacteria is necessary for PD-L1 induction, potentially indicating that Salmonella strains are delivering mediators from inside the host cell that trigger the increased PD-L1 expression. Using knockout mutants, we determined that this effect largely originates from the Salmonella pathogenicity island 2. We also show for the first time in any cell type that Salmonella combined with gamma interferon (IFN-γ) causes a synergistic induction of PD-L1. Finally, we show that Salmonella plus IFN-γ induction of PD-L1 decreased the cytokine production of activated T cells. Understanding Salmonella immune evasion strategies could generate new therapeutic targets and help to manipulate PD-L1 expression in other diseases.

[Galenic and analytic development of tacrolimus 0.06% eye drops]. / Développement galénique et analytique d'un collyre à base de tacrolimus 0,06 %

PURPOSE: The administration of topical tacrolimus (FK506) eye drops or ointment is effective in treating certain immunologic corneal diseases and in the prevention of rejection of high-risk corneal grafts. The purpose of this study is to determine the optimal formulation of tacrolimus 0.06% eye drops. A procedure for preparation is presented and discussed. METHODS: Tacrolimus monohydrate powder and virgin castor oil are used in this new formulation. The manufacturing process guarantees consistency of product sterility. Measurement by high-performance liquid chromatography allows precise control of the concentration of tacrolimus. RESULTS: The manufacture and packaging of tacrolimus 0.06% eye drops involve numerous controls allowing for guaranteed sterility and stability. The drops remained sterile and stabile for 28 days after opening regardless of storage conditions and can be stored for 3 months after manufacture. Tolerability studies are currently being performed.

A novel method for overexpression of peroxisome proliferator-activated receptor-γ in megakaryocyte and platelet microparticles achieves transcellular signaling.

BACKGROUND: Microparticles are submicrometer vesicles that contain RNA and protein derived from their parent cells. Platelet and megakaryocyte microparticles represent 80% of circulating microparticles, and their numbers are elevated in diseases such as cancer and type 2 diabetes. The ability of microparticles to transport protein, lipid and RNA to target cells, as a means of transcellular communication, remains poorly understood. Determining the influence that microparticles have on circulating cells is essential for understanding their role in health and in disease. OBJECTIVES: To develop a novel approach to modify the composition of platelet microparticles, and understand how such changes impact their transcellular communication. METHODS: This novel model utilizes a lentiviral technology to alter the transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) content of megakaryoblastic cell lines and primary megakaryocytes, and also the protein composition of generated platelets and microparticles. The subsequent microparticles were isolated and added to target cells for assessment of uptake and resultant signaling events. RESULTS: We successfully engineered microparticles to contain green fluorescent protein and elevated levels of PPARγ. We found that these altered microparticles could be internalized by the monocytic cell line THP-1 and primary human microvascular endothelial cells. Importantly, microparticle-delivered PPARγ was shown to increase the expression of fatty acid-binding protein 4 (FABP4), which is a known PPARγ target gene in THP-1 cells. CONCLUSION: This proof-of-concept modification of megakaryocyte, platelet and microparticle composition and subsequent change in target cell physiology is an important new tool to address transcellular communication of microparticles.

CD40 ligand (CD154) involvement in platelet transfusion reactions.

Platelet transfusions are commonly used treatments that occasionally lead to adverse reactions. Clinical trials, in vitro and animal studies have been performed to try to understand the causes of such reactions. Multiple studies have shown that the supernatant fraction of platelet concentrates contain prothrombotic and pro-inflammatory mediators. The origin of these mediators was first ascribed to white blood cells contaminating the platelet preparation. However, the accumulation of bioactive mediators after leukoreduction focused attention on platelets themselves during storage. Numerous cytokines, chemokines and prostaglandins are released in stored platelet concentrates. We have focused on a powerful mediator called soluble CD40 ligand (sCD40L, formally known as CD154) as a seminal contributor to adverse reactions. sCD40L can bind and signal the surface receptor, CD40, which is present on various types of human cells including white blood cells, vascular cells and fibroblasts. Downstream results of sCD40L/CD40 signaling include pro-inflammatory cytokine and chemokine production, prothrombotic mediator release, adherence and transmigration of leukocytes to endothelium and other undesirable vascular inflammatory events. Increased plasma levels of sCD40L can be detected in conditions such as myocardial infarction, stroke, unstable angina, high cholesterol, or other cardiovascular conditions. In retrospective studies, correlations were made between increased sCD40L levels of platelet concentrates and adverse transfusion reactions. We hypothesize that transfusion of partially activated, CD40L-expressing platelets along with sCD40L into a recipient with damaged or dysfunctional vascular tissue results in a "double-hit", thus inciting inflammation and vascular damage in the recipient.