Delayed Fracture Healing in Diabetics with Distal Radius Fractures.

PURPOSE OF THE STUDY Diabetics may have an increased fracture risk, depending on disease duration, quality of metabolic adjustment and extent of comorbidities, and on an increased tendency to fall. The aim of this retrospective one-centre study consisted in detecting differences in fracture healing between patients with and without diabetes mellitus. Data of patients with the most common fracture among older patients were analyzed. MATERIAL AND METHODS Classification of distal radius fractures was established according to the AO classification. Inital assessment and followup were made by conventional x-rays with radiological default settings. To evaluate fracture healing, formation of callus and sclerotic border, assessment of the fracture gap, and evidence of consolidation signs were used. RESULTS The authors demonstrated that fracture morphology does not influence fracture healing regarding time span, neither concerning consolidation signs nor in fracture gap behavior. However, tendency for bone remodeling is around 70% lower in investigated diabetics than in non-diabetics, while probability for a successful fracture consolidation is 60% lower. CONCLUSIONS To corroborate the authors hypothesis of delayed fracture healing in patients with diabetes mellitus, prospective studies incorporating influencing factors like duration of metabolic disease, quality of diabetes control, medical diabetes treatment, comorbidities and secondary diseaseas, like chronic nephropathy and osteoporosis, have to be carried out. Key words: diabetes, delayed fracture healing, distal radius fractures, callus formation, blood glucose level, osteoblasts.

[Malnutrition in Elderly Trauma Patients - Comparison of Two Assessment Tools]. / Die Erfassung des Ernährungsstatus alterstraumatologischer Patienten ­ ein Vergleich zweier etablierter Screeningmethoden.

Background: The prevalence of malnutrition in hospitalised patients is reported to be between 16 and 55 % across disciplines. Within hospital care, screening for malnutrition is required. However, in orthopaedics and trauma surgery, there is still no generally accepted recommendation for the methods for such a data survey. In the present study, the following aspects are to be investigated with the help of two established scores: (1) the prevalence of malnutrition in the patient population of geriatric trauma care, and (2) the correlation between methods of data survey. Material and Methods: Between June 2014 and June 2015, a consecutive series of hospitalised trauma patients were studied prospectively with two validated screening instruments to record nutritional status. The study was carried out at a municipal trauma surgery hospital, which is a first level interregional trauma centre as well as a university hospital. The Nutritional Risk Screening (NRS) and the Mini Nutritional Assessment (MNA Short and Long Form) were used. All patients were divided into three age groups: < 65 years, 65-80 years, and > 80 years. The prevalence of malnutrition in geriatric trauma patients and the correlation between the screening instruments were determined. For a better comparison, prescreening and main assessment were applied to all patients. For statistical evaluation, both quantitative and semi-quantitative parameters were used. Furthermore, the Kolmogorov-Smirnov test, Spearman's correlation analysis and the chi-square test were applied. These tests were two-sided and had a level of significance of 5 %. The present study was partially funded by the Oskar-Helene-Heim Foundation. Results: 521 patients (43.8 % women, 56.2 % men), with a mean age of 53.96 ± 18.13 years, were statistically evaluated within the present study. Depending on the method of the data survey, malnutrition (NRS≥3) in geriatric trauma patients varied from 31.3 % (65-80 years) to 60 % (> 80 years). With MNA, 28.8 and 54.3 % of patients were at risk of malnutrition (MNA 17-23.5), while the fractions of patients already suffering from malnutrition (MNA < 17) were 5.4 and 8.6 %, respectively. The correlation between the NRS and MNA total scores increases with the age of the patients. The correlation coefficient for patients under 65 years is r = - 0.380, while among patients aged between 65 and 80, it is r = - 0.481, and for patients over 80 years, there is a medium to strong correlation of r = - 0.638 (each with a Spearman correlation of p < 0.001). For the total population as well as the different age groups, statistically significant correlations were recorded between the categorised scores (chi-square test for linear trend, p < 0.001). Summary: The present study demonstrates high prevalence of malnutrition among the geriatric trauma patients. Because of its easy and rapid application, the NRS has an advantage in clinical use. It was shown that the two methods of data survey were highly correlated.

[Retrospective Analysis of Diabetics with Regard to Treatment Duration and Costs]. / Retrospektive Analyse von Diabetikern im Hinblick auf Behandlungsdauer und Behandlungskosten in einem überregionalen Traumazentrum.

Background: The increasing incidence of diabetes mellitus is also reflected in the patient population of a trauma and orthopaedic centre. Diabetics also exhibit more comorbidities than non-diabetics. In addition to surgical problems in these patients, hospitalisation is often accompanied by complications, which can prolong treatment and increase costs. The aim of this retrospective study is to analyse hospitalisation of diabetics compared to non-diabetics, as well as differences in treatment costs, depending on associated age and comorbidities. Patients/Material and Methods: 17,185 patients were treated at a transregional trauma and orthopaedic centre and were included in this retrospective analysis between 2012 and 2015. Comorbidities and hospitalisation of diabetics and non-diabetics were recorded. All costs charged by DRG were evaluated to calculate the cost per day and per patient, on the basis of the specific case rate. In this calculation, patient-related case rates were divided by the average residence time and the means of the calculated daily rates were calculated. Inclusion criteria were treatment within the various departments and a minimum hospitalisation of one day. Statistical analysis was performed with the SPSS program (version 22.0, SPSS Inc., Chicago, USA). Results: In comparison to non-diabetics (ND), diabetics (D) exhibited significantly more comorbidities, including: obesity, arterial hypertension, coronary heart disease, myocardial infarction (in the history), peripheral arterial disease, chronic kidney disease and hyperlipidaemia. Pneumonia in hospital was considerably commoner in diabetics (2.45 % [D] vs. 1.02 % [ND], p < 0.001). Time in hospital was significantly longer in diabetics (endoprosthetics 13.52 days [D] vs. 12.54 days [ND], p < 0.001; septic surgery 18.62 days [D] vs. 16.31 days [ND], p = 0.007; traumatology 9.82 days [D] vs. 7.07 days [ND], p < 0.001). For patients aged under 60 years, time in hospital was significantly longer for diabetics than for non-diabetics (9.98 days [D] vs. 6.43 days [ND] p < 0.001). Because of the longer time in hospital, treatment costs were higher by € 1,932,929.42 during the investigated time period. Conclusion: Because of their comorbidities, diabetics need to be categorised at an early stage as high-risk patients in traumatological and orthopaedic departments. Hospitalisation and the associated increased treatment costs, as well as postoperative complications, could be minimised in patients with diabetes by implementing an interdisciplinary treatment concept.

Delayed Fracture Healing in Diabetics with Distal Radius Fractures.

PURPOSE OF THE STUDY: Diabetics may have an increased fracture risk, depending on disease duration, quality of metabolic adjustment and extent of comorbidities, and on an increased tendency to fall. The aim of this retrospective one-centre study consisted in detecting differences in fracture healing between patients with and without diabetes mellitus. Data of patients with the most common fracture among older patients were analyzed. MATERIAL AND METHODS: Classification of distal radius fractures was established according to the AO classification. Inital assessment and follow-up were made by conventional X-rays with radiological default settings. To evaluate fracture healing, formation of callus and sclerotic border, assessment of the fracture gap, and evidence of consolidation signs were used. RESULTS: The authors demonstrated that fracture morphology does not influence fracture healing regarding time span, neither concerning consolidation signs nor in fracture gap behaviour. However, tendency for bone remodeling is around 70% lower in investigated diabetics than in non-diabetics, while probability for a successful fracture consolidation is 60% lower. CONCLUSIONS: To corroborate the authors hypothesis of delayed fracture healing in patients with diabetes mellitus, prospective studies incorporating influencing factors like duration of metabolic disease, quality of diabetes control, medical diabetes treatment, comorbidities and secondary diseases, like chronic nephropathy and osteoporosis, have to be carried out.

[Stem cell-based therapy in orthopaedics and trauma surgery - current concepts]. / Aktueller Stand der Stammzelltherapie in Orthopädie und Unfallchirurgie.

Over the last few years, numerous new treatment methods have been developed for musculoskeletal diseases. Some of these new methods are based on the targeted use of stem cells to initiate healing processes, to compensate for deficits or to activate the regeneration of tendons, muscles, bones and cartilage. This goal can be achieved through the direct use of stem cells on or in a carrier material or through a combination with tissue engineering. In this article, we give a short overview of the possible fields of application of inducible pluripotent haematopoietic, and adult stem cells as well as on their use in musculoskeletal tissue. Furthermore, we provide a summary of the current legal situation concerning the application of stem cells in humans.

[Use of Bone Morphogenetic Proteins (BMPs) for the Treatment of Pseudarthroses - Efficiency and Therapy Failure]. / Einsatz der Bone morphogenetic Proteins (BMPs) zur Behandlung von Pseudarthrosen - Effizienz und Therapieversagen.

In the therapy for pseudarthroses of the proximal tibia, the human recombinant bone morphogenetic proteins (BMP-2 and BMP-7) have been used for several years. Despite their limited and specified use as local mediators of bone healing, no conclusions regarding the therapeutic success can be made beforehand. The regulatory mechanisms have turned out to be much more complex and patient-specific than had been assumed before. To help understand the cell biological processes (signalling) and the current possibilities of predicting a successful use of BMP, this article summarises the relevant findings.

Gene expression changes in cancellous bone of type 2 diabetics: a biomolecular basis for diabetic bone disease.

PURPOSE: Diabetes mellitus type 2 (2DM) is associated with altered bone quality. In order to analyze associated changes on a molecular level, we investigated the gene expression of key factors of osteoblast metabolism in type 2 diabetics. METHODS: Total mRNA and protein of bone samples from 2DM patients and non-diabetic patients were isolated, and subsequently, reverse transcription polymerase chain reaction (RT-PCR) or Western blot was performed. Furthermore, pro- and anti-inflammatory serum cytokine levels were determined using a cytokine array. RESULTS: Expression of runt-related transcription factor 2 (RUNX2) was increased by 53 %. Expression of the bone sialoproteins, secreted phosphoprotein 1 (SPP1; osteopontin), and integrin-binding sialoprotein (IBSP), was elevated by more than 50 %, and activating transcription factor 4 (ATF4) expression was 13 % lower in the investigated diabetes group compared to the control group. Similarly, the expression of versican (VCAN) and decorin (DCN) was upregulated twofold in the diabetic group. At the same time, 2DM patients and controls show alterations in pro- and anti-inflammatory cytokine levels in the serum. CONCLUSIONS: This study identifies considerable changes in the expression of transcription factors and extracellular matrix (ECM) components of bone in 2DM patients. Furthermore, the analysis of key differentiation factors of osteoblasts revealed significant alterations in gene expression of these factors, which may contribute to the dysregulation of energy metabolism in 2DM.

Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity.

Primary human hepatocytes (PHH) are the "gold standard" for in vitro toxicity tests. However, 2D PHH cultures have limitations that are due to a time-dependent dedifferentiation process visible by morphological changes closely connected to a decline of albumin production and CYP450 activity. The 3D in vitro culture corresponds to in vivo-like tissue architecture, which preserves functional characteristics of hepatocytes, and therefore can at least partially overcome the restrictions of 2D cultures. Consequently, several drug toxicities observed in vivo cannot be reproduced in 2D in vitro models, for example, the toxic effects of acetaminophen. The objective of this study was to identify molecular differences between 2D and 3D cultivation which explain the observed toxicity response. Our data demonstrated an increase in cell death after treatment with acetaminophen in 3D, but not in 2D cultures. Additionally, an acetaminophen concentration-dependent increase in the CYP2E1 expression level in 3D cultures was detected. However, during the treatment with 10 mM acetaminophen, the expression level of SOD gradually decreased in 3D cultures and was undetectable after 24 h. In line with these findings, we observed higher import/export rates in the membrane transport protein, multidrug resistance-associated protein-1, which is known to be specific for acetaminophen transport. The presented data demonstrate that PHH cultured in 3D preserve certain metabolic functions. Therefore, they have closer resemblance to the in vivo situation than PHH in 2D cultures. In consequence, 3D cultures will allow for a more accurate hepatotoxicity prediction in in vitro models in the future.

Decrease of global methylation improves significantly hepatic differentiation of Ad-MSCs: possible future application for urea detoxification.

Hepatocyte transplantation is considered to be an alternative to orthotopic liver transplantation. Cells can be used to bridge patients waiting for a donor organ, decrease mortality in acute liver failure, and support metabolic liver diseases. The limited availability of primary human hepatocytes for such applications has led to the generation of alternative hepatocyte-like cells from various adult stem or precursor cells. The aim of this study was to generate hepatocyte-like cells from adipose-derived mesenchymal stem cells (Ad-MSCs) for clinical applications, which are available "off the shelf." Epigenetic changes in hepatocyte-like cells were induced by 5-azacytidine, which, in combination with other supplements, leads to significantly improved metabolic and enzymatic activities compared to nontreated cells. Cells with sufficient hepatic features were generated with a four-step protocol: 5-azacytidine (step 1); epidermal growth factor (step 2); fibroblast growth factor-4, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 3); and hepatocyte growth factor, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 4). Generated differentiated cells had higher phase I (CYP1A1/2, CYP2E1, CYP2B6, CYP3A4) and phase II activities compared to the undifferentiated cells. A strong expression of CYP3A7 and a weak expression of 3A4, as well as the important detoxification markers α-fetoprotein and albumin, could also be detected at the mRNA level. Importantly, urea metabolism (basal, NH4-stimulated, NH4- and ornithine-stimulated) was comparable to freshly isolated human hepatocytes, and unlike cryopreserved human hepatocytes, this activity was maintained after 6 months of cryopreservation. These findings suggest that these cells may be suitable for clinical application, especially for treatment of urea cycle disorders.

Differential effects of clinically used derivatives and metabolites of artemisinin in the activation of constitutive androstane receptor isoforms.

BACKGROUND AND PURPOSE: Widespread resistance to antimalarial drugs requires combination therapies with increasing risk of pharmacokinetic drug-drug interactions. Here, we explore the capacity of antimalarial drugs to induce drug metabolism via activation of constitutive androstane receptors (CAR) by ligand binding. EXPERIMENTAL APPROACH: A total of 21 selected antimalarials and 11 major metabolites were screened for binding to CAR isoforms using cellular and in vitro CAR-coactivator interaction assays, combined with in silico molecular docking. Identified ligands were further characterized by cell-based assays and primary human hepatocytes were used to elucidate induction of gene expression. KEY RESULTS: Only two artemisinin derivatives arteether and artemether, the metabolite deoxyartemisinin and artemisinin itself demonstrated agonist binding to the major isoforms CAR1 and CAR3, while arteether and artemether were also inverse agonists of CAR2. Dihydroartemisinin and artesunate acted as weak inverse agonists of CAR1. While arteether showed the highest activities in vitro, it was less active than artemisinin in inducing hepatic CYP3A4 gene expression in hepatocytes. CONCLUSIONS AND IMPLICATIONS: Artemisinin derivatives and metabolites differentially affect the activities of CAR isoforms and of the pregnane X receptor (PXR). This negates a common effect of these drugs on CAR/PXR-dependent induction of drug metabolism and further provides an explanation for artemisinin consistently inducing cytochrome P450 genes in vivo, whereas arteether and artemether do not. All these drugs are metabolized very rapidly, but only artemisinin is converted to an enzyme-inducing metabolite. For better understanding of pharmacokinetic drug-drug interaction possibilities, the inducing properties of artemisinin metabolites should be considered.

Superior antitumoral activity of dimerized targeted single-chain TRAIL fusion proteins under retention of tumor selectivity.

Although targeting of the death receptors (DRs) DR4 and DR5 still appears a suitable antitumoral strategy, the limited clinical responses to recombinant soluble TNF-related apoptosis inducing ligand (TRAIL) necessitate novel reagents with improved apoptotic activity/tumor selectivity. Apoptosis induction by a single-chain TRAIL (scTRAIL) molecule could be enhanced >10-fold by generation of epidermal growth factor receptor (EGFR)-specific scFv-scTRAIL fusion proteins. By forcing dimerization of scFv-scTRAIL based on scFv linker modification, we obtained a targeted scTRAIL composed predominantly of dimers (Db-scTRAIL), exceeding the activity of nontargeted scTRAIL ∼100-fold on Huh-7 hepatocellular and Colo205 colon carcinoma cells. Increased activity of Db-scTRAIL was also demonstrated on target-negative cells, suggesting that, in addition to targeting, oligomerization equivalent to an at least dimeric assembly of standard TRAIL per se enhances apoptosis signaling. In the presence of apoptosis sensitizers, such as the proteasomal inhibitor bortezomib, Db-scTRAIL was effective at picomolar concentrations in vitro (EC(50) ∼2 × 10(-12) M). Importantly, in vivo, Db-scTRAIL was well tolerated and displayed superior antitumoral activity in mouse xenograft (Colo205) tumor models. Our results show that both targeting and controlled dimerization of scTRAIL fusion proteins provides a strategy to enforce apoptosis induction, together with retained tumor selectivity and good in vivo tolerance.

Effects of atorvastatin metabolites on induction of drug-metabolizing enzymes and membrane transporters through human pregnane X receptor.

BACKGROUND AND PURPOSE: Atorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). EXPERIMENTAL APPROACH: Ligand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation. KEY RESULTS: All atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors. CONCLUSIONS AND IMPLICATIONS: Atorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound.

Further characterization of autologous NeoHepatocytes for in vitro toxicity testing.

Gold standard for in vitro toxicity tests and drug screenings is primary human hepatocytes (hHeps). Because of their limited availability efforts have been made to provide alternatives, e.g., monocyte-derived NeoHepatocytes. In the past years it has been critically discussed if gaining hepatocyte features is associated with trans-differentiation of monocytes or their activation towards a macrophage phenotype. Generating NeoHepatocytes in the presence of six different human AB sera, fetal calf serum (FCS) or autologous serum showed that yield and quality of NeoHepatocytes is inversely correlated to macrophage activation. Using autologous serum constantly the highest amount of cells with the best metabolic capacity was obtained. Focus of this study was to further analyze bio-transformation capacity of the optimized NeoHepatocytes for use as in vitro toxicity test-system. Treatment of the optimized NeoHepatocytes with two different pro-teratogenic substances with corresponding metabolites and eight known hepatotoxins showed comparable toxicity to hHeps. Bio-transformation rates, assessed by testosterone metabolism, were comparable in both cell types. Our data reveal that use of autologous serum reduced macrophage activation which improved yield and function of NeoHepatocytes resulting in bio-transformation and toxicity profiles comparable to hHeps. Thus, their easy accessibility makes them an ideal candidate for in vitro toxicity studies.

Autologous serum improves yield and metabolic capacity of monocyte-derived hepatocyte-like cells: possible implication for cell transplantation.

Hepatocyte-transplantation is a therapeutic approach for diverse acute and chronic liver diseases. As availability of primary cells is limited, there is an increasing demand for hepatocyte-like cells (e.g., neohepatocytes generated from peripheral blood monocytes). The aim of this study was to evaluate the effects of six different human AB sera, fetal calf serum, or autologous serum on production of neohepatocytes. The yield and quality of neohepatocytes varied considerably depending on the different sera. Using autologous sera for the whole production process we constantly generated the highest amount of cells with the highest metabolic activity for phase I (e.g., CYP1A1/2, CYP3A4) and phase II enzymes (e.g., glutathione-S-transferase). Moreover, similar effects were seen examining glucose and urea metabolism. Especially, glucose-6-phosphatase and PAS staining showed distinct serum-dependent differences. The role of macrophage activation was investigated by measuring the secretion of TNF-α, TGF-ß, and RANKL, MMP activity, as well as mRNA levels of different interleukins in programmable cells of monocytic origin (PCMO). Our data clearly demonstrate that the use of autologous serum reduced initial macrophage activation in PCMOs and subsequently improved both yield and function of differentiated neohepatocytes. The autologous approach presented here might also be useful in other stem cell preparation processes where cell activation during generation shall be kept to a minimum.

The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: a comparison of primary human hepatocytes and OATP-transfected HEK293 cells.

Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and -LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.

Protective role of HO-1 for alcohol-dependent liver damage.

BACKGROUND/AIMS: Alcoholic liver disease is continuously increasing in developed countries being a leading cause of death worldwide. Chronic ethanol consumption induces oxidative stress by accumulation of reactive oxygen intermediates (ROI) while reducing the cellular antioxidant defense. Induction of heme oxygenase-1 (HO-1) may protect primary human hepatocytes (hHeps) from such damage. Thus, the aim of this study was to investigate the potential of polyphenols to protect hHeps from ethanol-dependent oxidative damage. METHODS: hHeps were isolated by collagenase perfusion. ROI and cellular glutathione (GSH) were measured by fluorescent-based assays. Cellular damage was determined by lactate dehydrogenase (LDH) leakage and staining for apoptosis and necrosis. Nuclear translocation of Nrf2 and HO-1 expression were analyzed by Western blot. RESULTS: Ethanol and TGF-ß rapidly increase ROI and reduce GSH in hHeps, causing apoptosis with a release of approximately 40% total LDH after 72 h. Similar to incubation with hemin preincubation and co-incubation of cells with nifedipine, verapamil and quercetin significantly reduce oxidative stress and resulting cellular damage, in a dose-dependent manner, by initiating nuclear translocation of Nrf2 which in turn induces HO-1 under the control of p38 and ERK. Blocking of HO-1 activity with ZNPP9 reverses the protective effect of all three substances. CONCLUSION: Our results suggest that increasing HO-1 activity in hHeps protects them from oxidative stress-dependent damage. As polyphenols have great potential to induce HO-1 expression, they may play an important role for future therapeutic strategies to protect liver from oxidative stress-dependent damage observed during chronic alcohol consumption.

Intrasplenic or subperitoneal hepatocyte transplantation to increase survival after surgically induced hepatic failure?

BACKGROUND: As a basis for future clinical questions, we evaluated the efficacy of hepatocyte transplantation in a surgical model using a subperitoneal or intrasplenic approach for cell implantation. METHODS: In rats, acute liver failure was induced by subtotal hepatectomy. Series of allogenic hepatocyte transplantations were performed by varying cell number, site, and sequence of cell transplantation. RESULTS: Following subperitoneal or intrasplenic cell implantation subsequent to liver surgery, no survival benefit was achieved when compared to the control groups. However, intrasplenic cell implantation 24 h prior to liver surgery revealed a statistically significantly higher animal survival (72 vs. 29%). CONCLUSION: According to our experience, both timing and site of cell implantation played an important role in hepatocyte transplantation. Intrasplenic hepatocyte transplantation 1 day before liver surgery showed the best results in terms of survival. Consequently, we were able to establish a model of hepatocyte transplantation which may be the basis for further investigations evaluating potential treatment modalities to overcome deleterious postoperative liver insufficiency.

Blood monocyte-derived neohepatocytes as in vitro test system for drug metabolism.

The gold standard for human drug metabolism studies is primary hepatocytes. However, availability is limited by donor organ scarcity. Therefore, efforts have been made to provide alternatives, e.g., the hepatocyte-like (NeoHep) cell type, which was generated from peripheral blood monocytes. In this study, expression and activity of phase I and phase II drug-metabolizing enzymes were investigated during transdifferentiation of NeoHep cells and compared with primary human hepatocytes. Important drug-metabolizing enzymes are cytochrome P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, and 3A4), microsomal epoxide hydrolase 1, glutathione S-transferase A1 and M1, N-acetyltransferase 1, NAD(P)H menadione oxidoreductase 1, sulfotransferase 1A1, and UDP-glucuronosyltransferase 1A6. Monocytes and programmable cells of monocytic origin expressed only a few of the enzymes investigated. Throughout differentiation, NeoHep cells showed a continuously increasing expression of all drug-metabolizing enzymes investigated, resulting in stable basal activity after approximately 15 days. Fluorescence-based activity assays indicated that NeoHep cells and primary hepatocytes have similar enzyme kinetics, although the basal activities were significantly lower in NeoHep cells. Stimulation with 3-methylcholanthrene and rifampicin markedly increased CYP1A1/2 or CYP3A4 activities, which could be selectively inhibited by nifedipine, verapamil, ketoconazole, and quercetin. Our data reveal similarities in expression, activity, induction, and inhibition of drug-metabolizing enzymes between NeoHep cells and primary human hepatocytes and hence suggest that NeoHep cells are useful as an alternative to human hepatocytes for measuring bioactivation of substances.

Selective induction of human hepatic cytochromes P450 2B6 and 3A4 by metamizole.

The pyrazolone drug metamizole is a widely used analgesic. Analysis of liver microsomes from patients treated with metamizole revealed selectively higher expression of cytochromes P450, CYP2B6 and CYP3A4 (3.8- and 2.8-fold, respectively), and 2.9-fold higher bupropion hydroxylase activity compared with untreated subjects. Further investigation of metamizole and various derivatives on different potential target genes in human primary hepatocytes demonstrated time- and concentration-dependent induction by metamizole of CYP2B6 (7.8- and 3.1-fold for mRNA and protein, respectively, at 100 muM) and CYP3A4 (2.4- and 2.9-fold, respectively), whereas other genes (CYP2C9, CYP2C19, CYP2D6, NADPH:cytochrome P450 reductase, ABCB1, constitutive androstane receptor (CAR), pregnane X receptor (PXR)) were not substantially altered. Using reporter gene assays, we show that metamizole is not acting as a direct ligand to either PXR or CAR, suggesting a phenobarbital-like mechanism of induction. These data warrant further studies to elucidate the drug-interaction potential of metamizole, especially in patients with long-term treatment.

Cobalt-protoporphyrin induced heme oxygenase overexpression and its impact on liver regeneration.

Cobalt-protoporphyrin (CoPP)-dependent induction of heme oxygenase (HO)-1 has been shown to protect from ischemia-reperfusion injury, which remains a major source of graft loss after liver transplantation. The impact of HO-1 on liver regeneration, especially in reduced-size grafts, has not yet been evaluated. Using an experimental model, we investigated HO-1 induction by CoPP treatment on postoperative recovery of ischemically injured livers following partial (70%) hepatectomy. Wistar rats underwent partial hepatectomy under temporary inflow occlusion (30 minutes). One group of animals received CoPP (5 mg/kg body weight i.p.) 24 hours prior to surgery to induce high levels of HO-1 at the time of surgery, and the second group served as nontreated controls. At postoperative days 1, 4, 7, and 10, animals were exsanguinated, and blood and liver samples were stored for enzymatic (serum AST and ALT levels) and histologic (mitotic index) analyses (n = 5 each day). Additionally, postoperative body weight and weight of the remnant liver were measured. Although serum AST and ALT levels as well as remnant liver weight were comparable between both groups, CoPP-treated animals recovered from surgery more quickly as indicated by postoperative body weight. Moreover, the number of mitotic cells was significantly increased in this group at day 1 (33 +/- 5 versus 20 +/- 5 per 2000 hepatocytes) as compared with nontreated animals. Liver regeneration of ischemically injured livers following partial hepatectomy was improved by HO-1 overexpression following preoperative CoPP administration. Thus, it is conceivable that prevention of ischemia-reperfusion injury by HO-1 overexpression also might be beneficial for reduced-size liver grafts without affecting their proliferative capacity.