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.

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.

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.

Multipotent mesenchymal stem cells from human placenta: critical parameters for isolation and maintenance of stemness after isolation.

OBJECTIVE: This study was undertaken to isolate and characterize multipotent mesenchymal stem cells from term human placenta (placenta-derived mesenchymal stem cells, PD-MSCs). STUDY DESIGN: Sequential enzymatic digestion was used to isolate PD-MSCs in which trypsin removes the trophoblast layer, followed by collagenase treatment of remaining placental tissue. Karyotype, phenotype, growth kinetics, and differentiability of PD-MSC isolates from collagenase digests were analyzed. RESULTS: PD-MSC isolation was successful in 14 of 17 cases. Karyotyping of PD-MSC isolates from deliveries with a male fetus revealed that these cells are of maternal origin. Flow cytometry and immunocytochemistry confirmed the mesenchymal stem cell phenotype. Proliferation rates of PD-MSCs remained constantly high up to passage 20. These cells could be differentiated toward mesodermal lineage in vitro up to passage 20. Nonconfluent culture was critical to maintain the MSC stemness during long-term culture. CONCLUSION: Term placenta constitutes a rich, very reliable source of maternal mesenchymal stem cells that remain differentiable, even at high passage numbers.

Protocols for hematopoietic stem cell expansion from umbilical cord blood.

The reconstitution of adult stem cells may be a promising source for the regeneration of damaged tissues and for the reconstitution of organ dysfunction. However, there are two major limitations to the use of such cells: they are rare, and only a few types exist that can easily be isolated without harming the patient. The best studied and most widely used stem cells are of the hematopoietic lineage. Pioneering work on hematopoietic stem cell (HSC) transplantation was done in the early 1970s by ED. Thomas and colleagues. Since then HSCs have been used in allogenic and autologous transplantation settings to reconstitute blood formation after high-dose chemotherapy for various blood disorders. The cells can be easily harvested from donors, but the cell number is limited, especially when the HSCs originate from umbilical cord blood (UCB). It would be desirable to set up an ex vivo strategy to expand HSCs in order to overcome the cell dose limit, whereby the expansion would favor cell proliferation over cell differentiation. This review provides an overview of the various existing HSC expansion strategies-focusing particularly on stem cells derived from UCB-of the parameters that might affect the outcome, and of the difficulties that may occur when trying to expand such cells.

The magic behind stem cells.

This review article summarizes historical development of stem cell research, presents current knowledge on the plasticity potential of both embryonic and adult stem cells and discusses on the future of stem cell based therapies.

Embryonic stem cells: similarities and differences between human and murine embryonic stem cells.

The derivation of murine embryonic stem (mES) cell lines was reported for the first time in 1981 (Nature, 1981; 292:154-156; Proc Natl Acad Sci U S A, 1981; 78:7634-7638), and they have since proved to be a very useful tool with which to study mammalian development, which is characterized by pluripotency and differentiation. About 20 years later, the successful generation of human embryonic stem (hES) cell lines was described (Science, 1998; 282:1145-1147). Although mES and hES are derived from mammals, they cannot be looked at as being one and the same. While basic information for hES can be derived from mES, such information does not correspond on a one-to-one basis. This review gives an overview of the characteristics of embryonic stem cells with the main focus on the similarities and differences between human and mES cells.