Pharmacologic targeting of renal ischemia-reperfusion injury using a normothermic machine perfusion platform.

Normothermic machine perfusion (NMP) is an emerging modality for kidney preservation prior to transplantation. NMP may allow directed pharmacomodulation of renal ischemia-reperfusion injury (IRI) without the need for systemic donor/recipient therapies. Three proven anti-IRI agents not in widespread clinical use, CD47-blocking antibody (αCD47Ab), soluble complement receptor 1 (sCR1), and recombinant thrombomodulin (rTM), were compared in a murine model of kidney IRI. The most effective agent was then utilized in a custom NMP circuit for the treatment of isolated porcine kidneys, ascertaining the impact of the drug on perfusion and IRI-related parameters. αCD47Ab conferred the greatest protection against IRI in mice after 24 hours. αCD47Ab was therefore chosen as the candidate agent for addition to the NMP circuit. CD47 receptor binding was demonstrated by immunofluorescence. Renal perfusion/flow improved with CD47 blockade, with a corresponding reduction in oxidative stress and histologic damage compared to untreated NMP kidneys. Tubular and glomerular functional parameters were not significantly impacted by αCD47Ab treatment during NMP. In a murine renal IRI model, αCD47Ab was confirmed as a superior anti-IRI agent compared to therapies targeting other pathways. NMP enabled effective, direct delivery of this drug to porcine kidneys, although further efficacy needs to be proven in the transplantation setting.

Standardisation of flow cytometry for whole blood immunophenotyping of islet transplant and transplant clinical trial recipients.

Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Immunophenotyping of whole peripheral blood (WPB) by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. Healthy individuals and patients with type 1 diabetes (T1D) enrolled in islet transplantation were recruited and WPB was collected. 46 fluorochrome-conjugated mouse-anti-human antibodies were used (43 of 46 antibodies were titrated). BD cytometer setup and tracking beads were used to characterize and adjust for cytometer performance. Antibody cocktails were pre-mixed <60 minutes before staining. Multicolour panels were designed based on fluorochrome brightness, antigen density, co-expression, and fluorochrome spillover into non-primary detectors in each panel on a 5 laser flow cytometer. WPB sample staining used 50-300 µl WPB for each panel and was performed within 2 hours of blood sample collection. Samples were acquired on a BD-LSRFortessa. The operating procedures, including specimen collection, antibody cocktails, staining protocol, flow-cytometer setup and data analysis, were standardized. The staining index of 43 antibodies and the spillover spreading matrix for each panel was calculated. The final concentrations for the 46 antibodies used was determined for staining of WPB samples. Absolute cell-count and 7 leukocyte profiling panels consisting of subsets and/or status of granulocytes, monocytes, dendritic, B, NK, and T cells including regulatory T cells (Tregs) and NKT were designed and established on a 5 laser BD-LSR Fortessa. 13 T1D patients, including 4 islet transplant recipients and 8 healthy controls, were evaluated. The ability to reproducibly measure immune subsets and immune-profiles of islet transplant patients up to 18 months post transplantation has been established as a tool to measure immune cell reconstitution after transplantation.

From Ocean to Bedside: the Therapeutic Potential of Molluscan Hemocyanins.

Hemocyanins are large and versatile glycoproteins performing various immunological and biological functions in many marine invertebrates including arthropods and molluscs. This review discusses the various pharmacological applications of mollusc hemocyanin such as antiviral activity, immunostimulatory and anticancer properties that have been reported in the literature between the years 2000 and 2016. Emphasis is placed on a better mechanistic understanding of hemocyanin as a therapeutic agent. Elucidation of the mechanism of action is essential to improve the clinical efficacy and for a better understanding of some endogenous immunological functions of this complex glycoprotein.

Formulation of abalone hemocyanin with high antiviral activity and stability.

Hemocyanin has been shown to have potential antiviral activity against herpes simplex virus type-1. However, current liquid formulations have short shelf life and high risk of bacterial contamination. The aim of our study was to develop a stable functional formulation. Analytical techniques (nano-differential scanning calorimetry and spectroscopy) and biological assays (cytotoxicity and plaque reduction) were employed to measure the effect of sugar addition on the physical properties and shelf life of the solid formulated hemocyanin. Sucrose improved thermal stability significantly by both increasing the aggregation onset temperature (70°C to>78 °C) and enhancing the activation energy (18%). Lyophilisation without trehalose caused degradation and unfolding of the α-helices of hemocyanin. However, the addition of an optimal proportion of trehalose:protein (5:1 by weight) prevented the degradation and unfolding during lyophilisation, hence maintained the protein solubility. The estimated ED50 values of the formulated solid (0.43±0.1) and liquid samples (0.37±0.06) were similar in magnitude, and were significantly lower than the respective controls; thus, confirming enhanced antiviral activity of the formulation. Formulated compounds were stable for six months at 5 °C storage. The enhanced shelf life and stable antiviral activity of the formulation offers its significant potential as effective therapeutic agent in future clinical applications.