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.

GATA6+ Peritoneal Resident Macrophage: The Immune Custodian in the Peritoneal Cavity.

Peritoneal resident macrophages (PRMs) have been a prominent topic in the research field of immunology due to their critical roles in immune surveillance in the peritoneal cavity. PRMs initially develop from embryonic progenitor cells and are replenished by bone marrow origin monocytes during inflammation and aging. Furthermore, PRMs have been shown to crosstalk with other cells in the peritoneal cavity to control the immune response during infection, injury, and tumorigenesis. With the advance in genetic studies, GATA-binding factor 6 (GATA6) has been identified as a lineage determining transcription factor of PRMs controlling the phenotypic and functional features of PRMs. Here, we review recent advances in the developmental origin, the phenotypic identity, and functions of PRMs, emphasizing the role of GATA6 in the pathobiology of PRMs in host defense, tissue repairing, and peritoneal tumorigenesis.

Neutrophils Extracellular Traps Inhibition Improves PD-1 Blockade Immunotherapy in Colorectal Cancer.

Immune checkpoint inhibitors can improve the prognosis of patients with advanced malignancy; however, only a small subset of advanced colorectal cancer patients in microsatellite-instability-high or mismatch-repair-deficient colorectal cancer can benefit from immunotherapy. Unfortunately, the mechanism behind this ineffectiveness is unclear. The tumor microenvironment plays a critical role in cancer immunity, and may contribute to the inhibition of immune checkpoint inhibitors and other novel immunotherapies in patients with advanced cancer. Herein, we demonstrate that the DNase I enzyme plays a pivotal role in the degradation of NETs, significantly dampening the resistance to anti-PD-1 blockade in a mouse colorectal cancer model by attenuating tumor growth. Remarkably, DNase I decreases tumor-associated neutrophils and the formation of MC38 tumor cell-induced neutrophil extracellular trap formation in vivo. Mechanistically, the inhibition of neutrophil extracellular traps with DNase I results in the reversal of anti-PD-1 blockade resistance through increasing CD8+ T cell infiltration and cytotoxicity. These findings signify a novel approach to targeting the tumor microenvironment using DNase I alone or in combination with immune checkpoint inhibitors.

Pre-operative exercise therapy triggers anti-inflammatory trained immunity of Kupffer cells through metabolic reprogramming.

Pre-operative exercise therapy improves outcomes for many patients who undergo surgery. Despite the well-known effects on tolerance to systemic perturbation, the mechanisms by which pre-operative exercise protects the organ that is operated on from inflammatory injury are unclear. Here, we show that four-week aerobic pre-operative exercise significantly attenuates liver injury and inflammation from ischaemia and reperfusion in mice. Remarkably, these beneficial effects last for seven more days after completing pre-operative exercising. We find that exercise specifically drives Kupffer cells toward an anti-inflammatory phenotype with trained immunity via metabolic reprogramming. Mechanistically, exercise-induced HMGB1 release enhances itaconate metabolism in the tricarboxylic acid cycle that impacts Kupffer cells in an NRF2-dependent manner. Therefore, these metabolites and cellular/molecular targets can be investigated as potential exercise-mimicking pharmaceutical candidates to protect against liver injury during surgery.

Maresin 1 protects the liver against ischemia/reperfusion injury via the ALXR/Akt signaling pathway.

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury can be a major complication following liver surgery contributing to post-operative liver dysfunction. Maresin 1 (MaR1), a pro-resolving lipid mediator, has been shown to suppress I/R injury. However, the mechanisms that account for the protective effects of MaR1 in I/R injury remain unknown. METHODS: WT (C57BL/6J) mice were subjected to partial hepatic warm ischemia for 60mins followed by reperfusion. Mice were treated with MaR1 (5-20 ng/mouse), Boc2 (Lipoxin A4 receptor antagonist), LY294002 (Akt inhibitor) or corresponding controls just prior to liver I/R or at the beginning of reperfusion. Blood and liver samples were collected at 6 h post-reperfusion. Serum aminotransferase, histopathologic changes, inflammatory cytokines, and oxidative stress were analyzed to evaluate liver injury. Signaling pathways were also investigated in vitro using primary mouse hepatocyte (HC) cultures to identify underlying mechanisms for MaR1 in liver I/R injury. RESULTS: MaR1 treatment significantly reduced ALT and AST levels, diminished necrotic areas, suppressed inflammatory responses, attenuated oxidative stress and decreased hepatocyte apoptosis in liver after I/R. Akt signaling was significantly increased in the MaR1-treated liver I/R group compared with controls. The protective effect of MaR1 was abrogated by pretreatment with Boc2, which together with MaR1-induced Akt activation. MaR1-mediated liver protection was reversed by inhibition of Akt. CONCLUSIONS: MaR1 protects the liver against hepatic I/R injury via an ALXR/Akt signaling pathway. MaR1 may represent a novel therapeutic agent to mitigate the detrimental effects of I/R-induced liver injury.

Mechanical Ventilation with Moderate Tidal Volume Exacerbates Extrapulmonary Sepsis-Induced Lung Injury via IL33-WISP1 Signaling Pathway.

ABSTRACT: IL-33 and WNT1-inducible secreted protein (WISP1) play central roles in acute lung injury (ALI) induced by mechanical ventilation with moderate tidal volume (MTV) in the setting of sepsis. Here, we sought to determine the inter-relationship between IL-33 and WISP1 and the associated signaling pathways in this process.We used a two-hit model of cecal ligation puncture (CLP) followed by MTV ventilation (4 h 10 mL/kg) in wild-type, IL-33-/- or ST2-/- mice or wild-type mice treated with intratracheal antibodies to WISP1. Macrophages (Raw 264.7 and alveolar macrophages from wild-type or ST2-/- mice) were used to identify specific signaling components.CLP + MTV resulted in ALI that was partially sensitive to genetic ablation of IL-33 or ST2 or antibody neutralization of WISP1. Genetic ablation of IL-33 or ST2 significantly prevented ALI after CLP + MTV and reduced levels of WISP1 in the circulation and bronchoalveolar lung fluid. rIL-33 increased WISP1 in alveolar macrophages in an ST2, PI3K/AKT, and ERK dependent manner. This WISP1 upregulation and WNT ß-catenin activation were sensitive to inhibition of the ß-catenin/TCF/CBP/P300 nuclear pathway.We show that IL-33 drives WISP1 upregulation and ALI during MTV in CLP sepsis. The identification of this relationship and the associated signaling pathways reveals a number of possible therapeutic targets to prevent ALI in ventilated sepsis patients.

A road map from single-cell transcriptome to patient classification for the immune response to trauma.

Immune dysfunction is an important factor driving mortality and adverse outcomes after trauma but remains poorly understood, especially at the cellular level. To deconvolute the trauma-induced immune response, we applied single-cell RNA sequencing to circulating and bone marrow mononuclear cells in injured mice and circulating mononuclear cells in trauma patients. In mice, the greatest changes in gene expression were seen in monocytes across both compartments. After systemic injury, the gene expression pattern of monocytes markedly deviated from steady state with corresponding changes in critical transcription factors, which can be traced back to myeloid progenitors. These changes were largely recapitulated in the human single-cell analysis. We generalized the major changes in human CD14+ monocytes into 6 signatures, which further defined 2 trauma patient subtypes (SG1 vs. SG2) identified in the whole-blood leukocyte transcriptome in the initial 12 hours after injury. Compared with SG2, SG1 patients exhibited delayed recovery, more severe organ dysfunction, and a higher incidence of infection and noninfectious complications. The 2 patient subtypes were also recapitulated in burn and sepsis patients, revealing a shared pattern of immune response across critical illness. Our data will be broadly useful to further explore the immune response to inflammatory diseases and critical illness.

Hepatocyte high-mobility group box 1 protects against steatosis and cellular stress during high fat diet feeding.

BACKGROUND: Circulating high-mobility group box 1 (HMGB1) plays important roles in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Intracellular HMGB1 is critical for the biology of hepatocytes. However, the intracellular role of HMGB1 in hepatocellular steatosis is unknown. Therefore, we aimed to investigate the role of hepatocyte-specific HMGB1 (HC-HMGB1) in development of hepatic steatosis. METHODS: Wild type (WT) C57BL/6 and HC-HMGB1-/- mice were fed high-fat diet (HFD) or low-fat diet (LFD) for up to 16 weeks. RESULTS: As expected, HMGB1 translocated from nuclear into cytoplasm and released into circulation after HFD treatment. HC-HMGB1 deficiency significantly reduced circulating HMGB1, suggesting that hepatocyte is a major source of circulating HMGB1 during NAFLD. Unexpectedly, HC-HMGB1 deficiency promoted rapid weight gain with enhanced hepatic fat deposition compared with WT at as early as 4 weeks after HFD treatment. Furthermore, there was no difference between WT and HC-HMGB1-/- mice in glucose tolerance, energy expenditure, liver damage or systemic inflammation. Interestingly, hepatic gene expression related to free fatty acid (FFA) ß-oxidation was significantly down-regulated in HC-HMGB1-/- mice compared with WT, and endoplasmic reticulum (ER) stress markers were significantly higher in livers of HC-HMGB1-/- mice. In vitro experiments using primary mouse hepatocytes showed absence of HMGB1 increased FFA-induced intracellular lipid accumulation, accompanied by increased ER-stress, significant downregulation of FFA ß-oxidation, and reduced oxidative phosphorylation. CONCLUSIONS: Our findings suggest that hepatocyte HMGB1 protects against dysregulated lipid metabolism via maintenance of ß-oxidation and prevention of ER stress. This represents a novel mechanism for HMGB1-regulation of hepatocellular steatosis, and suggests that stabilizing HMGB1 in hepatocytes may be effective strategies for prevention and treatment of NAFLD.

Immune-Responsive Gene 1/Itaconate Activates Nuclear Factor Erythroid 2-Related Factor 2 in Hepatocytes to Protect Against Liver Ischemia-Reperfusion Injury.

BACKGROUND AND AIMS: Itaconate, a metabolite of the tricarboxylic acid cycle, plays anti-inflammatory roles in macrophages during endotoxemia. The mechanisms underlying its anti-inflammatory roles have been shown to be mediated by the modulation of oxidative stress, an important mechanism of hepatic ischemia-reperfusion (I/R) injury. However, the role of itaconate in liver I/R injury is unknown. APPROACH AND RESULTS: We found that deletion of immune-responsive gene 1 (IRG1), encoding for the enzyme producing itaconate, exacerbated liver injury and systemic inflammation. Furthermore, bone marrow adoptive transfer experiments indicated that deletion of IRG1 in both hematopoietic and nonhematopoietic compartments contributes to the protection mediated by IRG1 after I/R. Interestingly, the expression of IRG1 was up-regulated in hepatocytes after I/R and hypoxia/reoxygenation-induced oxidative stress. Modulation of the IRG1 expression levels in hepatocytes regulated hepatocyte cell death. Importantly, addition of 4-octyl itaconate significantly improved liver injury and hepatocyte cell death after I/R. Furthermore, our data indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) is required for the protective effect of IRG1 on mouse and human hepatocytes against oxidative stress-induced injury. Our studies document the important role of IRG1 in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that the IRG1/itaconate pathway activates Nrf2-mediated antioxidative response in hepatocytes to protect liver from I/R injury. CONCLUSIONS: Our data expand on the importance of IRG1/itaconate in nonimmune cells and identify itaconate as a potential therapeutic strategy for this unfavorable postsurgical complication.

TSLP protects against liver I/R injury via activation of the PI3K/Akt pathway.

Thymic stromal lymphopoietin (TSLP) is a cytokine mainly released by epithelial cells that plays important roles in inflammation, autoimmune disease, and cancer. While TSLP is expressed in the liver at high levels, the role of TSLP in liver ischemia/reperfusion (I/R) injury remains unknown. Experiments were carried out to determine the role of TSLP in liver I/R injury. Wild-type (WT) and TSLP receptor-knockout (TSLPR-/-) mice were subjected to liver partial warm I/R injury. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) level, necrotic areas by liver histology, hepatocyte death, and local hepatic inflammatory responses. Signal pathways were explored in vivo and in vitro to identify possible mechanisms for TSLP in I/R injury. TSLP and TSLPR protein expression increased during liver I/R in vivo and following hepatocyte hypoxia/reoxygenation in vitro. Deletion of TSLPR or neutralization of TSLP with anti-TSLP antibody exacerbated liver injury in terms of serum ALT levels as well as necrotic areas in liver histology. Administration of exogenous recombinant mouse TSLP to WT mice significantly reduced liver damage compared with controls, but failed to prevent I/R injury in TSLPR-/- mice. TSLP induced autophagy in hepatocytes during liver I/R injury. Mechanistically, Akt was activated in WT mice during liver I/R injury. The opposite results were observed in TSLPR-/- mice. In addition, TSLP could directly induce Akt activation in hepatocytes independent of nonparenchymal cells in vitro. Furthermore, the Akt agonist, insulin-like growth factor-1 (IGF-1), prevented I/R injury in TSLPR-/- mice and an Akt inhibitor, LY294002, blocked the protective effects of TSLP in WT mice subjected to I/R. Our data indicate that TSLP protects against liver I/R injury via activation of the PI3K/Akt pathway. Through this pathway, TSLP induces autophagy in hepatocytes. Thus, TSLP is a potent inhibitor of stress-induced hepatocyte necrosis.

TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity.

Fibroblastic reticular cells (FRCs), a subpopulation of stromal cells in lymphoid organs and fat-associated lymphoid clusters (FALCs) in adipose tissue, play immune-regulatory roles in the host response to infection and may be useful as a form of cell therapy in sepsis. Here, we found an unexpected major role of TLR9 in controlling peritoneal immune cell recruitment and FALC formation at baseline and after sepsis induced by cecal ligation and puncture (CLP). TLR9 regulated peritoneal immunity via suppression of chemokine production by FRCs. Adoptive transfer of TLR9-deficient FRCs more effectively decreased mortality, bacterial load, and systemic inflammation after CLP than WT FRCs. Importantly, we found that activation of TLR9 signaling suppressed chemokine production by human adipose tissue-derived FRCs. Together, our results indicate that TLR9 plays critical roles in regulating peritoneal immunity via suppression of chemokine production by FRCs. These data form a knowledge basis upon which to design new therapeutic strategies to improve the therapeutic efficacy of FRC-based treatments for sepsis and immune dysregulation diseases.

Dichotomous Role of Plasmin in Regulation of Macrophage Function after Acetaminophen Overdose.

Kupffer cells and monocyte-derived macrophages are critical for liver repair after acetaminophen (APAP) overdose. These cells produce promitogenic cytokines and growth factors, and they phagocytose dead cell debris, a process that is critical for resolution of inflammation. The factors that regulate these dynamic functions of macrophages after APAP overdose, however, are not fully understood. We tested the hypothesis that the fibrinolytic enzyme, plasmin, is a key regulator of macrophage function after APAP-induced liver injury. In these studies, inhibition of plasmin in mice with tranexamic acid delayed up-regulation of proinflammatory cytokines after APAP overdose. In culture, plasmin directly, and in synergy with high-mobility group B1, stimulated Kupffer cells and bone marrow-derived macrophages to produce cytokines by a mechanism that required NF-κB. Inhibition of plasmin in vivo also prevented trafficking of monocyte-derived macrophages into necrotic lesions after APAP overdose. This prevented phagocytic removal of dead cells, prevented maturation of monocyte-derived macrophages into F4/80-expressing macrophages, and prevented termination of proinflammatory cytokine production. Our studies reveal further that phagocytosis is an important stimulus for cessation of proinflammatory cytokine production as treatment of proinflammatory, monocyte-derived macrophages, isolated from APAP-treated mice, with necrotic hepatocytes decreased expression of proinflammatory cytokines. Collectively, these studies demonstrate that plasmin is an important regulator of macrophage function after APAP overdose.

Location is the key to function: HMGB1 in sepsis and trauma-induced inflammation.

High mobility group box 1 (HMGB1) is a multifunctional nuclear protein, probably known best as a prototypical alarmin or damage-associated molecular pattern (DAMP) molecule when released from cells. However, HMGB1 has multiple functions that depend on its location in the nucleus, in the cytosol, or extracellularly after either active release from cells, or passive release upon lytic cell death. Movement of HMGB1 between cellular compartments is a dynamic process induced by a variety of cell stresses and disease processes, including sepsis, trauma, and hemorrhagic shock. Location of HMGB1 is intricately linked with its function and is regulated by a series of posttranslational modifications. HMGB1 function is also regulated by the redox status of critical cysteine residues within the protein, and is cell-type dependent. This review highlights some of the mechanisms that contribute to location and functions of HMGB1, and focuses on some recent insights on important intracellular effects of HMGB1 during sepsis and trauma.

The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis.

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

iNOS promotes CD24+CD133+ liver cancer stem cell phenotype through a TACE/ADAM17-dependent Notch signaling pathway.

The inducible nitric oxide synthase (iNOS) is associated with more aggressive solid tumors, including hepatocellular carcinoma (HCC). Notch signaling in cancer stem cells promotes cancer progression and requires Notch cleavage by ADAM (a disintegrin and metalloprotease) proteases. We hypothesized that iNOS/NO promotes Notch1 activation through TACE/ADAM17 activation in liver cancer stem cells (LCSCs), leading to a more aggressive cancer phenotype. Expression of the stem cell markers CD24 and CD133 in the tumors of patients with HCC was associated with greater iNOS expression and worse outcomes. The expression of iNOS in CD24+CD133+ LCSCs, but not CD24-CD133- LCSCs, promoted Notch1 signaling and stemness characteristics in vitro and in vivo, as well as accelerating HCC initiation and tumor formation in the mouse xenograft tumor model. iNOS/NO led to Notch1 signaling through a pathway involving the soluble guanylyl cyclase/cGMP/PKG-dependent activation of TACE/ADAM17 and up-regulation of iRhom2 in LCSCs. In patients with HCC, higher TACE/ADAM17 expression and Notch1 activation correlated with poor prognosis. These findings link iNOS to Notch1 signaling in CD24+CD133+ LCSCs through the activation of TACE/ADAM17 and identify a mechanism for how iNOS contributes to progression of CD24+CD133+ HCC.

cGAS-mediated autophagy protects the liver from ischemia-reperfusion injury independently of STING.

Liver ischemia-reperfusion (I/R) injury occurs through induction of oxidative stress and release of damage-associated molecular patterns (DAMPs), including cytosolic DNA released from dysfunctional mitochondria or from the nucleus. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor known to trigger stimulator of interferon genes (STING) and downstream type 1 interferon (IFN-I) pathways, which are pivotal innate immune system responses to pathogen. However, little is known about the role of cGAS/STING in liver I/R injury. We subjected C57BL/6 (WT), cGAS knockout (cGAS-/-), and STING-deficient (STINGgt/gt) mice to warm liver I/R injury and that found cGAS-/- mice had significantly increased liver injury compared with WT or STINGgt/gt mice, suggesting a protective effect of cGAS independent of STING. Liver I/R upregulated cGAS in vivo and also in vitro in hepatocytes subjected to anoxia/reoxygenation (A/R). We confirmed a previously published finding that hepatocytes do not express STING under normoxic conditions or after A/R. Hepatocytes and liver from cGAS-/- mice had increased cell death and reduced induction of autophagy under hypoxic conditions as well as increased apoptosis. Protection could be restored in cGAS-/- hepatocytes by overexpression of cGAS or by pretreatment of mice with autophagy inducer rapamycin. Our findings indicate a novel protective role for cGAS in the regulation of autophagy during liver I/R injury that occurs independently of STING. NEW & NOTEWORTHY Our studies are the first to document the important role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

Oestrogen sulfotransferase ablation sensitizes mice to sepsis.

Sepsis is the host's deleterious systemic inflammatory response to microbial infections. Here we report an essential role for the oestrogen sulfotransferase (EST or SULT1E1), a conjugating enzyme that sulfonates and deactivates estrogens, in sepsis response. Both the caecal ligation and puncture (CLP) and lipopolysaccharide models of sepsis induce the expression of EST and compromise the activity of oestrogen, an anti-inflammatory hormone. Surprisingly, EST ablation sensitizes mice to sepsis-induced death. Mechanistically, EST ablation attenuates sepsis-induced inflammatory responses due to compromised oestrogen deactivation, leading to increased sepsis lethality. In contrast, transgenic overexpression of EST promotes oestrogen deactivation and sensitizes mice to CLP-induced inflammatory response. The induction of EST by sepsis is NF-κB dependent and EST is a NF-κB-target gene. The reciprocal regulation of inflammation and EST may represent a yet-to-be-explored mechanism of endocrine regulation of inflammation, which has an impact on the clinical outcome of sepsis.

Shedding of the tumor necrosis factor (TNF) receptor from the surface of hepatocytes during sepsis limits inflammation through cGMP signaling.

Proteolytic cleavage of the tumor necrosis factor (TNF) receptor (TNFR) from the cell surface contributes to anti-inflammatory responses and may be beneficial in reducing the excessive inflammation associated with multiple organ failure and mortality during sepsis. Using a clinically relevant mouse model of polymicrobial abdominal sepsis, we found that the production of inducible nitric oxide synthase (iNOS) in hepatocytes led to the cyclic guanosine monophosphate (cGMP)-dependent activation of the protease TACE (TNF-converting enzyme) and the shedding of TNFR. Furthermore, treating mice with a cGMP analog after the induction of sepsis increased TNFR shedding and decreased systemic inflammation. Similarly, increasing the abundance of cGMP with a clinically approved phosphodiesterase 5 inhibitor (sildenafil) also decreased markers of systemic inflammation, protected against organ injury, and increased circulating amounts of TNFR1 in mice with sepsis. We further confirmed that a similar iNOS-cGMP-TACE pathway was required for TNFR1 shedding by human hepatocytes in response to the bacterial product lipopolysaccharide. Our data suggest that increasing the bioavailability of cGMP might be beneficial in ameliorating the inflammation associated with sepsis.

Limited correlation between conventional pathologist and automatic computer-assisted quantification of hepatic steatosis due to difference between event-based and surface-based analysis.

Computer-assisted automatic quantification (CAQ) was developed as an alternative method for the diagnosis of hepatic steatosis in order to compensate for observer-dependent bias. Here, we aim to demonstrate that CAQ can provide an accurate and precise result in analysis of fatty content, but that it is inappropriate to validate CAQ by comparison with conventional pathologist estimation (PE). Male rats were fed with a methionine-choline-deficient plus high-fat diet for three days, one week, or two weeks to induce mild, moderate, or severe steatosis. Samples were collected from all liver lobes. Severity of hepatic steatosis was assessed by an experienced pathologist who estimated the percentage of hepatocytes containing lipid droplets. Fatty content was quantified by PE, CAQ, and biochemical analysis (BA). CAQ, PE, and BA can correctly reflect severe fatty change. However, in the case of mild and moderate steatosis, PE could not reflect the true fatty content ( r between PE and BA was <0). The result of CAQ correlated well with that of BA among the various degrees of severity of hepatic steatosis. In conclusion, due to a difference between event-based and surface-based analysis, it is inappropriate to validate the CAQ of hepatic steatosis by comparison with PE.

Preventing intra-abdominal adhesions with a sodium hyaluronate carboxymethylcellulose membrane enabled visualization of hepatic microcirculation.

We aimed to evaluate whether using sodium hyaluronate carboxymethylcellulose membrane (Seprafilm™) can facilitate assessment of hepatic microcirculation via orthogonal polarization spectroscopy (OPS) by preventing intra-abdominal adhesions and whether Seprafilm™ as a foreign material can evoke local or systemic inflammatory reactions. After the right median hepatic vein was ligated, rats received either placement of Seprafilm™ or untreated with observation of 1 or 4 weeks (n = 6/group). Hepatic microcirculation was visualized. Systemic and local inflammatory reactions were evaluated by blood count, histology and immunohistochemical staining for CD68. Seprafilm™ significantly (P < 0.05) prevented intra-abdominal adhesion formation compared to non-Seprafilm™ groups (adhesion score: 0 vs 1.3 ± 0.5 at POW1 and 0.3 ± 0.5 vs 3.5 ± 1.4 at POW4). Placement of Seprafilm™ provided sufficient liver surface for acquisition of OPS videos during the harvest procedure. Adhesiolysis in non-Seprafilm™ groups prevented visualization of hepatic microcirculation. A severe local foreign body reaction with formation of a "fibrin-like" membrane containing CD68-positive inflammatory histiocytic cells and mesothelial cells was observed in Seprafilm™ groups even at POW4. Use of Seprafilm™ conferred visualization of hepatic microcirculation after long term observation in experimental setting. In clinical situation, we would suggest being very cautious in immuno-compromised patients because of an ongoing local foreign body reaction caused by Seprafilm™.