Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. Escherichia coli, has been essential for the simple low-cost expression of the majority of proteins investigated to date. However, a number of biomedically relevant proteins fail to express and fold properly in prokaryotic expression hosts. For example, mammalian secreted proteins and membrane bound receptors often contain obligate post-translational modifications including disulfide bonds and unique glycosylation patterns that are required for proper folding and/or biological activity, preventing their expression in prokaryotes. Alternatively, a number of powerful eukaryotic expression systems are available for expression of challenging proteins including those that contain unique post-translational modifications. The most common over-expression platforms currently include yeast , baculovirus expression vector systems , and mammalian cell systems (including a variety of transformed cell lines such as CHO and HEK293). It should be noted that each expression platform has its own merits and disadvantages, and one may be more suitable for a particular protein over others.In the post-genomic era, the need for highly purified recombinant protein for biophysical and biochemical studies is greater than ever. In this regard, the laboratory workhorse, Pichia and Saccharomyces construct high-mannose type N-glycosylations on proteins and are incapable of producing the more complex patterns often seen in mammalian proteins.Yeast over-expression systems have a number of advantages. In many cases, yeast systems have provided exceptionally high yields of secreted recombinant protein while also being relatively inexpensive and time efficient.The baculovirus expression vector system is the principal method for production of challenging cytosolic proteins that are unable to be effectively synthesized in prokaryotic hosts.in vivo and are relatively homogeneous in nature, albeit with minor differences between different species of cell hosts.Mammalian cell culture is the prevailing method for biopharmaceutical protein production and its use is growing in popularity among academic laboratories.In this review, we discuss the many choices and possibilities encountered when planning protein over-expression experiments in mammalian cells including transient versus stable expression, the selection of expression vector elements, signal sequences, cell lines, media formulations, and methods for large-scale growth.The type of over-expression system used is primarily dictated by the ultimate downstream application of the protein under investigation, and even then, the investigators requirements may change over time. Preliminary expression experiments for structural analysis, for example, commonly explore a number of protein variants including homologues from different species, truncations at the amino or carboxyl-terminus, or specific mutations known to influence structure or function. To analyze a large number of protein variants quickly, transient expression experiments are most advantageous to obtain small amounts of protein for initial biochemical characterization . Alternatively, once an appropriate variant(s) has been identified, large quantities of homogeneous protein is required for setting crystallization trials, which may include the addition of binding partners and ligands. Under these conditions, the investment required to generate a stable clonal cell line would be warranted, and perhaps, even be ideal.cis-acting elements, or when integrated into the host genome. Stable expression of the transgene is generally desirable for large-scale production and control over protein quality and homogeneity. Alternatively, large-scale transient transfection experiments have grown in popularity over the past few years, primarily for the capacity to analyze a number of protein constructs rapidly.Stable mammalian cell expression can be driven either when the transgene (expression cassette) is replicated extra-chromosomally using viral proteins and cis-acting element  and trans-acting virally encoded protein.25As mentioned above, stable transgene expression can be maintained using one of two distinct strategies. Episomal vectors based on viral elements promote autonomous replication and retention in the nucleus . Episomal vectors rely on a via physical isolation with cloning cylinders. A noted drawback of this method is that the process of chromosomal integration is generally thought to be a random event, which in some cases results in gene silencing. Indeed, integration “position effects” are profound and result from differential chromatin modifications to loci DNA. Thus, its anticipated that levels of protein expression will vary from clonal lines produced in this manner necessitating clonal screening. Nonetheless, in general, stable clonal cell lines are an attractive expression platform that provides a variety of advantages, though most notably, a reliable and consistent yield and quality of recombinant protein.In contrast to episomal expression, stable chromosomal integration and expression relies only on the presence of a suitable selection marker for isolating and screening clonal cell lines . A wide Vectors used for stable transfection studies include a number of essential elements that promote optimal transcription, processing, RNA export, and translation. Here, we briefly discuss issues relating to the choice of transcription regulatory sequences , as well as the selection of signal sequence for protein secretion, use of fusion tags, choice of drug selection marker, and reporters of gene expression.cis-acting elements both within and outside the coding region are recognized to greatly effect cellular expression. For example, the Kozak sequence, which includes the initiating methionine, and in higher vertebrates is the consensus sequence gccRccAUGG, plays a role in translation initiation.38Mammalian expression vectors share in common a robust promoter/enhancer element for high levels of transcription. Most widespread are the strong viral promoters from cytomegalovirus (CMV) and SV40. Many commercial vectors also utilize the human EF-1α promoter although expression from viral promoters, at least in some cases, is considerably higher.Secretory peptides include residues at the amino-terminus of a protein that destine the nascent peptide for translocation into the ER. Following co-translational insertion of the growing peptide chain into the ER lumen, a signal peptidase cleaves the signal peptide from the protein. Perhaps not surprisingly, the proper selection of a signal peptide can have dramatic consequences on protein over-expression with some investigators reporting up to fourfold enhanced levels of expression.Fusion tags are frequently used to optimize protein folding and stability but can also provide a convenient means for single-step purification with high yield. Several fusion proteins are used extensively for cytoplasmic protein expression experiments, yet significantly fewer are available for secreted protein expression. Indeed, the only commonly used fusion partners for secreted proteins include human serum albumin and the crystallizable fragment, or constant domain of IgG, Fc.N-acetyltransferase) is also frequently used with good results.43For stable or episomal protein expression, an efficient drug selection marker is required. The most common selection reagents for mammalian cells are neomycin/G418, hygromycin, blastocidin S, zeocin, and puromycin. Neomycin and hygromycin resistance, coded for by an aminoglycoside phosphotransferase and hygromycin B phosphotransferase, respectively, are the most frequently cited drug markers and are present in a number of commercially available vectors.in vivo. For membrane proteins, for example, the fluorescence signal from GFP when introduced as a carboxyl-terminal fusion is useful for following the extent of protein expression and potential for aggregation.46Reporters of expression can provide a facile and non-invasive means to monitor protein expression A number of transformed mammalian cell lines have been explored for protein over-expression studies. By far the most extensively utilized cell line is the dihydrofolate reductase deficient Chinese hamster ovary cell line CHO-DG44.49Academic labs have embraced the use of HEK293 cells in protein over-expression studies for a number of reasons. HEK293 cells grow robustly and are easily transfected by a number of reagents with efficiencies routinely exceeding 50% using linear polyethylenimine, or approaching 100% with commercial lipid formulations.The methods used for large-scale cell growth primarily depend on the properties of the expression host. For example, HEK293 cells are a particularly attractive expression host because they readily adapt from adherent culture to suspension, thus a variety of options are available for their culture. For example, HEK293 cells grown as an adherent monolayer in disposable plastic tissue culture vessels are ideal for single cell cloning experiments, while growth in suspension in shake flasks is beneficial for pilot expression experiments and can be invaluable for large-scale protein production trials. Adherent cells are also adaptable to a wide variety of culture conditions in large volumes using either plastic or glass roller bottles, on hollow fiber reactors, on the surface of microcarriers, or polyester fiber disks as seen in packed bed bioreactors.For production of milligram quantities of secreted protein from 1 to 10 L of culture media, we favor growing adherent monolayers in disposable plastic roller bottles [® polyester fiber disks, for example, have the capacity to grow adherent cells to exceptionally high cell density (estimated at approximately 1 × 108 cells/mL), far greater than that which can be obtained in suspension.For expression studies, bioreactors are unrivaled for optimal mammalian cell growth. Typical bioreactors are sophisticated equipment capable of monitoring and optimizing numerous environmental variables simultaneously for improved cell growth [A number of manufacturers produce both classical and serum free media for most commonly used expression cell lines. For example, the media of choice for routine adherent culture of HEK293 cells is Dulbecco's modified Eagle's media containing >4.5 g/L glucose, sodium pyruvate, and fetal bovine serum (FBS) at 5–10%.To further enhance protein expression during expression experiments, expression media can be supplemented with small molecules such as sodium butyrate or valproic acid.Finally, seleno-methionine incorporation is an attractive means to prepare a heavy atom derivative for phase determination by the multiwavelength anomalous diffraction method in X-ray crystallography. Cell culture media specifically lacking methionine is readily available and has recently been used to incorporate the unnatural amino acid seleno-methionine into secreted proteins produced in mammalian cells at labeling efficiencies approaching 90%.Mammalian cells provide an attractive alternative to prokaryotic hosts for difficult to express proteins including secreted proteins. Purification of large quantities of secreted mammalian proteins is readily attainable, though the methods required differ significantly from those used for membrane and cytosolic proteins. Unfortunately, many of the tools that have been developed for optimizing expression and tracking of cell bound molecules are not helpful when screening for secreted proteins. Here, we have discussed some of the techniques that make it possible to maximize secreted protein quantity in a facile manner, including roller bottle or bioreactor culture and secretion specific fusion tags. Additionally, with careful design of the expression cassette and minimal optimization of parameters including signal sequence, cell type, and media, robust over-expression can be readily obtained for most secreted proteins.


We derive a two-layer multiplex Kuramoto model from Wilson-Cowan type physiological equations that describe neural activity on a network of interconnected cortical regions. This is mathematically possible due to the existence of a unique, stable limit cycle, weak coupling, and inhibitory synaptic time delays. We study the phase diagram of this model numerically as a function of the inter-regional connection strength that is related to cerebral blood flow, and a phase shift parameter that is associated with synaptic GABA concentrations. We find three macroscopic phases of cortical activity: background activity (unsynchronized oscillations), epileptiform activity (highly synchronized oscillations) and resting-state activity (synchronized clusters/chaotic behaviour). Previous network models could hitherto not explain the existence of all three phases. We further observe a shift of the average oscillation frequency towards lower values together with the appearance of coherent slow oscillations at the transition from resting-state to epileptiform activity. This observation is fully in line with experimental data and could explain the influence of GABAergic drugs both on gamma oscillations and epileptic states. Compared to previous models for gamma oscillations and resting-state activity, the multiplex Kuramoto model not only provides a unifying framework, but also has a direct connection to measurable physiological parameters. Fast electrochemical processes taking place on a complicated cytoarchitectural network structure render the human brain a highly complex dynamical system. Brain activity, as measured directly via EEG or MEG, or indirectly by means of MRI recordings, reveals characteristic macroscopic patterns such as oscillations in various frequency bands23Two physiological phenomena received much attention lately in terms of a mathematical understanding: resting-state activity, and gamma oscillations. Resting-state activity is spontaneous, highly structured activity of the brain during rest, and can be described in terms of networks of simultaneously active brain regions8101112Many existing network models for resting-state activity and gamma oscillations are based on single-neuron local dynamics1011161718In this work we derive a simple two-layer multiplex model from classical physiological equations that is able to capture the main features of cortical activity such as oscillations, synchronization and chaotic dynamics. This model unifies the roles of neural network topology, synaptic time delays, and excitation/inhibition. It provides a closed framework for simultaneously understanding the origin of resting-state activity and gamma oscillations.We consider i) Homogeneity. Cortical regions exhibit nearly identical dynamical behavior. We therefore assume the following parameters to be constant across regions,(ii) Stable local oscillations. We choose the parameters t) is an arbitrary solution of Eq. (iii) Weak coupling. Interactions between adjacent regions are weak, and inhibitory-inhibitory interactions are very weak in the sense that,.


Cancer development is dependent on intrinsic cellular changes as well as inflammatory factors in the tumor macro and microenvironment. The inflammatory milieu nourishes the tumor and contributes to cancer progression. Numerous studies, including ours, have demonstrated that the tumor microenvironment is immunosuppressive, impairing the anticancer immune responses. Chronic inflammation was identified as the key process responsible for this immunosuppression via induction of immature myeloid-derived suppressor cells (MDSCs). Upon a prolonged immune response, MDSCs are polarized toward immunosuppressive cells meant to control the exacerbated immune response. In cancer, the chronic inflammatory response renders the MDSCs harmful. Polarized MDSCs suppress T-cells and natural killer cells, as well as antigen-presenting cells, abrogating the beneficial immune response. These changes in the immunological milieu could also lead to high frequency of mutations, enhanced cancer cell stemness, and angiogenesis, directly supporting tumor initiation, growth, and spreading. The presence of MDSCs in cancer poses a serious obstacle in a variety of immune-based therapies, which rely on the stimulation of antitumor immune responses. Cumulative data, including our own, suggest that the selection of an appropriate and effective anticancer therapy must take into consideration the host’s immune status as well as tumor-related parameters. Merging biomarkers for immune monitoring into the traditional patient’s categorization and follow-up can provide new predictive and diagnostic tools to the clinical practice. Chronic inflammation and MDSCs could serve as novel targets for therapeutic interventions, which can be combined with conventional cancer treatments such as chemotherapy, radiotherapy, and cancer cell-targeted and immune-based therapies. Intervention in environmental and tumor-specific inflammatory mechanisms will allow better clinical management of cancer toward more efficient treatment. While some cancers are triggered by intrinsic cellular changes such as germ-line mutations, the majority of tumors are caused by acquired somatic mutations due to alterations in homeostatic environmental factors. In many cases, tumor development is associated with nutritional and environmental factors, generated by chronically occurring pathologies such as infections, autoimmune disorders, metabolic diseases, and more. The common denominator of these processes is chronic inflammation, which is an abnormal and sustained form of a protective response. It starts as an acute inflammation; a beneficial response resolving insults such as newly encountered pathogens or transformed cells. However, when the stimuli that induce the immune response could not be cleared, a continued inflammatory response evolves. The prolonged response leads to loss of tissue homeostasis, resulting in a condition similar to unhealed wounds . This tyIn this review, we highlight the regulatory checkpoints controlled by immunosuppressive cells and factors in the tumor micro and macro environments, focusing on MDSCs: immunosuppressive cells which play a critical role in maintaining normal homeostasis and when polarized under chronic inflammatory conditions could skew the environment toward supporting tumor development and spreading.Immune homeostasis is the body’s tightly regulated network of physiological checkpoints and balances that enables controlled defensive responses against pathogens or altered self-cells. In healthy individuals, an immune challenge by foreign or self-created insults initiates a cascade of appropriate responses that restore the tissue to its steady state. Homeostasis is transiently imbalanced when an acute inflammatory response is initiated to counteract a danger signal. In this regard, the acute inflammation is beneficial, as innate and adaptive immune cells are recruited and activated as long as the insult exists. Once the insult has been removed, the response is resolved and homeostasis is reconstituted, while enriching the host with a reservoir of memory and regulatory cells. These immune response cycles depend on a subtle balance between the effector and regulatory arms of the immune system and their capacity to clear the initial stimulus. The intricate cross talk between immune cells and soluble factors (cytokines and chemokines) ensues execution of pro- and anti-inflammatory responses at the appropriate time and location, thus preventing excessive and harmful immune activation. When the immune homeostasis is continuously disrupted, the host’s immune response can become hypo- or hyperactive. A compromised immune response poses severe immune deficiency-based complications, such as opportunistic infections and cancer. In contrast, autoimmune disorders are on the overactive side of the homeostatic spectrum. Thus, the maintenance of fine-tuned regulatory and effector immune functions is at the forefront of a balanced immune homeostasis.+CD25highFoxp3+ regulatory T cells (Tregs) are essential for the maintenance of immune homeostasis, as their dysfunction leads to the appearance of widespread, fatal, immune-mediated diseases , which lack DC and macrophage markers  (+Ly6G+Ly6Clow) cells. The identity of human MDSCs is more intricate and based on several markers (CD11b+CD33+HLA-DR−), which could be further divided into monocytic (CD14+CD11b+CD33+HLA-DR−) and granulocytic (CD15+CD11b+CD33+HLA-DR−) phenotypes. Both granulocytic and monocytic subpopulations have been shown to be suppressive, especially the latter. However, the majority of MDSCs are granulocytic, making up in numbers for their milder suppressive ability , also termed non-polarized or “resting” myeloid-derived suppressor cells (MDSCs). These cells were demonstrated to be tolerogenic and capable of suppressing immune system activities. In healthy individuals, these immature myeloid progenitors are retained in the bone marrow (BM) where they maintain a suppressive environment , most lictively)  and granammation . Under n CD11b+CD3+HLA-DR−Chronic inflammation develops when pathogenic or self-aberrant antigenic insults  are not resolved. The inflammatory immune response proceeds, albeit failing to eliminate the antigenic stimuli. The generated conditions are characterized by an influx of proinflammatory cells of the innate and adaptive immune systems. Such cells secrete cytokines, chemokines, and soluble factors that eventually create micro and macro environments that support tumor initiation and development. In parallel, the generated chronic inflammatory environment constantly recruits the regulatory arm of the immune system to resolve and control the inflammatory response. The continuous activity of the immune system together with the recruitment of inhibitory cells/factors eventually induces a general immunosuppression of T-cells and natural killer (NK) cells and in some cases of DCs. These indirectly support the tumor by enabling its escape from the immune effector functions .Helicobacter pylori  and inducible nitric oxide synthase (iNOS), along with enhanced production of nitric oxide (NO) and reactive oxygen species (ROS). These effects are further enhanced by TNF-α , 26. TheThe suppressive environment generated in the course of chronic inflammation is foremost generated by MDSCs. These cells were shown to directly abrogate the function of various effector cells of the innate and adaptive arms of the immune system while increasing the activity of regulatory mechanisms Figures E,F.+ and CD8+ T-cells to proliferate and produce IFN-γ in response to T-cell receptor (TCR) stimulation both in vivo and in vitro when cocultured with MDSCs , a plasma membrane molecule necessary for the homing of naive T-cells to lymph nodes. This reduces the activation of CD4+ and CD8+ T-cells and inhibits homing to tumor draining lymph nodes , and proteinases, which can induce genetic and epigenetic changes of cancer-associated genes and pathways. An increased risk for cancer has been linked with various pathologies characterized by chronic inflammation such as IBD, papillomavirus, hepatitis C virus, and ectively –48. Chroectively , 28, 49.H. pylori infection, the generated conditions lead to genomic instability in the mucosal layer and epithelial fractions of the colon and stomach, respectively, leading to tumor initiation , tumor-infiltrating lymphocytes (TIL), and cancer-associated fibroblasts (CAF) secrete several factors that contribute to tumor growth and metastasis while maintaining the immunosuppressive milieu  recognize endogenous danger signals as well as pathogen-associated molecular patterns. High-mobility group box protein 1 (HMGB1) and heat shock protein 60 (HSP60) have been shown to act as adjuvants activating APCs and inducing immune response –66. HMGBechanism , suggestH1 response, leading to increased production of proinflammatory cytokines by tumor-infiltrating leukocytes. The generated inflammation persists as long as the tumor is present, perpetuating a vicious cycle of chronic inflammation, MDSC accumulation, and immunosuppression, which in turn drive disease progression  also diminished MDSC number and activity, recuperating T-cell function. Addition of TNF-α and GM-CSF to MDSC cultures blocked their differentiation toward DCs and macrophages compared to addition of GM-CSF alone (2) produced by this enzyme supports proliferation and survival of tumor cells by a direct interaction and serve as a key inflammatory mediator. PGE2 drives cell proliferation and cytokine production, contributing to overall inflammation. In the case of colon cancer, COX-2 activity has been shown to increase production of angiogenic factors and correlated with immunosuppression mediated by Tregs  in the tumor microenvironment and expression of CXCR4 on MDSCs, mediating their recruitment in human ovarian cancer  reduces MDSC levels and restores the desired cytotoxic activity (Cancer vaccines are based on presentation of tumor antigens by autologous DCs to resident T-cells in order to enhance antitumor cytotoxic activity. Dysfunctional T-cells evident in a tumor-associated immunosuppressive environment will abrogate the efficacy of such treatments. Moreover, several studies have reported difficulties to generate mature DCs blastoma –90. Ex vactivity . Immune activity , 93. Detin vivo (The immunosuppression detected in chronic inflammation is a reversible phenomenon. Eliminating MDSCs by inducing their differentiation, inhibiting inflammatory signals that lead to their accumulation, or blocking their suppressive activity may lead to alleviation of chronic inflammation and restoration of immune functions. Various drugs and compounds, some of which are FDA approved, have been shown to target MDSCs in mouse models, attenuating chronic inflammation and inhibiting tumor growth , 94. Eliin vivo . Sildenain vivo . AlthougAs the immune system and tumor cells intimately interact and create a complex and dynamic multifactorial micro and macro inflammatory milieu, which eventually supports tumor initiation, growth, and invasion, it is mandatory to monitor in parallel the tumor features alongside with the immune status. The cumulative data depicting the capacity of effector and regulatory immune cells to shape the environment toward supporting tumor growth highlight the critical role of chronic inflammation and associated immunosuppression, mediated by MDSCs, as a major obstacle in the success of chemo- and immune-based anticancer therapies. Only very recently, it has become clear that in order to increase the success rates of anticancer immunotherapies, a combination between monitoring the tumor responses and the patient’s immune system functionality is critical for the design of optimal treatments. Therefore, it is suggested that prior to an applied immune-based therapy, the patients must be evaluated for their immune status, using biomarkers such as CD247and MDSC levels and suppressive features. If immunosuppression is detected, it must be combated either by targeting MDSCs or by neutralizing the inflammatory environment. Upon recuperation of the immune status, immune-based therapies could then be applied. Furthermore, as described by various studies including ours, changes in the patients’ immune status also occur in response to various chemotherapies. As adverse effects of chemotherapies were obtained on the immune system functionality, some enhancing MDSC-mediated immunosuppression and others inhibiting MDSC accumulation and suppressive activity, it is mandatory to follow up chemotherapy effects not only on the tumors but also on the innate and adaptive immune system arms. Combating MDSCs and chronic inflammation, while monitoring MDSCs and CD247 in T-cells and NK cells, may provide valuable tools for choosing the correct therapeutic regiments and timing while taking into account both the cancer and immune factors. By combating MDSCs, their direct and indirect harmful effects could be alleviated on the one hand and allow reconstitution of the immune response on the other hand, thus gaining better therapy results. Moreover, monitoring the immune system functionality in the course of a given anticancer treatment could have an added value on the evaluation of tumor regression or progression as chronic inflammation and immunosuppression will decrease or increase accordingly. Therefore, by combining a monitoring system to follow in parallel changes in tumor and immune system function based on the described biomarkers, an increase in the efficacy of both immune and non-immune based anticancer therapies could be achieved and enable the design of optimal personalized treatments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lung diseases are described by a wide variety of developmental mechanisms and clinical manifestations. Accurate classification and diagnosis of lung diseases are the bases for development of effective treatments. While extensive studies are conducted toward characterization of various lung diseases at molecular level, no systematic approach has been developed so far. Here we have applied a methodology for pathway-centered mining of high throughput gene expression data to describe a wide range of lung diseases in the light of shared and specific pathway activity profiles. We have applied an algorithm combining a Pathway Signal Flow (PSF) algorithm for estimation of pathway activity deregulation states in lung diseases and malignancies, and a Self Organizing Maps algorithm for classification and clustering of the pathway activity profiles. The analysis results allowed clearly distinguish between cancer and non-cancer lung diseases. Lung cancers were characterized by pathways implicated in cell proliferation, metabolism, while non-malignant lung diseases were characterized by deregulations in pathways involved in immune/inflammatory response and fibrotic tissue remodeling. In contrast to lung malignancies, chronic lung diseases had relatively heterogeneous pathway deregulation profiles. We identified three groups of interstitial lung diseases and showed that the development of characteristic pathological processes, such as fibrosis, can be initiated by deregulations in different signaling pathways. In conclusion, this paper describes the pathobiology of lung diseases from systems viewpoint using pathway centered high-dimensional data mining approach. Our results contribute largely to current understanding of pathological events in lung cancers and non-malignant lung diseases. Moreover, this paper provides new insight into molecular mechanisms of a number of interstitial lung diseases that have been studied to a lesser extent. High-throughput gene expression profiling has found wide applications in many areas of lung pathology research, diagnostics, and treatment  public repository were used  and were brought to a linear scale. Then we have converted all values to fold change (FC) respective to the mean expression value of the controls  included in the same dataset. These steps cast the data in all datasets to a common scale and type.For each dataset, microarray probe IDs were converted into Entrez Gene IDs. Microarray probes that did not match any known Entrez ID were discarded. Multiple probe sets for the same gene were averaged. Next, gene expression values in each dataset were inspected for data transformation type  using a modified version of the “GEO2R log autocheck” script  signaling pathway gets input signals from ligands and provides output signals via the sinks e Figure . Differee Figure  and highe Figure . In tota-omics data. In oposSOM package typical SOM runs require from minutes to, at maximum a few hours of computer time. SOM-space was optimized in extensive pre-studies  method was developed in the early 1980'ties by T. Kohonen Kohonen, . First aWe used SOM machine learning implemented in “som” R package as a core for oposSOM package . They are arranged according to the underlying SOM grid and visualized by an appropriate color gradient. The color patterns emerge as smooth textures representing the fingerprint of pathway activity perturbation for each disease. The individual portraits are mutually comparable. The SOM algorithm arranges similar meta-PSF profiles in neighboring tiles of the map whereas more different ones are located more distantly. Meta-PSFs located in the same spot-like region of the map are concertedly deregulated across the diseases. Overexpression (underexpression) spots in each “personal SOM” portrait were determined as clusters of meta-PSFs, which have values above (below) the predefined threshold (90 percent of meta-PSF extremes), or by applying K-means or correlation clustering to the meta-PSF profiles as described in detail elsewhere  estimation for multiple test correction as implemented in oposSOM and described in detail elsewhere , representing log fold difference of pathway deregulation in diseased over healthy state. A pathway was considered as significantly deregulated, if its p value was less than 0.05 and FDR qvalue was less than 0.2.A separate part of analyses focused on assessment of pathway activity changes in lung diseases compared to healthy lungs. For this, the difference of mean-centered PSF values between diseases and healthy controls was computed , and hierarchical clustering methods implemented in oposSOM ,” “FoxO signaling pathway” and “Dopaminergic synapse” were found to be significantly associated with all the overexpression spots and thus all the investigated lung conditions; however, with disease-specific molecular processes associated with each sink in these pathways  of non-cancer lung diseases contained COPD, sarcoidosis (SARC), and tubercolosis (TB). Numerous experimental studies, including our own, implicated immune/inflammatory pathways such as Toll-like receptor signaling, phagocytosis, and chemokine signaling in sarcoidosis, tuberculosis, and COPD  joined respiratory bronchiolitis interstitial lung disease (RB-ILD) Leslie, , hyperseThe third and the biggest community of interstitial lung diseases (Cluster 3—light green) included interstitial pneumonias, such as UIP/IFP, NSIP, and unclassified interstitial lung diseases (ILDs). This cluster was characterized by activation of various cytokine-cytokine receptor interactions, and upregulation of WNT signaling pathway. Moreover, it has been noticed that this cluster serves as a link between cancer and other ILD diseases  were clustered into one highly homogeneous community characterized by downregulation of immune system related pathways and upregulation of cell cycle, proliferation, and metabolism pathways, as expected  in the similarity graph. Though they are not included in any cluster, they provide links between different clusters.The community detection analysis confirmed the diverse nature of cancer and non-malignant lung diseases, but it also provided additional information that wasn't obvious using other similarity analysis methods, such as clustering, second level SOM or ICA. The main advantage here was that the graph object allowed for direct assessment of similarly deregulated pathways between all pairs of studied diseases, with reference to the healthy state. We have noticed that pathway deregulation states in fibrotic diseases (cluster 3) serve as a central hub that shares considerable similarities with inflammatory  and cancer diseases (cluster 4). Moreover, our results suggest that interstitial lung diseases seem to constitute a more heterogeneous group of diseases in terms of molecular mechanisms underlying their pathology. Even if it is well known that inflammation and fibrosis are the main characteristics of interstitial lung diseases Bourke, , our datUsing our novel PSF-SOM method we have evaluated similarities of pathway deregulation profiles in a large spectrum of lung pathologies by mining high-dimensional gene expression data and topologies of signaling and metabolic pathways. We pursued a systems biology view to identify most prominent properties of groups of lung diseases in terms of pathway deregulation patterns. Our results revealed considerable differences in pathway deregulations implicated in cancer and non-cancer lung diseases. While lung cancers were characterized by pathways implicated in cell proliferation, metabolism, non-malignant lung diseases were characterized by deregulations in pathways involved in immune/inflammatory response and fibrotic tissue remodeling. Moreover, we observed considerable heterogeneity in terms of pathway deregulation in interstitial lung diseases (ILD). We were able to identify three groups of ILDs and showed that the development of characteristic pathological processes such as fibrosis can be initiated by deregulations in different signaling pathways. These results raise a question whether it is appropriate to combine ILDs into single group of diseases. Finally, we also detected substantial similarities between cancers and a subset of interstitial lung diseases characterized by fibrosis development, which suggests the presence of shared molecular mechanisms involved in their pathogenesis.Our results replicate existing knowledge on pathological processes in lungs. The inflammation and fibrosis related pathways identified to be deregulated in non-malignant chronic lung diseases considerably overlapped with the ones reported by Cho et al.  using geFrom methodological point of view this paper demonstrates the power of our pathway centered analysis. While usual gene-centered studies provide lists of genes and associated functional categories, our approach more closely refers to systems level definitions and provides a rich outlook on actual molecular events associated with studied conditions. We have combined PSF and SOM algorithms that previously have been extensively benchmarked (Wirth et al., This study has a number of limitations that is worth to discuss here. First, the data analyzed in this study refer to endpoint pathologies, and not to initial steps of disease development. While the latter is important for assessing the causes of a disease, the former describes the state at which most therapeutic interventions are targeted. Thus, assessment of disease similarities based on end-point phenotypes is not suited to study initiation and early genesis of lung diseases. Second, our study was limited to KEGG pathways that contain previously curated information (Ogata et al., In conclusion, this paper describes the pathobiology of lung diseases from systems viewpoint using a pathway centered high-dimensional data mining approach. Our results largely contribute to current understanding of pathological events in lung cancers and non-malignant lung diseases. Moreover, our results provide new insight into molecular mechanisms of a number of interstitial lung diseases that have been studied to a lesser extent compared to interstitial pulmonary fibrosis, COPD, and sarcoidosis.AA, LN, and HB initiated the study. AA and LN performed calculations with contribution from HB and HL. AA, LN, HB, HL, and MP contributed to results interpretation and manuscript writing. All authors read edited and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Spatial stochastic reaction-diffusion simulations have become an important component of molecular modeling in Computational Neuroscience, as shown in a growing number of recent studies including our previous work in which we show that stochastic effects, in particular stochastic calcium dynamics, contribute to Purkinje cell calcium burst variability . HoweverThere are common challenges of parallelization for reaction-diffusion simulators as well as some unique challenges for STEPS, in which space is discretized into irregular tetrahedrons and not cubes that are commonly used by other reaction-diffusion simulators. Exact solutions are not beneficial, where stochastic diffusion across geometry partitions result in frequent conflict between nodes, requiring regular costly rollbacks. Therefore it is essential that an approximation algorithm is applied. Such an approximation must minimise loss of accuracy whilst maximising performance gain.Existing solutions such as the Gillespie-Multi-Particle method (GMP) ,5 have bOur method is implemented on an MPI framework, and we present performance gains under two scenarios- global application of the method, and application only at partition boundaries. We discuss the potential of this method for future large-scale parallel simulations in STEPS.


Pericytes are specialized mural cells located at the abluminal surface of capillary blood vessels, embedded within the basement membrane. In the vascular network these multifunctional cells fulfil diverse functions, which are indispensable for proper homoeostasis. They serve as microvascular stabilizers, are potential regulators of microvascular blood flow and have a central role in angiogenesis, as they for example regulate endothelial cell proliferation. Furthermore, pericytes, as part of the neurovascular unit, are a major component of the blood-retina/brain barrier. CNS pericytes are a heterogenic cell population derived from mesodermal and neuro-ectodermal germ layers acting as modulators of stromal and niche environmental properties. In addition, they display multipotent differentiation potential making them an intriguing target for regenerative therapies. Pericyte-deficiencies can be cause or consequence of many kinds of diseases. In diabetes, for instance, pericyte-loss is a severe pathological process in diabetic retinopathy (DR) with detrimental consequences for eye sight in millions of patients. In this review, we provide an overview of our current understanding of CNS pericyte origin and function, with a special focus on the retina in the healthy and diseased. Finally, we highlight the role of pericytes in de- and regenerative processes. Pericytes are specialized cells located at the abluminal surface of capillary blood vessels with key functions in vascular homoeostasis , focusing on retinal pericytes and their vascular functions as well as their contribution to retinal pathology progression. Because of its identical embryological origin, the mammalian retina is considered a part of the CNS. The nutrition of the retina is provided by two different vascular beds: the outer retina (photoreceptors) is passively supplied by the choroidal vasculature, whereas the inner retina is supplied by the retinal vasculature. The latter is comparable to CNS vasculature, constituting a tightly regulated cellular barrier. Retinal pericytes are essential constituents of the BRB and fulfill important functions to maintain vessel homeostasis. To provide a more comprehensive vision, this review will also include findings of other, non-retinal CNS pericytes. Finally, next to describing the vascular function of CNS pericytes the emerging hypothesis arguing in favor of a regenerative function and hence a therapeutic use of pericytes in tissue repair will be discussed.Pericytes are generated during embryonic and postnatal life  has widely been used to identify pericytes and vSMCs in vitro and in vivo, however capillary pericytes do not express aSMA in vivo  angiogenesis and vessel stabilization; (b) participate in blood flow regulation and neurovascular coupling; and (c) are an essential constituent of the BBB/BRB. As mentioned above, next to their vascular functions they possess a multipotent differentiation potential Figure .Pericytes play a major role in angiogenesis, participating in vessel formation, remodeling and stabilization , resulting in subsequent binding of PDGFRb expressing pericytes to the endothelial tip cells  involves the interaction between neurons, glia and vascular cells (neurovascular coupling). Although Fernández-Klett et al. , Bell et al.  indicating a regulatory role of pericytes on the gene expression profile of endothelial cells and astrocytes , Daneman et al. , a loss of neuronal layers and folding of the photoreceptor layer in the retina was associated with reduced pericyte coverage, resembling signs of diabetic retinopathy  and the outer BRB . Although the BBB is structurally similar to the inner BRB and both express several common transporters/receptors  mouse, developing retinal leakage, neovascularization with hyperglycemia and signs of advanced clinical DR  might represent a suitable model  in glaucomatous patients , as well as investigating histological samples of human stroke brains, Fernández-Klett et al.  has been proven in a multitude of studies , in terms of their expression profile and their differentiation potential as well as of their stromal capability, CNS pericytes have been considered to be the “CNS-resident MSCs” . The injection of ASCs after oxygen-induced retinopathy improved microvascular regrowth, and further prevented retinal capillary dropout in case of pre-injury injection, therefore providing proof for functional vascular protection . These cells represent an alternative type of adult MSCs expressing pericyte specific markers in vivo CNS (injury) models. As pericyte dysfunction and reduced pericyte coverage plays a crucial role in disease progression and as pericytes exhibit similar characteristics as MSCs, they seem to be highly suitable for cell-based therapies. Moreover, with increasing knowledge concerning CNS pericyte modulation and differentiation, future attempts may involve molecular therapies to modulate these cells in vivo.Although the differentiation potential of CNS pericytes, including retinal pericytes, has been demonstrated (Farrington-Rock et al., Pericytes play a crucial role in maintaining tissue homeostasis, and pericyte dysfunctions as well as a decrease in number underlie distinct pathologies. Their involvement in numerous diseases and their multipotency qualifies them as promising targets for future therapeutic regenerative approaches. CNS degenerative diseases and especially retinal degenerations often involve pathologies of the vasculature resulting in impaired vascular functions. As pericytes are crucially involved in vascular physiology as well as degenerative/regenerative processes, they are possible targets for therapeutic interventions. However, to fully harness pericytes as molecular “drug stores”, we need to increase our understanding of pericyte function in health and disease, leaving ample room for future studies.AT, SL and FJR wrote the manuscript. FS, DB, KAM, BB, AK-E, CS, CR, LA and HAR critically reviewed the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Due to a lack of sufficiently accurate and effective tools, allergy is often badly diagnosed. Moreover, its treatment is expensive, long, uncomfortable and of little effectiveness. In this context, less than a quarter of allergic patients receive a treatment currently. Allergies have therefore become an important public health issue, highlighting the many unmet medical needs. ANGANY Genetics has brought together expertise in protein genomics and in therapeutic protein production in plant expression systems, to produce recombinant allergens of an unprecedented quality, offering the following solutions to allergy sufferers: (1) a precise identification of each allergen involved rather than the current approximate diagnosis, (2) pure and certified recombinant allergens rather than imperfect and incomplete allergen extracts and (3) an effective treatment at the best cost to replace symptomatic-only or low-level curative allergy treatments.In this context, ANGANY Genetics has developed AllergoPur™ for production of high quality natural-like recombinant allergens needed for high efficiency of allergy treatment. AllergoPur™ is a plant-based production platform using transient expression in N. benthamiana. This strategy offers a rapid, flexible and high-yielding production of recombinant allergens. Typically, high rates of recombinant allergens can be recovered twelve days after gene delivery. Furthermore, in contrast to bacterial cells, plants can produce recombinant allergens that require complex post-translational modifications and, obviously, plants are also the best system for expression of plant allergens requiring plant-specific maturations.Using AllergoPur™, ANGANY Genetics has already produced high quality recombinant allergens from mites, pollens, mammals or mold. This is illustrated here with eight recombinant allergens required for an accurate diagnosis and an efficient immunotherapy of dust mite allergy. The detailed biological and structural characterizations of these recombinant allergens illustrate their quality and conformity to natural products.AllergoPur™ is a powerful tool for the rapid production of high quality natural like recombinant allergens. The capacity of this platform is illustrated with the successful expression of complex allergens impossible to produce in prokaryotic systems, such as Der p 1 or Hev b 13. Some of these allergens are currently tested in preclinical studies.


The tumor microenvironment (TME) is being increasingly recognized as a key factor in multiple stages of disease progression, particularly local resistance, immune-escaping, and distant metastasis, thereby substantially impacting the future development of frontline interventions in clinical oncology. An appropriate understanding of the TME promotes evaluation and selection of candidate agents to control malignancies at both the primary sites as well as the metastatic settings. This review presents a timely outline of research advances in TME biology and highlights the prospect of targeting the TME as a critical strategy to overcome acquired resistance, prevent metastasis, and improve therapeutic efficacy. As benign cells in TME niches actively modulate response of cancer cells to a broad range of standard chemotherapies and targeted agents, cancer-oriented therapeutics should be combined with TME-targeting treatments to achieve optimal clinical outcomes. Overall, a body of updated information is delivered to summarize recently emerging and rapidly progressing aspects of TME studies, and to provide a significant guideline for prospective development of personalized medicine, with the long term aim of providing a cure for cancer patients. Cancer is a systemic disease, and it is not a solo production but rather an ensemble performance . Cancer Physiologically, the stroma in healthy individuals is a physical barrier against tumorigenesis; however, neoplastic cells elicit various changes to convert the adjacent TME into a pathological entity. The orchestration of such an event implicates migration of stromal cells, remodeling of matrix, and expansion of vasculature . RegionaThe structurally and functionally essential elements in the stroma of a typical TME include fibroblasts, myofibroblasts, neuroendocrine cells, adipose cells, immune and inflammatory cells, the blood and lymphatic vascular networks, and the extracellular matrix (ECM). The naive stroma is a critical compartment in maintaining physiological homeostasis of normal tissue, and recent studies strengthened the concept that some stromal components have anticancer activities by regulating immunosuppression and restraining carcinogenesis, which is particularly the case of pancreatic ductal adenocarcinoma ,9. The a+ CSCs, including interleukin (IL)-6 and interferon-γ receptor 1 [Progression of organ-specific tumors is also reliant on infiltration of immune cells and occurrence of angiogenesis, which generates a stash for cancer stem cells (CSCs) and provides a complex signaling environment. CSCs, also known as tumor-initiating cells, have been intensively explored within the recent decade. Many tumor types involve CSCs in the TME milieu, which are characterized with the potential to cause resistance against various cytotoxicities due to intrinsic mechanisms, including genetic changes and epigenetic alterations. Both CAFs and CSCs are implicated in the TME-mediated signaling to remodel cancer cells; for instance, CAFs express high levels of extracellular factors including chemokine CXC motif ligand (CXCL)12, chemokine CC motif ligand (CCL)2, CCL8, and insulin-like growth factor binding protein 7, thereby forming an inflammatory niche -15. Furtceptor 1 .in vivo conditions both the innate and adaptive immune systems influence homeostasis, in particular the recruitment of immune cells into the tumor-adjacent milieu is active and forms distinct immune contextures, thereby exerting profound impacts on clinical outcome. For example, T cell activation involves both positive and negative checkpoint signals to finely tune responses to prevent excessive pathological changes [Under  changes ,18. The  changes ,20. Some changes . Indepen changes . Further changes .in vitro studies that prove the complex role of the TME cell lineages, experimental animal models with genetically modified stroma further presented convincing data of the biological importance of the TME. Genetic alterations in stromal fibroblasts caused pathologies in the adjacent glandular epithelium, as demonstrated by FGF10 overexpression in a tissue recombination model and TGF-β type II receptor conditional elimination in transgenic mice [In addition to many nic mice ,25. Thusnic mice . Simultade novo tumorigenesis, a permissive TME modifies treatment responses by affecting cell sensitivity to anticancer agents. The TME-induced resistance to interventions applied for multiple tumor types, as well as its magnitude, varies depending on the cancer cells, stroma properties, and therapeutic regimens. Further, drug resistance mediated by the TME is not limited to classical agents such as those administered in genotoxic chemotherapies; rather, it covers diverse pharmaceuticals including targeted agents [Therapeutic resistance remains a major problem in clinical oncology. In addition to fueling d agents . Recent d agents -29. TME-d agents ,30.Although numerous reports elaborated the biological role of TME-derived factors in tumor growth or metastasis, relatively few have delineated the impact of an agent-activated TME to the therapeutic outcome. Recent studies using targeted drugs or conventional chemotherapeutics have filled the gaps to show that treatment-induced alterations to the microenvironment can generate a protective niche or shielding reservoir for the remnant cancer cell population, which is termed minimal residual disease as the occult site to prime tumor relapse . ParticuTME-exerted protection for cancer cells apply to multiple therapeutic situations. Upon melanoma treatment by mitogen activated protein kinase (MAPK) pathway inhibitors, TAMs expand and release tumor necrosis factor (TNF)-α as a crucial growth factor that provides resistance to the targeted therapy through the microphthalmia transcription factor . InhibitAlthough dominant anticancer regimens, including chemotherapy and targeted therapy, provide major options for cancer patients, so far, mounting data pinpoints to an intricate link between epithelial-mesenchymal transition (EMT) and therapeutic resistance. Gain of function as resistance for cancer cells can be regulated by diverse mechanisms, and it may arise as a direct consequence of EMT triggered by a large array of the TME-derived molecules through activation of intracellular networks that cover hepatocyte growth factor/c-met, epidermal growth factor (EGF)/EGF receptor (EGFR), Wnt/beta-catenin axes, and several cytokine/chemokine-mediated pathways such as TGF-β/Smad signaling -51. In tTME-conferred resistance is not limited to solid tumors. A new study of leukemia identified a therapy-induced niche in the bone marrow, which empowers the resident leukemia propagating cells (LPCs) to survive with antiapoptotic properties . Upon trThroughout the course of tumor evolution, a vast group of host cells, ranging from fibroblasts to macrophages, sustain a supportive TME for disease progression, specifically by interfering immunosurveillance against cancer cells . Among t+ T cells and B cells when co-injected with tumor cells in mice, thus promoting tumor growth, highlighting the important interaction between MSCs and other components of the TME as well as the possibility of restoring tumor immunity in humans by therapeutic targeting IDO activity. Tumor-resident MSCs seem to be pathologically educated to favor tumor growth; inflammatory cytokines may act as a major driver for the change of local microenvironments, but other factors are also likely to be implicated in TME-exerted modifications. As supporting evidence, several studies demonstrated that exosomes derived from BM-MSCs can induce progenitor cells to undergo mesenchymal-to-epithelial transition (MET), indicating active message delivery between cancer cells and the bone marrow TME [MSCs are capable of modulating immune status; however, the immunoregulatory function of MSCs is not intrinsic but depends on their cytokine milieu . MSCs isrrow TME ,73.Of note, growth factors and cytokines released by MSCs invoke proliferative signaling of cancer cells and protect them against cell death, a function that can be exerted passively. Chemotherapy to leukemia elicits resistance by rebuilding an microenvironmental niche that allows cancer-propagating cells to evade apoptosis, and MSCs generate replatable mesenspheres and express CD29, CD51, and chemokine receptor CCR1 . In ovar+ CD25Hi CD4+ Tregs proliferation and maintains the immunosuppressive activity by reducing T lymphocytes and NK functions for an extended period upon co-culture in vitro [The contribution of MSCs to tumor progression and resistance is well established, while the MSC-mediated Tregs expansion and immunosuppression has recently attracted increasing interest. In particular, human leukocyte antigen-G5 secreted by MSCs stimulates FoxP3in vitro ,77. Tregin vitro .th century [Metastasis accounts for approximately 90% of overall mortality among solid tumor patients . The met century ,80.+ T-lymphocyte instigation in the local microenvironment [Local invasion is the physical entry of cancer cells resident within a well-confined primary tumor into the surrounding stroma. Cancer cells first breach the basement membrane, a specialized ECM structure in the TME, by co-opting the EMT program, which allows dissolution of tight junctions, loss of cell polarity, and acquisition of multiple mesenchymal attributes . Stromalironment . Thus, Tironment ,83. In aironment  and hypoironment , each caIntravasation is a critical step that allows cancer cells to cross pericyte and endothelial cell barriers before they gain access to other organs ,87. For + cells [0), tumor mass dormancy (proliferation refrained by apoptosis), or immune dormancy (an equilibrium maintained by immunosurveillance) [Either at primary sites or in vasculature vessels, cancer cells can release microvesicles or soluble factors to adapt incipient metastatic sites into ‘pre-metastatic niches’; for example, systemic factors attract bone marrow-derived macrophages and hematopoietic progenitor cells that are accompanied by CAFs and endothelial cells to remodel tissue and eventually cause lung metastasis . However+ cells ,91. Inteillance) . Unfortuillance) . BioavaiMicro RNAs (miRNAs) are circulated in cancer patient serum and can serve as important biomarkers for many cancer types . New stuAltogether, it is increasingly evident that distinct stages of tumor advancing are subject to continuous and comprehensive influence of the TME in a special and temporal manner, underscoring the necessity to consider the TME in the context of clinical management Figure .Figure 1Innate resistance to clinical therapeutics is a hallmark of cancer; however, acquired resistance has also emerged as a daunting challenge to anticancer treatments by minimizing the efficacy of otherwise successful regimens. The vast majority of mainstay therapeutic strategies against human tumors are designed to target intrinsic traits of cancer cells. In contrast, stromal cells within the TME are generally stable in genetics and/or epigenetics and thus less likely to be susceptible to diverse mechanisms of therapeutic resistance. Further, given the accumulating evidence of overwhelming heterogeneity at each aspect of tumor evolution which is significantly subject to functional influence of cancer cell extrinsic compartments, targeting the TME turns out to be quite urgent and should be given enough priority -99.In the tumor, continuous interactions between cancer cells and the surrounding TME actively occur via direct intercellular contact or through secreted signaling molecules. To date, a handful of targeted therapeutics against specific stromal compartments is successfully implemented, which shows decent promise in substantially minimizing pathological contributions of the TME in clinical settings Table . Based oin vivo has established translational avenues and paved the road for continued inputs into clinical frontiers. Despite the preliminary success of TME-targeted therapies, there remain several important issues that must be clearly addressed. Conventional anticancer treatments frequently cause structural and functional alterations of the TME, which contribute to acquired resistance and severely compromise clinical outcomes by generation of cancer-protective niches, emphasizing the necessity to consider the global TME response in future clinical intervention. First, novel biomarkers that indicate the treatment consequence and image the extent of TME damage through examination of patient bio-specimens (particularly serum samples) will allow for real time surveillance, therapeutic regimen optimization, and drug design innovation – each case is eagerly desired. Identification and selection of these molecules to establish a diagnostic panel applicable to clinical conditions would significantly accelerate the advancement of translational medicine. Second, the TME-stimulated cancer resistance and disease resilience may be technically prevented by rational administration of agents between therapeutic cycles to periodically retard key regulators of the TME signaling network, a feasible approach to minimize the influence of tumor-promoting factors from activated stromal cells that either develop a secretory phenotype or exert other adverse actions to enhance pathologies [in vitro cell culture studies meet major difficulty as specific elements of a typical TME, such as immune cells and vasculature, cannot be easily integrated. The recently emerging model of patient-derived xenograft reflects the complexity of tumors including the structural and functional heterogeneity of TME, but the host is immunodeficient [To date, the mainstay of therapeutic strategies that target the TME hologies ,102. Thieficient . In contTumors evolve in a complex, dynamic, and functionally multifaceted microenvironment, which they rely upon for sustained growth, invasion, and metastasis. Unlike cancer cells, stromal populations within the TME are genetically stable, and thus represent an attractive therapeutic target with minimal risk of treatment resistance and disease relapse. TME-oriented research is increasingly encouraged and advocated, including the endeavors made in basic, clinical, and translational medicine. In such an exciting era of TME biology, experimental data have led to new scientific concepts and identified novel therapeutic targets to control the TME-related pathologies. However, there are not only major advances but daunting challenges, the latter including how to uncover and restrain susceptible nodes in the structurally complex and functionally intertwined TME system. Given that key signaling pathways frequently crosstalk and mutually interact in an intricate network, insights into how to solve the tortuous maze in a wider landscape and how tumor type-specific TMEs may respond differently to current standard-of-care therapies remain as important issues to tackle with intelligence. Fortunately, with the wealth of data accumulated so far, we now have a roadmap to convert these challenges into opportunities. For instance, when defining predictive markers that will eventually aid in the selection of patients who most likely benefit from intervention, analysis based on the entire TME is an essential step of utmost importance to determine specific therapies to employ ,103,104.


Dermatomyositis is an autoimmune disease characterized by proximal myopathy, cutaneous Gottron papules and heliotrope rash; intestinal involvement associated to acute vasculitis is less common but could be a life-threatening condition.A 21-year-old woman, affected by dermatomyositis, presented to our attention with a three-day story of severe abdominal pain, no bowel movement and biliary vomit. She was diagnosed with acute abdomen. A CT scan with bowel contrast demonstrated the presence of a leakage from the retroperitoneal aspect of duodenum. The surgical and clinical management in the light of literature review is presented.Our first approach consisted in primary repair of the duodenal perforation with omentopexy. Post-operative course was complicated by hemorrhage. A reintervention showed a new perforation associated with multiple ischemic intestinal areas. We performed a gastroenteric anastomosis with functional exclusion of the damaged duodenum and positioning of drainages to create a biliary fistula. A nutritional enteric tube and an open abdomen vacuum-assisted closure system to monitor the fistula creation and to prevent abdominal contamination and collections were positioned. To reduce the amount of biliary leakage, a percutaneous transhepatic biliary drainage was placed, with progressive fistula flow disappearance in four months.In patients with dermatomyositis, when clinical findings and symptoms suggest abdominal vasculitis, it is very important to be aware of the risk of bowel and particularly duodenal perforations. Open abdomen treatment favors control of contamination by gastrointestinal contents, offers temporary abdominal closure, helps ICU care and delays definitive surgery. Dermatomyositis (DM) is an autoimmune disease characterized by cutaneous heliotropic rash, Gottron papules and proximal myopathy associated to dysphagia, dysphonia, Raynaud phenomenon, fatigue and non-erosive inflammatory polyarthritis . VasculiWe performed a literature review by searching on PubMed : only few cases of bowel perforation associated to dermatomyositis are described in literature, and surgical approach is not always mentioned or specified -19. In lWe report the case of a 21-year-old patient affected by DM presenting with rapid onset acute abdomen associated to severe vasculitis and complicated duodenal perforation, and discuss the surgical and clinical management in the light of literature review.A 21-year-old female diagnosed with DM in 2008, on treatment with prednisone and cyclosporine with moderate disease activity until December 2012, presented to our Emergency Department (ED) with a three day history of diffuse, acute abdominal pain, no bowel movement and biliary vomit. She underwent laparoscopic cholecystectomy in 2010 for symptomatic calculosis. The patient was admitted to our Department with a bowel perforation suspect. An oral follow-through was negative but a CT scan with oral contrast demonstrated a small leakage from the posterior aspect of the third duodenal portion . Argatroban was administered obtaining progressive increase in PLT count (354.000/μm3). Three months after surgery she had seizures with MRI scan positive for vasculitic diffuse encephalic lesions, treated with levetiracetam and metilprendisone. During hospitalization we observed nasal regurgitation of fluids, nasal speech and hoarseness probably due to loss of pharyngoesophageal muscle tone and increase and reduction in hepatic stasis values of unknown origin. After 8 months of follow-up, no signs or symptoms of abdominal disease were reported.During her ICU stay, the patient presented signs of renal vasculitis, therefore she underwent cycles of continuous veno-venous hemodialysis (CCVHD), plasmapheresis and intravenous immunoglobulin (IVIG), showing clear improvement of her renal function and negative immunological test. Low molecular weight heparin (LMWH) treatment was complicated by heparin induced thrombocytopenia (HIT) with low platelet (PLT) count (99.000/μmDM is an autoimmune disease characterized by cutaneous heliotropic rash, Gottron papules and proximal myopathy associated to dysphagia, dysphonia, Raynaud phenomenon, fatigue and non-erosive inflammatory polyarthritis . VasculiSurgical treatment is not standardized because of the rarity and variety of the gastrointestinal DM presentations that can affect the entire gastrointestinal tract. In literature we found few descriptions of ischemic gastrointestinal perforation in DM. Surgical treatments and outcomes reported were anecdotic and varied, including resection of the affected tract or sutures of the lesions Table . High reWhen clinical findings and symptoms suggest possible abdominal vasculitis in a young subject known for DM, it is very important to consider bowel and particularly duodenal perforation. We found very helpful CT scan with oral contrast to support diagnosis and we had to face the more life-threatening condition of multiple ischemic intestinal ulcerations conditioning duodenal multiple perforations. To manage this challenging condition we used open abdomen treatment with exclusion of the duodenal ischemic perforated tract through a gastroenteroanastomosis and PTBD with the creation of a guided fistula to decrease the flow and obtaining progressive healing with improvement of patient’s general conditions. This surgical treatment must always be accompanied by DM specific medical treatment to avoid further vasculitic complications and to obtain control of the disease activity.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CAPS: Anti-phospholipidic catastrophic syndrome; CT: Computed tomography; CVVHD: Continuous veno-venous haemodialysis; DM: Dermatomyositis; ED: Emergency Department; HIT: Heparin induced thrombocytopenia; ICU: Intensive care unit; IVIG: IntraVenous immunoGlobulin; LAC: Lupus anti coaugulant; LMWH: Low molecular weight heparin; MRI: Magnetic resonance imaging; NGT: Naso-gastric tube; PTBD: Percutaneous transhepatic biliary drainage; PLT: Platelet.The authors declare that they have no competing interests.RV made substantial contributions to acquisition and interpretation of data, was involved in conception and drafting of the manuscript. SC contributed to interpretation of data, was involved in conception, drafting and revision of the manuscript. SF and CC contributed to acquisition of data, drafted the manuscript. MV was involved in revising the manuscript critically for important intellectual content. ECA contributed to interpretation of data, gave final approval of the version to be published. All authors read and approved the final manuscript.


It is estimated that over 200 million adults worldwide have osteoporosis, a disease that has increasing socioeconomic impact reflected by unsustainable costs associated with disability, fracture management, hospital stays, and treatment. Existing therapeutic treatments for osteoporosis are associated with a variety of issues relating to use, clinical predictability, and health risks. Consequently, additional novel therapeutic targets are increasingly sought. A promising therapeutic candidate is sclerostin, a Wnt pathway antagonist and, as such, a negative regulator of bone formation. Sclerostin antibody treatment has demonstrated efficacy and superiority compared to other anabolic treatments for increasing bone formation in both preclinical and clinical settings. Accordingly, it has been suggested that sclerostin antibody treatment is set to achieve market approval by 2017 and aggressively compete as the gold standard for osteoporotic treatment by 2021. In anticipation of phase III trial results which may potentially signify a significant step in achieving market approval here, we review the preclinical and clinical emergence of sclerostin antibody therapies for both osteoporosis and alternative applications. Potential clinical challenges are also explored as well as ongoing developments that may impact on the eventual clinical application of sclerostin antibodies as an effective treatment of osteoporosis.  The identification of sclerostin as a therapeutic target and the optimisation of anti-sclerostin antibodies (Scl-mAb) have led to a vast array of preclinical studies documenting its ability to enhance bone formation, strength, and density , 2. DeveOsteoporosis is a metabolic bone disease characterised by a significant decrease in bone mineral density (BMD) and structural changes to the bone that greatly increase the risk of fracture . The conβ-catenin dependent pathway came to light specifically through the study of sclerosteosis and van Buchem disease, two rare genetic disorders associated with high levels of BMD and an associated low risk of fracture . Mo. Mo52].  ex vivo capacity of marrow cells to differentiate into osteoclasts. Moreover, an 80% reduction in surface erosion within the vertebrae and trabecular and tibial endocortical surfaces compared to ovx-vehicle samples was noted. At 26 weeks, however, the serum TRACP-5b and reduced ex vivo osteoclastic differentiation capacity of marrow cells were no longer evident. The observation that eroded surfaces remained over 80% lower in control groups nonetheless persisted.Some clarification may be sought in a recent study which assessed Scl-mAb (25 mg/kg) treatment administered once weekly for 6 or 26 weeks in ovx-rats . It shouInterestingly, at this time, the authors did not observe changes in RANKL or osteoprotegerin expression. As expected, Scl-mAb treatment induced significant increases in bone formation reflected by augmentations in serum P1NP and osteocalcin. Additionally, increases in the bone formation rate within trabecular, endocortical, and periosteal regions were observed. By week 26, however, only changes within the endocortical regions persevered while increases in skeletal mRNA of osteocytic genes were evident. In particular, SOST appeared to be markedly induced within the tibia and vertebral bone tissue . One can™) appears to increase, albeit not to statistically significant levels, the occurrence of atrial fibrillation and stroke in treated versus placebo groups [Generally, the safety profile of Scl-mAb treatment is positive, especially when compared to competitor osteoporotic therapeutics in development. For instance, the cathepsin K inhibitor MK 0822 (Merck, Odanacatibo groups . These sCompared to current anabolic market competitors such as teriparatide which is limited to 2 years' lifetime use, the substantial improvements in bone formation and microarchitecture subsequent to Scl-mAb treatment have been unrivalled. Consequently, the results of clinical studies demonstrating the therapeutic potential of romosozumab in phase III trials are eagerly awaited. While the clinical data relating to efficacy appears clear, there remain significant questions relating to optimal treatment interval. For instance, given its apparent superiority to increase BMD, should Scl-mAb be the first line of treatment for osteoporotic patients? However, it remains to be established if Scl-mAb can be used for a long term in a cyclical manner either alone or in a complementary manner with antiresorptive options. Many studies have already been performed following Scl-mAb therapy with an antiremodelling agent. It is likely, therefore, that this regimen will receive regulatory approval. This approach may help address questions attesting to the tolerability of Scl-mAb and whether the emergence of neutralising antibodies may become more of an issue with increased exposure.The emergence and clinical success of Scl-mAb have reinvigorated the therapeutic market for osteoporosis. Despite the many questions that remain, preclinical studies and published clinical trial results would imply that Scl-mAb will emerge as a dominant first-line treatment in the management of osteoporosis. Moreover, Scl-mAb is set to become a valuable tool in the development of international strategies to address the rise of osteoporosis and osteoporotic fracture management on a global scale.