Exogenous hydrogen sulfide gas does not induce hypothermia in normoxic mice

Hydrogen sulfide (H2S, 80 ppm) gas in an atmosphere of 17.5% oxygen reportedly induces suspended animation in mice; a state analogous to hibernation that entails hypothermia and hypometabolism. However, exogenous H2S in combination with 17.5% oxygen is able to induce hypoxia, which in itself is a trigger of hypometabolism/hypothermia. Using non-invasive thermographic imaging, we demonstrated that mice exposed to hypoxia (5% oxygen) reduce their body temperature to ambient temperature. In contrast, animals exposed to 80 ppm H2S under normoxic conditions did not exhibit a reduction in body temperature compared to normoxic controls. In conclusion, mice induce hypothermia in response to hypoxia but not H2S gas, which contradicts the reported findings and putative contentions

A Clinical Perspective on the Criteria for Liver Resection and the Use of Liver Function Tests

Ó The Author(s) 2009. This article is published with open access at Springerlink.com To the Editor, In a recently published survey of 100 liver centers, Breitenstein et al. [1] reported that on a global scale, (1) the average minimal remnant liver volume for resection is 25% (range = 15-40%) for normal liver parenchyma and 50% (range = 25–90%) for cirrhotic livers, (2) portal vein occlusion is employed in 89 % of the centers for purposes of augmenting liver volume before surgery, and that (3) 38 % of the centers employed liver function tests as part of their clinical routine, of which 76 % used the ICG clearance test. The interesting survey provoked a few issues that we feel obliged to address. The authors contend that ‘‘below a certain volume, a remnant liver cannot sustain metabolic, synthetic, and detoxifying functions’ ’ [1]—a statement that is unequivocal and uncontested. However, it should be born in mind that liver volume is not a directly proportional measure of liver function. We have demonstrated a few fundamental aspects of the volume-function relationship that support this notion: (i) Whereas liver function correlates with volume in uncompromised livers [2], there is significantly less correlation between liver volume an

The Role of Farnesoid X Receptor in Accelerated Liver Regeneration in Rats Subjected to ALPPS

Background: the role of bile acid (BA)-induced farnesoid X receptor (Fxr) signaling in liver regeneration following associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) was investigated in a rat model. Methods: Male Wistar rats underwent portal vein ligation (PVL) (n = 30) or ALPPS (n = 30). Animals were sacrificed pre-operatively and at 24, 48, 72, or 168 h after intervention. Regeneration rate, Ki67 index, hemodynamic changes in the hepatic circulation, and BA levels were assessed. Transcriptome analysis of molecular regulators involved in the Fxr signaling pathway, BA transport, and BA production was performed. Results: ALLPS induced more extensive liver regeneration (p < 0.001) and elevation of systemic and portal BA levels (p < 0.05) than PVL. The mRNA levels of proteins participating in hepatic Fxr signaling were comparable between the intervention groups. More profound activation of the intestinal Fxr pathway was observed 24 h after ALPPS compared to PVL. Conclusion: Our study elaborates on a possible linkage between BA-induced Fxr signaling and accelerated liver regeneration induced by ALPPS in rats. ALPPS could trigger liver regeneration via intestinal Fxr signaling cascades instead of hepatic Fxr signaling, thereby deviating from the mechanism of BA-mediated regeneration following one-stage hepatectomy

The Impact of Nanobody Density on the Targeting Efficiency of PEGylated Liposomes

Nanoparticles (NPs) are commonly modified with tumor-targeting moieties that recognize proteins overexpressed on the extracellular membrane to increase their specific interaction with target cells. Nanobodies (Nbs), the variable domain of heavy chain-only antibodies, are a robust targeting ligand due to their small size, superior stability, and strong binding affinity. For the clinical translation of targeted Nb-NPs, it is essential to understand how the number of Nbs per NP impacts the receptor recognition on cells. To study this, Nbs targeting the hepatocyte growth factor receptor (MET-Nbs) were conjugated to PEGylated liposomes at a density from 20 to 800 per liposome and their targeting efficiency was evaluated in vitro. MET-targeted liposomes (MET-TLs) associated more profoundly with MET-expressing cells than non-targeted liposomes (NTLs). MET-TLs with approximately 150-300 Nbs per liposome exhibited the highest association and specificity towards MET-expressing cells and retained their targeting capacity when pre-incubated with proteins from different sources. Furthermore, a MET-Nb density above 300 Nbs per liposome increased the interaction of MET-TLs with phagocytic cells by 2-fold in ex vivo human blood compared to NTLs. Overall, this study demonstrates that adjusting the MET-Nb density can increase the specificity of NPs towards their intended cellular target and reduce NP interaction with phagocytic cells

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion.

OBJECTIVE AND BACKGROUND: Activation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice. METHODS: Plasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury. RESULTS: In patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects. CONCLUSION: HMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage

New insight into the catalytic mechanism of bacterial MraY from enzyme kinetics and docking studies

Phospho-MurNAc-pentapeptide translocase (MraY) catalyzes the synthesis of Lipid I, a bacterial peptidoglycan precursor. As such, MraY is essential for bacterial survival and therefore is an ideal target for developing novel antibiotics. However, the understanding of its catalytic mechanism, despite the recently determined crystal structure, remains limited. In the present study, the kinetic properties of Bacillus subtilis MraY (BsMraY) were investigated by fluorescence enhancement using dansylated UDP-Mur-NAc-pentapeptide and heptaprenyl phosphate (C35-P, shortchain homolog of undecaprenyl phosphate, the endogenous substrate ofMraY)as second substrate. Varying the concentrations of both of these substrates and fitting the kinetics data to two-substrate models showed that the concomitant binding of both UDPMurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I andUMP.We built a model of BsMraY and performed docking studies with the substrate C35-P to further deepen our understanding of how MraY accommodates this lipid substrate. Based on these modeling studies, a novel catalytic role was put forward for a fully conserved histidine residue inMraY(His-289 in BsMraY), which has been experimentally confirmed to be essential for MraY activity. Using the current model of BsMraY, we propose that a small conformational change is necessary to relocate the His-289 residue, such that the translocase reaction can proceed via a nucleophilic attack of the phosphate moiety of C35-P on bound UDP-MurNAc-pentapeptide

An Overview of Three Promising Mechanical, Optical, and Biochemical Engineering Approaches to Improve Selective Photothermolysis of Refractory Port Wine Stains

During the last three decades, several laser systems, ancillary technologies, and treatment modalities have been developed for the treatment of port wine stains (PWSs). However, approximately half of the PWS patient population responds suboptimally to laser treatment. Consequently, novel treatment modalities and therapeutic techniques/strategies are required to improve PWS treatment efficacy. This overview therefore focuses on three distinct experimental approaches for the optimization of PWS laser treatment. The approaches are addressed from the perspective of mechanical engineering (the use of local hypobaric pressure to induce vasodilation in the laser-irradiated dermal microcirculation), optical engineering (laser-speckle imaging of post-treatment flow in laser-treated PWS skin), and biochemical engineering (light- and heat-activatable liposomal drug delivery systems to enhance the extent of post-irradiation vascular occlusion)

Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy

Background and aim: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. Methods: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. Results: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. Conclusions: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization

Evaluation of multi-exponential curve fitting analysis of oxygen-quenched phosphorescence decay traces for recovering microvascular oxygen tension histograms

Although it is generally accepted that oxygen-quenched phosphorescence decay traces can be analyzed using the exponential series method (ESM), its application until now has been limited to a few (patho)physiological studies, probably because the reliability of the recovered oxygen tension (pO2) histograms has never been extensively evaluated and lacks documentation. The aim of this study was, therefore, to evaluate the use of the ESM to adequately determine pO2 histograms from phosphorescence decay traces. For this purpose we simulated decay traces corresponding to uni- and bimodal pO2 distributions and recovered the pO2 histograms at different signal-to-noise ratios (SNRs). Ultimately, we recovered microvascular pO2 histograms measured in the rat kidney in a model of endotoxemic shock and fluid resuscitation and showed that the mean microvascular oxygen tension, 〈pO2〉, decreased after induction of endotoxemia and that after 2 h of fluid resuscitation, 〈pO2〉 remained low, but the hypoxic peak that had arisen during endotoxemia was reduced. This finding illustrates the importance of recovering pO2 histograms under (patho)physiological conditions. In conclusion, this study has characterized how noise affects the recovery of pO2 histograms using the ESM and documented the reliability of the ESM for recovering both low- and high-pO2 distributions for SNRs typically found in experiments. This study might therefore serve as a frame of reference for investigations focused on oxygen (re)distribution during health and disease and encourage researchers to (re-)analyze data obtained in (earlier) studies possibly revealing new insights into complex disease states and treatment strategies

Unaltered liver regeneration in post‐cholestatic rats treated with the fxr agonist obeticholic acid

In a previous study, obeticholic acid (OCA) increased liver growth before partial hepatectomy (PHx) in rats through the bile acid receptor farnesoid X‐receptor (FXR). In that model, OCA was administered during obstructive cholestasis. However, patients normally undergo PHx several days after biliary drainage. The effects of OCA on liver regeneration were therefore studied in post‐cholestatic Wistar rats. Rats underwent sham surgery or reversible bile duct ligation (rBDL), which was relieved after 7 days. PHx was performed one day after restoration of bile flow. Rats received 10 mg/kg OCA per day or were fed vehicle from restoration of bile flow until sacrifice 5 days after PHx. Liver regeneration was comparable between cholestatic and non‐cholestatic livers in PHx‐subjected rats, which paralleled liver regeneration a human validation cohort. OCA treatment induced ileal Fgf15 mRNA expression but did not enhance post‐PHx hepatocyte proliferation through FXR/SHP signaling. OCA treatment neither increased mitosis rates nor recovery of liver weight after PHx but accelerated liver regrowth in rats that had not been subjected to rBDL. OCA did not increase biliary injury. Conclusively, OCA does not induce liver regeneration in post‐cholestatic rats and does not exacerbate biliary damage that results from cholestasis. This study challenges the previously reported beneficial effects of OCA in li