European Journal of Molecular & Clinical Medicine

Chronic liver disease (e.g. cirrhosis) affects brain function. There is a high incidence of mild cognitive impairment and psychomotor slowing in patients with cirrhosis. This condition, known as minimal hepatic encephalopathy (MHE) affects more than 2 million people in the European Union and has serious health, social and economic consequences. There are no effective treatments for MHE. Rat models of MHE reproduce cognitive and motor alterations seen in patients, showing reduced performance in different types of cognitive tests, including learning a conditional discrimination task in a Y maze. We have shown that reduced ability to learn the Y maze task is due to reduced function of the glutamate-nitric oxide (NO)-cGMP pathway in cerebellum, assessed in vivo by microdialysis. This results in reduced formation of cGMP in response to activation of NMDA receptors and impairment of learning ability. We have found that both hyperammonemia and neuroinflammation contribute to impair this pathway. The effect is mediated by enhanced tonic activation of NMDA and GABAA receptors and of MAP-kinase p38. Based on this mechanistic studies, we have designed and tested new therapeutic strategies acting on specific targets in the brain, which have successfully restored the function of the glutamate-NO-cGMP pathway in vivo and learning ability in rats with

Invasive fungal diseases (IFDs) are an increasingly common complication in critically ill patients in Europe and are frequently fatal. Because of changes in treatment strategies and the increased use of antifungal prophylaxis, the epidemiology of IFDs has changed substantially in recent years and infections due to Candida species are no longer the majority in many institutions. In contrast, the emergence of non-Candida IFDs such as aspergillosis, ucrmycosis and fusariosis has increased. Rates of IFD-related mortality in Europe depend on the pathogen, geographical location and underlying patient characteristics, with rates ranging from 28 to 59% for Candida infections and from 38 to 80% for invasive aspergillosis. Early initiation of antifungal therapy is critical for improving outcomes;

Breast cancer remains the leading cause of new cancer cases in women and is responsible for the most cancer-related deaths in women worldwide. The goals of breast cancer treatment are to maintain or improve quality of life, prolong survival, and increase disease-free progression. The majority of breast cancer cases are estrogen receptor (ER)-positive and human epidermal growth factor receptor-2 (HER-2)-negative, and current treatment guidelines recommend multiple lines of endocrine therapy followed by chemotherapy in patients with locally recurrent or metastatic disease. Resistance to current therapies adds to the need for new therapeutic options. Translational research and preclinical data have provided insight into the identification of emerging signaling pathways for novel drug targets, and the development of a growing number of biologic targeted agents is currently underway to identify novel treatments. An alternative approach to improve patient benefit is to boost the efficacy and safety of existing agents by modifying their delivery or pharmacokinetics (ie, adding albumin to paclitaxel) as well as identifying new combination therapies. One combination therapy of interest is the addition of the 130 nm albumin-bound formulation of paclitaxel (nab-paclitaxel) to currently approved therapies or targeted agents in development. This review focuses on a number of key agents that are being investigated for the treatment of HER-2-negative breast cancer and the utilization of these agents as combination therapy to achieve prolonged disease control. Focal points • Bedside ○ New therapeutic options are necessary for breast cancer patients with HER-2-negative and either hormone receptor positive or negative disease who develop resistance to current therapies. Recent insights into molecular pathways may soon expand the treatment options for all patients with HER-2-negative breast cancer. • Bench ○ Several rationally designed combinations of biologic targeted agents and next generation chemotherapeutic agents are currently under investigation to prolong disease control and overcome treatment resistance in patients with HER-2-negative breast cancer.

Chronic liver disease (e.g. cirrhosis) affects brain function. There is a high incidence of mild cognitive impairment and psychomotor slowing in patients with cirrhosis. This condition, known as minimal hepatic encephalopathy (MHE) affects more than 2 million people in the European Union and has serious health, social and economic consequences. There are no effective treatments for MHE. Rat models of MHE reproduce cognitive and motor alterations seen in patients, showing reduced performance in different types of cognitive tests, including learning a conditional discrimination task in a Y maze. Reduced ability to learn the Y maze task is due to reduced function of the glutamate–nitric oxide (NO)–cGMP pathway in cerebellum, assessed in vivo by microdialysis. This results in reduced formation of cGMP in response to activation of NMDA receptors and impairment of learning ability. Both hyperammonemia and neuroinflammation contribute to impair this pathway. The effect is mediated by enhanced tonic activation of NMDA and GABAA receptors and of MAP-kinase p38. Based on these mechanistic studies new therapeutic strategies acting on specific targets in the brain have been designed and tested, which have successfully restored the function of the glutamate-NO-cGMP pathway in vivo and learning ability in rats with MHE. This can be achieved by therapeutic treatments using: a) phosphodiesterase 5 inhibitors (sildenafil, zaprinast), that increase cGMP levels by reducing its degradation b) extracellular cGMP c) antagonists of type A GABA receptors (bicuculline) d) neurosteroids that modulate GABAergic tone (pregnenolone sulfate) e) inhibitors of cyclooxygenase (ibuprofen) which reduce neuroinflammation f) inhibitors of MAP- kinase p38 (SB239063), that reduce microglial activation and neuroinflammation Translation of some of these treatments to clinical practice would improve cognitive function, quality of life and life span of patients with cirrhosis and MHE and reduce health systems costs. Focal points • Benchside The mechanisms underlying cognitive and motor alterations in minimal hepatic encephalopathy (MHE) are beginning to be clarified in animal models. A number of therapeutic targets have been identified to improve cognitive and motor function in MHE. Also, serum level of 3-nitrotyrosine is the first peripheral biomarker identified for diagnosis of MHE in cirrhotic patients, with high diagnostic accuracy, high sensitivity and specificity. • Bedside In the European Union more than 2 million patients with liver cirrhosis show MHE with mild cognitive impairment. MHE is an important, until now underestimated, health, social and economic problem. Early diagnosis and treatment of MHE will significantly improve quality of life and life span of the patients and reduce costs of hospitalization and treatment • Industry There are no specific treatments for the neurological alterations in MHE. A number of therapeutic targets have been identified in animal models to improve cognitive and motor function in MHE. This is a new market waiting for development of appropriate therapeutic treatments which would improve quality of life and survival of patients. Development of a kit for diagnosis of MHE in clinical practice is pending. • Governments – regulatory agencies Early diagnosis and treatment of MHE will significantly improve quality of life and life span of the patients and reduce costs of hospitalization and treatment. Screening of the presence of MHE in patients with liver diseases will reduce costs.

Everybody is at risk for cancer yet environmental factors, life style and diet as well as genetic factors influence the individual cancer risk. Targeted or personalized cancer prevention is based on the knowledge of the molecular characteristics of the tumor to be prevented, the molecular mechanisms of action of the compounds to be used and the genetic make-up of the person who opts for prevention medicine. Genetic factors are to a certain extent specific for cancer types or even subtypes as it has been shown for breast cancer. The growing knowledge of such genotype cancer risk associations will allow for the definition of personalized prevention strategies. Prevention in intermediate risk populations requires non-toxic, well tolerated and cheap compounds, such as Curcumin. Its main activity is the inhibition of nuclear factor kappa B (NFkB) activation. NFkB is involved in many cancers where it acts through the generation of chronic inflammation that can be contrasted with anti-inflammatory drugs such as Curcumin. Targeted prevention of cancer also increases the possibility to conduct serious clinical experimentation with target based patient selection. Focal points: • Bedside Prevention might retard cancer development for years if specifically targeted. Growing knowledge of genetic determinants of cancer risk allows for the selection of individuals for targeted prevention. • Benchside Genetic variants associated with the risk for specific cancer (sub-)types have been identified and additional research is needed in this promising field. The molecular mechanisms of cancer development and their relation with risk variants must be investigated and compounds that can interfere with these mechanisms must be identified. • Industry A major effort in cancer prevention is needed and justified by a large potential market. The major challenge is the design of non-toxic compounds suitable for preventive treatments of healthy people who are at risk of developing cancer • Community Active cancer prevention as opposed to early detection of cancer must become a focus of communication. • Governments More effort in preventive cancer medicine is needed and justified by accumulating evidence for targeted prevention. Cancer prevention can add years of healthy life and can reduce cancer therapy associated costs.

Over the past few decades public health has been immensely improved by preventing various types of diseases using vaccination, a method implying attenuated, killed or part of a microorganism to activate the immune system against it. Recently, nanovaccines have attracted a lot of attention as a new approach for enhancing the immune responses against immunogenic molecules. A wide variety of nanomaterials are reported as proper candidates for nanovaccination. Currently, the focus is the development of new formulations that trigger strong anti-cancer responses by presenting tumor antigens either directly to T immune cells or indirectly to antigen-presenting cells, holding great promise for safe, non-invasive, and cost-effective therapy of cancer. This review focuses on the critical aspects in the design of biomaterial based nanovaccines and reviews the state of the art of the formulations in clinical development and those currently available in the market. Focal points • Bedside Understanding the complex potential of the immune system to prevent and treat various diseases such as cancer, will contribute to the scientific advance in the development of new therapeutic strategies and will allow for the discovery of alternative treatments for deadly diseases. • Benchside Many of the immunostimulative pathways involved in the suppression of cancer tumor growth have been identified and additional research is still needed to recognize the molecular mechanisms of the immunotherapeutic strategies. • Industry The discovery of new non-toxic immunotherapeutic compounds may pave the way towards developing new cancer tumor therapeutic approaches. A major effort in the identification of preventing approaches is also needed to develop preventive nanoformulations for healthy people who are at the risk of cancer. • Community The treatment of cancer by cheap, efficient and as less invasive as possible methodologies will have a great impact on the quality of life of the patients. • Governments Since the development of anticancer nanoformulations needs extensive support from the authorities for success in clinical translation, the financial investments of the governments is necessary to translate the research in the lab to the bedside. The governmental support can also increase the years of healthy life of the patients and minimize the associated costs overtime.

Translational studies conducted in the Center for Cell Biology & Cancer Research at the Albany Medical College integrate the discovery of basic mechanisms underlying the development of human fibrotic disease with in vivo interventional strategies and tissue repair outcomes in animal models. This structured research program is expected to lead to the clinical adaptation of novel therapies specifically directed to the control of pathologically-relevant profibrotic genes in several organ systems. Perhaps the most mature, clinically-relevant, multidisciplinary effort focuses on molecular events underlying the pathophysiology of the renal fibrotic response to tissue injury. This program involves a network of collaborating urological surgeons, nephrologists, graduate students, pathologists, residents, transplant surgeons, basic scientists and molecular biologists and is built on a highly-collaborative framework that fosters translational interactions. This cooperative enterprise resulted in a new appreciation for the complexity of the TGF-β1-activated pathways leading to fibrotic gene expression in an in vivo model of renal injury that mimics obstructive uropathy in humans. Moreover, we have uncovered new, translationally-relevant and therapeutically-accessible, molecular targets. These are the focus of current pre-clinical studies with the goal being to assess their potential utility in the therapy of human renal fibrotic disease. Focal points: Interstitial fibrosis is a progressive disorder that frequently results in organ failure; current treatments are limited and largely ineffective. The majority of fibrotic diseases are irreversible and eventually fatal. Regardless of etiology, elevated tissue TGF-β1 levels and transcription of TGF-β1-responsive genes are linked to the activation of profibrotic signaling pathways. Plasminogen activator inhibitor-1(PAI-1) is a major causative factor in several clinically significant fibrotic syndromes. Translational research in the Center for Cell Biology & Cancer Research at Albany Medical College focuses on molecular events underlying transcriptional control of the profibrotic PAI-1 gene using an animal model of obstructive uropathy. The need for novel targeted approaches for the treatment of fibrosis highlights the clinical potential in the current probe of molecular mechanisms underlying TGF-β1-regulated PAI-1 gene control.

The Space Environment provides many challenges to the human physiology and therefore to extended habitation and exploration. Translational research and medical strategies are meeting these challenges by combining Earth based medical solutions with innovative and developmental engineering approaches. Translational methodologies are currently applied to spaceflight related dysregulations in the areas of: (1) cardiovascular fluid shifts, intracranial hypertension and neuro-ocular impairment 2) immune insufficiency and suppression/viral re-expression, 3) bone loss and fragility (osteopenia/osteoporosis) and muscle wasting, and finally 4) radiation sensitivity and advanced ageing. Over 40 years of research into these areas have met with limited success due to lack of tools and basic understanding of central issues that cause physiologic maladaptation and disrupt homeostasis. We will discuss the effects of living in space (reduced gravity, bone and muscle loss, increased radiation and varying atmospheric conditions [EVA]) during long-duration, exploration-class missions and how translational research has benefited not only space exploration but also Earth based medicine. Modern tools such as telemedicine advances in genomics, proteomics, and metabolomics (Omics-sciences) has helped address syndromes,

hrough its varied instances, technologies and applications, biomedical imaging readily lends itself to translational approaches from in-vitro all the way to clinical. Whereas the disciplines, technologies, scales and scopes vary throughout the translational pipeline, they tend to coalesce when reaching the in-vivo context (e.g., through animal models), which ideally then leads to direct evaluation, validation and application in the human. This presentation will focus on demonstrating such a potential through a few in-vivo examples using imaging in a translational context, for providing and exploiting new biomarkers as well as to affect clinical workup, from diagnosis to therapy planning and follow-up. Suggested implementation strategy for suitably supporting such a multidisciplinary effort in the scope of cancer as an example will also be presented.

Light Microscopy is one of the most valuable and versatile tools in biomedical research. Imaging “infiltrated” all kinds of natural science categories, serving cell and developmental biologists, pathobiologists, physicists, material scientists, chemicals, pharmaceutical specialists and clinicians, thereby acting as THE “translational” method. In recent years we observed fast and revolutionary development of new biooptical technologies, as well as a boom in fluorophore engineering towards advanced functionalities.

Acute kidney injury (AKI) is a very relevant and increasing health and socioeconomic problem worldwide. In critically ill patients, mortality to AKI may reach up to 50-80% of the cases. Even mild, spontaneously reversible episodes of AKI have a significant impact on medium and long term morbidity and mortality. Drug nephrotoxicity is among the most important causes of AKI, with 25% of the 100 most used drugs in intensive care units being nephrotoxic. AKI has been traditionally diagnosed when extensive renal damage gave way to signs derived from renal dysfunction, such as the elevation of plasma creatinine concentration, according to well established methods of stratification, including the AKIN and RIFLE criteria. In the last decade, further advance was provided by the identification of earlier markers of kidney injury, including KIM-1 and NGAL. Yet, more sensitive and specific markers, or combinations of markers, are needed to improve AKI diagnosis. However, new facets of AKI diagnosis emerge for a more personalized and preventive handling of this disease. In the last years, our group has been working on two new diagnostic concepts, namely: i) pre-emptive detection of drug-induced predisposition to AKI, as differentiated from early diagnosis; and ii) aetiological (drug-specific) diagnosis of AKI, potentially applicable to polymedicated patients. Drug-induced predisposition to acute renal failure (ARF) is a facet of nephrotoxicity hitherto mostly uncharacterized, quite underestimated, and impossible to diagnose, which potentially has a high human and socioeconomic impact. Our study has identified urinary GM2AP as the first of a new class of biomarkers of the enhanced risk of suffering an acute renal failure after a subnephrotoxic treatment with gentamicin. Gentamicin-predisposed animals with no sign of renal injury develop ARF when exposed to a second potentially nephrotoxic drug, also given at subnephrotoxic doses that are harmless to non-predisposed individuals. Subnephrotoxic gentamicin did not alter renal GM2AP gene expression or protein levels, determined by RT-PCR and Western blot and immunostaining, respectively, nor was its serum level modified. Further experiments indicate that, likely, the origin of the increased level of GM2AP in the urine might be a defective tubular handling of this protein as a consequence of gentamicin action. Markers of risk may revolutionize the prevention of ARF by enhancing our monitoring capacity of acquired predisposition to ARF, in a pre-emptive manner. With regard to the aetiological diagnosis of drug nephrotoxicity, we have identified regenerating isletderived protein III beta (reg IIIb) and gelsolin as potentially differential urinary markers of gentamicin’s nephrotoxicity. Indeed, both reg IIIb and gelsolin urinary levels differentiate the nephrotoxicity caused by gentamicin from that caused by cisplatin.

Hepatocellular carcinoma (HCC) remains a significant unmet medical need with very limited therapeutic options available. Although microRNA-21 (miR-21) has been shown to be upregulated in HCC, its contribution as an onco-miR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miR-21) and used them to interrogate dependency on miR-21 in a panel of 20 commercially available HCC cell lines in vitro. Upon lipid-mediated transfection, anti-miR-21, but not its mismatched (MM) control, caused significant de-repression of known direct targets of miR-21, inhibited survival of a large subset of HCC cell lines. Sensitive HCC cell lines showed dose- and time-dependent induction of caspase 3/7 activity upon treatment with anti-miR-21. In contrast, non-responder HCC cell lines failed to significantly upregulate caspase activity and maintained viability in the presence of anti-miR compound. To better understand the consequences of miR-21 suppression in HCC,

Background
Ionizing radiation induces lipid peroxidation and forms reactive oxygen species (ROS) within the cell. Due to their highly reactive state, ROS have short diffusion distances and quickly transfer unbalanced electrons to neighboring molecules. This transfer cycle continues until it finally culminates in the generation of reactive alkenals such as 4-hydroxynonenal (4HNE). Under normal conditions, intracellular 4HNE levels are controlled by conjugation to glutathione and are actively transported from the cell; RLIP76 protein has been determined to be the major transport protein involved in the efflux of 4HNE-conjugates. Radiation induces significant oxidative stress, and the increased levels of 4HNE conjugates overwhelm transport capacity. When this occurs, further conjugation is inhibited and free 4HNE levels rise, triggering apoptosis. It was hypothesized that adding exogenous RLIP76 protein would reduce 4HNE levels and correspondingly increase the recovery from acute radiation syndrome (ARS) or completely protect individuals exposed to lethal doses of radiation. Methods The National Institutes of Health has developed animal models of ARS so that medical countermeasures can be tested in accordance with the FDA Animal Rule, since testing in people is clearly not ethical. One of these models, the C57BL/6 mouse, has been successfully utilized by Terapio to evaluate the efficacy of recombinant human RLIP76 encapsulated in liposomes (RLIP76-PL). These studies were 30-day survival studies of mice exposed to total body irradiation of 7.45-8.75 Gy. Administration of RLIP76-PL was subcutaneous or intramuscular on a prophylactic (administered up to 20 h prior to irradiation) or therapeutic (administered up to 36 h after irradiation) schedule.
Results
As a prophylactic, there was 100% survival compared to 33% of the controls when mice were given three doses and exposed to 8.1 Gy. As a therapeutic, the drug is not administered until 24hrs after irradiation and remarkably, there is 92% survival compared to 8% of the controls.

In this presentation quantitative as well as qualitative analyses are reported regarding the communication strategies in the field of cancer prevention in the English-speaking world as compared to Italian-speaking world. The quantitative analyses were based on the occurrence of specific keywords in the global literature accessible via internet as well as on the chromatic -complexity investigated in a total of 1400 images. Such images refer to the “prevention” field and were taken from internet English-domains and Italian-domains.

Ewing sarcoma is the second most frequent primary tumor of bone, preferentially occurring in children and adolescents. Despite significant improvements have been achieved in localized tumors thanks to dose intensification of chemotherapy, outcome of patients with metastasis at diagnosis remains grim. In addition, even in the most favorable situations, patients must face with important side effects which significantly impact their quality of life. Either scientific and patient communities are now very sensitive on the need of new drugs, which may reduce chemotherapy toxicity while maintaining effectiveness of current regimens. We present here a new engeneered human bivalent single chain fragment variable diabody (C7 scFv diabody) directed against CD99, a transmembrane protein whose high expression characterizes Ewing sarcoma. The triggering of CD99 with C7scFv diabody induces rapid and massive Ewing sarcoma cell death through MDM2 ubiquitination and p53 reactivation. Accordingly, the most CD99-responsive Ewing sarcoma cells have transcriptional active p53 and greatly benefit from MDM2 degradation. CD99 triggering also potentiates the cytotoxic effect of doxorubicin in vitro and in vivo and reactivates p53 to a much greater degree, which in turn markedly increased expression of pro-apoptosis genes. Evaluation of Ki-67 labelling and apoptosis rate by TUNEL confirms the efficacy of the treatment in xenografts.

Metabolic phenotyping comprises the quantification of endogenous metabolites in biofluids, cells, and tissues. It provides insights into normal as well as aberrant metabolic pathways and biological processes, which is important for the understanding of disease phenotypes. It also allows the identification of biological markers, which can serve as early disease indicators and therapeutic markers for the evaluation of treatment effects. As metabolic markers are not species-restricted, the concept of metabolic phenotyping is highly applicable for translational research. Species independence allows the use of established animal and cell culture models for various diseases within a preclinical context. However, differences in the metabolic set-up of study organisms compared to humans needs to be taken into consideration to prevent misleading conclusions from otherwise valid experimental designs. To determine species-related metabolic differences, a targeted metabolomics approach was applied using a mass spectrometry platform for the quantification of a predefined set of endogenous metabolites, i.e. amino acids, biogenic amines, phosphatidylcholines, sphingomyelins, hexoses, steroid hormones and others. Results from this species comparison on the metabolic level will be presented.

Next generation sequencing (NGS) technologies are now widely used in medical research. NGS provides an unprecedented opportunity for high throughput analysis of genetic variations warranting their use in molecular diagnostics. However, among other obstacles, their adoption in clinics poses challenges in the provision for accurate and timely data analysis. A bioinformatics workflow for the analysis of the large amount of data from raw reads to final annotated variants suggesting their functional consequences with clinically actionable or reportable sensitivities and specificities is important. We have developed a number of targeted panels using NGS for the diagnosis of a number of haematological abnormalities and cancer.

The appearance of malignant cells in body fluids like urine, blood or body-cavity fluids are a clear indication for the existence of a tumor and urine or body-cavity fluid cytology are routine diagnostics today. Cytologic examination of the cellular features of fluids is a valuable adjunct to patient diagnosis and the staging and management of tumors. The German-language literature contains the earliest references to the cytology of malignant cells in fluid specimens. Preparation of the specimen has evolved from unstained wet smears to protocols that generally include centrifugation and the generation of stained smears and a cell block. The smears may be alcohol-fixed direct smears, cytospins, or a liquid-based preparation, and they are usually stained with the Papanicolaou method. Additional techniques, such as immunocytochemistry and flow cytometry, provide significant help in this differential diagnosis. Evaluation of microscopic images after Papanicolaou staining eluded digital pathology, an image-based information environment enabled by computer technology that allows for extracting information from a digital slide. With the advent of full-slide scanning digital methods are regarded as promising way to achieve better, faster and cheaper diagnosis, prognosis and prediction of cancer and other important diseases. One important feature are combinations with immunostaining, FISH technology etc., to elude additional information from the specimen. Circulating tumor cells (CTCs) can be found in the bloodstream and are always ready to attach to endothelial cells lining blood vessels and extravasate to enter tissues and organs to form a metastatic site. They show plastic phenotype and a small number of these cells undergo the epithelial-to-mesenchymal (EMT) program. De-differentiation and dissemination from the primary tumor is a basic prerequisite for colonization and growth of distant metastasis. Phenotypic and functional plasticity of cancer cells and the ability to adapt permanently to demanding conditions provide great challenge for identification and characterization of CTC´s from blood

Over the last two decades, prognostic and predictive biomarker studies in clinical and translational research settings have become synonymous with tissue microarrays (TMAs). TMAs are essentially “tissue archives” created by repeated transfer of small tissue cores from formalin-fixed paraffin-embedded tissues (“donor” blocks) into empty paraffin blocks (“recipient” blocks). In this manner, more than 500 different tissue spots can be arrayed onto a single TMA. TMA applications include the investigation of morphology, protein and gene expression or chromosomal aberrations. These can be visualized using H&E stains, immunohistochemistry (IHC), fluorescence and chromogenic mRNA or miRNA in situ hybridization (ISH). Despite many advantages such as cost-effectiveness, conventional tissue microarraying has several major drawbacks: it is a laborious, time-consuming and does not allow for precise tissue regions/structures to be captured. For studies aiming to investigate specific histological regions (e.g. interface between tumor and stroma) or particular cell types, conventional TMAs fall short. Our Translational Research Unit at the Institute of Pathology, University of Bern has developed next-generation TMAs (ngTMAs). ngTMA represents a process of TMA consulting, followed by slide scanning and digital pathology, as well as rapid, precise and automated TMA construction. Briefly, consulting in all aspects of TMA design/construction is discussed including histopathology and statistical considerations. Next, case review is performed and the best slide and paraffin block for subsequent TMA annotation. The selected slides (H&E or other staining) of all cases are digitally scanned. Digital images of each slide are then uploaded onto the slide management platform

European Regulation 726/2004/EC (Article 83) and Directive 2001/83 (Article 5), provide a regulatory framework for access to investigational medicines outside the context of the clinical trial, allowing physicians to access potentially life-saving medicines that would otherwise be unavailable for their patients. Treatment with an investigational product represents an important option for patients suffering from serious or life threatening conditions where licensed alternatives are either unavailable or unsuitable for the patient. They can often be the only treatment option for disease areas of high unmet need such as rare diseases and orphan indications. Whilst the pharmaceutical industry has continued to focus on accelerating access to innovative new treatments by shortening the development timelines; increased regulatory challenges and delays due to pricing & reimbursement negotiations can result in delays of many years between positive phase III trials and commercial availability. Access Programs hence provide an important mechanism to bridge the time between clinical development, marketing authorisation and product launch. Although this EU framework exists, each member state has decided independently how and when to allow such access, and developed national rules and legislation to reflect this.

Personalized Medicine currently focuses on the right treatment for the right patient, but its long-term goal includes personalized risk assessment and prevention. Current emphasis focuses on advances in genetic testing and biomarker to enhance patient care. The considerable data generated by such approaches and access to patient EHR’s has led to many statistically-based studies to predict disease risk and prognosis, e.g. the Gail model for breast cancer risk assessment; evaluation of BRCA mutation profiles; and expression level analysis of her2/neu for Herceptin response. Such correlative analysis has been used to enhance clinical-decision making but is limited in its potential for understanding mechanisms of risk and disease. We have extended these correlative approaches to include systems-based process modeling, extending from pre-disease risk to early detection, treatment and outcome, in an effort to develop models for testing and validation against both existing data and enhanced data collection. Development and testing of this approach in breast cancer will be presented. Risk Assessment We have begun to model risk assessment by including specific aspects of a patient’s physiological development to help identify risk factors that can lead to improved guidelines for risk prevention. This can also identify specific causes for risk and disease through the course of normal breast development. Risk from any specific factor is something that changes over a person’s lifetime and is not likely to be constant. Simple statistical correlation of a risk factor, i.e. do you smoke? do you smoke more than 1 pack of cigarettes per day? needs to evolve to show how risk from each factor varies over a person’s lifetime/stage of development. This is because the molecular processes underlying physiological change also vary. Methods Risk factors were identified from the literature (from RR=1.0 to >4.0) and compared with those in the Gail model and then a physiological model of breast development, from pre-menarche to menopause, was drafted. These included both those included in the Gail model as well as other biomarkers, e.g. breast-feeding history, radiation exposure, oral contraceptive use, etc. A data set that represented a combination of actual and simulated patients, with 1458 patients in each, was used for the analysis. Univariate analysis was performed and comparison between the Gail model results and our models was performed using ROC analysis. Subsequent refinement eliminated several variables from consideration in the model.

Conventional cancer therapies are limited by their toxicity and lack of specificity. To achieve targeted immunotherapy of cancer, we have chosen Wilm's Tumour antigen (WT1) as a target as it is over-expressed in most leukemia and many solid cancers. Using sophisticated WT1-TCR retroviral constructs, we have performed in vivo engraftment studies with CD34+ leukemic progenitor cells. In our model, treatment with WT1-TCR engineered patiens' T cells had cleared patients own leukemic cells. As the analysis of bone marrow indicated that control group showed evident engraftment of human leukemia cells, while the WT1-TCR treated group had none detectable.

Introduction Autoantibodies specific for malondialdehyde modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, the generation of MDA-LDL results in the formation of many different epitopes with high variability. Because it is not known, which MDA epitope is biological important, the aim of this study was to identify and characterize peptide mimotopes of MDA-LDL that could be used as antigens to improve the reproducible detection of MDA-specific autoantibodies. Methods and
Results
Peptide phage display libraries were screened for phages binding to the MDA-LDL specific natural IgM antibody LR04. After biopanning two consensus sequences (P1 & P2) of binding phages were synthesized. P1 and P2 were specifically bound by LR04, as the binding of LR04 to coated peptides was fully competed by MDA-LDL but not native LDL. P1 and P2 were also bound by other MDA specific murine (EN1) and human (IK17) antibodies. Furthermore, the binding of LR04 to late apoptotic cells was completely inhibited by both peptides, identifying them as mimotopes of naturally occurring epitopes on dying cells. Immunization of C57BL/6 mice with P2 conjugated to BSA, but not BSA alone, resulted in the robust induction of IgG1 and IgM antibodies against MDA-LDL. Moreover, serum IgG of immunized mice specifically stained epitopes in atherosclerotic lesion of rabbits and humans. Finally, we measured anti-mimotope antibody titers in serum samples previously collected from healthy subjects (n=17) and from patients (n=140) with stable angina pectoris undergoing percutaneous coronary intervention (Tsimikas, 2004). In patients a significant positive correlation was observed between anti-MDA-LDL and anti-mimotope IgM (P1, r=0.8; P2, r=0.6; p

Rare diseases are designated as affecting less than 200,000 individuals (US) and of the approximately 7000 designated rare diseases, the majority of these occur in pediatric patients, and across international boundaries. An example is pediatric ARDS (Acute Respiratory Distress Syndrome) that is not diagnosed until a previously healthy child presents in the pICU with severe symptoms and in which more children die each year than from cystic fibrosis and leukemia, combined. The Nathaniel Adamczyk Foundation (NAF) is focused on identifying risk factors and opportunities for prevention of this devastating disease. Both the diagnosis and patient management are challenged by having to deal with a syndrome in a critical care situation in a heterogeneous patient population. NAF has undertaken the development of an (inter) national tissue and data repository to support both clinical research and enhanced clinical decision support for patient management. Creation of an analytical platform to integrate, access and analyze temporal clinical data ranging from the pICU to also incorporate neo-natal ICU and pregnancy history is underway with a prototype already in testing. Analytical methods are being evaluated in collaboration with Dr. Mike Quasney (Medical College of Wisconsin) and the Virtual Pediatric ICU and PALISI (Pediatric Acute Lung Injury and Sepsis Investigators). This effort is exploring expanded international partnerships in both Europe and China to increase the accessible data for analysis and to further participate in the development of better diagnostic standards. We will present the initial state of both the analytics and platform development to encourage extension of this international effort to interested clinicians and clinical researchers.

The last two decades has seen a significant increase in the development of medicinal products to treat rare “orphan” diseases, largely due to the EU Orphan Medicinal Product Regulation (2000), but also because of the consistent advocacy by patient groups prior to this regulation. These groups from across Europe joined forces under the umbrella of EURORDIS, to secure the implementation of the regulation and ensure that there was patient representation on the committee at EMA (the European Medicines Agency) which would be responsible for assessing and giving opinions on applications, namely COMP (The Committee for Orphan Medicinal Products). As its Vice-Chair I have the privilege to ensure the patient perspective is always considered during these assessments.

The use of biomarkers is becoming increasingly integral to to the contemporary practice of medicine and continues to play a central role in preventive, predictive and personalized medicine. However, the limited number of FDA-approved, in use biomarkers, on one hand, and an increasing number of published potential biomarkers, on the other hand, calls for an accelerated approach to translating biomarker research to clinical application.

The environment of space results in a multitude of challenges to the human physiology that present barriers to extended habitation and exploration. Over 40 years of investigation to define countermeasures to address space flight adaptation has left gaps in our knowledge regarding mitigation strategies partly due to the lack of investigative tools, monitoring strategies, and real time diagnostics to understand the central causative agent (s) responsible for physiologic adaptation and maintaining homeostasis. Spaceflight-adaptation syndrome is the combination of space environmental conditions and the synergistic reaction of the human physiology. Our work addresses the role of oxidative stress and damage (OSaD) as a negative and contributing Risk Factor (RF) in the following areas of combined spaceflight related dysregulation: i) radiation induced cellular damage [1,2] ii) immune impacts and the inflammatory response [3,4] and iii) varicella zoster virus (VZV) reactivation [5]. Varicella-zoster (VZV)/Chicken Pox virus is a neurotropic human alphaherpesvirus resulting in varicella upon primary infection, suppressed by the immune system becomes latent in ganglionic neurons, and reactivates under stress events to re-express in zoster and possibly shingles.

The talk will start from a brief review of the physical basis of microwave imaging for medical diagnostics and of the challenges that have to be faced in this technology, to present three areas which are possibly the most promising ones for a fruitful application of microwave imaging in the medical arena. The first one is the monitoring of brain injuries, which is a topic of increasing importance for its impact on the European health system in the ageing society. In particular, it will be discussed how microwave imaging can play a role both in the detection of the diseases in the early stage and in their clinical follow-up, by filling the gap between current diagnostic modalities and the need of continuous monitoring at the patient's bed. The second one is the potential of enhancing the capabilities of microwave imaging by means of contrast agents. Indeed, while contrast enhancement is a common practice to improve performances in medical imaging,

Breast cancer is the most common cancer in women worldwide, with nearly 1.7 million new cases diagnosed in 2012. This represents about 12% of all new cancer cases and 25% of all cancers in women. The current standard method for detecting non-palpable early stage breast cancer is X-ray mammography. Despite the fact that X-rays provide high-resolution images at low radiation doses, its limitations are well documented. In the U.S., up to 75% of all malignancies identified by X-ray mammography are later found to be benign after biopsies. These false positive conclusions result in unnecessary biopsies, causing considerable distress to the patient and an unnecessary financial burden on the health service. Much more worryingly, up to 15% of all breast cancers present at the time of screening are missed by conventional mammography, often delaying treatment to the point where it’s no longer effective.

Short introduction to microwave hyperthermia from the point of view of biology and physics will be given firstly. The physical basis of microwave thermotherapy for cancer treatment and for other medical microwave therapeutic purposes (e.g. in cardiology, urology, surgery, physiotherapy, etc.) will be described in this talk. Different kinds of hyperthermia clinical applications will be mentioned (i.e. local, deep local, regional and intracavitary treatment). Different physical and technological approach to describe these above given different cases will be discussed. For each of these mentioned cases a different type of electromagnetic (EM) wave should be used: EM plane wave for local treatment, converging cylindrical EM wave for regional treatment and finally diverging cylindrical EM wave for intracavitary treatment. Then different types of applicators (resp. antennas) for microwave hyperthermia clinical applications will be discussed (e.g. waveguide, waveguide horn, evanescent mode, planar, array, lens, metamaterial etc. applicators). Each of these microwave technologies has its specific advantages in creation of the optimal SAR and temperature distribution in the area to be treated. It is given by its specific EM field distribution in the aperture of these applicators. And the importance of the so called treatment planning will be discussed. It is based on several different numerical methods (e.g. FDTD, FEM, MOM etc.) for calculation of the SAR in the treated area firstly and afterwards for calculation of the temperature distribution in the treated area with respect to the time, blood perfusion, etc.

This talk addresses the development of imaging techniques for the early detection of breast cancer, based on Ultra Wideband (UWB) radar, a promising emerging technology that exploits the dielectric contrast between normal and tumour tissues at microwave frequencies. Of particular interest in this work are issues related to techniques for classification of potential breast tumours into benign and malignant. This is particularly important given the results from recent studies of the dielectric properties of breast and tumour tissue, which have found that strong similarities exist between the dielectric properties of malignant, benign and normal fibroglandular breast tissue. This creates a more challenging imaging scenario and motivates the development of enhanced signal processing techniques for UWB imaging systems. Tumour growth and development patterns are modelled using Gaussian Random Spheres, using four discrete sizes and four different shapes. 62 Abstracts / New Horizons in Translational Medicine 2 (2015) 55–71 Feature extraction methods including Principal Component Analysis (PCA), Independent Component Analysis (ICA) and Discrete Wavelet Transform (DWT), are used to extract the most relevant features from the detailed Radar Target Signatures of the tumours, which are then classified with a number of different classification techniques: Linear Discriminant Analysis (LDA), Quadratic Discriminant Analysis (QDA) and Support Vector Machines (SVM). In addition to these techniques, a number of different multi-stage classification architectures are considered. The feature extraction and classification algorithms are evaluated for both homogeneous and heterogeneous breast tissue models, for a range of different tumour sizes and shapes.

Worldwide, nephrotoxicity poses a considerable health and economic burden. Nearly 25% of the top 100, most used drugs in intensive care units are potentially nephrotoxic. Moreover, nephrotoxicity causes 10-20% of the acute renal failure cases (ARF). ARF is a very serious condition with high incidence and mortality rate, which is estimated at approximately 50% of the cases despite dialysis application, especially within critically ill patients. Mortality increases to 80% when ARF courses with multi-organ damage. The clinical handling of renal injury and ARF is difficult and expensive because, other than dialysis, there are no available treatments. For this reason the search for strategies to prevent nephrotoxicity constitute an active area of investigation. In addition to drug targeting and medical chemistry for new and safer molecules, a line of interest is the identification of renoprotective adjuvants for co-administration along with potentially nephrotoxic drugs. At the preclinical level, many chemically unrelated antioxidants have been shown to protect the kidneys from cisplatin nephrotoxicity, especially in experimental animal models. They include curcumin, N-acetylcysteine, naringenin, selenium, vitamin C, vitamin E and other dietary components that scavenge free radicals formed by exposure to cisplatin. Although promising, antioxidants have not yet demonstrated a clear benefit in the clinical research conducted so far, which requires further investigation. In this line, a pre-clinical selection of candidates to be assayed at the clinical level must be pursued in order to (i) improve the efficacy of the preclinical-to-clinical transition; and (ii) to reduce early failure rate in clinical assays through the drug discovery process. One of the main problems identified in the translation of antioxidants to the clinical practice is their very low bioavailability derived from a very low absorption upon oral administration. Our research line has been focused on the effect of the natural antioxidants resveratrol and quercetin, and the antidiabetic metformin, at preventing drug nephroxicity.

Translational medicine science and the volume of information generated in this field have grown exponentially in the last decade and continue to grow faster every day. This has generated a huge amount of data. The application of Modeling and Simulation (M&S) in drug development has also grown in the last two decades, but mostly has been limited to analysis of single studies or to analysis of pooled data from several studies. Such application traditionally has been used either to support a new drug application or to make Go/No Go decisions about a given development program. However, rarely M&S has been integrated as a tool in portfolio management based on a quantitative evaluation of all the data in hand (e.g., translational Medicine data). In other words, many organizations utilize M&S still as a tool aiding study data analysis or at best a tool to guide a given development program, but not use M&S in portfolio management systematically (the large dashed box shown in the Fig. 1).

Special focus and aim of our research activities at AIT, the Austrian Institute of Technology, is to define reliable biomarkers suitable for early and non-invasive disease diagnosis from body fluids such as serum/plasma and saliva. Along a selection of research projects, which are described in more detail underneath, we will present and introduce the broad portfolio of high throughput technologies we successfully apply for diagnostic biomarker discovery and validation. As a first show case of successful non-invasive disease biomarker discovery we will present a study where we investigated and compared the genome wide methylation levels of lung cancer patients, patients suffering from lung fibrosis, patients with COPD (chronic obstructive pulmonary disease), and DNA samples derived from healthy lungs. Along this study we could identify specific methylation patterns for each of these lung diseases. After quantitative PCR validation of 240 disease specific methylation markers in the discovery sample set, the 90 top markers were picked and applied for serum testing (n=204). When we applied gradient boosting classification for differential diagnosis of tested lung diseases and healthy controls an AUC value of 0.95 was reached here to separate cancer from all other non-cancer samples whereas in differential diagnosis of healthy-, COPD and fibrosis patients AUC values of 0.71 and 0.49 were obtained for fibrosis, respectively COPD. Thus in case of COPD the presented method may be used to monitor cancer risk within COPD patients.

The differential diagnosis of biliary stenosis is a critical problem for gastroenterologists. An early identification of malignant lesions would enable the rapid resort to surgical resection which currently represents the only potentially curative option. Unfortunately, the diagnostic value of all available methods (e.g. imaging technics, standard serum biomarkers) is limited by relatively poor accuracy and negative predictive value. Recently, our group and others highlighted new potential cancer biomarkers in bile by using comparative proteomic analysis. Nevertheless, to date, only a few candidates have been verified for their diagnostic performances in discriminating between malignant and non-malignant stenoses. In addition, no data have yet been collected on the simultaneous measurement of these proteins with the intent of evaluating the diagnostic interest of a panel of biomarkers.

Function of autoantibodies from patients with autoimmune diseases in malignancies development is not clear yet. It has been reported that a cell-penetrating lupus autoantibody, 3E10, which was isolated from a mouse model of systemic lupus erythematosus (SLE), has been a potential targeted therapy for DNA-repair deficient malignancies. We have got four human monoclonal antinuclear antibodies from patients with autoimmune disease, 3B5, 3C1, 3E8 and 4F3. Our data showed that four antibodies could combine HEp-2 cells and display different nuclear types as antinuclear antibody (ANA). Also, these four ANAs can inhibit HEp-2 cells proliferation. We think these antibodies may be potential antibody drugs to cancer therapy. However, the function and mechanism are not clear. Further study, we want to clarify the effects of four ANAs on proliferation of various cancers cells and to investigate the mechanism of four ANAs affecting various cancers cells proliferation and their targets.

Varicella zoster virus (VZV) is a ubiquitous human herpesvirus typically causing childhood varicella (chickenpox) at which time a life-long latent infection is established in ganglionic neurons throughout the neuraxis. Reactivation of latent virus, typically in the elderly and immunocompetent usually causes zoster (shingles) but can also result in serious neurologic disease. In cases of vasculopathy, meningoencephalitis and myelitis where VZV is suspected, diagnosis requires detection of virus DNA or antibody in CSF. In collaboration with NASA, VZV DNA was found in saliva of health astronauts suggesting asymptomatic virus reaction due to the stress of spaceflight. This lead to a series of studies indicating virus DNA can be found in saliva of patients with VZV associated neurologic disease.

Sepsis and sepsis-associated multiple organ failure are associated with extensive tissue damage caused by over-activation of the innate immune system and by the excessive release of inflammatory mediators. The development of targeted therapies for sepsis remains a major challenge due to the complex network of inflammatory mediators involved in the septic process. Early detection and timely therapeutic intervention are crucial for improved outcome of patients with sepsis. Currently however, the diagnosis of sepsis mostly relies on general symptoms. Taking into account the extreme heterogeneity of septic patients, the application of supportive extracorporeal therapies to modulate the concentration of inflammatory mediators requires diagnostic tools to monitor the inflammatory profile of the patients in order to identify the optimal time window for application of supportive therapies. Here, we report on the development of extracorporeal adsorption systems for cytokine modulation and on the development and validation of a novel array technology to detect markers of inflammation (interleukins 6 and 10, Abstracts / New Horizons in Translational Medicine 2 (2015) 55–71 65 C-reactive protein, procalcitonin, serum amyloid A) in a bedside-approach (detection from whole blood samples within 30 min)

Polycystic kidney disease (PKD), in its autosomal recessive (AR) or autosomal dominant (AD) form, is characterized by the formation and expansion of numerous fluid-filled cysts within the kidneys. Quite often, the disease spreads to extrarenal territories including the liver. In addition to cyst formation, interstitial collagen deposition or scarring is sometimes observed in both kidney and liver. Progressive enlargement of the kidneys via replacement of the renal parenchyma with cysts and decreasing renal function makes ADPKD the leading genetic cause of renal transplantation. Highly aggressive fibrocystic kidney and liver disease in ARPKD means that many children with this form of disease do not live past the age of ten years. Using the PCK rat model of PKD, we have identify a minimally invasive biomarker cluster with high correlative value for fibrocystic disease progression. These results are important in that patient compliance, disease prognosis, interventional decisions and outcomes can be further and vastly improved by identification of minimally invasive or non-invasive biomarkers that are prognosticative of disease progression. Furthermore, rather than rely on a single biomarker, clinical outcomes may be better predicted by identification of a cluster of disease-relevant biomarkers which would bring increased correlation with disease progression. Clinical trials of therapeutics for chronic fibrotic diseases would also benefit from identification of such biomarkers given Big Pharma’s reluctance to invest in trials wherein endpoints could be years away with no interim hint of success/failure. Identification of minimally invasive or non-invasive biomarkers in proliferative fibrocystic disease can better stratify children waitlisted for scarce kidneys and/or livers. The tangible outcome/technology/product that will result from the proposed research is biomarker-cluster chips designed to read urine or serum samples to determine disease progress or remission from disease.

Bacterial meningitis is usually fatal without treatment and prompt and accurate diagnosis coupled with the timely administration of parenteral antibiotics are necessary in order to save lives. Despite the availability of highly effective antibiotics, the complications from bacterial meningitis (such as deafness, hydrocephalus, seizures and cerebral palsy) remain high. In areas with a high incidence of human immunodeficiency virus infection, Streptococcus pneumoniae is the commonest cause of bacterial meningitis. The diagnosis of bacterial meningitis can sometimes be delayed whilst samples are analysed in a laboratory using traditional methods of microscopy and antigen testing. We used cutting-edge high definition and quantitative mass spectrometry to identify specific protein signatures in cerebrospinal fluid associated with Streptococcus pneumoniae infection which could lead to the development of assays or point-of-care devices to improve the speed and accuracy of diagnosis, and consequently to enhance the prognosis of adults and children with bacterial meningitis. A range of samples (cases and controls, n=12) from Malawian children has been analysed. Our data indicate some clear trends, and confirm that quantitative proteomics analysis will be successful in generating a comprehensive protein list from which markers might be nominated. We identified a total of 519 proteins in data dependent discovery proteomics and obtained quantitative data for 161 proteins using data independent Hi3 quantification. Using Progenesis LCMS we obtained a list of 202 potential candidates using data dependent acquisition approach and 109 using data independent acquisition, 82 proteins being common to both workflows.

Claudins are transmembrane proteins constituting one of three tight junction protein families. There are 24 members of the claudin family identified, differently expressed in various cells/tissues and among species. In patients with inflammatory bowel disease (IBD), site and disease activity dependant changes in expression of certain claudins has been noted. The aim of the study was to explore expression of claudins in the mouse models of IBD and to compare it to claudin expression in human disease. The expression of several sealing claudins that are present in colon of humans and rodents has been evaluated by immunohistochemistry. A decrease in claudin 1 expression was observed in a chronic mouse DSS model and adoptive transfer model of colitis, as it has been reported in human disease. Claudin 3 expression was not altered in the non-inflamed mucosa. Nevertheless, a subset of claudin 3 was internalized into cytoplasm of absorptive cells in inflamed mucosa in a chronic DSS model. In an adoptive transfer model of IBD, 8 weeks post-transfer, a reduction in claudin 3 expression was noted in surface colon epithelium as noticed in patients with Crohn's disease and ulcerative colitis. Claudin 8 expression decreased in the upper part of crypts, as is reported in patients with Crohn’s disease. A subset of crypt base cells became strongly positive. Finally, a decrease in claudin expression in inflamed mucosa of human biopsies from patients with ulcerative colitis (UC) was observed.

Calcium pumps and channels modulate cell proliferation and apoptosis by regulating intracellular calcium (Ca2+). The plasma membrane Ca2+ ATPase isoform, PMCA2, is a calcium efflux mechanism that extrudes Ca2+ from the cytosol into the extracellular space. PMCA2 has a restricted expression, including expression in cochlear hair cells and cerebellar Purkinje cells. PMCA2 expression is increased in mouse mammary glands during lactation where it plays a major role in the excretion of Ca2+ into milk; however, PMCA2 expression has not been assessed in human breast tissue exhibiting lactational changes. Our previous studies have shown that PMCA2 mRNA levels are elevated in some breast cancer cell lines and that pan-PMCA antisense attenuates the proliferation of MCF-7 breast cancer cells. However, the consequences of silencing PMCA2 in breast cancer cells are still not well understood. Our study assessed PMCA2 expression in breast tissue exhibiting lactational change and in human malignant breast tissue samples. The role of PMCA2 in the proliferation of breast cancer cells was also evaluated. Immunohistochemistry using a rabbit anti-PMCA2 antibody showed membranous PMCA2 expression in the luminal epithelium of breast tissue exhibiting lactational change. PMCA2 expression was assessed in human breast tumor samples assembled into tissue microarrays. Nine of 96 breast tumours (9.4%) showed membranous PMCA2 staining. PMCA2 expression did not significantly correlate with the breast cancer pathological markers, estrogen, progesterone or HER2 receptor status. High-content imaging demonstrated that PMCA2 silencing in MDA-MB-231 breast cancer cells is associated with a reduction in cell number and an inhibition of the percentage of S-phase positive cells. The effect of PMCA2 silencing combined with various cytotoxics (cisplatin, doxorubicin or mitomycin C) on cell proliferation was assessed in MDA-MB-231 cells using a kinetic imaging system (IncuCyte). The results showed that PMCA2 silencing promotes the effects of some cytotoxics.

The tumor necrosis factor (TNF) ligand and cognate TNF receptor superfamily constitute an important immunoregulatory axis pivotal for the correct execution of immune responses. TNF ligand and receptor family members among others are involved in induction of cell death in malignant cells as well as in providing co-stimulatory signals that help mount effective anti-cancer immune responses. This diverse and important regulatory role in immunity has sparked great interest in the development of TNFL/TNFR-targeted cancer immunotherapeutics.

The ease of culture expansion of unrestricted somatic stem cells (USSCs) represents one of their primary advantages in clinical strategies. However, genetic alterations during culture expansion undoubtedly affect their therapeutic potential. Telomere shortening with aging is another factor that leads to aberrant stem cell functioning, interfering with potential therapeutic designs. This study evaluates the effect of cell density versus passaging number on the proliferation rate of cord blood (CB)-USSCs, reflected on the telomere length, pluripotent transcription factors expression, and differentiation potential. Methodology: CB-USSCs were cultured at seeding densities of 5000, 500, 50, 5 cells/cm2. Cells from different passages of each seeding density were subjected to pluripotency genes (Oct4, Sox2, Nanog, klf4, c-Myc) and PDGFRa gene expression analysis, measurement of absolute telomere length by real-time PCR, and induction of differentiation into osteogenic, adipogenic, and chondrogenic lineages. Proliferation rate was expressed as population doubling (PD) and cumulative PD (CPD). Results: USSCs from earlier passages (P7) cultured at 5000 cells/cm2 showed the highest telomere length with high expression of pluripotency, and proliferation genes which decreased gradually with passaging till reaching their lowest level at P11 Moreover, their PD at P7 was 3 and CPD (P7-P11) was 12.8. USSCs cultured at 5 cells/cm2 showed PD 12.9 at P7, with a higher expression of gene that plays an important role in proliferation (PDGFRa) than that of 5000 cells/cm2 at P7. Differentiation potentiality of 5000 cells/ cm2 at P7 was high with loss of differentiation at P11, while differentiation potentiality of 5 cells/ cm2 at P7 was much lower than that of 5000 cells/ cm2 at same passage.

Transcatheter valve intervention are more advanced and also successful evolution of conventional cardiac surgery. The first step in cardiac surgery was performed on the beating hearts in the case of digital or instrumental mitral commisurotomy. Although open heart surgery stays always the “Gold Standard” procedure, because it is crucial in understanding via direct “Visus” the valve diseases. Transcatheter technologies takes advantages from the open heart technique using catheter based instruments for elderly and high risk patients. Transcatheter mitral valve commisurotomy was the first surgical therapy converted to transcatheter one in the 1980. Today with a new art of transcatheter technology, with more clinical efficacy and safety is this “The Procedure of Choice” with a faster recovery and less peri-operative pain. But although it seems the TAVI procedure is more effective at elderly high risk or non-operable patients. The German TAVR registry shows that at low risk population, the observed mortality & morbidity is higher than that population by the EUROSCORE. The key and the crucial point at the introduction a new clinical technology is the optimal “TRANSLATION” to the daily human practice. The new interventional technology have to be supported – after previous excellent results of animal and all clinical phase studies - by “Clinical Evidence!!!”. In this technology imaging is the crucial factor in the selection & in the screening process, to guide patients to the right size and art of device selection, as well as in playing a fundamental role during procedures to guide the implant safely and effectively.

The tumor suppressor protein Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN) is a member of the protein tyrosine phosphatase family that can negatively regulate the serine/threonine kinase Akt to exert its tumour suppressor function. In addition to its normal functions such as neuronal migration and neuronal size control, PTEN protein is involved in pathological processes surrounding neuronal injury such as those associated with brain ischemia, neurological and mental disorders. It has been shown that modulation of the PTEN/mTOR pathway promotes axon regeneration in the adult CNS. We have previously shown that down-regulating the expression of PTEN protects against ischaemic neuronal death in vitro and in vivo (Ning et al. 2014). Recently, we showed that PTEN knockdown via siRNA increases motor neuron survival in Amyotrophic lateral sclerosis (ALS) (Kirby et al. 2011) in vitro and spinal muscular atrophy (SMA) in vivo (Ning et al. 2010, Little et al., unpublished). Our preliminary data show that the PTEN inhibitor, bpV, promotes cell survival in NSC34 G93A motor neuronal cell line.

The differential diagnosis of biliary stenosis is a critical problem for gastroenterologists. An early identification of malignant lesions would enable the rapid resort to surgical resection which currently represents the only potentially curative option. Unfortunately, the diagnostic value of all available methods (e.g. imaging technics, standard serum biomarkers) is limited by relatively poor accuracy and negative predictive value. Recently, our group and others highlighted new potential cancer biomarkers in bile by using comparative proteomic analysis. Nevertheless, to date, only a few candidates have been verified for their diagnostic performances in discriminating between malignant and non-malignant stenoses. In addition, no data have yet been collected on the simultaneous measurement of these proteins with the intent of evaluating the diagnostic interest of a panel of biomarkers. To overcome the limitation of classical verification tools and give a new impetus to the translation of bile biomarkers into clinical diagnostics, mass spectrometry-based quantification could represent a rapid and cost-effective opportunity thanks to its capacity for multiplexed, high-throughput analysis, combined with its analytical specificity and reliable quantification. Here we developed the first Selected Reaction Monitoring (SRM) assay for the multiplexed measurement of cancer biomarkers in human bile. For this purpose, 8 potential biomarker candidates previously highlighted by proteomic analysis were selected. Equal volumes of bile collected from patients presenting with malignant and non-malignant biliary stenosis were stacked on the top of a SDS-PAGE gel. Proteins were then digested in-gel with trypsin and proteotypic peptides of each candidate biomarker were quantified by nanoLC-SRM on a 5500-QTrap mass spectrometer (ABSciex) using heavy synthetic peptides as standards (PEPotecTM, Thermofisher). SRM data were finally analysed using Skyline software and manual validation. The developed assay proved to be valuable and reliable to quantify all the selected candidates. Moreover, the results confirmed the simultaneous overexpression of some of the proteins in bile samples from malignant stenoses.

In the last years, the new concept of predisposition to acquire acute kidney injury (AKI) is emerging. This concept was observed in our group when experimental animals exposed to an absolutely subnephrotoxic acute treatment with certain drugs (e.g. gentamicin and cisplatin) developed AKI when they were treated with a second insult with another drug, while control animals exposed to the same second drug experimented no toxicity. On these grounds, we decide to study if chronic exposure to nephrotoxicants might induce this predisposition to AKI and investigate how to detect this condition by the search of predisposition biomarkers. To this end, rats (Sprague-Dawley) were treated with a subtoxic dosage of the experimental nephrotoxin uranyl nitrate (UN) in the drinking water for 22 weeks, or plain water (as control). After 21 weeks both groups were treated with subtoxic regime of getamicin during 7 days. Renal function was monitored by means of serum creatinine, serum urea, proteinuria, N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase excretion measurement. After and before gentamicin treatment a subset of rats were sacrificed and their kidneys used for histology. With the purpose of identifying biomarker of predisposition, proteomic studies were performed before gentamicin administration at week 21. Chronic administration of UN in drinking water during 21 weeks did not modify renal function or renal tissue integrity. However, when rats exposed to UN during 21 weeks where challenged with low doses of gentamicin, they developed an overt renal failure as shown by an increase in creatinine, urea and by histological alterations. These alterations were not observed in the control group. Using a proteomic analysis, hemopexin was detected which was validated by Western blot. Urinary excretion of hemopexin was statically higher in the exposed group than in the control group. Our results suggest that chronic exposure to UN,

High-resolution volumetric optical imaging modalities, such as confocal microscopy, two-photon microscopy, and optical coherence tomography, have become increasing important in biomedical imaging fields. However, due to strong light scattering, the penetration depths of these imaging modalities are limited to the optical transport mean free path (1 mm) in biological tissues. Photoacoustic imaging, an emerging hybrid modality that can provide strong endogenous and exogenous optical absorption contrasts with high ultrasonic spatial resolution, has overcome the fundamental depth limitation while keeping the spatial resolution. The image resolution, as well as the maximum imaging depth, is scalable with ultrasonic frequency within the reach of diffuse photons. In biological tissues the imaging depth can be up to a few centimeters deep. In this presentation,

Natural products remain the best sources of drugs and drug leads. Various anticancer drugs are derived from the natural products. In our previous studies, we found that methanol extract of Pterocarpus santalinus exhibited potent cytotoxicity and anti-inflammatory activities. Among the series of compounds isolated from Pterocarpus santalinus, melanoxoin demonstrated the highest cytotoxic effects with a IC50 of 1.98 μg/ml in human non-small cell lung cancer H1299 cells. The results showed that melanoxoin regulated the transcription of several cell cycle regulators in H1299 cells. Melanoxoin induced tubulin depolymerization, suggesting that melanoxoin causes G2/M arrest thus increasing apoptosis in tumor cell. The results of neutral comet assay showed that treatment with melanoxoin induced significant DNA damage in H1299 cells, while the in vivo efficacy of melanoxoin was revealed by the mice xenograft models. Clinical data showed that an increase (61%) of MET expression was observed in non-small cell lung cancer samples compared with adjacent normal tissues.

Tubulointerstitial fibrosis –a common end-stage feature of chronic kidney disease- is characterized by the presence of renal myofibroblasts and excessive accumulation of extracellular matrix proteins (ECM) in the renal tubular interstitium. The cytokine transforming growth factor beta 1 (TGF-β1) promotes myofibroblast activation and ECM proteins expression through intracellular Smads activation, having an important role in renal fibrosis. We have recently reported that the heterozygous disruption of the TGF-β1 receptor activin receptor-like kinase 1 (ALK1) leads to an increase in TGF-β induced renal fibrosis after ureteral obstruction. Thus, we analyzed the effect of ALK1 heterozygosity in TGF-β1 induced signaling and its consequent fibrotic response in mouse embryonic fibroblasts (MEFs). We have analyzed Smad signaling pathaways in ALK1 heterozygous MEFs (ALK1+/-) and their respective controls (ALK1+/+) in basal conditions and after TGF-β1 treatment by Western blot and immunofluorescence. Moreover, we have analyzed collagen I, fibronectin and connective tissue growth factor (CTGF) expression in basal conditions, after TGF-β1 stimulation and after ALK5 inhibition –with SB431542- and Smad3 inhibition –with SIS3-. TGF-β1 stimulation induced Smad2 and Smad3 phosphorylation and Smad2/3 translocation into the nucleus in ALK1+/+ and ALK1+/- MEFs, being this increase higher in ALK1+/- MEFs. Basal Smad2 and Smad3 phosphorylation was higher in ALK1 heterozygous fibroblasts. TGF-β1 stimulation did not induce Smad1 phosphorylation in ALK1+/- MEFs and a very small Smad1 phosphorylation in ALK1+/- MEFs. ALK1 heterozygous MEFs expressed more collagen I, fibronectin and CTGF than their respective controls in basal conditions. Stimulation with TGF-β1 lead to an increase in collagen I, fibronectin and CTGF, being the increase in fibronectin and CTGF higher in ALK1+/- MEFs. Inhibition of ALK5 and Smad3 reversed the ALK1+/- phenotype.

Among the wide range of antibiotic available, beta-lactam antibiotics are used widely in bacterial pathogenic infections. The most common cause of bacterial resistance against beta-lactam antibiotics is the production of beta-lactamases by many members of Enterobacteriaceae family, including especially, E. col. (Harwalkar, Sataraddi et al. 2013). Extended-spectrum β-lactamases (ESBLs) are resistant to the third generation antibiotics, cephaloosporins and monobactum, additionally, these ESBL producing organisms™ exibit co-resistant to many other classes of antibiotics resulting limited treatment alternatives (Paterson, Bonomo 2005, Lartigue, Zinsius et al. 2007, Lewis, Herrera et al. 2007). Over the past decade, it has been observed dramatic increase of these ESBL producing E. coli strains, especially, CTX-M family of ESBL producers, to the third-generation cephalosporin antibiotic, Cefotaxime (Bonnet 2004, Lartigue, Zinsius et al. 2007, Paterson, Hujer et al. 2003, Paterson, Bonomo 2005, Ruppe, Lixandru et al. 2013). Although studies have published the diagnosis of various ESBL producers in worldwide, only few reports of molecular identification of CTX-M family of genes have been published(Al-Mayahie 2013, Harwalkar, Sataraddi et al. 2013, Literacka, Bedenic et al. 2009). This study aims to identify the DNA segments of CTX-M genes unique to E. coli, and analyse the surrounding genetic makeup of CTX-M genes. Collectively, understanding the genetic makeup of these resistance genes will shed a light on their mechanistic of antimicrobial resistance. Unlike other conventional clinical diagnostic methods, molecular diagnostics seeks evidence of a disease at the very basic causative level by detecting the nucleic acid identity. The main advantage of this diagnostic method is that the diagnosis carried out in the molecular level, and the method will identify the microbial pathogens at the early stages of the infectious diseases (Versalovic, Lupski 2002). Different types of rapid detection systems such as Real-Time PCR have been used to identify the different CTX-M family target genes (Birkett, Ludlam et al. 2007, Li, Chen 2012, Versalovic, Lupski 2002, Woodford, Fagan et al. 2006, Woodford, Sundsfjord 2005). The identified genes so far, has some degree of potency for identification, but the majority of these genes lacks the characteristics of a strong and unique genetic marker that can used as a specific target solely for identification of any CTX-M family of ESBL producing E. coli strains.

Wound repair is a quiescent mechanism to restore barriers in multicellular organisms upon injury. In chronic wounds, however, this program prematurely stalls. It is known that patterns of extracellular signals within the wound fluid are crucial to healing. Extracellular pH (pHe) is precisely regulated and potentially important in signaling within wounds due to its diverse cellular effects. Additionally, sufficient oxygenation is a prerequisite for cell proliferation and protein synthesis during tissue repair. It was, however, impossible to study these parameters in vivo due to the lack of imaging tools. Here, we present luminescent biocompatible sensor foils for dual imaging of pHe and oxygenation in vivo. To visualize pHe and oxygen, we used time-domain dual lifetime referencing (tdDLR) and luminescence lifetime imaging (LLI), respectively. With these dual sensors, we discovered centripetally increasing pHe-gradients on human chronic wound surfaces. In a therapeutic approach, we identify pHe-gradients as pivotal governors of cell proliferation and migration, and show that these pHe-gradients disrupt epidermal barrier repair, thus wound closure. Parallel oxygen imaging also revealed marked hypoxia, albeit with no correlating oxygen partial pressure (pO2)-gradient. This highlights the distinct role of pHe-gradients in perturbed healing. We also found that pHe-gradients on chronic wounds of humans are predominantly generated via centrifugally increasing pHe-regulatory Na+/H+-exchanger-1 (NHE1)-expression. We show that the modification of pHe on chronic wound surfaces poses a promising strategy to improve healing.

In order to assess the occupational health hazard of intermediates (IM), hazard data were traditionally generated through experimental animal testing, which are more and more replaced by alternative methods considering animal welfare and costs. Indeed, there are a number of other ways to assess the health hazards: comparing substances with similar structures, grouping them together into logical categories, doing specialized computer modelling, using weight of evidence approach (WoE) and integrated testing strategy (ITS). We have evaluated the current state of scientific knowledge from the literature and from in silico evaluation tools.

Background
The stroke population needing physical rehabilitation of the upper extremity is constantly increasing. To solve this problem, robot devices for improving motor performance are developed to assist physical rehabilitation in Korea. The aim of this study was to develop the robot-assisted wrist training system and to apply this to the chronic hemiplegic stroke participants. Methods Bilateral wrist robotic device was developed and we applied this to four chronic hemiplegic stroke participants. Robot-assisted wrist training system was assessed by Hand grip force, Medical Research Council, Range of motion, Upper extremity Fugl-Meyer Assessment, and Motor Activity Log. In addition, we measured functional magnetic Resonance Imaging (fMRI) analysis for cortical reorganization during both hand movement. Participants received 60 min, 20 sessions, five days a week, for four consecutive weeks. The assessments were done before and after 20 training sessions
Results
Robot-assisted wrist training system was built with the bilateral wrist flexion/extension and pronation/supination performance that provides a repetitive active-active, active-passive, and passive-passive mode. Muscle strength, Motion of wrist, and motor function were enhanced after 4-week training of wrist. And the cortical activity change was associated with inducing reorganization of motor cortex networks

Background and Aims Gentamicin is an aminoglycoside antibiotic widely used for the treatment of many infectious diseases. Its main side effect is its nephrotoxicity, which occurs in 10-25% of therapeutic courses, despite proper monitoring and hydration of patients, can lead to acute kidney injury (AKI). We have previously demonstrated that urinary damage markers like albuminuria increased as a consequence of the addition of gentamicin. In the present work we aimed at specifically studying the renal handling of albuminuria in a nephrotoxicity model, through in situ renal perfusion experiments. Methods Male Wistar rats were administrated by a single dose of gentamicin (150 mg/kg), or not. After 5 days, rats were anesthetized and an extracorporeal circuit for kidney perfusion was set up. The renal artery, vein and ureter of the right kidney were ligated. The renal artery of the left kidney and the urinary bladder were canulated. A catheter was placed in the right carotid artery and connected directly to the renal artery. Urine was continuously collected from a catheter placed in the urinary bladder at 10 min intervals. After 1 h of renal perfusion with blood from the carotid artery, oxygenated and warm (37 °C) Krebs-dextran (40 g/L of dextran) was perfused through the renal artery at 3 mL/min, and was discarded through the renal vein. Albuminuria was measured in the different urine fractions.
Results
From the second day after gentamicin administration, albuminuria was significantly increased, as compared to control rats, in which urinary markers were undetectable. When exogenous Krebs solution prefunding the kidney, neither gentamicin rats nor control rats excreted albuminuria. As a control of the perfusion experiments, urinary markers still appeared in the urine in gentamicin rats whose kidney was perfused with its own blood. However, albuminuria was undetectable in control rats.