[High-mobility group protein 1 promotes diethylnitrosamine-induced liver cancer formation in mice by activating mitochondrial biogenesis].

Objective: To study the role of high-mobility group protein 1 (HMGB1) in the promotion of diethylnitrosamine-induced liver cancer formation in C57BL/6 mice and its mechanism. Methods: HMGB1(loxp/loxp)/Alb-Cre(+/-) were used as a liver-specific knockout (KO) of HMGB1 gene in mice. HMGB1(loxp/loxp)/Alb-Cre(-/-), HMGB1(loxp/WT)/Alb-Cre(+/-) and HMGB1(loxp/WT)/Alb-Cre(-/-) born in the same litter were wild-type mice. Six 12-day-old male WT and KO mice were separated and given a single intraperitoneal injection of diethylnitrosamine (25 mg/kg). Six months later, HE staining was used to evaluate the histopathological changes and then the incidence of liver cancer in each mice group was calculated. Serum samples were taken from each mice group to determine alanine aminotransferase levels. Immunohistochemical staining was used to detect the expression and intracellular localizations of HMGB1 protein status in tumor tissue of the two groups of mice. Western blot was used to detect the expressional condition of mitochondrial biogenesis in tumor tissue of the two groups of mice. RT-PCR was used to detect mitochondrial DNA copy number of tumor tissue and normal liver tissue in the two groups of mice. Intra and inter group data comparison was compared using t-tests and one one-way analysis of variance. Results: Compared with WT mice, the liver/body weight ratio of KO mice was decreased significantly (t = 2.634, P = 0.0225). Serum alanine aminotransferase levels in both groups of mice were increased, and the difference was not statistically significant (t = 0.4062, P = 0.6932). There were many visible gray-white nodules of different sizes on the liver surface of WT mice, and the histological type was hepatocellular carcinoma. There was no statistically significant difference in the incidence of liver cancer among different genotypes of WT mice (P > 0.05). The incidence rate of liver cancer in KO mice was significantly reduced (t = 8.521, P < 0.001). Compared with WT mice, the expression levels of HMGB1 and mitochondrial biogenesis (PGC-1α and NRF1) was significantly reduced (t = 6.238, 4.852, P = 0.0335, 0.041) in tumor tissue of KO mice. Mitochondrial DNA copy number was decreased significantly (t = 9.211, P < 0.01). Mitochondrial DNA copy number in tumor tissue of WT mice was significantly higher than that in normal liver tissue (t = 8.305, P = 0.0142). Conclusion: HMGB1 promotes the formation of diethylnitrosamine-induced liver cancer by inducing mitochondrial biogenesis.

Propensity Score-Matched Analysis of Pure Laparoscopic Versus Hand-Assisted/Hybrid Major Hepatectomy at Two Western Centers.

BACKGROUND: Laparoscopic major hepatectomy is expanding, but little data exist comparing surgical approaches. The aim of this study was to test the hypothesis that pure laparoscopic liver resection (PLAP) has advantages over hand-assisted (HALS) or hybrid (HYB) resection for major hemi-hepatectomy at two western centers. METHODS: Using propensity score matching, 65 cases of HALS + HYB (18 hand-assisted and 47 hybrid) were matched to 65 cases of PLAP. Baseline characteristics were well matched for gender, age, ASA score, Childs A cirrhosis, right/left hepatectomy, malignancy, tumor size, and type between the groups. RESULTS: The HALS + HYB group had 27 right and 38 left major hepatectomies (n = 65) versus 29 right and 36 left (n = 65) in the PLAP group (p = NS). The median number of lesions resected was 1 in each group, with median size 5.6 cm (HALS + HYB) versus 6.0 cm (PLAP), (p = NS). The HALS + HYB group had shorter OR time (240 versus 330 min, p < 0.01), and less blood loss (EBL 150 ml vs. 300 ml, p < 0.01) versus the PLAP group, respectively. Median length of stay (LOS) was 4 days with HALS + HYB versus 5 days in the PLAP group (p = 0.02). There were no significant differences in use of the Pringle maneuver, transfusion rate, ICU stay, post-op morbidity, liver-specific complications, or R0 resection. Pain regimen/usage in each group is provided. There were no 30/90-day deaths in either group. CONCLUSION: This is the first reported series of propensity score matching of HALS + HYB versus PLAP for major hepatectomy. The HALS + HYB group had non-inferior OR time, blood loss, and LOS versus the PLAP group, while the other perioperative parameters were comparable. We conclude that minimally invasive liver resection with either PLAP or HALS + HYB technique yields excellent results.

High-mobility group box-1 in sterile inflammation.

High-mobility group box 1 (HMGB1) was originally defined as a ubiquitous nuclear protein, but it was later determined that the protein has different roles both inside and outside of cells. Nuclear HMGB1 regulates chromatin structure and gene transcription, whereas cytosolic HMGB1 is involved in inflammasome activation and autophagy. Extracellular HMGB1 has drawn attention because it can bind to related cell signalling transduction receptors, such as the receptor for advanced glycation end products, Toll-like receptor (TLR)2, TLR4 and TLR9. It also participates in the development and progression of a variety of diseases. HMGB1 is actively secreted by stimulation of the innate immune system, and it is passively released by ischaemia or cell injury. This review focuses on the important role of HMGB1 in the pathogenesis of acute and chronic sterile inflammatory conditions. Strategies that target HMGB1 have been shown to significantly decrease inflammation in several disease models of sterile inflammation, and this may represent a promising clinical approach for treatment of certain conditions associated with sterile inflammation.

Laparoscopic major hepatectomy: pure laparoscopic approach versus hand-assisted technique.

Laparoscopic liver resections are being performed with increasing frequency, with several groups having reported minimally invasive approaches for major anatomic hepatic resections. Some surgeons favor a pure laparoscopic approach, while others prefer a hand-assisted approach for major laparoscopic liver resections. There are clear advantages and disadvantages to a hand-assisted technique. The purpose of this study is to summarize the literature comparing pure laparoscopic and hand-assisted approaches for minimally invasive hepatic resection, and to describe our approach in 432 laparoscopic liver resections.

A current update on the evolution of robotic liver surgery.

Laparoscopic liver surgery has evolved significantly over the past decade. Increasing understanding of hepatic anatomy and advancements in technology have extended the scope of the minimally invasive approach. Robotic-assisted technology offers solutions to the fundamental limitations of conventional laparoscopic liver resection. Several centers have begun to utilize robotic technology to perform complex liver surgeries. The purpose of this review is to provide a comprehensive analysis of published literature about the role of robotic-assisted laparoscopic technology in liver surgery. A literature search of Pubmed was used to identify all English publications about robotic liver surgery. Publications were selected to examine all unique patient series. Outcomes analyzed included operative time, estimated blood loss, length of stay, complication rate, conversion rate to open, cost, and oncologic outcomes. A total of eight series containing 134 unique patients were selected for review. Sixty-nine percent of patients had malignant lesions resected, while 31% had benign lesions. Segmentectomy/wedge (36%) was the most common resection performed, followed by left lateral sectionectomy (28%) right hepatectomy (16%) and left hepatectomy (9%). A meta-analysis of the remaining data was not possible due to heterogeneity in methods for reporting. Outcomes varied widely between studies. Based on analysis of early published series, robotic liver surgery is a feasible and safe tool for the minimally invasive resection of hepatic lesions. Further evaluation is required to assess for improvement in outcomes, and long-term oncologic outcomes are still pending.

Safety of liver resection in the elderly: how important is age?

BACKGROUND: With the aging population, more elderly patients are being considered for hepatic resection. We investigated whether advanced age was associated with higher rate and severity of postoperative complications. METHODS: A total of 75 patients aged ≥70 years (group E) were matched with 75 patients aged <70 years (group Y) by the extent of liver resection and by operative indications. Primary outcome measures were rates and severity of complications. Secondary outcome measures were length of hospital stay and discharge destination. Univariate analysis was also performed to identify variables associated with higher surgical risk. RESULTS: Male-to-female ratio was 43:32 in both groups. Overall complication rates were 44 and 33.3% in group E and Y, respectively (P = 0.241; odds ratio = 1.57; 95% confidence interval [95% CI], 0.81-3.05). There was no mortality in both groups. The only postoperative age-related morbidity was confusion in the elderly. There was no difference in the rates of severe complications (grade ≥3) between group E and group Y (16 vs. 14.7%; P = 0.744; odds ratio = 1.11; 95% CI, 0.46-2.70). Median length of hospital stay were 7 and 6 days, respectively (P = 0.01). Nineteen percent and 1% of patients in group E and group Y were discharge to rehabilitation facilities, respectively (P = 0.001). Univariate analysis showed that preoperative systemic chemotherapy and longer operative time were associated with higher morbidity in the elderly. CONCLUSIONS: Liver resection can be performed in patients aged ≥70 years as safely as in younger patients. Duration and timing of systemic chemotherapy before liver resection should be optimized to minimize postoperative morbidity.

Adenovirus-mediated transfer of siRNA against MMP-2 mRNA results in impaired invasion and tumor-induced angiogenesis, induces apoptosis in vitro and inhibits tumor growth in vivo in glioblastoma.

Invasive tumors, including gliomas, utilize proteinases to degrade extracellular matrix components and diffuse into the adjacent tissues or migrate toward distant ones. In addition, proteinase activity is required for the formation of new blood vessels within the tumor. Levels of the proteinase matrix metalloproteinase-2 (MMP-2) are highly increased in gliomas. In this study, we examined the effect of the downregulation of MMP-2 via adenovirus-mediated siRNA in gliomas. Here, we show that siRNA delivery significantly decreased levels of MMP-2 in the glioblastoma cell lines U-87 and U-251. U-87 and U-251 cells showed impaired invasion through matrigel as well as decreased migration from tumor spheroids transfected with adenoviral vector expressing siRNA against MMP-2. Additionally, tumor-induced angiogenesis was decreased in in vitro experiments in cultured human microvascular endothelial cells (HMECs) in serum-free conditioned medium of glioblastoma cells transfected with these constructs and co-cultures of glioma cells with HMECs. We also observed decreased angiogenesis in the in vivo dorsal skin-fold chamber model. Moreover, MMP-2 inhibition induced apoptotic cell death in vitro, and suppressed tumor growth of preestablished U-251 intracranial xenografts in nude mice. Thus, specific targeting of MMP-2 may provide a novel, efficient approach for the treatment of gliomas and improve the poor outcomes of patients with these brain tumors.

HMGB1 preconditioning: therapeutic application for a danger signal?

High mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as a late mediator of lethality in sepsis and as an early mediator of inflammation following injury. In contrast to the proinflammatory role of HMGB1, recent evidence suggests beneficial applications of HMGB1 in injury states. One such application is the use of HMGB1 as a preconditioning stimulus. Preconditioning is a phenomenon whereby a low level of stressful stimuli confers protection against subsequent injury. Preconditioning has been demonstrated in multiple species, can be induced by various stimuli, and is applicable in different organ systems. Only with the recent introduction of the concept of endogenous molecules, such as HMGB1, as signals and mediators for inflammation during injury states has the use of endogenous molecules been investigated for this use. This review will focus on the use of endogenous molecules, specifically HMGB1, as a preconditioning stimulus and its mechanism of protection, as well as other protective applications for HMGB1.

Carbon monoxide protects rat lung transplants from ischemia-reperfusion injury via a mechanism involving p38 MAPK pathway.

Carbon monoxide (CO) provides protection against oxidative stress via anti-inflammatory and cytoprotective actions. In this study, we tested the hypothesis that a low concentration of exogenous (inhaled) CO would protect transplanted lung grafts from cold ischemia-reperfusion injury via a mechanism involving the mitogen-activated protein kinase (MAPK) signaling pathway. Lewis rats underwent orthotopic syngeneic or allogeneic left lung transplantation with 6 h of cold static preservation. Exposure of donors and recipients (1 h before and then continuously post-transplant) to 250 ppm CO resulted in significant improvement in gas exchange, reduced leukocyte sequestration, preservation of parenchymal and endothelial cell ultrastructure and reduced inflammation compared to animals exposed to air. The beneficial effects of CO were associated with p38 MAPK phosphorylation and were significantly prevented by treatment with a p38 MAPK inhibitor, suggesting that CO's efficacy is at least partially mediated by activation of p38 MAPK. Furthermore, CO markedly suppressed inflammatory events in the contralateral naïve lung. This study demonstrates that perioperative exposure of donors and recipients to CO at a low concentration can impart potent anti-inflammatory and cytoprotective effects in a clinically relevant model of lung transplantation and support further evaluation for potential clinical use.

Linking proximal and downstream signalling events in hepatic ischaemia/reperfusion injury.

Hepatic I/R (ischaemia/reperfusion) injury occurs in a variety of clinical settings including transplantation, elective liver resections and trauma. One of the challenges in studying the pathophysiology of I/R injury is the fact that the liver plays a central role in a variety of metabolic pathways in addition to governing aspects of immune surveillance and tolerance. The pathways activated in response to insults as varied as toxins, microbial and endogenous ligands and I/R may share common elements. The multiple intracellular signalling cascades involved in this process and the initiating events are still under investigation. Recent work on the role of TLRs (Toll-like receptors) in I/R injury has elucidated some of the more proximal signalling events in the pathway. In addition to the well-established role of signalling molecules such as NO (nitric oxide) in mediating damage or protection following hepatic I/R, more recent studies have focused on the participation of endogenous danger signals or DAMPs (damage-associated molecular patterns) such as HMGB1 (high-mobility group box 1). The complex interplay between HMGB1, TLRs and the many intracellular signalling molecules and pathways is illustrative of how our understanding of hepatic I/R injury is continually evolving.

Ex vivo application of carbon monoxide in University of Wisconsin solution to prevent intestinal cold ischemia/reperfusion injury.

Carbon monoxide (CO), a byproduct of heme catalysis, was shown to have potent cytoprotective and anti-inflammatory effects. In vivo recipient CO inhalation at low concentrations prevented ischemia/reperfusion (I/R) injury associated with small intestinal transplantation (SITx). This study examined whether ex vivo delivery of CO in University of Wisconsin (UW) solution could ameliorate intestinal I/R injury. Orthotopic syngenic SITx was performed in Lewis rats after 6 h cold preservation in control UW or UW that was bubbled with CO gas (0.1-5%) (CO-UW). Recipient survival with intestinal grafts preserved in 5%, but not 0.1%, CO-UW improved to 86.7% (13/15) from 53% (9/17) with control UW. At 3 h after SITx, grafts stored in 5% CO-UW showed improved intestinal barrier function, less mucosal denudation and reduced inflammatory mediator upregulation compared to those in control UW. Preservation in CO-UW associated with reduced vascular resistance (end preservation), increased graft cyclic guanosine monophosphate levels (1 h), and improved graft blood flow (1 h). Protective effects of CO-UW were reversed by ODQ, an inhibitor of soluble guanylyl cyclase. In vitro culture experiment also showed better preservation of vascular endothelial cells with CO-UW. The study suggests that ex vivo CO delivery into UW solution would be a simple and innovative therapeutic strategy to prevent transplant-induced I/R injury.

gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand.

Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation antigens. Their weak immunogenicity may be due to "self"-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6 mice with the rVV encoding the human gp100 elicited a specific CD8(+) T cell response. These lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2Db, that was recognized by the T cells. The ability to induce specific T cells with human but not mouse gp100 resulted from differences within the major histocompatibility complex (MHC) class I-restricted epitope and not from differences elsewhere in the molecule, as was evidenced by experiments in which mice were immunized with rVV containing minigenes encoding these epitopes. Although the human (hgp10025-33) and mouse (mgp10025-33) epitopes were homologous, differences in the three NH2-terminal AAs resulted in a 2-log increase in the ability of the human peptide to stabilize "empty" Db on RMA-S cells and a 3-log increase in its ability to trigger interferon gamma release by T cells. Thus, the fortuitous existence of a peptide homologue with significantly greater avidity for MHC class I resulted in the generation of self-reactive T cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation antigens expressed by tumors.

Participation of parasite surface glycoproteins in antibody-mediated protection of epithelial cells against Trichinella spiralis.

The L1 stage of the parasitic nematode Trichinella spiralis displays on its surface glycoproteins that are immunologically cross-reactive with several larval excretory-secretory (ES) products. The basis for the cross-reactivity is tyvelose, the terminal residue on the complex glycans shared by these surface and ES glycoproteins. In neonatal rats, tyvelose-specific monoclonal antibodies mediate the expulsion of larvae from the intestine. The aim of the studies described in this report was to determine how antibody binding to larval surfaces contributes to expulsion. In these experiments, which involve an in vitro assay of epithelial cell invasion, surface proteins on living larvae were biotinylated to distinguish them from ES products. Biotinylated and nonbiotinylated larvae were cocultured with avidin, biotin-specific antibodies, or anti-tyvelose monoclonal antibodies. Biotinylated larvae cultured with avidin or biotin-specific antibodies invaded Madin-Darby canine kidney (MDCK) cells equally as well as biotinylated larvae cultured with medium alone. Anti-tyvelose monoclonal antibodies were highly protective in this assay; however, biotinylation of larval surfaces hindered the ability of anti-tyvelose monoclonal antibodies to prevent larval invasion of epithelial cells. This correlated with a reduction in the binding of anti-tyvelose antibody to biotinylated larval surfaces. Our results indicate that antibody binding to surface glycoproteins contributes to protection against T. spiralis invasion but that surface binding alone is not sufficient for protection. Our findings support the notion that protection is effected by cross-linking of ES products to surface antigens.

In vivo murine tumor gene delivery and expression by systemic recombinant vaccinia virus encoding interleukin-1beta.

PURPOSE: This study investigates the feasibility of systemic gene delivery in a tumor-bearing host using a vaccinia virus-based in vivo gene delivery and expression system. METHODS: A recombinant vaccinia virus encoding human interleukin-1beta (hIL-1beta) was constructed with a strong synthetic vaccinia virus late promoter driving hIL-1beta gene expression. C57BL/6 mice bearing established subcutaneous pancreatic tumors were injected intravenously in a blinded, randomized fashion with different doses of either the recombinant vaccinia virus(vMJ601hIL-Ibeta), control vaccinia (wild-type or TK-deficient), or saline. Toxicity was assessed, serial tumor sizes were measured, and viral titers and the amount of hIL-1beta in tumor, liver, and spleen were determined. RESULTS: High viral titers (10(6) PFU/g) were detected in tumors for all three viruses on postinjection day 3, and tumor viral titers persisted at high levels until day 9. In contrast, viral titers were initially 104-fold lower in nontumor tissues and decreased to undetectable levels by day 9. vMJ60hIL-1beta was rapidly cleared from liver and spleen by day 3 (titer levels < 100 PFU/g), while tumor titer levels persisted at 8.5 x 10(6) PFU/g. hIL-1beta was measurable in three of three tumors from vMJ601hIL-1beta treated mice on postinjection day 3, one of three on day 6, and one of three on day 9; no hIL-1beta was detected in any other tumors or normal tissues. Wild-type vaccinia had no antitumor effects. Treatment with two different doses of vMJ601hIL-1beta resulted in a consistent and significant decrease in tumor size in repeatable experiments as compared to controls. Histologic analysis revealed tumor cell necrosis with a surrounding neutrophil infiltrate in the vMJ601hIL-1beta treated tumor. CONCLUSION: These data show that recombinant vaccinia virus encoding hIL-1beta given intravenously preferentially localizes and amplifies in tumor tissue, is rapidly cleared from liver and spleen, produces measurable hIL-1beta in tumor but not normal tissues, and inhibits growth of established solid tumors in mice. Recombinant vaccinia virus encoding therapeutic genes may be a practical, efficient vehicle for direct in vivo gene transfer and expression in the treatment of cancer.