Published by MDPI AG
Online ISSN: 2073-4409
Sequence information and predicted IC50 (nM) binding values for ICP27, VP22 and VP13/14 selected peptides.
HLA-A2 stabilization by exogenously loaded peptides on T2 cells. T 2 cells were plated out in a 96 well plate. Peptide (50 μg/mL) was added and the cells were left to incubate overnight. (A) Stabilized expression of HLA-A2 was detected using a FITC conjugated BB7.2 antibody. The HLA-A2 restricted Flu matrix M1 (58-66) (GILGFVFTL) 
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.
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.
The involvement of T-helper (Th)1, Th17 and Th22 cell subsets, in immunity, as well as in pathological inflammatory reactions, makes it important to determine their relative proportion. A triple FluoroSpot detecting the hallmark cytokines of Th1 (IFN-γ), Th17 (IL-17A) and Th22 (IL-22) was developed and evaluated using human peripheral blood mononuclear cells from healthy donors incubated with tetanus toxoid, Candida albicans extract, mycobacterial purified protein derivative or medium only. Antigen stimulation yielded mainly cells secreting IFN-γ, IL-17A or IL-22 alone but lower proportions of double-secreting cells were also found; triple-secreting cells were rare. The response to C. albicans contrasted in that higher proportions of IL-17A single secreting as well as co-secreting cells, in particular IL-17A/IL-22, were found. The FluoroSpot analysis correlated well with single cytokine ELISpot assays ran in parallel and the methods displayed a comparable sensitivity. The results demonstrate the functionality of the FluoroSpot assay for simultaneous analysis of distinct Th1, Th17, Th22 as well as intermediate cell populations. The method provides a mean for a simple and rapid analysis of the involvement of these cells in immunity and disease.
Identical IFN- γ spot sizes and size distributions in the 96- and 384-well format plates. ( A , B ) Respective images from a 96-well and a 384-well plate were captured with identical optical and digital resolution; ( C , D ) The spot sizes from both wells are plotted as a histogram with a fitted Log Normal curve overlaid in red. 
PBMC were stained with fluorescent Calcein AM dye and the specified numbers of cells were plated into respective wells of a 96-well plate. The cells were visualized on an ImmunoSpot ® S6 Ultimate Reader by using 480 nm as the excitation wavelength, and detecting fluorescence with a 520 ± 20 nm emission wavelength filter.
T cell functional affinity measurements in 384- and 96-well formats provide identical results. HCMV peptide pp65-induced IFN- γ production by CD8+ cells was measured in PBMC in both 96-well and 384-well plate types. The highest peptide concentration tested was 10 μ g/mL with subsequent 1:10 serial dilutions. The numbers of PBMC plated in the 96-well format was 3 × 10 5 /well (green line), and 1 × 10 5 /well for the 
The variability in spot counts among replicate wells is higher in 384-well plates than in 96-well plates when low frequency T cells are being detected. PBMC from three donors were serially diluted and tested, in quadruplicate wells for each donor and cell dilution, for antigen-elicited IFN- γ response. The PBMC were plated in serial dilution between 1.0 × 10 6 and 1.0 × 10 5 cells per well in the 96-well plate, and at 3 × 10 5 and 
Spot counts in 96-well format were approximately three times the spot count in 384-well plate format. For 11 test subjects, the HCMVpp65-induced response was measured in triplicate wells for each plate format with the mean of the triplicate spot counts shown. The ratio between these mean spot counts in 96-well format and 384-well format is shown in the right column.
Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor immunity, it is also advisable to account for determinant spreading by testing multiple epitopes. Efforts for comprehensive immune monitoring would require substantial numbers of PBMC to run the above tests systematically, which in most test cases is limiting. Immune monitoring with ELISPOT assays have been performed, thus far, in a 96-well format. In this study we show that one can increase cell utilization by performing the assay in 384-well plates whose membrane surface area is one third that of 96-well plates. Systematic testing of PBMC for antigen-specific T cell response in the two formats demonstrated that the 384-well assay corresponds to a one-in-three miniaturization of the 96-well assay. The lowest number of cells that can be used in the 384-well format, while allowing for sufficient contact with APC, is 33,000 PBMC/well. Therefore, with one million PBMC typically obtained from 1 mL of blood, a 30 well T cell ELISPOT assay can be performed in a 384-well format.
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.
The two pathways of apoptosis. There are two major pathways of apoptosis in mammalian cells, the intrinsic and extrinsic pathways. The intrinsic pathway is activated by cellular stresses (such as high glucose concentrations or growth factor deprivation) and results in activation of the BH3-only members of the Bcl-2 family (such as Bim and Puma) that initiate apoptosis signaling by binding to the Bcl-2-like pro-survival proteins (including Bcl-2 and Bcl-xL) and release of Bax and/or Bak to promote loss of mitochondrial outer membrane potential, cytochrome c release and activation of caspase-9, caspase-3 and apoptosis. The extrinsic pathway is initiated by activation of death receptors, such as Fas, that have an intracellular death domain. This results in formation of a death-inducing signaling complex in which the initiator caspase, caspase-8 is activated by its adaptor FAS-associated death domain (FADD). This results in activation of the caspase cascade and apoptosis. The BH3-only protein Bid is essential for death receptor-mediated apoptosis in beta cells, thereby providing cross-talk between the two apoptotic pathways.
Apoptotic pathways induced in beta cells in type 2 diabetes. Islet amyloid polypeptide, glucose and palmitate have been proposed to induce cellular stress through activation of endoplasmic reticulum (ER) stress, oxidative stress or NLRP3 inflammasome activation and IL-1β production. These stresses induce the intrinsic apoptosis pathway through activation of Bcl-2 family molecules. Islet amyloid poly-peptide (IAPP) has also been shown to directly activate the extrinsic apoptosis pathway.
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.
As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.
Fluorescence imaging of human cell lines used to study actin cytoskeleton function. The actin network (red) is labeled with fluorescently-conjugated phalloidin, and the nuclei (blue) are labeled with Hoechst 33342. Actin is seen to have a very complex localization pattern that differs across the various cell lines. In some cells it is highly concentrated close to the plasma membrane and in filopodial protrusions, in other cells stress fibers can be seen, and in some instances small punctate actin accumulations are present. The figure also shows the differing ratios of cytoplasm-to-nucleus area that exist across different cell lines, thereby highlighting the importance of making the most suitable choice of cell for actin imaging experiments. 
Schematic representation of how various imaging modalities compare in terms of spatial resolution, temporal resolution, and sample number; note that the temporal resolution of HCS is largely determined by the number of samples analyzed in parallel. 
Examples of imaging techniques used to visualize and analyze the cytoskeleton and focal adhesions in cells. 
The cytoskeleton plays several fundamental roles in the cell, including organizing the spatial arrangement of subcellular organelles, regulating cell dynamics and motility, providing a platform for interaction with neighboring cells, and ultimately defining overall cell shape. Fluorescence imaging has proved to be vital in furthering our understanding of the cytoskeleton, and is now a mainstay technique used widely by cell biologists. In this review we provide an introduction to various imaging modalities used to study focal adhesions and the actin cytoskeleton, and using specific examples we highlight a number of recent studies in animal cells that have advanced our knowledge of cytoskeletal behavior.
Bone marrow-derived mesenchymal stromal cells (BM-MSCs) stimulate proliferation of muscle progenitor cells. Representative superimposed differential interference contrast (DIC) and confocal fluorescence images showing the pyrimidine analogue EdU (5-ethynyl-2'-deoxyuridine) staining (green) in the nuclei of proliferating murine C2C12 skeletal myoblasts or murine neonatal cardiomyocytes in single and co-culture for 24 h with mouse Dil-labeled BM-MSCs (red). Note the higher number of proliferating cells in the co-culture systems. 
Clinical trials using BM-MSC therapy for cardiac repair/regeneration. 
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.
Glucocorticoid Receptor (GR) Gene Structure.  
Tissue-specific effects of glucocorticoids on apoptosis in the human body.  
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.
(a) Analysis of Influenza A (H1N1)-specific IgG- and IgA-secreting cells in ELISpot and in FluoroSpot. PBMC were collected before and seven days after vaccination with the influenza vaccine Pandemrix®. Ag-specific IgG- and IgA-ASC were detected using 0.75 μg/well of coated Ag. After overnight culturing of PBMC (250,000 cells/well), detection mAbs specific for IgG and IgA, and labeled with different fluorophores, were mixed together and added to the wells (FluoroSpot). In ELISpot, ASC were detected by adding biotin-labeled anti-IgG or anti-IgA detection mAbs followed by SA-ALP and a precipitating substrate. The figure shows representative examples of the vaccine-induced responses in donor 1; (b) Analysis of Influenza A (H1N1)-specific IgG- and IgA-secreting B cells in ELISpot and in FluoroSpot in six donors (ASC/ 250,000 PBMC). No Ag-specific ASC were seen before vaccination. Grey bars represent ELISpot whereas colored bars represent FluoroSpot. The diagram shows mean values with SD (n = 3). Each set of bars corresponds to data from donor 1–6 in numerical order from left to right.
(a) Analysis of the total number of B cells secreting IgG and IgA after vaccination with Pandemrix®. The wells were coated with mAbs specific for IgG and/or IgA. After an overnight culture of PBMC (100,000 cells/well) the detection procedure was identical to that used for detection of Ag-specific ASC in Figure 1a. The images are representative examples from donor 1. (b) ELISpot and FluoroSpot analysis of the frequency of B cells secreting IgG and IgA before and after vaccination with Pandemrix® (ASC/100,000 PBMC). The diagram shows mean values with SD (n = 3). Each set of bars corresponds to data from donor 1–6 in numerical order from left to right.
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.
Induction of AVD followed by persistent shrinkage (top), activation of caspase-3 (middle) and induction of cell death (bottom) by treatment with anti-Fas antibody (anti-Fas), as well as their prevention by simultaneous treatment with a Cl г channel blocker 
Induction of AVD (top) in both WEHI-231 ( A ) and WEHI-231/Bcl-2 ( B ) cells, as well as activation of caspase-3 (middle) and induction of DNA fragmentation (bottom) in WEHI-231 (A), but not in WEHI-231/Bcl-2 (B) cells treated with STS. The mean cell volume of STS-treated cells was normalized to that of non-treated (control) cells. Each data point represents the mean ± SEM (vertical bar) of 16 observations. * p < 0.05 vs. corresponding control. 
Induction of AVD followed by persistent shrinkage (top), activation of caspase-8 (middle) and activation of caspase-9 (bottom) in HeLa cells treated with anti-Fas antibody (anti-Fas) ( A ) and STS ( B ) in the absence (filled circles), but not in the presence (other filled symbols), of a channel blocker. The Cl г channel blocker was 0.5 mM DIDS, 0.5 mM 
Effects of pan-caspase inhibitors, zVAD- IPN ȝ0 DQG]' - GFE ȝ0  , on the AVD induction (top) and caspase-3 activation (bottom) in HeLa cells treated with anti-Fas antibody (anti-Fas) ( A ) and STS ( B ). The mean cell volume of anti-Fas- or STS-treated cells in the absence (filled circles) and presence (filled triangles) of a pan-caspase blocker was normalized to that of non-treated (control: open squares) cells. Each data point represents the mean ± SEM (vertical bar) of 10 observations. * p < 0.05 vs. corresponding control. It is noted that either pan-caspase blocker abolished caspase-3 activation, but not AVD induction in both anti-Fas- and STS-treated cells, and also that the late- SKDVH FHOO VKULQNDJH REVHUYHG DW K DIWHU VWLPXODWLRQ ZLWK DQWL -Fas or STS was significantly ( p < 0.05) suppressed by pan-caspase blockers. 
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.
Schematic structure of the intracellular truncated isoforms of VEGFR-1. Amino acid numbers correspond to the full length transmembrane receptor. JM, juxtamembrane domain; TK1, kinase domain, ATP binding; KI, Kinase insert; TK2, kinase domain, phosphotransferase; CT, C-terminal tail region. 
Schematic representation of Src in the low activity state (left) and the active state (right). In the low activity configuration, the SH2 domain binds the phosphorylated C-terminal Tyr 530 , while the SH3 domain interacts with the linker domain, promoting a relative "closed" conformation. In the active configuration, SH2 and SH3 domains are released from the intramolecular interactions with autophosphorylation of Tyr 419 , which enhances the catalytic activity of Src. Activation of Src is mediated by activation of transmembrane tyrosine receptors (RTK) upon binding the corresponding ligands or, alternatively, by the C-terminal intracellular truncated isoforms.
Src signaling pathways and function. Binding of ligands to the corresponding transmembrane tyrosine kinase receptors (RTK) or intracellular truncated C-terminal isoforms of RTKs can activate Src. Activation of Src is involved in different signaling pathways. Particularly important are the disruption of adherens junctions stabilized by E-cadherin and the disruption of focal adhesions, which promotes migration, invasion and metastasis. 
Expression of i21VEGFR-1 is positively regulated by the Notch pathway and negatively regulated by the micro RNA family miR-200. Addition of retinoic acid to the culture medium of MDA-MB-231 breast cancer cells inhibits the expression of the protein i21VEGFR-1. The effect of retinoic acid on the expression of the intracellular truncated isoform of VEGFR-1 is mediated by downregulation of Notch-3 expression. Moreover, retinoic acid increases the expression of the miR-200 family of micro RNAs in MDA-MB-231 breast cancer cells. The expression of the truncated intracellular protein i21VEGFR-1 decreases when the Notch signaling pathway is interfered with γ-secretase inhibitors.
One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.
Domain organization of RTKs. The following abbreviations are used: L, leucine-rich; CR, cysteine-rich; Ig, immunoglobulin-like; FnIII, fibronectin type III; ID, insert domain. The L1, CR1, L2 and CR2 domains of the ErbB family are alternatively termed Domains I-IV. The ErbB and Trk families are drawn as a monomer, but might be present as non-covalently formed dimers at the cell surface prior to ligand binding (see the main text). Not drawn to scale.
Schematic representations of the structures of the extracellular regions of the ErbB family. EGFR, ErbB3 and ErbB4 adopt the tethered conformation in the absence of ligand, while ErbB2 adopts an extended, or untethered, conformation that resembles the ligand-activated, dimerization-competent EGFR protomer in the ligand-bound form of the EGFR dimer, shown at the right. The 'dimerization arm' and 'tethering arm' are shown by an asterisk and an open triangle, respectively. Ligands are shown in red. Domains I–IV correspond to the domains shown in Figure 1. Not drawn to scale.  
Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights.
TA-65 and HTA treatment of CD4 T cells increases telomerase activity in response to primary (A) and secondary (B) cell stimulations. This figure shows representative results for cultures of purified CD4 T cells from a single donor that were exposed to either TA-65, HTA or DMSO (triplicate cultures for each condition). Top portion of Figure 1A,B illustrate the band products of telomerase activity indicated as TPG (TPG is total product generate, a measure of telomerase activity). Dark bands indicate high telomerase activity. The bottom portions of panels A and B represent quantization bar graph values for the enzyme activity. The average DMSO TPG was used to normalize telomerase activity between treatments. The total dilution of compounds in grams/mL is indicated above the lanes and below the bar graphs. Statistical significance was determined by student paired t test (p < 0.05).
Average telomerase activity for a primary stimulation for (A) CD4 (n = 6) and (B) CD8 (n = 6) T cells. TPG is total product generated for telomerase activity. DMSO treated samples were used to normalized the TPG between the donors, which allow direct donor’s variability comparisons.
TA-65 activates telomerase via MAPK pathway. CD8 T cells stimulated for a second time and treated with MAPK inhibitor (as described in materials and methods) blocked the increase telomerase activity from TA-65, but no significant inhibition was observed using AKT inhibitors. Statistical significance was determined by student paired t-test (p < 0.05).
Proliferation effects of TA-65 and HTA: Cell counts were determined on day 3 after stimulation. A, represents results for CD4 T cells and B represents results for CD8 T cells (concentrations are 106/mL). A and B top, represents actual cell concentration after first T cell stimulation, A and B bottom represents total cell concentration after second stimulation. Statistical significance was determined by student paired t test (p < 0.05).
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.
Kinetics of cytokine spot-forming cells (SFCs) in patients with acute graft-versus-host disease (aGVHD). Analysis of time course of the numbers of cytokine SFCs. Numbers of samples in IFN-at 3, 6, and 10 weeks after transplantation were 41, 41 and 35 on grade 0 aGVHD, 11, 12 and 9 on grade I, and 17, 15 and 15 on grade II-IV, respectively. Similarly, numbers of samples in IL-4 were 39, 40 and 33 on grade 0; 10, 11 and 8 on grade I; 16, 14 and 14 on grade II-IV. Numbers of samples in IL-10 were 25, 23 and 21 on grade 0; 5, 6 and 4 on grade I; 10, 9 and 10 on grade II-IV. Numbers of samples in IL-17 were 12, 10 and 9 on grade 0; 5, 5 and 3 on grade I; 4, 3 and 3 on grade II-IV. Data are expressed as means r SD. Each shaded area was determined from healthy donor PBMCs as the cut-off value (n = 35; mean + 2SD). 
Patient characteristics. 
IFN-and IL-4 SFCs indicate severity of aGVHD. The numbers of cytokine SFCs were analyzed according to the severity of aGVHD. Numbers of samples in IFN-were 151, 17, 13 and 15 on grade 0, I, II and III-IV, respectively. Similarly, numbers of samples in IL-4 were 141, 17, 13, and 15; in IL-10, 87, 10, 9 and 7; in IL-17, 38, 9, 4 and 3. 
IFN-and IL-4 SFCs were elevated in patients with aGVHD, irrespective of CMV infection. Two representative cases developed stage 2 diarrhea with positive antigenemia for CMV. One case (UPN#51) developed intestinal aGVHD, the other case (UPN#70) did not. Shaded area is shown as cut-off value for IFN-SFCs. 
Discrimination of aGVHD from CMV infection by IFN-and IL-4 SFCs. The numbers of IFN-and IL-4 SFCs in patients with grade 0 and I-IV aGVHD were analyzed by comparing with negative or positive CMV infection. Data are expressed as means r SD. 
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.
Regulation of cellular stress-induced cell death pathways by IAPs. Cellular stress likely activates the intrinsic pathway of cell death involving the release from the mitochondria, of pro-apoptotic factors including cytochrome c and Smac. Once in the cytoplasm, cytochrome c induces an ATP-dependent conformational change and oligomerization of APAF-1 (apoptotic peptidase activating factor-1) in the Apoptosome. APAF-1 then recruits and promotes the activation, through homodimerization, of the initiator caspase-9. Active caspase-9 is then stabilized by auto-processing and detached from apoptosome. DNA damage can also induce a depletion of cIAP1 and XIAP, allowing the formation of a RIP-1 containing platform named Ripoptosome. Ripoptosome can trigger either caspase-8 or -10 activation and apoptosis, or caspase-independent cell death referred to as Necroptosis. Initiator caspases-9, -8 or -10 induce the activating proteolytic processing of effector caspase-3 and/or -7 responsible for apoptotic cell death. IAPs can inhibit apoptotic pathways at several levels: 1) cIAPs and XIAP are potent inhibitors of Ripoptosome assembly; 2) XIAP can directly inhibit the activity of processed forms of caspase-9, -3 or -7; cIAPs;3) XIAP can induce the K48 ubiquitination and proteasome-mediated degradation of processed forms of caspase-9, -3 or -7; 4) NAIP is able to block the proteolytic processing of caspase-9 at the Apoptosome level; 5) cIAPs, ML-IAP and Apollon can bind to Smac, preventing it from neutralizing XIAP. SMs: Smac mimetics; UPS: ubiquitin-proteasome system. 
Regulation of TNFRI-signaling pathways by IAPs.TNF-R1 stimulation induces the recruitment to the receptor, of cIAPs and RIP1 via TRADD and TRAF2 into complex-I. cIAPs trigger K63 self-ubiquitination and K11 and K63 RIP1 polyubiquitination. Ub chains are recognized by Ub-binding domain of TAB2, IKKȖ and HOIP and connect TAB2/TAB3/TAK1, IKK complex and LUBAC, resulting to LUBAC-mediated linear ubiquitination of IKKȖ and TAK1-mediated phosphorylation of IKKȕ. Once activated, IKK complex triggers phosphorylation of IțB-Į, that is then degraded by UPS (ubiquitin proteasome system). Released NF-țB dimer translocates to the nucleus and promotes the transcription of target genes. Secondary cytoplasmic complex leading to cell death can be formed in absence of IAPs (complex-IIB) or when NF- N B signaling is defective (complex-IIA). 
Role of IAPs in DNA damage response. Genotoxic stress-induced DNA strand breaks are very quickly detected by sensor molecules that activate cell cycle checkpoints and DNA repair mechanisms. They also induce IRES-dependent up-regulation of XIAP and cIAP1. MDM2 is dephosphorylated and exported from the nucleus to the cytoplasm where it physically interacts and stimulates XIAP IRES. cIAP1 and XIAP are regulators of DNA damage-mediated NF-țB activating pathway. The sensor protein ATM is translocated from the nucleus to the cytoplasm where it interacts with TRAF6 and favors its Ubc13-mediated K63 ubiquitination. These Ub chains are a signal for the recruitment of cIAP1, TAB2/TAK1 complex and IKK complex. TAK1 induces the phosphylation of IKKȕ, cIAP1 mediates IKKȖ mono-ubiquitination while LUBAC induces its linear ubiquitination. All of these post-translational modifications are required for NF- N B activation. XIAP controls this process by regulating TAK1 activation and association with the IKK complex. An auto-ubiquitination and degradation of cIAPs and XIAP have also been described, allowing the assembly of Ripoptosome leading to caspase-dependent or independent cell death. UPS: ubiquitin-proteasome system. 
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.
S-shaped curve for compensatory and prioritized differentiation with a reverse S-shaped curve for stem cell accumulation. As the dose of hyperosmolar stress (sorbitol) increases, the stem cell accumulation rate decreases (C.) but the stress enzyme (SAPK) activity level (B) and differentiation marker (Hand 1) (A) level rise. Standard error of the mean was between 1%8% of the mean. Red bars in (A) and (C) show range of stress-induced differentiation. 
S-shaped curve for low dose stem cell survival and high dose organismal survival through compensatory differentiation. At low concentrations of Sorbitol, stem cell population size (D) and Inhibitor of differentiation (ID2) level (A) are not significantly changed but AMPK-dependent phosphorylation of ACC and inactivation of its anabolic activity do occur at low doses (C). At higher doses the accumulation rate decreases (D) and AMPK-dependent (B) decrease in Inhibitor of differentiation (ID2) protein level leads to induction of differentiation. Standard error of the mean was between 1%8% of the mean. Red bars in (A) and (D) show range of stress-induced differentiation. 
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.
Sources of intracellular ROS generation. Among Nox isoforms (Nox1 to Nox5 and Duox1 and Duox 2), Nox4 is the only isoform that primarily generates hydrogen peroxide (H 2 O 2 ) instead of superoxide (O 2 x ). Superoxide is converted to H 2 O 2 by 
Intracellular sources of ROS implicated in modulating adipocyte differentiation
Potential redox-sensitive pathways involved in regulation of adipogenesis. &(%3ȕ&&$$7 /enha QFHUELQGLQJSURWHLQȕ,*) -R, insulin-like growth factor receptor; PTP, protein tyrosine phosphatases; PGC- Į 33$5Ȗ FRDFWLYDWRU Į AMPK, AMP-activated protein kinase. 
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.
The spinal substantia gelatinosa (SG) plays a pivotal role in modulating nociceptive transmission through dorsal root ganglion (DRG) neurons from the periphery. TRP channels such as TRPV1 and TRPA1 channels expressed in the SG are involved in the regulation of the nociceptive transmission. On the other hand, the TRP channels located in the peripheral terminals of the DRG neurons are activated by nociceptive stimuli given to the periphery and also by plant-derived chemicals, which generates a membrane depolarization. The chemicals also activate the TRP channels in the SG. In this review, we introduce how synaptic transmissions in the SG neurons are affected by various plant-derived chemicals and suggest that the peripheral and central TRP channels may differ in property from each other.
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.
Detection by immunofluorescence of LC3 protein on whole-mount embryos of sea urchin. Equatorial optical sections captured by confocal laser scanning microscopy. In green, LC3 protein detection; in red, nuclei stained with propidium iodide. (A) Control embryo, after 18 h of growth; (B) Cd-treated embryo for 18 h; (C) Control embryo, after 24 h of growth; (D) Cd-treated embryo for 24 h; (E, F) Enlargements of a section of (B, D), respectively. Bar = 50 μm.
Diagram showing an overview of the most representative cellular, biochemical, and molecular events activated as defense strategies in embryos and larvae of P.
lividus at different times post fertilization and various concentrations of CdCl2.
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.
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.
Schematic showing a postulated mechanism for maturation stage ameloblast sensitivity to fluoride 
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.
Different techniques used during isolation of mesenchymal stem or stromal cells (MSC) from umbilical cord (UC) tissue. Basically enzymatic digestion or explant culture approaches are used. 
Primary UC-derived cell cultures can contain broad cell size distributions (I < II < III). 
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.
Regeneration in metazoans. 7KH LOOXVWUDWHG PHWDSKRU RI WKH 86$ 3VWDUV DQG VWULSHV ́ IODJ ZLWK four assigned distinct regeneration turning points outlined along the PXOWLFHOOXODU RUJDQLVPV 3UHJHQHUDWLRQ FRQWLQXXP ́ 7 DQG 7 RUJDQLVPV VSHFLI\ WKH 3:KROH ERG\ UHJHQHUDWLRQ ́ DQG 32UJDQ UHJHQHUDWLRQ ́ VWUDWHJLHV ZLWK PRGHO FDVHV WKH botryllid ascidians regeneration and the murine digit tip regeneration, respectively. 
Whole body regeneration in Botrylloides leachi from the Mediterranean coast of Israel. ( a ) A colony growing on glass slide, just before zooids  ́ dissection; ( b ) Immediately after zooids  ́ dissection. A piece of the tunic containing marginal ampullae is left; ( c ± f ) Whole body regeneration within two weeks. ( c ) Morphological changes, three days after dissection, the tunic fragment with ampullae constructions. This is follows by the formation of bud rudiment at the middle of the fragment ( d = day 7), well developed bud ( e = day 10) and the establishment of fully functional, filtering organism (the two extending siphons are seen), containing a single zooid ( f = day 15). 
Regeneration of the mouse digit tip (DTR). ( A ) Hindlimb digits showing complete regrowth following 3-months from amputations. ( A ¶ ) Absence of regrowth in digits amputated at more proximal planes (black bracket). ( B , B ¶ ) Pentachrome staining; bone in yellow, endothelium and epidermis in red. ( B ) A section through the digit tips, two days following amputation. ( B ¶ ) A section through a representative case for DTR, three months after amputation. Regrowth is visible within bone, nail, epidermis and vasculature (white arrowheads). ( B  ́ ) Alcian-blue stain of a regenerated digit; bone and tendons are colored light purple, dermis is colored dark purple. ( C ¶ ± C  ́ ) Genetic lineage tracing of tissues within the digit reveals germ-layer and lineage restriction throughout regeneration. K14 CreER R26 mT/mG transgenic mice show lineage restriction of epidermis (GFP expression, C ) 
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.
Cell architecture and organization of subcellular compartments depend on the experimental model 2 human submandibular glands (HSG) cells were cultured either on glass (2D, upper panels) or on 5 mg/mL matrigel (3D, center panels), as described in Material and Methods. ( A ) Phase contrast images of HSG cells grown in 2D (upper panel) or in 3D (center panel). In 3D cells form acinar-like structures that resemble the acini in intact SGs. In the lower panel an intact rat submandibular gland was imaged by two-photon microscopy (Excitation 740 nm) as previously described [17]. An acinus is highlighted (red broken line). ( B ) HSG cells grown in 2D or 3D and intact salivary glands were labeled with Hoechst (blue) and Texas-Red Phallodin (red) to highlight nuclei and actin respectively. Z-scans were acquired by confocal microsopy (excitation 405 nm and 562 nm) and maximal projections of the stacks are shown. In 2D, actin is mainly localized in stress fibers (upper panels, arrow), whereas in 3D is localized at the plasma membrane (center panel, arrow). In intact SGs, actin is localized primarily at the apical plasma membrane and decorates the acinar canaliculi (lower panel, arrows). Bars, 10 ȝ m. ( C ) HSG cells and intact SGs were labeled with antibodies against VAMP8 (red) and TfnR (green). HSG cells were also labeled with Hoechst (blue), whereas SGs were labeled with phalloidin (Cyan). Note that in SGs VAMP8 is localized close to the apical plasma membrane and does not colocalize with TfnR (lower panel). Bars, 10 ȝ m. 
Kinetics of internalization of dextran and transferrin in vivo. (A) The submandibular SGs of an anesthetized rat were exposed and imaged in time-lapse by two-photon microscopy (excitation wavelength 840 nm). A mixture of 10 g/mL TXR-Tfn and Alexa 488-dextran (70 kDa) was injected into the tail artery of the animal (time 0:00). After few seconds both probes appeared in the vasculature (time 0:03) and diffused into the stroma (time 0:40, arrow). After few min (time 3:30 and 7:00) only dextran (green) was internalized by stromal cells (arrowheads). (B) Left panel. Diagram showing the procedure for intra-organ injection. Right panel. A mixture of 10 mL TXR-Tfn and Alexa 488-dextran (70 kDa) was injected into the stroma of the rat SGs and the internalization was imaged in time-lapse by intravital two-photon microscopy (Excitation wavelength, 840 nm). Snapshots were taken at 6, 20 and 30 min. Tfn in red and dextran in green. Bar, 20 m. (C) Rat submandibular SGs were either fixed immediately (upper panel) and labeled for TfnR (green) and the nuclei (blue) or injected with TXR-Tfn (lower panel, red), and then processed for immunocytochemistry to reveal clathrin (green) and the nuclei (blue). Note, that at steady state TfnR1 is primarily localized in endosomal structures. The levels at the plasma membrane are low and not easily detectable by antibody staining. Bar 5 m.
Kinetics of internalization of dextran and transferrin in vitro . ( A ) Diagram showing the procedure to label and isolate stromal cells from rat submandibular SGs. Anesthetized animals were injected with Cy5-dextran and euthanized after 1 h. The submandibular SGs were removed and imaged by two-photon microscopy (excitation 930 nm) to detect the internalization of the dextran (red) in stromal cells (upper panel, arrows). Collagen fibers were also imaged (cyan). The glands were minced and dispersed by enzymatic digestion. Cells were sorted by FACS and cultured on glass coverslips. To check whether cy5-dextran was retained in cultures, cells were labeled with Hoechst (blue) imaged by confocal microscopy (lower panel). ( B ) Rat derived stromal cells were grown on coverslips for 24 h and incubated with a mixture of 10 ȝJP/ TXR-Tfn and Alexa-488 dextran (70 kDa). Cells were imaged in time-lapse by using confocal microscopy (excitation 488 nm and 561 nm). Snapshots were taken at 2, 15 and 30 min. Tfn (red) was internalized within the first 2 min (left panels) whereas dextran (green) appeared after 10 ± 15 min (center and right panels). Bar, 5 ȝ m . 
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.
Molecular mechanisms of NT on apoptosis regulation. NT acts directly through its receptors to block the apoptosis ( 1 ) or indirectly by competition to reverse the proneurotrophin-induced apoptosis ( 2 ). PI3K; phosphatidyl inositol 3 kinase, NT; neurotensin. 
Summary of the central and peripheral effects of tridecapeptide neurotensin (NT) on the regulation of cell apoptosis (n.d.: not determined).
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.
Crystal structure of human neuroglobin showing the heme group (red) and iron bound histidine side chains (blue). The partially dissociated histidine 64 is indicated.  
Cartoon showing the molecular events following a stroke. Neuron damage (top left) leads to mitochondrial release of cytochrome c (magenta) into the cytosol. In the cytosol cytochrome c can bind to either Apaf-1 (cyan) or neuroglobin (green). Binding to Apaf-1 leads to apoptosome formation and apoptotic cell death. Binding of cytochrome c to neuroglobin and subsequent reduction prevents cell death.
Native blue gel showing Apaf-1 binding to oxidized cytochrome c in the presence of ATP to give the apoptosome complex.  
A hypothetical interpretation of the know reactivities of neuroglobin in the suppression of apoptotic activity. C3+ and c2+ represent the oxidized and reduced forms of cytochrome c respectively.
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.
BAFF has a critical role in B-cell survival, maturation and function, which makes its pathway a prime therapeutic target for various autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjögren's syndrome. A cell-based assay that measures the functional activity of BAFF is required for many high throughput purposes, such as lead target screening and BAFF quantification. We report here the development of a sensitive BAFF responsive cell line via stable transfection of the BAFFR-TNFR1 hybrid receptor into monkey kidney epithelial COS-1 cells. The cellular response to BAFF can be detected by measuring the secretion of IL-8. This BAFF bioassay is not only reproducible and sensitive, but also responsive to a wide concentration range of BAFF stimulation in sera from various species. This cell line is useful in the development of sensitive bioassays to measure the levels of bioactive BAFF, inhibition of BAFF and neutralizing antibodies against any BAFF pathway-mediated therapeutic proteins.
Comparison of detection of Borrelia-specific T cells in peripheral blood by the iSpot Lyme assay and conventional ELISPOT assay. (A) The frequency of rBorrelia antigen-induced IFN-γ spot was established under both conditions in peripheral blood mononuclear cells (PBMC) of Borrelia positive patients. Data points obtained from the same donor with the iSpot Lyme assay and conventional ELISPOT assay are connected by a line. Each data point represents the mean spot forming unit (SFU) of triplicate antigen-stimulated wells minus the mean SFU of the corresponding medium control wells. A non-parametric Mann-Whitney U test was used to compare the matched results with a p-value of <0.05 considered statistically significant. (B) Representative well images for test results obtained from one healthy control run in triplicate and (C) from a Borrelia positive patient run in triplicate. (D) Size distribution of IFN-γ ELISPOTs obtained from the iSpot Lyme assay vs. the conventional ELISPOT assay, as specified by the closed and open circles, respectively.  
Evaluation of the iSpot Lyme assay as a diagnostic test. (A) The results of iSpot Lyme assays performed on 80 healthy controls (HC), 25 clinically diagnosed Lyme disease patients (LD) and 23 non-Lyme patients (NLP) are shown. Each symbol represents the mean SFU obtained in triplicate rBorrelia-stimulated wells of a test subject after subtraction of the mean SFU in triplicate medium control wells. Non-parametric Mann-Whitney U test was used to compare the results from LD vs. HC and LD vs. NLP. A p-value < 0.05 was considered statistically significant. The dotted line represents the cutoff value for positivity at 25 SFU. (B) Receiver Operating Characteristics analysis was used to determine the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), area under the curve value (AUC) and cutoff value for the iSpot Lyme assay. (C) Receiver Operating Characteristics analysis was used to determine the sensitivity, specificity, PPV, NPV, AUC and cutoff value for the conventional ELISPOT assay.  
Optimization and validation of the iSpot Lyme assay. (A) Intra-assay precision. Five PBMC samples with different rBorrelia-triggered SFU response levels were tested in triplicate wells each. Bars with the specified shades show the reactivity for the three individual wells, and the mean of the three. The coefficient of variation for the replicate wells was calculated. (B) Inter-assay precision. Cryopreserved PBMC aliquots of the specified three Lyme patients were tested for rBorrelia reactivity on five consecutive days. The coefficient of variation for inter assay variation was calculated. (C) Relationship between PBMC numbers plated in each well and the IFN-γ SFU in a Borrelia positive subject, and (D), in a healthy control. Open symbols represent the mean of triplicate antigen-stimulated (treated) wells, the closed symbols represent the mean of the corresponding medium (control) wells. The Standard Deviation (SD) for the triplicate is smaller than the symbol when not visible. (E) Dose response curve for rBorrelia antigen-stimulated PBMC. (F) Correlation of the frequency of IFN-γ secreting Borrelia-specific T cells assessed by the iSpot Lyme assay and the concentrations of IFN-γ in the culture supernatant as measured by Bio-Plex suspension array. The nonparametric Spearman's test was used to determine the correlation. The results showed a p-value <0.0001.  
Comparison of sensitivity and specificity for detecting Borrelia infection via measuring T cell immunity by ELISPOT vs. serum antibodies by Western Blot. (A) Lyme ELISPOT assays and Western Blot assay were performed on PBMC and serum of 22 clinically diagnosed Borrelia patients. The percentage of individuals who scored positive for each assay is shown. (B) Cross-reactivity was assessed in 23 subjects with other clinical conditions, as defined in Materials and Methods, using the iSpot Lyme, conventional ELISPOT and Western Blot assay.
Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-g as a biomarker, we developed a new enzyme-linked immunospot method (iSpot Lyme™) to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.
IgG and IgM antibody-secreting cells enumerated in two independent experiments using the multiplexed B cell fluorospot assay.
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.
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.
Herpes simplex virus type 1 (HSV-1) is a significant human pathogen that infects a large portion of the human population. Cells deploy a variety of defenses to limit the extent to which the virus can replicate. One such factor is the promyelocytic leukemia (PML) protein, the nucleating and organizing factor of nuclear domain 10 (ND10). PML responds to a number of stimuli and is implicated in intrinsic and innate cellular antiviral defenses against HSV-1. While the role of PML in a number of cellular pathways is controlled by post-translational modifications, the effects of phosphorylation on its antiviral activity toward HSV-1 have been largely unexplored. Consequently, we mapped phosphorylation sites on PML, mutated these and other known phosphorylation sites on PML isoform I (PML-I), and examined their effects on a number of PML's activities. Our results show that phosphorylation at most sites on PML-I is dispensable for the formation of ND10s and colocalization between PML-I and the HSV-1 regulatory protein, ICP0, which antagonizes PML-I function. However, inhibiting phosphorylation at sites near the SUMO-interaction motif (SIM) of PML-I impairs its ability to respond to HSV-1 infection. Overall, our data suggest that PML phosphorylation regulates its antiviral activity against HSV-1.
A novel axis of innate antiviral immunity, in which hsp70 is released from virus infected cells (neuron, left) to stimulate innate immune responses of macrophages (microglia, right). Virus core particles (purple nucleocapsids) in the neuron mediate gene expression that is stimulated by hsp70 and results in further hsp70 induction, setting into motion a positive feedback loop. The extracellular hsp70 is ligand for TLR2 and TLR4, activating signal transduction pathways that drive type 1 IFN expression (IFN-β in brain) and expression of antigen presenting complexes (MHC). IFN-β expression by brain macrophages is key to immunity against virus infected neurons.
Virus-hsp70 interaction in support of viral replication.
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.
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.
Schematic representation of some of the proteins and lipids localized to the MAMs. Example of proteins localized to the MAMs (such as Mfn2, Sigma 1R, BiP, IP3R1, VDAC, and GRP75) that have been shown to regulate Ca 2+ signaling and the 
The dynamic interplay among intracellular organelles occurs at specific membrane tethering sites, where two organellar membranes come in close apposition but do not fuse. Such membrane microdomains allow for rapid and efficient interorganelle communication that contributes to the maintenance of cell physiology. Pathological conditions that interfere with the proper composition, number, and physical vicinity of the apposing membranes initiate a cascade of events resulting in cell death. Membrane contact sites have now been identified that tether the extensive network of the endoplasmic reticulum (ER) membranes with the mitochondria, the plasma membrane (PM), the Golgi and the endosomes/lysosomes. Thus far, the most extensively studied are the MAMs, or mitochondria associated ER membranes, and the ER-PM junctions that share functional properties and crosstalk to one another. Specific molecular components that define these microdomains have been shown to promote the interaction in trans between these intracellular compartments and the transfer or exchange of Ca2+ ions, lipids, and metabolic signaling molecules that determine the fate of the cell.
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.
Structure of the Inhibitors of apoptosis. ( a ) Structure of the cellular IAP1 (cIAP1)-baculoviral IAP repeat (BIR)3 bound to the caspase-9 N -terminal peptide [30]. BIR3 is organized in four Į-helices (red) and 3 ȕ-strand sheets (yellow) maintained by zinc ion (grey). The interaction involved the surface hydrophobic groove of cIAP1 and the N -terminal peptide (ATPFQ) of the caspase 9 sub-unit (Constructed using the PyMOL Molecular Graphics System). ( b ) Representation of IAPs involved in the regulation of apoptosis. The type I baculoviral IAP repeats (BIR, blue) of cIAPs and X-linked IAP (XIAP) can bind to cell signalling intermediates TNFR associated factor 2 (TRAF2) and Transforming Growth Factor beta-activated kinase 1-binding protein 1 (TAB1), respectively. The type II BIRs (brown) contain a surface hydrophobic groove allowing the interaction with IBM found in caspase sub-units and IAP antagonists. The ubiquitin Associated (UBA) domain binds ubiquitin chains. The caspase recruitment domain (CARD) is a module of regulation of the RING E3-ubiquitin ligase activity. The RING domain confers to IAPs an E3-ubiquitin ligase activity. NACHT (domain present in NAIP, CIITA, HETE and TP1). LRR: Leucine Rich Repeat. 
Regulation of the apoptosome and caspase activity by IAPs. The release of cytochrome-c from mitochondria which occurs during intrinsic pathway of apoptosis triggers an ATP-dependent conformational change and oligomerisation of the adaptor apoptotic protease activating factor 1 (Apaf-1) in a heptameric complex apoptosome. Apaf-1 then recruits Caspase-9 via its pro-domain through a homotypic CARD-CARD interaction. Caspase-9 is activated by homodimerisation and promotes the activating cleavage of effector caspase-3 leading to apoptosis. Caspase-9 undergoes autocatalytic processing and is then quickly disconnected from the apoptosome which is free to recruit a new pro-caspase-9. XIAP can control caspase activating pathway at several steps. First, XIAP is present in the apoptosome where it directly binds processed caspase-9 and inhibits its activity. The inhibition of caspase-9 by XIAP could stabilize the caspase-9 apoptosome complex and block the cycle of caspase-9 activation. Second, XIAP can directly bind and inhibit active effector caspase-3. XIAP can inhibit caspase activity by hindering substrate accessibility or hiding the protease catalytic residue, and/or by promoting ubiquitination or neddylation. Although unable to inhibit their activity, cIAP1 can bind to processed caspases and promote their ubiquitination. An IBM-dependent binding of IAP antagonists such as Smac/Diablo prevents XIAP-mediated caspase inhibition while cIAPs could interfere with neutralizing binding XIAP-Smac/Diablo.
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.
TRPS1 is a GATA-type transcription factor that is closely related to human tricho-rhino-phalangeal syndrome (TRPS) types I and III, variants of an autosomal dominant skeletal disorder. During embryonic development, Trps1 represses Sox9 expression and regulates Wnt signaling pathways that determine the number of hair follicles and their normal morphogenesis. In the growth plate, Trps1 regulates chondrocytes condensation, proliferation, and maturation and phalangeal joint formation by functioning downstream of Gdf5 signaling and by targeting at Pthrp, Stat3 and Runx2. Also, Trps1 protein directly interacts with an activated form of Gli3. In embryonic kidneys, Trps1 functions downstream of BMP7 promoting the mesenchymal-to-epithelial transition, and facilitating tubule morphogenesis and ureteric bud branching. Moreover, Trps1 has been found to be closely related to tumorigenesis, invasion, and metastasis in prostate and breast cancers. It is interesting to note that during the development of hair follicles, bones, and kidneys, mutations in Trps1 cause, either directly or through crosstalk with other regulators, a notable change in cell proliferation and cell death. In this review, we will summarize the most recent studies on Trps1 and seek to elucidate the role for Trps1 in apoptotic regulation.
Mechanism of apoptosis. Activation of initiators caspase 8 and 10 are mediated in response to extrinsic stimuli and apoptosis signaling is controlled by anti-and pro-apoptotic protein interactions. 
Mechanisms of DNA hypermethylation and apoptosis-related genes inactivation (a) Inactivation of RASSF1A by Dnmt/P53 cooperation. (b) Inactivation of DAPK by the ternary complex Dnmt/RelB/DAXX. (c) Specific methylation of BAX leading to BAX ψ or BAX silencing. 
DNA methylation and anti-apoptotic strategies (a) Effects of both DNA demethylating agents and pro-methylation, mediated by folate, on apoptosis-related genes and apoptosis. (b) Conflicting results of demethylated agents and specific strategies against targeted DNA methylation on apoptosis. 
Apoptosis is a cell death programme primordial to cellular homeostasis efficiency. This normal cell suicide program is the result of the activation of a cascade of events in response to death stimuli. Apoptosis occurs in normal cells to maintain a balance between cell proliferation and cell death. A deregulation of this balance due to modifications in the apoptosic pathway leads to different human diseases including cancers. Apoptosis resistance is one of the most important hallmarks of cancer and some new therapeutical strategies focus on inducing cell death in cancer cells. Nevertheless, cancer cells are resistant to treatment inducing cell death because of different mechanisms, such as DNA mutations in gene coding for pro-apoptotic proteins, increased expression of anti-apoptotic proteins and/or pro-survival signals, or pro-apoptic gene silencing mediated by DNA hypermethylation. In this context, aberrant DNA methylation patterns, hypermethylation and hypomethylation of gene coding for proteins implicated in apoptotic pathways are possible causes of cancer cell resistance. This review highlights the role of DNA methylation of apoptosis-related genes in cancer cell resistance.
Neuroblastoma (NB) is one of the most common malignant solid tumors in childhood, which derives from the sympathoadrenal lineage of the neural crest and exhibits extremely heterogeneous biological and clinical behaviors. The infant patients frequently undergo spontaneous regression even with metastatic disease, whereas the patients of more than one year of age who suffer from disseminated disease have a poor outcome despite intensive multimodal treatment. Spontaneous regression in favorable NBs has been proposed to be triggered by nerve growth factor (NGF) deficiency in the tumor with NGF dependency for survival, while aggressive NBs have defective apoptotic machinery which enables the tumor cells to evade apoptosis and confers the resistance to treatment. This paper reviews the molecules and pathways that have been recently identified to be involved in apoptotic cell death in NB and discusses their potential prospects for developing more effective therapeutic strategies against aggressive NB.
Apoptotic cell death is coordinated through two distinct (type 1 and type 2) intracellular signaling pathways. How the type 1/type 2 choice is made remains a central problem in the biology of apoptosis and has implications for apoptosis related diseases and therapy. We study the problem of type 1/type 2 choice in silico utilizing a kinetic Monte Carlo model of cell death signaling. Our results show that the type 1/type 2 choice is linked to deterministic versus stochastic cell death activation, elucidating a unique regulatory control of the apoptotic pathways. Consistent with previous findings, our results indicate that caspase 8 activation level is a key regulator of the choice between deterministic type 1 and stochastic type 2 pathways, irrespective of cell types. Expression levels of signaling molecules downstream also regulate the type 1/type 2 choice. A simplified model of DISC clustering elucidates the mechanism of increased active caspase 8 generation and type 1 activation in cancer cells having increased sensitivity to death receptor activation. We demonstrate that rapid deterministic activation of the type 1 pathway can selectively target such cancer cells, especially if XIAP is also inhibited; while inherent cell-to-cell variability would allow normal cells stay protected.
Schematic representation of the FACS-based cell purification technique. ( A ) Structure of the retroviral construct (VICD8) used to co-express VEGF (mVEGF 164 ) and a 
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.
Quantification of caspase-8 in different gel slices and total amounts in the six DISC IPs.
Quantification of the DISC. (A-C) Workflow for the quantification as exemplified for caspase-8. (A) Base peak chromatogram of the mass spectrometric analysis of gel slice 8. A two hour gradient was applied. (B) Extracted ion chromatograms of the endogenous light and spiked-in heavy peptide NLYDIGEQLDSEDLASLK (A light and A heavy) and endogenous light and spiked-in heavy peptide FLSLDYIPQR. (B light and B heavy). RT: retention time; MA: marked area (AUC). The ratios of light and heavy areas are calculated and used for quantification of the light peptides. (C) Database search of MS2 data confirmed the sequence of the two light and heavy peptide pairs of procaspase-8. (D) Extracted ion chromatograms of corresponding peptide pairs for FADD in gel slice 13. A1 heavy/light and A2 heavy/light represent the same peptide sequence in two different charge states, 2+ and 3+, respectively, with the same retention time of 46 min. The second (B heavy/light) and third peptide (C heavy/light) are more hydrophilic and elute earlier from the reverse phase column. (E) Stoichiometry of the CD95 DISC. Total amounts of proteins were calculated from the extracted ion chromatograms and normalized to FADD. The stoichiometry was determined to be 1:2:0.1:0.3 for FADD, procaspase-8, c-FLIP and procaspase-10.
Contemporary quantitative mass spectrometry provides fascinating opportunities in defining the stoichiometry of high-molecular weight complexes or multiprotein platforms. The composition stoichiometry of multiprotein platforms is a key to understand the regulation of complex signaling pathways and provides a basis for constructing models in systems biology. Here we present an improved AQUA technique workflow that we adapted for the quantitative mass spectrometry analysis of the stoichiometry of the CD95 (Fas/APO-1) death inducing signaling complex (DISC). The DISC is a high-molecular weight platform essential for the initiation of CD95-mediated apoptotic and non-apoptotic responses. For protein quantification, CD95 DISCs were immunoprecipitated and proteins in the immunoprecipitations were separated by one-dimensional gel electrophoresis, followed by protein quantification using the AQUA technique. We will discuss in detail AQUA analysis of the CD95 DISC focusing on the key issues of this methodology, i.e., selection and validation of AQUA peptides. The application of this powerful method allowed getting new insights into mechanisms of procaspase-8 activation at the DISC and apoptosis initiation [1]. Here we discuss the AQUA methodology adapted by us for the analysis of the CD95 DISC in more detail. This approach paves the way for the successful quantification of multiprotein complexes and thereby delineating the intrinsic details of molecular interactions.
Antibodies and their immunohistochemical detection methods.
Programmed cell death (PCD)-related molecules in a case of Epstein-Barr virus (EBV)-related cytotoxic T-cell lymphoma (×40, Olympus BX50, FUJIFILM HC-300). (a) Cellular area (Hematoxylin-eosin staining). (b) In situ hybridization of EBV-encoded small RNA-1 (EBER-1). (c) Cleaved caspase-3. (d) Bcl-2. (e) Flice (caspase-8) inhibitory protein (Flip). (f) Survivin. (g–i) Beclin-1. (j–l) LC3. (m–o) 8-hydroxydeoxyguanosine (8-OHdG). (g, j and m) Cellular area. (h, k and n) Degenerative area. (i, l and o) Necrotic area. Diffuse proliferation of lymphoma cells is observed with sinusoidal patterning (a). Most lymphoma cells show EBER-1 signals in their nuclei (b). There are no cleaved caspase-3- positive lymphoma cells (c), and the lymphoma cells express Bcl-2 scantly (d), Flip moderately (e) and survivin strongly (f), suggesting that neoplastic expression of survivin suppresses cleaved caspase-3. From the cellular area to the necrotic area in peculiar necrosis, lymphoma cells do not express beclin-1-autophagic vesicle nucleation complex (g–i) but show enhanced autophagy labeled by LC3. Lymphoma cells exhibit macrogranular staining of LC3 with sporadic distribution of LC3-densely labeled cell debris or naked nuclei in the cellular (j) and degenerative areas (k) and aggregation of LC3-densely labeled cell-debris in the necrotic area (l). In the necrotic area the cell-debris is positive for 8-OHdG (o).  
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.
In vitro chondrogenesis using human ES and iPScells. 
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.
Application of GrB ELISPOT assay for monitoring antitumor response in cancer patients. 
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.
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.
Top-cited authors
Yang-Hsin Shih
  • Taipei Veterans General Hospital
Kunwoo Lee
  • Seoul National University
Jin-Hoi Kim
  • Konkuk University
Yu-Feng Luo
  • Hohai University
Min-Hee Kang
  • CHA Group