The state of the immune system is reflected, in part, by the cell populations present in individual tissues which reflect the tumor microenvironment (TME). Several studies have suggested that understanding the TME constituents is useful for predicting drug response outcomes. Despite the biological significance of various inflammatory cell types in the TME, a widely accepted quantitative paradigm that allows the comparison of multiple subtypes of inflammatory cells in tissues is lacking. This is largely due to our inability to integrate key spatial information for multiple biomarkers across whole tissue sections, understand the concordance of individual biomarkers/cell types, account for heterogeneity in the region of analysis, and the absence of validated correlative scoring paradigms to measure against drug response. Finally, if these hurdles of understanding are met, a system needs to be created which can efficiently and reproducibly asses these readouts in a clinical-use environment. In this study, we revealed by immunohistochemistry distinct populations of immune system cells on serial sections of clinical lung cancer tissues. The immune cell markers evaluated included CD68, CD33, CD11b, FoxP3, CD4, and CD8. Whole tissue image analysis (tIA) was performed using Flagship's CellMap™ algorithm tool for each biomarker. Biomarker expression was analyzed on cell by cell basis for the tumor microenvironment and tabulated across the whole tissues. In order to integrate spatial information of CD68, CD33, CD11b, FoxP3, CD4, and CD8 biomarker expression from each slide onto a single tissue section, we utilized Flagship's patented FACTS™ tIA tool. This tool allows for an overlay of biomarker content information on to a single reference slide (H&E), and thus can provide key spatial information on biomarker expression relationships and presence of distinct cell populations in relation to each other. These studies directly demonstrate an effective utilization of combination of IHC with a set of Flagship tIA-based tools to score and analyze immune cell biomarker content in whole clinical tissue samples. Importantly, these tools can spatially correlate multiple biomarkers across serial sections of a single tissue. These approaches will lead to the development of standardized quantitative paradigms with predictive power in evaluating patient drug response.


Inorganic nanoparticles usually provide novel and unique physical properties as their size approaches nanometer scale dimensions. The unique physical and optical properties of nanoparticles may lead to applications in a variety of areas, including biomedical detection. Therefore, current research is now increasingly focused on the use of the high surface-to-volume ratios of nanoparticles to fabricate superb chemical- or biosensors for various detection applications. This article highlights various kinds of inorganic nanoparticles, including metal nanoparticles, magnetic nanoparticles, nanocomposites, and semiconductor nanoparticles that can be perceived as useful materials for biomedical probes and points to the outstanding results arising from their use in such probes. The progress in the use of inorganic nanoparticle-based electrochemical, colorimetric and spectrophotometric detection in recent applications, especially bioanalysis, and the main functions of inorganic nanoparticles in detection are reviewed. The article begins with a conceptual discussion of nanoparticles according to types, followed by numerous applications to analytes including biomolecules, disease markers, and pharmaceutical substances. Most of the references cited herein, dating from 2010 to 2015, generally mention one or more of the following characteristics: a low detection limit, good signal amplification and simultaneous detection capabilities. Among the various nanomaterials, inorganic nanoparticles are extremely important in the development of sensors. Not only can they be easily synthesized, but also cheaply mass produced. For this reason, they can also be more readily integrated into a variety of applications. Recently, inorganic nanoparticles of different kinds and dimensions have become widely exploited as versatile and sensitive sensors or probes. The main objective of designer inorganic nanoparticles, enhanced sensitivity in bio-sensing applications, greatly benefits from their small size, where their properties are strongly influenced by increasing their surface area. Thus, the combination of inorganic nanoparticles and sensors is one of the most exciting areas in modern analytical detection development because they offer excellent prospects for designing highly sensitive and selective sensors. Electrochemical sensing and spectrophotometric detection based on the modification or use of a particular variety of inorganic nanoparticles cosntitute a fascinating research area in biomedical applications. The remarkable features resulting from the use of inorganic nanoparticles are now widely employed in various detection systems. Presently, inorganic nanoparticle electrochemical sensors and optical spectrophotometric sensors used for bioanalysis purposes are collected. In this review the electrochemical and optical spectrophotometric characteristics of biomedical probes based on inorganic nanoparticles and the sensing applications possible with these materials are discussed, and their advantages and weaknesses are also explored. We also summarize the promising future anticipated for the use of these inorganic nanoparticles in both electrochemical and spectrophotometric detection as shown in the following sections.Gold nanoparticles (AuNPs), one of the most commonly used metal nanoparticles, have been applied in various fields. The range of applications for AuNPs is growing rapidly and includes: electronics, photodynamics, therapeutic agent delivery, sensors, probes, diagnostics and catalysis. AuNPs possess several advantages: 1) AuNPs can be synthesized in a straightforward manner and can be made highly stable; (2) AuNPs possess unique optoelectronic properties; (3) AuNPs provide high surface-to-volume ratio with excellent biocompatibility; (4) these properties of AuNPs can be readily tuned by varying their size, shape, and the surrounding chemical environment and (5) AuNPs offer a suitable platform for multifunctionalization with a wide range of organic or biological ligands for the selective binding and detection of small molecules and biological targets −3/−4 as a redox probe PF6 complex onto a NiOx(NP) modified GCE was reported. NiOx(NP) provided strong affinity for phosphate groups, thus oligonucleotide probes with a terminal phosphate group can be attached to the surface of the modified electrode for the detection of DNA. The detection limit was 6.8 × 10−11 M. The Ru-complex only responded to the complementary sequence of DNA. The technique has the advantages of good selectivity, good sensitivity, excellent reproducibility, stability, and simplicity 3− [2NPs) covered on a porous Ni foam electrode was successfully applied for the quantification of glucose by an amperometric method [Nickel nanoparticles (NiNPs) are another type of metal nanoparticles that exhibit excellent electrocatalytic ability toward glucose broadly used in glucose sensors. Nie et al.  reported(CN)6]3− . Further(CN)6]3− . In ordec method .in situ ultrasonication process at room temperature. The PdNPs could help increase the current signal due to their high surface area and the physical adsorption of the glucose molecules onto the large surface area of the electrode. More importantly, the electrode is highly resistant against poisoning by the interference from the oxidation of common interfering agents [Palladium nanoparticles (PdNPs) are also of interest as a great potential catalyst for improving the performance of glucose sensors. For example, PdNPs modified on functional carbon nanotubes (FCNTs) have been reported. Based on the electrochemical results, PdNPs efficiently catalyzed the oxidation of glucose and showed excellent resistance towards poisoning from interfering species such as ascorbic acid and uric acid . Similarg agents .Colloidal gold is extensively used for molecular sensing because of the flexibilities it offers in terms of modification of the gold nanoparticles (AuNPs) surface with a variety of functional groups. To enable glucose detection, the naked eye detection of glucose in urine has been reported. Thiol-capped AuNPs were functionalized with glucose oxidase (GOD) using carbodiimide chemistry. A visible color change of the GOD-functionalized AuNPs from red to blue was observed , as show3 magnetic nanoparticles (BFO MNPs) has been reported. The authors found that BFO MNPs can catalyze the decomposition of H2O2 to produce OH radicals, which in turn oxidize the weakly fluorescent benzoic acid to a strongly fluorescent hydroxylated product with a maximum emission at 405 nm [3O4 nanoparticles, then adhered to a solid parafin carbon paste electrode by magnetic force to fabricate a working electrode. H2O2 was produced by the enzymatic reaction of GOD and electrochemiluminescence (ECL) could be obtained by the reaction between luminol and H2O2 [2O4  MNPs has been proposed. This nanomaterial exhibited catalytic activities similar to those of biological enzymes that could catalyze H2O2 to produce hydroxyl radicals, which oxidized peroxidase substrate to produce a color [A fluorometric method for the determination of glucose and hydrogen peroxide using BiFeOt 405 nm . Moreoveand H2O2 . Further a color .cis-diols and 5,6-cis-diols (for the furanose form) or 4,6-cis-diols (for the pyranose form), respectively, of a glucose molecule to two FPBA-AgNPs. Furthermore, the glucose level variations associated with a model biological reaction process can be monitored by using FPBA-AgNPs, whilst the reaction mechanism remains nearly unchanged [Colorimetric detection based on assemblies of 5-amino-2-fluorophenylboronic acid-modified silver nanoparticles (FPBA-AgNPs) has been demonstrated. The glucose-modulated assembly of the FPBA-AgNPs occurred by the regulable formation of interparticle linkages via the bridge binding of 1,2-nchanged . A new anchanged .Analytical performance of selected samples are listed in tables that catagorized according to their detection methods including electrochemical , and colDue to the good properties of inorganic nanoparticles, they have been prevalently used as powerful sensors and probes. Each type of nanoparticle exhibits its own fascinating properties. However, they shared a similarity of enhancing probe sensitivity owing to their high surface area-to-volume ratios. Metal particles mostly synthesized from their bulk material display remarkably properties and exceptional features due to their miniature size, while the excellent behavior of semiconductor and nanocomposites results from their combinations of various materials. They have been utilized in a variety of applications for numerous analytes as described in this review for both electrochemical and optical spectrophotometric detection. The use of nanoparticles is an excellent approach for biomedical detection. Various groups of examples as discussed clarify the efficiency of nanoparticles by an increment of signal. In addition to modification, nanoparticles are capable of providing selectivity against interferences and complicated matrix effects. Many benefits of nanoparticles have been revealed, although more feasible practices still need to be developed to overcome difficulties such as the tendency to agglomeration and precision in size-control. Moreover, differences in the synthesized products from day to day led to uncertain properties which stimulate the search for solutions. Applications of nanoparticles in biomedical detection are abundant as well. Among essential substances, small clusters such as single molecules or cells as early stage disease markers are recently of interest. Therefore, improved comprehension of the advantages resulting from the use of nanoparticles and curiosity is challenging many research groups and should lead to novel discoveries in the future.


Oxidants play an important role in homeostatic function, but excessive oxidant generation has an adverse effect on health. The manipulation of Reactive Oxygen Species (ROS) can have a beneficial effect on various lung pathologies. However indiscriminate uses of anti-oxidant strategies have not demonstrated any consistent benefit and may be harmful. Here we propose that nuanced strategies are needed to modulate the oxidant system to obtain a beneficial result in the lung diseases such as Acute Lung Injury (ALI) and Chronic Obstructive Pulmonary Disease (COPD). We identify novel areas of lung oxidant responses that may yield fruitful therapies in the future. Oxygen is essential for complex biologic life. While unicellular organisms existed on this planet for nearly 4.5 billion years, a dramatic increase in the earth's oxygen 2.3 billion years ago permitted the emergence of multicellular organisms. This evolutionary step was fueled by the bio-energetic capabilities and signaling pathways that were enabled by this oxygen surge. A second increase in atmospheric oxygen 1.5 billion years later led to life forms of increasing complexity and the phylogeny of modern plant and animal species [Reactive Oxygen Species (ROS) are a class of molecules that contain oxygen and readily react with bio-organic compounds. Many ROS possess one or more unpaired valence electrons , and all ROS can participate in reduction-oxidation (Redox) reactions .These molecules are routinely generated from endogenous sources such as mitochondrial respiration and enzymatic reactions and the inherent instability of ROS allow for fast interactions with other molecules. Physiologic concentrations of ROS participate in major signaling pathways. As an example, the canonical transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and activator protein 1 (AP-1) have redox sensitive cysteine residues that regulate their activity. When ROS occur in excess, acondition called oxidative stress, can lead to detrimental consequences.These reactive molecules can form adducts with enzymes that result in protein misfolding and loss of function. ROS can also react with DNA with mutagenic consequences or oxidize lipids that can lead to the generation of carcinogenic compounds and the activation of inflammatory and apoptotic signaling pathways. Since ROS have the capacity to react in an indiscriminate manner with cellular components, an extensive range of antioxidant defenses have evolved to protect the cell from damage.Antioxidant enzymes, non-enzymatic antioxidants, and transition metal binding proteins all interact to minimize collateral oxidation reactions that can lead to cellular dysfunction . The disThe lung is unique as it is constantly exposed to both exogenous and endogenous oxidating agents. Endogenous sources of free radical generation include mitochondrial leak, respiratory burst through NADPH oxidase, some enzymatic reactions like xanthine oxidation, and auto-oxidation reactions . The lun2·−) even under normal circumstances (As the site of oxidative phosphorylation (OXPHOS), mitochondria are a major source of ROS in the cell. ROS are generated as electrons constantly escape from the transport chain to generate superoxide -driven cleavage of H2O2 in a Fenton reaction, generates the highly reactive and toxic hydroxyl radical (.OH). Furthermore, monoamine oxidase (MAO), a flavin containing amine oxidoreductase localized on the outer mitochondrial membrane, is also an important mitochondrial source of ROS, in particular of H2O2 [2O2 crosses through mitochondrial membranes, thus MAO can contribute to an increase in the concentrations of ROS within the mitochondrial matrix and as well as cytosol. Mitochondria contribute 20%–30% of the cytosolic steady-state concentration of H2O2 [66(Shc), α-ketoglutarate dehydrogenase, besides redox cycling of redox-active molecules [The steady-state concentration of Oar space . Most ofmembrane . Additioepressed . O2·− prplex III . The Q-c of H2O2 . H2O2 cr of H2O2 . There aolecules .If the mitochondria are healthy and functional then ROS generation is low. However, as the components of the electron transport chain age or sustain damage from excessive ROS they will become dysfunctional leading to the vicious cycle of increased ROS production. To prevent this, mitochondria are constantly cleared by a process called mitophagy; while new mitochondria are simultaneously produced by mitochondrial biogenesis. Thus mitochondrial turnover is necessary to keep mitochondria young and healthy. While mitochondrial ROS has been frequently viewed as a deleterious by-product of the electron transport chain, there is an alternative view that a certain level of mitochondrial ROS is actually beneficial in promoting health, longevity, and stress resistance. This new concept of mitohormesis  postulat91 phox and p22 phox attached to either the plasma membrane or the endosome membrane in neutrophils and macrophages. On stimulation the membrane bound units associate with a complex of Rac1, p67 phox, p40 phox and p47 phox, which then can transfer electrons from NADPH to oxygen to form the superoxide (In the cytoplasm the NADPH oxidase (Nox) complex is another important source of ROS. The Nox complex is a cluster of proteins that donate an electron from NADPH to molecular oxygen to produce superoxide. The Nox system consists of 2 membrane bound subunits, gpperoxide .91phox) [There are different isoforms of Nox  depending on the variations of the major subunit (gp91phox) . The pro91phox) ,16.The mitochondria, and Nox oxidant systems function cooperatively as initial mitochondrial ROS in necessary to prime the Nox system for sustained ROS production. Mitochondrial ROS achieves this by increasing the expression of the Nox isoforms and also by up regulating the Nox activity by activating the Rac1 component of the Nox complex ,18. ThisAcute lung injury (ALI), or its severe form, acute respiratory distress syndrome (ARDS), are devastating clinical syndromes that are characterized by diffuse inflammation and consolidation. There is vascular permeability due to loss of the alveolar-capillary barrier and accumulation of protein-rich lung edema. The clinical manifestations include hypoxemia, bilateral infiltrates, and loss of lung compliance. The pathologic correlation of lung injury is diffuse alveolar damage, which involves various cell types such as inflammatory cells (neutrophils and macrophages), epithelial cells and endothelial cells. This process occurs as a result of dysregulated inflammation, activation of the coagulation cascade, altered permeability of alveolar endothelial and epithelial barriers, and oxidant stress. ALI and ARDS represent a broad clinical definition that encompasses complex types of injury from parenchymal and extra parenchymal etiology . In addiThere are increased levels of oxidants in the broncho-alveolar lavage fluid (BAL) of patients with ARDS . The souSepsis and lung injury may be viewed as mitochondrial disorders. Mitochondria are essential hubs of innate immune signaling and are thought to be mediators of inflammatory responses –29 and tIn addition dysfunctional mitochondria are sources of damage-associated molecular patterns (DAMPs) such as ROS, Mitochondrial transcription factor A (TFAM), which is a structural and functional homologue of high-mobility group protein B1 (HMGB1), ATP and mitochondrial DNA that initiate an inflammatory response further exacerbating organ injury,38–40. MTo date the isoforms Nox1, 2 and 4 have been implicated in the pathogenesis of ARDS. Hyperoxia increases Nox1 expression in both epithelial and endothelial cells of the lung and Nox1 deficiency is protective against epithelial and endothelial cell death . Nox2 isNox4 is expressed in lung myofibroblasts and epithelial cells after bleomycin induced injury and inhibition of Nox4 suppressed the fibrogenic process after lung injury ,51. Nox4There is hope that use of antioxidants will have a therapeutic impact in lung injury as the evidence suggests that oxidant stress plays a central role in the pathogenesis. However study results have been mixed, consistent with the poor performance of antioxidants in other diseases such as cancer and heart disease . N-acetyNAC can reduce disulfide bonds and therefore neutralize oxidant species and is a widely used antioxidant. While animal studies using NAC have shown some effect in reducing lung injury, the use in human subjects was harmful with worse mortality when given 24hrs after admission in critically ill patients ,56.In another study of critically ill surgical trauma patients, antioxidant supplementation using alpha-tocopherol and ascorbic acid resulted in decreased incidence of multi organ failure and ICU length of stay . Other aIn the most comprehensive study to date administration of glutamine and antioxidants to critically ill patients with organ failure showed that no difference with the antioxidants but increased mortality in the group receiving glutamine .Considering the fact that some of the signaling pathways set into motion by ROS may be beneficial, it is not surprising that blanket reduction of oxidants has given equivocal results so far. Furthermore, the optimal timing of anti-oxidant administration is not clear. Clearly in some situations ROS is necessary, as the oxidative burst of phagocytes is required to clear infection and transient, low levels of mitochondrial ROS are advantageous, as in the case of mitohormesis. Further research will be needed to delineate the timing and strategies to optimize antioxidant therapies.Recent research has highlighted the importance of the mitochondrial and Nox pathways in generation of ROS in the lung ,61. Use HO-1 is a critical lung defense mechanism against oxidative stress and inflammation. In animal studies, CO seems to mediate the antioxidant effect of HO-1 and clinical trials to increase the protective effects of HO-1 through CO inhalation are underway . IntriguOxidative stress underlies the pathologic consequences of cigarette smoke. There are over 1016 oxidants per puff of cigarette smoke generated from over 4500 compounds ,66. Ciga2O2, or indirect measurements, of by-products of oxidative stress including 8-isoprostane and 4HNE, correlate with COPD severity and increase during acute exacerbations [The use of exhaled breath condensates and other post-mortem studies have confirmed the presence of oxidative stress in the lungs from patients with COPD . Notablyrbations ,77.Simultaneously, an inability to mount an appropriate antioxidant defense is also associated with COPD .The overwhelming evidence confirms the pathogenic role of oxidative stress in COPD. While the oxidative stress induced by cigarette smoke is well known, far less is known about the interaction between the exogenous oxidants of cigarette smoke and cellular sources of oxidant such as mitochondria and NADPH oxidases.As compared to nuclear DNA, mitochondrial DNA is particularly sensitive to the effects of oxidative stress generated by cigarette smoke . MitochoCigarette smoke has been demonstrated to disrupt the electron transport chain leading to increased ROS production and mitochondrial dysfunction  CongruenStudies of lung tissue from smokers have exhibited elevated measures of both mitochondrial DNA damage and mitochondrial DNA mutations when compared with nonsmokers. Evidence of mitochondrial dysfunction is not limited to the lungs. In platelets, smoking inhibits oxidative phosphorylation leading to increased mitochondrial ROS production .Dysregulated mitochondrial ROS production has also been measured in both diaphragm and skeletal muscle of cigarette smokers and may explain the sarcopenia evident in patients with COPD ,84. The Additionally, ROS generated as a consequence of mitochondrial dysfunction may compound tobacco smoke mediated oxidative stress ,89. ReceA recent publication suggests that the transfer of mitochondria from mesenchymal stem cells could attenuate cigarette smoke induced emphysema in rats . AdditioIncreased airway inflammation is a hallmark of COPD and linked, in part, to airway recruitment of neutrophils . Given tDifferential regulation of Duox1 and Duox2 has also been demonstrated in the airway epithelium of individuals with COPD, healthy smokers and non-smokers . HoweverMoreover, Nox activation may be interwoven with innate immune signaling pathways. TLRs are the canonical pattern recognition receptor triggering the innate immune response. While increased airway inflammation is a hallmark of COPD, there is a paradoxical decrease in innate host defense in COPD .Decreased TLR2 and TLR4 functioning has been demonstrated in patients with COPD which in part may underlie the increased susceptibility to airway colonization by pathogenic bacteria ,98.Concurrently, Nox isoforms have been demonstrated to regulate TLRs while simultaneously, certain TLR signaling cascades are Nox-dependent. An example of this complex interplay is the role of TLR4 and Nox3 in COPD. Nox3 is an isoform that is usually absent in normal, adult lungs, but can be induced in murine adult lung and lung endothelial cells, with the unexpected finding that Nox3 is regulated by TLR4. We found that TLR4 knockout mice develop spontaneous COPD due to increased synthesis of Nox 3, a phenotype that is partially reversed by concomitant knockdown of the Nox3 enzyme .Biologic responses to oxidative stress have been extensively studied. Glutathione, via its role as a reducing agent, is a critical mediator of host antioxidant defense in the lung airway. While total glutathione levels are not decreased in COPD, their levels may be inadequate given the increased presence of oxidants as a result of cigarette smoke . GeneticMany antioxidant responses are controlled by Nuclear factor erythroid-2-related factor 2 (Nrf2). Nrf2 is an evolutionary conserved transcription factor that is sequestered and targeted for proteosomal degradation under basal conditions but results in the transcription of genes collectively associated with a cis acting binding site known as the antioxidant response element(ARE) ,107. DurDespite the pathogenic role of oxidative stress in COPD, pharmacologic utilization of antioxidants in COPD has not demonstrated dramatic improvements in disease outcomes. Results from clinical trials with NAC have been mixed. A Cochrane meta-analysis has suggested a decrease in exacerbation with the use of NAC. While the large scale BRONCHUS trial demonstrated no benefit, a recent study of high dose NAC demonstrated a modest decrease in exacerbations and improvement in Forced Expiratory Flow 25% to 75% but no improvement in various subjective dyspnea scores ,112. AttThe disappointing results from therapeutic drug trials in ALI and COPD should not dissuade us from recognizing the importance oxidative stress plays in the pathogenesis of pulmonary disease. Rather, it highlights an incomplete understanding of a crucial but complex biologic process. The classical paradigm of oxidative stress as a balance between oxidants and antioxidants is incomplete as this model fails to account for a multitude of subtleties in oxidant signaling. Oxidants and anti-oxidants are generated by a variety of systems and likely effect cellular functioning in diverse ways. Some of these systems contribute to homeostatic functioning, making blanket antioxidant therapy ineffective, at best, and possibly deleterious. Alternatively, varied cell types and biologic compartments have dissimilar regulation and responses to ROS. Further understanding of this complexity is necessary to determine how to deliver anti-oxidant therapy. Finally, while oxidant generation is critical to host defense, there is a limited understanding of how immunity regulates the oxidant/antioxidant balance and the subsequent pathologic consequence this imbalance. Our discoveries of TLR4-mediated Nox3 suppression and MKK3-induced mitochondrial oxidants provide some insight into previously unappreciated links between the immune system and oxidant generation but a myriad of questions remain. As a fundamentally protective response, signaling pathways downstream of innate immune activation may prime cells for the anticipatory increase in oxidative stress as a result of impending inflammation. Consequently, anti-apoptotic and anti-oxidant responses linked to innate immune functioning may be utilized to combat oxidant related diseases. The indiscriminant use of antioxidant therapy reinforces an incorrect belief that oxidants are uniformly deleterious. Instead we should focus on therapies to modulate oxidants in a highly targeted and controlled manner. While overwhelming evidence demonstrates that excessive oxidative stress contributes to the development of pulmonary disease, further understanding of this complex system will be required before effective therapy can be administered.


Cyanobacteria and phototrophic Proteobacteria, the column interior was dominated by Proteobacteria and putative anaerobic representatives of the Firmicutes and Bacteroidetes. The results suggest a stratification of the ikaite columns similar to that of classical soda lakes, with a light-exposed surface inhabited by primary producers and an anoxic subsurface. This was further supported by identification of major taxonomic groups with close relatives in soda lake environments, including members of the genera Rhodobaca, Dethiobacter, Thioalkalivibrio and Tindallia, as well as very abundant groups related to uncharacterised environmental sequences originally isolated from Mono Lake in California.The submarine ikaite columns located in the Ikka Fjord in Southern Greenland represent a unique, permanently cold (less than 6°C) and alkaline (above pH 10) environment and are home to a microbial community adapted to these extreme conditions. The bacterial and archaeal community inhabiting the ikaite columns and surrounding fjord was characterised by high-throughput pyrosequencing of 16S rRNA genes. Analysis of the ikaite community structure revealed the presence of a diverse bacterial community, both in the column interior and at the surface, and very few archaea. A clear difference in overall taxonomic composition was observed between column interior and surface. Whereas the surface, and in particular newly formed ikaite material, was primarily dominated by  Extremophiles are organisms that grow and reproduce optimally at or near the extreme ranges of environmental variables. This can be extremes of temperature, pressure, pH, salinity, aridity or radiation and environments representing one or more of these variables are common on earth. The largest proportion of such extreme habitats is comprised of permanently cold areas, including polar regions, permafrost, deep oceans, and alpine regions. Similarly, naturally occurring stable alkaline environments such as soda lakes and deserts, and alkaline ground water are distributed globally, although these occur much less frequently. A large number of bacteria have been isolated from these environments and they often show adaptations to optimal growth under the prevailing conditions –3.Clostridia and hydrogen-utilising Betaproteobacteria related to the genus Hydrogenophaga are abundant in serpentinite water [Soda lakes are the most thoroughly studied natural alkaline environment in terms of microbial diversity. They are mainly confined to arid regions at temperate and tropical latitudes and are characterised by high concentrations of sodium carbonate and sodium chloride, which is reflected in the adaptations of microorganisms isolated from these environments –6. Primate water –11, 13.Rhodonellum psychrophilum [Permanently cold, stable alkaline environments are a very rare occurrence and only a few such environments have been described: The upper layers of the permanently ice-covered Lake Untersee in east Antarctica , a serierophilum , 28, androphilum –32.There is significant biotechnological interest in microorganisms and enzymes from cold and alkaline environments and numerous studies have focussed on the isolation and characterisation of novel enzymes for low temperature and/or high pH applications –36. In aSamples of ikaite columns were collected from two locations in the Ikka Fjord, the Atoll Field and the Camp Field , during http://www.arb-silva.de/; database version SSU r121), was 70.7% and 68.8% for a complete match and 77.6% and 76.3% allowing one mismatch (excluding the three nucleotides in the 3’ end of the primers) for domain Bacteria and Archaea, respectively. The PCR amplification and preparation of amplicon libraries were performed essentially as described [A total of 70 ikaite samples, 3 seawater samples, and 6 sediment samples were used for DNA extraction. DNA was extracted from 0.5 g ikaite or sediment material using a ceramic bead beating procedure with the MO-BIO Powersoil DNA Extraction Kit  modified with G1 blocker . DNA from seawater samples were extracted using the same method after homogenisation of filters in liquid nitrogen. A 466 bp fragment covering the V3 and V4 hypervariable regions of the 16S rRNA gene from bacteria and archaea was PCR amplified using the primers 341F (5’-CCTAYGGGRBGCASCAG-3’) and 806R (5’-GGACTACNNGGGTATCTAAT-3’) . The ampescribed . BrieflyAdapters and tags for pyrosequencing were added in a second 10-cycle PCR on 5 ng of purified PCR product using the conditions described above with primers 341F and 806R carrying sequencing adapters and tags for multiplexing. The amplified fragments were gel-purified, quantified, and mixed in equal amounts before sequencing on a Genome Sequencer FLX pyrosequencing system .www.biopieces.org). Initially, tags for multiplexing and primer sequences used in the initial PCR (341F and 806R) were removed, discarding any sequences that did not contain both a multiplexing tag and the forward primer sequence. Sequences were then trimmed from both ends at the first high-quality base  and sequences with an average Phred quality score lower than 25 were discarded. Finally, sequences shorter than 250 bases and containing more than one ambiguous nucleotide were discarded. The complete dataset of quality-filtered sequences in QIIME-compatible format is available from the MG-RAST server .Trimming and quality-filtering of 16S sequences was performed using the software suite Biopieces (http://greengenes.lbl.gov/) [OTU clustering was performed using USEARCH , which ibl.gov/)  clusterewww.qiime.org) [Phylogenetic analysis was performed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline version 1.7 (ime.org) . The USETaxonomy was assigned using the RDP classifier with a confidence threshold of 50%  and a trSections of individual ikaite columns were collected during the summer from two locations in the Ikka Fjord . Columns2S) when above water immediately after collection. This was also noted on a previous expedition [Some columns gave of a distinctive odour of hydrogen sulphide . A total of 30 interior and 18 surface samples, as well as three seawater and two sediment samples, were obtained for pyrosequencing. Details of individual columns and samples used for pyrosequencing are given in http://greengenes.lbl.gov/). An overview of the combined frequency of all phylogenetic groups and of each identified OTU in both individual samples and in combinations of related samples can be found in Partial 16S rRNA gene sequences were amplified using primers flanking the V3 and V4 hypervariable regions of both bacteria and archaea. Two technical triplicates where prepared as sequencing controls by separate amplification and pyrosequencing of samples I11 and I39. Pyrosequencing of the 57 samples generated 654,635 high-quality sequences after trimming and quality filtering. Filtering of chimeric sequences and clustering at 97% identity resulted in 452,477 sequences in 18,410 operational taxonomic units (OTUs). Of these, 9,407 OTUs were represented by only a single sequence (singletons) and were discarded before further analysis leaving 9,003 OTUs in the dataset covering ikaite, seawater and sediment samples . PhylogeThe generated sequences and OTUs were used to estimate the richness and diversity of the ikaite community. It is worth noting that sequence data alone does not take into account the proportion of dead or dormant microorganisms and may thus overestimate the number of active bacteria participating in community functions. Bacteria have previously been cultivated directly from both new and old ikaite , 29, butRarefaction analysis of species (OTU) richness in a combined dataset of all samples taken from the ikaite columns resulted in a curve close to saturation at the 8,590 OTUs identified, suggesting that further sequencing effort is unlikely to significantly increase the number of observed OTUs in these samples. Rarefaction curves of interior and surface samples were approaching saturation at the 7,798 and 4,252 OTUs identified, respectively, indicating a significant OTU overlap between these two datasets . A totalThe total microbial diversity was estimated using the Shannon diversity index for various combinations of samples . As suggDifferences in overall community composition (beta-diversity) were investigated using average linkage cluster analysis (UPGMA) and the weighted UniFrac distance metric . The resDespite the clear differences in overall community structure, rarefaction analysis (see above) suggested a significant overlap in the presence of OTUs between surface and interior samples. This was confirmed by an analysis of the OTU distribution between these two sample types, which identified 3,460 shared OTUs representing 81.4% and 44.4% of all OTUs from the surface and interior datasets, respectively. While this does not take in to account the abundances of individual OTUs, it is in agreement with the observation that ikaite columns can grow up to 50 cm per year , which wProteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Cyanobacteria, whereas the microbial diversity in seawater and sediment samples were mainly restricted to Proteobacteria, Bacteroidetes, and Actinobacteria , which include numerous phototrophic genera , orders Natranaerobiales (12.8%), Clostridiales (7.7%), and Thermoanaerobacterales (2.9%), most of which grow anaerobically [Bacteroidetes identified in the column interior, primarily represented by the class Bacteroidia, order Bacteroidales (16.5%), are mostly anaerobic. The abundance of these orders suggests that the column interior is a largely anoxic environment, much like the lower layers and sediment of soda lakes, which are also abundant in anaerobic Firmicutes and Bacteroidetes [Natranaerobiales were first identified in soda lakes of the Wadi An Natrun in Egypt and the abundant Natranaerobiales in the ikaite columns may represent low-saline, cold-adapted relatives of this newly described order [Proteobacteria were abundant in all samples, although with significant differences in the distribution between classes , an abundant phototroph and a key primary producer in African soda lakes [Rhodobaca, Arthrospira and other free-living Cyanobacteria were almost absent from seawater samples (Rhodobaca and the Cyanobacteria (including chloroplast sequences) accounted for 33.5% and 36.6% of all sequences, respectively  were abundant both internally and at the surface of columns , suggesting that alkali-tolerant members of this genera are less prominent in high-saline environments. The closest characterised relative was Azoarcus taiwanensis (97.6%), a denitrifying, sulphide-oxidising facultative alkaliphile [OTUs classified to the genus  columns . Azoarcuda lakes  and the Helicobacteraceae in the Epsilonproteobacteria was very abundant in the column interior and in particular in sediment samples, but whereas a significant number of sequences from the columns were assigned to the genus Sulfurimonas, this genus was virtually absent from sediment samples  were identified in the ikaite column interior and additionally, in very high numbers in sediment samples  and subsequent evaporative concentration of CO32- [2+ from seawater and CO32- from spring water precipitate as ikaite and the high pH is maintained by continuous influx of alkaline spring water. In both cases, a high concentration of carbonate ions leads to an elevated pH. The similarities in geochemistry and community structure suggest that community function and nutrient cycling in these two environments could be overlapping. A notable exception is the absence of the organotrophic haloalkaliphilic archaea in the ikaite columns, which can develop to high densities in soda lakes [The separation between column surface and interior is similar to the stratification or water/sediment interface seen for soda lakes with an upper light-exposed, oxygenated layer and a lower anoxic layer rich in sulphur compounds. Furthermore, the geochemical environment encountered by the microorganisms inhabiting the ikaite columns is, in some respects, similar to that of soda lakes where the concentration of COf CO32- . In the da lakes .The significant overlap with soda lake communities would suggest that the high pH and perhaps general geochemical environment is the main selective driver, meaning that the ikaite columns are primarily inhabited by cold-adapted alkaliphiles rather than alkaline-adapted psychrophiles. This makes the columns an obvious target for comparative studies of cold-adaptation and how bacteria adapt to survive combinations of extreme environmental factors.Cyanobacteria, the interior appeared to be a largely anoxic environment populated by anaerobic heterotrophs and chemolithotrophs participating in cycling of sulphur compounds. The functional separation between surface and subsurface is also present in classical soda lakes and many of the most abundant phylogenetic groups identified in the ikaite columns were related to common groups found in soda lake environments. The apparent analogy to a cold soda lake implies that isolation of new alkaliphilic low-temperature species could be a valuable tool for studying the evolution of cold-adaptation. In addition, the unique combination of low temperature and high pH makes the columns a prime target for bioprospecting studies aimed at identifying cold- and alkaline-active enzymes.In-depth pyrosequencing of 16S rRNA genes from the ikaite columns and the surrounding Ikka Fjord revealed a surprisingly diverse, distinct bacterial community inhabiting the ikaite columns. A clear difference between the phylogenetic groups identified at the surface and in the interior of the columns was observed. While the surface was dominated by primary producers, such as anoxygenic phototrophic bacteria and S1 TableColumns were classified as ‘old’ or ‘new’ based on the observations described in the main text. Samples were further classified as ‘interior sample (is)’ when taken from a cross-section, ‘surface sample (ss)’ when taken from the top 5 mm at the surface, and ‘surface drilling (sd)’ when taken by drilling 1–2 cm in from the surface.(DOCX)Click here for additional data file.S2 TableSamples are named by column number (#), sample type  and sample number. See (DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


The adrenal cortex is critical for physiological function as the central site of glucocorticoid and mineralocorticoid synthesis. It possesses a great degree of specialized compartmentalization at multiple hierarchical levels, ranging from the tissue down to the molecular levels. In this paper, we discuss this functionalization, beginning with the tissue zonation of the adrenal cortex and how this impacts steroidogenic output. We then discuss the cellular biology of steroidogenesis, placing special emphasis on the mitochondria. Mitochondria are classically known as the “powerhouses of the cell” for their central role in respiratory adenosine triphosphate synthesis, and attention is given to mitochondrial electron transport, in both the context of mitochondrial respiration and mitochondrial steroid metabolism. Building on work demonstrating functional assembly of large protein complexes in respiration, we further review research demonstrating a role for multimeric protein complexes in mitochondrial cholesterol transport, steroidogenesis, and mitochondria–endoplasmic reticulum contact. We aim to highlight with this review the shift in steroidogenic cell biology from a focus on the actions of individual proteins in isolation to the actions of protein assemblies working together to execute cellular functions. The cortex of the adrenal is the principal site of synthesis of vertebrate glucocorticoid and mineralocorticoid steroid hormones . These hThe adrenal gland is composed of two anatomically and functionally distinct compartments: the cortex and the medulla. The adrenal medulla is innervated with chromaffin cells and plays a key role in catecholamine synthesis and the sympathetic stress response , while tvia a multienzyme pathway particular to each steroidogenic tissue, resulting in successive modifications to the sterol backbone . The. Thevia e Figure . Ultraste Figure . The laye Figure  10). Th. Thvia ae Figure .Developmentally, the adrenal cortex arises from the adrenocortical primordium, itself derived from the urogenital ridge, a specialized region of the embryonic coelomic epithelium that also serves as the developmental precursor of the kidneys and hematopoietic progenitors . Cells iEukaryotic cells are characteristically compartmentalized, containing numerous membrane-bounded organelles, each with specialized functions. These organelles achieve their specialization through non-uniform segregation of molecules, whether they are nucleic acids, proteins, lipids, or carbohydrates. The mitochondria are famously known as the “powerhouse of the cell” for their respiratory capacity and synthesis of adenosine triphosphate (ATP). Although mitochondrial energetics have traditionally served an ancillary role in steroidogenic research , the rec2). Ubiquinol transfers its electrons to a cytochrome-containing IMM protein complex, Complex III (ubiquinol/cytochrome c oxidoreductase), which further shuttles the electrons to cytochrome c. From cytochrome c, the electrons flow to the cytochrome-containing Complex IV , the terminal IMM protein complex of the mitochondrial respiratory chain, which uses the electrons to reduce O2 to yield H2O. The free energy of electron movement through the ETC is used to pump protons (H+) out of the mitochondrial matrix into the mitochondrial intermembrane space (IMS), creating a capacitance across the IMM. This potential energy is utilized to drive ATP synthesis by the IMM Complex V (ATP synthase), which condenses ADP + Pi to form ATP while pumping protons back into the matrix. Matrix ATP is then exchanged with cytosolic ADP by the adenine nucleotide translocator (ANT/SLC25), which works in conjunction with the outer mitochondrial membrane (OMM) voltage-dependent anion channel (VDAC) to form an energy-transducing mitochondrial contact site (The mitochondria generate ATP by oxidizing hydrogens derived from carbohydrates (through the tricyclic acid cycle) and fats (through fatty acid β-oxidation). Electrons from nicotinamide adenine dinucleotide (NADH) are donated to the iron–sulfur (Fe–S) clusters of mitochondrial complex I (NADH dehydrogenase), a multimeric inner mitochondrial membrane (IMM) protein complex. From Complex I, the electrons are sequentially shuttled to ubiquinone (coenzyme Q/CoQ), giving rise to ubiquinol  and hydroxysteroid dehydrogenase/ketosteroid reductase (HSD/KSR) enzymes. The CYP enzymes represent the majority of the steroidogenic enzymes and will receive the greatest attention here as they are the most abundant steroidogenic enzymes of mitochondria. A highly diverse superfamily of enzymes, the CYPs are characterized by a single heme prosthetic group and the ability to absorb light at 450 nm when reduced with carbon monoxide. Enzymatically, P450 enzymes exhibit an extraordinary ability to insert oxygen into non-activated carbon–hydrogen bonds at the same time exhibiting high structural selectively. They accomplish this feat through their ability to activate molecular oxygen, although the iron atom of their heme tetrapyrrole prosthetic group with the aid of an ancillary redox partner .Thousands of CYPs have been identified, in all domains of life, suggesting that this class of protein has ancient roots. There are 57 CYP genes encoding CYP enzymes in humans, subdivided into 18 clades. The majority of the CYP enzymes are localized to the ER, but one clade is localized to the mitochondria. While ER-localized CYPs obtain their electrons from a single P450 oxidoreductase, the mitochondrial CYPs utilize an electron transport chain containing the ferredoxin reductase (FDXR) and ferredoxin proteins . There aCYPA11A1 is absolutely essential for the synthesis of all vertebrate steroids, which are all characterized by the CYP11A1 reaction, namely cleavage of the cholesterol aliphatic side chain. The proposed reaction mechanism of CYP11A1 involves three sequential modifications of cholesterol. In the first step, CYP11A1 hydroxylates cholesterol at carbon 22 of the aliphatic tail; in the second step, cholesterol is hydroxylated at the carbon 20 of the aliphatic tail; finally, in the third step, oxidative scission of the C20–22 bond of the subsequent 20,22-dihydroxycholesterol yields the steroid pregnenolone and the reactive aldehyde, isocaproaldehyde . This re2+ and protein kinase C (PKC) can suppress CYP11A1 transactivation  influx and stimulation of PKC and calmodulin-dependent protein kinase kinase (CAMKK) intracellular signaling  and FDXR – as intermediates in electron donation from NADPH Figure . These ac and ubiquinol have been described previously . Surprisingly, StAR was found to stimulate steroid production in cell model systems without its leader sequence and import into the mitochondria . This le failure . Moreove failure , collect failure  and can  failure  supports+ family of ATPases, a broadly conserved family of ATPases implicated in various cellular processes (+-type ATPase domain (The ATAD3A protein belongs to the AAArocesses , 3–5. ATe domain . Severale domain , 144, whe domain , 145.The ER is a complex cellular organelle, formed by an interconnected network of cisternae , is distin vitro membrane association assays and cell models (Almost 30 years ago, Vance demonstrated phospholipid synthesis in cellular fractions enriched in mitochondrial and ER markers . Electrol models . A seconl models . Of notel models , and cAMl models , collectl models , and thil models , suggestSteroidogenesis begins with the mobilization and movement of cholesterol from intracellular stores into mitochondria. Control of steroidogenic output is organized at two levels: substrate availability and targeting, and enzyme expression and localization. Past and recent studies in hormone-inducible steroidogenic cells showed that cholesterol trafficking and targeting into mitochondria is rate limiting and driven by intracellular protein networks, referred to as the transduceosome, which amplifies the cAMP signal at the OMM, and the steroidogenic metabolon. This mitochondrial metabolon prevents unwanted crosstalk of the substrate cholesterol with other pathways, optimizing substrate concentration and targeting to CYP11A1. Deciphering the organization and regulation of intracellular protein assemblies that interact with the steroidogenic machinery will provide insight into the intracellular events involved in normal and disease states, facilitating diagnosis and treatment. These studies suggest a shift in focus in steroidogenic cell biology from the actions of individual proteins in isolation to the actions of protein assemblies working together to execute specialized cellular functions, in this case adrenal steroid formation.All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.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.


In the investigation of food allergy syndromes, serum assays detecting IgE antibodies (Ab) to food allergens use whole extracts on the solid phase, which contain specific allergen components in varying amounts. The interpretation of results assumes that the causative allergen components are adequately represented in the extract and that the presence and level of specific IgE titer to the whole extract of the relevant food(s) are diagnostic of the syndrome. IgE assays for beef or pork can underestimate the IgE Ab to a minor component of mammalian meat, such as galactose-α-1,3-galactose . The inciting food allergens in Eosinophilic Esophagitis (EoE) remain unclear, although IgE Ab to milk, wheat, soy, and peanut are frequently present in low titer.IgE Ab to relevant allergens and their components were measured in the sera of adults and children with peanut allergy, delayed anaphylaxis to mammalian meat, or esophageal biopsy-diagnosed EoE. Assay methods included: ImmunoCAP (CAP) using whole and component extracts, CAP assays on serial dilutions (1:2 to 1:8) of sera, and ImmunoCAP ISAC .Analysis with ISAC correlated well with results using component-specific CAP assays for both peanut allergens  and inhalant allergens (EoE). By contrast, no positive results for food allergens were found by ISAC in EoE sera that were positive for milk, wheat, or soy by CAP. Dilution assays showed no change  in either peanut allergy sera or EoE sera positive for aeroallergens (dust mite and cat); in contrast, calculated titers up to six times the undiluted value were noted for foods in EoE sera  and in mammalian meat allergy sera (beef and pork). CAP assays for 5 milk components revealed positivity to minor components in 50% of EoE sera. CAP assays for α-gal revealed positivity in 100% of patients presenting with delayed anaphylaxis to mammalian meat.Differences in the results of the dilution assays demonstrate that assaying undiluted sera can significantly underestimate IgE Ab levels if the IgE Ab are directed against a quantitatively minor component of the extract on the solid phase. These results strongly suggest that the IgE Ab to milk and other foods in EoE sera are directed against a minor component that has not yet been identified.


Allergic responses are dependent on the highly specific effector functions of IgE antibodies. Conversely, antibodies that block the activity of IgE can mediate tolerance to allergen. Technologies that harness the unparalleled specificity of antibody responses have revolutionized the way that we diagnose and treat human disease. This area of research continues to advance at a rapid pace and has had a significant impact on our understanding of allergic disease. This review will present an overview of humoral responses and provide an up-to-date summary of technologies used in the generation of human monoclonal antibodies. The impact that monoclonal antibodies have on allergic disease will be discussed, with a particular focus on allergen immunotherapy, which remains the only form of treatment that can modulate the underlying immune mechanisms and induce long-term clinical tolerance. Allergen immunotherapy (AIT) was introduced over a century ago with the first clinical trial conducted by Noon and Freeman in 1911 . The aimAntibodies are secreted glyco-proteins that recognize and bind to antigen with exquisite specificity through the highly variable fragment antigen binding (Fab) region Fig. . The varH, D, JH, and CH), the kappa (κ) light chain immunoglobulin locus on chromosome 2 , and the lambda (λ) light chain locus on chromosome 22 . Antibodies are produced by B cells which develop in the bone marrow and express a membrane version of the antibody in the form of a B cell receptor or membrane immunoglobulin. Following activation by antigen in the periphery, specific B cells undergo a series of processes that involve clonal proliferation, isotype switching (or class switch recombination), and affinity maturation, whereby somatic hypermutation of variable region genes leads to changes in the affinity of the BCR. B cells that bind to antigen with high affinity receive survival signals and may differentiate into memory B cells or plasma cells, the latter of which gives rise to secretion of high affinity antibodies. Long-lived, terminally differentiated plasma cells migrate to the bone marrow where they are thought to produce in excess of 108 antibody molecules per hour [Human antibodies are encoded within the heavy chain immunoglobulin locus on chromosome 14  has remained the only form of treatment that can modulate the underlying mechanisms of allergic disease. Currently, AIT is used to treat allergic rhinitis, allergic conjunctivitis, allergic asthma, and insect venom allergy. Both subcutaneous (SCIT) and sublingual (SLIT) forms of treatment are approved, though alternative routes  region of IgE  . It is a+ T cells as well as by basophils. The importance of IL-4 in Th2-mediated inflammation and the induction of IgE class switching prompted the generation of monoclonal antibodies designed to neutralize IL-4. Like other mAbs-targeting single cytokines , the results of clinical trials were disappointing, with variable levels of efficacy as a standalone treatment [The combination of monoclonal antibody therapy with AIT has been explored with other targets of the allergic response. IL-4 is a prototypic T helper 2 (Th2) cytokine produced primarily by CD4reatment . More rereatment . DespiteIn 1937, Robert Cooke and Mary Loveless demonstrated that co-administration of serum from subjects who had received multiple subcutaneous injections of ragweed pollen over 3–12 months, could inhibit immediate allergic responses in the skin, elicited by intradermal injection of ragweed with allergic serum . These eLater, similar studies , 65 provThe availability of well-characterized allergen-specific antibodies could have broad applications for our understanding and treatment of allergic diseases, from direct therapeutic application as passive immunotherapy to the standardization of allergen extracts for AIT. In addition, allergen-specific monoclonal antibodies can facilitate the design of novel forms of allergen immunotherapy. Inhibiting the activity of IgE has clear benefits in terms of improving the safety and efficacy of AIT, exemplified by the concomitant use of anti-IgE. It follows that hypoallergens  would make excellent candidates for allergen immunotherapy. Early attempts to alter the allergenicity of immunotherapy vaccines through chemical modification  had limited success due to lack of immunogenicity or residual IgE activity, but re-evaluation of this approach has led to a number of recent initiatives, most notably the EU-funded FAST project ••, 70.www.allergen.org), protein database (PDB) structures are available for 92 (11 %) of these [. demonstrated that a single point mutation in the epitope of Bet v 1, abolishes binding of BV16 and reduces binding of serum IgE by 50 % [Understanding the molecular determinants of IgE recognition would allow the rational design of recombinant hypoallergens for AIT. Epitopes formed by discreet continuous regions of polypeptide (“linear” epitopes) can be deduced by epitope arrays . Howeverof these . In turnof these –79 , more s


Intrahepatic cholestasis in the form of paraneoplastic phenomena is an uncommon presentation of Hodgkin's lymphoma (HL). Herein we report the diagnosis of mixed type HL-related idiopathic intrahepatic cholestasis in a 73-year-old man presenting with jaundice, after the inguinal lymph node biopsy indicative of mixed cellular type HL and liver biopsy consistent with intrahepatic cholestasis, following several diagnostic interventions including surgery for suspected extrahepatic obstructive cholestasis. Our findings emphasize the value of early liver biopsy in the diagnostic algorithm along with consideration of HL-related idiopathic intrahepatic cholestasis as a diagnosis of exclusion in cholestatic jaundice of obscure origin. The incidence of hepatic involvement at the time of diagnosis of Hodgkin's lymphoma (HL) was reported to range from 3% to 14%, along with much higher range of 38%–66% of incidence documented in postmortem series , whereasDirect intrahepatic parenchymal or biliary epithelial involvement of HL has been considered to be the most common reason for hepatic dysfunction in patients with HL , while eTo date, most cases of HL-related idiopathic cholestasis have been associated with VDBS in the literature, while HL-related idiopathic cholestatic hepatitis without a paucity of small intrahepatic bile ducts has been considered as a rare phenomenon being described only in a few reports , 7. A clA 73-year-old man was admitted to our department with a 4-month history of pruritus and 2-month history of jaundice, alcoholic stools, dark urine associated with malaise, fatigue, and 8 kg weight loss. He had been admitted to the hospital for the complaint of pruritus in January 2012, while he was on routine follow-up for hypothyroidism but otherwise healthy. In March 2012, he had been hospitalized in general surgery department for further investigation upon findings of elevated liver enzymes and hyperbilirubinemia on laboratory tests . No pancOn admission to our department, physical examination findings revealed body temperature of 38.2°C along with 1 × 2 cm left inguinal lymphadenopathy and hepatomegaly. Liver size by percussion was determined to be 10 cm with no evidence of ascites. The spleen was not palpable and the abdomen was not tender with normal bowel sounds and an operational scar on the right abdominal quadrant. Cardiac and chest examination revealed normal findings.There was no history of hepatitis, hepatotoxin exposure, intravenous (IV) drug abuse, or blood transfusion. Other than receiving thyroid hormone therapy for the last 10 years and oral secondary bile acid replacement for 2 months, he was free of any other medication or herbal remedies. As shown in The diagnosis of Hodgkin's disease in our patient presenting with the complaint of jaundice required extensive work-up including surgical procedures until the exclusion of bile duct obstruction or extrahepatic causes of cholestasis and identification of inguinal lymph node biopsy findings consistent with classical HL with mixed cellularity and liver biopsy findings consistent with intrahepatic cholestasis.In our case, jaundice developed in the absence of any medication or herbal remedy and all alternative diagnoses have been ruled out. Besides, pathological findings were compatible with the cases previously defined in literature  and in aGiven that idiopathic intrahepatic cholestasis as a presenting symptom of HL has been rarely documented in the medical literature , 5–7, ouWhen cholestasis-related clinical signs and symptoms are observed in patients with HL, our findings suggest considering possibility of either VBDS or idiopathic cholestasis in the diagnostic procedures.The exact pathogenesis of paraneoplastic syndrome-related cholestasis has not yet been clarified, while the emission of a cholestatic cytokine or elaboration of hormones such as androgens or 17-alkylated estrogens from the tumor cells has been proposed amongst the possible mechanisms . NonetheNotably, diagnosis of both VBDS and idiopathic intrahepatic cholestasis seems to be crucial given that these two paraneoplastic phenomena are distinct not only in terms of histological characteristics but also in their clinical course with better prognosis in case of idiopathic intrahepatic cholestasis .In this regard, given that patients with idiopathic cholestasis generally have a better prognosis when presenting with stage I/II HL and when treated with radiation , consideIn conclusion, given that jaundice is a commonly faced sign indicative of a large list of diseases in the clinical practice, our findings suggest the consideration of HL-related idiopathic intrahepatic cholestasis as a diagnosis of exclusion in patients presenting with jaundice of indefinite origin and signify the value of early liver biopsy in the differential diagnosis of the jaundice.


To describe our initial experience with radiofrequency ablation (RFA) of Bosniak IV renal cysts. From 2010 to 2014, 154 renal tumor cases were treated with percutaneous thermal ablation, of which 10 cases (6.4%) from nine patients were complex renal cysts and were treated with radiofrequency ablation. All complex cysts were classified as Bosniak IV . One patient had a single kidney. Lesion size ranged from 1.5 to 4.1cm (mean: 2.5cm) and biopsy was performed on four cysts immediately before the procedure, all of which were malignant (two clear cell and two papillary carcinoma). Mean volume reduction of complex cysts was 25% (range: 10–40%). No patients required retreatment with RFA and no immediate or late complications were observed. The follow-up of Bosniak IV cysts had a median of 27 months  and no recurrence or significant loss of renal function were observed. Mid-term follow-up of the cases in our database suggests that image-guided percutaneous RFA can treat Bosniak IV cysts with very low complication rates and satisfactorily maintain renal function. Renal cell carcinoma (RCC) is the third most common malignancy of the genitourinary tract and accounts for approximately 3% of all cancers in adults. The incidence and mortality rates of RCC have increased over the past years  and apprThe Bosniak classification of renal cysts classifies cysts as either benign or potentially malignant (complex) and is widely used to assist clinical decisions . NephronA retrospective study that compared perioperative, renal functional, and oncologic outcomes of renal cryoablation and partial nephrectomy reported that postoperative glomerular filtration rate (GFR) was 6% lower than preoperative GFR in the percutaneous cryoablation group and 13% lower in the partial nephrectomy group. Additionally, this difference persisted in the multivariate analysis, but cryoablation was associated with an increased recurrence risk .Percutaneous thermal ablation is an efficient and safe procedure for the treatment of small solid renal tumors -8, but tHowever, few studies have investigated the use of RFA for the treatment of suspected malignant renal cysts with only short- or medium-term follow-up after RFA , 13.This study describes our experience with RFA of Bosniak IV cysts with an emphasis on its feasibility, safety, and local control.After institutional review board approval, we assessed our radiological database and all Bosniak IV cyst cases treated with percutaneous radiofrequency ablation (RFA) were retrospectively analyzed.Percutaneous RFA procedures were performed in an interventional suite with CT (computer tomography) fluoroscopy capabilities under general endotracheal anesthesia using a Cool-Tip 200-W RF generator  with single (ACT1530/ACT2030) or cluster (ACC1525) 17-gauge needle RF electrode kits.In four cases, biopsies were conducted immediately before RFA under CT guidance, which was essential for the radiologist to position the needle inside the solid component or the septa of the lesion. An on-site pathologist confirmed that the biopsy had been adequately performed and that a sufficient amount of cells had been collected.Prior to needle insertion, the point of entry, safe trajectory, and end position of the needle were planned with the aid of CT and the percutaneous electrode was placed inside the tumor with needle progression guided by real-time CT fluoroscopy. RFA energy was applied for 12min. Overlapping sessions were required to encompass the entire volume of the lesion with a safety margin and a CT scan was performed immediately after RFA to assess potential immediate complications.Patients were evaluated with magnetic resonance imaging (MRI) or CT three, six, and 12 months after the procedure, and annually thereafter, to identify residual tumor and tumor recurrence. Treatment success was based on post-ablation axial imaging: increased attenuation in non-enhanced CT, reduced ablation zone size, and no enhancement on enhanced CT (<10HU) or MRI  were indicative of a successful ablation. Recurrence was defined as postablation enhancement in the ablation zone or enhancement growth on subsequent follow-up imaging.The Clavien-Dindo classification  was usedFrom 2010 to 2014, percutaneous thermal ablation was performed in 154 renal tumor cases. Of these, 10 renal cysts (6.4%) from nine patients were Bosniak IV  and were treated with radiofrequency ablation (RFA), and one was from a single kidney patient; other comorbidities are summarized in Lesion size ranged from 1.5 to 4.1cm (mean: 2.5cm) and biopsies were performed on four cysts, all of which were malignant (two clear cell and two papillary carcinomas). The others were treated based on the risk of malignancy of Bosniak IV lesions.The location of the lesions are summarized in The follow-up of Bosniak IV renal cysts had a median of 27 months .A reduction in tumor size was observed in all Bosniak IV cyst cases immediately after the first RFA session . Mean voThe procedure was successful in all cases, with CT revealing no lesion enhancement in the ablation zone immediately after the RFA, indicating that there was no residual tumor. No patients required a second RFA and no immediate or late complications were observed in this cohort.The creatinine serum levels of each patient before and after the procedure were retrospectively assessed and the estimative of the glomerular filtration rate (GFR) was done using the Modification of Diet in Renal Disease (MDRD) formula. There was an mean variation of 10mL/min (range: 1.3–20.7) of the GFR, and no patients displayed a decrease in renal function.Patients were evaluated with cross-sectional exams (contrast-enhanced CT or MRI) after the procedure, and no recurrence was found .This study describes our experience with percutaneous RFA of Bosniak IV renal cysts with an emphasis on its feasibility, safety, and local control. We showed that the procedure was effective for the treatment of Bosniak IV renal cysts. The success rate of RFA was 100%, which was confirmed by a lack of enhancement on CT and MRI scans of the treated areas within the median follow-up time of 27 months . Our findings are in line with previous case reports and preliminary studies , 13, 15.Even though Bosniak III and IV complex renal cysts are uncommon renal tumors, they are an important subgroup due to their risk of malignancy, which ranges from 16–100% and 90–100%, respectively , 13.The ablative treatment of Bosniak IV cysts is a minimally-invasive procedure that has not been yet associated with major complications to the best of our knowledge, and it leads to preservation of the renal parenchyma.Among 10 Bosniak IV cysts of our study, four were histologically proven to be renal cell carcinomas (two clear cell subtypes and two papillary subtypes). Some theoretical questions about the treatment of Bosniak IV renal cysts still remain unanswered, especially the concern about neoplastic implants and cyst rupture caused by biopsies and therapeutic punctures. Anecdotal cases of implants related to biopsy of solid lesions have been reported, but there are no studies to date of cystic lesion implants after percutaneous biopsies or RFA treatments. We biopsied four lesions that proved to be malignant and found no neoplastic implants at follow-up. Felker et al.  also repThere were no procedure-related complications, showing that percutaneous RFA is a safe method, especially in selected cases. In our sample, almost all cases were classified as clinical stage T1a tumors, and most were predominantly exophytic, with a safe distance to the collector system and in polar location. Although there is no adequate information on the selection criteria for RFA mentioned above, previous studies have not reported clinically relevant complications either , 13, 15.All biopsies and RFA procedures in our study were done under CT fluoroscopy. We believe that CT provides good visual control of the procedure. Additionally, needle trajectory was planned and controlled with Multiplan views. The radiologist was able to identify and prevent possible complications by planning the best access and performing hydrodisplacement of bowel or other structures when those were adjacent to the renal lesion. This supports the use of CT guidance over other imaging modalities such as ultrasound.Needle positioning was CT-guided, which enabled three-dimensional visualization of the lesion. Thus, the interventional radiologist was able to plan the needle trajectory, detect possible injuries in adjacent structures, and prevent complications such as lesions in intestinal or vascular loops. Because no acute or late complications were observed in our sample, the success rate of RFA can be partly attributed to CT-planning.To our knowledge, only three retrospective series evaluated percutaneous RFA for the treatment of renal cysts, and only one included just biopsy-proven malignant cystic disease  12, 13,,13, 15.The procedure was successful in all cases, with no lesion enhancement in the ablation zone on CT immediately after percutaneous RFA and on follow-up CT or MR exams, indicating that there was no residual tumor after the median of 27-months follow-up.In our study, we found a shrink of tumor size in all Bosniak IV cyst cases with the mean volume reduction of approximately 25% in all lesions immediately after the RFA procedure. Because there was no recurrence during the follow-up period, the mean volume reduction could be used as an intraoperative measure of treatment success. Nevertheless, a larger sample and more studies are needed to support this finding.Although the “heat sink effect” caused by undesired RF energy dispersion is less frequent in small cystic lesions and more evident in large, highly vascularized tissues, it represents one of the most important limitations of the RFA technique. In our sample, we treated small lesions  and our results were not affected by this limitation. Interestingly, microwave ablation (MWA) may minimize thermal dispersion and reduce the “heat sink effect”, resulting in a positive outcome in larger cystic lesions, but only a single study to date has investigated the efficacy of MWA in only seven cystic renal lesions, which was not enough to adequately assess the efficacy of MWA .The relatively small sample size, intermediate follow-up time and selection biases due to the retrospective series are limitations of our study. However, even though more comprehensive studies are needed to support the effectiveness of RFA for the treatment of Bosniak IV renal lesions, its efficacy and safety in our study were similar to those reported in the few studies available.Another limitation of our study was the percentage of lesions submitted to biopsy prior to the RFA (40%). However, the technical difficulties of performing percutaneous biopsies of predominantly cystic lesions should be considered, because some lesions have a small solid component where the needle should be positioned for collecting material for pathological analysis. Moreover, even if no malignant cells are found in the biopsy, Bosniak IV renal cysts still can be excised based on the presumed risk of malignancy based on the Bosniak classification , and basIn conclusion, despite the limitations of our study, the results suggest that image-guided percutaneous RFA can properly treat Bosniak IV renal cysts with very low complication rates and satisfactorily maintain renal function.


The classic neurotrophins Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF) and Neurotrophins NT-3 and NT-4 are well known to regulate various aspects of neuronal differentiation, survival and growth. They do this by binding to their cognate receptors, members of the Tropomyosin-related kinase (Trk) receptor tyrosine kinase family, namely TrkA, TrkB, and TrkC. These receptors are then internalized and localized to different cellular compartments, where signal transduction occurs. Conversely, members of the suppressor of cytokine signaling (SOCS) family are best known as negative regulators of signaling via the JAK/STAT pathway. Some members of the family, and in particular SOCS2, have roles in the nervous system that at least partially overlap with that of neurotrophins, namely neuronal differentiation and neurite outgrowth. Recent evidence suggests that SOCS2 is a novel regulator of NGF signaling, altering TrkA cellular localization and downstream signaling to affect neurite growth but not neuronal survival. This review first discusses regulation of Trk receptor signaling, followed by the role of SOCS2 in the nervous system and finishes with a discussion of possible mechanisms by which SOCS2 may regulate TrkA function. A great deal of interest has focused on the therapeutic potential of neurotrophins to prevent neurodegeneration and promote neural regeneration in a range of neurological diseases and neurotrauma   signaling cascades to effect changes in transcription. The neurotrophins are also known to activate members of the Rho GTPase family such as Cdc42 and Rac that are regulators of the cytoskeletal rearrangements required for cell motility and the formation of growth cones  and phospholipase C- γ1 (PLC-γ1) Figure  Obermei. These sA less established pathway downstream of the Trk receptors is the activation of the signal transducer and activator of transcription-3 (STAT3) transcription factor. Early studies demonstrated that dominant negative STAT3 can promote PC12 neurite outgrowth in the absence of NGF  has been shown to have a transient association with the Trk receptors upon neurotrophin binding  Chao, . The juxThe retrograde transport of neurotrophic stimuli has been most extensively studied in the developing peripheral nervous system and is critical for the formation and refinement of neuronal circuits during development and for the regulation of synaptic plasticity and neuronal survival in the mature nervous system  family have recently emerged as ubiquitin ligases regulating expression of a variety of receptors and their signaling pathways. One member of this family in particular, SOCS2, has a variety of functions in the nervous system, many of which overlap with the biological effects of TrkA signaling, suggesting a possible regulatory role for SOCS2 in TrkA function.The SOCS proteins are a family of intracellular proteins implicated in the negative regulation of a variety of cytokine, growth factor and hormone signals, particularly those mediated by the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway   comprise a variable N-terminal region, a central Src Homology 2 (SH2) domain and a highly conserved C-terminal SOCS box Figure . SH2 domThe SOCS proteins couple specific phosphotyrosine-motif recognition with ubiquitin-ligase activity and are thus potent inhibitors of a variety of cytokine signaling pathways. As previously mentioned, the SOCS proteins can participate in “negative feedback” inhibition or “cross-talk” suppression of independent cytokine stimuli. This cross-talk has been shown to extend to the cross regulation amongst the SOCS proteins. SOCS2, SOCS6, and SOCS7 appear to form a subgroup of the SOCS family that have been shown to interact with the SOCS box of all members of the SOCS family and may be required to regulate the stability of other SOCS proteins   receptor was used as bait to screen a cDNA library of proteins expressed in the human fetal brain  signaling  in vitro  neuroepithelial cells and E17 cortical neurons but not cortical astrocytes or adult multipotent stem cells , SOCS2TG (hypo-responsive) and GH receptor null (GHRKO) (non-responsive) mice, exhibit a variety of alterations in neural architecture  contain a large population of TrkA+ neurons that require NGF for survival and promotion of neurite outgrowth. Analysis of DRG neuron cultures derived from SOCS2TG and SOCS2KO mice showed that increased or no expression SOCS2 respectively, regulated NGF-mediated neurite outgrowth, with SOCS2 overexpression increasing and lack of SOCS2 decreasing neurite length and complexity (Uren et al., Co-immunoprecipitation of SOCS2 and TrkA mutant proteins in 293T cells showed that the juxtamembrane region of TrkA was required for binding of SOCS2, with possible involvement of the kinase domain as well. Overexpression of SOCS2 increased total TrkA levels in transfected 293T cells and PC12 cells and increased cell surface expression of TrkA in PC12 cells and SOCS2TG DRG neurons (Uren et al., While the mechanism by which SOCS2 regulates TrkA signaling and functional outcome remains to be determined, it most likely revolves around altered TrkA localization induced by SOCS2-mediated ubiquitination. This could regulate the amount of TrkA receptor at the cell surface and influence the rate at which it is internalized, recycled, or transported to different intracellular compartments, thereby modulating sensitivity of a cell to NGF and altering downstream signal transduction pathways. Ubiquitination of the lysine in the TrkA juxtamembrane KFG motif regulates TrkA expression levels and localization, with deletion of this domain leading to decreased ubiquitination, increased TrkA levels and increased phosphorylation of the AKT and MAP kinase pathways (Kiris et al., Neurotrophins and their receptors are expressed in specific areas throughout the nervous system. Their expression levels, signal transduction pathways and localization are tightly regulated by a range of different mechanisms, including dephosphorylation and degradation. SOCS proteins are best known for their role as negative feedback inhibitors of cytokine signaling but have been shown to have a range of other roles, depending on cell type and receptor under study. Their structure, which includes an SH2 interaction domain and the SOCS box ubiquitin ligase interaction domain, suggests that they may interact with many more proteins. This certainly appears to be the case for the TrkA receptor and SOCS2. However, given that SOCS2 has also been shown to bind to the BDNF and NT-3 neurotrophin receptors (Uren et al., 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.


Hepatocellular Carcinoma (HCC) is a growing sanitary problem worldwide. This tumor is indeed the third most deadly and the fifth most common cancer all over the word, with an incidence that, at least in the United States, has doubled in the past two decades .Current studies suggest that HCC can derive from liver progenitor cells or cancer stem cells (CSCs) in which signaling pathway similar to those identified in liver cancer , are activated. These findings support the hypothesis that CSCs contribute to the molecular heterogeneity and chemoresistance of this tumor . In addiCancer stem cells are neoplastic cells that possess distinct survival mechanisms and stem cell properties responsible for tumor formation and progression. Although the origin of CSCs in liver cancer is not fully elucidated, has been recently demonstrated that any hepatic lineage cell  can be reprogrammed into CSC by activating diverse cell type-specific pathways .in vivo growth and malignant progression of hepatic cancer progenitor cells and tumor metastasis [The role of preneoplastic lesions in HCC remains still controversial. A recent study  has demotastasis .In the liver, the ov-serpin SERPINB3 (or SCCA1) and its isoforms SERPINB4 (or SCCA2) are undetectable in normal hepatocytes, but their expression progressively increases in chronic liver diseases, dysplastic nodules and HCC, suggesting that they may be involved in relatively early events of hepatocarcinogenesis, although their specific role has not been defined yet .in vitro, demonstrating that SERPINB3 can enhance TGF-β expression and induce epithelial-mesenchymal transition, with increased invasiveness and cell proliferation [In the article “SERPINB3 is associated with TGF-β1 and cytoplasmic β-catenin expression in hepatocellular carcinomas with poor prognosis”  Turato eferation .The presence of SERPINB3 in human progenitor cells compartment has been recently demonstrated by our group. This serpin was indeed detected in EpCAM + cell fractions sorted from human foetal and adult livers and in the same ductular structures that express stem/progenitor cell markers. Moreover, the mouse homologous, Serpinb3b, was induced in a mouse model of liver stem/progenitor cell activation .Expression of SERPINB3 protein was also observed in hepatoblastoma, considered an embryonal tumour of the liver, especially in tumors with more advanced stage. The intensity of the protein signal was higher in less differentiated components of the tumor and in the clusters of undifferentiated variant, named small undifferentiated cell (SCUD) pattern, considered as primitive uncommitted progenitor cells .Recent findings revealed that SERPINB3/4 isoforms are RAS-responsive factors that can lead to inflammatory protein production and tumorigenesis. They are transcriptionally upregulated via MAPK and the ETS family transcription factors PEA3. The increased expression of these serpin family members determines NF-kB activation, essential for RAS-mediated cytokine production and tumor growth. The positive correlation between Ras mutation, enhanced SERPINB3 expression and IL-6 expression was documented in human colorectal and pancreatic tumour samples, reflecting the inflammatory and protumorigenic role of this serpin .These last results, taken together with the finding that high SERPINB3 expression was detected in liver progenitor cells and in the most aggressive liver tumors ,7 sugges


Broadly linked to domains such as attention  followed by a 6-s delay (phase 1), a fully informative task cue (1.5-s) followed by a 1.5-s delay (phase 2), and the target stimulus followed by some time to respond (phase 3)Wisniewski and colleagues explored the brain areas underlying associations between tasks (parity/magnitude) and rewards (high/low). The information required for task execution gradually unfolded across a trial: Participants subsequently received a symbolic Phase 1 required the symbolically cued task-reward associations to be maintained for 6-s until task cue presentation. Robust coding was observed in bilateral IPC, covering parts of both the supramarginal gyrus (SMG) and the angular gyrus (AG). The current literature links these areas to semantic and phonological processing; for example, left SMG supports verbal working memory . More generally, with the heterogeneity of inferior parietal cortex in mind, what does it mean to conclude that “the inferior parietal cortex flexibly changed its content of representation on a short timescale within trials”  attentional templates  and the inferior frontal gyrus (IFG)—initially linked to attentional reorientation And there is more. Using endogenous Posner tasks, Doricchi and colleagues (Doricchi et al., In sum, our comment is twofold. First, findings by Wisniewski and colleagues fit well with existent knowledge on parietal cortex, thereby questioning the aspect of novelty. Second, and more importantly, their study further prompts comprehensive exploration of TPJ in terms of prediction-outcome comparison—surpassing the level of specific domains, and considering the potentially distinct contributions of left and right TPJ.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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. The reviewer GA and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


B receptor agonists/antagonists have been shown to be allosteric modulators of CXCR4. The interplay between CXCL12/CXCR4-CXCR7 and GABA/GABAA-GABAB receptors systems could have many physiological implications in neurotransmission, cancer and inflammation. In addition, the GABAB agonist baclofen is currently used in medicine to treat spasticity in patients with spinal cord injury, cerebral palsy, traumatic brain injury, multiple sclerosis, and other disorders. More recently it has also been used in the treatment of alcohol dependence and withdrawal. The allosteric effects of this agent on CXCR4 could contribute to these beneficial effects or at the opposite, to its side effects.Since CXCL12 and its receptors, CXCR4 and CXCR7, have been found in the brain, the role of this chemokine has been expanded from chemoattractant in the immune system to neuromodulatory in the brain. Several pieces of evidence suggest that this chemokine system could crosstalk with the GABAergic system, known to be the main inhibitory neurotransmitter system in the brain. Indeed, GABA and CXCL12 as well as their receptors are colocalized in many cell types including neurons and there are several examples in which these two systems interact. Several mechanisms can be proposed to explain how these systems interact, including receptor–receptor interactions, crosstalk at the level of second messenger cascades, or direct pharmacological interactions, as GABA and GABA The chemokine CXCL12/SDF1 has been found to play important roles in several processes involved in ischemic stroke and its’ subsequent repair , brain ti family of proteins activating multiple G-protein dependent pathways (+ (GIRK), a voltage-gated K channel Kv2.1 associated to neuronal survival, and to increase high voltage activated (HVA) Ca2+ currents (CXCR4 is a G protein-coupled receptor (GPCR) widely expressed in a variety of cell types including leucocytes, where it promotes migration, recruitment and activation , neuronspathways . In neurcurrents .CXCR7, contrary to CXCR4, could not be demonstrated to be coupled to G proteins. Despite its phylogenic relation and ligand binding properties, CXCR7 does not mediate typical chemokine receptor responses such as leukocyte trafficking. It was first believed to be mainly involved in ligand sequestration . Howeverγ-aminobutyric acid (GABA) is the chief neuro-inhibitory neurotransmitter in mammalian systems but it also plays important roles in CNS development by regulating neurogenesis and synaptogenesis . In contA) receptors are ionotropic receptors. In response to binding GABA, their chloride-selective pore open resulting in hyperpolarization of the neuron. This causes an inhibitory effect on neurotransmission by diminishing the chance of a successful action potential occurring. The protein contains a number of different allosteric binding sites which modulate the activity of the receptor indirectly and are the targets of various other drugs, including the benzodiazepines, barbiturates, ethanol, neuroactive steroids, inhaled anesthetics, and picrotoxin, among others  required for functioning  and CXCR4 receptors tagged with GFP (green) in the membrane of Xenopus oocytes, data obtained using TIRF microscopy showed that CXCR4 and GABAB receptors did not co-localize in the membrane  were recently found to act pharmacologically directly on CXCR4 in an allosteric manner. Using electrophysiology in Xenopus oocytes and human embryonic kidney (HEK293) cells in which Rat CXCR4 and the GIRK channel were co-expressed, it could be demonstrated that GABAB antagonist and agonist modify the CXCL12-evoked activation of GIRK channels , which were in a similar range to the affinities of the ligands for GABAB receptors themselves, thus confirming that GABA and GABAB receptor ligands directly interact allosterically with the CXCR4 receptor (B receptor ligands on CXCR7.While somewhat unexpected, GABA and the agonists/antagonists of GABAchannels . By exprdulation . Finallyreceptor . In the There are many pathways by which GABA and CXCL12 systems can interact. GABA is able to block the effect of CXCL12 on CXCR4. Thus, it is likely that when the GABAergic system is activated, GABA released in the brain will antagonize the effect of CXCL12 on its receptor CXCR4, and thus could influence the chemokine neurotransmission as well as the inflammatory response in the central nervous system. Conversely, it has previously been shown that CXCR4 stimulation by CXCL12 can increase GABA release . TherefoCXCR4 activation by CXCL12 has been shown to increase presynaptic neurotransmitter release and particularly GABA release in several neuronal populations . If GABAB receptor stimulation by GABA spilling over following CXCL12 presynaptic stimulation and increase in GABAB release. However, GIRK currents might have been activated by the stimulation of postsynaptic CXCR4 by CXCL12, which was then blocked by CGP55845A.In dopaminergic neurons of the rat substantia nigra, CXCR4 stimulation by CXCL12 induces an increase of release of presynaptic neurotransmitter, particularly of GABA . CGP5584B receptors on cells of the immune system has recently been described, as well as a possible link to the inflammatory response (B receptor agonist, reduces chemotaxis from human mononuclear cells toward CXCL12 (B and CXCR4 receptors, the finding that an agonist of one receptor alters the response to an agonist of the other receptor was interpreted to indicate a heterologous desensitization between chemokine and GABAB receptors. This observation along with our own observations on the chemotaxis of cancer cell lines expressing CXCR4 can also be reinterpreted as a direct allosteric action of baclofen on CXCR4.Expression of GABAresponse . Along td CXCL12 . Given tBaclofen treatment was demonstrated to reduce the incidence of some carcinogen-induced gastrointestinal cancers in rats  as well Similarly, it has been shown that GABA can affect the cell proliferation and have anti-inflammatory properties through inhibition of fibroblast proliferation, although the mechanism of action of GABA was not elucidated . We suggB receptor agents to affect the CXCR4–GP-120 interaction.Fusion of HIV-1 with the host cell membrane is initiated by the binding of the viral envelope glycoprotein gp120 to both the CD4 cell surface receptor and one of the CXCR4 or CCR5 chemokine receptors . It has spasticity in patients with spinal cord injury, cerebral palsy, traumatic brain injury, multiple sclerosis and other disorders (B receptors. At the opposite, it could be responsible for its side effects. Overall, the effect of GABAergic agents on CXCR4 suggests new therapeutic potentials for neurological and immune diseases.As a conclusion, agents interacting at CXCR4 could be useful to treat cancer as well as HIV infection. Baclofen is currently approved for the treatment of isorders . Recentlisorders . The allThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.