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Abstract Background Signal transduction processes mediated by phosphatidyl inositol phosphates affect a broad range of cellular processes such as cell cycle progression, migration and cell survival. The protein kinase AKT is one of the major effectors in this signaling network. Chronic AKT activation contributes to oncogenic transformation and tumor development. Therefore, analogs of phosphatidyl inositol phosphates (PIAs) were designed as new small drugs to block AKT activity for cancer treatment. Here we characterize the biological effects of the PIAs SH-5 and SH-6 in colorectal cancer cell lines. Methods Serum-starved or serum-supplemented human colorectal cancer cell lines SW480, HT29 and HCT116 were exposed to SH-5 and SH-6. AKT activation was determined by western blotting. Cell viability was assessed using a colorimetric XTT-based assay, apoptosis and cell cycle changes were monitored by FACS analysis. The dynamics of cell morphology alterations was evaluated by confocal and time-lapse microscopy. Transcriptional changes due to inhibitor treatment were analyzed using Affymetrix HG-U133A microarrays and RT-PCR. Results While the PIAs clearly reduce AKT phosphorylation in serum starved cells, we did not observe a significant reduction under serum supplemented conditions, giving us the opportunity to analyze AKT independent effects of these compounds. Both inhibitors induce broadly the same morphological alterations, in particular changes in cell shape and formation of intracellular vesicles. Moreover, we observed the induction of binucleated cells specifically in the SW480 cell line. Gene expression analysis revealed transcriptional alterations, which are mostly cell line specific. In accordance to the phenotype we found a gene group associated with mitosis and spindle organization down regulated in SW480 cells, but not in the other cell lines. A bioinformatics analysis using the Connectivity Map linked the gene expression pattern of the inhibitor treated SW480 cells to PKC signaling. Using confocal laser scanning microscopy and time lapse recording we identified a specific defect in the last step of the cytokinesis as responsible for the binucleation. Conclusions The PIAs SH-5 and SH-6 impinge on additional cellular targets apart from AKT in colorectal cancer cells. The effects are mostly cell line specific and have an influence at the outcome of the treatment. In view of potential clinical trials it will be necessary to take these diverse effects into consideration to optimize patient treatment.

Background: Signal transduction processes mediated by phosphatidyl inositol phosphates affect a broad range of cellular processes such as cell cycle progression, migration and cell survival. The protein kinase AKT is one of the major effectors in this signaling network. Chronic AKT activation contributes to oncogenic transformation and tumor development. Therefore, analogs of phosphatidyl inositol phosphates (PIAs) were designed as new small drugs to block AKT activity for cancer treatment. Here we characterize the biological effects of the PIAs SH-5 and SH-6 in colorectal cancer cell lines.Methods: Serum-starved or serum-supplemented human colorectal cancer cell lines SW480, HT29 and HCT116 were exposed to SH-5 and SH-6. AKT activation was determined by western blotting. Cell viability was assessed using a colorimetric XTT-based assay, apoptosis and cell cycle changes were monitored by FACS analysis. The dynamics of cell morphology alterations was evaluated by confocal and time-lapse microscopy. Transcriptional changes due to inhibitor treatment were analyzed using Affymetrix HG-U133A microarrays and RT-PCR.Results: While the PIAs clearly reduce AKT phosphorylation in serum starved cells, we did not observe a significant reduction under serum supplemented conditions, giving us the opportunity to analyze AKT independent effects of these compounds. Both inhibitors induce broadly the same morphological alterations, in particular changes in cell shape and formation of intracellular vesicles. Moreover, we observed the induction of binucleated cells specifically in the SW480 cell line. Gene expression analysis revealed transcriptional alterations, which are mostly cell line specific. In accordance to the phenotype we found a gene group associated with mitosis and spindle organization down regulated in SW480 cells, but not in the other cell lines. A bioinformatics analysis using the Connectivity Map linked the gene expression pattern of the inhibitor treated SW480 cells to PKC signaling. Using confocal laser scanning microscopy and time lapse recording we identified a specific defect in the last step of the cytokinesis as responsible for the binucleation.Conclusions: The PIAs SH-5 and SH-6 impinge on additional cellular targets apart from AKT in colorectal cancer cells. The effects are mostly cell line specific and have an influence at the outcome of the treatment. In view of potential clinical trials it will be necessary to take these diverse effects into consideration to optimize patient treatment. [ABSTRACT FROM AUTHOR]

Background: The increasing number of care-dependent individuals requires approaches to prevent care dependency or reduce the loss of independence. Long-term care assessments can provide valuable insights into this., Objective: The aim of this article is to describe initial applicants with an identified need for long-term care as well as to provide a differentiated analysis of care-related diagnoses by age, gender, care level and federal state., Material and Methods: The nationwide database consists of long-term care assessments conducted by the Medical Service (MD) of individuals insured with the AOK aged 60 years and above who received a care level (PG) for the first time in 2021. Information relevant to long-term care was analyzed descriptively., Results: In this study 339,486 individuals with an average age of 79.6 years (±8.4 years) and a female proportion of 59.0% were analyzed. Approximately one half received care level 2 and 32.4% received care level 1. Care levels 3-5 were assessed less frequently (16.2% vs. 4.8% vs. 1.7%, respectively). Individuals living alone were represented more strongly in lower care levels, while individuals not living alone had a higher proportion in care levels 3-5. The most frequent care-relevant diagnoses were senility (R54), polyarthritis (M15) and dementia (F03) with significant differences observed between federal states (ICD-10 R chapter: 0.8% Berlin and Brandenburg vs. 37.9% Saxony; M chapter: 13.6% Bavaria and Hamburg vs. 39.9% Mecklenburg-Western Pomerania)., Conclusion: Social determinants, such as age, gender, living alone, and region can play a role in the classification into a care level. Significant differences in care-related diagnoses between federal states warrant further investigation in future research., (© 2024. The Author(s).)

Cardiovascular diseases are a significant cause of illness and death worldwide, often resulting in myofibroblast differentiation, pathological remodeling, and fibrosis, characterized by excessive extracellular matrix protein deposition. Treatment options for cardiac fibrosis that can effectively target myofibroblast activation and ECM deposition are limited, necessitating an unmet need for new therapeutic approaches. In recent years, microcurrent therapy has demonstrated promising therapeutic effects, showcasing its translational potential in cardiac care. This study therefore sought to investigate the effects of microcurrent therapy on cardiac myofibroblasts, aiming to unravel its potential as a treatment for cardiac fibrosis and heart failure. The experimental design involved the differentiation of primary rat cardiac fibroblasts into myofibroblasts. Subsequently, these cells were subjected to microcurrent (MC) treatment at 1 and 2 µA/cm 2 DC with and without polarity reversal. We then investigated the impact of microcurrent treatment on myofibroblast cell behavior, including protein and gene expression, by performing various assays and analyses comparing them to untreated myofibroblasts and cardiac fibroblasts. The application of microcurrents resulted in distinct transcriptional signatures and improved cellular processes. Gene expression analysis showed alterations in myofibroblast markers, extracellular matrix components, and pro-inflammatory cytokines. These observations show signs of microcurrent-mediated reversal of myofibroblast phenotype, possibly reducing cardiac fibrosis, and providing insights for cardiac tissue repair.

The development of brain metastases hallmarks disease progression in 20-40% of melanoma patients and is a serious obstacle to therapy. Understanding the processes involved in the development and maintenance of melanoma brain metastases (MBM) is critical for the discovery of novel therapeutic strategies. Here, we generated transcriptome and methylome profiles of MBM showing high or low abundance of infiltrated Iba1 high tumor-associated microglia and macrophages (TAMs). Our survey identified potential prognostic markers of favorable disease course and response to immune checkpoint inhibitor (ICi) therapy, among them APBB1IP and the interferon-responsive gene ITGB7. In MBM with high ITGB7/APBB1IP levels, the accumulation of TAMs correlated significantly with the immune score. Signature-based deconvolution of MBM via single sample GSEA revealed enrichment of interferon-response and immune signatures and revealed inflammation, stress and MET receptor signaling. MET receptor phosphorylation/activation maybe elicited by inflammatory processes in brain metastatic melanoma cells via stroma cell-released HGF. We found phospho-MET Y1234/1235 in a subset of MBM and observed a marked response of brain metastasis-derived cell lines (BMCs) that lacked druggable BRAF mutations or developed resistance to BRAF inhibitors (BRAFi) in vivo to MET inhibitors PHA-665752 and ARQ197 (tivantinib). In summary, the activation of MET receptor in brain colonizing melanoma cells by stromal cell-released HGF may promote tumor self-maintenance and expansion and might counteract ICi therapy. Therefore, therapeutic targeting of MET possibly serves as a promising strategy to control intracranial progressive disease and improve patient survival., (© 2024. The Author(s).)

Background: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses., Methods: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry., Results: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA-expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair., Conclusions: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Hospital admissions due to acute cardiovascular events dropped during the COVID-19 pandemic in the general population; however, evidence for residents of long-term care facilities (LTCF) is sparse. We investigated rates of hospital admissions and deaths due to myocardial infarction (MI) and stroke in LTCF residents during the pandemic. Our nationwide cohort study used claims data. The sample comprised 1,140,139 AOK-ensured LTCF residents over 60 years of age (68.6% women; age 85.3 ± 8.5 years) from the largest statutory health insurance in Germany (AOK), which is not representative for all LTCF residents. We included MI and stroke admission and compared numbers of in-hospital deaths from January 2020 to end of April 2021 (i.e., during the first three waves of the pandemic) with the number of incidences in 2015-2019. To estimate incidence risk ratios (IRR), adjusted Poisson regression analyses were applied. During the observation period (2015-2021), there were 19,196 MI and 73,953 stroke admissions. MI admissions declined in the pandemic phase by 22.5% (IRR = 0.68 [CI 0.65-0.72]) compared to previous years. This decline was slightly more pronounced for NSTEMI than for STEMI. MI fatality risks remained comparable across years (IRR = 0.97 [CI95% 0.92-1.02]). Stroke admissions dropped by 15.1% (IRR = 0.75 [CI95% 0.72-0.78]) in the pandemic. There was an elevated case fatality risk for haemorrhagic stroke (IRR = 1.09 [CI95% 1.03-1.15]) but not for other stroke subtypes compared to previous years. This study provides first evidence of declines in MI and stroke admissions and in-hospital deaths among LTCF residents during the pandemic. The figures are alarming given the acute nature of the conditions and the vulnerability of the residents., (© 2023. The Author(s).)

According to official statistics from the German long-term care insurance funds, around six out of every one hundred people with statutory health insurance are considered to be in need of long-term care. In this context, "need of long-term care" is defined according to the Eleventh Book of the Social Code (SGB XI) and therefore follows a demand-driven understanding of care, which also aligns with public discourse.In order to meet the increasing number of people in need of long term care - mostly caused by demographic change - with needs-based service structures, knowledge and evaluation of several factors are necessary: the prevalence of care dependency as defined by the SGB XI, the different degrees of severity, and the utilisation of long-term care and healthcare services.In this respect, the article presents findings and calculations based on currently available administrative data from German health and long-term care insurance funds and states its limitations. In terms of an actual epidemiological approach to the topic of long-term care, the aim should be broader reporting based on primary surveys., (© 2023. The Author(s).)

Background: Exercise exerts many health benefits by directly inducing molecular alterations in physically utilized skeletal muscle. Molecular adaptations of subcutaneous adipose tissue (SCAT) might also contribute to the prevention of metabolic diseases., Aim: To characterize the response of human SCAT based on changes in transcripts and mitochondrial respiration to acute and repeated bouts of exercise in comparison to skeletal muscle., Methods: Sedentary participants (27 ± 4 yrs) with overweight or obesity underwent 8-week supervised endurance exercise 3×1h/week at 80% VO2peak. Before, 60 min after the first and last exercise bout and 5 days post intervention, biopsies were taken for transcriptomic analyses and high-resolution respirometry (n = 14, 8 female/6 male)., Results: In SCAT, we found 37 acutely regulated transcripts (FC > 1.2, FDR < 10%) after the first exercise bout compared to 394, respectively, in skeletal muscle. Regulation of only 5 transcripts overlapped between tissues highlighting their differential response. Upstream and enrichment analyses revealed reduced transcripts of lipid uptake, storage and lipogenesis directly after exercise in SCAT and point to β-adrenergic regulation as potential major driver. The data also suggest an exercise-induced modulation of the circadian clock in SCAT. Neither term was associated with transcriptomic changes in skeletal muscle. No evidence for beigeing/browning was found in SCAT along with unchanged respiration., Conclusions: Adipose tissue responds completely distinct from adaptations of skeletal muscle to exercise. The acute and repeated reduction in transcripts of lipid storage and lipogenesis, interconnected with a modulated circadian rhythm, can counteract metabolic syndrome progression toward diabetes., (© 2023. The Author(s).)

Currently available COVID-19 vaccines include inactivated virus, live attenuated virus, mRNA-based, viral vectored and adjuvanted protein-subunit-based vaccines. All of them contain the spike glycoprotein as the main immunogen and result in reduced disease severity upon SARS-CoV-2 infection. While we and others have shown that mRNA-based vaccination reactivates pre-existing, cross-reactive immunity, the effect of vector vaccines in this regard is unknown. Here, we studied cellular and humoral responses in heterologous adenovirus-vector-based ChAdOx1 nCOV-19 (AZ; Vaxzeria, AstraZeneca) and mRNA-based BNT162b2 (BNT; Comirnaty, BioNTech/Pfizer) vaccination and compared it to a homologous BNT vaccination regimen. AZ primary vaccination did not lead to measurable reactivation of cross-reactive cellular and humoral immunity compared to BNT primary vaccination. Moreover, humoral immunity induced by primary vaccination with AZ displayed differences in linear spike peptide epitope coverage and a lack of anti-S2 IgG antibodies. Contrary to primary AZ vaccination, secondary vaccination with BNT reactivated pre-existing, cross-reactive immunity, comparable to homologous primary and secondary mRNA vaccination. While induced anti-S1 IgG antibody titers were higher after heterologous vaccination, induced CD4 + T cell responses were highest in homologous vaccinated. However, the overall TCR repertoire breadth was comparable between heterologous AZ-BNT-vaccinated and homologous BNT-BNT-vaccinated individuals, matching TCR repertoire breadths after SARS-CoV-2 infection, too. The reasons why AZ and BNT primary vaccination elicits different immune response patterns to essentially the same antigen, and the associated benefits and risks, need further investigation to inform vaccine and vaccination schedule development., Competing Interests: The authors UR, PH, ME, KS are employees, HW is the CEO of JPT. LL, LH, JB and AT are named on a filed patent application regarding the usage of CD3 downregulation as method for direct analysis of functional avidity of T cells and a patent application regarding the usage of iCope as method for the direct analysis of SARS-CoV-2 immune responses. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Henze, Braun, Meyer-Arndt, Jürchott, Schlotz, Michel, Grossegesse, Mangold, Dingeldey, Kruse, Holenya, Mages, Reimer, Eckey, Schnatbaum, Wenschuh, Timmermann, Klein, Nitsche, Giesecke-Thiel, Loyal and Thiel.)

The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports 24 h gene expression rhythms in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep-wake schedules. 10% of the expressed genes showed such diurnal rhythms at the population level, of which only a third differed between the two layers. Amplitude and phases of diurnal gene expression varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in their variability across subjects. We also identified small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin and presents a novel methodology to quantify sources of variability in human circadian rhythms., (© The Author(s) 2022. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Melanoma brain metastases (MBM) variably respond to therapeutic interventions; thus determining patient's prognosis. However, the mechanisms that govern therapy response are poorly understood. Here, we use a multi-OMICS approach and targeted sequencing (TargetSeq) to unravel the programs that potentially control the development of progressive intracranial disease. Molecularly, the expression of E-cadherin (Ecad) or NGFR, the BRAF mutation state and level of immune cell infiltration subdivides tumors into proliferative/pigmented and invasive/stem-like/therapy-resistant irrespective of the intracranial location. The analysis of MAPK inhibitor-naive and refractory MBM reveals switching from Ecad-associated into NGFR-associated programs during progression. NGFR-associated programs control cell migration and proliferation via downstream transcription factors such as SOX4. Moreover, global methylome profiling uncovers 46 differentially methylated regions that discriminate BRAF mut and wildtype MBM. In summary, we propose that the expression of Ecad and NGFR sub- classifies MBM and suggest that the Ecad-to-NGFR phenotype switch is a rate-limiting process which potentially indicates drug-response and intracranial progression states in melanoma patients., (© 2022. The Author(s).)

Stromal cells interact with immune cells during initiation and resolution of immune responses, though the precise underlying mechanisms remain to be resolved. Lessons learned from stromal cell-based therapies indicate that environmental signals instruct their immunomodulatory action contributing to immune response control. Here, to the best of our knowledge, we show a novel function for the guanine-exchange factor DOCK2 in regulating immunosuppressive function in three human stromal cell models and by siRNA-mediated DOCK2 knockdown. To identify immune function-related stromal cell molecular signatures, we first reprogrammed mesenchymal stem/progenitor cells (MSPCs) into induced pluripotent stem cells (iPSCs) before differentiating these iPSCs in a back-loop into MSPCs. The iPSCs and immature iPS-MSPCs lacked immunosuppressive potential. Successive maturation facilitated immunomodulation, while maintaining clonogenicity, comparable to their parental MSPCs. Sequential transcriptomics and methylomics displayed time-dependent immune-related gene expression trajectories, including DOCK2, eventually resembling parental MSPCs. Severe combined immunodeficiency (SCID) patient-derived fibroblasts harboring bi-allelic DOCK2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated fibroblasts. Conditional DOCK2 siRNA knockdown in iPS-MSPCs and fibroblasts also immediately reduced immunomodulatory capacity. Conclusively, CRISPR/Cas9-mediated DOCK2 knockout in iPS-MSPCs also resulted in significantly reduced immunomodulation, reduced CDC42 Rho family GTPase activation and blunted filopodia formation. These data identify G protein signaling as key element devising stromal cell immunomodulation., (© 2022. The Author(s).)

Background: SARS-CoV-2 mRNA vaccination of healthy individuals is highly immunogenic and protective against severe COVID-19. However, there are limited data on how disease-modifying therapies (DMTs) alter SARS-CoV-2 mRNA vaccine immunogenicity in patients with autoimmune diseases., Methods: As part of a prospective cohort study, we investigated the induction, stability and boosting of vaccine-specific antibodies, B cells and T cells in patients with multiple sclerosis (MS) on different DMTs after homologous primary, secondary and booster SARS-CoV-2 mRNA vaccinations. Of 126 patients with MS analysed, 105 received either anti-CD20-based B cell depletion (aCD20-BCD), fingolimod, interferon-β, dimethyl fumarate, glatiramer acetate, teriflunomide or natalizumab, and 21 were untreated MS patients for comparison., Results: In contrast to all other MS patients, and even after booster, most aCD20-BCD- and fingolimod-treated patients showed no to markedly reduced anti-S1 IgG, serum neutralising activity and a lack of receptor binding domain-specific and S2-specific B cells. Patients receiving fingolimod additionally lacked spike-reactive CD4 + T cell responses. The duration of fingolimod treatment, rather than peripheral blood B and T cell counts prior to vaccination, determined whether a humoral immune response was elicited., Conclusions: The lack of immunogenicity under long-term fingolimod treatment demonstrates that functional immune responses require not only immune cells themselves, but also access of these cells to the site of inoculation and their unimpeded movement. The absence of humoral and T cell responses suggests that fingolimod-treated patients with MS are at risk for severe SARS-CoV-2 infections despite booster vaccinations, which is highly relevant for clinical decision-making and adapted protective measures, particularly considering additional recently approved sphingosine-1-phosphate receptor antagonists for MS treatment., Competing Interests: Competing interests: PH and UR are employed by JPT Peptide Technologies., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Intestinal parasitic nematodes affect a quarter of the world's population, typically eliciting prominent effector Th2-driven host immune responses. As not all infected hosts develop protection against reinfection, our current understanding of nematode-induced memory Th2 responses remains limited. Here, we investigated the activation of memory Th2 cells and the mechanisms driving early recall responses to the enteric nematode Heligmosomoides polygyrus in mice. We show that nematode-cured mice harbor memory Th2 cells in lymphoid and non-lymphoid organs with distinct transcriptional profiles, expressing recirculation markers like CCR7 and CD62-L in the mesenteric lymph nodes (mLN), and costimulatory markers like Ox40, as well as tissue homing and activation markers like CCR2, CD69 and CD40L in the gut and peritoneal cavity (PEC). While memory Th2 cells persist systemically in both lymphoid and non-lymphoid tissues following cure of infection, peritoneal memory Th2 cells in particular displayed an initial prominent expansion and strong parasite-specific Th2 responses during early recall responses to a challenge nematode infection. This effect was paralleled by a significant influx of dendritic cells (DC) and eosinophils, both also appearing exclusively in the peritoneal cavity of reinfected mice. In addition, we show that within the peritoneal membrane lined by peritoneal mesothelial cells (PeM), the gene expression levels of cell adhesion markers VCAM-1 and ICAM-1 decrease significantly in response to a secondary infection. Overall, our findings indicate that the host peritoneal cavity in particular harbors prominent memory Th2 cells and appears to respond directly to H. polygyrus by an early recall response via differential regulation of cell adhesion markers, marking the peritoneal cavity an important site for host immune responses to an enteric pathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yordanova, Jürchott, Steinfelder, Vogt, Krüger, Kühl, Sawitzki and Hartmann.)

Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

The prevailing 'division of labor' concept in cellular immunity is that CD8 + T cells primarily utilize cytotoxic functions to kill target cells, while CD4 + T cells exert helper/inducer functions. Multiple subsets of CD4 + memory T cells have been characterized by distinct chemokine receptor expression. Here, we demonstrate that analogous CD8 + memory T-cell subsets exist, characterized by identical chemokine receptor expression signatures and controlled by similar generic programs. Among them, Tc2, Tc17 and Tc22 cells, in contrast to Tc1 and Tc17 + 1 cells, express IL-6R but not SLAMF7, completely lack cytotoxicity and instead display helper functions including CD40L expression. CD8 + helper T cells exhibit a unique TCR repertoire, express genes related to skin resident memory T cells (T RM ) and are altered in the inflammatory skin disease psoriasis. Our findings reveal that the conventional view of CD4 + and CD8 + T cell capabilities and functions in human health and disease needs to be revised.

The worldwide epidemic of overweight and obesity has led to an increase in associated metabolic comorbidities. Obesity induces chronic low-grade inflammation in white adipose tissue (WAT). However, the function and regulation of both innate and adaptive immune cells in human WAT under conditions of obesity and calorie restriction (CR) is not fully understood yet. Using a randomized interventional design, we investigated postmenopausal overweight or obese female subjects who either underwent CR for 3 mo followed by a 4-wk phase of weight maintenance or had to maintain a stable weight over the whole study period. A comprehensive immune phenotyping protocol was conducted using validated multiparameter flow cytometry analysis in blood and s.c. WAT (SAT). The TCR repertoire was analyzed by next-generation sequencing and cytokine levels were determined in SAT. Metabolic parameters were determined by hyperinsulinemic-euglycemic clamp. We found that insulin resistance correlates significantly with a shift toward the memory T cell compartment in SAT. TCR analysis revealed a diverse repertoire in SAT of overweight or obese individuals. Additionally, whereas weight loss improved systemic insulin sensitivity in the intervention group, SAT displayed no significant improvement of inflammatory parameters (cytokine levels and leukocyte subpopulations) compared with the control group. Our data demonstrate the accumulation of effector memory T cells in obese SAT and an association between systemic glucose homeostasis and inflammatory parameters in obese females. The long-standing effect of obesity-induced changes in SAT was demonstrated by preserved immune cell composition after short-term CR-induced weight loss., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Psoriasis is a very common chronic inflammatory skin disease characterized by epidermal thickening and scaling resulting from keratinocyte hyperproliferation and impaired differentiation. Pathomechanistic studies in psoriasis are often limited by using whole skin tissue biopsies, neglecting their stratification and cellular diversity. This study aimed at characterizing epidermal alterations in psoriasis at the level of keratinocyte populations. Epidermal cell populations were purified from skin biopsies of psoriasis patients and healthy donors using a novel cell type-specific approach. Molecular characterization of the transit-amplifying cells (TAC), the key players of epidermal renewal, was performed using immunocytofluorescence-technique and integrated multiscale-omics analyses. Already TAC from non-lesional psoriatic skin showed altered methylation and differential expression in 1.7% and 1.0% of all protein-coding genes, respectively. In psoriatic lesions, TAC were strongly expanded showing further increased differentially methylated (10-fold) and expressed (22-fold) genes numbers. Importantly, 17.2% of differentially expressed genes were associated with respective gene methylations. Compared with non-lesional TAC, pathway analyses revealed metabolic alterations as one feature predominantly changed in TAC derived from active psoriatic lesions. Overall, our study showed stage-specific molecular alterations, allows new insights into the pathogenesis, and implies the involvement of epigenetic mechanisms in lesion development in psoriasis. KEY MESSAGES: Transit amplifying cell (TAC) numbers are highly increased in psoriatic lesions Psoriatic TAC show profound molecular alterations & stage-specific identity TAC from unaffected areas already show first signs of molecular alterations Lesional TAC show a preference in metabolic-related alterations.

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo -expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank ( n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics., (Copyright © 2019 Andrzejewska, Catar, Schoon, Qazi, Sass, Jacobi, Blankenstein, Reinke, Krüger, Streitz, Schlickeiser, Richter, Souidi, Beez, Kamhieh-Milz, Krüger, Zemojtel, Jürchott, Strunk, Reinke, Duda, Moll and Geissler.)

Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, we evaluated their inductive and modulatory capacity regarding immune responses and underlying key mechanisms in vitro . For this, cryopreserved hAACs were either cultured in the presence of interferon-gamma (IFNγ) or left unstimulated. The expression of characteristic mesenchymal stromal cell markers (CD29, CD44, CD73, CD105, CD166) was revealed by flow cytometry that also highlighted a predominant negativity for CD90. A low immunogeneic phenotype in an inflammatory milieu was shown by lacking expression of co-stimulatory molecules and upregulation of the inhibitory ligands PD-L1 and PD-L2, despite de novo expression of HLA-DR. Co-cultures of hAACs with allogeneic peripheral blood mononuclear cells, proved their low immunogeneic state by absence of induced T cell proliferation and activation. Additionally, elevated levels of IL-1β, IL-33, and IL-10 were detectable in those cell culture supernatants. Furthermore, the immunomodulatory potential of hAACs was assessed in co-cultures with αCD3/αCD28-activated peripheral blood mononuclear cells. Here, a strong inhibition of T cell proliferation and reduction of pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-17A, IL-2) were observable after pre-stimulation of hAACs with IFNγ. Transwell experiments confirmed that mostly soluble factors are responsible for these suppressive effects. We were able to identify indolamin-2,3-dioxygenase (IDO) as a potential key player through a genome-wide gene expression analysis and could demonstrate its involvement in the observed immunological responses. While the application of blocking antibodies against both PD-1 ligands did not affect the immunomodulation by hAACs, 1-methyl-L-tryptophan as specific inhibitor of IDO was able to restore proliferation and to lower apoptosis of T cells. In conclusion, hAACs represent a cardiac-derived mesenchymal stromal-like cell type with a high potential for the application in an allogeneic setting, since they do not trigger T cell responses and even increase their immunomodulatory potential in inflammatory environments.

Viral infections have a major impact on morbidity and mortality of immunosuppressed solid organ transplant (SOT) patients because of missing or failure of adequate pharmacologic antiviral treatment. Adoptive antiviral T-cell therapy (AVTT), regenerating disturbed endogenous T-cell immunity, emerged as an attractive alternative approach to combat severe viral complications in immunocompromised patients. AVTT is successful in patients after hematopoietic stem cell transplantation where T-cell products (TCPs) are manufactured from healthy donors. In contrast, in the SOT setting TCPs are derived from/applied back to immunosuppressed patients. We and others demonstrated feasibility of TCP generation from SOT patients and first clinical proof-of-concept trials revealing promising data. However, the initial efficacy is frequently lost long-term, because of limited survival of transferred short-lived T-cells indicating a need for next-generation TCPs. Our recent data suggest that Rapamycin treatment during TCP manufacture, conferring partial inhibition of mTOR, might improve its composition. The aim of this study was to confirm these promising observations in a setting closer to clinical challenges and to deeply characterize the next-generation TCPs. Using cytomegalovirus (CMV) as model, our next-generation Rapamycin-treated (Rapa-)TCP showed consistently increased proportions of CD4 + T-cells as well as CD4 + and CD8 + central-memory T-cells (T CM ). In addition, Rapamycin sustained T-cell function despite withdrawal of Rapamycin, showed superior T-cell viability and resistance to apoptosis, stable metabolism upon activation, preferential expansion of T CM , partial conversion of other memory T-cell subsets to T CM and increased clonal diversity. On transcriptome level, we observed a gene expression profile denoting long-lived early memory T-cells with potent effector functions. Furthermore, we successfully applied the novel protocol for the generation of Rapa-TCPs to 19/19 SOT patients in a comparative study, irrespective of their history of CMV reactivation. Moreover, comparison of paired TCPs generated before/after transplantation did not reveal inferiority of the latter despite exposition to maintenance immunosuppression post -SOT. Our data imply that the Rapa-TCPs, exhibiting longevity and sustained T-cell memory, are a reasonable treatment option for SOT patients. Based on our success to manufacture Rapa-TCPs from SOT patients under maintenance immunosuppression, now, we seek ultimate clinical proof of efficacy in a clinical study.

Influenza vaccination is a common approach to prevent seasonal and pandemic influenza. Pre-existing antibodies against close viral strains might impair antibody formation against previously unseen strains-a process called original antigenic sin. The role of this pre-existing cellular immunity in this process is, despite some hints from animal models, not clear. Here, we analyzed cellular and humoral immunity in healthy individuals before and after vaccination with seasonal influenza vaccine. Based on influenza-specific hemagglutination inhibiting (HI) titers, vaccinees were grouped into HI-negative and -positive cohorts followed by in-depth cytometric and TCR repertoire analysis. Both serological groups revealed cross-reactive T-cell memory to the vaccine strains at baseline that gave rise to the majority of vaccine-specific T-cells post vaccination. On the contrary, very limited number of vaccine-specific T-cell clones was recruited from the naive pool. Furthermore, baseline quantity of vaccine-specific central memory helper T-cells and clonotype richness of this population directly correlated with the vaccination efficacy. Our findings suggest that the deliberate recruitment of pre-existing cross-reactive cellular memory might help to improve vaccination outcome.

Recent findings demonstrated proinflammatory functions of interleukin (IL)-9-producing T helper type (Th) 9 cells in the pathogenesis of intestinal bowel diseases (IBDs). However, also antiinflammatory properties have been ascribed to Th9 cells, pointing to a functional heterogeneity. To dissect the specific expression pattern and, especially, diversity of murine antigen-specific Th9 cells, we applied single cell transcription profiling. Th9 cells displayed reduced expression of typical activation markers, such as Cd40 ligand and Cd96 , whereas expression of Cd25 and Cd83 was increased compared with other Th subsets. Importantly, we identified two subsets of Th9 cells differing above all in their CD96 expression. The heterogeneous CD96 expression was specific for Th9 cells and not observed for other Th subtypes, such as Th1 cells. Lower CD96 expression was also observed in human IL-9 + compared with IFN-γ + T cells. Although Il9 was highly transcribed by all Th9 cells, IL-9 mRNA and protein expression was increased in CD96 low cells. Transfer of CD96 low Th9 cells into recombination activating gene 1-deficient ( Rag1 -/- ) mice caused severe weight loss, intestinal and colonic inflammation, and destruction of allogeneic skin grafts and thus showed high inflammatory potential. This was associated with their expansion and tissue accumulation. Contrastingly, CD96 high Th9 cells did not cause colitis and showed reduced expansion and migratory potential. Blockade of CD96 completely restored the expansion and inflammatory properties of CD96 high Th9 cells. Collectively, our data suggest an inhibitory role for the cosignaling receptor CD96 in Th9 cells, raising new opportunities in the treatment of IL-9-associated inflammations such as IBD., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Immunosenescence is a hallmark of the aging immune system and is considered the main cause of a reduced vaccine efficacy in the elderly. Although γδ T cells can become activated by recombinant influenza hemagglutinin, their age-related immunocompetence during a virus-induced immune response has so far not been investigated. In this study we evaluate the kinetics of γδ T cells after vaccination with the trivalent 2011/2012 northern hemisphere seasonal influenza vaccine. We applied multi-parametric flow cytometry to a cohort of 21 young (19-30 years) and 23 elderly (53-67 years) healthy individuals. Activated and proliferating γδ T cells, as identified by CD38 and Ki67 expression, were quantified on the days 0, 3, 7, 10, 14, 17, and 21. We observed a significantly lower number of activated and proliferating γδ T cells at baseline and following vaccination in elderly as compared to young individuals. The kinetics changes of activated γδ T cells were much stronger in the young, while corresponding changes in the elderly occurred slower. In addition, we observed an association between day 21 HAI titers of influenza A and the frequencies of Ki67+ γδ T cells at day 7 in the young. In conclusion, aging induces alterations of the γδ T cell response that might have negative implications for vaccination efficacy.

Circadian clocks are cell-autonomous oscillators regulating daily rhythms in a wide range of physiological, metabolic and behavioral processes. Feedback of metabolic signals, such as redox state, NAD + /NADH and AMP/ADP ratios, or heme, modulate circadian rhythms and thereby optimize energy utilization across the 24-h cycle. We show that rhythmic heme degradation, which generates the signaling molecule carbon monoxide (CO), is required for normal circadian rhythms as well as circadian metabolic outputs. CO suppresses circadian transcription by attenuating CLOCK-BMAL1 binding to target promoters. Pharmacological inhibition or genetic depletion of CO-producing heme oxygenases abrogates normal daily cycles in mammalian cells and Drosophila. In mouse hepatocytes, suppression of CO production leads to a global upregulation of CLOCK-BMAL1-dependent circadian gene expression and dysregulated glucose metabolism. Together, our findings show that CO metabolism is an important link between the basic circadian-clock machinery, metabolism and behavior.

Understanding the immune response after vaccination against new influenza strains is highly important in case of an imminent influenza pandemic and for optimization of seasonal vaccination strategies in high risk population groups, especially the elderly. Models predicting the best sero-conversion response among the three strains in the seasonal vaccine were recently suggested. However, these models use a large number of variables and/or information post- vaccination. Here in an exploratory pilot study, we analyzed the baseline immune status in young (<31 years, N = 17) versus elderly (≥50 years, N = 20) donors sero-negative to the newly emerged A(H1N1)pdm09 influenza virus strain and correlated it with the serological response to that specific strain after seasonal influenza vaccination. Extensive multi-chromatic FACS analysis (36 lymphocyte sub-populations measured) was used to quantitatively assess the cellular immune status before vaccination. We identified CD4+ T cells, and amongst them particularly naive CD4+ T cells, as the best correlates for a successful A(H1N1)pdm09 immune response. Moreover, the number of influenza strains a donor was sero-negative to at baseline (NSSN) in addition to age, as expected, were important predictive factors. Age, NSSN and CD4+ T cell count at baseline together predicted sero-protection (HAI≥40) to A(H1N1)pdm09 with a high accuracy of 89% (p-value = 0.00002). An additional validation study (N = 43 vaccinees sero-negative to A(H1N1)pdm09) has confirmed the predictive value of age, NSSN and baseline CD4+ counts (accuracy = 85%, p-value = 0.0000004). Furthermore, the inclusion of donors at ages 31-50 had shown that the age predictive function is not linear with age but rather a sigmoid with a midpoint at about 50 years. Using these results we suggest a clinically relevant prediction model that gives the probability for non-protection to A(H1N1)pdm09 influenza strain after seasonal multi-valent vaccination as a continuous function of age, NSSN and baseline CD4 count.

Odor discrimination behavior displays circadian fluctuations in mice, indicating that mammalian olfactory function is under control of the circadian system. This is further supported by the facts that odor discrimination rhythms depend on the presence of clock genes and that olfactory tissues contain autonomous circadian clocks. However, the molecular link between circadian function and olfactory processing is still unknown. To elucidate the molecular mechanisms underlying this link, we focused on the olfactory epithelium (OE), the primary target of odors and the site of the initial events in olfactory processing. We asked whether olfactory sensory neurons (OSNs) within the OE possess an autonomous circadian clock and whether olfactory pathways are under circadian control. Employing clock gene-driven bioluminescence reporter assays and time-dependent immunohistochemistry on OE samples, we found robust circadian rhythms of core clock genes and their proteins in OSNs, suggesting that the OE indeed contains an autonomous circadian clock. Furthermore, we performed a circadian transcriptome analysis and identified several OSN-specific components that are under circadian control, including those with putative roles in circadian olfactory processing, such as KIRREL2-an established factor involved in short-term OSN activation. The spatiotemporal expression patterns of our candidate proteins suggest that they are involved in short-term anabolic processes to rhythmically prepare the cell for peak performances and to promote circadian function of OSNs., (© 2015 The Author(s).)

Immunological competence declines progressively with age, resulting in increased susceptibility of the elderly to infection and impaired responses to vaccines. Underlying mechanisms remain largely obscure as they have been related to complex, individual systemic immune properties that are challenging to investigate. In this study, we explored age-related changes in human immunity during a primary virus infection experimentally induced by immunization with live-attenuated yellow fever (YF) vaccine. Applying detailed serology, advanced FACS analysis, and systems biology, we discovered that aged subjects developed fewer neutralizing Abs, mounted diminished YF-specific CD8(+) T cell responses, and showed quantitatively and qualitatively altered YF-specific CD4(+) T cell immunity. Among numerous immune signatures, low in vivo numbers of naive CD4(+) recent thymic emigrants and peripheral dendritic cells correlated well with reduced acute responsiveness and altered long-term persistence of human cellular immunity to YF vaccination. Hence, we reveal in this article that essential elements of immune responses such as recent thymic emigrants and dendritic cells strongly relate to productive immunity in the elderly, providing a conceivable explanation for diminished responsiveness to vaccination with neoantigens and infection with de novo pathogens in the aged population., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Glucocorticoids are indispensable anti-inflammatory and decongestant drugs with high prevalence of use at (~)0.9% of the adult population. Better holistic insights into glucocorticoid-induced changes are crucial for effective use as concurrent medication and management of adverse effects. The profiles of 214 metabolites from plasma of 20 male healthy volunteers were recorded prior to and after ingestion of a single dose of 4 mg dexamethasone (+20 mg pantoprazole). Samples were drawn at three predefined time points per day: seven untreated (day 1 midday - day 3 midday) and four treated (day 3 evening - day 4 evening) per volunteer. Statistical analysis revealed tremendous impact of dexamethasone on the metabolome with 150 of 214 metabolites being significantly deregulated on at least one time point after treatment (ANOVA, Benjamini-Hochberg corrected, q < 0.05). Inter-person variability was high and remained uninfluenced by treatment. The clearly visible circadian rhythm prior to treatment was almost completely suppressed and deregulated by dexamethasone. The results draw a holistic picture of the severe metabolic deregulation induced by single-dose, short-term glucocorticoid application. The observed metabolic changes suggest a potential for early detection of severe side effects, raising hope for personalized early countermeasures increasing quality of life and reducing health care costs.

Immunosenescence results from a continuous deterioration of immune responses resulting in a decreased response to vaccines. A well-described age-related alteration of the immune system is the decrease of de novo generation of T and B cells. In addition, the accumulation of memory cells and loss of diversity in antigen specificities resulting from a lifetime of exposure to pathogens has also been described. However, the effect of aging on subsets of γδTCR(+) T cells and Tregs has been poorly described, and the efficacy of the recall response to common persistent infections in the elderly remains obscure. Here, we investigated alterations in the subpopulations of the B and T cells among 24 healthy young (aged 19-30) and 26 healthy elderly (aged 53-67) individuals. The analysis was performed by flow cytometry using freshly collected peripheral blood. γδTCR(+) T cells were overall decreased, while CD4(+)CD8(-) cells among γδTCR(+) T cells were increased in the elderly. Helios(+)Foxp3(+) and Helios(-)Foxp3(+) Treg cells were unaffected with age. Recent thymic emigrants, based on CD31 expression, were decreased among the Helios(+)Foxp3(+), but not the Helios(-)Foxp3(+) cell populations. We observed a decrease in Adenovirus-specific CD4(+) and CD8(+) T cells and an increase in CMV-specific CD4(+) T cells in the elderly. Similarly, INFγ(+)TNFα(+) double-positive cells were decreased among activated T cells after Adenovirus stimulation but increased after CMV stimulation. The data presented here indicate that γδTCR(+) T cells might stabilize B cells, and functional senescence might dominate at higher ages than those studied here.