Intraperitoneal injection of class A TLR9 agonist enhances anti-PD-1 immunotherapy in colorectal peritoneal metastases.

Peritoneal metastases are associated with a low response rate to immune checkpoint blockade (ICB) therapy. The numbers of peritoneal resident macrophages (PRMs) are reversely correlated with the response rate to ICB therapy. We have previously shown that TLR9 in fibroblastic reticular cells (FRCs) plays a critical role in regulating peritoneal immune cell recruitment. However, the role of TLR9 in FRCs in regulating PRMs is unclear. Here, we demonstrated that the class A TLR9 agonist, ODN1585, markedly enhanced the efficacy of anti-PD-1 therapy in mouse models of colorectal peritoneal metastases. ODN1585 injected i.p. reduced the numbers of Tim4+ PRMs and enhanced CD8+ T cell antitumor immunity. Mechanistically, treatment of ODN1585 suppressed the expression of genes required for retinoid metabolism in FRCs, and this was associated with reduced expression of the PRM lineage-defining transcription factor GATA6. Selective deletion of TLR9 in FRCs diminished the benefit of ODN1585 in anti-PD-1 therapy in reducing peritoneal metastases. The crosstalk between PRMs and FRCs may be utilized to develop new strategies to improve the efficacy of ICB therapy for peritoneal metastases.

Biology and Metabolism of Sepsis: Innate Immunity, Bioenergetics, and Autophagy.

Sepsis is a complex, heterogeneous physiologic condition that represents a significant public health concern. While many insights into the pathophysiology of sepsis have been elucidated over the past decades of research, important questions remain. This article serves as a review of several important areas in sepsis research. Understanding the innate immune response has been at the forefront as of late, especially in the context of cytokine-directed therapeutic trials. Cellular bioenergetic changes provide insight into the development of organ dysfunction in sepsis. Autophagy and mitophagy perform crucial cell housekeeping and stress response functions. Finally, age-related changes and their potential impact on the septic response are reviewed.

Identification of pharmacological modulators of HMGB1-induced inflammatory response by cell-based screening.

High mobility group box 1 (HMGB1), a highly conserved, ubiquitous protein, is released into the circulation during sterile inflammation (e.g. arthritis, trauma) and circulatory shock. It participates in the pathogenesis of delayed inflammatory responses and organ dysfunction. While several molecules have been identified that modulate the release of HMGB1, less attention has been paid to identify pharmacological inhibitors of the downstream inflammatory processes elicited by HMGB1 (C23-C45 disulfide C106 thiol form). In the current study, a cell-based medium-throughput screening of a 5000+ compound focused library of clinical drugs and drug-like compounds was performed in murine RAW264.7 macrophages, in order to identify modulators of HMGB1-induced tumor-necrosis factor alpha (TNFα) production. Clinically used drugs that suppressed HMGB1-induced TNFα production included glucocorticoids, beta agonists, and the anti-HIV compound indinavir. A re-screen of the NIH clinical compound library identified beta-agonists and various intracellular cAMP enhancers as compounds that potentiate the inhibitory effect of glucocorticoids on HMGB1-induced TNFα production. The molecular pathways involved in this synergistic anti-inflammatory effect are related, at least in part, to inhibition of TNFα mRNA synthesis via a synergistic suppression of ERK/IκB activation. Inhibition of TNFα production by prednisolone+salbutamol pretreatment was also confirmed in vivo in mice subjected to HMGB1 injection; this effect was more pronounced than the effect of either of the agents administered separately. The current study unveils several drug-like modulators of HMGB1-mediated inflammatory responses and offers pharmacological directions for the therapeutic suppression of inflammatory responses in HMGB1-dependent diseases.

Preservation solution supplemented with biliverdin prevents lung cold ischaemia/reperfusion injury.

OBJECTIVES: Biliverdin (BV), one of the byproducts of heme catalysis through the heme oxygenase system, is a known scavenger of the reactive oxygen species. We hypothesized that adding BV to the perfusate and cold storage solution could protect rat lung grafts from oxidative injuries via its antioxidant efficacies. METHODS: Orthotopic left lung transplantation was performed in a syngenic Lewis-to-Lewis rat combination under 100% oxygen. Grafts were preserved in low-potassium dextran (LPD; Perfadex) at 4°C for 6 h with or without supplementation of 1 or 10 µM of BV into LPD. RESULTS: Prolonged cold storage and reperfusion resulted in a considerable deterioration of graft functions associated with massive apoptosis in the grafts after reperfusion. The untreated grafts exhibited the early up-regulations of mRNA for inflammatory mediators and an increase in a marker of lipid peroxidation, showing oxidative injuries. Although BV supplementation of LPD at a lower concentration (1 µM) did not improve the graft gas exchange, the grafts treated with BV (10 µM) showed a significant improvement of oxygenation and less inflammatory responses as well as reduced lipid peroxidation and apoptosis. Although the rapid activations of mitogen-activated protein kinases (MAPKs) were seen 30 min after reperfusion in the grafts stored in control LPD, BV treatment significantly reduced phosphorylated-MAPK protein expression. CONCLUSIONS: This study demonstrates that the exposure of the lung grafts to BV during cold storage can impart potent cytoprotective effects to lung cold ischaemia/reperfusion injury and significantly improve the lung graft function following extended cold preservation and transplantation by the mechanism of a reduction in oxidative injury and following inflammatory events.

Oral hydrogen water prevents chronic allograft nephropathy in rats.

Reactive oxygen species (ROS) contribute to the development of interstitial fibrosis and tubular atrophy seen in chronic allograft nephropathy (CAN). As molecular hydrogen gas can act as a scavenger of ROS, we tested the effect of treatment with hydrogen water (HW) in a model of kidney transplantation, in which allografts from Lewis rats were orthotopically transplanted into Brown Norway recipients that had undergone bilateral nephrectomy. Molecular hydrogen was dissolved in water and recipients were given HW from day 0 until day 150. Rats that were treated with regular water (RW) gradually developed proteinuria and their creatinine clearance declined, ultimately leading to graft failure secondary to CAN. In contrast, treatment with HW improved allograft function, slowed the progression of CAN, reduced oxidant injury and inflammatory mediator production, and improved overall survival. Inflammatory signaling pathways, such as mitogen-activated protein kinases, were less activated in renal allografts from HW-treated rats as compared with RW-treated rats. Hence, oral HW is an effective antioxidant and antiinflammatory agent that prevented CAN, improved survival of rat renal allografts, and may be of therapeutic value in the setting of transplantation.

The nitric oxide donor S-nitrosoglutathione reduces apoptotic primary liver cell loss in a three-dimensional perfusion bioreactor culture model developed for liver support.

INTRODUCTION: Artificial extracorporeal support for hepatic failure has met with limited clinical success. In hepatocytes, nitric oxide (NO) functions as an antiapoptotic modulator in response to a variety of stresses. We hypothesized that NO administration would yield improved viability and hepatocellular restructuring in a four-compartment, hollow fiber-based bioreactor with integral oxygenation for dynamic three-dimensional perfusion of hepatic cells in bioartificial liver support systems. METHODS: Isolated adult rat liver cells were placed in culture medium alone (control) or medium supplemented with various concentrations of an NO donor (S-nitrosoglutathione [GSNO]) in the bioreactors. Media samples were obtained from the cell perfusion circuit to monitor cellular response. After 24 and 72 h, histology biopsies were taken to investigate spontaneous restructuring of the cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to quantify apoptotic nuclei. RESULTS: Control bioreactors exhibited 47.9 +/- 2.9% (mean +/- standard error of the mean) apoptotic nuclei. In contrast, NO-treated bioreactors exhibited a biphasic response. Fewer apoptotic nuclei were seen in the 200 and 500 microM GSNO groups (14.4 +/- 0.4%). No effect was observed in the 10 microM GSNO group (47.3%), and increased TUNEL staining was observed in the 1000 microM GSNO group (82.6%). Media lactate dehydrogenase levels were lower in bioreactor groups treated with 200 or 500 microM GSNO (310 +/- 38 IU/L) compared with the control group (919 +/- 188 IU/L; p < 0.05). Protein synthesis was not affected, as measured by albumin levels in the media (115 +/- 19 microg/day/cell inoculum in GSNO-treated bioreactors at 24 h vs. 110 +/- 13 in controls; p = 0.851). Histologically, all of the bioreactor groups exhibited liver cell aggregates with some attached to the bioreactor capillaries. Increased numbers of cells in the aggregates and superior spontaneous restructuring of the cells were seen at 24 and 72 h in the bioreactor groups treated with either 200 or 500 microM GSNO compared with the control groups. CONCLUSION: Addition of an NO donor reduces adult rat liver cell apoptosis during the initial 24 h after cell inoculation within a three-dimensional perfusion bioreactor system for liver support and promotes liver cell aggregation and spontaneous restructuring of the cells at 24 and 72 h. GSNO-treated bioreactors remain metabolically active and show significantly lower levels of cellular injury as compared with controls. Further studies will be required to evaluate the impact of NO treatment of liver support bioreactors for clinical studies.

Parenteral calcium for intensive care unit patients.

BACKGROUND: Hypocalcemia is prevalent among critically ill patients requiring intensive care. Several epidemiological studies highlight a direct association between hypocalcemia and mortality. These data provide the impetus for current guidelines recommending parenteral calcium administration to normalize serum calcium. However, in light of the considerable variation in the threshold for calcium replacement, the lack of evidence to support a causal role of hypocalcemia in mortality, and animal studies illustrating that calcium supplementation may worsen outcomes, a systematic review is essential to evaluate whether or not the practice of calcium supplementation for intensive care unit (ICU) patients provides any benefit. OBJECTIVES: To assess the effects of parenteral calcium administration in ICU patients on the following outcomes: mortality, multiple organ dysfunction, ICU and hospital length of stay, costs, serum ionized calcium concentration, and complications of parenteral calcium administration. SEARCH STRATEGY: We searched The Cochrane Library, MEDLINE, EMBASE, Current Controlled Trials, and the National Research Register. We hand-searched conference abstracts from the Society of Critical Care Medicine, the European Society of Intensive Care Medicine, the American Thoracic Surgery, the American College of Surgeons, the American College of Chest Physicians, the American College of Physicians, and the International Consensus Conference in Intensive Care Medicine. We checked references of publications and attempted to contact authors to identify additional published or unpublished data. SELECTION CRITERIA: Randomised controlled and controlled clinical trials of ICU patients comparing parenteral calcium chloride or calcium gluconate administration with no treatment or placebo. DATA COLLECTION AND ANALYSIS: Two reviewers independently applied eligibility criteria to trial reports for inclusion and extracted data. MAIN RESULTS: There are no identifiable studies that have evaluated the association between parenteral calcium supplementation in critically ill ICU patients and the following outcomes: mortality, multiple organ dysfunction, ICU and hospital length of stay, costs, and complications of calcium administration. Serum ionized calcium concentration was reported in 5 studies (12 trial arms, 159 participants). These trials showed a small but significant increase in serum ionized calcium concentration after calcium administration. These trials showed considerable statistical heterogeneity and differed extensively in the population studied (adult versus neonate), the indication (hypocalcemia versus prophylaxis) and threshold of hypocalcemia for which parenteral calcium was administered, and the timing of subsequent measurement of serum ionized calcium concentration to the extent that we consider a pooled estimate almost inappropriate. AUTHORS' CONCLUSIONS: There is no clear evidence that parenteral calcium supplementation impacts the outcome of critically ill patients.

Blood soluble drag-reducing polymers prevent lethality from hemorrhagic shock in acute animal experiments.

Over the past several decades, blood-soluble drag reducing polymers (DRPs) have been shown to significantly enhance hemodynamics in various animal models when added to blood at nanomolar concentrations. In the present study, the effects of the DRPs on blood circulation were tested in anesthetized rats exposed to acute hemorrhagic shock. The animals were acutely resuscitated either with a 2.5% dextran solution (Control) or using the same solution containing 0.0005% or 5 parts per million (ppm) concentration of one of two blood soluble DRPs: high molecular weight (MW=3500 kDa) polyethylene glycol (PEG-3500) or a DRP extracted from Aloe vera (AVP). An additional group of animals was resuscitated with 0.0075% (75 ppm) polyethylene glycol of molecular weight of 200 kDa (PEG-200), which possesses no drag-reducing ability. All of the animals were observed for two hours following the initiation of fluid resuscitation or until they expired. We found that infusion of the DRP solutions significantly improved tissue perfusion, tissue oxygenation, and two-hour survival rate, the latter from 19% (Control) and 14% (PEG-200) to 100% (AVP) and 100% (PEG-3500). Furthermore, the Control and PEG-200 animals that survived required three times more fluid to maintain their blood pressure than the AVP and PEG-3500 animals. Several hypotheses regarding the mechanisms underlying these observed beneficial hemodynamic effects of DRPs are discussed. Our findings suggest that the drag-reducing polymers warrant further investigation as a potential clinical treatment for hemorrhagic shock and possibly other microcirculatory disorders.

The modulation of hepatic injury and heat shock expression by inhibition of inducible nitric oxide synthase after hemorrhagic shock.

The role of nitric oxide (NO) in maintaining homeostasis and regulating organ function during hemorrhagic shock is complex. The inducible NO synthase (iNOS) has been hypothesized to play a critical role in the pathophysiologic consequences of severe hemorrhage. Heat shock protein (HSP) expression is increased by hemorrhage and is a marker of the magnitude of ischemic injury in the liver. HSP induction is protective against injury in animal models of inflammation and is regulated by NO in hepatocytes. To clarify the role of iNOS in hepatic injury and its relationship to HSP expression in hemorrhagic shock, NOS was inhibited with L-N-6-(1-iminoethyl) lysine (L-NIL), which is reported to be a selective inhibitor of the inducible NOS isoform. Doses of 50 microg/kg or 150 microg/kg were infused over 1 h at the end of compensated shock. Plasma ornithine carbamoyltransferase (OCT), a specific marker of liver injury, was significantly reduced after hemorrhage with low-dose L-NIL (7.1+/-1.5 IU/L) compared to saline-treated control rats (13.0+/-1.5 IU/L, P < 0.005), while high-dose L-NIL significantly increased OCT release (35.9+/-7.2 IU/L, P< 0.05 versus shock alone) despite a greater MAP after resuscitation. HSP expression (HSP-72 and HSP-32) after hemorrhage was increased by L-NIL treatment at the highest dose. We conclude that excessive NO production from iNOS contributes to shock-induced hepatic injury. Our data suggest HSP expression may reflect the degree of ischemic injury after hemorrhage.