Beyond muscles: Investigating immunoregulatory myokines in acute resistance exercise - A systematic review and meta-analysis.

Myokines, released from the muscle, enable communication between the working muscles and other tissues. Their release during physical exercise is assumed to depend on immune-hormonal-metabolic interactions concerning mode (endurance or resistance exercise), duration, and intensity. This meta-analysis aims to examine the acute changes of circulating myokines inducing immunoregulatory effects caused by a bout of resistance exercise and to consider potential moderators of the results. Based on this selection strategy, a systematic literature search was conducted for resistance exercise intervention studies measuring interleukin (IL-) 6, IL-10, IL-1ra, tumor necrosis factor (TNF-) α, IL-15, IL-7, transforming growth factor (TGF-) ß1, and fractalkines (FKN) before and immediately after resistance exercise in healthy individuals. Random-effects meta-analysis was performed for each myokine. We identified a moderate positive effect of resistance exercise for IL-6 and IL-1ra. Regarding IL-15 and TNF-α, small to moderate effects were found. For IL-10, no significant effect was observed. Due to no data, meta-analyses for IL-7, TGF-ß1, and FKN could not be performed. No moderators (training status, type of exercise, risk of bias, age, sex, time of day, exercise volume, exercise intensity, exercise dose) of the results were detected for all tested myokines. Taken together, this systematic review and meta-analysis showed immediate positive effects of an acute resistance exercise session on IL-6, IL-1ra, TNF-α, and IL-15 levels.

A scoping review of regulatory T cell dynamics in convalescent COVID-19 patients - indications for their potential involvement in the development of Long COVID?

Background: Recovery from coronavirus disease 2019 (COVID-19) can be impaired by the persistence of symptoms or new-onset health complications, commonly referred to as Long COVID. In a subset of patients, Long COVID is associated with immune system perturbations of unknown etiology, which could be related to compromised immunoregulatory mechanisms. Objective: The objective of this scoping review was to summarize the existing literature regarding the frequency and functionality of Tregs in convalescent COVID-19 patients and to explore indications for their potential involvement in the development of Long COVID. Design: A systematic search of studies investigating Tregs during COVID-19 convalescence was conducted on MEDLINE (via Pubmed) and Web of Science. Results: The literature search yielded 17 relevant studies, of which three included a distinct cohort of patients with Long COVID. The reviewed studies suggest that the Treg population of COVID-19 patients can reconstitute quantitatively and functionally during recovery. However, the comparison between recovered and seronegative controls revealed that an infection-induced dysregulation of the Treg compartment can be sustained for at least several months. The small number of studies investigating Tregs in Long COVID allowed no firm conclusions to be drawn about their involvement in the syndrome's etiology. Yet, even almost one year post-infection Long COVID patients exhibit significantly altered proportions of Tregs within the CD4+ T cell population. Conclusions: Persistent alterations in cell frequency in Long COVID patients indicate that Treg dysregulation might be linked to immune system-associated sequelae. Future studies should aim to address the association of Treg adaptations with different symptom clusters and blood parameters beyond the sole quantification of cell frequencies while adhering to consensualized phenotyping strategies.

PRI: Re-Analysis of a Public Mass Cytometry Dataset Reveals Patterns of Effective Tumor Treatments.

Recently, mass cytometry has enabled quantification of up to 50 parameters for millions of cells per sample. It remains a challenge to analyze such high-dimensional data to exploit the richness of the inherent information, even though many valuable new analysis tools have already been developed. We propose a novel algorithm "pattern recognition of immune cells (PRI)" to tackle these high-dimensional protein combinations in the data. PRI is a tool for the analysis and visualization of cytometry data based on a three or more-parametric binning approach, feature engineering of bin properties of multivariate cell data, and a pseudo-multiparametric visualization. Using a publicly available mass cytometry dataset, we proved that reproducible feature engineering and intuitive understanding of the generated bin plots are helpful hallmarks for re-analysis with PRI. In the CD4+T cell population analyzed, PRI revealed two bin-plot patterns (CD90/CD44/CD86 and CD90/CD44/CD27) and 20 bin plot features for threshold-independent classification of mice concerning ineffective and effective tumor treatment. In addition, PRI mapped cell subsets regarding co-expression of the proliferation marker Ki67 with two major transcription factors and further delineated a specific Th1 cell subset. All these results demonstrate the added insights that can be obtained using the non-cluster-based tool PRI for re-analyses of high-dimensional cytometric data.

Destinations fostering older adults' walking for transport: a cross-sectional study from Germany.

BACKGROUND: Having destinations within walking distance can encourage older people to walk. Yet, not all destinations may be equally important. Little is known about the types of destinations fostering older adults' walking for transport in small and medium-sized towns and rural communities. The aim of this study was to explore the associations between the availability of different destinations and walking for transport among older adults living in communities with less than 100,000 inhabitants. METHODS: Between May and September 2019, self-reported data from 2242 older adults (≥ 65 years) living in the Metropolitan Region Northwest (Germany) were collected within the project AFOOT - Securing urban mobility of an aging population. Data from 2137 study participants were eligible for this analysis. Logistic regression models were used to investigate the relationship between the perceived destination availability of 19 different destinations within a 20-min or 10-min walk from home, respectively, and the engagement in walking for transport. Crude and adjusted models were run separately for each destination and distance category. Exploratory subgroup analyses examined the associations between the availability of destinations within a 20-min walk from home and walking for transport stratified by gender, use of a walking aid, and car availability. RESULTS: The availability of each of the investigated destinations within a 20-min walk and of nearly all of these destinations within a 10-min walk from home was significantly positively associated with walking for transport in crude models. Most associations remained significant after adjustment for covariates. The strongest associations were found for the availability of small stores, pharmacy, and bakery. The availability of a bus stop showed the weakest associations and was not significantly associated with walking for transport after adjustment for covariates. CONCLUSIONS: The provision of local amenities within walking distance may be a promising approach to foster older adults' walking for transport in smaller communities with less than 100,000 inhabitants and to enable active and healthy aging in place. Further quantitative and qualitative research is needed to validate these findings and to better understand older adults' walking behavior.

Persistent T-Cell Reactivity in a Seronegative Patient after SARS-CoV-2 Infection and One Vaccination.

We present here a 64-year-old male participant of the CoNAN study who experienced a PCR-confirmed mild SARS-CoV-2 infection but did not develop any measurable antibody response. Additionally, after vaccination with ChAdOx1 (AstraZeneca, Cambridge, UK) 11 months later, no antibodies were detected in six serological tests three weeks after the vaccination. When we assessed T-helper (Th) cell immunity, SARS-CoV-2-specific Th cells produced detectable amounts of IFNγ and TNF six weeks after the infection. A robust T-cell immunity remained detectable at least until six months after the infection and was boosted by the vaccination thereafter. This case report points out that an assessment of a prior infection or a vaccine response based solely on antibody detection might have limitations in individual patients.

Complicated Long Term Vaccine Induced Thrombotic Immune Thrombocytopenia-A Case Report.

BACKGROUND AND OBJECTIVES: Vaccine induced thrombotic thrombocytopenia (VITT) may occur after COVID-19 vaccination with recombinant adenoviral vector-based vaccines. VITT can present as cerebral sinus and venous thrombosis (CSVT), often complicated by intracranial hemorrhage. Today it is unclear, how long symptomatic VITT can persist. Here, we report the complicated long-term course of a VITT patient with extremely high titers of pathogenic anti-platelet factor 4 (PF4)-IgG antibodies. METHODS: Clinical and laboratory findings are presented, including the course of platelet counts, D-Dimer levels, clinical presentation, imaging, SARS-CoV-2-serological and immunological, platelet activating anti-PF4-IgG, as well as autopsy findings. RESULTS: The patient presented with extended superior sagittal sinus thrombosis with accompanying bifrontal intracerebral hemorrhage. Repeated treatment with intravenous immune globuline (IVIG) resolved recurrent episodes of thrombocytopenia. Moreover, the patient's serum remained strongly positive for platelet-activating anti-PF4-IgG over three months. After a period of clinical stabilization, the patient suffered a recurrent and fatal intracranial hemorrhage. CONCLUSIONS: Complicated VITT with extremely high anti-PF4-IgG titers over three months can induce recurrent thrombocytopenia despite treatment with IVIG and anticoagulation. Plasma exchange, immunoadsorption, and /or immunosuppressive treatment may be considered in complicated VITT to reduce extraordinarily high levels of anti-PF4-IgG. Long-term therapy in such cases must take the individual bleeding risk and CSVT risk into account.

Barriers, Facilitating Factors, and Intersectoral Collaboration for Promoting Active Mobility for Healthy Aging-A Qualitative Study within Local Government in Germany.

The promotion of walking and cycling to stay active and mobile offers great potential for healthy aging. Intersectoral collaboration for age-friendly urban planning is required in local government to realize this potential. Semi-structured interviews were conducted with the heads of planning and public health departments in city and district administrations of a Metropolitan Region in Germany to identify factors influencing action on the cross-cutting issue of active mobility for healthy aging. Although some administrations are working on the promotion of active mobility, they consider neither the needs of older people nor health effects. A lack of human resources and expertise, mainly due to the low priority placed on the issue, are described as the main barriers for further strategic collaboration. Furthermore, the public health sector often focuses on pathogens as the cause of morbidity and mortality, reducing their acceptance of responsibility for the topic. Facilitating factors include the establishment of new administrative structures, projects with rapid results that create awareness and credibility among citizens and politicians, additional staff with expertise in health promotion, and political commitment. In the future, new administrative structures for intersectoral collaboration are needed in order to consider various perspectives in complex developments, such as healthy aging, and to benefit from synergies.

Effect of Psycho-Regulatory Massage Therapy on Pain and Depression in Women with Chronic and/or Somatoform Back Pain: A Randomized Controlled Trial.

Chronic unspecific back pain (cBP) is often associated with depressive symptoms, negative body perception, and abnormal interoception. Given the general failure of surgery in cBP, treatment guidelines focus on conservative therapies. Neurophysiological evidence indicates that C-tactile fibers associated with the oxytonergic system can be activated by slow superficial stroking of the skin in the back, shoulder, neck, and dorsal limb areas. We hypothesize that, through recruitment of C-tactile fibers, psycho-regulatory massage therapy (PRMT) can reduce pain in patients with cBP. In our study, 66 patients were randomized to PRMT or CMT (classical massage therapy) over a 12-week period and tested by questionnaires regarding pain (HSAL= Hamburger Schmerz Adjektiv Liste; Hamburg Pain adjective list), depression (BDI-II = Beck depression inventory), and disability (ODI = Oswestry Disability Index). In all outcome measures, patients receiving PRMT improved significantly more than did those receiving CMT. The mean values of the HSAL sensory subscale decreased by -51.5% in the PRMT group compared to -6.7% in the CMT group. Depressive symptoms were reduced by -55.69% (PRMT) and -3.1% (CMT), respectively. The results suggest that the superiority of PRMT over CMT may rely on its ability to activate the C-tactile fibers of superficial skin layers, recruiting the oxytonergic system.

Deep Phenotyping of Urinary Leukocytes by Mass Cytometry Reveals a Leukocyte Signature for Early and Non-Invasive Prediction of Response to Treatment in Active Lupus Nephritis.

Non-invasive biomarkers are necessary for diagnosis and monitoring disease activity in lupus nephritis (LN) to circumvent risks and limitations of renal biopsies. To identify new non-invasive cellular biomarkers in the urine sediment of LN patients, which may reflect kidney inflammation and can be used to predict treatment outcome, we performed in-depth urinary immune cell profiling by mass cytometry. We established a mass cytometric workflow to comparatively analyze the cellular composition of urine and peripheral blood (PB) in 13 patients with systemic lupus erythematosus (SLE) with active, biopsy-proven proliferative LN. Clinical and laboratory data were collected at the time of sampling and 6 months after induction of therapy in order to evaluate the clinical response of each patient. Six patients with different acute inflammatory renal diseases were included as comparison group. Leukocyte phenotypes and composition differed significantly between urine and paired PB samples. In urine, neutrophils and monocytes/macrophages were identified as the most prominent cell populations comprising together about 30%-83% of nucleated cells, while T and B lymphocytes, eosinophils, and natural killer (NK) cells were detectable at frequencies of <10% each. The majority of urinary T cells showed phenotypical characteristics of activated effector memory T cells (EM) as indicated by the co-expression of CD38 and CD69 - a phenotype that was not detectable in PB. Kidney inflammation was also reflected by tissue-imprinted macrophages, which phenotypically differed from PB monocytes by an increased expression of HLA-DR and CD11c. The presence of activated urinary T cells and macrophages could be used for differential diagnosis of proliferative LN forms and other renal pathologies. Most interestingly, the amount of EM in the urine sediment could be used as a biomarker to stratify LN patients in terms of response to induction therapy. Deep immunophenotypic profiling of urinary cells in LN allowed us to identify a signature of activated T cells and macrophages, which appear to reflect leukocytic infiltrates in the kidney. This explorative study has not only confirmed but also extended the knowledge about urinary cells as a future non-invasive biomarker platform for diagnosis and precision medicine in inflammatory renal diseases.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Mass Cytometry Enabling Absolute and Fast Quantification of Silver Nanoparticle Uptake at the Single Cell Level.

In the last decades, significant efforts have been made to investigate possible cytotoxic effects of metallic nanoparticles (NPs). Methodologies enabling precise information regarding uptake and intracellular distribution of NPs at the single cell level remain to be established. Mass cytometry (MC) has been developed for high-dimensional single cell analyses and is a promising tool to quantify NP-cell interactions. Here, we aim to establish a new MC-based quantification procedure to receive absolute numbers of NPs per single cell by using a calibration that considers the specific transmission efficiency (TE) of suspended NPs. The current MC-quantification strategy accept TE values of complementary metal solutions. In this study, we demonstrate the different transmission behavior of 50 nm silver NPs (AgNP) and silver nitrate solution. We have used identical AgNPs for calibration as for in vitro-differentiated macrophages (THP-1 cell line) in a time- and dose-dependent manner. Our quantification relies on silver intensities measuring AgNPs in the same detection mode as the cells. Results were comparable with the TE quantification strategy using AgNPs but differed when using ionic silver. Furthermore, intact and digested cell aliquots were measured to investigate the impact of MC sample processing on the amount of AgNPs/cell. Taken together, we have provided a MC-specific calibration procedure to precisely calculate absolute numbers of NPs per single cell. Combined with its unique feature of multiplexing up to 50 parameters, MC provides much more information on the single cell level than single cell-inductively coupled plasma mass spectrometry (SC-ICPMS) and, therefore, offers new opportunities in nanotoxicology.

Stabilizing Antibody Cocktails for Mass Cytometry.

Mass cytometry is increasingly employed in larger immune profiling studies involving data acquisitions across several days and multiple sites. For gaining a maximum of information from respective data by computational analyses, several techniques have been developed to minimize noise in mass cytometric data sets, such as sample banking, standardized instrument setup, sample barcoding, and signal normalization. However, the repeated preparation of cocktails composed of isotope-tagged antibodies remained a significant source of error. We here show that premixed antibody cocktails fail to deliver expected staining patterns when stored at 4°C for 4 weeks. As a solution, we developed and tested a cryopreservation method for highly multiplexed antibody cocktails for mass cytometry including lanthanide, palladium, and platinum conjugates that yielded stable staining patterns for at least 9 months when stored at temperatures below -80°C. Using frozen aliquots of antibody cocktails is an economic and flexible approach to significantly improve data consistency in large mass cytometry studies with repetitive staining/measurement cycles spanning several days or involving multiple data acquisition sites. © 2019 International Society for Advancement of Cytometry.

Osmium-Labeled Microspheres for Bead-Based Assays in Mass Cytometry.

Polystyrene beads are broadly applied in flow cytometry. Implementing bead-based assays in mass cytometry is desired but hampered by the lack of an elemental label required for their detection. In this study, we introduce stable osmium tetroxide labeling as a universal approach for generating functionalized beads readily detectable by mass cytometry. We demonstrate the utility of osmium-labeled beads for signal spillover compensation in mass cytometry, and, strikingly, their application in quantitative Ab-binding capacity assays combined with high-dimensional profiling of human PBMC enabled the systematic assessment of receptor expression profiles across large numbers of cellular phenotypes. This analysis confirmed increased monocytic Siglec-1 expression in active systemic lupus erythematosus patients and, additionally, revealed interrelated reductions of CD4 expression by regulatory and memory CD4 T cells and HLA-DR expression by myeloid dendritic cells, pointing toward defective cross-talk at the immunological synapse that may limit immune responses in systemic lupus erythematosus. By converting conventional flow cytometry beads into beads suitable for mass cytometry, our approach paves the way toward the broad implementation of bead-based assays in high-dimensional cell profiling studies by mass cytometry in biomedical research.

Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level.

Background: Mass cytometry (Cytometry by Time of Flight, CyTOF) allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell. Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells' silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms. Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected. Conclusion: An effective mass cytometry protocol was developed for the detection and quantification of silver in single bacterial cells of different physiological states. The silver quantities were generally heterogeneously distributed among cells in a population, the degree of which was dependent on micro-environmental conditions and on silver applied either in ion or nanoparticle-aggregated form.

Dual-labelled antibodies for flow and mass cytometry: A new tool for cross-platform comparison and enrichment of target cells for mass cytometry.

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross-platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal-labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome-conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR® labelling protocol. After labelling with 141 Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4+ T-cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19-VioBlue-142 Nd, CD20-VioGreen-147 Sm, CD27-Cy5-167 Er and CD38-Alexa488-143 Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.

Silver nanoparticles for the detection of cell surface antigens in mass cytometry.

Mass cytometry has pioneered >40-parameter single-cell analyses that allow for the characterization of complex cellular networks at unprecedented depth. Up to 135 parameters can be simultaneously detected, but limited availability of metal tags suitable for labeling of specific probes prevents optimal exploitation of the analytical capacity of mass cytometers. To this end, we here establish the application of elemental silver nanoparticles (AgNP) of different size for reporting cell surface antigens on human leukocytes in mass cytometry assays. The mass channels at 107 Da and 109 Da are uniquely occupied by silver isotopes and do not interfere with other mass cytometry reagents. Streptavidin-coated AgNP (SA-AgNP) facilitated distinct and specific detection of various antigens, such as CD8, CD244 and CD294 on peripheral blood leukocytes pre-incubated with respective biotinylated primary antibodies. Signal intensities elicited by 40 nm-sized AgNP allowed specific detection of the low abundance antigen CD25 on both, peripheral blood regulatory T cells and CD25lo CD127+ CD4+ T cells, enabling their distinct clustering in viSNE plots. SA-AgNP were of high elemental purity, showed minor background binding to cells in immunoassays, and were compatible with previously established staining protocols for PBMC and leukocytes, facilitating their use in complex mass cytometry panels. Considering the synthesis of AgNP from isotopically purified silver, the usage of AgNP extends the analytical capacity of mass cytometry panels by one, prospectively two, additional parameters, suitable for the detection of cellular targets of low abundance. © 2016 International Society for Advancement of Cytometry.