[Tonometry and pachymetry to evaluate fluctuations of intraocular pressure in the context of SCUBA diving]. / Tonometrie und Pachymetrie zur Erfassung der Augeninnendruckschwankungen beim Scuba-Tauchen.

BACKGROUND: It is currently still not clarified whether diving using a self-contained breathing apparatus (SCUBA) is associated with intraocular pressure (IOP) fluctuations of clinical relevance and whether intensive diving could exacerbate the damage in glaucoma patients. OBJECTIVE: This study aimed to evaluate the effect of SCUBA diving on IOP in healthy volunteers without prior eye injuries or surgery. HYPOTHESIS: recreational diving does not lead to significant increases or fluctuations of the IOP. MATERIAL AND METHODS: The study included 16 divers (5 female) who performed a total of 96 dives with air or nitrox32 to a depth of 20-30 m for an average of 50 min. The central cornea thickness was measured using ultrasonic pachymetry Pocket IITM (Quantel Medical Pocket II™, Quantel Medical, Clermont-Ferrand, France), and the IOP was measured using an Icare® PRO (Icare® PRO, Icare Finland Oy, Espoo, Finland) directly before the dive and 10 min after surfacing. RESULTS: All data refer to the right eye. Average IOP values ranged from 15.6 to 19.2 mm Hg pre-dive and 16.8 to 18.2 mm Hg post-dive. The range of IOP values was 2.2-11.5 mm Hg pre-dive (∆ = 9.3 mm Hg) and 2.7-14.8 mm Hg post-dive (∆ = 12.1 mm Hg). Of the divers 11.5% vs. 18.8% had increased IOP values > 21 mm Hg (pre-dive vs. post-dive). CONCLUSION: This study found no significant differences in IOP values between pre-dive and post-dive measurements in healthy SCUBA divers. Therefore, recreational SCUBA diving is unlikely to affect the IOP in healthy individuals.

Evidence- and consensus-based (S3) Guidelines for the Treatment of Actinic Keratosis - International League of Dermatological Societies in cooperation with the European Dermatology Forum - Short version.

BACKGROUND: Actinic keratosis (AK) is a frequent health condition attributable to chronic exposure to ultraviolet radiation. Several treatment options are available and evidence based guidelines are missing. OBJECTIVES: The goal of these evidence- and consensus-based guidelines was the development of treatment recommendations appropriate for different subgroups of patients presenting with AK. A secondary aim of these guidelines was the implementation of knowledge relating to the clinical background of AK, including consensus-based recommendations for the histopathological definition, diagnosis and the assessment of patients. METHODS: The guidelines development followed a pre-defined and structured process. For the underlying systematic literature review of interventions for AK, the methodology suggested by the Cochrane Handbook for Systematic Reviews of Interventions, the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was adapted. All recommendations were consented during a consensus conference using a formal consensus methodology. Strength of recommendations was expressed based on the GRADE approach. If expert opinion without external evidence was incorporated into the reasoning for making a certain recommendation, the rationale was provided. The Guidelines underwent open public review and approval by the commissioning societies. RESULTS: Various interventions for the treatment of AK have been assessed for their efficacy. The consenting procedure led to a treatment algorithm as shown in the guidelines document. Based on expert consensus, the present guidelines present recommendations on the classification of patients, diagnosis and histopathological definition of AK. Details on the methods and results of the systematic literature review and guideline development process have been published separately. CONCLUSIONS: International guidelines are intended to be adapted to national or regional circumstances (regulatory approval, availability and reimbursement of treatments).

Erratum to: Resorbable osteosynthetic devices in pediatric traumatology: a prospective series of 24 cases.

Erratum to: Eur J Orthop Surg Traumatol DOI 10.1007/s00590-015-1656-8. The author would like to correct the errors in the publication of the original article. The corrected details are given below for your reading. Second and third authors' given names have been published incorrectly. The correct author names should be D. Popkov and H. Huber. The affiliations of the authors J. M. Poircuitte, D. Popkov, H. Huber, E. Polirsztok and P. Journeau are incorrect. The correct affiliations should be: J. M. Poircuitte, H. Huber, E. Polirsztok and P. Journeau: Service de chirurgie orthopedique pediatrique, Hopital d'enfant, Centre hospitalo-universitaire de Nancy, 5 allee du Morvan, 54500 Vandoeuvre les Nancy, France. D. Popkov: Russian Ilizarov Scientific Center for Restorative Traumatology and Orthopaedics, Kurgan, Russia. Corresponding author e-mail address should be p.journeau@ chu-nancy.fr.

Resorbable osteosynthetic devices in pediatric traumatology: a prospective series of 24 cases.

UNLABELLED: Bioresorbable devices are commonly used in traumatology. The biomechanical stability of these materials has improved in the past decade, and they have proven to be biologically non-hazardous, while their main advantage is that their use avoids reintervention for removal of the device. A prospective monocentric study was conducted: 24 patients presenting with a fracture that was amenable to osteosynthesis by small-diameter screws were included. These comprised ten tibial spine fractures, four osteochondritis dissecans of the distal femur, eight fractures of the medial epicondyle of the distal humerus, and two distal tibial apophyseal fractures. One or more screws were used that were made of a copolymer of poly-L-lactide-poly-D-lactide acid and trimethylene carbonate with a diameter of 2.8 mm. All patients were immobilized with a cast. Clinical and radiographic monitoring was conducted every month. The entire follow-up protocol had a duration of 24 months. One patient with osteochondritis dissecans presented with joint effusion. Joint stiffness at the time of cast removal resolved completely after 4 months, except for with three children (one epicondyle fracture, two tibial spine fractures). No subjective or objective instability could be detected by clinical examination. Radiographic follow-up revealed no secondary displacement, and all of the fractures had healed. No osteolysis was seen around the screws. No growth disturbances were noticed. Bioresorbable materials thus appear to be a suitable alternative approach for certain pediatric fractures. Their use resulted in outcomes similar to traditional techniques in terms of functional properties and bone healing. Although initial costs are presumably slightly higher, by avoiding a removal operation the total financial burden is most likely reduced. LEVEL OF EVIDENCE: III.

Low cytochrome oxidase 4I1 links mitochondrial dysfunction to obesity and type 2 diabetes in humans and mice.

OBJECTIVES: Cytochrome oxidase (COX) dysfunction is associated with mitochondrial oxidative stress. We determined the association between COX expression, obesity and type 2 diabetes. SUBJECTS/METHODS: COX4I1 and COX10 genes were measured in monocytes of 24 lean controls, 31 glucose-tolerant and 67 diabetic obese patients, and 17 morbidly obese patients before and after bariatric surgery. We investigated the effect of caloric restriction and peroxisome proliferator-activated receptor (PPAR) agonist treatment on Cox in obese diabetic mice, and that of diet-induced insulin resistance in Streptozotocin-treated mice. RESULTS: Low COX4I1 was associated with type 2 diabetes in obese patients, adjusting for age, gender, smoking, interleukin-6 and high-sensitivity C-reactive protein, all related to metabolic syndrome (MetS; odds ratio: 6.1, 95% confidence interval: 2.3-16). In contrast, COX10 was low in glucose-tolerant and diabetic obese patients. In morbidly obese patients, COX4I1 was lower in visceral adipose tissue collected at bariatric surgery. In their monocytes, COX4I1 decreased after bariatric surgery, and low COX4I1 at 4 months was associated with MetS at 7 years. In leptin-deficient obese diabetic mice, Cox4i1 was low in white visceral adipose tissue (n=13; P<0.001) compared with age-matched lean mice (n=10). PPARγ-agonist treatment (n=13), but not caloric restriction (n=11), increased Cox4i1 (P<0.001). Increase in Cox4i1 depended on the increase in glucose transporter 4 (Glut4) expression and insulin sensitivity, independent of the increase in blood adiponectin. In streptozotocin-treated mice (three groups of seven mice, diet-induced insulin resistance decreased Cox4i1 and Glut4 (P<0.001 for both). CONCLUSION: COX4I1 depression is related to insulin resistance and type 2 diabetes in obesity. In peripheral blood monocytes, it may be a diagnostically useful biomarker.

BCL2 protein signalling determines acute responses to neoadjuvant chemoradiotherapy in rectal cancer.

UNLABELLED: In locally advanced rectal cancer, neoadjuvant chemoradiotherapy is performed prior to surgery to downstage the tumour. Thirty to 40 % of patients do not respond. Defects in apoptotic machinery lead to therapy resistance; however, to date, no study quantitatively assessed whether B cell lymphoma 2 (BCL2)-dependent regulation of mitochondrial apoptosis, effector caspase activation downstream of mitochondria or a combination of both predicts patient responses. In a cohort of 20 rectal cancer patients, we performed protein profiling of tumour tissue and employed validated ordinary differential equation-based systems models of apoptosis signalling to calculate the ability of cancer cells to undergo apoptosis. Model outputs were compared to clinical responses. Systems modelling of BCL2-signalling predicted patients in the poor response group (p = 0.0049). Systems modelling also demonstrated that rectal cancers depended on BCL2 rather than B cell lymphoma-extra large (BCL(X)L) or myeloid cell leukemia 1 (MCL1) for survival, suggesting that poor responders may benefit from therapy with selective BCL2 antagonists. Dynamic modelling of effector caspase activation could not stratify patients with poor response and did not further improve predictive power. We deliver a powerful patient stratification tool identifying patients who will likely not benefit from neoadjuvant chemoradiotherapy and should be prioritised for surgical resection or treatment with BCL2 antagonists. KEY MESSAGES: Modelling BCL2-family proteins identifies patients unresponsive to therapy. Caspase activation downstream of mitochondria cannot identify these patients. Rectal tumours of poor responders are BCL2- but not BCL-XL-dependent. DR_MOMP allows clinicians to identify patients who would not benefit from therapy. DR_MOMP is also a useful patient stratification tool for BCL2 antagonists.

Expression of somatostatin receptor subtype 2 and subtype 5 in thyroid malignancies.

AIM: To retrospectively analyse the expression of somatostatin receptor subtypes 2 (SSTR 2) and 5 (SSTR 5) in thyroid malignancies, possibly the most relevant subtypes for targeted therapy with somatostatin peptide radioligands. In addition, findings were also correlated with the course of disease. PATIENTS, METHODS: 87 consecutive patients (59 women, 28 men) with thyroid malignancy were included; 52 had papillary carcinoma, 24 follicular carcinoma, six medullary carcinoma, two poorly differentiated carcinoma and three anaplastic carcinoma. After initial therapy 70 (80.5%) patients showed complete remission, 11 (12.6%) patients partial remission with clinical and biochemical signs of residual disease and six (6.9%) patients progressive disease. The immunohistochemical staining results of the primary malignancy for SSTR 2 and SSTR 5 were semiquantitatively assessed and correlated with various outcome parameters. RESULTS: In 10 of 87 (11.49%) thyroid cancer samples SSTR 2 showed positive immunohistochemical expression as compared to 75 of 87 (86.20%) for SSTR 5. All SSTR 2-positive cases expressed SSTR 5. Persistent or recurrent disease was found in 17 of 87 cases (19.54%). Fifty percent (6 /12) of SSTR 5-negative patients showed persistent disease as compared to 14.7 % (11 / 75) of SSTR 5-positive patients: seven of these were exclusively SSTR 5-positive, 4 showed dual expression of SSTR 5 and SSTR 2 (p = 0.01). No case showed only SSTR 2 expression. CONCLUSIONS: SSTR 5 was shown to be the main receptor subtype in the analysed differentiated or anaplastic thyroid malignancies, whereas SSTR 2 was found only in a small percentage. Deficient SSTR expression may indicate higher risk for persistent or recurrent disease after initial therapy. For this reason immunohistochemistry can be considered a prognostic marker which should be further validated in prospective studies.

[Cutaneous Rosai-Dorfman syndrome. Successful therapy with intrralesional corticosteroids]. / Kutanes Rosai-Dorfman-Syndrom. Erfolgreiche Therapie mit intraläsional applizierten Steroiden.

BACKGROUND: Cutaneous Rosai-Dorfman disease is a rare disorder belonging to the spectrum of non-Langerhans cell histiocytoses. It is characterized by dermal and subcutaneous infiltrates of histiocytes as well as accompanying lymphocytes, plasma cells and granulocytes. Because it is so rare, standard therapies have not been established. CASE REPORT: A 27-year-old man showed an excellent response to intralesional corticosteroids after unsuccessful prior treatment with methotrexate, systemic steroids and surgery as well as laser therapy.

Latrepirdine is a potent activator of AMP-activated protein kinase and reduces neuronal excitability.

Latrepirdine/Dimebon is a small-molecule compound with attributed neurocognitive-enhancing activities, which has recently been tested in clinical trials for the treatment of Alzheimer's and Huntington's disease. Latrepirdine has been suggested to be a neuroprotective agent that increases mitochondrial function, however the molecular mechanisms underlying these activities have remained elusive. We here demonstrate that latrepirdine, at (sub)nanomolar concentrations (0.1 nM), activates the energy sensor AMP-activated protein kinase (AMPK). Treatment of primary neurons with latrepirdine increased intracellular ATP levels and glucose transporter 3 translocation to the plasma membrane. Latrepirdine also increased mitochondrial uptake of the voltage-sensitive probe TMRM. Gene silencing of AMPKα or its upstream kinases, LKB1 and CaMKKß, inhibited this effect. However, studies using the plasma membrane potential indicator DisBAC2(3) demonstrated that the effects of latrepirdine on TMRM uptake were largely mediated by plasma membrane hyperpolarization, precluding a purely 'mitochondrial' mechanism of action. In line with a stabilizing effect of latrepirdine on plasma membrane potential, pretreatment with latrepirdine reduced spontaneous Ca(2+) oscillations as well as glutamate-induced Ca(2+) increases in primary neurons, and protected neurons against glutamate toxicity. In conclusion, our experiments demonstrate that latrepirdine is a potent activator of AMPK, and suggest that one of the main pharmacological activities of latrepirdine is a reduction in neuronal excitability.

Activation of executioner caspases is a predictor of progression-free survival in glioblastoma patients: a systems medicine approach.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. GBM cells are highly resistant to apoptosis induced by antitumor drugs and radiotherapy resulting in cancer progression. We assessed whether a systems medicine approach, analysing the ability of tumor cells to execute apoptosis could be utilized to predict the response of GBM patients to treatment. Concentrations of the key proapoptotic proteins procaspase-3, procaspase-9, Smac and Apaf-1 and the antiapopotic protein XIAP were determined in a panel of GBM cell lines and GBM patient tumor resections. These values were used as input for APOPTO-CELL, a systems biological based mathematical model built to predict cellular susceptibility to undergo caspase activation. The modeling was capable of accurately distinguishing between GBM cells that die or survive in response to treatment with temozolomide in 10 of the 11 lines analysed. Importantly the results obtained using GBM patient samples show that APOPTO-CELL was capable of stratifying patients according to their progression-free survival times and predicted the ability of tumor cells to support caspase activation in 16 of the 21 GBM patients analysed. Calculating the susceptibility to apoptosis execution may be a potent tool in predicting GBM patient therapy responsiveness and may allow for the use of APOPTO-CELL in a clinical setting.

[CD30-positive anaplastic large cell T-cell lymphoma developing during immunosuppressive therapy of pityriasis rubra pilaris with ustekinumab]. / CD30-positives anaplastisch großzelliges T-Zell-Lymphom unter immunsuppressiver Therapie einer Pityriasis rubra pilaris mit Ustekinumab.

The development of malignancies during therapy with biologics has been discussed controversially. A patient with extensive pityriasis rubra pilaris failed to respond to standard therapeutic approaches. While receiving immunomodulatory therapy, lastly with ustekinumab, the patient developed a CD30(+) anaplastic large cell lymphoma.

The PRIPS study: screening battery for subjects at risk for Parkinson's disease.

BACKGROUND AND PURPOSE: Screening batteries to narrow down a target-at-risk population are essential for trials testing neuroprotective compounds aiming to delay or prevent onset of Parkinson's disease (PD). METHODS: The PRIPS study focuses on early detection of incident PD in 1847 at baseline PD-free subjects, and assessed age, male gender, positive family history, hyposmia, subtle motor impairment and enlarged substantia nigra hyperechogenicity (SN+). RESULTS: After 3 years follow-up 11 subjects had developed PD. In this analysis of the secondary outcome parameters, sensitivity and specificity of baseline markers for incident PD were calculated in 1352 subjects with complete datasets (10 PD patients). The best approach for prediction of incident PD comprised three steps: (i) prescreening for age, (ii) primary screening for positive family history and/or hyposmia, and (iii) secondary screening for SN+. CONCLUSION: With this approach, one out of 16 positively screened participants developed PD compared to one out of 135 in the original cohort. This corresponds to a sensitivity of 80.0%, a specificity of 90.6% and a positive predictive value of 6.1%. These values are higher than for any single screening instrument but still too low for a feasible and cost-effective screening strategy which might require longer follow-up intervals and application of additional instruments.

Classification of complications after progressive long bone lengthening: proposal for a new classification.

INTRODUCTION: Long bone lengthening surgery using progressive surgical methods has been the source of frequent complications. Some authors have classified these complications either descriptively, according to the date of onset after the operation, or based on their severity. The Caton classification (1985) has had the virtue of contributing the notion of the treatment contract stipulating the objective to reach in treatment. Within the context of the preoperative information delivered to patients and their family, this contract can be improved by adding a notion of maximum treatment duration. The objective of this study was therefore to propose a classification that includes honoring a triple contract associating the planned gain in bone length, the duration of treatment, and the occurrence of sequelae. MATERIALS AND METHODS: The classification of complications proposed includes four grades: grade I: triple contract honored, including a few treatments without general anesthesia; grade II: triple contract fulfilled, but with unplanned interventions under general anesthesia; grade III: the time stipulated was not honored because the time to obtain bone union was too long or because the program was interrupted; grade IV: sequelae are present. This classification was assessed based on a consecutive series of 34 surgical procedures in 32 patients (two patients underwent two lengthening procedures during this period) at 43 bone segments associating progressive lengthening with external fixation or with nail lengthening. The grade of each complication was determined by each of the authors according to the classification proposed and other classifications reported in the literature (Caton, Paley, Popkov, and Donnan). RESULTS: Approximately one-third (10) of the 34 lengthening procedures did not present any complications. Two-thirds (24) presented 30 complications. Consensus was obtained between all the authors on the grades proposed for our classification and the Caton classification, but consensus was not reached with the other classifications in which part of the interpretation was subjective (Paley, Popkov, and Donnan). DISCUSSION: The classification proposed required respecting predetermined objectives during limb lengthening surgery based on a triple contract: gain, duration, and function. It is reliable and reproducible by different operators because the criteria are objective. It can also be applied to diverse surgical techniques, whether with external fixation and/or internal osteosynthesis. LEVEL OF EVIDENCE: Level IV: retrospective study or historical series.

Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate.

Cerebral ischemia and excitotoxic injury induce transient or permanent bioenergetic failure, and may result in neuronal apoptosis or necrosis. We have previously shown that ATP depletion and activation of AMP-activated protein kinase (AMPK) during excitotoxic injury induces neuronal apoptosis by transcription of the pro-apoptotic BH3-only protein, Bim. AMPK, however, also exerts pro-survival functions in neurons. The molecular switches that determine these differential outcomes are not well understood. Using an approach combining biochemistry, single-cell imaging and computational modeling, we here demonstrate that excitotoxic injury activated the bim promoter in a FOXO3-dependent manner. The activation of AMPK reduced AKT activation, and led to dephosphorylation and nuclear translocation of FOXO3. Subsequent mutation studies indicated that bim gene activation during excitotoxic injury required direct FOXO3 phosphorylation by AMPK in the nucleus as a second activation step. Inhibition of this phosphorylation prevented Bim expression and protected neurons against excitotoxic and oxygen/glucose deprivation-induced injury. Systems analysis and computational modeling revealed that these two activation steps defined a coherent feed-forward loop; a network motif capable of filtering any effects of short-term AMPK activation on bim gene induction. This may prevent unwanted AMPK-mediated Bim expression and apoptosis during transient or physiological bioenergetic stress.

[Quality assurance in coding expertise of hospital cases in the German DRG system. Evaluation of inter-rater reliability in MDK expertise]. / Qualitätssicherung in der Kodierungsbegutachtung von Krankenhausfällen im G-DRG-System. Prüfung der Interrater-Reliabilität bei MDK-Gutachtern.

AIM: The purpose of this study was to evaluate differences in the D-DRG results of a hospital case by 2 independently coding MKD raters. Calculation of the 2-inter-rater reliability was performed by examination of the coding of individual hospital cases. The reasons for the non-agreement of the expert evaluations and suggestions to improve the process are discussed. METHODS: From the expert evaluation pool of the MDK-WL a random sample of 0.7% of the 57,375 expertises was taken. Distribution equality with the basic total was tested by the χ² test or, respectively, Fisher's exact test. For the total of 402 individual hospital cases, the G-DRG case sums of 2 experts of the MDK were determined independently and the results checked for each individual case for agreement or non-agreement. The corresponding confidence intervals with standard errors were analysed to test if certain major diagnosis categories (MDC) were statistically significantly more affected by differing expertise results than others. RESULTS: In 280 of the total 402 tested hospital cases, the 2 MDK raters independently reached the same G-DRG results; in 122 cases the G-DRG case sums determined by the 2 raters differed (agreement 70%; CI 65.2-74.1). Different DRG results between the 2 experts occurred regularly in the entire MDC spectrum. No MDC chapter in which significant differences between the 2 raters arose could be identified. CONCLUSION: The results of our study demonstrate an almost 70% agreement in the evaluation of hospital cost accounts by 2 independently operating MDK. This result leaves room for improvement. Optimisation potentials can be recognised on the basis of the results. Potential for improvement was established in combination with regular further training and the expansion of binding internal code recommendations as well as exchange of code-relevant information among experts in internal forums. The presented model is in principle suitable for cross-border examinations within the MDK system with the advantage that further trends could be uncovered by more variety and larger numbers of the randomly selected cases.

Adolescent slipped capital femoral epiphysis treated by a modified Dunn osteotomy with surgical hip dislocation.

Between June 2001 and November 2008 a modified Dunn osteotomy with a surgical hip dislocation was performed in 30 hips in 28 patients with slipped capital femoral epiphysis. Complications and clinical and radiological outcomes after a mean follow-up of 3.8 years (1.0 to 8.5) were documented. Subjective outcome was assessed using the Harris hip score and the Western Ontario and McMaster Universities osteoarthritis index questionnaire. Anatomical or near-anatomical reduction was achieved in all cases. The epiphysis in one hip showed no perfusion intra-operatively and developed avascular necrosis. There was an excellent outcome in 28 hips. Failure of the implants with a need for revision surgery occurred in four hips. Anatomical reduction can be achieved by this technique, with a low risk of avascular necrosis. Cautious follow-up is necessary in order to avoid implant failure.

Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation.

Mitochondrial outer membrane permeabilisation (MOMP) during apoptosis is triggered by the activation and oligomerisation of Bax and Bak, but a quantification of these processes in individual cells has not yet been performed. Single-cell imaging of Bax translocation and oligomerisation in Bax-deficient DU-145 cells expressing CFP-Bax and YFP-Bax revealed that both processes started only minutes before or concomitantly with MOMP, with the majority of Bax translocation and oligomerisation occurring downstream of MOMP. Quantification of YFP-Bax concentrations at mitochondria revealed an increase of only 1.8 + or - 1.5% at MOMP onset. This was increased to 11.2 + or - 3.6% in bak-silenced cells. These data suggested that Bax activation exceeded by far the quantities required for MOMP induction, and that minimal Bax or Bak activation may be sufficient to trigger rapid pore formation. In a cellular automaton modelling approach that incorporated the quantities and movement probabilities of Bax and its inhibitors, activators and enablers in the mitochondrial membrane, we could re-model rapid pore formation kinetics at submaximal Bax activation.

Hepatic tumor-stroma crosstalk guides epithelial to mesenchymal transition at the tumor edge.

The tumor-stroma crosstalk is a dynamic process fundamental in tumor development. In hepatocellular carcinoma (HCC), the progression of malignant hepatocytes frequently depends on transforming growth factor (TGF)-beta provided by stromal cells. TGF-beta induces an epithelial to mesenchymal transition (EMT) of oncogenic Ras-transformed hepatocytes and an upregulation of platelet-derived growth factor (PDGF) signaling. To analyse the influence of the hepatic tumor-stroma crosstalk onto tumor growth and progression, we co-injected malignant hepatocytes and myofibroblasts (MFBs). For this, we either used in vitro-activated p19(ARF) MFBs or in vivo-activated MFBs derived from physiologically inflamed livers of Mdr2/p19(ARF) double-null mice. We show that co-transplantation of MFBs with Ras-transformed hepatocytes strongly enhances tumor growth. Genetic interference with the PDGF signaling decreases tumor cell growth and maintains plasma membrane-located E-cadherin and beta-catenin at the tumor-host border, indicating a blockade of hepatocellular EMT. We further generated a collagen gel-based three dimensional HCC model in vitro to monitor the MFB-induced invasion of micro-organoid HCC spheroids. This invasion was diminished after inhibition of TGF-beta or PDGF signaling. These data suggest that the TGF-beta/PDGF axis is crucial during hepatic tumor-stroma crosstalk, regulating both tumor growth and cancer progression.

Dynamics of outer mitochondrial membrane permeabilization during apoptosis.

Individual cells within a population undergo apoptosis at distinct, apparently random time points. By analyzing cellular mitotic history, we identified that sibling HeLa cell pairs, in contrast to random cell pairs, underwent apoptosis synchronously. This allowed us to use high-speed cellular imaging to investigate mitochondrial outer membrane permeabilization (MOMP), a highly coordinated, rapid process during apoptosis, at a temporal resolution approximately 100 times higher than possible previously. We obtained new functional and mechanistic insight into the process of MOMP: We were able to determine the kinetics of pore formation in the outer mitochondrial membrane from the initiation phase of cytochrome-c-GFP redistribution, and showed differential pore formation kinetics in response to intrinsic or extrinsic apoptotic stimuli (staurosporine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)). We also detected that the onset of mitochondrial permeabilization frequently proceeded as a wave through the cytosol, and that the frequency of wave occurrence in response to TRAIL was reduced by inhibition of protein kinase CK2. Computational analysis by a partial differential equation model suggested that the spread of permeabilization signals could sufficiently be explained by diffusion-adsorption velocities of locally generated permeabilization inducers. Taken together, our study yielded the first comprehensive analysis of clonal cell-to-cell variability in apoptosis execution and allowed to visualize and explain the dynamics of MOMP in cells undergoing apoptosis.

ILEI requires oncogenic Ras for the epithelial to mesenchymal transition of hepatocytes and liver carcinoma progression.

In human hepatocellular carcinoma (HCC), epithelial to mesenchymal transition (EMT) correlates with aggressiveness of tumors and poor survival. We employed a model of EMT based on immortalized p19(ARF) null hepatocytes (MIM), which display tumor growth upon expression of oncogenic Ras and undergo EMT through the synergism of Ras and transforming growth factor (TGF)-beta. Here, we show that the interleukin-related protein interleukin-like EMT inducer (ILEI), a novel EMT-, tumor- and metastasis-inducing protein, cooperates with oncogenic Ras to cause TGF-beta-independent EMT. Ras-transformed MIM hepatocytes overexpressing ILEI showed cytoplasmic E-cadherin, loss of ZO-1 and induction of alpha-smooth muscle actin as well as platelet-derived growth factor (PDGF)/PDGF-R isoforms. As shown by dominant-negative PDGF-R expression in these cells, ILEI-induced PDGF signaling was required for enhanced cell migration, nuclear accumulation of beta-catenin, nuclear pY-Stat3 and accelerated growth of lung metastases. In MIM hepatocytes expressing the Ras mutant V12-C40, ILEI collaborated with PI3K signaling resulting in tumor formation without EMT. Clinically, human HCC samples showed granular or cytoplasmic localization of ILEI correlating with well and poorly differentiated tumors, respectively. In conclusion, these data indicate that ILEI requires cooperation with oncogenic Ras to govern hepatocellular EMT through mechanisms involving PDGF-R/beta-catenin and PDGF-R/Stat3 signaling.