Optimal Cellular Responses Research Articles

Kidney Mesenchymal Stem Cell Differentiation: Effect of Scaffold and Basic Fibroblast Growth Factor.

Background: Chronic kidney disease (CKD) poses a global health challenge, and it needs alternative therapeutic approaches for patients with end-stage renal disease (ESRD). Although organ transplantation is effective, it faces challenges such as declining quality of life, immunological responses, transplant rejection, and donor shortages. Tissue engineering, by using suitable scaffolds, cells, and growth factors, emerges as a promising treatment option for kidney regeneration. Experiment: We precisely decellularized scaffold, derived from rat kidneys while maintaining its native three-dimensional (3D) architecture. The efficiency of decellularization was evaluated through histological examinations, including hematoxylin and eosin, periodic acid-Schiff, and DAPI staining, as well as scanning electron microscopy. The scaffolds were then recellularized with kidney mesenchymal stem cells (kMSCs), and their adhesion, proliferation, and differentiation were assessed over 1, 2, and 3 weeks. The expression of specific renal markers, including Wt-1, ZO-1, AQP-1, and ANG-1, was examined through quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in monolayer and 3D cultures. Results: The infiltration rate of cells into the scaffold increased in a time-dependent manner, and the expression of specific renal markers significantly increased, demonstrating successful differentiation of kMSCs within the scaffold. The application of basic fibroblast growth factor (bFGF) could intensify the expression of kidney-specific genes. Conclusions: The study highlighted the importance of preserving the 3D architecture of the scaffold during decellularization to achieve optimal cellular responses. Moreover, the capacity of mesenchymal stem cells in recellularized scaffolds facilitated tissue regeneration.

14-3-3 interaction with phosphodiesterase 8A sustains PKA signaling and downregulates the MAPK pathway

The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon external stimuli. Phosphodiesterase 8A (PDE8A) is an important regulator that inhibits signaling via cAMP-dependent PKA by hydrolyzing intracellular cAMP pool. Conversely, PDE8A activates the MAPK pathway by protecting CRAF/Raf1 kinase from PKA-mediated inhibitory phosphorylation at Ser259 residue, a binding site of scaffold protein 14-3-3. It still remains enigmatic as to how the cross-talk involving PDE8A regulation influences cAMP/PKA and MAPK signaling pathways. Here, we report that PDE8A interacts with 14-3-3ζ in both yeast and mammalian system, and this interaction is enhanced upon the activation of PKA, which phosphorylates PDE8A's Ser359 residue. Biophysical characterization of phospho-Ser359 peptide with 14-3-3ζ protein further supports their interaction. Strikingly, 14-3-3ζ reduces the catalytic activity of PDE8A, which upregulates the cAMP/PKA pathway while the MAPK pathway is downregulated. Moreover, 14-3-3ζ in complex with PDE8A and cAMP-bound regulatory subunit of PKA, RIα, delays the deactivation of PKA signaling. Our results define 14-3-3ζ as a molecular switch that operates signaling between cAMP/PKA and MAPK by associating with PDE8A.

Blood CD8+ Naïve T-Cells Identify MS Patients with High Probability of Optimal Cellular Response to SARS-CoV-2 Vaccine.

This single-center study included 68 multiple sclerosis (MS) patients who received the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination from one of several approved vaccine preparations in Spain. Blood samples were collected one to three months after the second dose of the vaccine had been administered. Cellular immune responses to the vaccine were assessed using QuantiFERON analysis, and peripheral blood mononuclear cell subsets were assayed using flow cytometry. Response associated with higher percentages of total lymphocytes, naïve CD4+ T-cells (p = 0.028), CD8+ T-cells (p = 0.013), and, mostly, naïve CD8+ T-cells (p = 0.0003). These results were confirmed by analyzing absolute numbers (p = 0.019; p = 0.002, and p = 0.0003, respectively). Naïve CD8 T-cell numbers higher than 17 cells/μL were closely associated with an optimal cellular response to SARS-CoV-2 vaccination (odds ratio: 24.0, confidence interval: 4.8-460.3; p = 0.0001). This finding clearly shows that independent of the treatment received, higher numbers of naïve CD8+ T-cells yield a strong cellular response to SARS-CoV-2 vaccines in MS patients. If this finding is validated with other viruses/vaccines, it could provide a good tool for identifying MS patients undergoing treatment who will develop strong cellular responses to anti-virus vaccines.

The Role of Ubiquitination and SUMOylation in DNA Replication.

DNA replication is a tightly regulated conserved process that ensures the faithful transmission of genetic material to define heritable phenotypic traits. Perturbations in this process result in genomic instability, mutagenesis, and diseases, including malignancy. Proteins involved in the initiation, progression, and termination of DNA replication are subject to a plethora of reversible post-translational modifications (PTMs) to provide a proper temporal and spatial control of replication. Among these, modifications involving the covalent attachment of the small protein ubiquitin or the small ubiquitin-like modifier (SUMO) to replication and replication-associated proteins are particularly important for the proper regulation of DNA replication as well as for optimal cellular responses to replication stress. In this article, we describe how the ubiquitination and SUMOylation processes impact DNA replication in eukaryotes and highlight the consequences of deregulated signals emanating from these two versatile regulatory pathways on cellular activities.

IL-4Rα-expressing CD11c+ cells contribute to driving optimal cellular responses during Schistosoma mansoni infection in mice.

Development of IL‐4 receptor alpha (IL‐4Rα)‐dependent cellular immunity regulates host protection against acute schistosomiasis. In this study, we investigated the importance of IL‐4Rα‐expressing CD11c+ cells in driving the development of optimal cellular responses to Schistosoma mansoni infection by using CD11ccreIL‐4Rα−/lox BALB/c mice, which lacked IL‐4Rα expression on dendritic cells and alveolar macrophages. Abrogation of IL‐4Rα expression on CD11c+ cells affected activation of CD4+ T cells, resulting in reduced numbers of effector CD4+ T cells and impaired production of Th1 and Th2 cytokines by CD4+ T cells ex vivo. However, secretion of both type 1 and type 2 Ab isotypes was unchanged in infected CD11c‐specific IL‐4Rα‐deficient mice compared to littermate controls. Together, these data demonstrate that IL‐4Rα‐expressing CD11c+ cells play an important role in maintaining cellular immunity during schistosomiasis in mice.

Cellular responses to radical propagation from ion-implanted plasma polymer surfaces

Biomolecule-functionalization, through the presentation of biological motifs that promote optimal cellular responses, has the capacity to improve the tissue integration of biomedical devices and hence patients’ quality of life. Radical-functionalized plasma polymer films (rPPFs) readily immobilize bioactive molecules on exposure to a biomolecule-containing aqueous solution without the need for chemical reagents. However, the potential for damage to cells and tissues due to the high local concentration of radicals in freshly deposited radical-functionalized plasma polymer films is of concern. In this study, we compared a fresh (4 h post-deposition) rPPF with one that had been aged for 11 days to explore the effect of the different radical fluxes on cellular responses. Primary osteoblasts and MG63 bone osteosarcoma cells were used to determine whether rPPFs at early aging times exhibited radical-induced cytotoxicity. The aging behavior of the rPPFs demonstrated a connection between the radical decay kinetics and the surface chemistry and wettability. Significant increases in cell attachment and spreading compared to bare Ti were observed for both cell lineages on the rPPF surfaces. The proliferation assays showed equivalent proliferation rates on both the fresh and aged surfaces, and no evidence of cytotoxicity was observed. Overall, we demonstrated that the high flux of radicals emerging to the surface has minimal influence on the biocompatibility of radical-functionalized plasma polymer films.

Age and the effect of exercise, nutrition and cognitive training on oxidative stress - The Vienna Active Aging Study (VAAS), a randomized controlled trial.

The purpose of this study was to investigated the effect of age - over or under life-expectancy (LE) - on six months resistance training alone or combined with a nutritional supplement, and cognitive training by analyzing markers for oxidative stress and antioxidant defense in institutionalized elderly, living in Vienna. Three groups (n = 117, age = 83.1 ± 6.1 years) - resistance training (RT), RT combined with protein and vitamin supplementation (RTS) or cognitive training (CT) - performed two guided training sessions per week for six months. Oxidative stress, antioxidant defense and DNA strand breaks were analyzed and transformed into an "antioxidant factor" to compare the total effect of the intervention. Physical fitness was assessed by the 6-min-walking, the chair-rise and the handgrip strength tests. We observed significant negative baseline correlations between 8-oxo-7.8-dihydroguanosine and handgrip strength (r = -0.350, p = 0.001), and between high sensitive troponin-T and the 6-min-walking test (r = -0.210, p = 0.035). RT and RTS groups, showed significant improvements in physical performance. Over LE, subjects of the RT group demonstrated a significant greater response in the "antioxidant factor" compared to RTS and CT (RT vs. RTS p = 0.033, RT vs. CT p = 0.028), whereas no difference was observed between the intervention groups under LE. Six months of elastic band resistance training lead to improvements in antioxidant defense, DNA stability and oxidative damage, summarized in the "antioxidant factor", however mainly in subjects over their statistical LE. Consuming a supplement containing antioxidants might inhibit optimal cellular response to exercise. The study was approved by the ethics committee of the City of Vienna (EK-11-151-0811) and registered at ClinicalTrials.gov, NCT01775111.

Multi-scale characterization of topographic modifications on metallic biomaterials induced by nanosecond Nd:YVO4 laser structuring

Laser surface structuring of biometals allows to achieve complex surface geometries at several scales since a rough surface is preferred to induce an optimal cellular response. A lack of standard measurement techniques to characterize implant surfaces was detected. This paper presents a comprehensive, multi-scale characterization of the topography of laser textured biometals with the ISO standard profile method; which allows to separate the contribution of the superimposed textures of the profile, the low frequency component (waviness) and the high frequency component (roughness), and to calculate the respective height parameters according to the standard definitions. Furthermore, the characterization was completed by the calculation of the spatial parameters obtained by FFT calculation in order to assess the horizontal characteristics of the surface. We focused on two common biometals, stainless steel 316L and titanium alloy Ti6Al4V, structured with an array of parallel grooves obtained by using a nanosecond Nd:YVO4 laser. Regarding waviness, some differences in symmetry and sharpness were found according to the metal. Conversely, the roughness of the topography revealed that the average height of the micro-structures developed inside the grooves is similar in both metals. The spatial periodicity of the micro-structures showed that the aspect ratio resulted higher in AISI 316L than in Ti6Al4V. The analytical approach presented in this paper allows to establish an elementary comparison of the results obtained in the scientific community, contributing to a better understanding of in vitro/vivo cellular growing on textured surfaces.

Ragulator and GATOR1 complexes promote fission yeast growth by attenuating TOR complex 1 through Rag GTPases

TOR complex 1 (TORC1) is an evolutionarily conserved protein kinase complex that promotes cellular macromolecular synthesis and suppresses autophagy. Amino-acid-induced activation of mammalian TORC1 is initiated by its recruitment to the RagA/B-RagC/D GTPase heterodimer, which is anchored to lysosomal membranes through the Ragulator complex. We have identified in the model organism Schizosaccharomyces pombe a Ragulator-like complex that tethers the Gtr1-Gtr2 Rag heterodimer to the membranes of vacuoles, the lysosome equivalent in yeasts. Unexpectedly, the Ragulator-Rag complex is not required for the vacuolar targeting of TORC1, but the complex plays a crucial role in attenuating TORC1 activity independently of the Tsc1-Tsc2 complex, a known negative regulator of TORC1 signaling. The GATOR1 complex, which functions as Gtr1 GAP, is essential for the TORC1 attenuation by the Ragulator-Rag complex, suggesting that Gtr1GDP-Gtr2 on vacuolar membranes moderates TORC1 signaling for optimal cellular response to nutrients.

Type III Intermediate Filaments Desmin, Glial Fibrillary Acidic Protein (GFAP), Vimentin, and Peripherin.

SummaryType III intermediate filament (IF) proteins assemble into cytoplasmic homopolymeric and heteropolymeric filaments with other type III and some type IV IFs. These highly dynamic structures form an integral component of the cytoskeleton of muscle, brain, and mesenchymal cells. Here, we review the current ideas on the role of type III IFs in health and disease. It turns out that they not only offer resilience to mechanical strains, but, most importantly, they facilitate very efficiently the integration of cell structure and function, thus providing the necessary scaffolds for optimal cellular responses upon biochemical stresses and protecting against cell death, disease, and aging.

IL2 production predicts Tfh differentiation

Abstract Naïve CD4 T cells differentiate into functionally distinct subsets in response to a set of extrinsic and intrinsic cues. Effector subsets, including Th1, Th2 and Th17 CD4s, are required for optimal cellular responses to invading pathogens, while class switched antibody responses depend on the differentiation of T follicular helper cells (Tfh). Development of both Tfh and non-Tfh occurs in parallel and involves two distinct but functionally related expression bifurcations. Upon formation of T cell receptor (TCR)/major histocompatibility (MHC) synapses with antigen-presenting cells (APC), a discrete subset of activated T cells secretes the cytokine IL2. Similarly, a portion of activated cells express the transcription factor Bcl6 and differentiate into Tfh cells, while the remainder express Blimp1 and develop into effector CD4s. However, the cellular, temporal and mechanistic relationships between these two bifurcations remain unclear. Here we show that the IL2 and Bcl6/Blimp1 expression bifurcations occur simultaneously, precede cell division, predict differentiation into Tfh and non-Tfh subsets, and are mechanistically related to antigen availability TCR density.

A Cationic Micelle Complex Improves CD8+ T Cell Responses in Vaccination Against Unmodified Protein Antigen.

Nanoscale carrier platforms promote immune responses to vaccination by facilitating delivery of vaccine components to immunologically relevant sites. The technique is particularly valuable for subunit vaccination, in which coadministration of immunostimulatory adjuvant is known to enhance immune responses to protein antigen. The fabrication of polymer-based nanoparticle vaccines commonly requires covalent attachment of vaccine components to the carrier surface. In contrast, we here describe a cationic micelle vaccination platform in which antigen and adjuvant loading is mediated by noncovalent molecular encapsulation and electrostatic complexation. Cationic micelles were generated through self-assembly of a polyarginine-conjugated poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-PPS) diblock copolymer amphiphile, with or without encapsulation of monophosphoryl lipid A (MPLA), an amphiphilic experimental vaccine adjuvant. Micelle complexes were subsequently formed by complexation of ovalbumin (OVA) and CpG-B oligodeoxynucleotide (a second experimental adjuvant) to the cationic micelles. In a 35-day study in mouse, micelle-mediated codelivery of OVA antigen and CpG-B enhanced cellular and humoral responses to vaccination. These outcomes were highlighted in spleen and lymph node CD8+ T cells, with significantly increased populations of IFNγ+, TNFα+, and polyfunctional IFNγ+ TNFα+ cells. Elevated cytokine production is a hallmark of robust cytotoxic T lymphocyte (CTL) responses sought in next-generation vaccine technologies. Increased production of OVA-specific IgG1, IgG2c, and IgG3 also confirmed micelle enhancement of humoral responses. In a subsequent 35-day study, we explored micelle-mediated vaccination against OVA antigen coadministered with MPLA and CpG-B adjuvants. A synergistic effect of adjuvant coadministration was observed in micelle-free vaccination but not in groups immunized with micelle complexes. This outcome underlines the advantage of the micelle carrier: we achieved optimal cellular and humoral responses to vaccination by use of this nanoparticle platform with a single adjuvant. In particular, enhanced CTL responses support future development of the cationic micelle platform in experimental cancer vaccines and for vaccination against reticent viral pathogens.

Protein Oxidation and DNA Repair Inhibition by 6-Thioguanine and UVA Radiation

Damage to skin DNA by solar UV is largely unavoidable, and an optimal cellular response to it requires the coordinated operation of proteins in numerous pathways. A fully functional DNA repair proteome for removing harmful DNA lesions is a prerequisite for an appropriate DNA damage response. Genetically determined failure to repair UV-induced DNA damage is associated with skin photosensitivity and increased skin cancer risk. Patients treated with immunosuppressant/anti-inflammatory thiopurines are also photosensitive and have high rates of sun-related skin cancer. Their DNA contains the base analog 6-thioguanine (6-TG), which acts as a UVA photosensitizer to generate reactive oxygen species (ROS), predominantly singlet oxygen ((1)O2). ROS damage both DNA and proteins. Here we show that UVA irradiation of cultured human cells containing DNA 6-TG causes significant protein oxidation and damages components of the DNA repair proteome, including the Ku, OGG-1, MYH, and RPA proteins. Assays of DNA repair in intact cells or in cell extracts indicate that this protein damage compromises DNA break rejoining and base and nucleotide excision repair. As these experimental conditions simulate those in the skin of patients taking thiopurines, our findings suggest a mechanism whereby UVA in sunlight may contribute to skin carcinogenesis in immunosuppressed patients.

Ultrafine calcium–titania–polyester dry powder coatings promote human mesenchymal cell attachment and biomineralization

An electrostatic ultrafine dry powder coating technique was developed, which utilizes nanoparticles to prevent agglomeration. When polyester resin, TiO2 (25%), nano-TiO2 (0.5%), filler, flow, degassing and curing agents were combined in a high-shear mixer, then sprayed (20kV) onto metal sheets and cured (200°C), biocompatible polymeric powder coatings (PPC) were created. This study's objective was to determine if these surface coatings could be successfully augmented with calcium-rich functional additives. Thus, formulations were enriched with CaO (1, 3 and 5%) and Ca3(PO4)2 (5%, CaP), and their coatings characterized. Particle size analysis of the powders confirmed their ultrafine dimensions. Volume diameters for 50% (D 0.5) were under 30μm, and 90% (D 0.9) under 50μm. Energy dispersive X-ray (EDX) analysis confirmed integration with minimal modification to the PPC base. Carbon (61–67%), oxygen (15–25%) and titanium (9–15%) dominated all surfaces, whereas calcium (0.5–1.0%) and phosphorus (0.3%) were only detected in the CaO and CaP enriched coatings respectively. Elemental mapping showed calcium clusters that were smaller but more abundant with more CaO (PPC+3%CaO, PPC+5%CaO), and colocalized to phosphorus in the PPC+5%CaP. Contact angle measurements indicated hydrophilic coatings (62–81°) and significantly (P<0.05) increased surface wetting with CaO. Optical microscopy showed human mesenchymal cell (HEPM, ATCC CRL-1486) attachment and spreading on all coatings within 24h, and increasing confluence with CaO incorporation. Alizarin Red-S staining detected the biomineralization of cultures on all coatings that increased with CaO and time. Ultimately, the PPC+5%CaO had the most cells and biomineralization. This ultrafine dry powder coating technology can create biocompatible surfaces from polyester resins and TiO2, which can be readily augmented with functional additives such as calcium, to titrate optimal cellular responses.

Second-Generation Langerhans Cells Originating from Epidermal Precursors Are Essential for CD8+ T Cell Priming

In vivo studies questioned the ability of Langerhans cells (LCs) to mediate CD8(+) T cell priming. To address this issue, we used intradermal immunization with plasmid DNA, a system in which activation of CD8(+) T cells depends on delayed kinetics of Ag presentation. We found that dendritic cells (DCs) located in the skin at the time of immunization have limited ability to activate CD8(+) T cells. This activity was mediated by a second generation of DCs that differentiated in the skin several days after immunization, as well as by lymph node-resident DCs. Intriguingly, CD8(+) T cell responses were not affected following treatment with clodronate liposomes, immunization of CCR2(-/-) mice, or local neutralization of CCL20. This suggests that local, rather than blood-derived, DC precursors mediate CD8(+) T cell priming. Analysis of DC differentiation in the immunized skin revealed a gradual increase in the number of CD11c(+) cells, which reached their maximum 2 wk after immunization. A similar differentiation kinetics was observed for LCs, with the majority of differentiating LCs proliferating in situ from epidermal precursors. By using B6/Langerin-diphtheria toxin receptor chimeric mice and LC ablation, we demonstrated that epidermal LCs were crucial for the elicitation of CD8(+) T cell responses in vivo. Furthermore, LCs isolated from lymph nodes 2 wk after immunization contained the immunization plasmid and directly activated Ag-specific CD8(+) T cells ex vivo. Thus, these results indicate that second-generation Ag-expressing LCs differentiating from epidermal precursors directly prime CD8(+) T cells and are essential for optimal cellular immune responses following immunization with plasmid DNA.

Tetraspanin CD37 contributes to the initiation of cellular immunity by promoting dendritic cell migration

Previous studies on the role of the tetraspanin CD37 in cellular immunity appear contradictory. In vitro approaches indicate a negative regulatory role, whereas in vivo studies suggest that CD37 is necessary for optimal cellular responses. To resolve this discrepancy, we studied the adaptive cellular immune responses of CD37(-/-) mice to intradermal challenge with either tumors or model antigens and found that CD37 is essential for optimal cell-mediated immunity. We provide evidence that an increased susceptibility to tumors observed in CD37(-/-) mice coincides with a striking failure to induce antigen-specific IFN-γ-secreting T cells. We also show that CD37 ablation impairs several aspects of DC function including: in vivo migration from skin to draining lymph nodes; chemo-tactic migration; integrin-mediated adhesion under flow; the ability to spread and form actin protrusions and in vivo priming of adoptively transferred naïve T cells. In addition, multiphoton microscopy-based assessment of dermal DC migration demonstrated a reduced rate of migration and increased randomness of DC migration in CD37(-/-) mice. Together, these studies are consistent with a model in which the cellular defect that underlies poor cellular immune induction in CD37(-/-) mice is impaired DC migration.

Relationship between ATM and Ribosomal Protein S6 Revealed by the Chemical Inhibition of Ser/Thr Protein Phosphatase Type 1

The optimal cellular responses to DNA damage are modulated by kinase and phosphatase. The ataxia telangiectasia mutated (ATM) is a Ser/Thr kinase which is the core of the DNA damage signaling apparatus. The Ser/Thr protein phosphatase type 1 (PP1) inhibitor, tautomycetin (TC) and an antibody to the phospho-(S/T)Q sites of the ATM substrate were used to identify the common substrates for PP1 and ATM in regulating the pathway for DNA damage response. Ribosomal protein S6 (RPS6) was first identified as a substrate for PP1 and ATM. The phosphorylation at Ser247 of RPS6 was then significantly decreased by PP1-mediated dephosphorylation immediately after UV irradiation. These results suggest that PP1 specifically dephosphorylated RPS6 at phospho-Ser247 in vivo. In response to DNA damage, ATM activity was finally required for the phosphorylation of RPS6 at Ser247. We propose from these results a novel mechanism for modulating the RPS6 function by PP1 and ATM which regulates cell growth and survival in response to DNA-damage stimuli.

Cancer cell response to nanoparticles: criticality and optimality

A stochastic variation in size and electrical parameters is common in nanoparticles. Synthesizing gold nanoparticles with a varying range of size and zeta potential, we show that there is clustering at certain regions of hydrodynamic diameter and zeta potentials that can be classified using k-clustering technique. A cluster boundary was observed around 50 nm, a size known for its optimal response to cells. However, neither size nor zeta potential alone determined the optimal cellular response (e.g., percentage cell survival) induced by such nanoparticles. A complex interplay prevails between size, zeta potential, nature of surface functionalization, and extent of adhesion of the cell to a solid matrix. However, it follows that the ratio of zeta potential to surface area, which scales as the electrical field (by Gaussian law), serves as an appropriate indicator for optimal cellular response. The phase plot spanned by fractional survival and effective electric field (charge density) indicates a positive correlation between mean cell survival and the magnitude of the electric field. The phase plot spanned by fractional survival and effective electric field (charge density) associated with the nanosurface shows a bifurcation behavior. Wide variation of cell survival response is observed at certain critical values of the surface charge density, whereas in other ranges the cellular response is well behaved and more predictable. Existence of phase points near the critical region corresponds to wide fluctuation of nanoparticle-induced response, for small changes in the nanosurface property. Smaller nanoparticles with low zeta potential (e.g., those conjugated with arginine) can have such an attribute (i.e., higher electrical field strength), and eventually they cause more cell death. The study may help in optimal design of nanodrugs. From the Clinical EditorIn this paper, important aspects of the background theory of optimizing gold nanoparticles for cancer therapy is discussed. Such nanoparticles have an enormous potential in future human applications once we fully understand the design parameters.

Forkhead Box O as a Sensor, Mediator, and Regulator of Redox Signaling

The forkhead box O (FOXO) family of transcription factors regulates a variety of cellular programs, including cell cycle arrest, reactive oxygen species (ROS) scavenging, and apoptosis, and are of key importance in the decision over cell fate. In animal model systems it has been shown that FOXO is involved in the regulation of long lifespan. FOXO activity is tightly controlled by the insulin signaling pathway and by a multitude of ROS-induced posttranslational modifications. In the cell, ROS levels can be sensed by virtue of stimulatory and inhibitory oxidative modification of cysteine residues within proteins that control various signaling cascades. Recently, it was shown that cysteines in FOXO can also act as sensors of the local redox state. In this review we have outlined the cysteine-dependent redox switches that regulate both the insulin and ROS signaling pathways upstream of FOXO. Further, we describe how FOXO controls ROS levels by transcriptional regulation of a multilayered antioxidant system. Finally, we will discuss how cysteine-based redox signaling to FOXO could play a role in fine-tuning the optimal cellular response to ROS to control organismal lifespan.

T Cell-dependent and -Independent IgA Responses: Role of TLR Signalling

Immunoglobulin A (IgA) represents the primary line of protection against incoming pathogens since it is the predominant isotype on mucosal surfaces. Mucosal surfaces are constantly exposed to inhaled, digested and sexually transmitted agents and therefore highly susceptible to infection by invading pathogens. Such pathogens typically carry pathogen-associated molecular patterns (PAMPs) which primarily signal through Toll-like receptors (TLRs). TLRs belong to a family of pattern-recognition receptors that link the innate and the acquired immune system. TLR stimulation in professional antigen-presenting cells (APCs) such as dendritic cells (DCs) is crucial for an optimal cellular and humoral immune response to be induced. Moreover TLRs have been shown to improve humoral responses by direct stimulation of B cells. Herein we review recent data, which points to a pivotal role of TLR signalling in controlling T-cell dependent and independent IgA responses both at mucosal and systemic levels. A better understanding of these mechanisms may facilitate the use of TLR agonists as adjuvants and consequently improve the development of effective mucosal vaccines.