Immunohistochemical and radiological characterization of wound healing in porcine liver after radiofrequency ablation.

BACKGROUND: Radiofrequency ablation (RFA) is a minimal invasive therapeutic option for patients with hepatocellular carcinoma or liver metastases. We investigated RFA-induced cellular changes in the liver of pigs. MATERIAL AND METHODS: Healthy pigs (n=18) were sacrificed between day 0 and 3 months after RFA. The wound healing process was evaluated by computed tomography (CT), chromotrope anilinblue (CAB) staining of large-scale and standard tissue sections. Immunohistochemistry (IHC) for heat shock protein 70, Caspase-3, Ki67, Reelin, Vinculin, Vimentin and α-SMA was perfomed. RESULTS: One day after RFA, CAB staining showed cell damage and massive hyperaemia. All IHC markers were predominantly expressed at the outer borders of the lesion, except Reelin, which was mainly detected in untreated liver regions. By staining for Hsp70, the heat stress during RFA was monitored, which was most distinct 1-2 days after RFA. CT revealed decreased lesion size after one week. Development of a Vimentin and α-SMA positive fibrotic capsule was observed. CONCLUSION: In the early phase signs of cell damage, apoptosis and proliferation are dominant. Reduced expression of Reelin suggests a minor role of hepatic stellate cells in the RFA zone. After one week myofibroblasts become prominent and contribute to the development of the fibrotic capsule. This elucidates the pathophysiology of RFA and could contribute to the future optimization of RFA procedures.

Energy status of pig donor organs after ischemia is independent of donor type.

BACKGROUND: Literature is controversial whether organs from living donors have a better graft function than brain dead (BD) and non-heart-beating donor organs. Success of transplantation has been correlated with high-energy phosphate (HEP) contents of the graft. METHODS: HEP contents in heart, liver, kidney, and pancreas from living, BD, and donation after cardiac death in a pig model (n=6 per donor type) were evaluated systematically. BD was induced under general anesthesia by inflating a balloon in the epidural space. Ten hours after confirmation, organs were retrieved. Cardiac arrest was induced by 9V direct current. After 10min of ventricular fibrillation without cardiac output, mechanical and medical reanimation was performed for 30min before organ retrieval. In living donors, organs were explanted immediately. Freeze-clamped biopsies were taken before perfusion with Celsior solution (heart) or University of Wisconsin solution (abdominal organs) in BD and living donors or with Histidine-Tryptophan-Ketoglutaric solution (all organs) in non-heart-beating donors, after perfusion, and after cold ischemia (4h for heart, 6h for liver and pancreas, and 12h for kidney). HEPs (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and phosphocreatine), xanthine, and hypoxanthine were measured by high-performance liquid chromatography. Energy charge and adenosine triphosphate-to-adenosine diphosphate ratio were calculated. RESULTS: After ischemia, organs from different donor types showed no difference in energy status. In all organs, a decrease of HEP and an increase in hypoxanthine contents were observed during perfusion and ischemia, irrespective of the donor type. CONCLUSION: Organs from BD or non-heart-beating donors do not differ from living donor organs in their energy status after average tolerable ischemia.

Effect of oxidative stress and endotoxin on human serum albumin in brain-dead organ donors.

Albumin, among other molecules, binds and detoxifies endotoxin in healthy people. Oxidative stress leads to protein oxidation and thus to the impaired binding properties of albumin. This property, in combination with increased gut permeability, leads to the appearance of endotoxin in the systemic circulation and to impaired organ function. We hypothesize that these processes occur in the serum of brain-dead organ donors. Endotoxin was determined with an adapted Limulus amoebocyte lysate assay. The albumin fractions and binding capacity were determined by high-performance liquid chromatography (HPLC). FlowCytomix (eBioscience, San Diego, Calif) was used to determine the cytokine levels. Carbonylated proteins (CPs) and myeloperoxidase (MPO) were measured by an enzyme-linked immunosorbent assay (ELISA). Eighty-four brain-dead organ donors were enrolled and categorized by the duration of intensive care unit (ICU) stay. The albumin-binding capacity for dansylsarcosine was reduced in brain-dead patients compared with controls. Endotoxin positivity in 16.7% of donors was associated with decreased binding capacity in donors and worse survival of recipients. The CP and MPO levels of organ donors were significantly higher than in healthy controls. The durations of ICU stay increased albumin oxidation. In addition, interleukin-6 (IL-6), IL-8, IL-10, and IL-1ß levels were increased in patients, whereas the interferon-γ (IFN-γ) levels were within the normal range. We conclude that oxidative stress and systemic endotoxemia are present in brain-dead organ donors, which might affect recipient survival. High endotoxin levels might be caused by increased gut permeability and decreased binding capacity of albumin influenced not just by higher albumin oxidation.

Impact of temperature on cell death in a cell-culture model of hepatocellular carcinoma.

BACKGROUND: Radiofrequency ablation (RFA) is one treatment option for hepatocellular carcinoma (HCC) where tumour cells are destroyed by heat. However, there is lack of knowledge about cellular reactions after heating. Therefore, we studied cell death after heat application in a cell-culture setting mimicking HCC. MATERIALS AND METHODS: Intracellularly stained hepatic stellate cells (LX-1) and HCC cells (HepG2) were cultivated in co-culture or alone. Apoptosis was determined by flow cytometry using AnnexinV-PE and eFluor®450. RESULTS: Heating resulted in early apoptosis for 20-30% of HepG2 cells and 10-15% of LX-1 cells. Late apoptosis was observed in a large percentage of cells 24 h after heating at 65°C for 15 min or 75°C for 5 min; 65°C for 10 min resulted in a moderate increase and 55°C for 15 min resulted in a minor percentage of late apoptotic cells. CONCLUSION: Heat-treated LX-1 and HepG2 cells die by apoptosis. This finding is important for future planning tools to ameliorate RFA outcome in clinic.

Cell to cell interactions influence sensitivity of liver cell lines during hyperthermia.

BACKGROUND: Thermal cancer therapy is used for hepatocellular carcinoma treatment. In this study we investigated the effect of hyperthermia on liver cells and compared data of our different cell culture fibrosis models (transwell vs. co-culture model). MATERIALS AND METHODS: The cell lines HepG2 and LX-1 were seeded in different numbers in transwells to simulate different grades of fibrosis and then heated from 55°C to 85°C for different time spans. Thereafter, metabolic activity was measured. RESULTS: Heating at 65°C showed that the greater the number of LX-1 cells treated together with HepG2 cells the lower the metabolic activity of HepG2 cells was. Compared to our previous co-culture study, there were significantly different results in cell survival from 55°C to 75°C. CONCLUSION: The co-culture fibrosis model is more physiological than the transwell model because it allows a higher seeding density and a higher degree of cell to cell interactions. Therefore, it is more efficient for investigating the effect of hyperthermia on liver cells.

Effect of hyperthermia on liver cell lines: important findings for thermal therapy in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma is the fifth most common malignant tumour, with a high mortality rate. This study aimed to investigate the effect of hyperthermia on HepG2 and LX-1 cell lines to gain more information on thermal treatment of liver tumours. MATERIALS AND METHODS: The cell lines HepG2, LX-1 and their co-cultures were heated from 55°C to 85°C for different time spans. After heat exposure, metabolic activity was measured immediately, and after 24 h and 48 h using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) test to assess how many cells had survived heating. RESULTS: Our results show highly significant differences between the temperature tolerance of HepG2 and LX-1 cells. Alone, HepG2 cells are most sensitive to heat-induced cell death, their sensitivity decreased with rising percentages of LX-1 cells in the co-culture. CONCLUSION: Our results suggest that the outcome of thermal cancer therapy is dependent on the temperature and the grade of fibrosis in the treated livers.

A three-state mathematical model of hyperthermic cell death.

Thermal treatments for tissue ablation rely upon the heating of cells past a threshold beyond which the cells are considered destroyed, denatured, or killed. In this article, a novel three-state model for cell death is proposed where there exists a vulnerable state positioned between the alive and dead states used in a number of existing cell death models. Proposed rate coefficients include temperature dependence and the model is fitted to experimental data of heated co-cultures of hepatocytes and lung fibroblasts with very small RMS error. The experimental data utilized include further reductions in cell viabilities over 24 and 48 h post-heating and these data are used to extend the three-state model to account for slow cell death. For the two cell lines employed in the experimental data, the three parameters for fast cell death appear to be linearly increasing with % content of lung fibroblast, while the sparse nature of the data did not indicate any co-culture make-up dependence for the parameters for slow cell death. A critical post-heating cell viability threshold is proposed beyond which cells progress to death; and these results are of practical importance with potential for more accurate prediction of cell death.

Vessel segmentation for ablation treatment planning and simulation.

In this paper, a novel segmentation method for liver vasculature is presented, intended for numerical simulation of radio frequency ablation (RFA). The developed method is a semiautomatic hybrid based on multi-scale vessel enhancement combined with ridge-oriented region growing and skeleton-based postprocessing. In addition, an interactive tool for segmentation refinement was developed. Four instances of three-phase contrast enhanced computed tomography (CT) images of porcine liver were used in the evaluation. The results showed improved accuracy over common approaches and illustrated the method's suitability for simulation purposes.

Myeloperoxidase and carbonyl proteins: promising markers for non-invasive monitoring of graft rejection after heart transplantation.

BACKGROUND: After heart transplantation (HTx), endomyocardial biopsy (EMB) is currently the standard method to diagnose acute graft rejection. A non-invasive marker of rejection would be desirable as an alternative or to permit more selective use of the costly and invasive EMB. METHODS: In this retrospective study, outcomes of routinely taken EMBs were used to select 28 patients after HTx EMB Grade 0R (8 patients), 1R (9 patients) or 2R (11 patients). For these patients, myeloperoxidase (MPO) and carbonyl proteins (CP) in serum were measured using enzyme-linked immunoassay (ELISA). RESULTS: MPO and CP levels in post-HTx patients with Grade 2R rejection were significantly (MPO: p < 0.01; CP: p < 0.001) elevated at the time of rejection compared with levels 1 month earlier. MPO and CP levels predicted Grade 2R rejection and the best cut-off point was 237.5 µg/l for MPO and 222.5 pmol/mg for CP, respectively. Clinically most important was the marked increase (doubling of basic values within 1 month) of MPO and CP levels in cases of Grade 2R rejection in post-HTx patients. CONCLUSIONS: MPO and CP seem to be appropriate parameters to monitor rejection events non-invasively and to minimize the application of EMBs after HTx.

Innate immune dysfunction in acute and chronic liver disease.

Liver cirrhosis is a common disease causing great public-health concern because of the frequent complications requiring hospital care. Acute liver failure is also prone to several complications but is rare. One of the main complications for both acute and chronic liver diseases is infection, which regularly causes decompensation of cirrhosis, possibly leading to organ failure and death. This review focuses on innate immune function in cirrhosis, acute-on-chronic liver failure and acute liver failure. The known defects of Kupffer cells, neutrophils and monocytes are discussed, together with the pathophysiological importance of gut permeability, portal hypertension and intrinsic cellular defects, and the role of endotoxin, albumin, lipoproteins and toll-like receptors. Based on these different pathomechanisms, the available information on therapeutic strategies is presented. Antibiotic and probiotic treatment, nutritional support, artificial liver support, and experimental strategies such as inhibition of toll-like receptors and use of albumin and colony-stimulating factors are highlighted.

Differential expression of GHRH receptor and its splice variant 1 in human normal and malignant mucosa of the oesophagus and colon.

Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as a growth factor for gastrointestinal (GI) tumours. The tumourigenic effects of GHRH appear to be mediated by the splice variant 1 (SV-1) of GHRH receptor as well as the full length pituitary type receptor for GHRH (GHRH-R). We examined the protein and mRNA expression of GHRH-R and SV-1 in normal human tissues and tumours of the gastrointestinal (GI-) tract by immunohistochemical staining and reverse transcriptase (RT)-PCR. Squamous cells and squamous cell carcinoma of the oesophagus were negative for GHRH-R and SV-1, while Barrett's mucosa and adenocarcinomas of the oesophagus showed a strong expression of both receptors. The expression of GHRH-R was absent in normal colonic mucosa other than neuroendocrine cells (NE) and lining epithelium (LE) but strong in tubular adenomas of the colon, while the staining for SV-1 was absent in cells other than NE. However, the expression of both receptors was significantly increased in tubulovillous adenomas and colorectal cancers. No differences were seen in protein levels for both receptors between normal and neoplastic tissues of the stomach, pancreas and liver. Because of low mRNA levels for both receptors in all samples tested, only a qualitative assessment could be made. However, mRNA for GHRH-R and SV-1 showed a near-perfect correlation with the assessment of receptor proteins by immunostaining. Our study shows that in contrast to normal mucosa, transformed mucosa of the oesophagus and the colon expresses GHRH-R and SV-1. This aberrant expression of GHRH-R and SV-1 in oesophageal and colorectal malignancies may provide a molecular target for a therapeutic approach based on GHRH antagonists.