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Aging is associated with cognitive decline and accumulation of senescent cells in various tissues and organs. Senolytic agents such as dasatinib and quercetin (D+Q) in combination have been shown to target senescent cells and ameliorate symptoms of aging-related disorders in mouse models. However, the mechanisms by which senolytics improve cognitive impairments have not been fully elucidated particularly in species other than mice. To study the effect of senolytics on aging-related multifactorial cognitive dysfunctions we tested the spatial memory of male Wistar rats in an active allothetic place avoidance task. Here we report that 8 weeks treatment with D+Q alleviated learning deficits and memory impairment observed in aged animals. Furthermore, treatment with D+Q resulted in a reduction of the peripheral inflammation measured by the levels of serum inflammatory mediators (including members of senescent cell secretome) in aged rats. Significant improvements in cognitive abilities observed in aged rats upon treatment with D+Q were associated with changes in the dendritic spine morphology of the apical dendritic tree from the hippocampal CA1 neurons and changes in the level of histone H3 trimethylation at lysine 9 and 27 in the hippocampus. The beneficial effects of D+Q on learning and memory in aged rats were long-lasting and persisted at least 5 weeks after the cessation of the drugs administration. Our results expand and provide new insights to the existing knowledge associated with effects of senolytics on alleviating age-related associated cognitive dysfunctions.

Major depressive disorder is a complex disease resulting from aberrant synaptic plasticity that may be caused by abnormal serotonergic signaling. Using a combination of behavioral, biochemical, and imaging methods, we analyze 5-HT7R/MMP-9 signaling and dendritic spine plasticity in the hippocampus in mice treated with the selective 5-HT7R agonist (LP-211) and in a model of chronic unpredictable stress (CUS)-induced depressive-like behavior. We show that acute 5-HT7R activation induces depressive-like behavior in mice in an MMP-9-dependent manner and that post mortem brain samples from human individuals with depression reveal increased MMP-9 enzymatic activity in the hippocampus. Both pharmacological activation of 5-HT7R and modulation of its downstream effectors as a result of CUS lead to dendritic spine elongation and decreased spine density in this region. Overall, the 5-HT7R/MMP-9 pathway is specifically activated in the CA1 subregion of the hippocampus during chronic stress and is crucial for inducing depressive-like behavior.

Aging of the brain can manifest itself as a memory and cognitive decline, which has been shown to frequently coincide with changes in the structural plasticity of dendritic spines. Decreased number and maturity of spines in aged animals and humans, together with changes in synaptic transmission, may reflect aberrant neuronal plasticity directly associated with impaired brain functions. In extreme, a neurodegenerative disease, which completely devastates the basic functions of the brain, may develop. While cellular senescence in peripheral tissues has recently been linked to aging and a number of aging-related disorders, its involvement in brain aging is just beginning to be explored. However, accumulated evidence suggests that cell senescence may play a role in the aging of the brain, as it has been documented in other organs. Senescent cells stop dividing and shift their activity to strengthen the secretory function, which leads to the acquisition of the so called senescence-associated secretory phenotype (SASP). Senescent cells have also other characteristics, such as altered morphology and proteostasis, decreased propensity to undergo apoptosis, autophagy impairment, accumulation of lipid droplets, increased activity of senescence-associated-β-galactosidase (SA-β-gal), and epigenetic alterations, including DNA methylation, chromatin remodeling, and histone post-translational modifications that, in consequence, result in altered gene expression. Proliferation-competent glial cells can undergo senescence both in vitro and in vivo, and they likely participate in neuroinflammation, which is characteristic for the aging brain. However, apart from proliferation-competent glial cells, the brain consists of post-mitotic neurons. Interestingly, it has emerged recently, that non-proliferating neuronal cells present in the brain or cultivated in vitro can also have some hallmarks, including SASP, typical for senescent cells that ceased to divide. It has been documented that so called senolytics, which by definition, eliminate senescent cells, can improve cognitive ability in mice models. In this review, we ask questions about the role of senescent brain cells in brain plasticity and cognitive functions impairments and how senolytics can improve them. We will discuss whether neuronal plasticity, defined as morphological and functional changes at the level of neurons and dendritic spines, can be the hallmark of neuronal senescence susceptible to the effects of senolytics.

SmartFlare™ RNA Detection Probes from Millipore is a novel technology to detect RNA in live cells based on the use of 12 nm gold nanoparticles coated with nucleotides. We proved that SmartFlares™ are internalized by human primary lymphocytes. However, fluorescence signals from target RNA detection can only be observed in the presence of Fetal Bovine Serum (FBS) in the medium, whereas it is not detectable without FBS or when medium is supplemented with human albumin. Image analysis of fluorescence generated from SmartFlare™ Uptake Control (gives constant signal regardless of contact with RNA) and RNA Specific Probes revealed further differences. In the presence of FBS, the fluorescence signal for both reagents was diffused within the cells, whereas in the absence of FBS, it was detected as single spots within the cells only when the Uptake Control was used. It is possible that FBS components are necessary for SmartFlare™ Probes to be released from cellular compartments into the cytoplasm where they can get into contact with target RNA. The exact mechanism of this phenomena should be further determined. However, for the first time, we present here that FBS in the cell culture medium is essential for RNA detection by SmartFlare™ technology in human lymphocytes.

The precise regulation of synaptic integrity is critical for neuronal network connectivity and proper brain function. Essential aspects of the activity and localization of synaptic proteins are regulated by posttranslational modifications. S-palmitoylation is a reversible covalent modification of the cysteine with palmitate. It modulates affinity of the protein for cell membranes and membranous compartments. Intracellular palmitoylation dynamics are regulated by crosstalk with other posttranslational modifications, such as S-nitrosylation. S-nitrosylation is a covalent modification of cysteine thiol by nitric oxide and can modulate protein functions. Therefore, simultaneous identification of endogenous site-specific proteomes of both cysteine modifications under certain biological conditions offers new insights into the regulation of functional pathways. Still unclear, however, are the ways in which this crosstalk is affected in brain pathology, such as stress-related disorders. Using a newly developed mass spectrometry-based approach Palmitoylation And Nitrosylation Interplay Monitoring (PANIMoni), we analyzed the endogenous S-palmitoylation and S-nitrosylation of postsynaptic density proteins at the level of specific single cysteine in a mouse model of chronic stress. Among a total of 813 S-PALM and 620 S-NO cysteine sites that were characterized on 465 and 360 proteins, respectively, we sought to identify those that were differentially affected by stress. Our data show involvement of S-palmitoylation and S-nitrosylation crosstalk in the regulation of 122 proteins including receptors, scaffolding proteins, regulatory proteins and cytoskeletal components. Our results suggest that atypical crosstalk between the S-palmitoylation and S-nitrosylation interplay of proteins involved in synaptic transmission, protein localization and regulation of synaptic plasticity might be one of the main events associated with chronic stress disorder, leading to destabilization in synaptic networks.

Ketamine is an N-methyl-d-aspartate receptor antagonist that has gained wide attention as a potent antidepressant. It has also been recently reported to have prophylactic effects in animal models of depression and anxiety. Alterations of neuroplasticity in different brain regions, such as the hippocampus, prefrontal cortex, and amygdala, are a hallmark of stress-related disorders, and such changes may endure beyond the treatment of symptoms. The present study investigated whether a prophylactic injection of ketamine has effects on structural plasticity in the brain in mice that are subjected to chronic unpredictable stress followed by an 8-day recovery period. Ketamine administration (3 mg/kg body weight) 1 h before stress exposure increased the number of resilient animals immediately after the cessation of stress exposure and positively influenced the recovery of susceptible animals to hedonic deficits. At the end of the recovery period, ketamine-treated animals exhibited significant differences in dendritic spine density and dendritic spine morphology in brain regions associated with depression compared with saline-treated animals. These results confirm previous findings of the prophylactic effects of ketamine and provide further evidence of an association between the antidepressant-like effect of ketamine and alterations of structural plasticity in the brain

Background We showed that T regulatory (Treg) cells can be attached to the surface of pancreatic islets providing local immunoprotection. Further optimization of the method can improve coating efficiency, which may prolong graft survival. In this study, we compared the effectiveness of two different molecules used for binding of the Tregs to the surface of pancreatic islets. Our aim was to increase the number of Treg cells attached to islets without compromising islets viability and function. Methods The cell surface of human Treg cells and pancreatic islets was modified using biotin-polyethylene glycol-N-hydroxylsuccinimide (biotin-PEG-NHS) or biotin-PEG-succinimidyl valeric acid ester (biotin-PEG-SVA). Then, islets were incubated with streptavidin as islet/Treg cells binding molecule. Treg cells were stained with CellTracker CM-DiL dye and visualized using a Laser Scanning Confocal Microscope. The number of Treg cells attached per islets surface area was analyzed by Imaris software. The effect of coating on islet functionality was determined using the glucose-stimulated insulin response (GSIR) assay. Results The coating procedure with biotin-PEG-SVA allowed for attaching 40% more Treg cells per 1 μm2 of islet surface. Although viability was comparable, function of the islets after coating using the biotin-PEG-SVA molecule was better preserved than with NHS molecule. GSIR was 62% higher for islets coated with biotin-PEG-SVA compared to biotin-PEG-NHS. Conclusion Coating of islets with Treg cells using biotin-PEG-SVA improves effectiveness with better preservation of the islet function. Improvement of the method of coating pancreatic islets with Treg cells could further facilitate the effectiveness of this novel immunoprotective approach and translation into clinical settings.

A peptidomimetic called A20 (Cystapep 1) structurally based upon the N-terminal fragment of human cystatin C is known to have strong antibacterial properties. A20 is characterized by high activity against several bacterial strains often isolated from infected wounds, including methicillin-resistant S. aureus (MRSA). In this work we wanted to explore the therapeutic potential of A20 in the treatment of wound infections. We examined, cytotoxicity, allergenicity and impact of A20 on the proliferation and viability of human keratinocytes. Furthermore, the previously described antimicrobial action of A20. has been confirmed here with reference strains of bacteria and extended by several other species. The A20 was highly active against Gram-positive bacteria with minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) between 8 and 128. μg/mL. A20 did not affect proliferation of primary human keratinocytes in concentrations up to 50. μg/mL. At the same time, it did not activate Peripheral Blood Mononuclear Cells (PBMCs), including basophils or neutrophils in vitro. Interestingly A20 was found to display immunomodulatory functions as it influences the production of Th2 cytokines (IL-4 and IL-13) by activated PBMCs. It was also resistant to degradation for at least 48. h in human plasma. The results indicate that A20 is effective against the multiantibiotic-resistant bacteria and has a high safety profile, which makes it a promising antimicrobial drug candidate. (Less)

T regulatory cells (Tregs) play a critical role in the immunologic tolerance to the graft in transplantation. Thus, due to their immunosuppressive capability, ex vivo expanded Tregs may be used as a cellular therapy and an attractive novel strategy to control chronic rejection and eliminate need for lifelong pharmacological immunosuppression. Since Treg therapy is still in its infancy, initially Tregs still need to be applied in combination with pharmacological agents to prevent rejection. Fortunately, some of the medications have been shown to enhance the function and number of Tregs. In the clinic, different immunosuppressive regimens are used for individual patients for different types of organ transplantation. In this review, we present the most commonly used pharmacological agents for immunosuppression and discuss how they affect the Treg population. It is extremely difficult to dissect the effect of single agent on Tregs population in clinical settings since usually the combination of several medications is applied at the same time for graft protection. Nevertheless, experimental and clinical data indicate that thymoglobulin as immunosuppressive induction and mTOR inhibitors as immunosuppressive maintenance agents have the most beneficial effect on Treg population in the blood. Among supplemental agents promoting Tregs, anti-TNFα preparations have been in clinical use (in autoimmune diseases) for many years, so they are optimal candidates for testing in transplant settings in combination with Treg based cellular therapy.

Regulatory T cells (Treg) play pivotal role in the maintenance of immune homeostasis due to their suppressive abilities. It is important to understand the nature of Treg and the mechanisms by which they function. From recent studies, we can conclude that the development and function of Treg cells is strongly dependent on gene expression. Furthermore, a variety of transcription factors have been proposed to either maintain or inhibit their properties. As it was demonstrated a decade ago, Forkhead box P3 transcription factor (FoxP3), a Treg marker, has the ability to keep them on the right immunosuppressive track. Whether the Treg lineage has the ability of being suppressive or not depends on up- or down-regulation of the foxp3 gene. It can be controlled by other factors present inside the cell. Two of them, Helios and SATB1, are considered to be important in proper Treg development. Helios, a member of the Ikaros family, has been shown to up-regulate expression of FoxP3 protein, whereas SATB1 is known to inhibit its expression. In this review, we will discuss the relations between these three factors, and how they affect Treg development and function.

Ex vivo expanded CD4+CD25highCD127- T regulatory cells (Tregs) are recognized as a promising candidate for immunosuppressive therapy in humans. However, due to the plasticity of Tregs lineage and artificial environment present during ex vivo expansion, Tregs easily lose suppressive activity. Here, we followed expanding CD4+CD25highCD127- Tregs and their naive (CD45RA+) and memory-like (CD45RA-) subsets in order to establish the best conditions of the expansion. We found that, regardless of the phenotype sorted, expanding Tregs were undergoing changes resembling homeostatic proliferation and transformed into effector memory-like cells which produced not only suppressive interleukin-10 (IL-10) but also IL-6, IL-17, and interferon-γ (IFN-γ). With the time ex vivo, Tregs were losing the expression of FoxP3 and suppressive activity both when stimulated and when at rest. The only variable that helped preserve suppressive abilities of Tregs was the limitation of the time of ex vivo cultures to 2 weeks only. According to our study, the highest number of highly suppressive Tregs could be yielded with CD4+CD25highCD127- Tregs cultured no longer than 2 weeks. Thorough quality check, preferentially with the assessment of FoxP3 expression and IFN-γ suppression assay, should be applied to assess suppressive activity of the cells.

Here, we describe a procedure and first-in-man clinical effects of adoptive transfer of ex vivo expanded CD4+CD25+CD127− T regulatory cells (Tregs) in the treatment of graft versus host disease (GvHD). The cells were sorted from buffy coats taken from two family donors, expanded ex vivo and transferred to respective recipients who suffered from either acute or chronic GvHD. The therapy allowed for significant alleviation of the symptoms and reduction of pharmacologic immunosuppression in the case of chronic GvHD, while in the case of grade IV acute GvHD it only transiently improved the condition, for the longest time within all immunosuppressants used nonetheless.

A series of analogues of trypsin inhibitor SFTI-1 were designed and synthesized to monitor peptide splicing. In the middle part of the SFTI-1 analogues, which is released upon incubation with proteinase, the RGD sequence or an acceptor of fluorescence for FRET was introduced. The results of studies with trypsin confirmed that the designed analogues underwent peptide splicing. Furthermore, we showed that a FRET displaying SFTI-1 analogue was internalized into the HaCaT keratinocytes, where it was degraded. Therefore, both proteolysis and the reduction of the disulfide bridge of the peptide took place. As a result, such analogues are a convenient tool to trace the proteolytic activity inside the cell. However, the cytotoxicity of SFTI-1 analogues grafted with the RGD sequence did not correlate with their susceptibility to peptide splicing. Nevertheless, these peptides were slightly more active than the reference peptide (GRGDNP). Interestingly, one of the analogues assigned as [desSer6]VI, under experimental conditions, appeared significantly more cytotoxic towards cancer cells U87-MG in contrast to the reference peptide. This article is protected by copyright. All rights reserved.

Purpose of review T regulatory cells (Tregs) play a central role in maintaining immune homeostasis and peripheral tolerance to foreign antigens in humans. The immune response to alloantigens and recurrence of autoimmunity contribute to pancreatic islet transplant dysfunction, hence the adoptive transfer of Tregs has the potential to significantly improve islet graft survival. In this review, we provide an in-depth analysis of challenges associated with the application of ex-vivo expanded Tregs therapy in pancreatic islet transplant. Recent findings Tregs administered systemically may poorly migrate to the site of transplantation, which is critical for tolerance induction and graft protection. Intraportal administration of pancreatic tissue exerts some limitations on the ability to cotransplant Tregs at the same site of islet transplantation. In order to maximize therapeutic potential of Tregs, islet transplantation protocols may need additional refinement. Further to this, the Tregs may require cryopreservation in order to make them readily available at the same time as islet transplant. Summary On the basis of current experience and technology, the combination of islet and Treg cotransplantation is feasible and has great potential to improve islet graft survival. The possibility to wean off, or withdraw, traditional immunosuppressive agents and improve patient quality of life makes it an interesting avenue to be pursued.

Pancreatic islet mass, represented by islet equivalent (IEQ), is the most important parameter in decision making for clinical islet transplantation. To obtain IEQ, the sample of islets is routinely counted under a microscope and discarded thereafter. Islet purity, another parameter in islet processing, is routinely assessed by estimation only. In this study, we validated our digital image analysis (DIA) system by using the software of Image Pro Plus and a custom-designed Excel template to assess islet mass and purity to better comply with current good manufacturing practice (cGMP) standards. Human islet samples (60 collected from a single isolation and 24 collected from 12 isolations) were captured as calibrated digital images for the permanent record. Seven trained technicians participated in determination of IEQ and purity by the manual counting method (manual image counting, Manual I) and DIA. IEQ count showed statistically significant correlations between the Manual I and DIA in all sample comparisons ( r > 0.819 and p < 0.0001). A statistically significant difference in IEQ between Manual I and DIA was not found in all sample groups ( p > 0.05). In terms of purity determination, statistically significant differences between assessment and DIA measurement were found in high-purity 100-μl samples ( p < 0.005) and low-purity 100-μl samples ( p < 0.001) of the single isolation. In addition, islet particle number (IPN) and the IEQ/IPN ratio did not differ statistically between Manual I and DIA. In conclusion, the DIA used in this study is a reliable technique to determine IEQ and purity. Islet sample preserved as a digital image and results produced by DIA can be permanently stored for verification, technical training, and information exchange among islet centers. Therefore, DIA complies better with cGMP requirements than the manual counting method. We propose DIA as a quality control tool to supplement the established standard manual method for islet counting and purity estimation.

A recently discovered population of lymphocytes, called T regulatory cells (Tregs), is characterized by expression of transcription factor Forkhead box P3 (FoxP3). These cells have been successfully used as therapeutic treatments and prophylaxis for graft-versus-host disease (GVHD) and diabetes and might become an attractive alternative to traditional immunotherapy. Here we evaluated how the type of culture medium and the type of serum can influence yield and quality of Tregs after in vitro expansion. We compared Treg fold of expansion and their phenotypical characteristics including expression of FoxP3, CD25, CD127, CD62L and CD45RA in three commercially available culture media (RPMI 1640 (Cellgro; Manassas VA, USA), SCGM (Cellgenix; Freiburg, Germany), and X-VIVO 20 (Lonza; Walkersville, MD, USA)) with addition of human serum (HS, 10%) or fetal bovine serum (FBS, 10%). Among the tested media, X-VIVO 20 supplemented with HS produced the highest yield after 17days of in vitro expansion (a median of 86-fold expansion, range 30-1365) and highest level of FoxP3 expression (a median of 66.8% of positive cells, range 56-84.8%) in CD4(+) CD25(hi)CD127(lo/neg) FACS sorted polyclonal Tregs. There was no difference in Tregs yield whether HS or FBS serum was used. In conclusion, the yield of the ex vivo expanded Tregs is related to the type of media applied. Supplementation of the culture with FBS or human serum is equally beneficial.

Promising results of initial studies applying ex-vivo expanded regulatory T cell (Treg) as a clinical intervention have increased interest in this type of the cellular therapy and several new clinical trials involving Tregs are currently on the way. Methods of isolation and expansion of Tregs have been studied and optimized to the extent that such therapy is feasible, and allows obtaining sufficient numbers of Tregs in the laboratory following Good Manufacturing Practice (GMP) guidelines. Nevertheless, Treg therapy could even more rapidly evolve if Tregs could be efficiently cryopreserved and stored for future infusion or expansions rather than utilization of only freshly isolated and expanded cells as it is preferred now. Currently, our knowledge regarding the impact of cryopreservation on Treg recovery, viability, and functionality is still limited. Based on experience with cryopreserved peripheral blood mononuclear cells (PBMCs), cryopreservation may have a detrimental effect on Tregs, can decrease Treg viability, cause abnormal cytokine secretion, and compromise expression of surface markers essential for proper Treg function and processing. Therefore, optimal strategies and conditions for Treg cryopreservation in conjunction with cell culture, expansion, and processing for clinical application still need to be investigated and defined.

OBJECTIVES To develop a novel approach for local immunoprotection using CD4(+)CD25(high)CD127(-) T regulatory cells (Tregs) attached to the surface of the islets before transplantation. BACKGROUND Tregs expanded ex vivo can control allo and autoreactivity, therefore, Treg-based therapy may offer more effective protection for transplanted islets from immunologic attack than currently used immunosuppression. Local application of Tregs can make such therapy more clinically feasible and efficient. METHODS Human islets were isolated and coated with allogeneic ex vivo expanded Tregs using biotin-poly(ethylene glycol)-N-hydroxysuccinimide ester (biotin-PEG-NHS) and streptavidin as binding molecules. RESULTS Coating pancreatic islets with Tregs did not affect islet viability (>90% fluorescein diacetate/propidium iodide) or the insulin secretion profile in dynamic islet perifusion assays. After in vitro incubation with allogeneic T effector cells, Treg-coated islets revealed preserved function with higher insulin secretion compared with controls-native islets, coated islets with T effector cells or when Tregs were added to the culture, but not attached to islets (P < 0.05). In addition, the Enzyme-linked immunosorbent spot (ELISPOT) assay revealed suppression of interferon (IFN)-γ secretion, when T effector cells were challenged with Treg-coated islets comparing to controls (99 ± 7 vs 151 ± 8 dots, respectively; P < 0.01). CONCLUSIONS We demonstrated, for the first time, the ability to bind immune regulatory cells to target cells with preservation of their viability and function and protective activity against immune attack. If successfully tested in an animal model, local delivery of immunoprotective Tregs on the surface of transplanted pancreatic islets may be an alternative or improvement to the currently used immunosuppression.