http://www.mdpi.com/journal/cells Latest open access articles published in Cells at http://www.mdpi.com/journal/cells http://www.mdpi.com/2073-4409/2/2/306 Building mathematical models of cellular networks lies at the core of systems biology. It involves, among other tasks, the reconstruction of the structure of interactions between molecular components, which is known as network inference or reverse engineering. Information theory can help in the goal of extracting as much information as possible from the available data. A large number of methods founded on these concepts have been proposed in the literature, not only in biology journals, but in a wide range of areas. Their critical comparison is difficult due to the different focuses and the adoption of different terminologies. Here we attempt to review some of the existing information theoretic methodologies for network inference, and clarify their differences. While some of these methods have achieved notable success, many challenges remain, among which we can mention dealing with incomplete measurements, noisy data, counterintuitive behaviour emerging from nonlinear relations or feedback loops, and computational burden of dealing with large data sets. Cells 2013-05-10 2 2 Review 10.3390/cells2020306 306 329 2073-4409 2013-05-10 doi: 10.3390/cells2020306 Alejandro Villaverde John Ross Julio Banga http://www.mdpi.com/2073-4409/2/2/294 An apoptotic program leading to controlled cell dismantling implies perturbations of nuclear dynamics, as well as changes affecting the organelle structure and distribution. In human cancer cells driven to apoptosis by different stimuli, we have recently investigated the morphological properties of several organelles, including mitochondria, lysosomes, endoplasmic reticulum and Golgi apparatus. In this review, we will discuss the body of evidence in the literature suggesting that organelles are generally relocated and/or degraded during apoptosis, irrespectively of the apoptogenic stimulus and cell type. Cells 2013-05-08 2 2 Review 10.3390/cells2020294 294 305 2073-4409 2013-05-08 doi: 10.3390/cells2020294 Maria Bottone Giada Santin Francesca Aredia Graziella Bernocchi Carlo Pellicciari Anna Scovassi http://www.mdpi.com/2073-4409/2/2/284 Mathematical modeling of signaling and gene regulatory networks has provided unique insights about systems behaviors for many cell biological problems of medical importance. Quantitative single cell monitoring has a crucial role in advancing systems modeling of molecular networks. However, due to the multidisciplinary techniques that are necessary for adaptation of such systems biology approaches, dissemination to a wide research community has been relatively slow. In this essay, I focus on some technical aspects that are often under-appreciated, yet critical in harnessing live cell imaging methods to achieve single-cell-level understanding and quantitative modeling of molecular networks. The importance of these technical considerations will be elaborated with examples of successes and shortcomings. Future efforts will benefit by avoiding some pitfalls and by utilizing the lessons collectively learned from recent applications of imaging in systems biology. Cells 2013-04-29 2 2 Essay 10.3390/cells2020284 284 293 2073-4409 2013-04-29 doi: 10.3390/cells2020284 Myong-Hee Sung http://www.mdpi.com/2073-4409/2/2/266 Pancreatic beta-cell apoptosis is an important feature of islets in type 2 diabetes. Apoptosis can occur through two major pathways, the extrinsic or death receptor mediated pathway, and the intrinsic or Bcl-2-regulated pathway. Hyperglycaemia, hyperlipidaemia and islet amyloid poly-peptide (IAPP) represent important possible causes of increased beta-cell apoptosis. Hyperglycaemia induces islet-cell apoptosis by the intrinsic pathway involving molecules of the Bcl-2 family. High concentrations of palmitate also activate intrinsic apoptosis in islets cells. IAPP oligomers can induce apoptosis by both intrinsic and extrinsic pathways. IL-1b produced through NLRP3 inflammasome activation can also induce islet cell death. Activation of the NLRP3 inflammasome may not be important for glucose or palmitate induced apoptosis in islets but may be important for IAPP mediated cell death. Endoplasmic reticulum (ER) and oxidative stress have been observed in beta cells in type 2 diabetes, and these could be the link between upstream metabolic abnormalities and downstream apoptotic machinery. Cells 2013-04-26 2 2 Review 10.3390/cells2020266 266 283 2073-4409 2013-04-26 doi: 10.3390/cells2020266 Jibran Wali Seth Masters Helen Thomas http://www.mdpi.com/2073-4409/2/2/244 The purpose of this study was to perform a number of preliminary in vitro evaluations on an array of modified gelatin gel sponge scaffolds for use in a bone graft application. The gelatin gels were modified through the addition of a number of components which each possess unique properties conducive to the creation and regeneration of bone: a preparation rich in growth factors (PRGF, a bioactive, lyophilized form of platelet-rich plasma), hydroxyapatite, and chitin whiskers. Platelet-rich plasma therapy is an emerging practice that has proven effective in a number of clinical applications, including enhancing bone repair through improved deposition of new bony matrix and angiogenesis. As such, the inclusion of PRGF in our gelatin scaffolds was intended to significantly enhance scaffold bioactivity, while the addition of hydroxyapatite and chitin whiskers were anticipated to increase scaffold strength. Additionally, the gelatin sponges, which readily dissolve in aqueous solutions, were subjected to 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) cross-linking, either during or post-gelation, to control their rate of degradation. Scaffolds were evaluated in vitro with respect to compressive strength, mass loss/degradation, protein release, and cellular interaction, with results demonstrating the potential of the gelatin gel sponge scaffold for use in the regeneration of bone. Cells 2013-04-26 2 2 Article 10.3390/cells2020244 244 265 2073-4409 2013-04-26 doi: 10.3390/cells2020244 Isaac Rodriguez Scott Sell Jennifer McCool Gunjan Saxena Andrew Spence Gary Bowlin http://www.mdpi.com/2073-4409/2/2/224 The gene expression kinetics for human cells exposed to hyperthermic stress are not well characterized. In this study, we identified and characterized the genes that are differentially expressed in human epidermal keratinocyte (HEK) cells exposed to hyperthermic stress. In order to obtain temporal gene expression kinetics, we exposed HEK cells to a heat stress protocol (44 °C for 40 min) and used messenger RNA (mRNA) microarrays at 0 h, 4 h and 24 h post-exposure. Bioinformatics software was employed to characterize the chief biological processes and canonical pathways associated with these heat stress genes. The data shows that the genes encoding for heat shock proteins (HSPs) that function to prevent further protein denaturation and aggregation, such as HSP40, HSP70 and HSP105, exhibit maximal expression immediately after exposure to hyperthermic stress. In contrast, the smaller HSPs, such as HSP10 and HSP27, which function in mitochondrial protein biogenesis and cellular adaptation, exhibit maximal expression during the “recovery phase”, roughly 24 h post-exposure. These data suggest that the temporal expression kinetics for each particular HSP appears to correlate with the cellular function that is required at each time point. In summary, these data provide additional insight regarding the expression kinetics of genes that are triggered in HEK cells exposed to hyperthermic stress. Cells 2013-04-10 2 2 Article 10.3390/cells2020224 224 243 2073-4409 2013-04-10 doi: 10.3390/cells2020224 Ibtissam Echchgadda Caleb Roth Cesario Cerna Gerald Wilmink http://www.mdpi.com/2073-4409/2/2/202 First described for their metabolic and immunosuppressive effects, glucocorticoids are widely prescribed in clinical settings of inflammation. However, glucocorticoids are also potent inducers of apoptosis in many cell types and tissues. This review will focus on the established mechanisms of glucocorticoid-induced apoptosis and outline what is known about the apoptotic response in cells and tissues of the body after exposure to glucocorticoids. Glucocorticoid-induced apoptosis affects the skeletal system, muscular system, circulatory system, nervous system, endocrine system, reproductive system, and the immune system. Interestingly, several cell types have an anti-apoptotic response to glucocorticoids that is cytoprotective. Lastly, we will discuss the pro- and anti-apoptotic effects of glucocorticoids in cancers and their clinical implications. Cells 2013-03-26 2 2 Review 10.3390/cells2020202 202 223 2073-4409 2013-03-26 doi: 10.3390/cells2020202 Amanda Gruver-Yates John Cidlowski http://www.mdpi.com/2073-4409/2/2/188 Phagocytosis involves the internalization of extracellular material by invagination of the plasma membrane to form intracellular vesicles called phagosomes, which have functions that include pathogen degradation. The degradative properties of phagosomes are thought to be conferred by sequential fusion with endosomes and lysosomes; however, this maturation process has not been studied in vivo. We employed Drosophila hemocytes, which are similar to mammalian professional macrophages, to establish a model of phagosome maturation. Adult Drosophila females, carrying transgenic Rab7-GFP endosome and Lamp1-GFP lysosome markers, were injected with E. coli DH5α and the hemocytes were collected at 15, 30, 45 and 60 minutes after infection. In wild-type females, E. coli were detected within enlarged Rab7-GFP positive phagosomes at 15 to 45 minutes after infection; and were also observed in enlarged Lamp1-GFP positive phagolysosomes at 45 minutes. Two-photon imaging of hemocytes in vivo confirmed this vesicle morphology, including enlargement of Rab7-GFP and Lamp1-GFP structures that often appeared to protrude from hemocytes. The interaction of endosomes and lysosomes with E. coli phagosomes observed in Drosophila hemocytes was consistent with that previously described for phagosome maturation in human ex vivo macrophages. We also tested our model as a tool for genetic analysis using 14-3-3e mutants, and demonstrated altered phagosome maturation with delayed E. coli internalization, trafficking and/or degradation. These findings demonstrate that Drosophila hemocytes provide an appropriate, genetically amenable, model for analyzing phagosome maturation ex vivo and in vivo. Cells 2013-03-26 2 2 Article 10.3390/cells2020188 188 201 2073-4409 2013-03-26 doi: 10.3390/cells2020188 Tetyana Shandala Chiaoxin Lim Alexandra Sorvina Douglas Brooks http://www.mdpi.com/2073-4409/2/1/163 Inhibitors of Apoptosis (IAPs) are a family of proteins with various biological functions including regulation of innate immunity and inflammation, cell proliferation, cell migration and apoptosis. They are characterized by the presence of at least one N-terminal baculoviral IAP repeat (BIR) domain involved in protein-protein interaction. Most of them also contain a C-terminal RING domain conferring an E3-ubiquitin ligase activity. In drosophila, IAPs are essential to ensure cell survival, preventing the uncontrolled activation of the apoptotic protease caspases. In mammals, IAPs can also regulate apoptosis through controlling caspase activity and caspase-activating platform formation. Mammalian IAPs, mainly X-linked IAP (XIAP) and cellular IAPs (cIAPs) appeared to be important determinants of the response of cells to endogenous or exogenous cellular injuries, able to convert the survival signal into a cell death-inducing signal. This review highlights the role of IAP in regulating apoptosis in Drosophila and Mammals. Cells 2013-03-14 2 1 Review 10.3390/cells2010163 163 187 2073-4409 2013-03-14 doi: 10.3390/cells2010163 Jean Berthelet Laurence Dubrez http://www.mdpi.com/2073-4409/2/1/136 Pulse power technology using nanosecond pulsed electric fields (nsPEFs) offers a new stimulus to modulate cell functions or induce cell death for cancer cell ablation. New data and a literature review demonstrate fundamental and basic cellular mechanisms when nsPEFs interact with cellular targets. NsPEFs supra-electroporate cells creating large numbers of nanopores in all cell membranes. While nsPEFs have multiple cellular targets, these studies show that nsPEF-induced dissipation of ΔΨm closely parallels deterioration in cell viability. Increases in intracellular Ca2+ alone were not sufficient for cell death; however, cell death depended of the presence of Ca2+. When both events occur, cell death ensues. Further, direct evidence supports the hypothesis that pulse rise-fall times or high frequency components of nsPEFs are important for decreasing ΔΨm and cell viability. Evidence indicates in Jurkat cells that cytochrome c release from mitochondria is caspase-independent indicating an absence of extrinsic apoptosis and that cell death can be caspase-dependent and –independent. The Ca2+ dependence of nsPEF-induced dissipation of ΔΨm suggests that nanoporation of inner mitochondria membranes is less likely and effects on a Ca2+-dependent protein(s) or the membrane in which it is embedded are more likely a target for nsPEF-induced cell death. The mitochondria permeability transition pore (mPTP) complex is a likely candidate. Data demonstrate that nsPEFs can bypass cancer mutations that evade apoptosis through mechanisms at either the DISC or the apoptosome. Cells 2013-03-06 2 1 Review 10.3390/cells2010136 136 162 2073-4409 2013-03-06 doi: 10.3390/cells2010136 Stephen Beebe Nova Sain Wei Ren http://www.mdpi.com/2073-4409/2/1/124 The neuropeptide, neurotensin, exerts numerous biological functions, including an efficient anti-apoptotic role, both in the central nervous system and in the periphery. This review summarizes studies that clearly evidenced the protective effect of neurotensin through its three known receptors. The pivotal involvement of the neurotensin receptor-3, also called sortilin, in the molecular mechanisms of the anti-apoptotic action of neurotensin has been analyzed in neuronal cell death, in cancer cell growth and in pancreatic beta cell protection. The relationships between the anti-apoptotic role of neurotensin and important physiological and pathological contexts are discussed in this review. Cells 2013-03-04 2 1 Review 10.3390/cells2010124 124 135 2073-4409 2013-03-04 doi: 10.3390/cells2010124 Christelle Devader Sophie Béraud-Dufour Thierry Coppola Jean Mazella http://www.mdpi.com/2073-4409/2/1/105 Recent improvements in the resolution of light microscopy, coupled with the development of a range of fluorescent-based probes, have provided new approaches to dissecting membrane domains and the regulation of membrane trafficking. Here, we review these advances, as well as highlight developments in quantitative image analysis and novel unbiased analytical approaches to quantitate protein localization. The application of these approaches to endosomal sorting and endosome-to-Golgi transport is discussed. Cells 2013-02-22 2 1 Review 10.3390/cells2010105 105 123 2073-4409 2013-02-22 doi: 10.3390/cells2010105 Pei Chia Paul Gleeson http://www.mdpi.com/2073-4409/2/1/83 Viruses have played an important role in human evolution and have evolved diverse strategies to co-exist with their hosts. As obligate intracellular pathogens, viruses exploit and manipulate different host cell processes, including cellular trafficking, metabolism and immunity-related functions, for their own survival. In this article, we review evidence for how autophagy, a highly conserved cellular degradative pathway, serves either as an antiviral defense mechanism or, alternatively, as a pro-viral process during virus infection. Furthermore, we highlight recent reports concerning the role of selective autophagy in virus infection and how viruses manipulate autophagy to evade lysosomal capture and degradation. Cells 2013-01-25 2 1 Review 10.3390/cells2010083 83 104 2073-4409 2013-01-25 doi: 10.3390/cells2010083 Abhilash Chiramel Nathan Brady Ralf Bartenschlager http://www.mdpi.com/2073-4409/2/1/67 The heart was initially believed to be a terminally differentiated organ; once the cardiomyocytes died, no recovery could be made to replace the dead cells. However, around a decade ago, the concept of cardiac stem cells (CSCs) in adult hearts was proposed. CSCs differentiate into cardiomyocytes, keeping the heart functioning. Studies have proved the existence of stem cells in the heart. These somatic stem cells have been studied for use in cardiac regeneration. Moreover, recently, induced pluripotent stem cells (iPSCs) were invented, and methodologies have now been developed to induce stable cardiomyocyte differentiation and purification of mature cardiomyocytes. A reprogramming method has also been applied to direct reprogramming using cardiac fibroblasts into cardiomyocytes. Here, we address cardiomyocyte differentiation of CSCs and iPSCs. Furthermore, we describe the potential of CSCs in regenerative biology and regenerative medicine. Cells 2013-01-15 2 1 Review 10.3390/cells2010067 67 82 2073-4409 2013-01-15 doi: 10.3390/cells2010067 Nanako Kawaguchi Toshio Nakanishi http://www.mdpi.com/2073-4409/2/1/57 Telomeres are structures at the ends of chromosomes that shorten during cell division and eventually signal an irreversible state of growth arrest known as cellular senescence. To delay this cellular aging, human T cells, which are critical in the immune control over infections and cancer, activate the enzyme telomerase, which binds and extends the telomeres. Several different extracts from the Astragalus membranaceus root have been documented to activate telomerase activity in human T cells. The objective of this research was to compare two extracts from Astragalus membranaceus, TA-65 and HTA, for their effects on both telomerase and proliferative activity of human CD4 and CD8 T cells. Our results demonstrate that, TA-65 increased telomerase activity significantly (1.3 to 3.3-fold relative to controls) in T cell cultures from six donors tested, whereas HTA only increased telomerase levels in two out of six donors. We also demonstrate that TA-65 activates telomerase by a MAPK- specific pathway. Finally, we determine that during a three-day culture period, only the T cells treated with the TA-65 extract showed a statistically significant increase in proliferative activity. Our results underscore the importance of comparing multiple telomerase activators within the same experiment, and of including functional assays in addition to measuring telomerase activity. Cells 2013-01-14 2 1 Article 10.3390/cells2010057 57 66 2073-4409 2013-01-14 doi: 10.3390/cells2010057 Brenda Molgora Riley Bateman Greg Sweeney Danielle Finger Taylor Dimler Rita Effros Hector Valenzuela http://www.mdpi.com/2073-4409/2/1/43 Tumorigenesis is often described as a result of accumulated mutations that lead to growth advantage and clonal expansion of mutated cells. There is evidence in the literature that cancer cells are influenced by the microenvironment. Our previous studies demonstrated that the mouse mammary gland is capable of redirecting mouse cells of non-mammary origins as well as Mouse Mammary Tumor Virus (MMTV)-neu transformed cells toward normal mammary epithelial cell fate during gland regeneration. Interestingly, the malignant phenotype of MMTV-neu transformed cells was suppressed during serial transplantation experiments. Here, we discuss our studies that demonstrated the potential of the regenerating mouse mammary gland to redirect cancer cells of different species into a functional tumor-free mammary epithelial cell progeny. Immunochemistry for human specific CD133, mitochondria, cytokeratins as well as milk proteins and FISH for human specific probe identified human epithelial cell progeny in ducts, lobules, and secretory acini. Fluorescent In Situ Hybridization (FISH) for human centromeric DNA and FACS analysis of propidium iodine staining excluded the possibility of mouse-human cell fusion. To our knowledge this is the first evidence that human cancer cells of embryonic or somatic origins respond to developmental signals generated by the mouse mammary gland microenvironment during gland regeneration in vivo. Cells 2013-01-10 2 1 Review 10.3390/cells2010043 43 56 2073-4409 2013-01-10 doi: 10.3390/cells2010043 Sonia Rosenfield Gilbert Smith http://www.mdpi.com/2073-4409/2/1/19 CD8+ T cells have the potential to control HSV-2 infection. However, limited information has been available on CD8+ T cell epitopes or the functionality of antigen specific T cells during infection or following immunization with experimental vaccines. Peptide panels from HSV-2 proteins ICP27, VP22 and VP13/14 were selected from in silico predictions of binding to human HLA-A*0201 and mouse H-2Kd, Ld and Dd molecules. Nine previously uncharacterized CD8+ T cell epitopes were identified from HSV-2 infected BALB/c mice. HSV-2 specific peptide sequences stabilized HLA-A*02 surface expression with intermediate or high affinity binding. Peptide specific CD8+ human T cell lines from peripheral blood lymphocytes were generated from a HLA-A*02+ donor. High frequencies of peptide specific CD8+ T cell responses were elicited in mice by DNA vaccination with ICP27, VP22 and VP13/14, as demonstrated by CD107a mobilization. Vaccine driven T cell responses displayed a more focused immune response than those induced by viral infection. Furthermore, vaccination with ICP27 reduced viral shedding and reduced the clinical impact of disease. In conclusion, this study describes novel HSV-2 epitopes eliciting strong CD8+ T cell responses that may facilitate epitope based vaccine design and aid immunomonitoring of antigen specific T cell frequencies in preclinical and clinical settings. Cells 2013-01-04 2 1 Article 10.3390/cells2010019 19 42 2073-4409 2013-01-04 doi: 10.3390/cells2010019 Rebecca Platt Tansi Khodai Tim Townend Helen Bright Paul Cockle Luis Perez-Tosar Rob Webster Brian Champion Timothy Hickling Fareed Mirza http://www.mdpi.com/2073-4409/2/1/1 A number of challenges have hindered the development of a unified theory for metazoan regeneration. To describe the full range of complex regeneration phenomena in Animalia, we suggest that metazoans that regenerate missing body parts exhibit biological attributes that are tailored along a morpho-spatial regeneration continuum, illustrated in its polar scenarios by the USA “stars and stripes” flag. Type 1 organisms (“T1, ‘stars’”) are typical colonial organisms (but contain unitary taxa) that are able to regenerate “whole new stars”, namely, whole bodies and colonial modules, through systemic induction and sometimes multiple regeneration foci (hollow regeneration spheres, resembling the blastula) that compete for dominance. They regenerate soma and germ constituents with pluripotent adult stem cells and exhibit somatic-embryogenesis mode of ontogeny. Type 2 organisms (“T2, ‘stripes’”) are capable of limited regeneration of somatic constituents via fate-restricted stem cells, and regenerate through centralized inductions that lead to a single regeneration front. T2 organisms are unitary and use preformistic mode of ontogeny. T1 and T2 organisms also differ in interpretation of what constitutes positional information. T2 organisms also execute alternative, less effective, regeneration designs (i.e., scar formation). We assigned 15 characteristics that distinguish between T1/T2 strategies: those involving specific regeneration features and those operating on biological features at the whole-organism level. Two model organisms are discussed, representing the two strategies of T1/T2 along the regeneration continuum, the Botrylloides whole body regeneration (T1) and the mouse digit-tip regeneration (T2) phenomena. The above working hypothesis also postulates that regeneration is a primeval attribute of metazoans. As specified, the “stars and stripes” paradigm allows various combinations of the biological features assigned to T1 and T2 regeneration strategies. It does not consider any concentration gradient or thresholds and does not refer to the “epimorphosis” and “morphallaxis” terms, regeneration types across phyla or across body plans. The “stars and stripes” paradigm also ignores, at this stage of analysis, cases of regeneration loss that may obscure biological trajectories. The main advantage of the “stars and stripes” paradigm is that it allows us to compare T1/T2 regeneration, as well as other modes of regeneration, through critical determining characteristics. Cells 2012-12-27 2 1 Concept Paper 10.3390/cells2010001 1 18 2073-4409 2012-12-27 doi: 10.3390/cells2010001 Baruch Rinkevich Yuval Rinkevich http://www.mdpi.com/2073-4409/1/4/1313 Wound healing results in the repair of injured tissues however fibrosis and scar formation are, more often than not the unfortunate consequence of this process. The ability of lower order vertebrates and invertebrates to regenerate limbs and tissues has been all but lost in mammals; however, there are some instances where glimpses of mammalian regenerative capacity do exist. Here we describe the unlocked potential that exists in mammals that may help us understand the process of regeneration post-injury and highlight the potential role of the actin cytoskeleton in this process. The precise function and regulation of the cytoskeleton is critical to the success of the healing process and its manipulation may therefore facilitate regenerative healing. The gelsolin family of actin remodelling proteins in particular has been shown to have important functions in wound healing and family member Flightless I (Flii) is involved in both regeneration and repair. Understanding the interactions between different cytoskeletal proteins and their dynamic control of processes including cellular adhesion, contraction and motility may assist the development of therapeutics that will stimulate regeneration rather than repair. Cells 2012-12-19 1 4 Review 10.3390/cells1041313 1313 1327 2073-4409 2012-12-19 doi: 10.3390/cells1041313 Xanthe Strudwick Allison Cowin http://www.mdpi.com/2073-4409/1/4/1293 Recent developments in stem cell research suggest that it may be time to reconsider the current focus of stem cell induction strategies. During the previous five years, approximately, the induction of pluripotency in somatic cells, i.e., the generation of so-called ‘induced pluripotent stem cells’ (iPSCs), has become the focus of ongoing research in many stem cell laboratories, because this technology promises to overcome limitations (both technical and ethical) seen in the production and use of embryonic stem cells (ESCs). A rapidly increasing number of publications suggest, however, that it is now possible to choose instead other, alternative ways of generating stem and progenitor cells bypassing pluripotency. These new strategies may offer important advantages with respect to ethics, as well as to safety considerations. The present communication discusses why these strategies may provide possibilities for an escape from the dilemma presented by pluripotent stem cells (self-organization potential, cloning by tetraploid complementation, patenting problems and tumor formation risk). Cells 2012-12-17 1 4 Review 10.3390/cells1041293 1293 1312 2073-4409 2012-12-17 doi: 10.3390/cells1041293 Hans-Werner Denker http://www.mdpi.com/2073-4409/1/4/1261 Liver regeneration is a complex phenomenon aimed at maintaining a constant liver mass in the event of injury resulting in loss of hepatic parenchyma. Partial hepatectomy is followed by a series of events involving multiple signaling pathways controlled by mitogenic growth factors (HGF, EGF) and their receptors (MET and EGFR). In addition multiple cytokines and other signaling molecules contribute to the orchestration of a signal which drives hepatocytes into DNA synthesis. The other cell types of the liver receive and transmit to hepatocytes complex signals so that, in the end of the regenerative process, complete hepatic tissue is assembled and regeneration is terminated at the proper time and at the right liver size. If hepatocytes fail to participate in this process, the biliary compartment is mobilized to generate populations of progenitor cells which transdifferentiate into hepatocytes and restore liver size. Cells 2012-12-13 1 4 Review 10.3390/cells1041261 1261 1292 2073-4409 2012-12-13 doi: 10.3390/cells1041261 Liang-I. Kang Wendy Mars George Michalopoulos http://www.mdpi.com/2073-4409/1/4/1246 A prerequisite for successful tissue engineering is adequate vascularization that would allow tissue engineering constructs to survive and grow. Angiogenic growth factors, alone and in combination, have been used to achieve this, and gene therapy has been used as a tool to enable sustained release of these angiogenic proteins. Cell-based therapy using endothelial cells and their precursors presents an alternative approach to tackling this challenge. These studies have occurred on a background of advancements in scaffold design and assays for assessing neovascularization. Finally, several studies have already attempted to translate research in neovascularization to clinical use in the blossoming field of therapeutic angiogenesis. Cells 2012-12-11 1 4 Review 10.3390/cells1041246 1246 1260 2073-4409 2012-12-11 doi: 10.3390/cells1041246 Jennifer Chung Dominique Shum-Tim http://www.mdpi.com/2073-4409/1/4/1225 Fluid flow has a great potential as a cell stimulatory tool for skeletal regenerative medicine, because fluid flow-induced bone cell mechanotransduction in vivo plays a critical role in maintaining healthy bone homeostasis. Applications of fluid flow for skeletal regenerative medicine are reviewed at macro and microscale. Macroflow in two dimensions (2D), in which flow velocity varies along the normal direction to the flow, has explored molecular mechanisms of bone forming cell mechanotransduction responsible for flow-regulated differentiation, mineralized matrix deposition, and stem cell osteogenesis. Though 2D flow set-ups are useful for mechanistic studies due to easiness in in situ and post-flow assays, engineering skeletal tissue constructs should involve three dimensional (3D) flows, e.g., flow through porous scaffolds. Skeletal tissue engineering using 3D flows has produced promising outcomes, but 3D flow conditions (e.g., shear stress vs. chemotransport) and scaffold characteristics should further be tailored. Ideally, data gained from 2D flows may be utilized to engineer improved 3D bone tissue constructs. Recent microfluidics approaches suggest a strong potential to mimic in vivo microscale interstitial flows in bone. Though there have been few microfluidics studies on bone cells, it was demonstrated that microfluidic platform can be used to conduct high throughput screening of bone cell mechanotransduction behavior under biomimicking flow conditions. Cells 2012-12-11 1 4 Review 10.3390/cells1041225 1225 1245 2073-4409 2012-12-11 doi: 10.3390/cells1041225 Brandon Riehl Jung Lim http://www.mdpi.com/2073-4409/1/4/1197 Cellular stress is the basis of a dose-dependent continuum of responses leading to adaptive health or pathogenesis. For all cells, stress leads to reduction in macromolecular synthesis by shared pathways and tissue and stress-specific homeostatic mechanisms. For stem cells during embryonic, fetal, and placental development, higher exposures of stress lead to decreased anabolism, macromolecular synthesis and cell proliferation. Coupled with diminished stem cell proliferation is a stress-induced differentiation which generates minimal necessary function by producing more differentiated product/cell. This compensatory differentiation is accompanied by a second strategy to insure organismal survival as multipotent and pluripotent stem cells differentiate into the lineages in their repertoire. During stressed differentiation, the first lineage in the repertoire is increased and later lineages are suppressed, thus prioritized differentiation occurs. Compensatory and prioritized differentiation is regulated by at least two types of stress enzymes. AMP-activated protein kinase (AMPK) which mediates loss of nuclear potency factors and stress-activated protein kinase (SAPK) that does not. SAPK mediates an increase in the first essential lineage and decreases in later lineages in placental stem cells. The clinical significance of compensatory and prioritized differentiation is that stem cell pools are depleted and imbalanced differentiation leads to gestational diseases and long term postnatal pathologies. Cells 2012-12-10 1 4 Review 10.3390/cells1041197 1197 1224 2073-4409 2012-12-10 doi: 10.3390/cells1041197 Ladan Mansouri Yufen Xie Daniel Rappolee http://www.mdpi.com/2073-4409/1/4/1182 Animal growth requires coordination of cell growth and cell cycle progression with developmental signaling. Loss of cell cycle control is extremely detrimental, with reduced cycles leading to impaired organ growth and excessive proliferation, potentially resulting in tissue overgrowth and driving tumour initiation. Due to the high level of conservation between the cell cycle machinery of Drosophila and humans, the appeal of the fly model continues to be the means with which we can use sophisticated genetics to provide novel insights into mammalian growth and cell cycle control. Over the last decade, there have been major additions to the genetic toolbox to study development in Drosophila. Here we discuss some of the approaches available to investigate the potent growth and cell cycle properties of the Drosophila counterparts of prominent cancer genes, with a focus on the c-Myc oncoprotein and the tumour suppressor protein FIR (Hfp in flies), which behaves as a transcriptional repressor of c-Myc. Cells 2012-12-05 1 4 Review 10.3390/cells1041182 1182 1196 2073-4409 2012-12-05 doi: 10.3390/cells1041182 Jue Lee Nicola Cranna Arjun Chahal Leonie Quinn http://www.mdpi.com/2073-4409/1/4/1168 Mechanical ablation of bone marrow in young rats induces rapid but transient bone growth, which can be enhanced and maintained for three weeks by the administration of parathyroid hormone (PTH). Additionally, marrow ablation, followed by PTH treatment for three months leads to increased cortical thickness. In this study, we sought to determine whether PTH enhances bone formation after marrow ablation in aged rats. Aged rats underwent unilateral femoral marrow ablation and treatment with PTH or vehicle for four weeks. Both femurs from each rat were analyzed by X-ray and pQCT, then analyzed either by microCT, histology or biomechanical testing. Marrow ablation alone induced transient bone formation of low abundance that persisted over four weeks, while marrow ablation followed by PTH induced bone formation of high abundance that also persisted over four weeks. Our data confirms that the osteo-inducive effect of marrow ablation and the additive effect of marrow ablation, followed by PTH, occurs in aged rats. Our observations open new avenues of investigations in the field of tissue regeneration. Local marrow ablation, in conjunction with an anabolic agent, might provide a new platform for rapid site-directed bone growth in areas of high bone loss, such as in the hip and wrist, which are subject to fracture. Cells 2012-12-05 1 4 Article 10.3390/cells1041168 1168 1181 2073-4409 2012-12-05 doi: 10.3390/cells1041168 Qing Zhang Christopher Miller Jesse Bible Jiliang Li Xiaoqing Xu Nozer Mehta James Gilligan Agnès Vignery Jodi Scholz http://www.mdpi.com/2073-4409/1/4/1156 Persistent cell shrinkage is a major hallmark of apoptotic cell death. The early-phase shrinkage, which starts within 30−120 min after apoptotic stimulation and is called apoptotic volume decrease (AVD), is known to be accomplished by activation of K+ channels and volume-sensitive outwardly rectifying (VSOR) Cl− channels in a manner independent of caspase-3 activation. However, it is controversial whether AVD depends on apoptotic dysfunction of mitochondria and activation of initiator caspases. Here, we observed that AVD is induced not only by a mitochondrial apoptosis inducer, staurosporine (STS), in mouse B lymphoma WEHI-231 cells, but also by ligation of the death receptor Fas in human B lymphoblastoid SKW6.4 cells, which undergo Fas-mediated apoptosis without involving mitochondria. Overexpression of Bcl-2 failed to inhibit the STS-induced AVD in WEHI-231 cells. These results indicate that AVD does not require the mitochondrial pathway of apoptosis. In human epithelial HeLa cells stimulated with anti-Fas antibody or STS, the AVD induction was found to precede activation of caspase-8 and caspase-9 and to be resistant to pan-caspase blockers. Thus, it is concluded that the AVD induction is an early event independent of the mitochondrial apoptotic signaling pathway and initiator caspase activation. Cells 2012-12-05 1 4 Article 10.3390/cells1041156 1156 1167 2073-4409 2012-12-05 doi: 10.3390/cells1041156 Emi Maeno Takeshi Tsubata Yasunobu Okada http://www.mdpi.com/2073-4409/1/4/1133 The small heme-protein neuroglobin is expressed at high concentrations in certain brain neurons and in the rod cells of the retina. This paper reviews the many studies which have recently identified a protective role for neuroglobin, in a wide range of situations involving apoptotic cell death. The origins of this protective mechanism are discussed in terms of both experimental results and computational modeling of the intrinsic pathway of apoptosis, which shows that neuroglobin can intervene in this process by a reaction with released mitochondrial cytochrome c. An integrated model, based on the various molecular actions of both neuroglobin and cytochrome c, is developed, which accounts for the cellular distribution of neuroglobin. Cells 2012-11-23 1 4 Article 10.3390/cells1041133 1133 1155 2073-4409 2012-11-23 doi: 10.3390/cells1041133 Thomas Brittain http://www.mdpi.com/2073-4409/1/4/1121 Intravital microscopy has enabled imaging of the dynamics of subcellular structures in live animals, thus opening the door to investigating membrane trafficking under physiological conditions. Here, we sought to determine whether the architecture and the environment of a fully developed tissue influences the dynamics of endocytic processes. To this aim, we imaged endocytosis in the stromal cells of rat salivary glands both in situ and after they were isolated and cultured on a solid surface. We found that the internalization of transferrin and dextran, two molecules that traffic via distinct mechanisms, is substantially altered in cultured cells, supporting the idea that the three dimensional organization of the tissue and the cues generated by the surrounding environment strongly affect membrane trafficking events. Cells 2012-11-16 1 4 Article 10.3390/cells1041121 1121 1132 2073-4409 2012-11-16 doi: 10.3390/cells1041121 Andrius Masedunskas Natalie Porat-Shliom Kamil Rechache Myo-Pale' Aye Roberto Weigert http://www.mdpi.com/2073-4409/1/4/1107 Cartilage tissue engineering is a promising field in regenerative medicine that can provide substantial relief to people suffering from degenerative cartilage disease. Current research shows the greatest chondrogenic potential for healthy articular cartilage growth with minimal hypertrophic differentiation to be from mesenchymal stem cells (MSCs) of synovial origin. These stem cells have the capacity for differentiation into multiple cell lineages related to mesenchymal tissue; however, evidence exists for cell surface markers that specify a greater potential for chondrogenesis than other differentiation fates. This review will examine relevant literature to summarize the chondrogenic differentiation capacities of tested synovium-derived stem cell (SDSC) surface markers, along with a discussion about various other markers that may hold potential, yet require further investigation. With this information, a potential clinical benefit exists to develop a screening system for SDSCs that will produce the healthiest articular cartilage possible. Cells 2012-11-16 1 4 Review 10.3390/cells1041107 1107 1120 2073-4409 2012-11-16 doi: 10.3390/cells1041107 Douglas Campbell Ming Pei http://www.mdpi.com/2073-4409/1/4/1089 Drought stress is one of the most important factors in the limitation of plant productivity worldwide. In order to cope with water deprivation, plants have adopted several strategies that produce major changes in gene expression. In this paper, the response to drought stress in the model legume Lotus japonicus was studied using a transcriptomic approach. Drought induced an extensive reprogramming of the transcriptome as related to various aspects of cellular metabolism, including genes involved in photosynthesis, amino acid metabolism and cell wall metabolism, among others. A particular focus was made on the genes involved in the cellular stress response. Key genes involved in the control of the cell cycle, antioxidant defense and stress signaling, were modulated as a consequence of water deprivation. Genes belonging to different families of transcription factors were also highly responsive to stress. Several of them were homologies to known stress-responsive genes from the model plant Arabidopsis thaliana, while some novel transcription factors were peculiar to the L. japonicus drought stress response. Cells 2012-11-13 1 4 Article 10.3390/cells1041089 1089 1106 2073-4409 2012-11-13 doi: 10.3390/cells1041089 Marco Betti Carmen Pérez-Delgado Margarita García-Calderón Pedro Díaz Jorge Monza Antonio Márquez http://www.mdpi.com/2073-4409/1/4/1061 Mesenchymal stem or stromal cells (MSC) have proven to offer great promise for cell-based therapies and tissue engineering applications, as these cells are capable of extensive self-renewal and display a multilineage differentiation potential. Furthermore, MSC were shown to exhibit immunomodulatory properties and display supportive functions through parakrine effects. Besides bone marrow (BM), still today the most common source of MSC, these cells were found to be present in a variety of postnatal and extraembryonic tissues and organs as well as in a large variety of fetal tissues. Over the last decade, the human umbilical cord and human amnion have been found to be a rich and valuable source of MSC that is bio-equivalent to BM-MSC. Since these tissues are discarded after birth, the cells are easily accessible without ethical concerns. Cells 2012-11-12 1 4 Review 10.3390/cells1041061 1061 1088 2073-4409 2012-11-12 doi: 10.3390/cells1041061 Andrea Lindenmair Tim Hatlapatka Gregor Kollwig Simone Hennerbichler Christian Gabriel Susanne Wolbank Heinz Redl Cornelia Kasper http://www.mdpi.com/2073-4409/1/4/1045 Mesenchymal stem cells (MSCs) are easily accessible and safe for regenerative medicine. MSCs exert trophic, immunomodulatory, anti-apoptotic, and tissue regeneration effects in a variety of tissues and organs, but their entity remains an enigma. Because MSCs are generally harvested from mesenchymal tissues, such as bone marrow, adipose tissue, or umbilical cord as adherent cells, MSCs comprise crude cell populations and are heterogeneous. The specific cells responsible for each effect have not been clarified. The most interesting property of MSCs is that, despite being adult stem cells that belong to the mesenchymal tissue lineage, they are able to differentiate into a broad spectrum of cells beyond the boundary of mesodermal lineage cells into ectodermal or endodermal lineages, and repair tissues. The broad spectrum of differentiation ability and tissue-repairing effects of MSCs might be mediated in part by the presence of a novel pluripotent stem cell type recently found in adult human mesenchymal tissues, termed multilineage-differentiating stress enduring (Muse) cells. Here we review recently updated studies of the regenerative effects of MSCs and discuss their potential in regenerative medicine. Cells 2012-11-08 1 4 Review 10.3390/cells1041045 1045 1060 2073-4409 2012-11-08 doi: 10.3390/cells1041045 Shohei Wakao Yasumasa Kuroda Fumitaka Ogura Taeko Shigemoto Mari Dezawa http://www.mdpi.com/2073-4409/1/4/1029 As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP's plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport. Cells 2012-11-05 1 4 Article 10.3390/cells1041029 1029 1044 2073-4409 2012-11-05 doi: 10.3390/cells1041029 Rajith N. Aturaliya Markus C. Kerr Rohan D. Teasdale http://www.mdpi.com/2073-4409/1/4/1010 Tendon healing is generally a time-consuming process and often leads to a functionally altered reparative tissue. Using degradable scaffolds for tendon reconstruction still remains a compromise in view of the required high mechanical strength of tendons. Regenerative approaches based on natural decellularized allo- or xenogenic tendon extracellular matrix (ECM) have recently started to attract interest. This ECM combines the advantages of its intrinsic mechanical competence with that of providing tenogenic stimuli for immigrating cells mediated, for example, by the growth factors and other mediators entrapped within the natural ECM. A major restriction for their therapeutic application is the mainly cell-associated immunogenicity of xenogenic or allogenic tissues and, in the case of allogenic tissues, also the risk of disease transmission. A survey of approaches for tendon reconstruction using cell-free tendon ECM is presented here, whereby the problems associated with the decellularization procedures, the success of various recellularization strategies, and the applicable cell types will be thoroughly discussed. Encouraging in vivo results using cell-free ECM, as, for instance, in rabbit models, have already been reported. However, in comparison to native tendon, cells remain mostly inhomogeneously distributed in the reseeded ECM and do not align. Hence, future work should focus on the optimization of tendon ECM decellularization and recolonization strategies to restore tendon functionality. Cells 2012-11-05 1 4 Review 10.3390/cells1041010 1010 1028 2073-4409 2012-11-05 doi: 10.3390/cells1041010 Gundula Schulze-Tanzil Onays Al-Sadi Wolfgang Ertel Anke Lohan http://www.mdpi.com/2073-4409/1/4/994 The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES) cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS) cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation. Cells 2012-11-05 1 4 Review 10.3390/cells1040994 994 1009 2073-4409 2012-11-05 doi: 10.3390/cells1040994 Atsuyuki Inui Takashi Iwakura A. Reddi http://www.mdpi.com/2073-4409/1/4/976 In this review, we summarize advances in our understanding of redox-sensitive mechanisms that regulate adipogenesis. Current evidence indicates that reactive oxygen species may act to promote both the initiation of adipocyte lineage commitment of precursor or stem cells, and the terminal differentiation of preadipocytes to mature adipose cells. These can involve redox regulation of pathways mediated by receptor tyrosine kinases, peroxisome proliferator-activated receptor γ (PPARγ), PPARγ coactivator 1α (PGC-1α), AMP-activated protein kinase (AMPK), and CCAAT/enhancer binding protein β (C/EBPβ). However, the precise roles of ROS in adipogenesis in vivo remain controversial. More studies are needed to delineate the roles of reactive oxygen species and redox signaling mechanisms, which could be either positive or negative, in the pathogenesis of obesity and related metabolic disorders. Cells 2012-11-05 1 4 Review 10.3390/cells1040976 976 993 2073-4409 2012-11-05 doi: 10.3390/cells1040976 Guei-Sheung Liu Elsa Chan Masayoshi Higuchi Gregory Dusting Fan Jiang http://www.mdpi.com/2073-4409/1/4/961 Despite encouraging preclinical results for therapeutic angiogenesis in ischemia, a suitable approach providing sustained, safe and efficacious vascular growth in the heart is still lacking. Vascular Endothelial Growth Factor (VEGF) is the master regulator of angiogenesis, but it also can easily induce aberrant and dysfunctional vascular growth if its expression is not tightly controlled. Control of the released level in the microenvironment around each cell in vivo and its distribution in tissue are critical to induce stable and functional vessels for therapeutic angiogenesis. The present review discusses the limitations and perspectives of VEGF gene therapy and of different cell-based approaches for the implementation of therapeutic angiogenesis in the treatment of cardiac ischemia. Cells 2012-11-05 1 4 Abstract 10.3390/cells1040961 961 975 2073-4409 2012-11-05 doi: 10.3390/cells1040961 Ludovic Melly Stefano Boccardo Friedrich Eckstein Andrea Banfi Anna Marsano http://www.mdpi.com/2073-4409/1/4/926 The endoplasmic reticulum (ER) is the site of secretory protein biogenesis. The ER quality control (QC) machinery, including chaperones, ensures the correct folding of secretory proteins. Mutant proteins and environmental stresses can overwhelm the available QC machinery. To prevent and resolve accumulation of misfolded secretory proteins in the ER, cells have evolved integral membrane sensors that orchestrate the Unfolded Protein Response (UPR). The sensors, Ire1p in yeast and IRE1, ATF6, and PERK in metazoans, bind the luminal ER chaperone BiP during homeostasis. As unfolded secretory proteins accumulate in the ER lumen, BiP releases, and the sensors activate. The mechanisms of activation and attenuation of the UPR sensors have exhibited unexpected complexity. A growing body of data supports a model in which Ire1p, and potentially IRE1, directly bind unfolded proteins as part of the activation process. However, evidence for an unfolded protein-independent mechanism has recently emerged, suggesting that UPR can be activated by multiple modes. Importantly, dysregulation of the UPR has been linked to human diseases including Type II diabetes, heart disease, and cancer. The existence of alternative regulatory pathways for UPR sensors raises the exciting possibility for the development of new classes of therapeutics for these medically important proteins. Cells 2012-11-01 1 4 Review 10.3390/cells1040926 926 960 2073-4409 2012-11-01 doi: 10.3390/cells1040926 Erik Snapp http://www.mdpi.com/2073-4409/1/4/905 Tooth morphogenesis is carried out by a series of reciprocal interactions between the epithelium and mesenchyme in embryonic germs. Previously clonal dental epithelial cell (epithelium of molar tooth germ (emtg)) lines were established from an embryonic germ. They were odontogenic when combined with a dental mesenchymal tissue, although the odontogenesis was quantitatively imperfect. To improve the microenvironment in the germs, freshly isolated dental epithelial cells were mixed with cells of lines, and germs were reconstructed in various combinations. The results demonstrated that successful tooth construction depends on the mixing ratio, the age of dental epithelial cells and the combination with cell lines. Analyses of gene expression in these germs suggest that some signal(s) from dental epithelial cells makes emtg cells competent to communicate with mesenchymal cells and the epithelial and mesenchymal compartments are able to progress odontogenesis from the initiation stage. Cells 2012-10-30 1 4 Article 10.3390/cells1040905 905 925 2073-4409 2012-10-30 doi: 10.3390/cells1040905 Akihiko Komine Yasuhiro Tomooka http://www.mdpi.com/2073-4409/1/4/886 The MST/Salvador-Warts-Hippo and mTOR/Akt/PI3K growth signaling pathways have been established as important modulators of cell growth, proliferation and cell survival in controlling organ size in Drosophila and mammals. Here, we sought to determine the role of the MST family of kinases, some of which are components of the Hippo pathway, and their closely related Sterile 20-like kinases (STK) as candidates for mediating cross-talk between the Hippo and mTOR pathways. Expression analysis in the HepG2 and MCF7 cell lines demonstrated common expression of MST1/2/4, MAP4K3/4/5, STK 24 (MST3), STK25, STK39, Pak1, SLK, Stradα/β and TAO2. All components of the Hippo signaling pathway are present in both cell lines except for YAP1 in MCF7 cells. mTOR inhibition via rapamycin decreases TAZ levels in HepG2 but not MCF7 cells and increases TEAD1 levels in MCF7 but not HepG2 cells, suggesting a selective role of the mTOR pathway in regulating these Hippo targets in a cell type-specific manner. Furthermore, the cellular localization of TAZ changes in response to mTORC1/2 inhibitors and Akt inhibition. These findings demonstrate the mTOR-dependent regulation of Hippo signaling at the level of the transcriptional regulators TAZ and TEAD1 and highlight the potential role for mTOR inhibitors in regulating Hippo-signaling dependent tumors. Cells 2012-10-19 1 4 Article 10.3390/cells1040886 886 904 2073-4409 2012-10-19 doi: 10.3390/cells1040886 Jonathan Chiang Julian A. Martinez-Agosto http://www.mdpi.com/2073-4409/1/4/874 Tissue resident mesenchymal stem cells (MSCs) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Bone marrow derived mesenchymal stem cells (BM-MSCs) and endothelial progenitor cells (EPC) are currently being considered and tested in clinical trials as a potential therapy in patients with such inflammatory lung diseases including, but not limited to, chronic lung disease, pulmonary arterial hypertension (PAH), pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD)/emphysema and asthma. However, our current understanding of tissue resident lung MSCs remains limited. This review addresses how environmental cues impact on the phenotype and function of this endogenous stem cell pool. In addition, it examines how these local factors influence the efficacy of cell-based treatments for lung diseases. Cells 2012-10-19 1 4 Review 10.3390/cells1040874 874 885 2073-4409 2012-10-19 doi: 10.3390/cells1040874 Robert F. Foronjy Susan M. Majka http://www.mdpi.com/2073-4409/1/4/851 Retinal degenerative diseases constitute a major cause of irreversible blindness in the world. Stem cell-based therapies offer hope for these patients at risk of or suffering from blindness due to the deterioration of the neural retina. Various sources of stem cells are currently being investigated, ranging from human embryonic stem cells to adult-derived induced pluripotent stem cells as well as human Müller stem cells, with the first clinical trials to investigate the safety and tolerability of human embryonic stem cell-derived retinal pigment epithelium cells having recently commenced. This review aims to summarize the latest advances in the development of stem cell strategies for the replacement of retinal neurons and their supportive cells, the retinal pigment epithelium (RPE) affected by retinal degenerative conditions. Particular emphasis will be given to the advances in stem cell transplantation and the challenges associated with their translation into clinical practice. Cells 2012-10-18 1 4 Review 10.3390/cells1040851 851 873 2073-4409 2012-10-18 doi: 10.3390/cells1040851 Silke Becker Hari Jayaram G. Astrid Limb http://www.mdpi.com/2073-4409/1/4/832 Bone marrow-derived mesenchymal stromal cells (BM-MSCs) represent the leading candidate cell in tissue engineering and regenerative medicine. These cells can be easily isolated, expanded in vitro and are capable of providing significant functional benefits after implantation in the damaged muscle tissues. Despite their plasticity, the participation of BM-MSCs to new muscle fiber formation is controversial; in fact, emerging evidence indicates that their therapeutic effects occur without signs of long-term tissue engraftment and involve the paracrine secretion of cytokines and growth factors with multiple effects on the injured tissue, including modulation of inflammation and immune reaction, positive extracellular matrix (ECM) remodeling, angiogenesis and protection from apoptosis. Recently, a new role for BM-MSCs in the stimulation of muscle progenitor cells proliferation has been demonstrated, suggesting the potential ability of these cells to influence the fate of local stem cells and augment the endogenous mechanisms of repair/regeneration in the damaged tissues. Cells 2012-10-17 1 4 Review 10.3390/cells1040832 832 850 2073-4409 2012-10-17 doi: 10.3390/cells1040832 Chiara Sassoli Sandra Zecchi-Orlandini Lucia Formigli http://www.mdpi.com/2073-4409/1/4/799 Damage to DNA is common and can arise from numerous environmental and endogenous sources. In response to ubiquitous DNA damage, Y-family DNA polymerases are induced by the SOS response and are capable of bypassing DNA lesions. In Escherichia coli, these Y-family polymerases are DinB and UmuC, whose activities are modulated by their interaction with the polymerase manager protein UmuD. Many, but not all, bacteria utilize DinB and UmuC homologs. Recently, a C-family polymerase named ImuC, which is similar in primary structure to the replicative DNA polymerase DnaE, was found to be able to copy damaged DNA and either carry out or suppress mutagenesis. ImuC is often found with proteins ImuA and ImuB, the latter of which is similar to Y‑family polymerases, but seems to lack the catalytic residues necessary for polymerase activity. This imuAimuBimuC mutagenesis cassette represents a widespread alternative strategy for translesion synthesis and mutagenesis in bacteria. Bacterial Y‑family and ImuC DNA polymerases contribute to replication past DNA damage and the acquisition of antibiotic resistance. Cells 2012-10-15 1 4 Review 10.3390/cells1040799 799 831 2073-4409 2012-10-15 doi: 10.3390/cells1040799 Paul Ippoliti Nicholas DeLateur Kathryn Jones Penny Beuning http://www.mdpi.com/2073-4409/1/4/774 The nucleolus has emerged as a cellular stress sensor and key regulator of p53-dependent and -independent stress responses. A variety of abnormal metabolic conditions, cytotoxic compounds, and physical insults induce alterations in nucleolar structure and function, a situation known as nucleolar or ribosomal stress. Ribosomal proteins, including RPL11 and RPL5, become increasingly bound to the p53 regulatory protein MDM2 following nucleolar stress. Ribosomal protein binding to MDM2 blocks its E3 ligase function leading to stabilization and activation of p53. In this review we focus on a number of novel regulators of the RPL5/RPL11-MDM2-p53 complex including PICT1 (GLTSCR2), MYBBP1A, PML and NEDD8. p53-independent pathways mediating the nucleolar stress response are also emerging and in particular the negative control that RPL11 exerts on Myc oncoprotein is of importance, given the role of Myc as a master regulator of ribosome biogenesis. We also briefly discuss the potential of chemotherapeutic drugs that specifically target RNA polymerase I to induce nucleolar stress. Cells 2012-10-15 1 4 Review 10.3390/cells1040774 774 798 2073-4409 2012-10-15 doi: 10.3390/cells1040774 Karl Holmberg Olausson Monica Nistér Mikael S. Lindström http://www.mdpi.com/2073-4409/1/4/754 14-3-3 proteins are implicated in the regulation of proteins involved in a variety of signaling pathways. 14-3-3-dependent protein regulation occurs through phosphorylation-dependent binding that results, in many cases, in the release of survival signals in cells. Autophagy is a cell digestion process that contributes to overcoming nutrient deprivation and is initiated under stress conditions. However, whether autophagy is a cell survival or cell death mechanism remains under discussion and may depend on context. Nevertheless, autophagy is a cellular process that determines cell fate and is tightly regulated by different signaling pathways, some of which, for example MAPK, PI3K and mTOR, are tightly regulated by 14-3-3 proteins. It is therefore important to understand the role of 14-3-3 protein in modulating the autophagic process. Within this context, direct binding of 14-3-3 to mTOR regulatory proteins, such as TSC2 and PRAS40, connects 14-3-3 with autophagy regulatory processes. In addition, 14-3-3 binding to human vacuolar protein sorting 34 (hVps34), a class III phosphatidylinositol-3-kinase (PI3KC3), indicates the involvement of 14-3-3 proteins in regulating autophagosome formation. hVps34 is involved in vesicle trafficking processes such as autophagy, and its activation is needed for initiation of autophagy. Chromatography and overlay techniques suggest that hVps34 directly interacts with 14-3-3 proteins under physiological conditions, thereby maintaining hVps34 in an inactive state. In contrast, nutrient starvation promotes dissociation of the 14-3-3–hVps34 complex, thereby enhancing hVps34 lipid kinase activity. Thus, 14-3-3 proteins are regulators of autophagy through regulating key components of the autophagic machinery. This review summarizes the role of 14-3-3 protein in the control of target proteins involved in regulating the master switches of autophagy. Cells 2012-10-15 1 4 Review 10.3390/cells1040754 754 773 2073-4409 2012-10-15 doi: 10.3390/cells1040754 Mercedes Pozuelo-Rubio http://www.mdpi.com/2073-4409/1/4/738 Stress in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a signaling mechanism that allows cellular adaptation to ER stress by engaging pro-adaptive transcription factors and alleviating protein folding demand. One such transcription factor, X-box binding protein (XBP1), originates from the inositol-requiring transmembrane kinase/endoribonuclease 1 (IRE1) UPR stress sensor. XBP1 up-regulates a pool of genes involved in ER protein translocation, protein folding, vesicular trafficking and ER- associated protein degradation. Recent data suggest that the regulation of XBP1 expression and transcriptional activity may be a tissue- and stress-dependent phenomenon. Moreover, the intricacies involved in “fine-tuning” XBP1 activity in various settings are now coming to light. Here, we provide an overview of recent developments in understanding the regulatory mechanisms underlying XBP1 expression and activity and discuss the significance of these new insights. Cells 2012-10-15 1 4 Review 10.3390/cells1040738 738 753 2073-4409 2012-10-15 doi: 10.3390/cells1040738 Andrew Byrd Joseph Brewer http://www.mdpi.com/2073-4409/1/4/711 Cells are constantly exposed to endogenous and exogenous cellular injuries. They cope with stressful stimuli by adapting their metabolism and activating various “guardian molecules.” These pro-survival factors protect essential cell constituents, prevent cell death, and possibly repair cellular damages. The Inhibitor of Apoptosis (IAPs) proteins display both anti-apoptotic and pro-survival properties and their expression can be induced by a variety of cellular stress such as hypoxia, endoplasmic reticular stress and DNA damage. Thus, IAPs can confer tolerance to cellular stress. This review presents the anti-apoptotic and survival functions of IAPs and their role in the adaptive response to cellular stress. The involvement of IAPs in human physiology and diseases in connection with a breakdown of cellular homeostasis will be discussed. Cells 2012-10-10 1 4 Review 10.3390/cells1040711 711 737 2073-4409 2012-10-10 doi: 10.3390/cells1040711 Arthur Marivin Jean Berthelet Stéphanie Plenchette Laurence Dubrez http://www.mdpi.com/2073-4409/1/4/699 High-mobility group box 1 protein (HMGB1), a member of highly conserved non-histone DNA binding protein family, has been studied as transcription factor and growth factor. Secreted extracellularly by activated monocytes and macrophages or passively released by necrotic or damaged cells, extracellular HMGB1 is a potent mediator of inflammation. Extracellular HMGB1 has apparently contrasting biological actions: it sustains inflammation (with the possible establishment of autoimmunity or of self-maintaining tissue damage), but it also activates and recruits stem cells, boosting tissue repair. Here, we focus on the role of HMGB1 in physiological and pathological responses, the mechanisms by which it contributes to tissue repair and therapeutic strategies base on targeting HMGB1. Cells 2012-09-28 1 4 Review 10.3390/cells1040699 699 710 2073-4409 2012-09-28 doi: 10.3390/cells1040699 Elia Ranzato Simona Martinotti Marco Pedrazzi Mauro Patrone http://www.mdpi.com/2073-4409/1/4/667 Current treatments for hearing loss, the most common neurosensory disorder, do not restore perfect hearing. Regeneration of lost organ of Corti hair cells through forced cell cycle re-entry of supporting cells or through manipulation of stem cells, both avenues towards a permanent cure, require a more complete understanding of normal inner ear development, specifically the balance of proliferation and differentiation required to form and to maintain hair cells. Direct successful alterations to the cell cycle result in cell death whereas regulation of upstream genes is insufficient to permanently alter cell cycle dynamics. The Myc gene family is uniquely situated to synergize upstream pathways into downstream cell cycle control. There are three Mycs that are embedded within the Myc/Max/Mad network to regulate proliferation. The function of the two ear expressed Mycs, N-Myc and L-Myc were unknown less than two years ago and their therapeutic potentials remain speculative. In this review, we discuss the roles the Mycs play in the body and what led us to choose them to be our candidate gene for inner ear therapies. We will summarize the recently published work describing the early and late effects of N-Myc and L-Myc on hair cell formation and maintenance. Lastly, we detail the translational significance of our findings and what future work must be performed to make the ultimate hearing aid: the regeneration of the organ of Corti. Cells 2012-09-25 1 4 Review 10.3390/cells1040667 667 698 2073-4409 2012-09-25 doi: 10.3390/cells1040667 Benjamin Kopecky Bernd Fritzsch http://www.mdpi.com/2073-4409/1/3/646 Virus infections induce heat shock proteins that in turn enhance virus gene expression, a phenomenon that is particularly well characterized for the major inducible 70 kDa heat shock protein (hsp70). However, hsp70 is also readily induced by fever, a phylogenetically conserved response to microbial infections, and when released from cells, hsp70 can stimulate innate immune responses through toll like receptors 2 and 4 (TLR2 and 4). This review examines how the virus-hsp70 relationship can lead to host protective innate antiviral immunity, and the importance of hsp70 dependent stimulation of virus gene expression in this host response. Beginning with the well-characterized measles virus-hsp70 relationship and the mouse model of neuronal infection in brain, we examine data indicating that the innate immune response is not driven by intracellular sensors of pathogen associated molecular patterns, but rather by extracellular ligands signaling through TLR2 and 4. Specifically, we address the relationship between virus gene expression, extracellular release of hsp70 (as a damage associated molecular pattern), and hsp70-mediated induction of antigen presentation and type 1 interferons in uninfected macrophages as a novel axis of antiviral immunity. New data are discussed that examines the more broad relevance of this protective mechanism using vesicular stomatitis virus, and a review of the literature is presented that supports the probable relevance to both RNA and DNA viruses and for infections both within and outside of the central nervous system. Cells 2012-09-11 1 3 Review 10.3390/cells1030646 646 666 2073-4409 2012-09-11 doi: 10.3390/cells1030646 Mi Young Kim Michael Oglesbee http://www.mdpi.com/2073-4409/1/3/631 Human enamel development of the permanent teeth takes place during childhood and stresses encountered during this period can have lasting effects on the appearance and structural integrity of the enamel. One of the most common examples of this is the development of dental fluorosis after childhood exposure to excess fluoride, an elemental agent used to increase enamel hardness and prevent dental caries. Currently the molecular mechanism responsible for dental fluorosis remains unknown; however, recent work suggests dental fluorosis may be the result of activated stress response pathways in ameloblasts during the development of permanent teeth. Using fluorosis as an example, the role of stress response pathways during enamel maturation is discussed. Cells 2012-08-30 1 3 Review 10.3390/cells1030631 631 645 2073-4409 2012-08-30 doi: 10.3390/cells1030631 Megan L. Sierant John D. Bartlett http://www.mdpi.com/2073-4409/1/3/617 Viruses commandeer regulatory pathways of their hosts to optimize their success as cellular parasites. The human tumor viruses, Epstein-Barr Virus (EBV), Kaposi’s Sarcoma Herpesvirus (KSHV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV) all affect autophagy for their own ends. EBV and KSHV regulate it during latent infections, a phase when no progeny virus is produced, while HBV and HCV use autophagy to promote their productive infections. Here we shall compare and contrast how these human tumor viruses regulate autophagy and what they gain by the appropriation of this cellular pathway. Cells 2012-08-27 1 3 Review 10.3390/cells1030617 617 630 2073-4409 2012-08-27 doi: 10.3390/cells1030617 Zachary L. Pratt Bill Sugden http://www.mdpi.com/2073-4409/1/3/597 In recent years, research on the autophagic process has greatly increased, invading the fields of biology and medicine. Several markers of the autophagic process have been discovered and various strategies have been reported studying this molecular process in different biological systems in both physiological and stress conditions. Furthermore, mechanisms of metalloid- or heavy metal-induced toxicity continue to be of interest given the ubiquitous nature and distribution of these contaminants in the environment where they often play the role of pollutants of numerous organisms. The aim of this review is a critical analysis and correlation of knowledge of autophagic mechanisms studied under stress for the most common arsenic (As) and cadmium (Cd) compounds. In this review we report data obtained in different experimental models for each compound, highlighting similarities and/or differences in the activation of autophagic processes. A more detailed discussion will concern the activation of autophagy in Cd-exposed sea urchin embryo since it is a suitable model system that is very sensitive to environmental stress, and Cd is one of the most studied heavy metal inductors of stress and modulator of different factors such as: protein kinase and phosphatase, caspases, mitochondria, heat shock proteins, metallothioneins, transcription factors, reactive oxygen species, apoptosis and autophagy. Cells 2012-08-27 1 3 Review 10.3390/cells1030597 597 616 2073-4409 2012-08-27 doi: 10.3390/cells1030597 Roberto Chiarelli Maria Carmela Roccheri http://www.mdpi.com/2073-4409/1/3/576 Experimental allergic encephalomyelitis (EAE) is mediated by neuroantigen-specific pro-inflammatory T cells of the Th1 and Th17 effector class. Th-17 cells can be clearly defined by expression of IL-17, but not IFN-γ, IL-2 or IL-3. Th1 cells do not express IL-17, but it is unclear presently to what extent they co-express the cytokines canonically assigned to Th1 immunity (i.e., IFN-γ, IL-2 and IL-3) and whether CD4 cells producing these cytokines indeed belong to a single Th1 lineage. It is also unclear to what extent the Th1 response in EAE entails polyfunctional T cells that co-express IFN-γ and IL-2. Therefore, we dissected the Th1 cytokine signature of neuroantigen-specific CD4 cells studying at single cell resolution co-expression of IFN-γ, IL-2 and IL-3 using dual color cytokine ELISPOT analysis. Shortly after immunization, in the draining lymph nodes (dLN), the overall cytokine signature of the neuroantigen-specific CD4 cells was highly type 1-polarized, but IFN-γ, IL-2, and IL-3 were each secreted by different CD4 cells in a mutually exclusive manner. This single cell – single cytokine profile was stable through the course of chronic EAE–polyfunctional CD4 cells co-expressing IL-2 and IFN-γ presented less than 5% of the neuroantigen-specific T cells, even in the inflamed CNS itself. The neuroantigen-specific CD4 cells that expressed IFN-γ, IL-2 and IL-3 in a mutually exclusive manner exhibited similar functional avidities and kinetics of cytokine production, but showed different tissue distributions. These data suggest that Th1 cells do not belong to a single lineage, but different Th1 subpopulations jointly mediate Th1 immunity. Cells 2012-08-24 1 3 Article 10.3390/cells1030576 576 596 2073-4409 2012-08-24 doi: 10.3390/cells1030576 Alexey Y. Karulin Stefan Quast Maike D. Hesse Paul V. Lehmann http://www.mdpi.com/2073-4409/1/3/558 All the therapeutic strategies for treating cancers aim at killing the cancer cells via apoptosis (programmed cell death type I). Defective apoptosis endow tumor cells with survival. The cell can respond to such defects with autophagy. Autophagy is a cellular process by which cytoplasmic material is either degraded to maintain homeostasis or recycled for energy and nutrients in starvation. A plethora of evidence has shown that the role of autophagy in tumors is complex. A lot of effort is needed to underline the functional status of autophagy in tumor progression and treatment, and elucidate how to tweak autophagy to treat cancer. Furthermore, during the treatment of cancer, the limitation for the cure rate and survival is the phenomenon of multi drug resistance (MDR). The development of MDR is an intricate process that could be regulated by drug transporters, enzymes, anti-apoptotic genes or DNA repair mechanisms. Reports have shown that autophagy has a dual role in MDR. Furthermore, it has been reported that activation of a death pathway may overcome MDR, thus pointing the importance of other death pathways to regulate tumor cell progression and growth. Therefore, in this review we will discuss the role of autophagy in MDR tumors and a possible link amongst these phenomena. Cells 2012-08-23 1 3 Review 10.3390/cells1030558 558 575 2073-4409 2012-08-23 doi: 10.3390/cells1030558 Priyank Kumar Dong-Mei Zhang Kurt Degenhardt Zhe-Sheng Chen http://www.mdpi.com/2073-4409/1/3/535 The intestine lies at the interface between the organism and its environment and responds to infection/inflammation in a multi-leveled manner, potentially leading to chronic inflammatory pathologies and cancer formation. Indeed, the immune response at the intestinal epithelium has been found to be involved in the origin and development of colorectal cancer, which is the third most commonly diagnosed neoplastic disease. Among the mechanisms induced upon inflammation, autophagy appears as a defensive strategy for the clearance of invading microbes and intracellular waste components. Autophagy has also been found to play an important role in colorectal cancer, where it seems to have a pro-survival or pro-death function depending on the stage of the neoplastic process. In this paper we discuss the dual role of autophagy in colorectal cancer and review evidence showing that modulation of autophagy affects the immune response and cancer biology. The study of key players involved in autophagy might contribute to the design of new approaches for colorectal cancer, consisting in combined therapies capable of modifying cancer-specific metabolism rather than simply evoking a generic apoptotic and/or autophagic response, thus enhancing the efficacy of currently used drugs and treatments. Cells 2012-08-21 1 3 Review 10.3390/cells1030535 535 557 2073-4409 2012-08-21 doi: 10.3390/cells1030535 Federica Madia Valentina Grossi Alessia Peserico Cristiano Simone http://www.mdpi.com/2073-4409/1/3/520 Autophagy is a housekeeping survival mechanism with a protective function against stress conditions. However, when stress severity or duration increases, it may promote cell death. Paradoxically, autophagy favors cancer development, since cancer cells could enhance their proliferation potential (thus becoming able to resist anticancer therapy) thanks to the energetic supply provided by organelle degradation typically driven by autophagy following a stepwise pathway. The main actors of the autophagic machinery as well as the features shared with apoptosis will be described. Special attention will be paid to the effects of autophagy manipulation. Cells 2012-08-13 1 3 Review 10.3390/cells1030520 520 534 2073-4409 2012-08-13 doi: 10.3390/cells1030520 Francesca Aredia Luis Miguel Guamán Ortiz Vincenzo Giansanti A. Ivana Scovassi http://www.mdpi.com/2073-4409/1/3/492 (Macro)-autophagy is a homeostatic process by which eukaryotic cells dispose of protein aggregates and damaged organelles. Autophagy is also used to degrade micro-organisms that invade intracellularly in a process termed xenophagy. Genome-wide association scans have recently identified autophagy genes as conferring susceptibility to Crohn’s disease (CD), one of the chronic inflammatory bowel diseases, with evidence suggesting that CD arises from a defective innate immune response to enteric bacteria. Here we review the emerging role of autophagy in CD, with particular focus on xenophagy and enteric E. coli strains with an adherent and invasive phenotype that have been consistently isolated from CD patients with ileal disease. Cells 2012-08-03 1 3 Review 10.3390/cells1030492 492 519 2073-4409 2012-08-03 doi: 10.3390/cells1030492 Paul Henderson Craig Stevens http://www.mdpi.com/2073-4409/1/3/464 Autophagy is an important cellular program with a “double face” role, since it promotes either cell survival or cell death, also in cancer therapies. Its survival role occurs by recycling cell components during starvation or removing stressed organelles; when damage becomes extensive, autophagy provides another programmed cell death pathway, known as Autophagic Cell Death (ACD). The induction of autophagy is a common outcome in PhotoDynamic Therapy (PDT), a two-step process involving the irradiation of photosensitizer (PS)-loaded cancer cells. Upon tissue oxygen interaction, PS provokes immediate and direct Reactive Oxygen Species (ROS)-induced damage to Endoplasmic Reticulum (ER), mitochondria, plasma membrane, and/or lysosomes. The main biological effects carried out in cancer PDT are direct cytotoxicity to tumor cells, vasculature damage and induction of inflammatory reactions stimulating immunological responses. The question about the role of autophagy in PDT and its putative immunological impact is hotly controversial and largely studied in recent times. This review deals with the induction of autophagy in PDT protocols and its dual role, also considering its interrelationship with apoptosis, the preferential cell death program triggered in the photodynamic process. Cells 2012-08-03 1 3 Review 10.3390/cells1030464 464 491 2073-4409 2012-08-03 doi: 10.3390/cells1030464 Valentina Inguscio Elisa Panzarini Luciana Dini http://www.mdpi.com/2073-4409/1/3/449 Macroautophagy is a non-selective, bulk degradation process conserved in eukaryotes. Response to starvation stress and/or regulation of nutrient breakdown/utilization is the major intracellular function of macroautophagy. Recent studies have revealed requirement for autophagy in diverse functions such as nutrient homeostasis, organelle degradation and programmed cell death in filamentous fungal pathogens, for proper morphogenesis and differentiation during critical steps of infection. In this review, we aim to summarize the physiological functions of autophagy in fungal virulence, with an emphasis on nutrient homeostasis in opportunistic human fungal pathogens and in the rice-blast fungus, Magnaporthe oryzae. We briefly summarize the role of autophagy on the host side: for resistance to, or subversion by, the pathogens. Cells 2012-08-02 1 3 Review 10.3390/cells1030449 449 463 2073-4409 2012-08-02 doi: 10.3390/cells1030449 Yizhen Deng Ziwei Qu Naweed I. Naqvi http://www.mdpi.com/2073-4409/1/3/428 Autophagy is an evolutionarily conserved catabolic process by which cells degrade their own components through the lysosomal machinery. In physiological conditions, the mechanism is tightly regulated and contributes to maintain a balance between synthesis and degradation in cells undergoing intense metabolic activities. Autophagy is associated with major tissue remodeling processes occurring through the embryonic, fetal and early postnatal periods of vertebrates. Here we survey current information implicating autophagy in cellular death, proliferation or differentiation in developing vertebrates. In developing systems, activation of the autophagic machinery could promote different outcomes depending on the cellular context. Autophagy is thus an extraordinary tool for the developing organs and tissues. Cells 2012-08-02 1 3 Review 10.3390/cells1030428 428 448 2073-4409 2012-08-02 doi: 10.3390/cells1030428 María R. Aburto Juan M. Hurlé Isabel Varela-Nieto Marta Magariños http://www.mdpi.com/2073-4409/1/3/409 T cell monitoring is increasingly performed using cryopreserved PBMC. It has been suggested that resting of PBMC after thawing, that is, culturing them overnight in test medium, produces higher antigen-induced spot counts in ELISPOT assays. To evaluate the importance of overnight resting, we systematically tested cryopreserved PBMC from 25 healthy donors. CEF peptides (comprising CMV, EBV and flu antigens) were used to stimulate CD8 cells and mumps antigen to stimulate CD4 cells. The data show that resting significantly increased antigen-elicited T cell responses only for CEF high responder PBMC. The maximal gain observed was doubling of spot counts. For CEF low responders, and for mumps responders of either low- or high reactivity levels, resting had no statistically significant effect on the observed spot counts. Therefore, resting is not a generally applicable approach to improve ELISPOT assay performance, but can be recommended only for clinical subject cohorts and antigens for which it has a proven benefit. Because resting invariably leads to losing about half of the PBMC available for testing, and because doubling the PBMC numbers plated into the assay reliably doubles the antigen-induced spot counts, we suggest the latter approach as a simple and reliable alternative to resting for enhancing the performance of ELISPOT assays. Our data imply that resting is not required if PBMC were cryopreserved and thawed under conditions that minimize apoptosis of the cells. Therefore, this study should draw attention to the need to optimize freezing and thawing conditions for successful T cell work. Cells 2012-07-30 1 3 Article 10.3390/cells1030409 409 427 2073-4409 2012-07-30 doi: 10.3390/cells1030409 Stefanie Kuerten Helena Batoulis Mascha S. Recks Edith Karacsony Wenji Zhang Ramu A. Subbramanian Paul V. Lehmann http://www.mdpi.com/2073-4409/1/3/396 Autophagy is an intracellular degradative process with a number of roles, one of which can be the protection of eukaryotic cells from invading microbes. Microtubule-associated protein light-chain 3 (LC3) is a key autophagy-related protein that is recruited to the double-membrane autophagosome responsible for sequestering material intended for delivery to lysosomes. GFP-LC3 is widely used as a marker of autophagosome formation as denoted by the formation of green puncta when viewed by fluorescence microscopy. Recently, it has been demonstrated that LC3 can be recruited to other membranes including single-membrane phagosomes, in a process termed LC3-associated phagocytosis (LAP). Thus, the observation of green puncta in cells can no longer, by itself, be taken as evidence of autophagy. This review will clarify those features of LAP which serve to distinguish it from autophagy and that make connections with host autophagic responses in terms of infection by microbial pathogens. More specifically, it will refer to concurrent studies of the mechanism by which LAP is triggered in comparison to autophagy. Cells 2012-07-30 1 3 Review 10.3390/cells1030396 396 408 2073-4409 2012-07-30 doi: 10.3390/cells1030396 Shu-chin Lai Rodney J. Devenish http://www.mdpi.com/2073-4409/1/3/372 Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focus on the signalling pathways by which environmental glucose directly, i.e., independently of insulin and glucagon, regulates autophagy in mammalian cells, but we will also briefly mention some data in yeast. Although glucose deprivation mainly induces autophagy via AMPK activation and the subsequent inhibition of mTORC1, we will also comment other signalling pathways, as well as evidences indicating that, under certain conditions, autophagy can be activated by glucose. A better understanding on how glucose regulates autophagy not only will expand our basic knowledge of this important cell process, but it will be also relevant to understand common human disorders, such as cancer and diabetes, in which glucose levels play an important role. Cells 2012-07-27 1 3 Review 10.3390/cells1030372 372 395 2073-4409 2012-07-27 doi: 10.3390/cells1030372 Félix Moruno Eva Pérez-Jiménez Erwin Knecht http://www.mdpi.com/2073-4409/1/3/346 Autophagy is a ubiquitous eukaryotic process that also occurs in trypanosomatid parasites, protist organisms belonging to the supergroup Excavata, distinct from the supergroup Opistokontha that includes mammals and fungi. Half of the known yeast and mammalian AuTophaGy (ATG) proteins were detected in trypanosomatids, although with low sequence conservation. Trypanosomatids such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for serious tropical diseases in humans. The parasites are transmitted by insects and, consequently, have a complicated life cycle during which they undergo dramatic morphological and metabolic transformations to adapt to the different environments. Autophagy plays a major role during these transformations. Since inhibition of autophagy affects the transformation, survival and/or virulence of the parasites, the ATGs offer promise for development of drugs against tropical diseases. Furthermore, various trypanocidal drugs have been shown to trigger autophagy-like processes in the parasites. It is inferred that autophagy is used by the parasites in an—not always successful—attempt to cope with the stress caused by the toxic compounds. Cells 2012-07-27 1 3 Review 10.3390/cells1030346 346 371 2073-4409 2012-07-27 doi: 10.3390/cells1030346 Ana Brennand Eva Rico Paul A. M. Michels http://www.mdpi.com/2073-4409/1/3/325 Skeletal muscles are the agent of motion and one of the most important tissues responsible for the control of metabolism. The maintenance of muscle homeostasis is finely regulated by the balance between catabolic and anabolic process. Macroautophagy (or autophagy) is a catabolic process that provides the degradation of protein aggregation and damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagy flux is fundamental for the homeostasis of skeletal muscles during physiological situations and in response to stress. Defective as well as excessive autophagy is harmful for muscle health and has a pathogenic role in several forms of muscle diseases. This review will focus on the role of autophagy in muscle homeostasis and diseases. Cells 2012-07-26 1 3 Review 10.3390/cells1030325 325 345 2073-4409 2012-07-26 doi: 10.3390/cells1030325 Paolo Grumati Paolo Bonaldo http://www.mdpi.com/2073-4409/1/3/313 Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments. Cells 2012-07-25 1 3 Article 10.3390/cells1030313 313 324 2073-4409 2012-07-25 doi: 10.3390/cells1030313 Hari Ramachandran Jessica Laux Ioana Moldovan Richard Caspell Paul V. Lehmann Ramu A. Subbramanian http://www.mdpi.com/2073-4409/1/3/284 Autophagy is an intracellular degradation process responsible for the delivery of cellular material to the lysosomes. One of the key mechanisms for control of autophagy is the modulation of the interaction between the autophagic protein Beclin 1 and the members of the anti-apoptotic Bcl-2 family (e.g., Bcl-2, Bcl-XL and Mcl-1). This binding is regulated by a variety of proteins and compounds that are able to enhance or inhibit the Bcl-2/Beclin 1 interaction in order to repress or activate autophagy, respectively. In this review we will focus on this interaction and discuss its characteristics, relevance and regulation. Cells 2012-07-06 1 3 Review 10.3390/cells1030284 284 312 2073-4409 2012-07-06 doi: 10.3390/cells1030284 Jean-Paul Decuypere Jan B. Parys Geert Bultynck http://www.mdpi.com/2073-4409/1/3/263 The budding yeast Saccharomyces cerevisiae responds to nutritional stress through the regulated activities of signaling pathways mediating autophagy and other conserved cellular processes. Autophagy has been studied intensely in yeast, where over 30 autophagy-related genes have been identified with defined roles enabling the formation of autophagic vesicles and their subsequent trafficking to the central yeast vacuole. Much less, however, is known regarding the regulatory mechanisms through which autophagy is integrated with other yeast stress responses. Nitrogen limitation initiates autophagy and pseudohyphal growth in yeast, the latter being a fascinating stress response characterized by the formation of multicellular chains or filaments of elongated cells. An increasing body of evidence suggests an interrelationship between processes responsive to nitrogen stress with cAMP-dependent PKA and the TOR kinase complex acting as key regulators of autophagy, pseudohyphal growth, and endocytosis. In this review, we will summarize our current understanding of the regulatory events controlling these processes. In particular, we explore the interplay between autophagy, polarized pseudohyphal growth, and to a lesser extent endocytosis, and posit that the integrated response of these processes in yeast is a critical point for further laboratory experimentation as a model of cellular responses to nitrogen limitation throughout the Eukaryota. Cells 2012-07-04 1 3 Review 10.3390/cells1030263 263 283 2073-4409 2012-07-04 doi: 10.3390/cells1030263 Qingxuan Song Anuj Kumar http://www.mdpi.com/2073-4409/1/3/248 Autophagy is a process of cellular self-degradation and is a major pathway for elimination of cytoplasmic material by the lysosomes. Autophagy is responsible for the degradation of damaged organelles and protein aggregates and therefore plays a significant role in cellular homeostasis. Despite the initial belief that autophagy is a nonselective bulk process, there is growing evidence during the last years that sequestration and degradation of cellular material by autophagy can be accomplished in a selective and specific manner. Given the role of autophagy and selective autophagy in several disease related processes such as tumorigenesis, neurodegeneration and infections, it is very important to dissect the molecular mechanisms of selective autophagy, in the context of the system and the organism. An excellent genetically tractable model organism to study autophagy is Drosophila, which appears to have a highly conserved autophagic machinery compared with mammals. However, the mechanisms of selective autophagy in Drosophila have been largely unexplored. The aim of this review is to summarize recent discoveries about the selectivity of autophagy in Drosophila. Cells 2012-06-29 1 3 Review 10.3390/cells1030248 248 262 2073-4409 2012-06-29 doi: 10.3390/cells1030248 Ioannis P. Nezis http://www.mdpi.com/2073-4409/1/3/204 Autophagy is an intracellular degradation pathway for long-lived proteins and organelles. This process is activated above basal levels upon cell intrinsic or environmental stress and dysregulation of autophagy has been linked to various human diseases, including those caused by viral infection. Many viruses have evolved strategies to directly interfere with autophagy, presumably to facilitate their replication or to escape immune detection. However, in some cases, modulation of autophagy appears to be a consequence of the virus disturbing the cell’s metabolic signaling networks. Here, we summarize recent advances in research at the interface of autophagy and viral infection, paying special attention to strategies that human tumor viruses have evolved. Cells 2012-06-25 1 3 Review 10.3390/cells1030204 204 247 2073-4409 2012-06-25 doi: 10.3390/cells1030204 Hildegard I. D. Mack Karl Munger http://www.mdpi.com/2073-4409/1/2/168 Macroautophagy has important physiological roles and its cytoprotective or detrimental function is compromised in various diseases such as many cancers and metabolic diseases. However, the importance of autophagy for cell responses has also been demonstrated in many other physiological and pathological situations. In this review, we discuss some of the recently discovered mechanisms involved in specific and unspecific autophagy related to mitochondrial dysfunction and organelle degradation, lipid metabolism and lipophagy as well as recent findings and evidence that link autophagy to unconventional protein secretion. Cells 2012-06-20 1 2 Review 10.3390/cells1020168 168 203 2073-4409 2012-06-20 doi: 10.3390/cells1020168 Stéphane Demine Sébastien Michel Kayleen Vannuvel Anaïs Wanet Patricia Renard Thierry Arnould http://www.mdpi.com/2073-4409/1/2/153 The aim of this prospective study was to investigate the diagnostic performance of Borrelia (Bb)-induced interferon (IFN)-γ secretion detected by ELISPOT modified to be feasible for clinical laboratories as a supplementary test to the laboratory diagnosis of Lyme neuroborreliosis (LNB) in an endemic setting. Between 2002 and 2004, patients with symptoms of suspected clinical LNB were included in a study conducted on the Åland islands in the Finnish archipelago, which is a hyper-endemic area for Lyme borreliosis (LB). Fourteen patients with confirmed LNB and 103 patients with non-LNB were included, and the numbers of spontaneous and Bb-induced IFN-γ-secreting cells were assayed by the ELISPOT test. The ELISPOT assay showed a weak diagnostic performance with a sensitivity of 36% and a specificity of 82%. The findings in this study show that this ELISPOT-assay modified to be feasible in clinical routine laboratories is not useful as a supplementary diagnostic tool in the laboratory diagnosis of patients with clinically suspected LNB. Cells 2012-06-08 1 2 Article 10.3390/cells1020153 153 167 2073-4409 2012-06-08 doi: 10.3390/cells1020153 Marika Nordberg Pia Forsberg Dag Nyman Barbro H. Skogman Clara Nyberg Jan Ernerudh Ingvar Eliasson Christina Ekerfelt http://www.mdpi.com/2073-4409/1/2/141 In order to provide better therapy we strive to increase our knowledge of how the immune system behaves and communicates in common pediatric immunological diseases, such as type 1 diabetes, allergic and celiac diseases. However, when dealing with pediatric diseases, where study subjects are almost exclusively children, blood volumes available for immunological studies are limited and as such must be carefully handled and used to their full extent. Single immune markers can easily be detected by a traditional Enzyme Linked Immunosorbent Assay (ELISA), whereas multiple markers can be detected by a fluorochrome (Luminex) or electrochemiluminescence (MSD) technique. These techniques however are sometimes not sensitive enough to detect low levels of secreted immune markers in limited sample sizes. To detect immune markers at the single-cell level, an Enzyme Linked Immuno-spot (ELISPOT) can be used to pin-point elusive immune markers in common pediatric immunological diseases. Cells 2012-05-29 1 2 Review 10.3390/cells1020141 141 152 2073-4409 2012-05-29 doi: 10.3390/cells1020141 Maria Faresjö http://www.mdpi.com/2073-4409/1/2/127 Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-g secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production) did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection. Cells 2012-05-10 1 2 Article 10.3390/cells1020127 127 140 2073-4409 2012-05-10 doi: 10.3390/cells1020127 Donald D. Anthony Kimberly A. Milkovich Wenji Zhang Benigno Rodriguez Nicole L. Yonkers Magdalena Tary-Lehmann Paul V. Lehmann http://www.mdpi.com/2073-4409/1/2/111 The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL) frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT) have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials. Cells 2012-05-10 1 2 Review 10.3390/cells1020111 111 126 2073-4409 2012-05-10 doi: 10.3390/cells1020111 Anatoli M. Malyguine Susan Strobl Kimberly Dunham Michael R. Shurin Thomas J. Sayers http://www.mdpi.com/2073-4409/1/2/100 Immunologic risk in kidney transplantation is typically minimized by avoiding, or at least limiting, the potential of donor specific humoral responses by testing for the presence of donor-specific antibodies (DSA). Additionally, selecting donor and recipient pairs with the least number of human leukocyte antigen (HLA) mismatches has been shown to play a role in transplant outcome. However, numerous other factors may play a role in the success of transplant outcome and patient health. Specifically, the use of T-cell allospecific ELISPOT assays have helped elucidate the role of pre-formed cellular responses as additional factors in post-transplant outcome. In this review, we will evaluate numerous uses of ELISPOT assays to assess the pre- and post-transplant immunologic risk of rejection episodes, graft survival and even viral susceptibility as well as the utility of ELISPOT assays in monitoring tolerance and withdrawal of immunosuppressive medications following kidney transplantation. Cells 2012-05-10 1 2 Review 10.3390/cells1020100 100 110 2073-4409 2012-05-10 doi: 10.3390/cells1020100 Jennifer R. Zitzner Anat R. Tambur http://www.mdpi.com/2073-4409/1/2/89 Mycobacterium tuberculosis (MTB) is a global heath epidemic, its threat amplified by HIV infection and the emergence of multidrug-resistant tuberculosis (MDR-TB). Interferon (IFN)-gamma release assays (IGRAs) have improved the accuracy of detection of MTB exposure in some subject groups as compared to the Tuberculin Skin Test (TST). However, as IFN-gamma is produced by both fully rested and more recently activated populations of memory T cells, it is not surprising that the measurement of this cytokine alone cannot accurately distinguish Latent TB Infected (LTBI) subjects from those with active (infectious) disease. Accurate and rapid diagnosis of infectious individuals would allow medication to be properly allocated and other actions taken to more effectively curtail MTB spread. Analysis of multi-cytokine profiles ex vivo after stimulation of PBMCs from LTBI and active MTB subjects indicate the real possibility of successfully discerning these two disease states within 24 hours of a subject’s blood draw. Due to the unparalleled sensitivity, low cost, and ease of use of Elispot assays, we propose that via a multiplex Elispot platform the accurate distinction of LTBI from active MTB-infected individuals is within reach. Cells 2012-05-07 1 2 Review 10.3390/cells1020089 89 99 2073-4409 2012-05-07 doi: 10.3390/cells1020089 Camilla Tincati Amedeo J. Cappione III Jennifer E. Snyder-Cappione http://www.mdpi.com/2073-4409/1/2/74 Immunohistochemistry (IHC) for detecting key signal molecules involved in programmed cell death (PCD) in archival human pathology specimens is fairly well established. Detection of cleaved caspase-3 in lymphocytes in rheumatoid arthritis (RA) and gastric surface foveolar glandular epithelia but not in synoviocytes in RA, gastric fundic glandular epithelia, or nasal NK/T-cell lymphoma (NKTCL) cells suggests anti-apoptotic mechanisms in cell differentiation and in oncogenesis such as the induction of survivin. Enzymatically pretreated and ultra-super sensitive detection of beclin-1 in synoviocytes in RA and gastric fundic glandular epithelia suggests enhanced autophagy. The deposition of beclin-1 in fibrinoid necrosis in RA and expression of beclin-1 in detached gastric fundic glandular cells suggest that enhanced autophagy undergoes autophagic cell death (ACD). NKTCL exhibited enhanced autophagy through LC3 labeling and showed densely LC3 labeled cell-debris in regions of peculiar necrosis without deposition of beclin-1, indicating massive ACD in NKTCL and the alternative pathway enhancing autophagy following autophagic vesicle nucleation. Autophagy progression was monitored by labeling aggregated mitochondria and cathepsin D. The cell-debris in massive ACD in NKTCL were positive for 8-hydroxydeoxyguanosine, suggesting DNA oxidation occurred in ACD. Immunohistochemical autophagy and PCD analysis in archival human pathology specimens may offer new insights into autophagy in humans. Cells 2012-05-07 1 2 Review 10.3390/cells1020074 74 88 2073-4409 2012-05-07 doi: 10.3390/cells1020074 Kazuhisa Hasui Taku Nagai Jia Wang Xinshan Jia Katsuyuki Aozasa Shuji Izumo Yoshifumi Kawano Takuro Kanekura Yoshito Eizuru Takami Matsuyama http://www.mdpi.com/2073-4409/1/2/61 Acute graft-versus-host disease (aGVHD) remains a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Infections may coexist and in certain circumstances aggravate aGVHD. It was described that type 1 as well as type 2 cytokines are important mediators of aGVHD. We measured spot-forming cells (SFCs) for interferon (IFN)-γ, interleukin (IL)-4, IL-10, and IL-17 in unstimulated peripheral blood from 80 patients with hematological disorders who underwent allogeneic hematopoietic stem cell transplantation by using the enzyme-linked immunospot (ELISPOT) assay that reflects the ongoing in vivo immune status. A serial monitoring showed that both type 1 and type 2 cytokine SFCs were correlated with aGVHD activity. The numbers of IFN-γ and IL-4 SFCs in patients with grade II-IV aGVHD were significantly higher than those in patients with grade 0 and/or I aGVHD. Elevation of IFN-γ and IL-4 SFCs was significantly correlated with the severity of aGVHD, but not with infection itself, e.g., cytomegalovirus infection. Cytokine SFCs are clinically relevant biomarkers for the diagnostic and therapeutic evaluation of aGVHD and concurrent infection. Cells 2012-04-30 1 2 Article 10.3390/cells1020061 61 73 2073-4409 2012-04-30 doi: 10.3390/cells1020061 Masahiro Hirayama Eiichi Azuma Yoshihiro Komada http://www.mdpi.com/2073-4409/1/2/51 In recent years we and others have used the ELISPOT assay successfully to identify novel tumor antigens by the characterization of spontaneous HLA class I restricted immune responses against a number of minimal 9–10 amino acid long peptide epitopes. In the present study, we examined the capability of using longer peptides when scrutinizing Peripheral Blood Mononuclear Cells (PMBC) from melanoma patients for spontaneous immunity by means of ELISPOT IFN-γ secretion assay. To this end, we examined PBMC for the presence of specific T-cell responses against long peptides derived from the tumor associated antigen BCL-X(L). The protein product of the larger BCL-X(L) differs from Bcl-X(S) protein by an inserted region (amino acids 126–188). Thus, we scrutinized eight long peptides covering this inserted region for spontaneous immunity. The peptides were overlapping and consisted of 20–23 amino acids. PBMC were pre-stimulated with peptide-pulsed autologous dendritic cells (DC) and subjected to the IFN-γ ELISPOT assay. Four of the BCL-X(L) derived peptides elicited very frequent responses in several patients. Additionally, in all patients responses against more than one of the peptides could be detected. In conclusion several long BCL-X(L) derived peptide epitopes exist, which may be used in anti-cancer immunity. Furthermore, the ELISPOT assay offers an attractive and sensitive method for the characterization of spontaneous immune reactivity against long peptides. Cells 2012-04-26 1 2 Article 10.3390/cells1020051 51 60 2073-4409 2012-04-26 doi: 10.3390/cells1020051 Stine Kiaer Larsen Morten Hansen Inge Marie Svane Per Thor Straten Mads Hald Andersen http://www.mdpi.com/2073-4409/1/2/35 The role of cytomegalovirus (CMV) infection in the pathogenesis and exacerbation of Inflammatory Bowel Disease (IBD) has been unresolved. Typically, the CMV genome remains dormant in infected cells, but a breakdown of immune surveillance can lead to re-activation of viral replication in the gut mucosa, which is not necessarily associated with viremia or changes in antibody titers. We hypothesized that the detection of CMV-specific CD8 effector T cells should permit the distinction between dormant and active CMV infection. As CD8 effector T cells, unlike memory CD8 T cells, have perforin (PFN) and granzyme B (GzB) preformed in their cytoplasmic granules, we employed single cell resolution ELISPOT assays to measure the CMV antigen-triggered release of these molecules by CD8 T cells isolated from subjects with IBD, and age-matched healthy controls. The frequencies of CMV-specific (GzB) and PFN-producing CD8 T cells were increased in IBD patients compared to healthy controls. Furthermore, the increased CMV reactivity was associated with active IBD disease and with longer disease duration. Notably, PCR on serum frequently failed to detect CMV DNA during flares. The data show that during active IBD there is a flare of CD8 T cell activity against CMV in a substantial proportion of IBD patients, suggesting CMV reactivation that serum PCR does not detect. While it remains open whether CMV reactivation is a cause or consequence of IBD, our data suggest that monitoring CMV antigen-specific effector CD8 T cells with GzB and PFN ELISPOT analysis can provide novel insights into the role of CMV infection in IBD. Additionally, our data have implications for the fields of transplantation, HIV, cancer, and autoimmune diseases, in all of which patient care critically depends on sensitive and reliable detection of a reactivation of CMV infection. Cells 2012-04-23 1 2 Article 10.3390/cells1020035 35 50 2073-4409 2012-04-23 doi: 10.3390/cells1020035 Tobias M. Nowacki Dominik Bettenworth Matthias Ross Jan Heidemann Paul V. Lehmann Andreas Lügering http://www.mdpi.com/2073-4409/1/2/27 We have evaluated a novel B-cell FluoroSpot assay for the analysis of antibody responses in healthy individuals vaccinated intramuscularly with Influenza A (H1N1) antigen (Pandemrix®, GlaxoSmithKline). Using the FluoroSpot assay and an ELISpot assay run in parallel for comparison, we measured the frequency of cells secreting antigen-specific as well as total IgG or IgA antibodies seven days post vaccination. The assays were based on high affinity monoclonal antibodies for capture and detection of human IgG and IgA. Whereas conventional ELISpot analyzes IgG- and IgA-secreting B cells separately, fluorescent detection enabled simultaneous enumeration of B cells secreting IgG or IgA in the same well. The FluoroSpot protocol was also simpler as the assay could be performed without the need for an amplifying detection step. While having all the advantages of a conventional ELISpot assay, including high sensitivity, robustness and ease of performance, the FluoroSpot assay adds further value in reducing costs, time and material. Cells 2012-04-20 1 2 Article 10.3390/cells1020027 27 34 2073-4409 2012-04-20 doi: 10.3390/cells1020027 Gun Kesa Per H. Larsson Niklas Ahlborg Bernt Axelsson http://www.mdpi.com/2073-4409/1/1/15 The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities have so far prevented their wide utilization as well as further expansion of their multicolor capability. An attractive solution is to replace the chromogenic reaction of the traditional ELISpot assay with a fluorescent detection system (fluorospot assay). Fluorospot assays using fluorophore-conjugated secondary antibodies in conjunction with fluorescence enhancers, FITC/anti-FITC and biotin/avidin amplification systems and dedicated equipment for spot detection have been developed to enumerate T-cells secreting two or three specific cytokines and, more recently, IgG and IgA antibody-secreting cells (ASCs). We hereby report a method for a multiplex B cell fluorospot assay that utilizes quantum-dot nanocrystals as reporters without further amplification systems or need of dedicated equipment. With this method we simultaneously enumerated HIV-1 gp41 envelope glycoprotein-specific IgG and IgM antibody-secreting cells with sensitivity comparable to that of the traditional ELISpot assay. Cells 2012-03-21 1 1 Article 10.3390/cells1010015 15 26 2073-4409 2012-03-21 doi: 10.3390/cells1010015 Mattia Bonsignori M. Anthony Moody http://www.mdpi.com/2073-4409/1/1/5 Tuberculosis is a global health problem. The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine has variable efficacy (0–80%) so there is a drive to develop novel vaccines. The cytokine, interferon gamma (IFNγ), is an essential component of the protective response to M. tuberculosis (M. tb) infection and is also produced in response to BCG vaccination. Induction of an IFNγ response is used as a biomarker of successful vaccination in the assessment of new tuberculosis (TB) vaccines. The IFNγ ELISPOT assay provides an important tool for TB research. It is used for both the diagnosis of infection (T.Spot assay), and for the evaluation of the immunogenicity of new TB vaccine candidates in human clinical trials, in the non-human primate (NHP) model of TB infection studies. The ELISPOT assay captures IFNγ produced by peripheral blood mononuclear cells (PBMCs) following specific stimulation, onto a membrane so individual cells can be enumerated and the frequency of responding cells determined. Hence spot forming units (SFU) per 106 cells provide the traditional measure for ELISPOT assays. The discriminatory power of SFU is limited. In some situations, the number of SFU in BCG vaccinated, and unvaccinated, subjects was found to be similar, although the spots were observed to be larger in vaccinated subjects. Spot size potentially provides a measure of the quantity of cytokine produced by individual cells. The AID ELISPOT plate reader software used to determine frequency of spots also has the capability to determine the size of each spot. Consideration of spot size in combination with spot forming units was investigated in our studies of BCG immunogenicity. This additional readout was found to enhance the discriminatory power of the ELISPOT assay, and provide more information on the immune response to BCG vaccination and infection with M.tb. Cells 2012-03-13 1 1 Article 10.3390/cells1010005 5 14 2073-4409 2012-03-13 doi: 10.3390/cells1010005 Laura S. Sibley Andrew D. White Alice Marriott Michael J. Dennis Ann Williams Philip D. Marsh Sally A. Sharpe http://www.mdpi.com/2073-4409/1/1/3 I am honored to introduce the new journal Cells, which has been created to serve as a hub for disseminating new findings and discoveries in cell biology to researchers worldwide. Cells is an international, peer-reviewed, open-access journal on cell biology, molecular biology, and biophysics. Much has been accomplished in molecular and cell biology in the past thirty years. We have unraveled the structure of many proteins, nucleic acids and lipids, and learned much about cell structures, including lysosomes, mitochondria, cytoskeletal elements, cell membranes, endoplasmic reticulum, and nuclei. Now, it is time to apply this knowledge to answering the age-old question: “How do cells work?” It appears there is a strong demand for a journal focused on various functional aspects of cell biology, including such fundamental ones as gametogenesis, embryonic development, tissue regeneration, tumorigenesis, and aging. [...] Cells 2011-09-21 1 1 Editorial 10.3390/cells1010003 3 4 2073-4409 2011-09-21 doi: 10.3390/cells1010003 Alexander E. Kalyuzhny http://www.mdpi.com/2073-4409/1/1/1 To expand the open access publishing project of our newly founded company MDPI [1,2] based in Basel, Switzerland, we are in the process of launching new journals. Based on our success in running journals that represent key areas in science and technology, such as Molecules [3], Sensors [4], Energies [5], Viruses [6], Pharmaceuticals [7], Cancers [8] and Toxins [9], we are launching a new journal entitled Cells. It is an open access journal combining cell biology, molecular biology and biophysics, toward an understanding of cell structure, function and interactions. [...] Cells 2011-01-03 1 1 Editorial 10.3390/cells1010001 1 2 2073-4409 2011-01-03 doi: 10.3390/cells1010001 Shu-Kun Lin