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The hormone and neurotransmitter serotonin regulates numerous physiological functions within the central nervous system and in the periphery upon binding to specific receptors. In the periphery, the serotonin receptor 7 (5-HT7R) is expressed on different immune cells including monocytes and macrophages. To investigate the impact of 5-HT7R-mediated signaling on macrophage properties, we used human THP-1 cells and differentiated them into pro-inflammatory M1- and anti-inflammatory M2-like macrophages. Pharmacological 5-HT7R activation with the specific agonist LP-211 especially modulates morphology of M1-like macrophages by increasing the number of rounded cells. Furthermore, 5-HT7R stimulation results in significantly reduced phagocytic and migratory ability of M1-like macrophages. Noteworthy, LP-211 treatment leads to changes in secretory properties of all macrophage types with the highest effects obtained for M0- and M2c-like macrophages. Finally, the importance of 5-HT7R for regulation of phagocytosis was confirmed in human primary CD14 + cells. These results indicate that 5-HT7R activation selectively impairs basic functions of macrophages and might thus be a new access point for the modulation of macrophage responses in the future treatment of inflammatory diseases., Competing Interests: Declarations. Ethical approval: Not applicable. Consent for publication: The authors hereby consent to publication of the work in Cellular and Molecular Life Sciences. Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2025. The Author(s).)

Immune-checkpoint-inhibitors (ICI) target key regulators of the immune system expressed by cancer cells that mask those from recognition by the immune system. They have improved the outcome for patients with various cancer types, such as melanoma. ICI-based therapy is frequently accompanied by immune-related adverse side effects (IRAEs). The reversible melanoma cancer mouse model (B16F10 cells stably expressing a ganciclovir (GCV)-inducible suicide gene in C57BL/6N mice: B16F10-GCV) allows chemotherapy-free tumor elimination in advanced disease stage and demonstrates almost complete recovery of the mouse heart from cancer-induced atrophy, molecular impairment and heart failure. Thus, enabling the study of anti-cancer-therapy effects. Here, we analyzed potential cardiac side effects of antibody-mediated PD-L1 inhibition in the preclinical B16F10-GCV mouse model after tumor elimination and 2 weeks recovery (50 days after tumor inoculation). Anti-PD-L1 treatment was associated with improved survival as compared to isotype control (Ctrl) treated mice. Surviving anti-PD-L1 and Ctrl mice showed similar cardiac function, dimensions and the expression of cardiac stress and hypertrophy markers. Although anti-PD-L1 treatment was associated with increased troponin I type 3 cardiac (TNNI3) blood levels, cardiac mRNA expression of macrophage markers and elevated cardiac levels of secreted inflammatory factors compared to Ctrl treatment, both groups showed a comparable density of inflammatory cells in the heart (using CXCR4-ligand 68 Ga-Pentixafor in PET-CT and immunohistochemistry). Thus, anti-PD-L1 therapy improved survival in mice with advanced melanoma cancer with no major cardiac phenotype or inflammation 50 days after tumor inoculation. Without a second hit that triggers the inflammatory response, anti-PD-L1 treatment appears to be safe for the heart in the preclinical melanoma mouse model., 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 © 2025 Schoenherr, Pietzsch, Barca, Müller, Bahr, Kasten, Zeug, Erschow, Falk, Ponimaskin, Thackeray, Hilfiker-Kleiner and Ricke-Hoch.)

Background and Aims: Systemic Inflammation (SI) is considered a key mechanism in disease progression and development of complications in decompensated liver cirrhosis. SI is mainly driven by portal hypertension and bacterial translocation. Transjugular intrahepatic portosystemic shunt (TIPS)-insertion represents an effective treatment for portal hypertension. This study aims to investigate the impact of TIPS-insertion on SI and bacterial translocation., Methods: We prospectively included 59 cirrhotic patients undergoing TIPS-insertion. Blood samples were collected at TIPS-insertion and follow-up 1, 3, 6, and 12 months (FU) thereafter. At all time points, we performed a comprehensive analysis of SI including 43 soluble inflammatory markers (SIMs), and surrogates of bacterial translocation (sCD14, sCD163). To investigate long-term kinetics of SI, C-reactive protein (CRP) and white blood cells (WBC) were retrospectively analyzed in a cohort of 177 patients up to 3-years after TIPS-insertion., Results: At TIPS-insertion, 30/43 SIMs, sCD14, and sCD163 measured significantly higher in cirrhotic patients compared to healthy controls. By FU6 25 SIMs and sCD14 measured at significantly lower levels compared to baseline. Interestingly, in patients with TIPS-indication refractory ascites IL-6 decreased to levels documented in earlier stages of cirrhosis. In long-term follow-up, CRP-levels significantly decreased after TIPS-insertion, which translated into improved liver-transplant-free survival in cox regression analysis (HR 0.968, p=0.042). Notably, patients with residual ascites post-TIPS showed significantly higher CRP- and IL-6 levels across all follow-ups compared to patients with resolved ascites., Conclusions: Decreasing portal hypertension via TIPS-insertion leads to a significant attenuation of SI and bacterial translocation over time.

Background & Aims: After pediatric liver transplantation (pLT), children undergo life-long immunosuppression since reliable biomarkers for the assessment of rejection probability are scarce. In the multicenter (n = 7) prospective clinical cohort "ChilSFree" study, we aimed to characterize longitudinal dynamics of soluble and cellular immune mediators during the first year after pLT and identify early biomarkers associated with outcome., Methods: Using a Luminex-based multiplex technique paired with flow cytometry, we characterized longitudinal dynamics of soluble immune mediators (SIMs, n = 50) and immune cells in the blood of 244 patients at eight visits over 1 year: before, and 7/14/21/28 days and 3/6/12 months after pLT., Results: The unsupervised clustering of patients based on SIM profiles revealed six unique SIM signatures associated with clinical outcome. From three signatures linked to improved outcome, one was associated with 1-year-long rejection-free survival and stable graft function and was characterized by low levels of pro-inflammatory SIMs (CXCL8/9/10/12, CCL7, SCGF-β, sICAM-1), and high levels of regenerative (SCF, TNF-β) and pro-apoptotic (TRAIL) SIMs (all, p <0.001, fold change >100). Of note, this SIM signature appeared 2 weeks after pLT and remained stable over the entire year, pointing towards its potential as a novel early biomarker for minimizing or weaning immunosuppression. In the blood of these patients, a higher frequency of CD56 bright natural killer cells (p <0.01), a known hallmark also associated with operationally tolerant pLT patients, was detected. The concordance of the model for prediction of rejection based on identified SIM signatures was 0.715, and 0.795, in combination with living-related transplantation as a covariate, respectively., Conclusions: SIM blood signatures may enable the non-invasive and early assessment of rejection risks in the first year after pLT, paving the way for improved clinical management., Impact and Implications: ChilSFree represents the largest pediatric liver transplant (pLT) cohort with paired longitudinal data on soluble immune mediators (SIMs) and immune phenotyping in the first year after pLT. SIM signatures allow for the selection of rejection-free patients 2 weeks after pLT independently of patient diagnosis, sex, or age. The SIM signatures may enable the non-invasive and early assessment of rejection risks, paving the way for minimization or withdrawal of immunosuppression after pLT., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: The Seraph 100 Microbind Affinity blood filter eliminate bacteria, viruses, fungi and toxins from blood stream., Methods: This is a prospective multicenter observational biomarker trial in PCR-positive SARS-CoV-2 patients with acute respiratory failure. Biomarkers were sequentially tested at three time points., Results: Forty-two patients with SARS-CoV-2 detected by PCR with acute respiratory failure were included. When receiving hemoperfusion treatment, 27 (64%) patients were on mechanical ventilation, 41 (98%) patients were treated in the ICU. The 3-month survival was 52%. After one hemoperfusion treatment cycle, D-dimer (p = 0.014), hemoglobin (p = 0.003) and LDH (p = 0.001) concentrations were significantly reduced 4 days after treatment. From the multiplex assay IL-1b, CXCL8/ IL-8, IL-10, IL-13, IL-15, CCL11/Eotaxin, G-CSF, and CXCL10/IP-10 were significantly reduced 1 h after treatment, however not 4 days later., Conclusion: Hemoperfusion with Seraph 100 Microbind Affinity Filter in patients with severe COVID-19 can transiently reduce several inflammatory biomarkers in the blood., (© 2024 International Society for Apheresis and Japanese Society for Apheresis.)

Background and Aims: Non-selective beta-blockers (NSBB) are a well-established treatment in patients with clinically significant portal hypertension. However, their potential role after insertion of a transjugular intrahepatic portosystemic shunt (TIPS) still needs to be determined. Of note, recent studies suggested that favourable anti-inflammatory effects of NSBB might be independent from pressure reduction. This study aimed to evaluate whether NSBB-treatment is associated with amelioration of systemic inflammation (SI), hepatic decompensation and survival after TIPS-insertion., Methods: In a retrospective study comprising 305 consecutive patients, we investigated the impact of NSBB-intake at TIPS-placement on periinterventional cirrhosis-associated complications and continued NSBB-treatment after discharge on complications including hepatic decompensation and mortality during 1-year follow-up, employing multivariable competing-risk-analyses. In a prospective cohort including 45 patients, we performed a comprehensive analysis of SI analysing 48 soluble inflammatory markers (SIMs) at baseline plus 3 and 6 months after TIPS-insertion., Results: Overall, 175 (57.4%) patients received NSBB-therapy prior to TIPS-insertion; upon discharge, this decreased to 131 (22.9%), with 36 (27.5%) discontinuing NSBB within 1-year follow-up. Neither NSBB-therapy at TIPS-insertion nor treatment-continuation after discharge were associated with lower risks for hepatic decompensation, individual cirrhosis-associated complications or mortality neither in the periinterventional period nor during follow-up. Similarly, in the prospective cohort NSBB-intake was not linked to lower levels or a more prominent change of WBC, CRP or any other SIM at any of the investigated time points., Conclusion: NSBB-therapy at the time of TIPS-insertion and its (dis-)continuation afterwards seems to have no significant impact on SI, development of hepatic decompensation and survival., (© 2024 The Author(s). Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd.)

Ex situ lung perfusion (ESLP) is used for organ reconditioning, repair, and re-evaluation prior to transplantation. Since valid preclinical animal models are required for translationally relevant studies, we developed a 17 mL low-volume ESLP for double- and single-lung application that enables cost-effective optimal compliance "reduction" of the 3R principles of animal research. In single-lung mode, ten Fischer344 and Lewis rat lungs were subjected to ESLP and static cold storage using STEEN or PerfadexPlus. Key perfusion parameters, thermal lung imaging, blood gas analysis (BGA), colloid oncotic pressure (COP), lung weight gain, histological work-up, and cytokine analysis were performed. Significant differences between perfusion solutions but not between the rat strains were detected. Most relevant perfusion parameters confirmed valid ESLP with homogeneous lung perfusion, evidenced by uniform lung surface temperature. BGA showed temperature-dependent metabolic activities with differences depending on perfusion solution composition. COP is not decisive for pulmonary oedema and associated weight gain, but possibly rather observed chemokine profile and dextran sensitivity of rats. Histological examination confirmed intact lung architecture without infarcts or hemorrhages due to optimal organ procurement and single-lung application protocol using our in-house-designed ESLP system., 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 © 2024 Katsirntaki, Hagner, Werlein, Braubach, Jonigk, Adam, Hidaji, Kühn, Falk, Ruhparwar and Wiegmann.)

Abstract: Allogeneic hematopoietic stem cell transplantation (alloSCT) is the only cure for many hematologic malignancies. However, alloSCT recipients are susceptible to opportunistic pathogens, such as human cytomegalovirus (HCMV). Letermovir prophylaxis has revolutionized HCMV management, but the challenge of late HCMV reactivations has emerged. Immunological surrogates of clinically significant HCMV infection (csCMVi) after discontinuation of letermovir remain to be defined. Therefore, we studied natural killer (NK)-cell reconstitution along with the global and HCMV pp65-specific T-cell repertoire of 24 alloSCT recipients at 7 time points before (day +90) and after (days +120-270) cessation of letermovir prophylaxis. Patients who experienced csCMVi had lower counts of IFN-γ+ HCMV-specific CD4+ and CD8+ T cells than HCMV controllers. Furthermore, patients with csCMVi displayed late impairment of NK-cell reconstitution, especially suppression of "memory-like" CD159c+CD56dim NK-cell counts that preceded csCMVi events in most patients. Moreover, several surrogates of immune reconstitution were associated with the severity of HCMV manifestation, with patients suffering from HCMV end-organ disease and/or refractory HCMV infection harboring least HCMV-specific T cells and "memory-like" NK cells. Altogether, our findings establish an association of delayed or insufficient proliferation of both HCMV-specific T cells and "memory-like" NK cells with csCMVi and the severity of HCMV manifestations after discontinuation of letermovir prophylaxis., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Purpose: Post-acute sequelae of COVID-19 (PASC) affect approximately 10% of convalescent patients. The spectrum of symptoms is broad and heterogeneous with fatigue being the most often reported sequela. Easily accessible blood biomarkers to determine PASC severity are lacking. Thus, our study aimed to correlate immune phenotypes with PASC across the severity spectrum of COVID-19., Methods: A total of 176 originally immunonaïve, convalescent COVID-19 patients from a prospective cohort during the first pandemic phase were stratified by initial disease severity and underwent clinical, psychosocial, and immune phenotyping around 10 weeks after first COVID-19 symptoms. COVID-19-associated fatigue dynamics were assessed and related to clinical and immune phenotypes., Results: Fatigue and severe fatigue were commonly reported irrespective of initial COVID-19 severity or organ-specific PASC. A clinically relevant increase in fatigue severity after COVID-19 was detected in all groups. Neutralizing antibody titers were higher in patients with severe acute disease, but no association was found between antibody titers and PASC. While absolute peripheral blood immune cell counts in originally immunonaïve PASC patients did not differ from unexposed controls, peripheral CD3 + CD4 + T cell counts were independently correlated with fatigue severity across all strata in multivariable analysis., Conclusions: Patients were at similar risk of self-reported PASC irrespective of initial disease severity. The independent correlation between fatigue severity and blood T cell phenotypes indicates a possible role of CD4 + T cells in the pathogenesis of post-COVID-19 fatigue, which might serve as a blood biomarker., (© 2023. The Author(s).)

Background: Sudden infant death syndrome (SIDS) has been considered to be triggered by a combination of underlying immune dysregulation and infections. The thymus is a crucial lymphatic organ responsible for T cell development in infancy. We hypothesized that an altered thymic immune status may be detectable by intrathymic cytokine profiling in SIDS., Methods: 27 cytokines in protein lysates of thymus tissue and thymus weights were assessed in 26 SIDS cases and 16 infants who died of other reasons., Results: Seventeen out of 27 cytokines were increased in thymic tissue of SIDS compared to controls without infections, and the most significant discrepancy was in infants younger than 20 weeks. The thymic cytokine profiles in SIDS cases were similar to those in controls with severe infection; however, the magnitude of the cytokine concentration elevation in SIDS was less pronounced, indicating sub-clinical infections in SIDS. In contrast to SIDS, intrathymic cytokine concentrations and thymus weight were increased with age in control children., Conclusions: Elevated thymic cytokine expression and thymus weight, as well as impaired age-related alterations in SIDS, may be influenced by subclinical infection, which may play a role in initiating SIDS in infants with a compromised immune response., Impact Statement: Increased thymic weight and cytokine concentration may suggest possible subclinical infection in SIDS. Elevated thymic weight and cytokine concentration mainly in SIDS cases aged <20 weeks. Age-related impairment in the thymic weight and cytokine expression may be impaired by subclinical infection in SIDS., (© 2023. The Author(s).)

Background: Macrophages have recently become attractive therapeutics in cancer immunotherapy. The potential of macrophages to infiltrate and influence solid malignancies makes them promising targets for the chimeric antigen receptor (CAR) technology to redirect their stage of polarization, thus enhancing their anticancer capacities. Given the emerging interest for CAR-macrophages, generation of such cells so far mainly depends on peripheral blood monocytes, which are isolated from the respective donor prior to genetic manipulation. This procedure is time-intensive and cost-intensive, while, in some cases, insufficient monocyte amounts can be recovered from the donor, thus hampering the broad applicability of this technology. Hence, we demonstrate the generation and effectiveness of CAR-macrophages from various stem cell sources using also modern upscaling technologies for next generation immune cell farming., Methods: Primary human hematopoietic stem and progenitor cells and induced pluripotent stem cells were used to derive anti-CD19 CAR-macrophages. Anticancer activity of the cells was demonstrated in co-culture systems, including primary material from patients with leukemia. Generation of CAR-macrophages was facilitated by bioreactor technologies and single-cell RNA (scRNA) sequencing was used to characterize in-depth response and behavior of CAR-macrophages., Results: Irrespective of the stem-cell source, CAR-macrophages exhibited enhanced and antigen-dependent phagocytosis of CD19 + target cancer cells with increased pro-inflammatory responses. Phagocytic capacity of CAR-macrophages was dependent on target cell CD19 expression levels with superior function of CAR-macrophages against CD19 + cancer cell lines and patient-derived acute lymphocytic leukemia cancer cells. scRNA sequencing revealed CAR-macrophages to be distinct from eGFP control cells after co-culture with target cells, which includes the activation of pro-inflammatory pathways and upregulation of chemokines and cytokines associated with adaptive immune cell recruitment, favoring the repolarization of CAR-macrophages to a pro-inflammatory state. Taken together, the data highlight the unique features of CAR-macrophages in combination with the successful upscaling of the production pipeline using a three-dimensional differentiation protocol and intermediate scale bioreactors., Conclusion: In summary, our work provides insights into the seminal use and behavior of CAR-macrophages which are derived from various sources of stem cells, while introducing a unique technology for CAR-macrophage manufacturing, all dedicated to the clinical translation of CAR-macrophages within the field of anticancer immunotherapies., Competing Interests: Competing interests: NL, MA and TM have filled a patent application on the generation of human iPSC-derived macrophages. All other authors declare no conflict of interest., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Following heart transplantation, a cascade of immunological responses is initiated influencing the clinical outcome and long-term survival of the transplanted patients. The anti-inflammatory cytokine interleukin-10 (IL-10) was shown to be elevated in the blood of heart transplant recipients directly after transplantation but the releasing cell populations and the composition of lymphocyte subsets following transplantation have not been thoroughly studied., Methods: We identified immune cells by immunophenotyping and analyzed intracellular IL-10 production in peripheral blood mononuclear cells (PBMC) of heart transplanted patients (n= 17) before, directly after and 24h post heart transplantation. The cells were stimulated with lipopolysaccharide or PMA/Ionomycin to enhance cytokine production within leukocytes in vitro ., Results and Discussion: We demonstrate that intermediate monocytes (CD14 high CD16 + ), but not CD8 + T cells, CD4 + T cells, CD56 + NK cells or CD20 + B cells appeared to be the major IL-10 producers within patients PBMC following heart transplantation. Consequently, the absolute monocyte count and the ratio of intermediate monocytes to classical monocytes (CD14 + CD16 - ) were specifically increased in comparison to pre transplant levels. Hence, this population of monocytes, which has not been in the focus of heart transplantation so far, may be an important modulator of clinical outcome and long-term survival of heart transplant recipients. Alteration of blood-circulating monocytes towards a CD14 high CD16 + phenotype could therefore shift the pro-inflammatory immune response towards induction of graft tolerance, and may pave the way for the optimization of immunosuppression., 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 © 2023 Ludwig, Chichelnitskiy, Kühne, Wiegmann, Iske, Ledwoch, Ius, Beushausen, Keil, Iordanidis, Rojas, Salman, Knoefel, Haverich, Warnecke and Falk.)

Background: Ex vivo lung perfusion (EVLP) uses continuous normothermic perfusion to reduce ischemic damage and to improve post-transplant outcomes, specifically for marginal donor lungs after the donation after circulatory death. Despite major efforts, the optimal perfusion protocol and the composition of the perfusate in clinical lung transplantation have not been identified. Our study aims to compare the concentration levels of cytokine/chemokine in different perfusion solutions during EVLP, after 1 and 9 h of cold static preservation (CSP) in a porcine cardiac arrest model, and to correlate inflammatory parameters to oxygenation capacities., Methods: Following cardiac arrest, the lungs were harvested and were categorized into two groups: immediate (I-EVLP) and delayed EVLP (D-EVLP), after 1 and 9 h of CSP, respectively. The D-EVLP lungs were perfused with either Steen or modified Custodiol-N solution containing only dextran (CD) or dextran and albumin (CDA). The cytokine/chemokine levels were analyzed at baseline (0 h) and after 1 and 4 h of EVLP using Luminex-based multiplex assays., Results: Within 4 h of EVLP, the concentration levels of TNF-α, IL-6, CXCL8, IFN-γ, IL-1α, and IL-1β increased significantly ( P  < 0.05) in all experimental groups. The CD solution contained lower concentration levels of TNF-α, IL-6, CXCL8, IFN-γ, IL-2, IL-12, IL-10, IL-4, IL-1RA, and IL-18 ( P  < 0.05) compared with those of the Steen solution. The concentration levels of all experimental groups have correlated negatively with the oxygenation capacity values ( P  < 0.05). Protein concentration levels did not reach statistical significance for I-EVLP vs. D-EVLP and CD vs. CDA solutions., Conclusion: In a porcine cardiac arrest model, a longer period of CSP prior to EVLP did not result in an enhanced protein secretion into perfusates. The CD solution reduced the cytokine/chemokine secretion most probably by iron chelators and/or by the protecting effects of dextran. Supplementing with albumin did not further reduce the cytokine/chemokine secretion into perfusates. These findings may help in optimizing the preservation procedure of the lungs, thereby increasing the donor pool of organs., 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. The handling editor CA declared a past co-authorship with the author CF., (© 2023 Radomsky, Koch, Olbertz, Liu, Beushausen, Keil, Rauen, Falk, Kühne and Kamler.)

Early kinetics of lymphocyte subsets involved in tolerance and rejection following heart transplantation (HTx) are barely defined. Here, we aimed to delineate the early alloimmune response immediately after HTx. Therefore, blood samples from 23 heart-transplanted patients were collected before (pre-), immediately (T0), 24 hours (T24), and 3 weeks (3 wks) after HTx. Immunophenotyping was performed using flow cytometry. A significant increase was detected for terminally differentiated (TEMRA) CD4 + or CD8 + T cells and CD56 dim CD16 + NK cells immediately after HTx linked to a decrease in naïve CD8 + and CM CD4 + T as well as CD56 bright CD16 - NK cells, returning to baseline levels at T24. More detailed analyses revealed increased CD69 + CD25 - and diminished CD69 - CD25 - CD4 + or CD8 + T-cell proportions at T0 associated with decreasing S1PR1 expression. Passenger T and NK cells were found at low frequencies only in several patients at T0 and did not correlate with lymphocyte alterations. Collectively, these results suggest an immediate, transient shift toward memory T and NK cells following HTx. Opposite migratory properties of naïve versus memory T and NK cells occurring in the early phase after HTx could underlie these observations and may impinge on the development of allo-specific immune responses., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1 + profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas., (© 2023. The Author(s).)

Introduction: Human cytomegalovirus (HCMV) causes significant morbidity and mortality in allogeneic stem cell transplant (alloSCT) recipients. Recently, antiviral letermovir prophylaxis during the first 100 days after alloSCT replaced PCR-guided preemptive therapy as the primary standard of care for HCMV reactivations. Here, we compared NK-cell and T-cell reconstitution in alloSCT recipients receiving preemptive therapy or letermovir prophylaxis in order to identify potential biomarkers predicting prolonged and symptomatic HCMV reactivation., Methods: To that end, the NK-cell and T-cell repertoire of alloSCT recipients managed with preemptive therapy (n=32) or letermovir prophylaxis (n=24) was characterized by flow cytometry on days +30, +60, +90 and +120 after alloSCT. Additionally, background-corrected HCMV-specific T-helper (CD4+IFNγ+) and cytotoxic (CD8+IFNγ+CD107a+) T cells were quantified after pp65 stimulation., Results: Compared to preemptive therapy, letermovir prophylaxis prevented HCMV reactivation and decreased HCMV peak viral loads until days +120 and +365. Letermovir prophylaxis resulted in decreased T-cell numbers but increased NK-cell numbers. Interestingly, despite the inhibition of HCMV, we found high numbers of "memory-like" (CD56dimFcεRIγ- and/or CD159c+) NK cells and an expansion of HCMV-specific CD4+ and CD8+ T cells in letermovir recipients. We further compared immunological readouts in patients on letermovir prophylaxis with non/short-term HCMV reactivation (NSTR) and prolonged/symptomatic HCMV reactivation (long-term HCMV reactivation, LTR). Median HCMV-specific CD4+ T-cell frequencies were significantly higher in NSTR patients (day +60, 0.35 % vs. 0.00 % CD4+IFNγ+/CD4+ cells, p=0.018) than in patients with LTR, whereas patients with LTR had significantly higher median regulatory T-cell (Treg) frequencies (day +90, 2.2 % vs. 6.2 % CD4+CD25+CD127dim/CD4+ cells, p=0.019). ROC analysis confirmed low HCMV specific CD4+ (AUC on day +60: 0.813, p=0.019) and high Treg frequencies (AUC on day +90: 0.847, p=0.021) as significant predictors of prolonged and symptomatic HCMV reactivation., Discussion: Taken together, letermovir prophylaxis delays HCMV reactivation and alters NK- and T-cell reconstitution. High numbers of HCMV-specific CD4+ T cells and low numbers of Tregs seem to be pivotal to suppress post-alloSCT HCMV reactivation during letermovir prophylaxis. Administration of more advanced immunoassays that include Treg signature cytokines might contribute to the identification of patients at high-risk for long-term and symptomatic HCMV reactivation who might benefit from prolonged administration of letermovir., 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 © 2023 Lauruschkat, Muchsin, Rein, Erhard, Grathwohl, Dölken, Köchel, Falk, Einsele, Wurster, Grigoleit and Kraus.)

Background: For sudden infant death syndrome (SIDS), an impaired immunocompetence has been discussed for a long time. Cytokines and chemokines are soluble immune mediators (SIM) whose balance is essential for the immune status. We hypothesized that an imbalanced immune response might contribute to the etiology of SIDS., Methods: We investigated 27 cytokines, chemokines, and growth factors in protein lysates of lungs derived from 29 SIDS cases and 15 control children deceased for other reasons., Results: Except for the CCL5, no significant differences were detected in the lungs between SIDS cases with and without mild upper respiratory tract infections. In contrast, IL-1RA, IL-7, IL-13, and G-CSF were decreased in the merged SIDS cases compared to control cases without evidence of infection. Plotting SIM concentrations against infant age resulted in increasing concentrations in control but not in SIDS lungs, indicating a disturbed immune maturation. Moreover, an age-dependent shift towards a Th2-related pattern was observed in SIDS., Conclusions: Our findings suggest that an impaired maturation of the immune system, an insufficient response to respiratory pathogens, and an immune response modulated by Th1/Th2 imbalance might play a possible role in triggering SIDS. These findings might in part be explained by chronic stress., Impact: Maturation of the cytokine and chemokine network may be impaired in SIDS. An imbalance between Th1- and Th2-related cytokines, which may reflect a state of chronic stress causing a more Th2 shift. An impaired immune maturation, an insufficient response to respiratory pathogens, and an immune response modulated by Th1/Th2 imbalance might play a possible role in SIDS., (© 2022. The Author(s).)

Introduction: SARS-CoV-2 vaccination is the leading strategy to prevent severe courses after SARS-CoV-2 infection. In our study, we analyzed humoral and cellular immune responses in detail to three consecutive homologous or heterologous SARS-CoV-2 vaccinations and breakthrough infections., Methods: Peripheral blood samples of n=20 individuals were analyzed in the time course of three SARS-CoV-2 vaccinations and/or breakthrough infection. S1-, RBD-, S2- and N-specific IgG antibodies were quantified using Luminex-based multiplex assays and electrochemiluminescence multiplex assays for surrogate neutralization in plasma. Changes in cellular immune components were determined via flow cytometry of whole blood samples., Results: All individuals (n=20) responded to vaccination with increasing S1-/RBD-/S2-specific IgG levels, whereas specific plasma IgA displayed individual variability. The third dose increased antibody inhibitory capacity (AIC) against immune-escape variants Beta and Omicron BA.1 independently of age. The mRNA-primed vaccination induced IgG and IgA immunity more efficiently, whereas vector-primed individuals displayed higher levels of memory T and B cells. Vaccinees showed SARS-CoV-2-specific T cell responses, which were further improved and specified after Omicron breakthrough infections in parallel to the appearance of new variant-specific antibodies., Discussion: In conclusion, the third vaccination was essential to increase IgG levels, mandatory to boost AIC against immune-escape variants, and induced SARS-CoV-2-specific T cells. Breakthrough infection with Omicron generates additional spike specificities covering all known variants., 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 © 2023 Ruhl, Kühne, Beushausen, Keil, Christoph, Sauer and Falk.)

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron (MN) disease characterized by progressive MN loss and muscular atrophy resulting in rapidly progressive paralysis and respiratory failure. Human mesenchymal stem/stromal cell (hMSC)-based therapy has been suggested to prolong MN survival via secretion of growth factors and modulation of cytokines/chemokines. We investigated the effects of hMSCs and a hMSC-conditioned medium (CM) on Cu/Zn superoxidase dismutase 1 G93A (SOD1 G93A ) transgenic primary MNs. We found that co-culture of hMSCs and MNs resulted in slightly higher MN numbers, but did not protect against staurosporine (STS)-induced toxicity, implying marginal direct trophic effects of hMSCs. Aiming to elucidate the crosstalk between hMSCs and MNs in vitro, we found high levels of vascular endothelial growth factor (VEGF) and C-X3-C motif chemokine 1 (CX3CL1) in the hMSC secretome. Co-culture of hMSCs and MNs resulted in altered gene expression of growth factors and cytokines/chemokines in both MNs and hMSCs. hMSCs showed upregulation of CX3CL1 and its receptor CX3CR1 and downregulation of interleukin-1 β (IL1β) and interleukin-8 (IL8) when co-cultured with SOD1 G93A MNs. MNs, on the other hand, showed upregulation of growth factors as well as CX3CR1 upon hMSC co-culture. Our results indicate that hMSCs only provide moderate trophic support to MNs by growth factor gene regulation and may mediate anti-inflammatory responses through the CX3CL1/CX3CR1 axis, but also increase expression of pro-inflammatory cytokines, which limits their therapeutic potential.

Mortality due to COVID-19 is not increased in immunosuppressed individuals after liver transplantation (OLT) compared to individuals without immunosuppression. Data on long-term protective immunity against SARS-CoV-2 in immunosuppressed convalescents, is limited. We prospectively measured immune responses against SARS-CoV-2 by quantifying antibodies against 4 different antigens (spike protein 1 and 2, receptor binding domain, nucleocapsid) and T cell responses by IFN-γ ELISPOT against 4 antigens (membrane, nucleocapsid, spike protein 1 and 2) in 24 OLT convalescents with immunosuppressive therapy longitudinally in the first year after COVID-19 including a booster vaccination in comparison to a matched cohort of non-immunosuppressed convalescents (non-IS-Con). Pre-pandemic OLT samples were retrieved from our prospective OLT biorepository (n = 16). No relevant T cell reactivity or immunoglobulin G (IgG) against SARS-CoV-2 were detectable in pre-pandemic samples of OLT recipients despite reactivity against endemic corona-viruses. OLT convalescents had a lower prevalence of IgG against nucleocapsid (54% vs. 90%) but not against spike protein domains (98-100% vs. 100%) after vaccination in the second half-year after COVID-19 compared to non-IS-Con. Also, concentrations of anti-nucleocapsid IgG were lower in OLT convalescents than in non-IS-Con. Concentration of IgG against spike protein domains was significantly increased by a booster vaccination in OLT convalescents. But concentration of IgG against two of three spike protein domains remains slightly lower compared to non-IS-Con finally. However, none of these differences was mirrored by the cellular immunity against SARS-CoV-2 that remained stable during the first year after COVID-19 and was not further stimulated by a corona vaccination in OLT convalescents. In conclusion, despite lower concentrations of anti-SARS-CoV-2 IgG in OLT convalescents anti-SARS-CoV-2 cellular immunity was as robust as in non-IS-Con., Competing Interests: The authors have declared that no competing interests exist.

In the era of organ machine perfusion, experimental models to optimize reconditioning of (marginal) liver grafts are needed. Although the relevance of cytokine signatures in liver transplantation has been analyzed previously, the significance of molecular monitoring during normothermic machine perfusion (NMP) remains elusive. Therefore, we developed a porcine model of cold ischemic liver graft injury after prolonged static cold storage (SCS) and subsequent NMP: Livers obtained from ten minipigs underwent NMP for 6 h directly after procurement (control group) or after 20 h of SCS. Grafts after prolonged SCS showed significantly elevated AST, ALT, GLDH and GGT perfusate concentrations, and reduced lactate clearance. Bile analyses revealed reduced bile production, reduced bicarbonate and elevated glucose concentrations after prolonged SCS. Cytokine analyses of graft perfusate simultaneously demonstrated an increase of pro-inflammatory cytokines such as Interleukin-1α, Interleukin-2, and particularly Interleukin-18. The latter was the only significantly elevated cytokine compared to controls, peaking as early as 2 h after reperfusion (11,012 ng/ml vs. 1,493 ng/ml; p = 0.029). Also, concentrations of High-Mobility-Group-Protein B1 were significantly elevated after 2 h of reperfusion (706.00 ng/ml vs. 148.20 ng/ml; p < 0.001) and showed positive correlations with AST ( r 2 = 0.846) and GLDH ( r 2 = 0.918) levels. Molecular analyses during reconditioning of liver grafts provide insights into the degree of inflammation and cell damage and could thereby facilitate future interventions during NMP reducing acute and chronic graft injury., 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 Beetz, Cammann, Weigle, Sieg, Eismann, Johanning, Falk, Krech, Oldhafer and Vondran.)

During the COVID-19 pandemic, vaccination is the most important countermeasure. Pharmacovigilance concerns however emerged with very rare, but potentially disastrous thrombotic complications following vaccination with ChAdOx1. Platelet factor-4 antibody mediated vaccine-induced immune thrombotic thrombocytopenia (VITT) was described as an underlying mechanism of these thrombotic events. Recent work moreover suggests that mechanisms of immunothrombosis including neutrophil extracellular trap (NET) formation might be critical for thrombogenesis during VITT. In this study, we investigated blood and thrombus specimens of a female patient who suffered severe stroke due to VITT after vaccination with ChAdOx1 in comparison to 13 control stroke patients with similar clinical characteristics. We analyzed cerebral thrombi using histological examination, staining of complement factors, NET-markers, DNase and LL-37. In blood samples at the hyper-acute phase of stroke and 7 days later, we determined cell-free DNA, myeloperoxidase-histone complexes, DNase activity, myeloperoxidase activity, LL-37 and inflammatory cytokines. NET markers were identified in thrombi of all patients. Interestingly, the thrombus of the VITT-patient exclusively revealed complement factors and high amounts of DNase and LL-37. High DNase activity was also measured in blood, implying a disturbed NET-regulation. Furthermore, serum of the VITT-patient inhibited reactive oxygen species-dependent NET-release by phorbol-myristate-acetate to a lesser degree compared to controls, indicating either less efficient NET-inhibition or enhanced NET-induction in the blood of the VITT-patient. Additionally, the changes in specific cytokines over time were emphasized in the VITT-patient as well. In conclusion, insufficient resolution of NETs, e.g. by endogenous DNases or protection of NETs against degradation by embedded factors like the antimicrobial peptide LL-37 might thus be an important factor in the pathology of VITT besides increased NET-formation. On the basis of these findings, we discuss the potential implications of the mechanisms of disturbed NETs-degradation for diagnostic and therapeutic approaches in VITT-related thrombogenesis, other auto-immune disorders and beyond., 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 de Buhr, Baumann, Werlein, Fingerhut, Imker, Meurer, Götz, Bronzlik, Kühnel, Jonigk, Ernst, Leotescu, Gabriel, Worthmann, Lichtinghagen, Tiede, von Köckritz-Blickwede, Falk, Weissenborn, Schuppner and Grosse.)

Human cytomegalovirus (HCMV) reactivation remains a relevant complication after hematopoietic stem cell transplantation (HSCT) despite the great progress made in prophylaxis and treatment. Adaptive Natural Killer (NK) cells undergo a persistent reconfiguration in response to HCMV reactivation however, the exact role of adaptive NK cells in HCMV surveillance is currently unknown. We studied the relationship between HCMV reactivation and adaptive NK cells in 70 patients monitored weekly until day +100 after HSCT. Absolute cell counts of adaptive NK cells increased significantly after resolution of HCMV-reactivation compared to patients without reactivation. Patients with HCMV-reactivation had an early reconstitution of adaptive NK cells ("Responders") and had mainly a single reactivation (75% Responders vs 48% Non-Responders). Adaptive NK cells eliminated HCMV-infected human foreskin fibroblasts (HFF) in vitro and recruited T cells in an in vitro transwell migration assay. An extensive cytokine/chemokine panel demonstrated strongly increased secretion of CXCL10/IP-10, IFN-α, IL-1α, IL-1β, IL-5, IL-7 and CCL4. Thus, adaptive NK cells may control viral spread and T cell expansion and survival during HCMV-reactivation. Taken together, we have demonstrated the potential of adaptive NK cells in the control of HCMV reactivation both by direct cytotoxicity and by recruitment of other immune cells., (© 2022. The Author(s).)

Background: Immunocompromised people (ICP) and elderly individuals (older than 80 years) are at increased risk for severe coronavirus infections. To protect against serious infection with SARS-CoV-2, ICP are taking precautions that may include a reduction of social contacts and participation in activities which they normally enjoy. Furthermore, for these people, there is an uncertainty regarding the effectiveness of the vaccination. The COVID-19 Contact (CoCo) Immune study strives to characterize the immune response to COVID-19 vaccination in immunocompromised, elderly people, and patients with hematological or oncological diseases. The study uses blood-based screenings to monitor the humoral and cellular immune response in these groups after vaccination. Questionnaires and qualitative interviews are used to describe the level of social participation., Methods: The CoCo Immune Study is a mixed methods prospective, longitudinal, observational study at two large university hospitals in Northern Germany. Starting in March 2021, it monitors anti-SARS-CoV-2 immune responses and collects information on social participation in more than 600 participants, at least 18 years old. Inclusion criteria and subcohorts: Participants with (1) regularly intake of immunosuppressive medication (ICP-cohort) or (2) age ≥ 80 years (80 + -cohort). Additionally, patients with current or former (3) myeloid, (4) lymphatic disease or (5) solid tumor under checkpoint inhibition (3-5: HO-cohort)., Exclusion Criteria: (1) refusal to give informed consent, (2) contraindication to blood testing, (3) inability to declare consent. Participants complete a questionnaire at four different time points: prior to full vaccination, and 1, 6 and 12 months after completed vaccination. In addition, participants draw blood samples themselves or through a local health care provider and send them with their questionnaires per post at the respective time points after vaccination. Patients of the HO cohort dispense additional blood samples at week 3 to 12 and at month 6 to 9 after 2nd vaccination to gain additional knowledge in B and T cell responses. Selected participants are invited to qualitative interviews about social participation., Discussion: This observational study is designed to gain insight into the immune response of people with weakened immune systems and to find out how social participation is affected after COVID-19 vaccination., Trial Registration: This study was registered with German Clinical Trial Registry (registration number: DRKS00023972) on 30th December 2020., (© 2022. The Author(s).)

Background: Cell-free DNA (cfDNA) and endogenous deoxyribonuclease activity are opposing mediators and might influence the inflammatory response following acute ischemic stroke. In this cohort study, we investigated the relation between these markers, circulating inflammatory mediators and clinical course including occurrence of stroke-associated infections (SAI) in patients with acute stroke., Methods: Ninety-two patients with stroke due to large vessel occlusion undergoing mechanical thrombectomy were prospectively recruited at Hannover Medical School from March 2018 to August 2019. Deoxyribonuclease activity, cfDNA, damage-associated molecular patterns, and circulating cytokines were measured in venous blood collected immediately before mechanical thrombectomy and 7 days later. Reperfusion status was categorized (sufficient/insufficient). Clinical outcome was evaluated using the modified Rankin Scale after 90 days, where a score of 3 to 6 was considered unfavorable. To validate findings regarding SAI, another stroke cohort (n=92) was considered with blood taken within 24 hours after stroke onset., Results: Patients with unfavorable clinical outcome had higher cfDNA concentrations. After adjustment for confounders (Essen Stroke Risk Score, National Institutes of Health Stroke Scale, and sex), 7-day cfDNA was independently associated with clinical outcome and especially mortality (adjusted odds ratio: 3.485 [95% CI, 1.001-12.134] and adjusted odds ratio: 9.585 [95% CI, 2.006-45.790]). No association was found between reperfusion status and cfDNA or deoxyribonuclease activity. While cfDNA concentrations correlated positively, deoxyribonuclease activity inversely correlated with distinct biomarkers. Baseline deoxyribonuclease activity was lower in patients who developed SAI compared with patients without SAI. This association was confirmed after adjustment for confounding factors (adjusted odds ratio: 0.447 [95% CI, 0.237-0.844]). In cohort 2, differences of deoxyribonuclease activity between patients with and without SAI tended to be higher with higher stroke severity., Conclusions: The interplay of endogenous deoxyribonuclease activity and cfDNA in acute stroke entails interesting novel diagnostic and potential therapeutic approaches. We confirm an independent association of cfDNA with a detrimental clinical course after stroke due to large vessel occlusion. This study provides first evidence for lower endogenous deoxyribonuclease activity as risk factor for SAI after severe stroke.

Mechanical thrombectomy (MT) is a highly efficient treatment in patients with acute ischemic stroke due to large vessel occlusion (LVO). However, in a relevant proportion of LVO, no sufficient recanalization can be achieved. The composition of cerebral thrombi is highly heterogeneous and may constitute a relevant factor for insufficient reperfusion. We hypothesized that circulating cytokines and growth factors involved in thromboinflammation and platelet activation may be associated with reperfusion status and thrombus composition in patients undergoing MT. An according biomarker panel was measured in plasma specimens taken prior to MT and at a 7-day follow-up. The reperfusion status was categorized into sufficient or insufficient. The composition of retrieved thrombi was histologically analyzed. Differences of baseline biomarker concentrations between insufficient and sufficient reperfusions were highest for interferon (IFN)-γ, epidermal growth factor, platelet-derived growth factor (PDGF)-AB/BB, and IFN-γ-induced protein 10 (IP-10/CXCL10). After applying correction for multiple comparisons and logistic regression analysis adjusting for stroke etiology, intravenous thrombolysis, and vascular risk factors, PDGF-AB/BB was identified as an independent predictor of reperfusion status (odds ratio: 0.403; 95% confidence interval: 0.199-0.819). Histological analysis revealed that the majority of thrombi had a mixed composition. In conclusion, this study provides the first evidence that cytokines and growth factors are potential effectors in patients undergoing MT for the treatment of acute ischemic stroke., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Chimeric antigen receptor (CAR) T-cell therapies have shown impressive results in patients with hematological malignancies; however, little success has been achieved in the treatment of solid tumors. Recently, macrophages (MΦs) were identified as an additional candidate for the CAR approach, and initial proof of concept studies using peripheral blood-derived monocytes showed antigen-redirected activation of CAR MΦs. However, some patients may not be suitable for monocyte-apheresis, and prior cancer treatment regimens may negatively affect immune cell number and functionality. To address this problem, we here introduce primary human hematopoietic stem and progenitor cells (HSPCs) as a cell source to generate functional CAR MΦs ex vivo. Our data showed successful CAR expression in cord blood (CB)-derived HSPCs, with considerable cell expansion during differentiation to CAR MΦs. HSPC-derived MΦs showed typical MΦ morphology, phenotype, and basic anti-bacterial functionality. CAR MΦs targeting the carcinoembryonic antigen (CEA) and containing either a DAP12- or a CD3ζ-derived signaling domain showed antigen redirected activation as they secreted pro-inflammatory cytokines specifically upon contact with CEA + target cells. In addition, CD3ζ-expressing CAR MΦs exhibited significantly enhanced phagocytosis of CEA + HT1080 cells. Our data establish human HSPCs as a suitable cell source to generate functional CAR MΦs and further support the use of CAR MΦs in the context of solid tumor therapy.

Nonalcoholic steatohepatitis (NASH) is induced by steatosis and metabolic inflammation. While involvement of the innate immune response has been shown, the role of the adaptive immune response in NASH remains controversial. Likewise, the role of regulatory T cells (Treg) in NASH remains unclear although initial clinical trials aim to target these regulatory responses. High-fat high-carbohydrate (HF-HC) diet feeding of NASH-resistant BALB/c mice as well as the corresponding recombination activating 1 (Rag)-deficient strain was used to induce NASH and to study the role of the adaptive immune response. HF-HC diet feeding induced strong activation of intrahepatic T cells in BALB/c mice, suggesting an antigen-driven effect. In contrast, the effects of the absence of the adaptive immune response was notable. NASH in BALB/c Rag1 -/- mice was substantially worsened and accompanied by a sharp increase of M1-like macrophage numbers. Furthermore, we found an increase in intrahepatic Treg numbers in NASH, but either adoptive Treg transfer or anti-cluster of differentiation (CD)3 therapy unexpectedly increased steatosis and the alanine aminotransferase level without otherwise affecting NASH. Conclusion: Although intrahepatic T cells were activated and marginally clonally expanded in NASH, these effects were counterbalanced by increased Treg numbers. The ablation of adaptive immunity in murine NASH led to marked aggravation of NASH, suggesting that Tregs are not regulators of metabolic inflammation but rather enhance it., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Cytokine profiles are often perturbed after infections of medical implants. With a non-invasive in vivo imaging system, we report in a mouse model that interferon expression after infection of subcutaneous implants with Streptococcus oralis , Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis , and Treponema denticola (alone or as a combination) was species-specific, persisted longer in the presence of implants, and notably decreased upon dual species infections. This type I interferon expression disappeared within two weeks; however, histology of implant-tissue interface indicated high recruitment of immune cells even after three weeks. This was suggestive that biomaterial-associated infections could have prolonged effects, including the systemic stimulation of inflammatory cytokines. The present study investigated the systemic impact of this chronic peri-implant inflammation on the systemic expression of inflammatory cytokines (23) using a multiplex assay. Initially, the cytokine measurement in murine fibroblasts exposed to periodontal pathogens remained limited to the expression of five cytokines, namely, IL-6, G-CSF, CXCL-1/KC, MCP-1 (MCAF), and IL-12 (p40). The systemic determination of cytokines in mice increased to 19 cytokines (IL-1α, IL-2, IL-3, IL-5, IL-6, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-17A, CCL-11/Eotaxin, G-CSF, IFN-γ, CXCL1/KC, MCP-1 (MCAF), MIP-1α/CCL3, MIP-1β/CCL4, CCL5/RANTES, and TNF-α). Systemic induction of cytokines was species-specific in the mouse model. The cytokine induction from infected implants differed significantly from sole tissue infections and sterile implants. Notably, systemic cytokine induction decreased after infections with dual species compared to single species infections. These findings describe the systemic effect of chronic peri-implant inflammation on the systemic induction of inflammatory cytokines, and this effect was strongly correlated to the type and composition of initial infection. Systemic modulations in cytokine expression upon dual species infections exhibit an exciting pattern that might explain the complications associated with biomaterial-related infection in patients. Moreover, these findings validate the requirement of multispecies infections for pre-clinical studies involving animal models.

Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies.