Clinical translation of immunomodulatory therapeutics.

Immunomodulatory therapeutics represent a unique class of drug products that have tremendous potential to rebalance malfunctioning immune systems and are quickly becoming one of the fastest-growing areas in the pharmaceutical industry. For these drugs to become mainstream medicines, they must provide greater therapeutic benefit than the currently used treatments without causing severe toxicities. Immunomodulators, cell-based therapies, antibodies, and viral therapies have all achieved varying amounts of success in the treatment of cancers and/or autoimmune diseases. However, many challenges related to precision dosing, off-target effects, and manufacturing hurdles will need to be addressed before we see widespread adoption of these therapies in the clinic. This review provides a perspective on the progress of immunostimulatory and immunosuppressive therapies to date and discusses the opportunities and challenges for clinical translation of the next generation of immunomodulatory therapeutics.

Percutaneous Hepatic Perfusion With Filtered Melphalan for Localized Treatment of Metastatic Hepatic Disease: A Risk Assessment.

Regional therapies for metastatic liver disease have garnered interest in recent years due to technological advances in drug delivery. A percutaneous hepatic perfusion (PHP) using a newly developed generation 2 (GEN2) filtration system was designed to mitigate systemic toxicity and cardiovascular risk associated with hepatic blood filtration during hepatic artery infusion of the chemotherapy drug melphalan. The GEN2 system was evaluated in healthy swine, and plasma samples were assessed for clinical chemistry, melphalan toxicokinetics (TK), inflammatory cytokines, catecholamines, hematological, and cardiac biomarkers. Cardiovascular safety was assessed by echocardiography, electrocardiogram, and telemetry. Toxicology parameters included clinical signs, body weight, gross pathology, and histopathology. There were no treatment-related deaths associated with the PHP procedure with GEN2 filtration, and all animals survived to scheduled necropsy. Assessment of the pharmacokinetic/TK plasma concentrations of melphalan demonstrated that the GEN2 filter was able to extract melphalan from blood with high efficiency and reduce melphalan exposure in the systemic circulation. The hemodynamic, immunosuppressive, immunotoxic, cardiotoxic, and histopathologic effects of melphalan were limited. The significant hemodynamic challenge imposed by filtration resulted in a compensatory tachycardia with supranormal left ventricular function, although no wall motion abnormalities were detected and left ventricular function remained normal. Catecholamines decreased and then quickly rebounded during washout. Transient and reversible effects of treatment on cardiac enzymes, catecholamines, and cytokines and reversible hemodynamic effects without cardiac damage indicated that PHP with melphalan was not cardiotoxic or immunotoxic under the conditions tested, due to high efficiency of the filtration system limiting exposure of melphalan to the systemic circulation.

Mechanisms of ER stress-induced apoptosis in atherosclerosis.

Endoplasmic reticulum (ER) stress is triggered by perturbations in ER function such as those caused by protein misfolding or by increases in protein secretion. Eukaryotic cells respond to ER stress by activating 3 ER-resident proteins, activating transcription factor-6, inositol requiring protein-1, and protein kinase RNA-like ER kinase (PERK). These proteins direct signaling pathways that relieve ER stress in a process known as the unfolded protein response (UPR). In pathological settings, however, prolonged UPR activation can promote cell death, and this process has recently emerged as an important concept in atherosclerosis. We review here the evidence for UPR activation and cell death in macrophages, smooth muscle cells, and endothelial cells in the context of advanced atherosclerosis as well as the existing literature regarding mechanisms of UPR-induced cell death. Knowledge in this area may suggest new therapeutic targets relevant to the formation of clinically dangerous atherosclerotic plaques.

Poly-N-acetylglucosamine fibers amplify the effectiveness of recombinant factor VIIA on clot formation in hemophilia B canine blood.

BACKGROUND: Achieving hemostasis in anticoagulated patients is an increasingly important clinical issue. Poly-N-acetylglucosamine (pGlcNAc) nanofibers activate platelets by ß3 subunit (CD61) and the von Willebrand receptor GP1b (CD42b) integrin signaling for generation of a prothrombotic surface membrane. Recombinant coagulation factor VIIa (rFVIIa) functions in hemophilia A and B by catalyzing formation of the Xa/Va complex on the surface of activated platelets. These observations suggest that pGlcNAc nanofibers may amplify the activity of rFVIIa in hemophilic blood. METHODS: The activity of rFVIIa on platelets was tested by performing thromboelastographic analysis with blood from hemophilia B dogs in the presence of pGlcNAc nanofibers and increasing concentrations of rFVIIa. Mechanisms for hemostatic system activation were investigated with inhibitors of tissue factor, factor XIIa, and platelet function. RESULTS: Recombinant FVIIa was observed to partially restore the ability of the hemophiliac blood to form fibrin clots in a dose-dependent manner with thromboelastographic analysis. The addition of pGlcNAc nanofibers amplified the rFVIIa effect. The activity of rFVIIa and the amplification effect of pGlcNAc were dependent on platelet integrin function but independent of FXIIa and tissue factor activities. CONCLUSIONS: The pGlcNAc nanofibers amplify rFVIIa activity in hemophilia B canine blood by activating platelets through integrin-dependent mechanisms.

NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis.

Endoplasmic reticulum (ER)-induced apoptosis and oxidative stress contribute to several chronic disease processes, yet molecular and cellular mechanisms linking ER stress and oxidative stress in the setting of apoptosis are poorly understood and infrequently explored in vivo. In this paper, we focus on a previously elucidated ER stress-apoptosis pathway whose molecular components have been identified and documented to cause apoptosis in vivo. We now show that nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) and NOX-mediated oxidative stress are induced by this pathway and that apoptosis is blocked by both genetic deletion of the NOX subunit NOX2 and by the antioxidant N-acetylcysteine. Unexpectedly, NOX and oxidative stress further amplify CCAAT/enhancer binding protein homologous protein (CHOP) induction through activation of the double-stranded RNA-dependent protein kinase (PKR). In vivo, NOX2 deficiency protects ER-stressed mice from renal cell CHOP induction and apoptosis and prevents renal dysfunction. These data provide new insight into how ER stress, oxidative stress, and PKR activation can be integrated to induce apoptosis in a pathophysiologically relevant manner.

Macrophage pro-inflammatory cytokine secretion is enhanced following interaction with autologous platelets.

BACKGROUND: Macrophages are the dominant phagocyte at sites of wound healing and inflammation, and the cellular and acellular debris encountered by macrophages can have profound effects on their inflammatory profile. Following interaction with apoptotic cells, macrophages are known to switch to an anti-inflammatory phenotype. Activated platelets, however, are also a major component of inflammatory lesions and have been proposed to be pro-inflammatory mediators. In the present study, we tested the hypothesis that macrophage interaction with activated platelets results in an inflammatory response that differs from the response following phagocytosis of apoptotic cells. METHODS: Human monocyte-derived macrophages (hMDMs) were co-incubated with autologous activated platelets (AAPs) and the platelet-macrophage interaction was examined by electron microscopy and flow cytometry. The cytokines TNF-α, IL-6, and IL-23 were also measured during LPS-activated hMDM co-incubation with AAPs, which was compared to co-incubation with apoptotic lymphocytes. Cytokine secretion was also compared to platelets pre-treated with the gluococorticoid dexamethasone. RESULTS: Macrophages trapped and phagocytized AAPs utilizing a mechanism that was significantly inhibited by the scavenger receptor ligand fucoidan. LPS-induced macrophage secretion of TNF-α, IL-6, and IL-23 was inhibited by co-incubation with apoptotic cells, but enhanced by co-incubation with AAPs. The platelet-dependent enhancement of LPS-induced cytokines could be reversed by pre-loading the platelets with the glucocorticoid dexamethasone. CONCLUSIONS: The interaction of human macrophages with autologous platelets results in scavenger-receptor-mediated platelet uptake and enhancement of LPS-induced cytokines. Therefore, the presence of activated platelets at sites of inflammation may exacerbate pro-inflammatory macrophage activation. The possibility of reversing macrophage activation with dexamethasone-loaded platelets is a promising therapeutic approach to treating unresolved inflammation.

The design and testing of a dual fiber textile matrix for accelerating surface hemostasis.

The standard treatment for severe traumatic injury is frequently compression and application of gauze dressing to the site of hemorrhage. However, while able to rapidly absorb pools of shed blood, gauze fails to provide strong surface (topical) hemostasis. The result can be excess hemorrhage-related morbidity and mortality. We hypothesized that cost-effective materials (based on widespread availability of bulk fibers for other commercial uses) could be designed based on fundamental hemostatic principles to partially emulate the wicking properties of gauze while concurrently stimulating superior hemostasis. A panel of readily available textile fibers was screened for the ability to activate platelets and the intrinsic coagulation cascade in vitro. Type E continuous filament glass and a specialty rayon fiber were identified from the material panel as accelerators of hemostatic reactions and were custom woven to produce a dual fiber textile bandage. The glass component strongly activated platelets while the specialty rayon agglutinated red blood cells. In comparison with gauze in vitro, the dual fiber textile significantly enhanced the rate of thrombin generation, clot generation as measured by thromboelastography, adhesive protein adsorption and cellular attachment and activation. These results indicate that hemostatic textiles can be designed that mimic gauze in form but surpass gauze in ability to accelerate hemostatic reactions.

Non-classical processes in surface hemostasis: mechanisms for the poly-N-acetyl glucosamine-induced alteration of red blood cell morphology and surface prothrombogenicity.

It is well established that platelets and the intrinsic plasma coagulation pathway can be activated when blood contacts artificial surfaces. Experiments were performed to assess the effect of hemostatic poly-N-acetyl glucosamine (pGlcNAc) nanofibers on red blood cells. The pGlcNAc nanofibers, isolated from a marine diatom, interact with red blood cells (RBCs) to produce stomatocytes. The stomatocytes could be converted to echinocytes by treatment with echinocytic reagents, as measured by electron microscopy. Electrophoretic and Western blot analysis of RBC surface proteins demonstrated that pGlcNAc fibers were bound to band 3 of the RBC. An important and unique result of the interaction of RBCs with pGlcNAc fibers was the activation of the intrinsic coagulation cascade. This prothrombotic effect was associated with the presentation of phosphatidylserine on the outer layer of the surface membrane of nanofiber bound RBCs. The results demonstrate that RBCs can play a direct and important role in achieving surface hemostasis by accelerating the generation of thrombin, and add to the growing body of evidence that RBCs can strongly interact with hemostatic systems.